genalex 6: genetic analysis in Excel. Population genetic software for teaching and research

genalex is a user‐friendly cross‐platform package that runs within Microsoft Excel, enabling population genetic analyses of codominant, haploid and binary data. Allele frequency‐based analyses include heterozygosity, F statistics, Nei's genetic distance, population assignment, probabilities of identity and pairwise relatedness. Distance‐based calculations include amova , principal coordinates analysis (PCA), Mantel tests, multivariate and 2D spatial autocorrelation and twogener . More than 20 different graphs summarize data and aid exploration. Sequence and genotype data can be imported from automated sequencers, and exported to other software. Initially designed as tool for teaching, genalex 6 now offers features for researchers as well. Documentation and the program are available at http://www.anu.edu.au/BoZo/GenAlEx/     

POPULATION GENETIC STRUCTURE OF COASTAL BOTTLENOSE DOLPHINS (TURSIOPS TRUNCATUS) IN THE NORTHERN BAHAMAS

Population substructure has important implications for a species' ecology and evolution. As such, knowledge of this structuring is critical for the conservation and management of natural populations. Among marine mammals, many examples exist of species that enjoy a broad geographical distribution, yet are characterized by fine‐scale population subdivisions. Coastal bottlenose dolphins have been studied extensively in a few regions globally, and these studies have highlighted a great diversity in both social strategies and demographic isolation. Here we use molecular genetic markers to examine the degree of population subdivision among three study sites separated by less than 250 km on Little Bahama Bank in the northern Bahamas. Mitochondrial DNA (mtDNA) sequence variation and microsatellite genotypes were used to assess partitioning of genetic variance among 56 individually recognized coastal ecotype bottlenose dolphins. Although resolved levels of genetic differentiation suggest gene flow among the three study sites, both nuclear and mitochondrial data indicate a significant degree of subdivision within the Little Bahama Bank population, and sex‐based analyses suggest that patterns of dispersal may not be strictly biased toward males. These results corroborate the site fidelity documented through long‐term photo‐identification studies in the NE Bahamas, and highlight the need to consider independent subpopulation units for the conservation and management of coastal bottlenose dolphins in the Bahamas.     

Spatial genetic analysis reveals high connectivity of tiger (Panthera tigris) populations in the Satpura–Maikal landscape of Central India

We investigated the spatial genetic structure of the tiger meta‐population in the Satpura–Maikal landscape of central India using population‐ and individual‐based genetic clustering methods on multilocus genotypic data from 273 individuals. The Satpura–Maikal landscape is classified as a global‐priority Tiger Conservation Landscape (TCL) due to its potential for providing sufficient habitat that will allow the long‐term persistence of tigers. We found that the tiger meta‐population in the Satpura–Maikal landscape has high genetic variation and very low genetic subdivision. Individual‐based Bayesian clustering algorithms reveal two highly admixed genetic populations. We attribute this to forest connectivity and high gene flow in this landscape. However, deforestation, road widening, and mining may sever this connectivity, impede gene exchange, and further exacerbate the genetic division of tigers in central India.     

Application of a SSR‐GBS marker system on investigation of European Hedgehog species and their hybrid zone dynamics

By applying second‐generation sequencing technologies to microsatellite genotyping, sequence information is produced which can result in high‐resolution population genetics analysis populations and increased replicability between runs and laboratories. In the present study, we establish an approach to study the genetic structure patterns of two European hedgehog species Erinaceaus europaeus and E. roumanicus. These species are usually associated with human settlements and are good models to study anthropogenic impacts on the genetic diversity of wild populations. The short sequence repeats genotyping by sequence (SSR‐GBS) method presented uses amplicon sequences to determine genotypes for which allelic variants can be defined according to both length and single nucleotide polymorphisms (SNPs). To evaluate whether complete sequence information improved genetic structure definition, we compared this information with datasets based solely on length information. We identified a total of 42 markers which were successfully amplified in both species. Overall, genotyping based on complete sequence information resulted in a higher number of alleles, as well as greater genetic diversity and differentiation between species. Additionally, the structure patterns were slightly clearer with a division between both species and some potential hybrids. There was some degree of genetic structure within species, although only in E. roumanicus was this related to geographical distance. The statistically significant results obtained by SSR‐GBS demonstrate that it is superior to electrophoresis‐based methods for SSR genotyping. Moreover, the greater reproducibility and throughput with lower effort which can be obtained with SSR‐GBS and the possibility to include degraded DNA into the analysis, allow for continued relevance of SSR markers during the genomic era.     

