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.
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.
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.
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.
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.
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.
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.
While many bat species are known to function as suppressors of arthropod abundance, including several economically important agricultural pest taxa, the broader ecological consequences of disease‐related bat declines have not been fully assessed in many regions. In our study, we tested whether big brown bats (Eptesicus fuscus), which demonstrate lower mortality from white‐nose syndrome, could potentially fill the ecological role formerly occupied by the more severely affected little brown bat (Myotis lucifugus). Overall, we found that big brown bats did not adopt more of the prey formerly consumed by little brown bats and that the dietary niche overlap between the two bat species did not change over time.
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.
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.
Bats have been portrayed as important consumers of mosquitoes, but evidence supporting this claim is surprisingly scant. We collected the fecal material of 2 common North American bats at 22 sites in ...Wisconsin, United States and screened samples for mosquitoes using a recently improved molecular method for detecting arthropod DNA. Overall, we detected 17 discrete operational taxonomic units assigned to the mosquito family (Diptera: Culicidae), 15 of which were assigned at the species level. We detected mosquitoes in 71.9% of samples and at all sampling sites for little brown bats (Myotis lucifugus). By comparison, we detected mosquitoes in 33.3% of samples and one-half of the sampling sites for big brown bats (Eptesicus fuscus). Our results suggest that the incidence and taxonomic richness of mosquito prey consumed by bats is considerably higher than has been previously shown. In light of globally declining bat populations, we propose that future studies reassess the importance of trophic interactions between bats and mosquitoes.
PDF
