Wickerhamomyces corioli f.a., sp. nov., a novel yeast species discovered in two mushroom species.

Two yeast strains, designated as 19-39-3 and 19-40-2, obtained from the fruiting bodies of Trametes versicolor and Marasmius siccus collected in Yunwu Mountain Forest Park, PR China, have been identified as representing a novel asexual ascomycetous yeast species. From the results of phylogenetic analyses of the sequences of the D1/D2 domains of the large subunit (LSU) rRNA, small subunit (SSU) rRNA and translation elongation factor 1-α (TEF1) genes, it was determined that these strains represent a member of the genus Wickerhamomyces, with Wickerhamomyces alni and Candida ulmi as the closest relatives. The novel species exhibited 6.6 and 6.7% differences in the D1/D2 domains compared with W. alni and C. ulmi, respectively. Additionally, distinct biochemical and physiological differences were observed between the novel species and its related counterparts. No sexual reproduction was observed in these strains, leading to the proposal of the name Wickerhamomyces corioli f.a., sp. nov. for this newly discovered species.

Savitreea siamensis sp. nov., an ascomycetous yeast species in the family Saccharomycetaceae discovered in Thailand.

Four yeast strains, representing a novel anamorphic species, were isolated in Thailand. The two strains (ST-3660T and ST-3647) were obtained from two different estuarine water samples in a mangrove forest. Strain DMKU-FW1-37 was derived from a grease sample, and another strain (TSU57) was isolated from a fruiting body of Phallus sp. Pairwise sequence analysis showed that the four strains had identical or differed by only one nucleotide substitution in the D1/D2 domains of the large subunit (LSU) rRNA gene, and differed by one to three nucleotide substitutions in the internal transcribed spacer (ITS) regions. Savitreea pentosicarens is the most closely related species to the four strains, but with 9-10 (1.57-1.72 %) nucleotide substitutions in the D1/D2 domains of the LSU rRNA gene and 29-31 (4.22-4.45 %) nucleotide substitutions in the ITS regions. Phylogenetic analyses based on the concatenated sequences of the ITS regions and the D1/D2 domains of the LSU rRNA gene showed that the four strains form a well-separated lineage from S. pentosicarens with high bootstrap support, confirming that they represent a distinct species. Therefore, the four strains are assigned as representives of a novel species of the genus Savitreea, for which the name Savitreea siamensis sp. nov. is proposed. The holotype is TBRC 4481T and the ex-type is PYCC 9794T (=ST-3660T). The MycoBank number of the novel species is MB 851951.

Fungal diversity present in snow sampled in summer in the north-west Antarctic Peninsula and the South Shetland Islands, Maritime Antarctica, assessed using metabarcoding.

We assessed the fungal diversity present in snow sampled during summer in the north-west Antarctic Peninsula and the South Shetland Islands, maritime Antarctica using a metabarcoding approach. A total of 586,693 fungal DNA reads were obtained and assigned to 203 amplicon sequence variants (ASVs). The dominant phylum was Ascomycota, followed by Basidiomycota, Mortierellomycota, Chytridiomycota and Mucoromycota. Penicillium sp., Pseudogymnoascus pannorum, Coniochaeta sp., Aspergillus sp., Antarctomyces sp., Phenoliferia sp., Cryolevonia sp., Camptobasidiaceae sp., Rhodotorula mucilaginosa and Bannozyma yamatoana were assessed as abundant taxa. The snow fungal diversity indices were high but varied across the different locations sampled. Of the fungal ASVs detected, only 28 were present all sampling locations. The 116 fungal genera detected in the snow were dominated by saprotrophic taxa, followed by symbiotrophic and pathotrophic. Our data indicate that, despite the low temperature and oligotrophic conditions, snow can host a richer mycobiome than previously reported through traditional culturing studies. The snow mycobiome includes a complex diversity dominated by cosmopolitan, cold-adapted, psychrophilic and endemic taxa. While saprophytes dominate this community, a range of other functional groups are present.

Concordance of multigene genealogy along with morphological evidence unveils five novel species and two new records of boletoid mushrooms (fungi) from India.

