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Background Massively parallel sequencing of cDNA is now an efficient route for generating enormous sequence collections that represent expressed genes. This approach provides a valuable starting point for characterizing functional genetic variation in non-model organisms, especially where whole genome sequencing efforts are currently cost and time prohibitive. The large and complex genomes of pines ( Pinus spp.) have hindered the development of genomic resources, despite the ecological and economical importance of the group. While most genomic studies have focused on a single species ( P. taeda ), genomic level resources for other pines are insufficiently developed to facilitate ecological genomic research. Lodgepole pine ( P. contorta ) is an ecologically important foundation species of montane forest ecosystems and exhibits substantial adaptive variation across its range in western North America. Here we describe a sequencing study of expressed genes from P. contorta , including their assembly and annotation, and their potential for molecular marker development to support population and association genetic studies. Results We obtained 586,732 sequencing reads from a 454 GS XLR70 Titanium pyrosequencer (mean length: 306 base pairs). A combination of reference-based and de novo assemblies yielded 63,657 contigs, with 239,793 reads remaining as singletons. Based on sequence similarity with known proteins, these sequences represent approximately 17,000 unique genes, many of which are well covered by contig sequences. This sequence collection also included a surprisingly large number of retrotransposon sequences, suggesting that they are highly transcriptionally active in the tissues we sampled. We located and characterized thousands of simple sequence repeats and single nucleotide polymorphisms as potential molecular markers in our assembled and annotated sequences. High quality PCR primers were designed for a substantial number of the SSR loci, and a large number of these were amplified successfully in initial screening. Conclusions This sequence collection represents a major genomic resource for P. contorta , and the large number of genetic markers characterized should contribute to future research in this and other pines. Our results illustrate the utility of next generation sequencing as a basis for marker development and population genomics in non-model species.

Natural selection can drive the repeated evolution of reproductive isolation, but the genomic basis of parallel speciation remains poorly understood. We analyzed whole-genome divergence between replicate pairs of stick insect populations that are adapted to different host plants and undergoing parallel speciation. We found thousands of modest-sized genomic regions of accentuated divergence between populations, most of which are unique to individual population pairs. We also detected parallel genomic divergence across population pairs involving an excess of coding genes with specific molecular functions. Regions of parallel genomic divergence in nature exhibited exceptional allele frequency changes between hosts in a field transplant experiment. The results advance understanding of biological diversification by providing convergent observational and experimental evidence for selection's role in driving repeatable genomic divergence.

Hybrid zones are common in nature and can offer critical insights into the dynamics and components of reproductive isolation. Hybrids between diverged lineages are particularly informative about the genetic architecture of reproductive isolation, because introgression in an admixed population is a direct measure of isolation. In this paper, we combine simulations and a new statistical model to determine the extent to which different genetic architectures of isolation leave different signatures on genome-level patterns of introgression. We found that reproductive isolation caused by one or several loci of large effect caused greater heterogeneity in patterns of introgression than architectures involving many loci with small fitness effects, particularly when isolating factors were closely linked. The same conditions that led to heterogeneous introgression often resulted in a reasonable correspondence between outlier loci and the genetic loci that contributed to isolation. However, demographic conditions affected both of these results, highlighting potential limitations to the study of the speciation genomics. Further progress in understanding the genomics of speciation will require large-scale empirical studies of introgression in hybrid zones and model-based analyses, as well as more comprehensive modelling of the expected levels of isolation with different demographies and genetic architectures of isolation.

Chemically mediated plant–herbivore interactions contribute to the diversity of terrestrial communities and the diversification of plants and insects. While our understanding of the processes affecting community structure and evolutionary diversification has grown, few studies have investigated how trait variation shapes genetic and species diversity simultaneously in a tropical ecosystem. We investigated secondary metabolite variation among subpopulations of a single plant species, Piper kelleyi (Piperaceae), using high-performance liquid chromatography (HPLC), to understand associations between plant phytochemistry and host-specialized caterpillars in the genus Eois (Geometridae: Larentiinae) and associated parasitoid wasps and flies. In addition, we used a genotyping-by-sequencing approach to examine the genetic structure of one abundant caterpillar species, Eois encina, in relation to host phytochemical variation. We found substantive concentration differences among three major secondary metabolites, and these differences in chemistry predicted caterpillar and parasitoid community structure among host plant populations. Furthermore, E. encina populations located at high elevations were genetically different from other populations. They fed on plants containing high concentrations of prenylated benzoic acid. Thus, phytochemistry potentially shapes caterpillar and wasp community composition and geographic variation in species interactions, both of which can contribute to diversification of plants and insects.

