Phylogenomics reveals patterns of ancient hybridization and differential diversification that contribute to phylogenetic conflict in willows, poplars, and close relatives.

Despite the economic, ecological, and scientific importance of the genera Salix L. (willows) and Populus L. (poplars, cottonwoods, and aspens) Salicaceae, we know little about the sources of differences in species diversity between the genera and of the phylogenetic conflict that often confounds estimating phylogenetic trees. Salix subgenera and sections, in particular, have been difficult to classify, with one recent attempt termed a "spectacular failure" due to a speculated radiation of the subgenera Vetrix and Chamaetia. Here, we use targeted sequence capture to understand the evolutionary history of this portion of the Salicaceae plant family. Our phylogenetic hypothesis was based on 787 gene regions and identified extensive phylogenetic conflict among genes. Our analysis supported some previously described subgeneric relationships and confirmed the polyphyly of others. Using an fbranch analysis, we identified several cases of hybridization in deep branches of the phylogeny, which likely contributed to discordance among gene trees. In addition, we identified a rapid increase in diversification rate near the origination of the Vetrix-Chamaetia clade in Salix. This region of the tree coincided with several nodes that lacked strong statistical support, indicating a possible increase in incomplete lineage sorting due to rapid diversification. The extraordinary level of both recent and ancient hybridization in both Salix and Populus have played important roles in the diversification and diversity in these two genera.

Sex determination through X-Y heterogamety in Salix nigra.

The development of non-recombining sex chromosomes has radical effects on the evolution of discrete sexes and sexual dimorphism. Although dioecy is rare in plants, sex chromosomes have evolved repeatedly throughout the diversification of angiosperms, and many of these sex chromosomes are relatively young compared to those found in vertebrates. In this study, we designed and used a sequence capture array to identify a novel sex-linked region (SLR) in Salix nigra, a basal species in the willow clade, and demonstrated that this species has XY heterogamety. We did not detect any genetic overlap with the previously characterized ZW SLRs in willows, which map to a different chromosome. The S. nigra SLR is characterized by strong recombination suppression across a 2 MB region and an excess of low-frequency alleles, resulting in a low Tajima's D compared to the remainder of the genome. We speculate that either a recent bottleneck in population size or factors related to positive or background selection generated this differential pattern of Tajima's D on the X and autosomes. This discovery provides insights into factors that may influence the evolution of sex chromosomes in plants and contributes to a large number of recent observations that underscore their dynamic nature.

Phylogenomics of the genus Populus reveals extensive interspecific gene flow and balancing selection.

Phylogenetic analysis is complicated by interspecific gene flow and the presence of shared ancestral polymorphisms, particularly those maintained by balancing selection. In this study, we aimed to examine the prevalence of these factors during the diversification of Populus, a model tree genus in the Northern Hemisphere. We constructed phylogenetic trees of 29 Populus taxa using 80 individuals based on re-sequenced genomes. Our species tree analyses recovered four main clades in the genus based on consensus nuclear phylogenies, but in conflict with the plastome phylogeny. A few interspecific relationships remained unresolved within the multiple-species clade because of inconsistent gene trees. Our results indicated that gene flow has been widespread within each clade and also occurred among the four clades during their early divergence. We identified 45 candidate genes with ancient polymorphisms maintained by balancing selection. These genes were mainly associated with mating compatibility, growth and stress resistance. Both gene flow and selection-mediated ancient polymorphisms are prevalent in the genus Populus. These are potentially important contributors to adaptive variation. Our results provide a framework for the diversification of model tree genus that will facilitate future comparative studies.

Sex-biased gene expression in flowers, but not leaves, reveals secondary sexual dimorphism in Populus balsamifera.

