Studies on the taxonomy of honeybees
in the Sudan
By
Elhadi Adam Omer
BSc. (Science) University of El Azhar, Cairo, Egypt.
MSc. (Agric.) University of Khartoum.
A thesis submitted in fulfillment of the requirements for the degree of
Doctor of philosophy
Supervisor
Professor Mohamed Abd El Halim Siddig
Co supervisor
Professor Mohamed Saeed Ali El Sarrag
Department of Crop Protection
Faculty of Agriculture
University of Khartoum.
September 2007
I
ﻦ اﻟ ﱠﺮﺣِﻴﻢ
ﺣ َﻤ ِ
ﺴ ِﻢ اﻟّﻠ ِﻪ اﻟ ﱠﺮ ْ
ِﺑ ْ
ﻲ ٍء ُﺛﻢﱠ ِإﻟَﻰ َرﱢﺑ ِﻬ ْﻢ
ﺷ ْ
ب ﻣِﻦ َ
ﻃﻨَﺎ ﻓِﻲ اﻟ ِﻜﺘَﺎ ِ
ﻻ ُأ َﻣ ٌﻢ َأ ْﻣﺜَﺎُﻟﻜُﻢ ﻣﱠﺎ َﻓ ﱠﺮ ْ
ﺣ ْﻴ ِﻪ ِإ ﱠ
ﺠﻨَﺎ َ
ﻻ ﻃَﺎ ِﺋ ٍﺮ َﻳﻄِﻴ ُﺮ ِﺑ َ
ض َو َ
ﻷ ْر ِ
}َ {37وﻣَﺎ ﻣِﻦ دَﺁ ﱠﺑ ٍﺔ ﻓِﻲ ا َ
ن }{38
ﺸﺮُو َ
ﺤَ
ُﻳ ْ
ﺳﻮرة اﻷﻧﻌﺎم.
II
DEDICATION
I dedicate this work to those whom I love and admire particularly, to the
unassuming person, who lightened and still light many dark corners in this
life, to my mother and father for their relentless efforts towards my
education in spite of their very meager resources. To my wife to whom I am
greatly indebted for her patience towards my kids while, I was outside
country during the practical course of the study, I dedicate this work to her
with my cordial appreciation. To my kids, brothers and sisters with great
gratitude and love.
III
ACKNOWLEDGEMENT
My sincere gratitude is devoted to my supervisor professor Mohamed
Abd Elhalim Siddig for his professional guidance and sincere interest in this
study. Thanks are extended to my co-supervisor professor Mohamed S. A.
Elssarag for his advices and grateful helps.
I am greatly indebted to Dr. S. Fuchs for his deep interest, keen
supervision, patience and continuous advices and encouragement throughout
the practical and analysis part of the morphometric section of this study. My
gratitude’s are equally due to professor N. Koeniger (director) for availing
me every facilitates in the institute [Institute fuer Bienenkunde
(Polytechnische Gesellschaft) Fachbereich Biologie der J. W. GoetheUniversitaet Frankfurt am Main Karl-von-Frisch-Weg 2, D-61440
Oberursel, Germany, where I finished the biometric analysis]. Thus my deep
thanks are converted to all the staff members of the institute.
I feel pleased to express my deep thanks and sincere gratitude to
professor Dr. Moritz and professor Hans (Institut für Zoologie MartinLuther-Universitut Halle-Wittenberg Hoher Weg 4, D 06099 Halle/Saale,
Germany.) for their grateful supervision and criticism during the molecular
genetic practical part of this study at their institute.
My thanks and wishes are offered to Dr. Marina Meixner (Research
Associate Department of Entomology Washington State University Pullman,
WA 99164-6382. USA.) for her considerable assistance and advices during
the molecular genetic analysis part of this work.
At last and not least I would like to express my thanks to my colleges in
the Faculty of Natural Resources and Environmental Studies, University of
Juba and the department of crop protection, Faculty of Agriculture,
University of Khartoum for sparing good atmosphere to complete this study.
IV
ABSTRACT
A thorough morphometrical and some molecular genetic studies
(Mitochondrial DNA) were carried out on the most common honeybees in
the Sudan. These so far contribute in the identification of the Sudanese
honeybees.
Nineteen samples of honeybee workers Apis mellifera L. were collected
from four different geographical zones of the Sudan. Four samples of the
small Asian bee workers Apis florea obtained from Gerry, Khartoum,
Madani and El-Dender were also included in the study.
Biometric measurements and analysis were performed for all the
samples. The 19 colonies were subjected to morphometric measurements
plus another 8 different samples of Apis mellifera L. were further subjected
to Mitochondrial DNA investigation and analysis. Results were compared
with those of the biometric study.
The morphometric statistical analysis of the nineteen samples revealed a
wide range of differences in most discriminant characters among the
samples. In the principal component analysis (PCA), three clusters were
graphically formed. Furthermore, the presence of these three clusters was
confirmed by some modern discriminant analysis methods, and they were
geographically correlated.
The cluster with the smallest measurements of some discriminant
characters originated from the forest zone. Its average measurements were as
follows: forewing length 8.23 mm., width 2.82 mm.; proboscis length 5.55
mm.; hind-leg length 6.83 mm.; body size (T3+ T4) 3.88 mm., and cubital
index 1.85 mm.
The second cluster with medium measurements of some discriminant
characters, originated from the semi-desert zone. Its mean average
measurements were as follows: forewing length 8.27 mm., width 2.88 mm.;
V
proboscis length 5.63 mm.; hind-leg length 7.00 mm.; body size (T3+ T4)
3.88 mm.; and cubital index 2.04 mm.
The third cluster, with the highest measurements of some discriminant
characters, originated from the savannah zone; mainly towords the border
with Ethiopia. Its average measurements were as follows: forewing length
8.45 mm., width 2.95 mm.; proboscis length 5.59 mm.; hind-leg 7.05 mm.;
body size (T3+ T4) 4.00 mm., and comparatively the highest cubital index
of 2.24 mm.
Comparison between the 19 Sudanese honeybees samples and 242
banck samples (data banck, Institute für Bienenkunde, Oberursel, Germany
from a neighbouring countries) was done using PCA. The three clusters of
the Sudanese bees were like-wise distinguishable as subclusters. The same
results were also confirmed by the discriminant analysis.
Therefore, the smallest bees of Sudan were identified as Apis mellifera
sudanesis instead of Apis mellifera yemenitica which represent the bees of
the forest zone. The medium sized bees were identified as Apis mellifera
yemenitica instead of sudanesis., representing the semi-desert zone bees,
while the bigest bees retained the name Apis mellifera
bandasii.,
representing the Savannah zone bees.
The measurement of genetic variation in the Sudanese honeybees Apis
mellifera L., at the mitochondrial DNA level of the 27 samples revealed the
present of sex different haplotypes. The cluster with the smallest
measurements (forest zone colonies) had only haplotype A1 representing
100% of the whole measured colonies; the medium cluster (semi-desert zone
colonies) posses two different haplotypes O1 and Y2 with percentages 75%
and 25% respectively from the whole measured colonies of the zone, while
the cluster of highest measurements (savannah zone) showed four different
haplotypes, O1, O1`, A2 and A4, representing 54%, 13%, 13% and 20%
VI
respectively. These results partially confirmed the biometric measurements
of the PCA and discriminant analysis. The current study represent the first
record on the classification of the Sudanese honeybees according to
mitochondrial DNA variability.
The present study suggest that, the presence of the gene flow among the
Sudanese bees in the southern part of the semi-desert zone and almost all the
savannah zone of the Sudan is a result of heterogeneous blood mixture
between the Sudanese bees and the Ethiopian bees in the border between the
two countries and the gene flow direction might be from the low land of the
savannah zone of Ethiopia towards the western part of the Sudan in the area
between latitudes 9º N and 15º N. Also this study suggests that the origin
haplotype of the Sudanese bees is A1 and the pure Sudanese bees might be
the south Sudan race (A. m. sudanesis).
The four Apis florea samples were also treated by PCA and discriminant
analysis, the results obtained so far revealed that, colonies are not very
distinct indicating that, all of these colonies were similar and originally they
were descendent of the first recorded colony of Apis florea in Khartoum in
1985.
Treatment of the four Sudanese Florea samples together with 6 Florea
colonies of different origins [2 from Sudan “Moggas ones” and 4 from the
data bank, Institute fur Bienenkunde-Oberursel-Germany (Mogga 1988)], by
cluster column analysis (which compare values across categories); revealed
that, the four target Florea samples of Sudan might be brought from
Pakistan or South Iran.
VII
ﻣﻠﺨﺺ اﻷﻃﺮوﺣﺔ
ﺃﺠﺭﻴﺕ ﺩﺭﺍﺴﺔ ﺸﺎﻤﻠﺔ ﻓﻲ ﺍﻟﻘﻴﺎﺴـﺎﺕ ﺍﻟﺒﻴﻭﻟﻭﺠﻴـﺔ ) (Biomorphometricsﻭﺍﻟﻭﺭﺍﺜـﺔ
ﺍﻟﺠﺯﻴﺌﻴﺔ ) (Molecular Biologyﻷﻜﺜﺭ ﺃﻨﻭﺍﻉ ﻨﺤل ﺍﻟﻌﺴل ﺇﻨﺘﺸﺎﺭﹰﺍ ﻓﻰ ﺍﻟﺴﻭﺩﺍﻥ .ﻤﻤﺎ ﻴـﺴﺎﻋﺩ
ﻓﻲ ﺘﻌﺭﻴﻑ ﻨﺤل ﺍﻟﻌﺴل ﺍﻟﺴﻭﺩﺍﻨﻲ.
ﺠﻤﻌﺕ ﺘﺴﻌﺔ ﻋﺸﺭ ﻋﻴﻨﺔ ﻤﻥ ﻨﺤل ﺍﻟﻌﺴل ﺍﻟﺴﻭﺩﺍﻨﻲ Apis mellifera lﻤﻥ ﺃﺭﺒﻊ ﻤﻨـﺎﻁﻕ
ﺠﻐﺭﺍﻓﻴﺔ ﻤﺨﺘﻠﻔﺔ ﻓﻲ ﺍﻟﺴﻭﺩﺍﻥ .ﻜﻤﺎ ﺍﺸﺘﻤﻠﺕ ﺃﻴﻀﺎ ﺍﻟﺩﺭﺍﺴﺔ ﺃﺭﺒﻊ ﻋﻴﻨـﺎﺕ ﻤـﻥ ﺍﻟﻨﺤـل ﺍﻵﺴـﻴﻭﻱ
ل ﻤﻥ ﻗـﺭﻯ ،ﺍﻟﺨﺭﻁـﻭﻡ ،ﻤـﺩﻨﻲ ﻭ
ﺍﻟﺼﻐﻴﺭ Apis floreaﺍﻟﻤﻭﺠﻭﺩﺓ ﻓﻲ ﺍﻟﺴﻭﺩﺍﻥ ﺠﻤﻌﺕ ﻤﻥ ﻜ ٍ
ﺍﻟﺩﻨﺩﺭ .ﻜل ﻫﺫﻩ ﺍﻟﻌﻴﻨﺎﺕ ﺃﺨﻀﻌﺕ ﻟﻠﻘﻴﺎﺴﺎﺕ ﺍﻟﺒﻴﻭﻟﻭﺠﻴﺔ ﻜﻤﺎ ﺤﻠﻠﺕ ﻨﺘﺎﺌﺠﻬﺎ.
ﺴﺒﻌﺔ ﻭﻋﺸﺭﻭﻥ ﻋﻴﻨﺔ ﻤﻥ ﻨﺤل ﺍﻟﻌﺴل ﺍﻟﺴﻭﺩﺍﻨﻲ ﺃﺠﺭﻴﺕ ﻟﻬﺎ ﺩﺭﺍﺴﺎﺕ ﻓﻲ ﺍﻟﻭﺭﺍﺜﺔ ﺍﻟﺠﺯﻴﺌﻴـﺔ
) Mitochondrial DNAﺘﺴﻌﺔ ﻋﺸﺭ ﻋﻴﻨﺔ ﻤﻥ ﺍﻟﻤﻘﺎﺴﺔ ﺒﻴﻭﻟﻭﺠﻴﺎ ﺒﺎﻹﻀﺎﻓﺔ ﺁﻟﻲ ﺜﻤـﺎﻨﻲ ﻋﻴﻨـﺎﺕ
ﺃﺨﺭﻯ ( .ﻫﺫﺍ ﻭﻗﺩ ﻗﻭﺭﻨﺕ ﻨﺘﺎﺌﺞ ﺩﺭﺍﺴﺔ ﺍﻟﻭﺭﺍﺜﺔ ﺍﻟﺠﺯﻴﺌﻴﺔ ﺒﻨﺘﺎﺌﺞ ﺍﻟﻘﻴﺎﺴﺎﺕ ﺍﻟﺒﻴﻭﻟﻭﺠﻴﺔ.
ﺃﻅﻬﺭﺕ ﺍﻟﺘﺤﺎﻟﻴل ﺍﻹﺤﺼﺎﺌﻴﺔ ﻟﻠﺘﺴﻌﺔ ﻋﺸﺭ ﻋﻴﻨﺔ ﺍﺨﺘﻼﻓﹰﺎ ﻭﺍﻀﺤﹰﺎ ﻓﻲ ﻤﻌﻅﻡ ﺍﻟﺼﻔﺎﺕ ﺍﻟﻤﻤﻴﺯﺓ
ﺒﻴﻥ ﺍﻟﻌﻴﻨﺎﺕ .ﻭ ﺒﺘﺤﻠﻴل ﺍﻟﻤﻜﻭﻨﺎﺕ ﺍﻷﺴﺎﺴﻴﺔ ) (PCAﺘﻜﻭﻨﺕ ﺒﻴﺎﻨﺎﺕ ﺜﻼﺜﺔ ﺘﺠﻤﻌﺎﺕ ﻤﺘﻨﺎﺴﺒﺔ ﺠﻐﺭﺍﻓﻴﺎ.
ﻭﻜﺫﻟﻙ ﺒﺘﺤﻠﻴل ﻨﺘﻴﺠﺔ ﺍﻟﻤﻜﻭﻨﺎﺕ ﺍﻷﺴﺎﺴﻴﺔ ﺒﻭﺍﺴـﻁﺔ ﺍﻟﺘﺤﻠﻴـل ﺍﻟﻤﻤﻴـﺯ ﺍﻟﺤـﺩﻴﺙ Discriminant
analysisﺘﻡ ﺍﻟﺘﺄﻜﺩ ﻤﻥ ﻭﺠﻭﺩ ﺘﻠﻙ ﺍﻟﺘﺠﻤﻌﺎﺕ ﺍﻟﺜﻼﺜﺔ ﻭﻫﻰ ﻜﺎﻻﺘﻰ :
ﺃ -ﺍﻟﻤﺠﻤﻭﻋﺔ ﺫﺍﺕ ﺍﻟﻘﻴﺎﺴﺎﺕ ﺍﻟﺼﻐﺭﻯ ﻟﺒﻌﺽ ﺍﻟﺼﻔﺎﺕ ﺍﻟﻤﻤﻴﺯﺓ ﻭﻗﺩ ﻨﺸﺄﺕ ﻫـﺫﻩ ﺍﻟﻤﺠﻤﻭﻋـﺔ ﻤـﻥ
ﻤﻨﺎﻁﻕ ﺍﻟﻐﺎﺒﺎﺕ ﻭﻜﺎﻥ ﻤﺘﻭﺴﻁ ﻗﻴﺎﺴﺎﺘﻬﺎ ﻜﺂﻻﺘﻲ -:
)ﺃ( ﻁﻭل ﺍﻟﺠﻨﺎﺡ ﺍﻷﻤﺎﻤﻲ
8.23ﻤﻠﻡ
)ﺏ( ﻋﺭﺽ ﺍﻟﺠﻨﺎﺡ ﺍﻷﻤﺎﻤﻲ
2.82ﻤﻠﻡ
)ﺩ( ﻁﻭل ﺍﻟﺭﺠل ﺍﻟﺨﻠﻔﻴﺔ
6.83ﻤﻠﻡ
)ﻩ( ﻁﻭل ﺍﻟﺼﻔﺎﺌﺢ ﺍﻟﻅﻬﺭﻴﺔ ﺍﻟﺜﺎﻟﺜﺔ ﻭﺍﻟﺭﺍﺒﻌﺔ )ﺤﺠﻡ ﺠﺴﻡ(
3.88ﻤﻠﻡ
)ﺝ( ﻁﻭل ﺍﻟﺨﺭﻁﻭﻡ
5.55ﻤﻠﻡ
)ﻯ( ﻤﻌﺎﻤل ﺍﻟﺠﻨﺎﺡ ﺍﻷﻤﺎﻤﻲ )(Cubital index
1.85ﻤﻠﻡ
ﺏ -ﺍﻟﻤﺠﻤﻭﻋﺔ ﺫﺍﺕ ﺍﻟﻘﻴﺎﺴﺎﺕ ﺍﻟﻤﺘﻭﺴﻁﺔ ﻟﺒﻌﺽ ﺍﻟﺼﻔﺎﺕ ﺍﻟﻤﻤﻴﺯﺓ ﻭﻗﺩ ﻨﺸﺄﺕ ﻫﺫﻩ ﺍﻟﻤﺠﻤﻭﻋـﺔ ﻤـﻥ
ﻤﻨﻁﻘﺔ ﺸﺒﻪ ﺍﻟﺼﺤﺭﺍﺀ .ﻭﻜﺎﻥ ﻤﺘﻭﺴﻁ ﻗﻴﺎﺴﺎﺘﻬﺎ ﻜﺂﻻﺘﻲ -:
)ﺃ( ﻁﻭل ﺍﻟﺠﻨﺎﺡ ﺍﻷﻤﺎﻤﻲ
8.27ﻤﻠﻡ
)ﺏ( ﻋﺭﺽ ﺍﻟﺠﻨﺎﺡ ﺍﻷﻤﺎﻤﻲ
2.88ﻤﻠﻡ
)ﺝ( ﻁﻭل ﺍﻟﺨﺭﻁﻭﻡ
5.63ﻤﻠﻡ
VIII
)ﺩ( ﻁﻭل ﺍﻟﺭﺠل ﺍﻟﺨﻠﻔﻴﺔ
7.00ﻤﻠﻡ
)ﻩ( ﻁﻭل ﺍﻟﺼﻔﺎﺌﺢ ﺍﻟﻅﻬﺭﻴﺔ ﺍﻟﺜﺎﻟﺜﺔ ﻭﺍﻟﺭﺍﺒﻌﺔ )ﺤﺠﻡ ﺠﺴﻡ(
3.88ﻤﻠﻡ
)ﻯ( ﻤﻌﺎﻤل ﺍﻟﺠﻨﺎﺡ ﺍﻷﻤﺎﻤﻲ )(Cubital index
2.04ﻤﻠﻡ
ﺝ -ﺃﻤﺎ ﺍﻟﻤﺠﻤﻭﻋﺔ ﺫﺍﺕ ﺍﻟﻘﻴﺎﺴﺎﺕ ﺍﻟﻜﺒﺭﻯ ﻟﺒﻌﺽ ﺍﻟﺼﻔﺎﺕ ﺍﻟﻤﻤﻴﺯﺓ .ﻨﺸﺄﺕ ﻫﺫﻩ ﺍﻟﻤﺠﻤﻭﻋـﺔ
ﻓـﻲ
ﻤﻨﻁﻘﺔ ﺍﻟﺴﺎﻓﻨﺎ ﻭ ﻏﺎﻟﺒﺎ ﻤﻊ ﺍﻟﺤﺩﻭﺩ ﺍﻷﺜﻴﻭﺒﻴﺔ .ﻭﻜﺎﻥ ﻤﺘﻭﺴﻁ ﻗﻴﺎﺴﺎﺘﻬﺎ ﻜﺂﻻﺘﻲ -:
)ﺃ( ﻁﻭل ﺍﻟﺠﻨﺎﺡ ﺍﻷﻤﺎﻤﻲ
8.45ﻤﻠﻡ
)ﺏ( ﻋﺭﺽ ﺍﻟﺠﻨﺎﺡ ﺍﻷﻤﺎﻤﻲ
2.95ﻤﻠﻡ
)ﺝ( ﻁﻭل ﺍﻟﺨﺭﻁﻭﻡ
5.59ﻤﻠﻡ
)ﺩ( ﻁﻭل ﺍﻟﺭﺠل ﺍﻟﺨﻠﻔﻴﺔ
7.05ﻤﻠﻡ
)ﻩ( ﻁﻭل ﺍﻟﺼﻔﺎﺌﺢ ﺍﻟﻅﻬﺭﻴﺔ ﺍﻟﺜﺎﻟﺜﺔ ﻭﺍﻟﺭﺍﺒﻌﺔ )ﺤﺠﻡ ﺠﺴﻡ(
4.00ﻤﻠﻡ
)ﻯ( ﻤﻌﺎﻤل ﺍﻟﺠﻨﺎﺡ ﺍﻷﻤﺎﻤﻲ )(Cubital index
2.24ﻤﻠﻡ
ﺘﻤﺕ ﺃﻴﻀﺎ ﻤﻘﺎﺭﻨﺔ ﺘﺴﻌﺔ ﻋﺸﺭ ﻋﻴﻨﺔ ﻤﻥ ﺍﻟﻨﺤل ﺍﻟﺴﻭﺩﺍﻨﻲ ﻤﻊ ﻤﺌﺎﺘﻴﻥ ﺍﺜﻨﺎﻥ ﻭ ﺃﺭﺒﻌﻭﻥ ﻋﻴﻨـﺔ
ﺒﻨﻜﻴﺔ ) ﺒﻨﻙ ﺍﻟﻤﻌﻠﻭﻤﺎﺕ ﺒﻤﻌﻬﺩ ﻋﻠﻡ ﻨﺤل ﺍﻟﻌﺴل ﺒﻤﺩﻴﻨﺔ ﺍﻭﺒﺭﺍﺴﻭل ﺒﺎﻟﻤﺎﻨﻴـﺎ ،ﻋﻴﻨـﺎﺕ ﻤـﻥ ﺍﻟـﺩﻭل
ﺍﻟﻤﺠﺎﻭﺭﺓ( ﺒﺎﺴﺘﻌﻤﺎل ﺘﺤﻠﻴل ﺍﻟﻤﻜﻭﻨﺎﺕ ﺍﻷﺴﺎﺴﻴﺔ ﻭ ﺍﻟﺘﺤﻠﻴل ﺍﻟﻤﻤﻴﺯ ﺍﻟﺤـﺩﻴﺙ ﺃﻤﻜـﻥ ﺘﻤﻴﻴـﺯ ﺍﻟﺜﻼﺜـﺔ
ﺘﺠﻤﻌﺎﺕ ﺍﻟﺴﻭﺩﺍﻨﻴﺔ ﻜﺘﺤﺕ ﻤﺠﻤﻭﻋﺎﺕ .
ﻴﺴﺘﺨﻠﺹ ﻤﻥ ﺫﻟـﻙ ﺃﻥ ﺍﻟﻨﺤـل ﺍﻟـﺴﻭﺩﺍﻨﻲ ﺍﻟـﺼﻐﻴﺭ ﺭﺒﻤـﺎ ﻴﻜـﻭﻥ Apis mellifera
sudanesisﺒﺩﻻ ﻋﻥ A. m. yementicaﻭﻴﻤﺜل ﻨﺤل ﺇﻗﻠﻴﻡ ﺍﻟﻐﺎﺒﺎﺕ .ﺍﻟﻨﺤل ﺍﻟﺴﻭﺩﺍﻨﻲ ﺍﻟﻤﺘﻭﺴﻁ
ﺭﺒﻤﺎ ﻴﻜﻭﻥ A. m. yementicaﺒﺩﻻ ﻋـﻥ A. m. sudanesisﻭﻴﻤﺜـل ﻨﺤـل ﺍﻹﻗﻠـﻴﻡ ﺸـﺒﻪ
ﺍﻟﺼﺤﺭﺍﻭﻱ .ﺒﻴﻨﻤﺎ ﻴﺤﺘﻔﻅ ﺍﻟﻨﺤل ﺍﻟﺴﻭﺩﺍﻨﻲ ﺍﻟﻜﺒﻴﺭ ﺒﺎﺴﻡ A. m. bandasiiﻭﻴﻤﺜل ﻨﺤـل ﺇﻗﻠـﻴﻡ
ﺍﻟﺴﺎﻓﻨﺎ .
ﺘﻤﺨﻀﺕ ﺩﺭﺍﺴﺔ ﺍﻟﻔﺭﻭ ﻗﺎﺕ ﺍﻟﻭﺭﺍﺜﻴﺔ ﺒﺎﺴﺘﺨﺩﺍﻡ ﺠﻴﻥ mtDNAﻓﻰ ﺍﻟﺴﺒﻌﺔ ﻭﻋﺸﺭﻭﻥ ﻋﻴﻨﺔ ﻤـﻥ
ﻤﺠﻤﻭﻋﺎﺕ ﺍﻟﻨﺤل ﺍﻟﺴﻭﺩﺍﻨﻲ ﻋﻥ ﻅﻬﻭﺭ ﺴﺘﺔ ﻋﻴﻨﺎﺕ ﻤﺨﺘﻠﻔﺔ ﻤﻥ ﺤﻴـﺙ ﺍﻟﻨـﺴﺏ .ﺍﻟﻤﺠﻤﻭﻋـﺔ ﺫﺍﺕ
ﺍﻟﻘﻴﺎﺴﺎﺕ ﺍﻟﺼﻐﺭﻯ ﻟﺒﻌﺽ ﺍﻟﺼﻔﺎﺕ ﺍﻟﻤﻤﻴﺯﺓ ﺤﺎﺯﺕ ﻋﻠﻰ ﺍﻟﻤﺼﻨﻔﺔ ﺍﻟﺠﻴﻨﻴﺔ A1ﺒﻨﺴﺒﺔ 100%ﻤـﻥ
ﻜل ﺍﻟﻌﻴﻨﺎﺕ ﺍﻟﻤﺩﺭﻭﺴﺔ ﻓﻲ ﺍﻹﻗﻠﻴﻡ ﺃﻤﺎ ﺍﻟﻤﺠﻤﻭﻋﺔ ﺫﺍﺕ ﺍﻟﻘﻴﺎﺴﺎﺕ ﺍﻟﻤﺘﻭﺴﻁﺔ ﻟﺒﻌﺽ ﺍﻟﺼﻔﺎﺕ ﺍﻟﻭﺭﺍﺜﻴـﺔ
ﺤﺎﺯﺕ ﻋﻠﻰ ﻨﻭﻋﻴﻥ ﻤﻥ ﺍﻟﻤﺼﻨﻔﺎﺕ ﺍﻟﺠﻴﻨﻴﺔ O1ﻭ Y2ﺒﻨﺴﺒﺔ 75ﺇﻟﻰ 25%ﻋﻠـﻰ ﺍﻟﺘـﻭﺍﻟﻲ ﻤـﻥ
ﺍﻟﻌﻴﻨﺎﺕ ﺍﻟﻤﺩﺭﻭﺴﺔ ﻓﻲ ﺍﻹﻗﻠﻴﻡ .ﺒﻴﻨﻤﺎ ﺍﻟﻤﺠﻤﻭﻋﺔ ﺫﺍﺕ ﺍﻟﻘﻴﺎﺴﺎﺕ ﺍﻟﻜﺒﺭﻯ ﻟـﺒﻌﺽ ﺍﻟـﺼﻔﺎﺕ ﺍﻟﻤﻤﻴـﺯﺓ
)ﻤﺠﻤﻭﻋﺔ ﺇﻗﻠﻴﻡ ﺍﻟﺴﺎﻓﻨﺎ( ﺤﺎﺯﺕ ﻋﻠﻲ ﺃﺭﺒﻌﺔ ﻤﺼﻨﻔﺎﺕ ﺠﻴﻨﻴﺔ ﻤﺨﺘﻠﻔﺔ A4 ، A2 ، O1- ، O1ﺒﻨﺴﺏ
20% ، 13% ، 13% ، 54%ﻋﻠﻰ ﺍﻟﺘﻭﺍﻟﻲ ﻤﻥ ﺍﻟﻌﻴﻨﺎﺕ ﺍﻟﻤﺩﺭﻭﺴﺔ ﻓﻲ ﺍﻹﻗﻠﻴﻡ .ﺒﺭﻫﻨﺕ ﻫﺫﻩ ﺍﻟﻨﺘﺎﻴﺞ
IX
ﻨﻭﻋﹰﺎ ﻤﺎ ﻨﺘﺎﺌﺞ ﺍﻟﻘﻴﺎﺴﺎﺕ ﺍﻟﺒﻴﻭﻟﻭﺠﻴﺔ )ﺘﺤﻠﻴل ﺍﻟﻤﻜﻭﻨﺎﺕ ﺍﻷﺴﺎﺴﻴﺔ PCAﻭﺍﻟﺘﺤﻠﻴل ﺍﻟﻤﻤﻴﺯ ﺍﻟﺤـﺩﻴﺙ (
ﺍﻟﺴﺎﺒﻘﻴﻥ.
ﻜﺫﻟﻙ ﺘﻘﺘﺭﺡ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ﺃﻥ ﻭﺠﻭﺩ ﺍﻹﻨﺴﻴﺎﺏ ﺍﻟﺠﻴﻨﻰ ﺒﻴﻥ ﺍﻟﻨﺤل ﺍﻟﺴﻭﺩﺍﻨﻰ ﻓﻰ ﺍﻟﺠﺯﺀ ﺍﻟﺠﻨﻭﺒﻰ
ﻤﻥ ﺍﻹﻗﻠﻴﻡ ﺸﺒﻪ ﺍﻟﺼﺤﺭﺍﻭﻯ ﻭﻜل ﺇﻗﻠﻴﻡ ﺍﻟﺴﺎﻓﻨﺎ ﻴﻌﺯﻯ ﻟﻠﺘﺩﺍﺨل ﻭﺍﻟﺘﺯﺍﻭﺝ ﺒﻴﻥ ﺍﻟﻨﺤل ﺍﻟﺴﻭﺩﺍﻨﻰ ﻭﺍﻟﻨﺤل
ﺍﻻﺜﻴﻭﺒﻰ ﻓﻰ ﺍﻟﺤﺩﻭﺩ ﺒﻴﻥ ﺍﻟﺒﻠﺩﻴﻥ ﻭﻜﺫﻟﻙ ﺭﺒﻤﺎ ﻴﻜﻭﻥ ﻤﺩﻯ ﻫﺫﺍ ﺍﻹﻨﺴﻴﺎﺏ ﺍﻟﺠﻴﻨـﻰ ﻴـﺸﻤل ﺍﻟﻤﻨﻁﻘـﺔ
ﺍﻟﻤﻨﺨﻔﻀﺔ ﻤﻥ ﺍﻟﺴﺎﻓﻨﺎ ﺍﻻﺜﻴﻭﺒﻴﺔ ﺤﺘﻰ ﻏﺭﺏ ﺍﻟﺴﻭﺩﺍﻥ ﺒﻴﻥ ﺨﻁﻰ ﻋﺭﺽ 9°Nﻭ .15°N
ﺃﻴﻀﹰﺎ ﺘﻘﺘﺭﺡ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ﺃﻥ ﺍﻟﻨﺤل ﺍﻟﺴﻭﺩﺍﻨﻰ ﺍﻟﻨﻘﻰ ﺭﺒﻤﺎ ﻴﻜﻭﻥ ﻨﺤل ﺠﻨﻭﺏ ﺍﻟـﺴﻭﺩﺍﻥ A. m.
sudanesisﻜﺫﻟﻙ ﺃﻥ ﺍﻟﻤﺼﻨﻑ ﺍﻟﺠﻴﻨﻰ ﺍﻟﻨﻘﻰ ﻟﻠﻨﺤل ﺍﻟﺴﻭﺩﺍﻨﻰ ﻗﺩ ﻴﻜﻭﻥ A1ﻭﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ﺘﻤﺜل
ﺃﻭل ﺘﻘﺭﻴﺭ ﻓﻲ ﺩﺭﺍﺴﺔ ﺘﻘﺴﻴﻡ ﺍﻟﻨﺤل ﺍﻟﺴﻭﺩﺍﻨﻲ ﻤﻥ ﺤﻴﺙ ﺍﻟﻔﺭﻭ ﻗﺎﺕ ﺍﻟﻭﺭﺍﺜﻴﺔ ﺒﺎﺴﺘﺨﺩﺍﻡ
ﺠﻴﻥ .mt DNA
ﻜﺫﻟﻙ ﺃُﺨﻀﻌﺕ ﺃﺭﺒﻌﺔ ﻋﻴﻨﺎﺕ ﻤﻥ ﺍﻟﻨﺤل ﺍﻵﺴﻴﻭﻱ ﺍﻟﺼﻐﻴﺭ Apis floreaﻟﺘﺤﻠﻴل ﺍﻟﻤﻜﻭﻨﺎﺕ
ﺍﻷﺴﺎﺴﻴﺔ ) ( PCAﻭ ﺍﻟﺘﺤﻠﻴل ﺍﻟﻤﻤﻴﺯ ﺍﻟﺤﺩﻴﺙ ) (Discriminant analysisﺤﻴﺙ ﺃﻭﻀﺤﺕ ﺍﻟﻨﺘﺎﺌﺞ
ﺃﻥ ﺍﻟﻤﺴﺘﻌﻤﺭﺍﺕ ﺍﻷﺭﺒﻌﺔ ﻟﻴﺴﺕ ﻭﺍﻀﺤﺔ ﺍﻟﺘﻤﻴﻴﺯ ﻤﻤﺎ ﻴﺩل ﻋﻠﻰ ﺃﻥ ﻫﺫﻩ ﺍﻟﻤﺴﺘﻌﻤﺭﺍﺕ ﻤﺘﻤﺎﺜﻠـﺔ ﻭﻓـﻰ
ﺍﻷﺼل ﺘﺭﺠﻊ ﺇﻟﻰ ﻤﺼﺩﺭ ﻭﺍﺤﺩ ﻭﻫﻭ ﺃﻭل ﺨﻠﻴﺔ ﺍﻜﺘﺸﻔﺕ ﻓﻲ ﺍﻟﺨﺭﻁﻭﻡ ﺴﻨﺔ 1985ﻡ .
ﺃُﺨﻀﻌﺕ ﺃﻴﻀﺎ ﺍﻷﺭﺒﻌﺔ ﻋﻴﻨﺎﺕ ﻤﻥ ﺍﻟﻨﺤل ﺍﻟﺼﻐﻴﺭ Apis Florea .ﻟﻠﻤﻘﺎﺭﻨﺔ ﻤﻊ ﺴـﺕ
ﻋﻴﻨﺎﺕ ﺃﺨﺭﻯ )ﻋﻴﻨﺘﻴﻥ ﻤﻥ ﺍﻟﺴﻭﺩﺍﻥ ,Mogga 1988.ﻭﺃﺭﺒﻌﺔ ﻤﻥ ﻤﻌﻬﺩ ﻋﻠﻡ ﺍﻟﻨﺤل ﺒﺄﻟﻤﺎﻨﻴﺎ( ﻋـﻥ
ﻁﺭﻴﻕ ﺘﺤﻠﻴل ﺍﻟﻌﻤﻭﺩ ﺍﻟﻌﻨﻘﻭﺩﻱ )ﻤﻘﺎﺭﻨﺔ ﺍﻟﻘﻴﻡ ﻋﺒﺭ ﺍﻷﺼﻨﺎﻑ ﺍﻟﻤﺨﺘﻠﻔﺔ( .ﺃﻭﻀﺤﺕ ﺍﻟﻨﺘﺎﺌﺞ ﺃﻥ ﺍﻷﺭﺒﻌﺔ
ﻋﻴﻨﺎﺕ ﺍﻟﺘﻰ ﺩﺭﺴﺕ ﺭﺒﻤﺎ ﻴﻜﻭﻥ ﻤﺼﺩﺭﻫﺎ ﻤﻥ ﺒﺎﻜﺴﺘﺎﻥ ﺃﻭ ﺠﻨﻭﺏ ﺇﻴﺭﺍﻥ.
X
TABLE OF CONTENTS
Title
Page
DEDICATION
II
ACKNOWLEDGEMENT
III
ABSTRACT
VII
ARABIC ABSTRACT
VIII
TABLE OF CONTENTS
X
LIST OF TABLES
XVIII
LIST OF FIGURES
XXI
CHAPTER: ONE INTRODUCTION
1- Sudan climate and vegetation zones.
1
1
1- a- Climate
1
1- b- Vegetation
1
1- b- i- The desert zone
1
1- b- ii- The semi-desert zone
1
1- b- iii- The poor savannah zone
1
1- b- iv- The rich savannah zone
2
1- b- v- The forest zone
2
2- Historical background of the Sudanese honeybees (Apis mellifera L.)
morphometrics.
XI
4
1- 3 Apis florea (Fabricius) from Sudan
6
1- 4- Molecular biology development
7
1- 5- The objectives of the present study
8
CHAPTER TWO: LITERATURE REVIEW
9
2- a- History of bees evolution
9
2- b- Origins of honeybees
11
2- c- Differentiation of Apinae
12
2- c- i- Genus Apis
12
2- c- ii- Distribution of the genus Apis
13
2- d- The dwarf honeybee Apis florea and Apis andrenifomis taxa
13
2- d- i- Distribution and description of Apis florea.
15
2- d- ii- Classification of Apis florea.
16
2- d- iii- Historical background of Apis florea in Africa.
17
2- e- Apis dorsata and Apis laboriosa (the giant honeybees)
19
2- f- Apis cerana and Apis mellifera L.
19
2- f- i- Races of Apis cerana:
20
2- f- i- (1) Apis cerana cerana.
20
2- f- i- (2) A. c. indica.
20
2- f- i- (3) A. c. japonica
20
2- f- i- (4) A. c. himalaya.
20
2- f- ii- Races of Apis mellifera L.:
XII
20
2- f- ii- (1) European races:
23
2- f- ii- (1)- a- Apis mellifera mellifera (German dark bees). 23
2- f- ii- (1)- b- A. m. ligustica Spin. (Italian bees).
23
2- f- ii- (1)- c- A. m. carnica Poltman (carniolin bees).
24
2- f- ii- (1)- d- A. m. caucasica Gorb (Caucasian bees).
24
2 f- ii- (2)- African races:
25
2- f- ii- (2)- a- Apis mellifera intermissa "Tellian bees" (Maa,
1953).
25
2- f- ii- (2)- b- A. m. lamarkii (Cockerel, 1906).
25
2- f- ii- (2)- c- A. m. scutellata Lepeltier (East Africa bees).
26
2- f- ii- (2)- d- A. m. adansonii (Latreille, 1804).
26
2- f- ii- (2)- e- A. m. monticolla (Smith, 1961) "Mountain
bees".
26
2- f- ii- (2)- f- A. m. capensis (Escholtz, 1822).
26
2- f- ii- (3)- Oriental races:
27
2- f- ii- (4)- Northern and South America races.
28
2- g- Probability and identification of the honeybees
28
2- h- Morphometrics.
30
2- h- i- Historical background of bees morphometrics.
2- h- ii- Recent developed biometry of Apis mellifera L.
2- i- Historical background of African honeybees classification:
2- j- East Africa races of honeybees.
30
35
38
42
XIII
2- j- i- Apis mellifera scutellata.
42
2- j- ii- A. m. monticolla
42
2- j- iii- A. m. yemenitica L.
43
2- j- iv- A. m. litorea.
43
2- j- v- A. m. sudanesis
43
2- j- vi- A. m. bandasii.
43
2- k- Classification of the Sudanese honeybee races.
43
2- l- Some biologic and ecological aspects of honeybees.
46
2- l- i- Biological factors
47
2- l- i- (1)- Migration
47
2- l- i- (2)- Reproductive swarming
48
2- l- i- (3)- Seasonal cycles of honeybee colonies
50
2- l- i- (4)- Temperament
51
2- l- ii- Ecological factors
52
2- m- Genetic diversity and honeybees
54
2- m- i- Allozymes
55
2- m- ii- Nuclear DNA
60
2- m- iii- Mitochondrial DNA
64
2- n- Animal mitochondrial DNA
65
2- n- i- Size of animal mitochondrial DNA
65
2- n- ii- Apis mellifera L. mitochondrial DNA
67
XIV
CHAPTER THREE: MATERIALS AND METHODS
88
3- 1- Sampling
88
3- 2- Morphometric analysis
91
3- 2- a- Preparation and measurements records of bees
94
3- 2- b- Statistical analysis
106
3- 3- Mitochondrial DNA
107
3- 3- a- DNA extraction
108
3- 3- b- PCR amplification
109
3- 3- c- DNA purification
110
3- 3- d- Size category of the fragments
111
3- 3- e- Endonuclease digestion
111
CHAPTER FOUR: RESULTS
4- 1- Morphometric analysis (Apis mellifera L.)
4- 1- i- Uni-variate analysis
112
112
112
4- 1- i- a- Proboscis and hind leg measurements (Table 3).
112
4- 1- i- b- Forewing measurements (Table 4).
112
4- 1- i- c- Forewing venation angles measurements (Table 5).
114
4- 1- i- d- Body size (tergites 3+4) and sternite 3 measurements
(Table 6):
114
4- 1- i- e- Pilosity measurements (Table 7):
114
4- 1- i- f- Sternite 6 (Abdominal slenderness) measurements
(Table 8).
115
XV
4- 1- i- g- Pigmentation (coloration) measurements (Table 9).
115
4- 1- i- h- Means, minimum, maximum and standard
deviation of each morphometric character from the
285 individual bees measured (Table 10).
124
4- 1- i- i- Sum of squares, dF, mean square, F values and
significances for each phenotypic character from the
measured individuals. (Sudanese honeybee Apis
mellifera L.) [Appendix H].
124
4- 1- ii- Multi-variety analysis.
124
4- 1- ii- a- Principal Component Analysis (PCA).
124
4- 1- ii- b- Discriminant analysis.
133
4- 2- Mitochondrial DNA analysis (Apis mellifera L.).
150
4- 2- i- Amplified PCR analysis.
151
4- 2- ii- Restriction analysis.
151
4- 3- observations on some behavior and biology of the Sudanese
honeybees Apis mellifera L.
165
4- 3- a- Colored colonies.
165
4- 3- b- Defensive behavior.
165
4- 3- c- Swarming and migration.
166
4- 3- d- Nesting sites.
167
4- 4- Morphometric statistical analysis of Apis florea.
168
XVI
CHAPTER FIVE: DISCUSSION, SUMMARY AND
CONCLUSION
5- 1- DISCUSSION
182
182
5- 1- a- Morphometrics: Apis mellifera L.
182
5- 1- b- Mitochondrial DNA. Apis mellifera L.
198
5- 1- c- Apis florea.
203
5- 2- SUMMARY AND CONCLUSION
208
5- 2- a- Apis mellifera L.
208
5- 2- b- Apis florea.
215
CHAPTER SIX: LIST OF REFRENCES
216
CHAPTER SEVEN: APPENDIXES
263
Appendix (A): Abbreviations of the morphometric characters used in
263
the study.
