Indian Journal of Geo-Marine Sciences Vol. 44(1), January 2015, pp.

Classical and 16S rRNA gene marker confirmed first record of the Menippid rumphii (Fabricius, 1798) from the West coast of India

Vivek Rohidas Vartak1*, Rajendran N2 & Wazir S. Lakra3

1Khar Land Research Station, Panvel-410206, Maharashtra, India 2Vellore Institute of Technology, Vellore- 632 014, Tamil Nadu, India 3Central Institute of Fisheries Education, Andheri (E), Mumbai-400061, India * [E-mail: [email protected]]

Received 22 February 2014; revised 7 July 2014

The Menippe rumphii (Fabricius, 1798), marine crab, occurring along the rocky shores of coastal areas of east coast of India was located along the rocky shores of the Konkan coastal region of Maharashtra. Classical taxonomy is applied for identification of this crab. Taxonomic descriptions and morphometric analysis confirmed the species as Menippe rumphii. The 16S rRNA gene from Menippe rumphii adults was sequenced to corroborate species identification. Phylogenetic analysis showed ostensible between sequences of Menippe rumphii from NCBI and study crab. Classical taxonomy and 16S rRNA gene marker substantiated the record of the specimens from the Konkan coast.

[Key words: First record, Menippe rumphii, 16S rRNA gene, Classical taxonomy]

5 Introduction pictures of Chhapgar on the collections from this region also shored up this observation. After Menippid crab, Menippe rumphii (Fabricius, 4 1798) known as maroon stone crab is a coastal detailed taxonomic examination as per Alcock crab species distributed in the Pacific Ocean this species was identified as Menippe rumphii from Taiwan to Indonesia1. This crab is found in (Fabricius, 1798) and has a valid record from the Brunei Darsm, Cambodia, China, Indonesia, Chennai, east coast of India. To authenticate the Malaysia, Singapore, Taiwan, Thailand, Viet taxonomic identity of this crab we decided to Nam, Brazil, India (east coast), Sri Lanka, develop molecular marker for this species. Djibouti, Somalia, and Oman2, 3. Indian Museum Proper species identification is important for effective fisheries resource management and possesses 100 specimens of this crab from 6, 7, 8, 9, 10 Penang, Tavoy, Mergui, Chennai coast, Ceylon, commercialization . Taxonomic Lakshadweep, Karachi and Persian Gulf 4. This identification by DNA based methods is more crab is found along the rocky shores beneath sophisticated way which identifies specimens in stones, corals and other hiding spots. They have all stages. Molecular methods are more exact, large maroon colored oval body with red eyes. saves time and an advanced step of species This crab is edible and is captured with the help identification if utilized along with classical taxonomy. Earlier studies on molecular methods of gill net from rocky sea shores of Sindhudurg 9, district of coastal region of Maharashtra state, showed effectiveness in identifying 11, 12, 13, 14, 15, 16, 17. The use of 16S rRNA gene in India. This crab is a delicacy of the local people 18 of the Konkan coastal region of Maharashtra. crustaceans is reviewed by Schubart and Neigel This species falsely resembles to bashful crab suggesting molecular systematics as a highly Atergatis integerrimus and stone crab effective tool for documenting and classifying Myomenippe hardwickii. Preliminary the biodiversity of this world. The 16SrRNA gene is used for phylogenetic studies19 due to identification on the basis of pictures of related 20 species suggested that the species might be stone high conservancy between different species . crab Myomenippe hardwickii. Perusals of the Hence, we decided to make use of classical INDIAN J. MAR. SCI., VOL. 44, NO. 1 JANUARY 2015 taxonomy and 16S rRNA gene marker for The DNA extraction was carried out using the identification and validation of this species. standard SDS-phenolphenol/chloroform/chloroform method21 with slight modifications. Concentration of isolated Material and Methods DNA was estimated using a UV Sample collection and morphometrics spectrophotometer and diluted to a final concentration of 100 ng⁄µl. Amplification of TheThe first recorded Menippid crab Menippe 16S rRNA gene was carried out with universal rumphii caught in February of 2013 on the rocky primers:16Sar(5’-CCTGTTTATCAAAAACAT- shores of Kelus (15° 54' 12.0528'' N; 73° 35' 3’)and16Sbr(5’CCGGTCTGAACTCAGATCAC 35.7864'' E), located along the KonkanKonkan coastal GT-3’) 22. PCPCRR reaction includes 10X Taq region of MaharashtraMaharashtra State of West coast of polymerase buffer, 10 mM of dNTP mix; 0.6 India (Figure 1). unit of Taq polymerase; 0.01 mM of each primer and 100 ng of DNA template. General Lat: 15° 54' 23.28" thermocyclerthermocycler conditions were an initial step of 2 LLong:ong: 73° 35' 13.21" min at 95°C and 34 cycles of 30 s at 95°C, 40 s at 50°C and 1 min at 72°C, followed by a final extension at 72°C for 10 min. Amplified products were sequenced at Eurofins Genomics India Pvt. Ltd, Bangalore India. Sequences were aligned using CLUSTALW23 in BioEdit v. 7.2.3. For comparison and validation, the 16S rRNA bian Sea gene sequences for other sympatric species were Ara obtained from NCBI Gene Bank (Table 2). TheseThese sequences includes one sequence of Menippe rumphii ffromrom Singapore and 9 Figure 1 Location of Menippe rumphii crab record in SinSindhudurgdhudurg district (dark shade) of Maharashtra state of sequences of MenippMenippee adina, Menippe West coast of India mercenaria,mercenaria, Menippe nodifrons, Myomenippe hardwickii, Chlorodiella Cytherea, Xantho First specimen found was female with 90 mm poressa, Paraxanthus barbiger, Xantho pilipes carapace width (Figure 2). Total ten were and Atergatis integerrimus belonging to two found in catch and all were female. All crabs sympatric families, and have been carefully stored in absolute alcohol in of Brachyuran subsection Heterotremata. plastic bottles and taken to the laboratory for Evolutionary history was inferred using the identification. Crabs were ideidentifiedntified based on Neighbor-Joining (NJ) mmethodethod 24 with bootstrap phenotypic criteria such as morphology, colour support of 10000 replicates25. Branches and appendages. Voucher sspecimenspecimens were stored correspondingcorresponding to partitions reproduced in less 0 at -80 C in DNA barcoding laboratory. Hundred than 50% bootstrap replicates are collapsed. mgmg muscle tissue from chelipeds was removed Perercentagecentage of replicate trees in which the from both the crabs with sterile scissors and associated taxa clustered together in the stored in absolute alcohol at -20°C20°C for molecular bootstrap test is shown above the branches. All study. Nine morphometricmorphometric measurements with positionspositions containing gaps and missing data were five ratios were used in analysis (Table 1a). eliminated. Maximum Composite Likelihood method26 was ususeded for computing evolutionary Table 1a Measurements used for morphometric analysis distances, which are in tthehe units of the number of base substitutions per site. EvEvolutionaryolutionary analyses Sr. No. Measurements taken were conducted in MEGA528. 1 Carapace length (CL) 2 Carapace width (CW) Results 3 Major cheliped merus length (MEL) 4 Major cheliped merus width (MEW) Systematics 5 Major cheliped manus length (MAL) 6 Major cheliped dactylus length (DAL) Family: Menippidae Ortmann, 1893 7 Penultimate segment length (PL) Genus: Menippe De Haan, 1833 8 Penultimate segment width (PW) 9 Telson width (TW) Synonymized taxa: Alpheus rumphii Weber, 1795 (nomen nudum) DNA extraction, polymerase chain reaction and Cancer rumphii Fabricius, 1798 data analysis Pseudocarcinus bellangerii H. Milne Edwards, 1834 VARTAK et al.: FIRST RECORD OF MENIPPE RUMPHII FROM WEST COAST OF INDIA

