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    27 vues6 pages

    Origin and Phylogenetic Position of The PDF

    Transféré par

    John Gamesby

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    © All Rights Reserved
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    Bul not His us Lond, (Zo) 6802): 101-106 Issue Origin and phylogenetic position of the Lesser Antillean species of Bothrops (Serpentes, Viperidae): biogeographical and medical implications WOLFGANG WUSTER AND ROGER S. THORPE School of Biological Sciences, University of Wales, Bangor LLS7 2UW, UK MARIA DA GRACA SALOMAO Laboratério de Herpetologia, Instituto Butantan, Av. Vital Brazil 1500, 05503-900 Sao Paulo-SP. Brazil LAURENT THOMAS Service des Urgences, CHRU, 97200 Fort de France, Martinique (French West indies) GIUSEPPE PUORTO Museu Bioldgico Instituto Butonton, At Vital Braid 1500, 05503-900 Sto Paulo-SP. Brazil R. DAVID G. THEAKSTON Liverpool School of Tropical Medicine, Pembroke Place. Liverpool 13 5QA, UK DAVID A. WARRELL Centre for Tropical Medicine. University of Oxford, Jol Radcliffe Hospital, Headington, Oxford OX3 9DU, UK SYNOPSIS. We une mizochondsal DNA sequences offer the origin and phylogenetic positon othe Lesser Ansitean species the pitviper genus otra. Beaches an B.lanceolaas. Tetsvo species forma monophyletic group wich nur forms the sister clade tothe Borla capers couples. High lvels of sence divergence aang the Caribbean species, and beawccn shen andthe acafest mainland felaives,suBgss a eltvey ancient eign of these stakes. The hypothesis tha the LLeseeAnillan Boros are the rel of a resent colonisation even rom within the South American Bator complex is rejected, as i the hypothesis that they were inoduced to thet island habitats by aboriginal humans, The high Kvel of ‘morphologial pomorphy deplyed hy ln 8asteping stone colonisation, St. Lia being colonised fist and 28 November 2002 then Marnie from St.Lucia, The medica implications of thes Lica suggest tat Retro ari INTRODUCTION “The genus Bothrops: Wagler, 1824 contains most of the pitviper Fauna of South America. The genus (including the arboreal species sometimes assigned 10 Bothriopsis) contains approximately 36 species. witha wider variety of body shapes and natral history ats than in any other New World pitviper genus, This ereater diversity has been ascribed to the fact that Borhrops was the firs. group of pipers to reach the South American continent. thus giving ample ‘opportunity for adaptive radiation (Wistert eta. in pres) Two species of Boshrops occur inthe Lesser Antilles: Both 1 (Garman, 1887) on St Lucia, and Bothrops lanceolata 1789) on Martinique. The Status and origin of these n the subject af much debate. Long considered to be Cconspecifie with Boxlrops aivox, B Janceolanus was revalidated by Hoge (1952), and the validity of & lanceolarus and B, caribbaeus confirmed by Lavell (1964), This latter interpretation has bes followed by most authors since then (e-g.. Campbell & Lamar, 1989), However, Sandner Montilla (1981, 1990) regarded the Lesser Amtllean Bothrops as conspecific with each other, as well 3s with ‘mainland Borhirops asper and the northern Veneruelan populations ‘of the B. asper-atrox comples. The origin ofthe Antillean Bovkrops has speculation and mythology. This includes popular tales that the caribba dacepede Forms has bo the subject of much findings ae discussed a recent case of envenoming fromm Sunt deus causes te same thombotic syndrome of envenoning 3B, lancrolaas snakes were originally introduced by Carib Indians in ther attempts, to gain control of the islands from resident Arawaks (Dowling 1965), and the notion that dispersal from the South American ‘mainland is common and ongoing (Sandner Montilla, 981) The repile fauna of the Lesser Antilles is primarily the result of long-distance dispersal by individual species. as these islands have rot been linked to the South American mainland or any other landmass at any time in their history (Thorpe eal in