Different filtering strategies of genotyping‐by‐sequencing data provide complementary resolutions of species boundaries and relationships in a clade of sexually deceptive orchids

Ongoing hybridization and retained ancestral polymorphism in rapidly radiating lineages could mask recent cladogenetic events. This presents a challenge for the application of molecular phylogenetic methods to resolve differences between closely related taxa. We reanalyzed published genotyping‐by‐sequencing (GBS) data to infer the phylogeny of four species within the Ophrys sphegodes complex, a recently radiated clade of orchids. We used different data filtering approaches to detect different signals contained in the dataset generated by GBS and estimated their effects on maximum likelihood trees, global F_ST and bootstrap support values. We obtained a maximum likelihood tree with high bootstrap support, separating the species by using a large dataset based on loci shared by at least 30% of accessions. Bootstrap and F_ST values progressively decreased when filtering for loci shared by a higher number of accessions. However, when filtering more stringently to retain homozygous and organellar loci, we identified two main clades. These clades group individuals independently from their a priori species assignment, but were associated with two organellar haplotype clusters. We infer that a less stringent filtering preferentially selects for rapidly evolving lineage‐specific loci, which might better delimit lineages. In contrast, when using homozygous/organellar DNA loci the signature of a putative hybridization event in the lineage prevails over the most recent phylogenetic signal. These results show that using differing filtering strategies on GBS data could dissect the organellar and nuclear DNA phylogenetic signal and yield novel insights into relationships between closely related species.     

Phylogenetic community structure in Minnesota oak savanna is influenced by spatial extent and environmental variation

The relative importance of environmental filtering, biotic interactions and neutral processes in community assembly remains an openly debated question and one that is increasingly addressed using phylogenetic approaches. Closely related species may occur together more frequently than expected (phylogenetic clustering) if environmental filtering operates on traits with significant phylogenetic signal. Recent studies show that phylogenetic clustering tends to increase with spatial scale, presumably because greater environmental variation is encompassed at larger spatial scales, providing opportunities for species to sort across environmental gradients. However, if environmental filtering is the cause of species sorting along environmental gradients, then environmental variation rather than spatial scale per se should drive the processes governing community assembly. Using species abundance and light availability data from a long‐term experiment in Minnesota oak savanna understory communities, we explicitly test the hypothesis that greater environmental variation results in greater phylogenetic clustering when spatial scale is held constant. Concordant with previous studies, we found that phylogenetic community structure varied with spatial extent. At the landscape scale (~1000 ha), communities were phylogenetically clustered. At the local scale (0.375ha), phylogenetic community structure varied among plots. As hypothesized, plots encompassing the greatest environmental variation in light availability exhibited the strongest phylogenetic clustering. We also found strong correlations between species functional traits, particularly specific leaf area (SLA) and perimeter per area (PA), and species light availability niche. There was also a phylogenetic signal in both functional traits and species light availability niche, providing a mechanistic explanation for phylogenetic clustering in relation to light availability. We conclude that the pattern of increased phylogenetic clustering with increased environmental variation is a consequence of environmental filtering acting on phylogenetically conserved functional traits. These results indicate that the importance of environmental filtering in community assembly depends not on spatial scale per se, but on the steepness of the environmental gradient.     

Is RAD‐seq suitable for phylogenetic inference? An in silico assessment and optimization

Inferring phylogenetic relationships between closely related taxa can be hindered by three factors: (1) the lack of informative molecular variation at short evolutionary timescale; (2) the lack of established markers in poorly studied taxa; and (3) the potential phylogenetic conflicts among different genomic regions due to incomplete lineage sorting or introgression. In this context, Restriction site Associated DNA sequencing (RAD‐seq) seems promising as this technique can generate sequence data from numerous DNA fragments scattered throughout the genome, from a large number of samples, and without preliminary knowledge on the taxa under study. However, divergence beyond the within‐species level will necessarily reduce the number of conserved and non‐duplicated restriction sites, and therefore the number of loci usable for phylogenetic inference. Here, we assess the suitability of RAD‐seq for phylogeny using a simulated experiment on the 12 Drosophila genomes, with divergence times ranging from 5 to 63 million years. These simulations show that RAD‐seq allows the recovery of the known Drosophila phylogeny with strong statistical support, even for relatively ancient nodes. Notably, this conclusion is robust to the potentially confounding effects of sequencing errors, heterozygosity, and low coverage. We further show that clustering RAD‐seq data using the BLASTN and SiLiX programs significantly improves the recovery of orthologous RAD loci compared with previously proposed approaches, especially for distantly related species. This study therefore validates the view that RAD sequencing is a powerful tool for phylogenetic inference.     

