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Molecular Phylogenetic Relations of fulica Based on Partial Sequence of COI Gene

Article in National Academy Science Letters · January 2017 DOI: 10.1007/s40009-017-0538-5

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Molecular Phylogenetic Relations of Achatina fulica Based on Partial Sequence of COI Gene

1 1 1 1 C. Jena • S. Sarkar • N. Jalaja • S. Krupanidhi

Received: 11 July 2015 / Revised: 6 February 2016 / Accepted: 4 January 2017 Ó The National Academy of Sciences, India 2017

Abstract The Giant African (Achatina fulica)is Further, secondarily became asymmetrical and their known to be a highly widespread and invasive species. soft body is enveloped within the spirally coiled univalve However, none of the studies in India have shown whether shell [2, 3]. According to Bouchet and Rocroi [3], clade there is any phylogenetic structure within the African giant falls within the informal group Pul- snails of the Indian subcontinent. We have collected a monata which branched out of . sample of Achatina fulica from Odisha state, India and The clade Stylommatophora contains the subclades Elas- built a molecular phylogeny of achatinoid and non mognatha, Orthurethra and the informal group Sig- achatinoid taxa from across its distribution using a mito- murethra. The molecular phylogenetic relations further chondrial gene to infer whether the sample from Odisha is confirm the grouping of genera and species to their nested within the achatinoid taxa and also to notice whether respective families [4–6]. the A. fulica is more closely related to the conspecifics In particular, due to its large size and wide distribution from the Indian subcontinent or otherwise. Our results in the southern mesozoic supercontinent Gondwana land, indicate a strong support for the monophyly of achatinoid the Achatina is of biogeographical and phylogeo- taxa with our focal sample nested deep within the clade and graphical importance [7–9]. Achatina fulica ( branched with conspecifics from other regions of the Indian fulica, Bowdich, 1822) is commonly known as African subcontinent. In addition, we also show that the family Giant and reported to grow up to a maximum of Orthalicidae is paraphyletic with respect to Streptaxida, a 20 cm in length. These land snails were collected from pattern that needs to be explored further in future studies. Bharatpur (Bhubaneswar) in the State of Odisha, India, located in Lat.19°180N and Long.84°510E. The land snails Keywords Á COI Á Á being an evolutionarily successful taxa primarily due to Achatinoid taxa Á Non-Achatinoid taxa their adaptability to extreme environmental conditions they were chosen as models and indicators of biological evo- lution [10, 11]. The present analysis using partial sequence Among gastropods, land snails and constitute the of COI gene of A. fulica (Odisha, India) is an attempt to largest assemblage of species ranging in number between build its phylogenetic affinities with the members of clade 30,000 and 35,000 [1] adopting varied life styles, habits Stylommatophora. and habitats and withstanding adverse seasonal variations The snails were maintained in the wooden troughs in the [2]. Molluscs are unsegmented and soft-bodied coelomates laboratory to get them acclimatized before they were taken having a prominent ventral foot and dorsal visceral mass. for sampling. The diagnostic characteristics and morpho- logical markers to identify A. fulica inhabiting at Bharatpur (Bhubaneswar, India) are as follows: The shell of A. fulica & S. Krupanidhi is wider at its operculum and tapering at its apex, which [email protected] reveals that the shell is more conical rather than globose. 1 School of Biotechnology, Vignan’s University (VFSTRU), The size of its shell is 9.0–12.0 cm in length and Vadlamudi, AP 522213, India 4.0–5.0 cm in width revealing that the length of its height 123 C. Jena et al. is double to that of its width at opercular region. In addi- agarose gel along with 100 bp DNA molecular marker and tion, the number of whorls in the shell is six. The coloration images were taken under Gel Doc system (MediccareTM, and vertical stripes are dark brownish alternated by cream Chennai, India). tinge. The snail has two pairs of tentacles of which the PCR reactions were performed in duplicates and the upper pair at its terminal is having eye spots. Similar to Isolated PCR product was sequenced bidirectionally with the other gastropods, the body of the snail is moist and leaves same primers used for PCR amplification by M/S Eurofins, behind slimy mucus in its track and a flat foot with Bangalore. The sequencing was performed by Big Dye tubercles on its lateral upper surface. These snails prefer to Terminator 3.1 v sequencing kit. The annotated sequences dwell in habitats rich in lime stones. The species of A. were checked further by ORF finder. The sequences fulica is uniquely identified with its characteristically obtained were submitted to NCBI and the given accession twisted columella. number is KP259270. In the present study, our taxonomic The snail was narcotized using menthol [12] and dis- sampling was based on the least e-values (9e-179 to sected to isolate the foot muscle tissue. 0.5 g muscle tissue 1e-52) obtained in BLAST and the representative species was used for the DNA extraction. Total genomic DNA was of the clade Stylommatophora, informal group Sigmurethra extracted based on the phenol–chloroform method descri- and its families namely Rhytididae, Clausiliidae, Streptaxi- bed by Brown [13] with slight modification. The purity of dae, Orthalicidae, Partulidae, Chondrinidae, Charopidae, the extracted genomic DNA was found to be 1.78 ng/ll and the another subclade Orthurethra were [14, 15]. selected for phylogenetic analysis. The DNA sequences Primers for the COI gene given by Folmer [16] were alignment of stylommetophoran and non stylommetophoran adopted in this study. PCR reaction was performed in genera was performed with default parameters in MUSCLE Thermal Cycler (Agilent). The components of PCR mix- [17] using the program MEGA v5.2 [18]. The resulted ture contain template genomic DNA, gene specific primers, aligned sequences in FASTA format were converted to dNTPs, Taq buffer, MgCl2 buffer and Taq DNA poly- PHYLIP format. The ML phylogenetic analysis involving merase. The reaction volume for all PCR reactions was set the unpartitioned nucleotide sequences were analyzed to 25 ll. All the reagents required for PCR were purchased through raxmlIGUI v1.3 software package [19]inGTR? C from (HiMedia, Mumbai, India). The PCR cycles were DNA substitution model. The constructed phylogenetic tree repeated for 35 times followed by a final extension for nodal support values were obtained by performing with 10 min at 72 °C. The reaction mixture devoid of template 1000 bootstrapping replications. was run as a negative control. Amplified DNA fragment Molecular and bioinformatics tools are now providing 375 bp was separated by gel electrophoresis in 1.2% an opportunity to explore genomes of both extinct and

