Unravelling the hidden diversity of cave mycobiota in Thailand's Satun Geopark.

Karst caves are distinctive ecosystems that have limited nutrients, darkness, low to moderate temperatures, and high moisture levels, which allow for a diverse range of fungal communities to thrive. Despite their significance, little is understood about the fungi found in karst caves in Thailand. In 2019, we studied the cultured mycobiota from five substrate types (air, water, rock, soil/sediment, and organic debris) in two karst caves (Le Stegodon and Phu Pha Phet Caves) of the Satun UNESCO Global Geopark, southern Thailand. A cumulative count of 829 distinct fungal morphological types was identified, encompassing 319 fungal culturable were observed. Based on preliminary analyses of the internal transcribed spacer (ITS) sequence using BLAST searches, the most common phylum among the fungal morphotypes was Ascomycota, harboring 282 species in 91 genera, 93.4% of which were distributed in the classes Eurotiomycetes, Sordariomycetes, and Dothideomycetes. The most common fungal genera identified in the two karst caves were Aspergillus, Penicillium, Cladosporium, Talaromyces, Xylaria, and Trichoderma, with 45, 41, 24, 14, 14, and 6 species identified, respectively. Discovering fungi in Thai karst caves highlights the extensive fungal diversity in the Satun UNESCO Global Geopark, implying undiscovered species, and emphasizing the need for comprehensive investigations in other unexplored Thai karst caves.

Studies on the Genus Pyrenopolyporus (Hypoxylaceae) in Thailand Using a Polyphasic Taxonomic Approach.

Over the past two decades, hypoxylaceous specimens were collected from several sites in Thailand. In this study, we examined their affinity to the genus Pyrenopolyporus using macroscopic and microscopic morphological characters, dereplication of their stromatal secondary metabolites using ultrahigh performance liquid chromatography coupled to diode array detection and ion mobility tandem mass spectrometry (UHPLC-DAD-IM-MS/MS), and molecular phylogenetic analyses. We describe and illustrate five novel species and a new record for the country, present multi-locus phylogenetic analyses that show the distinction between the proposed species, and provide proteomic profiles of the fungi using matrix associated laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) for the first time. Based on our findings, this strategy is useful as a complementary tool to distinguish species between Daldinia and Pyrenopolyporus in a consistent way with the phylogenetic analysis.

Two Novel Species of Talaromyces Discovered in a Karst Cave in the Satun UNESCO Global Geopark of Southern Thailand.

Karst caves are oligotrophic environments that appear to support a high diversity of fungi. Studies of fungi in Thailand's caves are limited. During a 2019 exploration of the mycobiota associated with soil samples from a karst cave, namely, Phu Pha Phet in the Satun UNESCO Global Geopark in Satun Province, southern Thailand, two previously undescribed fungi belonging to Talaromyces (Trichocomaceae, Eurotiales, Eurotiomycetes) were studied using a polyphasic approach combining phenotypic and molecular data. Based on datasets of four loci (ITS, BenA, CaM, and RPB2), phylogenetic trees of the section Trachyspermi were constructed, and two new species-Talaromyces phuphaphetensis sp. nov. and T. satunensis sp. nov.-phylogenetically related to T. subericola, T. resinae, and T. brasiliensis, are described. Detailed descriptions and illustrations of the new species are provided. This study increases the number of cave-dwelling soil fungi discovered in Thailand's Satun UNESCO Global Geopark, which appears to be a unique environment with a high potential for discovering fungal species previously undescribed.

Comprehensive treatise of Hevansia and three new genera Jenniferia, Parahevansia and Polystromomyces on spiders in Cordycipitaceae from Thailand.

Collections of pathogenic fungi found on spiders from Thailand were selected for a detailed taxonomic study. Morphological comparison and phylogenetic analyses of the combined ITS, LSU, tef1, rpb1 and rpb2 sequence data indicated that these specimens formed new independent lineages within the Cordycipitaceae, containing two new genera occurring on spiders, i.e. Jenniferia gen. nov. and Polystromomyces gen. nov. Two new species in Jenniferia, J.griseocinerea sp. nov. and J.thomisidarum sp. nov., are described. Two strains, NHJ 03510 and BCC 2191, initially named as Akanthomycescinereus (Hevansiacinerea), were shown to be part of Jenniferia. By including sequences of putative Hevansia species from GenBank, we also revealed Parahevansia as a new genus with the ex-type strain NHJ 666.01 of Pa.koratensis, accommodating specimens previously named as Akanthomyceskoratensis (Hevansiakoratensis). One species of Polystromomyces, Po.araneae sp. nov., is described. We established an asexual-sexual morph connection for Hevansianovoguineensis (Cordycipitaceae) with ex-type CBS 610.80 and proposed a new species, H.minuta sp. nov. Based on characteristics of the sexual morph, Hevansia and Polystromomyces share phenotypic traits by producing stipitate ascoma with fertile terminal heads; however, they differ in the shape and colour of the stipes. Meanwhile, Jenniferia produces non-stipitate ascoma with aggregated superficial perithecia forming a cushion. A new morphology of ascospores in Jenniferia is described, illustrated and compared with other species in Cordycipitaceae.

