MALACOLOGIA, 1998, 40(1-2): 267-278 TESTING ALTERNATIVE HYPOTHESES OF NEOTRIGONIA (BIVALVIA:TRIGONIOIDA) PHYLOGENETIC RELATIONSHIPS USING CYTOCHROME OXIDASE SUBUNIT DNA SEQUENCES I Walter R. Hoeh^ ^ Michael B. Black\ R. Gustafson\ Arthur E. Bogan^, RichardA. Lutz\ & Robert 0. Vrijenhoek^ ABSTRACT Uncertainties regardingthephylogenetic historywithin the Bivalvia have impeded attemptsto understandevolutionwithinthegroup. Estimatingtheevolutionary relationshipssurroundingthe Trigonioida has been especially problematic and has led to disparate hypotheses regarding (1) the origin and subsequent diversification of unionoid bivalves and (2) autobranch gill character state transitions. In orderto test alternative hypotheses of trigonioid phylogeny, 613 base pairs of DNAsequenceofthecytochrome oxidase subunit I genewereanalyzedfrom 14speciesof bivalves (ingroup) andthree speciesof non-bivalve mollusks (outgroup).All phylogeneticanaly- ses, using either nucleotide or inferred amino acid sequences, produced trees that robustly placed Neotrigonia (Trigonioida) as the sistertaxon to a monophyletic Unionoida. Furthermore, the Autobranchia, Veneroida, and Mytiloida were supported as monophyletic groups in these analyses,whereasthe Bivalviawas not.Thesephylogenetic relationshipssuggestthat(1) there wasasingleinvasionoffreshwaterbyaunionoidbivalveancestor, (2)trigonioidratherthanven- eroid bivalves gave rise to the Unionoida, (3) eitherthe eulamellibranchous orfilibranchous gill condition has evolved multiple times within the Autobranchia, and (4) the molluscan bivalved body plan may have evolved more frequentlythantraditional phylogenetic hypotheses suggest. Key words: Bivalvia, Trigonioida, Unionoida, cytochrome oxidase I, mtDNA, phylogenetics, convergence. INTRODUCTION recently been proposed by Salvini-Plawen & Steiner (1996; Fig. 1A) and Waller (1990; The higher-level phylogenetic relationships Fig. IB). The former study is an exemplar in within the Bivalvia are poorly understood at that explicit data matrices and phylogenetic present (e.g., Allen, 1985). This statement is methodologies were presented. A major point corroborated by the many disparate hypothe- ofdisagreementbetweenthetwophylogenies ses of evolutionary relationships that have presented in Figure 1 is the placement of the been proposed for the higher taxa within the Trigonioida, a relatively ancient bivalve taxon Bivalvia (e.g., Purchon, 1990; Waller, 1990; that, althoughcurrentlydepauperate, wastax- Starobogatov, 1992; Cope, 1996; B. Morton, onomically diverse during the Mesozoic (Cox 1996; Salvini-Plawen & Steiner, 1996). This et al., 1969; Allen, 1985). The Salvini-Plawen plethora of phylogenetic hypotheses likely & Steiner hypothesis (Fig. 1A) indicates that stems, in part, from arbitrary choices to ex- thetrigonioids are mostclosely related to pte- clude certain types of characters and a gen- riomorph bivalves (represented herein by eral lack of explicit and rigorous phylogenetic mytiloids), with the Veneroida closely related analyses. Although major anatomical charac- to the Unionoida. Their proposed sistertaxon ter suites distinguish two of the nominal sub- relationshipforthetrigonioid and pteriomorph classes within the Bivalvia, that is, Proto- bivalves was supported by the shared pres- branchia and Autobranchia, the ordinal-level ence of (1) byssate larvae and adults and (2) relationships within each subclass are not abdominal sense organs in these taxa stronglysupported by multipleshared-derived (Salvini-Plawen & Steiner, 1996). Alterna- morphological features (e.g.. Waller, 1990). tively, the hypothesis of Waller (Fig. IB) indi- Two contrasting phylogenies of the higher- catesthattheTrigonioida isthe sistertaxon to level relationships within the