Does One Size Fit All? Variations in the DNA Barcode Gaps of Macrofungal Genera.

The nuclear ribosomal internal transcribed spacer (nrITS) region has been widely used in fungal diversity studies. Environmental metabarcoding has increased the importance of the fungal DNA barcode in documenting fungal diversity and distribution. The DNA barcode gap is seen as the difference between intra- and inter-specific pairwise distances in a DNA barcode. The current understanding of the barcode gap in macrofungi is limited, inhibiting the development of best practices in applying the nrITS region toward research on fungal diversity. This study examined the barcode gap using 5146 sequences representing 717 species of macrofungi from eleven genera, eight orders and two phyla in datasets assembled by taxonomic experts. Intra- and inter-specific pairwise distances were measured from sequence and phylogenetic data. The results demonstrate that barcode gaps are influenced by differences in intra- and inter-specific variance in pairwise distances. In terms of DNA barcode behavior, variance is greater in the ITS1 than ITS2, and variance is greater in both relative to the combined nrITS region. Due to the difference in variance, the barcode gaps in the ITS2 region are greater than in the ITS1. Additionally, the taxonomic approach of "splitting" taxa into numerous taxonomic units produces greater barcode gaps when compared to "lumping". The results show variability in the barcode gaps between fungal taxa, demonstrating a need to understand the accuracy of DNA barcoding in quantifying species richness. For taxonomic studies, variability in nrITS sequence data supports the application of multiple molecular markers to corroborate the taxonomic and systematic delineation of species.

Host Shift Speciation of the Ectomycorrhizal Genus Suillus (Suillineae, Boletales) and Biogeographic Comparison With Its Host Pinaceae.

Suillus is a genus of ectomycorrhizal fungi associated almost exclusively with Pinaceae. Lack of sample collections in East Asia and unresolved basal phylogenetic relationships of the genus are the major obstacles for better understanding the Suillus evolution. A resolved phylogeny of Suillus representing global diversity was achieved by sequencing multiple nuclear ribosomal and protein coding genes and extensive samples collected in East Asia. Fungal fossils are extremely rare, and the Eocene ectomycorrhizal symbiosis (ECM) fossil of Pinus root has been widely used for calibration. This study explored an alternative calibration scenario of the ECM fossil for controversy. Ancestral host associations of Suillus were estimated by maximum likelihood and Bayesian Markov chain Monte Carlo (MCMC) analyses, inferred from current host information from root tips and field observation. Host shift speciation explains the diversification of Suillus major clades. The three basal subgenera of Suillus were inferred to be associated with Larix, and diverged in early Eocene or Upper Cretaceous. In the early Oligocene or Paleocene, subgenus Suillus diverged and switched host to Pinus subgenus Strobus, and then switched to subgenus Pinus four times. Suillus subgenus Douglasii switched host from Larix to Pseudotsuga in Oligocene or Eocene. Increased species diversity occurred in subgenus Suillus after it switched host to Pinus but no associated speciation rate shifts were detected. Ancestral biogeographic distributions of Suillus and Pinaceae were estimated under the Dispersal Extinction Cladogenesis (DEC) model. Ancestral distribution patterns of Suillus and Pinaceae are related but generally discordant. Dispersals between Eurasia and North America explain the prevalence of disjunct Suillus taxa.

A strategy for the next decade to address data deficiency in neglected biodiversity.

Measuring progress toward international biodiversity targets requires robust information on the conservation status of species, which the International Union for Conservation of Nature (IUCN) Red List of Threatened Species provides. However, data and capacity are lacking for most hyperdiverse groups, such as invertebrates, plants, and fungi, particularly in megadiverse or high-endemism regions. Conservation policies and biodiversity strategies aimed at halting biodiversity loss by 2020 need to be adapted to tackle these information shortfalls after 2020. We devised an 8-point strategy to close existing data gaps by reviving explorative field research on the distribution, abundance, and ecology of species; linking taxonomic research more closely with conservation; improving global biodiversity databases by making the submission of spatially explicit data mandatory for scientific publications; developing a global spatial database on threats to biodiversity to facilitate IUCN Red List assessments; automating preassessments by integrating distribution data and spatial threat data; building capacity in taxonomy, ecology, and biodiversity monitoring in countries with high species richness or endemism; creating species monitoring programs for lesser-known taxa; and developing sufficient funding mechanisms to reduce reliance on voluntary efforts. Implementing these strategies in the post-2020 biodiversity framework will help to overcome the lack of capacity and data regarding the conservation status of biodiversity. This will require a collaborative effort among scientists, policy makers, and conservation practitioners.

