Molecular approach to the identification and phylogenetic biogeography of snail Telescopium telescopium using mt-COI gene sequences

We aimed to apply DNA barcoding tool for the molecular identification of horn snails T. telescopium using mitochondrial cytochrome oxidase I gene (mt-COI) and to investigate their evolutionary relationship along with location-specific bio-geographical variations. The molecular data sets of this study indicate that strong probability of T. telescopium species taxonomic confirmation using mt-COI sequences. Results of the phylogenetic analysis suggest that Telescopium sp. was monophyletic with disseminated nodes and the evolution of group II originated from group I. The substantial genetic distance among the mt-COI sequences (0.005 to 0.184) were noticed. Large divergence between the south-west coast of India and Australia region population indicates limited gene flow between the two continents. Our study suggests that the genera Telescopium is globally ubiquitous but genetically showing inter-region differentiation. We conclude that mt-COI gene can be used to identify gastropod T. telescopium species.


Introduction
Indian coastal waters are receiving more attention from the scientific community due to their enormous fishery and shellfish potential resources [1]. In the Indian Ocean, Bay of Bengal waters hosts an extent range of molluscan (bivalves and gastropods) faunal distribution, species diversity and they are ecologically important to many communities [2]. However, this group diversity is highly underestimated [3]. Molluscs are the most diverse marine phylum and represent higher species diversity has resulted in a considerable taxonomic problem [4]. Some species in Indian coast remain uncertain; there is a need to incorporate molecular methods.
Molecular markers especially mitochondrial DNA cytochrome oxidase I (mt-COI) are considered as a better choice for species discrimination due to non-recombinant and environment independent nature [4]. The application of DNA barcoding brings substantial benefits to other fields, such as border biosecurity [5], biodiversity conservation [6], ecological monitoring [7]. DNA barcoding has successfully utilized for the taxonomic confirmation of marine organisms, the molecular phylogeny of gastropod molluscs is extensively studied using morphometric characteristics and DNA sequences [8]. The standard pairwise distance method such as Neighbour-Joining tree (NJ) based on Kimura 2-parameter distance (K2P) and GC content divergence assessment is currently the principal approach used to analyze patterns of diversity with COI barcode region. It has been informative at the species-level discrimination across the range of several groups from terrestrial, freshwater and marine environment [9]. The accuracy of such results depends particularly on the delineation between intra-specific variation and inter-specific DNA sequence divergence [10]. The strong molecular identification of marine gastropods requires the creation of comprehensive reference library of DNA barcode matching of each specimen to a diagnostic barcode sequence [11].
Gastropod mollusc Telescopium telescopium lives in mud-flats of mangrove forest in throughout the coastlines of Indian Ocean waters. Mangrove horn snail or mud whelks T. telescopium is the only living organism of this genus from the family Potamidae (Gastropoda), found abundantly and frequently dominated in mud-flats of mangrove forest in West and Central Indo-Pacific regions due in part of its central locations at the meeting of oceanic waters has the greatest diversity of mangrove forest. The distribution of this species is likely impacted locally by the loss of habitat [e.g. mangrove deforestation, marine pollution] [12]. So far, no reliable information available to deal the estimated population of this species in Indian waters.
Houbrick et al. [13] was reported the revised systematics and phylogeny of this taxa, following that Bandel et al. [14] was recognized two sub families, Potamidinae and Telescopinae, the later on included all the living potamidids except the genus Potamides in the family Potamididae. With the recent development of molecular techniques, the evolutionary relationship of Potamididae and their ecological association with mangrove ecosystem has also been studied using mt-COI sequence data [15]. The further research of this genus will reveal to complete understanding of the evolutionary relationship of this species in family Potamidae, ecological association with mangrove forest and their potential application in pollution monitoring [16] and marine bio-discovery program [17,18]. The DNA sequence of mt-COI gene data facilitate not only the inference the phylogenetic relationships of fauna but also providing an efficient method for species-level identification under the term of molecular taxonomy or DNA Barcoding [19].