Agaricales, Russulales and Boletales are dominant orders among the wild mushrooms in Basidiomycota. Boletaceae, one of the major functional elements in terrestrial ecosystem and mostly represented by ectomycorrhizal symbionts of trees in Indian Himalaya and adjoining hills, are extraordinarily diverse and represented by numerous genera and species which are unexplored or poorly known. Therefore, their hidden diversity is yet to be revealed. Extensive macrofungal exploration by the authors to different parts of Himalaya and surroundings, followed by through morphological studies and multigene molecular phylogeny lead to the discovery of five new species of wild mushrooms: Leccinellum bothii sp. nov., Phylloporus himalayanus sp. nov., Phylloporus smithii sp. nov., Porphyrellus uttarakhandae sp. nov., and Retiboletus pseudoater sp. nov. Present communication deals with morphological details coupled with illustrations and phylogenetic inferences. Besides, Leccinellum sinoaurantiacum and Xerocomus rugosellus are also reported for the first time from this country.

Ectomycorrhizal fungal communities in natural and urban ecosystems: Quercus humboldtii as a study case in the tropical Andes.

Worldwide urban landscapes are expanding because of the growing human population. Urban ecosystems serve as habitats to highly diverse communities. However, studies focusing on the diversity and structure of ectomycorrhizal communities are uncommon in this habitat. In Colombia, Quercus humboldtii Bonpl. is an ectomycorrhizal tree thriving in tropical montane forests hosting a high diversity of ectomycorrhizal fungi. Q. humboldtii is planted as an urban tree in Bogotá (Colombia). We studied how root-associated fungal communities of this tree change between natural and urban areas. Using Illumina sequencing, we amplified the ITS1 region and analyzed the resulting data using both OTUs and Amplicon Sequence Variants (ASVs) bioinformatics pipelines. The results obtained using both pipelines showed no substantial differences between OTUs and ASVs for the community patterns of root-associated fungi, and only differences in species richness were observed. We found no significant differences in the species richness between urban and rural sites based on Fisher's alpha or species-accumulation curves. However, we found significant differences in the community composition of fungi present in the roots of rural and urban trees with rural communities being dominated by Russula and Lactarius and urban communities by Scleroderma, Hydnangium, and Trechispora, suggesting a high impact of urban disturbances on ectomycorrhizal fungal communities. Our results highlight the importance of urban trees as reservoirs of fungal diversity and the potential impact of urban conditions on favoring fungal species adapted to more disturbed ecosystems.

Year-round dynamics of arbuscular mycorrhizal fungi communities in the roots and surrounding soils of Cryptomeria japonica.

Arbuscular mycorrhizal fungi (AMF) live simultaneously inside and outside of host plant roots for a functional mycorrhizal symbiosis. Still, the year-round dynamics and relationships between soil properties and AMF communities of trees in forest ecosystems remain unclear. We collected paired root and soil samples of the same Cryptomeria japonica trees at two forest sites (five trees at each site) every 2 months over a year. Total DNA was extracted from roots and soil separately and soil physicochemical properties were measured. With Illumina's next-generation amplicon sequencing targeting the small subunit of fungal ribosomal DNA, we clarified seasonal dynamics of soil properties and AMF communities. Soil pH and total phosphorus showed significant seasonality while total carbon, nitrogen, and C/N did not. Only pH was a good predictor of the composition and dynamics of the AMF community. The total AMF community (roots + soil) showed significant seasonality because of variation from May to September. Root and soil AMF communities were steady year-round, however, with similar species richness but contained significantly different AMF assemblages in any sampling month. Despite the weak seasonality in the communities, the top two dominant OTUs showed significant but different shifts between roots and soils across seasons with strong antagonistic relationships. In conclusion, few dominant AMF taxa are dynamically shifting between the roots and soils of C. japonica to respond to seasonal and phenological variations in their microhabitats. AMF inhabiting forest ecosystems may have high environmental plasticity to sustain a functional symbiosis regardless of seasonal variations that occur in the soil.

Polymerase chain reaction-based methods for the rapid identification of Amanita exitialis.

Amanita exitialis, a deadly mushroom found in eastern Asia, causes the highest death rates among all poisonous mushrooms in China. The aim of the present study was to develop an efficient, accurate, and user-friendly PCR-based method for identifying A. exitialis that could facilitate the prevention, diagnosis, and treatment of associated food poisoning. A. exitialis-specific primers and probes were designed based on the internal transcribed spacer region variations of 27 mushroom species. Specificity was confirmed using conventional and real-time PCR for 23 non-target mushroom species, including morphologically similar and closely related species. Compared to conventional PCR, real-time PCR was more sensitive (detectable DNA concentration: 1.36 × 10-2 ng/µL vs. 1.36 × 10-3) and efficient (analysis time: 1 h vs. 40 min). Furthermore, the real-time PCR results could be immediately visualized using amplification curve analysis. The results present two robust PCR-based methods for A. exitialis identification that can facilitate food safety.