Una Estrategia para la Siguiente Década para Enfrentar la Deficiencia de Datos de la Biodiversidad Ignorada Resumen La medida del avance hacia los objetivos internacionales para la biodiversidad requiere información sólida sobre el estado de conservación de las especies, la cual proporciona la Lista Roja de Especies Amenazadas de la Unión Internacional para la Conservación de la Naturaleza (UICN). Sin embargo, los grupos más hiperdiversos, como los invertebrados, las plantas y los hongos, carecen de datos y capacidad, particularmente en regiones megadiversas o de endemismo alto. Las políticas de conservación y las estrategias de biodiversidad dirigidas hacia el cese de la pérdida de biodiversidad para el 2020 necesitan ser adaptadas para solucionar estas insuficiencias de información para después del año 2020. Diseñamos una estrategia de ocho puntos para cerrar las brechas existentes en los datos mediante la reactivación de la investigación exploratoria en el campo sobre la distribución, abundancia y ecología de las especies; la vinculación más cercana entre la investigación taxonómica y la conservación; la mejora a las bases de datos mundiales sobre biodiversidad mediante la presentación obligatoria de datos espacialmente explícitos para las publicaciones científicas; el desarrollo de una base mundial de datos espaciales sobre las amenazas para la biodiversidad para facilitar las valoraciones de la Lista Roja de la UICN; la automatización de las preevaluaciones mediante la integración de datos de distribución y datos de amenazas espaciales; el desarrollo de la capacidad en la taxonomía, la ecología y el monitoreo de la biodiversidad en países con una gran riqueza de especies o endemismos; la creación de programas de monitoreo de especies para los taxones menos conocidos; el desarrollo de suficientes mecanismos de financiamiento para reducir la dependencia de los esfuerzos voluntarios. La implementación de estas estrategias en el marco de trabajo para la biodiversidad posterior al 2020 ayudará a superar la falta de capacidad y datos con respecto al estado de conservación de la biodiversidad. Lo anterior requerirá de un esfuerzo colaborativo entre científicos, formuladores de políticas y practicantes de la conservación.

Novel Laccaria Species From Juglandaceae Forest in Panama With Notes on Their Ecology.

Since 2013 there have been 22 new species of Laccaria described worldwide. Only three of these represent species from the neotropics. In Panama, Laccaria is abundant in monodominant Oreomunnea mexicana (Juglandaceae) forests based on sporocarps and environmental sequencing of roots. This study uses a combination of morphological and phylogenetic evidence to document up to seven species of Laccaria from these forests, one previously described, three described as new, and three requiring more data before being formally described. Molecular data used for phylogenetic analysis include the nuclear ribosomal ITS and 28S regions, along with TEF1 and RPB2. Laccaria stellata, has previously been reported from O. mexicana cloud forests of Panama. Laccaria dallingii sp. nov., L. nitrophila sp. nov., and L. fortunensis sp. nov. are described as new based on morphology and phylogenetic analysis of multiple collections. A taxon referred to as "PAN sp3" is resolved sister to L. stellata. Phylogenetic analysis also resolved two separate clades of Panamanian Laccaria as sister to L. roseoalbescens, a species previously described from Mexico. These three taxa are not described in this paper as there is too little material from which to make effective morphological descriptions even though their placement in phylogenetic analysis identify them as being unique. Ecologically, all described species except for L. fortunensis were amplified from O. mexicana ectomycorrhizal root tips. L. nitrophila was one of the most recovered species from the roots of O. mexicana in a previous study, and it has been shown to respond positively to long term nitrogen addition. Our results expand the knowledge of Laccaria diversity for Central America and highlight that at least some species of Laccaria are nitrophilic in neotropical Juglandaceae forests as well as in temperate forests.

