Caught in the Act: Incipient Speciation at the Southern limit of Viburnum in the Central Andes.

A fundamental objective of evolutionary biology is to understand the origin of independently evolving species. Phylogenetic studies of species radiations rarely are able to document ongoing speciation; instead, modes of speciation, entailing geographic separation and/or ecological differentiation, are posited retrospectively. The Oreinotinus clade of Viburnum has radiated recently from north to south through the cloud forests of Mexico and Central America to the Central Andes. Our analyses support a hypothesis of incipient speciation in Oreinotinus at the southern edge of its geographic range, from central Peru to northern Argentina. Although several species and infraspecific taxa of have been recognized in this area, multiple lines of evidence and analytical approaches (including analyses of phylogenetic relationships, genetic structure, leaf morphology, and climatic envelopes) favor the recognition of just a single species, V. seemenii. We show that what has previously been recognized as V. seemenii f. minor has recently occupied the drier Tucuman-Bolivian forest region from Samaipata in Bolivia to Salta in northern Argentina. Plants in these populations form a well-supported clade with a distinctive genetic signature and they have evolved smaller, narrower leaves. We interpret this as the beginning of a within-species divergence process that has elsewhere in the neotropics resulted repeatedly in Viburnum species with a particular set of leaf ecomorphs. Specifically, the southern populations are in the process of evolving the small, glabrous, and entire leaf ecomorph that has evolved in four other montane areas of endemism. As predicted based on our studies of leaf ecomorphs in Chiapas, Mexico, these southern populations experience generally drier conditions, with large diurnal temperature fluctuations. In a central portion of the range of V. seemenii, characterized by wetter climatic conditions, we also document what may be the initial differentiation of the leaf ecomorph with larger, pubescent, and toothy leaves. The emergence of these ecomorphs thus appears to be driven by adaptation to subtly different climatic conditions in separate geographic regions, as opposed to parapatric differentiation along elevational gradients as suggested by Viburnum species distributions in other parts of the neotropics.

A phylogenomic analysis of Lonicera and its bearing on the evolution of organ fusion.

PREMISE: The ~140 species of Lonicera are characterized by variously fused leaves, bracteoles, and ovaries, making it a model system for studying the evolution and development of organ fusion. However, previous phylogenetic analyses, based mainly on chloroplast DNA markers, have yielded uncertain and conflicting results. A well-supported phylogeny of Lonicera will allow us to trace the evolutionary history of organ fusion. METHODS: We inferred the phylogeny of Lonicera using restriction site-associated DNA sequencing (RADSeq), sampling all major clades and 18 of the 23 subsections. This provided the basis for inferring the evolution of five fusion-related traits. RESULTS: RADSeq data yielded a well-resolved and well-supported phylogeny. The two traditionally recognized subgenera (Periclymenum and Chamaecerasus), three of the four sections (Isoxylosteum, Coeloxylosteum, and Nintooa), and half of the subsections sampled were recovered as monophyletic. However, the large and heterogeneous section Isika was strongly supported as paraphyletic. Nintooa, a clade of ~22 mostly vine-forming species, including L. japonica, was recovered in a novel position, raising the possibility of cytonuclear discordance. We document the parallel evolution of fused leaves, bracteoles, and ovaries, with rare reversals. Most strikingly, complete cupules, in which four fused bracteoles completely enclose two unfused ovaries, arose at least three times. Surprisingly, these appear to have evolved directly from ancestors with free bracteoles instead of partial cupules. CONCLUSIONS: We provide the most comprehensive and well-supported phylogeny of Lonicera to date. Our inference of multiple evolutionary shifts in organ fusion provides a solid foundation for in depth developmental and functional analyses.

Multiple origins of lipid-based structural colors contribute to a gradient of fruit colors in Viburnum (Adoxaceae).

Structural color is poorly known in plants relative to animals. In fruits, only a handful of cases have been described, including in Viburnum tinus where the blue color results from a disordered multilayered reflector made of lipid droplets. Here, we examine the broader evolutionary context of fruit structural color across the genus Viburnum. We obtained fresh and herbarium fruit material from 30 Viburnum species spanning the phylogeny and used transmission electron microscopy, optical simulations, and ancestral state reconstruction to identify the presence/absence of photonic structures in each species, understand the mechanism producing structural color in newly identified species, relate the development of cell wall structure to reflectance in Viburnum dentatum, and describe the evolution of cell wall architecture across Viburnum. We identify at least two (possibly three) origins of blue fruit color in Viburnum in species which produce large photonic structures made of lipid droplets embedded in the cell wall and which reflect blue light. Examining the full spectrum of mechanisms producing color in pl, including structural color as well as pigments, will yield further insights into the diversity, ecology, and evolution of fruit color.

Replicated radiation of a plant clade along a cloud forest archipelago.

