Molecular insights into the phylogenetics of spiny lobsters of Gulf of Mannar marine biosphere reserve based on 28S rdna

Indian Journal of Biotechnology Vol 11, April 2012, pp 182-186 Molecular insights into the phylogenetics of spiny lobsters of Gulf of Mannar marine biosphere reserve based on 28S rdna P Suresh*, G Sasireka and K A M Karthikeyan 1 Department of Zoology, Thiagarajar College (Autonomous), Madurai 625 009, India 1 Department of Zoology, N M S S V N College (Autonomous), Madurai 625 019, India Received 10 August 2010; revised 23 April 2011; accepted 21 May 2011 Four spiny lobster species, Panulirus versicolor, P. ornatus, P. homarus and P. polyphagus, and a mud lobster Thenus orientalis were collected from the Gulf of Mannar marine biosphere reserve. Partial 28S rrna gene sequences of the spiny lobsters were examined for their nucleotide diversity, pairwise genetic distances, transition/transversion rate and phylogenetic relationships. The spiny lobster species recorded higher transition over transversion; GC content was also higher as reported in many groups of animals. Pair-wise genetic distance analysis shows that all the spiny lobsters were distinctly distant from the outgroup species and had minimum distance among the ingroup species. A molecular phylogeny based on 28S rdna D2 sequences indicates that the four species of Panulirus form a monophyletic group. Keywords: Gulf of Mannar, marine biosphere reserve, molecular phylogeny, spiny lobsters, 28S rdna Introduction Life began in primordial seas around 3.6 billion years ago and arthropods evolved during the Cambrian. In the early period and still today, they comprise the most diverse group of animals and also constitute the most species amongst animal groups. Classical morphological studies have considered arthropods monophyletic 1,2, diphyletic 3 or polyphyletic 4,5. Several studies have attempted to solve the internal phylogenetic pattern of the arthropod groups using different molecular markers, different rrna sequences 6,7, histone H3 8, elongation factor 1-alpha 9 and mitochondrial gene order 10. Among the arthropods, pancrustaceans are the oldest and the crustaceans are monophyletic 11, mainly inhabiting marine ecosystems. Crustaceans are perhaps the most morphologically diverse group of arthropods, with huge variation in numbers and morphology of appendages, body organization, mode of development, and size 12. In 1864, Fritz Muller, in his famous book, Fur Darwin (English version: Facts and Arguments for Darwin ), summarized arguments in favour of the Darwinian descent with modification, based entirely on examples from crustaceans 13. An updated classification of the recent *Author for correspondence: Mobile: +91-9443807901 E-mail: suresh_63@yahoo.com crustacea distinguishes six classes and the Malacostraca includes lobsters 14. Lobsters are a polyphyletic group of crawling decapod crustaceans that have a cylindrical, usually laterally compressed carapace and prominent abdomen with tail fan. Within lobsters, evolutionary relationships also present some uncertainties 15. Patek and Oakley 16 analysed the phylogenetic relationships of Panulirus homarus, P. inflatus and P. versicolor and observed monophyletic origin based on 16S rdna, but those three species fell outside the genus with 18S data set. Palinurid genera are commonly divided into spiny lobsters with a stridulating organ (Linuparus, Palinustus, Puerulus, Palinurus, Panulirus & Justitia) and without a stridulating organ (Jasus, Sagmariasus & Projasus). Spiny lobsters are commonly found in rocky shores and are one of the most commercially important groups of the Paniluridae. The family Paniluridae, or spiny lobsters, consists of approximately 45 species and inhabit in all major oceans. Spiny or rock lobsters are common throughout tropical and subtropical seas 17 and are keystone predators playing an important role in community dynamics, as they hold populations of sea urchins and other herbivorous invertebrates in check 18. Of the fourteen littoral species and six species of deep sea forms occurring in India, only five species, viz., P. versicolor, P. ornatus, P. homarus,

