Phylogenetic Relationships of the Land Snail; Eobania Vermiculata (Mu¨Ller, 1774) from Egypt and Saudi Arabia

The Journal of Basic & Applied Zoology (2012) 65, 144–151

The Egyptian German Society for Zoology The Journal of Basic & Applied Zoology

www.egsz.org www.sciencedirect.com

Phylogenetic relationships of the land snail; Eobania vermiculata (Mu¨ller, 1774) from Egypt and Saudi Arabia. A combined morphological and molecular analysis

Mahmoud M.A. Desouky a,b,*, Salem Busais b,c a Department of Zoology, Faculty of Science, Zagazig University, Egypt b Biology Department, Faculty of Science, Ha’il University, Saudi Arabia c Department of Biology, Faculty of Education, Aden University, Yemen

Received 8 July 2012; accepted 21 July 2012 Available online 25 August 2012

KEYWORDS Abstract Eobania vermiculata is a well-known circum-Mediterranean land snail having a cosmo- Eobania vermiculata; politan distribution that makes it suitable for phylogenetic studies. The present work examines Shell morphometrics; the phylogenetic relationships of two populations of this land snail from Egypt and Saudi Arabia Molecular phylogeny; using mitochondrial markers (partial 16S rDNA and COI gene sequencing) in addition to tradi- 16S rDNA; tional methods of shell’s shape analysis. The study highlights the extraordinary morphological vari- COI ations between the two studied snail populations. This variation seems to be related to the geographic origin but not the colouration of the shell and may have caused the present changes in their mitochondrial genes. The molecular phylogenetic analysis of partial 16S rDNA and COI gene segments conﬁrms the morphological ﬁndings. The two monophyletic populations of Egyptian and Saudi Arabian E. vermiculata were found to represent two distinct groups. The concordance of morphological and molecular results, that produced very clear separation of both populations, leads us to conclude that the two separate groups could be considered two separate subspecies. ª 2012 The Egyptian German Society for Zoology. Production and hosting by Elsevier B.V. All rights reserved.

