Reduction of genetic diversity in 'Alala (Hawaiian crow; Corvus hawaiiensis) between the late 1800s and the late 1900s.

Genetic and genomic data are increasingly used to aid conservation management of endangered species by providing insights into evolutionary histories, factors associated with extinction risks, and potential for future adaptation. For the 'Alala, or Hawaiian crow (Corvus hawaiiensis), genetic concerns include negative correlations between inbreeding and hatching success. However, it is unclear if low genetic diversity and inbreeding depression are consequences of a historical population bottleneck, or if 'Alala had historically low genetic diversity that predated human influence, perhaps as a result of earlier declines or founding events. In this study, we applied a hybridization-based sequence capture to generate a genome-wide single nucleotide polymorphism (SNP) dataset for comparing historical specimens collected in the 1890s, when 'Alala were more numerous, to samples taken between 1973 and 1998, when 'Alala population densities were near the lowest documented levels in the wild, prior to all individuals being collected for captive rearing. We found low genome-wide diversity in both sample groups, however, the modern sample group (1973 to 1998 cohort) exhibited relatively fewer polymorphic alleles, a lower proportion of polymorphic loci, and lower observed heterozygosity, consistent with a population decline and potential bottleneck effects. These results combined with a current low population size highlight the importance of continued efforts by conservation managers to mitigate inbreeding and maintain founder representation to preserve what genetic diversity remains.

Linking avian malaria parasitemia estimates from quantitative PCR and microscopy reveals new infection patterns in Hawai'i.

Plasmodium parasites infect thousands of species and provide an exceptional system for studying host-pathogen dynamics, especially for multi-host pathogens. However, understanding these interactions requires an accurate assay of infection. Assessing Plasmodium infections using microscopy on blood smears often misses infections with low parasitemias (the fractions of cells infected), and biases in malaria prevalence estimates will differ among hosts that differ in mean parasitemias. We examined Plasmodium relictum infection and parasitemia using both microscopy of blood smears and quantitative polymerase chain reaction (qPCR) on 299 samples from multiple bird species in Hawai'i and fit models to predict parasitemias from qPCR cycle threshold (Ct) values. We used these models to quantify the extent to which microscopy underestimated infection prevalence and to more accurately estimate infection patterns for each species for a large historical study done by microscopy. We found that most qPCR-positive wild-caught birds in Hawaii had low parasitemias (Ct scores ≥35), which were rarely detected by microscopy. The fraction of infections missed by microscopy differed substantially among eight species due to differences in species' parasitemia levels. Infection prevalence was likely 4-5-fold higher than previous microscopy estimates for three introduced species, including Zosterops japonicus, Hawaii's most abundant forest bird, which had low average parasitemias. In contrast, prevalence was likely only 1.5-2.3-fold higher than previous estimates for Himatione sanguinea and Chlorodrepanis virens, two native species with high average parasitemias. Our results indicate that relative patterns of infection among species differ substantially from those observed in previous microscopy studies, and that differences depend on variation in parasitemias among species. Although microscopy of blood smears is useful for estimating the frequency of different Plasmodium stages and host attributes, more sensitive quantitative methods, including qPCR, are needed to accurately estimate and compare infection prevalence among host species.

Sequence capture identifies fastidious chytrid fungi directly from host tissue.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) was discovered in 1998 as the cause of chytridiomycosis, an emerging infectious disease causing mass declines in amphibian populations worldwide. The rapid population declines of the 1970s-1990s were likely caused by the spread of a highly virulent lineage belonging to the Bd-GPL clade that was introduced to naïve susceptible populations. Multiple genetically distinct and regional lineages of Bd have since been isolated and sequenced, greatly expanding the known biological diversity within this fungal pathogen. To date, most Bd research has been restricted to the limited number of samples that could be isolated using culturing techniques, potentially causing a selection bias for strains that can grow on media and missing other unculturable or fastidious strains that are also present on amphibians. We thus attempted to characterize potentially non-culturable genetic lineages of Bd from distinct amphibian taxa using sequence capture technology on DNA extracted from host tissue and swabs. We focused our efforts on host taxa from two different regions that likely harbored distinct Bd clades: (1) wild-caught leopard frogs (Rana) from North America, and (2) a Japanese Giant Salamander (Andrias japonicus) at the Smithsonian Institution's National Zoological Park that exhibited signs of disease and tested positive for Bd using qPCR, but multiple attempts failed to isolate and culture the strain for physiological and genetic characterization. We successfully enriched for and sequenced thousands of fungal genes from both host clades, and Bd load was positively associated with number of recovered Bd sequences. Phylogenetic reconstruction placed all the Rana-derived strains in the Bd-GPL clade. In contrast, the A. japonicus strain fell within the Bd-Asia3 clade, expanding the range of this clade and generating additional genomic data to confirm its placement. The retrieved ITS locus matched public barcoding data from wild A. japonicus and Bd infections found on other amphibians in India and China, suggesting that this uncultured clade is widespread across Asia. Our study underscores the importance of recognizing and characterizing the hidden diversity of fastidious strains in order to reconstruct the spatiotemporal and evolutionary history of Bd. The success of the sequence capture approach highlights the utility of directly sequencing pathogen DNA from host tissue to characterize cryptic diversity that is missed by culture-reliant approaches.

