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