Genomic Diversity and Phenotypic Variation in Fungal Decomposers Involved in Bioremediation of Persistent Organic Pollutants.

Fungi work as decomposers to break down organic carbon, deposit recalcitrant carbon, and transform other elements such as nitrogen. The decomposition of biomass is a key function of wood-decaying basidiomycetes and ascomycetes, which have the potential for the bioremediation of hazardous chemicals present in the environment. Due to their adaptation to different environments, fungal strains have a diverse set of phenotypic traits. This study evaluated 320 basidiomycetes isolates across 74 species for their rate and efficiency of degrading organic dye. We found that dye-decolorization capacity varies among and within species. Among the top rapid dye-decolorizing fungi isolates, we further performed genome-wide gene family analysis and investigated the genomic mechanism for their most capable dye-degradation capacity. Class II peroxidase and DyP-type peroxidase were enriched in the fast-decomposer genomes. Gene families including lignin decomposition genes, reduction-oxidation genes, hydrophobin, and secreted peptidases were expanded in the fast-decomposer species. This work provides new insights into persistent organic pollutant removal by fungal isolates at both phenotypic and genotypic levels.

Disease-related population declines in bats demonstrate non-exchangeability in generalist predators.

The extent to which persisting species may fill the functional role of extirpated or declining species has profound implications for the structure of biological communities and ecosystem functioning. In North America, arthropodivorous bats are threatened on a continent-wide scale by the spread of white-nose syndrome (WNS), a disease caused by the fungus Pseudogymnoascus destructans. We tested whether bat species that display lower mortality from this disease can partially fill the functional role of other bat species experiencing population declines. Specifically, we performed high-throughput amplicon sequencing of guano from two generalist predators: the little brown bat (Myotis lucifugus) and big brown bat (Eptesicus fuscus). We then compared changes in prey consumption before versus after population declines related to WNS. Dietary niches contracted for both species after large and abrupt declines in little brown bats and smaller declines in big brown bats, but interspecific dietary overlap did not change. Furthermore, the incidence and taxonomic richness of agricultural pest taxa detected in diet samples decreased following bat population declines. Our results suggest that persisting generalist predators do not necessarily expand their dietary niches following population declines in other predators, providing further evidence that the functional roles of different generalist predators are ecologically distinct.

Predator preferences shape the diets of arthropodivorous bats more than quantitative local prey abundance.

Although most predators are generalists, the majority of studies on the association between prey availability and prey consumption have focused on specialist predators. To investigate the role of highly generalist predators in a complex food web, we measured the relationships between prey consumption and prey availability in two common arthropodivorous bats. Specifically, we used high-throughput amplicon sequencing coupled with a known mock community to characterize seasonal changes in little brown and big brown bat diets. We then linked spatiotemporal variation in prey consumption with quantitative prey availability estimated from intensive prey community sampling. We found that although quantitative prey availability fluctuated substantially over space and time, the most commonly consumed prey items were consistently detected in bat diets independently of their respective abundance. Positive relationships between prey abundance and probability of consumption were found only among prey groups that were less frequently detected in bat diets. While the probability of prey consumption was largely unrelated to abundance, the community structure of prey detected in bat diets was influenced by the local or regional abundance of prey. Observed patterns suggest that while little brown and big brown bats maintain preferences for particular prey independently of quantitative prey availability, total dietary composition may reflect some degree of opportunistic foraging. Overall, our findings suggest that generalist predators can display strong prey preferences that persist despite quantitative changes in prey availability.

Relationships among wood-boring beetles, fungi, and the decomposition of forest biomass.

A prevailing paradigm in forest ecology is that wood-boring beetles facilitate wood decay and carbon cycling, but empirical tests have yielded mixed results. We experimentally determined the effects of wood borers on fungal community assembly and wood decay within pine trunks in the southeastern United States. Pine trunks were made either beetle-accessible or inaccessible. Fungal communities were compared using culturing and high-throughput amplicon sequencing (HTAS) of DNA and RNA. Prior to beetle infestation, living pines had diverse fungal endophyte communities. Endophytes were displaced by beetle-associated fungi in beetle-accessible trees, whereas some endophytes persisted as saprotrophs in beetle-excluded trees. Beetles increased fungal diversity several fold. Over forty taxa of Ascomycota were significantly associated with beetles, but beetles were not consistently associated with any known wood-decaying fungi. Instead, increasing ambrosia beetle infestations caused reduced decay, consistent with previous in vitro experiments that showed beetle-associated fungi reduce decay rates by competing with decay fungi. No effect of bark-inhabiting beetles on decay was detected. Platypodines carried significantly more fungal taxa than scolytines. Molecular results were validated by synthetic and biological mock communities and were consistent across methodologies. RNA sequencing confirmed that beetle-associated fungi were biologically active in the wood. Metabarcode sequencing of the LSU/28S marker recovered important fungal symbionts that were missed by ITS2, though community-level effects were similar between markers. In contrast to the current paradigm, our results indicate ambrosia beetles introduce diverse fungal communities that do not extensively decay wood, but instead reduce decay rates by competing with wood decay fungi.

