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 diered 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 dier 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 buer [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