Estimating microhaplotype allele frequencies from low-coverage or pooled sequencing data.

BACKGROUND: Microhaplotypes have the potential to be more cost-effective than SNPs for applications that require genetic panels of highly variable loci. However, development of microhaplotype panels is hindered by a lack of methods for estimating microhaplotype allele frequency from low-coverage whole genome sequencing or pooled sequencing (pool-seq) data. RESULTS: We developed new methods for estimating microhaplotype allele frequency from low-coverage whole genome sequence and pool-seq data. We validated these methods using datasets from three non-model organisms. These methods allowed estimation of allele frequency and expected heterozygosity at depths routinely achieved from pooled sequencing. CONCLUSIONS: These new methods will allow microhaplotype panels to be designed using low-coverage WGS and pool-seq data to discover and evaluate candidate loci. The python script implementing the two methods and documentation are available at https://www.github.com/delomast/mhFromLowDepSeq .

Interrelationships and biogeography of the New World pufferfish genus Sphoeroides (Tetraodontiformes: Tetraodontidae) inferred using ultra-conserved DNA elements.

Colonization of the New World by marine taxa has been hypothesized to have occurred through the Tethys Sea or by crossing the East Pacific Barrier. To better understand patterns and timing of diversification, geological events can be coupled with time calibrated phylogenetic hypotheses to infer major drivers of diversification. Phylogenetic relationships among members of Sphoeroides, a genus of four toothed pufferfishes (Tetraodontiformes: Tetraodontidae) which are found nearly exclusively in the New World (eastern Pacific and western Atlantic), were reconstructed using sequences from ultra-conserved DNA elements, nuclear markers with clear homology among many vertebrate taxa. Hypotheses derived from concatenated maximum-likelihood and species tree summary methods support a paraphyletic Sphoeroides, with Colomesus deeply nested within the genus. Analyses also revealed S. pachygaster, a pelagic species with a cosmopolitan distribution, as the sister taxon to the remainder of Sphoeroides and recovered distinct lineages within S. pachygaster, indicating that this cosmopolitan species may represent a species complex. Ancestral range reconstruction may suggest the genus colonized the New World through the eastern Pacific before diversifying in the western Atlantic, though date estimates for these events are uncertain due to the lack of reliable fossil record for the genus.

Implications of Large-Effect Loci for Conservation: A Review and Case Study with Pacific Salmon.

The increasing feasibility of assembling large genomic datasets for non-model species presents both opportunities and challenges for applied conservation and management. A popular theme in recent studies is the search for large-effect loci that explain substantial portions of phenotypic variance for a key trait(s). If such loci can be linked to adaptations, 2 important questions arise: 1) Should information from these loci be used to reconfigure conservation units (CUs), even if this conflicts with overall patterns of genetic differentiation? 2) How should this information be used in viability assessments of populations and larger CUs? In this review, we address these questions in the context of recent studies of Chinook salmon and steelhead (anadromous form of rainbow trout) that show strong associations between adult migration timing and specific alleles in one small genomic region. Based on the polygenic paradigm (most traits are controlled by many genes of small effect) and genetic data available at the time showing that early-migrating populations are most closely related to nearby late-migrating populations, adult migration differences in Pacific salmon and steelhead were considered to reflect diversity within CUs rather than separate CUs. Recent data, however, suggest that specific alleles are required for early migration, and that these alleles are lost in populations where conditions do not support early-migrating phenotypes. Contrasting determinations under the US Endangered Species Act and the State of California's equivalent legislation illustrate the complexities of incorporating genomics data into CU configuration decisions. Regardless how CUs are defined, viability assessments should consider that 1) early-migrating phenotypes experience disproportionate risks across large geographic areas, so it becomes important to identify early-migrating populations that can serve as reliable sources for these valuable genetic resources; and 2) genetic architecture, especially the existence of large-effect loci, can affect evolutionary potential and adaptability.

These aren't the loci you'e looking for: Principles of effective SNP filtering for molecular ecologists.