Because sexual dimorphism in plants is often less morphologically conspicuous than in animals, studies of sex-biased gene expression may provide a quantitative metric to better address their commonality, molecular pathways, consistency across tissues and taxa, and evolution. The presence of sex-biased gene expression in tissues other than the androecium or gynoecium, termed secondary sexual characters, suggests that these traits arose after the initial evolution of dioecy. Patterns of sequence evolution may provide evidence of positive selection that drove sexual specialization. We compared gene expression in male and female flowers and leaves of Populus balsamifera to assess the extent of sex-biased expression, and tested whether sex-biased genes exhibit elevated rates of protein evolution. Sex-biased expression was pervasive in floral tissue, but nearly absent in leaf tissue. Female-biased genes in flowers were associated with photosynthesis, whereas male-biased genes were associated with mitochondrial function. Sex-biased genes did not exhibit elevated rates of protein evolution, contrary to results from other studies in animals and plants. Our results suggest that the ecological and physiological constraints associated with the energetics of flowering, rather than sexual conflict, have probably shaped the differences in male and female gene expression in P. balsamifera.

Population genomic analysis suggests strong influence of river network on spatial distribution of genetic variation in invasive saltcedar across the southwestern United States.

Understanding the complex influences of landscape and anthropogenic elements that shape the population genetic structure of invasive species provides insight into patterns of colonization and spread. The application of landscape genomics techniques to these questions may offer detailed, previously undocumented insights into factors influencing species invasions. We investigated the spatial pattern of genetic variation and the influences of landscape factors on population similarity in an invasive riparian shrub, saltcedar (Tamarix L.) by analysing 1,997 genomewide SNP markers for 259 individuals from 25 populations collected throughout the southwestern United States. Our results revealed a broad-scale spatial genetic differentiation of saltcedar populations between the Colorado and Rio Grande river basins and identified potential barriers to population similarity along both river systems. River pathways most strongly contributed to population similarity. In contrast, low temperature and dams likely served as barriers to population similarity. We hypothesize that large-scale geographic patterns in genetic diversity resulted from a combination of early introductions from distinct populations, the subsequent influence of natural selection, dispersal barriers and founder effects during range expansion.

Population genetics of freeze tolerance among natural populations of Populus balsamifera across the growing season.

Protection against freeze damage during the growing season influences the northern range limits of plants. Freeze tolerance and freeze avoidance are the two major freeze resistance strategies. Winter survival strategies have been extensively studied in perennials, but few have addressed them and their genetic basis during the growing season. We examined intraspecific phenotypic variation in freeze resistance of Populus balsamifera across latitude and the growing season. To investigate the molecular basis of this variation, we surveyed nucleotide diversity and examined patterns of gene expression in the poplar C-repeat binding factor (CBF) gene family. Foliar freeze tolerance exhibited latitudinal and seasonal variation indicative of natural genotypic variation. CBF6 showed signatures of recent selective sweep. Of the 46 SNPs surveyed across the six CBF homologs, only CBF2_619 exhibited latitudinal differences consistent with increased freeze tolerance in the north. All six CBF genes were cold inducible, but showed varying patterns of expression across the growing season. Some Poplar CBF homologs exhibited patterns consistent with historical selection and clinal variation in freeze tolerance documented here. However, the CBF genes accounted for only a small amount of the variation, indicating that other genes in this and other molecular pathways likely play significant roles in nature.

Relocation, high-latitude warming and host genetic identity shape the foliar fungal microbiome of poplars.

Micro-organisms associated with plants and animals affect host fitness, shape community structure and influence ecosystem properties. Climate change is expected to influence microbial communities, but their reactions are not well understood. Host-associated micro-organisms are influenced by the climate reactions of their hosts, which may undergo range shifts due to climatic niche tracking, or may be actively relocated to mitigate the effects of climate change. We used a common-garden experiment and rDNA metabarcoding to examine the effect of host relocation and high-latitude warming on the complex fungal endophytic microbiome associated with leaves of an ecologically dominant boreal forest tree (Populus balsamifera L.). We also considered the potential effects of poplar genetic identity in defining the reactions of the microbiome to the treatments. The relocation of hosts to the north increased the diversity of the microbiome and influenced its structure, with results indicating enemy release from plausible pathogens. High-latitude warming decreased microbiome diversity in comparison with natural northern conditions. The warming also caused structural changes, which made the fungal communities distinct in comparison with both low-latitude and high-latitude natural communities, and increased the abundance of plausible pathogens. The reactions of the microbiome to relocation and warming were strongly dependent on host genetic identity. This suggests that climate change effects on host-microbiome systems may be mediated by the interaction of environmental factors and the population genetic processes of the hosts.

