Morphological and allozyme variation of Eidolon
helvum (Mammalia:Megachiroptera) in the
islands of the Gulf of Guinea
JAVIER JUSTE'.'*, CARLOS I B W E Z ? AND ANNIE MACHORDOM'
' Departamento de Bioquimica y Biologia Molecular I v Facultad de Veterinaria, Universidad
Cornplutense de Madrid, Madrid 28040, Spain; 2EstacidnBioldgica de DoKana, CSIC,
Apartado 1056, E-41080 Sevilla, Spain; 'Muse0 Nacional de Ciencias Naturales (CSIC),
JOS&
Gutither Abascal 2, 28006 Madrid, Spain
Rpcewd 6 August 1999; accepted jor publzratzon 28 March 2000
hlorphological and genetic variation is evaluated among populations of the bat, Eidolon
h e l r ~ m in
, the islands of the Gulf of Guinea (Central Africa). The populations from the islands
of Bioko, Principc, and Sao Tomb do not show significant phenetic differentiation, although
a trcnd towards a reduction of size is found in thc latter two islands. The low genetic
distances between populations, as well as their values of Wright's fixation indexes, suggest
that gene flow has hampered differentiation on these islands. In contrast, the population
from Xnnobon, the smallest and farthermost island, shows remarkable morphological and
genetic differentiation. On the mainland, E. heluurn displays unique migratory and dispersal
behaviours, but migratory behaviour was not found in any of the island populations. The
combination of selective forces in harsher oceanic environments and restricted gene flow
among populations appears to have favoured the high degree of morphological dlfferentiation
of E. heliburn on Annobbn. Due to the extended length of the dry season in Annobon, an
earlier achievement of sexual maturity-and consequently smaller size -may be advantageous
in the absence of migration. The differentiation is morc marked among females, which also
suggests that selection may be linked to the reproductive pattern. The population of the
island of Annobon is herein dcscribed as a new subspecies, Eidolon helvum annobonensis
subsp. nov.
0 2000 'Ihc Liiiiiran Socictv of Loiidon
ADDITIONAL KEY WORDS:-Genetic
Africa - fruit bats.
variation
-
island populations
~
taxonomy
CONI'ENTS
Introduction. . . . . .
hlaterial and methods
. .
Study area . . . . .
Morphological analyses
Allozyme analyses . .
Results . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
* Corresponding author: Javier Justc,
0021-1066/00/100359+ 20 f35.00/0
360
361
361
362
362
363
at Seville address. E-mail: [email protected]
559
0 2000 T h e Linnt nn Sorich uf London
360
.J. ,JCSlX ET dL.
Morphological analyses
. . . . . . .
Allozyme analyses . . . . . . . . .
Discussion . . . . . . . . . . . . . .
Morphological variation . . . . . . .
Gcnetic variation
. . . . . . . . .
Population-genetic structure . . . . . .
Variation patterns . . . . . . . . .
Effects of migration and dispersal . . . .
Evolutionary and taxonomic inferences . .
Eidolon helvum annobonensis subsp. nov.
. . . . . . . . . . .
Acknowledgements
References . . . . . . . . . . . . . .
Appendix 1 . . . . . . . . . . . . . .
Appendix2 . . . . . . . . . . . . . .
Appendix3 . . . . . . . . . . . . . .
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363
364
368
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37 1
372
372
373
374
374
376
377
378
INTRODUCTION
The African straw-coloured bat, Eidolon helvum (Kerr, 1792), is the second largest
fruit bat in Africa, with unique morphological (Andersen, 1912), ecological (Thomas,
1982)) and reproductive (Bernard & Cumming, 1997) characteristics. A recent
phylogenetic study suggests that Eidolon’s origin may be closer to the typically Asian
Pteropus group than to any extant African fruit bat (Juste et al., 1999). Like the
Pteropus bats, E, helvum has narrow and pointed wings, a morphology particularly
apt for long-distance flying (Rosevear, 1965). In fact, E. helvum is able to move in
seasonal migrations as far as 2500 km in Sudan (Kock, 1969) or 1500 km in West
Africa (Thomas, 1983). Possibly associated with its high vagility, Eidolon is a poorly
diversified genus and only two species are currently recognized: the nominative E.
helvum spread across the sub-Saharan continent (DeFrees & Wilson, 1988)) and the
endemic E. dupreanum from Madagascar (Bergmans, 1990). E. helvum is considered
morphologically uniform throughout its vast distribution range. Only a population
from Southern Arabia, E. h. sabaeum, is distinguished at the subspecific level
(Bergmans, 1990), on the basis of its smaller size and heavier dentition (Andersen,
1912).
Islands appear to be favoured by E. heluum along the African coast (Rosevear,
1965): it is the only fruit bat that has successfully colonized the four islands of the
Gulf of Guinea in West Central Africa (Fig. 1) (Juste & Ibafiez, 1994). Its presence
has been reported in all the islands (Bioko, Principe, S%o Tomb and Annobon),
since the earliest scientific expeditions to this group (Barboza du Bocage, 1897,
1903; Cabrera, 1908). The maximum distance between the islands-and to the
mainland-in the Gulf of Guinea is 350 km. This does not appear to present an
insurmountable barrier to contact between the various island populations, and
differentiation would not be expected. Nevertheless, Eisentraut (1964) found some
cranial differences between specimens from Bioko and the mainland. Bergmans
(1990) has suggested that the lack of migratory movements could have provided
sufficient isolation to allow for the differentiation of this population. The status of
E. helvum in the islands is still unknown because only a few scattered specimens from
these populations exist in collections. Consequently, relationships among them, and
the possible role that the migratory behaviour and high dispersal potential of E.
helvum could play in defining their characteristics, are still to be assessed.
,
I
I
(857 km2)
@.
..
,
I
I
I
>
. .
Equator
280km
. ......... ..... .
.=..
..
P
Figure 1. SIap of ttic Gulf of Guinea. Central \2'rst Africa.
The objectives of this work wcrc: (1) to characterize morphologically and genetically
the populations of E. helvum from all the islands; (2) to examine for patterns of
variation within these two sources of variability, and for congruence betwctn them;
(3) to infer evolutionary relationships, as well as possible colonization patterns among
these populations in the geographic context of the Gulf of Guinea.
Bioko, Principe, Sao Tome and Annobon stretch NE-SW across the Gulf of
Guinea, and arc the only insular system off the sub-Saharan Atlantic coast of Africa.
