Phylogenetic affinities and systematic position of Entomelas sylvestris Baker, 1982 (Nematoda: Rhabdiasidae), a parasite of Breviceps sylvestris FitzSimons (Amphibia: Brevicipitidae) in South Africa

The genus Entomelas Travassos, 1930 currently includes nine species of rhabdiasid nematodes, eight of them parasitic in lizards and only one, Entomelas sylvestris Baker, 1982, parasitic in amphibians. Entomelas sylvestris was originally described from the Forest Rain Frog Breviceps sylvestris FitzSimons in South Africa and was not reported since. It was placed in the genus Entomelas without any specific arguments for this taxonomic decision, presumably mainly based on details of the buccal capsule morphology. We have found this species in the same host in Limpopo province, South Africa. Molecular phylogenetic analysis based on the newly-obtained sequence of complete ITS region and partial nuclear large ribosomal subunit (28S) gene of E. sylvestris and previously published sequences of a variety of other rhabdiasid taxa, has convincingly demonstrated that this species does not belong in Entomelas. Instead, it clustered together with the members of Rhabdias Stiles & Hassall, 1905 from amphibian hosts. Therefore, we transfer E. sylvestris into Rhabdias as Rhabdias sylvestris (Baker, 1982) n. comb. In our analysis E. sylvestris appears, albeit with weak support, as a basal/sister taxon to the rest of Rhabdias spp. which explains to some extent the differences in the buccal capsule morphology between this species and other Rhabdias spp.     

A New Rhabdiasid Nematode, <Emphasis Type="Italic">Chabirenia cayennensis</Emphasis> n. g., n. sp., Parasitic in the Glands of the Buccal Mucosa of a South American Saurian

Chabirenia cayennensis n. g., n. sp. (Nematoda: Rhabdiasidae) is described from the teiid lizard Ameiva ameiva. A total of 139 worms were recovered, all females, from the mucous glands of the buccal cavity. The worm has a free-living phase in a homogonic life-cycle. Infective larvae are enclosed in a sheath with chequered ornamentation and composed of two exuviae. The new genus is distinct from the five known genera of the family, Pneumonema Johnston, 1916, Acanthorhabdias Pereira, 1927, Entomelas Travassos, 1930, Rhabdias Stiles & Hassall, 1905 and Neoentomelas Hasagawa, 1989, in the following characters: helical habitus, longitudinal cuticular crests, very tiny buccal cavity without thick walls and three oesophageal onchia. Several characters of this new rhabdiasid suggest the Strongylida.     

Diversity in the genus Rhabdias (Nematoda,Rhabdiasidae): Evidence for cryptic speciation

Lungworms from the genus Rhabdias are common parasites of amphibians and reptiles distributed worldwide. To assess the diversity of Rhabdias spp., we performed molecular analyses of 35 specimens sampled in different regions of Brazil. Molecular analyses were based on the internal transcribed spacer (ITS), large subunit (28S) ribosomal and the cytochrome oxidase I (COI) mitochondrial genes. DNA sequence divergence was compared among ribosomal and mitochondrial genes, analyses using the general mixed Yule‐coalescent (GMYC) method based on the COI gene were used to identify possible cryptic diversity, and phylogenetic analyses using concatenated ITS and 28S ribosomal genes were used to test the monophyly of Rhabdiasidae. We revealed five morphospecies: R. cf. stenocephala, R. breviensis, R. pseudosphaerocephala and two new species, Rhabdias sp.4 and Rhabdias sp.5. DNA sequence levels of divergence among genes ITS, 28S and COI were compared, and the efficiency of the molecular markers to identify species (ITS and COI) and lineages (COI) was tested. GMYC was assigned to 17 well‐supported clades (i.e., 17 species), and cryptic diversity was detected in the Neotropical region as evidenced by the multiple lineages in R. breviensis and R. pseudosphaerocephala. In addition, our results suggest evidence for host–parasite cophylogeny in the R. pseudosphaerocephala complex and dispersal events among their populations. Phylogenetic analyses supported the monophyly of Rhabdiasidae and improved the resolution of main clades. Rhabdias breviensis is closely related to Rhabdias cf. africanus, Rhabdias cf. stenocephala, R. pseudosphaerocephala, Rhabdias sp.4 and Rhabdias sp.5 grouping together in a main clade with Neotropical‐related species. The large geographical distribution appeared to be a phylogenetic pattern among the species of Rhabdias from the neotropics.     

