Development and application of three-tiered nuclear genetic markers for basal Hexapods using single-stranded conformation polymorphism coupled with targeted DNA sequencing

BACKGROUND: Molecular genetic approaches have much to offer population biology. Despite recent advances, convenient techniques to develop and screen highly-resolving markers can be limiting for some applications and taxa. We describe an improved PCR-based, cloning-free, nuclear marker development procedure, in which single-stranded conformation polymorphism (SSCP) plays a central role. Sequence-variable alleles at putative nuclear loci are simultaneously identified and isolated from diploid tissues. Based on a multiple allele alignment, locus-specific primers are designed in conserved regions, minimizing 'null' alleles. Using two undescribed endemic Australian Collembola as exemplars, we outline a comprehensive approach to generating and validating suites of codominant, sequence-yielding nuclear loci for previously unstudied invertebrates. RESULTS: Six markers per species were developed without any baseline genetic information. After evaluating the characteristics of each new locus via SSCP pre-screening, population samples were genotyped on the basis of either DNA sequence, restriction site, or insertion/deletion variation, depending on which assay was deemed most appropriate. Polymorphism was generally high (mean of nine alleles per locus), and the markers were capable of resolving population structuring over very fine spatial scales (<100 km). SSCP coupled with targeted DNA sequencing was used to obtain genotypic, genic and genealogical information from six loci (three per species). Phylogeographic analysis identified introns as being most informative. CONCLUSION: The comprehensive approach presented here feasibly overcomes technical hurdles of (i) developing suitably polymorphic nuclear loci for non-model organisms, (ii) physically isolating nuclear allele haplotypes from diploid tissues without cloning, and (iii) genotyping population samples on the basis of nuclear DNA sequence variation

Ecological drivers of species distributions and niche overlap for three subterranean termite species in the southern Appalachian mountains, USA

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. In both managed and unmanaged forests, termites are functionally important members of the dead-wood-associated (saproxylic) insect community. However, little is known about regional-scale environmental drivers of geographic distributions of termite species, and how these environmental factors impact co-occurrence among congeneric species. Here we focus on the southern Appalachian Mountains-a well-known center of endemism for forest biota-and use Ecological Niche Modeling (ENM) to examine the distributions of three species of Reticulitermes termites (i.e., R. flavipes, R. virginicus, and R. malletei). To overcome deficiencies in public databases, ENMs were underpinned by field-collected high-resolution occurrence records coupled with molecular taxonomic species identification. Spatial overlap among areas of predicted occurrence of each species was mapped, and aspects of niche similarity were quantified. We also identified environmental factors that most strongly contribute to among-species differences in occupancy. Overall, we found that R. flavipes and R. virginicus showed significant niche divergence, which was primarily driven by dry-season precipitation. Also, all three species were most likely to co-occur in the mid-latitudes of the study area (i.e., northern Alabama and Georgia, eastern Tennessee and western North Carolina), which is an area of considerable topographic complexity. This work provides important baseline information for follow-up studies of local-scale drivers of these species’ distributions. It also identifies specific geographic areas where future assessments of the frequency of true syntopy vs. micro-allopatry, and associated interspecific competitive interactions, should be focused

The effect of sampling density and study area size on landscape genetics inferences for the Mississippi slimy salamander (Plethodon mississippi)

In landscape genetics, it is largely unknown how choices regarding sampling density and study area size impact inferences upon which habitat features impede vs. facilitate gene flow. While it is recommended that sampling locations be spaced no further apart than the average individual\u27s dispersal distance, for low-mobility species, this could lead to a challenging number of sampling locations, or an unrepresentative study area. We assessed the effects of sampling density and study area size on landscape genetic inferences for a dispersal-limited amphibian, Plethodon mississippi, via analysis of nested datasets. Microsatellite-based genetic distances among individuals were divided into three datasets representing sparse sampling across a large study area, dense sampling across a small study area, or sparse sampling across the same small study area. These datasets were a proxy for gene flow (i.e., the response variable) in maximum-likelihood population effects models that assessed the nature and strength of their relationship with each of five land-use classes (i.e., potential predictor variables). Comparisons of outcomes were based on the rank order of effect, sign of effect (i.e., gene flow resistance vs. facilitation), spatial scale of effect, and functional relationship with gene flow. The best-fit model for each dataset had the same sign of effect for hardwood forests, manmade structures, and pine forests, indicating the impacts of these land-use classes on dispersal and gene flow in P. mississippi are robust to sampling scheme. Contrasting sampling densities led to a different inferred functional relationship between agricultural areas and gene flow. Study area size appeared to influence the scale of effect of manmade structures and the sign of effect of pine forests. Our findings provided evidence for an influence of sampling density, study area size, and sampling effort upon inferences. Accordingly, we recommend iterative subsampling of empirical datasets and continued investigation into the sensitivities of landscape genetic analyses using simulations

Genetic insights into family group co-occurrence in Cryptocercus punctulatus, a sub-social woodroach from the southern Appalachian Mountains

