Testing hypotheses of hybrid taxon formation in the shrubby beardtongues (Penstemon subgenus Dasanthera).

PREMISE: Hybridization is increasingly being identified in the genomes of species across the tree of life, leading to a general recognition that hybridization plays an important role in the generation of species diversity. While hybridization may increase species diversity directly via the formation of new taxa through hybrid speciation, it may also act indirectly via the exchange of phenotypic and genetic variance between species, which may in turn stimulate future speciation events. METHODS: Using high-throughput sequence data, we resolved phylogenetic relationships and investigated the role of hybridization as a diversification mechanism in the shrubby beardtongues (Penstemon subgenus Dasanthera), a group of North American wildflowers that has undergone a recent and rapid adaptive radiation. Specifically, we tested four hypotheses of hybrid taxon formation resulting from hybridization between P. davidsonii and P. fruticosus. RESULTS: Species tree inference supports the monophyly of subgenus Dasanthera and elucidates relationships between taxa distributed in the Cascades and Sierra Nevada Mountains. Results also provide evidence of gene flow between P. davidsonii and P. fruticosus and support at least one hybrid origin hypothesis (P. davidsonii var. menziesii) in a region of contemporary distributional overlap. Hybridization may have also been facilitated by historical overlap in geographic distribution caused by species' responses to climatic changes during the Pleistocene. CONCLUSIONS: Our results support a history of hybridization between focal taxa in a rapidly radiating clade of plants and more broadly contribute to our growing understanding of the role of hybridization as a diversification mechanism in plants.

Phylogeographic analysis of shrubby beardtongues reveals range expansions during the Last Glacial Maximum and implicates the Klamath Mountains as a hotspot for hybridization.

Quaternary glacial cycles often altered species' geographic distributions, which in turn altered the geographic structure of species' genetic diversity. In many cases, glacial expansion forced species in temperate climates to contract their ranges and reside in small pockets of suitable habitat (refugia), where they were likely to interact closely with other species, setting the stage for potential gene exchange. These introgression events, in turn, would have degraded species boundaries, making the inference of phylogenetic relationships challenging. Using high-throughput sequence data, we employed a combination of species distribution models and hybridization tests to assess the effect of glaciation on the geographic distributions, phylogenetic relationships, and patterns of gene flow of five species of Penstemon subgenus Dasanthera, long-lived shrubby angiosperms distributed throughout the Pacific Northwest of North America. Surprisingly, we found that rather than reducing their ranges to small refugia, most Penstemon subgenus Dasanthera species experienced increased suitable habitat during the Last Glacial Maximum relative to the present day. We also found substantial evidence for gene exchange between species, with the bulk of introgression events occurring in or near the Klamath Mountains of southwestern Oregon and northwestern California. Subsequently, our phylogenetic inference reveals blurred taxonomic boundaries in the Klamath Mountains, where introgression is most prevalent. Our results question the classical paradigm of temperate species' responses to glaciation and highlight the importance of contextualizing phylogenetic inference with species' histories of introgression.

Fluidigm2PURC: Automated processing and haplotype inference for double-barcoded PCR amplicons.

PREMISE OF THE STUDY: Targeted enrichment strategies for phylogenomic inference are a time- and cost-efficient way to collect DNA sequence data for large numbers of individuals at multiple, independent loci. Automated and reproducible processing of these data is a crucial step for researchers conducting phylogenetic studies. METHODS AND RESULTS: We present Fluidigm2PURC, an open source Python utility for processing paired-end Illumina data from double-barcoded PCR amplicons. In combination with the program PURC (Pipeline for Untangling Reticulate Complexes), our scripts process raw FASTQ files for analysis with PURC and use its output to infer haplotypes for diploids, polyploids, and samples with unknown ploidy. We demonstrate the use of the pipeline with an example data set from the genus Thalictrum (Ranunculaceae). CONCLUSIONS: Fluidigm2PURC is freely available for Unix-like operating systems on GitHub (https://github.com/pblischak/fluidigm2purc) and for all operating systems through Docker (https://hub.docker.com/r/pblischak/fluidigm2purc).