Eimeria trichosuri: Phylogenetic position of a marsupial coccidium, based on 18S rDNA sequences

Phylogenetic analysis of the genus Eimeria suggests that parasite and host have coevolved over broad evolutionary timescales. Here we extend this analysis by determining the 18S rDNA gene sequence of the marsupial coccidium, Eimeria trichosuri, and assessing its phylogenetic position relative to Eimeria from birds, reptiles and placental mammals. This analysis placed E. trichosuri clones in a clade that diverged before the major clade comprising species from placental mammals. The position of E.trichosuri is consistent with host phylogeny where marsupials represent an ancient evolutionary line that predates the placental mammal line.     

Phylogeography of Rhabdias spp. (Nematoda: Rhabdiasidae) collected from Bufo species in Honshu,Shikoku, and Kyushu,Japan including possible cryptic species

The genus Rhabdias Stiles & Hassall, 1905 comprises lung parasites of amphibians and reptiles worldwide. In Japan, 9 species have been recorded, including Rhabdias incerta Wilkie, 1930 which has been reported only in Bufo species. In this study, to assess the diversity of R. incerta, we performed molecular analyses of Rhabdias species sampled from three species/subspecies of Japanese toads namely Bufo japonicus, B. japonicus formosus, and B. torrenticola, collected in various regions of Honshu, Shikoku, and Kyushu, Japan. DNA sequence divergence was compared using mtDNA (COI) and nuclear DNA (28S) to identify possible cryptic species. Morphological analysis was performed through light microscopy and scanning electron microscopy (SEM). The results revealed that Bufo spp. serve as hosts for at least three Rhabdias species. Morphologically, most samples were identified as R. incerta but with a longer body and larger buccal cavity than originally described. Rhabdias incerta appears to be specific to the genus Bufo and is further subdivided into two or three phylogroups based on subspecies divisions and biogeography of their host. Some Rhabdias specimens collected in this study resemble R. tokyoensis Wilkie, 1930, parasitic in hosts from the order Caudata, which suggests host switching. Both molecular and morphological analyses suggested the presence of undescribed and cryptic Rhabdias species within toads collected in Japan. This study was the first to molecularly characterize Rhabdias species in Japan, including novel sequences of R. incerta and two undescribed species.     

Molecular characterization of major and minor rDNA repeats and genetic variability assessment in different species of mahseer found in North India

Relationship among the mahseer species (Family: Cyprinidae) has long been debated in fish systematics. Present study concentrates on the nature of the phylogenetic relationship among the five mahseer species using the sequence of major ribosomal DNA (45S rDNA). We have covered rDNA sequence of approximately 5.2 kb per individual, 26.0 kb per species and 130.0 kb as a whole. We also characterized the 45S and 5S rDNA regions with respect to their nucleotide composition. For phylogenetic analyses, nucleotide sequences were divided into four datasets. First and second datasets contained 18S rDNA and ITS1 sequence, whereas third and fourth datasets consisted of ITS2 and complete 18S-ITS1-5.8S-ITS2-28S, respectively. The NJ tree was constructed for all the datasets. The mahseer species under study formed a monophyletic group well separated from the outgroup species. Similarly, the individuals of Neolissochilus hexagonolepis form monophyletic group with Tor species, indicating Neolissochilus as a sister genus of Tor. The findings from the present study provide greater insights into taxonomic status of mahseer, and set the stage for future investigations dealing with phylo-geography, taxonomy, conservation and co-evolution within this interesting and important group of fish.     

