New species and emendations of Orphella: taxonomic and phylogenetic reassessment of the genus to establish the Orphellales, for stonefly gut fungi with a twist.

We consolidate and present data for the sexual stages of five North American species of Orphella, fungal members of trichomycetes previously classified within Harpellales. Three species emendations accommodate the newly recognized characters, including not only the coiled zygospores and accompanying cells but also other morphological traits not provided in the original descriptions for O. avalonensis, O. haysii, and O. hiemalis. We describe three new species, Orphella cataloochensis from both the Smoky Mountains in USA and two provinces in Canada as well as O. pseudoavalonensis and O. pseudohiemalis, both from the Cascade Range, in Oregon, USA. Key morphological features for all known species are summarized and reviewed, with illustrations of some of the North American taxa to update and supplement the literature. The entire suite of morphological characters is discussed, with emphasis on species relationships and hypotheses on possible vicariant origins. We also present a molecular phylogeny based on nuc rDNA 18S and 28S, which supports Orphella as a lineage distinct from Harpellales, and we establish a new order, Orphellales, for it. With the combination of sexual features, now known for 12 of the 14 species of Orphella, and new molecular data, the group is now better characterized, facilitating and hopefully also promoting future studies toward a better understanding of their relationships, origins, and evolutionary history as stonefly gut-dwelling fungi.

An eight-gene molecular phylogeny of the Kickxellomycotina, including the first phylogenetic placement of Asellariales.

Kickxellomycotina is a recently described subphylum encompassing four zygomycete orders (Asellariales, Dimargaritales, Harpellales, Kickxellales). These fungi are united by the formation of disciform septal pores containing lenticular plugs. Morphological diversification and life history evolution has made the relationships within and among the four orders difficult to resolve on those grounds alone. Here we infer the phylogeny of the Kickxellomycotina based on an eight-gene supermatrix including both ribosomal rDNA (18S, 28S, 5.8S) and protein sequences (MCM7, TSR1, RPB1, RPB2, ß-tubulin). The results of this study demonstrate that Kickxellomycotina is monophyletic and related to members of the Zoopagomycotina. Eight unique clades are distinguished in the Kickxellomycotina, including the four defined orders (Asellariales, Dimargaritales, Harpellales, Kickxellales) as well as four genera previously placed within two of these orders (Barbatospora, Orphella, Ramicandelaber, Spiromyces). Dimargaritales and Ramicandelaber are the earliest diverging members of the subphylum, although the relationship between these taxa remains uncertain. The remaining six clades form a monophyletic group, with Barbatospora diverging first. The next split divides the remaining members of the subphylum into two subclades: (i) Asellariales and Harpellales and (ii) Kickxellales, Orphella and Spiromyces. Estimation of ancestral states for four potentially informative morphological and ecological characters reveals that arthropod endosymbiosis might have been an important factor in the early evolution of the Kickxellomycotina.

Using a five-gene phylogeny to test morphology-based hypotheses of Smittium and allies, endosymbiotic gut fungi (Harpellales) associated with arthropods.

Smittium, one of the first described genera of gut fungi, is part of a larger group of endosymbiotic microorganisms (Harpellales) that live predominantly in the digestive tracts of aquatic insects. As a diverse and species-rich taxon, Smittium has helped to advance our understanding of the gut fungi, in part due to the relative success of attempts to culture species of Smittium as compared to other members of Harpellales. Approximately 40% of the 81 known species of Smittium have been cultured. This is the first Smittium multigene dataset and phylogenetic analysis, using the 18S and 28S rRNA genes, as well as RPB1, RPB2, and MCM7 translated protein sequences. Several well-supported clades were recovered within Smittium. One includes the epitype S. mucronatum (the "True Smittium" clade), and another contains many species including S. simulii and S. orthocladii (the "Parasmittium" clade). Ancestral states were reconstructed for holdfast shape, thallus branching type, as well as asexual (trichospore) and sexual (zygospore) spore morphology. Two of these characters, holdfast shape and trichospore morphology, supported the split of the two main clades revealed by the molecular phylogeny, suggesting these are natural clades and these traits may have evolutionary and perhaps ecological significance.

Typification of Smittium, an important genus in the taxonomy of Harpellales.

The Harpellales genus Smittium is based on a type species, S. arvernense, which was described by Poisson in 1937 without designation of a type specimen. Smittium arvernense has not been reported since its original publication. Because the other 79 species of Smittium cannot be compared to the type species, a lectotype is proposed as well as an epitype for that lectotype that is also the holotype of S. mucronatum. Because Smittium is believed to be polyphyletic these type designations will provide stable application of names and, as well resolved phylogenetic analyses of member species emerge based on morphological and DNA sequence characters, they will provide a foundation for a more robust and revised classification.

Klastostachys, a new genus of Harpellales in Chironomidae larvae.

