Indian Journal of Biotechnology Vol 11, April 2012, pp 182-186

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

P Suresh*, G Sasireka and K A M Karthikeyan1 Department of Zoology, Thiagarajar College (Autonomous), Madurai 625 009, 1Department 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 species, versicolor, P. ornatus, P. homarus and P. polyphagus, and a mud lobster 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 species recorded higher transition over transversion; GC content was also higher as reported in many groups of . 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 crustacea distinguishes six classes and the Life began in primordial seas around 3.6 billion includes lobsters14. Lobsters are a years ago and evolved during the polyphyletic group of crawling decapod . In the early period and still today, they that have a cylindrical, usually laterally compressed comprise the most diverse group of animals and also carapace and prominent abdomen with tail fan. constitute the most species amongst groups. Within lobsters, evolutionary relationships also Classical morphological studies have considered present some uncertainties15. Patek and Oakley16 arthropods monophyletic1,2, diphyletic3 or analysed the phylogenetic relationships of Panulirus polyphyletic4,5. Several studies have attempted to homarus, P. inflatus and P. versicolor and observed solve the internal phylogenetic pattern of the monophyletic origin based on 16S rDNA, but those groups using different molecular markers, three species fell outside the genus with 18S data set. different rRNA sequences6,7, histone H38, elongation Palinurid genera are commonly divided into spiny factor 1-alpha9 and mitochondrial gene order10. lobsters with a stridulating organ (Linuparus, Among the arthropods, pancrustaceans are the oldest Palinustus, Puerulus, Palinurus, Panulirus & Justitia) and the crustaceans are monophyletic11, mainly and without a stridulating organ (, inhabiting marine ecosystems. Crustaceans are & Projasus). Spiny lobsters are commonly found in perhaps the most morphologically diverse group of rocky shores and are one of the most commercially arthropods, with huge variation in numbers and important groups of the Paniluridae. The family morphology of appendages, body organization, mode Paniluridae, or spiny lobsters, consists of of development, and size12. In 1864, Fritz Muller, in approximately 45 species and inhabit in all major his famous book, ‘Fur Darwin’ (English version: oceans. Spiny or rock lobsters are common ‘Facts and Arguments for Darwin’), summarized throughout tropical and subtropical seas17 and are arguments in favour of the Darwinian ‘descent with keystone predators playing an important role in modification’, based entirely on examples from community dynamics, as they hold populations of sea crustaceans13. An updated classification of the recent urchins and other herbivorous invertebrates in ______check18. Of the fourteen littoral species and six *Author for correspondence: species of deep sea forms occurring in India, only five Mobile: +91-9443807901 E-mail: [email protected] species, viz., P. versicolor, P. ornatus, P. homarus, SURESH et al: PHYLOGENETICS OF THE SPINY LOBSTERS 183