Morphological and molecular differentiation of two new species of Pseudoacanthocephalus Petrochenko, 1958 (Acanthocephala: Echinorhynchidae) from amphibians and reptiles in the Philippines, with identification key for the genus

The genus Pseudoacanthocephalus Petrochenko, 1958 currently includes 14 species of acanthocephalans parasitic in amphibians and reptiles worldwide. This work describes two new species of Pseudoacanthocephalus from amphibians and reptiles collected in several localities on Luzon Island, Philippines. Pseudoacanthocephalus nickoli n. sp. was found in two species of frogs, Rana luzonensis Boulenger and Rana similis (Günther), and Pseudoacanthocephalus smalesi n. sp. was found in a scincid lizard, Sphenomorphus abdictus Brown & Alcala. Differential diagnoses of the two new species of Pseudoacanthocephalus from their congeners are provided. Comparative analysis of nuclear ribosomal rRNA sequences encompassing the 3′ end of 18S nuclear rDNA gene, internal transcribed spacer region (ITS1+5.8S+ITS2), and 5′ end of the 28S gene strongly corroborated the morphological evidence and demonstrated significant differences between the two new species as well as between these species and closely related species from continental China and Vietnam. No intraspecific sequence variability was detected among different individuals representing each of the examined species. This is the first report of Pseudoacanthocephalus in the Philippines. A key to known species of Pseudoacanthocephalus is provided.     

Evolutionary relationships of sibling tapeworm species (Cestoda) parasitizing teleost fishes

DNA/DNA hybridization and sequencing of rDNA (partial 18S rDNA and ITS1) were used to investigate phylogenetic relationships among seven host-specific Bothriocephalus parasites (Cestoda, Pseudophyllidae). The small nucleotide divergence between six of the seven bothriocephalids suggests that isolation and differentiation of Bothriocephalus lineages in the different host species probably occurred recently and over a short time span. Comparison of the molecular phylogeny of the parasite species to the phylogeny of their hosts (teleostean fishes) revealed little congruence between the branching patterns of hosts and parasites, suggesting that bothriocephalids have not cospeciated with their hosts.      

Mycobiota associated with the ambrosia beetle Scolytodes unipunctatus (Coleoptera: Curculionidae, Scolytinae)

Ambrosia beetles (Coleoptera: Scolytinae) are associated with strictly entomochoric and mutualistic fungi. We studied the mycobiota associated with Scolytodes unipunctatus, ambrosia beetles that infest Cecropia trees in Central America. Isolates were characterized using morphology and rDNA sequences (ITS region, LSU, and SSU rDNA). Four species are described here: Raffaelea scolytodis sp. nov. (Ophiostomatales), Gondwanamyces scolytodis sp. nov., Custingophora cecropiae sp. nov., and Graphium sp. (Microascales). The genus Custingophora is emended to include Knoxdaviesia anamorphs of Gondwanamyces based on uniformity of DNA sequences and phenotype.     

Phylogenetic Relationships of Globodera millefolii,G. artemisiae,and Cactodera salina Based on ITS Region of Ribosomal DNA

Globodera millefolii and G. artemisiae are interesting because their type localities (Estonia and Russia, respectively) are geographically distant from those of the potato cyst nematodes and other Globodera species that seem to have originated in the Western world, and because the type host for each is a member of Compositae rather than Solanaceae. Sequence data for ITS1, ITS2, and 5.8S ribosomal DNA (ITS rDNA) for G. millefolii and G. artemisiae were nearly identical to sequence data for Cactodera salina from the rhizosphere of the estuary plant Salicornia bigelovii in Sonora, Mexico. The ITS rDNA sequences of these three species were all about 94% similar to those of two other Cactodera species for which ITS rDNA data were obtained. Phylogenetic analysis indicated that, based on the ITS rDNA data, G. millefolii and G. artemisiae are more closely related phylogenetically to the Cactodera species than to other nominal Globodera species. The molecular data further suggest that the genus Cactodera may comprise two or more morphologically similar but separate groups.     

