Evolutionary transitions in broad tapeworms (Cestoda: Diphyllobothriidea) revealed by mitogenome and nuclear ribosomal operon phylogenetics.

Broad tapeworms (Diphyllobothriidea) are parasites whose adults are capable of infecting a wide range of freshwater, marine and terrestrial tetrapods including humans. Previous works examining the evolution of habitat and host use in this group have been hampered by the lack of a well-resolved phylogeny. In order to produce a robust phylogenetic framework for diphyllobothriideans, we sequenced the complete mitochondrial genome of 13 representatives, carefully chosen to cover the major clades, and two outgroup species representing the Spathebothriidea and Haplobothriidea. In addition, complementary data from the nuclear ribosomal operon was sequenced for 10 representative taxa. Mitogenomes and ssrDNA and lsrDNA were used towards elucidating the phylogenetic framework for the Diphyllobothriidea. The Cephalochlamydidae is confirmed as the earliest diverging diphyllobothriidean lineage, and Solenophoridae and Diphyllobothriidae are sister groups. We infer a probable freshwater origin of the diphyllobothriideans. The ancestral condition for life cycle complexity could not be unambiguously resolved. However, we infer exclusive use of a three-host life cycle following the origin of the Solenophoridae + Diphyllobothriidae. Regarding definitive host use, although we infer reptiles as the most likely ancestral condition, this result should be revisited with a more densely sampled phylogeny in future studies. Freshwater habitat is used by the early diverging lineages within the Solenophoridae + Diphyllobothriidae clade. For the latter, habitat use shifts between freshwater and marine environments, and definitive host use includes marine and terrestrial mammals and birds. We use mitochondrial genomes to distinguish Schistocephalus species occurring in different species of sticklebacks and demonstrate conspecificity of Ligula cf. intestinalis specimens collected from two Fennoscandian ringed seal subspecies.

Nanopore Sequencing Resolves Elusive Long Tandem-Repeat Regions in Mitochondrial Genomes.

Long non-coding, tandem-repetitive regions in mitochondrial (mt) genomes of many metazoans have been notoriously difficult to characterise accurately using conventional sequencing methods. Here, we show how the use of a third-generation (long-read) sequencing and informatic approach can overcome this problem. We employed Oxford Nanopore technology to sequence genomic DNAs from a pool of adult worms of the carcinogenic parasite, Schistosoma haematobium, and used an informatic workflow to define the complete mt non-coding region(s). Using long-read data of high coverage, we defined six dominant mt genomes of 33.4 kb to 22.6 kb. Although no variation was detected in the order or lengths of the protein-coding genes, there was marked length (18.5 kb to 7.6 kb) and structural variation in the non-coding region, raising questions about the evolution and function of what might be a control region that regulates mt transcription and/or replication. The discovery here of the largest tandem-repetitive, non-coding region (18.5 kb) in a metazoan organism also raises a question about the completeness of some of the mt genomes of animals reported to date, and stimulates further explorations using a Nanopore-informatic workflow.

Molecular circumscription of new species of Gyrocotyle Diesing, 1850 (Cestoda) from deep-sea chimaeriform holocephalans in the North Atlantic.

Chimaeras, or ratfishes, are the only extant group of holocephalan fishes and are the sole host group of gyrocotylidean cestodes, which represent a sister group of the true tapeworms (Eucestoda). These unique, non-segmented cestodes have been known since the 1850s and multiple species and genera have been erected despite a general agreement that the delineation of species on the basis of morphology is effectively impossible. Thus, in the absence of molecular studies, the validity of gyrocotylid taxa and their specific host associations has remained highly speculative. Here we report the presence of Gyrocotyle spp. from rarely-caught deep-sea chimaeras collected in the North-East Atlantic, and describe two new species: G. haffii n. sp. from the bent-nose chimaera, Harriota raleighana Goode & Bean, and G. discoveryi n. sp. from the large-eyed rabbit fish, Hydrolagus mirabilis (Collett). Nuclear ribosomal sequence data were generated for individual parasites taken from different host species collected on different dates and from different localities and were combined with previously published sequences. Phylogenetic analyses supported the recognition of independent lineages and clusters, indicative of species, but were indecisive in recovering the root of the tree in analyses that included non-gyrocotylid outgroup taxa. The molecular data reveal variation not reflected in morphology and point to a complex picture of genetic divergence shaped by both isolation and migration in the deep-sea environment.

Phylogeny of hymenolepidid cestodes (Cestoda: Cyclophyllidea) from mammalian hosts based on partial 28S rDNA, with focus on parasites from shrews.