Addressing the diversity of Xylodon raduloides complex through integrative taxonomy.

In this study, the taxonomic diversity of the Xylodon raduloides species complex (Hymenochaetales, Basidiomycota) is examined. Specimens were studied using an integrative taxonomic approach that includes molecular phylogenetic and morphological analyses, and environmental niche comparisons. Four different species were found inside the Xylodon raduloides complex, with a biogeographic distribution pattern bound by geographic regions: Europe, North America, Patagonia, and Australia-New Zealand. Molecular, morphological, and environmental evidences delimit two lineages within this complex: a Northern Hemisphere clade with longer basidiospores and wider ranges in temperature and precipitation tolerance, and a Southern Hemisphere clade with smaller and more spherical basidiospores, and an isothermal and more humid climate preference. The integrative taxonomic approach used in this study demonstrates congruence between data sets and shows how morphological and environmental characteristics contribute to the differentiation of fungal species complexes. By combining various sources of taxonomic information, three new species are described: Xylodon laurentianus, X. novozelandicus, and X. patagonicus.

A systematic revision of the ectomycorrhizal genus Laccaria from Korea.

Species of Laccaria (Hydnangiaceae, Basidiomycota) are important in forest ecosystems as ectomycorrhizal fungi. Nine of the 75 described Laccaria species worldwide been reported from Korea. Most of these have European and North American names, and their identities are based solely on morphological features. To evaluate the taxonomy of Korean Laccaria, we used 443 specimens collected between 1981 and 2016 in a phylogenetic analysis based on sequence data from nuc rDNA internal transcribed spacer ITS1-5.8S-ITS2 rDNA (ITS) region, nuc 28S rDNA (28S), RNA polymerase II subunit 2 (rpb2), and translation elongation factor 1-α (tef1). Ten Laccaria species were identified. Three of these were previously reported from Korea: L. bicolor, L. tortilis, and L. vinaceoavellanea. Laccaria alba, L. japonica, and L. murina are confirmed as new reports from Korea. Lastly, four new Laccaria species are described: L. araneosa, L. parva, L. torosa, and L. versiforma. This study supports the general contention that Asian species of ectomycorrhizal fungi may not be conspecific with morphologically similar species from Europe and North America. Furthermore, identification based on morphology alone is often unreliable in Laccaria due to considerable overlap of characters among species. Thus, use of molecular methods is necessary for effective identification. Illustrations of the four newly described species and a taxonomic key to species of Laccaria in Korea are provided.

Reshaping Darwin's Tree: Impact of the Symbiome.

Much of the undescribed biodiversity on Earth is microbial, often in mutualistic or pathogenic associations. Physically associated and coevolving life forms comprise a symbiome. We propose that systematics research can accelerate progress in science by introducing a new framework for phylogenetic analysis of symbiomes, here termed SYMPHY (symbiome phylogenetics).

Two new species in the Suillus spraguei complex from China.

Suillus spraguei, synonym S. pictus, has been reported from eastern North America and eastern Asia associated with Pinus subgenus Strobus. Published phylogenetic analyses of rRNA internal transcribed spacer (ITS) sequence and population genetic studies indicated that S. spraguei as currently circumscribed might contain several geographically distinct species. This study examined this possibility through a multigene analysis of S. spraguei specimens from eastern North America and eastern Asia. These specimens were associated with Pinus strobus, P. koraiensis, P. armandii, and P. kwangtungensis. The multigene analysis included three genomic regions: the genes for translation elongation factor 1α (TEF1) and RNA polymerase II largest subunit (RPB1), and the nuc rRNA segments ITS1-5.8S-ITS2 (ITS) and 28S D1-D2 domains (28S). This study confirms that the S. spraguei complex consists of at least three cryptic species: S. spraguei sensu stricto associated with P. strobus in eastern North America; S. phylopictus associated with multiple species in Pinus subgenus Strobus (5-needle pines) throughout China and Japan; and S. kwangtungensis, currently found only in P. kwangtungensis forests in southeastern China. A third new species from Japan and Korea was suggested based on ITS phylogeny. Morphologically, S. spraguei and S. phylopictus resemble each other, whereas S. kwangtungensis is covered with more floccose scales. The new species add to the knowledge of macrofungal diversity in eastern Asia and highlight the necessity of comparing broadly distributed species complexes using morphological, molecular, and ecological data.

