Phylogeny of

PHYLOGENETIC RELATIONSHIPS IN SOME SPECIES OF THE GENUS MACROBRACHIUM BASED ON NUCLEOTIDE SEQUENCES OF THE MITOCHONDRIAL GENE CYTOCHROME OXIDASE I

Guido Pereira, Hilda De Stefano, Joseph Staton and Brian Farrell

GP, Universidad Central de Venezuela, Instituto de Zoologia Tropical, Apartado 47058, Caracas 1041-A, Venezuela HDS, Universidad Nacional Experimental Simon Rodriguez, Instituto de Estudios Cientifwos y Tecnologicos, Centro de Estudios Biomedicos y Veterinarios, Apartado 47925 Caracas 1041-A, Venezuela JS, BF, Harvard University, Museum of Comparative Zoology, 26 Oxford Street, Cambridge, Massachusetts 02138, USA

ABSTRACT subfamily Palaemoninae in which the most obvious conclusion was that the subfamily shows different de­ A phylogeny for four species of the freshwater grees of paraphyly at the subfamily and generic levels jg genus Macrobrachium (M. carcinus, M. olfersii,M. (Fig. 1). However, further corroboration is necessary ^acanthurus andM rosenbergii) based on the partial nucle- in order to improve this phylogenetic hypothesis to­ | otide sequence of the mitochondrial cytochrome c oxidase wards a more stable model. Molecular Systematics may W'l subunit I gene is presented. The results are consistent with provide further evidence independent of previous mor­ ©•J previous studies based on morphological data. phological analysis that would be of great help in order to improve our understanding of phylogenetic relation­ I. INTRODUCTION ships among the group. The objective of the present study is to determine The genus Macrobrachium comprises a group the partial nucleotide sequence of the mitochondrial cyto­ I; of approximately 250 species distributed in the tropics chrome c oxidase subunit I gene (COI) of representative {&,worldwide, they are very common in freshwater rivers species of Macrobrachium to begin a genetic database of jvand one species, Macrobrachium rosenbergii (De Man) palaemonid and to establish phylogenetic hypoth­ -,is cultivated worldwide. The alpha of the ge- esis to compare it with previously published phytogenies jjnus as well as that of the whole subfamily Palaemoninae based on morphological characters. ihas remained without major changes after the mono­ graphic studies of Holthuis (1950, 1951, 1952), who II. MATERIALS AND METHODS fpcombined several subgenera within the single genus ^Macrobrachium. Regarding the phylogenetic relation- Four species of freshwater shrimps in the family |« ships within the genus, some authors have recognized (Macrobrachium carcinus (L), M. olfersii |,Jseveral species groups, but no formal action has been (Wiegmann), M. acanthurus (Wiegmann) and M. .;taken to reorganize the genus so that it reflects phylo- rosenbergii (De Man)) and one species in the family Atyidae %genetic relationships. Pereira (1997), based on previ­ (Potimirim potimirim (Miiller)) were selected. Addition­ ous work (Pereira 1989) showed a cladogram of the ally, five taxa whose sequences were obtained from

E. Escobar-Briones & F. Alvarez Eds. Kluwer Academic/Plenum Publishers MODERN APPROACHES TO THE STUDY OF CRUSTACEA PP. 319-322 G. Pereira, H. De Stefano, J. Staton & B. Farrell

GenBank were included (Utopenaeusvannamei (Boone), with the following thermal cycle protocol (40 cycles): Table 2. Sequence and alignm Artemia franciscana (L),Daphniapulex (Leidig),Anoph­ 30 sec at 94 °C (DNA denaturing); 30 sec at 50 °C eles gambie (Giles) and Drosophilayakuba). (primer annealing); 60 sec at 72 °C (primer exten­ Potimirim potimirim The laboratory methods used in this study sion) . The product of this reaction was purified af­ followed Hillis et al. (1996). DNAwas extracted from ter being run on an agarose gel (QIAquick columns, ??????????????????????????' ATGATCAAATTTACMCGTAWT muscle tissue and visualized in agarose gels. mtDNA Qiagen, Valencia, CA, USA) and visualized in agar­ GACTGGTCCCACTAATGCTAG( fragments were amplified >by the polymerase chain ose gel. Sequencing was carried out using DNA au­ TTACTATCCAGAGGAATAGTAG reaction (PCR) using tested primers for COI (Table 1) tomatic sequencing machine (ABI PRISM 370A). TCTGTCGACCTTGGTATrmT The sequences were edited, translated to amino ac­ AGAACAGGAATACTAATAGACCC ids of the COI, aligned and translated back to DNA Table 1. Sequence information for oligonucleotide primers GGAGCTATTACTATACTTTCAAI sequences. DNA sequences for related species were (Simon et al. 1994, Folmer et al. 1994). obtained fron GenBank. Phylograms were generated Macrobrachium acanthurus A2963 5' AGGTAGTTCTTCATTATAIGMTGTTC 3' using a distance method (Saitou and Nei 1987) and A2771 5' GGATAA/GTCAGAA/GTAACGTCGA/TGG/TGGTATA/C 3' maximum parsimony (MP). Several outgroups CTCATCTTGATCTTCGGAKCTI were used simultaneously in order to use the overall GAGAAATTATCAGATCTACAACC S1718 5' GGAGGATTTGGAAATTGATTAGTTCC 3' outgroup parsimony (Madison et al. 1984, Pereira ACTGACTACTGCCCCTAATACT S1991 5'GTAATTAATATACGACCTAAAGG 3' 1997). Phylogenetic trees were generated with the TCCTTTTATCGAGAGGTATGG1 LCO1490 5' GGTCMCAAATCATAAAGATATTG 3' computer program PAUP (Swofford 1998), us­ ATCAGTAGACCTCGGGATTTTC ing the Branch and Bound algorithm excluding 3rd GTCACCGGGCATAACCATGGAi HC02198 5' TAAACTTCAGGGTGACCAAAAAATCA 3' position. CCGGAGCAATTACCATG1TACT

