The University of Maine DigitalCommons@UMaine Marine Sciences Faculty Scholarship School of Marine Sciences 2-1-1998 Phylogenetic Relationships of Clawed Lobster Genera (Decapoda : Nephropidae) Based on Mitochondrial 16S rRNA Gene Sequences Y. K. Tam Irv Kornfield University of Maine - Main, [email protected] Follow this and additional works at: https://digitalcommons.library.umaine.edu/sms_facpub Repository Citation Tam, Y. K. and Kornfield, Irv, "Phylogenetic Relationships of Clawed Lobster Genera (Decapoda : Nephropidae) Based on Mitochondrial 16S rRNA Gene Sequences" (1998). Marine Sciences Faculty Scholarship. 89. https://digitalcommons.library.umaine.edu/sms_facpub/89 This Article is brought to you for free and open access by DigitalCommons@UMaine. It has been accepted for inclusion in Marine Sciences Faculty Scholarship by an authorized administrator of DigitalCommons@UMaine. For more information, please contact [email protected]. JOURNALOF CRUSTACEANBIOLOGY, 18(1): 138-146, 1998 PHYLOGENETIC RELATIONSHIPSOF CLAWED LOBSTER GENERA (DECAPODA: NEPHROPIDAE) BASED ON MITOCHONDRIAL 16S rRNA GENE SEQUENCES YanKit Tamand Irv Kornfield ABSTRACT Approximately350 base pairs(bp) of the mitochondrial16S rRNAgene were used to studythe phylogeneticrelationships among 5 generaof the clawed lobsterfamily Nephropidae(infraorder Astacidea), including Homarus, Homarinus, Metanephrops, Nephrops, and Nephropsis. Maximum- parsimonyanalysis, using a hermitcrab, Pagurus pollicaris (infraorder Anomura), as an outgroup, produceda tree topologyin whichHomarus and Nephrops formed a well-supportedclade thatex- cludedHomarinus. The sametree topology was obtainedfrom both neighbor-joining and maximum- likelihoodanalyses. Some morphologicalcharacters that appear synapomorphic for Nephropsand Metanephropsmay be due to convergencerather than symplesiomorphy.The currenttaxonomy, therefore,does not reflectthe phylogenyof this groupas suggestedby the moleculardata. More moleculardata and studiesusing homologousmorphological characters are neededto reacha bet- ter understandingof the phylogenetichistory of clawedlobsters. Clawed lobsters are marinedecapods be- ical charactersmay provide valuable infor- longing to the superfamily Nephropoidea mationfor taxonomicclassification, the char- (Decapoda: Astacidea). The Nephropidae acters used in distinguishingdifferent taxa Dana, 1852, comprisingthree subfamilies and may not be homologous structuresand thus eleven genera, contains most of the clawed may not containphylogenetic signals. Thus, lobstersin this superfamily(Holthuis, 1974). the taxonomic classification of clawed lob- Amongthem are the commerciallyimportant sters may not reflect the phylogenetic rela- genera Homarus Weber, 1795, Nephrops tionships of the group. Homologous struc- Leach,1814, and Metanephrops Jenkins, 1972. turesneed to be definedand appliedin mak- Holthuis(1991) presenteda comprehensivere- ing phylogeneticinferences (Tshudy, 1993); view of clawed lobsters, emphasizingthose homoplasyof characters,such as convergence that are of interestto global fisheries. and parallelism,produce noise and mislead The Nephropidaeis an old family which data analysis. Dependingon the degree that has a fossil recordextending from the Mid- some morphologicalcharacters are conver- dle Jurassicto the Recent (Glaessner,1969). gent, phylogenetic inferences may be com- Ornamentationson the carapaceof fossils are promised.This concernapplies to inferences well preserved;these patternsof grooves and aboutfossil nephropidsas well. eminencesmay provideclues to the evolution Molecularcharacters, particularly DNA se- of lobster lineages. Based on carapacemor- quencedata, provide an independentdata set phology,Glaessner (1969) proposeda hypo- with which to construct phylogenetic hy- theticalphylogeny of astacideans.In this, the potheses(Hillis et al., 1996). Molecularstud- fossil genusPalaeophoberus Glaessner, 1932, ies of phylogenetic relationships among gave rise to the genus Hoploparia McCoy, clawed lobstersare limited.Chu et al. (1990) 1849, one line of which developedinto cray- studiedenzyme polymorphismin three spe- fish (Astacus), and anotherline into Nephrops- cies of Metanephropsin Taiwan.Hedgecock like andHomarus-like lobsters. It hadbeen pro- et al. (1977) examinedthe genetic variation posed thatfossil and Recentnephropids were between Homarus americanus H. Milne Ed- composedof two subfamilies(Mertin, 1941), wards, 1837, and H. gammarus(Linnaeus, the Nephropinaeand the Homarinae.How- 1758) by using allozyme data. The present ever, based on the morphologyof living lob- study,using molecularcharacters as an alter- sters,Holthuis (1974) did not acceptthe idea. native approachto morphology,provides in- The currenttaxonomy of clawedlobsters is