Appendix (B): Climatologically and Rainfall averages for at least 30
years from Climatologically stations in or/near the sample collection
areas.
265
Appendix (C): Taxonomic relationships between bees in the family
Apidae.
284
Appendix (D): Different species of the Genus Apini (Institute Für
Bienenkunde, Oberursel, Germany).
285
Appendix (E): Natural distribution of Honeybee Species (Institute Für
Bienenkunde, Oberursel, Germany).
286
Appendix (F): Geographical distribution of Genus Apis (Institute Für
Bienenkunde, Oberursel, Germany).
287
XVII
Appendix (G): Distribution of geographical honeybee races and mean
of annual temperature (F. Ruttner, Institute Für Bienenkunde Oberursel,
Germany).
288
AAppendix (H): Multivariate ANOVA Table: Sum of squares, dF, mean
values and significances for each phenotypic character character from the
s. (Sudanese honeybees Apis melli Apis mellifera L.)
289
XVIII
LIST OF TABLES
Table
Page
1- Sampling localities, respective geographical zones, map reference
numbers and coordinates of honeybee localities
89
2- List of characters used for the analysis
92
3- Means of measurements of proboscis and hind- leg of the
Sudanese honeybee workers Apis mellifera (mm.)
113
4- Means of measurements of the forewing of the Sudanese honeybee
workers Apis mellifera (mm.)
116
5- Means of measurements of forewing venation angles of the
Sudanese honeybee workers Apis mellifera (degrees)
117
6- Means of measurements of some tergites and sternites of the
Sudanese honeybee Apis mellifera (mm.).
118
7- Means of measurements of pilosity of the Sudanese honeybee
workers Apis mellifera (mm.)
119
8- Means of measurements of sternite 6 of the Sudanese honeybee
workers Apis mellifera (mm.)
120
9- Means of measurements of coloration of the Sudanese honeybee
workers Apis mellifera.
121
10- Mean, minimum, maximum and standard deviation of each
morphometric character from the 285 individual bees Apis
mellifera L. measured (measurements in mm. Angels in degree)
122
12- Factor loadings in varimax rotation for each character in the
principal component analysis
127
13- Means of some discriminant characters for the Sudanese
honeybee workers Apis mellifera (mm.), from the semi desert
region
137
XIX
14- Means of some discriminant characters for the Sudanese
honeybee workers Apis mellifera (mm.), from the savannah
region
138
15- Means of some discriminant characters for the Sudanese
honeybee workers Apis mellifera (mm.), from the forest region
139
16- Classification matrices of colonies in cluster groups based on
step-wise discriminant analysis. (Sudan samples only)
140
17- Pair-wise Group comparison a, b, c, d, e. (Sudan samples)
142
18- Discriminant analysis probability
143
19- a- Discriminant Classification results (Sudan and others)
144
19- b- Discriminant Classification results (Sudan and others)
146
20- Proximities discriminant centroid distances between the groups
(Dissimilarity matrix)
147
21- a- Sudanese honeybee workers Apis mellifera L. different
haplotypes
159
21- b- Distribution of the Sudanese honeybee worker haplotypes
according to the three different geographical zones
160
22- Means of measurements of proboscis and hind- leg of the
honeybee workers Apis florea (mm.) from Sudan
169
23- Means of measurements of forewing venation angles of the
honeybee workers Apis florea (degrees.) from Sudan
170
24- Mean of measurements of coloration of the honeybee workers
Apis florea from Sudan
171
25- Means of measurements of the forewing of the honeybee workers
Apis florea from Sudan (mm.)
172
26- Means of measurements of some tergites and sternites of
honeybee workers Apis florea (mm.) from Sudan
XX
173
27- Means of measurements of pilosity of the honeybee workers Apis
florea (mm.) from Sudan
173
28- Means of measurements of sternite 6 of the honeybee workers
Apis florea (mm.) from Sudan
173
29- Means of some discriminant characters for the honeybee workers
Apis florea from Sudan
174
30- Means, minimum, maximum and standard deviation of each
morphometric character from the 40 individual bees measured
(Apis florea) [Measurements in mm. Angels in degree]
175
31- Proximities discriminant centroid distances between the groups
(dissimilarity matrix). Apis florae
177
32- Some characteristics measurements of Apis florea from different
origins
178
33- Means of some characters of African bees mm. (Ruttner, 1975)
183
34- The average values of measurements taken for the different
characters of the Sudanese honeybee workers (El Sarrag, 1977)
184
35- The average values of measurements taken for the different
characters of the Sudanese honeybee workers (Saeed 1981)
185
36- The average values of measurements taken for the different
characters of the Sudanese honeybee workers (Mohamed 1982)
186
37- Average means of some discriminant characters for the Sudanese
honeybee workers (Apis mellifera L.) From different
geographical zones Mogga (1988)
187
XXI
LIST OF FIGURES
Figure.
Page.
1- Sudan map showing the collection localities.
3
2- Abdomin of the honeybee workers Apis mellifera L.
95
3- Length of the proboscis of the honeybee workers A. mellifera L.
96
4- Hind-leg of the honeybee workers A. mellifera L.
97
5- Classes of pigmentation of tergites (2 to 4) of the ho8neybee workers
Apis mellifera L.
98
6- Longitudinal diameter of tergite 3 and 4 of honeybee workers A.
mellifra L.
99
7- Sternite 3 of honeybee workers Apis mellifera L.
99
8- Length and width of sternite 6, of honeybee workers Apis mellifera L.
100
9- Honeybee workers Apis mellifera L. forewing.
101
10- Scutellum of the honeybee workers Apis mellfera L.
102
11- a Bee workers Apis mellfera L. Labrum.
103
11-b Labrum pigmentation of the honeybee workers Apis mellifera L.
104
12- Angles of wing venation of the honeybee workers Apis mellifera L.
105
13- Scatter plot graph of factor scores of factor 1 and factor 2 from
principal components analysis of 19 colony means of all morphometric
data.
128
14- Scatter plot graph of factor scores of factor 1 and factor 3 from
principal components analysis of 19 colony means of all morphometric
data.
129
15- Scatter plot graph of factor analysis of 239 samples of worker
honeybees of different origin.
XXII
132
17- Canonical Discriminant Function (Sudan samples only).
140
18- Dendrogram using average linkage between the groups (Sudan
colonies only).
141
19- Canonical Discriminant Function (Sudan and others).
148
20- Dendrogram using average linkage between the groups (Sudan and
others).
149
21- a- Agarose gel (1.5%) with the amplified PCR products of the of the
Sudanese honeybee worker samples.
153
21- b- Agarose gel (1.5%) with the amplified PCR products of the
Sudanese honeybee worker samples.
153
22- a- Acrylamide gel (8%) showing the Dra 1 restriction patterns of the
Intergenic region of the Sudanese honeybee worker samples
154
22- b- Acrylamide gel (10%) showing the Dra 1 restriction patterns of the
Intergenic region of the Sudanese honeybee worker samples
155
22- c- Acrylamide gel (8%) showing the Dra 1 restriction patterns of the
Intergenic region of the Sudanese honeybee worker samples.
156
22- d- Acrylamide gel (8%) showing the Dra 1 restriction patterns of the
Intergenic region of the Sudanese honeybee worker samples
157
22- e- Acrylamide gel (8%) showing the Dra 1 restriction patterns of the
Intergenic region of the Sudanese honeybee worker samples
158
23- a- Pie with 3rd visual effects representing the distribution percentage
of the Sudanese honeybee worker haplotypes of the studied 27 colonies.
161
23- b- Cluster column with 3rd visual effects representing the distribution
percentage of the Sudanese honeybee worker haplotypes of the studied 27
colonies.
161
XXIII
24- a- Pie with 3rd visual effects representing the distribution percentage
of semi-desert zone Sudanese honeybee worker haplotypes.
24- b- Pie with 3rd visual effects representing the distribution
percentage of savannah zone Sudanese honeybee worker
haplotypes.
24- c- Pie with 3rd visual effects representing the distribution
percentage of forest zone Sudanese honeybee worker
haplotypes.
25- Scatter plot graph of factor scores of factor 1 and factor 2
from principal components analysis of 4 colonies means of all
morphometric data (Apis florea).
162
163
164
179
26- Canonical Discriminant Function (Apis florea).
180
27- Graphic representation of cluster column analysis of 10
samples of Florea bees of different origins.
181
XXIV
XXV
26
27
CHAPTER ONE: INTRODUCTION
1- 1- Sudan climate and vegetation zones:
The Sudan is a vast country in Africa exhibiting the heart of the
continent. The total area measurements approximately 2,505, 813
square kilometers.
1- 1- a- Climate:
The climate is the most important force that keeping different
population in check. The temperature, humidity and rainfall affect
directly or indirectly every phase of the life.
1- 1- b- Vegetation:
According to Schmutterer (1969), There are main five types of
vegetation regions or zones in Sudan, which in general follow each
other from north to south, namely: desert, semi–desert, poor savannah,
rich savannah and forest region.
1- 1- b- i- The desert zone:
Represent about one third of the entire country. The rainfall is
very light and irregular (0 to 50 mm. per year). Various species of
Acacia are found along the Nile and in depressions not too far from
the river, thus Date palms ”Dom”, palms and Halfa grass are found in
plenty along the banks of the Nile river.
1- 1- b- ii- The semi - desert zone:
It is much smaller than the latter; its vegetation is richer than that
of the desert, which somewhat due to the high rainfall of about 50 to
300 mm. per year. Numerous shrubs and small trees grow in this area;
most of them are members of Acacia genus, thus various species of
short grasses are also common.
1- 1- b- iii- The poor savannah zone:
This region comparatively forms narrow belt, which runs
approximately in an east - west direction across the Sudan. The
1
rainfall of this vegetation belt varies from 300 to 500 mm. per annum
and the dry season lasts 4 – 6 months. The dominant trees are still
Acacias, which in many areas of the zone form open woodland, the
variety of trees in general is greater than in the preceding belt; the
same applies to herbs and grasses.
1- 1- b- iv- The rich savannah zone:
Lies between the poor savannah and the forest region, and it is
more- or less triangular in shape with a broad extension to the west
where it runs up to the Bahr el Arab in the south. The annual rainfall
amounts between 500 and 1000 mm. per year.
Acacia spp. is still the predominant trees of the region, but there is
an obvious change in the flora in comparison to that of the preceding
vegetation belt. Broad-leaved trees of the family Combretaceae
become more and more abundant, thus ‘Tebeldi’ tree Adansonia
digitata may be commonly found and ‘Heglig’ Balanites aegyptiaca
is also abundant in many places.
1- 1- b- v- The forest zone:
This region forms the most southerly vegetation belt in the Sudan.
The annual rainfall of this area is as high as 1000 to 1500 mm. per
annum resulting in very thick vegetation in many places, especially
along water streams and in depressions where gallery forests and
dense depression forests occur. These forests lie mainly in the
southwestern parts of the region and are the link to the rain forests of
the neighboring Congo and Uganda.
2
Fig (1): Sudan map showing the collection localities.
3
1- 2- Historical background of the Sudanese honeybees (Apis mellifera
L.) morphometrics:
It was understood that the Sudanese honeybee A. mellifera are not
thoroughly examined by earlier workers but the morphometric studies,
which were carried out in the last years revealed significant differences
among the Sudanese honeybees. Many research workers have conducted
several studies of the honeybee Apis mellifera L. races based on modern
biometrics. These suggested systems aimed at characterizing and classifying
the different geographical types of the Sudanese honeybees.
In 1975, Ruttner designated the Sudanese honeybees as a separate group
and gave them the name Apis mellifera nubica (Ruttner), thus he
characterize it with very small size, covered with very short hairs, most
slender abdomen of all African bees, the largest area of yellow coloration on
abdomen, with a tongue length of 5.36 mm and a wing length of 7.98 mm.
He stated that the Sudanese honeybee is similar to litorea, but clearly
belongs to another group.
Later, Elsarrag (1977) indicated that, the limited numbers of samples
collected from Khartoum state and described by Ruttner (1975) do not
perfectly represent the Sudanese honeybees. This was confirmed in (1978)
by Rashad,
and Elsarrag who illustrated that, several foreign races of
honeybees were introduced to Khartoum province and honeybees in
Khartoum were definitely hybridized with foreign blood. Comparing the
data given by Elsarrag (1977) on Khartoum bees with those obtained for
their hybrids F1 (Khartoum X unknown drones), Khartoum X Carniolan
drones, they’re backcross reciprocals and their F2 hybrids. Later on Ruttner
(1986), found that the A. mellifera nubica is completely scattered among
samples of Yemen, Oman, Saudia Arabia, Somalia and Chad. Accordingly
he nominated these bees as Apis mellifera yemenitica. To concludes this
4
taxon is incorporated into the subspecies yemenitica as a Sudanese
population.
In 1988, on the basis of multivariate morphometric analysis Mogga was
able to distinguish three morphoclusters: Yemenitica, a small bee of the
semi-desert zone; Sudanesis, a medium size bee of the forest and richsavannah; and Bandasii, a large bee along the border between Sudan and
Ethiopia. Matters became somewhat more complicated with the work of
Elsarrag et al., (1992), who reported only two subspecies for the region:
Sudanesis distributed throughout Sudan south of the Nubian Desert, and
Nubica along the Sudan Ethiopia border.
The altitude may form an ecological barrier to the spread of bee races in
the tropics. This is most clear in East Africa, where the costal plain and the
adjacent Kilimanjaro-Meru mountains are inhabited by different bee races,
namely Apis mellifera litorea (Smith) and Apis mellifera monticola (Smith),
respectively. However, in the Sudan the difference is mainly climatic and
vegetative. The ecological barriers resulting from higher altitude are
expected at Jebel Marra mountain (3042 m) in the west and the Imatong
mountains (3187 m) in the south of Sudan. While there is continuous natural
crossing among the Sudanese honeybees, it is still inconceivable to expect
the different ecological zones to be inhabited by a single race of honeybees,
having such a high capacity for specialized adaptation.
Thousands years ago, within some areas in the Sudan honeybees were
found preserved for trade purpose (cylindrical and boot hives). It did not
receive real intensive investigation.
Early seventies professor, Dr. Mohammed Saeed Ali Elsarrag and his
colleagues, explored this field and presented commendable work on
Sudanese honeybees records.
Bees were imported to Khartoum province as early April 1928 (European
honeybee races) by King from the apiary of the Egyptian Ministry of
5
Agriculture. These races included Italian, Cyprian, Carniolan as well as F1
Carnio-Egyptian honeybees. The latest large importation of bees, which
received extensive distribution to east and northern regions, was in 1967.
But generally, all the pure line colonies soon lost vigor and gradually
dwindled, as they may not compete with the indigenous bees.
The natural variability among honeybees in general and particularly the
Sudanese honeybees which showed high degree of variables morphometrics
were reported by Ruttner (1975); Elsarrag (1977); Rashad and Elsarrag
(1978 and 1980); Saeed, (1981); Mohammed (1982), and Mogga (1988).
The present investigation attempts to clarify in a large context the
geographical variability and classification of the Sudanese honeybees
(morphometrically and genetically). It is thought, the results of such a study
would explain the geographical distribution of the Sudanese honeybees
Which would presumably help any future plan of biological study of A.
mellifera and subsequent selective breading to improve beekeeping in the
Sudan.
1- 3- Apis florea (Fabricius) from Sudan:
Some samples of Apis florea from four different towns were included
in this study. This is a native honeybee species of the Asian sub-continent. It
was first discovered in Khartoum at a garden near the international airport
by Lord and Nagi (1985). It was believed that the initial colony might have
entered the country as part of an air cargo. By January 1987, twenty
additional colonies had been found, in a distance of 12 km from the origin.
In 1988, morphometrical study was carried out by Mogga and Ruttner in an
attempt to distinguished the origin of this bee and they stated that this A.
florea was brought to Sudan from a country of western Asia and exactly
from Pakistan.
The current study, re-focus on the florea, is it the same samples of Lord
and Nagi (1985)? Or there were some new colonies entered the country?
6
1- 4- Molecular biology development:
In the last decade techniques for the measurement of genetic variation in
honeybees at the DNA level have been developed and are proving to be
extremely powerful probes for the analysis of genetic variation.
The general technique is to look for restriction fragment length
polymorphisms (RFLPs). Which have been applied to nuclear DNA (Hall
1986, 1988; Severson et al., 1988) as well as to mitochondrial DNA (Moritz
et al., 1986; Smith, 1988, 1991; Smith and Brown, 1988, 1990; Smith et al.,
1989, 1991), and by sequence analysis (Cornuet, Garnery & Solignac 1991.
Garnery et al., 1991. Koulianos & Crozier 1991).
Besides the potential discriminating power the mitochondrial DNA may
provide valuables information on the phylogenetic links between subspecies
or populations of the honeybees, data on mitochondrial DNA indicate that
the subspecies Apis mellifera can be grouped into three lineages, A, M, and
C, that are largely congruent with that ones put forward by Ruttner (1988),
on the bases of detailed morphometrical studies. In turn, each lineage
includes different types of variants of mtDNA (haplotypes) discovered after
sequencing and/ or digestion with endonucleases.
The variable regions best studied are the intergenic spacer between the
tRNAleu and the cytochrome oxidase II gene (Corneut and Garnery 1991;
Garnery et al., 1992, 1993, and 1995). This region shows a length
polymorphism due to a variable number of copies (1-3) of a 192-196 bp
sequence (Q) and second sequence P0 of 67 bp that may be partially deleted
(sequence P with 52 bp), or completely lacking. In addition, this spacer
shows restriction polymorphisms with Dra1, making it possible to
characterize different haplotypes with low sampling and methodological
efforts (Garnery et al., 1993).
Based on the above mentioned genetic techniques, mainly mtDNA, a
part of this study was directed to fined differences that characterize the
7
subspecies of the Sudanese honeybee and compare the results with the
morphometric ones.
1- 5- The objectives of the present study:
1- The aim of this study was to investigate and classify the Sudanese
honeybees, by using the Principal Component Analysis (the modern
biometric method) together with genetic techniques, mainly mtDNA
variation (to study the haplotype distribution of mtDNA in Sudan
honeybees). Its was particularly felt necessary to conduct this work
following morphometrical studies carried out by Ruttner (1975); Elsarrag
(1977); Rashad and Elsarrag (1978 and 1980); Saeed (1981); Mohammed
(1982); and Mogga (1988). All there results revealed significant differences
among the Sudanese honeybees.
2- Establishing the taxonomical status of the Sudanese honeybees from
different climate zones, in light of Ruttner (1986) classification of bees from
the Sudan.
3- Investigating the geographical variants and distribution of the Sudanese
honeybees.
4- To check up the origin of Apis florea, which was done before by Mogga
(1988); following biometric modern techniques.
8
CHAPTER TWO: LITERATURE REVIEW
2-a- History of bees Evolution:
The ancient of insect’s dates back to Upper Carboniferous period, which
is about 350 million years ago. Some changes in insect fauna were noticed in
the Permian, Mesozoic, Triassic and Jurassic periods that followed. Once
flowering plants become established in Cretaceous period, many insects
including specoid (predatory wasps) and ants with social behaviour were
found associated with the plants (Winston, 1978). Bees diverged from a
wasp ancestor approximately 100 million years ago (Michener, 1974;
Michener and Grimaldi, 1988), when the angiosperms were becoming the
dominant vegetation. The development of characteristics such as plumose
hair, broadened hind legs for pollen collection, and mouth parts capable for
ingesting nectar allowed an ancestral form to abandon predatory lifestyle
and make flowering plants its primary food source (Raven and Axelrod,
1974). Due to pollen-collecting structures and habits, taxonomists place bees
in their own super family, Apoidea (order: Hymenoptera), Culliney, (1983);
Winston (1987), with 10 or 11 families (Michener, 1979. Michener and
Greenberg, 1980), 700 genera (Malyshev, 1968), and 20.000 species
(Michener, 1969) described. The honeybee is a long-tongued bee, classified
in the family Apidae (Apinae: Apini) (reviewed in Raven and Axelord, 1974)
along with the bumble bees (Bombinae: Bombini), the Orchid bees
(Bombinae: Euglossini) and the sting less bees (Meliponinae) (Winston and
Michener, 1977. Kimsey, 1984), as in appendix (C). Modern honeybees
belong to a single genus, Apis, which contains at least seven species: A.
andreniformis, A. cerana, A. dorsata, A. florea, A. koschevnikovi, A.
labortiosa, and A. mellifera, (Alexander 1990; Otis, 1990 and Michener,
1990), appendix (D).
9
Honeybees are believed to have diverged as a separate genus about 40-50
mya (Michener and Grimaldi, 1988. Kelner-Pillaut, 1969. Zeuner et al.,
1976).
Apis dorsata and A. florea have been considered extend of ancestry. The
two are believed to have separated as far back as the Oligocene, because
dorsata-type bees are present in the early Miocene (22mya) and because
these two species are believed to be the most distantly related members of
the genus (Cockerell, 1908). Further Apis evolution is believed to have
occurred during the Pliocene (2-6 mya) or early Pleistocene (2 mya) when
the ability to thermo regulates its colonies resulted in an enormous increase
in Apis distribution, including the colonization of Europe and Africa.
Ecological and morphological diversification at the subspecies level
followed, and the group spread rapidly to various climatic zones of the New
World. The sister species A. cerana and A. mellifera are believed to be still
in an early stage of speciation (Ruttner, 1988; Ruttner and Maul, 1983),
having split immediately before or during the Pleistocene (1-2 mya).
A considerable confusion with respect to honeybee taxonomy has existed
since several decades. The earliest classification listed four species within
the tribe Apini: A. mellifera Linnaeus (1758: 576), A. cerana Fabricius
(1793: 327), A. dorsata Fabricius (1793: 328) and A. florea Fabricius (1787:
305). Various generic subdivisions have been created and discarded
(Ashmead, 1904; Buttel-Reepen, 1906; Maa, 1953).
Despite, these records more recent classification systems are also
ambiguous. Ruttner (1988), using morphometric analysis, favors a return to
the earlier system of four species. (A. florea, A. cerana, A. mellifera, A.
dorsata). In contrast, Sakagami et al., (1980) favor expansion of the
taxonomy to include A. laboriosa (a close relative of Dorsata), while Starr et
al., (1987) favor the addition of laboriosa as well as A. breviligula. The
designation of a new genus, Micrapis, has also be proposed (Wu and Kuang,
10
1986) in which Florea would be divided into two distinct species, Micrapis
florea and Micrapis andreniformis. To date however, the most accepted
classification system includes Florea, Andreniformis, Dorsata, Cerana,
Mellifera, and Koschevnikovi (Alexander, 1990), while Laboriosa will
probably be given species status in the near future (Otis, 1990).
The genus Apis has been studied using morphological (Alexander, 1990.
Smith, 1991), biogeographically (Kellner-Pillault, 1969. Smith, 1991), and
molecular methods (Smith, 1991. Garney et al., 1991. Shepard and
Berlocher, 1989; Smith, 1990).
The current accepted review of Apis (Alexander, 1990) places the A.
florea, A. andreniformis line as the most ancestral, giving rise to A. dorsata
followed by A. mellifera, A. cerana, and finally A. koschevnikovi.
2- b- Origins of honey bees:
The earliest Apini (Apidae) fossil specimens have been found in Baltic
Amber from Eocene layers approximately 40 million years old. A fossilized
honeybee comb has recently been discovered in Malaysia dating from the
late Tertiary or Quaternary period suggesting an earlier origin of the genus
(Stauffer, 1979).
More recently, in January 12, 2006 Micheael, S. Engel. Stated that, a
new fossil honeybee is described and figured from middle Miocene deposits
of Iki Island, Japan. Apis lithohermaea (new species) is the largest fossil
honeybee discovered, rivaling in size the modern giant honeybee, A. dorsata
Fabricius. Thus it is the first fossil of the Dorsata species group recorded.
Although the Dorsata group does not occur farther north than Tibet and
southern China and in the Philippines in the Pacific, this lineage occurred
near what is today southern Korea and Japan during the Miocene. This
findings and the fact that fossil honeybee specimens are generally found
with individuals grouped together suggest early evolution of social behavior
in the Apini (Apidae).
11
Specimens found from the Oligocene, when considerable change
occurred in external morphology show rapid evolution during the next 10
million years. Also, comparative biochemical studies of extent bees have
indicated a greater degree of amino acid substitution in Apis mellifera
compared with other bees, and therefore, a more rapid protein evolutionary
rate in the honey bee lineage (Carlson and Brosemer, 1971, 1973).
On the basis of morphological evidence however, there has been
relatively little change in honeybees during the last 30 million years
(Culliney, 1983), and the physical resemblance of fossil forms to modern
worker bees suggests that complex social behavior had already developed by
the Miocene, 27 million years ago. These bees have morphological
characteristics, which partially point to the present day Meliponini and
partially to the Apini. Eusociality in bees originated from solitary living and
later sub social living. In the evolutionary path bumblebees are next to
solitary bees. Meliponidae are eusocial bees, which evolved after
bumblebees. Mass provisioning is noticed in Meliponidae.
2- c- Differentiation of Apinae:
Honeybees belonging to Apidae are the most evolved bees, which have
sting for defense, progressive provisioning, division of labor and
reproductive casts.
2- c- i- Genus Apis :The modern honey bees (Apidae: Apini) are all classified in only one
genus, Apis that includes the following species:
A- Single comb open-air nest includes:
Apis florea, A. andreniformis (the dwarf honeybees) A. laboriosa and
A. dorsata (the giant honeybee).
B- Multiple-comb cavity nest includes: A. mellifera, A. koschevnikovi (the
Saban honeybee), A. nuluensis (the Bornean mountain honeybees) A.
nigrocincta, A. cerana (the Asian honeybee), appendix (D). But extinct
12
apini have been classified into their own genus Electrapis. (Manning, 1960;
Zeuner and Manning, 1976; Culliney, 1983).
The natural geographical distribution of the genus Apis shows its greatest
species diversity in India and adjacent regions, with all of the species except
Apis mellifera don’t found there; appendix (E) and appendix (F). Therefore,
these regions probably constitute the area of origin and early evolution of the
Apini (Doediker, Thakar, and Shaw, 1959; Michener, 1974; Doediker,
1978). A. mellifera is thought to have originated in the Africa tropics or sub
tropics during the Tertiary period, migrating to western Asia and colder
European climates somewhat later.
2- c- ii- Distribution of the genus Apis:
Until modern times Apis was not found anywhere in the western
hemisphere, Australia, or the pacific except for some of the continental
islands such as Japan, Formosa, Philippines, and Indonesia (Michener,
1974). But movement of bees by European settlers for beekeeping has
resulted in Apis mellifera now having worldwide distribution appendix (F
and G) thus, some of the other species being more widespread in Asia as the
dwarf honey bee A. florea.
2- d- The dwarf honeybee Apis florea and Apis andreniformis taxa:
Many years ago, several subspecies, varieties, and nations of Apis florea
were described, and specimens of Apis andreniformis identified as Apis
florea (Maa, 1953). The accuracy of separation between those two honeybee
species is often difficult to assess, because the worker bees are more
sympatric (Otis, 1997). The confusion and mixing of the characters of those
two species is evident in the monograph of Ruttner (1988).
However, the distinction between the two species as unequivocal biological
species nowadays, has been well established based on:
13
a- Drone morphology (Ruttner, 1975; Kuang and Li, 1985; Wu and
Kuang, 1986, 1987; Ruttner, 1988; Wongsiri et al., 1990; Chen,
1993).
b- Nest structure (Dung et al., 1996; Rinderer et al., 1996).
c- Morphometrics (Rinderer et al., 1995).
d- Allozyme polymorphism (Nunamaker et al., 1984; Li et al., 1986; Gan
et al., 1991).
e- Mitochondrial DNA sequence divergences (Smith, 1991; Nanork et
al., 2001; Takahashi et al., unpublished data, cited in H. Randall
Hepburn et al., 2005).
f- Differences in the timing of mating flights (Rinderer et al., 1993).
g- Several of the above mentioned differences contribute to complete
reproductive isolation between the two species (Koeniger and Koeniger,
1991, 2000, 2001; Dung et al., 1996).
The most accurate characters used for identification of Apis florea and
Apis andreniformis are:
The “thumb” of the bifurcated basitarsus of the hind leg of drones of A.
florea is much longer than that of A. andreniformis (Ruttner, 1988);
differences in the structure of the endophallus (Lavrekhin, 1935; Wongsiri et
al., 1990; Koeniger, 1991); in worker bees, the jugal-vannal ratio of the hind
wing of A. florea is greater (about 75) than that of A. andreniformis (about
65); and the cubital index of A. florea (about 3) is significantly less than in
A. andreniformis (about 6). Abdominal tergite 2 of A. andreniformis is
deeply punctuate, that of A. florea is not. The marginal setae on the hind
tibiae of A. florea are usually entirely white, those of A. andreniformis darkbrown to blackish in sclerotized individuals. Permeating the older literature
is the idea that abdominal tergites 1 and 2 of A. florea are reddish and other
segments at least partially reddish, while those of A. andreniformis are
uniformity black.
14
An inspection of several hundred workers from each of several different
colonies of each species quickly demonstrates the extreme variation in
pigmentation thus precluding this as a useful distinguishing trait, a point
recognized rather long ago (Drory, 1888). Finally the combs of the two
species are very different (Rinderer et al., 1996).
2- d- i- Distribution and description of A. florea (Fabricius):
Apis florea is widespread species and its prevalence extends some 7000
km from its eastern-most extreme in Vietnam and southeastern China, across
mainland Asia along and below the southern flanks of the Himalayas,
westwards to the Plateau of Iran and southerly into Oman. This constitutes
some 70 degrees of longitude (40°–110° East) and nearly 30 degrees of
latitude (6°–34° North). Variations in altitude range from sea level to about
2000 m. A. florea has also been reported in historical times in Saudi Arabia
and Sudan, and occurred on Java, Indonesia. Phenotypic variation among A.
florea is not well understood. This species refers to bees of the plains up to
500 m. and, seasonal migrations occur up to 1500 m. (Muttoo, 1956). These
species appear to maintain several ancestral characteristics of the genus Apis
and show aspects of descendancy of the earliest honeybees in which
Workers are small, approximately 7 mm in length, and colonies construct a
single comb supported from branches, frequently at sites surrounded by
dense vegetation (Seeley, and Akratanakul, 1982). In spite of its small size it
competes with other Apis species (Koniger, 1976). The author of this work
notified that in the Sudan seldom prevalence of Mellifera colonies around
areas with abundant Florea species assembly around. Moreover, in the
northern parts of the Sudan Florea replaced the abundance of Mellifera for
instance. Their communicative dance occur on horizontal platform built on
the top of the comb, and direction to flowers is indicated by straight runs
toward the food source. Colonies tend to be small, less than 5000
individuals, and the workers are relatively docile (Michener,1974).
15
2- d-ii- Classification of Apis florea:
Several regional univariate morphometric analysis on Apis florea have
appeared over the last two centuries, but did not affected the taxonomy of
this species. In the first multivariate morphometric analysis of A. florea,
Ruttner (1988) had few samples from geographically non-contiguous
regions. Although the data were insufficient for a comprehensive analysis,
Ruttner (1988) demonstrated geographic variability and obtained three
morphoclusters for A. florea. Tahmasebi et al. (2002) analyzed the A. florea
of Iran and defined two morphoclusters from a geographical continuum.
Combining their data with that of Ruttner (1988) and Mogga and Ruttner
(1988), the latter reported three morphoclusters for all A. florea but a lack of
geographical contiguity applies to this database as well. New collections of
A. florea from Myanmar, Nepal, Cambodia, Thailand, Vietnam, Iran, Iraq,
Afghanistan, Sri Lanka and Saudi Arabia have greatly augmented the
database of A. florea over a geographical continuum of about 7000 km. The
additional, new samples fill gaps to provide a population continuum over the
full range of the natural distribution of A. florea for the first time. Following
the previous studies on the morphometrics, classification and biography of
Apis florea, Hepburn, et al., (2005) by using multivariate morphometric
analysis studied 2923 individual worker bees from 184 colonies representing
103 localities across the full distributional area of Apis florea Fabricius 1787
from Vietnam and southeastern China to Iran and Oman (~7000 km),
reporting that, comparisons of geographically separated A. florea
populations resulting in morphoclusters that reflect sampling artifacts. These
morphoclusters change with latitude but overlap when the full database is
contained in the same principal component analysis. A cluster analysis based
on Euclidean distances suggests degrees of affinity between various
geographic groupings of A. florea. This species occupies a large area that
includes rainforests, savannas, subtropical stepes, and semi deserts. The
16
seasonality of reproductive swarming is temporally continuous allowing
gene flow throughout this panmictic species. Thus, Hepburn, et al., (2005),
recommended that, A. florea is a single species that comprise of three
discernible morphoclusters. In the northwestern-most bees comprise a
morphocluster (1) that is statistically quite distinct from that to the southeast
(3); but, they are not isolated, they are joined by large areas of intermediate
forms (2) resulting in a continuous cline in morphometric traits within this
panmictic species.
The confirmation stated by Hepburn, et al., (2005) results, in studies of
variation in mitochondrial DNA, Smith (1991) established that A. florea
were homogeneous in Thailand and also in southern India but had diverged
between the two countries. The homogeneity of mtDNA in A. florea from
Thailand was subsequently confirmed (Nanork et al., 2001). Smith also
observed that different mtDNA clusters occur in A. cerana from N. and S.
India, paralleling
the differences observed in A. florea. More recently,
Takahashi et al. (unpublished data, cited in Hepburn, et al., 2005) proposed
three distinct mtDNA lineages for A. florea from eastern Asia:
(1) China/Myanmar;
(2) Southeast Asia: Thailand, Vietnam, Cambodia, and part of China;
(3) India. While there are no inconsistencies among these three studies,
available information is insufficient to apply to the whole area of A. florea
distribution. Similarly, available data on enzyme polymorphism in A. florea
(Li et al., 1986; Sheppard and Berlocher, 1989; Gan et al., 1991) are
likewise geographically limited precluding extrapolation to the whole A.
florea population. The available genetic data are too regional in nature to be
informative for the species as a whole.
2- d- iii- Historical background of Apis florea in Africa:
The first colony of Apis florea in Africa was discovered in November
1985 in Khartoum. Mogga and Ruttner (1986) reported that, the colony was
17
cut from a lemon tree and brought to the beekeeping unit of the university of
Khartoum for determination.
On November 2, 1985 Siham Kamil of the university of Khartoum,
William Lord (beekeeping specialist working for the Near East Foundation
in Sudan) and J. Mogga were called to see a second colony of Apis florea
established in another Khartoum garden, and hence more colonies were
detected.
The city of Khartoum is surrounded by desert, but in the centre of the
city where these Apis florea have been found, the gardens are irrigated and
there is flowering vegetation-obviously sufficient to sustain colonies of Apis
florea. There are no colonies of Apis mellifera present at that time in
Khartoum, other than those maintained by the University Bee Unit, which
have to be provided with continual supplies of food and water to ensure their
survival.
That is the first record of Apis florea in Africa, and it raises the
interesting questions of how long these bees have been present in Khartoum,
and how they arrived there. All colonies found so far have been well
established and local people confirm that the colonies have been present for
at least sex months. The nearest place to Khartoum where Apis florea is
recorded is Oman, some 2700 km to the East. A clue to their arrival may be
the fact that all colonies found so far are near to Khartoum International
Airport, which is also a base for aid lorries carrying supplies to and from
Port Sudan town.
It is thought that accidental or deliberate, human intervention is in Apis
florea‚ introductory in Sudan. Bees disseminate or spread disease and pests
and rendered hard to eradiate. Introduced bees compete with the native
species, which was adversely affected.
18
2- e- Apis dorsata and A. laboriosa (the giant honey bees):
There are two other closely related species of honeybees that construct
nests consisting of a single comb in the open. They are known as the giant
honeybees. These are large, feisty bees 17-19 mm in length, with 20,000 or
more workers in a colony. Their nests are constructed high in trees or
suspended from open cliff faces, and nests do not need to be concealed
because of the workers aggressive nature. Nests are also frequently
aggregated and colonies may migrate up and down mountains to take
advantage of seasonal nectar sources. Communicative dances are more
advanced collective in A. florea since they occurred in the vertical comb
face, and the direction to flowers has to be translated by the workers from
the vertical dance angle to the direction of the sun (Michener,1974). A.
laboriosa is the larger of the two species, and its large size, dark colour, and
longhair coat are probably adaptations for its high altitude Himalayan habitat
(Sakagami, Matsumura, and Ito, 1980).
2- f- A. cerana and A. mellifera:
A. cerana and A. mellifera, are medium size bees (10-11 mm), which
generally build multiple comb nests inside cavities. Colonies of A. cerana
are relatively small, 6000-7000 workers (Seeley and Akratanakul, 1982), but
A. mellifera colonies can reach size of 100,000 or more individuals. These
species are similar in morphology and behaviour and frequently considered
distant races of the same species. However, Ruttner and Maul (1983),
demonstrated that, although Cerana and Mellifera queens and drowns
attempt to mate with each other, no offspring result, and instrumental
insemination of Mellifera and Cerana queens with heterospecific semen
revealed that the hybrid fertilized eggs would cease development at the
blastula stage. These results indicate that Cerana and Mellifera are indeed
separate species, although are closely related.
19
2- f- i- Races of Apis cerana:
The eastern cavity-nesting honeybee, Apis cerana F., is widespread
over Asia and occupies a distribution range extending from Afghanistan to
China and from Japan to southern Indonesia (Ruttner, 1988). This species
has been grouped based on morphometric analysis (Ruttner, 1988) in four
subspecies with different distribution ranges:
2- f- i- (1)- Apis cerana cerana, from Afghanistan, Pakistan, north India,
China and North Vietnam.
2- f- i- (2)- A. c. indica, from south India, Sri Lanka, Bangladesh, Burma,
Malaysia, Indonesia and the Philippines.
2- f- i- (3)- A. c. japonica, from Japan.
2- f- I- (4)- A. c. himalaya, from central and east Himalayan Mountains
(Smith, 1991).
These subspecies include many populations some of which are
geographically isolated, such as those in the Philippines archipelago. As on
other oceanic islands, these populations may have undergone evolutionary
changes giving rise to reproductively isolated populations.
2- f- ii- Races of Apis mellifera :
The concept of a widespread race of honeybees in sub-Saharan Africa that is
closely related to the European races can be found in most reviews of bee
classification.
Before 1958, however, opinions differed as to wither sub-Saharan bees
where distinct species (Smith, 1865; Goetze, 1930, 1940; Maa, 1953) or an
infraspecific form of A. mellifera (Buttel-Reepen, 1906; Enderlein, 1906;
Ruttner, Kerr and Laidlaw, 1956). The modern bees classification started
with Kerr and Portugal-Araüjo (1958), who applied the biological species
concept to the problem and cited genetic crosses among European and
African races to justify association to one species. All evidence up to date
20
supports their conclusion, but partial reproductive isolation may exist
between some races (Kerr and Bueno, 1970).
The endemic distribution of the honeybee Apis mellifera L. is generally
considered to encompass Africa, Europe and pockets of western Asia
appendix (F). Across this range, variation in behavior, morphology and
genetic markers supports an evolutionary history of the species that includes
differentiation into several major phylogenetic lineages (Ruttner, 1988;
Cornuet and Garnery, 1991; Garnery et al., 1992). Based primarily on
morphological characters, more than two dozen subspecies have been
described within the lineages (Ruttner, 1992; Sheppard et al., 1997). These
subspecies typically exhibit reduced gene flow with other such groups due to
water, mountain or desert barriers and have been called “geographic races”,
to reflect their adaptation to specific geographic areas (Ruttner, 1988).
Based on morphological similarities and sequence divergence among
described subspecies, the speciation event that produced Apis mellifera has
been estimated to occur between 0.7 to 1.3 million years ago (Ruttner, 1988;
Cornuet and Garnery, 1991; Arias and Sheppard, 1996). While Apis
mellifera occupies a large geographic distribution allopatric from the rest of
the genus, a number of the other Apis species reside sympatrically in Asia.
Western Afghanistan is considered to be the eastern limit of A. mellifera,
with the closest proximity between Apis mellifera and its congeners
occurring somewhere in central Afghanistan (Ruttner, 1988). Consistent
with the estimated antiquity of A. mellifera and the pale climate of the region
separating A. cerana and A. mellifera, Ruttner postulated that an A. mellifera
ancestor reached western Asia about one million years ago (ibid). While the
relative youth of a million-year old A. mellifera lineage has been supported
by molecular studies, cladogenesis of A. mellifera and A. cerana appears to
have occurred at a much earlier time (6–9 mya) based on allozyme and DNA
sequence differences (Sheppard and Berlocher, 1989; Cornuet and Garnery,
21
1991; Arias et al., 1996). The apparent discrepancy between the age of A.
mellifera subspecies and the A. mellifera /A. cerana cladogenesis suggests
that alternative hypotheses for the origin of A. mellifera should be
considered. These include the possibility that A. cerana and A. mellifera are
not related to each other as sister taxa, rather they may be so related to as yet
un described or extinct species. Unfortunately, perhaps the least studied area
within the distribution of Apis is in the region between the known
distribution of Apis mellifera and Apis cerana.
The natural habitat of the honeybee Apis mellifera extends from the
southern tip of Africa through savannah, rain forest, desert, and the mild
climate of the Mediterranean before reaching the limit of its range in
northern Europe and southern Scandinavia appendix (F).
With such a variety of habitats, climatic conditions, and floras, it is not
surprising to find numerous subspecies (races) of honey bees (Apis mellifera
L.), each with distinctive characteristic adapted to each region (Louveaux,
1966), appendix (G). Still, recognition of valid races has been difficult for a
number of reasons. The Most important reason has been the movement of
honey bees all over the world for beekeeping, which has changed the natural
range of each race and resulted in considerable hybridisation of races.
Selection by beekeepers characteristics useful in management may have
altered the natural genotype of races as well, particularly in areas of
intensive beekeeping where many feral colonies are descended from
swarms, which have escaped from hives. Further difficulty may be that
scientists and beekeepers don’t always use the same criteria for determining
what a “race” is. Scientists tend to use morphometric measurements of such
characteristics as wing veins, mouthparts, antennal length, and the size of
certain body parts (Ruttner, 1975a; Daly and Balling, 1978; Ruttner,
Tassencourt, and Luveaux, 1978), whereas beekeepers prefer characters like
colour and behavioural traits, such as tendency to swarm, good honey
22
production, and gentleness. Finally, even within a single race there can be
tremendous variation and where to divide races and determining what is
“typical” for a race have always been somewhat subjective.
Some general conclusions have emerged concerning the characteristics
and places of origin for many honey bee races, and these have been
summarized by Ruttner (1975b); Ruttner, Tassencourt, and Louveaux,
(1978). He divided honeybee races into three distinctive groups: European,
Oriental (Near Eastern), and African. Little is known about the Oriental
races, and studies of many African regions are based on few specimens. The
European races have been relatively well studied, and there appears to be
more general agreement on these than on the African races. The brief
descriptions below are based on Ruttner`s conclusions.