An improved method for utilizing high-throughput amplicon sequencing to determine the diets of insectivorous animals.

DNA analysis of predator faeces using high-throughput amplicon sequencing (HTS) enhances our understanding of predator-prey interactions. However, conclusions drawn from this technique are constrained by biases that occur in multiple steps of the HTS workflow. To better characterize insectivorous animal diets, we used DNA from a diverse set of arthropods to assess PCR biases of commonly used and novel primer pairs for the mitochondrial gene, cytochrome oxidase C subunit 1 (COI). We compared diversity recovered from HTS of bat guano samples using a commonly used primer pair "ZBJ" to results using the novel primer pair "ANML." To parameterize our bioinformatics pipeline, we created an arthropod mock community consisting of single-copy (cloned) COI sequences. To examine biases associated with both PCR and HTS, mock community members were combined in equimolar amounts both pre- and post-PCR. We validated our system using guano from bats fed known diets and using composite samples of morphologically identified insects collected in pitfall traps. In PCR tests, the ANML primer pair amplified 58 of 59 arthropod taxa (98%), whereas ZBJ amplified 24-40 of 59 taxa (41%-68%). Furthermore, in an HTS comparison of field-collected samples, the ANML primers detected nearly fourfold more arthropod taxa than the ZBJ primers. The additional arthropods detected include medically and economically relevant insect groups such as mosquitoes. Results revealed biases at both the PCR and sequencing levels, demonstrating the pitfalls associated with using HTS read numbers as proxies for abundance. The use of an arthropod mock community allowed for improved bioinformatics pipeline parameterization.

Non-biological synthetic spike-in controls and the AMPtk software pipeline improve mycobiome data.

High-throughput amplicon sequencing (HTAS) of conserved DNA regions is a powerful technique to characterize microbial communities. Recently, spike-in mock communities have been used to measure accuracy of sequencing platforms and data analysis pipelines. To assess the ability of sequencing platforms and data processing pipelines using fungal internal transcribed spacer (ITS) amplicons, we created two ITS spike-in control mock communities composed of cloned DNA in plasmids: a biological mock community, consisting of ITS sequences from fungal taxa, and a synthetic mock community (SynMock), consisting of non-biological ITS-like sequences. Using these spike-in controls we show that: (1) a non-biological synthetic control (e.g., SynMock) is the best solution for parameterizing bioinformatics pipelines, (2) pre-clustering steps for variable length amplicons are critically important, (3) a major source of bias is attributed to the initial polymerase chain reaction (PCR) and thus HTAS read abundances are typically not representative of starting values. We developed AMPtk, a versatile software solution equipped to deal with variable length amplicons and quality filter HTAS data based on spike-in controls. While we describe herein a non-biological SynMock community for ITS sequences, the concept and AMPtk software can be widely applied to any HTAS dataset to improve data quality.

Experimental evidence of a symbiosis between red-cockaded woodpeckers and fungi.

Primary cavity excavators, such as woodpeckers, are ecosystem engineers in many systems. Associations between cavity excavators and fungi have long been hypothesized to facilitate cavity excavation, but these relationships have not been experimentally verified. Fungi may help excavators by softening wood, while excavators may facilitate fungal dispersal. Here we demonstrate that excavators facilitate fungal dispersal and thus we report the first experimental evidence of a symbiosis between fungi and a cavity excavator, the red-cockaded woodpecker (RCW,Picoides borealis). Swab samples of birds showed that RCWs carry fungal communities similar to those found in their completed excavations. A 26-month field experiment using human-made aseptically drilled excavations in live trees, half of which were inaccessible to RCWs, demonstrated that RCWs directly alter fungal colonization and community composition. Experimental excavations that were accessible to RCWs contained fungal communities similar to natural RCW excavations, whereas inaccessible experimental excavations contained significantly different fungal communities. Our work demonstrates a complex symbiosis between cavity excavators and communities of fungi, with implications for forest ecology, wildlife management, and conservation.

Armillaria altimontana, a new species from the western interior of North America.

