rDNA Sequence Data Research Articles

About the Analysis of 18S rDNA Sequence Data from Trypanosomes in Barcoding and Phylogenetics: Tracing a Continuation Error Occurring in the Literature.

The variable regions (V1-V9) of the 18S rDNA are routinely used in barcoding and phylogenetics. In handling these data for trypanosomes, we have noticed a misunderstanding that has apparently taken a life of its own in the literature over the years. In particular, in recent years, when studying the phylogenetic relationship of trypanosomes, the use of V7/V8 was systematically established. However, considering the current numbering system for all other organisms (including other Euglenozoa), V7/V8 was never used. In Maia da Silva et al. [Parasitology 2004, 129, 549-561], V7/V8 was promoted for the first time for trypanosome phylogenetics, and since then, more than 70 publications have replicated this nomenclature and even discussed the benefits of the use of this region in comparison to V4. However, the primers used to amplify the variable region of trypanosomes have actually amplified V4 (concerning the current 18S rDNA numbering system).

Morphology, morphogenesis and molecular phylogeny of Lamtostyla granulifera sinensis subsp. nov. (Ciliophora, Hypotrichia) from a wetland in China

The morphology, morphogenesis and molecular phylogeny of a hypotrichous ciliate, Lamtostyla granulifera sinensis subsp. nov., isolated from northern China, were investigated. This population appeared highly similar in morphology to L. granulifera Foissner, 1997. However, on detailed investigation some non-overlapping features were identified, i.e., the body shape and the arrangement of the cortical granules. These differences suggested the separation at subspecies level. Furthermore, the morphogenesis of the new subspecies is described, which is characterized by: (1) the posterior part of the parental adoral zone of membranelles is renewed; (2) the amphisiellid median cirral row is formed from two anlagen; and (3) the frontoventral-transverse cirral anlagen II to VI generate one transverse cirrus each. Phylogenetic analyses based on SSU rDNA sequence data show that Lamtostyla species are scattered in different clades. The monophyly of the genus Lamtostyla is also rejected by the AU test in this study.

Group 2i Isochrysidales flourishes at exceedingly low growth temperatures (0 to 6 °C)

Group 2i Isochrysidales (Haptophyta) microalgae were recently suggested to be the main producer of the C37:4 alkenone in high latitude oceans with seasonal sea ice, enabling potential quantitative sea ice reconstructions using C37:4%. However, the conditions favoring the growth of Group 2i are currently unclear. Phylogenetically based on small subunit 18S rDNA sequence data, Group 2i is indistinguishable from OTU8 and Hap-A previously reported in numerous saline lakes around the world, and has been found to bloom in the early spring season in Lake George, North Dakota. These observations suggest Group 2i may have a preference for low growth temperatures and may grow within or underneath seasonal ice. Here we performed systematic culture experiments of Group 2i RCC5486 (isolated from sea ice of Baffin Bay) at exceedingly low temperatures (0, 3, 6, 9 °C at 31 ppt) and different salinities (15, 21, 26, 31, 38 ppt at 3 °C). We found Group 2i RCC5486 displays unusually fast growth from 0 to 6 °C, but failed to grow at 10.5 and 12 °C. These data confirm that Group 2i is explicitly adapted to low temperatures in both saline lakes and high latitude ocean regions (i.e., it is a psychrophile). Salinity, on the other hand, does not significantly influence the growth rates of Group 2i RCC5486. In addition, both growth temperature and salinity show minimal influence on C37:4%, which supports the use of C37:4% as a proxy for sea ice, rather than temperature or salinity in high latitude oceans.