As tree species vary extensively in genome size, complexity, and resource development, reduced representation methods have been increasingly employed for the generation of population genomic data. By allowing rapid marker discovery and genotyping for thousands of genomic regions in many individuals without requiring genomic resources, restriction site-associated DNA sequencing (RADseq) methods have dramatically improved our ability to bring population genomic perspectives to non-model trees. The rapid recent increase in studies of trees utilizing RADseq suggests that it is likely to become among the most common approaches for generating genome-wide data for a variety of applications. Here we provide a practical review of RADseq and its application to research areas of tree genetics. We briefly review RADseq laboratory methods and consider analytical approaches for assembly, variant calling, and bioinformatic processing. To guide considerations for study design, we use in silico analyses of eight available tree genomes to illustrate how expected marker number and density vary across laboratory approaches and genome sizes, and to consider the ability of RADseq designs to query coding regions. We review the empirical use of RADseq for different research objectives, considering its strengths and limitations. Many studies have used RADseq data to perform genome scans for selection, although limited marker density and linkage disequilibrium will often compromise its utility for such analyses. Regardless of this limitation, RADseq offers a powerful and inexpensive technique for generating genome-wide SNP data that can greatly contribute to research spanning phylogenetic and population genetic inference, linkage mapping, and quantitative genetic parameter estimation for tree genetics.

Trait–environment correlations can arise from local adaptation and can identify genetically and environmentally appropriate seeds for restoration projects. However, anthropogenic changes can disrupt the relationships between traits and fitness. Finding the best seed sources for restoration may rely on describing plant traits adaptive in disturbed and invaded environments, recognizing that while traits may differ among species and functional groups, there may be similarities in the strategies that increase seedling establishment. Focusing on three grass genera, two shrub species, and two forb genera, we collected seeds of all taxa from 16 common sites in the sagebrush steppe of the western United States. We measured seed and seedling characteristics, including seed size, emergence timing, and root and shoot traits, and compiled a suite of environmental variables for each collection site. We described trait–environment associations and asked how traits or environment of origin were associated with seedling survival in invaded gardens. Sampling seven taxa from the same sites allowed us to ask how trait–environment–performance associations differ among taxa and whether natural selection favors similar traits across multiple taxa and functional groups. All taxa showed trait–environment associations consistent with local adaptation, and both environment of origin and phenotypes predicted survival in competitive restoration settings, with some commonalities among taxa. Notably, rapid emergence and larger seeds increased survival for multiple taxa. Environmental factors at collection sites, including lower slopes (especially for grasses), greater mean annual temperatures (especially for shrubs and forbs), and greater precipitation seasonality were frequently associated with increased survival. We noted one collection site with high seedling survival across all seven taxa, suggesting that conditions within some sites may result in selection for traits that increase establishment for multiple species. Thus, choosing native plant sources with the most adaptive traits, along with matching climates, will likely improve the restoration of invaded communities. Using seven co‐occurring taxa from 16 collection sites, we described trait‐by‐environment associations and asked how traits or environment of origin were associated with seedling survival in invaded gardens. All taxa showed TBE associations consistent with local adaptation, and both environment and phenotypic traits predicted survival in competitive restoration settings. We found that choosing native plant sources with the most adaptive traits, along with matching climates, could improve the restoration of invaded communities.

The spatial structure of genomic and phenotypic variation across populations reflects historical and demographic processes as well as evolution via natural selection. Characterizing such variation can provide an important perspective for understanding the evolutionary consequences of changing climate and for guiding ecological restoration. While evidence for local adaptation has been traditionally evaluated using phenotypic data, modern methods for generating and analyzing landscape genomic data can directly quantify local adaptation by associating allelic variation with environmental variation. Here, we analyze both genomic and phenotypic variation of rubber rabbitbrush (Ericameria nauseosa), a foundational shrub species of western North America. To quantify landscape genomic structure and provide perspective on patterns of local adaptation, we generated reduced representation sequencing data for 17 wild populations (222 individuals; 38,615 loci) spanning a range of environmental conditions. Population genetic analyses illustrated pronounced landscape genomic structure jointly shaped by geography and environment. Genetic‐environment association (GEA) analyses using both redundancy analysis (RDA) and a machine‐learning approach (Gradient Forest) indicated environmental variables (precipitation seasonality, slope, aspect, elevation, and annual precipitation) influenced spatial genomic structure and were correlated with allele frequency shifts indicative of local adaptation at a consistent set of genomic regions. We compared our GEA‐based inference of local adaptation with phenotypic data collected by growing seeds from each population in a greenhouse common garden. Population differentiation in seed weight, emergence, and seedling traits was associated with environmental variables (e.g., precipitation seasonality) that were also implicated in GEA analyses, suggesting complementary conclusions about the drivers of local adaptation across different methods and data sources. Our results provide a baseline understanding of spatial genomic structure for E. nauseosa across the western Great Basin and illustrate the utility of GEA analyses for detecting the environmental causes and genetic signatures of local adaptation in a widely distributed plant species of restoration significance.