Klastostachys reflexa, a new genus and species combination in the Harpellales, is established herein based on Stachylina reflexa, which was described in 1988. This gut fungus was found attached to the peritrophic matrix of small bloodworms, Cryptochironomus sp. (Chironomidae), in the Rocky Mountains of Colorado, USA. Klastostachys resembles Stachylina, a genus common in Chironomidae, but the unbranched thalli of Klastostachys disarticulate and disperse with the trichospores remaining attached to their generative cells. This manner of dissemination is unusual among Harpellales, being noted also for Carouxella spp., but members of that genus have zygospores attached at one pole to the zygosporophore (Type IV), whereas Klastostachys zygospores are medially attached to the zygosporophore at right angles (Type I).

Dacryodiomyces, a new genus of Harpellales in Chironomidae larvae.

A new genus of gut fungus in the Harpellales (Trichomycetes) with the type species Dacryodiomyces oklahomensis, found in Chironomidae larvae, is described from northeastern Oklahoma, USA. The most distinguishing characters are the elongate-ovoid zygospores attached to their zygosporophore at one end (Type IV), together with trichospores bearing a collar and a single appendage.

Trichomycete insect symbionts in Great Smoky Mountains National Park and vicinity.

Collections of trichomycete symbionts of larval aquatic insects in Great Smoky Mountains National Park and vicinity in the southern Appalachian region of the USA resulted in finding many taxa of Harpellales, including an unusual new monotypic genus, Barbatospora ambicaudata in Simuliidae, and five new species in Thaumaleidae or Chironomidae, Harpellomyces montanus, Smittium lentaquaticum, Sm. minutisporum, Stachylina gravicaudata and St. stenospora. In addition a new species of Amoebidium (Amoebidiales), A. appalachense, attached to the anal tubules of bloodworms (Chironomidae) is described. Axenic cultures of three of the new taxa were obtained, plus Sm. culisetae. Fourteen identified species representing 13 genera of previously known Harpellales are recorded from Plecoptera, Ephemeroptera and Diptera, as well as a new Dipteran host record for an unidentified harpellid that was found in a Blephariceridae. Also identified were Paramoebidium corpulentum and many undetermined species of Paramoebidium (Amoebidiales) from four orders of aquatic insect larvae. The occurrence of an Enterobryus species in Diplopoda and another Eccrinales from an aquatic beetle is noted. Amoebidiales,

Confirmation and identification of parasitic stages of obligate endobionts (Harpellales) in blackflies (Simuliidae) by means of rRNA sequence data.

Over the last 35y, the life cycle of endosymbiotic gut fungi (Harpellales) has been expanded to include cyst stages associated with the developing ovaries. Ovarian cysts (chlamydospore stages) have been identified after germination and production of asexual trichospores in vitro, but germination is not always successful, and spores exhibit morphological variation. Sequence data (for partial 18S and 28S rRNA genes) were generated for these putative stages of Harpellales using ungerminated cysts from adult blackflies and germinated cysts associated with field-collected blackfly egg masses. Cladistic analyses of the 18S and 28S rRNA sequences confirmed that ovarian cysts are stages in the life cycle of Harpellales. Ungerminated cysts, from a blackfly collected from New York state were identified as Pennella simulii and two samples from Newfoundland Prosimulium mixtum adults were identified as Harpella melusinae. Cysts with bipolar germ tubes, associated with field-collected Simulium egg masses from Newfoundland, were also identified as H. melusinae. Two other samples of cysts could not be matched with available sequences of gut fungi from larval hosts. The potential use of this approach to identify pathogenic stages associated with adult ovaries or field-collected egg masses among other host groups is highlighted and promoted as a tool to test the hypothesis that ovarian cysts are a dispersal stage common to all genera of Harpellales.

Trichomycete fungi (Zygomycota) associated with mosquito larvae (Diptera: Culicidae) in natural and artificial habitats in Manaus, AM Brazil

Fungal species of the class Trichomycete fungi (Zygomycota) are associated with the digestive tracts of Arthropoda. The relationships between these fungi and their hosts are still little understood: they may be commensal, beneficial or deleterious. Knowledge of the community structure of parasites/ pathogens and of the habitats of each species of Culicidae larvae can be important in studies that intend to use combined approaches to population control. Larvae of Culicidae and their associated trichomycete fungi were collected in Manaus County, AM, Brazil; sampling habitats included plants (natural habitat) and anthropic containers (artificial habitats). The total of 1,518 larvae were collected, 913 of which were in natural habitats and 605 were in artificial habitats, distributed in 12 species of seven genera. The total of 661 individuals (4th instar) were dissected to verify the presence of trichomycete fungi in the mid and hindgut. Infection of trichomycete fungi in the hindgut was observed in 15 percent of Culex urichii Coquillett, 13 percent of Culex (Culex) sp1, 9 percent of Limatus spp., 49 percent of Aedes aegypti Linnaeus and 1 percent of Ochlerotatus argyrothorax Bonne-Wepster & Bonne. Only in Ae. aegypti were trichomycete fungi observed in the peritrophic matrix, in the midgut; however, this fact is probably, a result of abnormal development of the fungi.

