A phylogenetic study of the genus Haligena (Halosphaeriales, Ascomycota)

The genus Haligena (Halosphaeriales, Ascomycota), with two accepted species, is encountered frequently in marine habitats, especially on wood in temperate regions. Phylogenetic analyses of Haligena elaterophora (type species) and H. salina were undertaken, with partial large subunit ribosomal DNA sequences, to determine their relationships with other closely related genera in the order. The genus was shown to be polyphyletic within the Halosphaeriales with the type species forming a basal clade to the order. Haligena salina constituted a sister clade with weak support of Neptunella longirostris in all analyses. Haligena elaterophora and H. salina differ significantly in the nature of their ascospore appendages: wider, more sticky and strap-like in H. elaterophora and spoon-shaped at the point of attachment; in H. salina they are longer and narrower, finely drawn out filaments. A new genus, Morakotiella, is introduced to accommodate H. salina.     

Independent terrestrial origins of the Halosphaeriales (marine Ascomycota)

A phylogenetic study of marine ascomycetes was initiated to test and refine evolutionary hypotheses of marine-terrestrial transitions among ascomycetes. Taxon sampling focused on the Halosphaeriales, the largest order of marine ascomycetes. Approximately 1050 base pairs (bp) of the gene that codes for the nuclear small subunit (SSU) and 600 bp of the gene that codes for the nuclear large subunit (LSU) ribosomal RNAs (rDNA) were sequenced for 15 halosphaerialean taxa and integrated into a data set of homologous sequences from terrestrial ascomycetes. An initial set of phylogenetic analyses of the SSU rDNA from 38 taxa representing 15 major orders of the phylum Ascomycota confirmed a close phylogenetic relationship of the halosphaerialean species with several other orders of perithecial ascomycetes. A second set of analyses, which involved more intensive taxon sampling of perithecial ascomycetes, was performed using the SSU and LSU rDNA data in combined analyses. These second analyses included 15 halosphaerialean taxa, 26 terrestrial perithecial fungi from eight orders, and five outgroup taxa from the Pezizales. In these analyses the Halosphaeriales were polyphyletic and comprised two distinct lineages. One clade of Halosphaeriales comprised 12 taxa from 11 genera and was most closely related to terrestrial fungi of the Microascales. The second clade of halosphaerialean fungi comprised taxa from the genera Lulworthia and Lindra and was an isolated lineage among the perithecial fungi. Both the main clade of Halosphaeriales and the Lulworthia/Lindra clade are supported by the data as being independently derived from terrestrial ancestors.     

Halosarpheia unicellularis sp. nov. (Halosphaeriales, Ascomycota) based on morphological and molecular evidence

Halosarpheia unicellularis sp. nov. is described from decayed attached wood ofAvicennia marina collected from two mangrove sites in the Red Sea, Egypt. The ascomycete is compared with other marine taxa having ascospores with polar unfurling appendages. It is also compared with other marine genera with oval, round or ellipsoid hyaline, unicellular ascospores, but for which appendages have not been reported. Molecular data confirms the assignment of the fungus toHalosarpheia which forms a clade with the type speciesHalosarpheia fibrosa.     

Havispora longyearbyenensis gen. et sp. nov.: an arctic marine fungus from Svalbard, Norway

Information on the diversity and ecology of arctic marine fungi is lacking. During a short visit to Longyearbyen (78 degrees 13'N 15 degrees 33'E), Svalbard, Norway, a new marine fungus growing on driftwood collected at the shore was encountered. This taxon belongs to the Halosphaeriales (Ascomycota), a fungal order of mostly marine species. Havispora longyearbyenensis gen. et sp. nov. is morphologically similar to Nautosphaeria and Nereiospora, all with tufts of appendages at polar and equatorial positions of the ascospore but differing in color and septation of the ascospore and morphology and ontogeny of the ascospore appendage.     

