Annals of West University of Timişoara, ser. Biology, 2015, vol XVIII (2), pp. 95-106
MOLECULAR PHYLOGENY OF THREE SPECIES OF LAND
SNAILS (STYLOMMATOPHORA AND ACHATINIDAE),
ARCHACHATINA MARGINATA (SWAINSON, 1821), ACHATINA
ACHATINA (LINNAEUS, 1758), AND ACHATINA FULICA
(BOWDICH, 1822) IN SOME SOUTHERN STATES AND NORTH
CENTRAL STATES IN NIGERIA
Michael Olufemi AWODIRAN1*, Matthee Conrad ARTHUR2, Awopetu Joel IDOWU1
1
Department of Zoology, Obafemi Awolowo University, Ile-Ife, Nigeria
2
Stellenbosch University, South Africa
*Corresponding author e-mail: [email protected]
Received 11 October 2015; accepted 4 November 2015
ABSTRACT
Partial sequences of mitochondrial gene cytochrome oxidase sub unit 1 (CO1) and
ribosomal RNA 18S nuclear encoding gene of 43 individuals belonging to two genera
in order Stylommatophora and the family Achatinidae (Archachatina and Achatina)
were obtained to investigate molecular phylogeny in the family. The CO1 was found to
be highly variable while the 18S was found to be highly conserved yielding invariable
sequences. Several primers were also tested both for the mitochondrial and nuclear
genomes but CO1 produced the best results giving single and clear bands. Four main
genetic phylogroups/clades were identified within the Bayesian tree constructed and
all the four clades were supported by bootstrap values of 100% and also were
supported by bootstrap values above 79% in the NJ. Two unidentified species used in
the analysis were found in the basal clade which may mean that they were of ancient
origin. This study provides preliminary and novel insights on the molecular phylogeny
of the snails’ species, though there is still a need to collect samples of other species in
this family to provide a more robust phylogenetic relationship of achatinid snails in
Nigeria.
KEY WORDS: mtDNA,, cytochrome oxidase sub unit 1, phylogeny, taxonomy,
polymorphism.
.
INTRODUCTION
The family Achatinidae is generally believed to be made up of about 13 genera
among which are Archachatina and Achatina which are the most conspicuous (Mead,
2004). These two genera contain land snails that are generally referred to as Giant land
snails. There are three species of these giant snails that are native to Africa (Raut &
Barker, 2002) and are the largest of all snails on earth. These are Achatina fulica
(Bowdich, 1822), Achatina achatina (Linnaeus, 1758) and Archachatina marginata
(Swainson, 1821) (Stylommatophora, Achatinidae).
Traditional classification and taxonomic studies of these snails are based on
morphological and soft tissues data analysis. Generally, as it is true of many
95
AWODIRAN et al: Molecular phylogeny of three species of land snails (Stylommatophora and Achatinidae),
Archachatina marginata (Swainson, 1821), Achatina achatina (Linnaeus, 1758), and Achatina fulica (Bowdich, 1822)
in some southern states and north central states in Nigeria
invertebrates, mollusks include poorly known groups, for which delimitation and
relationships between species are not clear. Taxonomic inferences in mollusks are
often hindered by a lack of morphological diversification between different lineages,
as occurs in different cryptic species or species showing overlapping variability
(Wilding et al., 2000; Liu et al. 2003; Elejalde et al., 2007).
The taxonomy of the Achatinidae is presently in a state of flux (Owen & Reid,
1986), partly because so many species are named from shell characters only (Bequaert,
1950; Owen & Reid, 1986), and partly because few people have collected large
samples that enable the range of variation to be appreciated. This confusion is further
pronounced by the striking polymorphism in body colouration shown by the family
Achatinidae (Owen & Reid, 1986).
Though Wade et al. (2001) used DNA sequences from A. marginata collected
in Nigeria to investigate comprehensive molecular phylogeny of stylommatophoran
snails; there has not been much emphasis on the molecular investigation of the
Nigerian achatinids. While several workers have carried out extensive studies on the
phylogeny of the achatinid snails in other places (Mead, 1950; Tillier, 1989; Tillier et
al., 1996; Bequaert, 1950; 2004, Van Bruggen, 2004), there is a great dearth of
information on the achatinids snails of Nigeria.