press Malhotraand Thorpe 1999), This means tht some species present in these islands represent long-standing endemic lineages (Thorpe et dil. in press; Malhotra and Thorpe 2000) whereas others appear to be the result of relatively recent dispersal events ftom well-defined source populations or taxa in South America, as is the ease forthe genus Corals (Henderson & Hedges, 1995). Compared to morphological data, molecular markers such as ‘mitochondrial DNA (mtDNA) sequence data have the advantage that they can give an estimate of phylogeny reasonably free of the confounding effects of differing natural selection pressures on the external phenowype. Moreover, molecular sequence data also have the advantage chat they can give at least an approximate estimate of times of div although the interpretation of ‘molecular clocks is subject to sarious analytical and empirical problems (Hillis er. 1996) Several recent miDNA-bused phylogenetic analyses ofthe genus Botlvops have included the Antillean species. Salome esa. (1997, ce between lineage PLATE 1 Boutropscaribeus © RS. Thorpe 1999), using 580>.p.ofeytochromeb sequence, found B.caribbaeus and B. lanceolaus to the sister species of the South American populations ofthe B. airox complex. However, the study included ‘nly timited sampling of South American members ofthe B.atrox complex, and did not include representatives of B. asper from Central America “The aim of this papers explore in more depth the origin ofthe Antillean Bevdrops, and its implications for other fields, using an expanded dataset of more sequence information froma larger number of potentially related species MATERIALS AND METHODS We obtained tissue (ventral scale clippings o tail tps) andor blood samples ffom species representing the principal clades within the genus Bothrans (including Bothrops), well as the closely related dothrocophias ~ see Wistert al, Gin pres). We also included samples of the B. asper-atrox complex from around the coast of South and Central America as these have becn considered w be potential founder populations fom which the ancestor of Antillean Bothrops couldhave arisen. Far ougroup cootng, We wed Sequences of Bothrops alterntus and Bethrocophias microphuh: ‘mus. Tso regionsof the mitochondial DNA molecule were amplified using the polymerase chain reaction (PCR): a 767 base pait (bp) section ofthe gene fr cytochrome b (cSt), and a 900 bp eepion of the gene for NADH dehydrogenase subunit 4 (ND4). Details of timer and laboratory protocols are given in Pook eta. (200). Sequences were aligned by eye against published Bovirops sequences (Puoro et al, 2001). In order test foe the presence of saturation ofcenaincategoresofsubsiution, wecalulted Tamura Neidistancesberween all samples. This takes mo account deviations from equal base compositions and differences i substitution rates among nucleotides, We then pled unadjusted prisances for transitions and tansversions, and forthe three codon positions sepately, against Tamura-Nei distances. A decline in the rate of ‘ccumulation of individual categories of substitution with increased “amuso-Neidistnoesindcstessaturatonof that sbstiutoncategry ‘We checkedall sequences for insertion, deletions othe presence ofstopcodons. Any ofthese would ave indicated thatthe sequences represent nvclear insertions ofthe mitochondrial genes (Zhang and Hewit, 1996). The sequence data were assayed forthe presence of a significant phylogenetic signal by means of the gl ce skewness "| i the ‘of the two photos have been exchanged, The snake ee ot coe W. WOSTER ET AL. Boutrops lanceolatus © D. Ware statistic (ills and Hueisenbeck, 1992), calculated from 100,000 luees randomly generated by PAUPY 4.0b8 (Swofford, 2001), Sequence divergences between clades were estimated using the program Phyltest (Kumar, 1996). ‘We analysed our sequence data using both maximum parsimony (MP) and maximum likelihood (ML) as optimality criteria, Using ‘multiple optimality criteria should identify those pacts of & phylogenetic