The aims of the study are to enrich the partial 28S rDNA dataset for hymenolepidids by adding new sequences for species parasitic in the genera Sorex, Neomys and Crocidura (Soricidae) and to propose a new hypothesis for the relationships among mammalian hymenolepidids. New sequences were obtained for Coronacanthus integrus, C. magnihamatus, C. omissus, C. vassilevi, Ditestolepis diaphana, Lineolepis scutigera, Spasskylepis ovaluteri, Staphylocystis tiara, S. furcata, S. uncinata, Vaucherilepis trichophorus and Neoskrjabinolepis sp. The phylogenetic analysis (based on 56 taxa) confirmed the major clades identified by Haukisalmi et al. (Zool Scr 39:631-641, 2010) based on analysis of 31 species: Ditestolepis clade, Hymenolepis clade, Rodentolepis clade and Arostrilepis clade; however, the support was weak for the early divergent lineages of the tree and for the Arostrilepis clade. Novelties revealed include the molecular evidence for the monophyly of Coronacanthus, the non-monophyletic status of Staphylocystis and the polyphyly of Staphylocystoides. The analysis has confirmed the monophyly of Hymenolepis, the monophyly of hymenolepidids from glirids, the position of Pararodentolepis and Nomadolepis as sister taxa, the polyphyly of Rodentolepis, the position of Neoskrjabinolepis and Lineolepis as sister taxa, and the close relationship among the genera with the entire reduction of rostellar apparatus. Resolved monophyletic groups are supported by the structure of the rostellar apparatus. The diversification of the Ditestolepis clade is associated with soricids. The composition of the other major clades suggests multiple evolutionary events of host switching, including between different host orders. The life cycles of Coronacanthus and Vaucherilepis are recognised as secondarily aquatic as these taxa are nested in terrestrial groups.

Integrated Approach Reveals Role of Mitochondrial Germ-Line Mutation F18L in Respiratory Chain, Oxidative Alterations, Drug Sensitivity, and Patient Prognosis in Glioblastoma.

Glioblastoma is the most common and malignant primary brain tumour in adults, with a dismal prognosis. This is partly due to considerable inter- and intra-tumour heterogeneity. Changes in the cellular energy-producing mitochondrial respiratory chain complex (MRC) activities are a hallmark of glioblastoma relative to the normal brain, and associate with differential survival outcomes. Targeting MRC complexes with drugs can also facilitate anti-glioblastoma activity. Whether mutations in the mitochondrial DNA (mtDNA) that encode several components of the MRC contribute to these phenomena remains underexplored. We identified a germ-line mtDNA mutation (m. 14798T > C), enriched in glioblastoma relative to healthy controls, that causes an amino acid substitution F18L within the core mtDNA-encoded cytochrome b subunit of MRC complex III. F18L is predicted to alter corresponding complex III activity, and sensitivity to complex III-targeting drugs. This could in turn alter reactive oxygen species (ROS) production, cell behaviour and, consequently, patient outcomes. Here we show that, despite a heterogeneous mitochondrial background in adult glioblastoma patient biopsy-derived cell cultures, the F18L substitution associates with alterations in individual MRC complex activities, in particular a 75% increase in MRC complex II_III activity, and a 34% reduction in CoQ10, the natural substrate for MRC complex III, levels. Downstream characterisation of an F18L-carrier revealed an 87% increase in intra-cellular ROS, an altered cellular distribution of mitochondrial-specific ROS, and a 64% increased sensitivity to clomipramine, a repurposed MRC complex III-targeting drug. In patients, F18L-carriers that received the current standard of care treatment had a poorer prognosis than non-carriers (373 days vs. 415 days, respectively). Single germ-line mitochondrial mutations could predispose individuals to differential prognoses, and sensitivity to mitochondrial targeted drugs. Thus, F18L, which is present in blood could serve as a useful non-invasive biomarker for the stratification of patients into prognostically relevant groups, one of which requires a lower dose of clomipramine to achieve clinical effect, thus minimising side-effects.

Australian spiny mountain crayfish and their temnocephalan ectosymbionts: an ancient association on the edge of coextinction?

Australian spiny mountain crayfish (Euastacus, Parastacidae) and their ecotosymbiotic temnocephalan flatworms (Temnocephalida, Platyhelminthes) may have co-occurred and interacted through deep time, during a period of major environmental change. Therefore, reconstructing the history of their association is of evolutionary, ecological, and conservation significance. Here, time-calibrated Bayesian phylogenies of Euastacus species and their temnocephalans (Temnohaswellia and Temnosewellia) indicate near-synchronous diversifications from the Cretaceous. Statistically significant cophylogeny correlations between associated clades suggest linked evolutionary histories. However, there is a stronger signal of codivergence and greater host specificity in Temnosewellia, which co-occurs with Euastacus across its range. Phylogeography and analyses of evolutionary distinctiveness (ED) suggest that regional differences in the impact of climate warming and drying had major effects both on crayfish and associated temnocephalans. In particular, Euastacus and Temnosewellia show strong latitudinal gradients in ED and, conversely, in geographical range size, with the most distinctive, northern lineages facing the greatest risk of extinction. Therefore, environmental change has, in some cases, strengthened ecological and evolutionary associations, leaving host-specific temnocephalans vulnerable to coextinction with endangered hosts. Consequently, the extinction of all Euastacus species currently endangered (75%) predicts coextinction of approximately 60% of the studied temnocephalans, with greatest loss of the most evolutionarily distinctive lineages.

The molecular phylogeny of the digenean family Opecoelidae Ozaki, 1925 and the value of morphological characters, with the erection of a new subfamily.

