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Varying colour pattern, yet genetically similar: Pebble Crab Seulocia vittata (Stimpson, 1858) (Brachyura: Leucosiidae) from the southeastern coast of India

Five adult specimens of leucosiid crab Seulocia vittata (Stimpson, 1858) were recently collected off the coast of Palk Bay, southern India. Typical morphological examination revealed the presence of two colour patterns: grey and red. Interestingly, molecular analysis based on the barcoding gene cytochrome oxidase sub unit I (COI) revealed that both grey and red colour patterns in S. vittata showed 0% sequence divergence between the specimens. This indicates a situation of reverse cryptic behavior in this crab. Surprisingly, the evolutionary and ecological processes leading to the absence of genetic divergence and variation in morphology (colour pattern) in S. vittata complex remain to be addressed.


INTRODUCTION
Pebble crab or leucosiid crab belonging to the family Leucosiidae (Samouelle, 1819) is rich in diversity (Ng et al. 2008;Galil & Ng 2015). It mostly inhabits the sandy and silty areas adjacent to seagrass beds, coral reef flats, as well as intertidal areas usually buried in the sand (Naderloo & Apel 2012;Ng & Komatsu 2016). Leucosiid crabs of India have a long history where the key to the Indian Leucosia was first provided by Alcock (1896) during an "Investigator" expedition. At present, 97 species belonging to 35 genera of the family Leucosiidae have been reported from India (Trivedi et al. 2018). While revisiting the Leucosiidae classification, Galil (2005) proposed a new genus Seulocia which differs from other genera in the shared characters such as third to sixth abdominal somites fused in males and the straight shaft in the first pleopod of males twisted once on its axis. So far, 11 species have been described in this genus (Ng et al. 2008;WoRMS 2019), of which six species S. cristata (Galil, 2005), S. pubescens (Miers, 1884), S. pulchra (Galil, 2005), S. rhomboidalis (De Haan, 1841), S. truncata (Alcock, 1896), and S. vittata (Stimpson, 1858) have been recorded in Indian waters (Trivedi & Vachhrajani 2017;Trivedi et al. 2018). All of these above records were mainly based on the morphological characteristics and lack of information on genetic relatedness among them.
DNA barcoding along with morphological examination has been considered as a useful tool for the validation of species (Madhavan et al. 2020). This method can effectively identify cryptic species due to differences in their genetic character (Bucklin et al. 2007). No such study has been reported for the genus Seulocia. Hence, in the present study along with the detailed morphological examination, we used mitochondrial cytochrome oxidase subunit I (COI) gene to validate the taxonomy of S. vittata from the southeastern coast of India.

MATERIALS AND METHODS
The fish landing centre at Mandapam in Ramnad District of Tamil Nadu, India is one of the major landing sites in the southeastern coast of India (9.286 0 N & 79.153 0 E). A total of five crab specimens were handpicked from the freshly discarded by-catch of commercial trawlers at the fish landing during June-July 2019. Specimens were quickly cleaned to remove sediments and photographed (Cannon Powershot G16) in the field to record fresh colouration. The specimens were preserved in 95% ethanol and brought to Sathyabama Marine Research Station, Rameswaram for further detailed examination.

Morphological examination
The specimens were examined by comparing key morphological features and photographs described by Galil (2005). Four specimens (3 male, 1 female) were in red colouration (LR) and 1 male was in typical bluish-grey colour (LG) as described by Galil (2005). The carapace length (cl in mm) was measured from the tip of the rostrum in the anterior region to the posterior border of the carapace. The carapace width (cw in mm) was measured from the lateral margins of the carapace. The specimens were then deposited in the national zoological collection of the Marine Biological Regional Centre (MBRC), Zoological Survey of India (ZSI), Chennai, Tamil Nadu, India.