tree that are supported independently ofthe oprimality criterion used. Such nodes should inspire greater confidence than odes that are unstable and vary depending on method of analysis, Al analyses were cared out using the program PAUP* 4.058 (Swofford, 2080). For MP analyses, we selected Bothrops alternatus and B, micro phthalmus as outgroups. We employed the heuristic search algorithm fof PAUP® 4,008, using TBR branch swapping and 100 randon ‘addition sequence replicates. The analysis was carried out on the unweighted data only ‘The extent to which individual nodes on the tre were supported by the data was assessed using bootstrapping and Bremer (1994) branch support, Non-parametric bootstrap was implemented using heuristic searching, 1000 eplicaes, TBR branch swapping and 10 andom- addition sequence replicates per bootstrap replicate. Bremer ‘branch support for individval nodes was calculated through the use (of the converse constraint option of PAUPS For ML analyses, we identified the most appropriate model of Sequence evolution through the use of the MODELTEST software (Posada & Crandall, 1998). A first ML search was run, using heuristic searching, a neighbour joining starting tree and TBR branch ‘swapping, and the sequence evolution parameters identified by the Modeltest software, These parameters were then re-estimated from the resulting ML. wee, and « farther search run using these re: estimated parameters. This was repeated until further estimates yielded no further changes of parameter values or tre likelihood Scores, Bootstrap analysis involved [00 replicates, using NI starting twee and NNI branch swapping ‘An important consideration of any proposed scientific hypothesis is whether the data supporting it ean reject alternative hypotheres, wih statistical significance. In other words, do the data allow ws co reject the nll hypothesis that differences in ree optimity between the optimal tree and wees consistent with lvernative hypotheses are {due to random chance? In the ease of the Antillean Bothrops, we {ested the following alternative phylogenetic hypotheses: (i) non- monophyly ofthe Antillean Bothrops, ie. the Amtllean populations (of Borhrops result from separate colonisation events; (11) non. ANTILLEAN BOTHROPS 103 imicrophiaimus ‘temas tonsa ¢-— fararaca insuaris 73/6. taeniatus puichor S23 punctatus jararacussu gee rr a fanceoletus S82 1 caribbaeus caribbaous sours] ~—— asper - Bolze atx - Suriname atrox Fr. Guyana eal Jf aor - Guyana ‘eucunss- 0. Martins joorral fevcurus- Salvador 2612 leucurus - P. Seguro/T. Freitas [2 colombiensis - S. Francisco colombiensis - Guaibacoa colombiensis - A.d,Orituco atrox - Sta. Izabel ‘marajoensis - |. Marajo atrox - S. Bento 61% marajoonsis - S.C. Arar microphthalmus ‘atematus jararaca insularis 0 ————— trenianus pulcher nee ee brazin punctatus lanceolatus 100 eariobacus caribbaous asper - Belize cobmbiensis- A. d. Orituco atrox - Sta. Izabel ‘marsjoonsis - |, Marajo atrox - 8, Bento marajoensis - 8. C. Arati colombiensis - S. Francisco colombiensis - Guaibacoa atrox - Suriname atrox - Fr. Guyana ‘atrox - Guyana 100° eucurus - D. Martins feucurus- Savador Jeucurus - P. SegurolT. Freitas, 99, kel 100 100 a7] Fig. 1 Maximum parsimony (op) and maximum ikeinood (bottom) estinstes ofthe phylogeny of Bothops. Inthe MP tse, numbers fone the sash TefertoBoottrap suppon, number aftr the slash iicate Bremer supper. In the MI. re, numbers on nodes indicate bootstrap support 04 monophyly of the B. asper-atrax complex, ie. that the Antillean iginate rom within the B. asper-atrox complex; ii) non-monophyly ofthe South American B, arms complex, ie. that the Antillean species originate from within the cis-Andean B,atrox ‘complex, paralleling the phylogeogeaphy of Cavallus (Henderson & Hedges, 1995); and (iv) monophyly of B.caribbaeus,B.lanceolats, B. asper and northern Venezuelan populations ofthe B asper-airo complex to the exclusion ofthe cis-Andean B. arrox complex, 2s implied by the classification of Sandner