ABSTRACT The wood-feeding cockroach Cryptocercus punctulatus Scudder (Blattodea: Cryptocercidae) is an important member of the dead wood (saproxylic) community in montane forests of the southeastern United States. However, its population biology remains poorly understood. Here, aspects of family group co-occurrence were characterized to provide basic information that can be extended by studies on the evolution and maintenance of sub-sociality. Broad sampling across the species&apos; range was coupled with molecular data (mitochondrial DNA (mtDNA) sequences). The primary questions were

Nuclear gene phylogeography using PHASE: dealing with unresolved genotypes, lost alleles, and systematic bias in parameter estimation

<p>Abstract</p> <p>Background</p> <p>A widely-used approach for screening nuclear DNA markers is to obtain sequence data and use bioinformatic algorithms to estimate which two alleles are present in heterozygous individuals. It is common practice to omit unresolved genotypes from downstream analyses, but the implications of this have not been investigated. We evaluated the haplotype reconstruction method implemented by PHASE in the context of phylogeographic applications. Empirical sequence datasets from five non-coding nuclear loci with gametic phase ascribed by molecular approaches were coupled with simulated datasets to investigate three key issues: (1) haplotype reconstruction error rates and the nature of inference errors, (2) dataset features and genotypic configurations that drive haplotype reconstruction uncertainty, and (3) impacts of omitting unresolved genotypes on levels of observed phylogenetic diversity and the accuracy of downstream phylogeographic analyses.</p> <p>Results</p> <p>We found that PHASE usually had very low false-positives (i.e., a low rate of confidently inferring haplotype pairs that were incorrect). The majority of genotypes that could not be resolved with high confidence included an allele occurring only once in a dataset, and genotypic configurations involving two low-frequency alleles were disproportionately represented in the pool of unresolved genotypes. The standard practice of omitting unresolved genotypes from downstream analyses can lead to considerable reductions in overall phylogenetic diversity that is skewed towards the loss of alleles with larger-than-average pairwise sequence divergences, and in turn, this causes systematic bias in estimates of important population genetic parameters.</p> <p>Conclusions</p> <p>A combination of experimental and computational approaches for resolving phase of segregating sites in phylogeographic applications is essential. We outline practical approaches to mitigating potential impacts of computational haplotype reconstruction on phylogeographic inferences. With targeted application of laboratory procedures that enable unambiguous phase determination via physical isolation of alleles from diploid PCR products, relatively little investment of time and effort is needed to overcome the observed biases.</p

Weak spatial-genetic structure in a native invasive, the southern pine beetle (Dendroctonus frontalis), across the eastern United States

The southern pine beetle, Dendroctonus frontalis, is a native pest of pine trees that has recently expanded its range into the northeastern United States. Understanding its colonization, dispersal, and connectivity will be critical for mitigating negative economic and ecological impacts in the newly invaded areas. Characterization of spatial-genetic structure can contribute to this; however, previous studies have reached different conclusions about regional population genetic structure, with one study reporting a weak east-west pattern, and the most recent reporting an absence of structure. Here we systematically assessed several explanations for the absence of spatial-genetic structure. To do this, we developed nine new microsatellite markers and combined them with an existing 24-locus data matrix for the same individuals. We then reanalyzed this full dataset alongside datasets in which certain loci were omitted with the goal of creating more favorable signal to noise ratios. We also partitioned the data based on the sex of D. frontalis individuals, and then employed a broad suite of genotypic clustering and isolation-by-distance (IBD) analyses. We found that neither inadequate information content in the molecular marker set, nor unfavorable signal-to-noise ratio, nor insensitivity of the analytical approaches could explain the absence of structure. Regardless of dataset composition, there was little evidence for clusters (i.e., distinct geo-genetic groups) or clines (i.e., gradients of increasing allele frequency differences over larger geographic distances), with one exception: significant IBD was repeatedly detected using an individual-based measure of relatedness whenever datasets included males (but not for female-only datasets). This is strongly indicative of broad-scale female-biased dispersal, which has not previously been reported for D. frontalis, in part owing to logistical limitations of direct approaches (e.g., capture-mark-recapture). Weak spatial-genetic structure suggests long-distance connectivity and that gene flow is high, but additional research is needed to understand range expansion and outbreak dynamics in this species using alternate approaches

Two divergent genetic lineages within the horned passalus beetle, Odontotaenius disjunctus (Coleoptera: Passalidae): An emerging model for insect behavior, physiology, and microbiome research