Within-breed heterozygosity of canine single nucleotide polymorphisms identified by across-breed comparison

Identification of single nucleotide polymorphisms (SNPs) by DNA sequence comparison across breeds is a strategy for developing genetic markers that are useful for many breeds. However, the heterozygosity of SNPs identified in this way might be severely reduced within breeds by inbreeding or genetic drift in the small effective population size of a breed (population subdivision). The effect of inbreeding and population subdivision on heterozygosity of SNPs in dog breeds has never been investigated in a systematic way. We determined the genotypes of dogs from three divergent breeds for SNPs in four canine genes (ACTC, LMNA, SCGB, and TYMS) identified by across-breed DNA sequence comparison, and compared the genotype frequencies to those expected under Hardy-Weinberg equilibrium (HWE). Although population subdivision significantly skewed allele frequencies across breeds for two of the SNPs, the deviations of observed heterozygosities compared with those expected within breeds were minimal. These results indicate that across-breed DNA sequence comparison is a reasonable strategy for identifying SNPs that are useful within many canine breeds.     

Evaluating summary statistics used to test for incomplete lineage sorting: mito‐nuclear discordance in the reef sponge Callyspongia vaginalis

Conflicting patterns of population differentiation between the mitochondrial and nuclear genomes (mito‐nuclear discordance) have become increasingly evident as multilocus data sets have become easier to generate. Incomplete lineage sorting (ILS) of nucDNA is often implicated as the cause of such discordance, stemming from the large effective population size of nucDNA relative to mtDNA. However, selection, sex‐biased dispersal and historical demography can also lead to mito‐nuclear discordance. Here, we compare patterns of genetic diversity and subdivision for six nuclear protein‐coding gene regions to those for mtDNA in a common Caribbean coral reef sponge, Callyspongia vaginalis, along the Florida reef tract. We also evaluated a suite of summary statistics to determine which are effective metrics for comparing empirical and simulated data when testing drivers of mito‐nuclear discordance in a statistical framework. While earlier work revealed three divergent and geographically subdivided mtDNACOI haplotypes separated by 2.4% sequence divergence, nuclear alleles were admixed with respect to mitochondrial clade and geography. Bayesian analysis showed that substitution rates for the nuclear loci were up to 7 times faster than for mitochondrial COI. Coalescent simulations and neutrality tests suggested that mito‐nuclear discordance in C. vaginalis is not the result of ILS in the nucDNA or selection on the mtDNA but is more likely caused by changes in population size. Sperm‐mediated gene flow may also influence patterns of population subdivision in the nucDNA.     

A unified DNA sequence and non‐DNA sequence mapping model of complex traits

Increasing evidence shows that quantitative inheritance is based on both DNA sequence and non‐DNA sequence variants. However, how to simultaneously detect these variants from a mapping study has been unexplored, hampering our effort to illustrate the detailed genetic architecture of complex traits. We address this issue by developing a unified model of quantitative trait locus (QTL) mapping based on an open‐pollinated design composed of randomly sampling maternal plants from a natural population and their half‐sib seeds. This design forms a two‐level hierarchical platform for a joint linkage‐linkage disequilibrium analysis of population structure. The EM algorithm was implemented to estimate and test DNA sequence‐based effects and non‐DNA sequence‐based effects of QTLs. We applied this model to analyze genetic mapping data from the OP design of a gymnosperm coniferous species, Torreya grandis, identifying 25 significant DNA sequence and non‐DNA sequence QTLs for seedling height and diameter growth in different years. Results from computer simulation show that the unified model has good statistical properties and is powerful for QTL detection. Our model enables the tests of how a complex trait is affected differently by DNA‐based effects and non‐DNA sequence‐based transgenerational effects, thus allowing a more comprehensive picture of genetic architecture to be charted and quantified.     