Phylogeny and taxonomy of the genera of Erysiphaceae, part 1: Golovinomyces

ABSTRACT Powdery mildews are a monophyletic group of obligate plant pathogenic fungi in the family Erysiphaceae. Powdery mildews are economically important in that they cause damage to many agriculturally significant crops and plants in ecologically important habitats. In this contribution, we introduce a new series of publications focusing on the phylogeny and taxonomy of this group, with an emphasis on specimens collected from North America. The first part of the series focuses on the genus Golovinomyces and includes a section detailing the powdery mildew species concept. We conducted analyses of Golovinomyces spp. with available rDNA sequence data from GenBank and supplemented the data set with rDNA (ITS, 28S, IGS) as well as protein-coding (GAPDH) data from 94 North American collections. Many of the species evaluated are included in phylogenetic and morphological analyses for the first time, including the American species G. americanus, G. brunneopunctatus, G. californicus, G. greeneanus, G. hydrophyllacearum, and G. sparsus. A special emphasis was placed on acquiring ex-type or ex-epitype sequences or presenting reference sequences for phylogenetic-taxonomic purposes. Three new species, G. eurybiarum, G. galiorum, and G. malvacearum, are described, and the new combinations G. fuegianus, G. mutisiae, and G. reginae are introduced. Ex-holotype sequences of Erysiphe sparsa (≡ G. sparsus) reveal that it should be reduced to synonymy with G. ambrosiae, and ex-epitype sequences of G. valerianae reveal that it should be reduced to synonymy with G. orontii. Multiple epitypes are designated with ex-epitype sequences.

Integrative Studies on Three Epibiotic Epistylis Species (Protozoa, Ciliophora, Peritrichia) in Lake Weishan Wetland, Northern China, Including the Establishment of a New Species

Three epibiotic Epistylis species, i.e., Epistylis weishanensis sp. nov., Epistylis daphniae Fauré-Fremiet, 1905, and Epistylis pygmaeum (Ehrenberg, 1838) Foissner et al., 1999, were investigated based on their living morphology, infraciliature, and small subunit (SSU) rDNA sequence data. Epistylis weishanensis sp. nov. is characterized by its double-layered peristomial lip, contractile vacuole located on the dorsal wall of the infundibulum, infundibular polykinety 3 (P3) composed of three equal-length rows that terminate above infundibular polykinety 1 (P1), 50-62 silverlines between the peristome and the trochal band, and about 30 silverlines between the trochal band and the scopula. Based on previous and newly obtained data for E. daphniae and E. pygmaeum, improved diagnoses and redescriptions are provided including, for the first time, data on their infraciliature. Phylogenetic analyses reveal that all three species do not group within the major clade of Epistylis, supporting the assertion that the genus Epistylis should be an assemblage of morphospecies and therefore needs to be revised.

Four new monorchiids from marine teleost fishes of Moreton Bay and the Great Barrier Reef, Australia, including the proposal of a new genus

We report four new species of monorchiids infecting teleost fishes from Australian waters. Two new species of Paralasiotocus Wee, Cutmore, Pérez-del-Olmo & Cribb, 2020, Pa. abstrusus n. sp. and Pa. tectus n. sp., are described from haemulids of the Great Barrier Reef. The two species are morphologically cryptic and occur in sympatry but differ significantly in cox1 mtDNA and ITS2 rDNA sequence data. Paralasiotocus tectus n. sp. is found only in Plectorhinchus albovittatus (Rüppell) whereas Pa. abstrusus n. sp. infects Pl. albovittatus, Plectorhinchus flavomaculatus (Cuvier) and Plectorhinchus lineatus (Linnaeus). The two species differ from all known species of Paralasiotocus in the possession of a clear gap in the spines of the terminal organ. A new species is described from a mullid, Parupeneus spilurus (Bleeker), from off Heron Island and Moreton Bay. The new species is morphologically broadly consistent with the concept of Paralasiotocus in the possession of an unspined genital atrium, bipartite terminal organ, and lobed ovary. However, it possesses a highly lobed cirrus and is phylogenetically widely separated from the two species of Paralasiotocus characterized here, and thus we propose Lobucirruatus infloresco n. g., n. sp. Proctotrema prominens n. sp., is described from Pl. albovittatus. It is differentiated from all other species of Proctotrema in the combination of a prominent metraterm, slightly fusiform body, slightly funnel-shaped oral sucker, elongate cirrus-sac, unlobed ovary, and caeca that terminate in the post-testicular region.

