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Hesperomyces virescens parasitizing Harmonia axyridis. A group of thalli of Hesperomyces virescens attached to the elytral tips of a specimen of Harmonia axyridis (from Watermaal-Bosvoorde, Belgium). Image provided by Gilles San Martin. Inserted is a drawing of a single adult thallus (D. Haelew. 601c, FH 00313615, Byron, Georgia, USA), with the following structures annotated: haustorium; cells I, II, and III of the receptacle; the appendage (app.) with antheridia (anth.); and the perithecium (perith.) with its terminal projections (proj.). Drawing by André De Kesel.
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Our understanding of fungal diversity is far from complete. Species descriptions generally focus on morphological features, but this approach may underestimate true diversity. Using the morphological species concept, Hesperomyces virescens (Ascomycota, Laboulbeniales) is a single species with global distribution and wide host range. Since its descr...
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... However, drawbacks for their protocol include the need for custom-made components and the fact that DNA extractions are fully depleted during PCR amplification and thus cannot be stored. Haelewaters et al. (2018a) published another method to extract DNA of Laboulbeniales. Using the REPLI-g Single Cell Kit (Qiagen), a sufficient amount of DNA can be obtained from a single thallus. ...
... Currently, the nuclear small and large subunit ribosomal RNA (nrSSU and nrLSU), the internal transcribed spacer region (ITS), the minichromosome maintenance complex component 7 protein-coding gene (MCM7), the translation elongation factor 1α protein-coding gene (TEF1), and the mitochondrial small subunit rRNA (mtSSU) have been sequenced for several species of Laboulbeniales (Goldmann and Weir 2012;Goldmann et al. 2013;Haelewaters et al. 2015Haelewaters et al. , 2018aHaelewaters et al. , 2019bHaelewaters et al. , 2022Sundberg et al. 2018aSundberg et al. , 2018bLiu et al. 2020;Van Caenegem et al. 2023a, 2023b. General fungal primers designed by White et al. (1990) have mainly been used to amplify regions of ribosomal RNA (nrSSU, ITS, nrLSU). ...
... Haelewaters et al. (2015) developed a Laboulbeniomycetes-specific nrSSU primer pair. Additionally, recent research reported low amplification of the ITS region using general fungal primers, which resulted in the design of a Hesperomyces-specific ITS primer pair (ITShespL and ITShespR) and the Laboulbeniomycetesspecific LabITS1 forward primer (Haelewaters et al. 2018a(Haelewaters et al. , 2019b. ...
Molecular studies of fungi within the order Laboulbeniales (Ascomycota, Pezizomycotina) have been hampered for years because of their minute size, inability to grow in axenic culture, and lack of reliable and cost-efficient DNA extraction protocols. In particular, the genus Laboulbenia is notorious for low success with DNA extraction and polymerase chain reaction (PCR) amplification. This is attributed to the presence of melanin, a molecule known to inhibit PCR, in the cells. We evaluated the efficacy of a standard single cell-based DNA extraction protocol by halving the recommended amount of reagents to reduce the cost per extraction and adding bovine serum albumin (BSA) during the multiple displacement amplification step to reverse the effect of melanin. A total of 196 extractions were made, 111 of which were successful. We found that halving the reagents used in the single cell-based extraction kit did not significantly affect the probability of successful DNA extraction. Using the halved protocol reduces cost and resource consumption. Moreover, there was no significant difference in the probability of successfully extracting DNA based on whether BSA was added or not, suggesting that the amount of melanin present in cells of the thallus has no major inhibitory effect on PCR. We generated 277 sequences from five loci, but amplification of the internal transcribed spacer region, the mitochondrial small subunit rDNA, and protein-coding genes remains challenging. The probability of successfully extracting DNA from Laboulbeniales was also impacted by specimen storage methods, with material preserved in > 95% ethanol yielding higher success rates compared to material stored in 70% ethanol and dried material. We emphasize the importance of proper preservation of material and propose the design of Laboulbeniales-specific primers to overcome the problems of primer mismatches and contaminants. Our new insights apply not only to the genus Laboulbenia; Laboulbeniales generally are understudied, and the vast majority of species remain unsequenced. New and approachable molecular developments will benefit the study of Laboulbeniales, helping to elucidate the true diversity and evolutionary relationships of these peculiar microfungi.
... In particular, considerations regarding rarity apply to microscopic organisms of which the vast majority of species are rare, or at least rarely recorded. Microbial, and more specifically fungal, diversity records are generally dominatedin terms of abundance-by a small number of "common" species, suggesting that being common is not frequent in microbial groups, but "widely distributed" species of fungi often turn out to be species complexes following more rigorous analysis (e.g., Pringle et al. 2005;Haelewaters et al. 2018). ...