The present study therefore investigates the molecular relationships among
three known species and two unknown species of the family Achatinidae across the
southern and North central states of Nigeria (Fig 1). We complete the molecular
phylogeny of these snails towards carrying out the eventual investigation of the
phylogeographical distribution of these species of snails in Nigeria.
MATERIALS AND METHODS
Specimens examined. We examined a total of 53 individuals representing
three known species and two unknown species of the family Achatinidae. A.
marginata was collected from eight localities across the sampled areas (Fig.1) while
A. achatina was collected from two sampling localities due to its restricted
distribution. Specimens of A. fulica and the two unknown species were collected from
National Centre for Genetic Resources and Conservation NACGRAB, Ibadan,
Nigeria.
DNA extraction. DNA was extracted from foot muscle of each specimen
using the Promega Wizard SV Genomic DNA Purification System, following the
manufacturer’s protocol. Polymerase chain reaction (PCR) is used to amplify the
mitochondrial genes cytochrome oxidase subunit 1COI (using primers LCO1490 (5’GGT CAA CAA ATC ATA AAG ATA TTG-3’ and HCO2198(5’-TAA ACT TCA
GGG TGA CCA AAA AAT CA-3’) (Folmer et al., 1994).The PCR conditions
include denaturation at 95°C, 5 min.; 40 cycles in all, annealing at 48°C, 45s and 1min
amplification for 72 C with final extension step of 10 min at 72 C for COI. 18S 7R
96
Annals of West University of Timişoara, ser. Biology, 2015, vol XVIII (2), pp. 95-106
primers were also used for the amplification of partial nuclear DNA sequences while
the PCR conditions include denaturation at 95°C, 5 min.; 40 cycles in all, annealing at
50°C, 45s and 1min amplification for 72 C with final extension step of 10 min at 72
C. Two other nuclear DNA primers; for ATP Synthetase subunit α and ATP
Synthetase β with varying annealing temperatures were tested to see if they could be
used to generate sequences for analysis. Reaction products were run on 1.5% agarose
gels and stained with ethidium bromide for about two hours at 120V to verify positive
amplifications and products were visualized under ultraviolet (UV) light using a UV
transiluminator. The gel bands of DNA were excised and the DNA extracted and
purified using the Promega Wizard SV Gel and PCR Clean- Up System. Amplicons
were sequenced using ABM PRISM Dye Terminator Cycle Sequencing Reaction Kit,
Perkin-Elmer. Sequencing was performed on a capillary ABI 3100 automated
sequencer, housed in the Department of Genetics, University of Stellenbosch. The
sequences were deposited in GenBank with accession numbers KT290283 KT290319.
Figure 1: Sites at which mollusks were collected
97
AWODIRAN et al: Molecular phylogeny of three species of land snails (Stylommatophora and Achatinidae),
Archachatina marginata (Swainson, 1821), Achatina achatina (Linnaeus, 1758), and Achatina fulica (Bowdich, 1822)
in some southern states and north central states in Nigeria
Out-group selection. An outgroup Amphidromus species (family
Camaenidae) was selected from the GenBank with the accession number AYI48562
(NCBI). Other closely related species could have been used but their sequences were
not derived from CO1 primers.
Phylogenetic analysis. Sequences obtained were aligned using the Bio Edit
Sequence Alignment Editor -BioEdit v.5.09. (Hall, 1999) and adjusted manually. The
phylogenetic trees were constructed by both parsimony and Neighbor-Joining method
based on the maximum likelihood distance using PAUP* 4.0b10 (Swofford, 2002).
Sequence distance values were computed by the TrNEf model for the construction of
neighbor Joining (NJ) tree (Tamura & Nei, 1993). Reliability of the nodes was
ascertained by 1000 boot strap replicates. The parsimony analysis was performed with
a bootstrap with 1000 pseudoreplicates which included the following:
(a) heuristic search with 100 addition sequences, tree bisection reconnection (TBR)
with 100 random additions, character optimization criteria, gaps were treated as
missing, branches collapsed (creating polytomies) if maximum branch length is zero.
Multistate taxa interpreted as uncertainty while Starting trees were obtained via
stepwise addition.
(b) Character-status summary also included: Of 616 total characters: all characters
were of type 'unord' .All characters have equal weight while 345 characters were
constant, 63 variable characters were parsimony-uninformative. The numbers of
parsimony informative characters were 208.
(c) 'MulTrees' option in effect, topological constraints not enforced while trees were
unrooted.