HyDe: A Python Package for Genome-Scale Hybridization Detection.

The analysis of hybridization and gene flow among closely related taxa is a common goal for researchers studying speciation and phylogeography. Many methods for hybridization detection use simple site pattern frequencies from observed genomic data and compare them to null models that predict an absence of gene flow. The theory underlying the detection of hybridization using these site pattern probabilities exploits the relationship between the coalescent process for gene trees within population trees and the process of mutation along the branches of the gene trees. For certain models, site patterns are predicted to occur in equal frequency (i.e., their difference is 0), producing a set of functions called phylogenetic invariants. In this article, we introduce HyDe, a software package for detecting hybridization using phylogenetic invariants arising under the coalescent model with hybridization. HyDe is written in Python and can be used interactively or through the command line using pre-packaged scripts. We demonstrate the use of HyDe on simulated data, as well as on two empirical data sets from the literature. We focus in particular on identifying individual hybrids within population samples and on distinguishing between hybrid speciation and gene flow. HyDe is freely available as an open source Python package under the GNU GPL v3 on both GitHub (https://github.com/pblischak/HyDe) and the Python Package Index (PyPI: https://pypi.python.org/pypi/phyde).

SNP genotyping and parameter estimation in polyploids using low-coverage sequencing data.

Motivation: Genotyping and parameter estimation using high throughput sequencing data are everyday tasks for population geneticists, but methods developed for diploids are typically not applicable to polyploid taxa. This is due to their duplicated chromosomes, as well as the complex patterns of allelic exchange that often accompany whole genome duplication (WGD) events. For WGDs within a single lineage (autopolyploids), inbreeding can result from mixed mating and/or double reduction. For WGDs that involve hybridization (allopolyploids), alleles are typically inherited through independently segregating subgenomes. Results: We present two new models for estimating genotypes and population genetic parameters from genotype likelihoods for auto- and allopolyploids. We then use simulations to compare these models to existing approaches at varying depths of sequencing coverage and ploidy levels. These simulations show that our models typically have lower levels of estimation error for genotype and parameter estimates, especially when sequencing coverage is low. Finally, we also apply these models to two empirical datasets from the literature. Overall, we show that the use of genotype likelihoods to model non-standard inheritance patterns is a promising approach for conducting population genomic inferences in polyploids. Availability and implementation: A C ++ program, EBG, is provided to perform inference using the models we describe. It is available under the GNU GPLv3 on GitHub: https://github.com/pblischak/polyploid-genotyping. Contact: blischak.4@osu.edu. Supplementary information: Supplementary data are available at Bioinformatics online.

Accounting for genotype uncertainty in the estimation of allele frequencies in autopolyploids.

Despite the increasing opportunity to collect large-scale data sets for population genomic analyses, the use of high-throughput sequencing to study populations of polyploids has seen little application. This is due in large part to problems associated with determining allele copy number in the genotypes of polyploid individuals (allelic dosage uncertainty-ADU), which complicates the calculation of important quantities such as allele frequencies. Here, we describe a statistical model to estimate biallelic SNP frequencies in a population of autopolyploids using high-throughput sequencing data in the form of read counts. We bridge the gap from data collection (using restriction enzyme based techniques [e.g. GBS, RADseq]) to allele frequency estimation in a unified inferential framework using a hierarchical Bayesian model to sum over genotype uncertainty. Simulated data sets were generated under various conditions for tetraploid, hexaploid and octoploid populations to evaluate the model's performance and to help guide the collection of empirical data. We also provide an implementation of our model in the R package polyfreqs and demonstrate its use with two example analyses that investigate (i) levels of expected and observed heterozygosity and (ii) model adequacy. Our simulations show that the number of individuals sampled from a population has a greater impact on estimation error than sequencing coverage. The example analyses also show that our model and software can be used to make inferences beyond the estimation of allele frequencies for autopolyploids by providing assessments of model adequacy and estimates of heterozygosity.

