Wood Decay Characteristics and Interspecific Interactions Control Bacterial Community Succession in Populus grandidentata (Bigtooth Aspen).

Few studies have investigated bacterial community succession and the role of bacterial decomposition over a continuum of wood decay. Here, we identified how (i) the diversity and abundance of bacteria changed along a chronosequence of decay in Populus grandidentata (bigtooth aspen); (ii) bacterial community succession was dependent on the physical and chemical characteristics of the wood; (iii) interspecific bacterial interactions may mediate community structure. Four hundred and fifty-nine taxa were identified through Illumina sequencing of 16S rRNA amplicons from samples taken along a continuum of decay, representing standing dead trees, downed wood, and soil. Community diversity increased as decomposition progressed, peaking in the most decomposed trees. While a small proportion of taxa displayed a significant pattern in regards to decay status of the host log, many bacterial taxa followed a stochastic distribution. Changes in the water availability and chemical composition of standing dead and downed trees and soil were strongly coupled with shifts in bacterial communities. Nitrogen was a major driver of succession and nitrogen-fixing taxa of the order Rhizobiales were abundant early in decomposition. Recently downed logs shared 65% of their bacterial abundance with the microbiomes of standing dead trees while only sharing 16% with soil. As decay proceeds, bacterial communities appear to respond less to shifting resource availability and more to interspecific bacterial interactions - we report an increase in both the proportion (+9.3%) and the intensity (+62.3%) of interspecific interactions in later stages of decomposition, suggesting the emergence of a more complex community structure as wood decay progresses.

Development of nuclear microsatellite loci for Pinus albicaulis Engelm. (Pinaceae), a conifer of conservation concern.

Pinus albicaulis (whitebark pine) is a widely-distributed but rapidly declining high elevation western North American tree and a candidate for listing under the U.S. Endangered Species Act. Our objectives were to develop reliable nuclear microsatellite markers that can be used to assess within-population genetic diversity as well as seed and pollen migration dynamics, and to validate markers using two geographically proximal P. albicaulis populations. We identified 1,667 microsatellite-containing sequences from shotgun DNA libraries of P. albicaulis. Primer pairs were designed for 308 unique microsatellite-containing loci, and these were evaluated for PCR amplification success and segregation in a panel of diploid needle tissue. DNA was extracted with an SDS protocol, and primers were screened through gel electrophoresis. Microsatellites were genotyped through fluorescent primer fragment analysis. Ten novel and 13 transferred loci were found to be reproducible in analyses based on 20 foliage samples from each of two locations: Henderson Mountain, Custer Gallatin National Forest, Montana, and Mt. Washburn, Yellowstone National Park, Wyoming (USA). Transferred loci had higher numbers of alleles and expected heterozygosities than novel loci, but also revealed evidence for a higher frequency of null alleles. Eight of the 13 transferred loci deviated significantly from Hardy-Weinberg Equilibrium, and showed large positive FIS values that were likely inflated by null alleles. Mantel's tests of transferred and novel markers showed no correlation between genetic and geographic distances within or among the two sampled populations. AMOVA suggests that 91% of genetic variability occurs within populations and 9% between the two populations. Studies assessing genetic diversity using these microsatellite loci can help guide future management and restoration activities for P. albicaulis.

Evaluating allopolyploid origins in strawberries (Fragaria) using haplotypes generated from target capture sequencing.

BACKGROUND: Hybridization is observed in many eukaryotic lineages and can lead to the formation of polyploid species. The study of hybridization and polyploidization faces challenges both in data generation and in accounting for population-level phenomena such as coalescence processes in phylogenetic analysis. Genus Fragaria is one example of a set of plant taxa in which a range of ploidy levels is observed across species, but phylogenetic origins are unknown. RESULTS: Here, using 20 diploid and polyploid Fragaria species, we combine approaches from NGS data analysis and phylogenetics to infer evolutionary origins of polyploid strawberries, taking into account coalescence processes. We generate haplotype sequences for 257 low-copy nuclear markers assembled from Illumina target capture sequence data. We then identify putative hybridization events by analyzing gene tree topologies, and further test predicted hybridizations in a coalescence framework. This approach confirms the allopolyploid ancestry of F. chiloensis and F. virginiana, and provides new allopolyploid ancestry hypotheses for F. iturupensis, F. moschata, and F. orientalis. Evidence of gene flow between diploids F. bucharica and F. vesca is also detected, suggesting that it might be appropriate to consider these groups as conspecifics. CONCLUSIONS: This study is one of the first in which target capture sequencing followed by computational deconvolution of individual haplotypes is used for tracing origins of polyploid taxa. The study also provides new perspectives on the evolutionary history of Fragaria.