Genomics in a changing arctic: critical questions await the molecular ecologist.

Molecular ecology is poised to tackle a host of interesting questions in the coming years. The Arctic provides a unique and rapidly changing environment with a suite of emerging research needs that can be addressed through genetics and genomics. Here we highlight recent research on boreal and tundra ecosystems and put forth a series of questions related to plant and microbial responses to climate change that can benefit from technologies and analytical approaches contained within the molecular ecologist's toolbox. These questions include understanding (i) the mechanisms of plant acquisition and uptake of N in cold soils, (ii) how these processes are mediated by root traits, (iii) the role played by the plant microbiome in cycling C and nutrients within high-latitude ecosystems and (iv) plant adaptation to extreme Arctic climates. We highlight how contributions can be made in these areas through studies that target model and nonmodel organisms and emphasize that the sequencing of the Populus and Salix genomes provides a valuable resource for scientific discoveries related to the plant microbiome and plant adaptation in the Arctic. Moreover, there exists an exciting role to play in model development, including incorporating genetic and evolutionary knowledge into ecosystem and Earth System Models. In this regard, the molecular ecologist provides a valuable perspective on plant genetics as a driver for community biodiversity, and how ecological and evolutionary forces govern community dynamics in a rapidly changing climate.

Local adaptation in the flowering-time gene network of balsam poplar, Populus balsamifera L.

Identifying the signature and targets of local adaptation is an increasingly important goal in empirical population genetics. Using data from 443 balsam poplar Populus balsamifera trees sampled from 31 populations, we tested for evidence of geographically variable selection shaping diversity at 27 homologues of the Arabidopsis flowering-time network. These genes are implicated in the control of seasonal phenology, an important determinant of fitness. Using 335 candidate and 412 reference single nucleotide polymorphisms (SNPs), we tested for evidence of local adaptation by searching for elevated population differentiation using F(ST)-based outlier analyses implemented in BayeScan or a Hierarchical Model in Arelquin and by testing for significant associations between allele frequency and environmental variables using BAYENV. A total of 46 SNPs from 14 candidate genes had signatures of local adaptation-either significantly greater population differentiation or significant covariance with one or more environmental variable relative to reference SNP distributions. Only 11 SNPs from two genes exhibited both elevated population differentiation and covariance with one or more environmental variables. Several genes including the abscisic acid gene ABI1B and the circadian clock genes ELF3 and GI5 harbored a large number of SNPs with signatures of local adaptation-with SNPs in GI5 strongly covarying with both latitude and precipitation and SNPs in ABI1B strongly covarying with temperature. In contrast to several other systems, we find little evidence that photoreceptors, including phytochromes, play an important role in local adaptation. Our results additionally show that detecting local adaptation is sensitive to the analytical approaches used and that model-based significance thresholds should be viewed with caution.

The adaptive potential of Populus balsamifera L. to phenology requirements in a warmer global climate.

The manner in which organisms adapt to climate change informs a broader understanding of the evolution of biodiversity as well as conservation and mitigation plans. We apply common garden and association mapping approaches to quantify genetic variance and identify loci affecting bud flush and bud set, traits that define a tree's season for height growth, in the boreal forest tree Populus balsamifera L. (balsam poplar). Using data from 478 genotypes grown in each of two common gardens, one near the southern edge and another near the northern edge of P. balsamifera's range, we found that broad-sense heritability for bud flush and bud set was generally high (H(2) > 0.5 in most cases), suggesting that abundant genetic variation exists for phenological response to changes in the length of the growing season. To identify the molecular genetic basis of this variation, we genotyped trees for 346 candidate single nucleotide polymorphisms (SNPs) from 27 candidate genes for the CO/FT pathway in poplar. Mixed-model analyses of variance identified SNPs in 10 genes to be associated with variation in either bud flush or bud set. Multiple SNPs within FRIGIDA were associated with bud flush, whereas multiple SNPs in LEAFY and GIGANTEA 5 were associated with bud set. Although there was strong population structure in stem phenology, the geographic distribution of multilocus association SNP genotypes was widespread except at the most northern populations, indicating that geographic regions may harbour sufficient diversity in functional genes to facilitate adaption to future climatic conditions in many sites.