2- f- ii- (1)- European races:
2- f- ii- (1)- a- Apis mellifera mellifera L. (German dark bees):
Originated in northern Europe and west- central Russia, probably
extending down into the Iberian Peninsula. They are large bees, although
with relatively short tongues (5.7-6.4 mm), and their common name is
derived from their brown-black colour, with only a few lighter yellow spots
on the abdomen. They tend to be nervous and aggressive but winter well in
severs climates. Worker population expands slowly in the spring, although
these bees where once popular for export around the world, their aggressive
nature, poor spring and early summer performance, and difficult in working
with flowers with long corollas such as clover have resulted in diminished
use of Mellifera for beekeeping.
2- f- ii- (1)- b- A. mellifera ligustica Spin. (Italian bees):
Originated in Italy, has been the most popular honeybee for beekeeping
throughout the world. Although, somewhat smaller than A. mellifera
Ligustica have relatively long tongues (6.3-6.6) mm) and abdomens with
bright yellow bands. They tend to be docile, and colony populations build up
23
quickly in the spring and remain strong throughout the summer. They over
winter with strong worker populations, although with high consumption of
honey that causes some difficulties in northern latitudes. They also have a
reputation as rapid comb builders and seem to initiate robbing of honey from
other colonies more quickly than the other European races.
2- f- ii- (1)- c- A. mellifera carnica Pollman (Carniolin bees):
Originated in the southern Austrian Alps, northern Yugoslavia, and the
Danube Valley. They are of a similar size to Ligustica, but tend to be grey or
brown in colour. These bees have also been popular for beekeeping,
particularly with hobbyists because of their gentle disposition. They over
winter in small colonies with low food consumption but developed quickly
in the spring. They may not maintain this high population throughout the
summer and seem to swarm more readily than the Italian bees. They are also
slow to construct new comb.
2- f- ii- (1)- d- A. mellifera caucasica Gorb. (Caucasian bees):
Evolved in the high valleys of the central Caucasus. They appear
similar to Carnica, although perhaps with a more lead-grey colour. Although
their behaviour is not as well known, they are considered gentle, slow to
expand in the spring but capable of reaching adequate summer populations,
and poor at over wintering because of susceptibility to the adult disease
Nosema. They also used propolis extensively and are reported to have only a
weak disposition to swarm.
There are a number of other European races, either been insufficiently
studied or grouped within one of the other European groups.. The
Macedonian bee A. m. cecropia Kiesw., now appears to belong to Carnica
race, but the position of the Russian steppe bee A. m. acervorum and the
trans Caucasian A. m. remipes are not so clear.
24
2- f- ii- (2)- African races:
The species name for the African bee is Apis mellifera Linnaeus (1758).
Even this name has been subject to controversy. In 1761 Linneaus changed
the name to Apis mellifica because the original name means, “honey carrier”
rather than “honey maker” which he preferred. According to the principal of
priority (Art. 23, International Code of Zoological Nomenclature, hereafter
abbreviated as ICZN; Ride et al., 1985), the oldest available name is the
valid name of a taxon. In spite of this long-standing rule, the junior name
still appears in some European literature.
According to a recent review of the infraspecific nomenclature of Apis
mellifera (Engle, 1999). 10 valid subspecies are recognized in Africa:
2- f- ii- (2)- a- Apis mellifera intermissa “Tellian bees” (Maa, 1953):
It is a North African race, found north of the Sahara from Libya to
Morocco. Small, dark bee it is reputedly aggressive and swarms frequently,
rearing over 100 queens in each swarming period. During droughts, over
80% of colonies may die; owing to intensive swarming colony numbers
rebound when conditions improve ( Louveaux, cited in Ruttner, 1975b).
2- f- ii- (2)- b- A. melliferea lamarckii (Cockerell, 1906):
It is Egyptian bees, formerly named A. m. fasciata, found in northeast
Africa, primarily in Egypt a long the Nile valley. Like Intermissa, they rear
numerous queens, with one colony recorded as rearing 368-queen cell and
producing one small swarm with 30 queens. They appear to be more closely
related to the bees of Central Africa, however, based on similarities of dance
dialects between Lamarckii and Adansonii (von Frisch, 1967a). Thus Ruttner
(1988), showed that Lamarckii belongs statistically, to the sub-Saharan
rather than the North Africa races primarily because of its yellow
pigmentation and small slender body.
25
2- f- ii- (2)- c- A. mellifera scutellata Lepeletier (East Africa bees):
Though much of their range, were considered to be adansonii (Smith,
1961) until Rutner (1975b) proposed that these bees from the savannahs of
central and equatorial East Africa and most of South Africa were actually a
separate sub species, A. m. scutellata. This has created some confusion in
1956 were thought to be Adansonii, and all of the literature concerning these
bees prior to the mid-seventies refers to them as Adansonii are indeed
different subspecies, and also about which sub species was introduced to
Brazil. Since Ruttner´s 1975 study, is the most recent and complete
taxonomic evaluation of African bees, although the status of these
subspecies is currently being re-evaluated and additional evidence may
result in further changes.
A. m. scutellata, (Lepeletier, 1836), in south Africa; it is a small bee
with a relatively short tongue, it is highly aggressive, swarms and absconds
frequently ,and is able to nest in a broad range of cites from cavities to open
nests.
2- f- ii- (2)- d- A. mellifera adansonii (Latreille, 1804):
Are found in West Africa and are conspicuously yellow in colour.
They appear to be similar to Scutellata in many of their behaviours, but have
not been well studied.
2- f- ii- (2)- e- A. mellifera monticola (Smith, 1961) “Mountain bees”:
Found in southeastern Africa, are of interest because of the high
altitude at which they are found in Tanzania, from 1500 to 3100 m. It is a
large, dark, gentle race, with longer hairs than the other African bees.
2- f- ii- (2)- f- A. mellifera capensis (Escholtz, 1822):
In South Africa “Cape bees” are only found at the tip of South Africa
and are unique among Apis mellifera in the common occurrence of female-
26
producing laying workers. They are morphologically similar to Scutellata,
but the degree of ovariole development and ability to lay parthenogenesis
females regularly separates them from the Scutellata group.
Other African races are found in limited areas of Africa. These may be
morphometrically distinguishable from other races. Only a few specimens of
each have been examined, and their biology has not been studied well
enough to reach firm conclusions about their taxonomic status. These
subspecies include:
A. m. major Ruttner.
A. m. sahariensis (Baldensperger, 1932) in Maghreb.
A. m. jementica (Ruttner, 1976), in Northeast Africa.
A. m. litorea (Smith, 1961), in south-easernt Africa.
A. m. unicolor (Latreille, 1804) in Madagascar.
Hepburn & Radlo. (1998) Showed that these 10 subspecies have clearly
separated morpho-clusters and they precisely delineated their geographical
distribution.
They also indicated geographical zones of high morphological variance
within and between colonies, thus identifying hybrid zones among
subspecies.
2- f- ii- (3)- Oriental races :
A number of oriental races from Turkey west to Iran have been
proposed, including A. m. syriaca, A. m. anatolia and A. m. meda, which is
similar to ligostica (Ruttner, Pourasghar, and Kauhausen, 1985). The
relationships between these groups have not been studied. A thorough
evaluation of the systematic of oriental honey bees might be important, since
presumably transition forms between temperate and tropically evolved races
might be found, possibly between A. mellifera and A. cerana.
27
2- f- ii- (4)- Northern and South American races:
Although honeybees are not native to Northern or South America, both
European and African races have been introduced in the last few hundred
years. In North America racial lines of European origin have generally been
maintained, although extensive mating between races and different selective
criteria by queen breeders have undoubtedly modified some of the bee’s
original characteristics. In South America the introduction of African bees in
1956 has resulted in the establishment and spread of A. m. scutellata
throughout much of South and Central America. These bees will be referred
to as “Africanized“ to differentiate them from bees studied in Africa, but
they appear to be morphologically, behaviourally, and ecologically almost
identical to Scutellata, and thus don‘t constitute a separate race.
2- g- Probability and identification of the honeybees:
Identifications are made with various degree of assurance. When
specimens of a species have unique and clearly defined structural or other
characters, then the identifications are irrefutable within the context of the
current classification. For example A. mellifera is distinguished structurally
from its nearest relative, A. cerana. The latter species have two vein lets
extending distal from the large basal cell of the hind wing rather than one as
in A. mellifera. This and other key characters have proven to be consistent
and species specific. Specimens of A. mellifera, therefore, can be
conclusively identified (Daly, 1988).
The geographical races and other distinctive populations of A. mellifera,
however usually cannot be conclusively identified. They exhibit characters
that may overlap to some degree or may grade imperceptibly into adjacent
populations.
Based on comparison of samples known to be typical of two or more
populations, one can estimate how often an identification based on certain
characters is likely to be correct. If quantative characters are used, statistical
28
analysis can provide a statement about the probability that a new sample is
correctly identified. In this case, the identification is probable rather than
conclusive. Identification of subspecies, geographical races, genetic hybrid
swarms, ecotypes, or biotypes are usually of this nature.
The accuracy of probable identifications depends entirely on how
representative the initial samples are with respect to the total populations to
be identified.
The samples unit is usually a collection of bees from a colony and
identification is based on pooled extracts or averages of characters of the
collection. Some procedures can identify individual bees. Thus Daly (1991)
demonstrated that, probability statements of identification must be
interpreted within the context of the procedure. For example, with current
methods in morphometrics, the statement that a colony collection is
Africanized (Brazil bees race) at 0.7 or 70% probability also indicates the
sample is European at 0.3 or 30% probability. The samples could be of
normal Africanized bees or normal European bees, but it is more likely to be
the former based on the analysis of known Africanized or European bees.
The statement doesn‘t mean that the colony is composed of 70% Africanized
bees and 30% European bees or that worker have 70% Africanized genes
and 30% European genes. To make such statements, the procedure must be
able to distinguish individuals or be based on genetic analysis, respectively.
Furthermore the statement that a new sample is Africanized at 1.0 or 100%
probability is not conclusive identification; it is still a probable identification
based on the initial analysis of known Africanized and European bees.
Daly (1991) showed that, all probable identifications carry the risk of
actual
misidentification.
Any
method
(morphometric,
biochemical,
behavioural, genetics) that yields a probable samples are being identified,
even a small risk becomes an important consideration in terms of the
numbers of samples that may be misidentified.
29
2- h- Morphometrics:
Morphometrics is the measurement and analysis of form. In biology the
forms measured are morphological structures of organisms and analysis is
usually by statistics. It is widely used in the study of insect life history,
physiology, ecology, and systematic (Daly, 1985). Morphometric analysis is
very important because it deals with variations in phenotypic characters
which are induced both by genetic and environmental factors (Daly, 1991).
To be useful in identification, the genetically determine racial differences
between taxa must be large enough to provide distinguishing characters in
spite of environmentally induced and local genetic variation with each taxon.
Since morphological characters generally have a higher heritability than
physiological characters (Soller and Bar Cohen, 1968; Falconer, 1989), the
analysis of morphological characters is an important tool in the
discrimination of different populations of honeybees.
2- h- i- Historical background of bee’s morphometrics:
The first man who used the name Apis mellifera was Linnaeus in 1758,
(Ruttner, 1988).
Morphometric of bees Apis mellifera have been extensively analysed,
especially during the first third of this century by Russians scientists, when
they were searching for bees with a long proboscis for the efficient
pollination of red clover and when apiculturists thought bees with longer
tongues that could reach nectar in flowers with long corolla tubes (Daly,
1991, 1992). In 1992, Merril stated that the early studies of honeybees gave
us much information on geographic variation and inheritance of
morphometrics and environmental influences on morphometrics.
The early exact morphometric measurement on honeybees was started
by Koshevnikov (1900) and followed by Martynov (1901) and Kulagin
(1906) (Alpatov, 1929). Ruttner (1978) wrote that in 1906 H. von ButtelReepen, made the first attempt to organize the multiplicity of honeybee
30
types in a rational manner, by using trinomial system. First the genus and
species designation; Apis mellifica ( now A. mellifera) and geographical
races or variety as third name. He also wrote that between 1925 and 1940
Alpatov and Geotze, provided a more exact bases for describing bee races by
introducing
biometrics-exact
measurements.
However,
the
early
classification of honeybees was based on individual morphometric
characters without statistical analysis.
Since then various investigators at different times in different places
continued to improve the gradual development of morphometric analysis of
honey bees. The earliest apparently advanced morphometric measurement
with adequate honeybee samples for statistical analysis was done by
Chochlov (1916), Michailov (1924) and Alpatov (1929) who classified
honeybee races based on morphometric measurements. Beside their attempts
to classify honeybees, they were also able to recognize the effects of
environmental factors on geographical variation of honeybees. They
demonstrate the linear relationship between the honeybee tongue and hind
leg length with latitude in the area along a line from the Baltic Sea to the
Caucasus Mountains.
In addition to the length of tongue, Alpatov included more characters
like femur, tibia, metatarsus, length and width of wing and size of wax
mirror. As Alpatov (1929) took more characters, he found trends in
geographical variation, opposite to tongue length, which is decrease in body
size from north to south in the plains of Russia. Alpatov (1933) studied
geographical variation of the honeybees. He found that bees of southern
localities as compared with northern ones had smaller body size, are larger
number of hooks on the hind wings, a smaller relative surface of the first
wax glands, relatively broader wings and legs. They were also more yellow
coloured on the tergites with longer tongues. He further wrote that, the first
important difference between A. m. adansonii and A. m. unicolor was the
31
longer tongue of the former; 5.820± 0.011 and shorter one of the later 5.587
± 0.007. In summary of all the differences observed, Alpatov concluded that
the relation of the yellow and dark African bees to each other, were the same
as that of A. m. mellifera, the yellow Italian bee to A. m. mellifera, the dark
European bee.
Ruttner et al., (1952) concluded that, the colouration of the exoskeleton,
which at one time constituted one of the bases for races taxonomy, has
become, at least for European bee races of little importance. They identified
three specific statistically constant groups of characters:
(1) Hair characteristics.
(i)colour of hair.
(ii)length of over hairs on the 5th . tergite.
(iii) width and the thickness of the tomenta (tomentum index).
(2) Wing venation where the cubital index was most useful-defined as ratio
of distances ‘a‘ determined by the point at which the nervous recurrence Nr;
joins the lower vein of the third cubital cell ‘c‘ of the front wing.
(3) Length of the proboscis.
Thus Alpatov (1929), summarized that the following conditions have
pronounced effect on the body size of worker bee:
(1) the season of development.
(2) the temperature of the surroundings during the pupal stage.
(3) the size of the cell.
(4) feeding by nurse bee of different age.
(5) individuality of the colony.
Alpatov noted that absolute body size and changes is some proportions
could be related to reduced larval feeding.
Grout (1937) demonstrated that, workers reared from enlarged brood
cells were significantly larger than workers from normal brood cells.
Recently, Eischen et al., (1982, 1983) reared worker larvae with different
32
numbers of nurse bees, finding positive correlations between the number of
nurse bees and dry weight and life span of the progeny.
Moreover, in 1929, Alpatov tried to indicate the rules of genetics and
environment in the geographical variation of honeybees, though he reviewed
the pioneering studies of Russian scientists on the effect of genetics and
environment in geographical variation in bees. By transplanting colonies to
new localities in Russia and observing European races in the United State,
he concluded that, the races and the geographical variants within races has
specific characters, including morphometrics, that were genetically
determined. During the same early period in the United States, Kellogg and
Bell (1904), Casteel and Phillips (1903), and Phillips (1929) produced major
papers on bee biometry and showed that drones were more variable than
workers. This feature of drones was later explained by Brueckner (1976) to
be the consequence of reduced developmental homeostasis that arises from
their homozygous genome.
The genetic basis for seven morphometric characters in bees, was first
established by Roberts (1961) who estimated heritabilities at 0.28 (number
of hamuli) to 0.85 (wing width and tongue length). Morphometric characters
are usually used for breeding and certification in Europe (Ruttner, 1988a).
Wayne and Hendrickson (1973), wrote that, the source of data in biological
taxonomy is essentially always the phenotypic; defined by Dobzhansky as “
what is perceived by observation of the organisms structures and functions,
or what a living being appears to be to our sense organs unaided or assisted
by various devices”.
Mani (1973) stated that, whether a species occurs or not in a given
biotop, depends upon:
(i) historical grounds: the species must have had opportunities to reach the
given biotop and having reached it, it must also have actually penterated it.
33
(ii) topographical grounds: geographical barreries to dispersal were an
important factor.
(iii) ecological grounds: the present state of affairs that can either exclude
the existance or determine the density of population.
Ruttner (1988) proved the presence of gradual variations in quantative
characters of honeybees with geographical latitude changes along the east
coast of the Atlantic Ocean from Scandinavia to the Cape of Good Hope.
Alpatov (1929) also noted the difficulty of measuring the overall body
size of honeybees and substituted single parts of the abdomen (sternites and
tergites), which are closely correlated to the overall size of the honeybee
worker (Ruttner, 1988).
The early biometrics of honeybees was initially based exclusively on
characters related to size of body parts. Goetze (1930, 1940, 1964)
introduced two taxonomically significant quantative characters to Alpatove‘s
list like indices of venation of the forewing and length of hairs of the
abdominal tergites, which proved very efficient in discriminating European
races. Louis (1963) also made an extensive study on the geographical
variability on wing vein crossing points.
Goncalves (1972) using the number of hamuli, categorized A. mellifera
castes into two; low line with hamuli ranges of 11- 13 in queens and 12- 13
in drones, and high line with hamuli ranges of 21- 22 in queens and 11- 30 in
drones. He further stated that, queens and drones equally influence the
phenotypic development of the characteristics in descendants.
Smith (1972) stated that, geographical barriers or environmental factors
limited distribution of the African honeybees. He went on to say that all
colour forms might be produced by the same queen, although there is a
tendency towards the darker form and higher proportion of dark workers in
the more mountainous areas at high altitudes.
34
Along with the development of morphometric measurements in
1930‘s statistical methods such as mean, standard deviation, coefficient of
variation, ratio of differences and correlation coefficients were introduced to
compare the geographical variability between European races of honeybees.
Before 1964 morphometric analysis of honeybees entirely based on sample
statistics and univariate methods.
For the first time Dupraw (1964, 1965) used multivariate analysis to
classify honeybee races. Moreover, Dupraw (1965) used discriminant
function analysis of 15 variables, based on venation of 13 forewing angles
and length and wide of the forewing and was able to establish cluster group
of European, African and Asian honeybees, which are very similar to
Ruttner´s (1988) geographical races of honeybees.
2- h- ii- Recent developed biometry of Apis mellifera.
Wafa et al., (1965) presented mean values of biometrical measurements
of the Egyptian honeybee A. m. lamarckii. Tongue length 5.65 ± 0.009 mm
and range of 5.31- 5.83 mm. Fore wing length 8.36 ± 0.007 mm and range of
8.18- 8.56 mm. Fore wing width 2.84 ± 0.003 mm and range of 2.80- 2.93
mm. Cubital index 2.46 ± 0.016 mm and range of 2.23- 2.62 mm. No. of
hooks on hind wing 21.10 ± 0.001 and range of 20.54- 21.98. Length of
basitarsus 2.21 ± 0.005 mm and range of 2.17- 2.23 mm. Width of basitarsus
1.09 ± 0.005 mm and range of 1.04- 1.11 mm. Tomentum index 0.26 ±
0.022 mm and range of 0.18- 0.28 mm. Length of first wax gland 1.30 ±
0.003 mm and range of 1.23- 1.35 mm. Width of first wax glang 1.96 ±
0.004 mm and range of 1.90- 2.07 mm. They found that most differences
between the means were non-significant and the coefficients of variation for
all characters except cubital and tomentum indices were relatively low. They
concluded that the group of bees studied were homogenous.
Daly (1975) applied univariate and multivariate statistical analysis for
identification of the Africanized bees of South America. By univariate
35
analysis, a series of overlapping for each character was found without clear
separation. In multivariate analysis approach, he was able to identify three
distinct groups:
(i) bees from Africa and their hybrids.
(ii) samples from Colombia and Venezuela.
(iii) mixed bees of European origin from Costa Rica, Surinam and United
States.
Mitev et al., (1975) stated that, three varieties of adansonii existed in
Guina Republic: A. m. adansonii (Latr.), A. m. monticola (Smith.) and A. m.
litorea (Smith.). They further divided these honeybees by the colour of
chitin to five groups. The following morphological measurements for the
Guina bees were provided: length of forewing 8.457 ± 0.018 mm. and 8.551
± 0.02 mm. in 1971, and 8.510 ± 0.017 mm. and 8.794 ± 0.016 mm. in
1972. Cubital index, 2.035 ± 0.043 in 1971, and 1.936 ± 0.029 mm. and
2.304 ± 0.038 in 1972. length of proboscis 5.445 ± 0.015 mm. and 5.468 ±
0.020 mm. in 1971, and 5.17 ± 0.02 mm. and 5.422 ± 0.038 mm. in 1972.
Cornuet et al., (1975) classified ten worker samples according to the
average values of four indices:
(1) width of yellow band on the second tergite.
(2) Length of hairs on the 5th tergite.
(3) Width of tomentum on the 4th tergite.
(4) length of proboscis.
Then thirty workers samples by the average values of two wing indices: (5)
and (6); ‚a‚ and ‚b‚ components of cubital index.
Ruttner et al., (1978) established a standard biometry of honeybees
based on 40 morphometric characters, by screening the less significant ones
and induced more characters than Alpatov (1929), Goetze (1930, 1940,
1964), and DuPraw (1964, 1965), as in table ( 2).
36
Based on the standard biometry, Ruttner (1988) recognized 24 distinct
taxonomic groups or geographical races of Apis mellifera, 7 in the Near
East, 10 in Africa and 7 in North and southeast Europe.
However, by applying a step-wise discriminate analysis procedure,
Ruttner et al., (1978) ; Daly and Balling (1978) showed the possibilities of
discriminating one race from another using fewer numbers
of selected
characters based on the region under investigation.
Ruttner (1988), particularly suggested the possibilities of using one
third of the original selected characters to discriminate African races of
honeybees, however, he emphasised the inclusion of different categories of
characters such as size, hairs, colour and wing venation. Crewe et al.,
(1994), also showed that 10 characters are fully adequate to discriminate
honeybees of the southern Africa region. Moreover, Hepburn and Radolff
(1996, 1997); Radolff and Hepburn (1997a,b) using 11 morphometric
characters were able to classify African honeybee populations in distinct
geographical races.
Along with the development of morphometric measurements, the
introduction of different multivariate techniques like principal components
and factor analyses were used to detect clusters of colonies within
populations (Ruttner et al., 1978; Ruttner, 1988). Step-wise discriminate
analysis was used to confirm the separation of clusters, to detect the most
discriminatory variables and to calculate the percentage of correctly
classified colonies (Ruttner, 1988; Daly, 1992).
To depict the distances between clusters, dendrograms and mahalanobis
distances were introduced (Tomasson and Fresanaye, 1971; Cornuet et al.,
1975; Cornuet and Garnery, 1991a, b; Daly, 1992). The introduction of
different multivariate techniques proven to be powerful tools in the
discrimination of honeybee races, ecotypes or strains within a race and
37
between genetic lines (Louis et al., 1968) and even to the level of F1 hybrids
(Rinderer et al., 1990).
2- i- Historical background of African honeybees Classification:
In 1979, Pager stated that, the earliest information on African bees was
found on a copper plate engraved after a drawing by the Dutch author
Pierter de Marees, first published in 1602.
Baldensperger (1924) wrote that in Abyssinia (Ethiopia) east or in
Senegambia west of Africa, both the Adansonii and the Intermissa were
found; and that there were no really pure ones of the first one or the other.
He further stated that, Frere Jules in Abyssinia, found two colonies of black
and yellow bees in the same hive, daughters of the same mother.
Baldensperger (1926) concluded that, the bee was an outcome of natural
breeding and not been produced by beekeepers in their selection.
Harris (1932) writing on the bees in Tanganyika Territory (Tanzania)
listed three races; Apis m. unicolor (Latr.), Apis m. unicolor intermissa
(Butt-Reap.) and Apis m. unicolor adansonii ( Latr.). He further stated that,
Adansonii was the common bee of the Territory, while unicolor was
restricted to the highlands of the North. Intermissa was only recorded from
Mountain Kilimanjaro and the highlands in the vicinity of Lake Nyasa in the
Southwest. This nomenclature was later proven incorrect by the work of
Smith (1961), as follow: by reviewed the development of taxonomy of
African bees he listed seven races: A. m. unicolor of Madagascar, Mouritus
and Reunion Islands, A. fasciata of Egypt and A. m. adansonii of Senegal,
all named by Latreillein 1804. A. m. scutellata of South Africa, A. m.
nigritarium of Congo and A. m. caffra, which resembled A. m. capensis,
were named in 1932 by Lepeletier. The A. m. capensis of Cape of Good
Hope, named by Eschscholtzin 1922. A. m. fasciata was renamed in 1906 by
Cockerell. In the same publication, Smith presented biometrical data on
three African bee races. A. mellifera adansonii, which he stated inhabited
38
Africa from the Sahara desert in the north to the Kalahari and Karoo desert
in the south: length of forewing 8.1 to 8.7 mm. with colony average of 8.42
to 8.51 mm. and tonguth 5.8- 5.9 mm. with mean of 5.85 mm. A. m.
monticola, length of forewing 8.7- 9.3 mm. with colony average 9.0- 9.09
mm. and tongue length 5.9- 6.2 mm. with mean of 6.05 mm. A. m. litorea
length of forewings 7.9- 8.4 mm. with colony average 8.13- 8.23 mm. and
tongue length 5.7- 5.8 mm. with mean of 5.75 mm.
The presence of morphological variability within the honeybees of
Africa has been recognized since the 1920´s (Rotter, 1920, 1921;
Baldensperger, 1922, 1924, 1932; Rueher, 1926; Giavarini, 1937).
However, classification was mainly based on colour variations. Such
descriptive classification continued until 1950´s (Aurelien, 1950; de Roeck,
1950; Dubois and Collart, 1950; Alber, 1952: Hassanein and Elbanby, 1956;
Kaschhef, 1959). In this period all the honeybees of the sub-Saharan regions
of Africa were considered as one taxon due to the presence of common
behavioural characters and uniform yellow pigmentation.
Kerr and Portugal-Araujo (1958) by means of genetic crossing
confirmed that the honeybees of Africa, south of Sahara belong to the same
species of
Apis mellifera. For the first time they recognized five
morphological distinct races: A. mellifera scutellata in all area south of
Sahara ( except the Cape region), A. m. capensis in southwest parts of the
Cape region, A. m. lamarckii in Egypt along the Nile Valley, A. m. unicolor
in Madagascar and A. m. intermissa in northwest Africa between Libya and
Morocco, which are geographically separated in different regions of the
continent of Africa.
Smith (1961) classified the honeybees of East Africa based on univariate
analysis
of
morphometric
measurements.
Besides
morphometric
measurements he included behavioural and ecological characters and he
recognized three races, A. m. scutellata, A. m. litorea and A. m. monticola.
39
DuPraw (1964, 1965) in his multivariate methods of honeybees
morphometric study, tried to discriminate the honeybees of Africa as well on
the bases of size of forewing and venation of wing angles. Thus
DuPraw(1965) by using non-linear classifications, identified geographical
variants native to Africa south of the Sahara which included both the yellow
bees of Central Africa A. m. adansonii and the dark forms from Cape of
Good Hope A. m. capensis and from Madgascar A. m. unicolor. He however,
found it impossible to distinguish on the basis of wing variable, between
Central Africa bees and those from Cape of Good Hope. The body colour
might be a useful supplementary character for this purpose. The situation
was not entirely simple, Dupraw concluded.
In his comprehensive multivariate study of geographical variation in
African honeybees Ruttner (1975,1988) recognized 10 races of A. m.
honeybees in different regions of the continent: A. m. adanosii, A. m.
lamarkii, A. m. litorea, A. m. jemenitica, A. m. monticola, A. m. scutellata,
A. m. sahariensis, A. m. intermissa, A. m. unicolor, and A. m. capensis. He
found that climate is one of the major isolating factors for races of
honeybees in tropical Africa. However, he also noted that the race of
honeybees (A. m. adanosii in west Africa) occurred in distinct ecological
areas over vast geographical distances. He also observed the existents of
different races of honeybees (A. m. jemenitica and A. m. adanosii) without
substantial ecological differences.
The work of Ruttner was based on a macro level sampling at continent
level (Hepburn and Radloff, 1998) and, as Ruttner (1988) stated,
his
morphometric studies of all Africa honeybees doesn’t achieve a complete
analysis of all variability of the huge continent nor does it indicate the
borders for the identified geographical races.
Recently, different authors tried to classify African races of honeybees
based on morphometric, DNA and pheromone analyses and got a more
40
refined pictures for different regions of the continent (Saeed,1981;
Mohamed; 1982, Mogga 1988; Meixner et al., 1989, 1994; Kassaye,1990;
Smith et al., 1991; Lebdi-Grissa et al., 1991b; Cornuet and Garnery, 1991b;
Kerr, 1992; Elsarrag et al., 1992; Crewe et al., 1994; Moritz et al., 1994;
Hepbun et al., 1994; Garnery et al., 1995).
Moreover, morphometric classification of honeybees at the continental
level with large sample size across five major transects of the continent with
different multivariate procedures was conducted by Hepburn and Radloff
(1996, 1997, 1998) Radloff and Hepburn (1997a, b), Radloff et al., (1997)
and Radloff et al., (1998). Along with the morphometric data they also used
pheromone analysis to discriminate the cluster groups and were able to
recognized a number of variations and ecotypes, which were not detected
earlier (different morphoclusters of Scutellata, Jemenitica and Monticola).
They also observed variations in races across the different ecological and
climatologically zones of the continent and tried to locate zones of
introgression and hyperdization of natural populations in different regions.
However, the classification of honeybees into well-defined sub species still
remains a controversial issue (Hepburn and Radloff, 1998).
Nowadays there are three major thoughts reflected in presenting the
observed geographical variations between populations of honeybees..
These are:
(1) as sub species or geographical races (Ruttner 1988, 1992).
(2) as adaptive ecotypes derived from adjacent populations
( Kerr 1992).
(3) as product of asynchronous gene fluctuations within a contiguous met
population for which the term “subspecies” may not be appropriate
(Hepburn and Crewe,1991: Hepburn and Radllof 1998).
41
Due to high migration, absconding and swarming behaviour and
consequential genetic mixing, lower molecular differentiation is observed
among African subspecies (Franck et al., 2001).
Moreover, the percentage of gene flow among honeybee population;
lack of coherence between the distributing of the biological traits and
morphometrically defined subspecies of Africa (Hepburn and Radloff,1998)
and the existent of the same subspecies in distinct ecological areas and the
occur rents of differences (Ruttner,1988) make the classification of
honeybees of the continent more complex. Besides these general problems
of classification of African honeybee populations, certain regions of the
continent like the East North of Africa in general and Sudan in particular
have not yet been adequately studied.
2- j- East Africa races of honeybees:
According to Hepburn, et al., 1998 the honeybees reported from the
east Africa region include:
2- j- i- A. m. scutellata:
At mid-altitudes between 500-2400 m. in woodland and tall grass
savannah of Kenya and Tanzania (Smith, 1961). Ruttner (1988) and
Hepburn and Radloff (1998) indicated the wide distribution of A. m.
scutellata from Ethiopia doun to South Africa including countries Such as
Rwanda, Burundi, Uganda, Malawi and Zembabwe.
2- j- ii- A. m. monticola:
Reported to occur in East Africa mountains areas between altitiudes of
2400-3200 m. (Smith, Ruttner, 1988). The distribution of this subspecies is
thought to be unique and consist of disjunct areas, which are isolated by
ecological barriers (Ruttner, 1988). This bee is reported to occur in
Tanzania, Kenya, Burundi and Ethiopia.
42
2- j- iii- A. m. jementica:
Asmall yellow bee, reported from hot and arid zones of East Africa
(Ruttner, 1998).
However, the distribution of this bee is large, extending 4500 km from
Chad (Gadbin, et al., 1979), Sudan (Ruttner, 1975; Rashad and Elsarrag,
1980, Saeed, 1981; Mohamed, 1982; Mogga 1988), Somalia and Saudia
Arabia (Ruttner, 1988), Yemen (Ruttner, 1975) up to Oman (Dutton et al.,
1981).
2- j- iv- A. m. litorea:
Reported to occur in the warm and humid coastal plains of Kenya and
Tanzania at altitudes between 0-500 m. above sea level (Smith, 1961). This
bee is replaced by A. m. jemenitica in the arid coastal plain of Somalia, but it
extends south wards to the costal plains of Mozambique (Ruttner, 1988).
2- j- v- A. m. sudanesis
Reported to occur in Sudan (Mogga, 1988) and in Ethiopia (Radloff
and Hepburn, 1997a).
2- j- vi- A. m. bandasii
Reported from Sudan (Mogga, 1988) and Ethiopia (Radloff and
Hepburn, 1997a).
2- k- Classification of the Sudanese honeybee races:The first honeybee race reported to occur in Sudan was A. mellifera
nubica, by Ruttner (1976). He described eleven African honeybee races
including A. m. nubica (yemenitica), the Sudanese bee race. He gave the
average morphometric as follows: Length T3 +T4 3.965 mm., Tongue
length 5.45 mm., hindleg length 7.2 mm., length and width of forewing
8.219 mm., and 2.88 respectively, sternite 6(L/W) 89.31 mm., colour (cT3)
8.5, colour scutellum 7.28, cubital index 2.46 and wing venation B4 100.3
and J10 52.28. He also described A. m. yemenitica of southwest Arabian
Peninsula as being similar to the nubica and litorea races.
43
Elsarrag (1977), studied morphometrics of worker honeybees from
four provinces in Sudan. He recorded the following averages: tongue length
5.50 ± 0.019 mm., flagellum length 2.66 ± 0.006 mm., basitarsus length 2.20
± 0.006 mm., basitarsus width 1.11 ± 0.003 mm., number of hair rows on
inner surface of basitarsus 11.90 ± 0.014. forewing length 8.60 ± 0.014 mm.,
and width 3.02 ± 0.008 mm., cubital index 2.37 ± 0.019, number of hamuli
21.40 ± 0.009, length of T3+4 3.70 ± 0.008 mm., slenderness (L/W) 86.00 ±
0.003 and percentage of yellow colouration on T3 71.38 ± 0.003. He stated
that the results showed high significant differences between provinces for all
tewelve traits studied. He concluded that, this was an indication of the bee
samples not belonging to the same race.
Saeed (1980) and Mohamed (1982), both investigated morphometrics
of honeybee worker of Sudan. They both reported highly significant
differences between the samples in the twelve traits studied.
Dutton et al., (1981) find two widely seperated A. mellifera populations
in mountains of northern and southern Oman identical with each other and
with A. m. yemenitica from north Yemen. They also established
morphometrical relationship between them and the bees from Sudan. They
measured the following parameters: length of hairs, tergites 3+4, hindleg,
tongue, length and wide of the forewing, length of sternite 6, colour of
tergite 3 and scutellum and wing venation angles A4, D7 and G18 of worker
bees. They also measured drones parameters.
Mogga (1988) investigated on the taxonomy and the geographical
variability of the Sudanese honeybee A. mellifera from four different
geographical zones, namely semi-desert, poor savannah, rich savannah and
forest zones. He presented the means and average values of biometrical
measurements of the Sudanese bees as follow: The mean length of proboscis
4.89 ±
5.76 mm., with an average 5.33 mm.; femur 2.14 ± 2.42 mm.
44
average 2.28 mm.; tibia 2.75 ± 3.05mm. average 2.90mm.; metatarsus 1.75
± 1.94 mm., average 1.84 mm.; total hindleg 6.72 ± 7.41 mm., average 7.06
mm.; width metatarsus 0.98 ± 1.13 mm., average 1.05 mm.; metatarsal index
54.01 ± 59.77mm., average 56.89mm.; forewing length7.88 ± 8.64 mm.,
average 8.26 mm., forewing width 2.69 ± 3.00 mm., average 2.85 mm.;
cubital vein “a” length 3.75 ± 4.43 mm., average 4.09 mm., cubital vein “b”
1.55 ± 2.05 mm., average 1.80mm.; the cubital index a/b 1.93 ± 2.67 average
2.30; number of hooks in the hindwing 18.80 ± 22.40, average 20.60; The
angles of the forewing: A4 30.60 ± 34.40 average 32.50; B4 95.20 ± 104.33,
average 100.27; D7 97.13 ± 104.80, average 100.97; E9 17.87 ± 21.20.
average 19.53; G18 93.13 ± 102.07, with an average 97.60; J10 49.60 ±
55.60, average 52.60; J16 89.60 ± 98.33, average 93.97; K19 75.47 ± 86.73,
average 81.10; L13 12.33 ± 15.60, average 13.97; N23 85.40 ± 92.80,
average 89.10; O26 33.40 ± 43.07, average 34.07; length tergite3, 1.90 ±
2.11 mm., average 2.01 mm.; length tergite4, 1.84 ± 2.07 mm., average 1.96
mm; the body length (T3+ T4), 3.75 ± 4.19 mm., average 3.95 mm.; length
sternite3,2.34 ± 2.60 mm., average 2.47 mm.; length wax mirror on S3 0.97
± 1.26mm., average 1.12 mm.; width of wax mirror on S3 1.84 ± 2.06 mm.,
average 1.95 mm.; distance between waxmirror in S3 0.23 ± 0.40 mm.,
average 0.32 mm.; length of hair in tergite 5 0.17 ± 0.22 mm., average 0.22
mm.; width of tomentum on tergite4 0.42 ± 0.75 mm., average 0.50 mm.;
width of dark stripe on tergite 4 0.19 ± 0.35 mm., average 0.28 mm.;
tomentum index 1.61 ± 2.56, average 2.08; length sternite6 2.19 ± 2.41
mm., average 2.30 mm.; width sternite 6 2.51 ± 2.82 mm., average 2.67
mm.; the abdominal slenderness (L/W) 82.63 ± 87.92, average 85.28;
colouration on tergite2 5.80 ± 9.00, average 7.40; colouration on tergite3,
5.60 ± 9.00, average 7.30; colouration on tergite4 2.87 ± 6.80, average 4.83;
45
colouration on scutellum 3.80 ± 8.13, average 5.97; colouration of
metatargum 0.00 ± 5.40, average 2.70; colouration on labrum1, 2.53 ± 5.07,
average 3.80; and labrum2, 0.20 ± 2.87, average 1.53.
2- l- Some biological and ecological aspects of honeybees:
One of the most highly polytypic species of all honeybee races is Apis
mellifera, with large geographical variations not only in morphology but also
in behaviour and physiology.
Merrill (1922), noticed the geographic variations and the inheritance of
morphological characters and the influence of environment of the
morphological characters of honeybees. Winston et al., (1983), Stated that,
one of the most striking aspects of honeybee biology was the variability
found within and between races of A. mellifera. These included behavioural
variants, morphological and physiological characteristics as colour, size,
tongue length, defensive behaviour, amount of propolis and burr comb used,
dialects of dance language and susceptibility to diseases.
The biology of honeybees is variable within and between races of Apis
mellifera is a result of adaptive responses to diverse ecological conditions
like climate, patterns of resource abundance and predation pressure (Ruttner,
1988; Hepburn and Radloff, 1988). As a result, temperament, during the
development stages, patterns of seasonal variability like brood rearing,
migration, swarming and absconding of bees vary from one race to another.
Moreover, geographical variations in behaviour, like orientation,
defence, tendency towards propolis collection and utilisation, robbing and
drifting, vary from race to race (Lauer and Lindauer, 1971, 1973; Ruttner,
1975).
Understanding the biology and ecology of honey- bees of an area is
very important not only for classification purposes but also for the efficient
and profitable management of honeybees according to their biological
behaviour and respective ecology.
46
2- l- i- Biological factors:
Research and studying the biological characteristics of bees belonging
to different races were much more important from the point of view of
practical beekeeper. Ex:
2- l- i- (1)- Migration:
Migration of honeybee colonies in the tropics considered as an
evolutionary adaptation to escape darsh periods and also as a means of
exploiting resource available in different ecological habitats at different
times (Chandler, 1976; Castagne, 1983; Hepburn and Radloff, 1995).
In a sense of evolution, the honeybees of tropical and temperate
regions develop different means of surviving to escape harsh periods. The
honeybees of the tropics adapt by migrating to resource rich areas, while the
temperate bees survive by means of massive hoarding (Chandler, 1976).
Seasonal migration of honeybees is considered as a unique
characteristic of tropical honeybees (Ruttner, 1988). Honeybees of most subSaharan Africa are reported to migrate on a seasonal basis, following dry
periods: A. m. yemenitica (Rashad an El-Sarrag, 1978; Peterson, 1985;
Sawadogo, 1993; Woyke, 1993). A. m. litorea (Ntenga, 1976); A. m.
capensis (Hepburn and Radloff, 1998). A. m. adansonii (Woyke,1989 ;
Adjaloo, 1991 ; Adjare, 1990 ; Mutsaers,1991) ; A. m. scutellata (Smith,
1961; Chandler, 1976; Nightingale, 1993); A. m. monticola (Smith, 1961;
Ntenga, 1976).
However, the tendency of migration differs from ecotype to ecotype,
from one ecological zone to another and also depends on responses to
different stimuli (Chandler, 1976). Lack of food and water, over heating and
fire were reported to be the major causes of migration of tropical Africa
(Fletcher, 1978).
Hepburn and Radloff (1998), indicate that the migration of African
honeybees is not a completely fixed trait and can vary within and between
47
races depending on varying environmental conditions. In Kenya the
migration of A. m. scutellata is reported to be facultative depending on the
availability of resources (Nnightingale, 1983). Moreover, A. m. yemenitica is
reported to not migrate in north Oman and Yemen but commonly migrate in
Sudan and Chad ( Hepburn and Radloff, 1998).
In tropical Africa migration is a common phenomenon and generally
considered as an adaptation to the type of environment where bees live,
losing colonies partially or totally as a result of migration ever season could
be one of the discouraging factors in the development of beekeeping
programs in the region.
However, in Africa as a whole literature on the type or races of bees,
which commonly migrate, the nature of ecology where much migration takes
place, the periods and extent of migration, where the bees migrate to and the
possible causes of migration are very poor. Such information are very
valuable not only from the biological point of view but also from a practical
beekeeping point of view to understand the real causes and associated
factors contributing to the migration of bees. The information would be very
important to formulate possible recommendations to minimise the migration
of bees and to develop an appropriate system to manage bees with migratory
behaviour.
2- l- i- (2)- Reproductive swarming:
For temperate and Africa races of bees the general factor associated
with reproductive swarming are considered the same (Smith, 1961;
Anderson et al., 1983; Ruttner, 1983, 1988, 1992). Paterson (1977),
observed that colonies of bees showing aggressive tendencies were avoided,
thus leaving the more aggressive bees to propagate. This resulted in high
incidence of swarming for the bees to maintain their natural population.
