Calceolariaceae809: A bait set for targeted sequencing of nuclear loci.

Premise: The genus Calceolaria (Calceolariaceae) is emblematic of the Andes, is hypothesized to have originated as a recent, rapid radiation, and has important taxonomic needs. Additionally, the genus is a model for the study of specialized pollination systems, as its flowers are nectarless and many offer floral oils as a pollination reward collected by specialist bees. Despite their evolutionary and ecological significance, obtaining a resolved phylogeny for the group has proved difficult. To address this challenge, we present a new bait set for targeted sequencing of nuclear loci in Calceolariaceae and close relatives. Methods: We developed a bioinformatic workflow to use incomplete, low-coverage genomes of 10 Calceolaria species to identify single-copy loci suitable for phylogenetic studies and design baits for targeted sequencing. Results: Our approach resulted in the identification of 809 single-copy loci (733 noncoding and 76 coding regions) and the development of 39,937 baits, which we validated in silico (10 specimens) and in vitro (29 Calceolariaceae and six outgroups). In both cases, the data allowed us to recover robust phylogenetic estimates. Discussion: Our results demonstrate the appropriateness of the bait set for sequencing recent and historic specimens of Calceolariaceae and close relatives, and open new doors for further investigation of the evolutionary history of this hyperdiverse genus.

Reduced representation approaches produce similar results to whole genome sequencing for some common phylogeographic analyses.

When designing phylogeographic investigations researchers can choose to collect many different types of molecular markers, including mitochondrial genes or genomes, SNPs from reduced representation protocols, large sequence capture data sets, and even whole genomes. Given that the statistical power and accuracy of various analyses are expected to differ depending on both the type of marker and the amount of data collected, an exploration of the variance across methodological results as a function of marker type should provide valuable information to researchers. Here we collect mitochondrial Cytochrome b sequences, whole mitochondrial genomes, single nucleotide polymorphisms (SNP)s isolated using a genotype by sequencing (GBS) protocol, sequences from ultraconserved elements, and low-coverage nuclear genomes from the North American water vole (Microtus richardsoni). We estimate genetic distances, population genetic structure, and historical demography using data from each of these datasets and compare the results across markers. As anticipated, the results exhibit differences across marker types, particularly in terms of the resolution offered by different analyses. A cost-benefit analysis indicates that SNPs collected using a GBS protocol are the most cost-effective molecular marker, with inferences that mirror those collected from the whole genome data at a fraction of the cost per sample.

The role of multiple Pleistocene refugia in promoting diversification in the Pacific Northwest.

Pleistocene glacial cycles drastically changed the distributions of taxa endemic to temperate rainforests in the Pacific Northwest, with many experiencing reduced habitat suitability during glacial periods. In this study, we investigate whether glacial cycles promoted intraspecific divergence and whether subsequent range changes led to secondary contact and gene flow. For seven invertebrate species endemic to the PNW, we estimated species distribution models (SDMs) and projected them onto current and historical climate conditions to assess how habitat suitability changed during glacial cycles. Using single nucleotide polymorphism (SNP) data from these species, we assessed population genetic structure and used a machine-learning approach to compare models with and without gene flow between populations upon secondary contact after the last glacial maximum (LGM). Finally, we estimated divergence times and rates of gene flow between populations. SDMs suggest that there was less suitable habitat in the North Cascades and Northern Rocky Mountains during glacial compared to interglacial periods, resulting in reduced habitat suitability and increased habitat fragmentation during the LGM. Our genomic data identify population structure in all taxa, and support gene flow upon secondary contact in five of the seven taxa. Parameter estimates suggest that population divergences date to the later Pleistocene for most populations. Our results support a role of refugial dynamics in driving intraspecific divergence in the Cascades Range. In these invertebrates, population structure often does not correspond to current biogeographic or environmental barriers. Rather, population structure may reflect refugial lineages that have since expanded their ranges, often leading to secondary contact between once isolated lineages.

Phylogenomic analyses in Phrymaceae reveal extensive gene tree discordance in relationships among major clades.