Gene expression reveals immune response strategies of naïve Hawaiian honeycreepers experimentally infected with introduced avian malaria.

The unprecedented rise in the number of new and emerging infectious diseases in the last quarter century poses direct threats to human and wildlife health. The introduction to the Hawaiian archipelago of Plasmodium relictum and the mosquito vector that transmits the parasite has led to dramatic losses in endemic Hawaiian forest bird species. Understanding how mechanisms of disease immunity to avian malaria may evolve is critical as climate change facilitates increased disease transmission to high elevation habitats where malaria transmission has historically been low and the majority of the remaining extant Hawaiian forest bird species now reside. Here, we compare the transcriptomic profiles of highly susceptible Hawai'i 'amakihi (Chlorodrepanis virens) experimentally infected with P. relictum to those of uninfected control birds from a naïve high elevation population. We examined changes in gene expression profiles at different stages of infection to provide an in-depth characterization of the molecular pathways contributing to survival or mortality in these birds. We show that the timing and magnitude of the innate and adaptive immune response differed substantially between individuals that survived and those that succumbed to infection, and likely contributed to the observed variation in survival. These results lay the foundation for developing gene-based conservation strategies for Hawaiian honeycreepers by identifying candidate genes and cellular pathways involved in the pathogen response that correlate with a bird's ability to recover from malaria infection.

Immune priming prior to pathogen exposure sheds light on the relationship between host, microbiome and pathogen in disease.

Dynamic interactions between host, pathogen and host-associated microbiome dictate infection outcomes. Pathogens including Batrachochytrium dendrobatidis (Bd) threaten global biodiversity, but conservation efforts are hindered by limited understanding of amphibian host, Bd and microbiome interactions. We conducted a vaccination and infection experiment using Eastern hellbender salamanders (Cryptobranchus alleganiensis alleganiensis) challenged with Bd to observe infection, skin microbial communities and gene expression of host skin, pathogen and microbiome throughout the experiment. Most animals survived high Bd loads regardless of their vaccination status and vaccination did not affect pathogen load, but host gene expression differed based on vaccination. Oral vaccination (exposure to killed Bd) stimulated immune gene upregulation while topically and sham-vaccinated animals did not significantly upregulate immune genes. In early infection, topically vaccinated animals upregulated immune genes but orally and sham-vaccinated animals downregulated immune genes. Bd increased pathogenicity-associated gene expression in late infection when Bd loads were highest. The microbiome was altered by Bd, but there was no correlation between anti-Bd microbe abundance or richness and pathogen burden. Our observations suggest that hellbenders initially generate a vigorous immune response to Bd, which is ineffective at controlling disease and is subsequently modulated. Interactions with antifungal skin microbiota did not influence disease progression.

Microbiomes associated with avian malaria survival differ between susceptible Hawaiian honeycreepers and sympatric malaria-resistant introduced birds.

