Delineating metamorphic pathways in the ascidian Ciona intestinalis

In most ascidians, metamorphosis of tadpole-like swimming larvae is accompanied by dynamic changes in their shape to form sessile adults. The mechanisms underlying ascidian metamorphosis have been debated for a long time. Although recent molecular studies have revealed the presence of various molecules involving in this process, the basic mechanism of the metamorphic events is still unclear. For example, it has not been solved whether all metamorphic events are organized by the same single pathway or by multiple, independent pathways. In the present study, we approached this question using the ascidian Ciona intestinalis. When the papillae and preoral lobes of the larvae were cut off, the papillae-cut larvae initiated certain trunk metamorphic events such as the formation of an ampulla, body axis rotation and adult organ growth without other metamorphic events. This observation indicates that metamorphic events can be divided into at least two groups, events initiated in the papillae-cut larva and events not initiated in this larva. In addition to this observation, we have isolated a novel mutant, tail regression failed (trf), which shows similar phenotypes to those of papillae-cut larvae. The phenotypes of trf mutants are basically different from those of swimming juvenile mutants (Sasakura, Y., Nakashima, K., Awazu, S., Matsuoka, T., Nakayama, A., Azuma, J., Satoh, N., 2005. Transposon-mediated insertional mutagenesis revealed the functions of animal cellulose synthase in the ascidian Ciona intestinalis. Proc. Natl. Acad. Sci. U. S. A. 102, 15134-15139.), which also show abnormal metamorphosis. These findings suggest a model by which ascidian metamorphic events can be classified into four groups initiated by different pathways.     

Investigating sperm cryopreservation in a model tunicate, Ciona intestinalis sp. A

In cryopreservation procedures, the capacity to protect the cells from freezing and thawing processes is sensitive to the choice of the cryoprotective agent (CPA) and to its optimal concentration. The advancement of research on Tunicate model species has raised interest in liquid nitrogen cryopreservation for the storage and distribution of genetic resources. Ciona intestinalis (Linnè, 1767) consists of a complex of cryptic taxa that are central to several areas of investigation, from comparative genomics to invasive biology. Here we investigated how five CPAs, three chilling rates and two freezing rates influence semen cryopreservation in C. intestinalis sp. A. By using larval morphology and motility as endpoints, we estimated that long term semen storage requires 10% dimethyl sulfoxide as a protective agent, −1 °C/min chilling rate (18 °C to 5 °C) and −13 °C/min freezing rate (5 °C to −80 °C), followed by immersion in liquid nitrogen.     

Spatial and temporal patterns of recruitment of the tunicate Ciona intestinalis on a mussel farm in Nova Scotia, Canada

Although possibly indigenous to Nova Scotia, the ascidian Ciona intestinalis has become a problem for local mussel aquaculturists. The local population ecology of the ascidian was studied by recording the depth, distribution, and timing of recruitment of C. intestinalis at four different locations (high wave exposure, low wave exposure, recently fallowed, and historically heavily fouled with tunicates) on a mussel farm at Indian Point, Nova Scotia. Animals were collected weekly from 11 collectors deployed at a depth of 4.5 m over two seasons (June-December 2003 and 2004). Recruitment occurred in two peaks during both seasons: the first peak lasted from late June to late July while the second peak lasted from early September to mid-November. Recruitment was highest in the sheltered and historically fouled location, intermediate in the fallowed site, and lowest in the site with highest wave exposure. Collectors were also deployed at different depths. Recruitment was highest at 4.5 m and to a lesser extent at 8.5 m. Little recruitment was seen at the surface (0.5 m) or in deeper water (12.5 and 16.5 m), except during the second peak in 2004. Overall, these results seem to indicate an abbreviated larval dispersal phase with young recruits settling very close to the pool of adults located on the mussel lines.     