Figure I. Partialview of'the morphological based cladogram after Pereira (1997). (prefers to species ofMacrobrachium used in the analysis MacrobrctcMum olfersii

CTATCTTGGATCTTCGGAGCT M. carcinus * TGGGAATGACCAAATCTACAAC M. vollenhoveni M. lar TAATTGACTAGTCCCTCTAATAC M. heterochirus CTTCTTCTATCCAGAGGAATGC M. horstii CCTCAGTTGACCTCGGTATCT M. placidulum M. felicinum * GATCTCCTGGAATAACTATAGAi M. olfersii * TGGAGCTATCACTATGGTIATA/ r-T^ M. hirtimanus M. latimanus C. caementerius Macrobrachium carcinus M. ohione M. brasiliense ??????????????????????????' M. iheringi TGATCAAATCTACAACGTTATT' M. nattereri C. brasiliensis GCTAGTCCCCCTTATACTAGG/ M. aracamuni ACTATCTAGAGGAATAGTGGA* M. cortezi CAGTAGACCTTGGAATCTTTT( M. ferrierai M. potiuna TCACCAGGAATAACCATAGACC By. luscus GGAGCCATCACTATATTATTGA M. quelchi M. nepalense Macrobrachium rosenbergii M. dux M. rosenbergii * M. panamense CCCATCTTGGACYTCGGAGCC M. amazonicum TCGGAAATGACCAAATCTACAAi M. acanthurus * MTTGACTAGTACCCCTAATATI ^ M. macrobrachion . M. weberi TCTTCTCTCCAGAGGAATAGIA M. equidens TCGGTAGATCTAGGTATTTTn M. lujae CCCCAGGAATAACTATAGATCG M. raridens M« foai AGCCATTACCATACTCTEAACTC

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Phytogeny ofMacrobrachium

Table 2. Sequence and alignment of 651 nucleotide for the Cytochrome C Oxidase Subunit 1 for five species used in the analysis. Jcol (40 cycles). 30 sec at 50 °C (primer exten Potimirim potimirim .vas purified af > — , 7??''???????????????????????GAATAGTAGGMCTGCCCTAAGTCTCTTMTTCGAGCTGMCTAGGTCAGCCAGGCAGACTMTTGGGA .quick columns, \\rGATCAMTTTACMCGTAWTTGTTACAbcCCATO^ ualized in agar lACTGGTCCCACTMTGCTAGGAGCCCCAGATATGGCTTTTCCCCGGATAMCM^ using DNAau PRISM 370A) rTACTATCCAGAGGAATAGTAGAAAGGGGCGTGGGCACAGGATGAACTGTGTACCCACCCCTTGCTAGAGGAATTGCTCATGCCGGGGCT ed to amino ac rcTGTCGACCTTGGTATTTTTTCACTCCACTTAGCAGGAGTGTC^ :d back to DNA \GMCAGGMTACTMTAGACCGGATACCCCTCTTTGTGTGATCTGTCTTTATTACTGCCATTTTATTACTTCTCTCTCTCCCGGTAC^^ ed species were C,GAGCTATTACTATACTTTCAACCGACCGTA\TTTAMTACCTCATTCTTTGACCCCGCAGGAAGTGGTGACC???????????????????? were generated yacrobrackium acanthurus INei 1987)and jral outgroups tTCATCTTGATCTTCGGAKCTCGAGCTGGCATATTMGCACATCTCTTTMGACTTMTCCGGGCTGAACTAGGGCAGCCAGGCAGACTAAT 3 use the overall CAGAMTWCAGATCT\CMCGTCATTGTCACTGCCCACGCATTCGT^^^ I. 1984,Pereira ACTG^CX^CTGCCCCTAATACTAGGTGCCCCCGACATGGCCTTCCCCCGMTAMCMCATGAGATTCTGACTTTTACCCCCATCACTCACAC erated with the ICC'rTTTATCGAGAGGTATGGTAGAAAGCGGGGTAGGCACGGGGTGAACTGTATACCCCCCACTAGCAGCCGGGACTGCACATGCAGGAGC rd 1998), us- VCAGT^GACCTCGGGATTTTCTCATTACACCTCGCAGGTGT^ m excluding 3rt CTCACCGGGCATMCCATGGACCGACTCCCCCTATTTGTATGGGCGGTATTCCTGACAGCCATTCTTCTTnTATTTCCCTTCCTGTATTAG CCGGAGGAATTACCATGTTACTMCAGACCGAMCCTAMmCTTCCTTCTTTGACCCTGTTGGGGGAGGAGACCCA?????????????????? i used in the analysis , Vacmbrachiwn olfersii

