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Chronic nonhealing wounds have been heralded as a silent epidemic, causing significant morbidity and mortality especially in elderly, diabetic, and obese populations. Polymicrobial biofilms in the wound bed are hypothesized to disrupt the highly coordinated and sequential events of cutaneous healing. Both culture-dependent and -independent studies of the chronic-wound microbiome have almost exclusively focused on bacteria, omitting what we hypothesize are important fungal contributions to impaired healing and the development of complications. Here we show for the first time that fungal communities (the mycobiome) in chronic wounds are predictive of healing time, associated with poor outcomes, and form mixed fungal-bacterial biofilms. We longitudinally profiled 100, nonhealing diabetic-foot ulcers with high-throughput sequencing of the pan-fungal internal transcribed spacer 1 (ITS1) locus, estimating that up to 80% of wounds contain fungi, whereas cultures performed in parallel captured only 5% of colonized wounds. The “mycobiome” was highly heterogeneous over time and between subjects. Fungal diversity increased with antibiotic administration and onset of a clinical complication. The proportions of the phylum Ascomycota were significantly greater ( P = 0.015) at the beginning of the study in wounds that took >8 weeks to heal. Wound necrosis was distinctly associated with pathogenic fungal species, while taxa identified as allergenic filamentous fungi were associated with low levels of systemic inflammation. Directed culturing of wounds stably colonized by pathogens revealed that interkingdom biofilms formed between yeasts and coisolated bacteria. Combined, our analyses provide enhanced resolution of the mycobiome during impaired wound healing, its role in chronic disease, and impact on clinical outcomes. IMPORTANCE Wounds are an underappreciated but serious complication for a diverse spectrum of diseases. High-risk groups, such as persons with diabetes, have a 25% lifetime risk of developing a wound that can become chronic. The majority of microbiome research related to chronic wounds is focused on bacteria, but the association of fungi with clinical outcomes remains to be elucidated. Here we describe the dynamic fungal communities in 100 diabetic patients with foot ulcers. We found that communities are unstable over time, but at the first clinical presentation, the relative proportions of different phyla predict healing times. Pathogenic fungi not identified by culture reside in necrotic wounds and are associated with a poor prognosis. In wounds stably colonized by fungi, we identified yeasts capable of forming biofilms in concert with bacteria. Our findings illuminate the associations of the fungal mycobiome with wound prognosis and healing. Wounds are an underappreciated but serious complication for a diverse spectrum of diseases. High-risk groups, such as persons with diabetes, have a 25% lifetime risk of developing a wound that can become chronic. The majority of microbiome research related to chronic wounds is focused on bacteria, but the association of fungi with clinical outcomes remains to be elucidated. Here we describe the dynamic fungal communities in 100 diabetic patients with foot ulcers. We found that communities are unstable over time, but at the first clinical presentation, the relative proportions of different phyla predict healing times. Pathogenic fungi not identified by culture reside in necrotic wounds and are associated with a poor prognosis. In wounds stably colonized by fungi, we identified yeasts capable of forming biofilms in concert with bacteria. Our findings illuminate the associations of the fungal mycobiome with wound prognosis and healing.