... Morphological divergence is not always indicative of molecular divergence since the evolution of morphological characters may be faster than molecular characters and vice versa (Jargeat et al. 2010). Moreover, certain groups of fungi have few morphological characteristics, and some species may exhibit cryptic diversity or high phenotypic plasticity (e.g., Slepecky and Starmer 2009;Haelewaters et al. 2018;Hapuarachchi et al. 2019;Van Caenegem et al. 2023a). These observations highlight the need to combine morphological and molecular data whenever possible when dealing with singleton-based species-in line with using integrated methodologies to study fungal diversity (Cazabonne et al. 2022). ...
Fungi are among the least known organisms on earth, with an estimated number of species between 1.5 and 10 million. This number is expected to be refined, especially with increasing knowledge about microfungi in undersampled habitats and increasing amounts of data derived from environmental DNA sequencing. A significant proportion of newly generated sequences fail to match with already named species, and thus represent what has been referred to as fungal “dark taxa”. Due to the challenges associated with observing, identifying, and preserving sporophores, many macro- and microfungal species are only known from a single collection, specimen, isolate, and/or sequence—a singleton. Mycologists are consequently used to working with “rare” sequences and specimens. However, rarity and singleton phenomena lack consideration and valorization in fungal studies. In particular, the practice of publishing new fungal species names based on a single specimen remains a cause of debate. Here, we provide some elements of reflection on this issue in the light of the specificities of the fungal kingdom and global change context. If multiple independent sources of data support the existence of a new taxon, we encourage mycologists to proceed with formal description, irrespective of the number of specimens at hand. Although the description of singleton-based species may not be considered best practice, it does represent responsible science in the light of closing the Linnean biodiversity shortfall.
... PCR reactions (25 µL total) consisted of 13.3 µL of RedExtract Taq polymerase (Sigma-Aldrich, St. Louis, MO), 2.5 µL of each 10-µM primer, 5.7 µL of ddH 2 O, and 1.0 µL of DNA extract. PCR conditions were as follows: for ITS, initial denaturation at 94 °C for 3 min; 35 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 45 s, and extension at 72 °C for 90 s; and final extension at 72 °C for 10 min (Haelewaters et al. 2018a); for LSU, initial denaturation at 94 °C for 5 min; 35 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 45 s, and extension at 72 °C for 1 min; and final extension at 72 °C for 7 min (Liu et al. 2020). We purified PCR products using 1.5 µL of Exo-FAP (0.5 µL exonuclease I and 1 µL FAST alkaline phosphatase; Thermo Fisher Scientific, Waltham, MA) per 10 µL of PCR product at 37 °C for 15 min, followed by deactivation at 85 °C for 15 min. ...
A new saprotrophic species was discovered during our fungal inventory at the Boston Harbor Islands National Recreation Area (Massachusetts), which consists of 34 islands and peninsulas. Simocybe ramosa sp. nov. (Agaricales, Crepidotaceae) is described based on morphology and molecular phylogenetic data. The holotype collection was found in a Quercus (oak)–Carya (hickory) forest under bark of a dead oak tree on World’s End peninsula, the largest land mass of the archipelago. Phylogenetic reconstruction of a dataset of the internal transcribed spacer region (ITS) resolved S. ramosa and S. rhabarbarina as sister species. Simocybe rhabarbarina is here redescribed based on the holotype and newly reported material from the Netherlands, and its presence on the island of Jersey, off the coast of northern France, is confirmed based on an ITS sequence. Finally, we compare morphological features of S. ramosa with S. rhabarbarina and the 20 species in the genus that have thus far been recorded in Canada, the US, and Mexico.
... It produces tiny three-dimensional structures, or thalli, that grow externally on the integument of the host. Hesperomyces harmoniae is the most recorded species in Hesperomyces virescens sensu lato, a complex of species that are segregated by host and geographic location (Haelewaters et al. 2018a;Van Caenegem et al. 2023). Although the first records of He. harmoniae within the native range of Ha. axyridis date from the 1930s , these were previously referred to as He. ...