PREMISE: Phylogenomic datasets using genomes and transcriptomes provide rich opportunities beyond resolving bifurcating phylogenetic relationships. Monkeyflower (Phrymaceae) is a model system for evolutionary ecology. However, it lacks a well-supported phylogeny as a basis for a stable taxonomy and for macroevolutionary comparisons. METHODS: We sampled 24 genomes and transcriptomes in Phrymaceae and closely related families, including eight newly sequenced transcriptomes. We reconstructed the phylogeny using IQ-TREE and ASTRAL, evaluated gene tree discordance using PhyParts, Quartet Sampling, and a cloudogram, and carried out reticulation analyses using PhyloNet and HyDe. We searched for whole genome duplication (WGD) events using chromosome numbers, synonymous distances, and gene duplication events as evidence. RESULTS: Most gene trees support the monophyly of Phrymaceae and each of its tribes. Most gene trees also support tribe Mimuleae being sister to Phrymeae + Diplaceae + Leucocarpeae, with extensive gene tree discordance among the latter three. Despite the discordance, the monophyly of Mimulus s.l. is rejected, and no individual reticulation event among the Phrymaceae tribes is well-supported. Reticulation likely occurred among Erythranthe bicolor and closely related species. No ancient WGD was detected in Phrymaceae. Instead, small-scale duplications are among potential drivers of macroevolutionary diversification of Phrymaceae. CONCLUSIONS: We show that analysis of reticulate evolution is sensitive to taxon sampling and methods used. We also demonstrate that phylogenomic datasets using genomes and transcriptomes present rich opportunities to investigate gene family evolution and genome duplication events involved in lineage diversification and adaptation.

Genomic evidence of an ancient inland temperate rainforest in the Pacific Northwest of North America.

The disjunct temperate rainforests of the Pacific Northwest of North America (PNW) are characterized by late-successional dominant tree species Thuja plicata (western redcedar) and Tsuga heterophylla (western hemlock). The demographic histories of these species, along with the PNW rainforest ecosystem in its entirety, have been heavily impacted by geological and climatic changes the PNW has experienced over the last 5 million years, including mountain orogeny and repeated Pleistocene glaciations. These environmental events have ultimately shaped the history of these species, with inland populations potentially being extirpated during the Pleistocene glaciations. Here, we collect genomic data for both species across their ranges to test multiple demographic models, each reflecting a different phylogeographical hypothesis on how the ecosystem-dominating species may have responded to dramatic climatic change. Our results indicate that inland and coastal populations in both species diverged ~2.5 million years ago in the early Pleistocene and experienced decreases in population size during glacial cycles, with subsequent population expansion. Importantly, we found evidence for gene flow between coastal and inland populations during the mid-Holocene. It is likely that intermittent migration in these species during this time has prevented allopatric speciation via genetic drift alone. In conclusion, our results from combining genomic data and demographic inference procedures establish that populations of the ecosystem dominants Thuja plicata and Tsuga heterophylla persisted in refugia located in both the coastal and inland regions of the PNW throughout the Pleistocene, with populations expanding and contracting in response to glacial cycles with occasional gene flow.

Phylogeny, classification, and character evolution of tribe Citharexyleae (Verbenaceae).

PREMISE: As a family of Neotropical origin and primarily Neotropical distribution, the Verbenaceae are a good but understudied system with which to understand Neotropical evolution. Tribe Citharexyleae comprises three genera: Baillonia, Citharexylum-one of the largest genera in Verbenaceae-and Rehdera. A molecular phylogenetic approach was taken to resolve intergeneric relationships in Citharexyleae and infrageneric relationships in Citharexylum. The phylogeny is used to elucidate character evolution in a widespread, morphologically diverse Neotropical genus. METHODS: Seven plastid regions, two nuclear ribosomal spacers, and six low-copy nuclear loci were analyzed for 64 species of Citharexyleae. Phylogenetic analyses were conducted using maximum likelihood, Bayesian inference, and multispecies coalescent approaches. Habit, presence or absence of thorns, inflorescence architecture, flower color, fruit color, and geography were examined to identify diagnostic character states for clades within Citharexylum. RESULTS: Rehdera is resolved as sister to Citharexylum, and Baillonia nested within Citharexylum. Two species, C. oleinum and C. tetramerum, are not closely related to tribe Citharexyleae, but may be related to members of tribe Duranteae instead. Seven clades within Citharexylum are inferred, each characterized by a combination of geography, fruit color and/or maturation, and inflorescence architecture. There is evidence of correlated evolution between habit, axillary inflorescences, and flower number per inflorescence. Shrubs with reduced inflorescences have evolved repeatedly. CONCLUSIONS: A subgeneric classification for Citharexylum is proposed. Although suites of associated traits are found, character morphology has been labile throughout Citharexylum's evolutionary history. Morphological diversity may be related to adaptation to differing mesic and xeric habitats.