However, unlike temperate races, African races invest more in a
reproductive swarming than in a hoarding strategy (Hepburn and Radloff,
48
1998), which is believed to be a natural mechanism of balancing the loss of
myriad colonies annually due to various hazards in their environment (
Fletcher,1978; Schneider and Blyther, 1988).
The high reproductive swarming potential of African honeybees is
believed to be attributed to many of their adaptive characters such as
fecundity, short developmental period, high foraging efficiency and small
body size (Fletcher, 1978). Moreover it seems more influenced by within
nest conditions than genetic factors (Fletcher, 1978).
Georges (1912) indicate that bees kept in small volume hives show
more inclination to swarm than those in large hives. On the other hand, it
was observed that relatively weak colonies in large volume box hives also
swarm (Hepburn, 1993). Regardless of the population size of a colony and
volume of hives space it was also observed that in some seasons and years
most colonies tend to swarm while in some other seasons and years even
highly overcrowded and strong colonies don’t show signs of swarming
(Nuru and Dereje, 1999).
However, the tendency to swarm still differs from race to another
(Ruttner, 1975). The reproductive periods can also vary between races and
may also be biphasic depending on the agro climatic conditions of the
localities. In African honeybees the close correspondence of the phenology
of reproductive swarming with the local climates, weather and the
availability of forage has been reported (Hepburn, and Radloff, 1998).
Moreover, reproductive swarm time variation within the same
subspecies as the result of ecological and climate variations were observed
for A. m. scutellata, A. m. adansonii, and A. m. capensis ( Hepburn and Jacot
Guillarmod 1991; Hepburn and Radloff, 1988). The number of queens
produced by a reproductive swarm colony of African honeybee also varies
from 10-200 depending on race and agro ecological conditions (Hepburn
and Radloff, 1998).
49
The information about African honeybees on reproductive swarming
such as the period of swarming, the reproductive swarming tendency, and
the number of swarms per colony is some what very week. Moreover, the
types of honeybee populations with high swarming tendencies and the
ecological conditions at which honeybees must swarm, are not sufficient.
Such information is essential to develop an appropriate management system
for bees with a high swarming tendency and in the long run the information
would be important for selecting honeybee colonies with relatively high
hoarding strategy rather than emphasising extreme brood rearing and
subsequent swarming.
2- l- i- (3)- Seasonal cycles of honeybee colonies :Every honeybee race is specifically adapted to its environment of
origin through long periods of natural selection (Ruttner, 1976). The
development of seasonal cycles according to changes in the environmental
conditions is one of the fundamental evolutionary successes of honeybee
colonies (Hepburn and Radloff, 1998).
In tropical Africa, unlike temperate regions, seasonal cycles of the
honeybee colony are governed by dry and wet seasons and the associated
flowering patterns of honey plants (Hepburn and Radloff, 1998).
The close association of seasonal cycles of honeybee colonies (brood
rearing, population build-up, swarming and declining of population (dearth
period) with environmental conditions (rainfall pattern, flowering time and
dry periods) and the existence of time shifts in reproductive swarming within
and between races in different climatic zones of Africa are well established
(Hepburn and Radloff,1998). They were also observed significant
correlations between brood rearing and swarming and the phenology of bee
plants for some of major climatologically and ecological zones of Africa.
Moreover, depending on the rainfall and flowering patterns of an area the
honeybee colony cycle can be monophasic, short or long, depending on the
50
duration of flowering. Honeybee populations, which adapted well to
synchronise to the change in the environmental condition would have a
better chance to survive in the dearth periods and are believed to be more
productive (Hepburn and Radloff,1998).
Information on colony seasonal cycles is very important to develop a
seasonal colony management calendar for different agro-ecological zones of
the country. Moreover, the information would be valuable to select the types
of honeybee populations, which easily synchronise to the change of
environmental situations. Strains of honeybee populations with a fast colony
build-up ability and fast honey storing tendency would be important to meet
the conditions of most parts of the country where flowering periods are
short.
2- l- i- (4)- Temperament :Generally, all tropical African honeybees are considered as highly
defensive or aggressive. The defensive nature of tropical African honeybees
is believed to have arisen due to the extreme pressures of predators and
disturbance in their ecology. However, in some areas of tropical Africa,
beekeeping can be done without protective clothes (Clauss, 1983), while in
some other areas the bees are reported to be swift and violent (Fletcher,
1978).
Moreover, the presences of variation in the degree of aggressiveness
among different races of tropical Africa honeybees and its association with
genetic variations have often been reported (Chandler, 1976; Fletcher, 1978;
Hepburn and Radloff, 1998). Colline et al., demonstrated the presents of
significant variations in sting alarm pheromone levels between genetically
and behaviourally different bees. Moreover, Fletecher (1978) indicated the
existence of inter and intracolonial variations among different honeybees
population.
51
Besides the genetic factors, temperament is also believed to be
influenced largely by climate (Castagne, 1983). Temperature is considered
the most important environmental factor that lowers the threshold responses
of bees (Fletcher, 1978). As a result, honeybees believed to be more
aggressive at hot, low altitude areas than cool- higher ones (Corner, 1985).
On the other hand the same subspecies of honeybees A. mellifera. yemenitica
is reported to be docile in very hot North Oman and North Yemen, but
aggressive in Sudan and in Chad ( Rashad and El-sarrag, 1980; Dutton et al.,
1980; Field, 1980; Gadbin, 1976). Moreover, the presence of aggressive and
docile bees within apiary and its association with colony size and its
variations from season to season and within the day with the changes of
weather have also been reported (Hepburn and Radloff, 1998). In 1985,
Kigatiira stated that, aggressiveness of tropical honeybees seems to be
positively correlated within colony size.
Information of the relative defensive behaviour of different honeybee
populations of different ecological areas and factors associated with
temperament variation is somewhat very poor for Sudan. Along with
morphometric analysis of local honeybee populations, having information
on the degree of the aggressiveness of different honeybee populations would
be important to supplement the morphometric and genetic classification of
honeybees of the area with behavioural characters. The information would
be also important to select honeybee population with relatively gentle and
reasonably manageable behaviour.
2- l- ii- Ecological factors:Knowledge of the environmental conditions where the bees lived is
obviously important. Different authors have indicated that, with some
exceptions, the general morphometric and behavioural characters of
honeybees have been found to be influenced by environmental factors
(Daly,and Balling, 1978; Spivak et al., 1988; Coenuet and Garnery, 1991;
52
Daly and Morse, 1991; Nazzi, 1992). Alpatov (1929) and Ruttner (1988)
demonstrated morphological variations across latitude.
Ruttner (1988) stated that subspecies are a result of an adaptation in
physiology and behaviour to give types of environment, which are
associated with secondary variation in the external morphological characters.
Murphy (1973) and Falconer (1989) also indicated the effects of
environmental influence on morphological and behavioural characteristics of
honeybees.
Falconer (1989) documented that phenotypic characters in a population
are the result of the combined effects of genotypic variance, environmental
variances and gene-environment variances. Tsurata et al., (1989) and Spivak
et al., (1990) found that the colour patterns of A. mellifera queens depend on
the developmental temperature at the pupal stage; at lower temperature the
pigmentation becomes dark. Again Szabo and Lefkovitch (1992) showed
that the colour patterns of honeybees is less than 40% heritable, while more
than 60% is attributed to environmental, gene-environment variation and
error.
Ruttner and Kauhausen (1985) stated that, in tropical Africa,
significant geographical variability in honeybees occurs in spite of the
absence of physical isolating barriers. The mechanism, which brings about
isolation was the selective adaptation of races of bees to certain biotopes. In
tropical Africa the presence of clear correlations between climatic conditions
and morphometric characters like pigmentations, hair length and body size
are well recognized (Ruttner, 1988). Gradual variation in morphological
characters was also observed with change in altitude (Smith, 1961). Smith
observed three distinct geographical races A. m. litorea, A. m. scutallata and
A. m. monticola in Tanzania across a 300 km distance involving a change of
altitude of 3000 m. from the coast to the rain forest of Mt kilimanjaro.
Kigatiira (1984) stated that, each group of bees in Kenya were characterized
53
by a specific geographical distribution confined by natural barriers thus: A.
m. monticola the large black mountain bee was found between 2400-3100 m.
altitude and A. m. scutellata occupied the Acacia savannah plains. Mbaya
(1985) concluded that, the morphology and behaviour of African bees in
Kenya varied according to ecological zones. He went on to say that, special
biological modifications of some characteristics of honeybees were
necessary if they were to become adapted to certain ecological zone of
Kenya.
The effects of altitude and the existence of short distance ecoclines are
also well recognized ( Mattu and Verma, 1984; Ruttner, 1988; Meixner et
al., 1989, 1994).
In a country like Sudan with contrasting and agro climatically features,
it is very important to consider the environmental factors (like altitude,
temperature and rainfall), along with the morphometric characterization of
honeybees of the area to fully understand the variations, from the biographic
context.
Today in the classification of geographical races of honeybees, holistic
approaches, including all possible data such as morphometric, biological,
behavioural and ecological characters have become more important to
understand the interaction of various factors and to obtain clear pictures of
the geographical races of ecotypes of honeybees of a given area. Therefore,
in this work an attempt was made to classify the honeybee populations of the
area with greater sampling distance resolution by obtaining morphometric, ,
ecology and genetics characters of the honeybees of the Sudan.
2- m- Genetic diversity and Honeybees:
Bees (Genus: Apinae; Hymenoptera) have differentiated into numerous
geographic races or subspecies. The subspecies differed in various
characteristics such as morphology, behaviour, ecology, sensitivity to
diseases and biochemical components. Because most of those characteristics
54
have a genetic basis, the level of genetic diversity within the species as a
whole may be considered high. Thus one of the honeybee DNA research
goals is to find differences that characterized the subspecies.
Understanding the structure of natural populations of honeybees and
identifying the nature and dynamics of the selection processes that operate
on them a wide range of suitable traits of high heritability is required. Until
very recently morphometrical characters were the primary means of such
studies, even though environmental effects might modify the expression of
the genotype thus detracting from the discriminatory power of the
morphometric technique. It has long been recognized that because
morphometric characters are polygenic in origin, the genotype cannot be
directly established through phenotypes.
Nonetheless, the usefulness of morphometric studies, coupled to
multivariate techniques of analysis remains well established (Ruttner 1988,
1992).
Because of the limitations of morphometric analysis in the
measurement of genetic diversity, the development of more accurate
techniques such as, allozymic variation, nuclear DNA (micro satellites) and
mitochondrial DNA, variations are needed.
2- m- i- Allozymes:
Considering the evaluation and discrimination of more precisely bee’s
genetic diversity, the second approach used after morphometrics is the study
of allozymic variation.
Chemo taxonomic methods have been used to differentiate among
subspecies of Apis mellifera L. Sylvester (1976) and Cornuet (1979) found
malate dehydrogenase (MDH) to be polymorphism in Apis, while
Nunamaker & Wilson (1981) and Nunamaker et al., (1984) demonstrated
that this enzyme fulfills the diagnostic requirements established by Ayala &
55
Powell (1972), and can be used to identify the African honeybee, A.
mellifera scutellata Lepeletier.
Thus, in the last century allozyme analysis became a widely used
instrument to study honeybee racial relationships (Badino et al., 1983, 1984;
Cornuet, 1982; Sheppard and Berlocher, 1984, 1985; Sheppard and
Mcpheron, 1986).
Allozymic variation has proven more amenable to the determination of
genotypes in a population (Contel et al., 1977). Breeding experiments have
established that the phenotypic expression of the allozymes is Mendelian
and not susceptible to environmental effects. Of the forty-odd enzyme
systems that have been investigated in honeybees, only seven are known to
be polymorphism. Of these, cytoplasm malate dehydrogenase (MDH) has
proven to be especially useful in the study of honeybee populations (Lobo et
al., 1989; Cornuet and Garnery 1991a).
Ndiritu et al., 1986, demonstrated that, little experimental work has
been done on allozymes of African honeybees. Most of the knowledge
accrued to date comes from the studies of Africanized bees in South and
Central America (Sylvester, 1982; Del Lama et al., 1988, 1990; Sheppart et
al., 1991), or comparisons of these bees with colonies obtained from South
Africa (Nunamaker and Wilson, 1981), but no multi-locus study on a large
collections of honeybees from Africa has been conducted. Thus, the
allozymic variability of the honeybee in Africa and its potential to provide
an additional source of data for taxonomic studies is still very poor.
Investigation of the honeybees of Southern Africa have shown that, the
bees classified as Capensis and Scutellata are morphometric and
homozygous for the fast form of malate dehydrogenase, MDH-100
(Nunamarker & Wilson 1981; Sylvester 1982; Nunamarker et al., 1984,
1986; Sheppard & Huettel 1988).
56
Based on the fact that, the size of adult honeybees can be influenced by
certain environmental factors (Alpatov 1929), and that bees which are reared
in old comb (with relatively small diameter cells) are noticeably smaller in
size than bees reared in new comb (with relatively larger cells). Nunamaker
et al., (1986) tried to determine:
1- whether the morphological variation that results from brood
development in combs with different cell size is a companied by
enzyme variability.
2- whether the malate dehydrogenase (MDH) and nonspecific esterase
(EST) enzyme of A. m. capensis Escholtz are the same in specimens
collected from native populations in South Africa compared with specimens
reared in an altered environment within a hive in Federal Republic of
Germany where natural colonies do not exist. They studied isozymic
uniformity in the presence of environmentally induced morphological
variation in Apis mellifera capensis, they demonstrated that, Apis mellifera
capensis Escholtz specimens from South Africa and Federal Republic of
Germany
exhibited
identical
electrophoretic
patterns
for
malate
dehydrogenase and non-specific esterase.
However, adults from Federal Republic of Germany (which were
reared in cells of large diameter) were significantly larger than workers from
native populations in South Africa, and the same in three body
measurements (body length, head capsule width, and length of forewing) as
A. mellifera subspecies from Laramie, Woy. Certain environmental factors
appear to have at least as much influence as genetic components in
determining final body size of adult worker bees in A. m. capensi. Three
different studies of allozymes of the honeybees of east Africa (Kenya) have
been reported. In the first account, Ndiritu et al., (1986) showed that there is
natural variation in the distribution of malate dehydrogenase alleles in the
region.
57
58
Unfortunately, it is not quite possible to pinpoint the relevant
subspecies involved. Subsequently, Sheppard and Huettel (1988) confirmed
this malate hydrogenase polymorphism and established that there is variation
in aconitase and esterase as well. On the basis of the distribution of the bee
samples these results would apply to both Scutellata and Monticola
(Sheppard &Huettel 1988).
Thus, Meixner et al., (1994) in their attempts to determine allozymic
variability of honeybees in Kenya and to evaluate differences in Allozyme
patterns of the morphometrically classified Apis mellifera scutellata and A.
Mellifera monticola. They studied; Morphological and allozyme variability
in honeybees from Kenya. 43 samples of honeybees from three different
regions in Kenya were analyzed morphometrically and surveyed for
electrophoresis variation ( allozyme study) at five enzyme loci; Malate
dehydrogenase “ MDH-1”; Phosphoglucomutase “ PGM-1”; Malic enzyme
“ME2´”; Esterase “ES-1” and Hexokinase “HK” ( those enzymes are known
to be polymorphism in European bees); they found that discriminate analysis
of the morphometrical measurements classified the samples in three clusters;
samples from above 2000 m as Apis mellifera monticola, samples from
below as A. m. scutellata and the samples which collected from Ngong
region (200 m.) has intermediate between the two mentioned clusters. Also
all enzyme loci in the study were polymorphism, with Est. And HK showing
highest degree of polymorphism. For ME, PGM and Est., new alleles were
reported, thus the analysis of allozyme data by a Distance Wagner procedure
of the provost genetic distances was performed so as to visualize similarities
between population groups, using the BIOS’s program of Softwood and
Selander (1981), resulted in two main clusters, consisting of A. m. monticola
from Mt Elgon and Mt Kenya in one cluster and all other populations in a
59
second cluster. Savannah and mountain bees from Ngong formed a separate
cluster.
Meixner et al., (1994) concluded that the frequency distributions of the
allozymes coupled to the morphometric discrimination, support the
hypothesis that the currently disjunction Afromontane populations of
Monticola derive from some common ancestor different from the Scutellata
of the plains and Ngong Hills below them. Also, Sheppard (1988), reported
that allozyme analysis might be more useful for African bees than New
World genetic studies, as true for European bees.
Shifting further north of Africa in the Maghreb, several important and in
depth studies of allozymes have been performed. Firstly, Cornuet (1993),
established that the frequency distributions were 0.90 for MDH-100 and
only0.10 for MDH-80 among Intermissa populations.
The point of Cornuet’s study was to investigate the relationship of the
bees of North Africa and the Iberian Peninsula. As it turns out, frequency
distribution differences for the alleles of MDH differ sharply in the African
‘A’ and western Mediterranean ‘M’ lineages (Cornuet & Garnery 1991a).
In extension to the above studies, Smith et al., (1991), examined
numerous enzyme loci for bees of the Iberian peninsula and found that all
the bees of Southern Spain possess the same MDH alleles as occurs in the
Intermissa of Morocco. Those of Mellifera extending down from the
Pyrenees displace the same alleles in northern Spain. The direction of gene
flow from Africa into Europe is indicated by the gradual dilution and
eventual elimination of the Intermissa MDH alleles as one progress through
Spain (Smith & Glenn 1994).
Collectively, the three regions of Africa for which honeybee allozymes
have been investigated actually provide four entirely different kinds of
information, (Hepburn et al., 1998). Firstly, the frequency distributions of
60
the MDH allozymes are consistent with the separation of African and
European lineages previously established in morphometric and mtDNA
analyses (Ruttner 1988; Cornuet and Garnery 1991a,b).
In southern Africa the morphometrically defined honeybee races of this
region are allozymically indistinguishable (Nunamaker & Wilson 1981;
Sheppard and Huettel 1988; Hepburn 1990).
Entirely different results were obtained from East Africa. Meixner et al.,
(1994) were able to show that frequency differences in allozymes were
significantly different among the Afromontane and lowland populations that
correspond with the morphometrically defined clusters, Monticola and
Scutellata respectively. Finally, in the Maghreb, the direction of gene flow
from Intermissa across the Mediterranean Sea to the Iberica populations is
also indicated by changing enzyme loci frequencies (Smith et al., 1991).
2- m- ii- Nuclear DNA:
Nuclear DNA has also been used as genetic probe in honeybees, but most
investigations have been directed to analyses of patriline structure
(Blanchetot 1991; Moritz et al., 1991; Fondrk et al., 1993).
Micro satellites loci are regions of DNA composed of short sequences
repeated in tandem that can be amplified by a polymerase chain reaction
(PCR) and then separated by electrophoresis (Hughes and Queller, 1993).
Since micro satellites represent an abundant class of hyper variable markers,
also in the honeybee (Estoup et al., 1993), they became particularly suitable
tool in the analysis of an intracolonial genetic relationship and polyandry
(multiple queen mating).
Recently, Estoup et al., (1994) estimated 7 to 20 patirilines in Apis
mellifera, with the average of 12.40 males per queen. The results for other
Apis have also been reported. A high degree of polyandry in Apis dorsata
61
queens showed the average effective number of mating at 25.5 (Moritz et al.,
1995), while relatively lower value of 5.56 in A. florea was reported by
Oldroyd et al., (1995).
The first application of nuclear DNA analysis to the population genetics
of honeybees is one based on micro satellite variation (Estoup et al., 1995).
The principal conclusions reached in this study were two fold. Firstly, the
micro satellite analyses confirmed the distinctness of the three honeybee
lineages proposed on morphometric (Ruttner 1988) and mtDNA grounds
(Smith et al., 1991; Garnery et al., 1992). Secondly, Estoup et al., (1995)
showed that the average heterozygosity and numbers of alleles were greater
in subspecies of the ‘A’ lineage (Intermissa. Scutellata, Capensis) than in
those of the ‘C’ and ‘M’ lineages. Micro satellite loci are extremely
polymorphism in African populations compared to European honeybee
populations and this has been interpreted as a consequence of larger
effective population sizes in Africa (Estoup et al., 1995; McMichael & Hall,
1996; Franck et al., 1998).
African populations would have been less influenced by quaternary ice
episodes, which are considered to be the main cause of honeybee subspecies
differentiation in Europe (Ruttner, 1988).
Differences in allele frequencies have been found among Apis mellifera
subspecies at several polymorphism loci (e.g. Mestriner and Contel et al.,
1977; Martins et al., 1977; Sylvester 1982; Del Lama et al., 1985 1988;
Spivak et al., 1988). These Polymorphisms are useful in the study of
honeybee biography and population biology (e. g. Cornuet 1979; Sheppard
and Berlocher 1984, 1985; Cornuet et al., 1986; Sheppard and Mcpheron
1986) and in the study of Africanized bees (Nunamaker and Wilson 1981,
Sylvester 1982, Nunamaker et al., 1984).
62
Lionel Garney, Pierre Franck, Emmanuelle Baudry, Dominique Vautrin,
Jean- Marie Cornuet and Michel Solignac (1998), investigated the genetic
variability and differentiation of the west European honeybee populations
(Apis mellifera mellifera & A. m. iberica), through micro satellite loci, they
postulated that, these two subspecies are characterized by a lower genetic
variability than most other studied subspecies and several tests are indicative
of a recent increase of the population size. Moreover, the genetic profile is
rather homogenous from southern Spain to Scandinavia. French populations
are more or less introgressed (a few percent up to 57%) by genes from the
north Mediterranean lineage that provides most of the imported queens. The
inferred percentage of introgressed nuclear genes is generally well correlated
with the proportional of alien mitochondrial deoxyribonucleic acid (mtDNA)
haplotypes detected in the same populations. The level of introgression is the
main source of genetic distances among populations. When introgressed
genes are disregarded, however, population’s cluster is two groups, which
correspond to both subspecies (Iberica and Mellifera), giving full support to
the taxonomy of this lineage.
Thus, Lionel Garnery et al., 1998. Noticed that, the use of micro satellites
for population genetics studies is expanding exponentially. While these
markers are very useful for the study of polymorphism in a variety of
species.
According to the hypothesis that, the use of nuclear DNA (micro
satellite) and mitochondrial DNA markers often display discordant patterns
of differentiation in the honeybees, Franck et al., 2001, in their previously
mentioned study (genetic diversity of the honeybee in Africa: micro satellite
and mitochondrial data) micro satellite marker section, they demonstrated
that, furthermore, eight populations from Morocco, Guinea, Malawi and
63
South Africa have been assayed with six micro satellite loci and compared to
a set of eight additional populations from Europe and the Middle East.
The African populations display higher genetic variability than
European populations at all micro satellite loci studied thus far. This
suggests that African populations have larger effective sizes than European
ones. According to their micro satellite allele frequencies, the eight African
populations cluster together, but are divided in two subgroups. These are the
populations from Morocco and those from the other African countries.
De la Ru´a1, J. Galia´n1, Serrano1 and Moritz (2002), in their study
Micro satellite analysis of non-migratory colonies of Apis mellifera iberica
from southeastern Spain. Demonstrated that, forty-five unmanaged honeybee
colonies from the southeast of the Iberian Peninsula (Apis mellifera iberica)
were selected for analyzing their genetic structure using eight micro satellite
loci. These colonies were not subjected to management for queen
replacement, rearing or migratory movements and previous studies showed
that they had mitochondrial DNA (mtDNA) of African origin. Six of the
micro satellite loci show intermediate levels of polymorphism with a total
number of alleles detected per locus ranging from 4 to 10. Micro satellite
data relate these Iberian populations to the African A. m. intermissa,
although the presence of some alleles and the observed heterozygosity are
characteristic of the European A. m. mellifera, thus corroborating the
postulated hybrid origin of A. m. iberica. The results suggest that no recent
introgression from Africa has happened and that the populations of A. m.
iberica are differentiated in many demes.
Franck et al., (1998) have studied the distribution of eight micro satellite
loci along a transect of honeybee populations from France to Morocco. Their
results showed that there is no gradual modification or frequency cline of
alleles across the Iberian populations. The three studied Spanish populations
64
(located in the Basque Country, Castilla and Andalucia) were similar to the
French populations and did not show introgression of African alleles. These
authors concluded that the Iberian Peninsula does not seem to be an
intergradations zone between European and African subspecies.
An additional, unexpected finding in the study of Estoup et al., (1995)
was the discovery of certain predominantly European alleles of the micro
satellite genes in the sahariensis region of southern Morocco. This results
suggests the possible introgression of ‘M’ lineage nuclear genes through
introduced drones, via-`a-vis results of the mtDNA analyses of the same
region (Garnery et al., 1995). This example illustrates well the advantage of
combining nuclear and mtDNA probes to genetically characterize honeybee
populations. Similar observations elsewhere also confirmed that when two
distinct lineages or populations come into contact the distribution of the
nuclear and mt DNA markers may not coincide (Sheppard et al., 1991;
Rinderer et al., 1991; Oldroyd et al., 1995; Garnery et al., 1995).
2- m- iii- Mitochondrial DNA:
Although allozymes provide a straightforward genetic interpretation that
is not susceptible to environmental effects, it is now well established that
they exhibit a relatively low level of polymorphism in honeybees (Daly
1991). This is thought to be a consequence of the haplodiploidy system in
the species (Pamilo & Crozier 1981).
DNA analyses overcome the limitations of morphometrics characters
and the relatively low polymorphism of allozymes.
The ascendancy of the polymerase chain reaction (PCR) as a tool for
bypassing cloning into micro-organisms to obtain sufficient amounts of
specific DNA has made population comparisons or molecular biosystematics
analyses of insects much more feasible today than a few years ago. Of
particular utility are primer pairs that will amplify the same genes from a
65
wide variety of insects ( and possibly other organisms). An extensive,
though no longer complete, list of primers from a number of different
laboratories that amplify portions of insect mitochondrial DNA has been
reported (Simon et al,. 1991).
2- n- Animals mitochondrial DNA:
In animals many mitochondria are formed within the cells. During egg
maturation, the large number of mitochondria that accumulate give rise to
those of the embryo. Sperms have relatively few mitochondria, and in the
animal tested, including vertebrates and insects, no paternal contribution of
these to the
progeny has been detected. Compared to the biparental
transmission and recombination of the nuclear DNA, maternal inheritance of
mtDNA allows genetic divergence to be more easily followed. Nucleotide
changes in mtDNA accumulate at a constant rate, so that the times when
ancestral lineages diverged can be estimated.
Animals mt DNA generally, encodes 13 proteins, two ribosomal RNAs,
and 22 tRNAs and has a region controlling replication and devoid of other
known functions. One exception is the mtDNA of nematodes, in which the
ATPase 8 gene has been lost ( Wolstenholme et al., 1987). Nematodes also
present unusual tRNAs, all of which have been inferred to lack the TψC loop
(Wolstenholme et al., 1987).
2- n- i- Size of animal mitochondrial DNA:
In most animals species studied so far, the size of mtDNA is remarkably
homogenous. The mitochondrial DNA (mtDNA) of animals (multicultural
eukaryotes) are present in the extra nuclear cytoplasm and are relatively
small circular DNA molecule, usually 16 kb (1 kilo base = 1000 nucleotide
base pairs = 1kb) long, and has been most commonly used to determine
genetic relationship among organisms (Brown, 1985; Wilson et al., 1985;
Avise, 1986; Avise et al., 1987; Moritz et al., 1987), although it ranges from
66
14 kb to 39 kb in length (Snyder et al., 1987; Wolstenholme et al., 1987).
Obviously this is due first to its high conservation in gene contents, but also
to the absence or shortness of intergenic sequences and to the lack of introns
( Attardi 1985; Brown 1985).
Most of the large-scale size variation lies in a single region of the
molecule, (the control region), which contain most of the regulatory
sequences. As a rule, this variation is related to the existence of repeated
sequences, the different number of units of repetition being responsible for
the length variability observed within several species and between related
species, (Cnemidophorus sp., Dens-More, Wright and Brown (1985);
Drosophila sp., Solignac, Monnerot and Mounolou (1986); Triturus
cristatus, Wallis 1987; Grylus sp., Rand and Harrison (1989); Acipenser
transmontanus, Buroker et al., (1990); Oryctolagus cuniculus, Mignotte et
al., (1990)”.
The occurrence of repeated sequence also accounts for the unusual length
of the mitochondrial genome in some animal species: 26 kb for the
nematode Romanomermis culicivorax (Powers, Platzer and Hyman 1986),
42 kb for the scallop Placopecten magellanicus (Snyder et al., 1987; La
Roche et al., 1990), and 36 kb for several species of the bark weevil
Piossodes (Boyce, Zwick and Aquardo 1989).
The mtDNA of one insect, Drosophila yakuba, has been sequenced in full
(Clary and Wolstenholme 1985. ), and partial sequences are known from D.
virilis (Clary and Wolstenholme 1987), D. melanogaster (de Bruijn 1983;
Satta et al. 1987; Garesse 1988), the mosquito Aedes albopictus (HsuChen et
al., 1984a, 19843), the locust Locusta migratoria (McCracken et al., 1987;
Uhlenbusch et al., 1987), and the honeybee Apis mellifera (Vlasak et al.,
1987 ) . These studies have indicated that the order of major genes has
changed between phyla, that tRNA positions change between members of
67
the same insect order (Drosophila and Aedes), and that insect mtDNAs are
very A+T rich.
In vertebrates, mtDNA has long been regarded as evolving much more
rapidly than single-copy nuclear DNA (scDNA), as judged on the basis of
mtDNA sequence comparisons and scnDNA heteroduplex formation (Brown
et al., 1979; Moritz et al., 1987).
By contrast, studies on echinoids (Vawter and Brown 1986) and
Drosophila (Powell et al., 1986) indicate roughly similar rates of divergence
for scnDNA and mtDNA. Vawter and Brown ( 1986) and Moritz et al., (
1987) attribute these relative differences in evolutionary rates between
mtDNA and scnDNA to differences in scnDNA rates, suggesting that the
mtDNA rates are relatively invariant. Nuclear genes have indeed been found
to vary significantly between various groups in evolutionary rates ( Wu and
Li 1985; B&ten 1986; Li and Tanimura 1987; Lake 1988; also see Ochman
and Wilson 1987).
2- n- ii- Apis mellifera L. Mitochondrial DNA:
Honeybees (Apis mellifera L.) mt DNA was found to be, between 16.5
and 17 kb length, ( Smith and Brown 1988). This range is explained by
length variability in at least two regions (Smith and Brown 1990): one of
them, as usual, is the control region and another one, where size differences
are larger (ca. 450 bp between the longest and shortest types), is the COICOII junction. This region has been recently sequenced by Crozier, Crozier
and Mackinlay (1989)., between the tRNAleu and the COII genes, they
found an unassigned sequence of 194 bp. Where only five nucleotides are
present in the Drosophila yakuba mtDNA (Clary and Wolsten-Holme 1985).
The sequence obtained by Crozier, Crozier and Mackinlay (1989)
belongs to the shortest type found in Apis mellifra. Corneut, et al., (1991),
68
sequenced a domain encompassing this unassigned sequence in a genome of
the largest size class in Apis mellifera. In addition, the corresponding domain
has been sequenced in three other Apis species, and in two related species
Bombus lucorum and Xylocopa violacea (Anthophoridae).
Thus far, mitochondrial DNA has enjoyed most attention in the analysis
of honeybees for several important reasons: mitochondrial DNA of
honeybee A. mellifera L. is a small circular, double-stranded DNA molecule
of between 16 500 and 17 000 base pairs ( Smith and Brown 1988) and
whose sequence has been determine (Crozier & Crozier 1993). The gene
content is highly conserved, and there are many identical copies of the
chromosome in each cell. The mitochondrial genome is substantially smaller
than the nuclear genome and can be readily studied as an entity (Smith
1991).
Thus, although honeybees appear to have relatively low levels of
allozyme variability (e.g. Sheppard and Berlocher 1984, 1985), the level of
variation in their mtDNA is well within the rate found in other species
(Avise and Lansman 1983). Second, although of fixed differences in
allozymes have been found among honeybee species, preliminary studies of
the mtDNAs of European and African subspecies (Smith 1988) indicate that
at least some have unique cleavage site patterns. Thus, because all the
offspring of a queen inherit the same mtDNA, large quantities of mtDNA
from a single source can be prepared by pooling tissues from hive mates.
The mitochondrial DNA molecule of honeybees has been studied within
and between populations (Smith 1988; Cornuet & Gernery 1991b; Garnery
et al., 1992; Moritz 1994; Moritz et al., 1994).
Mitochondrial DNA is a particularly valuables probe because of its
maternal inheritance, which means that all offspring of a single queen will
have the identical mtDNA molecule (Meusel & Moritz 1992). Also, because
69
of the strict maternal inheritance, honeybees of hybrid origin do not carry a
mixture of mtDNA's, they show only the pattern of their queen (Cornuet and
Garnery 1991a,b; Smith 1991), making the molecule particularly powerful in
the study of hybrid zones (Smith et al., 1989; Moritz et al., 1994, 1998).
Similarly, the genetic details of swarming bees can be resolved with the
mtDNA, so it is a potential means for studying colonization processes
(Garnery et al., 1992).
Mitochondrial DNA variability has also been successfully used in the
analysis of phylogeographic variations (Smith et al.,1991; Garnery et al.,
1992). Many studies have used mitochondrial DNA polymorphisms to
identify mitochondrial lineages within a species, and to make inferences
about the phylogeny and biography of the organisms carrying them.
The honeybees “Apis” are a particularly rich group for study of both
biogeography and mitochondrial DNA variation. The biography of
honeybees is complex and moderately well documented (Ruttner, 1968,
1988, and 1992).
In addition, the mitochondrial genomes of Apis species include an
apparently unique non-coding intergenic sequence, which provides a source
of rapidly evolving characters (Cornuet et al., 1991).
MtDNA characters have been used to study phylogeny
and
biogeography of A. mellifera subspecies (Garnery et al., 1991, 1992; Smith
1991b; Smith et al., 1991), and the pattern of gene flow between introduced
European and African honeybees in the New World (Hall and Muralidharan,
1989; Hall and Smith, 1991; Sheppard et al., 1991; Smith et al., 1989).
The novel non – coding region of Apis mtDNA is of particular interest
for studies of infra–specific biogeography and phylogeny. Early studies of
restriction polymorphisms in honeybees showed a region of size variations
in the mitochondrial genome. This region was small and uniform in size in
70
east European A. mellifera subspecies and larger and variable in west
European and African subspecies. The size variations in the west European
and African A. mellifera restriction fragments occurred in discrete unites that
suggested a tandemly repeated element ( Hall and Smith, 1991; Smith,
1991a; Smith and Brown, 1988, 1990; Smith et al., 1989).
Cornuet et al., 1991, subsequently sequenced this region of the
mitochondrial genome in several A. mellifera subspecies and in one
individual each of A. cerana, Dorsata and Florea. All four of the Apis
species were examined (as well as A. koschevnikovi and A. anderniformis:
Smith, unpub. Data; cited in Deborah, et al., 1996) and all have a non –
coding intergenic region, which is A+T rich. This region is flanked by the
cytochrome oxidase 1 (CO1) and Lucien tRNA genes on the 5' end and the
cytochrome oxidase 11 gene (COII) on the 3' end. In A. mellifera this region
consists of two units, a P unit of 54 bp which is 100% A+T, and a more
complex “Q” unit of 196 bp (93.4% A+T), which is similar in sequence to
the 3' end of the COI gene ( “Q1” ), the Lucien tRNA gene (the “Q2”
portion) and the “P” sequence (“Q3”; Cornuet et al., 1991).
The size class found in east Mediterranean A. mellifera corresponds to a
single Q sequence; the different size classes found in west European and
African A. mellifera correspond to: PQ, PQQ, and PQQQ. Cornuet et al.,
(1991) suggest that these repeats arose as a result of a duplication of portions
of the CO1 and the adjacent Lucien tRNA gene, followed by additional
tandem duplications. The P and Q sequences of A. mellifera can be folded
into hairpin and cloverleaf loop structures (Cornuet et al., 1991).
This intergenic sequence is smaller and less complex in A. cerana and
other Apis. species. The intergenic region of all Apis species share a pair of
short sequences (“stem sequences”) at the beginning and end of the non –
coding region which appear to be capable of base – pairing with one another
71
(Cornuet et al., 1991); Smith, unpubl. Data; cited in Deborah, et al., 1996),
and which are identical to the stem sequences of Mellifera P and Q3
sequences.
Honeybees have been surveyed for mitochondrial DNA (Moritz et al.,
1986 Smith 1988; Smith and Brown 1988) and nuclear (Hall 1986) RFLPs.
In an attempt to extend the knowledge of honeybee mtDNA sequence,
providing information useful for general evolutionary studies on insects; R.
H. Oozier, Y. C. Oozier, and A. G. Mackinlay (1989), studied the CO-I and
CO-II Region of Honeybee Mitochondrial DNA: Evidence for Variation in
Insect Mitochondrial Evolutionary Rates. They demonstrated that, the
sequence of a region of honeybee (Apis mellifera ligustica) mitochondrial
DNA, which contains the genes for cytochrome C oxidase subunits I and II
(CO-I and CO-II) and inferred genes for tRNA Asp, tRNA
Leu
UUR, tRNA
Lys, and tRNA Trp, is presented. The region includes the segment
previously identified as incurring a length increase in some other bee strains,
including Africanized bees. The sequence information of this study and of
that by Vlasak et al., shows that several shifts of tRNA genes have occurred
between Apis and Drosophila, but shifts of other kinds of genes have yet to
be demonstrated. The CO-I and CO-II gene sequences are both more A+T
rich than the corresponding Drosophila genes.
Parsimony analyses using the mouse and Xenopus sequences as out
groups show significantly more amino acid substitutions on the branch to
Apis ( 120) than on that to Drosophila (44) indicating a difference in the
long-term evolutionary rates of Hymenopteran and Dipteran mtDNA. Thus
this study indicate that, the rates of mtDNA evolution have differed greatly
between these lineages.
Leslie G. Willis et al., (1992), studied phylogenetic relationships in the
honeybee (Genus Apis) as determined by the sequence of the cytochrome
72
oxidase II Region of Mitochondrial DNA, from which they determined the
complete nucleotide sequence of the mitochondrial cytochrom oxidase II
(COII) gene for five species of the honeybee (Genus: Apis): A.
andreniformis, A. cerana, A. dorsata, A. florea, and A. koschevnikovi, these
were then compared to the known sequence of the A. mellifera gene from
Crozier et al., (1989), and the wasp Excristes roborator, Liu and
Beckenbach, (1992). Phylogenetic relationships were derived using the
parsimony methods DNAPARS and PROTPARS of Felsensrein (PHYLIP
Manual Version 3.4. University Herbarium, Univ. Of California, Berkeley).
The results suggest that A. dorsata is the most ancestral species, followed by
the branching of A. florea /A. andreniformis and A. koschevnikovi, and then
A. mellifera and A. cerana. This inference differs from the currently
accepted view that considers the A. florea / A. andreniformis line to be the
most ancestral. The genus Apis has been studied using morphological
( Alexander, 1990. Smith, 1991), biogeographically (Kellner-Pillault, 1969.
Smith, 1991), and molecular methods ( Smith, 1991. Garney et al., 1991.
Shepard and Berlocher, 1989. Smith, 1990).
The currently accepted view of Apis (Alexander, 1990) places the A.
florea, A. andreniformis line as the most ancestral, giving rise to A. dorsata
followed by A. mellifera, A. cerana, and finally A. koschevnikovi.
Under the following title: A simple test using restricted PCR-amplified
mitochondrial DNA to study the genetic structure of Apis mellifera L.,
Garnery, M. Solignac, G. Celebrano and J.-M. Cornet, (1993), demonstrated
that, the COI-COII intergenic region of Apis mellifera mtDNA contains an
important length polymorphism based on a variable number of copies of a
192- 196 bp sequence (Q) and the complete or partial detection of 67 bp
sequence (P0). This length variability has been combined with a restriction
site polymorphism to produce a rapid and simple test for the characterization
73
of mtDNA haplotypes. This test includes the amplification by the
polymerase chain reaction of the COI-COII region followed by Dra1,
restriction of the amplified fragment. In a survey of 302 colonies belong to
12 subspecies, 21 different haplotypes have been unambiguously allocated
to one of the 3-mtDNA lineages of the species. Although all colonies of
lineage C exhibit the same pattern (C ), each one of lineage A and M present
up to 10 different haplotypes, opening the way to studies on the genetic
structure and the evolution of a large fraction of the species. This test also
differentiates Southern Spanish and South Africa colonies, which can bee of
great interest for the Africanized bee problem. Although they propose an
even more discriminatory test which requires the amplification and
restriction by a single enzyme of only one PCR fragment. This fragment
contains the COI- COII intergenic region, which exhibits at least 7 length
variants which can be explained by the combination of 3 related sequences
P0 (67bp), P (54bp) and Q (192- 196bp): P0, P0QQ, P0QQQ, PQ, PQQ,
PQQQ and Q. The three-mtDNA lineages corresponding to the
aforementioned three branches are characterized by P0 (A), P (M) and
neither P0 nor P (C). The examination of the available COI-COII sequences
led to the conclusion that Dra1, the recognition site of which is TTTAAA,
should show a significant amount of polymorphism (Grnery, et al., 1993).
Genetic diversity of the west European honeybee (Apis mellifera
mellifera and A. m. iberica). I. Mitochondrial DNA. under this title Lionel
Garney, Pierre Franck, Emmanuelle Baudry, Dominique Vautrin, JeanMarie Cornuet and Michel Solignac (1998), studied the variability of
mitochondrial deoxyribonucleic acid ( mtDNA) in 973 colonies from 23
populations of the West European honeybees ( lineages M) using restriction
profiles of a polymerase chain reaction (PCR) amplified DNA fragment of
the COI- COII intergenic region. Although populations are almost always
74
introgressed by two other mtDNA lineages (A and C), results confirmed
that, the original haplotypes in Western Europe are those of mtDNA lineage
M. Iberian populations ( Apis mellifera iberica) are characterized by a
extended cline between haplotypes A and M, the former being almost fixed
in South Spain and Portugal, and the latter almost pure in northeastern
populations. This introgression is most likely attributable to humans and is
probably ancient. French populations (A. m. mellifera) exhibit various levels
of introgression by the C mt DNA lineage. Introgression is rather low in
regions with a dominance of a mature bee keeping while it reaches very high
values in regions where professional beekeepers regularly import foreign
queens (mainly A. m. ligustica and A. m. caucasica). When discarding
introgressed haplotypes, French populations group in two clusters, one for
the northeastern part of France and the other one for all other populations,
including Swedish and northeastern Spanish populations.
Thus, Lionel Garney, Pierre Franck, Emmanuelle Baudry, Dominique
Vautrin, Jean- Marie Cornuet and Michel Solignac (1998), noted that in the
original distribution area of the (Europe, Africa and Near and Middle East),
honeybee populations are relatively homogeneous over wide territories
corresponding to the subspecies or the geographical race.