Of the estimated 55 Hawaiian honeycreepers (subfamily Carduelinae) only 17 species remain, nine of which the International Union for Conservation of Nature considers endangered. Among the most pressing threats to honeycreeper survival is avian malaria, caused by the introduced blood parasite Plasmodium relictum, which is increasing in distribution in Hawai'i as a result of climate change. Preventing further honeycreeper decline will require innovative conservation strategies that confront malaria from multiple angles. Research on mammals has revealed strong connections between gut microbiome composition and malaria susceptibility, illuminating a potential novel approach to malaria control through the manipulation of gut microbiota. One honeycreeper species, Hawai'i 'amakihi (Chlorodrepanis virens), persists in areas of high malaria prevalence, indicating they have acquired some level of immunity. To investigate if avian host-specific microbes may be associated with malaria survival, we characterized cloacal microbiomes and malaria infection for 174 'amakihi and 172 malaria-resistant warbling white-eyes (Zosterops japonicus) from Hawai'i Island using 16S rRNA gene metabarcoding and quantitative polymerase chain reaction. Neither microbial alpha nor beta diversity covaried with infection, but 149 microbes showed positive associations with malaria survivors. Among these were Escherichia and Lactobacillus spp., which appear to mitigate malaria severity in mammalian hosts, revealing promising candidates for future probiotic research for augmenting malaria immunity in sensitive endangered species.

Hawaiian songbird radiations.

Robert Fleischer and colleagues introduce the unique songbird fauna of Hawaii and the threats it faces.

Transcriptional response of individual Hawaiian Culex quinquefasciatus mosquitoes to the avian malaria parasite Plasmodium relictum.

BACKGROUND: Plasmodium parasites that cause bird malaria occur in all continents except Antarctica and are primarily transmitted by mosquitoes in the genus Culex. Culex quinquefasciatus, the mosquito vector of avian malaria in Hawai'i, became established in the islands in the 1820s. While the deadly effects of malaria on endemic bird species have been documented for many decades, vector-parasite interactions in avian malaria systems are relatively understudied. METHODS: To evaluate the gene expression response of mosquitoes exposed to a Plasmodium infection intensity known to occur naturally in Hawai'i, offspring of wild-collected Hawaiian Cx. quinquefasciatus were fed on a domestic canary infected with a fresh isolate of Plasmodium relictum GRW4 from a wild-caught Hawaiian honeycreeper. Control mosquitoes were fed on an uninfected canary. Transcriptomes of five infected and three uninfected individual mosquitoes were sequenced at each of three stages of the parasite life cycle: 24 h post feeding (hpf) during ookinete invasion; 5 days post feeding (dpf) when oocysts are developing; 10 dpf when sporozoites are released and invade the salivary glands. RESULTS: Differential gene expression analyses showed that during ookinete invasion (24 hpf), genes related to oxidoreductase activity and galactose catabolism had lower expression levels in infected mosquitoes compared to controls. Oocyst development (5 dpf) was associated with reduced expression of a gene with a predicted innate immune function. At 10 dpf, infected mosquitoes had reduced expression levels of a serine protease inhibitor, and further studies should assess its role as a Plasmodium agonist in C. quinquefasciatus. Overall, the differential gene expression response of Hawaiian Culex exposed to a Plasmodium infection intensity known to occur naturally in Hawai'i was low, but more pronounced during ookinete invasion. CONCLUSIONS: This is the first analysis of the transcriptional responses of vectors to malaria parasites in non-mammalian systems. Interestingly, few similarities were found between the response of Culex infected with a bird Plasmodium and those reported in Anopheles infected with human Plasmodium. The relatively small transcriptional changes observed in mosquito genes related to immune response and nutrient metabolism support conclusions of low fitness costs often documented in experimental challenges of Culex with avian Plasmodium.

Museum Genomics Provide Evidence for Persistent Genetic Differentiation in a Threatened Seabird Species in the Western Atlantic.

Connectivity among wildlife populations facilitates exchange of genetic material between groups. Changes to historical connectivity patterns resulting from anthropogenic activities can therefore have negative consequences for genetic diversity, particularly for small or isolated populations. DNA obtained from museum specimens can enable direct comparison of temporal changes in connectivity among populations, which can aid in conservation planning and contribute to the understanding of population declines. However, museum DNA can be degraded and only available in low quantities, rendering it challenging for use in population genomic analyses. Applications of genomic methodologies such as targeted sequencing address this issue by enabling capture of shared variable sites, increasing quantity and quality of recovered genomic information. We used targeted sequencing of ultra-conserved Elements (UCEs) to evaluate potential changes in connectivity and genetic diversity of roseate terns (Sterna dougallii) with a breeding distribution in the northwestern Atlantic and the Caribbean. Both populations experienced range contractions and population declines due to anthropogenic activity in the 20th century, which has the potential to alter historical connectivity regimes. Instead, we found that the two populations were differentiated historically as well as contemporaneously, with little evidence of migration between them for either time period. We also found no evidence for temporal changes in genetic diversity, although these interpretations may have been limited due to sequencing artifacts caused by the degraded nature of the museum samples. Population structuring in migratory seabirds is typically reflective of low rates of divergence and high connectivity among geographically segregated subpopulations. Our contrasting results suggest the potential presence of ecological mechanisms driving population differentiation, and highlight the value of targeted sequencing on DNA derived from museum specimens to uncover long-term patterns of genetic differentiation in wildlife populations.

