Multigene Phylogenetic Analyses Research Articles

Morphology and multi-gene phylogeny reveal a new Brunneofusispora species from coffee in Yunnan Province, China

Coffee is one of the most important cash crops native to north-central Africa, and the second most popular beverage ingredient worldwide. Coffee-associated fungal pathogens have been well studied, but few studies have reported saprobes of coffee. In China, Yunnan Province is the main region of coffee plantations and production. To investigate saprobic fungi of coffee plants, dead branches of Coffea with fungal fruiting bodies were collected in Baoshan City, Yunnan Province, China. Multi-gene phylogenetic analyses of SSU, ITS, LSU, RPB2, and TEF1-α sequence data indicated that our collections formed an independent branch within Brunneofusispora and sister to B. sinensis. The new species B. baoshanensis, characterized by uniloculate ascomata with an erumpent long neck, and inequilateral, ends acute, 1-septate, constricted at the septum ascospores with mucilaginous sheath, was identified based on morphology and multi-gene phylogeny. This is the first report of Brunneofusispora species isolated from Coffea in China. A complete description, micrographs, and a phylogenetic tree showing the placement of the new species are provided.

Piecing together the taxonomic puzzle of Sphaerulina

Sphaerulina species are plant pathogenic fungi causing leaf spot diseases of various hosts, including arboreous and herbaceous plants. The morphological characteristics of their asexual morphs and leaf spot symptoms are like those of Septoria spp. Due to their similar morphology, species of Sphaerulina have largely been subsumed under Septoria s. lat. A recent revision of the genus Septoria based on morphological characteristics and phylogeneticrelationships resulted in the separation of Sphaerulina from Septoria. This study reveals the diversity of the genus Sphaerulina in Japan, and the species relationships based on a multigene phylogenetic analysis. Moreover, resultsof our phylogenetic analysis revealed seven novel species (Sph. farfugii, Sph. hydrangeicola, Sph. idesiae, Sph. lapsanastri, Sph. miurae, Sph. styracis, and Sph. viburnicola) which are described, and two species (Sep. duchesnea and Sep. nambuana) which are transferred to the genus Sphaerulina.

Three new Melanogaster species (Boletales, Paxillaceae) from southwestern China based on morphological and molecular evidence.

Three newly discovered Melanogaster species, namely M.cyaneus, M.diqingensis, and M.truncatisporus, are introduced and illustrated based on both morphological and molecular data from Sichuan and Yunnan provinces in China. A multigene phylogenetic analysis (nrITS, nrLSU, and rpb2) was performed mainly to verify the placement of the new species in Melanogaster. A second, nrITS-only phylogenetic analysis comprising more Melanogaster species for which only ITS sequences were available, was used to infer the relationship between the new species and as many known Melanogaster species as possible. Specimens of M.cyaneus, M.diqingensis, and M.truncatisporus formed three independent clades in a phylogenetic tree inferred from the ITS data set. The robust support from ITS for these clades and genetic similarity with other species being lower than 93.2% suggest that these three species are indeed distinct from the other Melanogaster species in the phylogeny. Morphologically, M.cyaneus is characterized by its blue or bluish gleba, light brown to yellowish brown peridium, and subglobose to globose basidiospores, 6.2-15 × 4.6-9.0 μm. Melanogasterdiqingensis is distinguished from other Melanogaster species by its pale yellow to brown-yellow peridium and obovate to subglobose basidiospores, 3.0-5.1 × 2.0-4.0 μm. Melanogastertruncatisporus is diagnosed by its subglobose to globose or irregularly elongate-pyriform basidiomata, pale yellow to deeply orange-yellow peridium, and subglobose to globose or pyriform, truncate basidiospores. Additionally, infrageneric classification based on the number of peridium layers, the average thickness of the peridium, and the average length and width of basidiospores was tested with M.cyaneus, M.diqingensis, and M.truncatisporus. Orthogonal partial least squares discriminant (OPLS-DA) analysis placed the three new species within the Melanogaster, Rivulares, and Variegati sections, respectively. However, the morphologically circumscribed sections were not monophyletic in the phylogenetic tree. Therefore, the current infrageneric classification should be abandoned.