Bivalvia have the Unionoida. However, no evidential sup- 'CenterforTheoreticalandAppliedGenetics,CookCollege, RutgersUniversity, NewBrunswick, NewJersey08903, USA: hoehw@miavx1.muohio.edu ^NorthCarolinaStateMuseumofNaturalSciences, P.O. Box29555, Raleigh, NorthCarolina,27626, USA. ^Corresponding authorandcurrentaddress: Departmentof Biological Sciences, KentState University, Kent, Ohio44242, USA 267 268 HOEH ETAL. Veneroid 1 Bivalves ^ Unionoid 3 Bivalves Trigonioid Bivalves Q 3>2 Mytiloid Bivalves Protobranch Bivalves Non-bivalve Mollusks FIG. 1. Bivalve relationships, basedon morphological characteristics, accordingto Salvini-Plawen & Steiner (1996;A) andWaller(1990; B). Both hypothesesindicateasingleoriginforthe Bivalvia(1) andfilibranchous lamellibranch gills (2) butdifferintheirimplicationsforeulamellibranch gill evolution. HypothesisAindicates a single origin foreulamellibranch gills (3) while indicates eithertwo origins (solid bars) ora single origin followed by a reversal tothefilibranch condition in trigonioids (hatched bars). port for this relationship was provided by ple, disparate hypotheses of trigonioid evolu- Waller (1990). tionary affinities. According to the hypothesis TheevolutionaryaffinitiesoftheTrigonioida of Salvini-Plawen & Steiner (1996; Fig. 1A), have been a contentious subject for over a the placement of the trigonioids is consistent hundred years (e.g., Steinmann, 1888; Neu- with a single origin of eulamellibranch gills mayr, 1889; Cox, 1960; B. Morton, 1987; (tissue-linked gill filaments) from filibranch Healy, 1989, 1996) and are central to an un- gills. In contrast, the phylogenetic placement derstanding of bivalve mollusk character evo- of trigonioids in the hypothesis of Waller lution, since representatives of the single ex- (1990; Fig. IB) suggests that there were ei- tant genus (Neotrigonia) have a mixture of ther (1) two origins of eulamellibranch gills or seemingly primitive (e.g., filibranchous gills (2) a single origin of eulamellibranch gills fol- [possessing ciliary-linked gill filaments], lack lowed by a reversal to filibranch gills in the of posterior mantle fusion, nacreous shells; trigonioids. Cox, 1960;Allen, 1985; B. Morton, 1987) and The phylogenetic uncertainty within the Bi- derived features (e.g., multi-vesicular sperm valvia impedes the rigorous testing of evolu- acrosome; Healy, 1989). This mosaic of tionary hypotheses. Therefore, the establish- seemingly primitive and derived character ment of a robust phylogenetic hypothesis for states has contributed tothe erection of multi- highertaxawithin the Bivalviawill enablecriti- NEOTRIGONIA PHYLOGENETIC RELATIONSHIPS 269 cal evaluations of (1) trigonioid evolutionary example, Skibinski et al., 1994: Zouros et al., relationships and (2) hypotheses of auto- 1994: Hoeh et al., 1996). DNA was also iso- branch bivalve gill character state transitions. lated from representatives of three additional Molecular systematic analyses have been molluscan classes (i.e., Gastropoda, Polypla- useful in situations where morphological cophora, and Scaphopoda) for use in gener- analyses were inconclusive (e.g., Avise, ating outgroup sequences. Subsequently, a 1994).Therefore, mitochondrial DNA(mtDNA) 710bp fragment of COI was PCR amplified sequences, obtained from the cytochrome and cycle sequenced for each of the 12 taxa oxidase subunit I (COI) gene, were used to as deschbed elsewhere (Folmer et al., 1994). construct a phylogenetic hypothesis for the Both strands of the COI fragment were se- major bivalve lineages represented in Figure quenced from each of two individuals from 1.COIwaschosenforthisanalysisbecauseof each terminal taxon to guard against PCR- its slow rateofevolution relativetoother mito- based contamination artifacts. The resulting chondrial protein coding genes, relative ease 12 COI sequences, plus the five veneroid bi- of unambiguous sequence alignment (e.g., valve COI sequences from Baldwin et al. Brown, 1985: Simon etal., 1994; Russe etal., (1996), were readily aligned by eye using 1996), and demonstrated appropriateness for MacClade 3.05 (Maddison & Maddison, this particularanalysis. 