Using High-Throughput Amplicon Sequencing to Evaluate Intragenomic Variation and Accuracy in Species Identification of Cordyceps Species.

While recent sequencing technologies (third generation sequencing) can successfully sequence all copies of nuclear ribosomal DNA (rDNA) markers present within a genome and offer insights into the intragenomic variation of these markers, high intragenomic variation can be a source of confusion for high-throughput species identification using such technologies. High-throughput (HT) amplicon sequencing via PacBio SEQUEL I was used to evaluate the intragenomic variation of the ITS region and D1-D2 LSU domains in nine Cordyceps species, and the accuracy of such technology to identify these species based on molecular phylogenies was also assessed. PacBio sequences within strains showed variable level of intragenomic variation among the studied Cordyceps species with C. blackwelliae showing greater variation than the others. Some variants from a mix of species clustered together outside their respective species of origin, indicative of intragenomic variation that escaped concerted evolution shared between species. Proper selection of consensus sequences from HT amplicon sequencing is a challenge for interpretation of correct species identification. PacBio consensus sequences with the highest number of reads represent the major variants within a genome and gave the best results in terms of species identification.

Population genomics revealed cryptic species within host-specific zombie-ant fungi (Ophiocordyceps unilateralis).

The identification and delimitation of species boundaries are essential for understanding speciation and adaptation processes and for the management of biodiversity as well as development for applications. Ophiocordyceps unilateralis sensu lato is a complex of fungal pathogens parasitizing Formicine ants, inducing zombie behaviors in their hosts. Previous taxonomic works with limited numbers of samples and markers led to the "one ant-one fungus" paradigm, resulting in the use of ant species as a proxy for fungal identification. Here, a population genomics study with sampling on three ant species across Thailand supported the existence of host-specific species in O. unilateralis s.l. with no footprints of long term introgression despite occasional host shifts and first-generation hybrids. We further detected genetic clusters within the previously delimited fungal species, with each little footprints of recombination, suggesting high levels of inbreeding. The clusters within each of O. camponoti-leonardi and O. camponoti-saundersi were supported by differentiation throughout the genome, suggesting they may constitute further cryptic species parasitizing the same host, challenging the one ant-one fungus paradigm. These genetic clusters had different geographical ranges, supporting different biogeographic influences between the north/center and the south of Thailand, reinforcing the scenario in which Thailand endured compartmentation during the latest Pleistocene glacial cycles.

A genome scan of diversifying selection in Ophiocordyceps zombie-ant fungi suggests a role for enterotoxins in co-evolution and host specificity.

Identification of the genes underlying adaptation sheds light on the biological functions targeted by natural selection. Searches for footprints of positive selection, in the form of rapid amino acid substitutions, and the identification of species-specific genes have proved to be powerful approaches to identifying the genes involved in host specialization in plant-pathogenic fungi. We used an evolutionary comparative genomic approach to identify genes underlying host adaptation in the ant-infecting genus Ophiocordyceps, which manipulates ant behaviour. A comparison of the predicted genes in the genomes of species from three species complexes-O. unilateralis, O. australis and O. subramanianii-revealed an enrichment in pathogenesis-associated functions, including heat-labile enterotoxins, among species-specific genes. Furthermore, these genes were overrepresented among those displaying significant footprints of positive selection. Other categories of genes suspected to be important for virulence and pathogenicity in entomopathogenic fungi (e.g., chitinases, lipases, proteases, core secondary metabolism genes) were much less represented, although a few candidate genes were found to evolve under positive selection. An analysis including orthologs from other entomopathogenic fungi in a broader context showed that positive selection on enterotoxins was specific to the ant-infecting genus Ophiocordyceps. Together with previous studies reporting the overexpression of an enterotoxin during behavioural manipulation in diseased ants, our findings suggest that heat-labile enterotoxins are important effectors in host adaptation and co-evolution in the Ophiocordyceps entomopathogenic fungi.

Insights from the genome of Ophiocordyceps polyrhachis-furcata to pathogenicity and host specificity in insect fungi.