Dietary supplementation of fructooligosaccharide (FOS) improves the innate immune response, stress resistance, digestive enzyme activities and growth performance of Caspian roach (Rutilus rutilus) fry

The present study investigated the effects of prebiotic fructooligosaccharide (FOS) on the innate immune response, stress resistance, digestive enzyme activities, growth factors and survival of Caspian Roach (Rutilus rutilus) fry. After acclimation, fish (0.67 ± 0.03 g) were allocated into 12 tanks (50 fish per tank) and triplicate groups were fed a control diet or diets containing 1%, 2% or 3% FOS. At the end of the trial (7 weeks), humoral innate immune parameters (serum Ig levels, lysozyme activity and alternative complement activity (ACH50)), resistance to salinity stress (150 g L⁻¹), digestive enzyme activities (amylase, lipase and protease) and growth factors (final weight, weight gain, specific growth rate (SGR), food conversion ratio (FCR), and condition factor) were assessed. At the end of the study the innate immune responses (Ig levels, lysozyme activity and ACH50) were significantly higher in 2% and 3% FOS fed fish (P < 0.05), whereas, 1% dietary FOS only elevated serum lysozyme activity. All dietary FOS levels significantly increased resistance to a salinity stress challenge (P < 0.05) and highest survival was observed in the 3% FOS group. Similarly, digestive enzyme activities were significantly elevated with increasing levels of dietary FOS (P < 0.05). Subsequently, elevated growth performance (final weight, SGR and FCR) was observed in roach fed 2% and 3% FOS compared to the control group (P < 0.05). These results indicate that FOS can be considered as a beneficial dietary supplement for improving the immune response, stress resistance, digestive enzyme activities and growth performance of Caspian roach fry.     

Seasonal variation in the mode of reproduction of Ulva intestinalis in a brackish water environment

We explored the reproductive modes of Ulva intestinalis in the inner part of the Baltic Sea during three consecutive years by using five microsatellite loci to estimate the relative abundance of diploid sporophytes and haploid gametophytes. Our results suggest that both diploid sporophytes and haploid gametophytes occur regularly in the Baltic Sea. The ratio of haploid to diploid individuals changes with seasons. Sporophytes are more abundant than gametophytes throughout the year, but the proportion of haploids increases from 10% in early summer to 35% in September. The over-wintering takes primarily place as diploid spores released by sporophytes. The sporophytes appear to reproduce both sexually and asexually in the Baltic Sea, since clones were found for this life phase. The fraction of individuals which belonged to an apparent diploid clone was higher in spring (62%) than in autumn (33%). We also found evidence for asexual clones in haploid gametophytes. The presence of both diploid and haploid individuals and the pattern of genetic and genotypic diversity provide evidence of sexual reproduction in the Baltic Sea. Thus the sporophytes and gametophytes do not function as two reproductively separate units. Compared with many other algal species with a reduced reproductive cycle in low salinity, U. intestinalis differs by having a multitude of reproductive modes also in the brackish water Baltic Sea, which can in part explain the dynamic propagation and high adaptability of the species.     

Involvement of lectin pathway activation in the complement killing of Giardia intestinalis

Giardia intestinalis (syn. G. lamblia, G. duodenalis) is a flagellated unicellular eukaryotic microorganism that commonly causes diarrheal disease throughout the world. In humans, the clinical effects of Giardia infection range from the asymptomatic carrier state to a severe malabsorption syndrome possibly due to different virulence of the Giardia strain, the number of cysts ingested, the age of the host, and the state of the host immune system at the time of infection.The question about how G. intestinalis is controlled by the organism remains unanswered. Here, we investigated the role of the complement system and in particular, the lectin pathway during Giardia infections. We present the first evidence that G. intestinalis activate the complement lectin pathway and in doing so participate in eradication of the parasite. We detected rapid binding of mannan-binding lectin, H-ficolin and L-ficolin to the surface of G. intestinalis trophozoites and normal human serum depleted of these molecules failed to kill the parasites. Our finding provides insight into the role of lectin pathway in the control of G. intestinalis and about the nature of surface components of parasite.     