1992). Sixteen of the 17 OTUs produced sequences 613 bp in length, while that of Geukensiawas 616 bp. The increased length MATERIALSAND METHODS ofthe Geukensiasequencewasduetoan au- tapomorphic, three nucleotide (single codon) Organisms insertion event. These three contiguous nu- cleotides, which are phylogenetically uninfor- The molluscan taxa used in this study, with mative for the taxa considered herein, were GenBank accession numbers and primary lit- deleted prior to all phylogenetic analyses. No erature citation (where applicable), are as fol- additional hypothesized insertion or deletion lows: (1) ingroup. Class Bivalvia, Subclass events were necessary to obtain the align- Protobranchia, Order Solemyoida, Solemya ment utilized in the subsequent analyses. velum (U56852), Order Nuculoida, Nucula The suitabilityofthe COI data set for phylo- tenuis (U56851), Subclass Autobranchia, genetic analyses at the required hierarchical Order Unionoida, Mutela rostrata (U56849), level was evaluated by plotting the substitu- Amblemaplicata(U56841), Anodontacygnea tion pattern of transitions and transversions ( 56842), Margaritifera margarltifera for each codon position (e.g., Orti & Meyer, (U56847), Order Trigonioida, Neotrigonia 1996). Furthermore, the degree of phyloge- margarltacea (U56850), Order Mytiloida, netic signal within the COI data set was eval- Modiolus modiolus (U56848), Geukensia de- uated using the g^ statistic of a random tree missa (U56844), Order Veneroida (the five disthbution (from 100,000 random trees: e.g., veneroid COI sequences are from Baldwin et Hillis, 1991; Hillis & Huelsenbeck, 1992) as al. [1996]), Corbicula fluminea (U47647), implemented in PAUP (Swofford, 1993). Phy- Rangia cuneata (U47652), Mercenaria mer- logenetic analyses were carried out on the cenaria (U47648), Mytilopsis leucophaeata COI nucleotide sequences using the maxi- (U47649), Dreissena polymorpha (U47653), mum likelihood ([ML], DNAML in PHYLIP (2) outgroup. Class Scaphopoda Dentalium 3.5c: Felsenstein, 1993), neighbor-joining sp. (U56843), Class Gastropoda Lepetodrilus ([NJ], MEGA 1.02; Kumar et al., 1993), and elevatus (U56846), Class Polyplacophora maximum parsimony ([MP], PAUP 3.1.1; Katharinasp. (U56845). Swofford, 1993) algorithms. Katharina, the only non-conchiferan mollusk taxon, was Methods used to root the resulting topologies. Atransi- tion/transversion ratio of 2.0 was utilized in Total DNAwas isolated from somatic (man- the ML analyses and the gamma distance tle) tissues of nine species of bivalves. Male (alpha = 0.5, using the Tamura-Nei model of gonadaltissueswerespecificallyavoideddur- nucleotide sequence evolution) was used to ing dissections to prevent comparisons of generate the pair-wise genetic distances for non-orthologoussequences (duetotheactual the NJ analyses. This particular distance orpotential presence ofdoubly uniparental in- takes into consideration among-site substitu- heritance of mtDNA in some bivalve taxa: for tion rate variation (e.g., Yang, 1996). Further- 270 HOEH ETAL. more, MP and NJ (again using gamma dis- were invariant, while 315 (102from 1stcodon tances, alpina=0.5) analyseswereconducted positions; 59 from 2nd; 154 from 3rd) were on the inferred COI amino acid sequences phylogenetically informative using the parsi- (using the Drosophila mtDNA genetic code). mony criterion.Analysis ofthetree length dis- Multiple