Replicated radiations, in which sets of similar forms evolve repeatedly within different regions, can provide powerful insights into parallel evolution and the assembly of functional diversity within communities. Several cases have been described in animals, but in plants we lack well-documented cases of replicated radiation that combine comprehensive phylogenetic and biogeographic analyses, the delimitation of geographic areas within which a set of 'ecomorphs' evolved independently and the identification of potential underlying mechanisms. Here we document the repeated evolution of a set of leaf ecomorphs in a group of neotropical plants. The Oreinotinus lineage within the angiosperm clade Viburnum spread from Mexico to Argentina through disjunct cloud forest environments. In 9 of 11 areas of endemism, species with similar sets of leaf forms evolved in parallel. We reject gene-flow-mediated evolution of similar leaves and show, instead, that species with disparate leaf forms differ in their climatic niches, supporting ecological adaptation as the driver of parallelism. Our identification of a case of replicated radiation in plants sets the stage for comparative analyses of such phenomena across the tree of life.

Phylogenetic inference of where species spread or split across barriers.

SignificanceGeography molds how species evolve in space. Strong geographical barriers to movement, for instance, both inhibit dispersal between regions and allow isolated populations to diverge as new species. Weak barriers, by contrast, permit species range expansion and persistence. These factors present a conundrum: How strong must a barrier be before between-region speciation outpaces dispersal? We designed a phylogenetic model of dispersal, extinction, and speciation that allows regional features to influence rates of biogeographic change and applied it to the neotropical radiation of Anolis lizards. Separation by water induces a threefold steeper barrier to movement than equivalent distances over land. Our model will help biologists detect relationships between evolutionary processes and the spatial contexts in which they operate.

Reconstructing Dipsacales phylogeny using Angiosperms353: issues and insights.

PREMISE: Phylogenetic relationships within major angiosperm clades are increasingly well resolved, but largely informed by plastid data. Areas of poor resolution persist within the Dipsacales, including placement of Heptacodium and Zabelia, and relationships within the Caprifolieae and Linnaeeae, hindering our interpretation of morphological evolution. Here, we sampled a significant number of nuclear loci using a Hyb-Seq approach and used these data to infer the Dipsacales phylogeny and estimate divergence times. METHODS: Sampling all major clades within the Dipsacales, we applied the Angiosperms353 probe set to 96 species. Data were filtered based on locus completeness and taxon recovery per locus, and trees were inferred using RAxML and ASTRAL. Plastid loci were assembled from off-target reads, and 10 fossils were used to calibrate dated trees. RESULTS: Varying numbers of targeted loci and off-target plastomes were recovered from most taxa. Nuclear and plastid data confidently place Heptacodium with Caprifolieae, implying homoplasy in calyx morphology, ovary development, and fruit type. Placement of Zabelia, and relationships within the Caprifolieae and Linnaeeae, remain uncertain. Dipsacales diversification began earlier than suggested by previous angiosperm-wide dating analyses, but many major splitting events date to the Eocene. CONCLUSIONS: The Angiosperms353 probe set facilitated the assembly of a large, single-copy nuclear dataset for the Dipsacales. Nevertheless, many relationships remain unresolved, and resolution was poor for woody clades with low rates of molecular evolution. We favor expanding the Angiosperms353 probe set to include more variable loci and loci of special interest, such as developmental genes, within particular clades.

Resolved phylogenetic relationships in the Ocotea complex (Supraocotea) facilitate phylogenetic classification and studies of character evolution.

PREMISE: The Ocotea complex contains the greatest diversity of Lauraceae in the Neotropics. However, the traditional taxonomy of the group has relied on only three main floral characters, and previous molecular analyses have used only a few markers and provided limited support for relationships among the major clades. This lack of useful data has hindered the development of a comprehensive classification, as well as studies of character evolution. METHODS: We used RAD-seq data to infer the phylogenetic relationships of 149 species in the Ocotea complex, generating a reference-based assembly using the Persea americana genome. The results provide the basis for a phylogenetic classification that reflects our current molecular knowledge and for analyses of the evolution of breeding system, stamen number, and number of anther locules. RESULTS: We recovered a well-supported tree that demonstrates the paraphyly of Licaria, Aniba, and Ocotea and clarifies the relationships of Umbellularia, Phyllostemonodaphne, and the Old World species. To begin the development of a new classification and to facilitate precise communication, we also provide phylogenetic definitions for seven major clades. Our ancestral reconstructions show multiple origins for the three floral characters that have routinely been used in Lauraceae systematics, suggesting that these be used with caution in the future. CONCLUSIONS: This study advances our understanding of phylogenetic relationships and character evolution in a taxonomically difficult group using RAD-seq data. Our new phylogenetic names will facilitate unambiguous communication as studies of the Ocotea complex progress.

Dispersers and environment drive global variation in fruit colour syndromes.