Savoryellales (Hypocreomycetidae, Sordariomycetes): a novel lineage of aquatic ascomycetes inferred from multiple-gene phylogenies of the genera Ascotaiwania, Ascothailandia, and Savoryella

The taxonomic placement of freshwater and marine Savoryella species has been widely debated, and the genus has been tentatively assigned to various orders in the Sordariomycetes. The genus is characterized as possessing paraphyses that deliquesce early, elongate, clavate to cylindrical asci with a poorly developed apical ring and versicolored, three-septate ascospores. We performed two combined phylogenetic analyses of different genes: (i) partial small subunit rRNA (SSU), large subunit rRNA (LSU), DNA-dependent RNA polymerase II largest subunit (rpb2) dataset and (ii) SSU rDNA, LSU rDNA, DNA-dependent RNA polymerase II largest subunit (rpb1 and rpb2), translation elongation factor 1-alpha (tef1), the 5.8S ribosomal DNA (5.8S rDNA) dataset. Our results indicate that Savoryella species formed a monophyletic group within the Sordariomycetes but showed no affinity to the Hypocreales, Halosphaeriales (now Microascales), Sordariales and Xylariales, despite earlier assignments to these orders. Savoryella, Ascotaiwania and Ascothailandia (and its anamorph, Canalisporium) formed a new lineage that has invaded both marine and freshwater habitats, indicating that these genera share a common ancestor and are closely related. Because they show no clear relationship with any named order we erect a new order Savoryellales in the subclass Hypocreomycetidae, Sordariomycetes. The genera Savoryella and Ascothailandia are monophyletic, while the position of Ascotaiwania is unresolved. All three genera are phylogenetically related and form a distinct clade similar to the unclassified group of marine ascomycetes comprising the genera Swampomyces, Torpedospora and Juncigera (TBM clade: Torpedospora/Bertia/Melanospora) in the Hypocreomycetidae incertae sedis.     

Morphology evolution and molecular phylogeny of Pestalotiopsis (Coelomycetes) based on ITS2 secondary structure

The internal transcribed spacer 2 (ITS2) located between the 5.8S and 28S genes of the nuclear ribosomal gene cistron is conserved at the level of secondary structure rather than primary sequence. Within the fungal genus Pestalotiopsis, there were two types of ITS2 sequence patterns, and hence secondary structures, which were supported by high bootstrap values in phylogenies based on the ProfDist distance and Profile neighbor-joining algorithms. Pestalotiopsis consists of two groups that differ in color intensity of the spore as measured by optical density (OD) in three median cells of conidia comprised of five cells with one basal and two to four apical appendages. OD was quantified using a novel method with the publicly available software, Image J. OD values of species within one clade were high (dark OD >0.6), while OD values of species in the other clade were low (pale OD <0.6). However, knobbed-tipped appendages, which have been used to classify species of Pestalotiopsis, were observed in both clades. In the dark clade, knobbed-tipped appendage strains aggregated in one subclade, but in the pale group, these strains did not aggregate.     

Phylogeny, evolution, and taxonomy of vannellid amoebae

We sequenced 18S rRNA genes from 21 vannellid amoebae (Amoebozoa; Vannellidae), including nearly all available type cultures, and performed a comprehensive phylogenetic analysis for 57 Vannellidae sequences. The results show that species of Vannella and Platyamoeba are completely mixed and do not form distinct clades. Several very closely related species pairs exist, each with a Vannella and a Platyamoeba species differing in only a few nucleotides. Therefore, presence (Vannella) or absence (Platyamoeba) of glycostyles in the cell surface coat is an invalid generic distinction; the genera must be merged. As Vannella has priority, we formally transferred Platyamoeba species into Vannella, except for the non-vannellid P. stenopodia, here renamed Stenamoeba stenopodia gen. n. comb. n. and transferred to the family Thecamoebidae. Our trees show that Vannella glycostyles were probably easily and repeatedly evolutionarily lost. We have established a new genus Ripella, with distinct morphology and sequence signatures for Vannella platypodia and morphologically similar species that form a clearly separate clade, very distant from other Vannellidae. Vannellids form four well-separated single-genus clades: Vannella sensu stricto, Ripella, Clydonella, and Lingulamoeba. Species of the revised genus Vannella comprise four closely related, well-supported subclades: one marine and three freshwater. Here, we provide an illustrated checklist for all 40 known Vannellidae species.     