Targeted Capture Sequencing in Whitebark Pine Reveals Range-Wide Demographic and Adaptive Patterns Despite Challenges of a Large, Repetitive Genome.

Whitebark pine (Pinus albicaulis) inhabits an expansive range in western North America, and it is a keystone species of subalpine environments. Whitebark is susceptible to multiple threats - climate change, white pine blister rust, mountain pine beetle, and fire exclusion - and it is suffering significant mortality range-wide, prompting the tree to be listed as 'globally endangered' by the International Union for Conservation of Nature and 'endangered' by the Canadian government. Conservation collections (in situ and ex situ) are being initiated to preserve the genetic legacy of the species. Reliable, transferrable, and highly variable genetic markers are essential for quantifying the genetic profiles of seed collections relative to natural stands, and ensuring the completeness of conservation collections. We evaluated the use of hybridization-based target capture to enrich specific genomic regions from the 27 GB genome of whitebark pine, and to evaluate genetic variation across loci, trees, and geography. Probes were designed to capture 7,849 distinct genes, and screening was performed on 48 trees. Despite the inclusion of repetitive elements in the probe pool, the resulting dataset provided information on 4,452 genes and 32% of targeted positions (528,873 bp), and we were able to identify 12,390 segregating sites from 47 trees. Variations reveal strong geographic trends in heterozygosity and allelic richness, with trees from the southern Cascade and Sierra Range showing the greatest distinctiveness and differentiation. Our results show that even under non-optimal conditions (low enrichment efficiency; inclusion of repetitive elements in baits), targeted enrichment produces high quality, codominant genotypes from large genomes. The resulting data can be readily integrated into management and gene conservation activities for whitebark pine, and have the potential to be applied to other members of 5-needle pine group (Pinus subsect. Quinquefolia) due to their limited genetic divergence.

An empirical evaluation of two-stage species tree inference strategies using a multilocus dataset from North American pines.

BACKGROUND: As it becomes increasingly possible to obtain DNA sequences of orthologous genes from diverse sets of taxa, species trees are frequently being inferred from multilocus data. However, the behavior of many methods for performing this inference has remained largely unexplored. Some methods have been proven to be consistent given certain evolutionary models, whereas others rely on criteria that, although appropriate for many parameter values, have peculiar zones of the parameter space in which they fail to converge on the correct estimate as data sets increase in size. RESULTS: Here, using North American pines, we empirically evaluate the behavior of 24 strategies for species tree inference using three alternative outgroups (72 strategies total). The data consist of 120 individuals sampled in eight ingroup species from subsection Strobus and three outgroup species from subsection Gerardianae, spanning ∼47 kilobases of sequence at 121 loci. Each "strategy" for inferring species trees consists of three features: a species tree construction method, a gene tree inference method, and a choice of outgroup. We use multivariate analysis techniques such as principal components analysis and hierarchical clustering to identify tree characteristics that are robustly observed across strategies, as well as to identify groups of strategies that produce trees with similar features. We find that strategies that construct species trees using only topological information cluster together and that strategies that use additional non-topological information (e.g., branch lengths) also cluster together. Strategies that utilize more than one individual within a species to infer gene trees tend to produce estimates of species trees that contain clades present in trees estimated by other strategies. Strategies that use the minimize-deep-coalescences criterion to construct species trees tend to produce species tree estimates that contain clades that are not present in trees estimated by the Concatenation, RTC, SMRT, STAR, and STEAC methods, and that in general are more balanced than those inferred by these other strategies. CONCLUSIONS: When constructing a species tree from a multilocus set of sequences, our observations provide a basis for interpreting differences in species tree estimates obtained via different approaches that have a two-stage structure in common, one step for gene tree estimation and a second step for species tree estimation. The methods explored here employ a number of distinct features of the data, and our analysis suggests that recovery of the same results from multiple methods that tend to differ in their patterns of inference can be a valuable tool for obtaining reliable estimates.

Multilocus analyses reveal little evidence for lineage-wide adaptive evolution within major clades of soft pines (Pinus subgenus Strobus).