Interesting mycoparasites and Paradingleyomyceslepidopterorum gen. et sp. nov. (Hypocreales, Polycephalomycetaceae) from Yunnan Province, China.

A novel genus, Paradingleyomyces was introduced to accommodate Pa.lepidopterorum sp. nov., based on a multigene phylogenetic analysis and its distinct morphological characteristics. Maximum likelihood (ML) and Bayesian inference analyses (BI) of ITS, SSU, LSU, tef-1α, rpb1, and rpb2 sequence data shown that Pa.lepidopterorum formed an independent lineage nested between Perennicordyceps and Dingleyomyces. Morphologically, Paradingleyomyces is distinguished from Perennicordyceps by the presence of a white subiculum on the stromata of Ophiocordycepscf.cochlidiicola. Perithecia are produced sporadically from the base to the apex of the stromata, and the secondary ascospores exhibit a notable length-to-width ratio. These characteristics distinguish Paradingleyomyces from Perennicordyceps which exhibits tortuous, branched, clavate to cylindrical stromata with rhizomorphs, parasitism of coleopteran and hemipteran larvae, and colonizes a broader range of fungal hosts. Additionally, perithecia in Perennicordyceps typically arise from the middle to the upper regions of the stromata, with secondary ascospores displaying a comparatively lower length-to-width ratio. Paradingleyomyces is morphologically identical to Dingleyomyces in its direct production of superficial perithecia on the stromata of Ophiocordyceps species. However, the phylogenetic analysis indicates that Paradingleyomyces and Dingleyomyces are not congeneric. Moreover, this study introduces another novel species, Polycephalomycestengchongensis, and a novel sexual morph of Pleurocordycepsyunnanensis. Dimorphic phialides and conidia of Pleurocordycepsparvicapitata were observed and described for the first time based on a fresh collection from Tengchong County, Yunnan Province, China.

Multi-gene phylogenetic analyses revealed two novel species and one new record of Trichobotrys (Pleosporales, Dictyosporiaceae) from China.

The rotting wood in freshwater is a unique eco-environment favoring various fungi. During our investigation of freshwater fungi on decaying wood, three hyphomycetes were collected from Jiangxi and Guangxi Provinces, China. Based on the morphological observations and phylogenetic analysis of a combined DNA data containing ITS, LSU, SSU and tef1-α sequences, two new Trichobotrys species, T.meilingensis and T.yunjushanensis, as well as a new record of T.effusa, were introduced. Additionally, a comprehensive description of the genus with both morphological and molecular data was first provided.

Insights into the molecular phylogeny and morphology of three novel Dothiora species, along with a worldwide checklist of Dothiora.

Most species of Dothiora are known from the dead parts of various host plants as saprobic fungi in terrestrial habitats occurring in tropical and temperate regions. In the present study, samples of Dothiora were collected from dead twigs and branches of Capparis spinosa, Rhaponticum repens, and an unknown angiosperm plant from the Tashkent and Jizzakh regions of Uzbekistan. Multi-gene phylogenetic analyses based on a combined ITS, LSU, SSU, TEF1, and TUB2 sequence data revealed their taxonomic positions within the Dothideaceae. Three new species of Dothiora, namely, Dothiora capparis, Dothiora rhapontici, and Dothiora uzbekistanica were proposed by molecular and morphological data. Likewise, the phylogenetic relationship and morphology of Dothiora are discussed. In addition, we provide a list of accepted Dothiora species, including host information, distribution, morphology descriptions, and availability of sequence data, to enhance the current knowledge of the diversity within Dothiora.

Morpho-phylogenetic identification and characterization of new causal agents of Fusarium species for postharvest fruit rot disease of muskmelon in northern Thailand and their sensitivity to fungicides.