Classification Molecular analysis Kingdom: Animalia, Phylum: ArthropodaArthropoda Neighbour Joining (NJ) tree involving Class: , Order: 18 nucleotide sequences is given in Figure 3. Super family: , Sequences of Menippe rumphii obtained under Family: Menippidae this study (Haplotypes 1-5) showed distinctive Species: Menippe rumphii clade with sequence of its haplotype (6) from Singapore. Other species under genus Menippe Morphological description clusteredclustered separately from Menippe rumphii Gastric region is distinct and fairly subdivided haplotypes without ambiguity, which indicated into three lobes. There are four blunt tubercles robustness of 16S rRNA gene in phylogenetic positioned in a square towards front between studies. Apart from this, NJ tree resulted in lobes and eyes. Brachial region has two low comprehensiblecomprehensible clade between two families indistinct transverse granular elevations which supported with higher bootstrap values run nearly parallel with the curve of the antero- indicating proper molecular differences between lateral border. First elevation is the more distinct these species. Numumberber of base differences per and can generally be traced across the gastric site between sequences is shown in Table 3. region also. Cardiac region has transverse Base differences per site between sequences of uninterrupted ridge. Surface of the carapace is Menippe rumphii from NCBI and present study smooth except antero-laterallateral fine pitting. Front was zero. Divergence was 0.08 and 0.18 of the carapace has two prominent round pointed between Menippe rumphii with other falsely lobes which ends with a prominent rounded resembling species viz. Myomenippe hardwickii tooth separated from the supra orbital margin by and AtergatAtergatisis integerrimus respectively. Thus, groove (for eyes). Antero-lalateralteral border is fairly resultsresults of molecular data analysis supports that sharp and distinguished by the four-tooth which the species identified and recorded was Menippe divides it into four broad lobes. Last two lobes rumphii. are distinctly acuminate than first two lobes. Chelipedsipeds are big and unequal as left is smaller thanthan right. They are smooth with fine and distant pitting on outer surface. Inner angle of wrist is bluntly prominent. Fingers are stout and black with short thumb. Legs are stout and smooth, except the upper border which is granular. Upper border of carpopodites is sparsely without any hairs. Borders of propodites and dactylus are thicker with hairs. Color of chelipeds is brownish yellow with a fine pattern of dark markings. This evaluationevaluation of description was first step to identify Menippe rumphii with classical taxonomy. TheThe ratios for morphometric measurements for female of MMenippeenippe rumphii is given in Table 1b.