Viruses make up a major component of the human microbiota but are poorly understood in the skin, our primary barrier to the external environment. Viral communities have the potential to modulate states of cutaneous health and disease. Bacteriophages are known to influence the structure and function of microbial communities through predation and genetic exchange. Human viruses are associated with skin cancers and a multitude of cutaneous manifestations. Despite these important roles, little is known regarding the human skin virome and its interactions with the host microbiome. Here we evaluated the human cutaneous double-stranded DNA virome by metagenomic sequencing of DNA from purified virus-like particles (VLPs). In parallel, we employed metagenomic sequencing of the total skin microbiome to assess covariation and infer interactions with the virome. Samples were collected from 16 subjects at eight body sites over 1 month. In addition to the microenviroment, which is known to partition the bacterial and fungal microbiota, natural skin occlusion was strongly associated with skin virome community composition. Viral contigs were enriched for genes indicative of a temperate phage replication style and also maintained genes encoding potential antibiotic resistance and virulence factors. CRISPR spacers identified in the bacterial DNA sequences provided a record of phage predation and suggest a mechanism to explain spatial partitioning of skin phage communities. Finally, we modeled the structure of bacterial and phage communities together to reveal a complex microbial environment with a Corynebacterium hub. These results reveal the previously underappreciated diversity, encoded functions, and viral-microbial dynamic unique to the human skin virome. IMPORTANCE To date, most cutaneous microbiome studies have focused on bacterial and fungal communities. Skin viral communities and their relationships with their hosts remain poorly understood despite their potential to modulate states of cutaneous health and disease. Previous studies employing whole-metagenome sequencing without purification for virus-like particles (VLPs) have provided some insight into the viral component of the skin microbiome but have not completely characterized these communities or analyzed interactions with the host microbiome. Here we present an optimized virus purification technique and corresponding analysis tools for gaining novel insights into the skin virome, including viral “dark matter,” and its potential interactions with the host microbiome. The work presented here establishes a baseline of the healthy human skin virome and is a necessary foundation for future studies examining viral perturbations in skin health and disease. To date, most cutaneous microbiome studies have focused on bacterial and fungal communities. Skin viral communities and their relationships with their hosts remain poorly understood despite their potential to modulate states of cutaneous health and disease. Previous studies employing whole-metagenome sequencing without purification for virus-like particles (VLPs) have provided some insight into the viral component of the skin microbiome but have not completely characterized these communities or analyzed interactions with the host microbiome. Here we present an optimized virus purification technique and corresponding analysis tools for gaining novel insights into the skin virome, including viral “dark matter,” and its potential interactions with the host microbiome. The work presented here establishes a baseline of the healthy human skin virome and is a necessary foundation for future studies examining viral perturbations in skin health and disease.

Background The skin harbors complex communities of resident microorganisms, yet little is known of their physiological roles and the molecular mechanisms that mediate cutaneous host-microbe interactions. Here, we profiled skin transcriptomes of mice reared in the presence and absence of microbiota to elucidate the range of pathways and functions modulated in the skin by the microbiota. Results A total of 2820 genes were differentially regulated in response to microbial colonization and were enriched in gene ontology (GO) terms related to the host-immune response and epidermal differentiation. Innate immune response genes and genes involved in cytokine activity were generally upregulated in response to microbiota and included genes encoding toll-like receptors, antimicrobial peptides, the complement cascade, and genes involved in IL-1 family cytokine signaling and homing of T cells. Our results also reveal a role for the microbiota in modulating epidermal differentiation and development, with differential expression of genes in the epidermal differentiation complex (EDC). Genes with correlated co-expression patterns were enriched in binding sites for the transcription factors Klf4, AP-1, and SP-1, all implicated as regulators of epidermal differentiation. Finally, we identified transcriptional signatures of microbial regulation common to both the skin and the gastrointestinal tract. Conclusions With this foundational approach, we establish a critical resource for understanding the genome-wide implications of microbially mediated gene expression in the skin and emphasize prospective ways in which the microbiome contributes to skin health and disease.

This study uses a set of PCR-based methods to examine the putative microbiota associated with lichen thalli. In initial experiments, generalized oligonucleotide-primers for the 16S rRNA gene resulted in amplicon pools populated almost exclusively with fragments derived from lichen photobionts (i.e., Cyanobacteria or chloroplasts of algae). This effectively masked the presence of other lichen-associated prokaryotes. In order to facilitate the study of the lichen microbiota, 16S ribosomal oligonucleotide-primers were developed to target Bacteria, but exclude sequences derived from chloroplasts and Cyanobacteria. A preliminary microbiotic survey of lichen thalli using these new primers has revealed the identity of several bacterial associates, including representatives of the extremophilic Acidobacteria, bacteria in the families Acetobacteraceae and Brucellaceae, strains belonging to the genus Methylobacterium , and members of an undescribed lineage in the Rhizobiales. This new lineage was investigated and characterized through molecular cloning, and was found to be present in all examined lichens that are associated with green algae. There is evidence to suggest that members of this lineage may both account for a large proportion of the lichen-associated bacterial community and assist in providing the lichen thallus with crucial nutrients such as fixed nitrogen.