All are relati\dy small and share a common volcanic origin (Fuster, 1955). Bioko
is the closest to thc mainland shoreline, and as a 'land-bridge' island, its flora and
fauna are relatively species-rich and poor in endemisms. In contrast, Principe, S2o
Tomi: and Annob6n are typical oceanic islands, and as such, show low diversity
but high levels of endemism at the specific and generic levels (Exell, 1968; Jones,
1994). The island populations of E. helvum are compared with a population from
362
J. JUSTE: ET,iI,
Rio Muni, a small continental region southwest of Cameroon that is included in
the Central African rainforest, and is approximately equidistant to all thc islands.
Morphological anaQses
A total of 170 specimens was collected between 1984 and 1989: 46 from Rio
Muni, West Central Africa (27 males and, 19 females), 37 from Bioko (22 males
and 15 females), 34 from Principe (22 males and 12 females), 25 from S2o Tomi:
(12 males and 13 females), and 28 from Annobon (14 males and 14 females). All
specimens were deposited in the collections of the Estacion Biologica de Dofiana
(CSIC), Seville, Spain.
A total of 12 wing, 13 cranial, and 14 dental variables (Appendix 1) were measured
by the senior author (ClT) using a digital caliper with a precision of 0.1 mm.
Measurements follow Bergmans (1979), with the exception of the mandibular angle
(MA) defined as the angle between the mandibular ramus and the coronoid process,
and obtained using a protractor with a precision of 2". For each data set (wing,
cranial and dental), data were log-transformed to linearize possible allometric
relationships. Missing values were estimated using the expectation-maximization
method of Little & Rubin (1987), which optimizes the missing values as a set by
stabilizing the covariance matrix. Only full-grown individuals-according to wing
bones and cranial suture criteria (Anthony, 1988)-were considered in the following
statistical analyses. After inspecting the data for normality, overall geographic
variation and presence of sexual dimorphism in the populations were evaluated
using 2-way MANOVAs by data set. Further analyses dealt with the sexes separately.
Principal component analysis (PCA) on the covariance matrices was used to assess
the variation among populations, since it does not presume any grouping of data.
The relative discriminatory power of each variable for each data set was assessed
by a stepwise discriminant analysis (DFA) designed to introduce variables so as to
maximize at each step Lawley-Hotelling's trace (=Rae's V). This measure is
proportional to the mean Mahalanobis distance. For each data set, the four variables
with the highest discriminant power were pooled by sex into the final matrix of the
12 variables selected. PCAs on these matrices were used to confirm and visualize
the differences among populations. Mahalanobis' d measured the multivariate
distances among them, and the values were tested for significance (HG0' = 0) based
on an F statistic. Neighbour-joining trees on the o? distance matrices were checked
for patterns of geographic relationships. A sequential Bonferroni procedure (Rice,
1989) was used to adjust the statistical significance of multiple simultaneous tests to
a table-wide a level of 0.05. All statistical analyses were performed in Matlab for
Windows ver. 4 . 2 ~(Mathworks, Inc., 1994). Matlab statistical functions and script
files are available from the authors upon request.
Alloryme anaQses
Tissue samples (liver, kidney, heart and muscle) from a total of 74 specimens of
E. helvum (10 from Rio Muni, 10 from Bioko, 27 from Principe, 18 from SZo Tomt
and 9 from Annobon) were collected from 1987 to 1992 and immediately stored in
IiqLiid nitroqen. \70uc hcrs are ckpositcd in the collections of the Estacibii Riolbgicn
dc llofiann (CSIC), Se\ ilk. S p i n .
Honiog-cnatcs (of tlic smipl(.s \\ crc riiii in hor17ont,il starch gel electrophoresis.
xcordiiiy t o ~tantlnrdproc cdurcs (Pnstcur P / o/.. 1 W7), to L i s ~genetic
e ~ ~ \ nriation
<it 34 prcsLinipti\ c loci ciicoding 25 cmz) matic 5) stems (Appendix 3). The loci Ert
1 and ,Sdh coiild not I,e scored and were cxcluclcd Allclcs nt each locus wcre
distinquislicd
cliff crential mohility in sidc-h) -side co~iiparisons,a i d designated
nlplinheticall) 111 their iiicrc<ising niohilit) riith the most common allclc designated
11) %a>.
DCita\\ere nnal>scd \\ i t h tlic BIOSYS-1 pncl..ige (S\$offord & Selmdcr, 1989).
Grnctic Iariabilit) iias estimated as the mean 1iuiiiI)crof alleles per locus, percentaqe
of pol5 morphic (93%)c i itei ion). loci (4
ol)srr\ cd ( H ) nnd expected heter o
per locus. Since no conipirisons 5 icldcd ohsen cd 1 alues of Iietcroq gosit) deviating
iignificnntlk fi om expected Hard1 \Yciiil)erg equilibrium, onl) \ alues for obscmecl
Iietcroz) gosit) arc p r r s c n t d . (hirtic rr1,itediics nrnong populations rids e\ aiudtcd
using Nci's (1 '378)uiiliised cltstanccs (D,)
m d modified (\\'riglit, 1978) Rogers'
( 1972) distance (I),?).
Diffcrenccs in genctic 3triicture nmong populations \L ere rstimnted from F,, \ alues
(\Yright. 19135). Gene Ao\\ hrtwcrn populations (\in) \\as cstimated direct15 from
the F, I \ alucs using \.\'riqlit's nictliod under the islnnd niodel ikithout selection or
mutation (IYriglit, 1965). and from Slatkin's ( 1 985) pril ntc alleles method after
correctin? for sample sizes. ;
i mid-point I ootcd plienogram \bas ( onstructcd from
the matrix of the modified Kogci 5 ' gciic+c distnnccs urinq the distancc-\Vnyner
p ~ ~ c c d i i i c\ \.l i i c h allo\\s to1 thc dctcmion of different rates of genetic evolution.
distances, of niorphologicnl
ciation het\\ccn the tiintrit cs of genetic (DN)
, aiid of the ininiinuni gcogrnpliic tliitniiccs (Am) among populntions.
ivcre nsseswd h hlantel's tests.