Table 1b Ratios derived from morphometric measurements of the maroon Stone crab from thethe west coast of India

Ratios Menippe rumphii (♀) CW/CL 1.64±0.01

MEL/MEW 1.66±0.08 Discussion Identifying and recognizing a species MAL/DAL 2.18±0.07 without an adequate morphological description is 29 unfeasible . The wrong species identification PL/PW 0.45±0.05 shores up threats for biodiversity and food

PL/TW 0.48±0.09 safety. Sea food labeling is modern thought emerging worldwide to avoid fraud associated with the mislabeled sea food. In such cases, the proper identification of species becomes vital for INDIAN J. MAR. SCI., VOL. 44, NO. 1 JANUARY 2015

Table 2 List of crabs used for phylogenetic analysis with Gene Bank accession numbers

Name of species Family Common Name Country Gene Bank acc. numbers Menippe rumphii Menippidae Maroon Stone crab Maharashtra, India KF220514 to KF220518* Menippe rumphii Menippidae Maroon Stone crab Singapore HM637976 Menippe adina Menippidae Gulf stone crab Singapore HM637973 Menippe mercenaria Menippidae Florida stone crab Singapore HM637974 Menippe nodifrons Menippidae Cuban stone crab Singapore HM637975 Menippe nodifrons Menippidae Cuban stone crab Mexico AJ130817 Myomenippe hardwickii Menippidae Musca stone crab Singapore HM637977 Chlorodiella cytherea Xanthidae Venus green crab USA JQ277186 Xanthidae Purple crab USA JQ277185 Paraxanthus barbiger Xanthidae Not available Chile FJ031225 Xantho pilipes Xanthidae Risso's Crab Singapore HM637956 Xantho pilipes Xanthidae Risso's Crab Germany AM076775 Atergatis integerrimus Xanthidae Bashful crab Singapore HM798423 Atergatis integerrimus Xanthidae Bashful crab Maharashtra, India KF220494 *- Submitted to NCBI under this study

Menippe rumphii Haplotype1

Menippe rumphii Haplotype2

Menippe rumphii Haplotype3 A 87 Menippe rumphii Haplotype4

97 Menippe rumphii Haplotype5

Menippe rumphii Haplotype6 B Menippidae

Menippe nodifrons Haplotype1 99 Menippe nodifrons Haplotype2

Myomenippe hardwickii

Menippe mercenaria 99 Menippe adina

Paraxanthus barbiger

Atergatis integerrimus Haplotype1 97 100 86 Atergatis integerrimus Haplotype2

Xanthidae Chlorodiella cytherea

Xantho poressa 50 Xantho pilipes Haplotype1 99 Xantho pilipes Haplotype2 Figure 3 The Neighbour joining tree of sequences of Menippe rumphii with other nine species belonging to Xanthidae and Menippidae family. Bootstrap values are presented above the branch. Bootstrap values below 50% are not shown on the tree. ‘A’ indicates haplotypes for Menippe rumphii from present study and ‘B’ indicates haplotypes for Menippe rumphii obtained from NCBI Gene Bank. The separate for families are represented by right brace. VARTAK et al.: FIRST RECORD OF MENIPPE RUMPHII FROM WEST COAST OF INDIA

Table 3 Evolutionary divergence estimates between sequences of Menippe rumphii and other nine species