Gene prediction and annotation in Penstemon (Plantaginaceae): A workflow for marker development from extremely low-coverage genome sequencing.

PREMISE OF THE STUDY: Penstemon (Plantaginaceae) is a large and diverse genus endemic to North America. However, determining the phylogenetic relationships among its 280 species has been difficult due to its recent evolutionary radiation. The development of a large, multilocus data set can help to resolve this challenge. • METHODS: Using both previously sequenced genomic libraries and our own low-coverage whole-genome shotgun sequencing libraries, we used the MAKER2 Annotation Pipeline to identify gene regions for the development of sequencing loci from six extremely low-coverage Penstemon genomes (∼0.005×-0.007×). We also compared this approach to BLAST searches, and conducted analyses to characterize sequence divergence across the species sequenced. • RESULTS: Annotations and gene predictions were successfully added to more than 10,000 contigs for potential use in downstream primer design. Primers were then designed for chloroplast, mitochondrial, and nuclear loci from these annotated sequences. MAKER2 identified longer gene regions in all six Penstemon genomes when compared with BLASTN and BLASTX searches. The average level of sequence divergence among the six species was 7.14%. • DISCUSSION: Combining bioinformatics tools into a workflow that produces annotations can be useful for creating potential phylogenetic markers from thousands of sequences even when genome coverage is extremely low and reference data are only available from distant relatives. Furthermore, the output from MAKER2 contains information about important gene features, such as exon boundaries, and can be easily integrated with visualization tools to facilitate the process of marker development.

Sequence-related amplified polymorphism (SRAP) markers: A potential resource for studies in plant molecular biology(1.).

In the past few decades, many investigations in the field of plant biology have employed selectively neutral, multilocus, dominant markers such as inter-simple sequence repeat (ISSR), random-amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP) to address hypotheses at lower taxonomic levels. More recently, sequence-related amplified polymorphism (SRAP) markers have been developed, which are used to amplify coding regions of DNA with primers targeting open reading frames. These markers have proven to be robust and highly variable, on par with AFLP, and are attained through a significantly less technically demanding process. SRAP markers have been used primarily for agronomic and horticultural purposes, developing quantitative trait loci in advanced hybrids and assessing genetic diversity of large germplasm collections. Here, we suggest that SRAP markers should be employed for research addressing hypotheses in plant systematics, biogeography, conservation, ecology, and beyond. We provide an overview of the SRAP literature to date, review descriptive statistics of SRAP markers in a subset of 171 publications, and present relevant case studies to demonstrate the applicability of SRAP markers to the diverse field of plant biology. Results of these selected works indicate that SRAP markers have the potential to enhance the current suite of molecular tools in a diversity of fields by providing an easy-to-use, highly variable marker with inherent biological significance.

Phylogeny and origins of holoparasitism in Orobanchaceae.

PREMISE: Orobanchaceae are a family of angiosperms that range from fully autotrophic and free-living to completely heterotrophic and dependent on their hosts (holoparasites). Most of the ca. 2060 species are hemiparasites that photosynthesize throughout all or part of their life cycles. Certain family members are ecologically important due to direct impacts on community biomass and diversity, plant-herbivore interactions, and nutrient cycling. Other members are among the most economically damaging weeds in the world. Multiple trophic transitions within this family make it ideal for studying molecular evolutionary and physiological changes that accompany the evolution of parasitism. • METHODS: To establish a phylogenetic framework for such work, we substantially increased taxonomic sampling at loci for which a significant amount of data already existed (nuclear ITS and PHYA, plastid matK and rps2) and added data from the low-copy nuclear locus, PHYB. • KEY RESULTS: The data provide strong support for relationships among six major clades and for the position of Brandisia hancei Hook. f. The positions of Boschniakia himalaica Hook. f. & Thomson, Centranthera cochinchinensis (Lour.) Merr., Mannagettaea hummelii Harry Sm., and Pterygiella nigrescens Oliv. are confirmed or suggested for the first time. • CONCLUSIONS: There is a single origin of parasitism, and from within the hemiparasites, holoparasitism has originated three times. Moving from the base to the tips of the Orobanchaceae tree, the successive major splits within the parasitic clade are: Cymbarieae + the rest; Orobancheae + the rest; Brandisia + the rest; Rhinantheae + the rest; and Pedicularideae + Buchnereae.