BACKGROUND: Ophiocordyceps unilateralis is an outstanding insect fungus for its biology to manipulate host ants' behavior and for its extreme host-specificity. Through the sequencing and annotation of Ophiocordyceps polyrhachis-furcata, a species in the O. unilateralis species complex specific to the ant Polyrhachis furcata, comparative analyses on genes involved in pathogenicity and virulence between this fungus and other fungi were undertaken in order to gain insights into its biology and the emergence of host specificity. RESULTS: O. polyrhachis-furcata possesses various genes implicated in pathogenicity and virulence common with other fungi. Overall, this fungus possesses protein-coding genes similar to those found on other insect fungi with available genomic resources (Beauveria bassiana, Metarhizium robertsii (formerly classified as M. anisopliae s.l.), Metarhizium acridum, Cordyceps militaris, Ophiocordyceps sinensis). Comparative analyses in regard of the host ranges of insect fungi showed a tendency toward contractions of various gene families for narrow host-range species, including cuticle-degrading genes (proteases, carbohydrate esterases) and some families of pathogen-host interaction (PHI) genes. For many families of genes, O. polyrhachis-furcata had the least number of genes found; some genes commonly found in other insect fungi are even absent (e.g. Class 1 hydrophobin). However, there are expansions of genes involved in 1) the production of bacterial-like toxins in O. polyrhachis-furcata, compared with other entomopathogenic fungi, and 2) retrotransposable elements. CONCLUSIONS: The gain and loss of gene families helps us understand how fungal pathogenicity in insect hosts evolved. The loss of various genes involved throughout the pathogenesis for O. unilateralis would result in a reduced capacity to exploit larger ranges of hosts and therefore in the different level of host specificity, while the expansions of other gene families suggest an adaptation to particular environments with unexpected strategies like oral toxicity, through the production of bacterial-like toxins, or sophisticated mechanisms underlying pathogenicity through retrotransposons.

New species of Ophiocordyceps unilateralis, an ubiquitous pathogen of ants from Thailand.

Ophiocordyceps unilateralis is an ubiquitous pathogen of ants with hidden phylogenetic diversity associated with host specificity. In this study, we describe two new species to this species complex: Ophiocordyceps septa and Ophiocordyceps rami. Both were found on unidentified ants of the genus Camponotus (C. sp.1 and C. sp2 respectively). Ophiocordyceps septa is very similar to Ophiocordyceps polyrhachis-furcata, Ophiocordyceps camponoti-leonardi, and Ophiocordyceps camponoti-saundersi (found respectively on the ants Polyrhachis furcata, Camponotus leonardi, and Camponotus saundersi) but differs in the size, the shape and the septation of the ascospores, while O. rami is clearly identifiable with macro-morphological features including multiple stromata similar to Ophiocordyceps halabalaensis on Camponotus gigas. A thorough morphological examination was also provided for O. polyrhachis-furcata, O. camponoti-leonardi, and O. camponoti saundersi, showing that the first was apparently distinguishable from the others by the absence of septation of the ascospores. A combined molecular phylogeny also supports O. septa and O. rami as distinct new species.

Phylogeny and evolution of life-history strategies in the Sycophaginae non-pollinating fig wasps (Hymenoptera, Chalcidoidea).

BACKGROUND: Non-pollinating Sycophaginae (Hymenoptera, Chalcidoidea) form small communities within Urostigma and Sycomorus fig trees. The species show differences in galling habits and exhibit apterous, winged or dimorphic males. The large gall inducers oviposit early in syconium development and lay few eggs; the small gall inducers lay more eggs soon after pollination; the ostiolar gall-inducers enter the syconium to oviposit and the cleptoparasites oviposit in galls induced by other fig wasps. The systematics of the group remains unclear and only one phylogeny based on limited sampling has been published to date. Here we present an expanded phylogeny for sycophagine fig wasps including about 1.5 times the number of described species. We sequenced mitochondrial and nuclear markers (4.2 kb) on 73 species and 145 individuals and conducted maximum likelihood and Bayesian phylogenetic analyses. We then used this phylogeny to reconstruct the evolution of Sycophaginae life-history strategies and test if the presence of winged males and small brood size may be correlated. RESULTS: The resulting trees are well resolved and strongly supported. With the exception of Apocrytophagus, which is paraphyletic with respect to Sycophaga, all genera are monophyletic. The Sycophaginae are divided into three clades: (i) Eukoebelea; (ii) Pseudidarnes, Anidarnes and Conidarnes and (iii) Apocryptophagus, Sycophaga and Idarnes. The ancestral states for galling habits and male morphology remain ambiguous and our reconstructions show that the two traits are evolutionary labile. CONCLUSIONS: The three main clades could be considered as tribes and we list some morphological characters that define them. The same biologies re-evolved several times independently, which make Sycophaginae an interesting model to test predictions on what factors will canalize the evolution of a particular biology. The ostiolar gall-inducers are the only monophyletic group. In 15 Myr, they evolved several morphological adaptations to enter the syconia that make them strongly divergent from their sister taxa. Sycophaginae appears to be another example where sexual selection on male mating opportunities favored winged males in species with small broods and wingless males in species with large broods. However, some species are exceptional in that they lay few eggs but exhibit apterous males, which we hypothesize could be due to other selective pressures selecting against the re-appearance of winged morphs.