International Journal for Parasitology 29 (1999) 1465±1476

Evidence for host-speci®c clades of tetraphyllidean tapeworms (Platyhelminthes: Eucestoda) revealed by analysis of 18S ssrDNAp

P.D. Olson a, b,*, T.R. Ruhnke c, J. Sanney c, T. Hudson c

aThe Natural History Museum, Department of Zoology, Division of Parasitic Worms, Cromwell Road, London SW7 5BD, UK bUniversity of Connecticut, Ecology and Evolutionary Biology, U-43, 75 No. Eagleville Road, Storrs, CT 06269-3043, USA cWest Virginia State College, Department of Biology, Institute, WV 25112-1000, USA Received 12 April 1999; received in revised form 22 June 1999; accepted 22 June 1999

Abstract

Sequence data from the V4 and V7±V9 variable regions of the 18S small subunit ribosomal DNA (ssrDNA) gene were used to examine relationships among 26 tetraphyllidean and two lecanicephalidean taxa. Newly collected specimens of 21 of the tetraphyllidean species were used to generate ssrDNA sequences that were combined with sequences previously available, including those of two diphyllidean taxa used for outgroup rooting. The sequences were aligned by eye according to secondary structural motifs of the conserved core of the molecule. Of the 1520 sites in the alignment, 874 (58%) were excluded from analysis due to alignment gaps and lack of positional homology as inferred by manual inspection. Genetic variability of the ssrDNA gene regions compared was greater than would be expected, based on the present taxonomy of the ingroup species, and the genetic divergences among tetraphyllidean `families' and genera were comparable to that among tapeworm orders. Phylogenetic hypotheses were generated by the methods of maximum parsimony and maximum likelihood (GTR + I + G nucleotide substitution model). Four most parsimonious trees resulted from analysis by maximum parsimony. Strict consensus of the four trees supported the monophyly of the Tetraphyllidea, with the lecanicephalidean taxa forming a sister lineage. Among the tetraphyllidean taxa included in the analysis were three major clades: a basal clade including species of the phyllobothriid genera Anthocephalum, Echeneibothrium, Rhinebothrium, Rhodobothrium and Spongiobothrium; a clade uniting the phyllobothriids of the genus Duplicibothrium with the dioecotaeniid genus Dioecotaenia; and a larger sister clade to the Duplicibothrium + Dioecotaenia clade that included the phyllobothriid genera Caulobothrium, Ceratobothrium, Clistobothrium, Paraorygmatobothrium and Prosobothrium, the litobothriid genus Litobothrium and the onchobothriid genera Acanthobothrium, Calliobothrium, Phoreiobothrium and Platybothrium. Maximum likelihood analysis resulted in a topology that was congruent where nodes were strongly supported by parsimony analysis, but di€ered in the relative positions of the well-supported clades. In addition,

*Note: Newly generated nucleotide sequence data reported in this paper are available in the EMBL, GenBank2 and DDJB databases under the accession numbers AF126064±AF126098. The full alignment has been deposited with the EMBL (accession No. DS37932) and can be accessed by anonymous FTP from ftp.ebi.ac.uk in directory /pub/databases/embl/align. *Corresponding author. Present address: The Natural History Museum, Department of Zoology, Division of Parasitic Worms, Cromwell Road, London SW7 5BD, UK. Tel.: 44-(0)207-942-5676; fax: 44-(0)207-942-5151 E-mail address: [email protected] (P.D. Olson).

0020-7519/99/$20.00 # 1999 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S0020-7519(99)00106-X 1466 P.D. Olson et al. / International Journal for Parasitology 29 (1999) 1465±1476 maximum likelihood analysis grouped the lecanicephalidean taxa among the tetraphyllidean taxa, indicating paraphyly of the order Tetraphyllidea as currently de®ned. Relationships suggested by both methods of analysis re¯ected common host associations of the taxa better than their current classi®cation, suggesting that coevolution has had a signi®cant role in the evolution of the group. # 1999 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Keywords: Eucestoda; Host±parasite cospeciation; Lecanicephalidea; Litobothriidae; Onchobothriidae; Phyllobothriidae; Platyhel- minthes; ssrDNA; Tetraphyllidea