Constrained lability in floral evolution: counting convergent origins of hummingbird pollination in Penstemon and Keckiella.

In the clade of Penstemon and segregate genera, pollination syndromes are well defined among the 284 species. Most display combinations of floral characters associated with pollination by Hymenoptera, the ancestral mode of pollination for this clade. Forty-one species present characters associated with hummingbird pollination, although some of these ornithophiles are also visited by insects. The ornithophiles are scattered throughout the traditional taxonomy and across phylogenies estimated from nuclear (internal transcribed spacer (ITS)) and chloroplast DNA (trnCD/TL) sequence data. Here, the number of separate origins of ornithophily is estimated, using bootstrap phylogenies and constrained parsimony searches. Analyses suggest 21 separate origins, with overwhelming support for 10 of these. Because species sampling was incomplete, this is probably an underestimate. Penstemons therefore show great evolutionary lability with respect to acquiring hummingbird pollination; this syndrome acts as an attractor to which species with large sympetalous nectar-rich flowers have frequently been drawn. By contrast, penstemons have not undergone evolutionary shifts backwards or to other pollination syndromes. Thus, they are an example of both striking evolutionary lability and constrained evolution.

Anther evolution: pollen presentation strategies when pollinators differ.

Male-male competition in plants is thought to exert selection on flower morphology and on the temporal presentation of pollen. Theory suggests that a plant's pollen dosing strategy should evolve to match the abundance and pollen transfer efficiency of its pollinators. Simultaneous pollen presentation should be favored when pollinators are infrequent or efficient at delivering the pollen they remove, whereas gradual dosing should optimize delivery by frequent and wasteful pollinators. Among Penstemon and Keckiella species, anthers vary in ways that affect pollen release, and the morphology of dried anthers reliably indicates how they dispense pollen. In these genera, hummingbird pollination has evolved repeatedly from hymenopteran pollination. Pollen production does not change with evolutionary shifts between pollinators. We show that after we control for phylogeny, hymenopteran-adapted species present their pollen more gradually than hummingbird-adapted relatives. In a species pair that seemed to defy the pattern, the rhythm of anther maturation produced an equivalent dosing effect. These results accord with previous findings that hummingbirds can be more efficient than bees at delivering pollen.

Phylogeny, taxonomic affinities, and biogeography of Penstemon (Plantaginaceae) based on ITS and cpDNA sequence data.

The large and diverse genus Penstemon (ca. 271 species) is endemic to North America and has been divided into six subgenera primarily based on anther dehiscence patterns. Species of Penstemon are known to be pollinated by a variety of insects (hymenopterans, lepidopterans, dipterans) and hummingbirds. Nucleotide sequence data from ITS and two noncoding regions of chloroplast DNA were used to reconstruct the phylogeny of Penstemon. Trees generated from nuclear and chloroplast DNA sequences are incongruent, which is probably the result of hybridization, and not fully resolved, which is likely due to a rapid evolutionary radiation. Penstemon represents a recent continental radiation where speciation has resulted primarily from evolutionary adaptations to ecological niches such as pollinator specialization. The results from these analyses show that the current circumscription of subgenera and sections needs revision to reflect more closely the evolutionary relationships of species. Specifically, species in subgenera Saccanthera, Habroanthus, and Penstemon are polyphyletic. These results also confirm the independent origin of hummingbird floral morphology in 10 clades.

ISSR techniques for evolutionary biology.

