D. T. J. Littlewood

Phylogeny and classification of the Digenea (Platyhelminthes: Trematoda).

Complete small subunit ribosomal RNA gene (ssrDNA) and partial (D1-D3) large subunit ribosomal RNA gene (lsrDNA) sequences were used to estimate the phylogeny of the Digenea via maximum parsimony and Bayesian inference. Here we contribute 80 new ssrDNA and 124 new lsrDNA sequences. Fully complementary data sets of the two genes were assembled from newly generated and previously published sequences and comprised 163 digenean taxa representing 77 nominal families and seven aspidogastrean outgroup taxa representing three families. Analyses were conducted on the genes independently as well as combined and separate analyses including only the higher plagiorchiidan taxa were performed using a reduced-taxon alignment including additional characters that could not be otherwise unambiguously aligned. The combined data analyses yielded the most strongly supported results and differences between the two methods of analysis were primarily in their degree of resolution. The Bayesian analysis including all taxa and characters, and incorporating a model of nucleotide substitution (general-time-reversible with among-site rate heterogeneity), was considered the best estimate of the phylogeny and was used to evaluate their classification and evolution. In broad terms, the Digenea forms a dichotomy that is split between a lineage leading to the Brachylaimoidea, Diplostomoidea and Schistosomatoidea (collectively the Diplostomida nomen novum (nom. nov.)) and the remainder of the Digenea (the Plagiorchiida), in which the Bivesiculata nom. nov. and Transversotremata nom. nov. form the two most basal lineages, followed by the Hemiurata. The remainder of the Plagiorchiida forms a large number of independent lineages leading to the crown clade Xiphidiata nom. nov. that comprises the Allocreadioidea, Gorgoderoidea, Microphalloidea and Plagiorchioidea, which are united by the presence of a penetrating stylet in their cercariae. Although a majority of families and to a lesser degree, superfamilies are supported as currently defined, the traditional divisions of the Echinostomida, Plagiorchiida and Strigeida were found to comprise non-natural assemblages. Therefore, the membership of established higher taxa are emended, new taxa erected and a revised, phylogenetically based classification proposed and discussed in light of ontogeny, morphology and taxonomic history.

The phylogeny of the Schistosomatidae based on three genes with emphasis on the interrelationships of Schistosoma Weinland, 1858.

Schistosomes are digenean flukes, parasitic of birds, mammals and crocodiles. The family Schistosomatidae contains species of considerable medical and veterinary importance, which cause the disease schistosomiasis. Previous studies, both morphological and molecular, which have provided a good deal of information on the phylogenetics of this group, have been limited in the number of species investigated or the type or extent of molecular data used. This paper presents the most comprehensive phylogeny to date, based on the sequences of 3 genes, complete ribosomal small subunit rRNA and large ribosomal subunit rRNA, and mitochondrial cytochrome oxidase 1, sequenced from 30 taxa including at least 1 representative from 10 of the 13 known genera of the Schistosomatidae and 17 of the 20 recognized Schistosoma species. The phylogeny is examined using morphological characters, intermediate and definitive host associations and biogeography. Theories as to the origins and spread of Schistosoma are also explored. The principal findings are that Ornithobilharzia and Austrobilharzia form a sister group to the Schistosoma; mammalian schistosomes appear paraphyletic and 2 Trichobilharzia species, T. ocellata and T. szidati, seem to be synonymous. The position of Orientobilharzia within the Schistosoma is confirmed, as is an Asian origin for the Schistosoma, followed by subsequent dispersal through India and Africa.

Phylogenetics of the Monogenea – evidence from a medley of molecules

Nuclear ribosomal DNA sequences of Monogenea from both complete small and partial large (D1-D2) subunits were determined and added to previously published sequences in order to best estimate the molecular phylogeny of the group. A total of 35 ssrDNA, 100 D1 lsrDNA and 51 D2 lsrDNA monogenean sequences were used, representing a total of 27 families. From these sequences different data sets were assembled and analysed to make the best use of all available molecular phylogenetic information from the taxa. Maximum parsimony and minimum evolution trees for each data partition were rooted against published sequences from the Cestoda, forcing the Monogenea to appear monophyletic. There was broad agreement between tree topologies estimated by both methods and between genes. Well-supported nodes were restricted to deeply diverging major groupings and more derived taxa with the lsrDNA data but were at most nodes with ssrDNA. The Polyonchoinea showed the greatest resolution with a general pattern of ((Monocotylidae(Capsalidae(Udonellidae+Gyrodactylidea)))((Anoplodiscidae+Sundanonchidae)(Pseudomurraytrematidae+Dactylogyridae))). The Heteronchoinea readily split into the Polystomatoinea+Oligonchoinea, and Chimaericolidae and Hexabothriidae were successively the most basal of oligonchoinean taxa. Relationships within the Mazocraeidea, comprising 27 families of which 15 were sampled here, were largely unresolved and appear to reflect a rapid radiation of this group that is reflected in very short internal branches for ssrDNA and D1 lsrDNA, and highly divergent D2 lsrDNA. A reduced morphological matrix, employing only those families represented by molecules, contrasted sharply with respect to polyonchoinean interrelationships. Deep branches of the Heteronchoinea were similar for both classes of data but also showed that the interrelationships of the mazocraeidean families are labile and susceptible to sampling.