Can functional genomic diversity provide novel insights into mechanisms of community assembly? A pilot study from an invaded alpine streambed.

An important focus of community ecology, including invasion biology, is to investigate functional trait diversity patterns to disentangle the effects of environmental and biotic interactions. However, a notable limitation is that studies usually rely on a small and easy-to-measure set of functional traits, which might not immediately reflect ongoing ecological responses to changing abiotic or biotic conditions, including those that occur at a molecular or physiological level. We explored the potential of using the diversity of expressed genes-functional genomic diversity (FGD)-to understand ecological dynamics of a recent and ongoing alpine invasion. We quantified FGD based on transcriptomic data measured for 26 plant species occurring along adjacent invaded and pristine streambeds. We used an RNA-seq approach to summarize the overall number of expressed transcripts and their annotations to functional categories, and contrasted this with functional trait diversity (FTD) measured from a suite of characters that have been traditionally considered in plant ecology. We found greater FGD and FTD in the invaded community, independent of differences in species richness. However, the magnitude of functional dispersion was greater from the perspective of FGD than from FTD. Comparing FGD between congeneric alien-native species pairs, we did not find many significant differences in the proportion of genes whose annotations matched functional categories. Still, native species with a greater relative abundance in the invaded community compared with the pristine tended to express a greater fraction of genes at significant levels in the invaded community, suggesting that changes in FGD may relate to shifts in community composition. Comparisons of diversity patterns from the community to the species level offer complementary insights into processes and mechanisms driving invasion dynamics. FGD has the potential to illuminate cryptic changes in ecological diversity, and we foresee promising avenues for future extensions across taxonomic levels and macro-ecosystems.

Analysis of Paralogs in Target Enrichment Data Pinpoints Multiple Ancient Polyploidy Events in Alchemilla s.l. (Rosaceae).

Target enrichment is becoming increasingly popular for phylogenomic studies. Although baits for enrichment are typically designed to target single-copy genes, paralogs are often recovered with increased sequencing depth, sometimes from a significant proportion of loci, especially in groups experiencing whole-genome duplication (WGD) events. Common approaches for processing paralogs in target enrichment data sets include random selection, manual pruning, and mainly, the removal of entire genes that show any evidence of paralogy. These approaches are prone to errors in orthology inference or removing large numbers of genes. By removing entire genes, valuable information that could be used to detect and place WGD events is discarded. Here, we used an automated approach for orthology inference in a target enrichment data set of 68 species of Alchemilla s.l. (Rosaceae), a widely distributed clade of plants primarily from temperate climate regions. Previous molecular phylogenetic studies and chromosome numbers both suggested ancient WGDs in the group. However, both the phylogenetic location and putative parental lineages of these WGD events remain unknown. By taking paralogs into consideration and inferring orthologs from target enrichment data, we identified four nodes in the backbone of Alchemilla s.l. with an elevated proportion of gene duplication. Furthermore, using a gene-tree reconciliation approach, we established the autopolyploid origin of the entire Alchemilla s.l. and the nested allopolyploid origin of four major clades within the group. Here, we showed the utility of automated tree-based orthology inference methods, previously designed for genomic or transcriptomic data sets, to study complex scenarios of polyploidy and reticulate evolution from target enrichment data sets.[Alchemilla; allopolyploidy; autopolyploidy; gene tree discordance; orthology inference; paralogs; Rosaceae; target enrichment; whole genome duplication.].

Diet of a rare herbivore based on DNA metabarcoding of feces: Selection, seasonality, and survival.

In herbivores, survival and reproduction are influenced by quality and quantity of forage, and hence, diet and foraging behavior are the foundation of an herbivore's life history strategy. Given the importance of diet to most herbivores, it is imperative that we know the species of plants they prefer, especially for herbivorous species that are at risk for extinction. However, it is often difficult to identify the diet of small herbivores because: (a) They are difficult to observe, (b) collecting stomach contents requires sacrificing animals, and (c) microhistology requires accurately identifying taxa from partially digested plant fragments and likely overemphasizes less-digestible taxa. The northern Idaho ground squirrel (Urocitellus brunneus) is federally threatened in the United States under the Endangered Species Act. We used DNA metabarcoding techniques to identify the diet of 188 squirrels at 11 study sites from fecal samples. We identified 42 families, 126 genera, and 120 species of plants in the squirrel's diet. Our use of three gene regions was beneficial because reliance on only one gene region (e.g., only trnL) would have caused us to miss >30% of the taxa in their diet. Northern Idaho ground squirrel diet differed between spring and summer, frequency of many plants in the diet differed from their frequency within their foraging areas (evidence of selective foraging), and several plant genera in their diet were associated with survival. Our results suggest that while these squirrels are generalists (they consume a wide variety of plant species), they are also selective and do not eat plants relative to availability. Consumption of particular genera such as Perideridia may be associated with higher overwinter survival.