The surface of leaves and fruits of Peruvian cacao is home for several Hannaella yeast species, including the new species Hannaella theobromatis sp. nov.

As part of a long-term study aiming to isolate and identify yeast species that inhabit the surface of leaves and fruits of native fine-aroma cacao in the department of Amazonas, Peru, we obtained multiple isolates of Hannaella species. Yeasts of the genus Hannaella are common inhabitants of the phyllosphere of natural and crop plants. On the basis of morphological, and physiological characteristics, and sequence analysis of the D1/D2 domains of the large subunit rRNA gene (LSU) and the internal transcribed spacer region (ITS), we identified five species of Hannaella from the phyllosphere of Peruvian cacao. Four have been previously described: H. phyllophila (isolates KLG-073, KLG-091), H. pagnoccae (KLG-076), H. sinensis (KLG-121), and H. taiwanensis (KLG-021). A fifth, represented by eight isolates (KLG-034, KLG-063, KLG-074, KLG-078, KLG-79, KLG-082, KLG-084, KLG-085), is not conspecific with any previously described Hannaella species, and forms the sister clade to H. surugaensis in the phylogenetic analysis. It has 2.6-3.9% (18-27 substitutions, 2-4 deletions, and 1-3 insertions in 610-938 bp-long alignments), and 9.8-10.0% nucleotide differences (37 substitutions and 14 insertions in 511-520 bp-long alignments) in the LSU and ITS regions, respectively, to H. surugaensis type strain, CBS 9426. Herein, the new species Hannaella theobromatis sp. nov. is described and characterised. The species epithet refers to its epiphytic ecology on its host Theobroma cacao.

Validation of a real-time polymerase chain reaction for the detection and quantification of the nucleic acid of Histoplasma from equine clinical samples.

Histoplasma capsulatum var. farciminosum (HCF) is a dimorphic fungus that causes epizootic lymphangitis in equids. Current diagnostic approaches, including culture, microscopy, and clinical presentation, lack speed, sensitivity, and specificity when diagnosing clinical cases. In this study, equine blood and pus samples on Whatman FTA cards from Senegal (n = 3), The Gambia (n = 19), Ethiopia (n = 16), and Mali (n = 13) were tested using a real-time PCR (qPCR) protocol. The assay was optimized and tested for its suitability to detect and quantify HCF in blood and pus loaded onto Whatman FTA cards at sampling. Whatman FTA cards were tested for their suitability for use with qPCR and were found to recover DNA more efficiently than from direct extraction. Using TaqMan fluorescent probes and specific primers, the assay demonstrated 100% analytical specificity when detecting multiple strains of Histoplasma and no false positives with off-target organisms. The assay's diagnostic performance was measured against an existing nested internal transcribed spacer PCR protocol using a receiver operating characteristic curve. The test was found to have a diagnostic specificity and sensitivity of 100% and 71.4%, respectively, when analyzing pus samples using a cycle threshold (Ct) cutoff determined by Youden's index (27.75). Blood sample cutoff Ct value was proposed at 34.55. Further optimization is required to improve the performance of the protocol when applied to blood samples. This study has, for the first time, demonstrated the ability to detect and quantify the DNA of Histoplasma spp. in equine blood and pus samples with a high degree of accuracy, providing a platform to further investigate the pathogenesis and epidemiology of this disease. IMPORTANCE: Histoplasmosis is a neglected yet major cause of morbidity and mortality in both equids and people in resource-scarce settings. One of the major hindrances to the control of histoplasmosis is a lack of readily available diagnostic tests. Tests are needed to support clinical decision-making and to be applied in population-based research to further understand this disease in situ. This paper reports, for the first time, the validation and application of a qPCR to detect Histoplasma directly from equine clinical samples, bypassing the need to culture this notoriously difficult organism. We report and comment on the performance of the qPCR in comparison with our previously developed nested PCR.

Three new species in Russula subsection Xerampelinae supported by genealogical and phenotypic coherence.