The following corrections and amendments are made to the 2016 classification of lichenized fungi published in the previous issue of this journal. Four families are added: Harpidiaceae (Pezizomycotina incertae sedis), with the two genera Euopsis and Harpidium; Pleomassariaceae (Pleosporales), with the genus Splanchonema; Squamarinaceae (Lecanorales), with the two genera Herteliana (moved from Ramalinaceae) and Squamarina (moved from Stereocaulaceae); and Trichosphaeriaceae (Sordariomycetes: Trichosphaeriales), with the genus Cresporhaphis. The following previously overlooked genera are also added: Allophoron (Pezizomycotina incertae sedis), Cresporhaphis (Trichosphaeriaceae), Gabura (Arctomiaceae), Julella (Trypetheliaceae), Knightiella (Icmadophilaceae), Porpidinia (Lecideaceae), Protoroccella (Roccellaceae), Psoromidium (Pannariaceae) and Tremotylium (Arthoniales incertae sedis). The classification is adjusted for four genera: Asteroporum (moved from Pezizomycotina incertae sedis to Dothideomycetes incertae sedis), Eremastrella (moved from Psoraceae to Lecideaceae), Hosseusia (moved from Pannariaceae to Lecanoromycetes incertae sedis) and Joergensenia (moved from Lecanorales incertae sedis to Pannariaceae). Further, the following overlooked generic synonyms are listed: Buscalionia (= Marcelaria [nom. cons. prop.]), Degeliella (= Psoromaria), Dirinastrum (= Buellia), Gymnographa (= Phaeographis), Kroswia (= Fuscopannaria), Marfloraea (= Lepra), Medusulina (= Fissurina), and Phaeographina (= Pliariona); the genus Anapyrenium is discussed as a potential synonym of Thelomma. Species numbers are adjusted for nine genera: Austrella (Pannariaceae; 3 spp.), Icmadophila (Icmadophilaceae; 5 spp.), Lepidocollema (Pannariaceae; 23 spp.), Massalongia (Massalongiaceae; 6 spp.), Parmeliella (Pannariaceae; 70 spp.), Psoromidium (Pannariaceae; 1 spp.), Pyrgillus (Pyrenulaceae; 7 spp.), Siphula (Icmadophilaceae; 17 spp.) and Synarthonia (Arthoniales incertae sedis; 5 spp.). The fossil lichen Honeggeriella (complexa) is validated by adding MycoBank registration numbers, the validity of the genus name Pallidogramme (Graphidaceae) is discussed and confirmed, and the authorship of the name Thallinocarpon (Lichinaceae) is clarified. Several genera are (continued to be) considered non-lichenized, namely Chaenothecopsis (Eurotiomycetes: Mycocaliciales: Sphinctrinaceae), Limboria (newly lectotypified with L. constellata; Pezizomycotina incertae sedis), Naetrocymbe (Dothideomycetes: Pleosporales: Naetrocymbaceae), and Obryzum (Dothideomycetes incertae sedis: Obryzaceae); the status of the genus Pleurotrema (Dothideomycetes incertae sedis: Pleurotremataceae) is also discussed. Seven genera are corrected to have molecular data available: Adelolecia, Aspiciliopsis, Aspilidea, Crocodia, Parasiphula, Vezdaea and Xylopsora. With these corrections, the number of lichenized species is now tabulated at 19,409 and the number of fungal genera, families, and orders including lichens at 1,002, 119, and 40, respectively.