The colours of fleshy fruits play a critical role in plant dispersal by advertising ripe fruits to consumers. Fruit colours have long been classified into syndromes attributed to selection by animal dispersers, despite weak evidence for this hypothesis. Here, we test the relative importance of biotic (bird and mammal frugivory) and abiotic (wet season temperatures, growing season length and UV-B radiation) factors in determining fruit colour syndrome in 3163 species of fleshy-fruited plants. We find that both dispersers and environment are important, and they interact. In warm areas, contrastive, bird-associated fruit colours increase with relative bird frugivore prevalence, whereas in cold places these colours dominate even where mammalian dispersers are prevalent. We present near-global maps of predicted fruit colour syndrome based on our species-level model and our newly developed characterisations of relative importance of bird and mammal frugivores.

Modeling Phylogenetic Biome Shifts on a Planet with a Past.

The spatial distribution of biomes has changed considerably over deep time, so the geographical opportunity for an evolutionary lineage to shift into a new biome may depend on how the availability and connectivity of biomes has varied temporally. To better understand how lineages shift between biomes in space and time, we developed a phylogenetic biome shift model in which each lineage shifts between biomes and disperses between regions at rates that depend on the lineage's biome affinity and location relative to the spatial distribution of biomes at any given time. To study the behavior of the biome shift model in an empirical setting, we developed a literature-based representation of paleobiome structure for three mesic forest biomes, six regions, and eight time strata, ranging from the Late Cretaceous (100 Ma) through the present. We then fitted the model to a time-calibrated phylogeny of 119 Viburnum species to compare how the results responded to various realistic or unrealistic assumptions about paleobiome structure. Ancestral biome estimates that account for paleobiome dynamics reconstructed a warm temperate (or tropical) origin of Viburnum, which is consistent with previous fossil-based estimates of ancestral biomes. Imposing unrealistic paleobiome distributions led to ancestral biome estimates that eliminated support for tropical origins, and instead inflated support for cold temperate ancestry throughout the warmer Paleocene and Eocene. The biome shift model we describe is applicable to the study of evolutionary systems beyond Viburnum, and the core mechanisms of our model are extensible to the design of richer phylogenetic models of historical biogeography and/or lineage diversification. We conclude that biome shift models that account for dynamic geographical opportunities are important for inferring ancestral biomes that are compatible with our understanding of Earth history.[Ancestral states; biome shifts; historical biogeography; niche conservatism; phylogenetics].

Joint Phylogenetic Estimation of Geographic Movements and Biome Shifts during the Global Diversification of Viburnum.

Phylogeny, molecular sequences, fossils, biogeography, and biome occupancy are all lines of evidence that reflect the singular evolutionary history of a clade, but they are most often studied separately, by first inferring a fossil-dated molecular phylogeny, then mapping on ancestral ranges and biomes inferred from extant species. Here we jointly model the evolution of biogeographic ranges, biome affinities, and molecular sequences, while incorporating fossils to estimate a dated phylogeny for all of the 163 extant species of the woody plant clade Viburnum (Adoxaceae) that we currently recognize in our ongoing worldwide monographic treatment of the group. Our analyses indicate that while the major Viburnum lineages evolved in the Eocene, the majority of extant species originated since the Miocene. Viburnum radiated first in Asia, in warm, broad-leaved evergreen (lucidophyllous) forests. Within Asia, we infer several early shifts into more tropical forests, and multiple shifts into forests that experience prolonged freezing. From Asia, we infer two early movements into the New World. These two lineages probably first occupied warm temperate forests and adapted later to spreading cold climates. One of these lineages (Porphyrotinus) occupied cloud forests and moved south through the mountains of the Neotropics. Several other movements into North America took place more recently, facilitated by prior adaptations to freezing in the Old World. We also infer four disjunctions between Asia and Europe: the Tinus lineage is the oldest and probably occupied warm forests when it spread, whereas the other three were more recent and in cold-adapted lineages. These results variously contradict published accounts, especially the view that Viburnum radiated initially in cold forests and, accordingly, maintained vessel elements with scalariform perforations. We explored how the location and biome assignments of fossils affected our inference of ancestral areas and biome states. Our results are sensitive to, but not entirely dependent upon, the inclusion of fossil biome data. It will be critical to take advantage of all available lines of evidence to decipher events in the distant past. The joint estimation approach developed here provides cautious hope even when fossil evidence is limited. [Biogeography; biome; combined evidence; fossil pollen; phylogeny; Viburnum.].

Evolutionary dynamics of genome size in a radiation of woody plants.