MR1 MR2 MR3 MR4 MR5 MR6 MM MA MN1 MN2 MH CC XP PB XP1 XP2 AT1 AT2 Menippe rumphii (Haplotype-1) 0.00 0.00 0.00 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 Menippe rumphii (Haplotype-2) 0.00 0.00 0.00 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 Menippe rumphii (Haplotype-3) 0.00 0.00 0.00 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 Menippe rumphii (Haplotype-4) 0.00 0.00 0.00 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 Menippe rumphii (Haplotype-5) 0.00 0.00 0.00 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 Menippe rumphii (Haplotype-6) 0.00 0.00 0.00 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 Menippe mercenaria 0.08 0.08 0.08 0.08 0.08 0.07 0.00 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 Menippe adina 0.08 0.08 0.08 0.08 0.08 0.07 0.00 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 Menippe nodifrons (Haplotype-1) 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.00 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 Menippe nodifrons (Haplotype-2) 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.00 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 Myomenippe hardwickii 0.08 0.08 0.08 0.08 0.08 0.07 0.07 0.07 0.09 0.09 0.02 0.02 0.02 0.02 0.02 0.02 0.02 Chlorodiella cytherea 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.20 0.20 0.21 0.02 0.01 0.02 0.02 0.01 0.01 Xantho poressa 0.18 0.19 0.19 0.19 0.19 0.19 0.20 0.20 0.20 0.20 0.21 0.14 0.01 0.01 0.02 0.01 0.01 Paraxanthus barbiger 0.18 0.18 0.18 0.18 0.18 0.18 0.17 0.17 0.19 0.19 0.19 0.12 0.11 0.02 0.02 0.01 0.01 Xantho pilipes (Haplotype-1) 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.20 0.20 0.21 0.11 0.11 0.11 0.01 0.01 0.01 Xantho pilipes (Haplotype-2) 0.20 0.20 0.20 0.20 0.20 0.20 0.19 0.19 0.20 0.20 0.21 0.13 0.13 0.12 0.01 0.01 0.02 Atergatis integerrimus (Haplotype-1) 0.18 0.18 0.18 0.18 0.18 0.18 0.17 0.17 0.18 0.18 0.20 0.08 0.11 0.10 0.10 0.11 0.00 Atergatis integerrimus (Haplotype-2) 0.18 0.18 0.18 0.18 0.18 0.18 0.17 0.17 0.19 0.19 0.20 0.09 0.11 0.10 0.11 0.12 0.01

The number of base differences per site from between sequences is shown. Standard error estimate(s) are shown above the diagonal and were obtained by a bootstrap procedure (10000 replicates). Menippidae family is separated by vertical black bar. The short codes created with first letter of genus and species are used on upper horizontal matrix bar. Dark highlight: Evolutionary divergence between Menippe rumphii and falsely resembling species, Faint highlight: Evolutionary divergence within Menippe rumphii haplotype INDIAN J. MAR. SCI., VOL. 44, NO. 1 JANUARY 2015

labeling the sea food. Serious and sincere efforts differences per site ranged from 0.0 to 0.21 are the main backbone for correct species between sequences of 10 crabs of Brachyuran identification and inventorisation. Hence, we subsection Heterotremata. In the present study, tried to search the database for this crab which zero divergence within the Menippe rumphii suggested unavailability of this crab from the haplotypes suggested significance of studying west coast of India. We found twenty three the divergence based on the number of base occurrences of Menippe rumphii crab from substitutions between the sequences. These world3 of which two reported in the Arabian Sea results gave vital support to the phylogenetic from Oman30 and Socotra Island, Yemen31. To analysis. At this stage, we studied one gene date, one credible occurrence for this crab is marker due to the robustness and unambiguous available from India reported from Chennai, East results obtained with 16S rRNA gene marker. coast of India4. These above records are reliable; There are other markers like COI, 12S, Cyt-B etc as the specimens are retained after confirm available for sequencing and studying taxonomic identification. Trivedi et al.32 reported phylogenetic relationship. Further scope is open occurrence of this crab from Gulf of Kutch, for DNA barcoding and developing more Gujarat, India. This record is not credible as the markers for this species. photograph publicized by authors is different Conclusion from the Menippe rumphii suggesting error in Record of Menippe rumphii (Fabricius, taxonomic identification. To confirm and 1798) from the west coast of India is thus authenticate our record we studied the confirmed in the present study. systematics and taxonomical description of this crab. Lai et al.33 used morphometric Acknowledgements measurements and ratios for discriminating Authors would like to thank authorities divergence between Portunus pelagicus species of Central Institute of Fisheries Education complex. In present study, we found (ICAR) Mumbai and Vellore Institute of morphometric measurements along with ratios as Technology, Vellore India for their support for valid and supportive choice in differentiating the this work. species. Classical taxonomy with molecular References marker used in this study played important role 1. Palomares MLD and Pauly D. SeaLifeBase. World Wide Web electronic publication in identifying the species. For this species, we www.sealifebase.org, version (2013). first time reported the 16S rRNA gene sequence 2. Peter NKL. Crabs. In K.E. Carpenter and V.H. 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