Molecular phylogeny of the subterranean genus Niphargus (Crustacea: Amphipoda) in the Middle East: a comparison with European Niphargids

The subterranean genus Niphargus is one of the most species‐rich genera among freshwater amphipods in the world, distributed in the Western Palearctic. Thus far, taxonomic and phylogenetic research has focused mainly on the European half of the genus range. In this study, 25 populations of Niphargus from Iran, Lebanon and the Crimean Peninsula were investigated. Bayesian inference based on 28S, H3 and COI gene sequences suggests that populations from the area belong to four different clades. Three species from Crimea and one species from Iran are nested at basal nodes, indicating their rather ancient origin. The rest of the species are younger and belong to two separate clades. One Crimean species is a sister‐species to east Romanian species. The second clade includes one species from Lebanon and all but one population from Iran. The origin of this clade corresponds to marine transgression between the Black Sea and Mediterranean approximately 12 Mya. This clade was further investigated taxonomically. Revision of qualitative morphological traits and unilocus species delimitation methods using COI suggest that this clade comprises 12–16 species, of which only three have been described so far. Multilocus coalescence delimitation methods (using fragments of COI, 28S, H3 and ITS) strongly supported 11 of these species. The remaining populations comprise at least two species complexes that require further and more detailed taxonomic research. © 2015 The Linnean Society of London     

PHYLOGENETIC ANALYSIS OF THE GENUS EUGLENA (EUGLENOPHYCEAE) WITH PARTICULAR REFERENCE TO THE TYPE SPECIES EUGLENA VIRIDIS1

Euglena viridis (subgenus Euglena) serves as the type species for the genus Euglena. In this study, molecular phylogenetic analyses using a small subunit (SSU) and a combined SSU–partial large subunit rDNA data set for members of the genus Euglena showed that strains identified as E. viridis on the basis of morphology are distributed between two separate nonsister clades. Although all the E. viridis strains examined were morphologically indistinguishable and possessed spherical mucocysts and stellate chloroplasts with one paramylon center, there was a high degree of sequence divergence between the E. viridis strains in different clades, making this a cryptic species. Like E. viridis, all taxa from the subgenus Euglena are characterized by having one or more stellate chloroplasts with paramylon grains clustered around the center of the chloroplast. These additional taxa were divided into four clades in all the molecular analyses. Strains of Euglena stellata formed two nonsister clades whose members had a single aggregate chloroplast with paramylon center and spindle‐shaped mucocysts. A geniculata clade included species with one or two stellate chloroplasts with paramylon centers and spherical mucocysts, and the cantabrica clade had members with one stellate chloroplast with paramylon center and spherical mucocysts often arranged in spiral rows. Interspersed among these were three additional clades bearing taxa from the subgenus Calliglena that contains members with discoid plastids and pyrenoids that may or may not be capped with paramylon. These taxa formed a laciniata clade, mutabilis clade, and gracilis clade. This study demonstrates that E. viridis and E. stellata are cryptic species that can only be distinguished at the molecular level. Because E. viridis is the designated type species for the genus Euglena, we designated an epitype for E. viridis.     

Molecular phylogeny of terrestrial holocarpic endoparasitic peronosporomycetes, <Emphasis Type="Italic">Haptoglossa</Emphasis> spp., inferred from 18S rDNA

Phylogenetic relationships of seven isolates of the genus Haptoglossa parasitic on terrestrial nematodes within the Peronosporomycetes were analyzed using 18S rDNA sequence data with 21 peronosporomycetes, 2 marine stramenopilous flagellates, and 2 hyphochytridiomycetes. The marine stramenopilous flagellates and hyphochytridiomycetes were used as the outgroup. All Haptoglossa isolates formed a monophyletic clade and clustered with the marine genus Eurychasma. The clade of Haptoglossa and Eurychasma formed a sister-group to the clade that consisted of all other peronosporomycetes. These results suggest that the genus Haptoglossa and other terrestrial peronosporomycetes included in the two subclasses, the Saprolegniomycetidae and the Peronosporomycetidae, might have originally adapted to the terrestrial environment individually. In the maximum-likelihood (ML) analysis, the Haptoglossa clade was divided into three subclades, one aplanosporic species clade and two zoosporic species clades. Phylogenetic analyses of combined 18S rDNA and cox2 genes among five species of Haptoglossa supported the results of the ML analysis using 18S rDNA and suggested that zoosporic species may be separated into two lineages. This topology of the analysis may suggest that aplanosporic species diverged from zoosporic species.     