random terminal taxon addition order thbution of 100,000 randomly generated runs, combined with global branch rearrange- trees, using all 17 taxa, suggests that there is ment options, were employed to generate a significant amount of hierarchical structure topologies from ML and MP analyses. These within the transformed COI data s«et (g^ = options increasedtheprobabilityoffindingthe -0.794; with 384 variable sites, p 0.01; actual besttopology undereach ofthetwoop- Hillis & Huelsenbeck, 1992). This analysis timalitycriteria (e.g., Hendy et al., 1988: Mad- was repeated using only the Corbicula. Den- dison, 1991). The robustness of the resulting tallum. Katharina. Lepetodhlus. Modiolus, topologies was evaluated by bootstrap analy- Neothgonia. and Solemya COI sequences ses (1,000 replicates for MP and NJ, 100 (seven taxa) in order to minimize the number replicates for ML). of closely related taxa present in the hierar- The best COI-based topology derived from chical structure analysis. Significant structure the DNAML analysis was compared with the was still present in this truncated COI data phylogenetic hypotheses presented in Figure matrix (g^ = -0.527; with 295 variable sites, p 1 using the Kishino-Hasegawa test (paired z < 0.01), which suggests that the hierarchical test; Kishino & Hasegawa, 1989) as imple- structure present in the original data matrix mented in DNAML.Tothisend,thetopological was not solely due to the presence of closely constraints option in PAUP was used to gen- related taxa. The findings from the plots of erate82usertrees(alltrees^^ .N<» ^ ^.<>^ '^ jxo .<^ .d* x)"^ ej^" o"^ í^ á^ ® ^^ v^^ ^>J5v"^J;^^.<^^<^^/\tí^^' ^?^> ^4<ö< ris bivalveshell absent ^I HI present FIG. 5. Morphologicalcharacteroptimization using MacCladeonthebesttopologyproducedfrom maximum likelihood analysis of the transformed CO! nucleotide matrix using Katharina as the outgroup. A. The most parsimonious estimate of autobranch bivalve gill character state transitions for the taxa included in these analyses isthateitherthefilibranchousoreulamellibranchouscharacterstateevolved at leasttwice. B. The most parsimonious estimate of character state transitions forthe taxa included in these analyses suggests thatthe bivalved phenotype hasevolved at leasttwice. Justificationforthe useof Katharinaastheoutgroup forthemolluscantaxaincluded inthisanalysisisprovided by numerousstudies (e.g., Salvini-Plawen. 1980, 1985, 1988, 1990; Wingstrand, 1985; Eernisse, etal., 1992; Salvini-Plawen & Steiner, 1996). 276 HOEH ETAL. culoida had independent origins (Purchon, tural Experiment Station Contribution #D- 1978; Salvini-Plawen & Steiner, 1996), the bi- 32104-3-98andwassupported bystatefunds valvedconditionwould have hadtoevolvedat and NSFgrantsOCE9633131 OCE9302205, , leastfive times within the Mollusca. This strik- and OCE8917311 (to R. V. and R.A. L.). ing assessment is nonetheless not totally un- expected when evaluated in the context of (1) the great evolutionary mutability of body plan LITERATURE CITED exemplifiedbythephylum Mollusca(e.g.,J. E. Morton & Yonge, 1964; Allen, 1985; Willmer, ADAMKEWICZ, S. L., M. G. HARASEWYCH, J. 1990) and (2) the multiple origins of bivalved BLAKE, D. SAUDEK&C. J.BULT 1997,Amole- external shells in fourphyla (Thomas, 1988). cular phylogeny ofthe bivalve mollusks. Molecu- Under the hypothesis of a polyphyletic Bi- larBiologyandEvolution. 