Montilla (1990), We used Wilcoxon signed-ranks (WSR) tests (Templeton tests ~ Templeton 1983) to compare the optimal MP tree and MP trees depicting alternative hypotheses, and Shimodaira-Hasegawa (SH) tests (Shimodaira & Hasegawa, 1999) to compare the corresponding ML trees, RESULTS, We aligned a total of 1401 b.p. of mtDNA sequence information (ND4: 693 b.p. eyth: 708 p.). The sequences included no indels or stop or other nonsense codons, and contained the usual bias towards transitions and substitutions concentrated into tied codon positions typical of mitochondrial DNA, We conclude that our sequences represent mtDNA rather than nuclear insertions. Samples are listed Jn Appendix 1. The 100,000 random trees generated a skewness Salisticof g1=0,590403, rejecting the null Hypothesis tht the data contain no significant phylogenetic signal (P < O01; Hillis and Huelsenbeck, 1992), Levels of sequence divergence among the taxa included ranged from 0.34% to 13.65% (unadjusted p-distance) Borhropscaribbacus tnd B lanceolatus differ from each other by 4.34%, and from the B. “asper-ctrox group by an average of 5.77% and 6, 15% respectively, with an average divergence of 5.9% when the Antillean haplotypes ate weated aa single clade. Levels of sequence divergence within the B, asper-aiox clade range from 0.3% to 5.5% ‘The MP analysis resulted in a single most parsimonious tee of 1030 steps (C1 0.5398; HI 0.4602: RI 0.6465). In this tee, the wo Aantillean taxa formed aclade, which in turn forms the sister clade of all samples of the Bothrops asper-atrox complex (Fi. |) ‘The MODELTEST softwate identified the GTR+I+G model, 2 submodel of the general time-rversible model (Yang etal. 1994) as, ‘optimal forthe data at hand. A ML tree was constructed using the parameters calculated by MODELTEST, and the parameters were recalculated from the resulting tree, and used in further ML search, which resulted ina ree withthe likelihood score-In(L}= 6652 69122. Further estimates of sequence evolution parameters didnot result inany change of parameter values oF tree likelihood score (Fg. 1). ‘The MP and ML wees differ only in branching crder within the cis- Andean B. arvox complex, and in the relative position of the B. Jararacussu-bravili lade and B. punctatus ‘The results of our tess of allernative ree topologies ae shown in Tablet W..WOSTER ETAL. ‘Table 1. Neither the WSR nor the SH test significantly reject the possibilty that the wo Antillean species may be the result of separate colonizations of the Lesser Antilles, although the result of| the SH test was almost significant. They do, however, significantly rejeet the hypothesis that the Antillean species originate from within, the cis-Andean radiation of the B. asper-atrox complex, and also raject Sandner Montilla’ suggestion of conspecificty between B. anceolatus, B. caribbaeus, B. asper and northem Venezuelan Bothrops.1o the exclusion of other South American populations of the B. airox group. DISCUSSION (Our resalts confirm the position ofthe Antillean species of Bothrops asthe siserclade of the Bohrops asper-atrox complex, as suggested. by Salomdo er al. (1997, 1999) and Waster etal. (1997, 1999). The monophyly ofthe Antillean taxa is supported by high bootstrap and Bremer support values although a tee supporting this arangement is not significantly longer than the optimal tree constrained not 10 include this clade. The high level of sequence divergence between the Antillean Bothrops and their mainland relatives (5.9%) is consistent with a lineage split dating back to the Miocene or earliest Pliocene. Wster etal (in press) suggested arate of sequence evolution for cyt» and ND4 of between 0.66 and 1.4% My! in Neotropical pitvipers. Ti would date the timing ofthe split hewween the Antillean Bothrops clade and the B. asper-atros clade at 42-8. Mya, ie. the late Miocene or earliest Pliocene, Similarly, the split between B. caribbacus and B. lancealatus (sequence divergence: 4.3%) can be dated to 3.1-6.5 Mya, Hedges (1996) estimated