Evolution of ectomycorrhizas as a driver of diversification and biogeographic patterns in the model mycorrhizal mushroom genus Laccaria.

A systematic and evolutionary ecology study of the model ectomycorrhizal (ECM) genus Laccaria was performed using herbarium material and field collections from over 30 countries covering its known geographic range. A four-gene (nrITS, 28S, RPB2, EF1α) nucleotide sequence dataset consisting of 232 Laccaria specimens was analyzed phylogenetically. The resulting Global Laccaria dataset was used for molecular dating and estimating diversification rates in the genus. Stable isotope analysis of carbon and nitrogen was used to evaluate the origin of Laccaria's ECM ecology. In all, 116 Laccaria molecular species were identified, resulting in a near 50% increase in its known diversity, including the new species described herein: Laccaria ambigua. Molecular dating indicates that the most recent common ancestor to Laccaria existed in the early Paleocene (56-66 million yr ago), probably in Australasia. At this time, Laccaria split into two lineages: one represented by the new species L. ambigua, and the other reflecting a large shift in diversification that resulted in the remainder of Laccaria. L. ambigua shows a different isotopic profile than all other Laccaria species. Isotopes and diversification results suggest that the evolution of the ECM ecology was a key innovation in the evolution of Laccaria. Diversification shifts associated with Laccaria's dispersal to the northern hemisphere are attributed to adaptations to new ecological niches.

Cantharellus chicagoensis sp. nov. is supported by molecular and morphological analysis as a new yellow chanterelle in midwestern United States.

Recent molecular systematic studies of Cantharellus cibarius sensu lato have revealed previously unknown species in different regions of North America. This study investigates yellow chanterelles in the Midwest using phylogenetic analysis of three DNA regions: nuc rDNA internal transcribed spacer 2 (ITS2) and 28S sequences and translation elongation factor 1α gene (EF1α). This analysis reveals a locally common taxon Cantharellus chicagoensis sp. nov. as distinct from sympatric species present in northeastern Illinois, northwestern Indiana and Wisconsin. This chanterelle features a pileus that often has a greenish yellow margin when immature, a squamulose disk when mature, a yellow spore print and the absence of a fragrant odor. Multiple Cantharellus specimens group with C. flavus and C. phasmatis, expanding their known range, and others with C. roseocanus Our observations highlight the diversity of Cantharellus in midwestern USA and further document the need for additional systematic focus on the region's fungi.

Examining the efficacy of a genotyping-by-sequencing technique for population genetic analysis of the mushroom Laccaria bicolor and evaluating whether a reference genome is necessary to assess homology.

Given the diversity and ecological importance of Fungi, there is a lack of population genetic research on these organisms. The reason for this can be explained in part by their cryptic nature and difficulty in identifying genets. In addition the difficulty (relative to plants and animals) in developing molecular markers for fungal population genetics contributes to the lack of research in this area. This study examines the ability of restriction-site associated DNA (RAD) sequencing to generate SNPs in Laccaria bicolor. Eighteen samples of morphologically identified L. bicolor from the United States and Europe were selected for this project. The RAD sequencing method produced anywhere from 290 000 to more than 3 000 000 reads. Mapping these reads to the genome of L. bicolor resulted in 84 000-940 000 unique reads from individual samples. Results indicate that incorporation of non-L. bicolor taxa into the analysis resulted in a precipitous drop in shared loci among samples, suggests the potential of these methods to identify cryptic species. F-statistics were easily calculated, although an observable "noise" was detected when using the "All Loci" treatment versus filtering loci to those present in at least 50% of the individuals. The data were analyzed with tests of Hardy-Weinburg equilibrium, population genetic statistics (FIS and FST), and population structure analysis using the program Structure. The results provide encouraging feedback regarding the potential utility of these methods and their data for population genetic analysis. We were unable to draw conclusions of life history of L. bicolor populations from this dataset, given the small sample size. The results of this study indicate the potential of these methods to address population genetics and general life history questions in the Agaricales. Further research is necessary to explore the specific application of these methods in the Agaricales or other fungal groups.