'?die IZIA4NO\.':\s s h v e d significant diffcrciiccs in geographic variation for all data
sets, a s n ~ l as
l a \x-iable dcgrec of sexual dimorphism by data set (Table 1). Sexual
dimorphism ~ v a more
s
pronounced in skull \.ariatiles and less iioticcahlc in wings. 'The
first three principal components ol'tlic PCXs ti)- data set (not shown) e x p l a i d between
7 1.1 "/I (dental variables for females) arid 84.2"%(wing variables for males) of the
\.ariation of the data. ;lccording to the cumulative \ d u e s ofRao's 1:, the skull data set
slioivcd tlic largcst differences aiiiorig groups. The \ d u e s of Rao's I' were larger in
fvmalcs for all data sets ('Tahle 2). which indicates that hetwcrn-group differences were
consistently largcr among females than among males.
'Ibe first three components of thr PCAs on the 12 selected variahlcs explaiiicd
74.5% aiid 75.6"/;1
of the total \.ariation for males aiid fcnialcs respectively. For both
sexes, the plots of the scores mi the first tlvo axes placed the Xnriobtjn population
clearly apart from the rest (Fig. 2). 'The remaining populations overlapped to various
dcgrccs. Ne\w-thcless, diffcrciiccs in crntroid position indicate a certain degree of
tliffcrrntiation aniong them (Fig. 2). For both scxes?the largest Mahalanohis' distances
J. J U S l L ETAL.
364
TABLE
1. Two-way multivariate analyses of variance (MANOVA) based on 12 wing, 14 skull, and 14
dental measurements of Eidolon helvum, to test by data set for overall effects of population, sex, and
their interactions. (*) Significant after a Bonferroni adjustment of
P to a tablc-wide
Q = 0.05
level
\$‘ilks’ h.
t;
111;
I’
0.2 1 1
0.832
0.619
4.052
1.693
I .078
48/391.1
12/101
48/39 I . 1
<0.001*
0.0792
0.3424
Skull
Population
Sex
Population*Sex
0.122
0.638
0.587
4.950
3.998
1.014
56/387.2
14/99
56/387.2
<0.00 I *
Dentition
Population
Sex
Population*Sex
0.248
0.779
0.525
2.997
2.002
1.247
56/387.2
14/99
w3a7.2
<0.001*
Effect
Wing
Population
Sex
Population*Sex
<0.001*
0.4514
0.0249
0.1205
were between Annobon and all other populations; all these values were highly
significant (Table 3). The shortest distance was between the mainland population
and that of Bioko. The Neighbour-joining method produced identical topologies for
males and females. The mainland and Bioko clustered together, and were followed-in a geographically sound pattern-by the other islands, with the population
of Annobon branching away from the rest at the base of the tree (Fig. 3).
Allogme anabses
Of the 34 loci scored, 19 were monomorphic in all the populations (Aat-1, Aat-2,
Acp, Car, Ck-1, Ck-2, Fum, Cpi Hbb, Idh-2, Ldh-1, Ldh-2, Mdh-1, Mdh-2, Me-2, Mpi,
Np, 6-Pgd, Sod-2). Allele frequencies at the remaining 15 polymorphic loci are listed
in Table 4. All populations showed at least one unique allele except Principe’s, and
the loci Est-1, Pep-A, Pgm-2, and Sod-1 had alleles shared only by island populations.
Diagnostic alleles showed generally low frequencies ( a 0 5”0). Populations differed
mainly in their allele frequencies, the differences were significant (P<O.OO1) for five
alleles (Adh, aGpd-2, Pep-D, Pgm-1 and Sod-I). The slowest allele of Sod-1 increased
its frequency along the line of islands, and almost reached fixation in Annobon
(Table 4).
The percentages of polymorphic loci (4 were very similar among populations
and ranged from 23.53% (Principe) to 29.41 ‘/o (mainland and Bioko). Mean observed
heterozygosity (Ho)ranged from 0.045 in the mainland population to 0.066 in the
population of Principe (Table 5). There was no significant correlation between P
and H,. The mean Wright’s fixation index (FST) among populations was 0.153, and
the corresponding Nm was 1.38, suggesting relatively high overall gene flow among
populations. In painvise comparisons, the lowest FST values were between Bioko
and Rio Muni (FST=0.029),and between Sao Tomt: and Principe (e5T=0.033).
The theoretical migrants per generation varied from 8.37 between Bioko and Rio
Muni to 1.06 between Rio Muni and Annobon (Table 6). The populations of Bioko
and Rio Muni showed the lowest genetic divergence, and Annobon showed the
largest values with respect to the rest of the populations (Table 7). The Wagner’s
~
~~
IIhlC
1.33
1.67
2.01
2.32
2.48
2.63
2.75
2.8 I
2.87
2.91
2.96
3.0 1
~
IVMC
IIIMC
II1F2
FA
IVF 1
IIFl
VF2
IVF2
v R.1C
IIIF3
VF1
IIMC
\‘
-
-
I .45
1.86
2.43
2.69
2.93
3.17
3.46
3.73
3.92
4.00
4.07
4.09
Wing
Male,
Frmaks
Var
Rao’s V
Var
Rao’s
FA
IVFP
IVF 1
VMC
VF 1
IIF3
VFP
IIFP
IIFl
IVMC
IIIMC
~~
ZB
C1-CI
AIL
RI,
GSL
PL
hlH
C’-lLI1
CBL
BCB
hf ‘-M‘
IOB
hZA
C,-hl,
Var
1.92
2.45
2.96
3.40
3.80
4.15
4.59
4.98
5.28
5.59
5.85
6.03
6.1 I
6.19
Rao’s V
hlales
Skull
MH
ZB
C1-C)
BCB
C,-hr,
ML
hIA
CBI.
PI’
C’-M’
IOB
GSL
hl - M
RI.
2.57
3.49
4.04
4.53
5.11
5.65
6.17
6.49
6.96
7.62
7.85
8.08
8.23
8.49
Female
Var
Rae'\ V
See Appendix 1 for acronyms of the variables
Var
1.26
1.33
1.41
1.47
1.54
1.60
I .66
1.69
1.1.5
0.49
0.7 1
0.85
0.99
1.07
\.’ar
Dentition
Rao’s \’
hldlC‘.\
5.63
5.44
.i..i6
0.90
1.69
2.18
3.06
3.52
4.16
4.49
4.76
4.96
5.17
5.31
Females
Rdo’? \’
TABLE
2. Relative contribution of each variable p a r ) in step-wise discriminant analyses among populations, by sex, for wing,
skull, and dental variables. Relative importance measured as the accumulative value of the Lawley-Hotelling’s trace (= Rao’s V).