A significant global problem affecting muskmelon (Cucumis melo L.) is fruit rot caused by phytopathogenic fungi, which results in unsaleable products and substantial financial losses. In 2022 and 2023, fruit rot on muskmelon was found during the postharvest storage period in Phayao Province of northern Thailand. The aim of the current study was to isolate the species of fungi causing the fruit rot lesions. Out of the rot lesions on muskmelons, nine fungal isolates were received. All isolates of fungi were identified through a combination of morphological characteristics and molecular analyses. Based on their morphological traits, all isolated fungal isolate was assigned to the genus Fusarium. All the fungal isolates were determined to belong to the Fusarium incarnatum-equiseti species complex through multi-gene phylogenetic analysis employing the calmodulin (cam), RNA polymerase second largest subunit (rpb2), and translation elongation factor 1-alpha (tef1-α) genes. These isolates were identified as F. compactum (SDBR-CMU483), F. jinanense (SDBR-CMU484, SDBR-CMU485, and SDBR-CMU486), F. mianyangense (SDBR-CMU487 and SDBR-CMU488), and F. sulawesiense (SDBR-CMU489, SDBR-CMU490, and SDBR-CMU491). Moreover, pathogenicity tests were subsequently carried out, and the results indicated that all fungal isolates caused symptoms of fruit rot on inoculated muskmelon fruits. Notably, this result was consistent with the symptoms observed throughout the postharvest storage period. In the fungicide screening test, all fungal isolates showed sensitivity to copper oxychloride. However, all isolates showed insensitivity to benalaxyl-M + mancozeb, carbendazim, mancozeb, and metalaxy. To the best of our knowledge, the present study is the first to identify F. compactum, F. jinanense, and F. mianyangense as new causative agents of muskmelon fruit rot in Thailand and other regions globally. This is also the first report of postharvest fruit rot on muskmelons caused by F. sulawesiense in Thailand. Furthermore, the fungicide screening results indicate that fungicide resistance can be beneficial in developing potential management strategies against postharvest fruit rot disease of muskmelon caused by these four pathogenic Fusarium species.

Multi-Locus Phylogeny and Morphology Reveal Two New Species of Hypoxylon (Hypoxylaceae, Xylariales) from Motuo, China.

Hypoxylaceous fungi are abundant in China, but their discovery and report are uneven in various provinces, with more fungi in Yunnan and Hainan and fewer fungi in Tibet. During the investigation of macro-fungi in Motuo county, Tibet Autonomous Region, we collected a number of xylarialean specimens. Six hypoxylaceous specimens growing on dead angiosperm were collected from the forests of Motuo county, and they were described and illustrated as two new species in Hypoxylon based on a combination of morphological characters and molecular evidence. Hypoxylon diperithecium was characterized by its bistratal perithecia, purple-brown stromatal granules, citrine to rust KOH-extractable pigments, and light brown to brown ascospores ellipsoid-inequilateral with conspicuous coil-like ornamentation. Hypoxylon tibeticum was distinct from other species by having pulvinate and applanate stromata, surface vinaceous, with orange granules, orange KOH-extractable pigments, and brown ascospores with inconspicuous ornamentation. The multi-gene phylogenetic analyses (ITS-LSU-RPB2-TUB) supported the two new taxa as separate lineages in the genus Hypoxylon. A key to all known Hypoxylon taxa from China is provided.

Phylogenetic analyses allow species-level recognition of Leptographium wageneri varieties that cause black stain root disease of conifers in western North America.

Leptographium wageneri is a native fungal pathogen in western North America that causes black stain root disease (BSRD) of conifers. Three host-specialized varieties of this pathogen were previously described: L. wageneri var. wageneri on pinyon pines (Pinus monophylla and P. edulis); L. wageneri var. ponderosum, primarily on hard pines (e.g., P. ponderosa, P. jeffreyi); and L. wageneri var. pseudotsugae on Douglas-fir (Pseudotsuga menziesii). Morphological, physiological, and ecological differences among the three pathogen varieties have been previously determined; however, DNA-based characterization and analyses are needed to determine the genetic relationships among these varieties. The objective of this study was to use DNA sequences of 10 gene regions to assess phylogenetic relationships among L. wageneri isolates collected from different hosts. The multigene phylogenetic analyses, based on maximum likelihood and Bayesian inference, strongly supported species-level separation of the three L. wageneri varieties. These results, in conjunction with previously established phenotypic differences, support the elevation of L. wageneri var. ponderosum and L. wageneri var. pseudotsugae to the species level as L. ponderosum comb. nov. and L. pseudotsugae comb. nov., respectively, while maintaining L. wageneri var. wageneri as Leptographium wageneri. Characterization of the three Leptographium species, each with distinct host ranges, provides a baseline to further understand the ecological interactions and evolutionary relationships of these forest pathogens, which informs management of black stain root disease.

