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.

The Epidemiology of Sudden Oak Death Disease Caused by Phytophthora ramorum in a Mixed Bay Laurel-Oak Woodland Provides Important Clues for Disease Management.

Epidemiological models are important for the understanding of disease progression in plants and for the design of control strategies. Phytophthora ramorum, the pathogen responsible for the disease known as Sudden Oak Death, causes lethal infection on several oaks but relies on California bay laurels for transmission. Here, repeated surveys of bay laurels and oaks indicated that bay laurel disease incidence was positively correlated with rainfall, bay laurel density, and an eastern aspect, and negatively correlated with bay laurel basal area. Oak infection only occurred in years when rainfall was higher than the 30-year average, and although infection rates were greater among larger trees, mortality was greater among smaller trees. Additionally, larger oaks closer to infected bay laurels exhibited greater infection rates. Disease incidence differed among sites, and only a fraction of bay laurels were disease superspreaders, while even fewer individuals were refugial trees harboring active infections during dry periods. Based on this study, reducing bay laurel density in denser stands and the number of superspreaders or refugial trees in less dense stands may reduce disease incidence. However, the selective removal of bay laurel trees 0-10 m from oaks is likely to be more effective in preventing infection of specific oaks.

Whole-genome sequence analyses of Glaesserella parasuis isolates reveals extensive genomic variation and diverse antibiotic resistance determinants.

BACKGROUND: Glaesserella parasuis (G. parasuis) is a respiratory pathogen of swine and the etiological agent of Glässer's disease. The structural organization of genetic information, antibiotic resistance genes, potential pathogenicity, and evolutionary relationships among global G. parasuis strains remain unclear. The aim of this study was to better understand patterns of genetic variation, antibiotic resistance factors, and virulence mechanisms of this pathogen. METHODS: The whole-genome sequence of a ST328 isolate from diseased swine in China was determined using Pacbio RS II and Illumina MiSeq platforms and compared with 54 isolates from China sequenced in this study and 39 strains from China and eigtht other countries sequenced by previously. Patterns of genetic variation, antibiotic resistance, and virulence mechanisms were investigated in relation to the phylogeny of the isolates. Electrotransformation experiments were performed to confirm the ability of pYL1-a plasmid observed in ST328-to confer antibiotic resistance. RESULTS: The ST328 genome contained a novel Tn6678 transposon harbouring a unique resistance determinant. It also contained a small broad-host-range plasmid pYL1 carrying aac(6')-Ie-aph(2")-Ia and bla ROB-1; when transferred to Staphylococcus aureus RN4220 by electroporation, this plasmid was highly stable under kanamycin selection. Most (85.13-91.74%) of the genetic variation between G. parasuis isolates was observed in the accessory genomes. Phylogenetic analysis revealed two major subgroups distinguished by country of origin, serotype, and multilocus sequence type (MLST). Novel virulence factors (gigP, malQ, and gmhA) and drug resistance genes (norA, bacA, ksgA, and bcr) in G. parasuis were identified. Resistance determinants (sul2, aph(3")-Ib, norA, bacA, ksgA, and bcr) were widespread across isolates, regardless of serovar, isolation source, or geographical location. CONCLUSIONS: Our comparative genomic analysis of worldwide G. parasuis isolates provides valuable insight into the emergence and transmission of G. parasuis in the swine industry. The result suggests the importance of transposon-related and/or plasmid-related gene variations in the evolution of G. parasuis.

A Microsatellite Analysis Used to Identify Global Pathways of Movement of Phytophthora cinnamomi and the Likely Sources of Wildland Infestations in California and Mexico.