3
z
n
5
c
T
”
h
I
L
L
1
9.6
I
I
9.8
PC 1
10
10.2
-0.4
-0.5
6 -0.6
a
-0.7
-0.8
phcnogrmi bascd on these distances showed a cluster including the mainlancl aiid
Rioko populations; and another group comprising Principe and Sao 'Tomi,. ' I ~ l i c
.4nnoh6n population stood by itself at the base of the tree (Fig. 3). Alantcl's I W S
shot\-cdsignificant correlation bet\vcen the matrices of Rogers' genetic distance\ and
morphological distances (males: I - =0.84, P= 0.015; fcmalcs: r= 0.85. ='Z 0.0 1 i ) :
;ind hct\vem matrices of genetic and geographic distances (l-= 0.7.5. P= 0.0 IO'i.
( :orrelations wcre highly significant betlvcen geographic and morphological matricc.s
inalcs: ? - =
0.82. P= 0.005; females: r= 0.79, P= 0.006).
\‘:\RIATION OF EIDOLOK HEI,VU\f IN 1 H E GULF OF GUINEA
367
TABLE
3. Mahalanobis’ 0’ distances (upper semi-matrix) between populations of the fruit bat Eidolon
helourn from thc Gulf of Guinea, and their associated probabilities (lower semi-matrix). Abbreviations:
RhI, Rio Muni (mainland); B, Bioko; P, Principe; ST, Stio Tomt.; A, Annobbn. (*) Significant after
Bonferroni adjustmcut of thc significance level to a tahle-wide CI = 0.05
RRI
B
P
S‘I
h
-
2.361
8.578
8.931
10.601
42.070
47.553
25.432
24.985
hlah
RRI
R
P
ST
4
0.319
0.013
0.00 1 *
4.00I*
-
11.373
6.953
0.022
<0.00 I *
4.00I*
-
<0.001*
Ecmalcs
KhI
u
I’
S’I‘
‘1
2.098
0.793
0.025
0.001*
<0.00 I *
9.268
12.306
4.396.5
7.725
8.528
~
0.0 18
0.002*
4.00I *
~
0.21
<0.001*
<0.00 1*
66.270
78.715
55.816
39.734
~
Males
r
Bioko
L
Rio Muni
- Principe
-
SBoTome
Annobon
Females
r Bioko
Annobon
Fi<pre3. Neighbour-joining topologies based on selccted morphological characters, b>-sex, built from
Mahalanobis 0’ distances betwcen populations of Eidolon helcum from the Gulf of Guinea.
,J. JUSTE E T d L
368
TABLE
4. Allele frequencies at 15 polymorphic loci for the populations studied of the fruit bat Ezdolon
helvum in the Gulf of Guinea. Abbreviations as per Table 3
Populations
Locus
Aeon
Allele
RM
B
P
s’r
A
a
0.9
0.1
0.95
0.05
I
0.75
0.25
0.7
0.3
I
0.84
0.16
0.654
0.346
0.889
0.111
0.694
0.306
1
0.833
0.167
0.944
0.056
0.944
0.056
1
1)
Adh
a
b
Ak
a
Est-1
b
a
b
~
0.9
~~
ctGpd-2
a
b
a
b
Idh-l
a
h
C
Lap
Mc- 1
~
~
0.95
0.05
1
a
I>
d
h
~~
0.85
~
0.1
0.95
0.05
0.6
0.4
0.95
0.05
L
aGpd- 1
0.15
1
~
0.6
0.4
0.95
a
1
1
-
-
I’ep-B
b
1,a
0.95
0.05
0.833
0.167
0.95
0.05
t)
P p -1
a
Pgm-2
b
a
h
Sod- I
1
~~
1
a
1)
1
__
I
~~
0.265
0.735
1
0.96
0.04
1
~
0.46
0.54
0.741
0.259
0.98 1
0.019
0.685
0.315
1
~
1
~
0.938
0.063
0.833
0.167
~
0.94 I
0.059
0.972
0.028
0.933
0.067
1
~
0.95
0.05
0.95
0.05
0.95
0.05
0.95
0.05
1
~
~
~
1
0.833
0.028
0.139
~
-
~
a
0.846
0.096
0.058
0.896
0.104
0.28
0.72
0.05
0.95
0.05
1
Pcp-A
I’ep-D
1
~
0.944
0.056
1
~
0.938
0.063
1
~
0.806
0.194
0.889
0.1 I I
0.969
0.0s 1
0.528
0.472
0.667
0.333
1
1
0 167
0.833
TABLE
5. Genetic variability measurcs for Eidolon helvum populations studied in the Gulf of Guinea.
Abbreviations as per Table 3
Population
Mean sample size
per locus
Mean no. alleles
per locus
% polymorphic
loci (4
(4
A
9.7
9.8
25.5
17.4
8.6
1.29
1.29
1.32
1.35
1.26
29.41
29.41
23.53
26.47
26.47
0.045
0.056
0.066
0.055
0.053
Mean
14.2
1.3
27.06
0.055
Kk1
B
P
S’I
Average heterozygosity
DISCUSSION
Moqhological variation
Andersen (1912) and Rosevear (1965) considered sexual dimorphism as irrelevant
for E. helvum. However, in all the populations studied, females were significantly
\‘hKIATION O F EfDOLQ\.HELI’I:\fIN ‘THE: (;CLF O F GL’INE.\
:iti9
6. IVright’s (1 965) Fsl values (upper semi-matrix) of Eidolon Adrwn from the Gulf of Guinea arid
associated .bin ( h v e r semi-matrix) otitainrd using \\rigtit’s ( 1965) relation liet\vern popularions, and
Slatkin’s (1985) privatc allrles mrthod (tic.tivecnparentheses). At)breviations as per ‘Tal,le 3
‘rmLE
Rhl
B
f
ST
1’
sl‘
0.095
0.088
0.08‘3
0.067
0. I87
0.179
0.033
0.141
B
Khl
~
8.371
(7.nl)
2.381
2.590
(1.30)
(2.21)
2.558
( I .+4)
I .08fj
3.481
(.723)
1.146
(2.05)
(1.30;
(-1.18)
j
7.326
1.523
1.27)
0.115
1.921
(3.3I )
TABLE
7. Pairwise values of Sei’s (1978) genetic distances (upper semi-matrix) and modified (il‘right,
1978) Rogers’ (I 972) distances (lower semi-matrix) between insular populations studied of the Akican
fruit bat Eidolon heluum. Abbreviations as pcr Table 3
B
P
ss
A
0.000
0.0 13
0.0 I 3
0.01 I
o.oo8
0.022
0.02-10.023
0.0 16
RLI
RRI
B
P
ST
A
~
0.058
0. I 2 1
0 .I I I
0.1%
~
I).12 I
0.100
0.159
0.004
0.075
0.153
0 . I3 1
~
Principe
Rio Muni
-
Figure 4. b\’aper phcnogram based on niodified (JVriglit, 1978) and Rogers’ (1972) genetic distanccs
hetwccn populations of Eidolon hulzwn in the Gulf of Guinca. Cophcnetic corrclation = 0.989.
