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.

Variation in seasonal timing traits and life history along a latitudinal transect in Mimulus ringens.

Seasonal timing traits are commonly under recurrent, spatially variable selection, and are therefore predicted to exhibit clinal variation. Temperate perennial plants often require vernalization to prompt growth and reproduction; however, little is known about whether vernalization requirements change across the range of a broadly distributed species. We performed a critical vernalization duration study in Mimulus ringens, coupled with population genomic analysis. Plants from eight populations spanning the latitudinal range were exposed to varying durations of 4°C vernalization between 0 and 56 days, and flowering response was assessed. RADSeq was also performed to generate 1179 polymorphic SNPs, which were used to examine population structure. We found unexpected life history variation, with some populations lacking vernalization requirement. Population genomic analyses show that these life history variants are highly divergent from perennials, potentially revealing a cryptic species. For perennial populations, minimum vernalization time was surprisingly consistent. However, once vernalized, northern populations flowered almost 3 weeks faster than southern. Furthermore, southern populations exhibited sensitivity to vernalization times beyond flowering competency, suggesting an ability to respond adaptively to different lengths of winter. Mimulus ringens, therefore, reveals evidence of clinal variation, and provides opportunities for future studies addressing mechanistic and ecological hypotheses both within and between incipient species.

Evolutionary history and ecology shape the diversity and abundance of phytochemical arsenals across monkeyflowers.

We examine the extent to which phylogenetic effects and ecology are associated with macroevolutionary patterns of phytochemical defence production across the Mimulus phylogeny. We grew plants from 21 species representing the five major sections of the Mimulus phylogeny in a common garden to assess how the arsenals (NMDS groupings) and abundances (concentrations) of a phytochemical defence, phenylpropanoid glycosides (PPGs), vary across the phylogeny. Very few PPGs are widespread across the genus, but many are common to multiple sections of the genus. Phytochemical arsenals cluster among sections in an NMDS and are not associated with total concentration of PPGs. There is a strong phylogenetic signal for phytochemical arsenal composition across the Mimulus genus, whereas ecological variables such as growing season length, latitude, and elevation do not significantly influence arsenal. In contrast, there is little phylogenetic signal for total PPG concentration, and this trait is significantly influenced by several ecological factors. Phytochemical arsenals and abundances are influenced by plant life history form. Both phylogenetic effects and ecology are related to phytochemical patterns across species, albeit in different ways. The independence of phytochemical defence concentrations from arsenal compositions indicates that these aspects of defence may continue to evolve independently of one another.

Variation in ecophysiological traits might contribute to ecogeographic isolation and divergence between parapatric ecotypes of Mimulus aurantiacus.

Many forms of reproductive isolation contribute to speciation, and early-acting barriers may be especially important, because they have the first opportunity to limit gene flow. Ecogeographic isolation occurs when intrinsic traits of taxa contribute to disjunct geographic distributions, reducing the frequency of intertaxon mating. Characterizing this form of isolation requires knowledge of both the geographic arrangement of suitable habitats in nature and the identification of phenotypes involved in shaping geographic distributions. In Mimulus aurantiacus, red- and yellow-flowered ecotypes are incompletely isolated by divergent selection exerted by different pollinators. However, these emerging taxa are largely isolated spatially, with a hybrid zone occurring along a narrow region of contact. In order to assess whether responses to abiotic conditions contribute to the parapatric distribution of ecotypes, we measured a series of ecophysiological traits from populations along a transect, including drought sensitivity, leaf area and the concentrations of vegetative flavonoids. In contrast to the abrupt transitions in floral phenotypes, we found that ecophysiological traits exhibited a continuous geographic transition that largely mirrors variation in climatological variables. These traits may impede gene flow across a continuous environmental gradient, but they would be unlikely to result in ecotypic divergence alone. Nevertheless, we found a genetic correlation between vegetative and floral traits, providing a potential link between the two forms of isolation. Although neither barrier appears sufficient to cause divergence on its own, the combined impacts of local adaptation to abiotic conditions and regional adaptation to pollinators may interact to drive discontinuous variation in the face of gene flow in this system.

Geographic cline analysis as a tool for studying genome-wide variation: a case study of pollinator-mediated divergence in a monkeyflower.