RAD‐sequencing for estimating genomic relatedness matrix‐based heritability in the wild: A case study in roe deer

Estimating the evolutionary potential of quantitative traits and reliably predicting responses to selection in wild populations are important challenges in evolutionary biology. The genomic revolution has opened up opportunities for measuring relatedness among individuals with precision, enabling pedigree‐free estimation of trait heritabilities in wild populations. However, until now, most quantitative genetic studies based on a genomic relatedness matrix (GRM) have focused on long‐term monitored populations for which traditional pedigrees were also available, and have often had access to knowledge of genome sequence and variability. Here, we investigated the potential of RAD‐sequencing for estimating heritability in a free‐ranging roe deer (Capreolous capreolus) population for which no prior genomic resources were available. We propose a step‐by‐step analytical framework to optimize the quality and quantity of the genomic data and explore the impact of the single nucleotide polymorphism (SNP) calling and filtering processes on the GRM structure and GRM‐based heritability estimates. As expected, our results show that sequence coverage strongly affects the number of recovered loci, the genotyping error rate and the amount of missing data. Ultimately, this had little effect on heritability estimates and their standard errors, provided that the GRM was built from a minimum number of loci (above 7,000). Genomic relatedness matrix‐based heritability estimates thus appear robust to a moderate level of genotyping errors in the SNP data set. We also showed that quality filters, such as the removal of low‐frequency variants, affect the relatedness structure of the GRM, generating lower h² estimates. Our work illustrates the huge potential of RAD‐sequencing for estimating GRM‐based heritability in virtually any natural population.     

Phylogenetic community structure and phylogenetic turnover across space and edaphic gradients in western Amazonian tree communities

Ecological and evolutionary processes influence community assembly at both local and regional scales. Adding a phylogenetic dimension to studies of species turnover allows tests of the extent to which environmental gradients, geographic distance and the historical biogeography of lineages have influenced speciation and dispersal of species throughout a region. We compare measures of beta diversity, phylogenetic community structure and phylobetadiversity (phylogenetic distance among communities) in 34 plots of Amazonian trees across white‐sand and clay terra firme forests in a 60 000 square kilometer area in Loreto, Peru. Dominant taxa in white‐sand forests were phylogenetically clustered, consistent with environmental filtering of conserved traits. Phylobetadiversity measures found significant phylogenetic clustering between terra firme communities separated by geographic distances of <200–300 km, consistent within recent local speciation at the watershed scale in the Miocene‐aged clay‐soil forests near the foothills of the Andes. Although both distance and habitat type yielded statistically significant effects on both species and phylogenetic turnover, the patterns we observed were more consistent with an effect of habitat specialization than dispersal limitation. Our results suggest a role for both broad‐scale biogeographic and evolutionary processes, as well as habitat specialization, influencing community structure in Amazonian forests.     

Shared quantitative trait loci underlying the genetic correlation between continuous traits

We review genetic correlations among quantitative traits in light of their underlying quantitative trait loci (QTL). We derive an expectation of genetic correlation from the effects of underlying loci and test whether published genetic correlations can be explained by the QTL underlying the traits. While genetically correlated traits shared more QTL (33%) on average than uncorrelated traits (11%), the actual number of shared QTL shared was small. QTL usually predicted the sign of the correlation with good accuracy, but the quantitative prediction was poor. Approximately 25% of trait pairs in the data set had at least one QTL with antagonistic effects. Yet a significant minority (20%) of such trait pairs have net positive genetic correlations due to such antagonistic QTL 'hidden' within positive genetic correlations. We review the evidence on whether shared QTL represent single pleiotropic loci or closely linked monotropic genes, and argue that strict pleiotropy can be viewed as one end of a continuum of recombination rates where r=0. QTL studies of genetic correlation will likely be insufficient to predict evolutionary trajectories over long time spans in large panmictic populations, but will provide important insights into the trade-offs involved in population and species divergence.     

