Sequence clustering threshold has little effect on the recovery of microbial community structure

Analysis of microbial community structure by multivariate ordination methods, using data obtained by high‐throughput sequencing of amplified markers (i.e., DNA metabarcoding), often requires clustering of DNA sequences into operational taxonomic units (OTUs). Parameters for the clustering procedure tend not to be justified but are set by tradition rather than being based on explicit knowledge. In this study, we explore the extent to which ordination results are affected by variation in parameter settings for the clustering procedure. Amplicon sequence data from nine microbial community studies, representing different sampling designs, spatial scales and ecosystems, were subjected to clustering into OTUs at seven different similarity thresholds (clustering thresholds) ranging from 87% to 99% sequence similarity. The 63 data sets thus obtained were subjected to parallel DCA and GNMDS ordinations. The resulting community structures were highly similar across all clustering thresholds. We explain this pattern by the existence of strong ecological structuring gradients and phylogenetically diverse sets of abundant OTUs that are highly stable across clustering thresholds. Removing low‐abundance, rare OTUs had negligible effects on community patterns. Our results indicate that microbial data sets with a clear gradient structure are highly robust to choice of sequence clustering threshold.     

Geographic and host‐mediated population genetic structure in a cestode parasite of the three‐spined stickleback

Comparative studies of genetic diversity and population structure can shed light on the ecological and evolutionary factors that influence host–parasite interactions. Here we examined whether geography, time and genetic variation in Alaskan three‐spined stickleback (Gasterosteus aculeatus Linneaus) hosts shape the population genetic structure of the diphyllobothridean cestode parasite Schistocephalus solidus (Müller, 1776). Host lineages and haplotypes were identified by sequencing the mitochondrial cytochrome b gene, and host population structure was assessed by Bayesian clustering analysis of allelic variation at 11 microsatellite loci. Parasite population structure was characterized according to allelic variation at eight microsatellite loci. Mantel tests and canonical redundancy analysis were conducted to evaluate the proportion of parasite genetic variation attributable to time and geography vs. host lineage, haplotype, and genotypic cluster. Host and parasite population structure were largely discordant across the study area, probably reflecting differences in gene flow, environmental influences external to the host, and genomic admixture among host lineages. We found that geography explained the greatest proportion of parasite genetic variation, but that variation also reflects time, host lineage, and host haplotype. Associations with host haplotypes suggest that one parasite genotypic cluster exhibits a narrower host range, predominantly infecting the most common host haplotypes, whereas the other parasite cluster infects all haplotypes equally, including rare haplotypes. Although experimental infection trials might prove otherwise, distributional differences in hosts preferentially infected by S. solidus could underlie the observed pattern of population structure.     

Divergence thresholds and divergent biodiversity estimates: can metabarcoding reliably describe zooplankton communities?

DNA metabarcoding is a promising method for describing communities and estimating biodiversity. This approach uses high‐throughput sequencing of targeted markers to identify species in a complex sample. By convention, sequences are clustered at a predefined sequence divergence threshold (often 3%) into operational taxonomic units (OTUs) that serve as a proxy for species. However, variable levels of interspecific marker variation across taxonomic groups make clustering sequences from a phylogenetically diverse dataset into OTUs at a uniform threshold problematic. In this study, we use mock zooplankton communities to evaluate the accuracy of species richness estimates when following conventional protocols to cluster hypervariable sequences of the V4 region of the small subunit ribosomal RNA gene (18S) into OTUs. By including individually tagged single specimens and “populations” of various species in our communities, we examine the impact of intra‐ and interspecific diversity on OTU clustering. Communities consisting of single individuals per species generated a correspondence of 59–84% between OTU number and species richness at a 3% divergence threshold. However, when multiple individuals per species were included, the correspondence between OTU number and species richness dropped to 31–63%. Our results suggest that intraspecific variation in this marker can often exceed 3%, such that a single species does not always correspond to one OTU. We advocate the need to apply group‐specific divergence thresholds when analyzing complex and taxonomically diverse communities, but also encourage the development of additional filtering steps that allow identification of artifactual rRNA gene sequences or pseudogenes that may generate spurious OTUs.     