Molecular identification
One representative each of red and grey coloured specimens was subjected to molecular identification. Total genomic DNA from the propodus/meri region of the major cheliped of the crab was extracted using OMEGA BIO-TEK E.Z.N.A. Blood & Tissue DNA Kit, USA following the manufacturer's protocol. PCR amplification was done for the mitochondrial cytochrome oxidase subunit I (COI) gene using LCO-1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO-2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) primers (Folmer et al. 1994). Each PCR contained 12.5μL 2X PCR master mix (Ampliqon, Denmark), 2.5μL each of the two primers (10nM), and 2.5μL of template DNA (10-20 ng) and water to make a final volume of 25μL. PCR conditions were as follows: initial denaturation at 95°C for 10 min, 35 cycles of 95°C for 45 sec, 50°C for 45 sec, and 72°C for 45 min and final extension at 72°C for 10 min. PCR products were then visualized on 1% agarose and products with the high intensity band were sequenced with ABI Prism 3730 Genetic Analyzer based on BigDye Terminator Chemistry.
Chromatograms were visualized, edited, and contigs were prepared using consensus sequences from both the strands in the BioEdit (Hall 1999). Sequences obtained in the present study were deposited in NCBI GenBank. Sequences of COI from the present study were then compared with published COI sequences of related taxa from NCBI GenBank using BLASTn tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
COI sequences of species of the genus Seulocia and other J TT related genus belonging to the family Leucosiidae were downloaded and aligned in the web version of Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). The alignment consisted of two sequences obtained in the present study (MN786514, MN786515), two published sequences of the genus Seulocia: S. vittata (MH675982), S. latirostrata (MH675981) as ingroup terminals, as well as four sequences from other genera as outgroup terminal: Leucosia rubripalma (MH675986), L. craniolaris (MH675985), Euclosiana scitula (MH675980), and E. crosnieri (MH675978). The pairwise genetic distance between the species was determined by the Kimura 2-parameter method (Kimura 1980) using MEGA 7 (Kumar et al. 2016). The phylogenetic tree was constructed using maximum likelihood method based on the Tamura-Nei model in MEGA 7 . Bootstrap test was conducted using 1,000 replications to get the best topology from a 75% majority rule consensus tree (Felsenstein 1985).

Short description
Carapace sparsely punctate anteriorly, anterior margin tridenticulate with median denticle slightly larger than the adjacent ones. Anterolateral margin with minute beaded lines. Margin of epibranchial angle of carapace finely milled, epimeral margin evenly milled throughout. Posterior margin of carapace slightly rounded in male specimens (Both Grey and Red) and rounded in female specimens (Red). Thoracic sinus deep, pterygostomian region anteriorly defined by scalloped, overhanging and oblique margin. Fused abdominal segment bearing granule medially in male, smooth without granules in females. Merus of the major cheliped perliform, tubercles on the lateral margins, few tubercles (3-5) on the dorsal as well as ventral region. The upper margin of carpus and palm smooth and lower margins perliform, movable finger with upper margin carinate (both grey and red). Meri of the remaining pereiopods bearing beaded lines on the dorsal and ventral margin, carpi prominently carinate dorsally. Propodi of the pereiopods carinate in dorsal and ventral margins, dactyli flat and non-carinate on the lateral margins.

Colouration -Grey (Male)
Carapace bluish-grey, with median dorsal reddishbrown band becoming broader posteriorly (Image 1A-C). Presence of two oblique bands of reddish-brown on each side diverging from the front. Major cheliped meri, carpi and pal with a combination of reddish-brown and bluish-grey band, distal region reddish, fixed and movable finger whitish anteriorly and reddish posteriorly. Meri, carpi, propodi, and dactyli of the remaining pereiopods with a combination of reddish and white bands, tip of the dactyli brown to black. Abdominal region almost whitish (Image 1B). Terminal end of the maxillipeds dark bluish-grey (Image 1C).

Colouration -Red (Male and Female)
Anterior region of carapace bluish-grey, latter half of the carapace brick red to reddish-brown in colour in females (Image 2A-C). In males, carapace with bluishgrey extended to latter half, posterior region of carapace brick-red (Image 2D-F). Oblique bands are not visible on carapace (Image 2D). Dorsal and ventral region of the meri, carpi, and propodi of the major chelipeds brickred to reddish colour, dactyli of the movable dark red with a whitish tip (Image 2A,B). Meri of the remaining pereiopods brick-red in colour, carpi, propodi, and dactyli dark brown to black in colour. Abdomen brick red with big black patch at the centre in both females and males J TT (Image 2B,2E). Propodi and carpi of the major cheliped were black ventrally (Image 2B, 2E). Terminal end of the maxillipeds dark bluish-grey in both females and males (Image 2C, 2F).