Phylogeny, Divergence Time Estimation and Biogeography of the Genus Onnia (Basidiomycota, Hymenochaetaceae).

Species of Onnia are important tree pathogens and play a crucial role in forest ecosystems. The species diversity and distribution of Onnia have been studied, however, its evolutionary history is poorly understood. In this study, we reconstructed the phylogeny of Onnia using internal transcribed spacers (ITS) and large subunit (LSU) rDNA sequence data. Molecular clock analyses developed the divergence times of Onnia based on a dataset (ITS + LSU rDNA + rpb1 + rpb2 + tef1α). Reconstruct Ancestral State in Phylogenies (RASP) was used to reconstruct the historical biogeography for the genus Onnia with a Dispersal Extinction Cladogenesis (DEC) model. Here, we provide a robust phylogeny of Onnia, with a description of a new species, Onnia himalayana from Yunnan Province, China. Molecular clock analyses suggested that the common ancestor of Onnia and Porodaedalea emerged in the Paleogene period with full support and a mean stem age of 56.9 Mya (95% highest posterior density of 35.9–81.6 Mya), and most species occurred in the Neogene period. Biogeographic studies suggest that Asia, especially in the Hengduan-Himalayan region, is probably the ancestral area. Five dispersals and two vicariances indicate that species of Onnia were rapidly diversified. Speciation occurred in the Old World and New World due to geographic separation. This study is the first inference of the divergence times, biogeography, and speciation of the genus Onnia.

Metabarcoding of organic tea (Camellia sinensis L.) chronosequence plots elucidates soil acidification-induced shifts in microbial community structure and putative function

Tea (Camellia sinensis L.) agroecosystem soil acidification (TASA) is occurring at epidemic rates relative to other crops, however TASA remains poorly defined in its pathogenesis and effects on soil microbiota. The present study examined bulk soil physicochemical and bacterial 16S rDNA sequence data to elucidate TASA-associated changes through 10 tea agroecosystem sites, including an organic-certified tea field chronosequence of 3-year- (Young), 8-year- (Middle), and 13-year-old (Old) plots. Soil pH declined linearly with age through chronosequence plots. Hierarchical clustering based on community β-diversity revealed a tipping point in community composition as soil acidified below pH 4.5, which had occurred between Middle and Old plots. Tipping point-associated changes in predicted community functionality included a precipitous decline in nitrogenase-encoding communities and concurrent proliferations of Al-detoxifiers, anti-oxidative specialists, and pathogen virulence factor-encoding communities. PERMANOVA modeling revealed that Streptomyces was the genus with population abundance corresponding most significantly with whole microbiome composition at both OTU- (r2 = 0.460, p < 0.001) and genus-levels (r2 = 0.512, p < 0.001), despite ranking 74th in total abundance among 186 core genera, providing computationally-modeled evidence of a keystone role of Streptomyces under tea agroecosystem conditions. Correlations of soil pH with Rhizobiales (r = 0.713, p < 0.001), Acidibacter (r = −0.717, p < 0.001), and predicted β-glucosidase-encoding communities (r = −0.622, p < 0.001) across all samples indicated potential utility as community-level TASA biomarkers. Moreover, two phylogenetically diverse OTU co-abundance modules also corresponded (p < 0.01) with soil pH across all samples, suggesting that TASA-induced shifts were interfaced by networks of diverse and interactive taxa, or keystone guilds. Presently, organic management per se was unable to retain soil pH within the optimal range for tea plant cultivation (pH 4.5–5.5), therefore additional considerations not currently encompassed by organic management guidelines, such as lower tea planting density, may be necessary to avoid the detrimental effects of soil acidification described above. Moreover, future studies may seek to further elucidate the keystone functions of Streptomyces spp. under tea agroecosystem conditions.

First report on the morphological and molecular identification of Lactocollybia variicystis from Salahadin Governorate, North Central Iraq

A rare agaric, Lactocollybia variicystis D.A. Reid and Eicker (Family: Marasmiaceae) is identified as a new record to Iraqi macromycota, based on morphological characteristics and molecular analysis. Descriptions, color photographs, comments and phylogenetic analysis based on ITS rDNA sequence data are given.