1. Introduction Linton, 1890 and Rhinebothroides Mayes, Brooks and Thorson, 1981. Their trees were based on a The phylogenetic relationships among tetra- small suite of morphological characters and the phyllidean tapeworms are poorly understood, methodological approach was not explicitly cla- and this fact has resulted in considerable instabil- distic. Caira et al. [4] recently completed a more ity in their classi®cation. Schmidt [1] recognised comprehensive cladistic analysis of selected the family Onchobothriidae Braun, 1900, whose species representing the majority of known members possess hooks on their bothridia, as diphyllidean, lecanicephalidean and tetraphylli- well as two `non-hooked' families: dean genera. In total, their study included 56 Phyllobothriidae Braun, 1900 and Triloculariidae ingroup and seven outgroup species, and was Yamaguti, 1959. In addition, he accorded ordinal based on 120 morphological characters. Results status to the dioecious `tetraphyllidean' genus of their analysis supported the monophyly of the Dioecotaenia Schmidt, 1969. More recently, Onchobothriidae. The Phyllobothriidae, however, Euzet [2] divided the Tetraphyllidea into eight was found to be paraphyletic and consisted of families: Cathetocephalidae Dailey and two main lineages (in addition to a large polyt- Overstreet, 1973; Chimaerocestidae Williams and omy of unresolved taxa), one of which formed a Bray, 1984; Dioecotaeniidae Schmidt, 1969; trichotomy with the lecanicephalidean and onch- Disculiceptidae Joyeux and Baer, 1936; obothriid clades. Although many relationships Litobothriidae Dailey, 1969; Onchobothriidae; remained unresolved, their work represents a rig- Phyllobothriidae; and Prosobothriidae, Baer and orous initial hypothesis of the phylogenetic re- Euzet, 1955. With the exception of the families lationships of tetraphyllidean genera, and Onchobothriidae and Phyllobothriidae, the provides a comprehensive suite of morphological remaining families recognised by Euzet [2] are characters that may be later compared and com- species-poor, and are diagnosed by peculiar mor- bined with other classes of data for `total evi- phological features. For example, species of the dence' analysis. Cathetocephalidae exhibit one to 24 strobilae per Molecular systematic studies of cestodes are scolex [3]. Of the larger two tetraphyllidean currently few in number. Olson and Caira [6] and families, the presence of hooks provides evidence Mariaux [7] have addressed relationships at the for the monophyly of the Onchobothriidae, while ordinal level. However, most of the lower-level no character unites all species of the taxonomic studies have focused on species of the Phyllobothriidae, and this family, as presently order Cyclophyllidea; typically being compari- de®ned [2], is likely to be non-natural (see [4]). sons between species within a genus of medical The few phylogenetic studies of tetraphylli- or economic importance (e.g. [8, 9]). The primary deans that have been published di€er substan- purpose of the present study was to evaluate the tially in completeness. Brooks and McLennan [5] phylogenetic utility of the two most variable provided a phylogenetic summary of tetraphylli- regions of the 18S small subunit ribosomal DNA deans at the `generic' level, in addition to species- gene (ssrDNA), the V4 and V7, for resolving re- level phylogenies of the genera Rhinebothrium lationships among members of the order P.D. Olson et al. / International Journal for Parasitology 29 (1999) 1465±1476 1467

Tetraphyllidea. Although both the analyses by Gustincich et al. [13]. Prior to extraction, all spe- Olson and Caira [6] as well as Mariaux [7] cimens were rinsed thoroughly in 95% EtOH and included at least some representatives of the lyophilised to facilitate grinding of the tissue. order Tetraphyllidea, the current investigation The entire ssrDNA gene was ampli®ed by PCR represents the ®rst molecular systematic study to in two overlapping fragments: a 1100 bp frag- focus speci®cally on this group of elasmobranch ment using primers 18S-E (5 0-CCGAATTCGAC parasites. The phylogenetic information gener- AACCTGGTTGATCCTGCCAGT-3 0) and 18S- ated by this study is useful for examining the nat- A27 0 (5 0-CCATACAAACGTCCCCGCCTG-3 0), uralness of the present familial-level classi®cation and a 1500 bp fragment using primers 18S-8 (5 0- of the order [2]. In addition, because of the strict GCAGCCGCGGTAATTCCAGC-3 0) and 18S- host-speci®city common among many tetraphylli- Cestode-6 (5 0-ACGGAAACCTTGTTACGACT- dean species [10, 11], examination of their phylo- 3 0). Primers A27 0 and Cestode-6 were designed genetic relationships has the potential to uncover previously to better match the tapeworm interesting patterns of host±parasite associations. ssrDNA gene [6]. Robust, high-®delity double- Revealing such patterns is the ®rst step toward a stranded ampli®cations were obtained with a broader understanding of the evolutionary his- Perkin-Elmer 2400 thermocycler using 2.5 mM tory between the parasites and their hosts, and MgCl bu€er [14] and the following thermocycling can be used as a guide for future investigations. pro®le: 3 min denaturation hold at 978C; 36 cycles of 1 min at 968C, 1 min at 548C, 1 min at 728C; and 7 min extension hold at 728C. 2. Materials and methods Unincorporated PCR primers and nucleotides were removed from the PCR products prior to 2.1. Collection of specimens sequencing by use of Qiagen1 QIAQuick2 spin columns. Nucleotide sequences were determined Fresh specimens of 21 previously collected tet- directly from cleaned PCR products by auto- raphyllidean tapeworm species were used to gen- mated sequencing using ABI BigDye2 erate new ssrDNA sequences. Table 1 shows a Terminator Cycle Sequencing Ready Reaction taxonomic listing of these species, their hosts and Mix and an ABI PRISM2 377 automated collection localities, as well as the other species sequencer. Sequencing reactions were cleaned included in the analyses for which ssrDNA using Sephadex-®lled Centrisep2 spin columns sequences were available previously. In addition (Princeton Separations). Sequences were deter- to the tetraphyllidean taxa, two lecanicephalidean mined for two non-contiguous regions of the species were included in the analyses to examine ssrDNA gene: from stems 20±22 (encompassing the position of the Lecanicephalidea relative to the V4 region) and stems 40±47 (encompassing the Tetraphyllidea. All specimens were ®xed and the V7±V9 regions). Sequencing primers used stored in 95% EtOH prior to DNA extraction. were 18S-8, 18S-A-27 0, 18S-11F (5 0- Some of the species analysed in the present study GGGTGGTGGTGCATGGCCGTT-3 0) and were determined to be new to science, and taxo- 18S-Cestode-6. The sequences were determined nomic descriptions are currently in preparation for the majority of sites from both the sense and (T.R. Ruhnke). Voucher specimens of the new anti-sense strands. material have been deposited at the Connecticut State Museum of Natural History. 2.3. Selection of outgroup taxa and sequence 2.2. DNA isolation, PCR ampli®cation and gene alignment sequencing Two diphyllidean (Platyhelminthes: Eucestoda) Genomic DNA of whole worms was extracted taxa were selected as outgroups, based on the following the method of either Coen et al. [12] or results of Olson and Caira [6] which showed 1468 P.D. Olson et al. / International Journal for Parasitology 29 (1999) 1465±1476

