Global population structure and adaptive evolution of aflatoxin‐producing fungi

Aflatoxins produced by several species in Aspergillus section Flavi are a significant problem in agriculture and a continuous threat to human health. To provide insights into the biology and global population structure of species in section Flavi, a total of 1,304 isolates were sampled across six species (A. flavus, A. parasiticus, A. nomius, A. caelatus, A. tamarii, and A. alliaceus) from single fields in major peanut‐growing regions in Georgia (USA), Australia, Argentina, India, and Benin (Africa). We inferred maximum‐likelihood phylogenies for six loci, both combined and separately, including two aflatoxin cluster regions (aflM/alfN and aflW/aflX) and four noncluster regions (amdS, trpC, mfs and MAT), to examine population structure and history. We also employed principal component and STRUCTURE analysis to identify genetic clusters and their associations with six different categories (geography, species, precipitation, temperature, aflatoxin chemotype profile, and mating type). Overall, seven distinct genetic clusters were inferred, some of which were more strongly structured by G chemotype diversity than geography. Populations of A. flavus S in Benin were genetically distinct from all other section Flavi species for the loci examined, which suggests genetic isolation. Evidence of trans‐speciation within two noncluster regions, whereby A. flavus S_BG strains from Australia share haplotypes with either A. flavus or A. parasiticus, was observed. Finally, while clay soil and precipitation may influence species richness in Aspergillus section Flavi, other region‐specific environmental and genetic parameters must also be considered.     

Comparative analyses of clinical and environmental populations of Cryptococcus neoformans in Botswana

Cryptococcus neoformans var. grubii (Cng) is the most common cause of fungal meningitis, and its prevalence is highest in sub‐Saharan Africa. Patients become infected by inhaling airborne spores or desiccated yeast cells from the environment, where the fungus thrives in avian droppings, trees and soil. To investigate the prevalence and population structure of Cng in southern Africa, we analysed isolates from 77 environmental samples and 64 patients. We detected significant genetic diversity among isolates and strong evidence of geographic structure at the local level. High proportions of isolates with the rare MAT a allele were observed in both clinical and environmental isolates; however, the mating‐type alleles were unevenly distributed among different subpopulations. Nearly equal proportions of the MAT a and MATα mating types were observed among all clinical isolates and in one environmental subpopulation from the eastern part of Botswana. As previously reported, there was evidence of both clonality and recombination in different geographic areas. These results provide a foundation for subsequent genomewide association studies to identify genes and genotypes linked to pathogenicity in humans.     

Genome‐wide association studies reveal genetic loci associated with plasma cholinesterase activity in ducks

Plasma cholinesterase (PCHE) activity is an important auxiliary test in human clinical medicine. It can distinguish liver diseases from non‐liver diseases and help detect organophosphorus poisoning. Animal experiments have confirmed that PCHE activity is associated with obesity and hypertension and changes with physiological changes in an animal's body. The objective of this study was to locate the genetic loci responsible for PCHE activity variation in ducks. PCHE activity of Pekin duck × mallard F2 ducks at 3 and 8 weeks of age were analyzed, and genome‐wide association studies were conducted. A region of about 1.5 Mb (21.8–23.3 Mb) on duck chromosome 9 was found to be associated with PCHE activity at both 3 and 8 weeks of age. The top SNP, g.22643979C>T in the butyrylcholinesterase (BCHE) gene, was most highly associated with PCHE activity at 3 weeks (?logP = 21.45) and 8 weeks (?logP = 27.60) of age. For the top SNP, the strong associations of CC and CT genotypes with low PCHE activity and the TT genotype with high PCHE activity indicates the dominant inheritance of low PCHE activity. Problems with block inheritance or linkage exist in this region. This study supports that BCHE is a functional gene for determining PCHE levels in ducks and that the genetic variations around this gene can cause phenotypic variations of PCHE activity.     