Introduction et al., 2004). Their total number is expected to be about 150,000 species (Ponder and Lindberg, 1997). Subclass Pulmo- One of the most diverse animals outside the arthropods is nata, from class Gastropoda, contains the most described spe- gastropods with more than 100,000 described species (Steinke cies with approximately 35,000 species (Solem, 1984). Within Pulmonata, the superfamily Helicidae that includes the most * Corresponding author at: Department of Zoology, Faculty of widespread species worldwide except for a few regions in Science, Zagazig University, Egypt. Tel.: +20 966507573909. E-mail address: [email protected] (M.M.A. Desouky). southern South America and some islands in the Pacific (Scott, Peer review under responsibility of The Egyptian German Society for 1997). Zoology. Land snails are an interesting group of invertebrates for studying phylogeography due to their limited dispersal capacity and their particular habitat requirements (Pfenninger et al., 1996). Indeed, this low dispersal capacity tends to preserve pat- Production and hosting by Elsevier terns of genetic variation that arose in the past. These patterns 2090-9896 ª 2012 The Egyptian German Society for Zoology. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jobaz.2012.07.009 Phylogenetic relationships of the land snail; Eobania vermiculata (Mu¨ller, 1774) from Egypt and Saudi Arabia 145 are not blurred by contemporary gene flow, and they render a ments helped to discriminate groups (Chiu et al., 2002; Alonso species particularly amenable to phylogeographic studies et al., 2006). DFA was performed to generate a linear combi- (Cruzan and Templeton, 2000). Land snails are also consid- nation of variables that maximized the probability of correctly ered suitable subjects for studies in evolutionary biology, due assigning observations to their pre-determined groups and to to their highly polymorphic shell colour and variability classify new observations into one of the groups. Morphomet- (Reyes-Tur et al., 2001; Goodacre et al., 2006). Current snails’ ric data were included in principal component analyses (PCA) phylogenetic studies are mainly based on morphological fea- using the analysis program SPSS, version 16. To assess the sig- tures, such as conchological data (Madec et al., 2003; Chiba, nificance of differences among groups, One-Way-ANOVA test 2005; Fiorentino et al., 2008), genital gross anatomy (Nishi and independent-samples t-test (P < 0.05) were performed. and Sota, 2007; Fiorentino et al., 2010; Giusti et al., 2011) These results were confirmed by using the test of Mann– and/or radular morphology (Weaver et al., 2008; Ko¨hler and Whitney (P < 0.05). Dames, 2009). Such morphological studies are debatable, unreliable and should be supplemented by other analytical DNA extraction, PCR and sequencing methods such as DNA sequencing (Puente, 1994). Molecular techniques have proven to be useful in cases where morpholog- Total genomic DNA was extracted from the foot muscles of ical features failed to provide unequivocal phylogenetic signals ethanol preserved specimens, using the standard phenol chlo- by providing an independent set of characters. roform extraction method (Van Moorsel et al., 2000). A frag- Eobania vermiculata (Mu¨ller, 1774), one of the main repre- ment of COI was amplified using the primers ACO 50- sentatives of the helicid land snails, attained a worldwide cctattataattgggggttttgg-30 and BCO 50-gtatcggctgtaaaataagc-30 distribution via anthropogenic activities (Godan, 1979). The (Hatzoglou et al., 1995). A fragment of the mitochondrial gene species is native to the Mediterranean area, but found its coding for the large ribosomal subunit (16S rDNA) was also way to many other countries. Therefore, the present ap- amplified using the primer combination of LR-J-12887 50- proaches were to investigate phylogenetic relationship of this cgatttgaactcagatca-30 (Simon et al., 1994) and 50-gtgcaaaggtag- land snail from Egypt and Saudi Arabia and look for any cataatca-30 (Gantenbein et al., 1999). PCR reactions were run congruence between morphological and genetic data sets. To in a total volume of 50 ll under the following conditions: 95 C achieve these aims, we constructed a molecular phylogeny by for 20 s, 48 C for 30 s and 72 C for 30 s (repeated for 25 cy- means of sequence data of two commonly used mtDNA genes; cles), plus a final extension step at 72 C for 5 min. Amplified cytochrome oxidase I (COI) and the large ribosomal subunit products were examined on 1.5% agarose gels. DNA frag- (16S rDNA), in conjunction with conchological analysis. ments were then excised and purified with QIAquick Gel Information regarding the extent and current patterns of diver- Extraction Kit, cat no. 28704, Qiagen, Germany, following gence within species and among populations along with the the manufacturer’s instructions. Detecting the appropriate geographical distribution of genetic variation provides key amplification products was straightforward for both genes data for the conservation of molluscs. Moreover, they will because no secondary bands (due to nonspecific priming) oc- improve our understanding of speciation processes. curred. Approximately 30 lg of the purified products was then used as templates for direct sequencing of both forward and re- Materials and methods verse strands, using the same amplification primers in an auto- matic ABI 310 DNA Sequencer with Big Dye Terminator Sampling Cycle Sequencing Ready Reaction Kit, Perkin Elmer. Sequenc- ing data were analysed by two different computer alignment Two colour morphs (dark and light) of the land snail Eobania programs, DNAStar (DNASTAR, Inc., USA) and Sequence vermiculata were collected from Al-Azizia Farm, Ha’il, Saudi Navigator (Perkin, Corp., USA). All sequences generated in Arabia and from Zagazig City, Egypt, during September this study were deposited in the GenBank of the National 2010. Thirty specimens were collected from each colour morph Center for Biotechnology Information (NCBI) (Table 3). from either location. Only shells with a reflected lip were used as it indicates cessation of growth and maturity of the snail. Determination of phylogenetic relationships Five individuals from both colour morphs of the two studied populations were preserved in 96% ethanol for DNA The BLAST of NCBI database (Altschul et al., 1997) was used extraction. to compare resolved sequences with known 16S rDNA and COI sequences. Multiple alignments of 16S rDNA and COI Morphological analysis gene segments from the studied populations of E. vermiculata were carried out with other two Europian populations of the Several shell features (Fig. 1) were measured to the nearest same species retrieved from GenBank (Table 1). 0.01 mm, using a digital caliper. These features include: aper- Phylogenetic trees were created on each gene separately as ture height (AH), aperture width (AW), last whorl width well as the combined two genes using Maximum Likelihood (LWW), maximum diameter (MaxD), spire height (SpH) and (ML) and Maximum Parsimony (MP). Sequence of Helix as- shell height (ShH). For all statistical tests, relative measures persa from the GenBank (Accession Nos. AY741408 and were taken for morphometric characters (AH, AW and SpH) JN701920 for 16S rDNA and COI respectively) were used as by dividing these characters by ShH. an out-group to root the trees for the studied genes. Variables were log-transformed to obtain linear relation- The programs PHYLIP package (ww.atgc-montpellier.fr/ ships. Discriminate Function Analysis (DFA) was performed phyml/) and PAUP* 4.10b (Swofford, 2002) were used for on all the shell variables measured to discover which measure- determination of phylogenetic relationships. A Neighbour- 146 M.M.A. Desouky, S. Busais

Figure 1 Shell measurements. AH, aperture height; AW, aperture width; LWW, last whorl width; MaxD, maximum diameter; SpH, spire height; ShH, shell height.