Population history in Arabidopsis halleri using multilocus analysis

A. halleri is a psuedometallophyte with a patchy distribution in Europe and is often spread by human activity. To determine the population history and whether this history is consistent with potential human effects, we surveyed nucleotide variation using 24 loci from 12 individuals in a large A. halleri population. The means of total and silent nucleotide variation (θ_W) are within the range expected for the species. The population genetic neutrality tests Tajima’s D and Wall’s B had significant composite results rejecting panmixia, and Approximate Bayesian Computation analysis revealed that a subdivision model better explained the variation than the standard neutral model, refugia (or admixture), bottleneck or change of population size models. A categorical regression analysis further supports the subdivision model, and under the subdivision model, the neutrality tests are no longer significant. The best support was for two source populations, a situation consistent with the mixing of two populations possibly mediated by human activity. This scenario might limit the genetic diversity and adaptive potential of the population. The non‐neutral population variation described here should be considered in bioinformatic searches for adaptation.     

PGEToolbox: A Matlab toolbox for population genetics and evolution

Assessing genetic diversity within populations is vital for understanding the nature of evolutionary processes at the molecular level. PGEToolbox is a Matlab-based open-sourced software package for data analysis in population genetics. The main features of this software are as follows: 1) capability for handling both DNA sequence polymorphisms and single nucleotide polymorphisms (SNPs), which include genotype and haplotype data; 2) exhaustive population genetic analyses and neutrality tests based on the coalescent theory; 3) extendibility and scalability for complex and large genome-wide datasets; 4) simple yet effective graphic user interfaces and sophisticated visualization of data and results. For academic uses, PGEToolbox is available free of charge at http://bioinformatics.org/pgetoolbox.     

Quantitative evaluation of intraspecific genetic diversity in a natural fish population using environmental DNA analysis

Recent advances in environmental DNA (eDNA) analysis using high‐throughput sequencing (HTS) enable evaluation of intraspecific genetic diversity in a population. As the intraspecific genetic diversity provides invaluable information for wildlife conservation and management, there is an increasing demand to apply eDNA analysis to population genetics and the phylogeography by quantitative evaluation of intraspecific diversity. However, quantitative evaluations of intraspecific genetic diversity using eDNA is not straightforward because the number of eDNA sequence reads obtained by HTS may not be an index of the quantity of eDNA. In this study, to quantitatively evaluate genetic diversity using eDNA analysis, we applied a quantitative eDNA metabarcoding method using the internal standard DNAs. We targeted Ayu (Plecoglossus altivelis altivelis) and added internal standard DNAs with known copy numbers to each eDNA sample obtained from three rivers during the library preparation process. The sequence reads of each Ayu haplotype were successfully converted to DNA copy numbers based on the relationship between the copy numbers and sequence reads of the internal standard DNAs. In all rivers, the calculated copy number of each haplotype showed a significant positive correlation with the haplotype frequency estimated by a capture‐based survey. Furthermore, estimates of genetic indicators such as nucleotide diversity based on the eDNA copy numbers were comparable with those estimated based on a capture‐based study. Our results demonstrate that eDNA analysis with internal standard DNAs enables reasonable quantification of intraspecific genetic diversity, and this method could thus be a promising tool in the field of population genetics and phylogeography.     

The coalescent process in models with selection, recombination and geographic subdivision

A population genetic model with a single locus at which balancing selection acts and many linked loci at which neutral mutations can occur is analysed using the coalescent approach. The model incorporates geographic subdivision with migration, as well as mutation, recombination, and genetic drift of neutral variation. It is found that geographic subdivision can affect genetic variation even with high rates of migration, providing that selection is strong enough to maintain different allele frequencies at the selected locus. Published sequence data from the alcohol dehydrogenase locus of Drosophila melanogaster are found to fit the proposed model slightly better than a similar model without subdivision.     

Sampling affects the detection of genetic subdivision and conservation implications for fisher in the Sierra Nevada

The small population of fisher (Pekania pennanti) in the southern Sierra Nevada is completely geographically and genetically isolated putting it at increased risk of extinction. Previous research using a clustered sampling scheme found a high amount of genetic subdivision within the southern Sierra Nevada population hypothesized to be caused by the Kings River Canyon. In this study, we use a larger and more geographically continuous set of genetic samples (n = 127) than was previously available to test this hypothesis and evaluate the genetic structure of the population. Both spatial and non-spatial population assignment models found three primary genetic clusters with moderate divergence between the clusters (F _ST = 0.05–0.13) at 10 microsatellite loci. These clusters appear to be associated with areas around the Kings River and Mountain Home State Demonstration Forest. One model also detected additional fine scale subdivision north of the Kings River that may be evidence of founder effects from a recent population expansion. The amount of population subdivision detected in this study is lower than previously found and indicates that while certain landscape features may reduce gene flow, these landscape features may be less of a barrier than initially thought. In the previous work, samples were collected in clusters which can inflate estimates of population structure by increasing the likelihood of oversampling related individuals. This study demonstrates how clustered sampling from a continuously distributed population can affect the assessment of population subdivision and influence conservation implications.     