The honeybee species has thus been split into 24 races according to
morphometric criteria. These 24 species have been grouped into larger sets
corresponding to evolutionary branches. Three such branches have been
distinguished: an Africa (A) branch including all African subspecies, a north
Mediterranean (C) branch in the central and east Mediterranean and in
central Europe, and a West European (M) branch, from Spain to Sweden and
Poland. These three branches were first identified through morphometry (
Ruttner et al., 1978, Ruttner 1988) and were approximately confirmed
through deoxyribonucleic acid (DNA) studies (Cornuet and Garnery, 1991.
75
Smith, 1991a and b. Garnery et al., 1992. Estoup et al., 1995. Arias and
Sheppard, 1996).
Also, Lionel Garney, Pierre Franck, Emmanuelle Baudry, Dominique
Vautrin, Jean- Marie Cornuet and Michel Solignac (1998) reported that, the
French indigenous subspecies, Apis mellifera mellifera, belongs to branch
M. It extends widely beyond the country limits since it was naturally found
everywhere in Europe north of the Alpine Arc (Ruttner, 1988).
In the Iberian Peninsula, a related subspecies, A. m. iberica (Geotze,
1964), has been recognized, although it has long been considered as a local
form of A. m. mellifera. Only other subspecies that are naturally present on a
French boarder is the Italian A. m. ligustica, a member of branch C, with
which A. m. mellifera hybridizes over the Alps and beyond (Badino et al.,
1993. Sheppard and Berlocher, 1985).
However, another subspecies, A. m. carnica (also belong to branch C)
has been massively imported in Germany and has almost replaced the former
A. m. mellifera populations ( Kauhausen-Keller and Keller, 1994. Maul and
Hahnle, 1994). The consequence is that there is now a possibility of
hybridization of A. m. mellifera with A. m. carnica in French areas close to
the German border.
Although beekeepers have long been expert in managing hives to get the
best productions out of them, they generally do not control the entire
biological cycle of honeybees, especially the mating of queens and drones.
Hence, the current genetic biodiversity in the west part of the Old World, the
original distribution area of the species, is still structured in a way that
expresses its evolutionary heritage, i.e. it is determined by past demography,
adaptation to local conditions, duration of isolation and natural migrations.
However, one has to take into account several technical improvements
more or less recently introduced in hive management and which may have
76
inferred with the natural evolution of populations. First queen breeding,
when operated on a large scale, artificially reduces the effective size of
populations, and can result in a loss of genetic variability. Second,
importation of foreign queens can modify the genetic pool of local bees
through hybridization. This genetic pollution is mainly due to professional
bee-keepers who import foreign subspecies for their own qualities and/ or in
the hope of producing superior hybrids with the local subspecies (Fresnaye
et al., 1974. Cornuet and Fresnaye 1979).
In France, A. m. ligustica and A. m. caucasica are the most in ported
subspecies, mainly because their hybrids with A. m. mellifera have
demonstrated superior qualities for honey yield (Fresnaye et al., 1974.
Fresnaye and Lavie, 1976). Other synthetic strains as the English “buckfast”
(Adam Br, 1966) or the American “midnight” and “star line” (Witherell,
1976) have also been imported, but to a lesser extent. A third factor is the
practice of moving hives several times through the year to increase and
diversity the honey production. According to when and where the mating
season occurs, this practice may have effects similar to those of in porting
queens. In addition to change in the genetic pool of populations, this can
artificially increase the genetic diversity in two ways: by introduction new
alleles and by increasing the effective population size.
Deborah R. Smith and Robert H. Hagen (1996), studied the biography of
Apis cerana as Revealed by Mitochondrial DNA Sequence Data. They
reported that, the non-coding intergenic region of the Apis cerana
mitochondrial DNA genome provides a rapidly evolving source of
characters for study in intra-specific biography. They sequenced the noncoding intergenic region in bees from 110 colonies of Apis cerana collected
over most of the species range. They found two major forms of non-coding
sequence: a western form, occurring in bees from India, Sri Lanka and the
77
Andaman Islands, and an eastern form, occurring in bees from Nepal,
Thailand, Malaysia, Indonesia, the Philippines, Hong Kong, Korea, Japan,
and India. Thus the eastern and western haplotypes co-occur in India. Thus
they found that within the eastern form, phylogenetic analysis of sequence
variation indicated two well supported groups of haplotypes: a *Sundaland
group* which was found in bees from peninsular Malaysia, Borneo, Java,
Bali, Lombok, Timor, and Flores., and a *Philippine group* which was
found in bees from Luzon, Mindanao, and Sangihe. Haplotypes from both
the Sundaland group and the Philippine group were found on the island of
Sulawesi, suggesting that this island was colonized independently by two
groups of A. cerana. In addition, the bees of Taiwan and a third group of
Sulawesi mitochondrial haplotypes characterized by absence of most of the
non-coding sequence.
Delarua, J. Serrano and Galian (1998), investigated the mitochondrial
DNA variability in the Canary Islands honeybees (Apis mellifera L.), they
reported that, the mt DNA of individuals from 79 colonies of Apis mellifera
from five Canary Islands was studied using the Dra1 test based on the
restriction of PCR products of the tRNAleu-COII intergenic region. Five
haplotypes of the Africa (A) lineage and one of the west European (C)
lineage were found. The haplotypes A14 and A15 are described for the first
time. These haplotypes have a new P sequence named P1. The wide
distribution and high frequency of haplotype A15 suggest that it is
characteristic of the Canarian Archipelago.
Thirteen haplotypes for the A lineage and their distribution have been
described to date (Garnery et al., 1992, 1993, 1995. Moritz et al., 1994). One
of these haplotypes A2, belonging to the African lineage, is present in
southern Spain, Sicily and some Greek Islands, suggesting that its present
distribution has been influenced by human activities. This haplotype was
78
also found in a few colonies from the Canary Islands, leading Garnery et al.,
(1993) to speculate about the Spanish origin of Canarian bee populations.
The Canarian Archipelago is composed of a chain of volcanic islands
originated in the Atlantic Ocean, 110 km of the northwest Africa coast. The
ancestors of Canarian organisms might come from the close mainland and
also from southern Iberia via the northeast trade winds (Oromi et al., 1991).
The evolutionary of many of them into endemic species or subspecies and
their relationships to the continental fauna is becoming clearer with the
availability of DNA sequence data (Tenebrionidae: pimelia, Juan et al.,
(1995, 1994), which suggest a congruent pattern of colonization for different
kinds of animals.
P. De La Ru A, U. E. Simon, A. C. Tilde, R. F. A. Moritz & S. Fuchs,
(2000), in their study MtDNA variation in Apis cerana populations from the
Philippines, they reported that, The cavity-nesting honeybee Apis cerana
occurs in Asia, from Afghanistan to China and from Japan to southern
Indonesia. Based on morphometric values, this species can be grouped into
four subspecies: A. c. cerana, A. c. indica, A. c. japonica and A. c. himalaya.
In order to analyze the geographical variability of A. c. indica from the
Philippine Islands, 47 colonies from different locations in three of the larger
islands (Mindanao, Luzon and Palawan) and four of the Visayan Islands
(Panay, Negros, Cebu and Leyte) were studied. Genetic variation was
estimated by Dra1 restriction enzyme and sequence analysis of PCRamplified fragments of the tRNAleu-COII region. They found four different
haplotypes namely Ce1, Ce2, Ce3 and Ce4 that discriminate among the bee
populations from different islands. The Ce1 haplotype is present in
Mindanao and Visayan Islands, Ce2 is restricted to Luzon, and both Ce3 and
Ce4 are only present in Palawan. Phylogenetic analysis of the sequences
shows a great intraspecific variability, is in accordance with the geological
79
history of these islands and partially agrees with some previous
morphological and molecular studies.
A total of 738 colonies from 64 localities along the African continent
have been analyzed by P. Franck et al., 2001, in their study genetic diversity
of the honeybee in Africa: micro satellite and mitochondrial data. They used
the restriction enzyme Dra 1
(RFLP) of the COI-COII mitochondrial
region. Mitochondrial DNA of African honeybees appears to be composed
of three highly divergent lineages. The African lineage previously reported
(named A) is present in almost all the localities except those from
northeastern Africa. In this area, two newly described lineages (called O and
Y), putatively originating from the Near East, are observed in high
proportion. This suggests an important differentiation of Ethiopian and
Egyptian honeybees from those of other African areas. The A lineage is also
present in high proportion in populations from the Iberian Peninsula and
Sicily.
Hiroyuki Tanaka, et al., 2001, studied the genetic differentiation among
geographic groups of three honeybee species, Apis cerana, A. koschevnikovi
and A. dorsata in Boroneo. They sequenced the mitochondrial cytochrome
oxidase 1 (CO1) gene and carried out phylogenetic analysis in order to
examine genetic relationships among geographic groups of each species
(geographic genetic variation). They compared the sequence divergencegeographic distance relationships among the three species.
Estimated
genetic differentiation was an order of magnitude large in A. kosochevnikovi
than in A. cerana and A. dorsata. Migratory nesting behavior and cold
tolerance of each honeybee, and the pale climate of the Southeast Asian
tropics, are discussed as factors that produced these characteristics for
mitochondrial genetic markers and conservation priorities are recommended.
80
Also, in this study, they suggested that, the genetic variations within
plant-pollinator assemblages clarifies not only phylogenetic concepts
associated with biological species, but may also indicate evolutionary
potential and adaptive diversity in mutualisms (Thompson, 1994).
Walter S. Sheppard & Marina D. Meixner (2003) named a new honeybee
subspecies from Central Asia (Apis mellifera pomonella), demonstrating
that, endemic honeybees of the Tien Shan Mountains in Central Asia are
described as a new subspecies, Apis mellifera pomonella, on the basis of
morphometric analyses. Principal component and discriminant analysis of
the morphological characters measured clearly place these bees into the
oriental evolutionary branch of honeybees, but also show that they are
distinct from the other subspecies in this lineage. The existence of this newly
described honeybee subspecies extends the range of endemic A. mellifera
more than 2000 km eastward than previously estimated. Sequence analysis
of mitochondrial DNA places A. m. pomonella within the C mitochondrial
lineage (a group that is inclusive of both C and O morphological lineages).
These findings support the conclusion that A. m. pomonella has a
phylogeographic history shared with subspecies from the eastern limit of the
previously known range.
The Iberian honeybee, Apis mellifera iberica (Goetze, 1964; Ruttner,
1988) has been subject of numerous studies about its molecular diversity
(Smith et al., 1991; Garnery et al., 1995, 1998a, b; Sheppard et al., 1996;
Franck et al., 1998; De la Rْa et al., 1999, 2002; Canovas et al ; 2002 ,.
Hernandez-Garcia et al., 2002) and morphometrical variations (Izquierdo et
al., 1985; Cornuet and Fresnaye 1989; Orantes-Bermejo and GarciaFernandez, 1995; Padilla et al., 1998, 2001). Earlier analyses showed that
two out of the five evolutionary lineages of A. mellifera coexist on the
Iberian Peninsula (Smith et al., 1991; Garnery et al., 1995). In the North, the
81
Iberian honeybee populations are more similar to the West European lineage
as they bear particular sequences in the intergenic tRNAleu-COII region (so
called mitochondrial haplotypes), whereas in the South they show
predominantly African haplotypes. These results led Smith et al., (1991) to
postulate that A. mellifera iberica is the result of the hybridization between
the West European A. mellifera mellifera and the North African A. mellifera
intermissa.
In spite of the occurrence of this generalized pattern of mitochondrial
haplotypes across the Iberian Peninsula, the studies realized at the regional
level have demonstrated that every region holds a peculiar composition of
molecular markers.
Thus, the honeybees from Murcia show a homogeneous composition of
mitochondrial haplotypes (De la Rْa et al., 1999), and neither traces of recent
introgression events from African subspecies (De la Rua et al., 2002), nor
the effects of transhumance movements (Hernandez- Garcia et al., 2002).
In Galicia (NW Spain) the gradient of lineage distribution shows a rather
steep transition, as the Southern provinces of Pontevedra and Orense have
predominantly (>90 percent) African haplotypes, whereas La Coruna and
Lugo in the North show almost only (>90 percent) West European
haplotypes (Canovas et al., 2002). In contrast, in East Spain the percentage
of
African
haplotypes
smoothly
decreases
northwards
along
the
Mediterranean provinces of Valencia (De la Rua et al., submitted: cited in
De la Rua P., R. et al., 2004).
De la Rua P., R. Hernadez-Garcia, B.V. Pedersen, J. Galian and J.
Serrano (2004), in their study molecular diversity of the honey bee Apis
mellifera iberica from Western Andalusia, they tried to characterize its
populations according to the variability shown by mtDNA haplotypes and
micro satellite loci as previously mentioned . They analyzed
82
the
mitochondrial and nuclear DNA ; mitochondrial haplotype corresponding to
the intergenic region tRNAleu-COII, and six micro satellite loci has been
determined in hives distributed in 24 localities of the provinces of Malaga,
Seville, Cadiz and Huelva. Six different haplotypes have been found, five of
the African and one of the West European evolutionary lineage. These
results corroborate the hybrid nature of the subspecies Apis mellifera iberica,
which has a predominant influence of the African lineage in the South, that
is gradually or steeply replaced northwards by the West European lineage.
The variability of the micro satellite loci is similar to that found in African
populations in relation to the detected number of alleles and the values of
genetic diversity. These observations show the genetic relationship between
Andalusian honeybee populations and those ones from North Africa. Micro
satellite data vary notably between the studied provinces. In the province of
Cadiz the mitochondrial homogeneity contrasts with the micro satellite
variability, what suggests a recent introgression event from African-like
populations of unknown geographic origin.
In regard to the African honeybees, one must first consider the results of
mtDNA studies with respect to the major lineages erected by Ruttner (1988),
based on the multivariate analyses morphometric characters. The combined
data of several studies using restriction enzymes to obtain restriction
fragment length polymorphisms, as well as actual sequence data (Arias &
Sheppard 1996), clearly support the existence of three major mtDNA
lineages.
The African lineage (A) includes all mtDNA types from colonies of
African origin,; a north Mediterranean – Caucasian lineage (C) the bees of
eastern Europe and the Caucasus region; and a western Mediterranean
lineage (M) for the bees of that region (Smith et al., 1989, 1991; Garnery et
al., 1992, 1995).
83
Most African bees currently analyzed belong to the A mitochondrial
lineage (Smith, 1991; Garnery et al., 1992, 1993; Arias & Sheppard, 1996;
De la RuÁ a et al., 1998). Only two colonies from Egypt have been
recognized as belonging to lineage O (Arias & Sheppard, 1996; Franck et
al., 2000b), thus further more in 2001, P. Franck et al., described two newly
lineages
from north-eastern Africa called “O” and “Y”. Each of these
lineages has unique length fragment polymorphisms as well as intergenic
sequences.
Garnery et al., (1992) proposed an hypothesis for the origin and
evolution of the population of Apis mellifera based on their mtDNA types.
These interpretations are very similar to those of Ruttner et al., (1978), but
there are some slight discrepancies. In Ruttner's scheme, the north Africa
intermissia were associated with iberica and mellifera; the mtDNA
associates intermissia with the other African subspecies. In fact, the mtDNA
data clearly indicate that the Spain is a hybrid zone, the southern half of the
country dominated by the intermissa mitotypes, the northern half by
mellifera mitotypes (Cornuet & Garnery 1991b; Garnery et al., 1992; Smith
et al., 1991; Sheppard et al., 1996).
This interpretation is supported by allozymic data (Cornuet 1982) and by
more recent studies of morphometrics and phermonal variance for the region
(Hepburn & Radloff 1996a). However, that the Iberian peninsula is such a
transition zone is not consistent with recent micro satellite data (Frank et al.,
1998).
The high similarity of mtDNA profiles among the bees of northern and
southern Africa is somewhat surprising (Cornuet &Garnery 1991b) that,
they share a common mitotype defined by a combination of 22
polymorphism restriction sites (Smith 1991; Garnery et al., 1992). The
reason for relatively low within – lineage diversity of the African lineage
84
can be parsimoniously explained as the result of the isolation of individual’s
populations rather than multiple, highly polymorphism ancestral populations
(Garnery et al., 1992).
In two different studies, Smith (1988, 1991) analyzed restriction site
poly-morphisms and fragment length variations for several European
subspecies and others from Africa. She was able to separate Intermissa,
Scutellata, Capensis and Unicolor as a group from the European subspecies,
thus once again confirming the mitochondrial distinction of the A, C and M
lineages. However if her data are subjected to cluster analysis, none of the
subspecies within the Africa A lineage can be distinguished from one
another.
In a similar study, but using a slightly different suite of restriction
endonucleases, Garney et al., (1992, 1995) also confirmed the mitochondrial
distinctions between lineages. However, clusters analyses of their data do
not allow the discrimination of the African Monticola, Scutellata and
Capensis from one another. Thus, Moritz R. F. A. et al., (1994) in their
study: Mitochondrial DNA variability in South African honeybees (Apis
mellifera L), demonstrated that, the mtDNA size variability of honeybees
(Apis mellifera) in a sample of 102 colonies covering the area south of the
27th parallel of latitude in Africa was analyzed using PCR. A region
between the COI and COll genes revealed four different size variants one of
which being a novel mitotype for honeybees not fitting the previously
published repeat pattern in the region (a fragment po with 69 bp and varying
number of fragment Q of 196 bp length). This region, which has been shown
to be useful for the biogeography classification of Apis mellifera subspecies,
only partially corresponded to the known distribution of African subspecies
of honeybees based on morphometrical and physiological data. The PoQQtype was the most common with an overall frequency of 0.76. The region,
85
which has been addressed as the hybrid zone between A. m. capensis and A.
m. scuteltata showed no mitotype variability and was monomorphic for the
PoQQ type. A considerable length polymorphism was found north and east
of this region with a frequency of 0.57 for PoQQ type and 0.36 for the PoQ
type. Less common were the PoQQQ type (0.02) and a type not fitting the
known P and Q repeat system (0.02). Digestion of the region with the Dral
restriction enzyme revealed previously undetected mtDNA variability in
Apis mellifera populations.
More recently, Sheppard et al., (1996) performed a restriction enzyme
analysis of eight subspecies (three European and five African). With the
enzyme Hinf 1, twenty distinct haplotypes were obtained taxonomically
useful. Lamarkii exhibited a single unique variant. Intermissa, monticolla
and sahariensis each exhibited at least one haplotype not shared with one
another, and other haplotypes that were shared with each other and with
Scutellata. For example, while Sahariensis had one unique haplotype its
other were identical to those of the neighboring Intermissa; the same being
true for Monticola and its neighbor Scutellata. Similarly, Intermissa shared
three of eight haplotypes the European Iberica.
While such results hold promise for studies of honeybee population
discrimination, it’s likely that the use of multiple probes would enhance
discrimination.
Whatever suite of restriction enzymes chosen as probes, the origin of the
honeybees themselves must be carefully considered. In several of the above
studies, a particular combination of subspecies was acquired on the basis of
accessibility and typological or racial homogeneity (Meixner et al., 1994;
Arias and Sheppard 1996). They actually represent point or spot samples
taken from continuous populations. These kinds of classification constraints
may well introduce a bias in the final interpretation of the data.
86
Thus, far there have been only two studies in which mitochondrial DNA
analyses were performed on a transect basis without pre-grouping the data
into sub specific groups before analysis.
Garnery et al., 1995, study the mitochondrial DNA variation along a
transect through Morocco and Spain using a combination of particular
power: length polymorphism and restriction site variation, their conclusions
is that, gene flow in the region has primarily been from south to north, which
is from populations of Intermissa in the Maghreb to Iberica populations on
the Iberian peninsula. Referring only to Intermissa.
Garnery et al., (1995) also suggested that the bees in Morocco it self
probably represent two sub lineages of the past, one coming from the
northeast and other from the south , and that the contact points between them
actually extend on both sides of the Atlas mountains for the length of the
country. In this particular study, Garnery et al., (1995), were principally
concerned with a phylogenetic analysis of the bees of the region. Their
technique was essentially a cluster analysis modified to estimate
phylogenetic distance between samples.
However, these data (Garnery et al., 1995) can be analyzed in a very
different way. In order to compare the results of this mtDNA study with the
variance characteristics of morphometric and phermonal analyses for the
same region, but without imposing the limits of classification into
subspecies.
Hepburn and Radloff (1996, 1997) re-analysed their mtDNA data.
Applying Greenacre's method of analysis (1988), they found that the
mtDNA data used in the Garnery et al., (1995) study resolved into six
distinct and significantly different mtDNA clusters or groups. The three
mtDNA clusters of Spain matched the distributions of three iberica
87
morphometric clusters. The three mtDNA clusters obtained in Morocco did
not exactly match the two morphometric clusters corresponding to
Intermissa and Sahariensis (Hepburn &Radloff 1996a). In summary analysis
of mtDNA unequivocsally support the three major honeybee lineages;
however the technique has been less successful in defining mtDNA clusters
that correspond with morphoclusters derived from multivariate analysis.
88
CHAPTER THREE: MATERIALS AND METHODS
3- 1- Sampling:
In a number of survey trips (2005-2006), nineteen samples of the
native Sudanese honeybee Apis mellifera workers were collected.
Samples were collected within the latitude 3º N to 22º N and
longitude 23º E and 38º E. and from at least three different
geographical zones of the Sudan; namely, semi-desert, savannah and
forest zones.
Four samples were captured from the following locations at the
semi-desert region: Shendi, Khartoum, El-Hissahisa and Madani.
From the poor and rich savannah regions, eleven samples were
captured at the following locations: Doka, El-Galabat, El-Hawata,
Galla-Elnahal, Kosti, El-Dalng, Kadogli, Malakal, Doleib, Ganal and
Wau-sholok. Then from the forest region, four samples were captured
at the following locations: Juba, Liria, Bango and Khour-Maquire. See
Table (1) and fig. (1). {Sudan map sampling localities}.
The samples were collected from nests found in traditional
cylindrical hives, tree cavities, resting feral swarms, established wild
colonies on tree branches, in soil and rock crevices, crevice in a store
wall in a farm and Apiary (Khartoum sample only).
Samples were captured by using an ordinary insect catching net
placed round the entrance of established colonies which were
disturbed and then attacking bees were caught, or swarm clusters and
exposed colonies nesting on shrub branches were shaken into the net.
Sampling sites were at least over 25 miles from each other. Thus
300 to 1000 miles separated the geographical zones from each other.
89
Four samples of Apis florea from Gerry, Khartoum, Madani, and ElDender were also captured on tree branches for investigation.
The bees so collected were divided into two groups (100 to 150 bee
per a group) then one group killed by hot water (so as to obtain a fully
stretched proboscis) and the second one killed by chloroform. Both of
the two groups were separately preserved in Ethanol (75 to 90%).
Table (1):
Sampling localities, respective geographical zones, map refrence
numbers & coordinates of honeybee localities analysed in this
study.
No.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Sample
localities
Shendi
Gerry
Khartoum
El-Hissahisa
Madany
Erkawit
Gala Elnahal.
Elhawata
Dokka
Glabat
Semsim
El- Dender
Kosti.
Singga
Geographical
zones
Semi- desert
zone.
Semi- desert zone.
Semi- desert zone.
Semi- desert zone.
Semi- desert zone.
Semi- desert zone.
Poor savannah
zone.
Poor savannah
zone.
Poor savannah
zone.
Poor savannah
zone.
Poor savannah
zone.
Poor savannah
zone.
Poor savannah
zone.
Poor savannah
zone.
90
Map
reference
(1).
(2).
(3).
(4).
(5).
(6).
Coordinates
Longitude Latitude
33°45E
17°00N
32°48 E
32°30E
33°21E
34°00E
16°15 N
15°45N
14°45N
14°15N
34°45E
13°36N
34°30E
13°30N
(9).
35°54E
12°45N
(10).
36°6 E
13°00N
(11).
(12).
34° 45 E
(13).
32°24E
(14).
33° 95 E
(7).
(8).
12° 36 N
13°24N
13° 15 N
Table (1) continued
15.
16.
Rosseris
El- Damazin
17.
Om- Rawaba
18.
Eldalang
19.
Kadogly
20.
Wau-Sholok.
21.
Malakal
22.
Dolaib.
23.
Gannal.
24.
Raja
25.
26.
ShawishMahadi
Kafindabi
27.
Kubum
28.
Zalinge
29.
Juba
30.
Poor savannah
zone.
Poor savannah
zone.
Poor savannah
zone.
Poor savannah
zone.
Poor savannah
zone.
Rich savannah
zone.
Rich savannah
zone.
Rich savannah
zone.
Rich savannah
zone.
Forest zone.
(15).
34° 38 E
11° 85 N
(16).
34° 29 E 11° 78 N
(17).
31° 20 E 12° 80 N
(18).
30°00E
12°30N
(19).
29°45E
11°00N
(20).
31°48E
9° 45N
(21).
31°45E
9° 30 N
(22).
32°00E
9° 18N
(23).
31°12E
9° 15N
(24).
28.08° E 7.70° N
Rich savannah
zone.
Rich savannah
zone.
Poor savannah
zone.
Poor savannah
zone.
Forest zone.
(25).
(29).
31°30E
4° 48N
Bango
Forest zone.
(30).
31°15E
4° 30N
31.
Liria
Forest zone.
(31).
32°15E
4° 15N
32.
Forest zone.
(32).
31°45E
5° 15N
33.
KhourMaquire.
Yei
Forest zone.
(33).
34.
Latokka
Forest zone
(34).
91
(26).
(27).
(28).
23° 80 E 11°
80 N
34° 29 E 11° 78
N
30° 60 E 4° 00 N
35.
Kajiko
Forest zone
(35).
3- 2- Morphometric analysis:
Fifteen bee workers were randomly taken from each colony as
representive samples for morphometric measurements at the institute
für Bienekunde [Oberursel, Germany]. Thirty eight characters as
listed in Table (2), consist of the characters introduced and tested by
Goetze (1964) and Alpatov (1929), i.e. pilosity, size, colour, cubital
venation (Nos. 1, 3-17, 21, 22, 27-30), plus some of the angles in the
wing venation tested by Dupraw (1964) in collaboration with the
laboratory of the institute für Bienekunde [Oberursel, Germany (Nos.
31-41)] and some newly selected characters (No. 18-20; 23-26
Ruttner, 1978). Three of these characters were eliminated, either
because they added no new information (length of hairs between the
facets of the eye and hind wing hamulies) or because of they proved
redundancy (No. 27, 28, cubital veins left).
The above-mentioned 39 characters were considered as, quantifiable
number for the taxonomic values in the institute für Bienekunde,
Oberursel, Germany), Ruttner et al., (1978).
92
Table (2): List of characters used for the analysis:
93
Character
Nu
Fig
Author
mbe
ure
r
Length of hairs on tergite 5.
1
Goetze
(1964).
2
Width of the tomentum band on
the side of tergite 4.
3
Goetze
(1964).
Width of the dark stripe between
the tomentum and the posterior
2
2
Goetze
(1964).
2
rim of the tergite.
4
Length of the stretched proboscis
(glossa+ mentum+ submentum).
68
Alpatov
(1929).
Length of the hind leg (femur
No.6, tibia No. 7, metatarsus No.
3
Alpatov
(1929).
4
8).
9
Width of metatarsus 3.
Alpatov
(1929).
1012
Pigmentation of tergites 2, 3 &4,
4
Goetze
evaluated according to a scale of
(1964).
5
10 grades between the darkest (0)
and brightest (9).
13,
14
15
Diameter of tergites 3 and 4,
Alpatov
longitudinal*
(1929).
Sternite 3 longitudinal*
Alpatov
(1929).
16,
17
18
Wax
mirrior,
on
sternite
3,
longitudinal and transversal*
Distance between wax mirrors, of
sternite 3.
7
Alpatov
(1929).
7
Ruttner
(1978).
94
6
7
Sternite
19,
6,
longitudinal
and
transversal*
20
Ruttner
(1978).
8
Table (2) continued
Forewing length and width.
Alpatov
(1928).
21
9
,
22
Pigmentation
23
(coloration)
of
the
scutellum.
Ruttner
(1978).
,
1
0
24
Pigmentation of the labrum.
Ruttner
(1978).
25
1
,
1
26
b
Segment ‘a’ and ‘b’ of the cubital cell of
27
right forewing.
Alpatov
(1929).
9
,
28
11angles between lines connecting cross
31
41
points of the venation on the forewing
(No.31= angle A4, 32= B4, 33= D7, 34=
E9, 35= G18, 36= J10, 37= J16, 38=
K19, 39= L13, 40= N23, 41= O26).
*The term ''longitudinal & transversal''
are used instead of ''length'' and ''width''
which may be misleading.
95
Goetze
(1964).
1
2
In addition to the above primary measurements, there were some
secondary values calculated by summation and division. For instance,
No. 2 and 3 for tomentum index, No. 6, 7 and 8 for length of the hind
leg, No. 8 and 9 for metatarsus index, No. 13 and 14 for values of
body size, No. 19 and 20 for index of body slenderness and No. 29
and 30 for cubital index.
For the statistical analysis, only the Primary values were used, but
the indices, being independent of body size were very useful in
characterizing a race. Even in early times a race was characterized by
broad metatarsi (Apis m. remipes, Gertstacker 1860; Buttel- Reepen
1906) or high cubital index (A. m. carnica; Goetze 1940) [the cubital
index is the ratio of the distance “a” and “b”, determined by the point
at which the nervous recurrent Nr joints the lower vein of the third
cubital cell “C”. of the right forewing Ruttner & Mackensen, 1952.
The index of slenderness is an objective measure of a slender or broad
body distinguishable even with the naked eye, thus mathematically it
is the ratio of the length to the width of the abdomen sternite 6.
3- 2- a- Preparation and measurements records of the bees:
The preparation of the bees and recording of the measurements are
organized in such a way that, all the values for each individual bee are
registered en bloc.
The measurements of the pilosity (length of hairs on tergite 5 and
width of the tomentum band on the side of tergite 4) are made on the
un dissected bee and that of other characters of the parts of the body
are adjusted on a slide. The measurements of wing venation angles,
cubital veins and characters of size were measured using a CCD
camera combined with an on-screen measuring system (Leitz)
96
magnification 50X, (Meixner, 1994). Or for some characters (hairs,
tergites, colour) on a stereomicroscope, magnification 40X.
With roughly about 40 characters per bee nearly 600 data were
accumulated per one sample; 11,400 data per all the samples.
97
Fig (2): - Abdomen of the honeybee workers Apis
mellifera L.
a. Width of tomentum of tergite 4 (No.2); b. Width of
the dark stripe between tomentum and posterior rim of
the tergite (No. 3); h. Length of hairs on tergite 5
(No.1).
98
Fig (3): Length of Proboscis of honeybee Workers Apis mellifera L.
99
Fig (4): Hind leg of the honeybee workers (Apis
mellifera L.) Fe: length of femur (No. 6), Ti: length of
tibia (No. 7), ML: length of metatarsus (No. 8), MT:
width of metatarsus (No. 9).
100
Fig (5): Classes of pigmentation of tergites (2 to 4).
Evaluated by 10 classes (No. 10- 12).
101
Fig (6): Longitudinal diameter of tergite 3 and 4 of
honeybee workers Apis mellifera L. (No. 13, 14).
Fig. (7): Sternite 3 diameters of honeybee
workers Apis mellifera L.:
S3: longitudinal diameter (No. 15), WL: wax mirror,
longitudinal (No. 16), WT: wax mirror transversal (No. 17),
WD: distance between wax mirrors (No. 18). [Honeybee
workers Apis mellifera L.]
102
Fig (8): Length and Width of Sternite 6. of
honeybee workers Apis mellifera L.
L6: longitudinal (No. 19); T6: transversal (No.
20).
103
Fig (9):
Ho
ne
yb
104
ee
wo
rk
ers
Ap
is
me
llif
er
a
L.
for
ew
in
g;
FL: length (No. 21); FB:
Width (NO. 22) a:
cubital
vein
(No. 27); b: cubital
vein (No. 28).
105
Fig (10): Scutellum of the
honeybee workers Apis mellfera L. Sc =
Scutellum: Scale of pigmentation 0
(completely dark); 9 (yellow). B, K:
Metatergum and mesotergal sclerite (scale of
pigmentation 0-5). [No. 23 & 24].
106
Fig (11- a): Bee workers Apis mellifera L. Labrum.
107
Fig (11- b.): Labrum pigmentation of the honeybee
workers Apis mellifera L. (No.25 &26).
108
Fig (12): Wing venation Angles of Honeybee worker Apis
mellifera L.
109
3- 2- b- Statistical analysis: First of all the crude data was recorded in perforated cards. Two cards
were necessary per bee, per sample and each card was labelled with the
number of the sample, number of the bees, sex, country and region.
The first steps of the statistical analysis were the transformation of the
data into a uniform scale and computation of the means and standard
deviation.
The important next step was the elimination of errors by inspection of
the standard deviations. A high value than usual indicated that a mistake
probably was made in writing, reading or punching. The printouts were then
compared with the original records and the erroneous cards were replaced.
The statistical analysis of the data was performed with SPSS for
windows and Statistical computer programs.
Thus this statistical analysis has so far been made on 32 characters and 285
samples of worker bees from all zones with autochthonous mellifera bees
available, so the basic information subjected to statistical treatment was
represented by a 285 X 32 array, where each of the 285 lines was a sample
of 15 bees. The 32 components of this vector line were the means of
individually measured character. Multivariate analysis allowed a general
survey of these data.
As it was not easy to imagine a cloud of 285 points in 32 dimensional
space and in order to simplify this problem, an attempt was made to
determine a few number of privileged axes: This was the aim of the
Principal Component Analysis (PCA).
From all the correlation coefficients between the first variable two by
two, it was possible to obtain histograms on the new axes and pointprojections on the planes formed by two of these axes.
110
Interpretation of the results consisted of characterizing the clusters of points.
The contribution of each of the basic variables in the formation of the new
ones was estimated. In this way, some little interesting characters could be
discarded, since selection of the most discriminant criteria was the most
important goal. So in this initial study, the basic tool was the PCA: a
descriptive and statistical hypothesis-free method.
The Progressive Discriminant Analysis was also used. With this
technique, and using a progressive number of the first variables, it was
possible to obtain a borderline between two populations to be identified for
classification.
The last objective was to get a picture of each bee-type collected and locate
it geographically. The Factor Analysis of correspondences allows
representation on the same plane, the profiles of the variables as well as
those of the samples.
3- 3- Mitochondrial DNA: In addition to the captured samples there were 16 Sudanese honeybee
worker samples added to the mitochondrial DNA analysis (samples
collected from localities which I am not captured bees).
Those samples are: 5 samples from; Halfa- Elgadeda and Fau (semi-desert
zone). El- Damazin and Om-Rawaba (savannah zone), and Raja (forest
zone), collected by Mogbil (2004-2005).
Eleven samples were obtained from the data bank of the institute für
Bienekunde [Oberursel, Germany]; (Sudanese honeybee workers collected
by Mogga 20 years ago, for morphometric studies at that time, thus they
were preserved in ethanol at room temperature).
Those samples are: Yei, Lwatoka, Kafindapy, Shawish- Mahadi and
Kubbum (forest and rich savannah zone). Zalinge, Roseris, Singa and
111
Simsim (poor savannah zone). Salahap and Kassala (semi-desert zone).
Worker bees used for the mitochondrial DNA analysis were killed by
chloroform and immediately immersed in ethanol 90% for preservation at
room temperature.
3- 3- a- DNA extraction:
Before DNA extraction, a single bee from each preserved bees samples
in ethanol 90%, was taken in a separate tube (one bee from each group
representing the sample), and were carefully washed by distilled water (to
remove alcohol). The sample tubes were shaken by a thermo mixture
(shaking only) for more than one hour and the distilled water was changed
from time to time.
Each target bee mesotharax was dissected and the muscles were kept in a
separate tubes (1.5 ml Eppendorf tubes) and stored at –20º C. until they
were processed in the laboratory.
Total DNA was extracted from thoraces using Phenol- chloroform
extraction and ethanol precipitation protocols (Sambrook et al., 1989), with
slight modification as follow:
1- Some distal water was added to the muscles tubes, and shaked on the
thermomixture for 15 minutes.
2- Water was removed outside the tubes. 400 µL. Wilson Buffer (PH= 0,8)
was added to each tube.
3- Tissues inside tubes were crashed by an special plastic stick.
4- Ten µL. of Proteinase-K, was added to each tube and gentelly shaked (so
as to perform protein digestion).
5- Samples were put in the thermomixture (55º C and 6-7 cycles) for eight
hours (tubes were genttely shaked from time to time).
112
6- 410 µL. of Phenol-chloroform-isoamylalkohol (P C 1), 25: 24: 1. was
added to each sample tube (the same volume as in step 2 and 4), with
gentelly mixing for five minutes.
7- Samples were centrifuged for 15 minutes at 13000 rpm. speed.
8- The supernatant (270 µL.) was taked to a new tube, then an equal amount
of chlorofom-iso was added, with gentelly mixing for five minutes and
centrifuged for 15 minutes at 13000 rpm. speed.
9- The supernatant (about 180 µL.) was transformed into a new tube. 10%
from the volume of the supernatant sodium acetate (18 µL.) and 2 volumes
ethanol 100% (360 µL.) were added. The sample tubes were kept inside
freezer for overnight at –20º C.
10- Samples were centrifuged at 13000 rpm. speed for 15 minutes. The
supernatant were carfully thrown out the tubes. Two volumes of ethanol
70%, were added and centrifuged at 13000 rpm. speed for 5 minutes.
11- The supernatant were carfully thrown out and the remaining pellets
were dried at 55º C (on the thermomixture. The tubes were opened).
12- 30 µL of water or Buffer was added, then the DNA was preserved at
–20º C.
3- 3- b- PCR amplification:
The mitochondrial DNA fragment including the COI- COII intergenic
region (tRNAleu gene and the 5´-end of the COII gene), was amplified by
PCR (Biometra thermocycler), using the primers E2 (5´GGCAAGAATAAGTGCATTG-3´) and H2 (5´-CAATAT
CATTGATGACC-3´) (Garnery et al., 1992), as follow:
The PCR reaction was performed with the corresponding 1x buffer, 0.25
µL of each dNTPs, 0.1 µL Mgcl2, 0.1 µL Promega Taq polymerase, 0.25 µL
of primers E2 and H2, 1.0 µL of extracted DNA, in a total volume of 30 µL.
113
Reactions were submitted to an initial denaturation of 5 min at 96º C, 30.
Cycles of 95º C for 0.5 min, 50ºC for 1.5 min and 72ºC for 1.5 min, and a
final extension of 10 min at 72º C.
3- 3- c- DNA purification:
The extracted DNA was purified from the remaining of the chemicals
used in the PCR reaction by DNA Clean-up System (promega) following
the manufacturer’s instructions as follow:
1- 5 volume of Buffer P.B was added to 1 volume of the PCR product and
mixed (5× 30 µL or 150 µL to each).
2- The QLA spin quick column was placed in a provided 2 ml collection
tube.
3- The mixture (of step 1.) was transformed to the QLA quick column and
centrifuged for 30 to 60 seconds at 13.000 rpm.
4- The supernatant was discarded, and the QLA quick column was placed
back into the same tube.
5- 750 µL Buffer P.E was added into the QLA quick column and
centrifuged for 30 to 60 seconds at 13.000 rpm.
6- As in 4 plus the QLA quick column was centrifuged for an additional 1minute.
7- The QLA column was placed in a cleaned 1.5 ml microcentrifuge tube.
8- The DNA was diluted by adding 30 µL of sterilled water to the centre of
the QLA quick column membrane; and the QLA quick column was left
stand for 2 minutes and centrifuged for 1 minute.
114
4- 3- d- Size category of the fragments:
Five µL. of the PCR product was electrophoresed through 1.5% agarose gel
and
stained with ethidium bromide in order to determine the size of each
product.
3- 3- e- Endonuclease digestion:
Fifteen µL. of the PCR product was digested with the restriction enzyme
DraI at 37º C for over night. Restriction fragments were separated on 10%
and 8% acryl amide gel and stained with ethidume bromide.
115
CHAPTER FOUR: RESULTS
4- 1- Morphometric analysis (Apis mellifera L.):
4- 1- i - Univariate analyses:
The results obtained in the univariate analysis of the 19 colony samples of
Apis mellifera L. from at least four different geographical regions were shown
in Tables (3 to 9).
4- 1- i- a- The proboscis and hind- leg measurements (mm.), {Table 3}:
The mean length of the proboscis for the samples varied from 5.51 To
5.75, with an average of 5.63. Mean length of the different parts of the hind-leg
for the sample varied from: Femur 2.23 To 2.46, with an average of 2.34. Tibia
2.86 to 3.16, with an average of 2.98. The metatarsus 1.78 to 2.01, with an
average of 1.88. The mean total hind-leg length for the samples varied from
7.00 to 7.87, with an average of 7.26. The mean width of metatarsus for the
samples varied from 1.00 to 1.13, with an average of 1.07. The samples
metatarsal index varied from 55.91 to 60. 04 with an average of 57.39.
4- 1- i- b- Forewing measurements (mm.), {Table 4}:
The mean length of forewing for the samples varied from 8.16 to 8.63,
with an average of 8.36, while the mean width varied from 2.79 to 3.01, with an
average of 2.90. The mean cubital vein “a” for the samples varied from 4.19 to
5.25, with an average of 4.67, and vein “b” varied from 1.56 to 2.12, with an
average of 1.90. The mean cubital index a/b of the samples varied from 1.75 to
2.67, with an average of 2.13.
116
Table (3):
Means of measurements of proboscis and hind- leg of the Sudanese honeybee
workers Apis mellifera (mm.).
117
4- 1- i- c- Forewing venation angles measurements (degrees), (Table 5) :
The means of angles of the forewing venations for the samples varied
as follows:
A4: from 30.37 to 35.32 , with an average of 32.40. B4: from 97.84 to 105.70 ,
with an average of 101.86. D7: from 96.85 to 103.59 , with an average of
100.17. E9: from 18.17 to 20.96 , with an average of 19.73. G18: from 95.63
to 101.78 , with an average of 98.60. J10: from 49.32 to 56.13 , with an
average of 52.87. J16: from 88.61 to 95.57 , with an average of 93.01. K19:
from 74.58 to 83.54 , with an average of 80. 39. L13: from 12.66 to 23.60 ,
with an average of 15.30. N23: from 83.85 to 93.72 , with an average of 88.11.
O26: from 34.07 to 43.72 , with an average of 37. 77.
4- 1- i- d- Body size (tergites 3 and 4) and sternite 3 measurements (mm.),
{Table 6}:
The mean length of tergite 3 (T3) for the samples varied from 1.94 to 2.09,
with an average of 2.03. Tergite 4 (T4) varied from 1.90 to 2.05, with an
average of 1.97. The mean body length T3+4 varied from 3.79 to 4.06, with an
average of 4.00. Mean length of sternite (S3) for the samples varied from 2.36
to 2.58, with an average of 2.48.Mean length of wax merrior on S3 varied from
1.03 to 1.16, with an average of 1.11. Mean width of the wax merrior on S3
varied from 1.90 to 2.04, with an average of 1.98. Mean distance between wax
merriors on S3 varied from 0.30 to 0.38. with an average of 0.34.