The genetic structure of a sample of isolates of the oomycete plant pathogen Phytophthora cinnamomi from natural and agricultural outbreaks and the long-distance movement of individual genotypes were studied using four microsatellite markers to genotype 159 isolates of Californian, Mexican, and worldwide origins. Allelic profiles identified 75 multilocus genotypes. A STRUCTURE analysis placed them in three groups characterized by different geographic and host ranges, different genic and genotypic diversity, and different reproductive modes. When relationships among genotypes were visualized on a minimum spanning network (MSN), genotypes belonging to the same STRUCTURE group were contiguous, with rare exceptions. A putatively ancestral group 1 had high genic diversity, included all A1 mating type isolates and all Papuan isolates in the sample, was rarely isolated from natural settings in California and Mexico, and was positioned at the center of the MSN. Putatively younger groups 2 and 3 had lower genic diversity, were both neighbors to group 1 but formed two distinct peripherical sectors of the MSN, and were equally present in agricultural commodities and natural settings in Mexico and California. A few genotypes, especially in groups 2 and 3, were isolated multiple times in different locations and settings. The presence of identical genotypes from the same hosts in different continents indicated that long-distance human-mediated movement of P. cinnamomi had occurred. The presence of identical genotypes at high frequencies in neighboring wildlands and agricultural settings suggest that specific commodities may have been the source of recent wild infestations caused by novel invasive genotypes.

Comparative Genomic Analysis of a Multidrug-Resistant Listeria monocytogenes ST477 Isolate.

Listeria monocytogenes is an opportunistic human foodborne pathogen that causes severe infections with high hospitalization and fatality rates. Clonal complex 9 (CC9) contains a large number of sequence types (STs) and is one of the predominant clones distributed worldwide. However, genetic characteristics of ST477 isolates, which also belong to CC9, have never been examined, and little is known about the detail genomic traits of this food-associated clone. In this study, we sequenced and constructed the whole-genome sequence of an ST477 isolate from a frozen food sample in China and compared it with 58 previously sequenced genomes of 25 human-associated, 5 animal, and 27 food isolates consisting of 6 CC9 and 52 other clones. Phylogenetic analysis revealed that the ST477 clustered with three Canadian ST9 isolates. All phylogeny revealed that CC9 isolates involved in this study consistently possessed the invasion-related gene vip. Mobile genetic elements (MGEs), resistance genes, and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system were elucidated among CC9 isolates. Our ST477 isolate contained a Tn554-like transposon, carrying five arsenical-resistance genes (arsA-arsD, arsR), which was exclusively identified in the CC9 background. Compared with the ST477 genome, three Canadian ST9 isolates shared nonsynonymous nucleotide substitutions in the condensin complex gene smc and cell surface protein genes ftsA and essC. Our findings preliminarily indicate that the extraordinary success of CC9 clone in colonization of different geographical regions is likely due to conserved features harboring MGEs, functional virulence and resistance genes. ST477 and three ST9 genomes are closely related and the distinct differences between them consist primarily of changes in genes involved in multiplication and invasion, which may contribute to the prevalence of ST9 isolates in food and food processing environment.

WGS analysis of ST9-MRSA-XII isolates from live pigs in China provides insights into transmission among porcine, human and bovine hosts.

Objectives: To elucidate the phylogenetic relationships among ST9-MRSA-XII isolates from different sources and their genetic features in colonization of different hosts. Methods: We obtained whole-genome sequences of two ST9-MRSA-XII isolates from nasal swabs associated with live pigs in China, and compared them with 135 previously sequenced genomes of 78 human-associated, 39 bovine and 18 porcine Staphylococcus aureus consisting of 11 MRSA of SCCmecXII, 62 MRSA of other SCCmec types and 62 MSSA. The distribution of diverse mobile genetic elements (MGEs), resistance genes and virulence determinants was investigated in relation to isolate phylogeny. Comparisons of SNPs and small insertion/deletions (indels) were conducted to examine genome-level variation between porcine and bovine ST9-MRSA-XII. Results: Phylogenetic analysis revealed that both of our porcine ST9-MRSA-XII isolates clustered with porcine, bovine and human-associated ST9-MRSA-XII. All of these isolates possessed a novel type V pathogenicity island, νSaα, carrying the von Willebrand-binding protein gene vwb, the immune evasion complex gene scn, the aminoglycoside resistance gene aadE, staphylococcal superantigen-like genes (ssl1-ssl11) and lpl tandem genes. Compared with bovine ST9-MRSA-XII BA01611, our porcine isolates contain non-synonymous nucleotide substitutions in genes encoding adhesins and an indel located in a phosphonate ABC transporter pseudogene. Conclusions: The data suggest transmission of ST9-MRSA-XII among swine, cattle and humans. The extraordinary success of the ST9-MRSA-XII group in colonization of various hosts is likely due to acquisition of many MGEs harbouring functional antimicrobial resistance and virulence genes. Transmission of ST9-MRSA-XII between porcine and bovine hosts was accompanied by changes in binding profile and function in genes involved in metabolism.