Identifying models of trait-mediated community assembly using random forests and approximate Bayesian computation.

Ecologists often use dispersion metrics and statistical hypothesis testing to infer processes of community formation such as environmental filtering, competitive exclusion, and neutral species assembly. These metrics have limited power in inferring assembly models because they rely on often-violated assumptions. Here, we adapt a model of phenotypic similarity and repulsion to simulate the process of community assembly via environmental filtering and competitive exclusion, all while parameterizing the strength of the respective ecological processes. We then use random forests and approximate Bayesian computation to distinguish between these models given the simulated data. We find that our approach is more accurate than using dispersion metrics and accounts for uncertainty in model selection. We also demonstrate that the parameter determining the strength of the assembly processes can be accurately estimated. This approach is available in the R package CAMI; Community Assembly Model Inference. We demonstrate the effectiveness of CAMI using an example of plant communities living on lava flow islands.

Chloroplast primers for clade-wide phylogenetic studies of Thalictrum.

PREMISE: Chloroplast primers were developed for phylogenetic and comparative studies in Thalictrum (Ranunculaceae). METHODS AND RESULTS: We assembled and annotated the complete plastome sequence of T. thalictroides by combining multiple whole genome sequencing libraries. Using transcriptome-sequencing libraries, we also assembled a partial plastome of the related species T. hernandezii. From the newly assembled plastomes and one previously sequenced plastome, we designed and validated 28 primer pairs to target variable portions of the chloroplast genome in Thalictrum. Furthermore, we tested the validated primers in 62 species of Thalictrum. The total alignment length of the 28 regions was 15,268 bp with 2443 variable sites and 92% character occupancy. CONCLUSIONS: The newly developed chloroplast primer pairs improve the phylogenetic resolution (bootstrap support and tree certainty) in Thalictum and will be a useful resource for future phylogenetic and evolutionary studies for species in the genus and in close relatives in Thalictroideae.

Three new species of Lachemilla (Rosaceae) from South America.

Three new species of Lachemilla (Rosaceae), two from Colombia and one from Peru, are described and illustrated. Lachemillarothmaleriana is characterized by its stout stems, sericeous-villous indumentum, and wide ascending sheaths with trilobate lateral lobes. Lachemillaargentea presents a unique combination of tripartite basal leaves with an adaxial silvery villous indumentum, and decumbent branches with verticillate lobed sheaths. Finally, Lachemillacyanea has distinctly basal reniform leaves with a blue-green color and hirsute pubescence. Phylogenetic analyses of the nuclear ribosomal cistron and multiple regions of the plastid genome revealed the allopolyploid origin of the three new taxa.

ResumenTres nuevas especies de Lachemilla (Rosaceae), dos de Colombia y una de Perú, se describen e ilustran. Lachemillarothmaleriana se caracteriza por tener tallos robustos, indumento sericeo-villoso, y verticilos anchos ascendentes con lóbulos laterales trilobados. Lachemillaargentea presenta una combinación única de hojas basales tripartitas con indumento villoso plateado en el haz y ramas decumbentes con verticilos lobulados. Finalmente, Lachemillacyanea presenta hojas basales distintivas de forma reniforme, color azul­verdoso y pubescencia hirsuta. Análisis filogenéticos del cistrón ribosomal nuclear y múltiples regiones del genoma cloroplástico revelaron el origen alopoliploide de los tres nuevos taxones.

Quantifying morphological variation in the Castilleja pilosa species complex (Orobanchaceae).