Table 1 List of taxa analysed, their hosts and collection localities. Classi®cation follows that of Euzet [2]

Classi®cation Host Collection locality Species [GenBank No.] (common name)

OUTGROUP Or. DIPHYLLIDEA Echinobothrium fautleyae [AF124464] Rhinoptera steindachneri Gulf of California, Santa Rosalia, Mexico (Paci®c cownose ray) Macrobothridium sp. [AF124463] Rhinobatos typus Timor Sea, Shoal Bay, Darwin, NT Australia (Giant shovelnose ray) INGROUP Or. LECANICEPHALIDEA Cephalobothrium cf. aetobatidis [AF124464] Aetobatus narinari Gulf of Thailand, Bungsaray, Thailand (Spotted eagle ray) Eniochobothrium gracile [AF124465] Rhinoptera sp. Timor Sea, Fog Bay, NT Australia (Cownose ray) Or. TETRAPHYLLIDEA Fm. Litobothriidae Litobothrium alopias [AF124468] Alopias superciliosus Gulf of California, Santa Maria, Mexico (Bigeye thresher shark) Litobothrium ampli®ca [AF124467] Alopias pelagicus Gulf of California, Santa Maria, Mexico (Pelagic thresher shark) Fm. Prosobothriidae Prosobothrium armigerum [AF126068] Prionace glauca Atlantic Ocean, Montauk, New York, USA (Blue shark) Fm. Dioecotaeniidae Dioecotaenia cancellata [AF126074] Rhinoptera bonasus Chesapeake Bay, Cambridge, Maryland, USA (Cownose ray) Fm. Onchobothriidae Acanthobothrium sp. 1 [AF126065±66] Dasyatis longus Gulf of California, La Paz, Mexico (Longtail stingray) Acanthobothrium sp. 2 [AF126067] Dasyatis brevis Gulf of California, Bahia de Los Angelos, Mexico (Whiptailed stingray) Calliobothrium sp. [AF124469] Mustelus canis Long Island Sound, Connecticut, USA (Dusky smooth-hound) Calliobothrium violae [AF126064] Mustelus canis Long Island Sound, Connecticut, USA (Dusky smooth-hound) Platybothrium auriculatum [AF124470] Prionace glauca Atlantic Ocean, Montauk, New York, USA (Blue shark) Phoreiobothrium sp. [AF126095±96] Sphyrna mokarran Gulf of Mexico (Great hammerhead) Fm. Phyllobothriidae SbFm. Echeneibothriinae Echeneibothrium vernetae [AF126083±84] Raja erinacea Passamoquaddy Bay, New Brunswick, Canada (Little skate) SbFm. Phyllobothriinae Anthocephalum alicae [AF126091±92] Dasyatis americana Atlantic Ocean, Florida Bay, Florida, USA (Southern stingray) Anthocephalum n. sp. 1 [AF126087±88] Dasyatis longus Gulf of California, La Paz, Mexico (Longtail stingray) Anthocephalum n. sp. 2 [AF126089±90] Dasyatis brevis Gulf of California, Bahia de Los Angelos, Mexico (Whiptailed stingray) Ceratobothrium xanthocephalum [AF126085±86] Isurus oxyrinchus Atlantic Ocean, Montauk, New York, USA (Short®n mako shark) P.D. Olson et al. / International Journal for Parasitology 29 (1999) 1465±1476 1469

Table 1 (continued )

Classi®cation Host Collection locality Species [GenBank No.] (common name)