Systematics of the ectomycorrhizal genus Lactarius in the Rocky Mountain alpine zone.

Lactarius (Russulales) is an important component of ectomycorrhizal fungal communities in cold-dominated contiguous arctic and disjunct alpine habitats where it associates primarily with Betula, Dryas and Salix However, little is known of this genus in the central and southern Rocky Mountain alpine zone (3000-3900 m) of North America. Molecular phylogenetic analyses of nuc rDNA ITS1-5.8S-ITS2 (ITS barcode) and the second largest subunit of the RNA polymerase II gene (RPB2) partial sequences in conjunction with detailed morphological examination confirm at least six species occurring above treeline. Most have intercontinental distributions in North America and Eurasia according to molecular comparison with type material and collections from Europe, Fennoscandia, Svalbard and Alaska. Rocky Mountain collections of L. lanceolatus (subgenus Russularia), along with the type from Alaska are paraphyletic with respect to L. aurantiacus and North American taxa L. luculentus and L. luculentus v. laetus Rocky Mountain collections of L. nanus, L. glyciosmus, L. repraesentaneus and L. salicis-reticulatae (subgenus Piperites) all form clades with European material from type localities and other arctic-alpine habitats. The arctic-alpine L. pseudouvidus/L. brunneoviolaceus group appears to be a complex containing additional taxa. North American material originally described as part of this group is well-separated phylogenetically and is described here as L. pallidomarginatus sp. nov. Lactarius lanceolatus, L. nanus and L. salicis-reticulatae appear largely restricted to arctic-alpine habitats with Salix Lactarius glyciosmus and L. repraesentaneus occur in arctic-alpine, subalpine and boreal habitats with Betula and also Picea and possibly Salix for the latter. Species distributions are hypothesized to be shaped by host ranges, glaciation and long distance dispersal.

Filling gaps in biodiversity knowledge for macrofungi: contributions and assessment of an herbarium collection DNA barcode sequencing project.

Despite recent advances spearheaded by molecular approaches and novel technologies, species description and DNA sequence information are significantly lagging for fungi compared to many other groups of organisms. Large scale sequencing of vouchered herbarium material can aid in closing this gap. Here, we describe an effort to obtain broad ITS sequence coverage of the approximately 6000 macrofungal-species-rich herbarium of the Museum of Natural History in Venice, Italy. Our goals were to investigate issues related to large sequencing projects, develop heuristic methods for assessing the overall performance of such a project, and evaluate the prospects of such efforts to reduce the current gap in fungal biodiversity knowledge. The effort generated 1107 sequences submitted to GenBank, including 416 previously unrepresented taxa and 398 sequences exhibiting a best BLAST match to an unidentified environmental sequence. Specimen age and taxon affected sequencing success, and subsequent work on failed specimens showed that an ITS1 mini-barcode greatly increased sequencing success without greatly reducing the discriminating power of the barcode. Similarity comparisons and nonmetric multidimensional scaling ordinations based on pairwise distance matrices proved to be useful heuristic tools for validating the overall accuracy of specimen identifications, flagging potential misidentifications, and identifying taxa in need of additional species-level revision. Comparison of within- and among-species nucleotide variation showed a strong increase in species discriminating power at 1-2% dissimilarity, and identified potential barcoding issues (same sequence for different species and vice-versa). All sequences are linked to a vouchered specimen, and results from this study have already prompted revisions of species-sequence assignments in several taxa.

Australasian sequestrate fungi 18: Solioccasus polychromus gen. & sp. nov., a richly colored, tropical to subtropical, hypogeous fungus.

Solioccasus polychromus gen. & sp. nov., the most brightly colored hypogeous fungus known, is described from Papua New Guinea and tropical northern Australia south into subtropical forests along the Queensland coast and coastal mountains to near Brisbane. Phylogenetic analysis of molecular data places it as a sister genus to Bothia in the Boletineae, a clade of predominantly ectomycorrhizal boletes. Ectomycorrhizal trees, such as members of the Myrtaceae (Eucalyptus, Corymbia, Lophostemon, Melaleuca spp.) and Allocasuarina littoralis, were present usually in mixture or in some cases dominant, so we infer some or all of them to be among the ectomycorrhizal hosts of S. polychromus.