Table 1 Data for gene sequences of Eobania vermiculata retrieved from database. Gene Country References GenBank accession No 16S France Steinke et al. (2004) AY546357 Croatia Puizina et al. (unpublished) JF277380 COI France Steinke et al. (2004) AY546277 Croatia Puizina et al. (unpublished) JF277391

Joining (NJ) tree based on uncorrected p-distances was con- non-parametric bootstrap resampling was used (1000 repli- structed with MEGA vers. 5.0 and PAUP* 4.10b. Maximum cates). Genetic distances were calculated using PAUP* v. Likelihood (ML) and Maximum Parsimony (MP) were per- 4.0b10 (Swofford, 2002). formed in order to check for consistency of the results using different algorithms based on different assumptions of Results molecular evolution. The ML analysis was performed using PHYLIP package with model parameters fitted to the data Study sites by likelihood maximization. The AIC as implemented in Mr Model test ver. 2.3 (Nylander, 2004) was used to estimate the best-fit model of nucleotide substitution for each data The locations of the two collection sites are shown in Fig. 2. partition of each gene as follows: (GTR+G) was the best-fit The distance between the two sites is about 1045 km and they model for 16S rDNA gene and combined 16S rDNA and are separated by natural barrier; the Red Sea. Zagazig is a part COI genes, while (HKY+G) was the best-fit model for COI of deltoic plain lying in the Nile delta, Northern Egypt while gene. MP analyses were performed in PAUP* v. 4.0b10 Ha’il region is a desert plateau overlying the Arabian shield (Swofford, 2002) branch swapping with 1000 random stepwise in the northern part of Saudi Arabia. Data about the two stud- additions of taxa. All changes had the same weight and gaps ied sites and their average climatic parameters are shown in were included as a fifth state. To evaluate clade support, Table 2.

Figure 2 A map showing the two collection sites from Egypt and Saudi Arabia. Phylogenetic relationships of the land snail; Eobania vermiculata (Mu¨ller, 1774) from Egypt and Saudi Arabia 147

Table 2 Data about location and climatic parameters of the two studied sites (source: World Meteorological database, http:// weatherbase.com). Parameters Zagazig city Azizia farm, Ha’il Lat_Lon 30 35’ 2500 N; 3129’.4200E28 05’ 4500N; 4152’ 29’’ E Eleveation (m above sea level) 24 1012 Average annual temp. 21 22 Average annual humidity 56% 33% Average annual precipitation 30 mm 110 mm Mean wind speed 4 knots 6 knots

the dark and light colour morphs of Saudi Arabian groups and among the dark and light Egyptian specimens (Fig. 3). The principal component analyses (PCA) using the morphometric data extracted two principal components with an Eigen value 1 in the analysis of Egyptian and Saudi Arabian dark colour morphs. These factors demonstrated 51.1% and 30.6% of the total variance. The variables that mainly contributed to the ﬁrst component factor were ShH, AH, AW and SpH whereas those contributed to the second factor were Max D and LWW. The ﬁrst and second factors separated two main groups by using scatter plots, which completely agreed with their illustration in the phylogenetic tree. The separation among Egyptian and Saudi Arabian snails was clear. A limited overlap was observed between the Egyptian dark morph and the Saudi Arabian light morph (Fig. 4).