Revealing new records of hypocrealean fungicolous fungi from northern Thailand

Hypocreales comprise a diverse group of fungi that exhibit a wide range of lifestyles in both aquatic and terrestrial environments. A significant number of the identified fungicolous taxa belong to Hypocreales. Samples were collected from Chiang Rai and Chiang Mai Provinces during an investigation on fungicolous fungi in northern Thailand. Based on morphological characteristics and multi-gene phylogenetic analyses of ITS-LSU-tef1α-rpb2 and ITS-LSU-tub2-tef1α, two isolates were identified as Hypomyces sichuanensis (Hypocreaceae) and Mariannaea humicola (Nectriaceae) respectively. This study marks the first documentation of Hypomyces sichuanensis and Mariannaea humicola as fungicolous species from Thailand. Additionally, the study reveals a novel host record for M. humicola on Marasmius sp.

Eucalyptus leaf spot disease caused by Coniella eucalyptorum in Sri Lanka

Coniella has a wide distribution as a plant pathogen in Eucalyptus species. Here we report a leaf spot disease caused by Coniella eucalyptorum on Eucalyptus camaldulensis in Sri Lanka. The pathogen was isolated from symptomatic leaf samples of E. camaldulensis plants from Polonnaruwa District in Sri Lanka. The isolated fungus was identified as C. eucalyptorum based on morphological characteristics and multi-gene phylogenetic analysis of combined sequences of the internal transcribed spacer region (ITS), the partial translation elongation factor 1-α gene (tef1-α), and large subunit of nuclear ribosomal RNA (LSU). The pathogenicity test was performed to confirm its pathogenicity to the host, which produced symptoms similar to those observed in the field. This is the first report of C. eucalyptorum associated with Eucalyptus plantations in Sri Lanka. This finding is important to expand the knowledge of plant pathogen diversity in Eucalyptus species and other economically important crops in Sri Lanka.

First report of Alternaria arborescens causing white spot disease on Chinese chive in China

Chinese chive (Allium tuberosum) is a perennial plant in Liliaceae family with highly edible and medicinal value, cultivated in many areas of the globe. From 2022 to 2023, a white spot disease showing nearly circular white spots, with brown mold layers growing on the surface, was observed in Puding, Guiyang, China, with an estimated incidence of 20–35%. To determine this disease, morphological characters, 5.8S nrDNA (ITS), 18S nrDNA (SSU), 28S nrDNA (LSU), partial translation elongation factor 1-alpha (TEF), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (RPB2) and major allergen Alt-α1 (Alt-α1) multigene phylogenetic analyses and pathogenicity test were carried out, the results showed that the pathogen was A. arborescens. To our knowledge, this study firstly reported that A. arborescens was the causal agent of white spot disease on Chinese chive in China.

Tetraploa lignicola, a new freshwater fungal species from Yunnan Province, China

Two fungal strains were isolated from submerged decaying woods in the freshwater habitats, which are characterized by cylindrical, septate, verrucose, brown conidia, composed of four vertical columns of cells, 3-septate in each column, with four appendages at the base which are slightly tapering toward the apex and each with 5–10-septate. Multigene phylogenetic analyses of combined ITS, LSU, and SSU sequences data showed that the two isolates clustered into a separated clade in Tetraploa. Herein, we report it as a new hyphomycetous species, Tetraploa lignicola. The descriptions, illustrations, and a phylogenetic tree show the placement of T. lignicola, the comparisons between the new species and other morphologically similar taxa in Tetraploa are also provided.