larger than males, particularly the skull. Bergmans (1990) has shown this trend for
other populations of the species. The population from Bioko is on average slightly
larger in body size than that of the mainland, as Eisentraut (1964) had noticed, hut
these differences are not statistically significant. The populations from S%o Tomi.
and Principe are slightly smallcr than that of the mainland, and the individuals from
S%o Tomi: are a little larger than those from Principe, although the differenccs are
not statistically significant. It seems that there is a trend towards a reduction in size
associated with a reduction of island area, hut the small number of islands precludes
370
J. JUSTE E 7 , l L .
any statistical testing. In agreement with this trend, E. helvum from Annobon shows
particularly reduced measurements for all data sets (Appendix 2). The measurements
for this population are the smallest ever reported for E. helvum, including the
subspecies E. helvum sabaeum from the other extreme of the range, in the Arabian
Peninsula.
Sexes showed slight differences in their patterns of differentiation. Females are
more distinct among populations in wing, skull and dental measurements, as is
evident from their larger accumulative values of the Lawley-Hotelling trace. In the
PCAs, the separation of Annobon along the PC 1 for females points to size as the
main source of variation. Instead, the large percentage of explanation accounted
for by PC2 (25.1YO) for males, indicates relatively important differences among
populations not only in size but also in shape (Fig. 2).
Genetic variation
The average polymorphism (P= 0.27 1) among the populations of E. helvum studied
is similar to the value reported by Peterson & Heaney (1993) for the Asian fruit bat
Haplonycterisjscheri (P= 0.228).
E. helvum’s polymorphism value is intermediate between
the values obtained for the Asian Cynopterus brachyotis (P= 0.38 1; Peterson & Heaney,
1993) and for Rousettus egyptiacus (P=O.177) reported from the Guinea group of
islands (Juste, Machordom & Ibafiez, 1996). Average heterozygosity (H= 0.055) for
the populations studied was identical to the values found for Aethalops alecto (Kitchener
et al., 1993) and Cynoptems titthaecheilus (Schmitt, Kitchener & How, 1995). Eidolon’s
heterozygosity is higher than the values reported for other fruit bats, e.g. C. sphinx
(H: 0.028; Schmitt et al., 1995); H.jscheri (H=0.034; Peterson & Heaney, 1993), or
R. egyptiacus (H=0.038;Juste et al., 1996).
Although the island populations of E. helvum show slightly lower values of
polymorphism, the average heterozygosity was higher in these populations. It seems,
therefore, that there is no reduction of genetic variability in the islands with respect
to the mainland. The expected pattern of reduction of genetic variability in islands
does not hold for other fruit bats studied in the islands (Juste et al., 1996, 1997) nor
for other fruit bats from Indonesia (Kitchener et al., 1993). However, in a study
involving a larger number of islands, Peterson & Heaney (1993) found a trend (albeit
not significant) towards a reduction of variability in smaller islands, and Schmitt et
al. (1995) found a significant, negative relationship between heterozygosity and ‘level
of isolation’. In populations with limited gene flow, reduction of variability would
result mainly from genetic drift on low effective population sizes (Schmitt et al.,
1995). Effective population sizes of E. heluum are quite high in the Gulf islands (Juste
& Ibafiez, 1994). This fact would have helped maintain levels of genetic variability
relatively high, even in Annobon.
Population-genetic structure
The overall value (&=0.153) of genetic structure is similar to that found for
= 0.17 1). This is
between-island populations of the fruit bat Cynopterus nusatensgara (FST
considered an indication of overall differentiation across populations (Schmitt et al.,
1995). Pairwise comparisons show that this value is due mainly to the differences
brtat een Annobhn and all the other population$. The difference brtjt een .Znnoh6n
and the mninlaiid is (low to 20"o (F\, = 0.187). and o\ er 1 (1"o \I ith reipcc t to the chscst
population, thnt of Sjio Tonic. 'This pittern also 'ippcars in thc genetic distances, I\ hich
heti\een Annobon and all the other populations except Sjio ToniC.
are o\er 2"0 (D,)
The low values of genetic distance and &, het\\ecn the oceanic islands of Sgo Torn6
and Principe suggest little rcstric tiou to gene flon betwccn them.
The rate of theoretical migrants per generation \ aries considerably among islands.
Gene flow is highest between the 'land-bridgc' island of Bioko and the mainland,
\+ith a value (h
= 7.5 1) similar to the average (,Im= 7.53) reported for non-isolatedby-distance populations of Cjnolterui (Prterson & Heaney, 1993)..'hvalues between
the mainland and the oceanic islands decrease lcith distance, which suggests that
this factor is acting as a barrier to gene flow from the mainland. Particularl) for
= 1) for inAnnobon, the value of "lm approaches the niinimuin required (~im
dependent divergence by random drift under the neutral island niodel (IYright.
1965). Gene flow is high ( ~ l=h7.33) bet\\ecn Sao Tom6 and Principe. Nonetheless,
the value of JVm obtained by the pri\ ate alleles method is considerably smaller (,Ih=
2.05). Estimates of ,Vm obtained from F-statistics and pri\ ate allele methods also
show some differences for other pairwise comparisons (Table 6). These differences
could indicate the role of selection in determining diversity among islands (Schmitt
et al., 1995),and suggest some caution in the interpretation of the ialucs. Interestingl),
the most important differences for the t\<o estimates of .Zm valucs occur in betweenisland comparison$.
The highly significant correlations hrtween morphological and geographic distances for both sexes, as well as the phenograms, indicate that morphological
variation of E. helvum populations in the Gulf islands clearly fbllows a geographic
pattern. Mantel's test shows concordance of morphological and genetic patterns.
The fact that some alleles for Pgm-1 and PeF-11 arc shared only among island
populations, and the increasing frequency of the slowest allele of Sod-1 along the
oceanic islands, give support to a geographic pattern of differentiation. Genetic arid
geographic matrices show a weak correlation, which probably reflects the effect of
the short distance between Sao Tomi: and Principe on the genetic matrix. The
estimate of gene flow and the presence of a diagnostic allele at Es-1 in these two
populations sustain the inter-island genetic resemblance.