Genome‐wide association analyses reveal polygenic genomic architecture underlying divergent shell morphology in Spanish Littorina saxatilis ecotypes

Gene flow between diverging populations experiencing dissimilar ecological conditions can theoretically constrain adaptive evolution. To minimize the effect of gene flow, alleles underlying traits essential for local adaptation are predicted to be located in linked genome regions with reduced recombination. Local reduction in gene flow caused by selection is expected to produce elevated divergence in these regions. The highly divergent crab‐adapted and wave‐adapted ecotypes of the marine snail Littorina saxatilis present a model system to test these predictions. We used genome‐wide association (GWA) analysis of geometric morphometric shell traits associated with microgeographic divergence between the two L. saxatilis ecotypes within three separate sampling sites. A total of 477 snails that had individual geometric morphometric data and individual genotypes at 4,066 single nucleotide polymorphisms (SNPs) were analyzed using GWA methods that corrected for population structure among the three sites. This approach allowed dissection of the genomic architecture of shell shape divergence between ecotypes across a wide geographic range, spanning two glacial lineages. GWA revealed 216 quantitative trait loci (QTL) with shell size or shape differences between ecotypes, with most loci explaining a small proportion of phenotypic variation. We found that QTL were evenly distributed across 17 linkage groups, and exhibited elevated interchromosomal linkage, suggesting a genome‐wide response to divergent selection on shell shape between the two ecotypes. Shell shape trait‐associated loci showed partial overlap with previously identified outlier loci under divergent selection between the two ecotypes, supporting the hypothesis of diversifying selection on these genomic regions. These results suggest that divergence in shell shape between the crab‐adapted and wave‐adapted ecotypes is produced predominantly by a polygenic genomic architecture with positive linkage disequilibrium among loci of small effect.     

Surprisingly little population genetic structure in a fungus‐associated beetle despite its exploitation of multiple hosts

In heterogeneous environments, landscape features directly affect the structure of genetic variation among populations by functioning as barriers to gene flow. Resource‐associated population genetic structure, in which populations that use different resources (e.g., host plants) are genetically distinct, is a well‐studied example of how environmental heterogeneity structures populations. However, the pattern that emerges in a given landscape should depend on its particular combination of resources. If resources constitute barriers to gene flow, population differentiation should be lowest in homogeneous landscapes, and highest where resources exist in equal proportions. In this study, we tested whether host community diversity affects population genetic structure in a beetle (Bolitotherus cornutus) that exploits three sympatric host fungi. We collected B. cornutus from plots containing the three host fungi in different proportions and quantified population genetic structure in each plot using a panel of microsatellite loci. We found no relationship between host community diversity and population differentiation in this species; however, we also found no evidence of resource‐associated differentiation, suggesting that host fungi are not substantial barriers to gene flow. Moreover, we detected no genetic differentiation among B. cornutus populations separated by several kilometers, even though a previous study demonstrated moderate genetic structure on the scale of a few hundred meters. Although we found no effect of community diversity on population genetic structure in this study, the role of host communities in the structuring of genetic variation in heterogeneous landscapes should be further explored in a species that exhibits resource‐associated population genetic structure.     

Geography,seasonality, and host‐associated population structure influence the fecal microbiome of a genetically depauparate Arctic mammal

The Canadian Arctic is an extreme environment with low floral and faunal diversity characterized by major seasonal shifts in temperature, moisture, and daylight. Muskoxen (Ovibos moschatus) are one of few large herbivores able to survive this harsh environment. Microbiome research of the gastrointestinal tract may hold clues as to how muskoxen exist in the Arctic, but also how this species may respond to rapid environmental changes. In this study, we investigated the effects of season (spring/summer/winter), year (2007–2016), and host genetic structure on population‐level microbiome variation in muskoxen from the Canadian Arctic. We utilized 16S rRNA gene sequencing to characterize the fecal microbial communities of 78 male muskoxen encompassing two population genetic clusters. These clusters are defined by Arctic Mainland and Island populations, including the following: (a) two mainland sampling locations of the Northwest Territories and Nunavut and (b) four locations of Victoria Island. Between these geographic populations, we found that differences in the microbiome reflected host‐associated genetic cluster with evidence of migration. Within populations, seasonality influenced bacterial diversity with no significant differences between years of sampling. We found evidence of pathogenic bacteria, with significantly higher presence in mainland samples. Our findings demonstrate the effects of seasonality and the role of host population‐level structure in driving fecal microbiome differences in a large Arctic mammal.     