Sequencing reduced-representation libraries of restriction site-associated DNA (RADseq) to identify single nucleotide polymorphisms (SNPs) is quickly becoming a standard methodology for molecular ecologists. Because of the scale of RADseq data sets, putative loci cannot be assessed individually, making the process of filtering noise and correctly identifying biologically meaningful signal more difficult. Artefacts introduced during library preparation and/or bioinformatic processing of SNP data can create patterns that are incorrectly interpreted as indicative of population structure or natural selection. Therefore, it is crucial to carefully consider types of errors that may be introduced during laboratory work and data processing, and how to minimize, detect and remove these errors. Here, we discuss issues inherent to RADseq methodologies that can result in artefacts during library preparation and locus reconstruction resulting in erroneous SNP calls and, ultimately, genotyping error. Further, we describe steps that can be implemented to create a rigorously filtered data set consisting of markers accurately representing independent loci and compare the effect of different combinations of filters on four RAD data sets. At last, we stress the importance of publishing raw sequence data along with final filtered data sets in addition to detailed documentation of filtering steps and quality control measures.

Molecular phylogenetics of New World searobins (Triglidae; Prionotinae).

Phylogenetic relationships among members of the New World searobin genera Bellator and Prionotus (Family Triglidae, Subfamily Prionotinae) and among other searobins in the families Triglidae and Peristediidae were investigated using both mitochondrial and nuclear DNA sequences. Phylogenetic hypotheses derived from maximum likelihood and Bayesian methodologies supported a monophyletic Prionotinae that included four well resolved clades of uncertain relationship; three contained species in the genus Prionotus and one contained species in the genus Bellator. Bellator was always recovered within the genus Prionotus, a result supported by post hoc model testing. Two nominal species of Prionotus (P. alatus and P. paralatus) were not recovered as exclusive lineages, suggesting the two may comprise a single species. Phylogenetic hypotheses also supported a monophyletic Triglidae but only if armored searobins (Family Peristediidae) were included. A robust morphological assessment is needed to further characterize relationships and suggest classification of clades within Prionotinae; for the time being we recommend that Bellator be considered a synonym of Prionotus. Relationships between armored searobins (Family Peristediidae) and searobins (Family Triglidae) and relationships within Triglidae also warrant further study.

Multi-locus species tree for the Amazonian peacock basses (Cichlidae: Cichla): emergent phylogenetic signal despite limited nuclear variation.

The inference of phylogenies of closely related species is obstructed by phenomena such as porous species boundaries and deep coalescence, and is often exacerbated by low levels of nucleotide variation among most loci surveyed in phylogenetic studies. We investigated the utility of twenty-one nuclear loci that had a range of 5-40 (median of 14) variable sites per locus to estimate the phylogeny of the genus Cichla, a group of 15 Neotropical cichlid fishes that began to diverge in the early to mid Miocene. We found that under a concatenated approach, the least variable loci, while contributing less to the overall phylogenetic signal (posterior node support), nevertheless provided information that increased support for the final tree. Moreover, this was not a result of misdirection by mutational noise, as the inference from all data was far superior to those from reduced datasets (those with more variable loci) in terms of the relative precision of posterior tree space. Phylogenetic methods that allowed each locus to have a separate genealogy, including Bayesian concordance analysis and a multispecies coalescent model, provided phylogenies that were also compatible with the concatenated tree in terms of the eight recently delimited species of Cichla, albeit with somewhat diminished support for some branches. In contrast, described species that still regularly exchange genes showed unstable relationships among analyses: not a surprising result from analyses that assume that gene tree heterogeneity results from incomplete lineage sorting and not gene flow. Importantly, we also observed that the confidence intervals for node ages in the coalescent analyses were quite wide, and likely susceptible to influence of the prior on node density (e.g. birth-death).

Simultaneous delimitation of species and quantification of interspecific hybridization in Amazonian peacock cichlids (genus cichla) using multi-locus data.