Detection of individual ploidy levels with genotyping‐by‐sequencing (GBS) analysis

Ploidy levels sometimes vary among individuals or populations, particularly in plants. When such variation exists, accurate determination of cytotype can inform studies of ecology or trait variation and is required for population genetic analyses. Here, we propose and evaluate a statistical approach for distinguishing low‐level ploidy variants (e.g. diploids, triploids and tetraploids) based on genotyping‐by‐sequencing (GBS) data. The method infers cytotypes based on observed heterozygosity and the ratio of DNA sequences containing different alleles at thousands of heterozygous SNPs (i.e. allelic ratios). Whereas the method does not require prior information on ploidy, a reference set of samples with known ploidy can be included in the analysis if it is available. We explore the power and limitations of this method using simulated data sets and GBS data from natural populations of aspen (Populus tremuloides) known to include both diploid and triploid individuals. The proposed method was able to reliably discriminate among diploids, triploids and tetraploids in simulated data sets, and this was true for different levels of genetic diversity, inbreeding and population structure. Power and accuracy were minimally affected by low coverage (i.e. 2×), but did sometimes suffer when simulated mixtures of diploids, autotetraploids and allotetraploids were analysed. Cytotype assignments based on the proposed method closely matched those from previous microsatellite and flow cytometry data when applied to GBS data from aspen. An R package (gbs2ploidy) implementing the proposed method is available from CRAN.     

Patterns of morphological variation among populations of the widespread annual killifish Nothobranchius orthonotus are independent of genetic divergence and biogeography

Populations of annual killifish of the genus Nothobranchius occur in patchily distributed temporary pools in the East African savannah. Their fragmented distribution and low dispersal ability result in highly structured genetic clustering of their populations. In this study, we examined body shape variation in a widely distributed species, Nothobranchius orthonotus with known phylogeographic structure. We tested whether genetic divergence of major mitochondrial lineages forming two candidate species is congruent with phenotypic diversification, using linear and geometric morphometry analyses of body shape in 23 wild populations. We also conducted a common‐garden experiment with two wild‐derived populations to control for the effect of local environmental conditions on body shape. We identified different allometric trajectories for different mitochondrial lineages and candidate species in both sexes. However, in a principal components analysis of population‐level body shape, the separation among mitochondrial lineages was incomplete. Higher similarity of mitochondrial lineages belonging to different candidate species than that of same candidate species prevented distinction of the two candidate species on the basis of body shape. Analysis at the individual level demonstrated that N. orthonotus express high intrapopulation variability, with major overlap among individuals from all populations. In conclusion, we suggest that N. orthonotus be considered as a single species with an extensive geographic range, strong population genetic structure and high morphological variability.     

Relationships among tetraploid wheat (Triticum turgidum L.) landrace populations revealed by isozyme markers and agronomic traits

Diversity and relationships among ten tetraploid wheat landrace populations, collected from different localities in the central highlands of Ethiopia, were studied using isozyme markers and agronomic traits. This type of analysis in crop species is fundamental for designing optimal germ plasm collection, management practices and for developing an index for parental selection. The populations differed in allelic frequencies. Gene-diversity estimates showed that the populations encompass an appreciable amount of variation. However, differentiation between them was low, as was also confirmed by the presence of gene flow. Much of the diversity (85%), was attributable to the within-population level. The genetic distances were mostly small with the exception of those between a few pairs of populations. Thus, the relationships discerned among the populations were more of a similarity nature which could be ascribed to sharing a common ancestral population and/or adaptation to similar climatic conditions. The pattern of genetic divergence appeared to be independent of geographic distance. Considerable divergence in the agronomic traits was observed for certain populations. Cluster analyses of the isozyme and agronomic data produced different patterns and memberships of groupings. This lack of agreement could be ascribed to the different forces of evolution acting on isozyme markers and agronomic traits since agronomic traits, are the prime target of artificial selection. The clustering based on agronomic traits resulted in grouping together populations with similar agronomic performance. The results of this study suggest that taking more samples within a locality or population would be a better approach to capture the range of variation in the landrace populations of the central highlands of Ethiopia.     