Integrating genomic and phenotypic data to evaluate alternative phylogenetic and species delimitation hypotheses in a recent evolutionary radiation of grasshoppers

Although resolving phylogenetic relationships and establishing species limits are primary goals of systematics, these tasks remain challenging at both conceptual and analytical levels. Here, we integrated genomic and phenotypic data and employed a comprehensive suite of coalescent‐based analyses to develop and evaluate competing phylogenetic and species delimitation hypotheses in a recent evolutionary radiation of grasshoppers (Chorthippus binotatus group) composed of two species and eight putative subspecies. To resolve the evolutionary relationships within this complex, we first evaluated alternative phylogenetic hypotheses arising from multiple schemes of genomic data processing and contrasted genetic‐based inferences with different sources of phenotypic information. Second, we examined the importance of number of loci, demographic priors, number and kind of phenotypic characters and sex‐based trait variation for developing alternative species delimitation hypotheses. The best‐supported topology was largely compatible with phenotypic data and showed the presence of two clades corresponding to the nominative species groups, one including three well‐resolved lineages and the other comprising a four‐lineage polytomy and a well‐differentiated sister taxon. Integrative species delimitation analyses indicated that the number of employed loci had little impact on the obtained inferences but revealed the higher power provided by an increasing number of phenotypic characters and the usefulness of assessing their phylogenetic information content and differences between sexes in among‐taxa trait variation. Overall, our study highlights the importance of integrating multiple sources of information to test competing phylogenetic hypotheses and elucidate the evolutionary history of species complexes representing early stages of divergence where conflicting inferences are more prone to appear.     

Habitat filtering influences the phylogenetic structure of avian communities across a coastal gradient in southern Brazil

The evolution of a particular trait or combination of traits within lineages may affect subsequent evolutionary outcomes, leading closely related species to exhibit higher phenotypic similarity than expected under a simple Brownian‐motion evolutionary model. Niche theory postulates that phenotypes determine species distribution across environmental gradients, leading to a phylogenetic signature in the community assembly. Thus, the incorporation of species phylogeny in the analysis of community ecology structure allows one to link broader environmental, spatial and temporal factors to local, small‐scale ecological processes, thus enabling understanding of community assembly patterns in a broader context. We used the net relatedness index to assess phylogenetic structure within avian communities across a harshness gradient in coastal habitats in southern Brazil. We also evaluated phylogenetic beta diversity, to test whether closely related species exploit habitats with similar environmental conditions. In order to do so, we scaled up phylogenetic information from the species to site level using phylogenetic fuzzy weighting. We found a pattern of phylogenetic clustering in less‐vegetated habitats, namely sandy beach and dunes, which are subject to harsher conditions because of proximity to the ocean. Basal lineages were associated with the more structurally homogeneous sandy beach, while late‐divergence clades occurred in more complex habitats, which were positively related to vegetation cover and height. The observed pattern of phylogenetic clustering suggested the importance of harsh conditions in constraining the distribution of avian lineages. Furthermore, contrasting environmental features between habitats influenced phylogenetic variation, demonstrating the prevalence of phylogenetic habitat filtering. From an applied point of view, such as planning and management of biological reserves, we showed that the full array of habitat patches embedded within coastal ecological gradients must be included in order to preserve distinct evolutionary lineages.     