Clistobothrium montaukensis [AF126069] Isurus oxyrinchus Atlantic Ocean, Montauk, New York, USA (Short®n mako shark) Paraorygmatobothrium sp. [AF126081±82] Mustelus californicus Gulf of California, Puertecitos, Mexico (Grey smoothhound) Rhodobothrium sp. [AF126097±98] Rhinoptera bonasus Gulf of Mexico, Ocean Springs, Mississippi, USA (Cownose ray) Spongiobothrium sp. [AF126079±80] Dasyatis brevis Gulf of California, Puertecitos, Mexico (Whiptailed stingray) SbFm. Rhinebothriinae Caulobothrium sp. [AF126093±94] Myliobatis californicus Gulf of California, Bahia de Los Angelos, Mexico (Bat eagle ray) Duplicibothrium minutum [AF126070±71] Rhinoptera bonasus Gulf of Mexico, Ocean Springs, Mississippi, USA (Cownose ray) Duplicibothrium n. sp. 1 [AF126072] Rhinoptera steindachneri Gulf of California, Puertecitos, Mexico (Paci®c cownose ray) Duplicibothrium n. sp. 2 [AF126073] Rhinoptera steindachneri Gulf of California, Puertecitos, Mexico (Paci®c cownose ray) Rhinebothrium sp. [AF126075±76] Dasyatis americana Atlantic Ocean, Florida Bay, Florida, USA (Southern stingray) Rhinebothrium maccallumi [AF124476] Dasyatis americana Gulf of Mexico (Southern stingray) Rhinebothrium n. sp. [AF126077±78] Dasyatis longus Gulf of California, La Paz, Mexico (Longtail stingray) them to be close, but clearly outside of the by anonymous FTP (accession No. DS37932) Tetraphyllidea. Newly generated sequences were from ftp.ebi.ac.uk in directory/pub/databases/ assembled using Sequencher 3.0 (GeneCodes). embl/align. Contiguous sequences, together with ssrDNA sequences retrieved from GenBank, were aligned 2.4. Phylogenetic analyses by reference to the conserved core of the ssrDNA secondary structural model of Neefs et All analyses were performed with PAUP* (DL al. [15]. Application of this model to the ssrDNA Swo€ord, 1998, version 4.0b1a), and the resulting gene of cestodes can be found in Olson and networks were rooted with the outgroup taxa. Caira ([6], their Appendix A). Alignments were Analysis by maximum parsimony (MP) was per- handled using GDE [16] for a SUN workstation formed using the heuristic search (1000 search and exported to a Macintosh personal computer replicates), random-addition sequence and TBR for analysis. Regions in which homologies could branch-swapping options. All characters were not be unambiguously determined or where gaps run unordered and with equal weights. Gaps exceeded 2 bp in length were excluded from ana- were treated as missing data. Nodal support was lyses. This resulted in 874 (58%) of the 1520 assessed by bootstrap resampling (1000 bootstrap total sites in the alignment (including alignment replicates with three heuristic searches/replicate), gaps) being excluded. The sites included in the and by decay analysis [17] using AutoDecay (T analysis encompassed the conserved 3 0 end of the Eriksson, N WikstroÈ m, version 3.0.3). V4 region combined with the last quarter of the Alternative topologies consistent with current gene (from stem 40 to 47, encompassing the con- classi®cation [2] or strict patterns of host associ- served 3 0 end of the V7 region as well as the V8 ation were analysed in order to compare their and V9 regions). The full alignment is available di€erences in tree length. Nexus tree ®les were 1470 P.D. Olson et al. / International Journal for Parasitology 29 (1999) 1465±1476 generated with MacClade (WP Maddison, DR and a gamma model (I + G). Log-likelihood Maddison, 1997, version 3.07) such that di€erent scores for the 16 possible combinations were groups of taxa (i.e. Onchobothriidae, compared by chi-square analysis (see [18]). Phyllobothriidae, Phyllobothriinae, Rhine- Because it was computationally infeasible to use bothriinae, Lecanicephalidea within the even a heuristic search strategy for the ML Tetraphyllidea, ray-hosted taxa, and shark-hosted analysis, it was necessary to limit the tree space taxa) were constrained to form monophyletic, in which topologies were evaluated under the but unresolved clades. Maximum parsimony ana- optimality criterion of ML. This was achieved by lyses were performed using these constraints and saving all trees one step longer than the most the signi®cance of the di€erence in the lengths of parsimonious tree, and using the most likely of the resulting strict consensus trees evaluated by these as the starting tree for the ML analysis. comparison to the unconstrained most parsimo- Saving all trees at this length resulted in the nious trees (MPTs) using the Kishino±Hasegawa evaluation of topologies which di€ered among all and Templeton compatibility tests implemented basal nodes subtending the ingroup taxa, as evi- in PAUP* (Table 2). dent by Bremer support values (Fig. 1A). The Sequences were also analysed by the method of possibility of a more likely topology was then maximum likelihood (ML) in order to examine examined by re-arranging the starting tree via the potentially misleading e€ect of multiple sub- TBR branch swapping. stitutions on analysis by MP. A model of nucleo- tide substitution was chosen in the following way: each model implemented by PAUP* [Jukes± 3. Results Cantor, Kimura two-parameter, Felsenstein, 84/ Hasegawa, Kishino and Yano, 85 and General 3.1. Analysis by parsimony Time-Reversible (GTR)] was used to generate a log-likelihood value based on the strict consensus Of the 663 sites included, 440 (66%) were con- topology from analysis by parsimony. In addition stant, 62 (10%) were autapomorphic and 161 to testing each of the four models alone, esti- (24%) were parsimony informative. Empirical mates of among-site rate variation were incorpor- nucleotide frequencies showed a bias toward gua- ated using: (1) an invariant sites model (I), (2) a nine and thymine relative to adenine and cytosine gamma model (G), and (3) both an invariant sites (29%, 26%, 21% and 24%, respectively).