Bayesian analysis was performed using the MR BAYES v3.0 package
(Huelsenbeck & Ronquist, 2001). In order to get the best model of nucleotide
substitution, AIC criteria was carried out using MODEL TEST, Version 3.06 (Posada
and Crandall, 1998) with the programs from PAUP* 4.0b 10 (Swofford, 2002).
HKY+G model was used on the data matrix and rate variation across sites was
modeled using gamma distribution, with a proportion of sites being variants.
RESULTS AND DISCUSSIONS
A 615 base pair fragment of the CO1 locus was successfully amplified and
sequenced for 43 specimens while 450 base pair of the 18S 7R locus for the nuclear
gene was also sequenced. Table 1 shows the number of specimens of each species used
for DNA analysis.
Phylogenetic relationships among the snails species under this study were
analyzed by both the maximum-parsimony and neighbour joining methods and the
trees were found to be almost similar (Fig 2). The most appropriate substitution model
with an estimate of invariable sites (I = 0) and a discrete approximation of the gamma
distribution shape parameter (α = 0.4119)
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Annals of West University of Timişoara, ser. Biology, 2015, vol XVIII (2), pp. 95-106
The Bayesian analysis recovered the same tree as the maximum parsimony and
is shown in Fig 3.
The results of various primers tested are shown in Table 2. Cytochrome
oxidase sub unit 1 (CO1) is the only primer tested that produces data that are
analyzable. 18S primers produced good PCR results with single bands but the
sequences were invariable. Other primers did not yield any appreciable results in PCR
products and amplification quality (Table 2).
TABLE 1: Snail populations Achatinidae used for DNA analysis collected in some southern states and north central
states in Nigeria.
SPECIES
LOCALITY
ZONE
NO OF
SPECIMEN
USED
Archachatina marginata
ASEJIRE (AS)
RAIN FOREST
RAIN FOREST
1*
IBADAN(IB)
RAIN FOREST
5
IFE (IF)
RAIN FOREST
7
GBONGAN (GB)
4
OKADA (ED)
NIGER DELTA (LOW LAND RAIN
1*
SABONGIDA ORA (EDO L)
FOREST)
NIGER DELTA (LOW LAND RAIN
5
ILORIN (IL)
FOREST)
1*
LOKOJA
DERIVED SAVANNA
4
GUINEA SAVANNA
Achatina achatina
IBADAN (IB)
RAIN FOREST
3
IFE (IF)
RAIN FOREST
3
Achatina fulica
IBADAN (NACGRAB)
RAIN FOREST
3
UNKNOWN SPECIES I
IBADAN (NACGRAB)
RAIN FOREST
3
UNKNOWN SPECIES II
IBADAN (NACGRAB)
RAIN FOREST
3
•
Other specimens were sequenced but were short er in length and so could not be used.
TABLE 2: List of primers tested and their results of populations Achatinidae used for DNA analysis collected in some southern
states and north central states in Nigeria.
PCR
amplification
sequence
PRIMER
FORWARD PRIMER
REVERSE PRIMER
REFERENCE
result
quality
product
LCO 1490
GGT CAA CAA ATC ATA
TAA ACT TCA GGG
FOLMER, et al.,
+
single band
good
HCO 2198 AAG ATA TTG
TGA CCA AAA AAT
1994
CA
18 S
ATPS Α
ATPS Β
GCGAAAGCATTTGCCAA
GAA
GAGCCMATGCAGACTGG
TATTAAGGCYGT
CGTGAGGGHAAYGATTT
HTACCATGAGATGAT
GCATCACAGACCT
GTTATTGC
CTGTGGTAGTAGTT
GGTCTTCKCNAAGT
T
TACTTGCTTGGNGG
DCCRCGGGCACGG
GC
99
WHITING et al.,
1997
JARMAN et al.,
2002
JARMAN et al.,
2002
+
single band
good
-
no band
none
-
no band
none
AWODIRAN et al: Molecular phylogeny of three species of land snails (Stylommatophora and Achatinidae),
Archachatina marginata (Swainson, 1821), Achatina achatina (Linnaeus, 1758), and Achatina fulica (Bowdich, 1822)
