Int. J. Aquat. Biol. (2022) 10(5): 364-369 ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2022 Iranian Society of Ichthyology Original Article New distribution record of Crassostreine oyster Magallana gryphoides (Schlotheim, 1820) in Kerala, India Vineetha Vijayan Santhi, Smrithi Sreekanth, Mugdha Sukumaran, Mano Mohan Antony Department of Zoology, (Research Centre, University of Kerala), University College, Palayam, Thiruvananthapuram, Kerala, India. s Article history: Received 12 June 2022 Accepted 14 August 2022 Available online 2 5 October 2022 Keywords: Morphology Dharmadom estuary Magallana bilineata Gene sequencing Phylogeny Abstract: The Crassostreine oyster Magallana gryphoides (Bivalvia: Ostreidae) has been recorded for the first time on the Kerala coasts from Dharmadom estuary, Kannur, Kerala, India. The report indicates the range extension of M. gryphoides on the South-west coast of India. The external morphological characters were phenotypic and insufficient for species identification as it resembles Magallana bilineata. However, internal shell characters gave important information, especially the adductor muscle scar. The accurate species determination was achieved from the mitochondrial COI and 16S gene sequencing, followed by molecular phylogenetic analysis. The native oyster M. bilineata and M. gryphoides were found to co-exist in the same habitat sharing similar ecological conditions, sharing a sister group relationship. Introduction The true oysters of the family Ostreidae Rafinesque, 1851 have a cosmopolitan distribution and encompass species with high economic importance. They are considered the most efficient ‘keystone niches’ among all the keystone species known for making unique micro-ecosystems (Sanjeeva, 2008). In India, oysters are widely distributed in estuaries, bays, harbours, and backwaters. However, comprehensive data on the distribution of Oysters dated way back to 1987 by Rao (1987), who reported Crassostrea (Magallana) gryphoides from Gujarat, Maharashtra and Goa. Subsequently, M. gryphoides had been reported from different regions on Indian coasts, from Konkan coast (Sawant, 1997), Maharashtra (Tibile and Singh, 2003), Sunderbans (Trivedi et al., 2015), Goa (Reece et al., 2008; Nagi et al., 2010) and from nearby coasts of Karachi (Siddiqui and Ahmed, 2002), Myanmar (Li et al., 2017) and Bangladesh (Chowdury et al., 2021). Initially, the Indian oysters were assigned to the genus Ostrea by Awati and Rai (1931), later revised Correspondence: Mano Mohan Antony DOI: https://doi.org/10.22034/ijab.v10i5.1672 E-mail: manomohanantony@universitycollege.ac.in DOR: 20.1001.1.23830956.2022.10.5.2.3 and re-assigned under the genus Crassostrea by Rao (1956, 1958) and Durve (1967). Recently, Salvi et al. (2014) and Salvi and Mariottini (2017) proposed a new genus, Magallana Salvi & Mariottini, 2016, for the Asian-Pacific clade of true oysters of the subfamily Crassostreinae, thereby, Asian-Pacific species of Crassostrea were re-assigned into a new genus Magallana gen. nov (Salvi et al., 2014). The genus Magallana was valid based on the 2016 description and all the species in the genus were included in WoRMS in 2017, which makes M. gryphoides (Schlotheim, 1820), the accepted name for the Asian-Pacific species of C. gryphoides (Schlotheim, 1820) and M. bilineata (Roding, 1798) for the native Indian backwater oyster, C. madrasensis, Preston 1916. However, it is noteworthy to observe the molecular data deficiency of oysters from the Indian sub-continent in the above-mentioned papers. Therefore, in the current study, we attempted to establish the new distribution record of M. gryphoides in the South-West coast of India https://ij-aquaticbiology.com/index.php/ijab/article/view/1672 364 Santhi et al./ New record of Crassostreine oyster, Magallana gryphoides based on the mitochondrial COI and 16S gene sequencing and phylogenetic analysis along with ecological and morphological information. Materials and Methods Oysters were collected from Dharmadom Estuary (11.796918N, 75.462153E), Kannur, Kerala, India, by hand-picking from shallow coastal waters. Tissue from the adductor muscle was used for molecular analysis. The ecological factors such as depth, nature of Substrate, salinity, pH, hardness, dissolved oxygen, temperature, and population density were recorded (APHA, 1989; Peters et al., 2017). The collected species were primarily identified based on published papers (Siddiqui and Ahmed, 2002; Li et al., 2017; Chowdhury et al., 2021). The classification and Scientific names followed WoRMS (www.marinespecies.org). For species confirmation, the mitochondrial gene sequencing method was employed using mitochondrial cytochrome oxidase I (COI) and 16S ribosomal RNA (16S rRNA). The genomic DNA was