4- 1- i- e- Pilosity measurements (mm.), {Table 7}:
The mean length of hairs on tergite (T5) for the samples varied from 0.17 to
0.26, with an average of 0.20. Mean width of tomentum on tergite (T4) varied
from 0.59 to 1.05, with an average of 0.89. The mean width of the dark stripe
118
on T4 varied from 0.26 to 0.66, with an average of 0.44. The mean tomentum
index for the samples varied from 2.21 to 5.90, with an average of 3.14.
4- 1- i- f- Sternite 6 (Abdominal slenderness) measurements (mm.),
{Table 8}:
The mean length of sternite (S6) for the samples varied from 2.22 to 2.49,
with an average of 2.19. While the mean width varied from 2.59 to 2.83, with
an average of 2,72. The abdominal slenderness (L/W) for the samples varied
from 81.31 to 87.31, with an average of 84.49.
4- 1- i- g- Pigmentation (Colouration) measurements {Table 9}:
The mean colouration on T2 for the samples varied from 5.16 to 8.94,
with an average of 8.66; Mean colouration on T3 varied from 5.96, to 9.54 ,
with an average of 8.51.
The mean colouration on tergie 4 (T4) varied from 3.29 to 5.25, with an
average of 4.47. The mean colouration of the scutellum for the samples varied
from 4.48 to 7.44, with an average of 6.89; while the mean colouration of
metatergum varied from 1.03 to 6.44, with an average of 3.82.
The mean colouration of the labrum for the samples varied from 1.60 to 8.10,
with an average of 4.10, and from 0.13 to 3.02 with an average of 1.36.
119
Table (4): Means of measurements of the forewing of
the
Sudanese honeybee workers Apis mellifera L.
(mm.)
Length
Width
Cubital
Vein a
Cubital
Vein b
Gala Elnahal
8.38
2.86
4.35
1.76
2.12
Madani
8.19
2.79
4.52
1.95
2.02
Khartoum
8.22
2.86
4.61
2.05
1.93
Shendi
8.19
2.94
4.64
2.03
1.96
Kadogli
8.42
2.93
4.75
1.95
2.09
Eldalang
8.45
2.95
4.66
1.96
2.04
Wau- Sholok
8.51
2.95
5.17
1.79
2.49
Malakal
8.39
2.93
5.25
1.73
2.62
Doleib.
8.63
2.99
5.15
1.90
2.34
Gannal.
8.45
3.01
4.87
1.56
2.67
Elhawata
8.54
2.98
5.20
1.83
2.49
Doka
8.16
2.87
4.63
1.78
2.24
Glabat
8.52
3.00
4.61
2.12
1.88
Kosti
8.49
2.93
4.52
2.03
1.93
Hissahisa
8.48
2.92
4.93
1.87
2.26
Juba
8.23
2.81
4.19
1.91
1.89
Bango
8.27
2.85
4.21
2.06
1.75
Liria
8.18
2.82
4.38
1.89
1.99
KhourMaqure
8.24
2.82
4.25
2.03
1.80
Sample
120
C. Index
A/b
Table (5): Means of measurements of forewing venation angles
of the Sudanese honeybee workers Apis mellifera L. (degrees)
Forewing venation angles
Sample
A4
B4
D7
E9
G18
J10
J16
K19
L13
N23
O26
GalaElnahal
35.32
97.84
102.82
20.08
97.14
49.87
94.62
83.22
12.66
89.22
39.17
Madani
33.96
98.36
97.74
18.17
96.76
49.32
95.30
78.42
15.11
87.55
35.06
Khartoum
Shendi
31.38
32.23
103.62
101.33
100.39
99.79
20.56
20.02
95.86
95.63
52.24
51.94
93.00
94.28
79.49
80.19
14.80
15.12
87.33
87.42
37.94
36.84
Kadogli
32.21
101.65
97.78
20.56
100.89
51.19
90.56
82.91
14.24
85.98
38.62
Eldalang
Wau-Sholok
32.00
31.71
103.06
100.63
96.85
99.03
20.96
19.91
99.55
100.19
51.90
53.78
88.96
91.11
82.83
83.08
14.33
16.03
84.48
83.85
38.37
36.07
Malakal
Doleib.
31.90
30.37
100.37
105.00
97.43
101.81
20.26
20.08
101.78
96.30
54.74
55.73
92.73
93.78
82.43
79.69
15.22
15.86
86.45
90.25
34.75
38.55
Gannal.
34.05
100.99
99.53
20.62
99.41
55.78
88.61
74.58
15.93
85.24
36.59
Elhawata
32.23
99.70
99.03
18.76
98.28
54.75
93.18
81.29
15.78
90.22
34.07
Doka
33.47
101.34
102.86
19.89
98.53
51.68
91.99
82.32
14.72
87.44
35.01
Glabat
31.67
103.34
102.89
20.03
99.97
50.78
92.92
83.54
13.63
88.18
37.38
Kosti
33.39
100.36
101.29
19.82
99.12
50.83
95.13
80.31
15.65
88.15
38.32
Hissahisa
31.96
101.05
98.96
19.00
97.05
53.03
91.70
80.60
15.09
88.97
35.00
Juba
31.67
105.70
100.27
19.36
99.32
53.23
95.34
78.83
14.73
91.05
43.72
Bango
31.83
104.53
100.42
18.88
99.13
56.13
95.51
74.66
23.60
93.72
40.92
Liria
32.02
102.98
100.67
18.66
99.60
53.19
95.57
79.76
14.46
89.15
40.44
KhourMaqure
32.22
103.46
103.59
19.35
98.84
54.40
92.86
79.22
13.76
89.49
40.87
121
Table (6): Means of measurements of some tergites and
sternites of the Sudanese honeybee Apis mellifera L.
(mm.)
Sample
Length
T3
Length
T4
Length
T3 +4
Length
S3
Length
Wax
Mirror
S3
Width
wax
Mirror
S3
Distance
Between
W/mirror
S3
Gala- Elnahal
2.07
2.05
4.06
2.55
1.16
2.04
0.31
Madani
1.96
1.91
3.82
2.45
1.13
2.02
0.34
Khartoum
1.94
1.9
3.79
2.4
1.11
1.96
0.32
Shendi
2.06
2.02
4.03
2.42
1.11
1.94
0.32
Kadogli
2.09
1.98
4.02
2.5
1.13
2
0.33
Eldalang
2.09
2.02
4.05
2.53
1.14
1.97
0.34
Wau-Sholok
2.03
1.99
3.97
2.48
1.11
2.03
0.34
Malakal
2.03
2
3.98
2.47
1.11
2.01
0.35
Doleib.
2.06
2.01
4.01
2.53
1.12
2.02
0.32
Gannal.
2.05
2.03
4.02
2.57
1.16
1.99
0.36
Elhawata
2.01
2
3.96
2.51
1.07
1.99
0.38
Doka
2.06
1.98
3.98
2.48
1.12
2
0.31
Glabat
2.05
2.01
4.01
2.58
1.15
2.03
0.33
Kosti
1.99
1.94
3.88
2.52
1.11
1.98
0.3
Hissahisa
1.99
1.95
3.88
2.56
1.11
2.02
0.36
Juba
2.07
1.95
3.96
2.42
1.08
1.94
0.34
Bango
1.99
1.94
3.87
2.36
1.08
1.9
0.34
Liria
1.98
1.91
3.84
2.36
1.03
1.9
0.35
Khour-Maqure
1.97
1.92
3.84
2.44
1.07
1.92
0.36
122
Table (7): Means of measurements of
pilosity of the Sudanese honeybee
workers Apis mellifera L. (mm.)
Samples
Length of
hair
Width of
tomentum
Width of
Tomentum
Dark
Index
stripe
Gala Elnahal
Madani
Khartoum
Shendi
Kadogli
Eldalang
Wau- Sholok
Malakal
Doleib.
Gannal.
Elhawata
Doka
Glabat
Kosti
Hissahisa
Juba
Bango
Liria
Khour-Maqure
0.21
0.88
0.45
2.91
0.21
0.98
0.26
5.68
0.21
0.93
0.45
3.04
0.21
0.92
0.50
2.74
0.26
0.87
0.53
2.44
0.19
0.94
0.53
2.66
0.18
0.79
0.45
2.62
0.19
0.99
0.43
3.43
0.22
0.91
0.46
2.93
0.20
0.87
0.41
3.10
0.22
0.85
0.49
2.61
0.18
0.96
0.51
2.82
0.17
0.98
0.66
2.21
0.21
1.05
0.26
5.90
0.17
0.92
0.36
3.74
0.17
0.83
0.41
3.03
0.23
0.83
0.49
2.54
0.22
0.59
0.38
2.31
0.24
0.81
0.40
3.00
123
Table (8): Means of measurements of sternite 6 of the
Sudanese honeybee workers Apis mellifera L. (mm.)
Samples
Length of
Width of
Abdominal
S6
S6
Slenderness
L/W.
Gala Elnahal
2.37
2.76
85.20
Madani
2.23
2.72
81.31
Khartoum
2.22
2.59
84.94
Shendi
2.22
2.64
83.26
Kadogli
2.34
2.75
84.18
Eldalang
2.33
2.79
82.96
Wau- Sholok
2.37
2.77
84.66
Malakal
2.31
2.76
82.92
Doleib.
Gannal.
Elhawata
2.38
2.49
2.37
2.75
2.83
2.74
85.94
87.31
85.64
Doka
Glabat
Kosti
2.28
2.37
2.32
2.72
2.77
2.68
82.95
84.82
86.01
Hissahisa
2.36
2.75
84.80
Juba
Bango
2.27
2.26
2.66
2.67
84.89
83.80
Liria
2.24
2.61
85.16
Khour-Maqure
2.24
2.63
84.51
124
Table (9): Means of measurements of colouration
of the Sudanese honeybee workers
125
Table (10):
Mean, minimum, maximum and standard deviation of each morphometric
character from the 285
Individual bees Apis mellifera L. measured (measurements in mm. Angels in
degree).
126
Table (10) continued:
127
4-1- i- h- Mean, minimum, maximum and standard deviation of each
morphometric character from the 285 individual bees measured:
The minimum, maximum and standard deviation values of each
morphometric character for the 285 individual worker bees indicated the
presence of a wide range of variability within the honeybees collected in this
study., (Table 10).
4- 1- i- i- Sum of squares, dF, mean square, F values and significances
for each phenotypic character from the measured individuals.
(Sudanese honeybees Apis mellifera L.).
The analysis of variance means for the phenotypic characters of 19
colonies also revealed the existence of extensive variability in most of the
measured characters across all localities (p ≤ 0.005).
Characters which had high F values are as follow (from the highest
values to the lowers ones), width of the wax mirror on sternite3 (17), width
of the forewing (22), length of the sternite3 (15), length of the cubital vein a
(27), wing venation of the angle O26 (41), length of the proboscis (4), width
of the sternite6 (20), length of the wax mirror on sternite3 (16), length of the
forewing (21), length of the tergite3 (13), width of the metatarsus (8), length
of the sternite6 (19), pigmentation of the labrum 1 (25), and the wing
venation of the angle G18 (35) [Numbers between brackets are the code
numbers of the character measured as in Table (2) ]. But generally, they
were all had high F values than the rest of the other characters, the summary
of the analysis of variance of all the characters with their respective F values
As shown in Appendix (H).
4- 1- ii - Multivariate analyses:
4- 1- ii- a- Principal components analysis (PCA):
128
The principal components analysis of morphometric characters of 19
colony means data was used to detect the presence of possible cluster groups
of colonies. According to this analysis three factors with eigenvalues greater
than 1 were exracted. The a cumulative eigenvalue of these factors was
18.179. Factor 1 had the highest eigenvalue of 12.347 followed by factors 2
and 3 with eigenvalues of 3.458 and 2.374 respectively (Table 12). Except
some wing venation angles as D7, G18, K19, L13, and some body size
characters as cubital vein 2 length, distance between wax mirror in sternite 3
and hair length which had absolute value of factor loadings less than 0.50;
the remaining characters had absolute value of factor loading between 0.51 –
0.90.
Based on varimax rotation factor loadings analysis, characters such as
cubital vein 1, forewing venation angle E9, hind leg femur length, forewing
venation angle J16, forewing length, sternite3 length, sternite 6 length, tergite
3 length, tergite 4 length, hind leg metatarsus length, forewing venation
angle N23, forewing venation angle O26, hind leg tibia length, forewing
width, sternite 6 width and hind leg metatarsus width all had high loading
values in factor 1 and 37.414 % of the variance in the data is attributed to
this factor. While characters such as forewing venation angles A4, B4, and
J10; wax mirror in sternite 3 length, tomentum 1 length and wax mirror in
sternite 3 width all had high loading values in factor 2, this accounted for
10.479 % of the variance in the data. Thus colouration characters such as
pigmentation on tergites 2, 3 and 4; scutellum 1 and 2 plus tomentum 2
length all had high loading values in factor 3 and accounted 7.194 % of the
variance in the data.
Generally, these three factors accounted for 55.087 % of the variance
from the data used in this analysis. The values of factor loading of each
character in the respective group of extracted factors were shown in Table
129
(12). The scatter plot graph of factor scores of factor 1 and 2 and that of
factor 1 and 3, of the principal components analysis of the 19 colony means
of all morphometric characters revealed the presence of three possible
groups of clusters of colonies (Figure 13 & 14).
Three clear scattered clusters of samples were formed in this graph
figure (13). The first cluster (dark green colour) was completely separated
from the others, consisted of the forest zone samples. While the middle dark
orange colour cluster composed of the semi-desert zone samples and the last
largest cluster (light blue colour) consisted of savannah zone samples, in
which three samples; Doka No. 9, Kosti No. 13 and Galla- El Nahal No. 7
were considered to be within the semi- desert zone samples.
130
Table (12) Factor loadings in varimax rotation for
each character in the principal components analysis.
Component
Characters Factor 1 Factor 2 Factor 3
Wing angle A4.
-0,736
Wing angle B4.
0,766
Cubital vein 1 length
0,769
Cubital vein 2 length
Wing angle D7.
Distance between wax
mirror in sternite 3.
Wing angle E9.
0,610
Hind leg femur length
0,652
Wing angle G18
Hair length.
Wing angle J10
0,679
16
Wing angle J
-0,765
Wing angle K19
Wing angle L13
Forewing length
0,752
Sternite 3 length
0,696
Sternite 6 length
0,728
Tergite 3 length
0,544
-0,516
Tergite 4 length
0,716
Metatarsus length
0,721
Wax mirror length
0,554
-0,610
Wing angle N23.
-0,587
Wing angle O26.
-0,507
Tergite 2 pigment
0,754
Tergite 3 pigment
0,750
Tergite 4 pigment
0,876
Scutellum 1 pigment
0,796
Scutellum 2 pigment
0,564
Hind leg tibia length
0,667
Width of tomentum 1
-0,512
Width of tomentum 2
-0,582
Width of forewing
0,899
Width of sternite 6
0,792
Width of metatarsus
0,772
Width of wax mirror
0,559
-0,680
Explained variance
12.347
3.458
2.374
Extraction Method: Principal Component Analysis. Rotation Method:
Varimax with Kaiser Normalization. A Rotation converged in 6 iterations.
131
Fig (13): Scatter plot graph of factor scores of
factor 1 and factor 2 from principal components
analysis of 19 colony means of all morphometric
data. Horizontal axis: factor 1; Vertical axis: factor
2. (Numbers in the scatter plot graph indicate the
locality code).
1- Shendi, 3- Khartoum, 4- El-Hissahisa, 5- Madani, 7- Galla- Elnahal, 8- ElHawata, 9- Dokka, 10- El-Galabat, 13- Kosti, 18- El-Dalang, 19- Kadogli, 20- WauSholok, 21- Malakal, 22- Doleib, 23- Ganal, 29- Juba, 30- Bango, 31- Lirria, 32Kour- Maquire.
132
Fig (14): Scatter plot graph of factor scores of factor 1 and factor 3
from principal components analysis of 19 colony means of all
morphometric data. Horizontal axis: factor 1; Vertical axis: factor 3.
(Numbers in the scatter plot graph indicate the locality code).
1- Shendi, 3- Khartoum, 4- El-Hissahisa, 5- Madani, 7- Galla- Elnahal, 8El- Hawata, 9- Dokka, 10- El-Galabat, 13- Kosti, 18- El-Dalang, 19Kadogli, 20- Wau- Sholok, 21- Malakal, 22- Doleib, 23- Ganal, 29- Juba,
30- Bango, 31- Lirria, 32- Kour- Maquire.
133
From side of view of the morphometric methods of
honeybees classification an attempt was made to establish the
taxonomic states of the target ninteen colonies of Sudanese
honeybee Apis mellifera in relation to the rest of the African
Apis mellifera subspecies. The samples were analysed by PCA
with 239 samples (from the data bank, University of Frankfurt,
Institut für Bienenkunde, Oberursel, Germany). These included
18 samples: Ethwest (west of Ethiopia Mellifera bees), 19
samples Ethmount (Ethiopia Mountain Mellifera bees), 24
samples Lamarkii, 26 samples Jemenitica, 9 samples Litorea,
47 samples Scutellata, 27 samples Monticola, 50 samples
Adansoni and 22 samples Jemenitica- Sudan (Mogga 1987).
Atotal of 258 samples were processed. Graphical presentation
of the results were shown in Figure (15).
In figure (15), ninteen of the Sudanese honeybees samples
showed three distinguishable clusters on the uper side of the
scatter plot graph among the Jemenitica and Jemenitica-Sudan
samples. These three clusters were composed as follows:
Samples originating from the forest zone to the left uper side
of the graph; samples originating from the savannah zone to the
left uper side of the graph, closing to Jemenitica-Sudan and
somewhat near to some Scutellata samples; while cluster
originating from the semi-desert zone to the left uper side of the
graph within Jemenitica-Sudan and between Jemenitica and
savannah zone sampls.
Lamarkii and Adansoni samples formed two different
clusters but, near to each other at the right uper side of the
graph. Ethmount samples formed a clear seperate clusters to the
134
right downwards side of the graph, also Ethwest samples were
scattered in the downwards midle side of the graph between
Ethmount and Scutellata samples. Most of Monticola samples
were clustering at the left downwards side of the graph.
In figure (15), most Sudanese samples were distributed
among and arround Jemenitica and Jemenitica-Sudan races.
However, Sudanese clusters were distinguishable. The forest
and semi-desert samples were distributed in between Jemenitica
and Jemenitica-Sudan clusters at the left side of the graph, thus
they were near to some Monticola and Scutellata samples;
while the savannah samples were distributed near to the centre
at the uper side of the graph.
Ethmount, Ethwest and Lamarkii samples were distributed
at the downwards side of the graph, quite seperate from Apis
mellifera jemenitica samples.
135
Fig (15): Scatter plot graph of factor analysis of 239 samples of
worker honeybees of different origin. Horizontal axis: factor 1;
Vertical axis: factor 2.
136
4- 1- ii- b- Discriminant analysis:
In a further inspection and scrutiny on the three cluster
groups observed in the principal components analysis, some
step-wise discriminant analyses tests were done. In this case the
colony means data were also used.
There are some discriminant characters for each of the
three clusters which analysed as shown in Tables (13, 14 and
15). The three clusters showed some clear morphological and
geographical relationship. Table (13) represented samples from
the semi-desert zone, Table (14) represented samples from the
savannah zone and Table (15) represented samples from the
forest zone.
The variables used in the discriminant analysis are: L. Hair,
L. Femur, and L.Tib. L.Tar., W.Tar., L. Probo., P.T2., P.T3.,
P.T4., L.T3., L.T4., L.ST3., L.WM., W.WM., D.WM., L.ST6.,
W.ST6., L.FW.,W.FW., SCUT1., SCUT2., Cub1., Cub2.
Angles A4. B4. D7., E9., G18., J10., J16., K19., L13.,
N23., and O26.
In this analysis each colony was assumed to have a priori probability of
being in any particular cluster. Based on this analysis as shown in Table
(16), out of 4 colonies grouped in the semi-desert cluster all of them or
100% were correctly classified in this cluster. Forest cluster had the same
results as semi-desert in which out of the 4 colonies grouped in the cluster or
100% were all correctly classified in the forest cluster. Considering
savannah cluster out of 11 colonies grouped in this cluster 10 of them were
correctly classified as savannah cluster. The remaining one colony from
kosti was in semi-desert cluster, the PC rather thinks Kosti is semi-desert
137
with P= 0.509. Generally a 94,7% of original grouped cases correctly
classified.
By using the pair-wise group comparison test Table (17),
the separation of cluster groups was highly significant for all
cluster groups. Between the clusters semi-desert and savannah,
the separations of F values obtained were: 10.435 (the highest
one) and 0.618 (the lower one). While between semi-desert and
forest the separations of F values obtained were: 21.812 (the
highest one) and 13.521 (the lower one). Also the separation of
the F value between savannah clusters and forest obtained were
45.647 (the highest one) and 22.087 (the lower one) as shown
in Table (17).
The discriminant analysis of the colony means data also
confirmed the presence of three morphoclusters of colonies as
shown in figure (17).
Thus average linkage between the group centroids analysis
Table (20), shows the different distances between the groups
(semi-desert cluster to savannah 4.336; semi-desert to forest
6.108 and savannah to forest 5.941). In more details using the
average linkage between the groups as in figure (18), colonies
Juba, Lirria, Kour- Maquire were very closed to each other and
Bango close to them too, (the forest cluster). Also colonies
Shendi, Khartoum, Kosti and Madani appeared to be one cluster
(semi-desert cluster) than the rest of the clusters. Thus colonies
El-Dalang, Kadogli, Malakal, Wau- Sholok, El-Hissahisa, ElHawata, then El-Galabat, Doleib and Ganal were all appeared
to be one cluster (the savannah cluster). Only one colony
138
“Dokka” was far away from semi-desert and forest clusters
though somewhat near to savannah cluster.
For more clarification to the previous results obtained by
the PCA analysis of the relationship between the target 19
Sudanese Apis mellifera colonies and some other African
mellifera subspecies. A discriminant analysis was carried out in
Table (18) demonstrates a discriminant analysis probability
(regarding the individual colonies) in which colonies Gala- El
nahal, El-Dalang, Wau- Sholok, Malakal, Ganal, El- Hawata,
then El-Galabat, Kosti, El-Hissahisa, Juba and Lirria were all
had posterior probability of 1.00 for being in cluster jemeniticaSudan. Colony Bango had 78% posterior probability for being
in cluster jemenitica-Sudan. Only one colony “Kadogly” had a
low posterior probability (46%) for being in cluster jemeniticaSudan. The previous results were summarized in table (19b) in
which all the 19 colonies considered as one group (ungrouped
cases), 18 colonies or 94.7% had posterior probability for being
in cluster jemenitica-Sudan. One colony had a very low
posterior probability (5.3%) for being in cluster Adansoni.
Thus in Table (18), the probabilities without JemeniticaSudan (with other Mellifera subspecies); colonies Wau-Sholok,
El-Hissahisa, El-Hawata and Doleib had a high posterior
probability (1.00) for being in cluster Adansoni. Only one
colony “Shendi” had a high posterior probability (1.00) for
being in cluster Lamarkii. Dokka colony had a high posterior
probability (1.00) for being in cluster Scutellata, while ElDalang, El-Galabat, Gala-Elnahal, had 98%, 92%, and 85%
posterior probability for being in Scutellata cluster respectively.
139
Thus colonies Khartoum, Lirria, Madani and Kosti had 67%,
66%, 63% and 50% respectively probability for being in cluster
yemenitica. The rest probabilities were somewhat weak.
The discriminant analysis probability between the data
bank African mellifera subspecies (Apis mellifera jemeniticaSudan, A. m. jemenitica, A. m. scutellata, A. m. monticola, and
A. m. litorea) was shown in Table 19a. Out of 22 colonies
grouped in cluster Jemenitica-Sudan 19 or 86.4% of them were
correctly classified as jemenitica-Sudan cluster, the remaining
three colonies one (4.5%) was in cluster Jemenitica and one
(4.5%) was in cluster Litorea and the last one (4.5%) in cluster
Scutellata. Out of 26 colonies grouped in cluster Jemenitica 24
or 92.3% of them were correctly classified as Jemenitica cluster
whereas, the remaining two colonies were, one (4.5) was in
cluster Litorea and the second (4.5) in Scutellata cluster. Out of
50 colonies grouped in cluster Scutellata 44 or 88% of them
were correctly classified in cluster Scutellata while, the
remaining 6 colonies were classified 2 colonies (4.0%) to the
following
clusters:
Jemenitica,
Litorea
and
Monticola
respectively. In the same Table out of 9 colonies grouped in
cluster Litorea 8 or 88.9% of them were correctly classified in
cluster Litorea the rest one or 11.1% was classified in cluster
Jemenitica. Also out of 27 colonies grouped in cluster
Monticolla 26 or 96.3% of them were correctly classified in
cluster Monticola, while the remaining one (3.7%) was in
cluster Scutellata.
Discriminant analysis of the distances between the group
centroids (the three predicted clusters: semi-desert; savannah;
140
forest and the data bank African Mellifera subspecies) Table
(20) and figure (19), in which the semi-desert cluster colonies
centre was far away from the following clusters: Ethmount,
Ethwest, Monticola, Lamarkii, Scutellata, Litorea, Adansoni,
Jemenitica,
and
Jemenitica-Sudan
respectively.
Cluster
savannah colonies centre was far away from Ethmount, Ethwest
Monticola, Lamarkii, Litorea, Jemenitica, Adansoni, Scutellata,
and Jemenitica-Sudan clusters respectively. While forest cluster
colonies centre was far away from clusters; Ethmount, Ethwest,
Monticola,Lamarkii, Scutellata, Adansoni, Litorea, Jemenitica,
and Jemenitica-Sudan respectively.
The discriminant analysis of the distances between the
group’s centroids Table (20) and figure (20) demonstrated the
independences of the different clusters.
141
Table (13):
Means of some discriminant characters for the Sudanese
Honeybee workers Apis mellifera (mm.). From
The semi desert region.
142
Table (14):
Means of some discriminant characters for
The Sudanese honeybee workers Apis mellifera (mm.). From
The Savannah region.
143
Table (15):
Means of some discriminant characters for
The Sudanese honeybee workers Apis mellifera (mm.). From
The Forest region.
144
Originalcount
% Of
classification
Table (16): Classification matrices of colonies in
cluster groups based on step-wise discriminant
analysis. (Sudan samples only).
Predicted Group
Total No. of
Colonies
Samples Membership
SemiSavan
desert
nah
Forest
Semi4
0
0
4
desert
Savanna
1
10
0
11
Forest
0
0
4
4
Semi100,0
,0
,0
100,0
desert
Savanna
9,1
90,9
,0
100,0
Forest
,0
,0
100,0
a 94,7% of original grouped cases correctly classified.
Fig (17): Canonical Discriminant Function (Sudan
samples only).
145
100,0
Fig (18 ): Dendrogram using average linkage
Between the groups (Sudan colonies only).
146
Table (17): Pair-wise Group comparison
Step Samples
1
Semidesert
a,b,c,d,e
Semi-desert
F
Sig.
F
, 618
Sig.
, 443
Forest
F
15,093
Sig
, 001
Semi-desert F
Sig
Savannah
F
2,501
Sig.
, 116
Forest
F
18,685
Sig
, 000
Semi-desert F
Sig
Savannah
F
7,183
Sig
, 004
Forest
F
13,521
Sig
, 000
Semi-desert F
Sig
Savannah
F
5,823
Sig
, 007
Forest
F
19,799
Sig
, 000
Semi-desert F
Sig
Savannah
F
10,435
Sig
, 000
Forest
F
21,812
Sig
, 000
1, 16 degrees of freedom for step 1
2, 15 degrees of freedom for step 2.
3, 14 degrees of freedom for step 3.
4, 13 degrees of freedom for step 4.
5, 12 degrees of freedom for step 5.
. (Sudan samples).
Savannah
, 618
Forest
15,093
, 443
, 001
30,154
, 000
Savannah
2
3
4
5
a.
b.
c.
d.
e.
147
30,154
, 000
2,501
, 116
45,647
, 000
7,183
, 004
28,900
, 000
5,823
, 007
29,105
, 000
10,435
, 000
22,087
, 000
18,685
, 000
45,647
, 000
13,521
, 000
28,900
, 000
19,799
, 000
29,105
, 000
21,812
, 000
22,087
, 000
Table (18) Discriminant analysis probability:
With
Sample
Jemenitica- Without
Sudan.
Probability
Assigned
Locality
Gala Elnahal
Madani
Khartoum
Shendi
Kadogli
Eldalang
Wau- Sholok
Malakal
Doleib.
Gannal.
Elhawata
Doka
Glabat
Kosti
Hissahisa
Juba
Semi- desert
Semi- desert
Semi- desert
Semi- desert
Savannah
Savannah
Savannah
Savannah
Savannah
Savannah
Savannah
Savannah
Savannah
Savannah
Savannah
Forest
Bango
Liria
KhourMaqure
Forest
Forest
Forest
Assigned
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
1.000
0.850
0.920
0.720
0.460
0.990
0.990
0.990
0.980
0.990
0.990
0.990
0.990
0.990
0.990
1.000
6
4
4
3
4
6
8
8
8
8
8
6
6
4
8
8
0.855
0.639
0.677
0.999
0.300
0.988
1.000
0.979
0.992
0.606
0.996
0.991
0.929
0.506
1.000
0.862
9
9
0.780
0.990
8
4
0.973
0.660
9
0.850
5
0.673
3 = Lamarkii; 4 = Jemenitica; 5 = Litorea; 6 = Scutellata; 7 = Monticola;
8Adansoni; 9 = Jemenitica- Sudan.
148
Jemenitica
Sudan.
Probability
Table (19a): Discriminant Classification results (Sudan and others).
149
Table (19a) continued: Discriminant Classification results
(Sudan and others). A 92,1% of original grouped cases correctly
classified.
150
le (19b): Discriminant Classification results (Sudan and others).
151
ble (20): Proximities discriminant centroid distances between the groups
(dissimilarity matrix).
152
Fig (19): Canonical Discriminant Function (Sudan and others).
153
Fig (20 ): Dendrogram using average linkage between the groups (Sudan
and others).
154
4- 2- Mitochondrial DNA analysis of Apis mellifera L.:
PCR- amplified DNA can be used for direct analysis (length variation),
restriction (Hall and Smith 1991; Mortiz et al., 1994) or sequence (Cornuet
et al., 1991) analysis.
The intergenic region analysed in this work is located between the
tRNAleu gene and the COII gene.
The variability of this region results from:
a- The intergenic region amplified PCR products size in which The PCR
product was electrophoretically separated in a 1.5% agarose gel at 100 V for
2.5 h. The gels were stained with ethidium bromide and photographed over
a UV light screen. then later from the gel photo the fragment total size will
estimated in comparison with the marker (added on the gel).
b- The Dra 1 restriction patterns length variation of the PCR products which
rune in 8% and 10% Acryl amide gel, then stained with ethidium bromide.
As in the above the restricted patterns lengths was estimated from the gel
photo depending on the specific marker added.
c- Sequenced intergenic region PCR products; in which the variability
results from the superimposition of length variation (presence / absence of
the P sequence, number of reiterated Q sequences, possible small deletions
in both) and nucleotide substitutions. Sequence P0 = 67 bp. (only found in
African lineages); sequence Q has variable number of copies (192 – 196 bp.)
about 200 bp. (Garnery et al., 1993.)
Structure and part of the nucleotide changes are accessible through
PCR amplification of the entire region and analysis of the restriction profiles
obtained with the enzyme Dra 1 (TTTAAA). In this work restriction method
was applied.
155
4- 2- i- Amplified PCR analysis:
Due to size and Dra l restriction site polymorphisms, a total of six
products of different size of the tRNAleu – CO II intergenic region were
observed after PCR amplification as shown in Table (21a); Figure (21a) and
(21b). One corresponds to the lineage A1 and the others to, A2, A4, Y2, O1,
or O1` lineages.
In Figure (21a) and (21b) according to Cornuet et al., (1991), the
amplified PCR products demonstrated two length categories of fragments
corresponding to the following combinations; Lane 25, P0QQ (A4); Lane
24, P0Q (A1); Lane 3, P0QQ (Y2); Lane 16, P0Q (O1); Lane 11, P0QQ
(O1`); Lane 13, P0Q (O1); Lane 9, P0QQ (O1`); Lane 23, P0Q (O1); Lane 8,
P0QQ (A2); Lane 33, P0Q (A1); Lane 19, P0Q (O1); Lane 31, P0Q (A1);
Lane 17, P0QQ (A4); Lane 5, P0Q (O1) and Lane 20, P0Q (O1). [Lane M in
all the gels is the molecular weight markers (50 bp ladder, Gibco BRL). The
bands at ~ 640 bp are of size P0Q, and the bands of ~ 820 bp are of size
P0QQ.].
4- 2- ii- Restriction analysis:
Dra 1 (RFLP) of the tRNAleu - COII intergenic region exhibit from 3 to 5
bands, provided a total of 6 different haplotypes among the 27 Sudanese
honeybee workers colonies analysed in this study as shown in the 8% and
10% Acryl amide gels in figures (22a), (22b), (22c), (22d) and (22e), those
haplotypes are: A1 (Lane 31, 24, 33, 32, 34, 29, 30, and Lane 35); A2 (Lane
8 and Lane 7); A4 (Lane 25, 17, 26 and Lane 25); O1 (Lane 19, 23, 5, 20,
13, 16, 6, 18, 21, 22, 23 and Lane 4); Y2 (Lane 3); O1` (Lane 9 and Lane
11). Lane M in all the gels is the molecular weight markers (50 bp ladder,
Gibco BRL). Results of the different haplotypes obtained from the Sudanese
honeybee workers were summarized in Table (21a).
156
For more scrutiny to the results obtained in Table (21a), another analysis
was taken, by using the cluster column with a 3rd visual effect and pie with a
3rd visual effect analysis methods the percentage distribution of the
haplotypes (O1, A1, A4, A2, O1`and Y2) of the studied colonies as a whole
were detected as shown in figures (23a and 23b).
In regards to the three target geographical zones individually haplotypes
distribution were depicted in the semi-desert zone colonies, as shown in
Table (21b) and Figure (24a). In the savannah zone colonies demonstrated
in Table (21b) and figure (24b). For the forest zone colonies as shown in
Table (21b) and figure (24c).
157
Fig (21a): Agarose gel (1.5%) with the amplified
PCR products of the Sudanese honeybee workers
(samples 25, 24, 3, 16, 11, 13, 9, 23, 8, 33, 19, and
31).
Fig (21b): Agarose gel (1.5%) with the amplified PCR
products of the
Sudanese honeybee workers
(samples 17, 5, 20, and 25).
158
Fig ( 22a): Acrylamide gel (8%) showing
the Dra 1 restriction patterns of the
Intergenic region of the Sudanese
honeybee worker samples. (samples 31,
19, 24, 23, 5, 20, 33, 8, 13, 16, 9, 25, 11, and
17 Big fragments).
159
Fig (22b): Acrylamide gel (10%) showing the
Dra 1 restriction patterns of the Intergenic
region of the Sudanese honeybee worker
samples. (samples 31, 19, 24, 23, 5, 20, 33, 8, 13,
16, 9, 25, 11, and 17 Small fragments).
160
Fig (22c ): Acrylamide gel (8%)
showing the Dra 1 restriction patterns
of the Intergenic region of the
Sudanese honeybee worker samples.
(Samples 26, 25, 6, 32, and 34).
161
Fig (22d): Acrylamide gel (8%) showing the Dra 1 restriction
patterns of the Intergenic region of the Sudanese honeybee
worker samples.
(Samples 31, 18, 21, 22, 23, 7, 5, and 3).
162
Fig (22e):Acrylamide gel (8%) showing the
Dra 1 restriction patterns of the Intergenic
region of the Sudanese honeybee worker
samples. (Samples 29, 4, 5, 30, 23, 13, 35, and
6).
163
Table (21a): Sudanese honeybee workers Apis mellifera L.
different haplotypes.
Colony
ID.
25
17
26
8
7
29
30
31
35
24
33
32
34
19
23
4
5
20
13
16
6
18
21
22
9
11
Location
Sha- Mahadi
Om-Rawaba.
Kafindapi
Hawata
Galla-Elnahal
Juba
Bango
Lirria
Kajiko
Raja
Yei
KourMaquire
Latokka
Kadogli
Ganal
El-Hissahisa
Madani
Wau-Sholok
Kosti
Damazin
Erkawit
El- Dalang
Malakal
Doleib
Dokka
Semsim
PCR
size
Dra 1 fragment sizes (bp).
P0QQ
P0QQ
P0QQ
P0QQ
P0QQ
P0Q
P0Q
P0Q
P0Q
P0Q
P0Q
47
47
47
47
47
47
47
47
47
47
47
108
108
108
108
108
108
108
108
108
108
108
P0Q
P0Q
P0Q
P0Q
P0Q
P0Q
P0Q
P0Q
P0Q
P0Q
P0Q
P0Q
P0Q
P0QQ
P0QQ
47
47
47
47
47
47
47
47
47
47
47
47
47
47
47
108
108
108
108
108
108
108
108
108
108
108
108
108
108 130
108 130
164
65
65
65
65
65
65
65
65
65
65
65
65
65
Haplotypes
193 483
193 483
193 483
670
670
483
483
483
483
483
483
A4
A4
A4
A2
A2
A1
A1
A1
A1
A1
A1
483
483
420
420
420
420
420
420
420
420
420
420
420
420
420
A1
A1
"O1"
"O1"
"O1"
"O1"
"O1"
"O1"
"O1"
"O1"
"O1"
"O1"
"O1"
"O1'“
"O1'"
3
Khartoum
P0QQ 47 65 92
165
130
482
"Y2"
Table (21b):Distribution of the sudanese honeybee
worker haplotypes according to the three different
geographical zones.
Geographical
Haplotypes
Time
zone
found
numbers of
the
haplotypes
Semi-desert
O1
3
Y2
1
A4
3
A2
2
O1
8
O1`
2
A1
8
zone
Savannah
zone
Forest zone
166
Fig (23a ): Pie with 3rd visual effects representing the
distribution percentage of the Sudanese honeybee worker
haplotypes of the studied 27 colonies.
Fig (23b): Cluster column with 3rd visual effects
representing the distribution percentage of the Sudanese
honeybee worker haplotypes of the studied 27 colonies.
167
Fig (24a): Pie with 3rd visual effects representing the
distribution percentage of semi-desert zone of Sudanese
honeybee worker haplotypes.
168
Fig (24b): Pie with 3rd visual effects representing the
distribution percentage of savannah zone of Sudanese
honeybee worker haplotypes.
169
Fig (24c): Pie with 3rd visual effects representing the distribution
percentage of forest zone of Sudanese honeybee worker
haplotypes.
170
4- 3- observations on some behaviour and biology of the Sudanese
honeybees Apis mellifera L.:
Understanding the biology and ecology of honeybees of an area is very
important not only for classification purposes but also for the efficient and
profitable management of honeybees according to their biological behaviour
and respective ecology. Fore this reason, the following observations were
included.
4- 3- a- Coloured colonies:
In the east region of semi-desert and savannah zones, towards the SudanEthiopia border, mixed colonies of black and yellow honeybees were
common. The ratio of black to yellow; however from Dokka ‘ one of the
studied sample areas; 100 bees were taken randomly. The numbers of black
to yellow bees were counted. A ratio of 59: 41 black to yellow bees was
found. This colony was one of the most defensive and nervous colonies.
Worker bees started attacking about 6 to 8 meters away from the hive. By
the time the hive was opened, almost 95% or the whole hive population was
out knocking the intruders and each other nervously. Another colony at ElGalabat from the same region has somewhat, the same ratio of black to
yellow bees. Also colonies of mixed bees were also reported by Mohamed
(1982) and Mogga (1988). Thus colonies of all yellow bees were also found
in this zone as in El-Hawata and Galla-Elnahal regions.
4- 3- b- Defensive behaviour:
Generally all colonies from which samples were collected defended
themselves effectively. Feral bee colonies of the semi-desert zones, Madani,
El-Hissahisa, and Shendi, attacked at distances ranging between 150 m. to
about half kilometre away from their colonies, Shendi colony was the most
defensive and nervous colony in this region, we have been told from some
natives that, this colony killed a man few months ago.
171
Samples from savannah zone specially that from rock crevice as in
Kadogly was very defensive. A large bee population persisted followed us
nearly one and half kilometre away and the natives believed that this colony
is a magic colony and its 20 years old. Equally, samples from the forest zone
were very defensive once disturbed.
In the central region of the semi-desert zone at Khartoum, the only
collection trip from a colony bees kept in Langstroth hive; were equally very
defensive, with which a day before our visit we told the surrounded natives
that, tomorrow they have to take care and kept inside doors. The same with
Kosti colony in which we dig a half-meter under the ground so as to reach
the colony combs. So with this experience, it could be concluded that, the
Sudanese honeybees were very defensive and aggressive.
4- 3- c- Swarming and migration:
Reproductive swarms or seasonal migration of honeybees in Sudan
occurred in the different geographical zones at different times of the year,
depending on the climatic conditions.
Ruttner, 1988 demonstrated that, seasonal migration of honeybees is
considered as a unique characteristic of tropical honeybees. Honeybees of
most sub-Saharan Africa are reported to migrate on a seasonal basis,
following dry periods: A. m. yemenitica (Rashad and El-Sarrag, 1978;
Peterson, 1985; Sawadogo, 1993; Woyke, 1993).
In the semi-desert and most of savannah regions, swarming took place
between August and October. In forest zone it occurred between February
and April, and to a lesser extent around June and August to September.
Migration swarms seem to be common in areas devoid of water and forage
during dry months of the year. This occurred in the poor savannah zone. In
the rain season migration swarms move from the riverbanks and established
172
away from the Niles, while in the dry season they established on the Niles
banks.
4- 3- d- Nesting sites:
Honeybees in Sudan construct nests in different areas with different
conditions, some times within one area you may find two different nesting
sites. For instance, in Kosti (savannah region) we found a colony nest
constructed in the ground crevices to a depth of about half meter or more;
another colony nesting in a tree cavity. In the forest and some savannah
zones they built nests mostly in tree cavities, rock crevices, and evacuated
termite mounds. Beekeepers in south Kordofan (savannah region) believed
there were two honeybee strains in their area. One strain nested mainly in
termite mounds, while the second nested in tree cavities. The former was
generally larger in size, docile and when found could be harvested during the
day with relative ease. The later, nesting in cavities other than termite
mounds, was smaller, very defensive and only accustomed beekeepers may
harvest this strain at daytime.
173
4- 4- Morphometric statistical analysis of Apis florea.