© 2019 by the authors. The horned passalus (Odontotaenius disjunctus) is one of the most extensively studied saproxylic beetles in the eastern United States. For several decades this species has been the subject of investigations into the behaviors associated with subsociality as well as physiological responses to stress, and, most recently, the composition of its gut microbiome has been closely examined. However, no published study to date has characterized this beetle’s broad-scale population genetic structure. Here, we conducted intensive geographic sampling throughout the southern Appalachian Mountains and surrounding areas and then assessed mitochondrial DNA (mtDNA) sequence variation among individuals. Unexpectedly, we discovered two divergent, yet broadly sympatric, mtDNA clades. Indeed, the magnitude of divergence between- vs. within-clades ranged from 5.9 to 7.5×, depending on the dataset under consideration, and members of the two lineages were often syntopic (i.e., found in the same rotting log). Given the potential implications for past and future studies on behavior, physiology, and the gut microbiome, we developed a simple cost-efficient molecular assay (i.e., polymerase chain reaction restriction fragment length polymorphism; PCR-RFLP) to rapidly determine mtDNA clade membership of O. disjunctus individuals. We suggest that the evolutionary processes that gave rise to the emergence and persistence of divergent sympatric lineages reported here warrant investigation, as this type of spatial-genetic pattern appears to be rare among southern Appalachian forest invertebrates

Identification of Eastern United States Reticulitermes Termite Species via PCR-RFLP, Assessed Using Training and Test Data

Reticulitermes termites play key roles in dead wood decomposition and nutrient cycling in forests. They also damage man-made structures, resulting in considerable economic loss. In the eastern United States, five species (R. flavipes, R. virginicus, R. nelsonae, R. hageni and R. malletei) have overlapping ranges and are difficult to distinguish morphologically. Here we present a molecular tool for species identification. It is based on polymerase chain reaction (PCR) amplification of a section of the mitochondrial cytochrome oxidase subunit II gene, followed by a three-enzyme restriction fragment length polymorphism (RFLP) assay, with banding patterns resolved via agarose gel electrophoresis. The assay was designed using a large set of training data obtained from a public DNA sequence database, then evaluated using an independent test panel of Reticulitermes from the Southern Appalachian Mountains, for which species assignments were determined via phylogenetic comparison to reference sequences. After refining the interpretive framework, the PCR-RFLP assay was shown to provide accurate identification of four co-occurring species (the fifth species, R. hageni, was absent from the test panel, so accuracy cannot yet be extended to training data). The assay is cost- and time-efficient, and will help improve knowledge of Reticulitermes species distributions

Identification of Genetically Important Individuals of the Rediscovered Floreana Galápagos Giant Tortoise (Chelonoidis elephantopus) Provide Founders for Species Restoration Program

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.Species are being lost at an unprecedented rate due to human-driven environmental changes. The cases in which species declared extinct can be revived are rare. However, here we report that a remote volcano in the Galápagos Islands hosts many giant tortoises with high ancestry from a species previously declared as extinct: Chelonoidis elephantopus or the Floreana tortoise. Of 150 individuals with distinctive morphology sampled from the volcano, genetic analyses revealed that 65 had C. elephantopus ancestry and thirty-two were translocated from the volcano’s slopes to a captive breeding center. A genetically informed captive breeding program now being initiated will, over the next decades, return C. elephantopus tortoises to Floreana Island to serve as engineers of the island’s ecosystems. Ironically, it was the haphazard translocations by mariners killing tortoises for food centuries ago that created the unique opportunity to revive this “lost” species today. CORRECTION PUBLISHED 13 SEPT 2017: The original version of this Article contained an error in the title of the paper, where the word “Provides” was incorrectly given as “Provide”. This has now been corrected in the PDF and HTML versions of the Article and in the accompanying Supplementary Information file. The attached full-text article has been updated to incorporate this change

Genomic regions associated with pseudorabies virus infection status in naturally infected feral swine (Sus scrofa)

Pseudorabies virus (PRV)—the causative agent of Aujeszky’s disease—was eliminated from commercial pig production herds in the United States (US) in 2004; however, PRV remains endemic among invasive feral swine (Sus scrofa). The circulation of PRV among abundant, widespread feral swine populations poses a sustained risk for disease spillover to production herds. Risk–based surveillance has been successfully implemented for PRV in feral swine populations in the US. However, understanding the role of host genetics in infection status may offer new insights into the epidemiology and disease dynamics of PRV that can be applied to management strategies. Genetic mechanisms underlying host susceptibility to PRV are relatively unknown; therefore, we sought to identify genomic regions associated with PRV infection status among naturally infected feral swine using genome–wide association studies (GWAS) and gene set enrichment analysis of single nucleotide polymorphism data (GSEA–SNP). Paired serological and genotypic data were collected from 6,081 feral swine distributed across the invaded range within the contiguous US. Three complementary study populations were developed for GWAS: 1) comprehensive population consisting of feral swine throughout the invaded range within the contiguous US; 2) population of feral swine under high, but temporally variable PRV infection pressure; and 3) population of feral swine under temporally stable, high PRV infection pressure. We identified one intronic SNP associated with PRV infection status within candidate gene AKAP6 on autosome 7. Various gene sets linked to metabolic pathways were enriched in the GSEA–SNP. Ultimately, improving disease surveillance efforts in feral swine will be critical to further understanding of the role host genetics play in PRV infection status, helping secure the health of commercial pork production