Table 2 Tree scores and tests of taxonomic and host-association constraint clades

Constraint clade N a Tree length No. of trees Steps longer Kishino±Hasegawa Templeton's

Taxonomic Lecanicephlidea within Tetraphyllideab 19 730 16 39 < 0.0001* < 0.0001* Fm. Onchobothriidae 6 714 8 23 0.0004* 0.0007* Fm. Phyllobothriidae 16 696 2 5 0.6686 0.6353 SbFm. Phyllobothriinae 8 717 30 26 0.0019* 0.0087* SbFm. Rhinebothriinae 7 710 6 19 0.0172* 0.0171*

Host association Ray-hosted tetraphyllideans 14 698 6 7 0.2975 0.2632 Shark-hosted tetraphyllideans 12 694 4 3 0.7817 1.000

aNumber of taxa in constraint clade. bConstrained node indicated by an asterisk in Fig. 1B. *Indicates a signi®cant di€erence (P < 0.05) in tree length between the strict consensus topologies of the constrained and most parsimonious trees. P.D. Olson et al. / International Journal for Parasitology 29 (1999) 1465±1476 1471

Macrobothridium sp. Diphyllidea (OUTGROUP) Echinobothrium fautleyae 7 Cephalobothrium cf. aetobatidis Lecanicephalidea 85 Eniochobothrium gracile n Ech Echeneibothrium vernetae v Rhi Rhinebothrium maccallumi 2 6 v Rhi Rhinebothrium n. sp. 15 6 90 v Rhi Rhinebothrium sp. A 100 95 2 v Phy Spongiobothrium sp. 2 53 u Phy Rhodobothrium sp. 1 v Phy Anthocephalum sp. 2 19 1 56 v Phy Anthocephlaum alicae 100 v Phy Anthocephalum sp. 1 2 u Dio Dioecotaenia cancellata 8 u Rhi Duplicibothrium n. sp. 2 u 98 6 Rhi Duplicibothrium minutum u 1 95 Rhi Duplicibothrium n. sp. 1 3 ¨ Rhi Caulobothrium sp. 1 73 l Phy Ceratobothrium xanthocephalum 11 l Lit Litobothrium alopias 1 100 l Lit Litobothrium amplifica l Phy Clistobothrium montaukensis 1 l Phy Paraorygmatobothrium sp. 1 l Onc Calliobothrium violae 1 l Onc Calliobothrium sp. l Onc Phoreiobothrium sp. 4 8 l Onc Platybothrium auriculatum 66 1 100 l Pro Prosobothrium armigerum l 8 Onc Acanthobothrium sp. 1 + rays 99 l Onc Acanthobothrium sp. 2 Host associations Macrobothridium sp. Diphyllidea (OUTGROUP) .005 Echinobothrium fautleyae .013 v .064 Phy Anthocephalum sp. 1 .059 .022 .004 v Phy Anthocephalum sp. 2 .019 v Phy Anthocephlaum alicae .078 .025 v Rhi Rhinebothrium maccallumi .027 v Phy Spongiobothrium sp. .034 .016 .018 u Phy Rhodobothrium sp. B .015 .079 .027 v Rhi Rhinebothrium sp. .038 .004 v Rhi Rhinebothrium n. sp. .042 n Ech Echeneibothrium vernetae .051 Cephalobothrium cf. aetobatidis .006 .119 Eniochobothrium gracile Lecanicephalidea .004 .034 l Lit Litobothrium alopias .010 .014 l Lit Litobothrium amplifica * .180 u Dio Dioecotaenia cancellata .017 .010 u .002 .035 Rhi Duplicibothrium n. sp. 2 .002 .007 .020 u Rhi Duplicibothrium minutum .005 u Rhi Duplicibothrium n. sp. 1 .029 .014 ¨ Rhi Caulobothrium sp. .004 .021 l Phy Ceratobothrium xanthocephalum .005 l Phy Clistobothrium montaukensis .010 .037 l Phy Paraorygmatobothrium sp. .018 .007 .006 l Onc Calliobothrium violae .011 n l Onc Calliobothrium sp. Rajidae (Skates) .002 .036 l v Onc Phoreiobothrium sp. Dasyatidae (Diamond stingrays) .023 .012 .027 l Onc Platybothrium auriculatum u Rhinopteridae (Cownose rays) .010 .012 l Pro Prosobothrium armigerum ¨ Myliobatidae (Eagle rays) .019 .030 l Onc Acanthobothrium sp. 1 l .018 + rays Sharks l Onc Acanthobothrium sp. 2 Fig. 1. Results of phylogenetic analysis by maximum parsimony (A) and maximum likelihood (B) showing patterns of host associ- ation. (A) Strict consensus of four most parsimonious trees. Tree-length = 691 steps (max./min. steps = 347/1113), CI = 0.5, RI = 0.55, RC = 0.23, HI = 0.5. Numbers above nodes are decay indices; numbers below nodes are bootstrap indicese50% (1000 replicates). (B) Most likely topology based on a GTR + I + G nucleotide substitution model. Numbers show estimates of genetic distance. An asterisk (*) indicates the constraint node used to test the position of the Lecanicephalidea (see text). Dio, Dioecotaniidae; Ech, Echeneibothriinae; Lit, Litobothriidae; Phy, Phyllobothriinae; Pro, Prosobothriidae; Onc, Onchobothriidae; Rhi, Rhinebothriinae. 1472 P.D. Olson et al. / International Journal for Parasitology 29 (1999) 1465±1476