Armillaria altimontana, previously considered North American biological species (NABS) X, is described as new. To date, it appears that A. altimontana prefers higher-elevation, mesic sites within the dry, conifer forest zone of western interior North America. This species has been found on hardwoods and conifers and is associated most commonly with Abies-dominated forest types in southern British Columbia, Washington, Oregon, Idaho and northern California. Partial elongation factor 1-alpha (tef1) sequences were generated from six isolates of A. altimontana originating from three locations in northern Idaho. Phylogenetic analyses of all 10 North American Armillaria species were carried out with maximum parsimony and maximum likelihood. Results indicate that isolates of A. altimontana formed a monophyletic group and clustered with A. calvescens, A. cepistipes, A. gallica and A. nabsnona, which is in agreement with recent phylogenetic studies of Armillaria.

Intragenomic variation in the ITS rDNA region obscures phylogenetic relationships and inflates estimates of operational taxonomic units in genus Laetiporus.

Regions of rDNA are commonly used to infer phylogenetic relationships among fungal species and as DNA barcodes for identification. These regions occur in large tandem arrays, and concerted evolution is believed to reduce intragenomic variation among copies within these arrays, although some variation still might exist. Phylogenetic studies typically use consensus sequencing, which effectively conceals most intragenomic variation, but cloned sequences containing intragenomic variation are becoming prevalent in DNA databases. To understand effects of using cloned rDNA sequences in phylogenetic analyses we amplified and cloned the ITS region from pure cultures of six Laetiporus species and one Wolfiporia species (Basidiomycota, Polyporales). An average of 66 clones were selected randomly and sequenced from 21 cultures, producing a total of 1399 interpretable sequences. Significant variation (≥ 5% variation in sequence similarity) was observed among ITS copies within six cultures from three species clades (L. cincinnatus, L. sp. clade J, and Wolfiporia dilatohypha) and phylogenetic analyses with the cloned sequences produced different trees relative to analyses with consensus sequences. Cloned sequences from L. cincinnatus fell into more than one species clade and numerous cloned L. cincinnatus sequences fell into entirely new clades, which if analyzed on their own most likely would be recognized as "undescribed" or "novel" taxa. The use of a 95% cut off for defining operational taxonomic units (OTUs) produced seven Laetiporus OTUs with consensus ITS sequences and 20 OTUs with cloned ITS sequences. The use of cloned rDNA sequences might be problematic in fungal phylogenetic analyses, as well as in fungal bar-coding initiatives and efforts to detect fungal pathogens in environmental samples.

DNA-based detection of the fungal pathogen Geomyces destructans in soils from bat hibernacula.

White-nose syndrome (WNS) is an emerging disease causing unprecedented morbidity and mortality among bats in eastern North America. The disease is characterized by cutaneous infection of hibernating bats by the psychrophilic fungus Geomyces destructans. Detection of G. destructans in environments occupied by bats will be critical for WNS surveillance, management and characterization of the fungal lifecycle. We initiated an rRNA gene region-based molecular survey to characterize the distribution of G. destructans in soil samples collected from bat hibernacula in the eastern United States with an existing PCR test. Although this test did not specifically detect G. destructans in soil samples based on a presence/absence metric, it did favor amplification of DNA from putative Geomyces species. Cloning and sequencing of PCR products amplified from 24 soil samples revealed 74 unique sequence variants representing 12 clades. Clones with exact sequence matches to G. destructans were identified in three of 19 soil samples from hibernacula in states where WNS is known to occur. Geomyces destructans was not identified in an additional five samples collected outside the region where WNS has been documented. This study highlights the diversity of putative Geomyces spp. in soil from bat hibernacula and indicates that further research is needed to better define the taxonomy of this genus and to develop enhanced diagnostic tests for rapid and specific detection of G. destructans in environmental samples.

Relationships among North American and Japanese Laetiporus isolates inferred from molecular phylogenetics and single-spore incompatibility reactions.

Relationships were investigated among North American and Japanese isolates of Laetiporus using phylogenetic analysis of ITS sequences and single-spore isolate incompatibility. Single-spore isolate pairings revealed no significant compatibility between North American and Japanese isolates. ITS analysis revealed 12 clades within the core Laetiporus clade, seven of which are known to occur in North America (including Hawaii and the Caribbean), three in Japan, two in South America, three in Europe and one in South Africa. The identity of L. sulphureus s.s. has yet to be determined and could be either L. "sulphureus" (clade C), which appears to be restricted to Europe and occurs on angiosperms and gymnospersm, or L. "sulphureus" (clade E), which is found in Europe, North America and South America exclusively on angiosperms. Three clades, one from the Caribbean, one from Hawaii and one from South Africa, have yet to be named formally. Of the three Laetiporus species found in Japan two have been named recently (L. cremeiporus and L. montanus) and one has been epitypified (L. versisporus). The single-spore incompatibility and ITS data support recognition of the three Japanese taxa as distinct biological and evolutionary species.