LTATCTTGGATCTrCGGAGCTrGAGCAGGmmGTCGGCACATCCCTMGACTCTTMTTCGAGCTGAATTAGGTCAACCCGGGAGACWGAT -cinus * TGGGMTGACCAMTCTACMCGTCATWGTCACCGSWCACGCTTTCGXAATMTTTTCTTCATGGTMTGCCTATCATMTTGGAGGATTCGG lenhoveni IWTTGACTAGTCCCTCTMTACTAGGAGCACCTGAmTGGCCTTCCCCCGAATGMTMCATMGATTCTGACTCCTGCCCCCCTCTCTAAAT terochirus tTTCTrCTATCCAGAGGAATGGTAGAAAGAGGTGTAGGAACCGGGTGAACCGTGTATCCCCCCCTAGCTGCAGGAACCGCCCACGCCGGAG rstii CCTCAGTTGACCTCGGTATCTTITCCCTCCACTTAGCCG icidulum icinum * (.ATCTCCTGGMTMCTATAGACCGACTACCCCTATTCGTTTGAGCGGTATTATTMCCGCCATTATTGTCGTTCTTTCC^^ ersii * IGGAGCTATCACXWGGTTATMCTGACCGAMCCTAAAX^CATCCTTCTTTGACCCTGCCGGGGGTGGAGACCCMTTTTAT??????????? timanus imanus menterius Macrobrachium cardrms ione isiliense ,'r'?????????????????????????????????GCACATCTCTGANACTCTTAATTCGTGCTGAGTTAGGACAACCGGGCAGACTAATCGGAAA :ringi 1GATCAMTCTACMCGTTATTGTCACAGCTCACGCGTTCGTMTAATTTTCTTCATGGTAATGCCMTTATMTTGGAGGCTTT ttereri siliensis (CTAGTCCCCCTmTACTAGGAGCCCCAGACATGGCCTTCCCGCGMTAAATMTATMGATTCTGGCTCTTACCTCCCTCTCTAACTCTCCT icamuni \CTATCTAGAGGAATAGTGGAAAGAGGGGTGGGGACAGGATGAACTGTGTACCCCCCTCTAGCAGCAGGAACTGCTCACGCGGGACCTG rtezi IAGTAGACCTTGGMTCTTTTCCCTTCACCTTGCCGGTGTCTCATCTATCCTGGGTGCCGTCMTTTCATCACCACTGTAATTAACATGCGA rierai tiuna I'CACCAGGMTAACCATAGACCGGCTACCCCTATTAGTGTGAGTC 3CUS (IGACICCATCACTATATTATTGACAGACCGAMCTCAMX\CTGANNNTTTYGACCCAGCAGGNGGAGGCGACCTCAAT??????????????? elchi palense Macrobrachium rosenbergii x senbergii * namense rCCATCTTGGACYTCGGAGCGTGAGCAGGCATGGTAGGTACGTCACTMGACTCTTMTTCGAGCAGAATTAGGGCAGCCGGGCAGACTGA lazonicum I'CGGAMTGACCAMTCTACMCGTWTGTCACTGCCCACGC^^ inthurus * icrobrachion MTrGACmGTACCCCTMmTTAGGGGCCCCAGACATAGCATTCCCACGCATAMCMCAXVVGATTCTGACTCCTACCCCCATCTCTAACACT beri I'C'nrTCTCCAGAGGMTAGTAGAMGAGGGGTTGGCACAGGATGMCTGTTTATCCACCACTAGCGGCCGGTACCGCCCACGCCGGGGCA uidens rcGGX\GATCTAGGTATTTTTTCCCTCCACCTAGCAGGAGTTTCTTCMTCTIAGGGGCTGTCMCTTTATTACC^^ ae i:CCCAGGMTMCWAGATCGACTGCCCCTATTCGTATGAGCCGTAT^ idens \GCCATTACCATACTCTTMCTGATCGAMCCTVV\TAC^^ li 321 G. Pereira, H. De Stefano, J. Staton & B. Farrell