Back to basics: an evaluation of NaOH and alternative rapid DNA extraction protocols for DNA barcoding, genotyping, and disease diagnostics from fungal and oomycete samples.

The ubiquity, high diversity and often-cryptic manifestations of fungi and oomycetes frequently necessitate molecular tools for detecting and identifying them in the environment. In applications including DNA barcoding, pathogen detection from plant samples, and genotyping for population genetics and epidemiology, rapid and dependable DNA extraction methods scalable from one to hundreds of samples are desirable. We evaluated several rapid extraction methods (NaOH, Rapid one-step extraction (ROSE), Chelex 100, proteinase K) for their ability to obtain DNA of quantity and quality suitable for the following applications: PCR amplification of the multicopy barcoding locus ITS1/5.8S/ITS2 from various fungal cultures and sporocarps; single-copy microsatellite amplification from cultures of the phytopathogenic oomycete Phytophthora ramorum; probe-based P. ramorum detection from leaves. Several methods were effective for most of the applications, with NaOH extraction favored in terms of success rate, cost, speed and simplicity. Frozen dilutions of ROSE and NaOH extracts maintained PCR viability for over 32 months. DNA from rapid extractions performed poorly compared to CTAB/phenol-chloroform extracts for TaqMan diagnostics from tanoak leaves, suggesting that incomplete removal of PCR inhibitors is an issue for sensitive diagnostic procedures, especially from plants with recalcitrant leaf chemistry. NaOH extracts exhibited lower yield and size than CTAB/phenol-chloroform extracts; however, NaOH extraction facilitated obtaining clean sequence data from sporocarps contaminated by other fungi, perhaps due to dilution resulting from low DNA yield. We conclude that conventional extractions are often unnecessary for routine DNA sequencing or genotyping of fungi and oomycetes, and recommend simpler strategies where source materials and intended applications warrant such use.

Sutorius: a new genus for Boletus eximius.

Sutorius is described as a new genus of Boletaceae to accommodate Boletus robustus originally named illegitimately by C.C. Frost from eastern North America. The legitimate name, Boletus eximius, provided by C.H. Peck, has been used since for a dark purple to chocolate brown bolete with finely scaly stipe and reddish brown spore deposit. This iconic taxon has been documented on five continents. Despite the straightforward species identification from morphology, the interpretation of stipe macro-morphology and spore color has led to equivocal generic placement. Phylogenetic analyses of genes encoding large subunit rRNA and translation elongation factor 1α confirm Sutorius as a unique generic lineage in the Boletaceae. Two species are recognized based on multiple accessions: S. eximius, represented by collections from North America, Costa Rica, Guyana, Indonesia and Japan (molecular data are lacking for only the Guyanan and Japanese material); and S. australiensis, represented by material from Queensland, Australia. Additional collections from Zambia and Thailand represent independent lineages, but sampling is insufficient to describe new species for these entities.

Incorporating DNA barcodes into a multi-year inventory of the fishes of the hyperdiverse Lower Congo River, with a multi-gene performance assessment of the genus Labeo as a case study.

BACKGROUND AND AIMS: Here we describe preliminary efforts to integrate DNA barcoding into an ongoing inventory of the Lower Congo River (LCR) ichthyofauna. The 350 km stretch of the LCR from Pool Malebo to Boma includes the world's largest river rapids. The LCR ichthyofauna is hyperdiverse and rich in endemism due to high habitat heterogeneity, numerous dispersal barriers, and its downstream location in the basin. MATERIALS AND METHODS: We have documented 328 species from the LCR, 25% of which are thought to be endemic. In addition to detailing progress made to generate a reference sequence library of DNA barcodes for these fishes, we ask how DNA can be used at the current stage of the Fish Barcode of Life initiative, as a work in progress currently of limited utility to a wide audience. Two possibilities that we explore are the potential for DNA barcodes to generate discrete diagnostic characters for species, and to help resolve problematic taxa lacking clear morphologically diagnostic characters such as many species of the cyprinid genus Labeo, which we use as a case study. RESULTS: Our molecular analysis helped to clarify the validity of some species that were the subject of historical debate, and we were able to construct a molecular key for all monophyletic and morphologically recognizable species. Several species sampled from across the Congo Basin and widely distributed throughout Central and West Africa were recovered as paraphyletic based on our molecular data. CONCLUSION: Our study underscores the importance of generating reference barcodes for specimens collected from, or in close proximity to, type localities, particularly where species are poorly understood taxonomically and the extent of their geographical distributions have yet to be established.