Ninety years after Zahlbruckner, we present the most recent update to the classification of lichen fungi in the Ascomycota and Basidiomycota to genus level, with species numbers and references to changes compared to the 2010 Outline of Ascomycota and other recent classifications. Updated statistics on global species richness of lichen fungi and species richness at family, order and class level are given. The number of accepted species is 19,387 in 995 genera, 115 families, 39 orders and eight classes. Lichenized Basidiomycota amount to 172 species (0.9% of the total), 15 genera (1.5%), five families (4.3%), five orders (12.8%) and one class (12.5%). The most speciose genera are Xanthoparmelia, Lecanora, Arthonia, Cladonia, Pertusaria, Ocellularia, Graphis, Caloplaca, Usnea and Buellia. The average number of species per genus is 19.5 and 256 genera are monospecific. Using newly defined categories, two genera (Xanthoparmelia, Lecanora) are ultradiverse (more than 500 species), 17 hyperdiverse (201–500 species) and 12 megadiverse (101–200). The largest family is Parmeliaceae, with 2,765 species and 77 genera, followed by Graphidaceae (2,161; 79), Verrucariaceae (943; 43), Ramalinaceae (916; 43) and Lecanoraceae (791; 25). The largest order is Lecanorales, with 6,231 species and 234 genera, followed by Ostropales (3,261; 138), Arthoniales (1,541, 103), Peltigerales (1,301; 67) and Caliciales (1,276; 55). The largest class is Lecanoromycetes, with 15,131 species and 701 genera, followed by Arthoniomycetes (1,541; 103), Eurotiomycetes (1,203; 63), Dothideomycetes (812; 39) and Lichinomycetes (390; 50). A total of 751 out of 995 genera (75%) have molecular data. Fifty-nine genera remain in unresolved positions at the family, order or class level. The phylogenetic position of the 39 orders containing lichenized fungi suggests 20–30 independent lichenization events during the evolution of higher Fungi, 14–23 in the Ascomycota and 6–7 in the Basidiomycota. The following names are validated: Candelariomycetidae Miądl. et al. ex Timdal & M.Westb. subcl. nov., Cystocoleaceae Locq. ex Lücking, B.P.Hodk. & S.D.Leav. fam. nov, Letrouitineae Gaya & Lutzoni subordo nov., Rhizocarpales Miądl. & Lutzoni ordo nov. and Teloschistineae Gaya & Lutzoni subordo nov. Lectotypes are designated for Clathroporinopsis M.Choisy and Protoschistes M.Choisy, making both synonyms of Gyalecta Ach., and Stromatothelium Trevis., making it a synonym of Pyrenula Ach. Members of Cyphobasidiales, which are here interpreted as hyperlichenized fungi, as well as fossil lichen fungi, are added in additional classifications in two appendices.

Background We present a novel method to encode ambiguously aligned regions in fixed multiple sequence alignments by 'Pairwise Identity and Cost Scores Ordination' (PICS-Ord). The method works via ordination of sequence identity or cost scores matrices by means of Principal Coordinates Analysis (PCoA). After identification of ambiguous regions, the method computes pairwise distances as sequence identities or cost scores, ordinates the resulting distance matrix by means of PCoA, and encodes the principal coordinates as ordered integers. Three biological and 100 simulated datasets were used to assess the performance of the new method. Results Including ambiguous regions coded by means of PICS-Ord increased topological accuracy, resolution, and bootstrap support in real biological and simulated datasets compared to the alternative of excluding such regions from the analysis a priori. In terms of accuracy, PICS-Ord performs equal to or better than previously available methods of ambiguous region coding (e.g., INAASE), with the advantage of a practically unlimited alignment size and increased analytical speed and the possibility of PICS-Ord scores to be analyzed together with DNA data in a partitioned maximum likelihood model. Conclusions Advantages of PICS-Ord over step matrix-based ambiguous region coding with INAASE include a practically unlimited number of OTUs and seamless integration of PICS-Ord codes into phylogenetic datasets, as well as the increased speed of phylogenetic analysis. Contrary to word- and frequency-based methods, PICS-Ord maintains the advantage of pairwise sequence alignment to derive distances, and the method is flexible with respect to the calculation of distance scores. In addition to distance and maximum parsimony, PICS-Ord codes can be analyzed in a Bayesian or maximum likelihood framework. RAxML (version 7.2.6 or higher that was developed for this study) allows up to 32-state ordered or unordered characters. A GTR, MK, or ORDERED model can be applied to analyse the PICS-Ord codes partition, with GTR performing slightly better than MK and ORDERED. Availability An implementation of the PICS-Ord algorithm is available from http://scit.us/projects/ngila/wiki/PICS-Ord . It requires both the statistical software, R http://www.r-project.org and the alignment software Ngila http://scit.us/projects/ngila .