Cucullosporella mangrovei, ultrastructure of ascospores and their appendages

The ultrastructure ofCucullosporella mangrovei ascospores is described. Mature ascospores possess two wall layers, an outer electron-dense episporium and an innermost tripartite mesosporium. Episporial elaborations form electrondense spore wall ornamentations from which extend fibrils that may constitute a highly hydrated exosporium which was not visualised at either the scanning electron microscope or light microscope level. Ascospores possess a hamate appendage at each pole which unfolds in seawater to form a long thread. Ultrastructurally the polar appendage comprises folded fibro-granular electron-dense material and fine fibrils. The fibrils form a matrix around and within the fibro-granular appendage and around the entire unreleased ascospore. These fibrils have not been observed associated with the ascospore appendages in other species of the Halosphaeriales and are a discrete and new appendage component. The fibro-granular appendage and fibrils are bounded by the outer delimiting membrane which is absent around released ascospores. The nature of the spore appendage is compared with that of other marine and freshwater ascomycetes and the taxonomic assignment of the species is discussed.     

A cladistic analysis of Asarum (Aristolochiaceae) and implications for the evolution of herkogamy

A cladistic analysis of Asarum was conducted to examine relationships among species within the genus and to test the monophyly of several groups of taxa that have often been treated as segregate genera. Thirty-two species were drawn from throughout the range of the genus, representing a broad sample of sections and all segregate genera. The data matrix included 37 characters derived from various aspects of vegetative and floral morphology. A strict consensus of all most parsimonious trees suggests that Asarum s.l. is monophyletic and consists of two main clades: an Asarum clade, which is characterized by connate styles and inferior ovaries, and an Asiasarum-Hexastylis-Heterotropa clade, which is characterized by ridges on the inner perianth surface, dorsal stigmas, and bifid style extensions. The latter is a large and morphologically diverse clade that includes the North American segregate Hexastylis and two Asiatic segregates. Examination of pollination mechanisms in the context of this phylogeny supports the conclusion that herkogamy, and thus obligate insect pollination, is derived from a plesiomorphic condition of autonomous self-pollination. Associated with herkogamy are characters such as glandular trichomes and other ornamentation of the surface of the calyx that probably represent increased specialization to attract insect pollinators. This study also indicates that chromosomal evolution has occurred via aneuploid decrease from an ancestral chromosome number of 2n = 26 to 2n = 24 in Heterotropa. The recognition of two subgenera, subgenus Asarum and subgenus Heterotropa, corresponding to the two clades in the cladistic analysis, is recommended.     

Three new genera representing novel lineages of Sordariomycetidae (Sordariomycetes, Ascomycota) from tropical freshwater habitats in Costa Rica

Three new genera are established in the Sordariomycetidae based on morphological and molecular data (SSU and LSU nrDNA) to accommodate five ascomycete species collected from submerged woody debris in freshwater habitats from Costa Rica. The genus Bullimyces contains three new species, B. communis, B. costaricensis and B. aurisporus. Bullimyces is characterized by globose to subglobose, membranous, black, ostiolate ascomata; deliquescent, hyaline, globose cells that fill the center of the centrum; unitunicate asci that deliquesce early in some species; and septate, thick-walled ascospores with or without gelatinous sheaths or appendages. Bullimyces species form a well supported clade with 100% bootstrap support, but the position of the genus in the Sordariomycetidae remains unclear. The second genus, Riomyces, is represented by a single species, R. rotundus. Riomyces is characterized by globose to subglobose, membranous, black, ostiolate ascomata, unitunicate, cylindrical asci, hyaline, globose cells that fill the hamathecium and septate, thick-walled ascospores with a gelatinous sheath. Although Riomyces is morphologically similar to Bullimyces, the two genera did not group together with support in any analysis. The third genus, Hydromelitis, is represented by a single species, H. pulchella. Hydromelitis is characterized by pyriform, membranous, black, ostiolate ascomata, unitunicate asci lacking an apical structure, simple, thin-walled, septate paraphyses and hyaline to golden yellow, multiseptate, thick-walled ascospores with a gelatinous sheath. Bullimyces, Riomyces and Hydromelitis were nested within an unsupported clade consisting of members of the Ophiostomatales, Magnaporthales and freshwater Annulatacaceae sensu lato and sensu stricto.     