... 88 from NDFF, 9 from Flickr, 2 from Ukrainian Biodiversity Information Network, and 1 from Projekt LIFE ARTEMIS and National Biodiversity Network each. In addition, 92 collections of Ha. axyridis infected with He. harmoniae were retrieved from the literature, excluding records covered in the sources above (Garcés and Williams 2004;Riddick and Schaefer 2005;Riddick 2006Riddick , 2010Harwood et al. 2006a, b;Nalepa and Weir 2007;Riddick and Cottrell 2010;Steenberg and Harding 2010;De Kesel 2011;Haelewaters and De Kesel 2011;Cottrell and Riddick 2012;Herz and Kleespiel 2012;Haelewaters et al. 2012a, b;, 2017, 2018a, b, 2019, 2022aCeryngier and Twardowska 2013;Ceryngier et al. 2013;Pfliegler 2014;Raak-van den Berg et al. 2014;Cornejo and González 2015;Gorczak et al. 2016;van Wielink 2017;López-Arroyo et al. 2018;Orlova-Bienkowskaja et al. 2018;Fiedler and Nedvěd 2019;Crous et al. 2021;Knapp et al. 2022). Those collections consisted of one to hundreds of infected ladybirds each, together accounting for 4,559 individual records. ...
... New records will be quality-screened based on the criteria presented above before being added to the Beetlehangers dataset. With Beetlehangers.org, we hope to (1) promote research on this common ectoparasite of Ha. axyridis (Haelewaters et al. 2018a(Haelewaters et al. , 2022b, (2) increase public knowledge and interest in the utility of CS records of parasitic fungi, and (3) continue to increase records including in poorly sampled regions with the help of targeted social media campaigns, such as the one we did in Europe in collaboration with local researchers. ...
Citizen science is an increasingly powerful tool to gather large quantities of biological data and to engage the public. The number of citizen science initiatives has rapidly increased in the last 15 years. Invasive alien species such as the harlequin ladybird, Harmonia axyridis (Coleoptera, Coccinellidae), provide a particularly good opportunity for broad-scale use of such initiatives. Harmonia axyridis is parasitized by a fungus, Hesperomyces harmoniae (Ascomycota, Laboulbeniales), that can be found throughout the range of its host. Here we present Beetlehangers.org, a website and data repository where we combine observations of He. harmoniae from literature, online databases, and citizen science initiatives, including new records of both Ha. axyridis and He. harmoniae. Whereas Ha. axyridis is now present in 86 countries across six continents (including seven new country records), the distribution of its parasite He. harmoniae comprises 33 countries in five continents (including two new country records since its description in 2022). We explore spatiotemporal trends of He. harmoniae in light of these records. Finally, we discuss challenges and new opportunities for citizen science in relation to species interactions such as these and provide future perspectives for the website as a home for future Laboulbeniales research and outreach.
... This Laboulbeniales parasite associated with Ha. axyridis has for a long time been referred to Hesperomyces virescens, until it was recognized as one of several species in the He. virescens species complex (Haelewaters et al. 2018). It was only recently formally described as He. ...
... These two species, however, should be re-examined as to whether they actually belong to Hesperomyces. Of the ladybird-associated species of Hesperomyces, ten have been formally described (Thaxter 1931;Majewski and Sugiyama 1985;Goldmann et al. 2013;Das et al. 2018;Haelewaters and De Kesel 2020;Crous et al. 2021;Haelewaters et al. 2022b) while several others have been recognized as distinct species, but not yet described (Haelewaters et al. 2018;Van Caenegem et al. 2023b). Studies supported by molecular phylogenetic data have shown that species of Hesperomyces are highly host specific: individual species are usually associated with a single host genus or species (Haelewaters et al. 2018). ...
... Of the ladybird-associated species of Hesperomyces, ten have been formally described (Thaxter 1931;Majewski and Sugiyama 1985;Goldmann et al. 2013;Das et al. 2018;Haelewaters and De Kesel 2020;Crous et al. 2021;Haelewaters et al. 2022b) while several others have been recognized as distinct species, but not yet described (Haelewaters et al. 2018;Van Caenegem et al. 2023b). Studies supported by molecular phylogenetic data have shown that species of Hesperomyces are highly host specific: individual species are usually associated with a single host genus or species (Haelewaters et al. 2018). ...
Between 2016 and 2022, the ladybird (Coleoptera: Coccinellidae) fauna was surveyed on seven main islands of the Canary Islands archipelago. Adult specimens were collected and screened for thalli of Laboulbeniales (Ascomycota: Laboulbeniomycetes). Altogether, 9,807 ladybird specimens belonging to 39 species were screened, of which 103, representing 10 species, were infected by Laboulbeniales of the genus Hesperomyces. Morphological and molecular examination allowed to distinguish at least seven host-specific Hesperomyces species belonging to three groups: He. virescens species complex (associated with Adalia decempunctata, Harmonia axyridis, Hyperaspis vinciguerrae, Novius cruentatus, Parexochomus nigripennis, and P. quadriplagiatus), He. coccinelloides species complex (Scymnus nubilus, Sc. subvillosus durantae, and Stethorus tenerifensis), and a species (on Nephus flavopictus) that cannot be assigned to either of the above species complexes or the currently described species in the genus. The most widespread and abundant species was Hesperomyces ex St. tenerifensis; it was recorded on all seven islands with prevalence rates ranging between 0.04 and 0.19.