BACKGROUND: Introgression likely plays a significant role in evolution, but understanding the extent and consequences of this process requires a clear identification of species boundaries in each focal group. The delimitation of species, however, is a contentious endeavor. This is true not only because of the inadequacy of current tools to identify species lineages, but also because of the inherent ambiguity between natural populations and species paradigms. The result has been a debate about the supremacy of various species concepts and criteria. Here, we utilized multiple separate sources of molecular data, mtDNA, nuclear sequences, and microsatellites, to delimit species under a polytypic species concept (PTSC) and estimate the frequency and genomic extent of introgression in a Neotropical genus of cichlid fishes (Cichla). We compared our inferences of species boundaries and introgression under this paradigm to those when species are identified under a diagnostic species concept (DSC). RESULTS: We find that, based on extensive molecular data and an inclusive species concept, 8 separate biological entities should be recognized rather than the 15 described species of Cichla. Under the PTSC, fewer individuals are expected to exhibit hybrid ancestry than under the DSC (~2% vs. ~12%), but a similar number of the species exhibit introgression from at least one other species (75% vs. 60%). Under either species concept, the phylogenetic breadth of introgression in this group is notable, with both sister species and species from different major mtDNA clades exhibiting introgression. CONCLUSIONS: Introgression was observed to be a widespread phenomenon for delimited species in this group. While several instances of introgressive hybridization were observed in anthropogenically altered habitats, most were found in undisturbed natural habitats, suggesting that introgression is a natural but ephemeral part of the evolution of many tropical species. Nevertheless, even transient introgression may facilitate an increase in genetic diversity or transfer of adaptive mutations that have important consequences in the evolution of tropical biodiversity.

Species-level phylogeny of 'Satan's perches' based on discordant gene trees (Teleostei: Cichlidae: Satanoperca Günther 1862).

Neotropical rivers are home to the largest assemblage of freshwater fishes, but little is known about the phylogeny of these fishes at the species level using multi-locus molecular markers. Here, we present a phylogeny for all known species of the genus Satanoperca, a widespread group of Neotropical cichlid fishes, based on analysis of six unlinked genetic loci. To test nominal and proposed species limits for this group, we surveyed mtDNA sequence variation among 320 individuals representing all know species. Most nominal species were supported by this approach but we determined that populations in the Xingu, Tapajós, and Araguaia+Paraná Rivers are likely undescribed species, while S. jurupari and S. mapiritensis did not show clear genetic distinction. To infer a phylogeny of these putative species, we conducted maximum likelihood and Bayesian non-clock and relaxed clock analyses of concatenated data from three genes (one mitochondrial, two nuclear). We also used a multi-species coalescent model to estimate a species tree from six unlinked loci (one mitochondrial, five nuclear). The topologies obtained were congruent with other results, but showed only minimal to moderate support for some nodes, suggesting that more loci will be needed to satisfactorily estimate the distribution of coalescent histories within Satanoperca. We determined that this variation results from topological discordance among separate gene trees, likely due to differential sorting of ancestral polymorphisms.

Genomic region associated with run timing has similar haplotypes and phenotypic effects across three lineages of Chinook salmon.

Conserving life-history variation is a stated goal of many management programs, but the most effective means by which to accomplish this are often far from clear. Early- and late-migrating forms of Chinook salmon (Oncorhynchus tshawytscha) face unequal pressure from natural and anthropogenic forces that may alter the impacts of genetic variation underlying heritable migration timing. Genomic regions of chromosome 28 are known to be strongly associated with migration variation in adult Chinook salmon, but it remains unclear whether there is consistent association among diverse lineages and populations in large basins such as the Columbia River. With high-throughput genotyping (GT-seq) and phenotyping methods, we examined the association of genetic variation in 28 markers (spanning GREB1L to ROCK1 of chromosome 28) with individual adult migration timing characteristics gleaned from passive integrated transponder recordings of over 5000 Chinook salmon from the three major phylogeographic lineages that inhabit the Columbia River Basin. Despite the strong genetic differences among them in putatively neutral genomic regions, each of the three lineages exhibited very similar genetic variants in the chromosome 28 region that were significantly associated with adult migration timing phenotypes. This is particularly notable for the interior stream-type lineage, which exhibits an earlier and more constrained freshwater entry than the other lineages. In both interior stream-type and interior ocean-type lineages of Chinook salmon, heterozygotes of the most strongly associated linkage groups had largely intermediate migration timing relative to homozygotes, and results indicate codominance or possibly marginal partial dominance of the allele associated with early migration. Our results lend support to utilization of chromosome 28 variation in tracking and predicting run timing in these lineages of Chinook salmon in the Columbia River.