A standardizable protocol for infection of Rhodnius prolixus with Trypanosoma rangeli, which mimics natural infections and reveals physiological effects of infection upon the insect

Trypanosoma rangeli is a protozoan parasite that shares hosts - mammals and triatomines - with Trypanosoma cruzi, the etiological agent of Chagas disease. Although T. rangeli is customarily considered to be non-pathogenic to human hosts, it is able to produce pathologies in its invertebrate hosts. However, advances are hindered by a lack of standardization of infection procedures and these pathologies need documentation. To establish a suitable, and standardizable, infection protocol, the duration of the fourth instar was evaluated in nymphs infected by injection into the thorax with different concentrations of parasites, and compared with nymphs infected naturally (i.e. orally). We demonstrate that delays in moult were attributable to the presence of the parasite in the haemolymph (vs. the gut) and propose that the protocol presented here simulates closely natural infections. This methodology was then used for the evaluation of physiological parameters and several hitherto unreported effects of T. rangeli infection on Rhodnius prolixus were revealed. Haemolymph volume was greater in infected than uninfected nymphs but this alteration could not be attributed to water retention, since infected insects lost the same amount of water as controls. However, we found that lipid content and fat body weight were both increased in insects infected by T. rangeli. We propose that this is due to the parasite’s sequestration of host blood lipids and carrier proteins. With these findings, we have taken a few first steps to unravelling physiological details of the host-parasite interaction. We suggest future directions towards a fuller understanding of mechanistic and adaptive aspects of triatomine-trypanosomatid interactions.     

Nitrogen uptake and assimilation in Enteromorpha intestinalis (L.) Link (Chlorophyta): using N to determine preference during simultaneous pulses of nitrate and ammonium

We investigated the ability of Enteromorpha intestinalis (L.) Link to take up pulses of different species of nitrogen simultaneously, as this would be an important mechanism to enhance bloom ability in estuaries. Uptake rates and preference for NH₄⁺ or NO₃⁻ following 1, 3, 6, 9, 12 or 24 h of exposure to either ¹⁵NH₄NO₃ or NH₄¹⁵NO₃ were determined by disappearance of N from the medium. Differences in assimilation rates for NH₄⁺ or NO₃⁻ were quantified by the accumulation of NH₄⁺, NO₃⁻, and atom % ¹⁵N in the algal tissue. NH₄⁺ concentration was reduced more quickly than water NO₃⁻ concentration. Water column NH₄⁺ concentration after the longest time interval was reduced from 300 to 50 μM. Water NO₃⁻ was reduced from 300 to 150 μM. The presence of ¹⁵N or ¹⁴N had no effect on uptake of either NH₄⁺ or NO₃⁻. ¹⁵N was removed from the water at an almost identical rate and magnitude as ¹⁴N. Differences in accumulation of ¹⁵NH₄⁺ and ¹⁵NO₃⁻ in the tissue reflected disappearance from the water; ¹⁵N from NH₄⁺ accumulated faster and reached an atom % twice that of ¹⁵N from NO₃⁻. This outcome suggested that when NH₄⁺ and NO₃⁻ were supplied in equal concentrations, more NH₄⁺ was taken up and assimilated. The ability to take up high concentrations of NH₄⁺, and NO₃⁻ simultaneously is important for bloom-forming species of estuarine macroalgae subject to multiple nutrient species from various sources.     

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.     

Insight into the role of cetaceans in the life cycle of the tetraphyllideans (Platyhelminthes: Cestoda)

Four types of tetraphyllidean larvae infect cetaceans worldwide: two plerocercoids differing in size, ‘small’ (SP) and ‘large’ (LP), and two merocercoids referred to as Phyllobothrium delphini and Monorygma grimaldii. The latter merocercoid larvae parasitize marine mammals exclusively and exhibit a specialised cystic structure. Adult stages are unknown for any of the larvae and thus the role of cetaceans in the life cycle of these species has been a long-standing problem. The SP and LP forms are thought to be earlier stages of P. delphini and M. grimaldii that are presumed to infect large pelagic sharks that feed on cetaceans. A molecular analysis of the D2 variable region of the large subunit ribosomal DNA gene based on several individuals of each larval type collected from three Mediterranean species of cetaceans showed consistent and unique molecular signatures for each type regardless of host species or site of infection. The degree of divergence suggested that LP, P. delphini and M. grimaldii larvae may represent separate species, whereas SP may be conspecific with M. grimaldii. In all host species, individuals of SP accumulated in the gut areas in which the lymphoid tissue was especially developed. We suggest therefore that these larvae use the lymphatic system to migrate to the abdominal peritoneum and mesenteries where they develop into forms recognizable as M. grimaldii. The plerocercoid stage of P. delphini remains unknown. In a partial phylogenetic tree of the Tetraphyllidea, all larvae formed a clade that included a representative of the genus Clistobothrium, some species of which parasitize sharks such as the great white which is known to feed on cetaceans. A bibliographic examination of tetraphyllidean infections in marine mammals indicated that these larvae are acquired mostly offshore. In summary, the evidence suggests that cetaceans play a significant role in the life cycle of these larvae. In addition, it seems clear that cetaceans act as natural intermediate hosts for P. delphini and M. grimaldii, as within these hosts they undergo development from the plerocercoid stage to the merocercoid stage. Because tetraphyllidean species use fish, cephalopods and other marine invertebrates as intermediate hosts, the inclusion of cetaceans in the life cycle would have facilitated their transmission to apex predators such as the large, lamnid sharks. The biological significance of infections of LP in cetaceans is unclear, but infections do not seem to be accidental as such larvae show high prevalence and abundance as well as a high degree of site specificity, particularly in the anal crypts and bile ducts.     