P. polyphagus and Thenus orientalis, of littoral and dGTP, 100 ng each of the forward and reverse species contribute to the fishery. primers, 3 U of Taq DNA polymerase and 1× Taq Molecular tools have been proved more DNA polymerase assay buffer (10×), and the authenticated and powerful in resolving controversies, remaining volume with glass distilled water even, of deep phylogenetic levels. Ribosomal RNA (Bangalore Genei, India). The PCR reaction cycles genes provided useful information in inferring the consisted of initial denaturation for 5 min at 94°C, phylogeny of crustaceans at different systematic 35 cycles of 94°C for 30 sec, 60°C for 40 sec and hierarchies. Ribosomal genes of different regions 72°C for 40 sec, and followed by the final extension have evolved at varying rates, making them useful in of 72°C for 10 min. The PCR products were gel phylogenetic analysis across a broad taxonomic purified with Gel extraction kit (Bangalore Genei, spectrum. 28S rDNA is very large and its D2 region is India) and directly sequenced by dideoxy chain comparatively conserved, so it is chosen for termination method19 using the BigDye terminator phylogenetic studies. The present paper presents the kits in the ABI 3130 Genetic Analyzer, according to results of a phylogenetic analysis of partial sequences manufacturer’s protocols. The sequences were then of 28S rDNA to establish monophyly of the genus deposited in the EMBL/DDBJ/GenBank data libraries Panulirus, using the closely related of the NCBI and aligned using CLUSTAL X20. T. orientalis as an outgroup species. Following the sequence variation estimation, neighbor-joining (NJ), minimum evolution (ME) and Materials and Methods maximum parsimony (MP) based phylogenetic Single male specimen for each lobster species, viz., analysis of the 28S rDNA were conducted using P. versicolor, P. ornatus, P. homarus, P. polyphagus MEGA version 421. Bootstrap analyses of 1000 and T. orientalis were procured alive from fisher replications were also conducted on the trees inferred man’s catch from Mandapam (9.17° N & 79.22° E), from the NJ, ME and MP methods. Gulf of Mannar marine biosphere reserve and brought to the laboratory in ice-cold containers. Genomic Results and Discussion DNA was extracted from a single male individual of The 28S rDNA-D2 sequences were submitted to each species. Muscle tissues (15 mg) from carapace the NCBI-GenBank under accession numbers: were taken with liquid nitrogen and ground in a glass P. versicolor-FJ966368, P. ornatus-FJ966369, homogeniser containing lysis buffer with 1% SDS, P. homarus-FJ966370, P. polyphagus-FJ966371 and proteinase K (500 µg/mL) and RNAse, and kept at T. orientalis-FJ966372. Table 1 provides basic 37°C for 3 h. The DNA was extracted in statistics on nucleotide information for the D2 region phenol/chloroform. 28S rDNA-D2 sequences were sequences. The sequence length varied from 463 bp amplified on a MJ Mini-Bio Rad gradient thermal (P. polyphagus) to 575 bp (P. ornatus). The average cycler. Primer sequences for the amplification of the contents of T, C, A and G were 22.3, 25.4, 18.6 and 28S rDNA region were: Forward primer 33.7%, respectively. The GC content was higher 5'-TACCGTGAGGGAAAGTTGAAA-3' and reverse compared to AT in a narrow range in almost all the primer 5'-AGACTCCTTGGTCCGTGTTT-3'. The species, including the outgroup species. The D2 primer set resulted in the amplification of the sequences of the lobster species appeared most homologous fragment from all the five lobster informative; 325 were conserved, 141 were variable, species. The PCR reaction mix was prepared in a total 122 were singleton and 18 were parsimony volume of 50 µL with 100 ng of genomic DNA, informative. Pairwise genetic distance across the D2 2.5 mM concentration each of dATP, dTTP, dCTP 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 A T G C GC (bp) 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 and the outgroup species T. orientalis is separated out species (0.343 to 0.379). P. polyphagus was recorded in all the phylogenetic trees. Within the major clade, more close with the outgroup species than all other P. homarus and P. polyphagus are grouped in the first Panulirus spp. studied. P. versicolor and P. ornatus inner branch and P.versicolor and P.ornatus are were highly distant (0.078) than all other ingroup placed in separate inner branches. The bootstrap species combinations, and P. homarus and values recorded among the four ingroup species was P. polyphagus were closest (0.016) (Table 2). The above 50 in the neighbor joining and minimum transition/transversion rate ratios are k1=1.282 evolution trees. Monophyletic relationship within the (purines) and k2=2.041 (pyrimidines). Based on the genus Panulirus is strongly maintained in all our nucleotide substitution analysis, the nucleotide phylogenetic analyses. frequencies were 0.181 (A), 0.225 (T), 0.26 (C) and The knowledge of phylogeny is an important 0.334 (G). The overall transition/transversion bias prerequisite to understand the evolution of was R=1.002. Maximum Composite Likelihood morphological, ecological and behavioural estimate of the pattern of nucleotide substitution adaptations. This study corroborates the divergence recorded the transitional substitutions: A→G=11.74, pattern and phylogenetic relationship among the four T→C=14.51, C→T=12.6 and G→A=6.37, and species in the genus Panulirus spp. based on their 28S transversional substitutions: A→T=6.17, A→C=7.11, rDNA D2 sequences. Comparisons of the DNA T→A=4.96, T→G=9.15, C→A=4.96, C→G=9.15, sequences of metazoa show an excess of transitional G→T=6.17 and G→C=7.11 (Table 3). over transversional substitutions22. It is generally Several methods have been developed to assess assumed that the ratio of transitions to transversions is phylogenetic relationships among species by using higher in animal genomes, possibly as a result of the their nucleotide sequence variations of specific underlying chemistry of mutation. The spiny lobster regions in genome. Following the sequence variation species also recorded higher transition over estimation of 28S rDNA of the lobster species, transversion and GC content was also higher as neighbor joining, minimum evolution and maximum reported in many groups of animals. Pair-wise genetic parsimony implemented in MEGA4 were used for the distance analysis shows that all the spiny lobsters phylogenetic reconstruction. Phylogenetic tree distinctly distant from the outgroup species and had constructions showed a congruent and identical tree minimum distance among the ingroup species. Prior topology for all types of trees constructed, namely studies on evolution of the spiny lobsters23, and neighbor joininig, minimum evolution and maximum molecular studies based on 16S rDNA and parsimony (Figs 1 to 3), including bootstrap method cytochrome oxidase subunit I, have also reported the (Figs 4 to 6). In the analyses, all the four species of monophyletic origin of Panulirus24. The results of our the genus Panulirus are clustered into one major clade phylogenetic analyses consistently suggest that the

Table 2—Pair-wise distance of 28S rDNA sequence of the spiny lobster genus Panulirus

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 --- Table 3—Maximum composite likelihood estimate of the pattern of nucleotide substitution in 28S rDNA of the spiny lobsters

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 --- Fig. 1—Neighbor joining tree showing relationship amongst the Only entries within a row be compared 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.

Acknowledgement The present work was supported by the University Grants Commission, New Delhi (F.No. 32-492/2006–SR). Authors are grateful to the

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