Phylogenetic relationships of Echinostoma Rudolphi, 1809 (Digenea: Echinostomatidae) and related genera re-assessed via DNA and morphological analyses

In order to investigate the relationships within the Echinostomatidae two data sets of gene sequences were analysed. The first consisted of all previously published ND1 sequences (20) together with 17 new sequences. The latter represented six species from the cosmopolitan genera Echinostoma, Echinoparyphium, Hypoderaeum and Isthmiophora. The second data-set of ITS sequences again included all previously published sequences (12) and three new sequences from species of Echinostoma, Echinoparyphium and Isthmiophora. All new isolates, as well as voucher material from five previously sequenced isolates, were identified on the basis of morphological characters. The phylogenetic trees inferred from the ND1 data set helped to clarify the generic affiliation of all isolates and confirmed the morphological identifications. The only exception was Echinoparyphium aconiatum, whose current position in the genus Echinoparyphium was not supported by the sequence data. Although the ITS data provided insufficient resolution for an unequivocal solution to the relationships within the genus Echinostoma, it supported the identification of Echinoparyphium ellisi and the distinct species status of three isolates of Echinostoma revolutum as predicted from the ND1 data.

The phylogeny of the Lepocreadioidea (Platyhelminthes, Digenea) inferred from nuclear and mitochondrial genes: Implications for their systematics and evolution

The phylogenetic relationships of representative species of the superfamily Lepocreadioidea were assessed using partial lsrDNA and nad1 sequences. Forty-two members of the family Lepocreadiidae, six putative members of the Enenteridae, six gyliauchenid species and one Gorgocephalidae, were studied along with 22 species representing 8 families. The Lepocreadioidea is found to be monophyletic, except for the two species of the putative enenterid genus Cadenatella, which are found to be only distantly related to the lepocreadioids. The Lepocreadioidea is formed of five clades in a polytomy, the Gorgocephalidae, a clade containing the Enenteridae and Gyliauchenidae, a small clade of atypical lepocreadiines and the deep-sea lepidapedine lepocreadiids, a small clade consisting of a freshwater form and a group of shallow-water putative lepidapedines and the final clade includes the remaining lepocreadiids. Thus, the generally accepted concept of the Lepocreadiidae is polyphyletic. The Enenteridae (minus Cadenatella) and the Gyliauchenidae are jointly and individually monophyletic, and are sister groups. The nad1 gene on its own places a deep-sea lepocreadiine with the deep-sea lepidapedines, whereas lsrDNA, combined sequences and morphology place this deep-sea lepocreadiine within a group of typical lepocreadiids. It could not be demonstrated that a significant proportion of sites in the nad1 gene evolved under positive selection; this anomalous relationship therefore remains unexplained. Most deep-sea species are in a monophyletic group, a few of which also occur in shallow waters, retaining some characters of the deep-sea clade. Many lepocreadioid species infect herbivorous fish, and it may be that the recently discovered life-cycle involving a bivalve first intermediate host and metacercariae encysted on vegetation is a common life-cycle pattern. The host relationships show no indication of co-speciation, although the host-spectrums exhibited are not random, with related worms tending to utilize related hosts. There are, however, many exceptions. Morphology is found to be of limited value in indicating higher level relationships. For example, even with the benefit of hindsight the gyliauchenids show little morphological similarity to their sister group, the Enenteridae.

A molecular phylogeny of bryozoans.

We present the most comprehensive molecular phylogeny of bryozoans to date. Our concatenated alignment of two nuclear ribosomal and five mitochondrial genes includes 95 taxa and 13,292 nucleotide sites, of which 8297 were included. The number of new sequences generated during this project are for each gene:ssrDNA (32), lsrDNA (22), rrnL (38), rrnS (35), cox1 (37), cox3 (34), and cytb (44). Our multi-gene analysis provides a largely stable topology across the phylum. The major groups were unambiguously resolved as (Phylactolaemata (Cyclostomata (Ctenostomata, Cheilostomata))), with Ctenostomata paraphyletic. Within Phylactolaemata, (Stephanellidae, Lophopodidae) form the earliest divergent clade. Fredericellidae is not resolved as a monophyletic family and forms a clade together with Plumatellidae, Cristatellidae and Pectinatellidae, with the latter two as sister taxa. Hyalinella and Gelatinella nest within the genus Plumatella. Cyclostome taxa fall into three major clades: i. (Favosipora (Plagioecia, Rectangulata)); ii. (Entalophoroecia ((Diplosolen, Cardioecia) (Frondipora, Cancellata))); and iii. (Articulata ((Annectocyma, Heteroporidae) (Tubulipora (Tennysonia, Idmidronea)))), with suborders Tubuliporina and Cerioporina, and family Plagioeciidae each being polyphyletic. Ctenostomata is composed of three paraphyletic clades to the inclusion of Cheilostomata: ((Alcyonidium, Flustrellidra) (Paludicella (Anguinella, Triticella)) (Hislopia (Bowerbankia, Amathia)) Cheilostomata); Flustrellidra nests within the genus Alcyonidium, and Amathia nests within the genus Bowerbankia. Suborders Carnosa and Stolonifera are not monophyletic. Within the cheilostomes, Malacostega is paraphyletic to the inclusion of all other cheilostomes. Conopeum is the most early divergent cheilostome, forming the sister group to ((Malacostega, Scrupariina, Inovicellina) ((Hippothoomorpha, Flustrina) (Lepraliomorpha, Umbonulomorpha))); Flustrina is paraphyletic to the inclusion of the hippothoomorphs; neither Lepraliomorpha nor Umbonulomorpha is monophyletic. Ascophorans are polyphyletic, with hippothoomorphs grouping separately from lepraliomorphs and umbonulomorphs; no cribrimorphs were included in the analysis. Results are discussed in the light of molecular and morphological evidence. Ancestral state reconstruction of larval strategy in Gymnolaemata revealed planktotrophy and lecithotrophy as equally parsimonious solutions for the ancestral condition. More comprehensive taxon sampling is expected to clarify this result. We discuss the extent of non-bryozoan contaminant sequences deposited in GenBank and their impact on the reconstruction of metazoan phylogenies and those of bryozoan interrelationships.