A new species of Leptobacillim,L. symbioticum,isolated from mites and sori of soybean rust

A verticillium-like fungus forming a whitish colony and mainly solitary phialides that produced fusiform to cylindrical conidia in chains was isolated from uredinia of Phakopsora pachyrhizi, a causal agent of soybean rust. In addition, two similar looking isolates were obtained from Prostigmata mites. Our taxonomic study based on morphology and phylogenetic analysis using ITS rDNA and LSU rDNA D1/D2 region sequences revealed that the three isolates were the same species and assigned to the genus Leptobacillium. These isolates represent a new species, morphologically and phylogenetically distinguished from the type species, L. leptobactrum, and we propose L. symbioticum sp. nov. In addition, Simplicillium chinense and S. coffeanum are assigned to the genus Leptobacillium.     

A large 28S rDNA-based phylogeny confirms the limitations of established morphological characters for classification of proteocephalidean tapeworms (Platyhelminthes,Cestoda)

Proteocephalidean tapeworms form a diverse group of parasites currently known from 315 valid species. Most of the diversity of adult proteocephalideans can be found in freshwater fishes (predominantly catfishes), a large proportion infects reptiles, but only a few infect amphibians, and a single species has been found to parasitize possums. Although they have a cosmopolitan distribution, a large proportion of taxa are exclusively found in South America. We analyzed the largest proteocephalidean cestode molecular dataset to date comprising more than 100 species (30 new), including representatives from 54 genera (80%) and all subfamilies, thus significantly improving upon previous works to develop a molecular phylogeny for the group. The Old World origin of proteocephalideans is confirmed, with their more recent expansion in South America. The earliest diverging lineages are composed of Acanthotaeniinae and Gangesiinae but most of the presently recognized subfamilies (and genera) appear not to be monophyletic; a deep systematic reorganization of the order is thus needed and the present subfamilial system should be abandoned. The main characters on which the classical systematics of the group has been built, such as scolex morphology or relative position of genital organs in relation to the longitudinal musculature, are of limited value, as demonstrated by the very weak support for morphologically-defined subfamilies. However, new characters, such as the pattern of uterus development, relative ovary size, and egg structure have been identified, which may be useful in defining phylogenetically well-supported subgroups. A strongly supported lineage infecting various snakes from a wide geographical distribution was found. Although several improvements over previous works regarding phylogenetic resolution and taxon coverage were achieved in this study, the major polytomy in our tree, composed largely of siluriform parasites from the Neotropics, remained unresolved and possibly reflects a rapid radiation. The genus Spasskyellina Freze, 1965 is resurrected for three species of Monticellia bearing spinitriches on the margins of their suckers.     

Molecular phylogeny of the Cyathocotylidae (Digenea,Diplostomoidea) necessitates systematic changes and reveals a history of host and environment switches

The Cyathocotylidae is a globally distributed family of digeneans parasitic as adults in fish, reptiles, birds and mammals in both freshwater and marine environments. Molecular phylogenetic analysis of interrelationships among cyathocotylids is lacking with only a few species included in previous studies. We used sequences of the nuclear 28S rRNA gene to examine phylogenetic affinities of 11 newly sequenced taxa of cyathocotylids and the closely related family Brauninidae collected from fish, reptiles, birds and dolphins from Australia, Southeast Asia, Europe, North America and South America. This is the first study to provide sequence data from adult cyathocotylids parasitic in fish and reptiles. Our analyses demonstrated that the members of the genus Braunina (family Brauninidae) belong to the Cyathocotylidae, placing the Brauninidae into synonymy with the Cyathocotylidae. In addition, our DNA sequences supported the presence of a second species in the currently monotypic Braunina. Our phylogeny revealed that Cyathocotyle spp. from crocodilians belong to a separate genus (Suchocyathocotyle, previously proposed as a subgenus) and subfamily (Suchocyathocotylinae subfam. n.). Morphological study of Gogatea serpentum indicum supported its elevation to species as Gogatea mehri. The phylogeny did not support Holostephanoides within the subfamily Cyathocotylinae; instead, Holostephanoides formed a strongly supported clade with members of the subfamily Szidatiinae (Gogatea and Neogogatea). Therefore, we transfer Holostephanoides into the Szidatiinae. DNA sequence data revealed the potential presence of cryptic species reported under the name Mesostephanus microbursa. Our phylogeny indicated at least two major host switching events in the evolutionary history of the subfamily Szidatiinae which likely resulted in the transition of these parasites from birds to fish and snakes. Likewise, the transition to dolphins by Braunina represents another major host switching event among the Cyathocotylidae. In addition, our phylogeny revealed more than a single transition between freshwater and marine environments demonstrated in our dataset by Braunina and some Mesostephanus.     