Morphological and multi-gene phylogenetic analyses reveal five new hyphomycetes from freshwater habitats.

During the survey on freshwater hyphomycetes in Guangxi, Guizhou and Hainan Provinces, China, five fresh collections were encountered. Based on their morphology, these five isolates were identified as belonging to Hermatomyces, Kirschsteiniothelia, Paramonodictys, Pleopunctum and Sparticola. Multi-gene phylogenetic analyses were performed for each genus, which resulted in the identification of five new species, namely Hermatomyces hainanensis, Kirschsteiniothelia ramus, Paramonodictys globosa, Pleopunctum guizhouense, and Sparticola irregularis. Detailed descriptions and illustrations of the morphological characteristics of these new taxa were provided. This research enriches the biodiversity of freshwater dematiaceous hyphomycetes.

Anteaglonium saxicola (Anteagloniaceae, Pleosporales), a new species isolated from rocks in northern Thailand

A new species of rock-inhabiting fungi, described herein as Anteaglonium saxicola, was isolated from rock samples collected from natural forests in Lamphun Province, Thailand. This species was introduced based on morphological characteristics and multi-gene phylogenetic analyses. Multi-gene phylogenetic analyses based on a combination of the 28S large subunit (nrLSU), 18S small subunit (nrSSU), and internal transcribed spacer (ITS) of the nuclear ribosomal DNA (rDNA) along with the translation elongation factor (tef1-α) genes revealed that A. saxicola belongs to the clade of the genus Anteaglonium and is clearly distinct from other known Anteaglonium species. This paper provides a description, illustrations, and phylogenetic position of this novel species.

First Report of Curvularia hominis as a Causal Agent of Riceberry Rice Leaf Spot in Northern Thailand

L eaf spot disease was observed on Riceberry rice (Oryza sativa L.) in the San Pa-Tong District of Chiang Mai Province, northern Thailand during the rainy season in September 2015. A single strain of Curvularia was isolated from disease symptoms. To confirm its pathogenicity, the isolated fungus was used to inoculate healthy Riceberry rice leaves. Subsequently, the same fungus was re-isolated from the leaves, displaying similar symptoms to those observed in the field. Morphological characteristics and multi-gene phylogenetic analyses of the internal transcribed spacers (ITS) and large subunit (LSU) of nuclear ribosomal DNA, glyceraldehyde-3-phosphate dehydrogenase (GPDH), and the RNA polymerase II second largest subunit (RBP2) genes were applied to identify the pathogen as Curvularia hominis. This finding represents the first report of C. hominis as the causal agent of leaf spot disease on Riceberry rice in Thailand.

Blackwellomyces kaihuaensis and Metarhizium putuoense (Hypocreales), Two New Entomogenous Fungi from Subtropical Forests in Zhejiang Province, Eastern China

Entomogenous fungi are one of the most diverse fungal groups among ascomycetes. During investigations of macro-fungi biodiversity in Kaihua and Putuo in Zhejiang Province, China, in 2021, several entomopathogenic fungal specimens were collected from subtropical forests. After a careful examination of morphology and multi-gene phylogenetic analyses applying nrSSU, nrLSU, tef1, rpb1 and rpb2, two novel species were discovered, described herein as Blackwellomyces kaihuaensis and Metarhizium putuoense, and further compared with other similar species. Detailed descriptions, color photographs of fresh specimen and figures illustrating microscopic features of the two new species are provided.

Additions to the Xylariomycetidae (Sordariomycetes) Fungal Flora from Northern Thailand: Amphisphaeria chiangmaiensis sp. nov. and A. hydei sp. nov.