Sequence capture using AFLP‐generated baits: A cost‐effective method for high‐throughput phylogenetic and phylogeographic analysis

Target sequence capture is an efficient technique to enrich specific genomic regions for high‐throughput sequencing in ecological and evolutionary studies. In recent years, many sequence capture approaches have been proposed, but most of them rely on commercial synthetic baits which make the experiment expensive. Here, we present a novel sequence capture approach called AFLP‐based genome sequence capture (AFLP Capture). This method uses the AFLP (amplified fragment length polymorphism) technique to generate homemade capture baits without the need for prior genome information, thus is applicable to any organisms. In this approach, biotinylated AFLP fragments representing a random fraction of the genome are used as baits to capture the homologous fragments from genomic shotgun sequencing libraries. In a trial study, by using AFLP Capture, we successfully obtained 511 orthologous loci (>700,000 bp in total length) from 11 Odorrana species and more than 100,000 single nucleotide polymorphisms (SNPs) in four analyzed individuals of an Odorrana species. This result shows that our method can be used to address questions of various evolutionary depths (from interspecies level to intraspecies level). We also discuss the flexibility in bait preparation and how the sequencing data are analyzed. In summary, AFLP Capture is a rapid and flexible tool and can significantly reduce the experimental cost for phylogenetic studies that require analyzing genome‐scale data (hundreds or thousands of loci).     

Structural determinants in GABARAP required for the selective binding and recruitment of ALFY to LC3B‐positive structures

Several autophagy proteins contain an LC3‐interacting region (LIR) responsible for their interaction with Atg8 homolog proteins. Here, we show that ALFY binds selectively to LC3C and the GABARAPs through a LIR in its WD40 domain. Binding of ALFY to GABARAP is indispensable for its recruitment to LC3B‐positive structures and, thus, for the clearance of certain p62 structures by autophagy. In addition, the crystal structure of the GABARAP‐ALFY‐LIR peptide complex identifies three conserved residues in the GABARAPs that are responsible for binding to ALFY. Interestingly, introduction of these residues in LC3B is sufficient to enable its interaction with ALFY, indicating that residues outside the LIR‐binding hydrophobic pockets confer specificity to the interactions with Atg8 homolog proteins.     

Acidic lipase Lip I.3 from a Pseudomonas fluorescens‐like strain displays unusual properties and shows activity on secondary alcohols

Aims

Identification, cloning, expression and characterization of a novel lipase – Lip I.3 – from strain Pseudomonas CR‐611.

Methods and Results

The corresponding gene was identified and isolated by PCR‐amplification, cloned and expressed in Escherichia coli, and purified by refolding from inclusion bodies. Analysis of the deduced amino acid sequence revealed high homology with members of the bacterial lipase family I.3, showing 97% identity to a putative lipase from Pseudomonas fluorescens Pf0‐1, and 93% identity to a crystallized extracellular lipase from Pseudomonas sp. MIS38. A typical C‐terminal type I secretion signal and several putative Ca²⁺ binding sites were also identified. Experimental data confirmed that Lip I.3 requires Ca²⁺ ions for correct folding and activity. The enzyme differs from the previously reported family I.3 lipases in optimal pH, being the first acidophilic lipase reported in this family. Furthermore, Lip I.3 shows a strong preference for medium chain fatty acid esters and does not display interfacial activation. When tested for activity on secondary alcohol hydrolysis, Lip I.3 displayed higher efficiency on aromatic alcohols rather than on alkyl alcohols.

Conclusions

A new family I.3 lipase with unusual properties has been isolated, cloned and described. This will contribute to a better knowledge of family I.3 lipases, a family that has been scarcely explored, and that might provide a novel source of biocatalysts.