Colonization of the Gulf islands, according to a stepping-stone model, is the most
parsimonious explanation for this geographic pattern. Independent colonization
events from the mainland are expected to have occurred due to the relatively long
distances between islands and their small size. Howexrer, numerous examples,
including plants (P'igueiredo, 1994), mollusks (Gascoigne, 1994), birds (Peet &
Atkinson, 1994) and shrews (Heim de Balzac & Hutterer, 1982), indicate close
evolutionary relationships between the Gulf islands. The association is particularly
tight between Sao Tomi. and Principe, which share diverse cndemisms wen of land
snails or amphibians. Like E. helvum, the endemic populations of the fruit bat R.
egptiacus from Sao Tomi: and Principe show close affinities (Juste et al., 1996).
However, the two populations of R. e,ptiacus-a less \,agile species--are more
differentiated morphologically than those of E. helvum.
Ejf2c.t.s qf rnigration and di,qersul
F,’./10/7~rinis commoiily acknowledged as a migratory species. 4 seasonal rnovcmcnt
rakes p l a c ~at thr beginning of the rainy season following births which is accomplished
I)!. I;ii-ge groups of’ males and lactating females (Thomas, 1983). \.Ve found adult
iudi\~idualsof E. helmm in both wet and dry seasons in all our visits t o the islands.
111 addition. lactating females Lvere also found in all the islands. These obser\.ations.
togc.tlic%r\vith information gathered from the locals, strongly suggest that none of
I l i t . itisul;tr populations of E. helzwn undertakes seasonal migrations to the mainland.
; i s I’,ixc11triiut ( 1964) and Bergmans ( 1 990) had suggested for the population of Bioko.
Zt%\.ertliclcss.\vv ltave detected important differences in colony size between dry
a i i ( 1 \ w t scasons in the islands (e.g. ‘Cacahual’ colony in Bioko drops from tens of
thousands of indi\.iduals to a febv hundred during the rainy season). This indicates
intr:r-island tno~xmients,which are probably associated with fruit availability, the
triggcr factor suggested for the migration in the mainland (Thomas, 1983). In the
abscncc of migration, the quite high levels of gene flow found among the islands
\I oultl result from dispersing indi\.iduals. I?. hdzwn’s wing morphology shoivs the
largest aspect ratio among ilfricari fruit bats (.Jute & Ibaiiez, 1994), Lvliich al1oLl-s
fijr ils high dispersal capacity. Vagrant specimens of E. hehum have been reported
‘15 h r as 100 kin off Africa (Varona, 1975). In the Gulf of Guinea, tlie dominant
wtitli\vesterly mo\.ement of wind (Wauthy, 1983) would favour inter-island contact.
Sc\xwlieless, the role of dispersal in structuring the populations (Thomas, 1983), or
in dctcrmining the distribution patterns of this species (Bergmans, 1990), is still
LIW and remains to be investigated.
Ezlolutionap and taxonomic inferences
‘l’hc \.ariation found in the populations of Bioko, Principe and Siio Tom6 can be
rx)iisitlcrcd \vithin the range of the nominate subspecies, E. hehum helzum. The le\.cls
(,I’ gcnc flo\z I,ct\vcen the mainland and the islands---and among the latter---~swm
1 0 prevent tlif-feretitiation. Unlike those populations, E. helrium shows remarkable
morphological and genetic differentiation in Annobon. Founder effect is considcred
th(, major e\-olutionary force for determining gene pool differentiation in insular
populations (Kilpatrick, 1981), and that may be the case for Annobhn. In addition,
i l i c ohscrvcd imorphological trend towards a reduction of size on the thrce oceanic
i.;laiitls suggcsts the existence of a selective pressure in this direction. The role of
itaturnl selection is also suggested by the diffcrcnccs in estimates of 3 j n l y tlic. F\tatistics and pri\.stc allele methods, and the gradient of frequencies for Sod. AA
rccluctiou ill size in island populations seems to he a general pattern for hats
.Krzatio\z.ski. 1967), and it has been explained as a result of change in selective
~ i r e 011
s tropliic conditions (Palmeirim, 1991) or flight environments (IliopoulouWoi-gudaki, 1986).111the Gulf islands, tlie combination of selective forces in harsher
oceanic cn\~ironmcntsand restricted gene flow would have favoured morphological
difkrcntiation of E. helzluni in Annobon, which undergoes a pronounced long dry
w i s o t i (H(~rnlindez
Pacheco, 1943) arid seasonal fruit shortage. In these conditions,
;ui earlier achie\.ernrnt of sexual maturity- ---andconsequently smaller size-may
be
;id\.;uitagcous in the absence of migratory behaviour. The more marked differt m t i a t ioii among females also sugests that selection could be linked to the reproducti\,r
pattern. Similar erir ironmental pressures ma) be acting on the small subspccicb E.
helvum Jabaeum, which cxpcricnces comparable isolation and harsh conditions in the
Arabian Peninsula.
hvarez (1961) noticed the particularly small size of the population of E. helzurn
from Annobon, and suggested its recognition at thc subespecific 1eLel. Thc cxtcnt
of D, genetic distances for this population (0.016-0.024) is within the range of
distances described for conspecific subspecies in other fruit bats like R. eg,ptzatus
(D,=0.019; Juste et al., 1997), or .-lethalop, alecto (D,=0.011; Kitchener et al.,
1993), or the vespertilionid L@otzs lucfugus (D,
=0.011; Herd, 1987). Therefore, the
population of Annobon is described hereafter as Ezdolon heloum annobonenszs subsp.
no\'.
Eidolon heluum annobonensis subsp. nov.
Holoppe. Female adult (EBD 17603) (skin and skull), collected January 12, 1987 in
San Antonio de Pal6 (1"24'S, 5"38'E), Annobon island, by Javier Juste.
Geographical distribution. The subspecies is endemic to the island of Annobon (\Yest
Central Africa).
Diagnosis. Small size combined with weak dentition.
Description. Eidolon helvum annobonensis is a large fruit bat with a large fox-like head.
Externally, it resembles the nominate subspecies, and shows the typical colour
pattern of E. helvum in both sexes: light brown on the back turning to greenish grey
on the rump. The flanks arc yellowish. This colour extends along the dorsal side of
arms and forearms, contrasting against the darkricss of thc rving membrane. Vcntrally,
the fur is yellowish. The subspecies shows marked individual variability and some
specimens have a darker appearance. The holotype presents 4 + 3 + 3 palatal ridges.