Comparative performance of double‐digest RAD sequencing across divergent arachnid lineages

Next‐generation sequencing technologies now allow researchers of non‐model systems to perform genome‐based studies without the requirement of a (often unavailable) closely related genomic reference. We evaluated the role of restriction endonuclease (RE) selection in double‐digest restriction‐site‐associated DNA sequencing (ddRADseq) by generating reduced representation genome‐wide data using four different RE combinations. Our expectation was that RE selections targeting longer, more complex restriction sites would recover fewer loci than RE with shorter, less complex sites. We sequenced a diverse sample of non‐model arachnids, including five congeneric pairs of harvestmen (Opiliones) and four pairs of spiders (Araneae). Sample pairs consisted of either conspecifics or closely related congeneric taxa, and in total 26 sample pair analyses were tested. Sequence demultiplexing, read clustering and variant calling were performed in the pyRAD program. The 6‐base pair cutter EcoRI combined with methylated site‐specific 4‐base pair cutter MspI produced, on average, the greatest numbers of intra‐individual loci and shared loci per sample pair. As expected, the number of shared loci recovered for a sample pair covaried with the degree of genetic divergence, estimated with cytochrome oxidase I sequences, although this relationship was non‐linear. Our comparative results will prove useful in guiding protocol selection for ddRADseq experiments on many arachnid taxa where reference genomes, even from closely related species, are unavailable.     

Phylogenomic analysis suggests Coreidae and Alydidae (Hemiptera: Heteroptera) are not monophyletic

Next‐generation sequencing technologies (NGS) allow systematists to amass a wealth of genomic data from non‐model species for phylogenetic resolution at various temporal scales. However, phylogenetic inference for many lineages dominated by non‐model species has not yet benefited from NGS, which can complement Sanger sequencing studies. One such lineage, whose phylogenetic relationships remain uncertain, is the diverse, agriculturally important and charismatic Coreoidea (Hemiptera: Heteroptera). Given the lack of consensus on higher‐level relationships and the importance of a robust phylogeny for evolutionary hypothesis testing, we use a large data set comprised of hundreds of ultraconserved element (UCE) loci to infer the phylogeny of Coreoidea (excluding Stenocephalidae and Hyocephalidae), with emphasis on the families Coreidae and Alydidae. We generated three data sets by including alignments that contained loci sampled for at least 50%, 60%, or 70% of the total taxa, and inferred phylogeny using maximum likelihood and summary coalescent methods. Twenty‐six external morphological features used in relatively comprehensive phylogenetic analyses of coreoids were also re‐evaluated within our molecular phylogenetic framework. We recovered 439–970 loci per species (16%–36% of loci targeted) and combined this with previously generated UCE data for 12 taxa. All data sets, regardless of analytical approach, yielded topologically similar and strongly supported trees, with the exception of outgroup relationships and the position of Hydarinae. We recovered a monophyletic Coreoidea, with Rhopalidae highly supported as the sister group to Alydidae + Coreidae. Neither Alydidae nor Coreidae were monophyletic; the coreid subfamilies Hydarinae and Pseudophloeinae were recovered as more closely related to Alydidae than to other coreid subfamilies. Coreinae were paraphyletic with respect to Meropachyinae. Most morphological traits were homoplastic with several clades defined by few, if any, synapomorphies. Our results demonstrate the utility of phylogenomic approaches in generating robust hypotheses for taxa with long‐standing phylogenetic problems and highlight that novel insights may come from such approaches.     

Phylogenetic tests of community assembly across regional to continental scales in tropical and subtropical rain forests

Aim To measure and quantify community phylogenetic structure to evaluate how evolutionary, ecological and biogeographic processes have shaped the distributions and assemblage of tropical and subtropical rain forest tree species across local, regional and continental scales. Location Australia. Methods We used 596 assemblage‐level samples and 1137 woody species in rain forest vegetation sampled across two latitude regions (tropics and sub‐tropics) and five distinct areas. Based on this dataset, we obtained and analysed species‐level trait values (for leaf size, seed size, wood density and maximum height at maturity), measures of community phylogenetic structure and species turnover across space (beta) and evolutionary time (phylobeta). Results Phylobeta values showed that at continental scales (i.e. across the latitude regions combined) species replacement, as turnover in assemblages through time, was by more phylogenetically distant (i.e. less closely related) taxa. Within latitude regions replacement was by more closely related taxa. Assemblages of species were more phylogenetically clustered across the whole phylogeny (net relatedness index) and with respect to more recent divergences (nearest related taxon index) where the effects of historic disturbance (climatic oscillations) had been greater, and less clustered in long‐term stable (refugial) locations. Local species composition in the stable wet tropics showed significant phylogenetic evenness, but there was no corresponding evenness in distributions of the ecological traits measured. Main conclusions Despite a shared evolutionary and biogeographic history, the two regions diverged from each other before the development of internal divergences. Phylogenetic evenness is more evident in long‐term stable habitats (refugia) where species interact in conserved niches. Phylogenetic clustering is more evident where recolonization of more highly disturbed areas from historically reduced species pools reflects filtering of species into phylogenetically preferred habitats.     