Discovery of the parasite Marteilia cocosarum sp. nov. In common cockle (Cerastoderma edule) fisheries in Wales, UK and its comparison with Marteilia cochillia

Diseases of bivalve molluscs caused by paramyxid parasites of the genus Marteilia have been linked to mass mortalities and the collapse of commercially important shellfish populations. Until recently, no Marteilia spp. have been detected in common cockle (Cerastoderma edule) populations in the British Isles. Molecular screening of cockles from ten sites on the Welsh coast indicates that a Marteilia parasite is widespread in Welsh C. edule populations, including major fisheries. Phylogenetic analysis of ribosomal DNA (rDNA) gene sequences from this parasite indicates that it is a closely related but different species to Marteilia cochillia, a parasite linked to mass mortality of C. edule fisheries in Spain, and that both are related to Marteilia octospora, for which we provide new rDNA sequence data. Preliminary light and transmission electron microscope (TEM) observations support this conclusion, indicating that the parasite from Wales is located primarily within areas of inflammation in the gills and the connective tissue of the digestive gland, whereas M. cochillia is found mainly within the epithelium of the digestive gland. The impact of infection by the new species, here described as Marteilia cocosarum n. sp., upon Welsh fisheries is currently unknown.

Water Quality and Microbial Community Assessment in Artisanal Mining- Affected Sediments of Cikidang River, Banten, Java, Indonesia

The total microbial diversity and community in submerged sediments near mining sites, transition and intact sites at Cikidang River, Banten were assessed using 16S rDNA sequence data and biodiversity indices. Assessed water quality parameters in the river were water current, dissolved oxygen (DO), pH, redox potential, salinity, temperature and turbidity. Microbial alpha diversity used were Shannon diversity index, ChaoI index and Operational Taxonomic Units (OTUs). These analyses indicated a total of 50 taxa of sediment microbes. Based on Shannon diversity index, the highest value was observed at mining site. High abundant microbes in sediments at mining sites for phylum, class, order, family and genera levels were represented by Proteobacteria, Gammaproteobacteria, Pseudomonadales, Moraxellaceae, and Acinetobacter, respectively. In contrast, high abundant microbes in the sediment of the intact site for each taxon consisted of Firmicutes, Alphaproteobacteria, Erysipelotrichales, Erysipelotrichaceae, and Erysipelothrix. The water quality of mining sites was characterised by alkaline pH (8.807, 95% CI: 8.624 - 8.990) and lower redox potential (59.000 mV, 95% CI: 36.233 - 81.767) that differed significantly from the intact sites (P&lt;0.05). This study also confirmed that mining sites have a higher genus diversity. Research on potential microbes of related genus as bioremediators could be recommended for further study.

Identification of fungi found on desiccated human remains in an arid outdoor environment.

Three fungi not previously reported on desiccated human remains were identified on cadavers at the Forensic Investigation Research Station (FIRS) in Whitewater, Colorado. The location of the FIRS provides the unique opportunity to observe the stages of decomposition in a high desert environment. The two cadavers used in the study were in the late stages of decomposition (PMI of approximately 1520 and 1820 days) to the point of desiccation and had developed an extensive black crust on the skin that remained. Skin samples of the two cadavers were taken and plated onto potato dextrose agar to determine whether fungi were present on the desiccated tissues. Three different fungi consistently dominated cultures grown from numerous samples taken from each cadaver. Based on morphological observations, nuclear rDNA sequence data, and phylogenetic analyses, two fungi were identified to species (Aureobasidium melanogenum and Didymella glomerata) and one fungus was identified to the genus level (Alternaria). These results will contribute to the understanding of the role that fungi might play in late-stage decomposition and the extended postmortem period.