Xerampelinae is a subsection composed of species of ectomycorrhizal fungi belonging to the hyperdiverse and cosmopolitan genus Russula (Russulales). Species of Xerampelinae are recognized by their fishy or shrimp odor, browning context, and a green reaction to iron sulfate. However, species delimitation has traditionally relied on morphology and analysis of limited molecular data. Prior taxonomic work in Xerampelinae has led to the description of as many as 59 taxa in Europe and 19 in North America. Here we provide the first multilocus phylogeny of European and North American members based on two nrDNA loci and two protein-coding genes. The resulting phylogeny supports the recognition of 17 species-rank Xerampelinae clades; however, higher species richness (~23) is suggested by a more inclusive nuclear rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS barcode) analysis. Phylogenetic and morphological analyses support three new species with restricted geographic distributions: R. lapponica, R. neopascua, and R. olympiana. We confirm that the European species R. subrubens is present in North America and the North American species R. serissima (previously known as R. favrei) is present in Europe. Most other Xerampelinae appear restricted to either North America or Eurasia, which indicates a high degree of regional endemism; this includes R. xerampelina, a name widely applied to North American taxa, but a species restricted to Eurasia.

Chionobium takahashii, gen. et sp. nov., associated with snow blight of conifers in Japan.

Gremmenia abietis (Dearn.) Crous (syn: Phacidium abietis) was originally described in North America to accommodate the species associated with snow blight of Abies and Pseudotsuga spp. In Japan, this species was first observed on the dead needles on Abies sachalinensis and Picea jezoensis var. jezoensis in 1969. However, the identity of Japanese species was unclear due to the lack of molecular data and the absence of anamorph description. In this study, we collected fresh specimens from various conifer species (A. sachalinensis, A. veitchii, Pic. jezoensis var. jezoensis, Pic. jezoensis var. hondoensis, Pinus koraiensis, and Pin. pumila) in Japan and revised the taxonomy based on morphological and phylogenetic analyses. Phylogenetic analyses based on nuc rDNA internal transcribed spacer ITS1-5.8S-ITS2 (ITS), nuc 28S rDNA (28S), and RNA polymerase II second largest subunit (RPB2) regions indicated that the species belongs to Phacidiaceae. Conidiomata formed in vitro produced pyriform, hyaline conidia without mucoid appendage, which distinguished the species from phylogenetically related genera. Consequently, we established Chionobium takahashii to accommodate the snow blight fungus in Japan. Further phylogenetic analyses also indicated that C. takahashii includes several distinct clades corresponding to the host genera (Abies, Picea, Pinus). Morphological differences among those clades were unclear, suggesting that C. takahashii may contain host-specific cryptic species.

Ogataea nonmethanolica f.a, sp. nov., a novel yeast species isolated from rotting wood in Brazil and Colombia.

Three yeast isolate candidates for a novel species were obtained from rotting wood samples collected in Brazil and Colombia. The Brazilian isolate differs from the Colombian isolates by one nucleotide substitution in each of the D1/D2 and small subunit (SSU) sequences. The internal transcribed spacer (ITS) and translation elongation factor 1-α gene sequences of the three isolates were identical. A phylogenetic analysis showed that this novel species belongs to the genus Ogataea. This novel species is phylogenetically related to Candida nanaspora and Candida nitratophila. The novel species differs from C. nanaspora by seven nucleotides and two indels, and by 17 nucleotides and four indels from C. nitratophila in the D1/D2 sequences. The ITS sequences of these three species differ by more than 30 nucleotides. Analyses of the sequences of the SSU and translation elongation factor 1-α gene also showed that these isolates represent a novel species of the genus Ogataea. Different from most Ogataea species, these isolates did not assimilate methanol as the sole carbon source. The name Ogataea nonmethanolica sp. nov. is proposed to accommodate these isolates. The holotype of Ogataea nonmethanolica is CBS 13485T. The MycoBank number is MB 851195.

Phylogenomic delineation of two new species of ascomycetous yeasts, Wickerhamiella koratensis sp. nov. and Wickerhamiella limtongiae sp. nov., and proposal of two synonyms, Wickerhamiella infanticola and Wickerhamiella tropicalis.

Two novel ascomycetous yeast species of the genus Wickerhamiella are proposed based on isolates obtained in Thailand from food waste and the fruiting body of a polypore fungus, and on a combination of conventional DNA-barcode sequence analyses and whole-genome phylogenies. We focus on a particular subclade of the genus Wickerhamiella that contains species found in anthropic environments and describe Wickerhamiella limtongiae sp. nov. (DMKU-FW31-5T=PYCC 9022T=TBRC 15055T), found on food waste samples. In an adjacent clade, we describe Wickerhamiella koratensis sp. nov. (DMKU-KO16T=PYCC 8908T=TBRC 14869T), which represents the closest relative of Wickerhamiella slavikovae and was isolated from the fruiting body of Bjerkandera sp. In the subclade of W. limtongiae sp. nov., we propose that Wickerhamiella infanticola should be regarded as a synonym of Wickerhamiella sorbophila and that Wickerhamiella tropicalis should be regarded as a synonym of Wickerhamiella verensis.