Pacific boletes: implications for biogeographic relationships.

The obligate association of boletes with their plant partners is critical to understanding biogeographic distribution of these fungi. Only in rare instances are boletes not obligatory associates of plants; the majority are presumed or proven partners in obligate symbioses with a variety of plants. The array of plant-associated distributions provides a potential handle for evaluating bolete distribution on a global scale. However, migration processes remain unclear and distributions are often disjunct. As an illustration of phylogeographic studies of putatively widespread bolete taxa, we present preliminary analyses for Tylopilus ballouii using LSU rDNA and RPB1 sequence data. The LSU data suggest geographic structuring of the tested accessions. However, RPB1 data indicate that long-distance dispersal events (possibly mediated by humans) are possible, or that selection or other factors have obscured geographical patterns. Molecular divergence between samples in RPB1 argues against panmixis, and indicates that populations have been isolated for long periods.

Morphological and molecular evidence supporting an arbutoid mycorrhizal relationship in the Costa Rican páramo.

This study examines evidence for a particular arbutoid mycorrhizal interaction in páramo, a high-altitude neotropical ecosystem important in hydrological regulation but poorly known in terms of its fungal communities. Comarostaphylis arbutoides Lindley (Ericaceae) often forms dense thickets in Central American páramo habitats. Based on phylogenetic classification, it has been suggested that C. arbutoides forms arbutoid mycorrhizae with diverse Basidiomycetes and Ascomycetes; however, this assumption has not previously been confirmed. Based on field data, we hypothesized an arbutoid mycorrhizal association between C. arbutoides and the recently described bolete Leccinum monticola Halling & G.M. Mueller; in this study, we applied a rigorous approach using anatomical and molecular data to examine evidence for such an association. We examined root samples collected beneath L. monticola basidiomes for mycorrhizal structures, and we also compared rDNA internal transcribed spacer (ITS) sequences between mycorrhizal root tips and leaf or basidiome material of the suspected symbionts. Root cross sections showed a thin hyphal sheath and intracellular hyphal coils typical of arbutoid mycorrhizae. DNA sequence comparisons confirmed the identity of C. arbutoides and L. monticola as the mycorrhizal symbionts. In addition, this paper provides additional evidence for the widespread presence of minisatellite-like inserts in the ITS1 spacer in Leccinum species (including a characterization of the insert in L. monticola) and reports the use of an angiosperm-specific ITS primer pair useful for amplifying plant DNA from mycorrhizal roots without co-amplifying fungal DNA.

Morphological and molecular systematics of Rocky Mountain alpine Laccaria.

The alpine zone is comprised of habitats at elevations above treeline, and macromycetes play important ecological roles as decomposers and mycorrhizal symbionts here as elsewhere. Laccaria is an important group of ectomycorrhizal basidiomycetes widely used in experimental and applied research. A systematic study of alpine Laccaria species using morphological, cultural and molecular (ribosomal DNA internal transcribed spacer) data revealed five taxa in the Rocky Mountain alpine zone: L. laccata var. pallidifolia, L. nobilis (the first published report for arctic-alpine habitats), L. pumila, L. montana and L. pseudomontana (a newly described taxon similar to L. montana with more ellipsoidal, finely echinulate basidiospores). All occur in the southern Rocky Mountains of Colorado; however, only L. pumila and L. montana were found on the Beartooth Plateau in the northern Rocky Mountains of Montana and Wyoming. All are associated with dwarf and shrub Salix species, with L. laccata var. pallidifolia also associated with Dryas octopetala and Betula glandulosa. Maximum-parsimony phylogenetic analysis of rDNA-ITS sequences for 27 Laccaria accessions supports the morphological species delineations.