SURESH et al: PHYLOGENETICS OF THE SPINY LOBSTERS 183 P. polyphagus and Thenus orientalis, of littoral species contribute to the fishery. Molecular tools have been proved more authenticated and powerful in resolving controversies, even, of deep phylogenetic levels. Ribosomal RNA genes provided useful information in inferring the phylogeny of crustaceans at different systematic hierarchies. Ribosomal genes of different regions have evolved at varying rates, making them useful in phylogenetic analysis across a broad taxonomic spectrum. 28S rdna is very large and its D2 region is comparatively conserved, so it is chosen for phylogenetic studies. The present paper presents the results of a phylogenetic analysis of partial sequences of 28S rdna to establish monophyly of the genus Panulirus, using the closely related slipper lobster T. orientalis as an outgroup species. Materials and Methods Single male specimen for each lobster species, viz., P. versicolor, P. ornatus, P. homarus, P. polyphagus and T. orientalis were procured alive from fisher man s catch from Mandapam (9.17 N & 79.22 E), Gulf of Mannar marine biosphere reserve and brought to the laboratory in ice-cold containers. Genomic DNA was extracted from a single male individual of each species. Muscle tissues (15 mg) from carapace were taken with liquid nitrogen and ground in a glass homogeniser containing lysis buffer with 1% SDS, proteinase K (500 µg/ml) and RNAse, and kept at 37 C for 3 h. The DNA was extracted in phenol/chloroform. 28S rdna-d2 sequences were amplified on a MJ Mini-Bio Rad gradient thermal cycler. Primer sequences for the amplification of the 28S rdna region were: Forward primer 5'-TACCGTGAGGGAAAGTTGAAA-3' and reverse primer 5'-AGACTCCTTGGTCCGTGTTT-3'. The primer set resulted in the amplification of the homologous fragment from all the five lobster species. The PCR reaction mix was prepared in a total volume of 50 µl with 100 ng of genomic DNA, 2.5 mm concentration each of datp, dttp, dctp and dgtp, 100 ng each of the forward and reverse primers, 3 U of Taq DNA polymerase and 1 Taq DNA polymerase assay buffer (10 ), and the remaining volume with glass distilled water (Bangalore Genei, India). The PCR reaction cycles consisted of initial denaturation for 5 min at 94 C, 35 cycles of 94 C for 30 sec, 60 C for 40 sec and 72 C for 40 sec, and followed by the final extension of 72 C for 10 min. The PCR products were gel purified with Gel extraction kit (Bangalore Genei, India) and directly sequenced by dideoxy chain termination method 19 using the BigDye terminator kits in the ABI 3130 Genetic Analyzer, according to manufacturer s protocols. The sequences were then deposited in the EMBL/DDBJ/GenBank data libraries of the NCBI and aligned using CLUSTAL X 20. Following the sequence variation estimation, neighbor-joining (NJ), minimum evolution (ME) and maximum parsimony (MP) based phylogenetic analysis of the 28S rdna were conducted using MEGA version 4 21. Bootstrap analyses of 1000 replications were also conducted on the trees inferred from the NJ, ME and MP methods. Results and Discussion The 28S rdna-d2 sequences were submitted to the NCBI-GenBank under accession numbers: P. versicolor-fj966368, P. ornatus-fj966369, P. homarus-fj966370, P. polyphagus-fj966371 and T. orientalis-fj966372. Table 1 provides basic statistics on nucleotide information for the D2 region sequences. The sequence length varied from 463 bp (P. polyphagus) to 575 bp (P. ornatus). The average contents of T, C, A and G were 22.3, 25.4, 18.6 and 33.7%, respectively. The GC content was higher compared to AT in a narrow range in almost all the species, including the outgroup species. The D2 sequences of the lobster species appeared most informative; 325 were conserved, 141 were variable, 122 were singleton and 18 were parsimony informative. Pairwise genetic distance across the D2 sequence data among the ingroup species was very Table 1 Nucleotide base composition of 28S rdna sequence of the spiny lobster genus Panulirus Species Acc. no. Length (bp) A T G C GC content (%) Panulirus versicolor FJ966368 461 89 103 152 117 58.35 P. ornatus FJ966369 575 111 124 199 141 59.13 P. homarus FJ966370 464 81 105 157 121 59.91 P. polyphagus FJ966371 463 85 101 157 120 59.82 Thenus orientalis FJ966372 407 75 96 134 102 57.99