Halosarpheia japonica sp. nov. (Halosphaeriales, Ascomycota) from marine habitats in Japan

A new Halosarpheia species, collected from driftwood from Hakkeijima beach, Yokohama, Japan, is described and illustrated and is compared with other species of the genus. The new fungus was growing together with its anamorph on a piece of decaying wood. SSU and LSU rDNA sequences for both morphs were 99% similar. Phylogenetic analyses of SSU and LSU rDNA sequences of the both morphs confirm their anamorph–teleomorph relationship and placed the new fungus in the Halosarpheia sensu stricto clade with high statistical support. Halosarpheia japonica is characterized by its polar appendage that is initially enclosed in a cellular sheath and dissolves in water, the appendage then swells to form a huge tree-like structure. The other three species currently included in Halosarpheia sensu stricto differ from H. japonica by having two polar appendages that uncoil in water to form long filaments.     

Molecular phylogeny of Sinocyclocheilus (Cypriniformes: Cyprinidae) inferred from mitochondrial DNA sequences

More than 10 species within the freshwater fish genus Sinoncyclocheilus adapt to caves and show different degrees of degeneration of eyes and pigmentation. Therefore, this genus can be useful for studying evolutionary developmental mechanisms, role of natural selection and adaptation in cave animals. To better understand these processes, it is indispensable to have background knowledge about phylogenetic relationships of surface and cave species within this genus. To investigate phylogenetic relationships among species within this genus, we determined nucleotide sequences of complete mitochondrial cytochrome b gene (1140 bp) and partial ND4 gene (1032 bp) of 31 recognized ingroup species and one outgroup species Barbodes laticeps. Phylogenetic trees were reconstructed using maximum parsimony, Bayesian, and maximum likelihood analyses. Our phylogenetic results showed that all species except for two surface species S. jii and S. macrolepis clustered as five major monophyletic clades (I, II, III, IV, and V) with strong supports. S. jii was the most basal species in all analyses, but the position of S. macrolepis was not resolved. The cave species were polyphyletic and occurred in these five major clades. Our results indicate that adaptation to cave environments has occurred multiple times during the evolutionary history of Sinocyclocheilus. The branching orders among the clades I, II, III, and IV were not resolved, and this might be due to early rapid radiation in Sinocyclocheilus. All species distributed in Yunnan except for S. rhinocerous and S. hyalinus formed a strongly supported monophyletic group (clade V), probably reflecting their common origins. This result suggested that the diversification of Sinocyclocheilus in Yunnan may correlate with the uplifting of Yunnan Plateau.     

An ITS-based phylogenetic framework for the genus Vorticella: finding the molecular and morphological gaps in a taxonomically difficult group