Population genetic analyses can evaluate how evolutionary processes shape diversity and inform conservation and management of imperiled species. Taimen ( Hucho taimen ), the world’s largest freshwater salmonid, is threatened, endangered, or extirpated across much of its range due to anthropogenic activity including overfishing and habitat degradation. We generated genetic data using high throughput sequencing of reduced representation libraries for taimen from multiple drainages in Mongolia and Russia. Nucleotide diversity estimates were within the range documented in other salmonids, suggesting moderate diversity despite widespread population declines. Similar to other recent studies, our analyses revealed pronounced differentiation among the Arctic (Selenge) and Pacific (Amur and Tugur) drainages, suggesting historical isolation among these systems. However, we found evidence for finer-scale structure within the Pacific drainages, including unexpected differentiation between tributaries and the mainstem of the Tugur River. Differentiation across the Amur and Tugur basins together with coalescent-based demographic modeling suggests the ancestors of Tugur tributary taimen likely diverged in the eastern Amur basin, prior to eventual colonization of the Tugur basin. Our results suggest the potential for differentiation of taimen at different geographic scales, and suggest more thorough geographic and genomic sampling may be needed to inform conservation and management of this iconic salmonid.

Foundational hypotheses addressing plant–insect codiversification and plant defense theory typically assume a macroevolutionary pattern whereby closely related plants have similar chemical profiles. However, numerous studies have documented variation in the degree of phytochemical trait lability, raising the possibility that phytochemical evolution is more nuanced than initially assumed. We utilize proton nuclear magnetic resonance ( 1 H NMR) data, chemical classification, and double digest restriction-site associated DNA sequencing (ddRADseq) to resolve evolutionary relationships and characterize the evolution of secondary chemistry in the Neotropical plant clade Radula ( Piper ; Piperaceae). Sequencing data substantially improved phylogenetic resolution relative to past studies, and spectroscopic characterization revealed the presence of 35 metabolite classes. Metabolite classes displayed phylogenetic signal, whereas the crude 1 H NMR spectra featured little evidence of phylogenetic signal in multivariate tests of chemical resonances. Evolutionary correlations were detected in two pairs of compound classes (flavonoids with chalcones; p -alkenyl phenols with kavalactones), where the gain or loss of a class was dependent on the other’s state. Overall, the evolution of secondary chemistry in Radula is characterized by strong phylogenetic signal of traditional compound classes and weak phylogenetic signal of specialized chemical motifs, consistent with both classic evolutionary hypotheses and recent examinations of phytochemical evolution in young lineages.

Species delineation has a long and contentious history, yet most agree that sympatric populations exhibiting high levels of reproductive isolation and evolving independently are species. In an opinion piece, Hill and Powers (2021; hereafter H&P) claim that several recognized species of crossbills (Loxia spp.) do not represent species because by no measure are they discrete, the vocalizations used to categorize crossbills are learned, modified and can switch to that of a different species, and reproductive isolation is incomplete and weak. We argue that the behavioral and genetic evidence indicate that Cassia crossbills L. sinesciuris, which we focus on because the data relevant to species status are more diverse and extensive, are genetically discrete; call modification rarely leads to crossbill misclassification and overwhelmingly results in call divergence and enhanced discrimination; and are nearly completely reproductively isolated with little evidence of introgression from sympatric red crossbills. The differences in our conclusions result in part from H&P mischaracterizing and misconstruing the ecology of Cassia crossbills, geographic context of their divergence, and evidence for reproductive isolation. H&P seemingly require that crossbills must adhere to the typical model of bird speciation–protracted divergence in allopatry, followed by a gradual increase in sympatry if reproductive isolation and ecological divergence allow–and require evidence such as initial long periods of allopatry, FST values > 0.2, divergent mtDNA and intrinsic postzygotic isolation. Although such evidence commonly distinguishes bird species, an increasing number of studies show that such criteria are not necessary to indicate sympatric, evolutionarily independent lineages.