Estimates from molecular data for the fraction of new nonsynonymous mutations that are adaptive vary strongly across plant species. Much of this variation is due to differences in life history strategies as they influence the effective population size (Ne ). Ample variation for these estimates, however, remains even when comparisons are made across species with similar values of Ne . An open question thus remains as to why the large disparity for estimates of adaptive evolution exists among plant species. Here, we have estimated the distribution of deleterious fitness effects (DFE) and the fraction of adaptive nonsynonymous substitutions (α) for 11 species of soft pines (subgenus Strobus) using DNA sequence data from 167 orthologous nuclear gene fragments. Most newly arising nonsynonymous mutations were inferred to be so strongly deleterious that they would rarely become fixed. Little evidence for long-term adaptive evolution was detected, as all 11 estimates for α were not significantly different from zero. Nucleotide diversity at synonymous sites, moreover, was strongly correlated with attributes of the DFE across species, thus illustrating a strong consistency with the expectations from the Nearly Neutral Theory of molecular evolution. Application of these patterns to genome-wide expectations for these species, however, was difficult as the loci chosen for the analysis were a biased set of conserved loci, which greatly influenced the estimates of the DFE and α. This implies that genome-wide parameter estimates will need truly genome-wide data, so that many of the existing patterns documented previously for forest trees (e.g. little evidence for signature of selection) may need revision.

Targeted enrichment strategies for next-generation plant biology.

PREMISE OF THE STUDY: The dramatic advances offered by modern DNA sequencers continue to redefine the limits of what can be accomplished in comparative plant biology. Even with recent achievements, however, plant genomes present obstacles that can make it difficult to execute large-scale population and phylogenetic studies on next-generation sequencing platforms. Factors like large genome size, extensive variation in the proportion of organellar DNA in total DNA, polyploidy, and gene number/redundancy contribute to these challenges, and they demand flexible targeted enrichment strategies to achieve the desired goals. METHODS: In this article, we summarize the many available targeted enrichment strategies that can be used to target partial-to-complete organellar genomes, as well as known and anonymous nuclear targets. These methods fall under four categories: PCR-based enrichment, hybridization-based enrichment, restriction enzyme-based enrichment, and enrichment of expressed gene sequences. KEY RESULTS: Examples of plant-specific applications exist for nearly all methods described. While some methods are well established (e.g., transcriptome sequencing), other promising methods are in their infancy (hybridization enrichment). A direct comparison of methods shows that PCR-based enrichment may be a reasonable strategy for accessing small genomic targets (e.g., ≤50 kbp), but that hybridization and transcriptome sequencing scale more efficiently if larger targets are desired. CONCLUSIONS: While the benefits of targeted sequencing are greatest in plants with large genomes, nearly all comparative projects can benefit from the improved throughput offered by targeted multiplex DNA sequencing, particularly as the amount of data produced from a single instrument approaches a trillion bases per run.

Interspecific phylogenetic analysis enhances intraspecific phylogeographical inference: a case study in Pinus lambertiana.

Pinus lambertiana (sugar pine) is an economically and ecologically important conifer with a 1600-km latitudinal range extending from Oregon, USA, to northern Baja California, Mexico. Like all North American white pines (subsect. Strobus), sugar pine is highly susceptible to white pine blister rust, a disease caused by the fungus Cronartium ribicola. We conducted a chloroplast DNA (cpDNA) survey of Pinus subsect. Strobus with comprehensive geographical sampling of P. lambertiana. Sequence analysis of 12 sugar pine individuals revealed strong geographical differentiation for two chloroplast haplotypes. A diagnostic restriction site survey of an additional 72 individuals demarcated a narrow 150-km contact zone in northeastern California. In the contact zone, maternal (megagametophtye) and paternal (embryo) haplotypes were identified in 31 single seeds, demonstrating bidirectional pollen flow extending beyond the range of maternal haplotypes. The frequencies of the Cr1 allele for white pine blister rust major gene resistance, previously determined for 41 seed zones, differ significantly among seed zones that are fixed for the alternate haplotypes, or contain a mixture of both haplotypes. Interspecific phylogenetic analysis reveals that the northern sugar pine haplotype belongs to a clade that includes Pinus albicaulis (whitebark pine) and all of the East Asian white pines. Furthermore, there is little cpDNA divergence between northern sugar pine and whitebark pine (dS = 0.00058). These results are consistent with a Pleistocene migration of whitebark pine into North America and subsequent chloroplast introgression from whitebark pine to sugar pine. This study demonstrates the importance of placing phylogeographical results in a broader phylogenetic context.

Widespread genealogical nonmonophyly in species of Pinus subgenus Strobus.