Morphology, Morphogenesis and Molecular Phylogeny of a New Soil Ciliate, Holostichides eastensis nov. spec. (Ciliophora, Hypotrichia)

A new bakuellid ciliate, Holostichides eastensis nov. spec. isolated from China, is investigated in terms of its morphology, ontogenesis, and molecular phylogenesis. It is characterized by size 150-180 µm × 45-60 µm in vivo; 27-46 macronuclear nodules; cortical granules dark brown, spherical, about 0.5 µm across, clustered in groups and then arranged longitudinally; three frontal cirri with one to four smaller cirri below the middle one, a buccal cirrus, nine to 15 frontoterminal cirri, and midventral complex composed of five to eight cirral pairs and two long midventral rows. The main events during binary fission are as follows: (1) in the proter, the undulating membrane anlage is formed from both the dedifferentiation of old undulating membranes and the basal bodies developing de novo, and the proximal part of the parental adoral zone of membranelles is renewed by a field of basal bodies that originates de novo; (2) in the opisthe, the oral primordium is formed intrakinetally; (3) besides the middle frontal cirrus and buccal cirrus, the FVT-anlagen II produces the extra one to four cirri. Phylogenetic analyses based on SSU rDNA sequence data suggest a close relationship between Holostichides eastensis nov. spec. and its congeners, indicating that the genus Holostichides is monophyletic.

A new species, new host records and life cycle data for lepocreadiids (Digenea) of pomacentrid fishes from the Great Barrier Reef, Australia

A new species of lepocreadiid, Opechonoides opisthoporusn. sp., is described infecting 12 pomacentrid fish species from the Great Barrier Reef, Australia, with Abudefduf whitleyi Allen & Robertson as the type-host. This taxon differs from the only other known member of the genus, Opechonoides gure Yamaguti, 1940, in the sucker width ratio, cirrus-sac length, position of the testes, position of the pore of Laurer’s canal, and relative post-testicular distance. The new species exhibits stenoxenic host-specificity, infecting pomacentrids from seven genera: Abudefduf Forsskål, Amphiprion Bloch & Schneider, Neoglyphidodon Allen, Neopomacentrus Allen, Plectroglyphidodon Fowler & Ball, Pomacentrus Lacépède and Stegastes Jenyns. Phylogenetic analyses of 28S rDNA sequence data demonstrate that O. opisthoporusn. sp. forms a strongly supported clade with Prodistomum orientale (Layman, 1930) Bray & Gibson, 1990. The life cycle of this new species is partly elucidated on the basis of ITS2 rDNA sequence data; intermediate hosts are shown to be three species of Ctenophora. New host records and molecular data are reported for Lepocreadium oyabitcha Machida, 1984 and Lepotrema amblyglyphidodonis Bray, Cutmore & Cribb, 2018, and new molecular data are provided for Lepotrema acanthochromidis Bray, Cutmore & Cribb, 2018 and Lepotrema adlardi (Bray, Cribb & Barker, 1993) Bray & Cribb, 1996. Novel cox1 mtDNA sequence data showed intraspecific geographical structuring between Heron Island and Lizard Island for L. acanthochromidis but not for L. adlardi or O. opisthoporusn. sp.

Morphological and Molecular Characterization of a New Myxozoan, Myxobolus grassi sp. nov. (Myxosporea), Infecting the Grass Carp, Ctenopharyngodon idella in the Gomti River, India.

Myxosporeans are well-known parasites infecting food fishes in fresh and marine water around the globe. Grass carp (Ctenopharyngodon idella), a freshwater food fish commonly cultured in India with has significant economic importance. Herein, the study focuses on the description of a new myxosporean species, Myxobolus grassi sp. nov. from the gills as primary site and liver as secondary site of infection in grass carp. Both organs (gill and liver) were infected concurrently in the host and the prevalence of grass carp infection was 4.05% in gill filaments and liver, respectively. Identification of species was based on the morphological and morphometric features of the myxospore as well as 18S rDNA sequence data. A smear from gill and liver exhibited hundreds of morphologically similar myxospores. BLAST search revealed 98% sequence similarity and 0.03 genetic distance with M. catlae (KM029967) infecting gill lamellae of mrigal carp (Cirrhinus cirrhosus) from India and 98–84% sequence similarity with other myxobolids in India, China, Japan, Malaysia, Turkey and Hungary. Phylogenetically, it clustered with other myxobolids infecting gills and related organs (i.e., vital organ) of Indian cyprinid carp species. On the basis of myxospore morphology and 18S sequence, we propose M. grassi sp. nov.