Conservation genomics and systematics of a near-extinct island radiation.

Conservation benefits from incorporating genomics to explore the impacts of population declines, inbreeding, loss of genetic variation and hybridization. Here we use the near-extinct Mariana Islands reedwarbler radiation to showcase how ancient DNA approaches can allow insights into the population dynamics of extinct species and threatened populations for which historical museum specimens or material with low DNA yield (e.g., scats, feathers) are the only sources for DNA. Despite their having paraphyletic mitochondrial DNA (mtDNA), nuclear single nucleotide polymorphisms (SNPs) support the distinctiveness of critically endangered Acrocephalus hiwae and the other three species in the radiation that went extinct between the 1960s and 1990s. Two extinct species, A. yamashinae and A. luscinius, were deeply divergent from each other and from a third less differentiated lineage containing A. hiwae and extinct A. nijoi. Both mtDNA and SNPs suggest that the two isolated populations of A. hiwae from Saipan and Alamagan Islands are sufficiently distinct to warrant subspecies recognition and separate conservation management. We detected no significant differences in genetic diversity or inbreeding between Saipan and Alamagan, nor strong signatures of geographical structuring within either island. However, the implications of possible signatures of inbreeding in both Saipan and Alamagan, and long-term population declines in A. hiwae that pre-date modern anthropogenic threats require further study with denser population sampling. Our study highlights the value that conservation genomics studies of island radiations have as windows onto the possible future for the world's biota as climate change and habitat destruction increasingly fragment their ranges and contribute to rapid declines in population abundances.

An efficient method for simultaneous species, individual, and sex identification via in-solution single nucleotide polymorphism capture from low-quality scat samples.

Understanding predator population dynamics is important for conservation management because of the critical roles predators play within ecosystems. Noninvasive genetic sampling methods are useful for the study of predators like canids that can be difficult to capture or directly observe. Here, we introduce the FAECES* method (Fast and Accurate Enrichment of Canid Excrement for Species* and other analyses) which expands the toolbox for canid researchers and conservationists by using in-solution hybridization sequence capture to produce single nucleotide polymorphism (SNP) genotypes for multiple canid species from scat-derived DNA using a single enrichment. We designed a set of hybridization probes to genotype both coyotes (Canis latrans) and kit foxes (Vulpes macrotis) at hundreds of polymorphic SNP loci and we tested the probes on both tissues and field-collected scat samples. We enriched and genotyped by sequencing 52 coyote and 70 kit fox scats collected in and around a conservation easement in the Nevada Mojave Desert. We demonstrate that the FAECES* method produces genotypes capable of differentiating coyotes and kit foxes, identifying individuals and their sex, and estimating genetic diversity and effective population sizes, even using highly degraded, low-quantity DNA extracted from scat. We found that the study area harbours a large and diverse population of kit foxes and a relatively smaller population of coyotes. By replicating our methods in the future, conservationists can assess the impacts of management decisions on canid populations. The method can also be adapted and applied more broadly to enrich and sequence multiple loci from any species of interest using scat or other noninvasive genetic samples.

Independent evolutionary transitions to pueriparity across multiple timescales in the viviparous genus Salamandra.