Pleistocene speciation in the genus Populus (salicaceae).

The macroevolutionary consequences of recent climate change remain controversial, and there is little paleobotanical or morphological evidence that Pleistocene (1.8-0.12 Ma) glacial cycles acted as drivers of speciation, especially among lineages with long generation times, such as trees. We combined genetic and ecogeographic data from 2 closely related North American tree species, Populus balsamifera and P. trichocarpa (Salicacaeae), to determine if their divergence coincided with and was possibly caused by Pleistocene climatic events. We analyzed 32 nuclear loci from individuals of P. balsamifera and P. trichocarpa to produce coalescent-based estimates of the divergence time between the 2 species. We coupled the coalescent analyses with paleodistribution models to assess the influence of climate change on species' range. Furthermore, measures of niche overlap were used to investigate patterns of ecological differentiation between species. We estimated the divergence date of P. balsamifera and P. trichocarpa at approximately 75 Ka, which corresponds closely with the onset of Marine Isotope Stage 4 (∼76 Ka) and a rapid increase in global ice volume. Significance tests of niche overlap, in conjunction with genetic estimates of migration, suggested that speciation occurred in allopatry, possibly resulting from the environmental effects of Pleistocene glacial cycles. Our results indicate that the divergence of keystone tree species, which have shaped community diversity in northern North American ecosystems, was recent and may have been a consequence of Pleistocene-era glaciation and climate change.

Evolution of a ZW sex chromosome system in willows.

Transitions in the heterogamety of sex chromosomes (e.g., XY to ZW or vice versa) fundamentally alter the genetic basis of sex determination, however the details of these changes have been studied in only a few cases. In an XY to ZW transition, the X is likely to give rise to the W because they both carry feminizing genes and the X is expected to harbour less genetic load than the Y. Here, using a new reference genome for Salix exigua, we trace the X, Y, Z, and W sex determination regions during the homologous transition from an XY system to a ZW system in willow (Salix). We show that both the W and the Z arose from the Y chromosome. We find that the new Z chromosome shares multiple homologous putative masculinizing factors with the ancestral Y, whereas the new W lost these masculinizing factors and gained feminizing factors. The origination of both the W and Z from the Y was permitted by an unexpectedly low genetic load on the Y and this indicates that the origins of sex chromosomes during homologous transitions may be more flexible than previously considered.

Intercontinental divergence in the Populus-associated ectomycorrhizal fungus, Tricholoma populinum.

The ectomycorrhizal fungus Tricholoma populinum is host-specific with Populus species. T. populinum has wind-dispersed progagules and may be capable of long-distance dispersal. In this study, we tested the hypothesis of a panmictic population between Scandinavia and North America. DNA sequences from five nuclear loci were used to assess phylogeographic structure and nucleotide divergence between continents. Tricholoma populinum was composed of Scandinavian and North American lineages with complete absence of shared haplotypes and only one shared nucleotide mutation. Divergence of these lineages was estimated at approx. 1.7-1.0 million yr ago (Ma), which occurred after the estimated divergence of host species Populus tremula and Populus balsamifera/Populus trichocarpa at 5 Ma. Phylogeographic structure was not observed within Scandinavian or North American lineages of T. populinum. Intercontinental divergence appears to have resulted from either allopatric isolation; a recent, rare long-distance dispersal founding event followed by genetic drift; or the response in an obligate mycorrhizal fungus with a narrow host range to contractions and expansion of host distribution during glacial and interglacial episodes within continents. Understanding present genetic variation in populations is important for predicting how obligate symbiotic fungi will adapt to present and future changing climatic conditions.

Classic clover cline clues.

