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

Building the climate resilience of arid zone freshwater biota

Abstract This report describes the research undertaken to develop national guidelines for climate adaptation planning for arid zone aquatic ecosystems and freshwater biodiversity. The guidelines focus on the protection of habitats and processes that support the persistence of freshwater biota under a changing climate. They support policy development, planning and on-ground actions. The major climate adaptation goal is to reduce the risk of the loss of aquatic habitats, deteriorating water quality and the extinction of aquatic and water-dependent species. A portfolio of adaptation approaches to maintaining aquatic habitats, the water resources that support them, and the species that depend upon them, is proposed within a framework of strategic adaptive management. This approach best addresses the uncertainty that exists as to how climatic changes will play out across the arid zone with respect to water availability and ecological processes.&nbsp; Recommended climate adaptation actions include: combining a national mapping program that identifies the major types of arid zone aquatic ecosystems, their biological assets and the surface water and groundwater resources that sustain them, with vulnerability assessments that determine the climate sensitivity and likely persistence of key habitats; recognising the importance of evolutionary refugia and ecological refuges as priority sites for arid zone climate adaptation planning and policy; protecting a dynamic (spatial and temporal) mosaic of perennial, temporary and ephemeral waterbodies to provide the range of conditions needed to support aquatic and water-dependent species with varying life history traits and dispersal abilities; maintaining the integrity of the dry sediments of temporary and ephemeral waters to ensure the persistence of viable seed and egg banks; recognising the importance of key hydrological and ecological processes, particularly connectivity and dispersal; reducing the existing stressors on aquatic ecosystems and aquatic biota; identifying new and novel waterbodies created by arid zone industries (e.g. mining, pastoralism) that could provide valuable offsets for aquatic systems lost through climatic drying; implementing climate adaptation actions within a strategic adaptive management framework accompanied by a dedicated program for indigenous, industry and local community engagement and education.&nbsp; Please cite this report as: Davis, J, Sunnucks, P, Thompson, R, Sim, L, Pavlova, A, Morán-Ordóñez, A, Brim Box, J, McBurnie, G, Pinder, A, Choy, S, McNeil D, Hughes, J, Sheldon, F, Timms, B, 2013,&nbsp;Building the climate resilience of arid zone freshwater biota, National Climate Change Adaptation Research Facility, Gold Coast, pp. 30. This report describes the research undertaken to develop national guidelines for climate adaptation planning for arid zone aquatic ecosystems and freshwater biodiversity. The guidelines focus on the protection of habitats and processes that support the persistence of freshwater biota under a changing climate. They support policy development, planning and on-ground actions. The major climate adaptation goal is to reduce the risk of the loss of aquatic habitats, deteriorating water quality and the extinction of aquatic and water-dependent species. A portfolio of adaptation approaches to maintaining aquatic habitats, the water resources that support them, and the species that depend upon them, is proposed within a framework of strategic adaptive management. This approach best addresses the uncertainty that exists as to how climatic changes will play out across the arid zone with respect to water availability and ecological processes.&nbsp; Recommended climate adaptation actions include: combining a national mapping program that identifies the major types of arid zone aquatic ecosystems, their biological assets and the surface water and groundwater resources that sustain them, with vulnerability assessments that determine the climate sensitivity and likely persistence of key habitats; recognising the importance of evolutionary refugia and ecological refuges as priority sites for arid zone climate adaptation planning and policy; protecting a dynamic (spatial and temporal) mosaic of perennial, temporary and ephemeral waterbodies to provide the range of conditions needed to support aquatic and water-dependent species with varying life history traits and dispersal abilities; maintaining the integrity of the dry sediments of temporary and ephemeral waters to ensure the persistence of viable seed and egg banks; recognising the importance of key hydrological and ecological processes, particularly connectivity and dispersal; reducing the existing stressors on aquatic ecosystems and aquatic biota; identifying new and novel waterbodies created by arid zone industries (e.g. mining, pastoralism) that could provide valuable offsets for aquatic systems lost through climatic drying; implementing climate adaptation actions within a strategic adaptive management framework accompanied by a dedicated program for indigenous, industry and local community engagement and education.&nbsp; Please cite this report as: Davis, J, Sunnucks, P, Thompson, R, Sim, L, Pavlova, A, Morán-Ordóñez, A, Brim Box, J, McBurnie, G, Pinder, A, Choy, S, McNeil D, Hughes, J, Sheldon, F, Timms, B, 2013, Building the climate resilience of arid zone freshwater biota, National Climate Change Adaptation Research Facility, Gold Coast, pp. 30