the divergence of the B, axper-atrox complex to have taken place within the last 4 My, and assumed dispersal tothe Antilles to have taken place during that timeframe, whereas our data suggest a slightly earlier ineage spit In any case it can be concluded thatthe two Antillean Bothrops speciesrepresenttwo relatively old, independent lineages. Obviously, in view of the errors inherent in any attempt at molecular lock usage, these estimates should be treated as approximations rather than exact timings, ‘The notion that these populations are the result of a recent dispersal event from within South America, 2s is the case in West Indian Corallus (Henderson & Hedges, 1995), is refuted by both tree topology and statistical tee comparison tests. Equally, the notion thatthe presence of these snakes in the Lesser Antilles isthe result of a primitive form of biological warfare among aboriginal, people (Dowling, 1965) will have 10 be abandoned, despite is, romantic appeal, ‘The colonisation sequence of the «wo species can be resolved {rom morphological data, panicularly sealation, In terms of dorsal and ventral scale counts, B. cavibbaeus is indistinguishable from ‘many populations of the B. asper-atrox complex. On the other hand, Differences in tre length or lkcihood, statistics and he significance, between the most parsimonious or the mos key ers, and ees ‘constrained tobe compte with lle phylogenetic o bogcegraphicel hypotheses, ‘Wileoxon signed ranks ‘Shimodra-Hasegawa segs) -2 P tla) P Now-monophyly off. caribbacus and B. tanceolatus 7 61s Is0sms 008s Non-monophly of 8. ayper-aox complex 5 oust 7-02515 SusK66 OS) Nonemonophyly of ee-Adean B. aor complex 5 016s" 20623 o.atse Monophly of & cariBbaeus, anceolats.asper Is oon-ommas® 24321305 ft northern Venevvelan populations ANTILLEAN BOTHROPS 2. lanceolars hs highes ventral and dorsal eae row counts than practically all populations of the B. jararacusu-punctats-airox clade. This suggests that he exteme scale couns found in B. Janceolams represent an autapemorphy compared 1 B,caribbacus and mainiand Bothrops. This makes a hypothesis of dispersal from the mainland to St. Lucia, and then a further dispersal event to Martinique, more parsimonious than dispersal t0 Martinique followed by funber dispersal to St. Livia. Since St. Lucia lies between South America and Martinique hs scenario is also more seographically parsimonious than the alternative. The slightly eater length ofthe branch leading to B lancealats is also consist ent wththis hypothesis (De Salle & Templeton 988; Thorpe eral. 1994), An understanding of the phylogenetic position of Bothrops caribhaeus and Blanceolatus may also have implications for their ‘eno composition and the teatment of stakebit inthe Caribbean Bothrops lanceolatus envenoming has been documented to produce a.uniguesyrome different fom that of ether species of Bors Inaddition to loal symptoms suchas pao, swelling. bleding athe siteof the bite, eechy mosis and necrosis, which are common to mest ‘roaline envenomings, the systemic botheopic syndrome observed inCentral and South Americaischaracterisdby the development of ‘consumption eoagulopathies and spontaneous systemic bleeding ‘depending on venom components which affect clotting factors a8 well as haemorshagins which damage vascular endotheliums (Barranes eral, 1985; Kamiguti eal, 1991). On the other hand, pat from similar local signs, the severiy of systemic envenoming by Bothrops lanceolatus in Martinigue was correlated with the ‘development of multiple cerebral infarctions and/or other major ‘vessel occlusion that may appear within 8 hours to7 days after the bite in approximately 30t040% of cases (Thomas eral 199, 198), Infarction can develop inpatients who present nally with signs ‘of moderate envenoming with normal bloc clotting and ow serum levels of venom antigens. The infarction process can involve several small vascular teritories aliopeter. and is associated With the

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