A major goal of speciation research is to reveal the genomic signatures that accompany the speciation process. Genome scans are routinely used to explore genome-wide variation and identify highly differentiated loci that may contribute to ecological divergence, but they do not incorporate spatial, phenotypic or environmental data that might enhance outlier detection. Geographic cline analysis provides a potential framework for integrating diverse forms of data in a spatially explicit framework, but has not been used to study genome-wide patterns of divergence. Aided by a first-draft genome assembly, we combined an FCT scan and geographic cline analysis to characterize patterns of genome-wide divergence between divergent pollination ecotypes of Mimulus aurantiacus. FCT analysis of 58 872 SNPs generated via RAD-seq revealed little ecotypic differentiation (mean FCT  = 0.041), although a small number of loci were moderately-to-highly diverged. Consistent with our previous results from the gene MaMyb2, which contributes to differences in flower colour, 130 loci have cline shapes that recapitulate the spatial pattern of trait divergence, suggesting that they may reside in or near the genomic regions that contribute to pollinator isolation. In the narrow hybrid zone between the ecotypes, extensive admixture among individuals and low linkage disequilibrium between markers indicate that most outlier loci are scattered throughout the genome, rather than being restricted to one or a few divergent regions. In addition to revealing the genomic consequences of ecological divergence in this system, we discuss how geographic cline analysis is a powerful but under-utilized framework for studying genome-wide patterns of divergence.

Divergent selection drives genetic differentiation in an R2R3-MYB transcription factor that contributes to incipient speciation in Mimulus aurantiacus.

Identifying the molecular genetic basis of traits contributing to speciation is of crucial importance for understanding the ecological and evolutionary mechanisms that generate biodiversity. Despite several examples describing putative "speciation genes," it is often uncertain to what extent these genetic changes have contributed to gene flow reductions in nature. Therefore, considerable interest lies in characterizing the molecular basis of traits that actively confer reproductive isolation during the early stages of speciation, as these loci can be attributed directly to the process of divergence. In Southern California, two ecotypes of Mimulus aurantiacus are parapatric and differ primarily in flower color, with an anthocyanic, red-flowered morph in the west and an anthocyanin-lacking, yellow-flowered morph in the east. Evidence suggests that the genetic changes responsible for this shift in flower color have been essential for divergence and have become fixed in natural populations of each ecotype due to almost complete differences in pollinator preference. In this study, we demonstrate that a cis-regulatory mutation in an R2R3-MYB transcription factor results in differential regulation of enzymes in the anthocyanin biosynthetic pathway and is the major contributor to differences in floral pigmentation. In addition, molecular population genetic data show that, despite gene flow at neutral loci, divergent selection has driven the fixation of alternate alleles at this gene between ecotypes. Therefore, by identifying the genetic basis underlying ecologically based divergent selection in flower color between these ecotypes, we have revealed the ecological and functional mechanisms involved in the evolution of pre-mating isolation at the early stages of incipient speciation.

Ecogeographic isolation and speciation in the genus Mimulus.

Despite a long history of examining the geographic context of speciation, differences in geographic range have rarely been considered a legitimate isolating mechanism. This likely results from the complex relationship between historical and ecological processes in determining the spatial distribution of species. Ecogeographic isolation is the proportion of geographic isolation that results from genetically based ecological differences between taxa and should therefore be measured as an isolating mechanism under the biological species concept. In this study, species distribution modeling was used to evaluate the potential ranges of 12 recently diverged pairs of species in the genus Mimulus. Variation in the distribution models showed that these species differ significantly in the niches they occupy. These differences result in substantial ecogeographic isolation, with an average strength of 0.67, revealing that, on average, Mimulus species exhibit only 33% overlap in the extent of suitable habitat with their closest relatives. Because prezygotic barriers act early in the life cycle of organisms, this strong barrier has the potential to contribute greatly to the total isolation experienced between diverging species. Therefore, ecogeographic isolation appears to play an important role in Mimulus, and estimating the strength of this barrier is essential to our general understanding of speciation.

Transposition, duplication, and divergence of the telomerase RNA underlies the evolution of Mimulus telomeres.

Telomeres are nucleoprotein complexes with a crucial role of protecting chromosome ends. It consists of simple repeat sequences and dedicated telomere-binding proteins. Because of its vital functions, components of the telomere, for example its sequence, should be under strong evolutionary constraint. But across all plants, telomere sequences display a range of variation and the evolutionary mechanism driving this diversification is largely unknown. Here, we discovered in Monkeyflower (Mimulus) the telomere sequence is even variable between species. We investigated the basis of Mimulus telomere sequence evolution by studying the long noncoding telomerase RNA (TR), which is a core component of the telomere maintenance complex and determines the telomere sequence. We conducted total RNA-based de novo transcriptomics from 16 Mimulus species and analyzed reference genomes from 6 species, and discovered Mimulus species have evolved at least three different telomere sequences: (AAACCCT)n, (AAACCCG)n, and (AAACCG)n. Unexpectedly, we discovered several species with TR duplications and the paralogs had functional consequences that could influence telomere evolution. For instance, M. lewisii had two sequence-divergent TR paralogs and synthesized a telomere with sequence heterogeneity, consisting of AAACCG and AAACCCG repeats. Evolutionary analysis of the M. lewisii TR paralogs indicated it had arisen from a transposition-mediate duplication process. Further analysis of the TR from multiple Mimulus species showed the gene had frequently transposed and inserted into new chromosomal positions during Mimulus evolution. From our results, we propose the TR transposition, duplication, and divergence model to explain the evolutionary sequence turnovers in Mimulus and potentially all plant telomeres.