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

Two behavioural traits promote fine-scale species segregation and moderate hybridisation in a recovering sympatric fur seal population

Background: In systems where two or more species experience secondary contact, behavioural factors that regulate interspecific gene flow may be important for maintaining species boundaries and reducing the incidence of hybridisation. At subantarctic Macquarie Island, two species of fur seal breed in close proximity to one another, hybridise at very high levels (up to 21% of hybrid pups are born annually), yet retain discrete gene pools. Using spatial and genetic information collected for pups and adults over twelve years, we assessed two behavioural traits – interannual site fidelity and differences in habitat use between the species - as possible contributors to the maintenance of this species segregation. Further, we explored the breakdown of these traits in pure-species individuals and hybrids. Results: We found virtually complete spatial segregation of the parental species, with only one exception; a single territory that contained adults of both species and also the highest concentration of hybrid pups. The spatial distribution of each species was closely linked to habitat type (pebbled vs boulder beaches), with members of each species breeding almost exclusively on one type or the other but hybrids breeding on both or at the junction between habitats. Inter-annual site fidelity was high for both sexes of pure-species adults, with 66% of females and all males returning to the same territory or a neighbouring one in different years. An important consequence for pure females of breeding on the 'wrong' habitat type, and thus in a heterospecific aggregation, was the production of hybrid pups. Low habitat fidelity of hybrid females facilitated bi-directional backcrossing, resulting in more diverse hybrid offspring. Conclusion: In a disturbed system where two sympatric fur seal species breed in close proximity, discrete gene pools are retained by extremely fine-scale and strong spatial segregation of the species. Two behavioural traits were found to be important in maintaining this stable population structure, and habitat type was a strong indicator of where species locate and a potentially powerful predictor of future directions of hybridisation. A direct consequence of the breakdown of this trait was the production of hybrid offspring, which may have severe implications if hybrids have reduced fitness.Melanie L Lancaster, Simon D Goldsworthy and Paul Sunnuck

Accounting for cryptic population substructure enhances detection of inbreeding depression with genomic inbreeding coefficients: an example from a critically endangered marsupial

Characterizing inbreeding depression in wildlife populations can be critical to their conservation. Coefficients of individual inbreeding can be estimated from genome-wide marker data. The degree to which sensitivity of inbreeding coefficients to population genetic substructure alters estimates of inbreeding depression in wild populations is not well understood. Using generalized linear models, we tested the power of two frequently used inbreeding coefficients that are calculated from genome-wide SNP markers, FH and F^III, to predict four fitness traits estimated over two decades in an isolated population of the critically endangered Leadbeater's possum. FH estimates inbreeding as excess observed homozygotes relative to equilibrium expectations, whereas F^III quantifies allelic similarity between the gametes that formed an individual, and upweights rare homozygotes. We estimated FH and F^III from 1,575 genome-wide SNP loci in individuals with fitness trait data (N = 179–237 per trait), and computed revised coefficients, FHby group and F^IIIby group, adjusted for population genetic substructure by calculating them separately within two different genetic groups of individuals identified in the population. Using FH or F^III in the models, inbreeding depression was detected for survival to sexual maturity, longevity and whether individuals bred during their lifetime. F^IIIby group (but not FHby group) additionally revealed significant inbreeding depression for lifetime reproductive output (total offspring assigned to each individual). Estimates of numbers of lethal equivalents indicated substantial inbreeding load, but differing between inbreeding estimators. Inbreeding depression, declining population size, and low and declining genetic diversity suggest that genetic rescue may assist in preventing extinction of this unique Leadbeater's possum population. © 2020 John Wiley & Sons Lt