in some southern states and north central states in Nigeria
Bootstrap
57
54
92
53
75
64
99
100
75
61
99
69
74
100
100
53
100
(a)
100
AmAs03
AmEd02
AmIb02
AmIf05
AmIf07
AmIf06
AmGB04
AmIb01
AaIf03
AmGb01
AmIf02
AmIf04
AmIb04
UN02
AmLk01
AmIf03
AmLK05
AaIf01
AmLk02
AmEd101
AmEd104
AmEdi03
AmEdi02
AmIL04
AmEd105
AmGb03
AmGB02
AmLk03
UN03
UNI03
AmIb03
AmIb05
AmIf01
AaIf04
AaIb03
AaIb01
AaIb04
Aaf02
UNI02
Aaf01
Aaf03
UN01
UN101
Amp
Annals of West University of Timişoara, ser. Biology, 2015, vol XVIII (2), pp. 95-106
Bootstrap
57
54
92
53
75
64
99
100
75
61
99
69
74
100
100
(b)
53
100
AmAs03
AmEd02
AmIb02
AmIf05
AmIf07
AmIf06
AmGB04
AmIb01
AaIf03
AmGb01
AmIf02
AmIf04
AmIb04
UN02
AmLk01
AmIf03
AmLK05
AaIf01
AmLk02
AmEd101
AmEd104
AmEdi03
AmEdi02
AmIL04
AmEd105
AmGb03
AmGB02
AmLk03
UN03
UNI03
AmIb03
AmIb05
AmIf01
AaIf04
AaIb03
AaIb01
AaIb04
Aaf02
UNI02
Aaf01
Aaf03
UN01
UN101
Amp
Fig 2: Molecular phylogeny of five species of achatinid snails (a) Neighbour joining tree (b) parsimony tree
101
AWODIRAN et al: Molecular phylogeny of three species of land snails (Stylommatophora and Achatinidae),
Archachatina marginata (Swainson, 1821), Achatina achatina (Linnaeus, 1758), and Achatina fulica (Bowdich, 1822)
in some southern states and north central states in Nigeria
Majority rule
92
90
97
86
83
84
100
88
68
100
100
100
100
100
AmAs03
AmEd02
AmIb02
AmIf05
AmIf07
AmIf06
AmGB04
AmIb01
AaIf03
AmGb01
AmIf02
AmIf04
AmIb04
UN02
AmIL04
AmEd105
AmLk01
AmIf03
AmLk02
AmLK05
AaIf01
AmEd101
AmEd104
AmEdi03
AmEdi02
AmGb03
AmGB02
AmLk03
UN03
UNI03
AmIb03
AmIb05
AmIf01
AaIf04
AaIb03
AaIb01
AaIb04
Aaf01
Aaf02
Aaf03
UN01
UN101
UNI02
Amp
Fig 3: Phylogenetic relationships (Bayesian tree) among five species of achatinid snails (three species are known
while the other two are unknown) from four different vegetation zones in Nigeria.
Four main genetic phylogroups/clades were identified within the Bayesian tree
constructed and all the four clades were supported by bootstrap values of 100% and
also were supported by bootstrap values above 79% in the NJ. Clade 1 contains the
Archachatina sub populations (8 sub-clades a-h). 1a and b contains A.marginata
mostly of Ife while 1c cannot be defined as it is made up of A.marginata from two
102
Annals of West University of Timişoara, ser. Biology, 2015, vol XVIII (2), pp. 95-106
populations (Ife and Ibadan), while the unknown species is also contained in this sub
clade. 1d is made up of A.marginata from Ilorin and Sabongida Ora (Edo 1). 1e sub
clade contains three individuals from Lokoja and one from Ife and with this is also
Achatina achatina from Ife. 1f sub clade is wholly made up of two specimens from
Sabongida Ora and 1g also of two individuals from Sabongida Ora population.
From the foregoing, clade 1 obviously shows homogeneity of the A.marginata
populations of the rainforest zone of the south west Nigeria. Lokoja and Sabongida
Ora (Edo1) populations have potential to separate. Clade 2 has two individuals of two
different unidentified species which were also found in clade 4. Clade 3 has both A.
marginata (from Ife and Ibadan) and Achatina achatina from both Ife and Ibadan.
The two unidentified species which were found in clade 4 (basal) probably means that
they are the most ancient in evolution among the species understudy.