Large and small rDNA sequences of 41 species of the family Opecoelidae are utilised to produce phylogenetic inference trees, using brachycladioids and lepocreadioids as outgroups. Sequences were newly generated for 13 species. The resulting Bayesian trees show a monophyletic Opecoelidae. The earliest divergent group is the Stenakrinae, based on two species which are not of the type-genus. The next well-supported clade to diverge is constituted of three species of Helicometra Odhner, 1902. Based on this tree and the characters of the egg and uterus, a new subfamily, the Helicometrinae, is erected and defined to include the genera Helicometra, Helicometrina Linton, 1910 and Neohelicometra Siddiqi et Cable, 1960. The subfamily Opecoelinae is found to be monophyletic, but the Plagioporinae is paraphyletic. The single representative of the Opecoelininae (not of the type genus) is nested within a group of deep-sea 'plagioporines'. The two representatives of the Opistholebetidae are embedded within a group of shallow-water 'plagioporine' species. The Opistholebetidae is reduced to subfamily status pro tem as its morphological and biological characteristics are distinctive. This implies that as opecoelid systematics develops with more molecular evidence, several further subfamilies will be recognised. Many of the morphological characters were found to be homoplasious, but the characters defining the Helicometrinae and Opecoelinae, such as filamented eggs, reduced cirrus-sac and uterine seminal receptacle, are closely correlated with the inferred phylogeny.

The first multi-gene phylogeny of the Macrostomorpha sheds light on the evolution of sexual and asexual reproduction in basal Platyhelminthes.

The Macrostomorpha-an early branching and species-rich clade of free-living flatworms-is attracting interest because it contains Macrostomum lignano, a versatile model organism increasingly used in evolutionary, developmental, and molecular biology. We elucidate the macrostomorphan molecular phylogeny inferred from both nuclear (18S and 28S rDNA) and mitochondrial (16S rDNA and COI) marker genes from 40 representatives. Although our phylogeny does not recover the Macrostomorpha as a statistically supported monophyletic grouping, it (i) confirms many taxa previously proposed based on morphological evidence, (ii) permits the first placement of many families and genera, and (iii) reveals a number of unexpected placements. Specifically, Myozona and Bradynectes are outside the three classic families (Macrostomidae, Microstomidae and Dolichomacrostomidae) and the asexually fissioning Myomacrostomum belongs to a new subfamily, the Myozonariinae nov. subfam. (Dolichomacrostomidae), rather than diverging early. While this represents the first evidence for asexuality among the Dolichomacrostomidae, we show that fissioning also occurs in another Myozonariinae, Myozonaria fissipara nov. sp. Together with the placement of the (also fissioning) Microstomidae, namely as the sister taxon of Dolichomacrostomidae, this suggests that fissioning is not basal within the Macrostomorpha, but rather restricted to the new taxon Dolichomicrostomida (Dolichomacrostomidae+Microstomidae). Furthermore, our phylogeny allows new insights into the evolution of the reproductive system, as ancestral state reconstructions reveal convergent evolution of gonads, and male and female genitalia. Finally, the convergent evolution of sperm storage organs in the female genitalia appears to be linked to the widespread occurrence of hypodermic insemination among the Macrostomorpha.

Mating behavior and the evolution of sperm design.

Sperm are the most diverse of all animal cell types, and much of the diversity in sperm design is thought to reflect adaptations to the highly variable conditions under which sperm function and compete to achieve fertilization. Recent work has shown that these conditions often evolve rapidly as a consequence of multiple mating, suggesting a role for sexual selection and sexual conflict in the evolution of sperm design. However, very little of the striking diversity in sperm design is understood functionally, particularly in internally fertilizing organisms. We use phylogenetic comparative analyses covering 16 species of the hermaphroditic flatworm genus Macrostomum to show that a complex sperm design is associated with reciprocal mating and that this complexity is lost secondarily when hypodermic insemination--sperm injection through the epidermis--evolves. Specifically, the complex sperm design, which includes stiff lateral bristles, is likely a male persistence trait associated with sexual conflicts over the fate of received ejaculates and linked to female resistance traits, namely an intriguing postcopulatory sucking behavior and a thickened epithelium of the sperm-receiving organ. Our results suggest that the interactions between sperm donor, sperm, and sperm recipient can change drastically when hypodermic insemination evolves, involving convergent evolution of a needle-like copulatory organ, a simpler sperm design, and a simpler female genital morphology. Our study documents that a shift in the mating behavior may alter fundamentally the conditions under which sperm compete and thereby lead to a drastic change in sperm design.

Molecular evidence that the genus Cadenatella Dollfus, 1946 (Digenea: Plagiorchiida) belongs in the superfamily Haploporoidea Nicoll, 1914.

A re-examination of published lsrDNA sequence data of haploporid digeneans has shown that the genus Cadenatella Dollfus, 1946, hitherto considered a lepocreadioid, is correctly placed within the superfamily Haploporoidea Nicoll, 1914, although its relationships within the superfamily are not resolved. The morphological similarities and differences between Cadenatella and other haploporoids are discussed, and the subfamily Cadenatellinae Gibson & Bray, 1982 is considered the best repository for Cadenatella spp. at present.