Novel microsatellite DNA markers indicate strict parthenogenesis and few genotypes in the invasive willow sawfly Nematus oligospilus

Invasive organisms can have major impacts on the environment. Some invasive organisms are parthenogenetic in their invasive range and, therefore, exist as a number of asexual lineages (=clones). Determining the reproductive mode of invasive species has important implications for understanding the evolutionary genetics of such species, more especially, for management-relevant traits. The willow sawfly Nematus oligospilus Förster (Hymenoptera: Tenthredinidae) has been introduced unintentionally into several countries in the Southern Hemisphere where it has subsequently become invasive. To assess the population expansion, reproductive mode and host-plant relationships of this insect, microsatellite markers were developed and applied to natural populations sampled from the native and expanded range, along with sequencing of the cytochrome-oxidase I mitochondrial DNA (mtDNA) region. Other tenthredinids across a spectrum of taxonomic similarity to N. oligospilus and having a range of life strategies were also tested. Strict parthenogenesis was apparent within invasive N. oligospilus populations throughout the Southern Hemisphere, which comprised only a small number of genotypes. Sequences of mtDNA were identical for all individuals tested in the invasive range. The microsatellite markers were used successfully in several sawfly species, especially Nematus spp. and other genera of the Nematini tribe, with the degree of success inversely related to genetic divergence as estimated from COI sequences. The confirmation of parthenogenetic reproduction in N. oligospilus and the fact that it has a very limited pool of genotypes have important implications for understanding and managing this species and its biology, including in terms of phenotypic diversity, host relationships, implications for spread and future adaptive change. It would appear to be an excellent model study system for understanding evolution of invasive parthenogens that diverge without sexual reproduction and genetic recombinatio

A revision of brain composition in Onychophora (velvet worms) suggests that the tritocerebrum evolved in arthropods

<p>Abstract</p> <p>Background</p> <p>The composition of the arthropod head is one of the most contentious issues in animal evolution. In particular, controversy surrounds the homology and innervation of segmental cephalic appendages by the brain. Onychophora (velvet worms) play a crucial role in understanding the evolution of the arthropod brain, because they are close relatives of arthropods and have apparently changed little since the Early Cambrian. However, the segmental origins of their brain neuropils and the number of cephalic appendages innervated by the brain - key issues in clarifying brain composition in the last common ancestor of Onychophora and Arthropoda - remain unclear.</p> <p>Results</p> <p>Using immunolabelling and neuronal tracing techniques in the developing and adult onychophoran brain, we found that the major brain neuropils arise from only the anterior-most body segment, and that two pairs of segmental appendages are innervated by the brain. The region of the central nervous system corresponding to the arthropod tritocerebrum is not differentiated as part of the onychophoran brain but instead belongs to the ventral nerve cords.</p> <p>Conclusions</p> <p>Our results contradict the assumptions of a tripartite (three-segmented) brain in Onychophora and instead confirm the hypothesis of bipartite (two-segmented) brain composition. They suggest that the last common ancestor of Onychophora and Arthropoda possessed a brain consisting of protocerebrum and deutocerebrum whereas the tritocerebrum evolved in arthropods.</p

Testing the neutral theory of molecular evolution using genomic data: a comparison of the human and bovine transcriptome