Adaptive clines are striking examples of natural selection in action, yet few have been studied in depth. In this issue of Molecular Ecology, Kooyers & Olsen (2012) introduce modern analyses and thinking towards studies of a classical example of the rapid and repeated evolution of latitudinal and altitudinal clines in cyanogenesis in white clover, Trifolium repens L. Recognizing that adaptive clines represent trade-offs in the selective benefits of traits at different ends of a geographical transect, these researchers focus on whether evidence for selection can be found at regional (coarse) and local (fine) scales. After adjusting for population genetic patterns generated by demographic processes, Kooyers and Olsen provide evidence that the cyanogenesis cline is adaptive across a transect from Louisiana to Wisconsin, USA. Within local populations, divergent selection on coupling dominant and recessive alleles that underlie cyanogenesis is predicted to drive populations to gametic phase disequilibrium (LD), a pattern that has been found in several other studies reviewed by Kooyers and Olsen. The absence of LD within any sampled populations in this study leads the authors to suggest that selective patterns within these clines may be more complex than previously proposed, perhaps even following theoretical predictions of a geographic mosaic.

Climate-driven local adaptation of ecophysiology and phenology in balsam poplar, Populus balsamifera L. (Salicaceae).

PREMISE OF THE STUDY: During past episodes of climate change, many plant species experienced large-scale range expansions. Expanding populations likely encountered strong selection as they colonized new environments. In this study we examine the extent to which populations of the widespread forest tree Populus balsamifera L. have become locally adapted as the species expanded into its current range since the last glaciation. METHODS: We tested for adaptive variation in 13 ecophysiology and phenology traits on clonally propagated genotypes originating from a range-wide sample of 20 subpopulations. The hypothesis of local adaption was tested by comparing among-population variation at ecologically important traits (Q(ST)) to expected variation based on demographic history (F(ST)) estimated from a large set of nuclear single nucleotide polymorphism loci. KEY RESULTS: Evidence for divergence in excess of neutral expectations was present for eight of 13 traits. Bud phenology, petiole length, and leaf nitrogen showed the greatest divergence (all Q(ST) > 0.6), whereas traits related to leaf water usage showed the least (all Q(ST) ≤ 0.30) and were not different from neutrality. Strong correlations were present between traits, geography, and climate, and they revealed a general pattern of northern subpopulations adapted to shorter, drier growing seasons compared with populations in the center or eastern regions of the range. CONCLUSIONS: Our study demonstrates pronounced adaptive variation in ecophysiology and phenology among balsam poplar populations. These results suggest that as this widespread forest tree species expanded its range since the end of the last glacial maximum, it evolved rapidly in response to geographically variable selection.

Integrative genomics reveals paths to sex dimorphism in Salix purpurea L.

Sex dimorphism and gene expression were studied in developing catkins in 159 F2 individuals from the bioenergy crop Salix purpurea, and potential mechanisms and pathways for regulating sex development were explored. Differential expression, eQTL, bisulfite sequencing, and network analysis were used to characterize sex dimorphism, detect candidate master regulator genes, and identify pathways through which the sex determination region (SDR) may mediate sex dimorphism. Eleven genes are presented as candidates for master regulators of sex, supported by gene expression and network analyses. These include genes putatively involved in hormone signaling, epigenetic modification, and regulation of transcription. eQTL analysis revealed a suite of transcription factors and genes involved in secondary metabolism and floral development that were predicted to be under direct control of the sex determination region. Furthermore, data from bisulfite sequencing and small RNA sequencing revealed strong differences in expression between males and females that would implicate both of these processes in sex dimorphism pathways. These data indicate that the mechanism of sex determination in Salix purpurea is likely different from that observed in the related genus Populus. This further demonstrates the dynamic nature of SDRs in plants, which involves a multitude of mechanisms of sex determination and a high rate of turnover.

Transcription profiles of mitochondrial genes correlate with mitochondrial DNA haplotypes in a natural population of Silene vulgaris.