Phylogenetic analysis of correlation structure in stalk-eyed flies (Diasemopsis,Diopsidae)

Morphological divergence among species may be constrained by the pattern of genetic variances and covariances among traits within species. Assessing the existence of such a relationship in nature requires information on the stability of intraspecific correlation and covariance structure and the correspondence of this structure to the pattern of evolutionary divergence within a lineage. Here, we investigate these issues for nine morphological traits and 15 species of stalk-eyed flies in the genus Diasemopsis. Within-species matrices for these traits were generated from phenotypic data for all the Diasemopsis species and from genetic data for a single Diasemopsis species, D. dubia. The among-species pattern of divergence was assessed by calculating the evolutionary correlations for all pairwise combinations of the morphological traits along the phylogeny of these species. Comparisons of intraspecific matrices reveal significant similarity among all species in the phenotypic correlations matrices but not the covariance matrices. In addition, the differences in correlation structure that do exist among species are not related to their phylogenetic placement or change in the means of the traits. Comparisons of the phenotypic and phylogenetic matrices suggest a strong relationship between the pattern of evolutionary change among species and both the intraspecific correlation structure and the stability of this structure among species. The phenotypic and the phylogenetic matrices are significantly similar, and pairs of traits whose intraspecific correlations are more stable across taxa exhibit stronger coevolution on the phylogeny. These results suggest either the existence of strong constraints on the pattern of evolutionary change or a consistent pattern of correlated selection shaping both the phenotypic and phylogenetic matrices. The genetic correlation structure for D. dubia, however, does not correspond with patterns found in the phenotypic and phylogenetic data. Possible reasons for this disagreement are discussed.     

Phylogenetic diversity of macromycetes and woody plants along an elevational gradient in Eastern Mexico

Phylogenetic information provides insight into the ecological and evolutionary processes that organize species assemblages. We compared patterns of phylogenetic diversity among macromycete and woody plant communities along a steep elevational gradient in eastern Mexico to better understand the evolutionary processes that structure their communities. Macrofungi and trees were counted and identified in eight sites from 100 to 3500 m asl, and sequence data retrieved from GenBank for the same or closely related species were used to reconstruct their phylogenies. Patterns of species richness and phylogenetic diversity were similar for both macrofungi and trees, but macromycete richness and diversity peaked at mid‐elevations, whereas woody plant richness and diversity did not show significant trends with elevation. Phylogenetic similarity among sites was low for both groups and decreased as elevational distance between sites increased. Macromycete communities displayed phylogenetic overdispersion at low elevations and phylogenetic clustering at high elevations; the latter is consistent with environmental filtering at high elevation sites. Woody plants generally exhibited phylogenetic clustering, consistent with the potential importance of environmental filtering throughout the elevational gradient.     

Neutral lipid fatty acid composition as trait and constraint in Collembola evolution

Functional traits determine the occurrence of species along environmental gradients and their coexistence with other species. Understanding how traits evolved among coexisting species helps to infer community assembly processes. We propose fatty acid composition in consumer tissue as a functional trait related to both food resources and physiological functions of species. We measured phylogenetic signal in fatty acid profiles of 13 field‐sampled Collembola (springtail) species and then combined the data with published fatty acid profiles of another 24 species. Collembola fatty acid profiles generally showed phylogenetic signal, with related species resembling each other. Long‐chain polyunsaturated fatty acids, related to physiological functions, demonstrated phylogenetic signal. In contrast, most food resource biomarker fatty acids and the ratios between bacterial, fungal, and plant biomarker fatty acids exhibited no phylogenetic signal. Presumably, fatty acids related to physiological functions have been constrained during Collembola evolutionary history: Species with close phylogenetic affinity experienced similar environments during divergence, while niche partitioning in food resources among closely related species favored species coexistence. Measuring phylogenetic signal in ecologically relevant traits of coexisting species provides an evolutionary perspective to contemporary assembly processes of ecological communities. Integrating phylogenetic comparative methods with community phylogenetic and trait‐based approaches may compensate for the limitations of each method when used alone and improve understanding of processes driving and maintaining assembly patterns.     