bottlesim: a bottleneck simulation program for long‐lived species with overlapping generations

Population bottlenecks reduce genetic diversity and thus cause great concern in conservation biology. Previous theoretical studies often assume discrete generations in projecting declines in genetic diversity caused by bottlenecks. This assumption creates complexities when applying the models to long‐lived species with overlapping generations. bottlesim is a program for simulating bottlenecks to estimate the impact on genetic diversity; the novelties include an overlapping‐generation model, a wide range of reproductive systems, and flexible population size settings. With these features, bottlesim will be a useful tool for estimating the genetic consequences of bottlenecks, evaluating conservation plans, and performing power analysis.     

Loss of genetic variability in social spiders: genetic and phylogenetic consequences of population subdivision and inbreeding

The consequences of population subdivision and inbreeding have been studied in many organisms, particularly in plants. However, most studies focus on the short‐term consequences, such as inbreeding depression. To investigate the consequences of both population fragmentation and inbreeding for genetic variability in the longer term, we here make use of a natural inbreeding experiment in spiders, where sociality and accompanying population subdivision and inbreeding have evolved repeatedly. We use mitochondrial and nuclear data to infer phylogenetic relationships among 170 individuals of Anelosimus spiders representing 23 species. We then compare relative mitochondrial and nuclear genetic variability of the inbred social species and their outbred relatives. We focus on four independently derived social species and four subsocial species, including two outbred–inbred sister species pairs. We find that social species have 50% reduced mitochondrial sequence divergence. As inbreeding is not expected to reduce genetic variability in the maternally inherited mitochondrial genome, this suggests the loss of variation due to strong population subdivision, founder effects, small effective population sizes (colonies as individuals) and lineage turnover. Social species have < 10% of the nuclear genetic variability of the outbred species, also suggesting the loss of genetic variability through founder effects and/or inbreeding. Inbred sociality hence may result in reduction in variability through various processes. Sociality in most Anelosimus species probably arose relatively recently (0.1–2 mya), with even the oldest social lineages having failed to diversify. This is consistent with the hypothesis that inbred spider sociality represents an evolutionary dead end. Heterosis underlies a species potential to respond to environmental change and/or disease. Inbreeding and loss of genetic variability may thus limit diversification in social Anelosimus lineages and similarly pose a threat to many wild populations subject to habitat fragmentation or reduced population sizes.     

Effects of the Tanaka Line on the genetic structure of Bombax ceiba (Malvaceae) in dry‐hot valley areas of southwest China

Southwest China is an important biodiversity hotspot. The interactions among the complex topography, climate change, and ecological factors in the dry‐hot valley areas in southwest China may have profoundly affected the genetic structure of plant species in this region. In this study, we determined the effects of the Tanaka Line on genetic variation in the wild Bombax ceiba tree in southwest China. We sampled 224 individuals from 17 populations throughout the dry‐hot valley regions. Six polymorphic expressed sequence tag–simple sequence repeat primers were employed to sequence the PCR products using the first‐generation Sanger technique. The analysis based on population genetics suggested that B. ceiba exhibited a high level of gene diversity (H_E: 0.2377–0.4775; I: 0.3997–0.7848). The 17 populations were divided into two groups by cluster analysis, which corresponded to geographic characters on each side of the Tanaka Line. In addition, a Mantel test indicated that the phylogeographic structure among the populations could be fitted to the isolation‐by‐distance model (r² = .2553, p < .001). A barrier test indicated that there were obstacles among populations and between the two groups due to complex terrain isolation and geographic heterogeneity. We inferred that the Tanaka Line might have promoted the intraspecific phylogeographic subdivision and divergence of B. ceiba. These results provide new insights into the effects of the Tanaka Line on genetic isolation and population differentiation of plant species in southwest China.     