The ability to bear live offspring, viviparity, has evolved multiple times across the tree of life and is a remarkable adaptation with profound life-history and ecological implications. Within amphibians the ancestral reproductive mode is oviparity followed by a larval life stage, but viviparity has evolved independently in all three amphibian orders. Two types of viviparous reproduction can be distinguished in amphibians; larviparity and pueriparity. Larviparous amphibians deliver larvae into nearby ponds and streams, while pueriparous amphibians deliver fully developed juveniles and thus do not require waterbodies for reproduction. Among amphibians, the salamander genus Salamandra is remarkable as it exhibits both inter- and intraspecific variation in the occurrence of larviparity and pueriparity. While the evolutionary relationships among Salamandra lineages have been the focus of several recent studies, our understanding of how often and when transitions between modes occurred is still incomplete. Furthermore, in species with intraspecific variation, the reproductive mode of a given population can only be confirmed by direct observation of births and thus the prevalence of pueriparous populations is also incompletely documented. We used sequence capture to obtain 1,326 loci from 94 individuals from across the geographic range of the genus, focusing on potential reproductive mode transition zones. We also report additional direct observations of pueriparous births for 20 new locations and multiple lineages. We identify at least five independent transitions from the ancestral mode of larviparity to pueriparity among and within species, occurring at different evolutionary timescales ranging from the Pliocene to the Holocene. Four of these transitions occurred within species. Based on a distinct set of markers and analyses, we also confirm previous findings of introgression between species and the need for taxonomic revisions in the genus. We discuss the implications of our findings with respect to the evolution of this complex trait, and the potential of using five independent convergent transitions for further studies on the ecological context in which pueriparity evolves and the genetic architecture of this specialized reproductive mode.

Transcriptome assembly and differential gene expression of the invasive avian malaria parasite Plasmodium relictum in Hawai'i.

The malaria parasite Plasmodium relictum (lineage GRW4) was introduced less than a century ago to the native avifauna of Hawai'i, where it has since caused major declines of endemic bird populations. One of the native bird species that is frequently infected with GRW4 is the Hawai'i 'amakihi (Chlorodrepanis virens). To achieve a better understanding of the transcriptional activities of this virulent parasite, we performed a controlled challenge experiment of 15 'amakihi that were infected with GRW4. Blood samples containing malaria parasites were collected at two time points (intermediate and peak infection stages) from host individuals that were either experimentally infected by mosquitoes or inoculated with infected blood. We then used RNA sequencing to assemble a high-quality blood transcriptome of P. relictum GRW4, allowing us to quantify parasite expression levels inside individual birds. We found few significant differences (one to two transcripts) in GRW4 expression levels between host infection stages and between inoculation methods. However, 36 transcripts showed differential expression levels among all host individuals, indicating a potential presence of host-specific gene regulation across hosts. To reduce the extinction risk of the remaining native bird species in Hawai'i, genetic resources of the local Plasmodium lineage are needed to enable further molecular characterization of this parasite. Our newly built Hawaiian GRW4 transcriptome assembly, together with analyses of the parasite's transcriptional activities inside the blood of Hawai'i 'amakihi, can provide us with important knowledge on how to combat this deadly avian disease in the future.

Comparative Analysis of Annotation Pipelines Using the First Japanese White-Eye (Zosterops japonicus) Genome.

Introduced into Hawaii in the early 1900s, the Japanese white-eye or warbling white-eye (Zosterops japonicus) is now the most abundant land bird in the archipelago. Here, we present the first Z. japonicus genome, sequenced from an individual in its invasive range. This genome provides an important resource for future studies in invasion genomics. We annotated the genome using two workflows-standalone AUGUSTUS and BRAKER2. We found that AUGUSTUS was more conservative with gene predictions when compared with BRAKER2. The final number of annotated gene models was similar between the two workflows, but standalone AUGUSTUS had over 70% of gene predictions with Blast2GO annotations versus under 30% using BRAKER2. Additionally, we tested whether using RNA-seq data from 47 samples had a significant impact on annotation quality when compared with data from a single sample, as generating RNA-seq data for genome annotation can be expensive and requires well preserved tissue. We found that more data did not significantly change the number of annotated genes using AUGUSTUS but using BRAKER2 the number increased substantially. The results presented here will aid researchers in annotating draft genomes of nonmodel species as well as those studying invasion success.

Identification of novel bacterial biomarkers to detect bird scavenging by invasive rats.