Molecular studies

The PCR products of the studied sequences are shown in Fig. 5. 339–341 bp and 219–253 bp fragments were sequenced from 16S rDNA and COI gene respectively from the studied snail populations. No insertion or deletion was observed in all studied gene segments, but as expected mitochondrial genes Figure 3 Classification results by discriminate Factor analysis showed an excess of A+T nucleotides. The GenBank Acces- (DFA) on morphological differentiation among Egyptian dark (1) sion Numbers of these sequences are shown in Table 3. and light (2) colour morphs and Saudi Arabian dark (3) and light The Maximum Likelihood (ML) and Maximum Parsimony (4) colour morphs of Eobania vermiculata. (MP) for a combination of the two studied genes (564 bp) gave very similar results and showed only slight differences between ML and MP trees in 16S rDNA gene where their relationships have a small support value. Morphological studies The COI phylogenetic tree revealed two maximally supported clades (Fig. 6), one containing Egyptian and Saudi- ANOVA of the six examined morphological characters re- Arabian sequences and the other containing two European vealed that LWW (p = 0.004), AW (P = 0.000) and Max D sequences with 93% bootstrap value in ML and 100% in (P = 0.000) show significant differences between the two MP analysis. The Egyptian sequences of dark and light colour studied populations of E. vermiculata. On the other hand, no morphs clustered together in a separate clade with 95% boot- significant differences were recorded between any morpholog- strap value in ML and 100% in MP analysis. The sequences ical characters within either the Egyptian or Saudi Arabian from Saudi Arabia formed weakly supported cluster in ML dark and light colour morphs. analysis with 62%. On the contrary, it has a strong bootstrap The discriminate factor analysis (DFA) using the morpho- value (100%) in MP analysis. The genetic distance between the metric data extracted two main groups of E. vermiculata dis- clades of Egyptian and Saudi Arabian sequences was relatively tributed in Egypt and Saudi Arabia. DFA analyses were high (16.8%) in the studied COI gene segment. obtained; the first account was 90.7% of the total group’s var- Similarly, 16S rDNA phylogenetic tree revealed two maxi- iability, the second account was 7.6% while the third was mally supported clades (Fig. 7), one containing Egyptian and 1.7%. The separation between the Egyptian and Saudi Ara- Saudi Arabian sequences and the other containing two Euro- bian groups by the first and second functions, which have a pean sequences with strong bootstrap value. The 16S rDNA high score, was clear as no overlap was observed among the sequences from Saudi Arabia were more heterogeneous. The populations. However, a limited overlap was detected between light colour morph of Saudi Arabian snails was clearly 148 M.M.A. Desouky, S. Busais

distinguished from the dark colour morph of Saudi snails and other Egyptian snails with strong bootstrapping value in both analyses of ML and MP. The Egyptian sequences of dark and light colour morphs formed a separate clade with 92% bootstrap value in ML and 100% in MP. The genetic distance between the clades of Egyptian and Saudi Arabian sequences was 14.5% in the studied 16S rDNA gene segment. In the combined two mtDNA gene fragments of E. vermiculata, the European snails from Croatia and France formed a monophyletic group and sister to the Middle East group (Fig. 8). The clade of Saudi Arabian snails was clearly distinguished from the Egyptian clade with a strong bootstrapping value in both analyses of MP and ML. The genetic distance of the combined mtDNA genes between Saudi Arabian and Egyptian clades was approximately 16%.

Discussion

Land snails display extraordinary variations in their colour, banding pattern, decoration and form of the shell (Schilthuizen et al., 2003) which can be relied on for taxonomic and phylogenetic purposes. Eobania vermiculata is a well-known circum- Mediterranean species (Neubert, 1998). This species is well known for its survival capability and has been introduced by Figure 4 Morphological differentiation among land snails E. human activity to many countries ranging from USA (Roth vermiculata. The scatter grams show (1- dark morph and 2- light and Sadeghian, 2006) to Japan (Ueshima et al., 2004). It was morph) of the Egyptian population and (3- dark morph and 4- recorded for the ﬁrst time in central Saudi Arabia by Mordan light morph) of the Saudi Arabian population, ordered along ﬁrst (1980). As hypothesized by Neubert (1998), the invasion and second principal components of PCA. pathway of this species to the Arabian Peninsula started in

Figure 5 PCR products of 16S rDNA and COI segments veriﬁed by electrophoresis in agarose gel using 100 bp ladder. Lane H2O: negative control; Lane 1: dark Saudi Arabian morph; Lane 2: light Saudi Arabian morph; Lane 3: dark Egyptian morph; Lane 4: light Egyptian morph.