Evolution and the latitudinal diversity gradient: speciation, extinction and biogeography.

A latitudinal gradient in biodiversity has existed since before the time of the dinosaurs, yet how and why this gradient arose remains unresolved. Here we review two major hypotheses for the origin of the latitudinal diversity gradient. The time and area hypothesis holds that tropical climates are older and historically larger, allowing more opportunity for diversification. This hypothesis is supported by observations that temperate taxa are often younger than, and nested within, tropical taxa, and that diversity is positively correlated with the age and area of geographical regions. The diversification rate hypothesis holds that tropical regions diversify faster due to higher rates of speciation (caused by increased opportunities for the evolution of reproductive isolation, or faster molecular evolution, or the increased importance of biotic interactions), or due to lower extinction rates. There is phylogenetic evidence for higher rates of diversification in tropical clades, and palaeontological data demonstrate higher rates of origination for tropical taxa, but mixed evidence for latitudinal differences in extinction rates. Studies of latitudinal variation in incipient speciation also suggest faster speciation in the tropics. Distinguishing the roles of history, speciation and extinction in the origin of the latitudinal gradient represents a major challenge to future research.

Strong premating reproductive isolation drives incipient speciation in Mimulus aurantiacus.

Determining which forms of reproductive isolation have the biggest impact on the process of divergence is a major goal of speciation research. These barriers are often divided into those that affect the potential for hybridization (premating isolation), and those that occur after mating (postmating isolation), and much debate has surrounded the relative importance of these categories. Within the species Mimulus aurantiacus, red- and yellow-flowered ecotypes occur in the southwest corner of California, and a hybrid zone occurs where their ranges overlap. We show that premating barriers are exclusively responsible for isolation in this system, with both ecogeographic and pollinator isolation contributing significantly to total isolation. Postmating forms of reproductive isolation have little or no impact on gene flow, indicating that hybrids likely contribute to introgression at neutral loci. Analysis of molecular variation across thousands of restriction-site associated DNA sequencing (RAD-seq) markers reveals that the genomes of these taxa are largely undifferentiated. However, structure analysis shows that these taxa are distinguishable genetically, likely due to the impact of loci underlying differentiated adaptive phenotypes. These data exhibit the power of divergent natural selection to maintain highly differentiated phenotypes in the face of gene flow during the early stages of speciation.

The geography of divergence with gene flow facilitates multitrait adaptation and the evolution of pollinator isolation in Mimulus aurantiacus.

Ecological adaptation is the driving force during divergence with gene flow and generates reproductive isolation early in speciation. Although gene flow opposes divergence, local adaptation can be facilitated by factors that prevent the breakup of favorable allelic combinations. We investigated how selection, genetic architecture, and geography have contributed to the maintenance of floral trait divergence and pollinator isolation between parapatric ecotypes of Mimulus aurantiacus. Combining greenhouse, field, and genomic studies, we show that sharp clines in floral traits are maintained by spatially varying selection. Although adaptation breaks down where the ecotypes co-occur, leading to the formation of a hybrid zone, the largely non-overlapping distributions of the ecotypes shield them from immigrant genes, facilitating divergence across most of the range. In contrast to the sharp genetic discontinuities observed across most hybrid zones, we observed a gradual cline in genome-wide divergence and a pattern of isolation by distance across the landscape. Thus, contrary to a long period of allopatry followed by recent re-contact, our data suggest that floral trait divergence in M. aurantiacus may have evolved with locally restricted, but ongoing gene flow. Therefore, our study reveals how the geographic distribution of an organism can contribute to the evolution of premating isolation in the early stages of divergence with gene flow.

Unification of methods for estimating the strength of reproductive isolation.

Understanding the evolution of reproductive isolation is tantamount to describing the origin of species. Therefore, a primary goal in evolutionary biology is to identify which reproductive barriers are most important to the process. To achieve this goal, the strength of multiple forms of isolation must be compared in an equivalent manner. However, a diversity of methods has been used to estimate barrier strength, falling into several mathematically distinct categories. This study provides a unified method for calculating isolation that relates the amount of gene flow experienced by taxa to random expectations in a simple linear framework. This approach has three distinct advantages over previous methods: (1) it is directly related to gene flow, (2) it is symmetrical, such that measures in both the positive and negative range are comparable, and (3) it is equivalent between broad categories of reproductive isolation, allowing for appropriate comparisons. This linear formulation can be adjusted for use in all forms of isolation, and can accommodate cases in which null expectations for con- and heterospecific gene flow differ. Additionally, this framework can be used to calculate total reproductive isolation and the relative contributions of individual barriers.