Testing the "one-log-one-genet" hypothesis: methodological challenges of population sampling for the Hawaiian wood-decay fungus Rhodocollybia laulaha.

We test our "one-log-one-genet" sampling method for the Hawaiian mushroom Rhodocollybia laulaha that posits all R. laulaha mushrooms collected from a single log represent a single genet. We also examine the potential expansion of single genets beyond the confines of one log and the temporal persistence of genets in nature. Finally, we estimate error rates in AFLP scoring. To our knowledge, this is one of few examinations of naturally occurring fungal genets in the tropics and a novel report of AFLP error rates in fungi. Forty-six mushrooms from seven logs were genotyped with the IGS1 locus, two microsatellite loci and 184 AFLP loci from three primer pair combinations. One hundred fifty-three mushroom collections representing the geographic range of R. laulaha were genotyped with the IGS1 and microsatellite loci. The probabilities of two genets sharing identical multilocus genotypes by chance (without actually being the same genet) were calculated for each genotype recovered. The data suggest that R. laulaha mushrooms from one log typically represent one genet, that genets might expand beyond the confines of a single log and that a single genet may persist in a collecting site for as much as 13 y. We offer initial evidence to support the "one-log-one genet" sampling method and the idea that R. laulaha vegetative expansion and persistence in nature might be common. In addition, we caution against exclusive use of AFLP loci for identifying fungal genets due to relatively high error rates in scoring.

Cantharellaceae of Guyana II: new species of Craterellus, new South American distribution records for Cantharellus guyanensis and Craterellus excelsus, and a key to the Neotropical taxa.

Craterellus olivaceoluteus sp. nov. and Craterellus cinereofimbriatus sp. nov. are described as new to science. These fungi were collected from Guyana in association with ectomycorrhizal host trees in the genera Dicymbe (Fabaceae subfam. Caesalpinioideae) and Pakaraimaea (Dipterocarpaceae). Cantharellus guyanensis Mont., originally described from French Guiana, is redescribed from recent collections from Guyana, with additional range extensions for the species provided based on material examined from French Guiana, Venezuela, and north central, northeastern and southern Brazil, circumscribing nearly the entire Guiana Shield region and beyond. A new distribution record from French Guiana is provided for Craterellus excelsus T.W. Henkel & Aime. Macromorphological, micromorphological and habitat data are provided for the new species and C. guyanensis as well as DNA sequence data from the nuclear ribosomal regions of the internal transcribed spacer (ITS) and 28S large subunit (LSU); additional sequence data is provided for C. guyanensis and C. excelsus specimens collected outside Guyana. The relationships of these taxa within the Cantharellaceae were evaluated with phylogenetic analyses of ITS and LSU sequence data. This work brings the total number of Cantharellaceae species known from Guyana to eight. A key to the Cantharellus and Craterellus species known from the lowland Neotropics and extralimital montane Central and South America is provided.

Cantharellaceae of Guyana I: new species, combinations and distribution records of Craterellus and a synopsis of known taxa.