Genotyping single nucleotide polymorphisms and inferring ploidy by amplicon sequencing for polyploid, ploidy-variable organisms.

Whole genome duplication is hypothesized to have played a critical role in the evolution of several major taxa, including vertebrates, and while many lineages have rediploidized, some retain polyploid genomes. Additionally, variation in ploidy can occur naturally or be artificially induced within select plant and animal species. Modern genetic techniques have not been widely applied to polyploid or ploidy-variable species, in part due to the difficulty of obtaining genotype data from polyploids. In this study, we demonstrate a strategy for developing an amplicon sequencing panel of single nucleotide polymorphisms for high-throughput genotyping of polyploid organisms. We then develop a method to infer ploidy of individuals from amplicon sequencing data that is generalized to apply to any ploidy and does not require prior identification of heterozygous genotypes. Combining these two techniques will allow researchers to both infer ploidy and generate ploidy-aware genotypes with the same amplicon sequencing panel. We demonstrate this approach with white sturgeon Acipenser transmontanus, a ploidy-variable (octoploid, decaploid and dodecaploid) imperiled species under conservation management in the Pacific Northwest and obtained a panel of 325 loci. These loci were validated by examining inheritance in known-cross families, and the ploidy inference method was validated with known ploidy samples. We provide scripts that adapt existing pipelines to genotype polyploids and an R package for application of the ploidy inference method. We expect that these techniques will empower studies of genetic variation and inheritance in polyploid organisms that vary in ploidy level, either naturally or as a result of artificial propagation practices.

Steelhead (Oncorhynchus mykiss) lineages and sexes show variable patterns of association of adult migration timing and age-at-maturity traits with two genomic regions.

As life history diversity plays a critical role in supporting the resilience of exploited populations, understanding the genetic basis of those life history variations is important for conservation management. However, effective application requires a robust understanding of the strength and universality of genetic associations. Here, we examine genetic variation of single nucleotide polymorphisms in genomic regions previously associated with migration phenology and age-at-maturity in steelhead (Oncorhynchus mykiss) from the Columbia River. We found chromosome 28 markers (GREB1L, ROCK1 genes) explained significant variance in migration timing in both coastal and inland steelhead. However, strength of association was much greater in coastal than inland steelhead (R 2 0.51 vs. 0.08), suggesting that genomic background and challenging inland migration pathways may act to moderate effects of this region. Further, we found that chromosome 25 candidate markers (SIX6 gene) were significantly associated with age and size at first return migration for inland steelhead, and this pattern was mediated by sex in a predictable pattern (males R 2 = 0.139-0.170; females R 2 = 0.096-0.111). While this encourages using these candidate regions in predicting life history characteristics, we suggest that stock-specific associations and haplotype frequencies will be useful in guiding implementation of genetic assays to inform management.

One species or four? Yes!...and, no. Or, arbitrary assignment of lineages to species obscures the diversification processes of Neotropical fishes.

Species are fundamental units in many biological disciplines, but there is continuing disagreement as to what species are, how to define them, and even whether the concept is useful. While some of this debate can be attributed to inadequate data and insufficient statistical frameworks in alpha taxonomy, an equal part results from the ambiguity over what species are expected to represent by the many who use them. Here, mtDNA data, microsatellite data, and sequence data from 17 nuclear loci are used in an integrated and quantitative manner to resolve the presence of evolutionary lineages, their contemporary and historical structure, and their correspondence to species, in a species complex of Amazonian peacock "bass" cichlids (Cichla pinima sensu lato). Results suggest that the historical narrative for these populations is more complex than can be portrayed by recognizing them as one, two, or four species: their history and contemporary dynamics cannot be unambiguously rendered as discrete units (taxa) at any level without both choosing the supremacy of one delimitation criterion and obscuring the very information that provides insight into the diversification process. This calls into question the utility of species as a rank, term, or concept, and suggests that while biologists may have a reasonable grasp of the structure of evolution, our methods of conveying these insights need updating. The lack of correspondence between evolutionary phenomena and discrete species should serve as a null hypothesis, and researchers should focus on quantifying the diversity in nature at whatever hierarchical level it occurs.