Inter-simple sequence repeat (ISSR) markers were originally devised for differentiating among closely related plant cultivars but have become extremely useful for studies of natural populations of plants, fungi, insects, and vertebrates. The markers are easily generated using minimal equipment and are hypervariable, yielding a large amount of data for a reasonable cost to the researcher. The methods for Miniprep DNA extraction and cleanup, polymerase chain reaction (PCR) amplification, optimization, data gathering and scoring, and data analyses are outlined.

The evolution and expression of RBCL in holoparasitic sister-genera Harveya and Hyobanche (Orobanchaceae).

The evolution of holoparasitism decreases the adaptive value of genes maintaining the photosynthetic apparatus. These may become pseudogenes through insertion or deletion events resulting in frameshift mutations, or by the evolution of premature stop codons. The holoparasitic sister genera Harveya and Hyobanche have undergone alternate pathways of evolution and expression at the plastid locus rbcL. An open reading frame in all but a single species of Harveya is maintained by purifying selection and is expressed. However, the function of Rubisco in this putative holoparasite is unknown. Conversely, Hyobanche has undergone rbcL pseudogene formation, and comparison of synonymous and nonsynonymous rates of evolution indicates that selection has not played a role in its evolution. This is complicated by the following findings: multiple pseudogene copies of rbcL exist in tissues of Hyobanche, rbcL transcripts also encode pseudogenes, and the large subunit is present in some tissues of Hyobanche. We hypothesize that the rbcL operon is in a state of degradation as may be expected in a holoparasite and is not endogenously expressed. Rather, the large subunit may be taken up from the host plants, and accumulate in tissues as a result of transpiration.

Hybridization and gene flow between a day- and night-flowering species of Zaluzianskya (Scrophulariaceae s.s., tribe Manuleeae).

Despite apparent ethological isolation based on specialized pollination systems, hybridization between day-flowering Zaluzianskya microsiphon and night-flowering Z. natalensis has been proposed due to intermediate individuals found in sympatric populations of these species. The extent of this putative hybridization was investigated using inter-simple sequence repeat (ISSR) markers and principal components analysis (PCA) of morphological traits. The species are genetically similar, but show some intra- and interspecific variation in band frequencies. Neighbor-joining analyses of the ISSR data demonstrated that although the species largely formed distinct groups, several individuals from the sympatric populations of each species and the "hybrids" clustered together rather than with members of their own species. These results are consistent with hybridization, although they could also indicate historical similarity. Nine loci were present only in individuals of Z. microsiphon, the "hybrids," and sometimes the sympatric individuals of Z. natalensis. In contrast, only one locus showed the reverse pattern. This suggests unidirectional gene flow from Z. microsiphon to Z. natalensis, which is also supported by population-level examinations of four loci. Ordination revealed separate phenotype clusters for each species, with hybrid individuals located in between but often closer to the Z. natalensis cluster. One hypothesis is that hybrids are backcrossing with Z. natalensis, leading to introgression of Z. microsiphon genetic material.

ORIGIN AND BIOGEOGRAPHY OF AESCULUS L. (HIPPOCASTANACEAE): A MOLECULAR PHYLOGENETIC PERSPECTIVE.

Sequences of chloroplast gene matK and internal transcribed spacers of nuclear ribosomal RNA genes were used for phylogenetic analyses of Aesculus, a genus currently distributed in eastern Asia, eastern and western North America, and southeastern Europe. Phylogenetic relationships inferred from these molecular data are highly correlated with the geographic distributions of species. The identified lineages closely correspond to the five sections previously recognized on the basis of morphology. Ancestral character-state reconstruction, a molecular clock, and fossil evidence were used to infer the origin and biogeographic history of the genus within a phylogenetic framework. Based on the molecular phylogenetic reconstruction of the genus, sequence divergence, and paleontological evidence, we infer that the genus originated during the transition from the Cretaceous to the Tertiary (~65 M.Y.B.P.) at a high latitude in eastern Asia and spread into North America and Europe as an element of the "boreotropical flora"; the current disjunct distribution of the genus resulted from geological and climatic changes during the Tertiary.