However, w 2 analysis did not reveal signi®cant Phyllobothriidae to be monophyletic did not heterogeneity in base frequencies among taxa, result in a signi®cantly longer tree length. and these data were thus not subject to this po- tential source of systematic error. Four MPTs 3.2. Analysis by maximum likelihood resulted from the analysis (691 steps, CI = 0.5, RI = 0.55, RC = 0.23 and HI = 0.5). A strict Chi-square analysis showed the GTR + I + G consensus of the MPTs is shown in Fig. 1A. model of nucleotide substitution to be a signi®- Strong nodal support was found for most of the cantly better ®t to the data than the less-complex distal nodes of the tree, while deeper-level nodes models, based on a strict consensus topology of were generally supported by decay indices of 1 the MPTs. The most likely topology (Fig. 1B) and bootstrap values below 50%. The lecanice- had a log-likelihood of 4201.0417; the proportion phalidean taxa were found to be the sister to the of sites estimated to be invariable (I) was 53% tetraphyllidean species, and monophyly of the (observed proportion = 66%) and the estimate of Tetraphyllidea was thus supported. Within the G was 0.74. Estimated branch lengths among in- Tetraphyllidea, three major clades were recov- ternal ingroup nodes ranged from 0.002 to 0.064, ered: (1) a basal clade including species in the although branch lengths of deep-level internal phyllobothriid genera Anthocephalum, Echenei- nodes ranged from 0.002 to only 0.016. Terminal bothrium, Rhinebothrium, Rhodobothrium and branch lengths ranged from 0.002 to 0.18. The Spongiobothrium; (2) a clade uniting the phyllo- topology based on ML analysis di€ered from the bothriid genus Duplicibothrium with the dioeco- MPTs, primarily in that the lecanicephalidean taeniid genus Dioecotaenia; and (3) a larger sister taxa were placed within the Tetraphyllidea, and clade to the Duplicibothrium + Dioecotaenia some members of basal clades were `ladderised' clade that included the phyllobothriid genera (Fig. 1B). The three Anthocephalum species Caulobothrium, Ceratobothrium, Clistobothrium, formed the most basal clade in the ingroup. Paraorygmatobothrium, the prosobothriid genus Prosobothrium, the litobothriid genus Lito- bothrium and the onchobothriid genera Acanthobothrium, Calliobothrium, Phoreio- 4. Discussion bothrium and Platybothrium. Based on current concepts of tetraphyllidean 4.1. Genetic variability and phylogenetic signal classi®cation [2], the family Phyllobothriidae was within the ssrDNA gene found to be paraphyletic and monophyly of the family Onchobothriidae was brought into ques- Variability in the V4 and V7 regions of the tion by the position of the prosobothriid species, ssrDNA gene observed among the tetraphylli- Prosobothrium armigerum, as the sister taxon to dean genera and families was comparable to that Platybothrium auriculatum. Of the genera for among at least some of the orders of tapeworms which multiple species were included in the (cf. [6]). Such variation was greater than would analysis, support of monophyly was found for be expected for comparisons at these levels of Acanthobothrium, Anthocephalum, Dupliciboth- taxonomic inference [19], suggesting that the taxa rium and Litobothrium. Species in the genus involved are indeed very old. As a result, most of Rhinebothrium were found to be paraphyletic, the sites within these regions had to be removed and monophyly of the two species in the genus prior to analysis; the divergence among sequences Calliobothrium was ambiguous. Constraint ana- was too great to reliably infer their positional lyses (Table 2) showed that forcing the family homologies. How the tetraphyllideans compare Onchobothriidae, and subfamilies Phyllo- with other orders of tapeworms with regard to bothriinae and Rhinebothriinae to be monophy- intra-ordinal variability of the ssrDNA gene can- letic, resulted in signi®cantly longer tree lengths not be readily determined at this time. In con- in each case. However, forcing the family trast to the present study, Mariaux's ordinal-level P.D. Olson et al. / International Journal for Parasitology 29 (1999) 1465±1476 1473 molecular analysis of tapeworms [7] targeted either particularly long or short terminal regions of the ssrDNA gene that show high levels branches relative to the other taxa (Fig. 1B). of conservation. Mariaux analysed nucleotide Evaluation of the tree lengths of 1 000 000 ran- positions from stem 6 to stem 16, encompassing dom topological arrangements of the taxa the V1±V2 regions, the conserved 3 0 end of the resulted in a g1-statistic of 0.878; a highly left- V4 region to stem 35, encompassing the V5 skewed distribution indicating a strong degree of region, and the highly conserved V8±V9 regions hierarchical structure in the data. However, left- of the 3 0 end of the ssrDNA gene. These areas skewness in the distribution of cladogram lengths are adjacent to the regions examined herein, with is in¯uenced, among other things, by characters the exception of the short V8±V9 regions com- that strongly support small clades of taxa within mon to both studies. He found almost no intra- a larger, and potentially poorly supported, ordinal variability among species of proteocepha- tree [24]. Thus, the value of the g1 statistic in this lideans, while the same conserved regions were case was likely in¯uenced by the support for found to be informative for inferring relation- well-resolved