The skull shows the 'typical' Eidolon morphology, although it is smaller than in
the nominate species. It shows a more delicate general appearance with narroLver
postorbital processes, slender rostrum, and a more marked interorbital constriction
than E. h. helvum. The jaw exhibits quite individual variation in the shape of the
coronoid and angular processes, but it is always less massive than in the nominate
subspecies. The teeth are similar in shape than those of E. h. helium but notably
smaller, particularly thc premolars and molars.
Measurements. Average values for the subspecies are given in Appendix 2 by sex.
Measurements (mm) of the holotype (EBD 17603) are:
Body measurements: FA: 109.6; Ear: 27.5; Total length: 172.0; Tail length:, 19.2;
IIMC 53.7; IIFl: 15.3; IIF2: 7.9; IIIWC 75.6; IIIFl: 48.6; IIIFZ: 70.0; IKVC 73.5;
IVFl: 40.2; IVF2: 43.9; VIZIC: 67.5; VFl: 31.3; VF2: 28.8.
Cranial measurements: GSL: 50.2; CBL 48.2; RL:19.9; PL: 24.7; ZR: 27.1; IOR:
8.1; POB 11.5; BCB: 19.3; C'-M': 17.3; C'-M2: 19.4; C'-C': 9.2; Af-M': 14.6; M L
39.5; MH. 15.1; Cl-LW2: 20.4; Cl-A43: 22.1; MA: 134".
Dental measurements (length and breadth respectively): P': 3.1-2.3; P':3.8- 2.4;
iV':3.8-2.4; 1W': 1.7-1.6; P3: 2.9-2.0; P4: 3.6-2.1; 12.1,: 4.3-1.8; AI2: 2.6-1.7; 121;:
1.3-1.0.
Comparisons. E. h. annobonensis is separable from the subspecies E. h. helvum by its
smaller body and wing size, smaller skull, and more delicate dentition; from E. h.
d m e u m 11) its liqhter fur colour and slender dentition and from E. duprennum b\i its
miallrr 47e and less elongated muzzle.
midi
\l'c ,ire yrntcful to Leandro Mbomio, former Culture hlinister of the Republic
Equatorial Guinea and to Jose Luis Xavier Mendes former hlinister of Agriculture
of ihc. Democratic Republic of Sao Tome and Principe. We thank the following:
K. E. Strauss for ,tatistical advice and for making available the hlatlah functions:
C' Lopc7-Gonzalez for her suggestions; A. Ayong Nguema and C. Ruiz for their
,t\slst,tncc n i t h the firld work. The work was supported by the Instituto dc Coop('1 ,icion para cl Desarrollo (ICD) of the Spanish Ministerio de Asuntos Exteriores.
\ \ ith the cooperation of the Estaci6n Biol6gica de Dofiana (CSIC), and Xsociac-i6n
dc . h i g o < dc Dofiana. Travel expenses were partially financed by the Xgencia
Ly)afiola dr Cooperaci6n Internacional, the Junta de Andalucia (RNhl-158), and
I)\ 1)GICJ'T (PBSO-014.3).
of
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zooldgica de la Academia de Cienrzas de Cuba 1: 3-4.
Wauthy B. 1983. Introduction a la climatologie du Golfe de Guint-e. Oclanographie Tropicale 18:
103- 138.
Wright S. 1965. The interpretation of population structure by F-statistics with special regard to
systems of mating. Evolution 19: 395-420.
'i
7 (i
J. JLXE
icr.4~
Wright S. 1978. Euoiution and the genetm oJpopu1ation.r: vol. 4. Ihriabilip Zuffhinand among natu~a(po~ulcItiotr,.
Chicago: Cniversity of Chicago Press.
iZPPENDIX 1
A l e a urements
Cling r,ariablr.J. Forearm (FA);second digit metacarpal (ILifG);first phalanx (ffF1);sccoiid phalanx (IlF28:
third digit metacarpal (IfZMC); first phalanx (ZffFl);second phalanx (fffF2);fourth digit metacarpal
(Il:\fC); first phalanx (IVFI);second phalanx (IVFZ);fifth digit metacarpal (?<\lC); first phalanx ' [ F / ,
srcond phalanx (LFZ).
C.'rnnzal Lwiabb. Greatest skull length (GSf,); condylo-basal length (C'BL);rostrum length IRLj; palatal
lcngth (PI.): zygomatic breadth (ZBj; interorbital breadth (104; braincase breadth (BC3);toothro\\
Icngth (C'-hf'); breadth across upper canines (C'-C'); breadth across M-hl' (hf'-Xf'); mandihlc lcngth
(.ZlL):mandible height ( M q ;mandibular toothrow length (Cl-M2); mandibular anglc ( 3 t l ) .
llental 11ariahle.s. Length of P3 (LP);length of P' (LP');length of M' (UP);
lcngth of M' (Lif?;breadtli
of P' (BPI;hreadth of P' ( B q ;breadth of M' (BM');breadth of M' (M3;length of P' (Let;length 01'
P'(LP');length of M' (U');
length of M' (Lbfq;length of M" (Lbf');breadth of' P' (BP');hrracltli 01'
P'(BP');hrcadth of M i (BM');breadth of A l ' ( B A f ) ;breadth of h4'' (BM').
P
c
dd
FA
dd
CBL
PL
ML
C'-M'
ZB
86
00
PO
dd
0 '?