Phylogenetic patterns differ for native and exotic plant communities across a richness gradient in Northern California

Aim Increasingly, ecologists are using evolutionary relationships to infer the mechanisms of community assembly. However, modern communities are being invaded by non‐indigenous species. Since natives have been associated with one another through evolutionary time, the forces promoting character and niche divergence should be high. On the other hand, exotics have evolved elsewhere, meaning that conserved traits may be more important in their new ranges. Thus, co‐occurrence over sufficient time‐scales for reciprocal evolution may alter how phylogenetic relationships influence assembly. Here, we examined the phylogenetic structure of native and exotic plant communities across a large‐scale gradient in species richness and asked whether local assemblages are composed of more or less closely related natives and exotics and whether phylogenetic turnover among plots and among sites across this gradient is driven by turnover in close or distant relatives differentially for natives and exotics. Location Central and northern California, USA. Methods We used data from 30 to 50 replicate plots at four sites and constructed a maximum likelihood molecular phylogeny using the genes: matK, rbcl, ITS1 and 5.8s. We compared community‐level measures of native and exotic phylogenetic diversity and among‐plot phylobetadiversity. Results There were few exotic clades, but they tended to be widespread. Exotic species were phylogenetically clustered within communities and showed low phylogenetic turnover among communities. In contrast, the more species‐rich native communities showed higher phylogenetic dispersion and turnover among sites. Main conclusions The assembly of native and exotic subcommunities appears to reflect the evolutionary histories of these species and suggests that shared traits drive exotic patterns while evolutionary differentiation drives native assembly. Current invasions appear to be causing phylogenetic homogenization at regional scales.     

Universal target‐enrichment baits for anthozoan (Cnidaria) phylogenomics: New approaches to long‐standing problems

Anthozoans (e.g., corals, anemones) are an ecologically important and diverse group of marine metazoans that occur from shallow to deep waters worldwide. However, our understanding of the evolutionary relationships among the ~7,500 species within this class is hindered by the lack of phylogenetically informative markers that can be reliably sequenced across a diversity of taxa. We designed and tested 16,306 RNA baits to capture 720 ultraconserved element loci and 1,071 exon loci. Library preparation and target enrichment were performed on 33 taxa from all orders within the class Anthozoa. Following Illumina sequencing and Trinity assembly, we recovered 1,774 of 1,791 targeted loci. The mean number of loci recovered from each species was 638 ± 222, with more loci recovered from octocorals (783 ± 138 loci) than hexacorals (475 ± 187 loci). Parsimony informative sites ranged from 26 to 49% for alignments at differing hierarchical taxonomic levels (e.g., Anthozoa, Octocorallia, Hexacorallia). The per cent of variable sites within each of three genera (Acropora, Alcyonium, and Sinularia) for which multiple species were sequenced ranged from 4.7% to 30%. Maximum‐likelihood analyses recovered highly resolved trees with topologies matching those supported by other studies, including the monophyly of the order Scleractinia. Our results demonstrate the utility of this target‐enrichment approach to resolve phylogenetic relationships from relatively old to recent divergences. Redesigning the baits with improved affinities to capture loci within each subclass will provide a valuable toolset to address systematic questions, further our understanding of the timing of diversifications and help resolve long‐standing controversial relationships in the class Anthozoa.     