Genetic structure among populations in the endemic Hawaiian Plantago lineage: insights from microsatellite variation

Endemic Hawaiian species in the genus Plantago show considerable morphological and ecological diversity. Despite their variation, a recent phylogenetic analysis based on DNA sequence data showed that the group is monophyletic and that sequence variation among species and morphotypes is low. This lack of sequence polymorphisms resulted in an inability to resolve species and population affinities within the most recently derived clade of this lineage. To assess species boundaries, population genetic structure and interpopulation connectivity among the morphologically and ecologically distinct populations within this clade, genetic variation was examined using eight microsatellite loci. Within‐population genetic diversity was found to be lowest in the Maunaiu, Hawai'i population of the endangered P. hawaiensis, and highest in the large P. pachyphylla population from 'Eke, West Maui. Isolation by distance across the range of populations was detected and indicated restricted dispersal. This result is likely to be attributable to few interisland dispersal events in the evolutionary history of this lineage. Genetic differentiation within islands tended to be higher among populations occurring in contrasting bog and woodland habitats, suggesting ecological barriers to gene flow and the potential role of ecological divergence in population diversification. Overall, these results are consistent with findings from phylogenetic analysis of the entire lineage. Our data bring new insights regarding patterns of dispersal and population genetic structure to this endemic and endangered group of island taxa. As island environments become increasingly fragmented, information of this type has important implications for the successful management of these fragile populations and habitats.     

Adaptation in temporally variable environments: stickleback armor in periodically breaching bar‐built estuaries

The evolutionary consequences of temporal variation in selection remain hotly debated. We explored these consequences by studying threespine stickleback in a set of bar‐built estuaries along the central California coast. In most years, heavy rains induce water flow strong enough to break through isolating sand bars, connecting streams to the ocean. New sand bars typically re‐form within a few weeks or months, thereby re‐isolating populations within the estuaries. These breaching events cause severe and often extremely rapid changes in abiotic and biotic conditions, including shifts in predator abundance. We investigated whether this strong temporal environmental variation can maintain within‐population variation while eroding adaptive divergence among populations that would be caused by spatial variation in selection. We used neutral genetic markers to explore population structure and then analysed how stickleback armor traits, the associated genes Eda and Pitx1 and elemental composition (%P) varies within and among populations. Despite strong gene flow, we detected evidence for divergence in stickleback defensive traits and Eda genotypes associated with predation regime. However, this among‐population variation was lower than that observed among other stickleback populations exposed to divergent predator regimes. In addition, within‐population variation was very high as compared to populations from environmentally stable locations. Elemental composition was strongly associated with armor traits, Eda genotype and the presence of predators, thus suggesting that spatiotemporal variation in armor traits generates corresponding variation in elemental phenotypes. We conclude that gene flow, and especially temporal environmental variation, can maintain high levels of within‐population variation while reducing, but not eliminating, among‐population variation driven by spatial environmental variation.     

Subspecies delineation amid phenotypic,geographic and genetic discordance in a songbird

Understanding the processes that drive divergence within and among species is a long‐standing goal in evolutionary biology. Traditional approaches to assessing differentiation rely on phenotypes to identify intra‐ and interspecific variation, but many species express subtle morphological gradients in which boundaries among forms are unclear. This intraspecific variation may be driven by differential adaptation to local conditions and may thereby reflect the evolutionary potential within a species. Here, we combine genetic and morphological data to evaluate intraspecific variation within the Nelson's (Ammodramus nelsoni) and salt marsh (Ammodramus caudacutus) sparrow complex, a group with populations that span considerable geographic distributions and a habitat gradient. We evaluated genetic structure among and within five putative subspecies of A. nelsoni and A. caudacutus using a reduced‐representation sequencing approach to generate a panel of 1929 SNPs among 69 individuals. Although we detected morphological differences among some groups, individuals sorted along a continuous phenotypic gradient. In contrast, the genetic data identified three distinct clusters corresponding to populations that inhabit coastal salt marsh, interior freshwater marsh and coastal brackish–water marsh habitats. These patterns support the current species‐level recognition but do not match the subspecies‐level taxonomy within each species—a finding which may have important conservation implications. We identified loci exhibiting patterns of elevated divergence among and within these species, indicating a role for local selective pressures in driving patterns of differentiation across the complex. We conclude that this evidence for adaptive variation among subspecies warrants the consideration of evolutionary potential and genetic novelty when identifying conservation units for this group.     

Cryptic species and co‐diversification in sand scorpions from the Karakum and Kyzylkum deserts of Central Asia

Little is known about species diversification within the deserts of Central Asia. For example, the degree of lineage divergence and timing of population differentiation, as well as potential biogeographic barriers driving diversification, are nearly unknown. Here, we analysed a multi‐locus data set for a widespread sand scorpion (Mesobuthus gorelovi) to evaluate cryptic species diversity and phylogeographic patterns across the Karakum and Kyzylkum deserts. We also combined these data with previously published sequence data to test for a signal of co‐diversification. A consensus species delimitation approach indicated that the widespread M. gorelovi is likely composed of up to five distinct species that began to diversify at the Miocene–Pliocene boundary. We observed shared patterns of lineage divergence across the Amu Darya River region in three scorpion taxa and found support for a shared history of assemblage diversification across this biogeographic barrier. Thus, major river systems appear to facilitate diversification among desert scorpions.     