Mitogenomics and phylogenomics reveal priapulid worms as extant models of the ancestral Ecdysozoan.

SUMMARY Research into arthropod evolution is hampered by the derived nature and rapid evolution of the best‐studied out‐group: the nematodes. We consider priapulids as an alternative out‐group. Priapulids are a small phylum of bottom‐dwelling marine worms; their tubular body with spiny proboscis or introvert has changed little over 520 million years and recognizable priapulids are common among exceptionally preserved Cambrian fossils. Using the complete mitochondrial genome and 42 nuclear genes from Priapulus caudatus, we show that priapulids are slowly evolving ecdysozoans; almost all these priapulid genes have evolved more slowly than nematode orthologs and the priapulid mitochondrial gene order may be unchanged since the Cambrian. Considering their primitive bodyplan and embryology and the great conservation of both nuclear and mitochondrial genomes, priapulids may deserve the popular epithet of “living fossil.” Their study is likely to yield significant new insights into the early evolution of the Ecdysozoa and the origins of the arthropods and their kin as well as aiding inference of the morphology of ancestral Ecdysozoa and Bilateria and their genomes.

Validity reassessment of Trichobilharzia species using Lymnaea stagnalis as the intermediate host

The systematics within the genus Trichobilharzia is complicated. After the description of the type species Trichobilharzia ocellata, the name was routinely used for nearly all European findings of ocellate furcocercariae. T. ocellata was also described from North America and Japan. However, the identity of T. ocellata remains questionable. Comparison of data from the literature showed differences among various T. ocellata isolates and led us to the conclusion that the North American and the Japanese findings are not identical with European T. ocellata. In addition, the description of T. szidati corresponds with the recently reported European T. ocellata isolates. Sequence analysis of the ITS region confirmed that they are identical.

Molecular phylogenetics of the four Schistosoma species groups determined with partial 28S ribosomal RNA gene sequences

Partial 28S ribosomal RNA (rRNA) gene sequences, including the variable domains D1, D2 and D3, were determined for representative species from the 4 Schistosoma species groups. On an alignment of 1345 bp from S. mansoni, S. haematobium, S. spindale and S. japonicum (with Heterobilharzia americana chosen as an outgroup), both maximum likelihood and maximum parsimony analyses provide a robust molecular phylogeny for the genus; ((((S. haematobium, S. spindale), S. mansoni), S. japonicum), H. americana). When analysed separately, both domain D1 and domain D2 yielded similarly informative data whereas D3 failed to resolve the phylogeny. These results confirm a phylogeny previously suggested by 18S rRNA gene sequences, corroborating the status of S. spindale as a sister taxon to S. haematobium, and demonstrate the utility of 28S rRNA gene sequence data for resolving phylogenies within the Schistosomatidae.

Digenean parasites of deep-sea teleosts: a review and case studies of intrageneric phylogenies.

Studies on the digenean parasites of deep-sea (> 200 m depth) teleosts are reviewed and two case study generic phylogenies are presented based on LSU rDNA and ND1 mtDNA sequences. The phylogeny of the lepocreadiid genus Lepidapedon, the most common deep-sea digenean genus, is not clearly resolved as the two gene trees are not compatible. It can be inferred, however, that the genus has radiated in the deeper waters off the continental shelf, mainly in fishes of the gadiform family Macrouridae. Steringophorus, a fellodistomid genus, is better resolved. In this case a deep-sea radiation is also indicated, but the pattern of host-specificity is not clear, with evidence of much host-switching. Results of studies of the parasites of the macrourid fish Coryphaenoides (Nematonurus) armatus from various depths have reinforced recent views on the lack of zoned depth-related communities in the deep-sea. The diversity of deep-sea digeneans is relatively low with only 18 families (of about 60) reported. Little, or nothing, is known from most deep-sea areas and nothing from trenches and mid-ocean ridge systems.