The results obtained in the analysis of the 4 colonies of Apis florea
collected from four different towns in Sudan were shown in Tables (22 –
28). Thus there are some discriminant characters for each of the four
samples analysed as demonstrated in Table (29) in which the four colonies
show some morphometrical and geographical relationship.
Table (30) represent the analysis of variance means for the phenotypic
characters from the measured individuals of the four colonies, which
revealed the existence of some variability in most of the measured characters
across all localities.
In a further treatment, the four colonies are analysed by PCA (cluster
analysis), the results obtained were shown in figure (25).
Also results were subjected to proximities discriminant centroid distances
between the group’s analysis as shown in Table (31) and figure (26). The
results of Khartoum and Madani are somewhat near to each other, and Gerry
is more closed to El-Dender, than the other distances.
In a further scrutiny, the average mean measurements of some characters
(forewing length, forewing width, hind-leg length, tergite 3 plus 4 length and
cubital index) of the four samples from Sudan were analysed by a cluster
column (compares values across categories) with 6 Florea samples [2 from
Sudan “Moggas ones” and 4 from the data bank, Institute fur BienenkundeOberursel-Germany (Mogga 1988)]: Khartoum south, Toti, South India,
Oman, South Iran and Pakistan. The results are presented in Table (32) and
figure (27), which indicated that, the colonies were somewhat big in size like
those from Pakistan and south Iran.
174
Table (22). Florea.
175
Table (23). Florea.
176
Table (24). Florea.
177
Table (25). Florea.
178
Tble (26 ): Means of measurements of some tergites and sternites of honeybee
worker Apis florea (mm.) from Sudan
Length
Wax
Mirror S3
Width
wax
Mirror
S3
Distan- bet
ween
Wax
Mirror
175.6699
147.6299
149.1466
7.8404
285.6069
176.6256
151.6191
146.6624
8.1349
132.1938
267.5583
169.5331
141.8922
141.0434
7.8131
140.9742
291.2166
174.0833
145.327
146.7718
6.7861
Length
T3
Length
T4
Length
T3 +4
Length
S3
El- Dender
150.7302
143.1693
293.8995
Gerry
145.3644
140.2425
Khartoum
135.3645
Madani
150.2424
Sample
Table (27): Means of measurements of pilosity of the honeybee workers
Apis florea (mm.) from Sudan
Samples
El- Dender
Gerry
Khartoum
Madani
Length of
hair
Width of
tomentum
Width of
Dark
stripe
Tomentum
Index
2.439
56.9208
32.691
1.7763
2.439
55.767
18.0762
3.7941
2.439
51.5364
20.7684
2.8279
2.439
54.9978
21.153
3.0317
Table (28): Means of measurements of sternite 6 of the
honeybee workers Apis florea (mm.) from Sudan
Samples
Length of
Width of
Abdominal Slenderness
El- Dender
Gerry
Khartoum
Madani
S6
S6
L/W. S6
147.6299
196.2996
75.2267
151.6191
200.2218
75.7551
141.8922
190.4472
74.51444
145.327
193.7276
75.02733
179
Table (29). Florea.
180
Table (30). Florea.
181
Table (30) continued. Florea.
182
Table (31). Florea.
183
Table (32). Florea.
184
Fig (25) Florea.
185
Fig (26) Florea.
186
Fig (27) Florea.
187
CHAPTER FIVE: DISCUSSION, SUMMARY AND
CONCLUSION.
5- 1- DISCUSSION
5- 1- a- Morphometrics (Apis mellifera L.):
Along with the development of morphometric measurements, the
introduction
of
different
multivariate
techniques
like
principal
components and factor analyses were used to detect clusters of colonies
within populations (Ruttner et al., 1978; Ruttner, 1988).
The first attempt in the racial studies of the Sudanese honeybees Apis
mellifera L. was conducted by Ruttner (1975), Table (33); El Sarrag
(1977), Table (34); Saeed (1981), Table (35); Mohamed (1982), Table
(36) and Mogga (1988), Table (37).
In the present work, analysis of morphometric characters of the
honeybees Apis mellifera L. of Sudan as in Tables (3- 10) revealed a wide
variation in the fourty five characteristics measured. Fourteen of these
formed the discriminant characters which include; forewing length and
width, wing venation angles G18 and O26, proboscis length, hind-leg
metatarsus length and width, sternite 3 length, sternite 6 length and
width, wax mirror length and width, cubital vein 1 length and labrium 1
colouration; Appendix (H) summerized the significances for each
phenotypic character from the measured individuals at P ≤ 0.005.
This wide variation might be taken as an indication that, these honeybee
samples do not belong to one race. Elsarrag (1977), Saeed (1981),
Mohamed (1982), all found highly significant differences for all the 12
traits studied and concluded that, this is an indication that the samples did
not belong to one race. Similarly Rashad and Elsarrag (1978, 1980), and
Rashad et al., 1984, Mogga (1988) and Elsarrag (1992), stated a high
degree of regional variation of the Sudanese honeybees.
188
Table (33) Ruttner:
189
Table (34): The average values of
measurements taken for the
different characters of the
S
Characters
u
Tongue length (mm.)
d
Flagellum length (mm.)
a
B. Tarsus iii length (mm.)
n
B. Tarsus iii width (mm.)
e
No. Hair rows
s
Forewing length (mm.)
e
Forewing width (mm.)
Cubital index
h
No. Hamuli
o
T3 + T4 length (mm.)
n
Slenderness S6 L/I
e
% Color T3
y
Mean
SE ±
5.50
0.019
2.66
0.006
2.20
0.006
1.11
0.003
11.90
0.014
8.60
0.014
3.02
0.014
2.37
0.008
21.40
0.019
3.70
0.009
86.00
0.008
71.36
0.003
bee workers Apis mellifera L. (El
Sarrag, 1977).
190
Table (35):
The avera ge values of measurements taken for the
different characters of the Sudanese honeybee
workers Apis mellifera L. (Saeed 1981).
Characters
Mean
SE ±
Tongue length (mm.)
5.18
0.379
Flagellum length (mm.)
2.71
0.104
B. Tarsus iii length (mm.)
8.29
0.165
B. Tarsus iii width (mm.)
3.13
0.156
No. Hair rows
1.99
0.234
Forewing length (mm.)
20.57
0.775
Forewing width (mm.)
2.02
0.097
Cubital index
1.03
0.073
No. Hamuli
10.72
0.859
T3 + T4 length (mm.)
69.36
12.160
Slenderness S6 L/I
85.54
4.105
% Color T3
3.74
0.136
191
192
Table (36): The average values of
measurements taken for the different
characters of the Sudanese honeybee
workers Apis mellifera L. (Mohamed
1982).
Characters
Mean
SE ±
Tongue length (mm.)
5.46
0.025
Flagellum length (mm.)
2.70
0.086
2.11
0.015
B. Tarsus iii width (mm.) 1.11
0.001
No. Hair rows
11.96
0.098
Forewing length (mm.)
8.12
0.034
Forewing width (mm.)
2.85
0.015
Cubital index
2.34
0.070
No. Hamuli
20.84
0.329
T3 + T4 length (mm.)
3.86
0.025
Slenderness S6 L/I
87.56
0.713
% Color T3
66.90
1.526
B. Tarsus iii length
(mm.)
194
Table (37): Average means of some discriminant characters for the Sudanese
honeybee workers (Apis mellifera L.)
From different geographical zones. Mogga (1988).
195
Similar variability was also observed for the neighbouring Ethiopia
honeybees (Smith, 1961; Ruttner, 1975, 1988; Kassaye, 1990; Radloff
and Hepburn, 1997a, 1988; and Hepburn and Radloff, 1998). Kenya
honeybees (Meixner et al., 1989, and 1994).
Winston et al., (1983) concluded that, the most striking aspects of
honeybee biology were the variability found within and between races of
Apis mellifera L. They mentioned that among these variations were
morphological and physiological characteristics as colour, body size and
tongue length. Ruttner and Kauhaser (1985) also found a very high
degree of variability among honeybees of tropical countries compared
with the total variability for Apis mellifera L. They concluded that for
body size, the African portion of the total variability was 63% and for the
length of proboscis was 55%.
In Africa, in the absent of geographical barriers, the occurence of
different subspecies are results of ecological factors was recognized
(Ruttner, 1981, 1988; Dietz et al., 1986). Although the Sudanese
honeybee samples for the present study originated from areas without
geographical barries, they showed very clear geographical distribution
pattern.
The dark green colour samples displayed in the left side of the
figures (13 and 14) orginated from forest zone. These samples presented
most of the least measurements and the most slender abdomin of the two
Sudanese honeybee subgroups. In 1982, Mohamed Ali Hussen had
described the Sudanese honeybees originated from south Sudan,
particulary Equatoria province (the same region of the forest zone
samples) as having the shortest proboscis measurements among Apis
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mellifera L. and they are very small in body size and they have the most
slender abdomen compared to the other African and/or European
honeybee strains. Samples from the forest zone were completely
seperated in morphometrics from the samples of the semi-desert and
savannah zones and even from Mogga (1988) samples but all of them
aggregated among the Yemenitica samples of Ruttner as shown in figure
(15).
The dark orange samples distributed around the middle of the figures
(13, 14) were those originating from semi-desert zone. These samples
presented most of the medium measurements of the two other subgroups
described in this study.
In 1976, Ruttner had described the Sudanese bees originating from the
semi-desert zones as having the most slender abdomen of all the African
bees examined. He concluded that the samples clearly belong to another
group, correctly to be considered as an independant race, and named
them Apis mellifera nubica. In 1986, Ruttner further found Sudanese bees
from the semi-desert morphometrically inseperated from the bees of
Saudia Arabia, Yemen, Oman and Chad, thus he renamed the Sudanese
bees Apis mellifera yemenitica. He then concluded that Apis mellifera
jemenitica was the African bees from dry thorny-bush zone. It is
occurence in Yemen, Oman, and Saudia Arabia was not astonishing since
this was part of the Ethiopia zoogeographic zone. Thus, the results of
Mogga (1988) agreed with Ruttner (1986) discription of the size and
slenderness of the Sudanese bees of the semi-desert zone.
The results of the semi-desert zone cluster of this study agreed with
the previous mentioned discription of Ruttner (1986) as compared to the
savannah zone cluster only, because forest zone bees were not considered
in Ruttner investigation.
However, samples from Shendi and Khartoum originated from the
197
semi-desert region along the Nile valley, in addition to their natural
vegetation of various small Acacia sp. and other flowering shrubs, there
are small irrigation native farms of vegetables and fruits, thus samples
Madani and El-Hissahisa in spite of their origin on the Nile valley they
possess the same mentioned natural vegetations, and they were among
one of the most bigest national irrigation schemes in Sudan “El-Gazirra
Scheme“ where various economic crops, field crops, vegetations and
fruits were grown.
Regarding most of the discriminant characters of the semi-desert zone
colonies (Shendi, Khartoum, El-Hissahisa and Madani); some up
normality in Khartoum colony measurements was found, in other wards
it had the least proboscis value, at the same time it had the largest or
medium values in the rest of the measurements. The large values of
characters for the Khartoum sample from the semi-desert zone might be
an infleunce of the imported European bees to Khartoum state. Elsarrag
(1977) reported that bees of Khartoum province were hybridized with
foreign honeybee races.
In the dearth season, bees resorted to forage on flowering plant
species growing along the Nile vallies. All of the four colonies of the
semi-desert zone were aggregated around Moggas and Yemenitica
samples as shown in figure (15).
Considering savannah zone (the widest geographical vegetation zone),
Nine samples out of eleven (light blue colour samples) were distributed
to the right side of the graph figures (13 & 14). Most of these samples
were from the eastern region, towards the border with Ethiopia. Samples
from Om-Rawaba, El-Dalang and Kadogli were from the far west part of
this region.
Bees from the eastern zone of Sudan might be infleunced by the
Ethiopia honeybees which were generally large. Most of these samples
198
were distributed to middle side of figure (15), among studied samples
Scutellata, Monticola and Ethwest (Ethiopia western bees).
Generally bees displayed to the right side of the graph in PCA consist of
large bees, while those distributed at the left side of PCA graph
comprises small bees.
The largest average of samples from the savannah zone mean values
for forewing length were 8.45 mm., width 2.95 mm.; hind-leg 7.05 mm.;
body size (T3+ T4) 4.00 mm., and comparatively largest cubital index
2.24 mm. These results agreed with those found by Mogga (1988), he
found honeybee samples originating from the north part of the savannah
zone (poor savannah zone), to have largest values for the forewing
length, 8.46 mm., width 2.94 mm.; hind-leg 7.26 mm.; body sixe 4.04
mm.; and cubital index 2.30 mm. Also the same results confirmed those
found by Rashad et al., (1982). Who found bee samples originating from
Nile province (savannah zone) to have largest values for the forewing
length 8.56 mm., and width 3.16 mm.
The present correlation between climatic zone and morphometric
characters also agreed with the finding of Ruttner and Kauhaser (1985).
who found in tropical Africa, significant geographical variability in
honeybees inspite of the absence of physical isolation barriers. They
stated that, the mechanism bringing about isolation was the selective
adaptation of the races bees to certain biotopes. This principal implied
permanent hybridization and existence of intermediates, reflecting the
intermediate ecological zones.
In the Sudan, the climatic conditions affecting the supporting
vegetative forage for bees seemed to play a greater role. Graded long
distance variations of quantative characters in bees referred to as
“geocling“ by Huxley (1938), while the graded variations over a short
distance on slopes of monutains as found in East Africa were termed as
199
“geocling“.
Considering the present results, it is tempting to suggest that, geocling
exist, among the Sudanese honeybees.
Samples from the forest zone exhibited the smallest mean average values
of forewing length 8.23 mm., and width 2.82 mm.; proboscis length 5.55
mm.; hind-leg length 6.83 mm.; body size 3.88 mm., and cubital index
1.85 mm. Values of these parameters were similar to the findings of
Mohamed Ali Hussen (1982), proboscis length 5.46 mm., forewing
length 8.12 mm., forewing width 2.85 and cubital index 2.34 mm. But
larger in samples from savannah zone to the north and west. Forewing
length 8.45 mm., and width 2.95 mm.; proboscis length 5.67 mm.; hindleg length 7.05 mm.; body size 4.00 mm.; and cubital index 2.26 mm.
Samples from the semi-desert zone gained medium mean average
values of forewing length 8.27 mm., and width 2.88 mm.; proboscis
length 5.63 mm.; hind-leg length 7.00 mm.; body size 3.88 mm.; and
cubital index 2.04 mm.
The smallest mean average values of the body different parameters in
the forest zone samples, may be attributed to the small size of the cells,
the honeybee workers build in their combs. Hence it is an adaptive
mechanism that honeybees utlize the maximum area for building up their
nests. Grout (1937) studied the influnce of cell size upon the size and
variability of the honeybees. He found that the size of the brood cell
affect the size of the adult worker bee and significantly larger bees were
obtained through the use of enlarged cell foundation. Hassanein and
Elbanby (1956) studied the biometrics of the Egyptian honeybees. They
obtained great differences in measurements of the worker appendages for
those reared in Langstroth hive compared to those reared in pipe-hive.
The former gave higher figures. From these facts it can be said that, the
small size of the forest zone honeybees refered to the small cell size they
200
build in their combs.
The smallest mean average values of body parameters which found in
the semi-desert zone bees (as compared to savannah zone bees). This
phenomenon might be due to the influnce of the high temperatures and
law humidity almost all year round in the semi-arid zone. This limits the
amount of forage in this zone, compared to the forage found in the
savannah zone. Nagi (1984) found that from April to June and from
September to November, there was insufficient food in Shambat area
(located in the semi-desert zone), resulting in construction of small
queens. Therefore, lack of nectar and insufficient forage might play a role
in the smallness size of the bees in the semi-desert zone.
Kigtiira (1984), wrote that, bees in Kenya were characterized by a
specific geographical distribution confined by natural barriers; namely
Apis mellifera monticola the mountain bees (2400- 3100), then A. m.
Scutellata and A. m. nubica (yemenitica) the Acacia savannah plain bees.
Ruttner and Kauhausen (1985) like wise stated that, the variation of bee
in tropical Africa showed new principle in bee taxonomy, hardly
considered before: diversification and isolation by ecological factors.
Thus it could be concluded from figure (13, 14 and 15) that, the
alignment of the Sudanese honeybee samples was not by coincidence. In
1985, Mbaya also found that, the morphology and behavior of bees in
Kenya varied according to ecological zone. He further said special
biological modifications of some characteristics of honeybees were
necessary if they were to become adapted to certain ecological zones of
Kenya.
The probability of the existance of at least three different ecotypes of
the Sudanese bees as mentioned above through the PCA analysis, was
confirmed by some advanced modern discriminant analysis methods.
Such as step-wise discriminate analysis which was used to confirm the
201
separation of clusters, detect the most discriminatory variables and
calculation of the percentage of correctly classified colonies (Ruttner,
1988; Daly, 1992). However, by applying a step-wise discriminate
analysis Procedure, Ruttner et al., (1978); Daly and Balling (1978)
showed the possibilities of discriminating one race from another using
fewer numbers of selected characters based on the region under
investigation.
In the step-wise discriminant analysis Table (16), 94.7% of the
original cases were correctly classified, in which all the semi-desert and
forest colonies were 100% correctly classified as different clusters, but in
case of the savannah colonies 10 out of 11 colonies were correcly
classified as one cluster but the remaining colony (Kosti), the PC rather
thinks it should be semi-desert (with P = 0.509) and this result was very
clear in the PCA analysis graph figure (14); this apparently can be
attributed to: Kosti colony might be migrated from the semi-desert zone
during the dry periods as honeybees of sub-Saharan Africa were reported
to be migrate on a seasonal basis, following dry periods: A. m. yemenitica
L. (Rashad and El-sarrg, 1978; Peterson, 1985, Sawadogo, 1993; and
Woyke, 1993) thus Kosti colony geographicaly is somewhat near to the
border between the semi-desert and savannah zones.
Based on pair-wise group comparison test (at different degrees of
freedoms) the seperation of cluster groups (semi-desert, savannh and
forest) was highly significant for all the clusters as shown in Table (17),
and it conformed with the PCA results of of the univariate analysis
Appendix (H).
On the other hand considering the relationship between our target three
clusters with Yemenitica Sudan and the other African mellifera
subspecies used in this study, the discriminsant analysis probability Table
(18), demonstrated that colonies Gala- Elnahal, El-Hawata, El-Galabat,
202
Kosti, El-Hissahisa, Juba and Lirria were all had posterior probability of
100% for being in cluster Yemenitica-Sudan. Colonies Khartoum, Doleib,
and Dokka had 92, 98, and 98% respectively posterior probability for
being in cluster Yemenitica-Sudan. While colonies Kour-Maquir, Bango,
and Shendi had 85, 78, and 72% respectively posterior probability for
being in cluster Yemenitica-Sudan. However, only one coloni (Kadogli)
had less than 50% (46%) for being in cluster Yemenitica-Sudan. The
discriminant analysis probabilities (Table 19b) confirmed and the above
mentioned results, in which out of the target 19 colonies 18 (94.7%)
colonies had posterior probabiliy for being in cluster Yemenitica-Sudan.
Only one colony had a very law probability for being in cluster Adansoni.
Considering the second choice of the discriminant analysis probability as
demonstrated in Tables (18, 19a and 19b) without Yemenitica-Sudan
cluster; there were some colonies showed a high posterior probability for
being in another African mellifera subspecies clusters; for instance in
Table (18) colony Dokka, had a high posterior probability of 100% for
being in cluster Scutellata.this may be attributed to: Dokka was one of
the coloured colonies in this study in which the percentage ratio of black
to yellow colour is 59: 41 thus it is located in the border between Sudan
and Ethiopia. Colonies Khartoum, Lirria, and Madani had a high
posterior probability for being in cluster Yemenitica. Away from Sudan
clusters, the probability between the other African mellifera clusters
revealed a high posterior probabilities for many clusters to be in the
other; for instance out of 50 colonies grouped in cluster Scutellata 44
colonies were correctly classified as Scutellata while the remaining 6
colonies were classified as: 2 colonies (4.0%) to each of the following
clusters: Yemenitica, Litorea and Monticola respectively (Table 19a).
To depict the distances between clusters, dendrograms and
mahalanobis distances were introduced (Tomasson and Fresanaye, 1971;
203
Cornuet et al., 1975; Cornuet and Garnery, 1991a, b; Daly, 1992).
By using the discriminant analysis, distances between the group
centroids of the 3 target clusters: semi-desert, savannah and forest were
depicted in Table (20) as: Semi-desert to savannah = 4.3; Semi-desert to
forest = 6,1; Savannah to forest = 6,0.(And vice versa); which
demonstrated: clusters semi-desert and savannah were more close to each
other and both of them were far away from forest cluster by semi equal
distances and these supported the independence of the forest cluster as it
is very clear in the PCA figures (13, 14 and 15). These results strongly
indicated the existence of at least three different honeybee ecotypes
among the Sudanese bees (see Fig 17).
The same results were confirmed in figure (19), in which the semidesert cluster colonies centre was far away from the following clusters:
Ethmount, Ethwest, Monticola, Lamarkii, Scutellata, Litorea, Adansoni,
Jemenitica, and Jemenitica-Sudan respectively. Cluster savannah
colonies centre was far away from Ethmount, Ethwest Monticola,
Lamarkii, Litorea, Jemenitica, Adansoni, Scutellata, and JemeniticaSudan clusters respectively. While forest cluster colonies centre was far
away from clusters; Ethmount, Ethwest, Monticola,Lamarkii, Scutellata,
Adansoni, Litorea, Jemenitica, and Jemenitica-Sudan respectively.
Using average linkage between the groups (Sudan samples and
others) a dendrogram figure (20) was issued, in which all the clusters can
be divided into five different groups as followed:
Group 1 include: Jemenitica, Adansoni, Litorea and Scutellata clusters,
in which Jemenitica and Adansoni were very closed to each other and
Litorea; Scutellata were closed to them too.
Group 2 include: Jemenitica-Sudan, savannah, and semi-desert clusters.
In which Jemenitica-Sudan and savannah were very closed to each other
and semi-desert was closed to them. Group 1 and 2 were closed to each
204
other.
Group 3 include: forest cluster, which is closed to group 1 and 2 together.
Group 4 include: Ethmonut, Ethwest and Monticola clusters; in which
Ethmonut, Ethwest and Monticola colonies were very closed to each
other. Group 5 include: Lamarkii, which was very close to group 1, 2,
and 3. Group 4 was close to all the groups. All the different 5 groups (fig.
20) had a very cleared relationship with each other as one species (Apis
mellifera.), thus it is strongly proved that Sudan colonies represent at
least three different clusters.
However, the above-mentioned relationship and the interaction
between the bee’s subspecies are not surprising between the African
honeybees clusters mainly Yemenitica, Scutellata and Monticola (NorthEast Africa races). The distribution and interaction between these
subspecies is predicted with the following assumptions: The gene flow
within African honeybees is very high due to high swarming and
migratory behaviour (Hepburn and Radloff, 1988) and subsequent law
molecular differentiation among African subspecies is well noted (Frank
et al., 2001).
Considering
all
the
above-mentioned
presentations,
which
confirmed the presence of three different clusters “ecotypes” among the
Sudanese honeybees, Elsarrag (1992) suggested that, two honeybee
subspecies exist in Sudan; namely A. m. sudanesis and A. m. nubica. The
former was described to be distributed all over the Sudan, between
latitude 3º N and
16º
20
N. The latter subspecies was described as mixed bees, distributed
along the international boundaries of Sudan, Ethiopia and Uganda. In the
present study, this last description corresponded in part with the samples
from savannah zone. While the former description covered most of the
forest and western parts of the savannah zones. Mogga (1988), by using
205
the first analysis method only (PCA) suggested that, three ecotypes of
honeybees exist in the Sudan; namely: A. m. yemenitica L. (the small bee
of semi-desert zone), A. m. sudanesis (the medium bee of forest and rich
savannah zone) and A. m. bandasii (the large bee along the border of
Sudan and Ethiopia). In the present study the description of the former
bees (Yemenitica) corresponded with the cluster of bee samples
originating mainly from the semi-desert zone; while Bandasii subspecies
partially corresponded with the samples from the poor savannah. The
race Sudanesis as in Mogga (1988), was medium in size and should be
distributed in both rich savannah and forest zones but, in the present
study the forest zone cluster was confirmed to be completely different
from both semi-desert and savannah clusters through the PCA analysis
(figures 13, 14 and 15) and the discriminant analysis (Tables 16 & 17and
figures 17, 18, 19 &20) thus it gained the smallest size among all the
Sudanese bees other clusters which conformed with Mohamed
investigations (1982).
5- 1- b- Mitochondrial DNA (Apis mellifer L.):
In the last decade techniques for the measurements of genetic
variations in honeybees at the DNA level have been developed and are
proving to be extremely powerful probes for the analysis of genetic
variation.
The mtDNA technique was based on examining restriction fragment
length polymorphisms (RFLPs). Such techniques have been applied to
honeybee’s mitochondrial DNA (Moritz et al., 1986, 1994; Smith, 1988,
1991; Smith and Brown, 1988, 1990; Smith et al., 1989, 1991). Thus
study of mitochondrial DNA is of particular application in honeybees as
it is the ideal marker of colony – since all individuals in the colony,
queen, workers and drones sharing the same haplotype (excluding
mutations). Therefore, studying 27 individuals obtained information on
206
27 colonies. This property, combined with the haploids of the genome
and the high variability of some of its regions, confers a high power of
resolution, as it enables precise detection of foreign haplotypes in
populations.
With the Dra 1 restriction enzyme test, it was possible to differentiate
6 different haplotypes (O1, A1, A4, A2, O1` and Y2) within the 27
colonies studied in the Sudan. (Table 21a) and figure 22 (a, b, c, d, and
e), confirms the potential use of this technique as a powerful tool in
population genetic studies of honeybees. Thus, Primarily these results
presented strong evidence for the existence of more than one subspecies
among the Sudanese honeybee populations.
Moritz et al., (1994) in their study, the Mitochondrial DNA
variability in South African honeybees (Apis mellifera L.), demonstrated
that, the variability of mtDNA size of honeybees (Apis mellifera L.) in a
sample of 102 colonies covering the area south of the 27th parallel of
latitude in Africa were analysed using PCR and Dra I restriction enzyme.
A region between the COI and COll genes revealed four different size
variant haplotypes, which has been shown to be useful for the bio
geographic classification of Apis mellifera subspecies, it is partially
corresponded to the known distribution of African subspecies of
honeybees based on morphometrical and physiological data. De la Rúa,
et al., (2000), investigated the mtDNA variation in Apis cerana
populations, 47 colonies were collected from different locations in
Philippines Islands, genetic variation was estimated by restriction
analysis of PCR-amplified fragments of the tRNALeu -COII region and
they found four different haplotypes (Ce1, Ce2, Ce3, and Ce4) that
discriminated among the bee population, from different Islands. Using
the restriction enzymes HpaII and AluI, Meixner et al., (2000), had a
strong correlation with mtDNA haplotype and morphology among
207
honeybees from Kenya.
The different mobility of the restricted fragments in the gel photos
compared to the marker is due to the skewed base-pair composition of the
mtDNA. (De la Rúa, et al., 1998). Thus Lane M in all the gels is the
molecular weight markers (50 bp ladder, Gibco BRL).
Using the cluster column and pie with a 3rd visual effects, the
percentage distribution of the haplotypes (O1, A1, A4, A2, O1`and Y2) of
the studied colonies as a whole were detected as: 41% (11 colonies), 30%
(8 colonies), 11% (3 colonies), 7% (2 colonies), 7% (2 colonies) and 4%
(one colony) for the following haplotypes O1, A1, A4, A2, O1` and Y2
respectively; as demonstrated in Table (21a) and figures (23a ; 23b).
The individual haplotype distribution in the three target geographical
zones were as follows: In the semi-desert zone colonies, haplotype O1 is the
common haplotype and represent 75% of the analysed colonies; while
haplotype Y2 represent only 25% and all the rest haplotypes (A1, A2, A4
and O1`) were nil as shown in Table (21b) and Figure (24a). The most
common haplotype in the savannah zone is O1 (54%) followed by A4
(20%), O1` (13%) and A2 (13%) respectively while haplotypes A1 and Y2
represent 0% in this region as shown in Table (21b) and figure (24b). For
the forest zone haplotype A1 is the only abundant haplotype in the region,
which represent 100% as, shown in Table (21b) and figure (24c) though the
rest of the haplotypes (A2, A4, O1, O1` and Y2) were nil.
The higher percentage of O1 haplotype in savannah zone(Table 21b and
figure 24b ), can be attributed to the fact that most of the analysed colonies
were collected from the far eastern part of this zone (the border of Sudan
with Ethiopia). Meixner., (2006) *personal communication* , in her study
about mtDNA of Ethiopia honey bees (unpublished paper), indicated that,
haplotype O is relatively common on the west rim of Ethiopia dome. Rashad
and Elsarrag (1978) demonstrated that, the honeybees in some parts of the
208
Sudan were hybridized with foreign honeybee races either through the
several importation of particular races to the country or through the
migratory swarms of honeybees from adjacent countries.
Therefore, it can be concluded that honeybees from the savannah zone
are mixture of many different strains and this is strongly confirmed by the
morphometric PCA analysis results (figure, 15), which showed the scatter of
the individuals from different subspecies particularly between the North-east
Africa races (Jemenitica, Scutellata, Monticola and Adansonii). This
justification can be attributed to the fact that Scutellata and Monticola were
the most abundant races on the border region between Sudan and Ethiopia.
The same results were confirmed by the proximities discriminant centroid
distances between the groups (Table 20), (figures 19a, 19b, and 20). There
were numerous publications, which reported that, A. m. scutellata was
highly mobile insect that easily absconds (Hepburn and Radloff 1988;
Ruttner 1976 and Ruttner 1988).
However,
colonies
Om-Rawaba,
Shawish-Mahadi
and
Kafindapi
(savannah zone) were from the extreme western part of the zone near to the
west border of Sudan. Their possession of A4 haplotype is not a surprise,
according to Franck et al., (2001) except North-east Africa all the rest of the
continental Mellifera races had only haplotype A. Also haplotype O1 was
the dominant haplotype in semi-desert zone as shown in Table (21b) and
figure (24a), and this might be due to the fact that clusters of semi-desert
and savannah were more close to each other as it was confirmed in the
discriminant analysis of distances between the group centroids, Table (20)
and figure (17).
Haplotype Y2 was found in Khartoum colony of semi-desert zone, this
phenomenon can be explained by assuming
that Khartoum state was
invaded with other foreign imported races. Elsarrag (1977), reported that,
bees of Khartoum province were hybridised with foreign honeybee races.
209
Haplotype A1 of the forest zone indicated that, the honeybee populations
from this zone are homogenous in their mtDNA haplotype (all of the
colonies gained A1 haplotype only), even though, the colonies were
sampled from different regions of the zone, as shown in Table (21b) and
figure (24c).
The molecular study results of mitochondrial DNA variability is
partially confirmed the previous morphometrical grouping. Undoubtedly the
forest zone colonies were confirmed 100% as a completely separate cluster
and this is a very strong evidence for the morphometric results of PCA and
discriminant analysis. Comparing these results with Mogga (1988), this
cluster should be A. m. sudanesis Rashad. In the case of savannah colonies,
the presence of different haplotypes is not a big deal, as this phenomenon
can be attributed to the heterogeneous mixture of the blood between the
previous mentioned subspecies. This was supported by Meixner et al.,
(2000), who demonstrated that, some honeybee colonies collected in
savannah environment of Kenya had both the morphology and the mtDNA
haplotype of
A. m. monticola or the morphology of one race and the
mtDNA of the other. They reported that, there was a colony, which
combined the morphology of A. m. monticola with the mtDNA haplotype of
A. m. scutellata. Thus all samples of A. m. litorea that they analysed share
the mitochondrial haplotype typical of A. m. scutellata. So it is just a matter
of heterogeneous mixture of the blood between the different races of the
zone. Comparing savannah colonies with the results of Mogga (1988)
regarding the classification of the Sudanese honeybees, this cluster should
be A. m. bandassi. While the semi-desert zone cluster as demonstrated
before corresponds to A. m. yemenitica race.
From the present results, such bee migration might have possibly
happened across the Sudan Ethiopia border, influencing the bee population
in the savannah zone of eastern region. It was also here that the mixed bees
210
of black and yellow individuals, of the same mother were found. Mixed
colonies were reported by Bal densperger (1924) to have found black and
yellow bees in the same hive in Ethiopia. The land also rises here in altitude
from the eastern region of Sudan towards the Ethiopia high lands. Thus at
higher altitudes, larger and darker bees were found as reported by Smith
(1961). Evidently, the Ethiopian honeybees influence on the bees of eastern
region of Sudan was most likely.
It could, however be mentioned at this point that, for a complete picture
of the Sudanese bee race and their distribution, more work is needed
specially with regards to the geographical variation. There is also a need to
investigate a possible existence of ecocline on the sloops of Imatong
mountains (3187 m) in the extreme southeast and Jebel Marra (3042 m) in
the western part of Sudan.
Considering the molecular genetic studies, this is the first record on the
classification of the Sudanese honeybees according to the mitochondrial
DNA variability. Mitochondrial DNA data alone are probably insufficient to
infer taxonomic and genetic status of honeybee colonies. Extending micro
satellite (nuclear DNA) analysis to honeybee subspecies will be useful in the
future for understanding the phylogeography of Apis mellifera and resolving
relationships among the African subspecies. Also more detailed analysis of
savannah cluster mitochondrial DNA are needed to better resolve the
relationship of this wide cluster populations with the neighbouring
subspecies.
5- 1- c- Apis florea.
Analysis of morphometric characters of the honeybees A. florea
from Sudan as in Tables (22- 28), revealed some morphometrical
and geographical relationship among the four measured colonis.
The results obtained from the principle component analysis (PCA)
figure (25), indicated that colonies are not very distinct. The plot shows
211
only two axes loaded with 18.2 and 12.8% of total variance. Factor
loadings seem not very clear, but apparently it is mostly sizes as factor 1
and colours as factor 2.
In the results obtained in Table (31), the proximity matrix indicates
colony discriminant analysis centroids as follows; close relationship
between Khartoum and madani colonies and again between Gerry and ElDender colonies, in comparison to the other distances. Also in figure
(26), the discriminant analysis plot demonstrated that most bees were
allocated to their correct colonies, except one of Madani group, which
was allocated to Khartoum group.
From the principal components and factor analysis of 20 characters
of 18 samples of A. florea, Ruttner (1988) obtained three morphoclusters
(1) South India and Sri Lanka, (2) Thailand and (3) Oman, Pakistan and
Iran. More recently Tahmasebi et al. (2002), analyzed the A. florea of
Iran and defined two morphoclusters. Combining their data with that of
Ruttner (1988) and Mogga and Ruttner (1988) a same groups of
countries, they also reported three morphoclusters for A. florea.
However, all the individuals of the four colonies were analyzed
through the PCA and factor analysis in which, colonies were not very
distinct. In the discriminant analysis of the centroid distances between the
groups apparently there was a clear relationship between colonies Gerry
and El-Dender and Madani and Khartoum colonies respectively. These
results may be due to, that all the analyzed colonies were from one
geographical zone (semi-desert zone). Radloff and Hepburn (1998,
2000), and Hepburn et al. (2001b) have established empirically that the
greater the sampling distances between localities the greater the
likelihood that factual morphoclusters will emerge in multivariate
analyses. Conversely, where between-group variation is larger than
within group variation, biometric subgroups within smaller geographic
212
domains may be swamped and obscured.
The average mean measurements of the forewing length, forewing
width, hind-leg length, tergites 3 +4 length and cubital index, of the four
colonies (El-Dender Gerry, Khartoum, and Madani) were analysed by a
cluster column (compares values across categories) for 6 florea colonies
from different regions [2 from Sudan “Moggas ones” and 4 from the data
bank, Institute fur Bienenkunde-Oberursel-Germany (Mogga 1988)]:
Khartoum south, Toti, South India, Oman, South Iran and Pakistan. As
shown in Table (32) and figure (27). Samples Madani, Khartoum, Tuti
and South Iran were very closed to each other in the average mean of
characters: cubital index, length of tergites 3+4, hind-leg length and
forewing width; thus colonies Gerry, Khartoum south, El- Dender and
Pakistan were closed to each other too, mainly in the average means of
the following characters: cubital index, length of tergites 3+4, hind-leg
length and forewing width.
This indicated that, the colonies were
somewhat big in size like those from Pakistan or south Iran. Apis florea
ecotypes of Pakistan and Iran, according to Ruttner (1988) were
considered as one geographical race; consequently our target four-florea
colonies of Sudan should be from the same origin.
Mogga (1988), indicated that, the florea bees in Khartoum originated
from countries of Western Asia; Pakistan, South Iran and Oman. The
biogeography of A. florea as demonstrated by (Hepburn, et al., 2005) is
extremely widespread, extending some 7000 km from its eastern-most
extreme in Vietnam and Southeastern China, across Mainland Asia along
and below the southern flanks of the Himalayas, Westwards to the
Plateau of Iran and Southerly into Oman. This constitutes some 70
degrees of longitude (40°–110° East) and nearly 30 degrees of latitude
(6°–34° North). Variations in altitude range from sea level to about 2000
m. A. florea has also been introduced in historical times in Saudi Arabia
213
and Sudan, and occurred on Java, Indonesia, since ~50 years ago.
Evidently the Florea bees in El-Dender Gerry, and Madani, have
been swarmed and migrated from Khartoum. The first discovery of Apis
florea in Sudan was in Khartoum at a garden near the international
airport by Lord and Nagi (1985). It was believed that the initial colony
might have entered the country as part of an air cargo. By January 1987,
twenty additional colonies had been found, the most distance being 12
km from the Khartoum airport. This was again a prove that no means can
prevent the distribution of honeybee species.
Twenty-two years ago is the age of the first Florea colony invaded
Sudan so it was expect the distribution of A. florea in Sudan would be
extremely widespread. Instead of the above mentioned four studied
colonies from the semi-desert zone. There are some evidences [personal
communications with some colleges from the field of honey beekeepers]
that some colonies of Florea had been found in the far north part of
Sudan (desert zone) particularly in Abu-Hamed town on the Nile valley.
Also it had been found in the far eastern part of the country at ElDamazin city near the border between Sudan and Ethiopia (Savannah
zone). Also in South Sudan at Malakal city (rich savannah zone, near to
the forest zone), few months ago it was found in “Kosti“ near the
author’s house.
It was quite significant also that Apis florea accepted any site for
nesting. Colonies of Florea had been found nesting in different areas
which the indigenous Apis mellifera L. that seeks well protected sites
away from any human activities. Florea therefore competes well for
nesting sites and most probably for the forage in urban areas even outside
its native habitat. This was confirmed with the rapid establishment of the
Florea populations in Khartoum city and the rest of the previous
mentioned towns in the Sudan. In 1984, Whitecombe stated that, Apis
214
florea were well adapted to a higher range of ambient temperature than
Apis mellifera yemenitica L. The rapid establishment of Apis florea was
also confirmed by Ruttner et al., (1985) who studied nesting sites for
Apis florea in Iran. It could, however be mentioned that, within the few
coming years Florea colonies are going to invade some African countries
from Sudan, specially Ethiopia at the eastern part of Sudan, Kenya and
Uganda at the southern part of the country even Egypt at the northern
part border.
215
5- 2- SUMMARY AND CONCLUSION
4-2- a- Apis mellifera L.
The longest North- South latitude 3°N to 22°N and the broadest
East- West longitude 23°E to 30°E, gives the Sudan a contrast in climate
and consequent variety of seasons and natural vegetation. This resulted in
five distinct climatically zones: desert, semi-desert, poor savannah, rich
savannah and forest zones. With such degree of variation, the Sudanese
honeybees found wherever climatic conditions permit, differed
accordingly.
This study focused on the taxonomic status and geographical
variations of the Sudanese honeybees using the biometrical and
mitochondrial DNA variation analysis, nineteen samples were collected
from four geographical zones of the country via: semi-desert, poor
savannah, rich savannah and forest zones. Further 8 other samples were
added in the molecular genetic (mtDNA variability) part of the study.
The 19 bee samples were collected from resting swarms, feral
established colonies and one hived colony. From these samples 39
morphometrical characteristics were measured.
The Sudanese honeybees were classified and named as Apis
mellifera nubica, by Ruttner (1975). He used samples originating from
the semi-desert zone. In 1976, he added that, besides the Sudanese’s
bee’s total small size, their short appendices were remarkable. Therefore,
clearly belong to another group, correctly to be considered as an
independent race. Rashad et al., (1984) suggested that, there were two
independent honeybee subspecies in the Sudan. Firstly Apis mellifera
sudanesis, distributed all over Sudan between latitudes 3° N and 16° 20`
N. The second was Apis mellifera nubica Ruttner, described as mixed
bees, distributed along the International boundaries of Sudan, Ethiopia
and Uganda. However, in 1986, Ruttner withdrew the name “Nubica” in
216
favor of Yemenitica, a name that included bees from the Saudia Arabia,
Yemen, Oman, Somalia and Chad.
Mogga (1988) by using the PCA analysis method suggested that,
three ecotypes of honeybees exist in the Sudan; namely: A. m. yemenitica
L. (the small bee of semi-desert zone), A. m. sudanesis (the medium bee
of forest and rich savannah zone) and A. m. bandasii (the large bee along
the border of Sudan and Ethiopia).
Importation of European honeybees to Khartoum state started since
colonial period; the earliest being in 1928 by King. It went on in 1987.
Owing to this, Elsarrag (1977), considered the Khartoum state bees as
being hybridized. Also Rashad and Elsarrag (1978) demonstrated that,
the honeybees in some parts of the Sudan were hybridized with foreign
honeybee races either through the several importations of particular races
to the country or through the migratory swarms of honeybees from
adjacent countries. This argument may apply to sample of Khartoum
from the semi-desert zone of the present study, particularly the
mitochondrial DNA variation part of the study. In general this sample
gained haplotype Y2, which was completely different from the
haplotypes of the other studied colonies of the semi-desert zone and even,
from the savannah and forest zone colonies too. Meanwhile, other
samples, El-Hissahisa, Madani, and Arkawit were all gained haplotype
O1. Fletcher (1978) reported that, compared to the quantity of genetic
material introduced from African into Brazil where, European bees were
already established, the scale of honeybee importations into Africa from
the United States and European may be regarded as massive. This was
particularly more so, when the Production of Italian drones by 30 or more
colonies were encouraged in Pretoria over several decades. Nevertheless,
the Adansonii population of Pretoria area had remained un affected by its
prolonged exposure to imported genetic materials, Fletcher concluded.
217
Buco et al., (1987) also found that, the Africanized bees in South
America were distinctly different and smaller than European bees. Thus,
the feral bees of South America clearly show the influence of both their
European and African parentage, although they were more similar to
their African parents. The up normal Y2 haplotype of the Khartoum
colony, despite its origin from semi-desert zone might most probably be
attributed to hybridization with foreign bees through the migratory
swarms of honeybees from adjacent countries. Morse et al., (1973) had
also stated that, the success of imported bees with an established success
in honey production and acceptable management behavior in the tropics,
with its well-adapted native bee was far from reach.