Rapid advances in genomic tools for use in ecological contexts and non-model systems allow unprecedented insight into interactions that occur beyond direct observation. We developed an approach that couples microbial forensics with molecular dietary analysis to identify species interactions and scavenging by invasive rats on native and introduced birds in Hawaii. First, we characterized bacterial signatures of bird carcass decay by conducting 16S rRNA high-throughput sequencing on chicken (Gallus gallus domesticus) tissues collected over an 11-day decomposition study in natural Hawaiian habitats. Second, we determined if field-collected invasive black rats (Rattus rattus; n = 51, stomach and fecal samples) had consumed birds using molecular diet analysis with two independent PCR assays (mitochondrial Cytochrome Oxidase I and Cytochrome b genes) and Sanger sequencing. Third, we characterized the gut microbiome of the same rats using 16S rRNA high-throughput sequencing and identified 15 bacterial taxa that were (a) detected only in rats that consumed birds (n = 20/51) and (b) were indicative of decaying tissue in the chicken decomposition experiment. We found that 18% of rats (n = 9/51) likely consumed birds as carrion by the presence of bacterial biomarkers of decayed tissue in their gut microbiome. One species of native bird (Myadestes obscurus) and three introduced bird species (Lophura leucomelanos, Meleagris gallopavo, Zosterops japonicus) were detected in the rats' diets, with individuals from these species (except L. nycthemera) likely consumed through scavenging. Bacterial biomarkers of bird carcass decay can persist through rat digestion and may serve as biomarkers of scavenging. Our approach can be used to reveal trophic interactions that are challenging to measure through direct observation.

Genetically modifying skin microbe to produce violacein and augmenting microbiome did not defend Panamanian golden frogs from disease.

We designed two probiotic treatments to control chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) on infected Panamanian golden frogs (Atelopus zeteki), a species that is thought to be extinct in the wild due to Bd. The first approach disrupted the existing skin microbe community with antibiotics then exposed the frogs to a core golden frog skin microbe (Diaphorobacter sp.) that we genetically modified to produce high titers of violacein, a known antifungal compound. One day following probiotic treatment, the engineered Diaphorobacter and the violacein-producing pathway could be detected on the frogs but the treatment failed to improve frog survival when exposed to Bd. The second approach exposed frogs to the genetically modified bacterium mixed into a consortium with six other known anti-Bd bacteria isolated from captive A. zeteki, with no preliminary antibiotic treatment. The consortium treatment increased the frequency and abundance of three probiotic isolates (Janthinobacterium, Chryseobacterium, and Stenotrophomonas) and these persisted on the skin 4 weeks after probiotic treatment. There was a temporary increase in the frequency and abundance of three other probiotics isolates (Masillia, Serratia, and Pseudomonas) and the engineered Diaphorobacter isolate, but they subsequently disappeared from the skin. This treatment also failed to reduce frog mortality upon exposure.

A genome-wide investigation of adaptive signatures in protein-coding genes related to tool behaviour in New Caledonian and Hawaiian crows.

Very few animals habitually manufacture and use tools. It has been suggested that advanced tool behaviour co-evolves with a suite of behavioural, morphological and life history traits. In fact, there are indications for such an adaptive complex in tool-using crows (genus Corvus species). Here, we sequenced the genomes of two habitually tool-using and ten non-tool-using crow species to search for genomic signatures associated with a tool-using lifestyle. Using comparative genomic and population genetic approaches, we screened for signals of selection in protein-coding genes in the tool-using New Caledonian and Hawaiian crows. While we detected signals of recent selection in New Caledonian crows near genes associated with bill morphology, our data indicate that genetic changes in these two lineages are surprisingly subtle, with little evidence at present for convergence. We explore the biological explanations for these findings, such as the relative roles of gene regulation and protein-coding changes, as well as the possibility that statistical power to detect selection in recently diverged lineages may have been insufficient. Our study contributes to a growing body of literature aiming to decipher the genetic basis of recently evolved complex behaviour.

Sustained immune activation is associated with susceptibility to the amphibian chytrid fungus.

The disease chytridiomycosis caused by the fungus Bd has devastated amphibian populations worldwide. Functional genomic contributions to host susceptibility remain enigmatic and vary between species and populations. We conducted experimental Bd infections in Rana yavapaiensis, a species with intraspecific variation in chytridiomycosis susceptibility, to assess the skin and spleen transcriptomic response to infection over time. We predicted that increased immune gene expression would be associated with a positive disease outcome, but we instead found that surviving frogs had significantly reduced immune gene expression compared to susceptible frogs and to uninfected controls. MHC class IIß gene expression was also significantly higher in susceptible frogs compared to surviving frogs. Furthermore, susceptible frogs expressed a significantly larger number of distinct class IIß alleles, demonstrating a negative correlation between class IIß expression, functional diversity, and survival. Expression of the MHC class IIß locus previously associated with Bd disease outcomes was a significant predictor of Bd infection intensity at early infection stages but not at late infection stages, suggesting initial MHC-linked immune processes are important for ultimate disease outcomes. We infer through disease association and phylogenetic analysis that certain MHC variants are linked to the immune expression that was negatively associated with survival, and we hypothesize that frogs that did not express these alleles could better survive infections. Our study finds that MHC expression at early and late infection stages predicts Bd infection intensity, and suggests that generating a sustained immune response against Bd may be counterproductive for surviving chytridiomycosis in this partially susceptible species.