Robustly delimited species are of paramount importance, the identification of which relies on our ability to discern boundaries between one species and the next. This is not difficult to do when species are very distinct from one another. However, in recently evolved lineages where putative species may have relatively few diagnostic features (e.g., species complexes composed of very similar species, the boundaries between which are often unclear), defining species boundaries can be more challenging. Hence, the field of species delimitation has widely advocated the use of multiple lines of evidence to delimit species, particularly in species complexes. Excessive taxonomic confusion, often the result of species descriptions that shift through time (e.g., during revisionary work and regional treatments), can further complicate the search for diagnostic features in species complexes. Here, as a first step in robustly delimiting species boundaries, we quantify and describe morphological variation in the Castilleja pilosa species complex. We first infer the morphospace of the species complex and use fuzzy-clustering techniques to explore the morphological variation in the system. Next, we hypothesize the position of type specimens within that morphospace. In so doing, we aim to visualize the impact that regional treatments have had on the conceptualization of taxa through time. We find that there is limited morphological variation among members of this complex, and we determine that the morphological concept of these species have shifted through time and are no longer accurately represented by species descriptions.

Increasing phylogenetic stochasticity at high elevations on summits across a remote North American wilderness.

PREMISE: At the intersection of ecology and evolutionary biology, community phylogenetics can provide insights into overarching biodiversity patterns, particularly in remote and understudied ecosystems. To understand community assembly of the high alpine flora in the Sawtooth National Forest, USA, we analyzed phylogenetic structure within and between nine summit communities. METHODS: We used high-throughput sequencing to supplement existing data and infer a nearly completely sampled community phylogeny of the alpine vascular flora. We calculated mean nearest taxon distance (MNTD) and mean pairwise distance (MPD) to quantify phylogenetic divergence within summits, and assessed whether maximum elevation explains phylogenetic structure. To evaluate similarities between summits, we quantified phylogenetic turnover, taking into consideration microhabitats (talus vs. meadows). RESULTS: We found different patterns of community phylogenetic structure within the six most species-rich orders, but across all vascular plants phylogenetic structure was largely not different from random. There was a significant negative correlation between elevation and tree-wide phylogenetic diversity (MPD) within summits: overdispersion degraded as elevation increased. Between summits, we found high phylogenetic turnover driven by greater niche heterogeneity on summits with alpine meadows. CONCLUSIONS: Our results provide further evidence that stochastic processes may also play an important role in the assembly of vascular plant communities in high alpine habitats at regional scales. However, order-specific patterns suggest that adaptations are still important for assembly of specific sectors of the plant tree of life. Further studies quantifying functional diversity will be important in disentangling the interplay of eco-evolutionary processes that likely shape broad community phylogenetic patterns in extreme environments.

Phylogeny of the Australian Solanum dioicum group using seven nuclear genes, with consideration of Symon's fruit and seed dispersal hypotheses.

The dioecious and andromonoecious Solanum taxa (the "S. dioicum group") of the Australian Monsoon Tropics have been the subject of phylogenetic and taxonomic study for decades, yet much of their basic biology is still unknown. This is especially true for plant-animal interactions, including the influence of fruit form and calyx morphology on seed dispersal. We combine field/greenhouse observations and specimen-based study with phylogenetic analysis of seven nuclear regions obtained via a microfluidic PCR-based enrichment strategy and high-throughput sequencing, and present the first species-tree hypothesis for the S. dioicum group. Our results suggest that epizoochorous trample burr seed dispersal (strongly linked to calyx accrescence) is far more common among Australian Solanum than previously thought and support the hypothesis that the combination of large fleshy fruits and endozoochorous dispersal represents a reversal in this study group. The general lack of direct evidence related to biotic dispersal (epizoochorous or endozoochorous) may be a function of declines and/or extinctions of vertebrate dispersers. Because of this, some taxa might now rely on secondary dispersal mechanisms (e.g. shakers, tumbleweeds, rafting) as a means to maintain current populations and establish new ones.

Extensive allopolyploidy in the neotropical genus Lachemilla (Rosaceae) revealed by PCR-based target enrichment of the nuclear ribosomal DNA cistron and plastid phylogenomics.