Toward a better understanding of the infrageneric relationships in Cortinarius (Agaricales, Basidiomycota)

Research on the molecular systematics of Cortinarius, a species-rich mushroom genus with nearly global distribution, is just beginning. The present study explores infrageneric relationships using rDNA ITS and LSU sequence data. One large dataset of 132 rDNA ITS sequences and one combined da-taset with 54 rDNA ITS and LSU sequences were generated. Hebeloma was used as outgroup. Bayesian analyses and maximum-likelihood (ML) analyses were carried out. Bayesian phylogenetic inference performed equally well or better than ML, especially in large datasets. The phylogenetic analysis of the combined dataset with species representing all currently recognized subgenera recovered seven well-supported clades (Bayesian posterior probabilities BPP > 90%). These major clades are: /Myxacium s.l., /subg. Cortinarius, the /phlegmacioid clade (including the subclades /Phlegmacium and /Delibuti), the /calochroid clade (/Calochroi, /Ochroleuci and /Allutus), the /telamonioid clade (/Telamonia, /Orellani, /Anomali), /Dermocybe s.l. and /Myxotelamonia. Our results show that Cortinarius consists of many lineages, but the relationships among these clades could not be elucidated. On one hand, the low divergence in rDNA sequences can be held responsible for this; on the other hand, taxon sampling is problematic in Cortinarius phylogeny. Because of the incredibly high diversity (~2000 Cortinarius species), our sampling included <5% of the known species. By choosing type species of subgenera and sections, our sampling is strongly biased toward Northern Hemisphere taxa. More extensive taxon sampling, especially of species from the Southern Hemisphere, is essential to resolve the phylogeny of this important genus of ectomycorrhizal fungi.     

Support for the coevolution of Neoparamoeba and their endosymbionts,Perkinsela amoebae-like organisms

Some of the species from the genus Neoparamoeba, for example N. perurans have been shown to be pathogenic to aquatic animals and thus have economic significance. They all contain endosymbiont, Perkinsela amoebae like organisms (PLOs). In this study we investigated phylogenetic ambiguities within the Neoparamoeba taxonomy and phylogenetic congruence between PLOs and their host Neoparamoeba to confirm the existence of a single ancient infection/colonisation that led to cospeciation between all PLOs and their host Neoparamoeba. DNA was extracted and rRNA genes from host amoeba and endosymbiont were amplified using PCR. Uncertainties in the Neoparamoeba phylogeny were initially resolved by a secondary phylogenetic marker, the internal transcribed spacer 2 (ITS2). The secondary structure of ITS2 was reconstructed for Neoparamoeba. The ITS2 was phylogenetically informative, separating N. pemaquidensis and N. aestuarina into distinct monophyletic clades and designating N. perurans as the most phylogenetically divergent Neoparamoeba species. The new phylogenetic data were used to verify the tree topologies used in cophylogenetic analyses that revealed strict phylogenetic congruence between endosymbiotic PLOs with their host Neoparamoeba. Strict congruence in the phylogeny of all PLOs and their host Neoparamoeba was demonstrated implying that PLOs are transmitted vertically from parent to daughter cell.     