Despite growing evidence of rapid evolution in protein coding genes, the contribution of positive selection to intra- and interspecific differences in protein coding regions of the genome is unclear. We attempted to see if genes coding for secreted proteins and genes with narrow expression, specifically those preferentially expressed in the mammary gland, have diverged at a faster rate between domestic cattle (Bos taurus) and humans (Homo sapiens) than other genes and whether positive selection is responsible. Using a large data set, we identified groups of genes based on secretion and expression patterns and compared them for the rate of nonsynonymous (dN) and synonymous (dS) substitutions per site and the number of radical (Dr) and conservative (Dc) amino acid substitutions. We found evidence of rapid evolution in genes with narrow expression, especially for those expressed in the liver and mammary gland and for genes coding for secreted proteins. We compared common human polymorphism data with human-cattle divergence and found that genes with high evolutionary rates in human-cattle divergence also had a large number of common human polymorphisms. This argues against positive selection causing rapid divergence in these groups of genes. In most cases dN/dS ratios were lower in human-cattle divergence than in common human polymorphism presumably due to differences in the effectiveness of purifying selection between long-term divergence and short-term polymorphism

Limited population structure, genetic drift and bottlenecks characterise an endangered bird species in a dynamic, fire-prone ecosystem

Fire is a major disturbance process in many ecosystems world-wide, resulting in spatially and temporally dynamic landscapes. For populations occupying such environments, fire-induced landscape change is likely to influence population processes, and genetic patterns and structure among populations. The Mallee Emu-wren Stipiturus mallee is an endangered passerine whose global distribution is confined to fire-prone, semi-arid mallee shrublands in south-eastern Australia. This species, with poor capacity for dispersal, has undergone a precipitous reduction in distribution and numbers in recent decades. We used genetic analyses of 11 length-variable, nuclear loci to examine population structure and processes within this species, across its global range. Populations of the Mallee Emu-wren exhibited a low to moderate level of genetic diversity, and evidence of bottlenecks and genetic drift. Bayesian clustering methods revealed weak genetic population structure across the species\u27 range. The direct effects of large fires, together with associated changes in the spatial and temporal patterns of suitable habitat, have the potential to cause population bottlenecks, serial local extinctions and subsequent recolonisation, all of which may interact to erode and homogenise genetic diversity in this species. Movement among temporally and spatially shifting habitat, appears to maintain long-term genetic connectivity. A plausible explanation for the observed genetic patterns is that, following extensive fires, recolonisation exceeds in-situ survival as the primary driver of population recovery in this species. These findings suggest that dynamic, fire-dominated landscapes can drive genetic homogenisation of populations of species with low-mobility and specialised habitat that otherwise would be expected to show strongly structured populations. Such effects must be considered when formulating management actions to conserve species in fire-prone systems

Mitochondrial DNA Indicates Late Pleistocene Divergence of Populations of Heteronympha merope, an Emerging Model in Environmental Change Biology

Knowledge of historical changes in species range distribution provides context for investigating adaptive potential and dispersal ability. This is valuable for predicting the potential impact of environmental change on species of interest. Butterflies are one of the most important taxa for studying such impacts, and Heteronympha merope has the potential to provide a particularly valuable model, in part due to the existence of historical data on morphological traits and glycolytic enzyme variation. This study investigates the population genetic structure and phylogeography of H. merope, comparing the relative resolution achieved through partial DNA sequences of two mitochondrial loci, COI and ND5. These data are used to define the relationship between subspecies, showing that the subspecies are reciprocally monophyletic. On this basis, the Western Australian subspecies H. m. duboulayi is genetically distinct from the two eastern subspecies. Throughout the eastern part of the range, levels of migration and the timing of key population splits of potential relevance to climatic adaptation are estimated and indicate Late Pleistocene divergence both of the Tasmanian subspecies and of an isolated northern population from the eastern mainland subspecies H. m. merope. This information is then used to revisit historical data and provides support for the importance of clinal variation in wing characters, as well as evidence for selective pressure acting on allozyme loci phosphoglucose isomerase and phosphoglucomutase in H. merope. The study has thus confirmed the value of H. merope as a model organism for measuring responses to environmental change, offering the opportunity to focus on isolated populations, as well as a latitudinal gradient, and to use historical changes to test the accuracy of predictions for the future