Vertebrate neural stem cell segmentation, tracking and lineaging with validation and editing

This protocol and the accompanying software program called LEVER (lineage editing and validation) enable quantitative automated analysis of phase-contrast time-lapse images of cultured neural stem cells. Images are captured at 5-min intervals over a period of 5-15 d as the cells proliferate and differentiate. LEVER automatically segments, tracks and generates lineage trees of the stem cells from the image sequence. In addition to generating lineage trees capturing the population dynamics of clonal development, LEVER extracts quantitative phenotypic measurements of cell location, shape, movement and size. When available, the system can include biomolecular markers imaged using fluorescence. It then displays the results to the user for highly efficient inspection and editing to correct any errors in the segmentation, tracking or lineaging. To enable high-throughput inspection, LEVER incorporates features for rapid identification of errors and for learning from user-supplied corrections to automatically identify and correct related errors.     

Comparative landscape genetics of two river frog species occurring at different elevations on Mount Kilimanjaro

Estimating population connectivity and species' abilities to disperse across the landscape is crucial for understanding the long‐term persistence of species in changing environments. Surprisingly, few landscape genetic studies focused on tropical regions despite the alarming extinction rates within these ecosystems. Here, we compared the influence of landscape features on the distribution of genetic variation of an Afromontane frog, Amietia wittei, with that of its more broadly distributed lowland congener, Amietia angolensis, on Mt. Kilimanjaro, Tanzania. We predicted high gene flow in the montane species with movements enhanced through terrestrial habitats of the continuous rainforest. In contrast, dispersal might be restricted to aquatic corridors and reduced by anthropogenic disturbance in the lowland species. We found high gene flow in A. wittei relative to other montane amphibians. Nonetheless, gene flow was lower than in the lowland species which showed little population structure. Least‐cost path analysis suggested that dispersal is facilitated by stream networks in both species, but different landscape features were identified to influence connectivity among populations. Contrary to a previous study, gene flow in the lowland species was negatively correlated with the presence of human settlements. Also, genetic subdivision in A. wittei did not coincide with specific physical barriers as in other landscape genetic studies, suggesting that factors other than topography may contribute to population divergence. Overall, these results highlight the importance of a comparative landscape genetic approach for assessing the influence of the landscape matrix on population connectivity, particularly because nonintuitive results can alter the course of conservation and management.     

Historical range contraction,and not taxonomy,explains the contemporary genetic structure of the Australian tree <Emphasis Type="Italic">Acacia dealbata</Emphasis> Link

Irrespective of its causes, strong population genetic structure indicates a lack of gene flow. Understanding the processes that underlie such structure, and the spatial patterns it causes, is valuable for conservation efforts such as restoration. On the other hand, when a species is invasive outside its native range, such information can aid management in the non-native range. Here we explored the genetic characteristics of the Australian tree Acacia dealbata in its native range. Two subspecies of A. dealbata have previously been described based on morphology and environmental requirements, but recent phylogeographic data raised questions regarding the validity of this taxonomic subdivision. The species has been widely planted within and outside its native Australian range and is also a highly successful invasive species in many parts of the world. We employed microsatellite markers to investigate the population genetic diversity and structure among 42 A. dealbata populations from across the species’ native range. We also tested whether environmental variables purportedly relevant for the putative separation of subspecies are linked with population genetic differentiation. We found no relationship between population genetic structure of A. dealbata in Australia and these environmental features. Rather, we identified two geographically distinct genetic clusters that corresponded with populations in the northeastern part of mainland Australia, and the southern mainland and Tasmanian range of the species. Our results do not support the taxonomic subdivision of the species into two distinct subspecies based on environmental features. We therefore assume that the observed morphological differences between the putative subspecies are plastic phenotypic responses. This study provides population genetic information that will be useful for the conservation of the species within Australia as well as to better understand the invasion dynamics of A. dealbata.     

widgetcon: A website and program for quick conversion among common population genetic data formats

One of the most tedious steps in genetic data analyses is the reformatting data generated with one program for use with other applications. This conversion is necessary because comprehensive evaluation of the data may be based on different algorithms included in diverse software, each requiring a distinct input format. A platform‐independent and freely available program or a web‐based tool dedicated to such reformatting can save time and efforts in data processing. Here, we report widgetcon , a website and a program which has been developed to quickly and easily convert among various molecular data formats commonly used in phylogenetic analysis, population genetics, and other fields. The web‐based service is available at https://www.widgetcon.net . The program and the website convert the major data formats in four basic steps in less than a minute. The resource will be a useful tool for the research community and can be updated to include more formats and features in the future.