Integrative taxonomy in the genus Rhabdias Stiles et Hassall, 1905 from anuran in Brazil,description of two new species and phylogenetic analyses

About 20 valid species of the genus Rhabdias are known in the Neotropical region. The present study aimed to describe two new species of Rhabdias parasitizing the lungs of Leptodactylus macrosternum and Leptodactylus podicipinus from Brazil. Distinctive characteristics between these species are numerous and based on body size, size of the buccal capsule, shape and size of the oesophagus, and position of the vulva. Molecular data based on ribosomal genes 28S and ITS region and mitochondrial COI of the two species are presented. Molecular analysis and comparison of the partial mitochondrial COI sequence of Rhabdias matogrossensis n. sp. and Rhabdias guaianensis n. sp. revealed a genetic divergence between these new species and the sequences of Rhabdias spp. previously deposited in GenBank. In the phylogenetic analysis, R. matogrossensis n. sp. was grouped with R. breviensis species complex, and R. guaianensis n. sp. was grouped as a sister group of R. cf. stenochepala. This study contributes to improving the diversity of known species of Rhabdias described in Brazilian anurans.     

Additional rDNA ITS sequences and its phylogenetic consequences for the genus Leveillula with emphasis on conidium morphology

Taxonomy of the genus Leveillula has long been considered as a challenge in powdery mildew systematics. The rDNA diversity has recently been used for phylogenetic analysis of several specimens of the genus Leveillula. In the present study, additional rDNA ITS sequences are provided and a new phylogenetic analysis is carried out aiming at a better understanding of the genetic diversity in the genus Leveillula. New analyses confirmed that L. taurica is unique in the genus, as it exhibits an intraspecific gene sequence diversity considerably higher than in other species. In several cases L. taurica s. lat. on a certain host plant species has a sequence different from L. taurica on other host plants. Moreover, DNA data indicated different lineages among L. taurica specimens which were hardly distinguishable by morphology. More than one genotype occurring on a single host is sometimes possible. According to these results, several races such as Leveillula on Artemisia, Acroptilon, Onobrychis, are molecularly well characterized. While there is enough molecular evidence to delimit such races as independent taxa, clear morphological delimitations between these new and already published taxa are very difficult or even impossible. However, ecological features, and above all, host specificity for biotrophic fungi such as powdery mildew, would be a good criterion to discriminate cryptic taxa along with rDNA sequences. In fact, many collections of Leveillula strains on different hosts show their own type of conidial morphology, which is usually consistent for a strain on a single host species. Hence, we have proposed to describe new species for Leveillula on some host plants such as Artemisia, Acroptilon, Echinops and Onobrychis.     

UPDATING THE GENUS DICTYOSPHAERIUM AND DESCRIPTION OF MUCIDOSPHAERIUM GEN. NOV. (TREBOUXIOPHYCEAE) BASED ON MORPHOLOGICAL AND MOLECULAR DATA1

Recent molecular analyses of Dictyosphaerium strains revealed a polyphyletic origin of this morphotype within the Chlorellaceae. The type species Dictyosphaerium ehrenbergianum Nägeli formed an independent lineage within the Parachlorella clade, assigning the genus to this clade. Our study focused on three different Dictyosphaerium species to resolve the phylogenetic position of remaining species. We used combined analyses of morphology; molecular data based on SSU and internally transcribed spacer region (ITS) rRNA sequences; and the comparison of the secondary structure of the SSU, ITS‐1, and ITS‐2 for species and generic delineation. The phylogenetic analyses revealed two lineages without generic assignment and two distinct clades of Dictyosphaerium‐like strains within the Parachlorella clade. One clade comprises the lineages with the epitype strain of D. ehrenbergianum Nägeli and two additional lineages that are described as new species (Dictyosphaerium libertatis sp. nov. and Dictyosphaerium lacustre sp. nov.). An emendation of the genus Dictyosphaerium is proposed. The second clade comprises the species Dictyosphaerium sphagnale Hindák and Dictyosphaerium pulchellum H. C. Wood. On the basis of phylogenetic analyses, complementary base changes, and morphology, we describe Mucidosphaerium gen. nov with the four species Mucidosphaerium sphagnale comb. nov., Mucidosphaerium pulchellum comb. nov., Mucidosphaerium palustre sp. nov., and Mucidosphaerium planctonicum sp. nov.