... Consequentially, some systematists have used the term "integrative taxonomy" to name and differentiate this more inclusive approach when addressing taxonomic questions (e.g., Padial & Riva, 2009;Moraes et al., 2021). Integrative taxonomy has been applied to a vast sort of different groups of organisms, including plants, fungi, and invertebrate and vertebrate animals (e.g., Bolotov et al., 2020;Haelewaters et al., 2018;He et al., 2022). However, regarding South American snakes, most studies have extensively focused on the family Dipsadidae (e.g., Abegg et al., 2022;Arredondo et al., 2020;Costa et al., 2022;Trevine et al., 2022;Zaher et al., 2018). ...
The widespread Tantilla melanocephala is a South American snake, which has a convoluted taxonomic history and a
poorly addressed systematics. The taxonomic identity and the phylogenetic affinities of their populations are unclear,
suggesting the existence of a complex of undescribed species. Here, we preliminarily address the morphological
diversity and genetic structure of the T. melanocephala complex through comparative morphological approaches and
phylogenetic analyses using maximum likelihood. Our results confirm the presence of hidden diversity within the T.
melanocephala complex and strongly indicate that the populations of T. melanocephala from southern South America
represent a distinct lineage, which is morphologically diagnosable under a unique combination of character states.
Therefore, we formally describe these populations as a new species and present a comparison among all other South
American species of Tantilla. We also estimate the divergence times of the main clades within the complex through
Bayesian analysis, suggesting a biogeographical scenario of lineage diversification. Finally, our study clarifies aspects
of the intricate taxonomy of the T. melanocephala complex, providing a framework for future studies to avoid the
description of synonyms.
... It is also relatively speciose, with 2454 described species in 164 genera (Data S1). The true diversity is likely much higher, particularly given the presence of cryptic speciation in the class (Haelewaters et al., 2018. Finally, while relatively poorly studied, sufficient research into Laboulbeniomycetes has been conducted to enable analyses of a range of biological shortfalls. ...
... Recent work has revealed a significant amount of cryptic diversity (Haelewaters et al., 2018, thus rendering the true diversity likely even higher. This is a common trend in mycology (Bickford et al., 2007;Hawksworth & Lücking, 2017) and other organismal fields (e.g. ...
... In addition, misapplication of names may have also played a factor in this observation; many species with disjunct distributions have not been sequenced and, based on the results of recent molecular phylogenetic studies (e.g. Crous et al., 2021;Haelewaters et al., 2018Haelewaters et al., , 2022, it may be that some of the disjunct records may represent different, cryptic or near-cryptic species. ...
Most empirical research on biological shortfalls has focused on vertebrate taxa. This is important given many species in poorly studied groups such as invertebrates, plants, and fungi are predicted to possess high conservation risk. Here, we focus on Laboulbeniomycetes: a class of microfungi that are understudied. We examined four shortfalls: Linnean (knowledge gaps in species diversity), Wallacean (knowledge gaps in distributions), Latimerian (knowledge gaps in species persistence), and the newly introduced Scottian (knowledge gaps in species conservation assessments) shortfalls. The Linnean shortfall in Laboulbeniomycetes is hard to predict due to inconsistent species description rates. Analysis of distribution patterns indicates Laboulbeniomycetes are likely to experience an extremely high Wallacean shortfall, with many species having highly disjunct known distributions. Latimerian shortfall analysis shows over half (51%) of Laboulbeniomycetes have not been recorded in >50 years, while the group has a collective Scottian shortfall of 100%, given none of the 2454 described species have received an IUCN threat assessment. We suggest continued study of natural history collections, expanded citizen science programmes, and machine-learning identification approaches as important tools for reducing knowledge shortfalls in both Laboulbeniomycetes and poorly studied taxa more generally.
... Applying various species delimitation methods and consistently estimating species diversity can increase credibility, and inconsistent results imply varying delineation powers of multiple methods. In the case of inconsistent findings, it is recommended that species should be designated conservatively so that they can genuinely represent evolutionary metapopulation lineages [103]. ...