Development of a novel mycobiome diagnostic for fungal infection.

BACKGROUND: Amplicon-based mycobiome analysis has the potential to identify all fungal species within a sample and hence could provide a valuable diagnostic assay for use in clinical mycology settings. In the last decade, the mycobiome has been increasingly characterised by targeting the internal transcribed spacer (ITS) regions. Although ITS targets give broad coverage and high sensitivity, they fail to provide accurate quantitation as the copy number of ITS regions in fungal genomes is highly variable even within species. To address these issues, this study aimed to develop a novel NGS fungal diagnostic assay using an alternative amplicon target. METHODS: Novel universal primers were designed to amplify a highly diverse single copy and uniformly sized DNA target (Tef1) to enable mycobiome analysis on the Illumina iSeq100 which is a low cost, small footprint and simple to use next-generation sequencing platform. To enable automated analysis and rapid results, a streamlined bioinformatics workflow and sequence database were also developed. Sequencing of mock fungal communities was performed to compare the Tef1 assay and established ITS1-based method. The assay was further evaluated using clinical respiratory samples and the feasibility of using internal spike-in quantitative controls was assessed. RESULTS: The Tef1 assay successfully identified and quantified Aspergillus, Penicillium, Candida, Cryptococcus, Rhizopus, Fusarium and Lomentospora species from mock communities. The Tef1 assay was also capable of differentiating closely related species such as A. fumigatus and A. fischeri. In addition, it outperformed ITS1 at identifying A. fumigatus and other filamentous pathogens in mixed fungal communities (in the presence or absence of background human DNA). The assay could detect as few as 2 haploid genome equivalents of A. fumigatus from clinical respiratory samples. Lastly, spike-in controls were demonstrated to enable semi-quantitation of A. fumigatus load in clinical respiratory samples using sequencing data. CONCLUSIONS: This study has developed and tested a novel metabarcoding target and found the assay outperforms ITS1 at identifying clinically relevant filamentous fungi. The assay is a promising diagnostic candidate that could provide affordable NGS analysis to clinical mycology laboratories.

Morphological and phylogenetic analyses reveal a new species of Anthracophyllum (Omphalotaceae, Agaricales) in Zhejiang Province, China.

During the investigations of macrofungi resources in Zhejiang Province, China, an interesting wood rot fungus was collected. Based on morphological and molecular phylogenetic studies, it is described as a new species, Anthracophyllum sinense. A. sinense is characterized by its sessile, charcoal black and pleurotoid pileus, sparse lamellae occasionally branching, clavate basidia with long sterigmata [(3-)6-7(-8) µm], and non-heteromorphous cystidia. A. sinense establishes a separate lineage close to A. archeri and A. lateritium in the phylogenetic tree.

Nakazawaea tricholomae f.a., sp. nov., a Novel Ascomycetous Yeast Species Isolated from Two Mushroom Species in China.

Two yeast strains designated as 20-27-1 and 20-28 were isolated from the fruiting bodies of Tricholoma gambosum and Marasmius maximus, respectively, which were collected in Wudaogou, Weichang county, Chengde area, Hebei Province, China. The multi-locus analysis of the sequences of the rDNA ITS, D1/D2 LSU, and SSU regions, together with partial sequences of two protein-coding genes RPB1 and TEF1 indicates that the two strains are closely related to Nakazawaea ernobii and Nakazawaea holstii, showing the similarity values of 99.3-98.7%, 97.2-97.1%, 91.9-92.5%, and 84.6% in D1/D2 LSU, ITS, TEF1, and RPB1, respectively. Physiologically, the two strains are different from N. ernobii and N. holstii in the assimilation of melibiose, inulin, and DL-lactic acid. Both the phenotypic and phylogenetic analyses indicate that those two strains represent a novel species in the genus Nakazawaea, for which the name Nakazawaea tricholomae f.a., sp. nov. (Fungal Names: FN 571492) is proposed.

Starmerella kisarazuensis f.a., sp. nov., a novel yeast isolated from Trifolium pratense flowers.