Significance and Impact of the Study

The unusual properties shown by Lip I.3 and the finding of activity and enantioselectivity on secondary alcohol esters may contribute to the development of new enzymatic tools for applied biocatalysis.     

The role of immigration and local adaptation on fine‐scale genotypic and phenotypic population divergence in a less mobile passerine

Dispersal and local patterns of adaptation play a major role on the ecological and evolutionary trajectory of natural populations. In this study, we employ a combination of genetic (25 microsatellite markers) and field‐based information (seven study years) to analyse the impact of immigration and local patterns of adaptation in two nearby (< 7 km) blue tit (Cyanistes caeruleus) populations. We used genetic assignment analyses to identify immigrant individuals and found that dispersal rate is female‐biased (72%). Data on lifetime reproductive success indicated that immigrant females produced fewer local recruits than their philopatric counterparts whereas immigrant males recruited more offspring than those that remained in their natal location. In spite of the considerably higher immigration rates of females, our results indicate that, in absolute terms, their demographic and genetic impact in the receiving populations is lower than that in immigrant males. Immigrants often brought novel alleles into the studied populations and a high proportion of them were transmitted to their recruits, indicating that the genetic impact of immigrants is not ephemeral. Although only a few kilometres apart, the two study populations were genetically differentiated and showed strong divergence in different phenotypic and life‐history traits. An almost absent inter‐population dispersal, together with the fact that both populations receive immigrants from different source populations, is probably the main cause of the observed pattern of genetic differentiation. However, phenotypic differentiation (P_ST) for all the studied traits greatly exceeded neutral genetic differentiation (F_ST), indicating that divergent natural selection is the prevailing factor determining the evolutionary trajectory of these populations. Our study highlights the importance of integrating individual‐ and population‐based approaches to obtain a comprehensive view about the role of dispersal and natural selection on structuring the genotypic and phenotypic characteristics of natural populations.     

Genotypic and phenotypic characterization of a large,diverse population of maize near‐isogenic lines

Genome‐wide association (GWA) studies can identify quantitative trait loci (QTL) putatively underlying traits of interest, and nested association mapping (NAM) can further assess allelic series. Near‐isogenic lines (NILs) can be used to characterize, dissect and validate QTL, but the development of NILs is costly. Previous studies have utilized limited numbers of NILs and introgression donors. We characterized a panel of 1270 maize NILs derived from crosses between 18 diverse inbred lines and the recurrent inbred parent B73, referred to as the nested NILs (nNILs). The nNILs were phenotyped for flowering time, height and resistance to three foliar diseases, and genotyped with genotyping‐by‐sequencing. Across traits, broad‐sense heritability (0.4–0.8) was relatively high. The 896 genotyped nNILs contain 2638 introgressions, which span the entire genome with substantial overlap within and among allele donors. GWA with the whole panel identified 29 QTL for height and disease resistance with allelic variation across donors. To date, this is the largest and most diverse publicly available panel of maize NILs to be phenotypically and genotypically characterized. The nNILs are a valuable resource for the maize community, providing an extensive collection of introgressions from the founders of the maize NAM population in a B73 background combined with data on six agronomically important traits and from genotyping‐by‐sequencing. We demonstrate that the nNILs can be used for QTL mapping and allelic testing. The majority of nNILs had four or fewer introgressions, and could readily be used for future fine mapping studies.     