Septal pore complex morphology in the Agaricomycotina (Basidiomycota) with emphasis on the Cantharellales and Hymenochaetales

The ultrastructure of septa and septum-associated septal pore caps are important taxonomic markers in the Agaricomycotina (Basidiomycota, Fungi). The septal pore caps covering the typical basidiomycetous dolipore septum are divided into three main phenotypically recognized morphotypes: vesicular-tubular (including the vesicular, sacculate, tubular, ampulliform, and globular morphotypes), imperforate, and perforate. Until recently, the septal pore cap-type reflected the higher-order relationships within the Agaricomycotina. However, the new classification of Fungi resulted in many changes including revision of existing and addition of new orders. Therefore, the septal pore cap ultrastructure of more than 325 species as reported in literature was related to this new classification. In addition, the septal pore cap ultrastructures of Rickenella fibula and Cantharellus formosus were examined by transmission electron microscopy. Both fungi have dolipore septa associated with perforate septal pore caps. These results combined with data from the literature show that the septal pore cap-type within orders of the Agaricomycotina is generally monomorphic, except for the Cantharellales and Hymenochaetales.It appears from the fungal phylogeny combined with the septal pore cap ultrastructure that the vesicular-tubular and the imperforate type both may have arisen from endoplasmic reticulum. Thereafter, the imperforate type eventually gave rise to the perforate septal pore cap-type.     

Diversimorbus metrosiderotis gen. et sp. nov. and three new species of Holocryphia (Cryphonectriaceae) associated with cankers on native Metrosideros angustifolia trees in South Africa

The Cryphonectriaceae includes important tree pathogens, especially on the Myrtales. During a routine disease survey in the Western Cape Province of South Africa, a fungus resembling the Eucalyptus pathogen Holocryphia eucalypti was observed on native Metrosideros angustifolia (Myrtales). The aims of this study were to identify the fungus and to expand surveys for fungi in the Cryphonectriaceae on M. angustifolia. Fungi were identified based on DNA sequence comparisons and morphological features, and their pathogenicity was tested on M. angustifolia under field conditions. Based on morphology and multigene phylogenetic analyses of DNA sequence data from six gene regions, we describe a new genus including a single species and three new species of Holocryphia (Cryphonectriaceae) from M. angustifolia. These fungi are provided with the names Diversimorbus metrosiderotis gen. et sp. nov., Holocryphia capensis sp. nov., Holocryphia gleniana sp. nov., and Holocryphia mzansi sp. nov. We also revise H. eucalypti, the type of the genus, to include only isolates from Eucalyptus in South Africa. Research results indicated that H. mzansi may undergo host shifts between different tree genera in the Myrtaceae. Inoculation tests showed that isolates of all the newly described species can cause lesions on the branches of M. angustifolia, indicating that they are all pathogens of this tree.     

Recrudescence of Toxoplasma gondii infection in chronically infected rats (Rattus norvegicus)

The kinetics of Toxoplasma gondii infection reactivation in the brain and muscles was analyzed in this study to determine the preferred tissue by the parasite during immunosuppression. Two groups of Wistar rats (G1 and G2) were inoculated with 10⁴ bradyzoites of BTU10 strain (genotype I), p.o., and other two groups (G3 and G4) were inoculated with 0.9% saline solution. G2 and G4 were immunosuppressed with dexamethasone (DXM) and hydrocortisone sodium succinate (HSS). The presence of antibodies was researched in all groups through modified agglutination test (MAT) on days 0 and 21 p.i., and brain and muscle tissues of the rats were bioassayed in mice. G2 rats died at approximately 19.2 days after drug treatment, while G1 rats survived. The reactivation was initially observed in G1 brain and G2 muscles. Thus, the initial reactivation in muscles after immunosuppression allows doctors to save precious time to control the evolution of reactivated infection, preventing brain damage to the host.     