Vorticella includes more than 100 currently recognized species and represents one of the most taxonomically challenging genera of ciliates. Molecular phylogenetic analysis of Vorticella has been performed so far with only sequences coding for small subunit ribosomal RNA (SSU rRNA); only a few of its species have been investigated using other genetic markers owing to a lack of similar sequences for comparison. Consequently, phylogenetic relationships within the genus remain unclear, and molecular discrimination between morphospecies is often difficult because most regions of the SSU rRNA gene are too highly conserved to be helpful. In this paper, we move molecular systematics for this group of ciliates to the infrageneric level by sequencing additional molecular markers—fast-evolving internal transcribed spacer (ITS) regions—in a broad sample of 66 individual samples of 28 morphospecies of Vorticella collected from Asia, North America and Europe. Our phylogenies all featured two strongly supported, highly divergent, paraphyletic clades (I, II) comprising the morphologically defined genus Vorticella. Three major lineages made up clade I, with a relatively well-resolved branching order in each one. The marked divergence of clade II from clade I confirms that the former should be recognized as a separate taxonomic unit as indicated by SSU rRNA phylogenies. We made the first attempt to elucidate relationships between species in clade II using both morphological and multi-gene approaches, and our data supported a close relationship between some morphospecies of Vorticella and Opisthonecta, indicating that relationships between species in the clade are far more complex than would be expected from their morphology. Different patterns of helix III of ITS2 secondary structure were clearly specific to clades and subclades of Vorticella and, therefore, may prove useful for resolving phylogenetic relationships in other groups of ciliates.     

Preliminary phylogenetic analysis of the fern genus Lomariopsis (Lomariopsidaceae)

A preliminary phylogenetic analysis is presented forLomariopsis based on sequence data from the chloroplast intergenic spacertrnL-trnF. The analysis includes 27 (60%) of the approximately 45 species in the genus. A strict consensus of six most parsimonious trees supports two main clades—theSorbifolia-group and theJapurensis-group—previously proposed based on heteroblastic leaf development. TheSorbifolia-group is entirely neotropical and includes all the Antillean species. The species in this clade had either smooth or crested spores, but the tree was ambiguous whether these spore types define two separate clades. TheJapurensis-group consists of two clades, one primarily neotropical and the other entirely paleotropical. Within the neotropical clade nests a clade of two African species, which have long-spiny spores typical of the neotropical clade and unlike those found in the African-Madagascan clade. The occurrence of these two species in Africa is best explained by longdistance spore dispersal of their ancestral species from the neotropics to Africa. Within the paleotropical clade of theJapurensis-group, a clade of three African species is nested among seven species from Madagascar (all the species from that island). Within the genus as a whole, a derived character—the abortion of the rachis apex and its replacement by the distal lateral pinna assuming a terminal position—was found to have evolved separately in each of the four species with this kind of leaf apex. A scanning electron microcope study of the spores revealed five types, and a transformation series for these different types is proposed. Characters of spore morphology and heteroblastic leaf development agreed with many of the clades in the phylogenetic tree. This study represents the first phylogeny for the genus.     

Macrochaete gen. nov. (Nostocales,Cyanobacteria), a taxon morphologically and molecularly distinct from Calothrix

Historically, the genus Calothrix included all noncolonial, tapered, heterocytous filaments within the cyanobacteria. However, recent molecular phylogenies show that “Calothrix” defined in this sense represents five distinct clades. The type species of Calothrix is marine, with solitary basal heterocytes, no akinetes, and distal ends tapering abruptly into short hairs. We examined the morphology and phylogeny of 45 tapering cyanobacteria in the Rivulariaceae, including freshwater and marine representatives of both Calothrix (35 strains) and its sister taxon Rivularia (10 strains). The marine Calothrix fall into two lineages, but we lack the generitype and so cannot identify the clade corresponding to the type species. The freshwater and soil Calothrix fall into the C. parietina clade and are characterized by having a basal heterocyte, no akinetes, and gradual tapering—but not into a long hyaline hair. Macrochaete gen. nov. is a freshwater taxon sister to the Calothrix lineages but clearly separated from Rivularia. The species in this genus differ morphologically from Calothrix by their ability to produce two heteromorphic basal heterocytes and specific secondary structures of the 16S–23S ITS. An additional feature present in most species is the presence of a distal, long hyaline hair, but this character has incomplete penetrance due to its expression only under specific environmental conditions (low phosphate), and in one species appears to be lost. We recognize three species: M. psychrophila (type species) from cold environments (high mountains, Antarctica), M. santannae from wet walls of subtropical South America, and M. lichenoides, a phycobiont of lichens from Europe.