PREMISE OF THE STUDY: Polyploidy has been long recognized as an important force in plant evolution. Previous studies had suggested widespread occurrence of polyploidy and the allopolyploid origin of several species in the diverse neotropical genus Lachemilla (Rosaceae). Nonetheless, this evidence has relied mostly on patterns of cytonuclear discordance, and direct evidence from nuclear allelic markers is still needed. METHODS: Here we used PCR target enrichment in combination with high throughput sequencing to obtain multiple copies of the nuclear ribosomal (nr) DNA cistron and 45 regions of the plastid genome (cpDNA) from 219 accessions representing 48 species of Lachemilla and to explore the allopolyploid origin of species in this group. KEY RESULTS: We were able to identify multiple nrDNA ribotypes and establish clear evidence of allopolyploidy in 33 species of Lachemilla, showing that this condition is common and widespread in the genus. Additionally, we found evidence for three autopolyploid species. We also established multiple, independent origins of several allopolyploid species. Finally, based solely on the cpDNA phylogeny, we identified that the monotypic genus Farinopsis is the sister group of Lachemilla and allied genera within subtribe Fragariinae. CONCLUSIONS: Our study demonstrates the utility of the nuclear ribosomal DNA cistron to detect allopolyploidy when concerted evolution of this region is not complete. Additionally, with a robust chloroplast phylogeny in place, the direction of hybridization events can be established, and multiple, independent origins of allopolyploid species can be identified.

Predicting plant conservation priorities on a global scale.

The conservation status of most plant species is currently unknown, despite the fundamental role of plants in ecosystem health. To facilitate the costly process of conservation assessment, we developed a predictive protocol using a machine-learning approach to predict conservation status of over 150,000 land plant species. Our study uses open-source geographic, environmental, and morphological trait data, making this the largest assessment of conservation risk to date and the only global assessment for plants. Our results indicate that a large number of unassessed species are likely at risk and identify several geographic regions with the highest need of conservation efforts, many of which are not currently recognized as regions of global concern. By providing conservation-relevant predictions at multiple spatial and taxonomic scales, predictive frameworks such as the one developed here fill a pressing need for biodiversity science.

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

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

Impact of whole-genome duplication events on diversification rates in angiosperms.

PREMISE OF THE STUDY: Polyploidy or whole-genome duplication (WGD) pervades the evolutionary history of angiosperms. Despite extensive progress in our understanding of WGD, the role of these events in promoting diversification is still not well understood. We seek to clarify the possible association between WGD and diversification rates in flowering plants. METHODS: Using a previously published phylogeny spanning all land plants (31,749 tips) and WGD events inferred from analyses of the 1000 Plants (1KP) transcriptome data, we analyzed the association of WGDs and diversification rates following numerous WGD events across the angiosperms. We used a stepwise AIC approach (MEDUSA), a Bayesian mixture model approach (BAMM), and state-dependent diversification analyses (MuSSE) to investigate patterns of diversification. Sister-clade comparisons were used to investigate species richness after WGDs. KEY RESULTS: Based on the density of 1KP taxon sampling, 106 WGDs were unambiguously placed on the angiosperm phylogeny. We identified 334-530 shifts in diversification rates. We found that 61 WGD events were tightly linked to changes in diversification rates, and state-dependent diversification analyses indicated higher speciation rates for subsequent rounds of WGD. Additionally, 70 of 99 WGD events showed an increase in species richness compared to the sister clade. CONCLUSIONS: Forty-six of the 106 WGDs analyzed appear to be closely associated with upshifts in the rate of diversification in angiosperms. Shifts in diversification do not appear more likely than random within a four-node lag phase following a WGD; however, younger WGD events are more likely to be followed by an upshift in diversification than older WGD events.

Incongruence in molecular species delimitation schemes: What to do when adding more data is difficult.

Using multiple, independent approaches to molecular species delimitation is advocated to accommodate limitations and assumptions of a single approach. Incongruence in delimitation schemes is a potential by-product of employing multiple methods on the same data, and little attention has been paid to its reconciliation. Instead, a particular scheme is prioritized, and/or molecular delimitations are coupled with additional, independent lines of evidence that mitigate incongruence. We advocate that incongruence within a line of evidence should be accounted for before comparing across lines of evidence that can themselves be incongruent. Additionally, it is not uncommon for empiricists working in nonmodel systems to be data-limited, generating some concern for the adequacy of available data to address the question of interest. With conservation and management decisions often hinging on the status of species, it seems prudent to understand the capabilities of approaches we use given the data we have. Here, we apply two molecular species delimitation approaches, spedeSTEM and BPP, to the Castilleja ambigua (Orobanchaceae) species complex, a relatively young plant lineage in western North America. Upon finding incongruence in our delimitation, we employed a post hoc simulation study to examine the power of these approaches to delimit species. Given the data we collected, we find that spedeSTEM lacks the power to delimit while BPP is capable, thus allowing us to address incongruence before proceeding in delimitation. We suggest post hoc simulation studies like this compliment empirical delimitation and serve as a means of exploring conflict within a line of evidence and dealing with it appropriately.