terminal clades, rather than strong ships among species of cyclophyllideans [7]. signal for the overall topology. Thus, we can only draw the conclusion that the relative ages of the currently recognised higher taxa (i.e. orders) may be more disparate than 4.2. Phylogenetic implications their equal taxonomic ranking [20] would imply. Estimates of branch lengths, based on both The multiloculate bothridial morphology MP and ML analyses, showed that most terminal exhibited by species in the genera Caulobothrium, branches were approximately equivalent in Dioecotaenia, Duplicibothrium, Echeneibothrium, length. However, the tetraphyllidean taxon, Rhinebothrium and Spongiobothrium (e.g. see Dioecotaenia cancellata, was found to be highly ®gures in [2]) was found to be a symplesio- divergent relative to the other study taxa (with a morphic condition of tetraphyllideans, rather branch length of 0.18 as estimated by the than a synapomorphy of these taxa. However, GTR + I + G nucleotide substitution model). the phylogenetic position of Caulobothrium sp. as The next terminal branch closest in length was the sister taxon to Ceratobothrium xanthocepha- that of the lecanicephalidean, Eniochobothrium lum was unexpected based on morphological gracile, which had an estimated branch length of di€erences between these two species. In addition 0.12. Lengths of nearly all other terminal to bothridial homologies, species in the genus branches were well below 0.05. This is notable Caulobothrium share derived morphological fea- because the genus Dioecotaenia is one of the only tures of the proglottid with species in the genera groups of tapeworms that exhibits strobila of Dioecotaenia, Duplicibothrium, Glyphobothrium separate sexes (dioecy). As a rule, tapeworms are and Serendip (see [25]). For example, members of strictly hermaphroditic and true dioecy is known these genera have testes distributed in the ovarian only among the members of one other unrelated ®eld, and marked protandric segment develop- group, the cyclophyllidean family Dioeco- ment, which are features not found in C. xantho- cestidae [21]. The possibility that the unique sex- cephalum. Nevertheless, the close anity of ual dimorphism of D. cancellata may be coupled Caulobothrium sp. to C. xanthocephalum was sup- with an increased rate of genetic divergence is ported by both methods of analysis, and showed intriguing and warrants further investigation. strong nodal support (Fig. 1A). Furthermore, it is interesting that the phylogen- The placement of Duplicibothrium spp. with D. etic position of D. cancellata was apparently not cancellata supports the contention of Brooks and a€ected by either long-branch attraction [22] or Barriga [25] that these genera are closely related. repulsion [23], as it was found by both MP and These species share the presence of bothridia that ML analyses to be closest to species in the genus are fused dorso-ventrally as well as the absence Duplicibothrium which were not found to have of the large class of microtriches on all bothridial 1474 P.D. Olson et al. / International Journal for Parasitology 29 (1999) 1465±1476 surfaces (T.R. Ruhnke, personal observation). In The unexpected grouping of the prosobothriid addition, the three species of Duplicibothrium and species Prosobothrium armigerum with the oncho- the species of Dioecotaenia are found exclusively bothriid species Platybothrium auriculatum was in cownose rays (Rhinoptera spp.). supported by both methods of analysis and The cruciform bothridial morphology of the showed among the highest levels of nodal sup- Litobothriidae is suciently unique among all port. This indicates that the Onchobothriidae, a known tapeworms to have been used as a basis tetraphyllidean family supported by several de- for the recognition of ordinal status of the rived morphological features, and shown to be group [26]. The position of these species in the monophyletic by Caira et al. [4], may include at present analysis, however, is consistent with their least one non-hooked species within it. An a- more recent taxonomic placement within the nity between these two taxa, albeit dicult to order Tetraphyllidea by Euzet [2], but not with explain morphologically, is consistent with the the molecular analysis of Olson and Caira [6] in fact that both are parasites of blue sharks which these same two representative species were (Prionace glauca). found to be the sister group of the lecanicephali- dean, proteocephalidean and tetraphyllidean taxa. Similarly, Caira et al. [4] showed the genus 4.3. Host±parasite associations Litobothrium to be the sister to all of the lecani- cephalidean, proteocephalidean and tetraphylli- Rigorous comparisons of the congruence dean taxa in their study. Although the position between independently derived host and parasite of the Litobothriidae within the Tetraphyllidea phylogenies (e.g. [27±29]) are few in number, and was supported by both methods of analysis, its have yet to be performed for members of the exact position di€ered between the methods and order Tetraphyllidea. However, tetraphyllidean did not show a high level of nodal support. species typically show a high degree of host- Similar to the Litobothriidae, ordinal status of speci®city [4, 10]. For example, species in the the Lecanicephalidea has not been universally genus Paraorygmatobothrium show a strong accepted. An alternative position of the order degree of speci®city