Pereira (1997), however more species should be in­ THE cluded in order to have a more robust hypothesis. We hope that this work will motivate further research on FRESHW^ molecular Systematics of this important group. (DEC REFERENCES THEL Folmer OM, Black W, Hoeh R, Lutz R, and Vrijenhoek R (1994) DNA primers for amplification of mitochon­ TH drial cytochrome c oxidase subunit I from diverse meta- DE L = 869 zoan invertebrates. Mol Mar Biol Biotech 3:294-299 IC = 0.612 Hillis MH, Moritz C, and Mable BK (1996) Molecular IC excluding uninformative Systematics. Second edition, Sinauer Associates 655 p characters = 0.553 Total characters = 651 Holthuis LB (1950) The of the Siboga-Expedition. PartX. The Palaemonidae collected Figure 2.A: distance based tree (Neighbor-joining). B: maxi­ by the Siboga and Snellius expeditions with remarks mum parsimony tree. on other species I. Subfamily Palaemoninae. Siboga Universii Expeditie, Monographie 39, 268 p III. RESULTS Holthuis LB (1951) A general revision of the Palaemonidae (Crustacea, Decapoda, Natantia) of We sequenced 651 DNA nucleotide bases at the Americas. I. The Subfamilies Euryrhynchinae the 5' end of the COI mitochondrial gene (Table 2), and Pontoniinae. Allan Hancock Foundation Pub­ for four species of freshwater shrimps in the family lications, Occasional Papers 11, 332 p Palaemonidae and one species in the Family Atyidae. Holthuis LB (1952) A general revision of the Palaemonidae The results of the distance and phylogenetic analyses (Crustacea, Decapoda, Natantia) of the Americas. II. are shown using neighbor-joining (Fig. 2A) and maxi­ The subfamily Palaemonidae. Allan Hancock Foun­ The descrrf mum parsimony (Fig. 2B). Maximum parsimony re­ dation Publications of the University of Southern Cali­ of Macrobrachium i sulted in a single tree of 191 steps and a consistency fornia, Ocasional Paper 12, 396 p rodriguezi, Pereira, 1? index (CI) of 0.6. The distance based (DB) tree (Fig. Maddison WP, Donoghue MJ and Maddison DR (1984) The origin and signif 2A) agrees more closely with morphological-based hy­ Outgroup analysis and parsimony. Syst Zool 33:83-03 in Macrobrachium is potheses. Regarding outgroups, insects cluster together Pereira G (1989) Cladistics, Taxonomy, Biogeography and and outside of , showing the progressive the evolutionary history of the Family I.tt splitting ofArtemia and Cladocera, both within the Class Palaemonidae (Crustacea, Decapoda, ). Branchiopoda and considered a primitive group; then Ph.D. Thesis, University of Maryland, College Park, The shrimp follow the Decapoda, •mthUtopenaeus appearing first, tra­ USA, 450 p resent a group of c ditionally considered a primitive genus. Next the represen­ Pereira G (1997) A cladistic analysis of the freshwater radiated into the fresl tatives of the two caridean families, RrstPotimirimpotimirim shrimps of the family Palaemonidae (Crustacea, Within the genus, m from the family Atyidae and then all the members of the Decapoda, Caridea). Acta Biol Venezuel 17, 68 p centration to complf Palaemonidae, M. olfersii andM. carcinus in a single clus­ Saitou N and Nei M (1987) The neighbor-joining others are totally inc ter and M. acanthurus and M. rosenbergii in another. method: a new method for reconstructing phyloge­ I; far from estuaries. E The MP tree (Fig. 2B) does not provide reso­ netic trees. Mol Biol Evol 6:514-525 small and numerous lution at higher levels, however results within caridean Simon C, Frati F, Beckenbach A, Crespi B, Liu H and as 13 larval stages, 1 species are almost identical using both methods. When Flook P (1994) Evolution, weighting and phyloge­ Inland freshwater si compared with previous phylogenetic trees based on netic utility of mitochondrial gene sequences and a eggs and have abbre morphological data (Fig. 1), the results are fully con­ compilation of polymerase chain reaction primers. 17 species of Macrol sistent. They agree in that Atyidae is the sister group of Ann Entom Soc Am 87:651-701 occur in South Amei the Palaemonidae, M. carcinus andM. olfersii are more Swofford D (1998) PAUP*. Phylogenetic analysis us­ of M. brasiliense (V closely related to each other as well as the pair M. ing parsimony (*and other methods). Version 4a, 1979), M. jelskii acanthurus-M. rosenbergii. This result agrees with Sinauer Associates, Sunderland, Massachussets (Magalhaes, 1989),

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