Using RDNA sequences to define dinoflagellate species

Dinoflagellate species are traditionally defined using morphological characters, but molecular evidence accumulated over the past several decades indicates many morphologically-based descriptions are inaccurate. This recognition led to an increasing reliance on DNA sequence data, particularly rDNA gene segments, in defining species. The validity of this approach assumes the divergence in rDNA or other selected genes parallels speciation events. Another concern is whether single gene rDNA phylogenies by themselves are adequate for delineating species or if multigene phylogenies are required instead. Currently, few studies have directly assessed the relative utility of multigene versus rDNA-based phylogenies for distinguishing species. To address this, the current study examined D1-D3 and ITS/5.8S rDNA gene regions, a multi-gene phylogeny, and morphological characters in Gambierdiscus and other related dinoflagellate genera to determine if they produce congruent phylogenies and identify the same species. Data for the analyses were obtained from previous sequencing efforts and publicly available dinoflagellate transcriptomic libraries as well from the additional nine well-characterized Gambierdiscus species transcriptomic libraries generated in this study. The D1-D3 and ITS/5.8S phylogenies successfully identified the described Gambierdiscus and Alexandrium species. Additionally, the data showed that the D1-D3 and multigene phylogenies were equally capable of identifying the same species. The multigene phylogenies, however, showed different relationships among species and are likely to prove more accurate at determining phylogenetic relationships above the species level. These data indicated that D1-D3 and ITS/5.8S rDNA region phylogenies are generally successful for identifying species of Gambierdiscus, and likely those of other dinoflagellates. To assess how broadly general this finding is likely to be, rDNA molecular phylogenies from over 473 manuscripts representing 232 genera and 863 described species of dinoflagellates were reviewed. Results showed the D1-D3 rDNA and ITS phylogenies in combination are capable of identifying 97% of dinoflagellate species including all the species belonging to the genera Alexandrium, Ostreopsis and Gambierdiscus, although it should be noted that multi-gene phylogenies are preferred for inferring relationships among these species. A protocol is presented for determining when D1-D3, confirmed by ITS/5.8S rDNA sequence data, would take precedence over morphological features when describing new dinoflagellate species. This protocol addresses situations such as: a) when a new species is both morphologically and molecularly distinct from other known species; b) when a new species and closely related species are morphologically indistinguishable, but genetically distinct; and c) how to handle potentially cryptic species and cases where morphotypes are clearly distinct but have the same rDNA sequence. The protocol also addresses other molecular, morphological, and genetic approaches required to resolve species boundaries in the small minority of species where the D1-D3/ITS region phylogenies fail.

Survey of Volvariella (Agaricales, Basidiomycota) including Two New Species, V. neovolvacea and V. thailandensis, from Northern Thailand

During the period from 2018 to 2020, a survey of macrofungi in northern Thailand was conducted and seven specimens of Volvariella were collected. The morphological characteristics and phylogenetic analyses of the internal transcribed spacers (ITS) and large subunit (nrLSU) of the nuclear ribosomal DNA (rDNA) sequence data indicated that three specimens belonged to V. bombycina and V. volvacea. Four other specimens were distinct from all other known species within the genus Volvariella. We described these specimens as two new species, namely V. neovolvacea and V. thailandensis. Comprehensive descriptions, illustrations, line drawings, and a phylogenetic tree are provided to show the position of the two new species. The comparisons between morphologically similar and phylogenetically related species are also discussed.