Phylogenetic relationships among Pinus species from subgenus Strobus remain unresolved despite combined efforts based on nrITS and cpDNA. To provide greater resolution among these taxa, a 900-bp intron from a late embryogenesis abundant (LEA)-like gene (IFG8612)was sequenced from 39 pine species, with two or more alleles representing 33 species. Nineteen of 33 species exhibited allelic nonmonphyly in the strict consensus tree, and 10 deviated significantly from allelic monophyly based on topology incongruence tests. Intraspecific nucleotide diversity ranged from 0.0 to 0.0211, and analysis of variance shows that nucleotide diversity was strongly associated (P < 0.0001)with the degree of species monophyly. Although species nonmonophyly complicates phylogenetic interpretations, this nuclear locus offers greater topological support than previously observed for cpDNA or nrITS. Lacking evidence for hybridization, recombination, or imperfect taxonomy, we feel that incomplete lineage sorting remains the best explanation for the polymorphisms shared among species. Depending on the species, coalescent expectations indicate that reciprocal monophyly will be more likely than paraphyly in 1.71 to 24.0 x 10(6) years, and that complete genome-wide coalescence in these species may require up to 76.3 x 10(6) years. The absence of allelic coalescence is a severe constraint in the application of phylogenetic methods in Pinus, and taxa sharing similar life history traits with Pinus are likely to show species nonmonophyly using nuclear markers.

Fossil calibration of molecular divergence infers a moderate mutation rate and recent radiations for pinus.

Silent mutation rate estimates for Pinus vary 50-fold, ranging from angiosperm-like to among the slowest reported for plants. These differences either reflect extraordinary genomic processes or inconsistent fossil calibration, and they have important consequences for population and biogeographical inferences. Here we estimate mutation rates from 4 Pinus species that represent the major lineages using 11 nuclear and 4 chloroplast loci. Calibration was tested at the divergence of Pinus subgenera with the oldest leaf fossil from subg. Strobus (Eocene; 45 MYA) or a recently published subg. Strobus wood fossil (Cretaceous; 85 MYA). These calibrations place the origin of Pinus 190-102 MYA and give absolute silent rate estimates of 0.70-1.31x10(-9) and 0.22-0.42x10(-9).site-1.year-1 for the nuclear and chloroplast genomes, respectively. These rates are approximately 4- to 20-fold slower than angiosperms, but unlike many previous estimates, they are more consistent with the high per-generation deleterious mutation rates observed in pines. Chronograms from nuclear and chloroplast genomes show that the divergence of subgenera accounts for about half of the time since Pinus diverged from Picea, with subsequent radiations occurring more recently. By extending the sampling to encompass the phylogenetic diversity of Pinus, we predict that most extant subsections diverged during the Miocene. Moreover, subsect. Australes, Ponderosae, and Contortae, containing over 50 extant species, radiated within a 5 Myr time span starting as recently as 18 MYA. An Eocene divergence of pine subgenera (using leaf fossils) does not conflict with fossil-based estimates of the Pinus-Picea split, but a Cretaceous divergence using wood fossils accommodates Oligocene fossils that may represent modern subsections. Because homoplasy and polarity of character states have not been tested for fossil pine assignments, the choice of fossil and calibration node represents a significant source of uncertainty. Based on several lines of evidence (including agreement with ages inferred using calibrations outside of Pinus), we conclude that the 85 MYA calibration at the divergence of pine subgenera provides a reasonable lower bound and that further refinements in age and mutation rate estimates will require a synthetic examination of pine fossil history.

Evolutionary relationships among Pinus (Pinaceae) subsections inferred from multiple low-copy nuclear loci.

Sequence data from nrITS and cpDNA have failed to fully resolve phylogenetic relationships among Pinus species. Four low-copy nuclear genes, developed from the screening of 73 mapped conifer anchor loci, were sequenced from 12 species representing all subsections. Individual loci do not uniformly support either the nrITS or cpDNA hypotheses and in some cases produce unique topologies. Combined analysis of low-copy nuclear loci produces a well-supported subsectional topology of two subgenera, each divided into two sections, congruent with prior hypotheses of deep divergence in Pinus. The placements of P. nelsonii, P. krempfii, and P. contorta have been of continued systematic interest. Results strongly support the placement of P. nelsonii as sister to the remaining members of sect. Parrya, suggest a moderately well-supported and consistent position of P. krempfii as sister to the remaining members of sect. Quinquefoliae, and are ambiguous about the placement of P. contorta. A successful phylogenetic strategy in Pinus will require many low-copy nuclear loci that include a high proportion of silent sites and derive from independent linkage groups. The locus screening and evaluation strategy presented here can be broadly applied to facilitate the development of phylogenetic markers from the increasing number of available genomic resources.