In this study, the phylogenetic relationships of three known species and two
unidentified species of land snails in the family achatinidae was carried out on the
basis of the nucleotide sequence of the mitochondrial gene for cytochrome oxidase sub
unit 1. Mitochondrial genes have been found to be useful as a molecular marker
because it provides easy access to an orthologous set of genes with little or no
recombination and rapid evolution (Ballard & Rand, 2005; Elejalde et al., 2008). The
sequence length of the mitochondrial CO1 gene used in the analysis was fairly long
(615 bp) and compared with what was obtained in some other studies of phylogenetic
relationships of snails (Kojima et al., 2001; used 516 bp; Jorgensen et al, 2008
employed the use of 626 bp; while Kojima et al., 2006 used 489 bp of CO1).
The nuclear sequences obtained from 18S primers could not be used in the
analysis because they were not variable being very highly conserved (Jorgensen et al.,
2008) while other nuclear primers tested did not yield any appreciable results. Though,
Jarman et al., (2002) used ATPS α and β nuclear primers for some molluscan species
these two primers could not work for achatinid snails’ used in this rstudy. This work
has therefore shown CO1 gene as a reliable phylogenetic marker for the land snails
employed in this study. Many previous studies have established cytochrome c oxidase
1 as a reliable molecular marker (Folmer et al., 1994, Zhang & Hewitt, 1997, Hershler
et al., 1999; Liu et al., 2001).
Also, the preliminary analysis of all the five species in the family achatinidae
revealed interspecific differences among nucleotide sequences within the analyzed
region of the mitochondrial DNA; the tree topologies showed the five species to be
distinct enough to be separate into different taxa. The Archachatina clade (Clade 1)
showed that all samples of this species from various localities across the sampled area
are monophyletically derived with very high bootstrap probabilities (100%). There is
probably a very low rate of divergence among the individuals of the different
localities. This may be due to very low rate of evolution among the species studied as a
103
AWODIRAN et al: Molecular phylogeny of three species of land snails (Stylommatophora and Achatinidae),
Archachatina marginata (Swainson, 1821), Achatina achatina (Linnaeus, 1758), and Achatina fulica (Bowdich, 1822)
in some southern states and north central states in Nigeria
result of little or no genetic differentiation and absence of environmental heterogeneity
of the sampled area.
The two unidentified snail species form a sister clade with the Archachatina
clade implies a close relationship with Archachatina marginata. The presence of
Achatina achatina in the clade 3 indicated that it is sufficiently different from
Archachatina to be placed in a different genus. This finding agrees with the traditional
classification based on morphological parameters and soft tissues (Mead, 1950;
Bequaert, 1950; Mead, 2004; Van Bruggen, 2004). Moreover, Wade et al. (2001)
showed Archachatina marginata and Achatina as sister relatives.
Similarly, Achatina fulica and the second unidentified snail species formed a
basal clade implying very ancient origin but distinct from others. However, it was not
possible to compare the findings of this study with those of previous workers because
their studies on the family Achatinidae were based on land snails peculiar to their
environment. Moreover, Fontinallia (2010) opined that despite huge amount of
morphological data available on the Achatinidae, no systematic molecular approach
has yet been attempted to correlate molecular data with the morphological data, in
particular, among various genera and species of the family Achatinidae. The present
study agrees with the finding of previous workers supporting monophyly of the family
Achatinidae (Wade et al, 2001, Wade et al., 2007; Fontinallia, 2010) .The limited
natural range of the Achatinidae to sub Saharan Africa (Raut & Barker, 2002) and the
existence of fossils no earlier than the Pleistocene (0.01-1.8 MYA) (Zilch, 1959-1960;
Solem, 1979) suggesed that the achatinids are a relatively recent group, although Raut
& Barker (2002) believed them to be much older than the fossil record.
Given the preliminary and novel insights on the molecular phylogeny of the
snails’ species studied in this work there is need to collect samples of other species in
this family to provide a more robust phylogenetic relationship of achatinid snails in
Nigeria. Further work also needs to be done using mtDNA barcode to identify the two
unidentified species.
ACKNOWLEDGEMENT
Our research group wants to acknowledge the financial support from African Network
of Scientific and Technological Institutions (ANSTI) for research visit to Stellenbosch
University, South Africa and the supply of three species of snails by the National Centre for
Genetic Resources and Conservation (NACGRAB), Ibadan, Nigeria. We also appreciate the
three anonymous reviewers of this manuscript for their constructive feedback and insightful
suggestions, which greatly improved this article.
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