BACKGROUND: Although rapid changes in copy number and gene order are common within plant mitochondrial genomes, associated patterns of gene transcription are underinvestigated. Previous studies have shown that the gynodioecious plant species Silene vulgaris exhibits high mitochondrial diversity and occasional paternal inheritance of mitochondrial markers. Here we address whether variation in DNA molecular markers is correlated with variation in transcription of mitochondrial genes in S. vulgaris collected from natural populations. RESULTS: We analyzed RFLP variation in two mitochondrial genes, cox1 and atp1, in offspring of ten plants from a natural population of S. vulgaris in Central Europe. We also investigated transcription profiles of the atp1 and cox1 genes. Most DNA haplotypes and transcription profiles were maternally inherited; for these, transcription profiles were associated with specific mitochondrial DNA haplotypes. One individual exhibited a pattern consistent with paternal inheritance of mitochondrial DNA; this individual exhibited a transcription profile suggestive of paternal but inconsistent with maternal inheritance. We found no associations between gender and transcript profiles. CONCLUSIONS: Specific transcription profiles of mitochondrial genes were associated with specific mitochondrial DNA haplotypes in a natural population of a gynodioecious species S. vulgaris.Our findings suggest the potential for a causal association between rearrangements in the plant mt genome and transcription product variation.

Nucleotide diversity and linkage disequilibrium in balsam poplar (Populus balsamifera).

*Current perceptions that poplars have high levels of nucleotide variation, large effective population sizes, and rapid decay of linkage disequilibrium are based primarily on studies from one poplar species, Populus tremula. *We analysed 590 gene fragments (average length 565 bp) from each of 15 individuals from different populations from throughout the range of Populus balsamifera. *Nucleotide diversity (theta(total) = 0.0028, pi = 0.0027) was low compared with other trees and model agricultural systems. Patterns of nucleotide diversity and site frequency spectra were consistent with purifying selection on replacement and intron sites. When averaged across all loci we found no evidence for decay of linkage disequilibrium across 750 bp, consistent with the low estimates of the scaled recombination parameter, rho = 0.0092. *Compared with P. tremula, a well studied congener with a similar distribution, P. balsamifera has low diversity and low effective recombination, both of which indicate a lower effective population size in P. balsamifera. Patterns of diversity and linkage indicate that there is considerable variation in population genomic patterns among poplar species and unlike P. tremula, association mapping techniques in balsam poplar should consider sampling single nucleotide polymorphisms (SNPs) at well-spaced intervals.

Genomic diversity, population structure, and migration following rapid range expansion in the Balsam poplar, Populus balsamifera.

Rapid range expansions can cause pervasive changes in the genetic diversity and structure of populations. The postglacial history of the Balsam Poplar, Populus balsamifera, involved the colonization of most of northern North America, an area largely covered by continental ice sheets during the last glacial maximum. To characterize how this expansion shaped genomic diversity within and among populations, we developed 412 SNP markers that we assayed for a range-wide sample of 474 individuals sampled from 34 populations. We complemented the SNP data set with DNA sequence data from 11 nuclear loci from 94 individuals, and used coalescent analyses to estimate historical population size, demographic growth, and patterns of migration. Bayesian clustering identified three geographically separated demes found in the Northern, Central, and Eastern portions of the species' range. These demes varied significantly in nucleotide diversity, the abundance of private polymorphisms, and population substructure. Most measures supported the Central deme as descended from the primary refuge of diversity. Both SNPs and sequence data suggested recent population growth, and coalescent analyses of historical migration suggested a massive expansion from the Centre to the North and East. Collectively, these data demonstrate the strong influence that range expansions exert on genomic diversity, both within local populations and across the range. Our results suggest that an in-depth knowledge of nucleotide diversity following expansion requires sampling within multiple populations, and highlight the utility of combining insights from different data types in population genomic studies.

A targeted sequence capture array for phylogenetics and population genomics in the Salicaceae.

PREMISE: The family Salicaceae has proved taxonomically challenging, especially in the genus Salix, which is speciose and features frequent hybridization and polyploidy. Past efforts to reconstruct the phylogeny with molecular barcodes have failed to resolve the species relationships of many sections of the genus. METHODS: We used the wealth of sequence data in the family to design sequence capture probes to target regions of 300-1200 bp of exonic regions of 972 genes. RESULTS: We recovered sequence data for nearly all of the targeted genes in three species of Populus and three species of Salix. We present a species tree, discuss concordance among gene trees, and present population genomic summary statistics for these loci. CONCLUSIONS: Our sequence capture array has extremely high capture efficiency within the genera Populus and Salix, resulting in abundant phylogenetic information. Additionally, these loci show promise for population genomic studies.