DNA barcode and phylogenetic relationship
BLAST analysis revealed that the sequences for LG and LR exhibited 99.67% and 99.83% similarity with existing COI sequence of Seulocia vittata sequence in the NCBI GenBank. The phylogenetic analysis based on ML tree constructed using single mitochondrial gene fragment (COI -653bp) resulted in tree topology that S. vittata (grey and red colour patterns) are closely related to S. vittata available in the GenBank (Figure 1). Out of 653 sites, 148 were parsimony informative sites. In the ML tree, all the S. vittata clustered together to form a monophyletic clade. In addition, S. vittata is sister to its congener comprising Seulocia latirostrata and other related genera Leucosia and Euclosiana (Figure 1). Furthermore, the calculated pairwise genetic distance of COI gene fragment using Kimura-2 parameter revealed that LR, LG, and published S. vittata in the GenBank have no genetic divergence. However, the genetic distance of 0.177 was calculated between S. vittata and Seulocia latirostrata, which is comparable to the genetic distance of more than 0.200 with other genera such as Leucosia and Euclosiana in the family Leucosiidae (Table 1).

DISCUSSION
A nomenclatural and taxonomical validation of the brachyuran crabs of the world includes over 6793 species belonging to 1,271 genera and 93 families (Ng et al. 2008). Interestingly, recent advances in the molecular techniques have received greater attention in

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understanding the evolutionary perspectives of marine brachyuran crabs (Hultgren & Stachowicz 2008;Lai et al. 2013;Fratini et al. 2018;Mantelatto et al. 2018;Chen et al. 2019). The integrative approach of both morphological and molecular analyses offers robust information not only on the taxonomic ambiguity of the species, including cryptic species (Baeza & Prakash 2019) but also in the monitoring of commercial crabs for seafood safety (Rath et al. 2018). In India, the molecular based study in brachyuran crabs was very limited (Vartak et al. 2015;Apreshgi et al. 2016;Ravichandran et al. 2017;Rath et al. 2018;Madhavan et al. 2020). In the present study, we performed the molecular phylogeny as well as identified the pairwise sequence divergence study of Seulocia vittata and its congeners based on the COI gene fragment. The ML tree suggests that S. vittata was sister to the S. latirostrata and other related species that are supported with high bootstrap values.
Seulocia vittata has a wide geographic distribution in the Indo-Pacific from Mauritius, India, Singapore, Malaysia, Thailand, Indonesia, China, and Philippines (Galil 2005). The present study represents a rediscovery of S. vittata in the south east coast of India after the original report of Alcock (1896) during the "investigator" expedition. Moreover, no information exists on its varying colour pattern. We observed that though the individuals of S. vittata differed in their colour pattern (grey and red variants), their genetic distance showed no variation between these two-colour forms. This

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indicates a situation of reverse cryptic behaviour in this crab. Surprisingly, the evolutionary and ecological processes leading to the absence of genetic divergence and variation in morphology (colour pattern) in S. vittata complex remain to be addressed. There could be several possible explanations: a) S. vittata possess the capacity to change colour and camouflage in nature as anti-predatory mechanisms (Stevens et al. 2014); b) the colour variation could be due to ecological adaptation to different depths and habitats such as reefs and open sand flats (Darnell 2012); c) morphological colour variation with low genetic structuring may indicate high dispersal capacities of S. vittata throughout the evolutionary history of this species. However, to validate the above hypotheses, extensive sampling efforts and detailed examinations at larger geographical scales are required.
Based on the outcome of this study, we recommend integrative taxonomic and phylogeographic approaches to demonstrate the extent and magnitude of species complexity in the leucosiid crabs. This goal needs to be prioritized as there is a recent increase in the trawl net operations in the south eastern coast of India that could lead to decline in the benthic biodiversity (Purohit 2017). This could cause profound implications in the conservation planning, stock assessment, biogeography, evolutionary as well as the natural history of leucosiids. Lastly, the species complexity in S. vittata will provide the opportunity to understand the important mechanisms of speciation among the leucosiid crabs.