Next‐generation sequencing (NGS)‐based identification of induced mutations in a doubly mutagenized tomato (Solanum lycopersicum) population

The identification of mutations in targeted genes has been significantly simplified by the advent of TILLING (Targeting Induced Local Lesions In Genomes), speeding up the functional genomic analysis of animals and plants. Next‐generation sequencing (NGS) is gradually replacing classical TILLING for mutation detection, as it allows the analysis of a large number of amplicons in short durations. The NGS approach was used to identify mutations in a population of Solanum lycopersicum (tomato) that was doubly mutagenized by ethylmethane sulphonate (EMS). Twenty‐five genes belonging to carotenoids and folate metabolism were PCR‐amplified and screened to identify potentially beneficial alleles. To augment efficiency, the 600‐bp amplicons were directly sequenced in a non‐overlapping manner in Illumina MiSeq, obviating the need for a fragmentation step before library preparation. A comparison of the different pooling depths revealed that heterozygous mutations could be identified up to 128‐fold pooling. An evaluation of six different software programs (camba , crisp , gatk unified genotyper , lofreq , snver and vipr ) revealed that no software program was robust enough to predict mutations with high fidelity. Among these, crisp and camba predicted mutations with lower false discovery rates. The false positives were largely eliminated by considering only mutations commonly predicted by two different software programs. The screening of 23.47 Mb of tomato genome yielded 75 predicted mutations, 64 of which were confirmed by Sanger sequencing with an average mutation density of 1/367 Kb. Our results indicate that NGS combined with multiple variant detection tools can reduce false positives and significantly speed up the mutation discovery rate.     

A multi‐parent advanced generation inter‐cross (MAGIC) population for genetic analysis and improvement of cowpea (Vigna unguiculata L. Walp.)

Multi‐parent advanced generation inter‐cross (MAGIC) populations are an emerging type of resource for dissecting the genetic structure of traits and improving breeding populations. We developed a MAGIC population for cowpea (Vigna unguiculata L. Walp.) from eight founder parents. These founders were genetically diverse and carried many abiotic and biotic stress resistance, seed quality and agronomic traits relevant to cowpea improvement in the United States and sub‐Saharan Africa, where cowpea is vitally important in the human diet and local economies. The eight parents were inter‐crossed using structured matings to ensure that the population would have balanced representation from each parent, followed by single‐seed descent, resulting in 305 F₈ recombinant inbred lines each carrying a mosaic of genome blocks contributed by all founders. This was confirmed by single nucleotide polymorphism genotyping with the Illumina Cowpea Consortium Array. These lines were on average 99.74% homozygous but also diverse in agronomic traits across environments. Quantitative trait loci (QTLs) were identified for several parental traits. Loci with major effects on photoperiod sensitivity and seed size were also verified by biparental genetic mapping. The recombination events were concentrated in telomeric regions. Due to its broad genetic base, this cowpea MAGIC population promises breakthroughs in genetic gain, QTL and gene discovery, enhancement of breeding populations and, for some lines, direct releases as new varieties.     

Fine‐scale population structure of Collichtys lucidus populations inferred from microsatellite markers

The spinyhead croaker Collichthys lucidus (Richardson) is a small sciaenid species distributed along the inshore waters of northwestern Pacific Ocean, and now has been listed as Key Protected Commercial Sources of Aquatic Animals and Plants in China. To delineate stock boundaries and inform conservation policy for its management, samples were collected from eight locations across the Chinese coastal waters and analyzed at nine microsatellite loci. C. lucidus populations showed low genetic diversity (expected heterozygosity = 0.445–0.542; observed heterozygosity = 0.392–0.539; Polymorphism Information Content = 0.268–0.684). Strong genetic fdifferentiation (F_st = 0.065–0.510, all significant after Bonferroni correction) among all populations and high levels of self‐recruitment (89.2%–91.5%) were observed, which suggested limited genetic exchange for this species. Clustering results of discriminant analysis of principal components and STRUCTURE found strong support for obvious genetic clusters (populations FZ, XM and SZ vs. populations SH, YRE, ZS, WZ and ND). The results of the present study not only supported the phylogeographic pattern of north‐south differentiation, but also suggested that C. lucidus populations may be predominantly sustained by self‐replenishment rather than by recruitment from distant populations.     