14: 619-629. valvia, the degree of morphological conver- ALLEN, J.A., 1985, The Recent Bivalvia:theirform gence on the bivalved body plan varies con- and evolution. Pp. 337-403, in E. R. trueman & M. R. CLARKE, eds.. TheMollusca, Vol. 10, Evolu- siderably within the extant mollusks. In the tion. Academic Press, Inc., Orlando, Florida, case of the opisthobranch gastropod genus U.S.A. Julia, the shell has converged on the mono- ANDREWS, E. ., 1988, Excretorysystemsofmol- myarian (= single adductor muscle) bivalve luscs. Pp. 381-448, in E. R. TRUEMAN & M. R. condition while the soft anatomical character- CLARKE, eds., The Mollusca. Vol. 11, Form and istics are clearly those of a gastropod (Kay, Function.Acaóemlc Press, Inc., San Diego, Cali- 1968). However, the degree of convergence fornia, U.S.A. in body plans between autobranch and proto- ATKINS, D., 1937, On the ciliary mechanisms and bbortahncshheblilvaalnvdesaisnamtuocmihcaglreacthearraacntderinsvtoaltvees.s iicnnrgtoesdrcreoevpliiaccteaislonSoschniipetsnhceoef.gill7las9m.:elQ3lu3ia9brr-ta3en7rcl3hy.s.JoPuarrntalII:ofsoMrit-- The phylogenetic hypothesisdisplayed in Fig- AVISE, J. C. 1994, Molecularmarkers, naturalhis- ure 3 suggeststhat in thedistinctevolutionary tory and evolution. Chapman & Hall, New York, histories of the autobranch and protobranch NewYork. U.S.A. 511 pp. mollusks, in addition to the convergent bi- BALDWIN, B. S.. M. BLACK, O. SANJUR, R. vofalhveeaddshaelnld, trhaedurleaw,e(r2e)ocroignivnesrgofenltab(i1al) lpoaslspes,s G19U9S6T.AAFSdOiaNg,nosR.tiAc.LmoUlTeZcu&laRr. marVkReIrJEfoNrHOzeEbKr,a (3) origins of the dimyarian adductor muscle mussels (Dreissenapolymorpha) and potentially condition, and (4) origins of fibrous ligament. cuola-rocMcaurrirniengBiboivlaolgvyesa:ndmiBtiooctheocnhdnroilaolgyC.OI5.:9M-o1l4e.c- Thus, the evolutionary mutability of body BERG-MADSEN, V., 1987, Tuarangia hom Born- planswithinthe Mollusca may begreaterthan holm (Denmark) and similaritiesin Baltoscandian that suggested bythe traditional classification and Australasian late Middle Cambrian faunas. schemes. Phylogenetic evaluations of addi- Alcheringa. 11: 245-259. tional molecular and morphological data sets BROWN, W. M., 1985, The mitochondrial genome are neededtotestthe hypothesized polyphyly of animals. Pp. 95-130, in R. j. macintyre, ed., ofthe Bivalviaandfurtherdecipherpatternsof Molecular evolutionary genetics. Plenum, New molluscan body plan evolution. York. NewYork, U.S.A. COPE,J. W., 1996,TheearlyevolutionoftheBi- valvia. Pp. 361-370, inJ. TAYLOR, ed.. Originand ACKNOWLEDGMENTS evolutionary radiation of the Mollusca. Oxford University Press. London, England. Wethank B. Baldwin for helpwith sequenc- COtXh,e LB.ivRa.l,vi1a9.60P,rTohceoeudgihntgssonofthteheclMasasliafciocaltoigoincaofl ing the veneroids used in this analysis. S. L. SocietyofLondon. 34: 60-88. Baldauf, D.J. Berg, S. I. Guttman, S. L. Neale, COX, L. R., R NUTTALL & E. R. TRUEMAN, M. O'Connell, P. O'Reilly, D. T Stewart, B. W. 1969, General features of the Bivalvia. Pp. Sutherland, and T. R. Waller provided helpful 2-129, in R. MOORE, ed.. Treatise on inverte- comments on earlier versions of this manu- bratepaleontology PartN,Vol. 1.TheGeological script. Neothgonia specimens were provided Society of America, Inc. and The University of by S. Boyd, and Mutela specimens were pro- Kansas, Lawrence, Kansas, U.S.A. DAVIS, G. M., 1994, Molecular genetics and taxo- vided byG. Soliman. R. Bielerprovided assis- tance with literature acquisition. We also wish 2n:o3m-i2c3d.iscrimination. The Nautilus. Supplement, to thanktwo anonymous reviewers and G. M. EERNISSE, D. J., J. S. ALBERT & F E. ANDER- Davis forcomments on earlierversions ofthe SON, 1992,AnnelidaandArthropodaare notsis- manuscript. This study is NewJerseyAgricul- ter taxa: a phylogenetic analysis of spiralian

Testing alternative hypothesis of Neotrigonia (Bivalvia: Trigonioida) phylogenetic relationships using cytochrome c oxidase subunit I DNA sequences

Similar Testing alternative hypothesis of Neotrigonia (Bivalvia: Trigonioida) phylogenetic relationships using cytochrome c oxidase subunit I DNA sequences