The genus Laetiporus (Basidiomycota, Polyporales) in East Asia.

Relationships among East Asian, North American and European Laetiporus sulphureus s. lat., a cosmopolitan brown rot species complex, were assessed with phylogenetic analyses and incompatibility tests. Three East Asian taxa, Laetiporus cremeiporus sp. nov., Laetiporus montanus and Laetiporus versisporus, are described and illustrated as well as compared with related taxa from Southeast Asia, North America and Europe. Phylogenetic analyses showed that L. cremeiporus and L. versisporus are clearly distinct species among Laetiporus taxa. The three conifer inhabiting species, Laetiporus conifericola, Laetiporus huroniensis and L. montanus, are closely related to each other. The European population of L. montanus exhibits two sequence variants of the EF1alpha: one is the same as observed in L. sulphureus in Europe and the other is that observed in East Asian population of L. montanus. A key to the known species of Laetiporus in the northern hemisphere is provided.

Effects of cloning and root-tip size on observations of fungal ITS sequences from Picea glauca roots.

To better understand the effects of cloning on observations of fungal ITS sequences from Picea glauca (white spruce) roots two techniques were compared: (i) direct sequencing of fungal ITS regions from individual root tips without cloning and (ii) cloning and sequencing of fungal ITS regions from individual root tips. Effect of root tip size was investigated by selecting 20 small root tips (SRT, 1.0-2.0 mm long) and 20 large root tips (LRT, 5.0-6.0 mm long). DNA was isolated from each tip and PCR-amplified with fungal-specific primers. PCR reactions were divided into two portions, one of which was sequenced directly and one of which was cloned first followed by sequencing of 12 random clones. With direct sequencing all 20 SRT produced an identifiable sequence, while only 13 of 20 LRT (65%) yielded an identifiable sequence. With cloning and sequencing all 40 tips produced identifiable fungal ITS sequences regardless of size. Failure of direct sequencing in LRT was associated with the presence of multispecies assemblages. Cloning identified 18 taxa overall while direct sequencing identified four. Cloning was not affected by tip size and identified more taxa relative to direct sequencing, although cost and probability of observing lab-based contaminants (e.g., airborne or reagent-based) were higher. We suggest that standardized controls be run whenever clones are sequenced from environmental samples, including positive controls derived from pure cultures and negative controls that cover the entire extraction, amplification and cloning process. Additional studies on larger root segments and bulked samples are needed to determine whether cloning can detect fungi accurately and cost-effectively in complex environmental samples.

Molecular phylogeny of Laetiporus and other brown rot polypore genera in North America.

Phylogenetic relationships were investigated among North American species of Laetiporus, Leptoporus, Phaeolus, Pycnoporellus and Wolfiporia using ITS, nuclear large subunit and mitochondrial small subunit rDNA sequences. Members of these genera have poroid hymenophores, simple septate hyphae and cause brown rots in a variety of substrates. Analyses indicate that Laetiporus and Wolfiporia are not monophyletic. All North American Laetiporus species formed a well supported monophyletic group (the "core Laetiporus clade" or Laetiporus s.s.) with the exception of L. persicinus, which showed little affinity for any genus for which sequence data are available. Based on data from GenBank, the southern hemisphere species L. portentosus also fell well outside the core Laetiporus clade. Wolfiporia dilatohypha was found to represent a sister group to the core Laetiporus clade. Isolates of Phaeolus, Pycnoporellus and members of the core Laetiporus clade all fell within the Antrodia clade of polypores, while Leptoporus mollis and Laetiporus portentosus fell within the phlebioid clade of polypores. Wolfiporia cocos isolates also fell in the Antrodia clade, in contrast to previous studies that placed W. cocos in the core polyporoid clade. ITS analyses resolved eight clades within Laetiporus s.s., three of which might represent undescribed species. A combined analysis using the three DNA regions resolved five major clades within Laetiporus s.s.: a clade containing conifer-inhabiting species ("Conifericola clade"), a clade containing L. cincinnatus ("Cincinnatus clade"), a clade containing L. sulphureus s.s. isolates with yellow pores ("Sulphureus clade I"), a clade containing L. sulphureus s.s. isolates with white pores ("Sulphureus clade II") and a clade containing L. gilbertsonii and unidentified isolates from the Caribbean ("Gilbertsonii clade"). Although there is strong support for groups within the core Laetiporus clade, relationships among these groups remain poorly resolved.