6$
0P
68
99
09
dd
00
RL
68
GSL
?P
Sex
Var
117.0
118.5
52.9
53.8
51.3
49.9
19.1
19.3
25.5
26.0
30.8
31.0
19.1
19.8
40.5
42.1
129.0
129.0
59.4
58.1
57.0
55.8
23.9
22.8
29.4
29.0
35.3
33.8
23.6
22.3
47.5
46.8
121.7
124.5
56.3
56.1
54.2
53.6
21.6
21.6
27.4
27.5
33.7
32.2
21.0
21.2
44.9
44.9
Rio Muni
d (13- 18), ? (1 1-1 3)
Min
Max
Mean
117.0
116.2
52.2
55.6
50.1
53.4
18.8
21.1
26.0
26.0
32.6
31.6
19.7
19.9
42.7
44.2
132.2
134.0
58.7
59.1
56.8
57.1
22.5
23.2
29.6
29.5
35.4
34.8
23.1
22.0
47.3
47.6
122.9
126.1
56.6
56.9
54.5
55.1
21.6
22.1
27.6
27.8
33.6
33.0
21.3
21.2
45.2
45.6
Bioko
d(13-15), ? ( I I )
hlin
Max
Mean
114.9
118.0
51.8
51.8
50.1
50.2
19.5
19.5
24.9
25.6
31.1
31.0
18.7
20.0
42.0
42.8
125.5
127.0
56.6
56.2
55.1
54.4
22.3
21.3
27.9
27.7
34.8
32.3
21.9
21.5
45.5
44.1
120.1
122.0
54.3
54.3
52.4
56.5
20.7
20.7
26.6
26.6
32.3
31.6
20.4
20.7
43.5
43.6
Principe
d (20-2 I), P (6-8)
Min
Max
Mean
116.4
114.3
51.3
51.9
49.9
50.3
19.8
19.6
25.5
25.0
29.8
28.9
20.1
19.1
41.4
40.7
124.0
125.0
57.9
57.0
56.4
54.9
22.8
21.3
28.5
27.6
33.2
32.9
22.2
21.5
46.5
45.5
121.0
120.9
54.5
54.0
52.8
51.8
21.0
20.3
27.1
26.2
32.0
31.4
20.9
20.4
43.8
43.0
SPo Tome
d(6-9), P(8-10)
hlin
Max
Mean
109.0
107.6
49.9
49.5
47.4
47.1
19.0
19.0
24.4
24.6
28.7
27.1
19.0
18.7
39.8
39.0
115.0
119.0
57.0
51.8
54.9
49.9
22.8
20.0
27.5
25.8
31.7
29.8
22.0
19.9
45.5
41.3
111.7
113.9
51.7
50.6
49.8
48.7
20.1
19.6
25.6
25.1
30.0
29.0
20.1
19.3
41.3
40.2
Annobh
d(6-91, O(8)
Min
Max
Mean
-
-
32.4
31.7
20.2
19.5
43
--
30.3
29.6
19.5
18.6
41.3
40.5
28.5
31.4
30.7
19.8
19.1
42
19.6
-
-
--
124.7
124.3
53.6
53.3
52.2
51.3
109.0
113.5
52.5
50.4
49.9
48.0
118.0
116.6
53.3
52.1
51.1
49.6
Saudi Arabia
d(3-5), P(3-4)
Min
Max hlean
2
8
2
4
E0
2
f
2
2
%
0
z
3
-
+
APPENDIX 2
P
Selected measurements in 6 d ( k r ) of adult Eidolon helvum from the Gulf of Guinea by sex and population. The last three columns correspond to the subspecies F
E. h. subaeum according to Bergmans (1990). Sample sizes are given in parentheses.
reins examined and staining procedurrs uscd in the study. Sam(,\ l?)llo\\ t h ( . KOnmitter of thc International nioii of Biochcmistry (1984). Each protciii is l i i l l o \ \ ( ~ 1
I)! its ;il,lti-c\,intion.its Erizymc Commission unibcr. rhc tissue cxamincci (H = l i e x r ; 1, = I k t c~ ~ ~ ~ d
thc buffer utilized. Buffers [TC= tris/citratc; hlE = tris/malratc/E1)'I'I-\; TBk; = w i d h i r a w / ED'I'.\
hllo~sPastcur P/ nl. (1987): except for 'TC 8 , 'ThlE 6.9 and I X H 8.3 Ishich arc hall' diliitctl h r y l \ ,
.\sparratc aminotransferasc (&-1, A,lo/-2)2.6.1. I ; H/I,: T h i E 6.9. Aconitatc h)dr.ar,isc ,.hoic 4 . 2 . 1 . ~ :
H: 'TC: 6.7. :kid phosphatase (Acp) 3.1.3.2: I,; TLlE 6.9/TC 6.4. Adenylatc kinas(, ,.I/;\ 2.7. 1.13: H:
'I.(:6.4. .2lcolioI dehydrogenase (A&) I . 1. I . I ; L; TC 6.4. Carbonic anhy3rasc ((,'wj 4.2.1.1: I.: -1'KE 2j.O.
C:rratine kiiiasc. (01-1, G-2)2.7.3.2; H; TC 6.4. Esterasr (E>t-1)3.1.1.1: 1,; LiOH 8.:1. F,stcra\i, I<\::'! 3 . I . I . 1: I,: I,iOH 8.3. Fumarate hydi-atasc (Fum) 4.2.1.2; H; TLIK 6.9. C ; I y c . c r o l - : i - ~ ~ t i o ~ ~ ~ l i ~ i ~ ~ ~
drhydrogetiasc (r-Cpd-I ) I . 1.1.8; H; '
I
T
8/'l'BE 8.6. G1~ccrol-3-pliosphatcdehydrogenasr I ~-G/nl-:.'~
1 . I . 1.8; L: THE 8.6. Glucose-6-phosphate isomcrase (Gpi) 5.3.1.9; H; T l I E 6.9. Hacmoglol)iiir ; t / M :
T H I : 8.6. Isocitrate dchydrogenase ( I d - 1 , Idh-2) 1. I . 1.42; H/L; 'K 6.7. Ixucinamidt, pcptida.
3 4 . 1 I . 1; I,; 'I'BE 8.6. Lactate dehydrogrnase (Lcih-1,U/i-2)1.1.1.27; H; TC: 6.7. hlalatc tlrli!-dl.cr~ci~;i\c.
;.lfd/i-l, .\fdh-2)1.1.1.37; HA,; TC 6.7. hlalic enzyme (L\le-l, Ah-2) 1.1.1.40; H: Tf: 6.7. l h i t i o s c ~ - ( ; phosphate isornrrase (,\lpz) 5.3.1.8; H/L; 'I'RE 8.6. Purine nucleoside phosphor)
'I'RE 8.6 arid I,; IdOH 8.3. Leucyl-tyrosinc pcpridase (Pep-A) 3.4.1 1; H; TC 8.
pcptidase jPtj~-B)3.1.1 I;H; TC 8. Phrnyl-proline pcptidase (PtpD) 3.4.1 1; H; TC: 8. ~i-l'liospho~lrici~iii~
;icicl dehydrogenasc (6Pgu) 1.1.1.44; H; 1'C 6.1.Phosphoglucomutase ( & ~ - lP; p - ? ; 2 . i . i . I : H/I,:
6.4/T3K 6.9. Sorbitol dchydrogcnase (Sdh) 1.1.1.14; L; TC 6.4. Supcroxidr dismutii
l . l . 5 . l . l : I,; TC6.4.
I):