Generalized genomic distance-based regression methodology for multilocus association analysis

Large-scale, multilocus genetic association studies require powerful and appropriate statistical-analysis tools that are designed to relate genotype and haplotype information to phenotypes of interest. Many analysis approaches consider relating allelic, haplotypic, or genotypic information to a trait through use of extensions of traditional analysis techniques, such as contingency-table analysis, regression methods, and analysis-of-variance techniques. In this work, we consider a complementary approach that involves the characterization and measurement of the similarity and dissimilarity of the allelic composition of a set of individuals' diploid genomes at multiple loci in the regions of interest. We describe a regression method that can be used to relate variation in the measure of genomic dissimilarity (or "distance") among a set of individuals to variation in their trait values. Weighting factors associated with functional or evolutionary conservation information of the loci can be used in the assessment of similarity. The proposed method is very flexible and is easily extended to complex multilocus-analysis settings involving covariates. In addition, the proposed method actually encompasses both single-locus and haplotype-phylogeny analysis methods, which are two of the most widely used approaches in genetic association analysis. We showcase the method with data described in the literature. Ultimately, our method is appropriate for high-dimensional genomic data and anticipates an era when cost-effective exhaustive DNA sequence data can be obtained for a large number of individuals, over and above genotype information focused on a few well-chosen loci.     

Influence of gene flow on divergence dating – implications for the speciation history of Takydromus grass lizards

Dating the time of divergence and understanding speciation processes are central to the study of the evolutionary history of organisms but are notoriously difficult. The difficulty is largely rooted in variations in the ancestral population size or in the genealogy variation across loci. To depict the speciation processes and divergence histories of three monophyletic Takydromus species endemic to Taiwan, we sequenced 20 nuclear loci and combined with one mitochondrial locus published in GenBank. They were analysed by a multispecies coalescent approach within a Bayesian framework. Divergence dating based on the gene tree approach showed high variation among loci, and the divergence was estimated at an earlier date than when derived by the species‐tree approach. To test whether variations in the ancestral population size accounted for the majority of this variation, we conducted computer inferences using isolation‐with‐migration (IM) and approximate Bayesian computation (ABC) frameworks. The results revealed that gene flow during the early stage of speciation was strongly favoured over the isolation model, and the initiation of the speciation process was far earlier than the dates estimated by gene‐ and species‐based divergence dating. Due to their limited dispersal ability, it is suggested that geographical isolation may have played a major role in the divergence of these Takydromus species. Nevertheless, this study reveals a more complex situation and demonstrates that gene flow during the speciation process cannot be overlooked and may have a great impact on divergence dating. By using multilocus data and incorporating Bayesian coalescence approaches, we provide a more biologically realistic framework for delineating the divergence history of Takydromus.     

Habitat loss and fragmentation reduce effective gene flow by disrupting seed dispersal in a neotropical palm

Habitat loss and fragmentation often reduce gene flow and genetic diversity in plants by disrupting the movement of pollen and seed. However, direct comparisons of the contributions of pollen vs. seed dispersal to genetic variation in fragmented landscapes are lacking. To address this knowledge gap, we partitioned the genetic diversity contributed by male gametes from pollen sources and female gametes from seed sources within established seedlings of the palm Oenocarpus bataua in forest fragments and continuous forest in northwest Ecuador. This approach allowed us to quantify the separate contributions of each of these two dispersal processes to genetic variation. Compared to continuous forest, fragments had stronger spatial genetic structure, especially among female gametes, and reduced effective population sizes. We found that within and among fragments, allelic diversity was lower and genetic structure higher for female gametes than for male gametes. Moreover, female gametic allelic diversity in fragments decreased with decreasing surrounding forest cover, while male gametic allelic diversity did not. These results indicate that limited seed dispersal within and among fragments restricts genetic diversity and strengthens genetic structure in this system. Although pollen movement may also be impacted by habitat loss and fragmentation, it nonetheless serves to promote gene flow and diversity within and among fragments. Pollen and seed dispersal play distinctive roles in determining patterns of genetic variation in fragmented landscapes, and maintaining the integrity of both dispersal processes will be critical to managing and conserving genetic variation in the face of continuing habitat loss and fragmentation in tropical landscapes.     