184 INDIAN J BIOTECHNOL, APRIL 2012 minimum (0.016 to 0.078) compared to the outgroup species (0.343 to 0.379). P. polyphagus was recorded more close with the outgroup species than all other Panulirus spp. studied. P. versicolor and P. ornatus were highly distant (0.078) than all other ingroup species combinations, and P. homarus and P. polyphagus were closest (0.016) (Table 2). The transition/transversion rate ratios are k 1 =1.282 (purines) and k 2 =2.041 (pyrimidines). Based on the nucleotide substitution analysis, the nucleotide frequencies were 0.181 (A), 0.225 (T), 0.26 (C) and 0.334 (G). The overall transition/transversion bias was R=1.002. Maximum Composite Likelihood estimate of the pattern of nucleotide substitution recorded the transitional substitutions: A G=11.74, T C=14.51, C T=12.6 and G A=6.37, and transversional substitutions: A T=6.17, A C=7.11, T A=4.96, T G=9.15, C A=4.96, C G=9.15, G T=6.17 and G C=7.11 (Table 3). Several methods have been developed to assess phylogenetic relationships among species by using their nucleotide sequence variations of specific regions in genome. Following the sequence variation estimation of 28S rdna of the lobster species, neighbor joining, minimum evolution and maximum parsimony implemented in MEGA4 were used for the phylogenetic reconstruction. Phylogenetic tree constructions showed a congruent and identical tree topology for all types of trees constructed, namely neighbor joininig, minimum evolution and maximum parsimony (Figs 1 to 3), including bootstrap method (Figs 4 to 6). In the analyses, all the four species of the genus Panulirus are clustered into one major clade and the outgroup species T. orientalis is separated out in all the phylogenetic trees. Within the major clade, P. homarus and P. polyphagus are grouped in the first inner branch and P.versicolor and P.ornatus are placed in separate inner branches. The bootstrap values recorded among the four ingroup species was above 50 in the neighbor joining and minimum evolution trees. Monophyletic relationship within the genus Panulirus is strongly maintained in all our phylogenetic analyses. The knowledge of phylogeny is an important prerequisite to understand the evolution of morphological, ecological and behavioural adaptations. This study corroborates the divergence pattern and phylogenetic relationship among the four species in the genus Panulirus spp. based on their 28S rdna D2 sequences. Comparisons of the DNA sequences of metazoa show an excess of transitional over transversional substitutions 22. It is generally assumed that the ratio of transitions to transversions is higher in animal genomes, possibly as a result of the underlying chemistry of mutation. The spiny lobster species also recorded higher transition over transversion and GC content was also higher as reported in many groups of animals. Pair-wise genetic distance analysis shows that all the spiny lobsters distinctly distant from the outgroup species and had minimum distance among the ingroup species. Prior studies on evolution of the spiny lobsters 23, and molecular studies based on 16S rdna and cytochrome oxidase subunit I, have also reported the monophyletic origin of Panulirus 24. The results of our phylogenetic analyses consistently suggest that the Table 2 Pair-wise distance of 28S rdna sequence of the spiny lobster genus Panulirus Table 3 Maximum composite likelihood estimate of the pattern of nucleotide substitution in 28S rdna of the spiny lobsters P. versicolor P. ornatus P. homarus P. polyphagus T. orientalis Panulirus versicolor --- P. ornatus 0.078 --- P. homarus 0.063 0.075 --- P. polyphagus 0.060 0.072 0.016 --- Thenus orientalis 0.379 0.352 0.344 0.343 --- A T C G A -- 6.17 7.11 11.74 T 4.96 --- 14.51 9.15 C 4.96 12.6 --- 9.15 G 6.37 6.17 7.11 --- Only entries within a row be compared Fig. 1 Neighbor joining tree showing relationship amongst the species of spiny lobster genus Panulirus