Flower color as a model system for studies of plant evo-devo.

Even though pigmentation traits have had substantial impacts on the field of animal evolutionary developmental biology, they have played only relatively minor roles in plant evo-devo. This is surprising given the often direct connection between flower color and fitness variation mediated through the effects of pollinators. At the same time, ecological and evolutionary genetic studies have utilized the molecular resources available for the anthocyanin pathway to generate several examples of the molecular basis of putatively adaptive transitions in flower color. Despite this opportunity to synthesize experimental approaches in ecology, evolution, and developmental biology, the investigation of many fundamental questions in evo-devo using this powerful model is only at its earliest stages. For example, a long-standing question is whether predictable genetic changes accompany the repeated evolution of a trait. Due to the conserved nature of the biochemical and regulatory control of anthocyanin biosynthesis, it has become possible to determine whether, and under what circumstances, different types of mutations responsible for flower color variation are preferentially targeted by natural selection. In addition, because plants use anthocyanin and related compounds in vegetative tissue for other important physiological functions, the identification of naturally occurring transitions from unpigmented to pigmented flowers provides the opportunity to examine the mechanisms by which regulatory networks are co-opted into new developmental domains. Here, we review what is known about the ecological and molecular basis of anthocyanic flower color transitions in natural systems, focusing on the evolutionary and developmental features involved. In doing so, we provide suggestions for future work on this trait and suggest that there is still much to be learned from the evolutionary development of flower color transitions in nature.

The biology of speciation.

Since Darwin published the "Origin," great progress has been made in our understanding of speciation mechanisms. The early investigations by Mayr and Dobzhansky linked Darwin's view of speciation by adaptive divergence to the evolution of reproductive isolation, and thus provided a framework for studying the origin of species. However, major controversies and questions remain, including: When is speciation nonecological? Under what conditions does geographic isolation constitute a reproductive isolating barrier? and How do we estimate the "importance" of different isolating barriers? Here, we address these questions, providing historical background and offering some new perspectives. A topic of great recent interest is the role of ecology in speciation. "Ecological speciation" is defined as the case in which divergent selection leads to reproductive isolation, with speciation under uniform selection, polyploid speciation, and speciation by genetic drift defined as "nonecological." We review these proposed cases of nonecological speciation and conclude that speciation by uniform selection and polyploidy normally involve ecological processes. Furthermore, because selection can impart reproductive isolation both directly through traits under selection and indirectly through pleiotropy and linkage, it is much more effective in producing isolation than genetic drift. We thus argue that natural selection is a ubiquitous part of speciation, and given the many ways in which stochastic and deterministic factors may interact during divergence, we question whether the ecological speciation concept is useful. We also suggest that geographic isolation caused by adaptation to different habitats plays a major, and largely neglected, role in speciation. We thus provide a framework for incorporating geographic isolation into the biological species concept (BSC) by separating ecological from historical processes that govern species distributions, allowing for an estimate of geographic isolation based upon genetic differences between taxa. Finally, we suggest that the individual and relative contributions of all potential barriers be estimated for species pairs that have recently achieved species status under the criteria of the BSC. Only in this way will it be possible to distinguish those barriers that have actually contributed to speciation from those that have accumulated after speciation is complete. We conclude that ecological adaptation is the major driver of reproductive isolation, and that the term "biology of speciation," as proposed by Mayr, remains an accurate and useful characterization of the diversity of speciation mechanisms.

Comparative approaches to the evolution of reproductive isolation: a comment on Scopece et al. 2007.

Speciation can be driven by the evolution of many forms of reproductive isolation. Comparative study is a powerful approach for elucidating the relative importance of individual isolating barriers in the speciation process. A recent contribution by Scopece and colleagues provides comparative data for two groups of deceptive pollination orchids and aims to test hypotheses about which forms of isolation are most important in the two clades. The authors compare pollinator isolation and postmating isolation between the two orchid groups, and conclude that food-deceptive orchid species have less isolation by pollinator specificity than sexually deceptive species, and that postmating isolation is more important in the food-deceptive clade. Although we find this approach to be novel and potentially powerful, these conclusions are called into question by the methods used to define and select species and quantify pollinator isolation. Definition and selection of taxa were performed in a biased manner that undermines the ability to infer general patterns of speciation. Furthermore, pollinator isolation was calculated inconsistently for the two groups under study, effectively nullifying the comparison.