Fig. 1 Maximum-likelihood cladogram based on RAxML analysis of the full concatenated dataset of partial gene COI of the chosen genera. The bootstrap values above 40% are shown

123 Molecular Phylogenetic Relations of Achatina fulica Based on Partial Sequence of COI Gene

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Popul Ecol logenetic analysis of 25 genera belonging to Stylomme- 44:129–136 tophora was shown in Fig. 1 and this tree was rooted on the 7. Dayrat B, Conrad M et al (2011) Phylogenetic relationships and evolution of pulmonate gastropods (Mollusca): new insights from non Stylommetophora pulmonate taxa namely Laemodonta increased taxon sampling. Mol Phylogenet Evol 59:425–437 (Order ) and Siphonaria (Order Basom- 8. Grande C, Templado J (2004) Molecular phylogeny of euthy- matophora) corresponding to the gastropod [21]. neura (Mollusca: Gastropoda). Mol Biol Evol 21(2):303–313 The nodes in the present cladogram, showed low support 9. Kurabayashi A, Usehima R (2000) complete sequence of mito- chondrial DNA of the primitive ophistobranch gastropod Pupa (Fig. 1), which is not surprising that the chosen ingroup strigosa: systematic implications of the genome organization. taxa are highly divergent from each other. However, our Mol Biol Evol 17(2):266–277 focal taxon is shown to be nested within achatinoid taxa 10. Solem A (1985) Camaenid land snails from Western and central and formed a monophyletic group with very high bootstrap Australia (Mollusca: Pulmonata:Camaenidae). Remaining Kim- berley genera and addenda to the Kimberley. Rec West Aust Mus support. These deeper aspects of taxonomy in the clade Suppl 20:707–981 Stylommotophora needs to be explored for the under- 11. Grande C, Templado J (2008) Evolution of gastropod mito- standing of their monophyly. The partial nucleotide chondrial genome arrangements. BMC Evol Biol 8(6):18–61 sequence of COI gene of A. fulica Odisha confirmed its 12. Inglis WG (1985) Evolutionary waves: patterns in the origins of phyla. Aust J Zool 33(153):178 phylogenetic affinities with achatinoid taxa and also cor- 13. Brown TA (1991) Essential molecular biology, A practical responded to all the chosen taxa (Fig. 1) as described in the approach. Oxford University Press, New York gastropodan classification [20]. 14. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a This study concludes that A. fulica Odisha is more laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor closely related to the conspecifics form the Indian sub- 15. Rueda LRJ, Vazquez AM (1998) Quantifying of DNA. In: Isaac continent and our result shows a strong support to the PG, Ingram DS (eds) Karp A., Molecular tools for screening monophyletic of achatiniod taxa with our focal sample biodiversity: plants and animalsChapman and Hall, London, nested and branched with conspecifics form other regions pp 18–21 16. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA of the Indian subcontinent. primers for amplification of Mitochondril cytochrome c oxidase subunit I from diverse metazoan invertebrates. 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