Systematics and evolution of the genus Torrubiella (Hypocreales, Ascomycota)

Torrubiella is a genus of arthropod-pathogenic fungi that primarily attacks spiders and scale insects. Based on the morphology of the perithecia, asci, and ascospores, it is classified in Clavicipitaceae s. lat. (Hypocreales), and is considered a close relative of Cordyceps s. 1., which was recently reclassified into three families (Clavicipitaceae s. str., Cordycipitaceae, Ophiocordycipitaceae) and four genera (Cordyceps s. str, Elaphocordyceps, Metacordyceps, and Ophiocordyceps). Torrubiella is distinguished morphologically from Cordyceps s. lat. mainly by the production of superficial perithecia and the absence of a well-developed stipitate stroma. To test and refine evolutionary hypotheses regarding the placement of Torrubiella and its relationship to Cordyceps s. lat., a multi-gene phylogeny was constructed by conducting ML and Bayesian analyses. The monophyly of Torrubiella was rejected by these analyses with species of the genus present in Clavicipitaceae, Cordycipitaceae, and Ophiocordycipitaceae, and often intermixed among species of Cordyceps s. lat. The morphological characters traditionally used to define the genus are, therefore, not phylogenetically informative, with the stipitate stromata being gained and/or lost several times among clavicipitaceous fungi. Two new genera (Conoideocrella, Orbiocrella) are proposed to accommodate two separate lineages of torrubielloid fungi in the Clavicipitaceae s. str. In addition, one species is reclassified in Cordyceps s. str. and three are reclassified in Ophiocordyceps. The phylogenetic importance of anamorphic genera, host affiliation, and stipitate stromata is discussed.     

Structure and development of the style base in Abildgaardia, Bulbostylis, and Fimbristylis (Cyperaceae,Cyperoideae, Abildgaardieae)

The Abildgaardieae tribe within the family Cyperaceae comprises six or seven genera, among which Abildgaardia, Bulbostylis and Fimbristylis pose a challenge regarding their morphological delimitation. Molecular phylogenetic analyses including species of Abildgaardieae are rare, but in most of those studies, Abildgaardia and Fimbristylis appear as more closely related to each other than to the Bulbostylis genus. Duration of the style base has been one of the most widely used characters for delimiting these three genera. The style base is a persistent structure in most species of Bulbostylis and deciduous in Abildgaardia and Fimbristylis. The reasons why the style base may persist or fall off have been scarcely discussed. The assumption that abscission layers are present in the style base of all three genera and the fact that tracheids have been observed in the style base of Bulbostylis suggest that this structure might have histological complexity. In view of this, a complete ontogenetic and anatomical study of the gynoecium has been carried out for all these three genera. It turned out that the style base is histologically simple in Abildgaardia, Bulbostylis and Fimbristylis and shows similar structure and development in all three genera. The fact that the style base has a shorter duration in Abildgaardia and Fimbristylis than in Bulbostylis might be related to the lower number of sclerotised cells that make up such structures in the mature fruit of the former two genera. Abscission of the style and style base may be the result of much simpler reasons than the differentiation of an abscission layer, resulting merely from mechanical shear force effects. Differences among genera have been observed in the shape of the style base and the development of the style. The histological simplicity of the style base is consistent with the homoplastic appearance of this structure in genera that are not closely related (e.g. Rhynchospora). Because of this, while the presence of the thickened style base seems to be a synapomorphy in species of Abildgaardieae, its persistence on or detachment from the fruit might have emerged repeatedly during this clade evolution and might not be a suitable character for genera delimitation.     