Table 3 Data for gene sequences of Egyptian and Saudi Arabian populations of Eobania vermiculata. Gene Colour morph Collected from Length GenBank accession Number 16S Dark Ha’il, KSA 339 bp JF807626 Light Ha’il, KSA 340 bp JF807627 Dark Zagazig, Egypt 340 bp JF807628 Light Zagazig, Egypt 341 bp JF807629 COI Dark Ha’il, KSA 253 bp JF807630 Light Ha’il, KSA 237 bp JF807631 Dark Zagazig, Egypt 219 bp JF807632 Light Zagazig, Egypt 220 bp JF807633 Phylogenetic relationships of the land snail; Eobania vermiculata (Mu¨ller, 1774) from Egypt and Saudi Arabia 149

Figure 6 Maximum liklihoode (ML) tree for the COI mtDNA sequences of Eobania vermiculata obtained with PHYML. ML bootstrap values (> 50%) are given above the nodes, and Maximum Parsimony (MP) are given below the nodes. D, dark morph; L, light morph.

Figure 7 Maximum likelihood (ML) tree for the 16S rDNA sequences of Eobania vermiculata obtained with PHYML. ML bootstrap values (>50%) are given above the nodes, and Maximum Parsimony (MP) are given below the nodes. D, dark morph; L, light morph.

Magnin, 2001; Madec and Bellido, 2007). Such variations were attributed to climatic conditions such as the temperature gra- dient co-varying with altitude (Welter-Schultes, 2000) and humidity (Goodfriend, 1986). It may have also arisen from selection in different environments (Engelhard and Silk, 1994; Welter-Schultes, 2000), as a product of population history (Gould and Woodruff, 1990; Davison, 2002) or from geographical isolation (Lazaridou-Dimitriadou et al., 1994). Further studies are needed to assess the association between shell shape of the studied species and climatic factors. However, the dependence on morphometric shell characters as taxonomic markers alone (or any single analysis) is insuffi- Figure 8 Maximum liklihoode (ML) tree for a combination of cient due to the geographical variations (Jordaens et al., 2009). the COI and 16S rDNA of Eobania vermiculata obtained with Molecular methods often simplify distinguishing the species PHYML. ML bootstrap values (>50%) are given above the that have similar problems thereby eliminating previously nodes, and Maximum Parsimony (MP) are given below the nodes. occurring variation problems (Carranza and Arnold, 2006; D, dark morph; L, light morph. Busais and Joger, 2011). For this purpose, a wide array of genetic techniques have been utilized to address species delimita- the lower Miocene and was supported by the Levantine branch tion for many snails, including PCR–RFLP analysis (Carvalho of the western Palaearctic fauna. The cosmopolitan distribution et al., 2004), RAPD analysis (Rybska et al., 2000) and DNA of this snail and the variation in its shell morphometric measure- sequencing (Stothard et al., 2000; Bargues et al., 2001; Bargues ments make it suitable for studying phenotypic differentiation et al., 2003; Bargues et al., 2007; Remigio, 2002; Pfenninger and phylogeny in relation to geographical distribution. et al., 2006). The study of the morphometric characteristics of the shell In concordance with morphometric analysis, the present showed that there was considerable variation between the molecular analysis (16S rDNA and COI sequence data) re- two studied snail populations from Saudi Arabia and Egypt vealed that the two studied populations of E. vermiculata were as revealed by the discriminate, principal component and t-test grouped according to their geographical origin but not due to analyses. On the other hand, no significant differences were the colouration of their shells. This differentiation arose possi- reported for shell morphometrics between light and dark col- bly due to the diversity in climatic conditions and variations in our morphs of either population. Similarly, substantial varia- topographic areas. When the same mitochondrial markers tion in quantitative and qualitative shell morphology among from two European populations of E. vermiculata (retrieved populations of geographically subdivided land snails was often from database) were included in the analysis, they were also observed (Lazaridou-Dimitriadou et al., 1994; Pfenninger and clearly separated from the two studied populations. Therefore, 150 M.M.A. Desouky, S. Busais the resulting significant morphological differences between the References two populations of E. vermiculata seem to be related to their geographic origin which may have caused present changes in Alonso, M.R., Goodacre, S.L., Emerson, B., Ibań˜ez, M., Hutterer, R., the mitochondrial genes. Groh, K., 2006. Canarian land snail diversity: conflict between According to Elejalde et al. (2008), the intraspecific diver- anatomical and molecular data on the phylogenetic placement of gence in molluscs is typically not higher than 5% for COI five new species of Napaeus (Gastropoda, Pulmonata, Enidae). and 16S rDNA genes. The present study revealed that the p-ge- Biol. J. Linn. Soc. 89 (1), 169–187. netic distances of COI and 16S rDNA genes are considerably Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: high and p-value of COI gene of the studied population was a new generation of protein database search programs. Nucl. Acids higher than 16S rDNA gene divergence values. 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