Haplotyping RAD loci: an efficient method to filter paralogs and account for physical linkage.

Next-generation sequencing of reduced-representation genomic libraries provides a powerful methodology for genotyping thousands of single-nucleotide polymorphisms (SNPs) among individuals of nonmodel species. Utilizing genotype data in the absence of a reference genome, however, presents a number of challenges. One major challenge is the trade-off between splitting alleles at a single locus into separate clusters (loci), creating inflated homozygosity, and lumping multiple loci into a single contig (locus), creating artefacts and inflated heterozygosity. This issue has been addressed primarily through the use of similarity cut-offs in sequence clustering. Here, two commonly employed, postclustering filtering methods (read depth and excess heterozygosity) used to identify incorrectly assembled loci are compared with haplotyping, another postclustering filtering approach. Simulated and empirical data sets were used to demonstrate that each of the three methods separately identified incorrectly assembled loci; more optimal results were achieved when the three methods were applied in combination. The results confirmed that including incorrectly assembled loci in population-genetic data sets inflates estimates of heterozygosity and deflates estimates of population divergence. Additionally, at low levels of population divergence, physical linkage between SNPs within a locus created artificial clustering in analyses that assume markers are independent. Haplotyping SNPs within a locus effectively neutralized the physical linkage issue without having to thin data to a single SNP per locus. We introduce a Perl script that haplotypes polymorphisms, using data from single or paired-end reads, and identifies potentially problematic loci.

Testing mitochondrial capture and deep coalescence in Amazonian cichlid fishes (Cichlidae: Cichla).

Hybridization and introgression have important consequences in evolution, such as increasing the genetic diversity and adaptive potential of a species. One of their most conspicuous footprints is discordance among gene trees or between genes and phenotypes. However, most studies that report introgression fail to disprove the null hypothesis that genetic incongruence may result from stochastic sorting of ancestral allelic polymorphisms. In the case of ancient introgression, these two processes may be especially difficult to distinguish topologically, but they make different predictions about the patterns of coalescence among loci. Here we apply three methods, molecular dating, multispecies coalescent models, and gene tree simulation under coalescence, to compare these two hypotheses that explain the polyphyletic mtDNA of the butterfly peacock bass, Cichla orinocensis. In comparison with a species tree based on 20 unlinked nuclear loci, we determined that mtDNA divergences were too recent to be explained by ancestral polymorphism. Similarly, coalescent species tree branches were significantly shorter when putative introgressed mtDNA was incorporated, and simulations showed the mtDNA topology to be unlikely under lineage sorting only. We conclude that introgression approximately 1.5 million years ago resulted in capture by C. orinocensis of an mtDNA lineage ancestral to the modern subspecies C. oc. monoculus.

Systematics, biogeography, and evolution of the Neotropical peacock basses Cichla (Perciformes: Cichlidae).

To investigate forces influencing diversification in Neotropical fishes, the phylogenetic relationships among species and populations of the cichlid genus Cichla were examined. Mitochondrial DNA was sequenced for 454 individuals of the 5 nominal Cichla species and several putative undescribed species. Phylogenetic analyses support the distinction of two major clades of Cichla. Clade A includes C. temensis and two undescribed species from the lower Amazonas and Xingu Rivers. Clade B includes C. orinocensis, C. monoculus, C. ocellaris. C, intermedia, and an undescribed species from the upper Madeira River. Species boundaries were relatively well-circumscribed for clade B, while incomplete lineage sorting was inferred for clade A. Three probable instances of introgression were observed, including a regional population of C. orinocensis from the Negro River that shows a history of introgression. Biogeographic patterns from Cichla are partially congruent with those seen in several other Neotropical fish clades, and the diversification of Cichla species is inferred to result from both vicariance and sympatric divergence.