Morphology, cell division, and phylogeny of Notohymena antarctica Foissner, 1996 and Engelmanniella mobilis (Engelmann, 1862) Foissner, 1982 (Ciliophora, Hypotrichia)

The morphogenesis of two hypotrich ciliates, Notohymena antarctica Foissner, 1996 and Engelmanniella mobilis (Engelmann, 1862) Foissner, 1982, were investigated using protargol staining. The main events of the oxytrichid Notohymena antarctica during binary fission are as follows: (1) the oral primordium of the opisthe is formed apokinetally left of transverse cirrus II/1; (2) six streaks of frontal-ventral-transverse cirral anlagen are segmented in the ordinary pattern 1: 3: 3: 3: 4: 4 from left to right, which form three frontal, four frontoventral, one buccal, three ventral, two pretransverse ventral and five transverse cirri; (3) marginal anlagen are formed intrakinetally and the right marginal anlagen develop much earlier than those on the left. Phylogenetic analyses based on SSU rDNA sequence data indicate that Notohymena is monophyletic. A remarkable morphogenetic feature in E. mobilis is the mode of formation of the right marginal rows. In this paper, we clarified that the right marginal anlagen of E. mobilis are formed and develop to the right of the parental outer right marginal row, and that the grandparental (inner) right marginal row and great-grand parental (the outermost) left marginal row are resorbed. It is the first time to report a population of E. mobilis with comprehensive information, i.e. the morphology, ontogeny and molecular sequence.

Supplementary studies on Myxobolus talievi Dogiel, 1957 (Cnidaria, Myxozoa) from the skeletal muscle of the cottoid fish Cyphocottus eurystomus in Lake Baikal: Morphological, histological and molecular characterisations.

Myxobolus talievi Dogiel, 1957 was originally described from the eyes, skeletal muscles and body cavity of endemic cottoid fish from Lake Baikal. In the present study, we supply new information on the myxospore morphology and histopathology of M. talievi; furthermore, we complete the original species description by Dogiel and Bogolepova (1957) with 18S ribosomal DNA (rDNA) sequence data. Histopathological analysis showed that the plasmodium was encapsulated by a thin layer of connective tissue and located in the intermuscular connective tissue among muscle cells. No inflammation was observed. Phylogenetic analysis revealed that M. talievi clustered with Myxobolus sp. 2 (NCBI Acc. No. U13830), an unidentified Myxobolus species from cottoid fish studied by Smothers et al. (1994), and located in the sister clade of Myxobolus spp. developing spores in the nerves of salmonids.

Three new species of Helicometroides Yamaguti, 1934 from Japan and Australia, with new molecular evidence of a widespread species.

We report specimens of monorchiids infecting Haemulidae from the waters off Japan and Australia; these specimens represent five species of Helicometroides Yamaguti, 1934, three of which are unambiguously new. Helicometroides murakamii n. sp. infects Diagramma pictum pictum from off Minabe, Japan; Helicometroides gabrieli n. sp. infects Plectorhinchus chrysotaenia from off Lizard Island, Australia; and Helicometroides wardae n. sp. infects Plectorhinchus flavomaculatus and Plectorhinchus multivittatus from off Heron Island, Australia. Helicometroides murakamii n. sp. and H. gabrieli n. sp. conform to the most recent diagnosis of Helicometroides in lacking a terminal organ, but H. wardae n. sp. possesses a terminal organ with distinct, robust spines; despite this morphological distinction, the three form a strongly-supported clade in phylogenetic analyses. We also report specimens morphologically consistent with Helicometroides longicollis Yamaguti, 1934, from D. pictum pictum from off Minabe, Japan, and Diagramma pictum labiosum on the Great Barrier Reef, Australia. Genetic analyses of ITS2 rDNA, 28S rDNA and cox1 mtDNA sequence data for the Japanese specimens reveal the presence of two distinct genotypes. Specimens of the two genotypes were discovered in mixed infections and are morphologically indistinguishable; neither genotype can be associated definitively with H. longicollis as originally described. We thus identify them as H. longicollis lineage 1 and 2, pending study of further fresh material. Genetic analyses of specimens from the Great Barrier Reef are consistent with the presence of only H. longicollis lineage 1. This species thus has a range that incorporates at least Australia and Japan, localities separated by over 7000 km.