with their shark hosts was the primary di€erence between the results of (see [30, 31]), and species in the genus the two methods of analysis; MP (Fig. 1A) sup- Calliobothrium are speci®c to various species of ported their position outside of a monophyletic the shark genus Mustelus (e.g. [32]). Additionally, Tetraphyllidea, while ML (Fig. 1B) supported species of the genus Echeneibothrium seem in gen- their position within the order. Constraining the eral to be speci®c to species of the skate genus node subtending the position of the Raja (see [1]). Species of the genus Lecanicephalidea in the ML analysis (denoted by Potamotrygonocestus are speci®c to their fresh- an asterisk in Fig. 1B) resulted in a signi®cantly water stingray hosts species (Potamotrygon spp.) longer tree length via MP analysis (Table 2). It is and may have co-speciated with them [33]. These interesting that while the ML analysis was con- and other examples indicate that parallel specia- sistent with the results of Caira et al. [4] in the tion between tetraphyllideans and their elasmo- inclusion of the Lecanicephalidea within the branch hosts is a feature common to the Tetraphyllidea, in their analysis the lecanicephali- evolution of these tapeworms, particularly at dean taxa formed a clade together with lower taxonomic levels (i.e. families and genera). Echeneibothrium vernetae, which bears an apical This pattern could be spurious, however, if the structure of the scolex potentially homologous morphological variation among worms is a result with those of lecanicephalideans. However, in the of host-induced variation. If such a phenomenon present study, the positions of both the lecanice- occurred, then parasite species within a genus phalidean taxa and E. vernetae moved together could be distributed among several host species. between the two methods of analysis, and in the The ssrDNA nucleotide variation among the con- case of ML were separated by only a single node. generic species sequenced in this study, however, P.D. Olson et al. / International Journal for Parasitology 29 (1999) 1465±1476 1475 does not support the idea that morphological Acknowledgements di€erences among parasite taxa is a result of such host induced variation. For example, The authors wish to thank the following Duplicibothrium minutum and Duplicibothrium n. people for their assistance with the collection and sp. 1 di€er primarily in bothridial morphology, identi®cation of specimens: J.N. Caira, P. Cislo, which could reasonably be postulated to be the S. Curran, C. Healy, B. Jacobs, and K. Jensen result of adaptation to di€erences in the mucosal (University of Connecticut). Specimen collections lining of the host intestine. However, these two were supported primarily by National Science species showed four nucleotide di€erences even in Foundation grants DEB-9300796 to J.N. Caira the highly conserved regions of the ssrDNA gene and G.W. Benz (Tennessee Aquarium), and DEB-9521943 to J.N.C. and C.S. Henry and many more in the variable regions excluded (University of Connecticut). We thank J.N.C. from the analysis. also for critical review of an earlier version of the Results of both methods of analysis showed manuscript. P.D.O. gratefully acknowledges that the relationships among the tetraphyllidean C.M. Simon for the use of her laboratory, and P. taxa included in the present study re¯ect com- Betts and X. Liu of the Macromolecular mon host associations better than their current Characterization Facility at the University of classi®cation (Fig. 1). Indeed, forced monophyly Connecticut for assistance with automated of taxa based on strict patterns of host associ- sequencing and primer synthesis. Laboratory ex- ation did not result in signi®cantly longer tree penses and labour were supported in part by lengths than the unconstrained MPTs, whereas NSF grant DEB-9701052 to J.N.C., C.M.S. and most constraints based on their present classi®- P.D.O., a University of Connecticut Research cation did (Table 2). In the MP analysis, the Foundation grant to C.M.S. and P.D.O., a phyllobothriid parasites of sharks formed a clade Sigma Xi grant to P.D.O., a West Virginia State together with the litobothriid, onchobothriid and College Faculty Development Award to T.R.R., prosobothriid parasites of sharks, rather than a and a WVSC Foundation Research Stipend to clade with the other phyllobothriid taxa included J.S. P.D.O. gratefully acknowledges the support in the analysis, all of which parasitise rays. of a Marshall±Sher®eld Fellowship (Marshall Similarly, a more restricted clade of shark para- Aid Commission, UK) during the writing of the sites was observed in the ML analysis, including manuscript. both phyllobothriid and onchobothriid taxa. Both methods of analysis united D. cancellata (Dioecotaniidae) with the three species of Duplicibothrium (Rhinebothriinae); all of which parasitise cownose rays (Rhinoptera spp.). The References other members of the subfamily Rhinebothriinae (Rhinebothrium spp.) were grouped with members [1] Schmidt GD. Handbook of tapeworm identi®cation. Boca Raton, FL: CRC Press, 1986. of the subfamily Phyllobothriinae, in a clade [2] Euzet L. Order Tetraphyllidea Carus, 1863. In: Khalil (MP analysis), or set of clades (ML analysis), in LF, Jones A, Bray RA, editors. Keys to the cestode which their host association (diamond stingrays; parasites of vertebrates. 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