SURESH et al: PHYLOGENETICS OF THE SPINY LOBSTERS 185 joining, minimum evolution, maximum parsimony and bootstrap in the present study. Fig. 2 Minimum evolution tree showing relationship amongst the species of spiny lobster genus Panulirus Fig. 3 Maximum parsimony tree showing relationship amongst the species of spiny lobster genus Panulirus Fig. 4 Neighbor joining tree (bootstrap) showing relationship amongst the species of spiny lobster genus Panulirus Fig. 5 Minimum evolution tree (bootstrap) showing relationship amongst the species of spiny lobster genus Panulirus Fig. 6 Maximum parsimony tree (bootstrap) showing relationship amongst the species of spiny lobster genus Panulirus spiny lobster genus Panulirus is monophyletic and all the four species studied are closely related with each other, this is supported in pairwise distance analysis and phylogenetic tree reconstruction using neighbor Acknowledgement The present work was supported by the University Grants Commission, New Delhi (F.No. 32-492/2006 SR). Authors are grateful to the Management for kind support and encouragement. References 1 Lankester E R, The structure and classification of the Arthropoda, Quart J Microscop Sci, 47 (1904) 523-582. 2 Snodgrass R, Evolution of the Annelida, Onychophora and Arthropoda, Smithson Misc Coll, 97 (1938) 1-159. 3 Tiegs O W & Manton S M, The evolution of the Arthropoda, Biol Rev, 33 (1958) 255-337. 4 Anderson D T, Embryology and phylogeny in annelids and arthropods (Pergamon Press, Oxford, UK) 1973. 5 Manton S M, Arthropod phylogeny A modern synthesis, J Zool, 171 (1973) 111-130. 6 Wheeler W, Molecular systematics and arthropods, in Arthropod fossils and phylogeny, edited by G D Edgecombe (Columbia University Press, New York, USA) 1998, 9-32. 7 Giribet G & Ribera C, A review of arthropod phylogeny: New data based on ribosomal DNA sequences and direct character optimization, Cladistics, 16 (2000) 204-231. 8 Colgar D J, McLauchlan A, Wilson G D F, Livingston, S P, Edgecombe G D et al, Histone H3 and U2 snrna DNA sequences and arthropod molecular evolution, Aust J Zool, 46 (1998) 419-437. 9 Regier J C & Shultz J W, Molecular phylogeny of arthropods and the significance of the Cambrian explosion for molecular systematic, Am Zool, 38 (1998) 918-928. 10 Boore J L, Lavrov D V & Brown W M, Gene translocation links insects and crustaceans, Nature (Lond), 392 (1998) 667-668. 11 Pisani D, Poling L L, Lyons-Weiler M & Hedges S B, The colonization of land by animals: Molecular phylogeny and divergence times among arthropods, BMC Evol Biol, 2 (2004) 1-10. 12 Wheeler W C, Giribet G & Edgecombe G D, Assembling the tree of life, in Arthropod systematics, edited by J Cracraft & M J Donoghue (Oxford University Press, New York) 2004, 281-295. 13 Richter S, Moller O S & Wirkner C S, Advances in Crustacean phylogenetics, Arthropod Syst Phyl, 67 (2009) 275-286. 14 Martin J W & Davis G E, An updated classification of recent Crustacea, in Science series 39 (Natural History Museum of Los Angeles County, California, USA) 2001. 15 Palero F, Crandall K A, Abello P, Macpherson E & Pascual M, Phylogenetic relationships between spiny, slipper and coral lobsters (Crustacea, Decapoda, Achelata), Mol Phyl Evol, 50 (2009) 152-162. 16 Patek S N & Oakley T H, Comparative tests of evolutionary trade-offs in a palinurid lobster acoustic system, Evolution, 57 (2003) 2082-2100. 17 Holthuis L B, Marine lobsters of the World, in FAO species catalogue, vol 13, FAO Fisheries Synopsis, No. 125 (Food

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