isolated from muscle tissue using NucleoSpin® Tissue Kit (Macherey-Nagel). Partial COI and 16S rRNA was amplified via Polymerase Chain Reaction using the primers of LCO1490 and HCO2198 (Folmer et al., 1994) for COI and 16Sar and 16Sbr (Palumbi et al., 1991) for 16S rRNA. All the retrieved sequences were deposited in NCBI-GenBank. For both 16S and COI datasets, Multiple Sequence Alignment (MSA) was done in MEGA 7 (Kumar et al., 2016) and the phylogenetic tree (Bayesian Inference tree) was constructed in MrBayes 3.2.7 (Ronquist et al., 2012). Bayesian analysis (GTR+G+I model as best fitting model for both 16S and COI dataset) was done and two independent runs were performed for 2×106 generations sampling per 1000 generations. The first 25% of the trees acquired were discarded as burn-in, and a 50% majority-rule consensus tree with posterior probability (PP) values were generated from the leftover trees. The phylogenetic tree was constructed using FigTree v1.4.2. Talonostrea salpinx was taken as the outgroup. Results The studied site showed a depth of 0.8-1.2 m with a muddy substratum. Individuals of M. gryphoides were collected from the beds of M. bilineata. Solitary individuals along with gregarious forms with M. bilineata were observed. Compared to M. bilineata (11-18/m2), the population density of M. gryphoides was very fewer ranges of 2-5/m2. The pH of the studied site was 7.55, surface water temperature 32°C, salinity 30.14 ppt, hardness-284 mg/L and dissolved oxygen 7.33 mg/L. All the external shell characters were similar for both M. gryphoides and M. bilineata since they are phenoplastic and share a common habitat. However, characteristic differences were observed in the internal shell characters, especially in the adductor muscle scar. Magallana gryphoides exhibited characteristic pearly white colouration, reniform or crescent-shaped adductor muscle scar, nearly straight dorsally and close to the posterodorsal margin and the shell interior was whitish and shiny devoid of any purplish or blackish markings. For M. bilineata, the adductor muscle scar was characteristically dark purplish-black in both valves. The white umbo cavity always had purple markings around the whole or some parts of the margin. In the ventral margin, deeper colouration was observed facing the posterior portion of the gill, especially in the lower valve (Fig. 1). The mean and standard deviation of shell length (SL), shell height (SH), shell width (SD), and total weight (TW) in M. gryphoides was 3.64±4.751, 79.68±6.215, 32.86±2.572, and 128.19±2.08, and for M. bilineata as 74.4±13.682, 100.74±14.504, 35.026±10.607, and 191.56±31.735, respectively. A total of eight gene sequences were obtained from M. gryphoides and M. bilineata and submitted to the NCBI-GenBank and acquired accession numbers (Table 1). The phylogenetic relationship of M. gryphoides with the other Asian-Pacific species was inferred from COI and 16S gene datasets. The BI tree was constructed for each gene dataset, using four original sequences and other sequences of the Asian-Pacific species retrieved from NCBI- 365 Int. J. Aquat. Biol. (2022) 10(5): 364-369 GenBank (Figs. 2 and 3). The topology of both the trees was similar with M. belcheri in the upper clade, M. dianbeinsis, M. bilineata and M. gryphoides placed in the middle clade and M. gigas, M. sikamea, M. hongonenesis, M. nippona, M. ariakensis and M. angulata in the bottom clade. The original sequences of the M. gryphoides shared the same clade with the other 3 sequences of M. gryphoides in the COI tree and showed more similarity with the sequences from Goa, India (EU007488 and Figure 1. Shell characteristics of Magallana gryphoides and M. bilineata. (a) Lower valve of M. gryphoides (b) Lower valve of M. bilineata (c) M. gryphoides among the cluster of M. bilineata (d) Adductor muscle scar of M. gryphoides (e) Adductor muscle scar of M. bilineata (f) Upper valve of M. gryphoides (g) Upper valve of M. bilineata. Species Species ID GenBank Accession numbers 16S COI Magallana gryphoides DB1 ON926958 ON920920 DB2 ON926959 ON921053 Magallana bilineata DU1 ON926953 ON912074 DU2 ON926954 ON920843 Table 1. Gene sequence data of collected oysters. 