ABSTRACT During the continuous investigation into inconspicuous xylarialean species in northern Thailand, two amphisphaeria-like taxa were found in samples collected between July and October 2022. Both taxa have solitary, immersed ascomata with a two-layered peridium and unitunicate asci that are morphologically similar to Amphisphaeria species. Macro–micro morphological comparisons with related taxa and multigene phylogenetic analyses based on combined internal transcribed spacer (ITS), partial 28S large subunit rDNA (LSU), partial RNA polymerase II second largest subunit (rpb2), and partial β-tubulin (tub2) sequences revealed that the two taxa are novel to science. Amphisphaeria chiangmaiensis sp. nov. is distinguished from known taxa by having larger ascomata, a J- apical ring in Melzer’s reagent, and hyaline ascospores. Amphisphaeria hydei sp. nov. differs from its sister taxa in having larger ascomata, a longer ostiolar canal, and longer asci. Morphological descriptions, illustrations, and phylogenetic analyses of two novel taxa are provided herein.

Broadening the Knowledge of Mexican Boletes: Addition of a New Genus, Seven New Species, and Three New Combinations.

Boletes are one of the most common groups of fungi in temperate, subtropical, and tropical ecosystems. In Mexico, the northern region has mainly been explored in terms of bolete diversity. This study describes a new genus and seven new species based on macromorphological, micromorphological, molecular, phylogenetic, and ecological data. Garcileccinum gen. nov. is typified with G. salmonicolor based on multigene phylogenetic analysis of nrLSU, RPB2, and TEF1, and it is closely related to Leccinum and Leccinellum. Garcileccinum viscosum and G. violaceotinctum are new combinations. Boletellus minimatenebris (ITS, nrLSU, and RPB2), Cacaoporus mexicanus (RPB2 and ATP6), Leccinum oaxacanum, Leccinum juarenzense (nrLSU, RPB2, and TEF1), Tylopilus pseudoleucomycelinus (nrLSU and RPB2), and Xerocomus hygrophanus (ITS, nrLSU, and RPB2) are described as new species. Boletus neoregius is reclassified as Pulchroboletus neoregius comb. nov. based on morphological and multigene phylogenetic analysis (ITS and nrLSU), and its geographic distribution is extended to Central Mexico, since the species was only known from Costa Rica. Furthermore, T. leucomycelinus is a new record from Mexico. This study contributes to increasing our knowledge of boletes and expands the diversity found in Mexican forests.

Gamete Recognition Gene Divergence Yields a Robust Eutherian Phylogeny across Taxonomic Levels

The extraordinary morphological diversity among extant mammals poses a challenge for studies of speciation, adaptation, molecular evolution, and reproductive isolation. Despite the recent wealth of molecular studies on mammalian phylogenetics, uncertainties remain surrounding both ancestral and more recent divergence events that have proven difficult to resolve. Multi-gene datasets, especially including genes that are highly divergent, often provide increased support for higher-level affinities within Mammalia; however, such analyses require vast amounts of genomic sequence data and at times, intensive, high-performance computational effort. Furthermore, despite the large-scale efforts dedicated to comprehensive, multi-gene phylogenetic analyses using a combination of mitochondrial, nuclear, and other sequences (e.g., tRNA, ultra-conserved elements, and transposable elements), many relationships across Mammalia remain highly controversial. To offer another approach and provide a phylogenetic solution to this longstanding issue, here we present a phylogenetic tool based on a single reproductive molecular marker, zonadhesin (gene: Zan), one of two known mammalian speciation genes, which encodes the rapidly evolving sperm protein zonadhesin that mediates species-specific adhesion to the egg and thereby promotes reproductive isolation among placental mammals (Eutheria). Topological comparison of Zan Maximum Likelihood phylogenies to a nearly complete mammalian supertree confirmed Zan’s striking phylogenetic utility and resolution at both deeper and more terminal nodes in the placental mammalian phylogeny. This single gene marker yielded an equivalent and/or superiorly supported topology in comparison to a supertree generated using DNA sequences from a supermatrix of 31 genes from 5911 species (extinct and extant). Resolution achieved with this new phylogenetic approach provides unique insights into the divergence of both early and recent mammalian radiations. Finally, and perhaps most importantly, the utility of zonadhesin as a singular molecular marker was especially useful in clades where sufficient taxon sampling is impossible to achieve, and where only a subset of members of the mammalian species tree is available. The eutherian relationships presented here provide a foundation for future studies in the reconstruction of mammalian classifications, including reproductive isolation, hybridization, and biodiversification of species.