Evolutionary significant of horn snail
Mangrove horn snail or mud whelks T. telescopium is the only living organism of this genus from the family Potamidae (Gastropoda), found abundantly and frequently dominated in mud-flats of mangrove forest in West and Central Indo-Pacific regions due in part of its central locations at the meeting of oceanic waters has the greatest diversity of mangrove forest. The distribution of this species is likely impacted locally by the loss of habitat [e.g. mangrove deforestation, marine pollution] [12]. So far, no reliable information available to deal the estimated population of this species in Indian waters. Houbrick et al. [13] was reported the revised systematics and phylogeny of this taxa, following that Bandel et al. [14] was recognized two sub families, Potamidinae and Telescopinae, the later on included all the living potamidids except the genus Potamides in the family Potamididae. With the recent development of molecular techniques, the evolutionary relationship of Potamididae and their ecological association with mangrove ecosystem has also been studied using mt-COI sequence data [15].
The further research of this genus will reveal to complete understanding of the evolutionary relationship of this species in family Potamidae, ecological association with mangrove forest and their potential application in pollution monitoring [16] and marine bio-discovery program [17,18]. The DNA sequence of mt-COI gene data facilitate not only the inference the phylogenetic relationships of fauna but also providing an efficient method for species-level identification under the term of molecular taxonomy or DNA Barcoding [19].
The aim of this present study, to improve the systematics of the genus Telescopium by the integration of morphometric characteristics and DNA barcoding of T. telescopium shells found in the Bay of Bengal waters. ii) to determine their evolutionary relationship by biogeographical variations based on mt-COI sequences.

Collection of samples
Horn snails used in this study were collected in Parangipettai coast, Bay of Bengal -India during March to August 2011 and their taxonomy was identified using [13]. All the specimens (n=10) were preserved in 95% ethanol for DNA molecular analysis.

DNA Isolation and Sequencing
Total DNA was extracted from the muscle tissue/eggs of each specimen using Qiagen DNA Easy blood and tissue kit (Hilden, Germany). Universal Primers LCO 1490 and HCO2198 as described in Folmer et al. [20] were used for the amplification of a fragment of mt-COI gene. PCRs were performed in a 12.5 µl volume with 6.25 µl of 10% trehalose, 1.25 µl PCR buffer [200 mM Tris-HCl (pH 8.4), 500 mM KCl], 2 .85 µl of nuclease free water, 0.625 µl MgCl2 (50 mM), 0.5 µl of each primer (10 µM), and 1 µl of DNA template. A single soak at 95°C for 2 min was followed by 35 cycles (denaturation at 94°C of 30s, annealing at specific temperature 52°C for 30s) and extension at 72°C for 1 min) and a final extension at 72°C for 10 min on a PCR Thermal Cycler. Further, the PCR products were purified using PCR Purification Kit (Qiagen, Hilden, Germany). The same primers were used for the sequencing reaction, and the PCR products were sequenced using an ICM version 3.1 automated sequencer (MegaBace) Bioserve DNA sequencer, BioServe Biotechnologies, Hyderabad, India.

Data analysis
DNA sequences were edited using Bioedit Sequence Alignment Editor V 1.0, 2005 [21], and an alignment of snail's sequences was performed using CLUSTAL W [22]. For the phylogenetic analysis, we were included 26 sequences of T. telescopium were retrieved from variable sites in the data set along with out-group of organisms. Phylogenetic analysis was conducted using Maximum Likelihood (ML) and Bayesian inference (BI). For ML analysis, the best fit jModelTest v 3.06 (Provo UT, USA) used for constructing the phylogenetic tree with genetic distance data were generated in MEGA6 [23]. For BI, general time-reversible model (GTR) of sequence evolution with a gamma distribution and variable set was selected and made with the use of MrBayes v 3.2.1 [24]. The Markov Chain Monte Carlo (MCMC) analysis was run for 50,000, which was sampled every 1000 generations and the first 25% of the tree were discarded as burn-in, the remaining trees were used to construct the final tree and estimate posterior probabilities (Pp).