Ecological genomics of local adaptation in Cornus florida L. by genotyping by sequencing

Discovering local adaptation, its genetic underpinnings, and environmental drivers is important for conserving forest species. Ecological genomic approaches coupled with next‐generation sequencing are useful means to detect local adaptation and uncover its underlying genetic basis in nonmodel species. We report results from a study on flowering dogwood trees (Cornus florida L.) using genotyping by sequencing (GBS). This species is ecologically important to eastern US forests but is severely threatened by fungal diseases. We analyzed subpopulations in divergent ecological habitats within North Carolina to uncover loci under local selection and associated with environmental–functional traits or disease infection. At this scale, we tested the effect of incorporating additional sequencing before scaling for a broader examination of the entire range. To test for biases of GBS, we sequenced two similarly sampled libraries independently from six populations of three ecological habitats. We obtained environmental–functional traits for each subpopulation to identify associations with genotypes via latent factor mixed modeling (LFMM) and gradient forests analysis. To test whether heterogeneity of abiotic pressures resulted in genetic differentiation indicative of local adaptation, we evaluated F_st per locus while accounting for genetic differentiation between coastal subpopulations and Piedmont‐Mountain subpopulations. Of the 54 candidate loci with sufficient evidence of being under selection among both libraries, 28–39 were Arlequin–BayeScan F_st outliers. For LFMM, 45 candidates were associated with climate (of 54), 30 were associated with soil properties, and four were associated with plant health. Reanalysis of combined libraries showed that 42 candidate loci still showed evidence of being under selection. We conclude environment‐driven selection on specific loci has resulted in local adaptation in response to potassium deficiencies, temperature, precipitation, and (to a marginal extent) disease. High allele turnover along ecological gradients further supports the adaptive significance of loci speculated to be under selection.     

Evaluating high-throughput sequencing as a method for metagenomic analysis of nematode diversity

Nematodes play an important role in ecosystem processes, yet the relevance of nematode species diversity to ecology is unknown. Because nematode identification of all individuals at the species level using standard techniques is difficult and time-consuming, nematode communities are not resolved down to the species level, leaving ecological analysis ambiguous. We assessed the suitability of massively parallel sequencing for analysis of nematode diversity from metagenomic samples. We set up four artificial metagenomic samples involving 41 diverse reference nematodes in known abundances. Two samples came from pooling polymerase chain reaction products amplified from single nematode species. Two additional metagenomic samples consisted of amplified products of DNA extracted from pooled nematode species. Amplified products involved two rapidly evolving ~400-bp sections coding for the small and large subunit of rRNA. The total number of reads ranged from 4159 to 14771 per metagenomic sample. Of these, 82% were > 199 bp in length. Among the reads > 199 bp, 86% matched the referenced species with less than three nucleotide differences from a reference sequence. Although neither rDNA section recovered all nematode species, the use of both loci improved the detection level of nematode species from 90 to 97%. Overall, results support the suitability of massively parallel sequencing for identification of nematodes. In contrast, the frequency of reads representing individual species did not correlate with the number of individuals in the metagenomic samples, suggesting that further methodological work is necessary before it will be justified for inferring the relative abundances of species within a nematode community.     

LINKAGE TO THE MATING‐TYPE LOCUS ACROSS THE GENUS MICROBOTRYUM: INSIGHTS INTO NONRECOMBINING CHROMOSOMES

Parallels have been drawn between the evolution of nonrecombining regions in fungal mating‐type chromosomes and animal and plant sex chromosomes, particularly regarding the stages of recombination cessation forming evolutionary strata of allelic divergence. Currently, evidence and explanations for recombination cessation in fungi are sparse, and the presence of evolutionary strata has been examined in a minimal number of fungal taxa. Here, the basidiomycete genus Microbotryum was used to determine the history of recombination cessation for loci on the mating‐type chromosomes. Ancestry of linkage with mating type for 13 loci was assessed across 20 species by a phylogenetic method. No locus was found to exhibit trans‐specific polymorphism for alternate alleles as old as the mating pheromone receptor, indicating that ages of linkage to mating type varied among the loci. The ordering of loci in the ancestry of linkage to mating type does not agree with their previously proposed assignments to evolutionary strata. This study suggests that processes capable of influencing divergence between alternate alleles may act at loci in the nonrecombining regions (e.g., gene conversion) and encourages further work to dissect the evolutionary processes acting upon genomic regions that determine mating compatibility.