Three yeast isolates, NBRC 115909T, NBRC 115910 and NBRC 116270, were isolated from Trifolium pratense (red clover) flowers collected from Kisarazu, Chiba, Japan. Analysis of the sequences of the D1/D2 domains of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) regions revealed that these isolates represent a single novel species within the genus Starmerella. Also, no ascospore formation was observed. The yeast isolates were closely related to Starmerella vitae UWOPS 00-107.2T and Starmerella bombi NRRL Y-17081T. They differed from S. vitae, the most closely related species with a validly published name, by ten nucleotide substitutions with two gaps in the D1/D2 domains and 20 nucleotide substitutions in the ITS region. Moreover, the three isolates exhibited distinct phenotypic characteristics from the closely related species. Therefore, we suggest that these three isolates represent a novel species, designated as Starmerella kisarazuensis f.a., sp. nov. The holotype is NBRC 115909T (isotype: CBS 18485T).

Phylogeny and taxonomy of the genera of Erysiphaceae, part 5: Erysiphe (the "Microsphaera lineage" part 1).

In this contribution, we offer the fifth installment of a series focusing on the phylogeny and taxonomy of powdery mildews. This paper is the second segment evaluating the genus Erysiphe. The first treatment of Erysiphe focused on phylogenetically basal species in the "Uncinula lineage." This research presents a phylogenetic-taxonomic assessment of species that form the group previously referred to as the "Microsphaera lineage." Given the size of the group, we split the treatment of this lineage of Erysiphe species into two parts based on their phylogenetic placement. Phylogenetic trees based on ITS+28S data are supplemented by sequences of additional markers (CAM, GADPH, GS, RPB2, and TUB). Included in the analysis of the Microsphaera lineage is the "Erysiphe aquilegiae complex" (group, clade, cluster), which encompasses sequences obtained from an assemblage of Erysiphe species with insufficient resolution in rDNA analyses. Attempts have been made to resolve this group at the species level by applying a multilocus approach. A detailed discussion of the "Erysiphe aquilegiae complex" is provided. Sequences are provided for the first time for several species, particularly North American species, such as Erysiphe aggregata, E. erineophila, E. parnassiae, and E. semitosta. Ex-type sequences for Microsphaera benzoin and M. magnusii have been retrieved. Alphitomorpha penicillata, Microsphaera vanbruntiana, and M. symphoricarpi are epitypified with ex-epitype sequences. The new species Erysiphe alnicola, E. deutziana, E. cornigena, E. lentaginis, and E. sambucina are described, the new combinations E. lauracearum, E. passiflorae, and E. sambucicola are introduced, and the new name E. santali is proposed.

Paraphyly and cryptic diversity unveils unexpected challenges in the "naked lichens" (Calvitimela, Lecanoromycetes, Ascomycota).

Molecular phylogenetics has revolutionized the taxonomy of crustose lichens and revealed an extensive amount of cryptic diversity. Resolving the relationships between genera in the crustose lichen family Tephromelataceae has proven difficult and the taxon limits within the genus Calvitimela are only partly understood. In this study, we tested the monophyly of Calvitimela and investigated phylogenetic relationships at different taxonomic levels using an integrative taxonomic approach. We performed a global sampling of all species currently assigned to Calvitimela and conducted additional sampling of C. melaleuca sensu lato across Norway. We included 108 specimens and produced more than 300 sequences from five different loci (ITS, LSU, MCM7, mtSSU, TEF1-α). We inferred phylogenetic relationships and estimated divergence times in Calvitimela. Moreover, we analyzed chemical and morphological characters to test their diagnostic values in the genus. Our molecular phylogenetic results show evolutionarily old and deeply divergent lineages in Calvitimela. The morphological characters are overlapping between divergent subgenera within this genus. Chemical characters, however, are largely informative at the level of subgenera, but are often homoplastic at the species level. The subgenus Calvitimela is found to include four distinct genetic lineages. Detailed morphological examinations of C. melaleuca s. lat. reveal differences between taxa previously assumed to be morphologically cryptic. Furthermore, young evolutionary ages and signs of gene tree discordance indicate a recent divergence and possibly incomplete lineage sorting in the subgenus Calvitimela. Phylogenetic analysis and morphological observations revealed that C. austrochilensis and C. uniseptata are extraneous to Calvitimela (Tephromelataceae). We also found molecular evidence supporting C. septentrionalis being sister to C. cuprea. In the subgenus Severidea, one new grouping is recovered as a highly supported sister to C. aglaea. Lastly, two fertile specimens were found to be phylogenetically nested within the sorediate species C. cuprea. We discuss the need for an updated classification of Calvitimela and the evolution of cryptic species. Through generic circumscription and species delimitation we propose a practical taxonomy of Calvitimela.