366 Santhi et al./ New record of Crassostreine oyster, Magallana gryphoides Figure 2. Phylogenetic tree based on BI analysis of COI sequences. Bayesian posterior probability is shown at the nodes. Original sequences are marked in Red. Figure 3. Phylogenetic tree based on BI analysis of 16S rRNA sequences. Bayesian posterior probability is shown at the nodes. Original sequences are marked in Red. 367 Int. J. Aquat. Biol. (2022) 10(5): 364-369 EU007492) (Reece et al., 2008). The clade of M. gryphoides exhibited a sister group relationship with M. bilineata and M. dianbaiensis in COI and 16S trees. However, 16S sequences were scarce in NCBI-GenBank. Discussion The new report of M. gryphoides from Dharmadom estuary represents the range extension of the species on the South-west coast as it was earlier reported from the coasts of Gujarat, Maharashtra and Goa (Rao, 1987; Tibile and Singh, 2003; Reece et al., 2008). Since, the Indian oyster taxonomy is mostly dependent on the morphological shell traits, which are highly plastic, the presence of M. gryphoides on the South-west coast might be masked earlier. Therefore, the presence of more oyster species and their distribution is highly anticipated on the Indian coasts. However, molecular identification methods should be encouraged and given priority as they can provide decisive outcomes in oyster taxonomy. All the ecological conditions observed were optimum for the growth and distribution of oysters, highlighting the scope of oyster culture in the Dharmadom estuary. Being benthic, shells cope with the external environmental conditions that result in ecophenotypic plasticity (Lam and Morton, 2004; Huber, 2010; Liu et al., 2011; Santhi et al., 2021). Therefore, species identification could not be achieved based on external shell characteristics. However, it is the adductor muscle scar that gave important information for species identification. A similar finding was also made by Siddiqui and Ahmed (2002) in C. madrasensis (=M. bilineata) and C. gryphoides (=M. gryphoides) with respect to the white colour of the adductor muscle scar in C. gryphoides (=M. gryphoides) and purpleness in C. madrasensis (=M. bilineata). However, molecular markers had proved to be efficient markers for species delineation. Nevertheless, the molecular database of Indian oysters is still lacking and only a few sequences are available in NCBI- GenBank. Though, accurate identification was achieved by mitochondrial COI gene sequencing and thereby BLAST analysis. The previous name of Crassostrea gryphoides was also used for a European fossil species as Crasssostrea gryphoides von Schlotheim, 1813 that existed during the Miocene and Pliocene, and became extinct over three million years ago (Harzhauser et al., 2016). However, it has been considered as not conspecific with the extant M. gryphoides (Harzhauser et al., 2016) differing with respect to the shape of adductor muscle scar (Durve, 1974), shell outline (Durve and Bal, 1961), and size (Chatterji et al., 1985; Nagi et al., 2011). Therefore, according to Huber (2010), Harzhauser et al. (2016), and Li et al. (2017), the same name should not be used also for an extant species. After the recent revision of Asia-Pacific oysters into the newly assigned genus Magallana Salvi and Mariottinni, 2016, the extant species of C. gryphoides from the India and Pakistan region were re-assigned as M. gryphoides (Schlotheim, 1820). The current study also provides molecular phylogenetic data that agrees with the recent revision in the Crassostreinae subfamily by Salvi and Mariottini (2017). The BI tree showed the monophyletic origin of Asia-Pacific true oysters (Subfamily: Crassostreinae) recently assigned to the genus Magallana in COI and 16S trees. Moreover, M. bilineata and M. gryphoides exhibited a sister group relationship with M. dianbaiensis in both trees with a high Bayesian probability of 1. This is in accordance with Melo et al. (2010), Wu et al. (2013), Xia et al. (2014), Li et al. (2017), Chowdhury et al. (2021), and Ghaffari et al. (2022). The sister group relationship of M. bilineata and M. dianbeiensis was also demonstrated by Salvi and Mariottini (2017, 2021), Al-Kandari et al. (2021), and Willan et al. (2021). However, the gene data of M. gryphoides is still scarce in NCBI-GenBank. Therefore, more molecular studies are required on this species and other Magallana species of the Indian waters to resolve their taxonomic uncertainties. Since, the systematics of true oysters is critical to developing the sustainable use of species and understanding the diversity of Oysters worldwide 368 Santhi et al./ New record of Crassostreine oyster, Magallana gryphoides (Sigwart et al., 2021), the gap areas should be immediately dealt with to document and conserve the biodiversity of Indian oysters. The aid of molecular taxonomy and evolutionary analyses via a phylogenetic approach would unmask the biodiversity and evolutionary reactions of Indian oysters that remained unnoticed and unknown to the world. Being the Keystone species, any conservation measures to this taxon will ultimately benefit the ecosystem. As the oyster population of India is radically decreasing (Laxmilatha, 2022), it has become an emergency issue to be dealt with. References Al-Kandari M., Oliver P.G., Salvi, D. (2021). 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