Variation in the reproductive potential of Schistocephalus infected male sticklebacks is associated with 11-ketotestosterone titre

Parasites can impact host reproduction by interfering with host endocrine systems, but the adaptive nature of such effects is disputed. Schistocephalus solidus plerocercoids are parasites of three-spined sticklebacks Gasterosteus aculeatus that are often associated with impaired host reproduction. Here, we relate reproductive behavior and physiology to levels of the androgen 11-ketotestosterone (11KT) in naturally infected and non-infected male sticklebacks from two UK populations. In one population infected males harbored heavy infections and showed uniformly reduced 11KT titres and kidney spiggin (nesting glue protein) content compared to non-infected fish. However in a second population infection levels were more variable and males with smaller infections recorded 11KT and spiggin titres that overlapped those of non-infected fish; among infected males from this population 11KT and kidney spiggin also both correlated negatively with infection severity. Male reproductive behavior correlated closely with 11KT titre in both populations, and infected males with high 11KT levels exhibited normal reproductive behavior. Our results suggest that Schistocephalus infection per se does not block reproductive development in male sticklebacks, and that some male fish may have the ability to breed whilst infected. Our results are not consistent with the hypothesis that Schistocephalus adaptively castrates male hosts via endocrine disruption; rather they support the hypothesis that reproductive disruption is a side effect of the energetic costs of infection.     

Local immune response against larvae of Rhipicephalus (Boophilus) microplus in Bos taurus indicus and Bos taurus taurus cattle

Bos taurus indicus cattle are less susceptible to infestation with Rhipicephalus (Boophilus) microplus than Bos taurus taurus cattle but the immunological basis of this difference is not understood. We compared the dynamics of leukocyte infiltrations (T cell subsets, B cells, major histocompatibility complex (MHC) class II-expressing cells, granulocytes) in the skin near the mouthparts of larvae of R. microplus in B. t. indicus and B. t. taurus cattle. Previously naïve cattle were infested with 50,000 larvae (B. t. indicus) or 10,000 larvae (B. t. taurus) weekly for 6 weeks. One week after the last infestation all of the animals were infested with 20,000 larvae of R. microplus. Skin punch biopsies were taken from all animals on the day before the primary infestation and from sites of larval attachment on the day after the first, second, fourth and final infestations. Infiltrations with CD3⁺, CD4⁺, CD8⁺ and γδ T cells followed the same pattern in both breeds, showing relatively little change during the first four weekly infestations, followed by substantial increases at 7 weeks post-primary infestation. There was a tendency for more of all cell types except granulocytes to be observed in the skin of B. t. indicus cattle but the differences between the two breeds were consistently significant only for γδ T cells. Granulocyte infiltrations increased more rapidly from the day after infestation and were higher in B. t. taurus cattle than in B. t. indicus. Granulocytes and MHC class II-expressing cells infiltrated the areas closest to the mouthparts of larvae. A large volume of granulocyte antigens was seen in the gut of attached, feeding larvae.     

Phylogenetic reassessment of Hyaloscyphaceae sensu lato (Helotiales, Leotiomycetes) based on multigene analyses

Hyaloscyphaceae is the largest family in Helotiales, Leotiomycetes. It is mainly characterized by minute apothecia with well-differentiated hairs, but its taxonomic delimitation and infrafamilial classification remain ambiguous. This study performed molecular phylogenetic analyses using multiple genes including the ITS-5.8S rDNA, the D1–D2 region of large subunit of rDNA, RNA polymerase II subunit 2, and the mitochondrial small subunit. The primary objective was to evaluate the phylogenetic utility of morphological characters traditionally used in the taxonomy of Hyaloscyphaceae through reassessment of the monophyly of this family and its genera. The phylogenetic analyses inferred Hyaloscyphaceae as being a heterogeneous assemblage of a diverse group of fungi and not supported as monophyletic. Among the three tribes of Hyaloscyphaceae only Lachneae formed a monophyletic lineage. The presence of hairs is rejected as a synapomorphy, since morphologically diversified hairs have originated independently during the evolution of Helotiales. The true- and false-subiculum in Arachnopezizeae are hypothesized to have evolved through different evolutionary processes; the true-subiculum is likely the product of a single evolutionary origin, while the false-subiculum is hypothesized to have originated multiple times. Since Hyaloscyphaceae sensu lato was not resolved as monophyletic, Hyaloscyphaceae sensu stricto is redefined and only applied to the genus Hyaloscypha.