The genus Lasiodiplodia, a member of the family Botryosphaeriaceae, is an important fungal disease genus in agriculture. However, the Lasiodiplodia species survey and genetic diversity in Taiwan remain unclear. This study aimed to investigate the Lasiodiplodia species associated with various fruit species to explore the cryptic Lasiodiplodia species diversity, validate species delimitation, and unveil cryptic genetic diversity. Overall, six Lasiodiplodia species were identified, with several new records of infection identified. Additionally, phylogenetic analyses indicated that the relations of all isolates of L. theobromae might be paraphyletic. They were grouped with L. brasiliense based on Automatic Barcode Gap Discovery (ABGD), Automatic Partitioning (ASAP) and structure-based clustering analyses. These analyses did not provide conclusive evidence for L. brasiliensis as a stable species. It may be necessary to gather more information to clarify the species delineation. The multiple new records of Lasiodiplodia species with high genetic diversity and differentiation revealed that the diversity of Lasiodiplodia in Taiwan was underestimated in the past. We found that L. theobromae has the highest number of haplotypes but the lowest number of haplotype and nucleotide diversities, indicating a recent population expansion. This was supported by the significant negative Tajima’s D and Fu and Li’s D* tests. The high genetic diversity, low gene flow, and host-associated differentiation of Lasiodiplodia species indicate that they might harbour powerful evolutionary potential in Taiwan. This study provided critical insights into genetic variation, host-associated differentiation, and demography of Lasiodiplodia species, which would be helpful for disease management of related pathogens.
... Given that it is difficult to delimitate these species based solely on morphological studies, integrative taxonomy is indispensable in recognizing species of the section. This method, which delimits and describes taxa by integrating information from different types of data and methodologies (e.g., phylogeny, comparative morphology, habitat and preference of hosts, and behavior), is proven to be useful for species recognition in plants, animals, and fungi [2,[60][61][62][63][64]. In this study, species with similar morphological characteristics are successfully recognized using this method. ...
Species of Amanita sect. Vaginatae (Fr.) Quél. are challenging to delimitate due to the morphological similarity or morphostasis among different taxa. In this study, a multi-locus (nuc rDNA region encompassing the internal transcribed spacers 1 and 2 with the 5.8S rDNA, the D1–D3 domains of nuc 28S rDNA, partial sequences of translation elongation factor 1-a, and the second largest subunit of RNA polymerase II) phylogeny was employed to investigate the species diversity of the section in eastern China. Sixteen species were recognized, including four new species; namely, A. circulata, A. multicingulata, A. orientalis, and A. sinofulva. They were documented with illustrated descriptions, ecological evidence, and comparisons with similar species. A key to the species of the section from eastern China is provided.
... Even those taxa that were once thought to have a wide host range, such as Hesperomyces virescens Thaxt. (Haelewaters et al. 2018a(Haelewaters et al. , 2022b and Laboulbenia flagellata Peyr. (De Weggheleire 2019; , have been shown to encompass multiple (pseudo-) cryptic species. ...
... Thaxter (1917Thaxter ( , 1931 described G. nycteribiidarum on M. aranea (as Pterellipsis aranea Coquillett 1899), which implies that the three-isolate clade may represent G. nycteribiidarum sensu stricto. More specimens and sequences are needed, but based on these preliminary results, species in the G. nycteribiidarum species complex may be segregated by host, possibly at the level of genus-as is the case within Hesperomyces virescens sensu lato (Haelewaters et al. 2018a(Haelewaters et al. , 2022b. ...
This paper describes and illustrates five new species of Gloeandromyces (Ascomycota, Laboulbeniales) associated with tropical American bat flies (Diptera, Streblidae). These are Gloeandromyces cusucoensis sp. nov. from Trichobius uniformis in Costa Rica and Honduras, G. diversiformis sp. nov. from Strebla wiedemanni in Costa Rica, G. plesiosaurus sp. nov. from Trichobius yunkeri in Panama, G. pseudodickii sp. nov. from Trichobius longipes in Ecuador and Panama, and G. verbekeniae sp. nov. from Strebla galindoi in Ecuador and Panama. The description of these five species doubles the number of known species in the genus. Morphological characteristics, host association, and a three-locus (18S nuc rDNA, 28S nuc rDNA, TEF1) phylogenetic reconstruction support placement of these taxa in the genus Gloeandromyces. Three of the new species are polymorphic; they have multiple morphotypes that grow in specific positions on the host integument: G. diversiformis f. diversiformis, f. musiformis, and f. vanillicarpiformis; G. plesiosaurus f. asymmetricus and f. plesiosaurus; and G. verbekeniae f. verbekeniae and f. inflexus. Finally, a dichotomous key to all species and morphotypes is presented.