Soil fungal communities differ between shaded and sun-intensive coffee plantations in El Salvador.

Coffea arabica is a highly traded commodity worldwide, and its plantations are habitat to a wide range of organisms. Coffee farmers are shifting away from traditional shade coffee farms in favor of sun-intensive, higher yield farms, which can impact local biodiversity. Using plant-associated microorganisms in biofertilizers, particularly fungi collected from local forests, to increase crop yields has gained traction among coffee producers. However, the taxonomic and spatial distribution of many fungi in coffee soil, nearby forests and biofertilizers is unknown. We collected soil samples from a sun coffee system, shade coffee system, and nearby forest from Izalco, Sonsonate, El Salvador. At each coffee system, we collected soil from the surface (upper) and 10 cm below the surface (lower), and from the coffee plant drip line (drip line) and the walkway between two plants (walkway). Forest soils were collected from the surface only. We used ITS metabarcoding to characterize fungal communities in soil and in the biofertilizer (applied in both coffee systems), and assigned fungal taxa to functional guilds using FUNGuild. In the sun and shade coffee systems, we found that drip line soil had higher richness in pathotrophs, symbiotrophs, and saprotrophs than walkway soil, suggesting that fungi select for microhabitats closer to coffee plants. Upper and lower soil depths did not differ in fungal richness or composition, which may reflect the shallow root system of Coffea arabica. Soil from shade, sun, and forest had similar numbers of fungal taxa, but differed dramatically in community composition, indicating that local habitat differences drive fungal species sorting among systems. Yet, some fungal taxa were shared among systems, including seven fungal taxa present in the biofertilizer. Understanding the distribution of coffee soil mycobiomes can be used to inform sustainable, ecologically friendly farming practices and identify candidate plant-growth promoting fungi for future studies.

Opening the door to greater phylogeographic inference in Southeast Asia: Comparative genomic study of five codistributed rainforest bird species using target capture and historical DNA.

Indochina and Sundaland are biologically diverse, interconnected regions of Southeast Asia with complex geographic histories. Few studies have examined phylogeography of bird species that span the two regions because of inadequate population sampling. To determine how geographic barriers/events and disparate dispersal potential have influenced the population structure, gene flow, and demographics of species that occupy the entire area, we studied five largely codistributed rainforest bird species: Arachnothera longirostra, Irena puella, Brachypodius atriceps, Niltava grandis, and Stachyris nigriceps. We accomplished relatively thorough sampling and data collection by sequencing ultraconserved elements (UCEs) using DNA extracted from modern and older (historical) specimens. We obtained a genome-wide set of 753-4,501 variable loci and 3,919-18,472 single nucleotide polymorphisms. The formation of major within-species lineages occurred within a similar span of time (0.5-1.5 mya). Major patterns in population genetic structure are largely consistent with the dispersal potential and habitat requirements of the study species. A population break across the Isthmus of Kra was shared only by the two hill/submontane insectivores (N. grandis and S. nigriceps). Across Sundaland, there is little structure in B. atriceps, which is a eurytopic and partially frugivorous species that often utilizes forest edges. Two other eurytopic species, A. longirostra and I. puella, possess highly divergent populations in peripheral Sunda Islands (Java and/or Palawan) and India. These species probably possess intermediate dispersal abilities that allowed them to colonize new areas, and then remained largely isolated subsequently. We also observed an east-west break in Indochina that was shared by B. atriceps and S. nigriceps, species with very different habitat requirements and dispersal potential. By analyzing high-throughput DNA data, our study provides an unprecedented comparative perspective on the process of avian population divergence across Southeast Asia, a process that is determined by geography, species characteristics, and the stochastic nature of dispersal and vicariance events.