Divergent population structure and climate associations of a chromosomal inversion polymorphism across the Mimulus guttatus species complex

Chromosomal rearrangement polymorphisms are common and increasingly found to be associated with adaptive ecological divergence and speciation. Rearrangements, such as inversions, reduce recombination in heterozygous individuals and thus can protect favourable allelic combinations at linked loci, facilitating their spread in the presence of gene flow. Recently, we identified a chromosomal inversion polymorphism that contributes to ecological adaptation and reproductive isolation between annual and perennial ecotypes of the yellow monkeyflower, Mimulus guttatus. Here we evaluate the population genetic structure of this inverted region in comparison with the collinear regions of the genome across the M. guttatus species complex. We tested whether annual and perennial M. guttatus exhibit different patterns of divergence for loci in the inverted and noninverted regions of the genome. We then evaluated whether there are contrasting climate associations with these genomic regions through redundancy analysis. We found that the inversion exhibits broadly different patterns of divergence among annual and perennial M. guttatus and is associated with environmental variation across population accessions. This study is the first widespread population genetic survey of the diversity of the M. guttatus species complex. Our findings contribute to a greater understanding of morphological, ecological, and genetic evolutionary divergence across this highly diverse group of closely related ecotypes and species. Finally, understanding species relationships among M. guttatus sp. has hitherto been stymied by accumulated evidence of substantial gene flow among populations as well as designated species. Nevertheless, our results shed light on these relationships and provide insight into adaptation in life history traits within the complex.     

Genetic patterns across an invasion's history: a test of change versus stasis for the Eurasian round goby in North America

Biological invasions comprise accidental evolutionary experiments, whose genetic compositions underlie relative success, spread and persistence in new habitats. However, little is known about whether, or how, their population genetic patterns change temporally and/or spatially across the invasion's history. Theory predicts that most would undergo founder effect, exhibit low genetic divergence across the new range and gain variation over time via new arriving propagules. To test these predictions, we analyse population genetic diversity and divergence patterns of the Eurasian round goby Neogobius melanostomus across the two decades of its North American invasion in the Laurentian Great Lakes, comparing results from 13 nuclear DNA microsatellite loci and mitochondrial DNA cytochrome b sequences. We test whether ‘genetic stasis’, ‘genetic replacement’ and/or ‘genetic supplement’ scenarios have occurred at the invasion's core and expansion sites, in comparison with its primary native source population in the Dnieper River, Black Sea. Results reveal pronounced genetic divergence across the exotic range, with population areas remaining genetically distinct and statistically consistent across two decades, supporting ‘genetic stasis’ and ‘founder takes most’. The original genotypes continue to predominate, whose high population growth likely outpaced the relative success of later arrivals. The original invasion core has stayed the most similar to the native source. Secondary expansion sites indicate slight allelic composition convergence towards the core population over time, attributable to some early ‘genetic supplementation’. The geographic and temporal coverage of this investigation offers a rare opportunity to discern population dynamics over time and space in context of invasion genetic theory vs. reality.     

A question of time: the land snail Murella muralis (Gastropoda: Pulmonata) reveals constraints on past ecological speciation

The lively debate about speciation currently focuses on the relative importance of factors driving population differentiation. While many studies are increasingly producing results on the importance of selection, little is known about the interaction between drift and selection. Moreover, there is still little knowledge on the spatial‐temporal scales at which speciation occurs, that is, arrangement of habitat patches, abruptness of habitat transitions, climate and habitat changes interacting with selective forces. To investigate these questions, we quantified variation on a fine geographical scale analysing morphological (shell) and genetic data sets coupled with environmental data in the land snail Murella muralis, endemic to the Mediterranean island of Sicily. Analysis of a fragment of the mitochondrial DNA cytochrome oxidase I gene (COI) and eight nuclear microsatellite loci showed that genetic variation is highly structured at a very fine spatial scale by local palaeogeographical events and historical population dynamics. Molecular clock estimates, calibrated here specifically for Tyrrhenian land snails, provided a framework of palaeogeographical events responsible for the observed geographical variations and migration routes. Finally, we showed for the first time well‐documented lines of evidence of selection in the past, which explains divergence of land snail shell shapes. We suggest that time and palaeogeographical history acted as constraints in the progress along the ecological speciation continuum. Our study shows that testing for correlation among palaeogeography, morphology and genetic data on a fine geographical scale provides information fundamental for a detailed understanding of ecological speciation processes.