There is increasing evidence that animals can acquire mate preferences through the use of public information, notably by observing (and copying) the mate preferences of others in the population. If females acquire preferences through social mechanisms, sexual selection could act very rapidly to spread the preference and drive elaboration of the preferred trait(s). Although there are reports of 'mate-choice copying' in polygynous species, there is no clear evidence for this process in monogamous species. Here, we investigated whether adult female zebra finches Taeniopygia guttata can socially acquire sexual preferences for individual males and, in a separate study, for a generalized trait (coloured leg bands) of males. In both studies, test females observed males in two simultaneous conditions: a ('chosen') mixed-sex situation in which a male was paired with a (model) female, and a ('unchosen') same-sex situation in which a male was paired with another male. In the first experiment, after two weeks of females observing males, test females significantly preferred individual males who had been paired with another female (i.e. chosen males). In the second experiment, test females significantly preferred novel males that were wearing the same leg band colour as the apparently chosen males. Our findings are consistent with the conclusion that female zebra finches' mate preferences are altered by public information. Our study implies that mate preferences can spread rapidly through populations by social mechanisms, affecting the strength of sexual selection in a monogamous species.

A combination of molecular phylogenetic analyses of ITS and mtSSU sequences, morphological and chemical analyses were used to investigate the lineages nominally included in the sterile lichen genus Lepraria. A core group (Lepraria s. str.) was resolved as sister to Stereocaulon. Species producing the secondary compounds argopsin, pannarin and usnic acid were found to belong to other lineages of lichen-forming ascomycetes. Study of Leprocaulon revealed that all species, except the type, likely represent members of Lepraria s. str. that have evolved a fruticose growth form. The correct name for the type species of Leprocaulon is shown to be L. quisquiliare, not L. microscopicum, and the genus is redefined to include several species previously placed in Lepraria. Leprocaulon quisquiliare is also shown to comprise two morphologically convergent species. The name is lectotypified and epitypified on material from the type region (Germany) and its application restricted to Old World populations. New World populations of L. quisquiliare are described as L. americanum. Leprocaulon, in its revised sense, is recognized in a new family (Leprocaulaceae) and order (Leprocaulales) sister to the Caliciales and including the genus Halecania. A new genus of Pilocarpaceae, Nelsenium, is introduced to accommodate Lepraria usnica. The status of Lepraria ecorticata is discussed in the context of usnic acid-producing Lecanora species. These nomenclatural novelties are proposed: (i) transfers from Leprocaulon to Lepraria: Lepraria albicans comb. nov., L. arbuscula comb. nov., L. congestum comb. nov., L. gracilescens comb. nov., L. pseudoarbuscula comb. nov., L. subalbicans comb. nov., L. tenellum comb. nov.; (ii) transfers from Lepraria to Leprocaulon: Leprocaulon adhaerens comb. nov., L. coriense, L. santamonicae comb. nov., L. terricola comb. nov. and L. textum comb. nov.; (iii) new taxa: Leprocaulales ord. nov., Leprocaulaceae fam. nov., Nelsenium gen. nov., Leprocaulon americanum sp. nov. and L. knudsenii sp. nov.

Although the Appalachian Mountains of southwestern Virginia, United States, are known to represent a major ‘hotspot’ of biodiversity for North America, no significant survey of overall lichen diversity has been conducted in the region thus far. Presented here is a list of 221 distinct taxa of lichens, lichenicolous fungi, and ‘lichen allies’ collected during the 2008 Hugo L. Blomquist Bryological and Lichenological Foray in the mountains of southwestern Virginia. Collections were made from diverse habitats, primarily in the Mount Rogers National Recreation Area (MRNRA), and yielded 41 potential state records. Particularly noteworthy collections include: Sphaerellothecium coniodes (a lichenicolous fungus that was not previously known to exist in North America), Hypotrachyna lividescens (a primarily neotropical macrolichen that has not previously been reported from North America), Pycnora praestabilis (a lignicolous crust not previously reported from any other location in eastern North America), Heterodermia erecta (a foliose lichen previously known in the world only from a single small region of Georgia/North Carolina), and Psilolechia clavulifera (a crustose lichen taxon previously reported from only one other location in eastern North America). The sheer diversity of lichens, along with the number of rare and/or potentially endangered taxa, highlights the need for continued preservation efforts in MRNRA and the southern Appalachian Mountains in general.