Taxonomic assignment
Description: Shell is very thick and solid, black coloured at first glance, originally reddish- The present study was designed to identify the mangrove mollusc T. telescopium using mt-COI gene sequences. Morphological data were compared to molecular results to illustrate major trends encountered. Morphologically identified taxa and corresponding molecular identifications using mt-COI gene of T. telescopium are listed in Table 1. Morphological assessments identified T. telescopium of ten samples are shown in Figure 1. The COI data of genera Telescopium was significantly correlated with morphological assessment. Our results confirmed the mt-COI based molecular identification of T. telescopium to support previous suggestions of [15].

Molecular Phylogenetic analysis
The phylogenetic tree was constructed along with twenty-six sequences retrieved from variable sites in the data set (Table 2). Indian mangrove snails exactly formed a phylogenetic group with same species referred from GenBank in the phylogenetic tree of mt-COI (Figure 2), and these results supported the validity of morphological identification of T. telescopium species. The topological tree derived from the best-fit models of nucleotide substitution for individual data based on AIC selected by jModel Test with criterion (GTR) was calculated.
The variation in nucleotide composition was studied using GC content analysis. GC content is also varies taxonomically within mitochondrial genome, although all mitochondrial genomes sequenced to date are poor, and widely applied in systematics [25,26] and 37.62 % in north western pacific molluscs varied substantially among mollusan orders [27]. Generally, it is higher in mammals, birds and teleost fish (range 32-46%), following that molluscs (29-40%) and found lowest in nematodes and insects ranging from 15-35% [25].
Results of this study showed three major strong clades and a significant distinction of this species diversified from all over the Indian ocean in the newly reconstructed phylogenetic tree. In Bayesian tree method, our molecular phylogenetic hypothesis showed genera  sites in Indian Ocean waters. In the previous study, Lydeard et al. [28] were reported moderate support (bootstrap 87%) for a sister relationship between Telescopium and Terebralia sp, later Reid et al. [15] have reported limited support of posterior probability (93%) using mt-COI, 16S and 28S sequence data sets. As well, Reid et al. [15] have reported the higher value of genetic distance up to 16% (used Kimura two-parameter model) of T. telescopium collected in various sites and mt-COI sequence of this species formed a complex phylogenetic structure. Similar to this previous study, we have observed the maximum value of genetic distance in between the species up to 18.7% after additionally adding our Indian snails mt-COI sequence data in data set. The results of this present study confirm that Telescopium snails reported in Australian and Indian waters are very close relatives or from same population stock. The individual snails were collected in Bay of Bengal waters formed many subclades with the higher value of genetic divergence (1.0-10.5%), these results demonstrated that more intra-subpopulation of horn snail was appeared and diversified in the new mangrove environment shortly and evolved. Similar results of this study were reported by [15], the results of this present study confirmed that the potamidae horn snail's origin and radiation were within the Indo-West Pacific region.
As presently understood, horn snail T. telescoipum is the only living organism from the Potamidae occupying a distinct range of microhabitats of mangrove ecosystem [13,29]. The detailed biogeographic history of Potamidae is not yet studied completely, the evolution of the Potamidae molluscs has been very well connected with that of mangrove ecosystem ever since, while few genera (Cerethedia spp, Terebralia sp) lost their dependence with mangrove fauna [15]. Based on the previous research report, T barcoding. As given the results in Table 3, the expansion of geographical coverage significantly increased intra-specific divergences. Intra-specific sequence divergences ranged from 0 to 18%, the evolutionary distance between the sequences of bio-geographic

Conclusions
The conclusions of this study are in agreement with the phylogenetic analysis conducted by [15], as well as with the latest morphological update of the genus [29]. The outcome of this study established a significant contribution to DNA barcode reference sequences of horn snails and demonstrated the inter-generic relationship for effectively monitoring their ecological association with mangrove fauna. In addition, we provided a newly constructed phylogenetic structure using mt-COI sequence data, which suggests that T. telescopium is monophyletic.
However, it is important to note that some of the cases of deep intra-species genetic divergence showed a biogeographic partition between lineages on the Indian Ocean waters, and mainly found in the Indo-Pacific and Indian Ocean waters, but genetically showing inter-region differentiation. Results of this study suggest that an integrative taxonomy of morphological and molecular methods represents a power tool, when used appropriately, and serve as a point of reference for future genetics research of T. telescopium, which will be vital for all consideration