In the principal component analysis together with the modern
discriminant analysis of our target 19-honeybee colonies (Apis mellifera
L.), the results revealed the present of three clusters of the Sudanese bees
and they were geographically identifiable. While with the Dra 1
restriction enzyme test, there were 6 different haplotypes had been
differentiated among the 27 studied colonies?
The dark green colour samples distributed to the left side of the PCA
graph consisted of the smallest bees. Their average value measurements
were: forewing length 8.23 mm., and width 2.82 mm.; proboscis length
5.55 mm.; hind-leg length 6.83 mm.; T
3+4
length (body size) 3.88 mm.,
wing venation angles G18, 99.2 and O26, 41.49 ;hind-leg metatarsus
length 1.80 mm.; hind-leg metatarsus width 1.04 mm.; Sternlte 3 length
2.39 mm.; sternite 6 length 2.25 mm.; sternite 6 width 2.64 mm.; wax
mirror length 1.06 mm.; wax mirror width 1.92 mm.; cubital vein a
length 4.26 mm.; labrum 1 colour 3.05; and cubital index 1.85 mm. The
mitochondrial DNA variation analysis of this cluster resulted the
appearance of haplotype A1 in all the studied 8 colonies of the zone. This
result confirmed the PCA and discriminant analysis of the biometrics,
218
that the forest zone cluster was completely seperated from both the
savannah and semi-desert zone clusters.
The second cluster (dark orange colour) samples distributed around
the middle of the PCA graph had medium measurement values. Their
mean average measurements were: forewing length 8.27 mm., and width
2.88 mm.; proboscis length 5.63 mm.; hind-leg length 7.00 mm.; T
3+4
length (body size) 3.88 mm.; wing venation angles G18, 96.33 and O26
36.21; hind-leg metatarsus length 1.91 mm.; hind-leg metatarsus width
1.09 mm.; Sternlte 3 length 2.46 mm.; sternite 6 length 2.26 mm.; sternite
6 width 2.68 mm.; wax mirror length 1.12 mm.; wax mirror width 1.99
mm.; cubital vein a length 4.68 mm.; labrum 1 colour 6.39; and cubital
index 2.04 mm. Mitochondrial DNA variation analysis of this cluster
colonies (4 colonies) resulted the appearance of two different haplotypes;
O1 and Y2 with representing percentages 75 and 25% respectively of all
the analysed colonies.
The third cluster of samples (light blue colour samples) distributed to
the right side of the PCA graph possessed most of the largest
measurements. Their average mean measurements were: forewing length
8.45 mm., width 2.95 mm.; hind-leg 7.05 mm.; T
3+4
length (body size)
4.00 mm., wing venation angles G18 99.20 and O26 36.99; hind-leg
metatarsus length 1.90 mm.; hind-leg metatarsus width 1.09 mm.;
Sternlte 3 length 2.52 mm.; sternite 6 length 2.36 mm.; sternite 6 width
2.76 mm.; wax mirror length 1.13mm.; wax
mirror width 2.01 mm.; cubital vein a length 4.83 mm.; labrum 1 colour
3.65 mm.; and comparatively largest cubital index 2.24 mm. While the
mitochondrial DNA variation analysis of this cluster revealed the
presence of four different haplotypes: O1, A4, O1` and A2. The most
common haplotype in the savannah zone is O1 (54%) followed by A4
(20%), O1` (13%) and A2 (13%) respectively.
219
Franck, et al., 2001, demonstrated that, honeybees from North-eastern
Africa contain three highly divergent mitochondrial lineages A, O, and Y.
While in the other parts of Africa, honeybees carry only mitotypes of
lineage A. Also the gene flow within African honeybees is very high due to
high swarming and migratory behaviour (Hepburn and Radloff, 1988).
Considering the mitochondrial DNA variation analysis of this study, It
seems that, Sudanese honeybees as a whole harbours a mixture of A, O and
Y haplotypes which is really not surprised from what is known about the
North-east African bees. Haplotype O (O1 and O1`) represent 47%; thus,
haplotype A (A1, A2, and A4) represent 47% while haplotype Y (Y2)
represent only 6% of the total haplotypes analysed in this study. Which
indicated that the abundant honeybee haplotypes of Sudan were haplotypes
O and A.
These three clusters of the Sudanese honeybees were geographically
correlated. The smallest bees originated from the forest zone. The
medium bees originated from the semi-desert zone. While the largest
Sudanese bees originated from the savannah zone, mainly along the
Sudan Ethiopia border. Where these bee samples originated in the three
zones, no physical isolation barriers exist. This conformed to Ruttner and
Kauhausen (1985) finding: existence of geographical variability of
honeybee in spite of the absence of physical isolating barriers in tropical
Africa.
Thus, the present of more than one haplotype in one cluster as in the
savannah cluster is not surprised and this conformed to Meixner et al.,
(200), they demonstrated that, some honeybee colonies that were collected
in savannah zone environment of Kenya had both the morphology and the
mtDNA haplotype of A. m. monticola or the morphology of one race and the
mtDNA of other, thus they reported that, there was a colony in their study
combined the morphology of A. m. monticola with the mtDNA haplotype of
220
A. m. scutellata. Thus all samples of A. m. litorea that they analysed shared
the mitochondrial haplotype typical of A. m. scutellata. More than this
Franck, et al., (2001) documented that; honeybees from North-eastern
Africa contain three highly divergent mitochondrial lineages A, O, and Y.
While in the other parts of Africa, honeybees carry only one mitotypes of
lineage A. thus, Meixner. (2006) *Personal communication, in her study
mtDNA variation of Ethiopian honey bees (unpublished paper) indicated
that, haplotype O is relatively common on the west rim of Ethiopia dome,
and this zone is the eastern border of the Sudan and Ethiopia, in the same
time most of the savannah zone colonies were collected from this area. So it
is just a matter of heterogeneous mixture of the blood between the different
races of the zone. Supporting this most of this zone colonies were coloured
(black and yellow).
Rainey (1963) proved that insects fly in the same direction of the
wind. Moreover, Papdopoulo (1975) stated that, the African honeybees
frequently migrate and they can migrate up to 28- 30 km. Again, Brown et
al., (1969) were able to demonstrate the influence of the Inter Tropical
Continental Zone (ITCZ, moist front) on the movement of some insects.
Elsarrag (1977) suggested that, ITCZ to some extent influences the
movements of the migratory honeybee swarms from the south to the north
with the moist south-westerly winds within the country (Sudan) as well as
across the south-eastern boarders. El Sarrag (1977) suggested the possibility
of migration of the African honeybee races, from the south with the moist
southwesterly wind. Observation around Kosti however, indicated migration
between March to May towards the Nile, when the green Vegetation and
water away from the Nile dried out with the advent of the dry season.
Along the Nile, the bees were able to find water and flowering plants,
fruits and some vegetables. During the rainy season, reproductive swarms
migrate back, away from the Nile. The above mentioned observation on the
221
bees migration around Kosti was conformed with the PCA result of Kosti
colony as it appeared to be more closed to semi-desert zone than its origin
savannah zone. Also Wille, H. (1979) reported that, bees swarms at Abu
Naama and Damazin areas, which had spend the dry season in the banks of
the Blue Nile, migrated during or shortly after the rainy season hundreds of
kilometres east and west wards. As drought became stronger, they went
again back to the Blue Nile in several steps. These migrations would be
admitted to be a response to lack or presence of water, nectar or honeydew
and pollen.
Considering the mitochondrial DNA results of the study plus the
presence of colored bees (black and yellow bees in one hive) of some
studied colonies, it could be assumed that there was a gene flow among the
honeybees of Sudan in the area between the latitudes 9º N and 15º N (the
southern part of semi-desert zone and almost all the savannah zone of
Sudan). The direction of the gene flow was from the low land of savannah
zone of Ethiopia towards the western part of the Sudan. Also as it was
confirmed that, the forest zone cluster was completely separated from the
other two clusters (semi-desert and savannah zone clusters), the present of
haplotype A1 in the forest zone colonies only, plus no evidences that, south
Sudan was invaded by a foreign honey bee races before, it could be assume
that the original Sudanese honey bees mitochondrial DNA haplotype may be
is haplotype A1, thus the pure Sudanese honey bee race may be Apis
mellifera sudanesis (South Sudan bees).
Based on both biometric and mitochondrial DNA results of the present
study which were some what conformed each other, the Sudanese
honeybees, through selective adaptation to certain biotopes consisted of
three ecotypes or races, showed some clear geographical distribution pattern
for the character measured, considering the biometrical analysis it is typical
of the tropical African bees (Ruttner and Kauhausen, 1985). While
222
regarding the mitochondrial DNA variation also it is typical of African bees
haplotypes distribution Franck, et al., (2001).
The medium size bees (O1 and Y2 haplotypes) distributed in the semidesert zone were inseparable from the Yemenitica race. Thus the bees from
this zone of Sudan maintained the name Apis mellifera yemenitica, Ruttner.
The small size bees (A1 haplotype) of Sudan were distributed in the forest
zone. These bees retained the name Apis mellifera sudanesis, Rashad.
The largest Sudanese honeybees (O1, A4, O1` and A2 haplotypes) were
distributed in the savannah zone. The distribution area covered most of the
country’s potential region for beekeeping, between 5° N to 16° N latitude.
Some bee colonies of this zone composed of black and yellow bees. These
bees retained the name Apis mellifera bandasii, Mogga.
Honeybee samples along Sudan Uganda and Kenya borders plus
samples of mountainous areas such as Imatong 3187 m. and Jebel Marra
3042 m. could not be included in this study. Inclusion of such samples in
future biometric and molecular genetics studies may clarify the honeybee
races and their geographical distribution in the Sudan.
5- 2- b- Apis florea:
This bee species, a native of Asian subcontinent has got established in
different towns or geographical zones of the Sudan, in fairly short period.
With this rapid established, it could be concluded that, Florea competed
well for nesting sites and forage with the indigenous Apis mellifera
yemenitica. Thus it can be concluded that, within the few coming years the
Florea bees will distribute from Sudan to the neighbouring countries
particularly Ethiopia, Kenya and Egypt.
Concluding from the present results, the four different Florea bee colonies
of Gerry, Khartoum, Madani and El-Dender originally were from the first
discovered Florea colony in Sudan (Khartoum) by Lord and Nagi (1985).
Thus their mother colony entered Sudan from Pakistan or South Iran.
223
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270
CHAPTER SEVEN: APPENDIXES
Appendix (A):
Abbreviations of the morphometric
characters used in the study:
Number
Measured character
Abbreviation
1-
Hair length (mm.)
Hair
2-
Tomentum 1. """"
Tom1
3-
"""""""""""" 2. """"
Tom2
4-
Proboscis. """""
prob
5-
Femur.
"""""
fem
6-
Tibia.
"""""
tib
7-
Tarsus length."""""
ltar
8-
"""“ Width. """""
wtar
9-
Tergite2 pigment.
pt2
10-
"""""3 """""""""
pt3
11-
"""""4 """""""""
pt4
12-
Tergite3 length (mm.).
lt3
13-
""""""''''4 '''''''''''''''' ''''''''''''''
lt4
14-
Sternite3 """"" """"""
lst3
15-
Wax mirror length.(mm.).
lwm
16-
""""" """""" width.
wwm
17-
Distance between wax
dwm
mirrors.(mm).
18-
Sternite 6 length. (mm.).
lst6
19-
""""""""""" width.
wst6
20-
Forewing length. """"""
lfw
21-
"""""""""" width. """"""
wfw
22-
Scutellum 1 pigment.
scut1
23-
""""""""""" 2 """""""""
scut2
271
Appendix (A) continued:
24-
Labrum
1 pigment
25-
""""""""
26-
Cubital vein1length. (mm.).
cub1
27-
Cubital vein2. """". """"""
cub2
28-
Angle A4. ( Degree.).
a4
29-
"""""" B4. """"""""""
b4
30-
"""""" D7. """"""""""
d7
31-
"""""" E9. """"""""""
e9
32-
"""""" G18. """""""""
g18
33-
""""""J10. """""""""
j10
34-
""""" J18. """""""""
j16
35-
""""" K19. """""""""
k19
36-
""""" L13. """""""""
l13
37-
"""" N23. """""""""
n23
38-
"""" O26. """""""""
o26
39-
Hind leg total length. (mm.).
Leg
40-
Tergite3 + Tergite 4. """""""
lt3lt4
41-
Metatarsal index L/W. """""
lw_mtar
42-
Wax mirror """"" """". """""
lw_wxm
43-
Sternite 6 """" """". """""
lw_st6
44-
Cubital vein index a/b. """""
Cind
45-
Tomentum index (mm.).
Toind
46-
Body size/Leg.
bs_leg
47-
Forewing Length/Width. (mm).
2 """"""""
""""""
272
plab1
plab2
Lwfw
Appendix (B):
Climatologically & Rainfall averages for at least 30 years from
climatological stations in or / near the samples collection areas.
KHARTOUM:
Weather station KHARTOUM is at about 15.60°N 32.50°E.
Height about 382m / 1253 feet above sea level.
Average Rainfall
Months Jan Feb Mar Apr May Jun Jul
mm
Inches
0
0
Aug Sep Oct Nov Dec Year
0 0.2 0.4 3.7 7.2 48.6 69.1 20.9 4.5 0.2 0 155.5
0 0 0 0.1 0.3 1.9 2.7 0.8 0.2
0 0 6.1
Source: KHARTOUM data derived from GHCN 1.
1088 months between 1899 and 1989.
Average Maximum Temperature
Months Jan Feb Mar Apr May Jun
°C
°F
Jul
Aug Sep Oct Nov Dec Year
31 33.2 36.9 40 41.8 41.2
38 36.7 38.6 39.2 35.2 31.8 37
87.8 91.8 98.4 104 107.2 106.2 100.4 98.1 101.5 102.6 95.4 89.2 98.6
Source: KHARTOUM data derived from GHCN 2 Beta. 444 months
between 1950 and 1987.
Average Minimum Temperature
Months Jan Feb Mar Apr May Jun Jul
°C
°F
Aug Sep Oct Nov Dec Year
15.6 17 20.3 23.5 26.7 27.2 25.8 25 25.7 25.3 20.9 17 22.5
60.1 62.6 68.5 74.3 80.1 81 78.4 77 78.3 77.5 69.6 62.6 72.5
Source: KHARTOUM data derived from GHCN 2 Beta.
443 months between 1950 and 1987.
SHENDI:
Weather station SHENDI is at about 16.70°N 33.40°E.
Height about 360m / 1181 feet above sea level.
273
MADANI:
Weather station WAD MEDANI is at about 14.40°N
33.40°E. Height about 408m / 1338 feet above sea level.
Average Maximum Temperature
Months Jan Feb Mar
°C
°F
Apr May Jun Jul Aug Sep Oct Nov Dec Year
33.1 35 38.3 40.8 41.5 39.6 35.9 34 35.5 37.9 36.3 33.5 36.8
91.6 95 100.9 105.4 106.7 103.3 96.6 93.2 95.9 100.2 97.3 92.3 98.2
Source: WAD MEDANI data derived from GHCN 2
Beta. 455 months between 1950 and 1987.
Average Minimum Temperature
Months Jan Feb Mar Apr May Jun Jul
Aug Sep Oct Nov Dec Year
14.1 15.8 18.9 21.4 24.3 24.7 23 22.3 21.9 21.7 18.2 15 20.1
57.4 60.4 66 70.5 75.7 76.5 73.4 72.1 71.4 71.1 64.8 59 68.2
°C
°F
Source: WAD MEDANI data derived from GHCN
2 Beta. 455 months between 1950 and 1987.
Average Rainfall
Months Jan Feb Mar Apr May Jun Jul
mm
inches
0
0
0
0
Aug Sep Oct Nov Dec Year
0 0.2 9.2 26.7 103.2 119 50.8 10
0 0 319.5
0 0 0.4 1.1 4.1 4.7
2 0.4
0 0 12.6
Source: BARAKAT data derived from GHCN 1. 444
months between 1929 and 1983.
EL-HISSAHISA:
It is about 14.75°N. and 33. 35°E.
274
Average Rainfall
Months Jan Feb Mar Apr May Jun Jul
Aug
Sep Oct Nov Dec Year
mm
0.0 0.0 0.2 0.1 5.4
23.1 88.8 115.6 46.1 6.1 0.0 0.0 282.7
inches
0.0 0.0 0.0 0.0 0.2
0.9 3.5 4.6
1.8 0.2 0.0 0.0 11.1
Source: RUFA'A data derived from GHCN 1. 437 months between 1913 and 1988.
PORT SUDAN:
Weather station PORT SUDAN is at about 19.57°N 37.20°E. Height
about 3m / 9 feet above sea level.
Average Maximum Temperature
Source: PORT SUDAN data derived from GHCN 2 Beta. 483 months
between 1906 and 1947.
Average Minimum Temperature
Source: PORT SUDAN data derived from GHCN 2 Beta. 488
months between 1906 and 1947.
Average Rainfall
Source: SUAKIN data derived from GHCN 1. 1092 months
between 1891 and 1987. 275
KOSTI:
Weather station KOSTI is at about 13.17°N 32.60°E.
Height about 381m / 1250 feet above sea level.
Average Rainfall
Months Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Year
mm
inches
0
0
0 0.3 2.8 16.1 45.8 106 137.4 61.1 18.4 1.3
0 0 0.1 0.6 1.8 4.2 5.4 2.4 0.7 0.1
0 391.2
0 15.4
Source: KOSTI data derived from GHCN 1. 967
months between 1909 and 1989.
Average Maximum Temperature
Months Jan Feb Mar Apr May Jun
°C
°F
Jul Aug Sep Oct Nov Dec Year
31.5 33.5 37.5 40 41.2 39.5 36.1 34.5 36.2
38 35.4 32.2 36.3
88.7 92.3 99.5 104 106.2 103.1 97 94.1 97.2 100.4 95.7 90 97.3
Source: ED DUEIM data derived from GHCN 2 Beta.
440 months between 1950 and 1987.
Average Minimum Temperature
Months Jan Feb Mar Apr May Jun Jul
°C
°F
Aug Sep Oct Nov Dec Year
16.4 17.7 20.5 22.7 24.9 25.2 23.9 23.3 23.4 23.9 20.9 17.6 21.7
61.5 63.9 68.9 72.9 76.8 77.4 75 73.9 74.1 75 69.6 63.7 71.1
Source: ED DUEIM data derived from GHCN 2 Beta.
436 months between 1950 and 1987.
276
DOKKA:
Weather station is at about 12.75°N 35.90°E. Height
about 764m / 2506 feet above sea level.
Average Maximum Temperature
Months Jan Feb Mar
°C
°F
Apr May Jun Jul Aug Sep Oct Nov Dec Year
34.7 36.3
39 40.6 40.3 37.2 33 31.8 33.7 36.7 37 35 36.3
94.5 97.3 102.2 105.1 104.5 99 91.4 89.2 92.7 98.1 98.6 95 97.3
Source: GEDAREF data derived from GHCN 2 Beta. 453 months between
1950 and 1987.
Average Minimum Temperature
Months Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Year
°C
17.1 18.4 21.5 23.8 24.9 23.1 21.4 21.0 21.2 21.9 20.9 18.1 21.1
°F
62.8 65.1 70.7 74.8 76.8 73.6 70.5 69.8 70.2 71.4 69.6 64.6 70.0
Source GEDAREF data derived from GHCN 2 Beta. 453 month
between 1950 – 1987.
GALLABAT:
Weather station GALLABAT is at about 12.80°N 36.17°E. Height
about 764m / 2506 feet above sea level.
Average Maximum Temperature
Months Jan Feb Mar
°C
°F
Apr May Jun Jul Aug Sep Oct Nov Dec Year
35.9 37 38.6 39.3 37.6 33.5 29.6 29.1 30.8 34 35.9 35.7 34.7
96.6 98.6 101.5 102.7 99.7 92.3 85.3 84.4 87.4 93.2 96.6 96.3 94.5
Source: GALLABAT data derived from GHCN 2 Beta. 337 months between
1906 and 1940.
Average Minimum Temperature
Months Jan Feb Mar Apr May Jun Jul
Aug Sep Oct Nov Dec Year
°C
16.3 18.2 20.3 22.5 22.5 20.2 19.2 18.9 18.8 18.3 16.8 15.7 18.9
°F
61.3 64.8 68.5 72.5 72.5 68.4 66.6 66.0 65.8 64.9 62.2 60.3 66.0
Source: GALLABAT data derived from GHCN 2 Beta. 301 months between
1906 and 1940.
277
El HAWATA:
Weather station HAWATA is at about 13.40°N 34.60°E. Height about 440m /
1443 feet above sea level.
Average Rainfall
Months Jan Feb Mar Apr May Jun
Mm
0.0 0.0 0.0
Inches 0.0 0.0 0.0
Jul
Aug
Sep Oct
Nov Dec Year
1.2 15.1 99.5 154.1 201.7 79.5 15.8 0.6
0.0
567.8
0.0 0.6
0.0
22.4
3.9
6.1
7.9
3.1
0.6
0.0
Source: HAWATA data derived from GHCN 1. 420 months between 1950
and 1988.
Average Maximum Temperature
Months Jan
Feb Mar
Apr
May
Jun Jul
Aug Sep Oct Nov Dec Year
°C
34.7 36.3 39.0
40.6
40.3
37.2 33.0 31.8 33.7 36.7 37.0 35.0 36.3
°F
94.5 97.3 102.2 105.1 104.5 99.0 91.4 89.2 92.7 98.1 98.6 95.0 97.3
Source: GEDAREF data derived from GHCN 2 Beta. 453 months
between 1950 and 1987.
Average Minimum Temperature
Months Jan
Feb Mar Apr May Jun
Jul
Aug Sep Oct
Nov Dec Year
°C
17.1 18.4 21.5 23.8 24.9 23.1 21.4 21.0 21.2 21.9 20.9 18.1 21.1
°F
62.8 65.1 70.7 74.8 76.8 73.6 70.5 69.8 70.2 71.4 69.6 64.6 70.0
Source: GEDAREF data derived from GHCN 2 Beta. 455
months between 1950 and 1987.
GALA El- NAHAL:
Weather station is at about 13.60°N 34.75°E. Height about 440m
/ 1443 feet above sea level.
Average Rainfall
Months Jan Feb Mar Apr May Jun Jul
Mm
Aug
Sep Oct Nov Dec Year
0.0 0.1 1.0 4.8 25.8 94.5 177.3 192.1 97.6 26.9 4.5 0.0 626.4
Inches 0.0 0.0 0.0 0.2 1.0
3.7 7.0
7.6
3.8 1.1 0.2 0.0 24.7
Source: GEDAREF data derived from GHCN 1. 1047 months
between 1903 and 1990.
278
Average Maximum Temperature
Months Jan Feb Mar
Apr
May Jun Jul
Aug Sep Oct Nov Dec Year
°C
34.7 36.3 39.0 40.6 40.3 37.2 33.0 31.8 33.7 36.7 37.0 35.0 36.3
°F
94.5 97.3 102.2 105.1 104.5 99.0 91.4 89.2 92.7 98.1 98.6 95.0 97.3
Source: GEDAREF data derived from GHCN 2 Beta. 453
months between 1950 and 1987.
Average Minimum Temperature
Months Jan Feb Mar Apr May Jun Jul
°C
Aug Sep Oct Nov Dec Year
17.1 18.4 21.5 23.8 24.9 23.1 21.4 21.0 21.2 21.9 20.9 18.1 21.1
°F
62.8 65.1 70.7 74.8 76.8 73.6 70.5 69.8 70.2 71.4 69.6 64.6 70.0
Source: GEDAREF data derived from GHCN 2 Beta. 455 months
between 1950 and 1987. SINGA:
Weather station SINGA is at about 13.15°N 33.95°E. Height about
436m / 1430 feet above sea level.
Average Maximum Temperature
Source: SINGA data derived from GHCN 2 Beta. 321 months
between 1914 and 1943.
Average Minimum Temperature
Source: SINGA data derived from GHCN 2 Beta. 338 months
between 1914 and 1943.
279
Average Rainfall
Source: SENNAR data derived from GHCN 1. 945 months
between 1907 and 1989.
ROSEIRES:
Weather station ROSEIRES is at about 11.85°N 34.38°E. Height
about 467m / 1532 feet above sea level.
Average Rainfall
Source: ROSEIRES data derived from GHCN 1. 796 months
between 1903 and 1987.
Average Maximum Temperature
Source: ROSEIRES data derived from GHCN 2 Beta. 483
months between 1905 and 1947.
280
Average Minimum Temperature
Source: ROSEIRES data derived from GHCN 2 Beta. 494
months between 1905 and 1947.
KADUGLI:
Weather station KADUGLI is at about 11.00°N
29.70°E. Height about 499m / 1637 feet above sea
level.
Average Maximum Temperature
Months Jan Feb Mar
Apr
May Jun Jul
Aug Sep Oct Nov Dec Year
°C
34.9 36.5 39.1
39.8
38.2
°F
94.8 97.7 102.4 103.6 100.8 94.8 89.2 88.2 90.5 94.8 97.7 95.0 95.7
34.9 31.8 31.2 32.5 34.9 36.5 35.0 35.4
Source: KADUGLI data derived from GHCN 2 Beta. 453
months between 1950
and 1987.
Average Minimum Temperature
Months Jan
Feb Mar Apr May Jun Jul
Aug Sep Oct Nov Dec Year
°C
17.2 19.2 21.8 23.2 23.6 22.4 21.5 21.1 20.6 20.0 18.4 17.7 20.5
°F
63.0 66.6 71.2 73.8 74.5 72.3 70.7 70.0 69.1 68.0 65.1 63.9 68.9
Source: KADUGLI data derived from GHCN 2 Beta. 450 months
between 1950 and
1987.
Average Rainfall
Months Jan Feb Mar Apr May Jun
mm
Jul
Aug Sep
Oct Nov Dec Year
0.0 0.8 1.8 14.4 74.9 112.7 150.4 152.8 140.0 73.3 2.9 0.0 725.5
281
inches 0.0 0.0 0.1 0.6 2.9 4.4
5.9
6.0
5.5
2.9 0.1 0.0 28.6
Source: KADUGLI data derived from GHCN 1. 950 months between
1910 and 1989.
El DALANG:
Weather station DILLING is at about 12.00°N 29.60°E.
Height about 670m / 2198 feet above sea level.
Average Rainfall
Months Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Year
Mm
0.0 0.0 1.8 5.6 33.8 80.5 143.5 159.4 119.4 37.9 0.4 0.0 565.1
inches 0.0 0.0 0.1 0.2 1.3 3.2 5.6 6.3 4.7 1.5 0.0 0.0 22.2
Source: DILLING data derived from GHCN 1. 439 months between 1950
and 1988.
Average Maximum Temperature
Months Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Year
°C
34.9 36.5 39.1 39.8 38.2 34.9 31.8 31.2 32.5 34.9 36.5 35.0 35.4
°F
94.8 97.7 102.4 103.6 100.8 94.8 89.2 88.2 90.5 94.8 97.7 95.0 95.7
Source: KADUGLI data derived from GHCN 2 Beta. 453 months between
1950 and 1987.
Average Minimum Temperature
Months Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Year
°C
17.2 19.2 21.8 23.2 23.6 22.4 21.5 21.1 20.6 20.0 18.4 17.7 20.5
°F
63.0 66.6 71.2 73.8 74.5 72.3 70.7 70.0 69.1 68.0 65.1 63.9 68.9
Source: KADUGLI data derived from GHCN 2 Beta.
450 months between 1950 and 1987.
UM RAWABA:
Weather station UMM RUWABA is at about 12.80°N
31.20°E. Height about 450m / 1476 feet above sea level.
Average Rainfall
282
Source: UMM RUWABA data derived from GHCN 1. 916
months between 1912 and 1989.
Average Maximum Temperature
Source: EL OBEID data derived from GHCN 2 Beta. 496
months between 1905 and 1947.
Average Minimum Temperature
Source: EL OBEID data derived from GHCN 2 Beta. 484
months between 1905 and 1947.
KUBBUM:
Weather station KUBBUM is at about 11.80°N 23.80°E.
Average Rainfall
283
Source: KUBBUM data derived from GHCN 1 484 months between
1943 and 1985.
ZALINGEI:
Weather station ZALINGEI is at about 12.90°N 23.30°E.
Height about 900m / 2952 feet above sea level.
Average Rainfall
Source: ZALINGEI data derived from GHCN 1. 620 months
between 1929 and 1986.
Average Maximum Temperature
Source: Source: NYALA data
derived from GHCN 2 Beta. 452
months (1950 - 1987.)
Average Minimum Temperature
284
Source: NYALA data derived from GHCN 2 Beta. 440 months
between 1950 and 1987.
WAU-SHOLOK:
Weather station is at about 9.75°N 31.80°E. Height about
390m / 1279 feet
above sea level.
Average Rainfall
Months Jan Feb Mar Apr May Jun
Mm
Jul
Aug
Sep
Oct Nov Dec Year
0.0 0.4 3.2 19.5 65.6 115.3 159.7 174.2 135.8 79.7 4.6
Inches 0.0 0.0 0.1 0.8
2.6
4.5
6.3
6.9
5.3
3.1
0.2
0.0 758.5
0.0 29.9
Source: KODOK data derived from GHCN 1. 816 months between
1903 and 1978.
Average Maximum Temperature
Months Jan
Feb Mar
Apr
May Jun Jul
Aug Sep Oct Nov Dec Year
°C
35.7 37.2 38.9
38.6
35.8 33.0 31.1 30.9 32.2 34.0 35.6 35.7 34.9
°F
96.3 99.0 102.0 101.5 96.4 91.4 88.0 87.6 90.0 93.2 96.1 96.3 94.8
Source: MALAKAL data derived from GHCN 2 Beta.
391 months between 1915 and 1947.
Average Minimum Temperature
Months Jan Feb Mar Apr May Jun Jul
Aug Sep Oct Nov Dec Year
°C
18.3 19.9 21.8 23.6 23.0 21.9 21.5 21.4 21.7 21.7 19.4 18.2 21.0
°F
64.9 67.8 71.2 74.5 73.4 71.4 70.7 70.5 71.1 71.1 66.9 64.8 69.8
285
Source: MALAKAL data derived from GHCN 2 Beta. 372
months between 1915 and 1947.
MALAKAL:
Weather station MALAKAL is at about 9.60°N 31.70°E.
Height about 388m / 1273 feet above sea level.
Average Maximum Temperature
Months Jan Feb Mar Apr
May Jun Jul Aug Sep Oct Nov Dec Year
°C
35.1 36.9 38.8 38.5 35.8 32.8 30.9 30.8 31.8 33.4 35.1 34.9 34.6
°F
95.2 98.4 101.8 101.3 96.4 91.0 87.6 87.4 89.2 92.1 95.2 94.8 94.3
Source: MALAKAL data derived from GHCN 2 Beta. 443
months between 1950 and 1987.
Average Minimum Temperature
Months Jan Feb Mar Apr May Jun Jul
Aug Sep Oct Nov Dec Year
°C
18.4 20.0 22.8 23.9 23.4 22.2 21.7 21.7 21.7 21.7 19.7 18.2 21.3
°F
65.1 68.0 73.0 75.0 74.1 72.0 71.1 71.1 71.1 71.1 67.5 64.8 70.3
Source: MALAKAL data derived from GHCN 2 Beta. 441
months between 1950 and 1987.
Average Rainfall
Months Jan Feb Mar Apr May Jun Jul
Mm
Aug Sep
Oct Nov Dec Year
0.0 0.3 2.1 14.6 40.3 98.3 139.3 154.4 106.0 71.9 2.8 0.0 647.6
Inches 0.0 0.0 0.1 0.6 1.6 3.9 5.5
6.1
4.2
2.8 0.1 0.0 25.5
Source: MELUT data derived from GHCN 1. 362 months
between 1951 and 1981.
DOLIEB:
Weather station DOLEIB is at about 9.30°N 31.63°E. Height
about 391m / 1282 feet above sea level.
Average Maximum Temperature
286
Months Jan Feb
Mar Apr
May Jun Jul Aug Sep Oct Nov Dec Year
°C
36.2 37.9 39.7 39.7 37.0 34.3 32.5 32.2 33.7 35.0 36.4 36.0 35.9
°F
97.2 100.2 103.5 103.5 98.6 93.7 90.5 90.0 92.7 95.0 97.5 96.8 96.6
Source: DOLEIB HILL data derived from GHCN 2 Beta.
410 months between 1906 and 1943.
Average Minimum Temperature
Months Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Year
°C
18.2 19.8 21.8 23.2 22.7 21.6 21.2 21.2 21.4 21.3 19.2 17.7 20.8
°F
64.8 67.6 71.2 73.8 72.9 70.9 70.2 70.2 70.5 70.3 66.6 63.9 69.4
Source: DOLEIB HILL data derived from GHCN 2 Beta.
416 months between 1907 and 194
Average Rainfall
Months Jan Feb Mar Apr May Jun
Mm
Jul
Aug Sep
Oct Nov Dec Year
0.0 0.3 5.9 28.0 98.5 134.9 204.9 211.7 155.6 92.4 6.9 0.0 944.1
inches 0.0 0.0 0.2 1.1 3.9 5.3
8.1
8.3
6.1
3.6 0.3 0.0 37.2
Source: FANGAK data derived from GHCN 1. 691
months between 1922 and 1981.
GANAL:
Weather station is at about 9.25.00°N 31.20°E. Height about
390m / 1279 feet above sea level.
Average Rainfall
Months Jan Feb Mar Apr May Jun Jul
Mm
Aug Sep
Oct Nov Dec Year
0.0 0.3 2.1 14.6 40.3 98.3 139.3 154.4 106.0 71.9 2.8 0.0 647.6
Inches 0.0 0.0 0.1 0.6 1.6
3.9 5.5
6.1
Source: MELUT data derived from GHCN 1. 362
months between 1951 and 1954.
287
4.2
2.8 0.1 0.0 25.5
Average Minimum Temperature
Months Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Year
°C
18.3 19.9 21.8 23.6 23.0 21.9 21.5 21.4 21.7 21.7 19.4 18.2 21.0
°F
64.9 67.8 71.2 74.5 73.4 71.4 70.7 70.5 71.1 71.1 66.9 64.8 69.8
Source: MALAKAL data derived from GHCN 2 Beta. 372
months between 1915 and 1947.
JUBA:
Weather station JUBA is at about 4.87°N
31.60°E. Height about 460m / 1509 feet
above sea level.
Average Maximum Temperature
Jan Feb Mar Apr May Jun Jul
Aug Sep Oct Nov Dec Year
°C 36.9 37.5 37.1 34.9 33.2 32.0 30.8 30.9 32.4 33.5 34.7 35.8 34.1
°F 98.4 99.5 98.8 94.8 91.8 89.6 87.4 87.6 90.3 92.3 94.5 96.4 93.4
Source: JUBA data derived from GHCN 2 Beta. 453 months
between 1950 and 1987.
Average Minimum Temperature
Jan Feb Mar Apr May Jun Jul
Aug Sep Oct Nov Dec Year
°C 19.6 21.3 23.1 23.0 22.3 21.4 20.7 20.6 20.7 20.9 20.5 19.5 21.1
°F 67.3 70.3 73.6 73.4 72.1 70.5 69.3 69.1 69.3 69.6 68.9 67.1 70.0
Source: JUBA data derived from GHCN 2 Beta. 452
months between 1950 and 1987.
Average Rainfall
Months Jan Feb Mar Apr
May Jun
288
Jul
Aug Sep
Oct
Nov Dec Year
Mm
3.5 11.9 42.1 104.7 155.0 114.4 128.3 137.9 113.7 107.5 41.8 9.7 971.4
inches 0.1 0.5 1.7 4.1
6.1
4.5
5.1
5.4
4.5
4.2
1.6 0.4 38.2
Source: JUBA data derived from GHCN 1. 1045
months between 1901 and 1988.
BANGO:
Weather station is at about 4.90°N 31.50°E.
Height about 457m / 1499 feet above sea level.
Average Maximum Temperature
Months Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Year
°C
36.9 37.5 37.1 34.9 33.2 32.0 30.8 30.9 32.4 33.5 34.7 35.8 34.1
°F
98.4 99.5 98.8 94.8 91.8 89.6 87.4 87.6 90.3 92.3 94.5 96.4 93.4
Source: JUBA data derived from GHCN 2 Beta. 453
months between 1950 and 1987.
Average Minimum Temperature
Months Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Year
°C
19.6 21.3 23.1 23.0 22.3 21.4 20.7 20.6 20.7 20.9 20.5 19.5 21.1
°F
67.3 70.3 73.6 73.4 72.1 70.5 69.3 69.1 69.3 69.6 68.9 67.1 70.0
Source: JUBA data derived from GHCN 2 Beta. 452
months between 1950 and 1987.
LIRRIA:
Weather station is at about 4.25°N 32.25°E. Height about
460m / 1509 feet above sea level.
Average Maximum Temperature
Months Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Year
°C
37.5 37.1 36.3 35.1 33.3 32.1 30.4 30.8 32.7 34
289
35.4 36.1 34.1
°F
99.5 98.8 97.3 95.2 91.9 89.8 86.7 87.4 90.9 93.2 95.7 97
93.4
Source: TORIT data derived
from GHCN 2 Beta. 138 months
(1922 - 1940.)
Average Minimum Temperature
Months Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Year
°C
19
20.2 21
20.8 20.3 19.5 19.1 18.9 18.7 18.9 18.3 18.1 19.3
°F
66.2 68.4 69.8 69.4 68.5 67.1 66.4 66
65.7 66
64.9 64.6 66.7
Source: TORIT data derived from GHCN 2 Beta. 192 months (1922 1940.)
Average Rainfall
Months Jan Feb Mar Apr May Jun Jul
Aug Sep Oct Nov Dec Year
mm
5.8 16.3 52.8 62 85.8 84.7 117.8 120 82.2 65.7 55.5 24.8 785
inches
0.2 0.6 2.1 2.4 3.4 3.3 4.6
4.7 3.2 2.6 2.2 1
30.9
Source: KAPOETA data derived from GHCN 1. 362 months (1951 -1981.)
KHOUR-MAQUIRE:
Weather station is at about 5.25°N 31.75°E. Height about 422m / 1384
feet above sea level.
Average Maximum Temperature
Months
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Year
°C
37.5 37.1 36.3 35.1 33.3 32.1 30.4 30.8 32.7
°F
99.5 98.8 97.3 95.2 91.9 89.8 86.7 87.4 90.9 93.2 95.7
34 35.4 36.1 34.1
97 93.4
Source: TORIT data derived from GHCN 2 Beta. 138 months (1922 -1940).
Average Minimum Temperature
Months Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Year
°C
19.0 20.2 21.0 20.8 20.3 19.5 19.1 18.9 18.7 18.9 18.3 18.1 19.3
°F
66.2 68.4 69.8 69.4 68.5 67.1 66.4 66.0 65.7 66.0 64.9 64.6 66.7
Source: TORIT data derived
from GHCN 2 Beta. 192
290
months between 1922 and 1940.
YEI:
Weather station YEI is at about 4.00°N 30.60°E.
Average Rainfall
Source: YEI data derived from GHCN 1. 362 months between 1951
and 1981.
RAJA:
Weather station WAU is at about 7.70°N 28.00°E. Height about
438m / 1437 feet above sea level.
Average Rainfall
Source: WAU data derived from GHCN 1. 1008 months between
1904 and 1987.
291
Appendix (C): Taxonomic relationships between bees in the
family Apidae.
292
Appendix (D): Different species of the Genus Apini
(Institute Für Bienenkunde, Oberursel, Germany).
293
Appendix (E): Natural distribution of
Honeybee Species (Institute Für
Bienenkunde, Oberursel, Germany).
294
Appendix (F): Geographical
distribution of Genus Apis
(Institute Für
Bienenkunde, Oberursel,
Germany).
295
Appendix (G): Distribution of
geographical honeybee races and
mean of annual temperature (F.
Ruttner, Institute Für
Bienenkunde, Oberursel,
Germany).
296
Appendix (H):
Multivariate ANOVA Table: Sum of squares, dF, mean square,
F values and significances for each phenotypic character from the
measured individuals. (Sudanese honeybee Apis mellifera L.)
No.
Character
123456789101112131415161718192021222324-
Hair
Tom1
Tom2
Prob
Fem
Tib
Ltar
Wtar
Pt2
Pt3
Pt4
Lt3
Lt4
Lst3
Lwm
Wwm
Dwm
Lst6
Wst6
Lfw
Wfw
Scut1
Scut2
Plab1
Sum of
squares
6.097
776.250
100.282
596.188
120.115
172.043
350.731
102.291
0.514
3.448
1.486
58.133
114.567
512.573
111.078
229.209
9.656
449.644
450.963
2276.432
571.699
0.105
1.894
25.219
df
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
297
Mean
square
3.048
388.125
50.141
298.094
60.057
86.022
175.365
51.146
0.257
1.724
0.743
29.066
57.283
256.287
55.539
114.605
4.828
224.822
225.481
1138.216
285.849
0.053
0.947
12.610
F
0.471
6.178
0.558
11.523
2.574
1.700
9.608
8.179
0.328
2.590
2.857
1.435
3.566
12.300
10.207
15.324
1.251
7.809
11.075
10.940
15.010
0.119
0.245
6.378
Significances
0.633
0.010
0.583
0.001
0.107
0.214
0.002
0.004
0.725
0.106
0.087
0.267
0.052
0.001
0.001
0.000
0.313
0.004
0.001
0.001
0.000
0.888
0.786
0.009
Appendix (H) continued:
No.
Character
2526272829303132333435363738394041424344454647-
Plab2
Cub1
Cub2
A4
B4
D7
E9
G18
J10
J16
K19
L13
N23
O26
Leg
Lt3lt4
Lw_mtar
Lw_wxm
Lw_st6
Cind
Toind
Bs_leg
Lwfw
Sum of
squares
2.511
127.136
4.329
3.769
32.003
6.317
2.545
25.617
24.535
33.840
28.469
10.506
39.370
71.139
1570.136
315.184
1.717
2.868
3.408
0.601
1.525
0.536
1.051
df
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
p ≤ 0.005
298
Mean
square
1.255
63.568
2.164
1.885
16.001
3.159
1.272
12.809
12.268
16.920
14.235
5.253
19.685
35.570
785.068
157.592
0.859
1.434
1.704
0.301
0.762
0.268
0.526
F
4.404
12.643
1.125
1.511
4.991
0.764
2.557
7.477
3.941
5.743
2.430
1.108
4.966
12.461
4.073
2.497
0.591
1.092
0.875
6.124
0.735
0.137
2.522
Significances
0.030
0.001
0.349
0.250
0.021
0.482
0.109
0.005
0.041
0.013
0.120
0.354
0.021
0.001
0.037
0.114
0.566
0.359
0.436
0.011
0.495
0.873
0.112
299
300
301
302
304
305
306
307
308
309
310
312
313
314
315
316
317
318
319
320
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
352
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