Espécies de fungos da classe Trichomycete (Zygomycota) estão associados com o trato digestivo dos Arthropoda. A relação entre esses fungos e seus hospedeiros ainda é pouco conhecida, podendo ser comensal, benéfica ou deletéria. Conhecimentos da estrutura de comunidades parasitas/ patógenos e os habitats de larvas de Culicidae podem ser importantes em estudos que utilizam medidas combinadas para o controle populacional. Larvas de Culicidae e os fungos Trichomycetes associados foram coletados no município de Manaus, AM; amostras de criadouros incluindo plantas (habitat natural) e reservatórios antrópicos (habitat artificial). O total de 1518 larvas foi coletado, 913 em criadouros naturais e 605 em criadouros artificiais, distribuídas em 12 espécies de sete gêneros. O total de 661 indivíduos (4° estádio) foi dissecado para verificar a presença de fungos Trichomycetes no intestino médio e posterior. Infecção de fungos Trichomycetes no intestino posterior foram observados em 15 por cento de Culex urichii Coquillett, 13 por cento de Culex (Culex) sp1, 9 por cento of Limatus spp., 49 por cento de Aedes aegypti Linnaeus e 1 por cento de Ochlerotatus argyrothorax Bonne-Wepster & Bonne. Somente em Ae. aegypti foram observados fungos Trichomycetes na matriz peritrófica, no intestino médio; porém este fato é provavelmente, um resultado de um desenvolvimento anormal deste fungo.

Symbiotic Harpellales (Trichomycetes) in Tasmanian aquatic insects.

Surveys for symbiotic fungi in the guts of aquatic insect larvae (Trichomycetes: Harpellales) in Tasmania, Australia, resulted in the discovery of four new species: two in Gripopterygidae (Plecoptera) nymphs, Plecopteromyces leptoperlarum and P. trinotoperlarum, and two associated with Diptera larvae, Smittium magnosporum in Thaumaleidae and Stachylina dolichospora in Chironomidae. Previously described species of Harpellales from other localities are reported and new host records summarized. A key to all Tasmanian species of Harpellales is provided.

Fungal symbionts (Harpellales) in Norwegian aquatic insect larvae.

Collections of aquatic insect larvae in Norway, over a 40 d period in May and Aug 2002, resulted in finding more than 25 species and one new genus of Harpellales (Trichomycetes). Nine new fungal symbionts are described and named: Ephemerellomyces aquilonius (a new monotypic genus), Glotzia stenospora and Legeriosimilis europaeus in mayfly nymphs (Ephemeroptera); Genistelloides amplispora and G. communis in stonefly nymphs (Plecoptera); and Smittium biforme, Sm. precipitiorum, Stachylina acutibasilaris and St. lentica in midge larvae (Diptera: Chironomidae). Two possibly new species of Smittium in Chironomidae larvae are described but not formally named. New hosts and biogeographical distributions are recorded for 14 previously described species, including the rare occurrence of Smittium simulii in mosquito larvae.

Trichomycetes (Zygomycota) in the digestive tract of arthropods in Amazonas, Brazil.

Eight species of Harpellales and three species of Eccrinales (Zygomycota: Trichomycetes) were found associated with the digestive tract of arthropods from terrestrial and aquatic environments in the central Amazon region of Brazil. New species of Harpellales include: Harpella amazonica, Smittium brasiliense, Genistellospora tropicalis in Simuliidae larvae and Stachylina paucispora in Chironomidae larvae. Axenic cultures of S. brasiliense were obtained. Probable new species of Enterobryus (Eccrinales), Harpella, and Stachylina (Harpellales) are described but not named. Also reported are the previously known species of Eccrinales, Passalomyces compressus and Leidyomyces attenuatus in adult Coleoptera (Passalidae), and Smittium culisetae and Smittium aciculare (Harpellales) in Culicidae and Simuliidae larvae, respectively. Comments on the distribution of some of these fungi and their hosts in the Neotropics are provided.

Trichomycetes (Zygomycota) in the digestive tract of arthropods in Amazonas, Brazil

Eight species of Harpellales and three species of Eccrinales (Zygomycota: Trichomycetes) were found associated with the digestive tract of arthropods from terrestrial and aquatic environments in the central Amazon region of Brazil. New species of Harpellales include: Harpella amazonica, Smittium brasiliense, Genistellospora tropicalis in Simuliidae larvae and Stachylina paucispora in Chironomidae larvae. Axenic cultures of S. brasiliense were obtained. Probable new species of Enterobryus (Eccrinales), Harpella, and Stachylina (Harpellales) are described but not named. Also reported are the previously known species of Eccrinales, Passalomyces compressus and Leidyomyces attenuatus in adult Coleoptera (Passalidae), and Smittium culisetae and Smittium aciculare (Harpellales) in Culicidae and Simuliidae larvae, respectively. Comments on the distribution of some of these fungi and their hosts in the Neotropics are provided

A new Coelomomyces pathogenic to mosquitoes in Costa Rica

A new species of fungus belonging to the lethal genus Coelomomyces, C. neotropicus, is described and illustrated. It was found parasitizing larvae of two species of Culicidae, Culex pilosus and Aedes sp., in a lowland tropical wet forest swamp in northeast Costa Rica.