Members of the Cantharellaceae (Cantharellales, Basidiomycota) are common ectomycorrhizal associates of the leguminous genus Dicymbe in the Pakaraima Mountains of Guyana. Eight distinct species or morphospecies currently are recognized in Craterellus Pers. or Cantharellus Adans. ex Fr. from Guyanese Dicymbe-dominated forests. We evaluated the systematics of these taxa with phylogenetic analyses of DNA sequence data from the nuclear ribosomal regions of the internal transcribed spacer (ITS) and 28S large subunit (LSU). The results of these analyses along with careful assessment of morphology let us described two new species, Craterellus atratoides sp. nov. and Craterellus strigosus sp. nov., redescribe Craterellus atratus (Corner) Yomyart et al. based on new material from Guyana, and propose a new combination in Craterellus for Cantharellus pleurotoides T.W. Henkel, Aime & S.L. Mill. Macroscopic illustrations are provided for two additional cantharelloid morphospecies confirmed in Craterellus, as well as the regionally endemic Cantharellus guyanensis Mont. Macromorphological, micromorphological and habitat data are provided for C. atratoides, C. strigosus and C. atratus, and ITS and LSU sequence data are provided for each of the eight known Guyanese taxa.

Investigating the allelic evolution of an imperfect microsatellite locus in the Hawaiian mushroom Rhodocollybia laulaha.

The evolutionary mechanisms that give rise to microsatellite alleles remain poorly understood in general and are especially understudied for fungal microsatellite loci. The unusual G28 microsatellite locus was developed from the Hawaiian mushroom Rhodocollybia laulaha. Here, we employ a novel approach to test for allele size homoplasy and examine competing mechanistic models of microsatellite evolution in the context of biogeographic expectations for this locus based on Hawaiian geologic history. Seven G28 alleles have been identified from a sampling of 153 individuals. The G28 locus is composed of a trinucleotide imperfect motif, which permits examination of the relationships between alleles and allows for detection of potential size homoplasy within the repetitive element. Alignment of G28 allele sequence data across multiple unrelated individuals suggests that alleles of like size are homologous within Hawaii. A variety of gap coding methods are explored in the inference of allele evolution. Length differences between alleles appear to be the result of polymerase slippage at multiple positions in the repetitive element, suggesting an intricate process of allelic evolution, which is not necessarily stepwise. Complex migration scenarios must be invoked to explain the current geographic distribution of alleles if their evolution was in fact sequential (from longest to shortest or from shortest to longest) as predicted by the "progression rule."

Phylogeography and biogeography of fungi.

The rigorous study of processes shaping geographic distributions of lineages is a relatively new and emerging field in mycology. While it was previously generally believed that most fungi have wide distributions and largely unstructured populations, recent studies have shown that this is not the case. The study of distributions in tandem with molecular approaches to phylogeny has recently made substantial advances to our understanding of the diversity and biogeography of fungi. Comprehensive species inventories have provided a better picture of the actual distribution of these organisms, while robust phylogenies based on molecular characters have provided both data that allow interpretation of current distributions and testable hypotheses regarding the processes responsible for distribution patterns. This commentary provides an introduction to five papers in this issue of Mycological Research that focus on fungal phylogeography. These papers are based on oral contributions given at two symposia at the International Mycological Congress (IMC8) held in Cairns (Australia) in August 2006.

A 'dirty' business: testing the limitations of terminal restriction fragment length polymorphism (TRFLP) analysis of soil fungi.

Terminal restriction fragment length polymorphism (TRFLP) is an increasingly popular method in molecular ecology. However, several key limitations of this method have not been fully examined especially when used to study fungi. We investigated the impact of spore contamination, intracollection ribosomal DNA internal transcribed spacer (ITS) region variation, and conserved restriction enzyme recognition loci on the results produced by TRFLP to characterize soil fungal communities. We find that (i) the potential for nontarget structures such as spores to contribute DNA to target sample extractions is high; (ii) multiple fragments (i.e. 'extra peaks') per PCR primer-restriction enzyme combination can be detected that are caused by restriction enzyme inefficiency and intracollection ribosomal DNA ITS variation; and (iii) restriction enzyme digestion in conserved vs. variable gene regions leads to different characterizations of community diversity. Based on these results, we suggest that studies employing TRFLP need to include information from known, identified fungi from sites within which studies take place and not to rely only on TRFLP profiles as a short cut to fungal community description.