Amphibian‐killing chytrid in Brazil comprises both locally endemic and globally expanding populations

Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is the emerging infectious disease implicated in recent population declines and extinctions of amphibian species worldwide. Bd strains from regions of disease‐associated amphibian decline to date have all belonged to a single, hypervirulent clonal genotype (Bd‐GPL). However, earlier studies in the Atlantic Forest of southeastern Brazil detected a novel, putatively enzootic lineage (Bd‐Brazil), and indicated hybridization between Bd‐GPL and Bd‐Brazil. Here, we characterize the spatial distribution and population history of these sympatric lineages in the Brazilian Atlantic Forest. To investigate the genetic structure of Bd in this region, we collected and genotyped Bd strains along a 2400‐km transect of the Atlantic Forest. Bd‐Brazil genotypes were restricted to a narrow geographic range in the southern Atlantic Forest, while Bd‐GPL strains were widespread and largely geographically unstructured. Bd population genetics in this region support the hypothesis that the recently discovered Brazilian lineage is enzootic in the Atlantic Forest of Brazil and that Bd‐GPL is a more recently expanded invasive. We collected additional hybrid isolates that demonstrate the recurrence of hybridization between panzootic and enzootic lineages, thereby confirming the existence of a hybrid zone in the Serra da Graciosa mountain range of Paraná State. Our field observations suggest that Bd‐GPL may be more infective towards native Brazilian amphibians, and potentially more effective at dispersing across a fragmented landscape. We also provide further evidence of pathogen translocations mediated by the Brazilian ranaculture industry with implications for regulations and policies on global amphibian trade.     

Sampling related individuals within ponds biases estimates of population structure in a pond‐breeding amphibian

Effective conservation and management of pond‐breeding amphibians depends on the accurate estimation of population structure, demographic parameters, and the influence of landscape features on breeding‐site connectivity. Population‐level studies of pond‐breeding amphibians typically sample larval life stages because they are easily captured and can be sampled nondestructively. These studies often identify high levels of relatedness between individuals from the same pond, which can be exacerbated by sampling the larval stage. Yet, the effect of these related individuals on population genetic studies using genomic data is not yet fully understood. Here, we assess the effect of within‐pond relatedness on population and landscape genetic analyses by focusing on the barred tiger salamanders (Ambystoma mavortium) from the Nebraska Sandhills. Utilizing genome‐wide SNPs generated using a double‐digest RADseq approach, we conducted standard population and landscape genetic analyses using datasets with and without siblings. We found that reduced sample sizes influenced parameter estimates more than the inclusion of siblings, but that within‐pond relatedness led to the inference of spurious population structure when analyses depended on allele frequencies. Our landscape genetic analyses also supported different models across datasets depending on the spatial resolution analyzed. We recommend that future studies not only test for relatedness among larval samples but also remove siblings before conducting population or landscape genetic analyses. We also recommend alternative sampling strategies to reduce sampling siblings before sequencing takes place. Biases introduced by unknowingly including siblings can have significant implications for population and landscape genetic analyses, and in turn, for species conservation strategies and outcomes.     

Climate‐associated population declines reverse recovery and threaten future of an iconic high‐elevation plant

Although climate change is predicted to place mountain‐top and other narrowly endemic species at severe risk of extinction, the ecological processes involved in such extinctions are still poorly resolved. In addition, much of this biodiversity loss will likely go unobserved, and therefore largely unappreciated. The Haleakalā silversword is restricted to a single volcano summit in Hawai‘i, but is a highly charismatic giant rosette plant that is viewed by 1–2 million visitors annually. We link detailed local climate data to a lengthy demographic record, and combine both with a population‐wide assessment of recent plant mortality and recruitment, to show that after decades of strong recovery following successful management, this iconic species has entered a period of substantial climate‐associated decline. Mortality has been highest at the lower end of the distributional range, where most silverswords occur, and the strong association of annual population growth rates with patterns of precipitation suggests an increasing frequency of lethal water stress. Local climate data confirm trends toward warmer and drier conditions on the mountain, and signify a bleak outlook for silverswords if these trends continue. The silversword example foreshadows trouble for diversity in other biological hotspots, and illustrates how even well‐protected and relatively abundant species may succumb to climate‐induced stresses.