dependentclues about the phylogeny of the based on morphologicalfeatures (Glaessner, clawed lobsters. By using universalprimers 1969; Holthuis,1991). Althoughmorpholog- within a conservative region in the mito- 138 TAMAND KORNFIELD:PHYLOGENETICS OF CLAWEDLOBSTER GENERA 139 Table 1. Species studied and the sampling localities. Species Infraorder Abbreviations Sampling locality Homarus americanus H. Milne Edwards, 1837 Astacidea HA Gulf of Maine, U.S.A. Homarus gammarus (Linnaeus, 1758) Astacidea HG Guernsey, U.K. Homarinus capensis (Herbst, 1792) Astacidea HC Cape Province, South Africa Metanephrops mozambicus Macpherson, 1990 Astacidea MM Natal, South Africa Nephrops norvegicus (Linnaeus, 1758) Astacidea NN Celtic Sea, Ireland Nephropsis aculeata Smith, 1881 Astacidea NA Massachusetts, U.S.A. Nephropsis stewarti Wood-Mason, 1872 Astacidea NS Natal, South Africa Panulirus longipes (A. Milne Edwards, 1868) Palinuridea PL South China Sea, Hong Kong Scyllarides nodifer (Stimpson, 1866) Palinuridea SN Gulf of Mexico, U.S.A. Pagurus pollicaris Say, 1817 Anomura PP Massachusetts, U.S.A. chondrial genome, a homologous DNA seg- Pagurus pollicaris Say, 1817 (infraorderAnomura), was ment from all the taxa can be amplified and used as an outgroup for parsimony analysis. DNA was prepared from samples of muscle from the compared. abdomen or pereiopods. MtDNA was prepared by phe- A now conventional source for DNA se- nol/chloroform extraction of proteinase-K-digested tis- quences is the mitochondrion (Avise, 1994), sues (Ausubel et al., 1989). The DNA templates were then an organelle that contains multiple copies of subjected to polymerase chain reaction (PCR) (Saiki et standard et a small, maternally inherited genome, mito- al., 1988), using protocols (Palumbi al., 1991). A 570-base-pair (bp) region within the mtl6S rRNA chondrial DNA (mtdna). MtDNA sequences (16S) gene was amplified using primers 16SA (5'CGC- were used to define taxonomic relationships CTGTTTATCAAAAACAT3') and 16SB (5'CTCCG- among species of Homarus and distinguish GTTTGAACTCAGATC3') (Xiong and Kocher, 1991). from them the genus Homarinus Komfield, PCR amplification was performed using 30 cycles of 30 s s / the lobster of South Africa 94?C, / 50?C, 60 72?C, 90 s; the initial denatu- 1995, Cape ration step at 94?C lasted 5 min and the final extension (Kornfield et al., 1995). The 16S ribosomal step at 72?C lasted 10 min. Double-stranded PCR prod- RNA (rRNA) gene in the mitochondrial ucts were subjected to asymmetric PCR from both 5'-di- genome contains conservative regions which rection to generate templates for dideoxy sequencing et have been used in many phylogenetic stud- (Sanger al., 1977), using 35 cycles of the same PCR conditions. Prior to sequencing, PCR products were pu- ies at generic and higher taxonomic levels for rified by filtration through Millipore Ultrafree-MC re- a wide variety of organisms (Hillis et al., generated cellulose membrane filters of 30,000 nominal 1996). Recently, Crandall and Fitzpatrick molecular weight limit (NMWL) (Millipore Corporation). (1996) used a mitochondrial 16S rRNA gene Sequences were aligned using ESEE (Cabot and Beck- to the of enbach, 1989). Secondary structures of the partial mtl6S sequence study relationships cray- rRNA gene sequences were inferredusing Mulford (Jaeger fishes. The present study examined relation- et al., 1989) to assure homologous sequence alignment. ships among five of the 11 extant genera of Sequences without 2 highly variable regions (Parker and the family Nephropidae by comparing par- Kornfield, 1996) were subjected to all data analyses (Fig. A matrix of tial sequences of the mtl6S rRNA gene. Our 1). sequence divergence using Kimura's two- parametermethod (Kimura, 1980) was generated and sub- objective was to compare phylogenetic hy- jected to neighbor-joining analysis using MEGA (Kumar potheses based on this molecular data set to et al., 1993). Five hundred bootstrap replicates were per- the current taxonomy based on morphologi- formed to access the confidence level at each branch cal features. (Felsenstein, 1985). Maximum-likelihood trees were con- structed using the program DNAML in PHYLIP, Version 3.5 was con- MATERIALSAND METHODS (Felsenstein, 1993). Parsimony analysis ducted using the exhaustive search option in PAUP Ver- Seven species in 5 genera of the clawed lobster fam- sion 3.1.1 (Swofford, 1993); alignment gaps were in- ily Nephropidae were studied: Homarus americanus, cluded as characters and only phylogenetically informa- Homarus gammarus, Homarinus capensis (Herbst, 1792), tive characters (Hillis et al., 1996) were used. To assess Metanephrops mozambicus Macpherson, 1990, Nephrops the heuristic confidence in the parsimony trees generated norvegicus (Linnaeus, 1758),