PREMISE: Plant genome size ranges widely, providing many opportunities to examine how genome size variation affects plant form and function. We analyzed trends in chromosome number, genome size, and leaf traits for the woody angiosperm clade Viburnum to examine the evolutionary associations, functional implications, and possible drivers of genome size. METHODS: Chromosome counts and genome size estimates were mapped onto a Viburnum phylogeny to infer the location and frequency of polyploidization events and trends in genome size evolution. Genome size was analyzed with leaf anatomical and physiological data to evaluate the influence of genome size on plant function. RESULTS: We discovered nine independent polyploidization events, two reductions in base chromosome number, and substantial variation in genome size with a slight trend toward genome size reduction in polyploids. We did not find strong relationships between genome size and the functional and morphological traits that have been highlighted at broader phylogenetic scales. CONCLUSIONS: Polyploidization events were sometimes associated with rapid radiations, demonstrating that polyploid lineages can be highly successful. Relationships between genome size and plant physiological function observed at broad phylogenetic scales may be largely irrelevant to the evolutionary dynamics of genome size at smaller scales. The view that plants readily tolerate changes in ploidy and genome size, and often do so, appears to apply to Viburnum.

Viburnum tinus Fruits Use Lipids to Produce Metallic Blue Structural Color.

Viburnum tinus is an evergreen shrub that is native to the Mediterranean region but cultivated widely in Europe and around the world. It produces ripe metallic blue fruits throughout winter [1]. Despite its limited fleshy pulp [2], its high lipid content [3] makes it a valuable resource to the small birds [4] that act as its seed-dispersers [5]. Here, we find that the metallic blue appearance of the fruits is produced by globular lipid inclusions arranged in a disordered multilayer structure. This structure is embedded in the cell walls of the epicarp and underlaid with a dark layer of anthocyanin pigments. The presence of such large, organized lipid aggregates in plant cell walls represents a new mechanism for structural coloration and may serve as an honest signal of nutritional content.

Diversification in evolutionary arenas-Assessment and synthesis.

Understanding how and why rates of evolutionary diversification vary is a key issue in evolutionary biology, ecology, and biogeography. Evolutionary rates are the net result of interacting processes summarized under concepts such as adaptive radiation and evolutionary stasis. Here, we review the central concepts in the evolutionary diversification literature and synthesize these into a simple, general framework for studying rates of diversification and quantifying their underlying dynamics, which can be applied across clades and regions, and across spatial and temporal scales. Our framework describes the diversification rate (d) as a function of the abiotic environment (a), the biotic environment (b), and clade-specific phenotypes or traits (c); thus, d ~ a,b,c. We refer to the four components (a-d) and their interactions collectively as the "Evolutionary Arena." We outline analytical approaches to this framework and present a case study on conifers, for which we parameterize the general model. We also discuss three conceptual examples: the Lupinus radiation in the Andes in the context of emerging ecological opportunity and fluctuating connectivity due to climatic oscillations; oceanic island radiations in the context of island formation and erosion; and biotically driven radiations of the Mediterranean orchid genus Ophrys. The results of the conifer case study are consistent with the long-standing scenario that low competition and high rates of niche evolution promote diversification. The conceptual examples illustrate how using the synthetic Evolutionary Arena framework helps to identify and structure future directions for research on evolutionary radiations. In this way, the Evolutionary Arena framework promotes a more general understanding of variation in evolutionary rates by making quantitative results comparable between case studies, thereby allowing new syntheses of evolutionary and ecological processes to emerge.

Fruit syndromes in Viburnum: correlated evolution of color, nutritional content, and morphology in bird-dispersed fleshy fruits.

PREMISE: A key question in plant dispersal via animal vectors is where and why fruit colors vary between species and how color relates to other fruit traits. To better understand the factors shaping the evolution of fruit color diversity, we tested for the existence of syndromes of traits (color, morphology, and nutrition) in the fruits of Viburnum. We placed these results in a larger phylogenetic context and reconstructed ancestral states to assess how Viburnum fruit traits have evolved across the clade. RESULTS: We find that blue Viburnum fruits are not very juicy, and have high lipid content and large, round endocarps surrounded by a small quantity of pulp. Red fruits display the opposite suite of traits: they are very juicy with low lipid content and smaller, flatter endocarps. The ancestral Viburnum fruit may have gone through a sequence of color changes before maturation (green to yellow to red to black), though our reconstructions are equivocal. In one major clade of Viburnum (Nectarotinus), fruits mature synchronously with reduced intermediate color stages. Most transitions between fruit colors occurred in this synchronously fruiting clade. CONCLUSIONS: It is widely accepted that fruit trait diversity has primarily been driven by the differing perceptual abilities of bird versus mammal frugivores. Yet within a clade of largely bird-dispersed fruits, we find clear correlations between color, morphology, and nutrition. These correlations are likely driven by a shift from sequential to synchronous development, followed by diversification in color, nutrition, and morphology. A deeper understanding of fruit evolution within clades will elucidate the degree to which such syndromes structure extant fruit diversity.

Accumulation over evolutionary time as a major cause of biodiversity hotspots in conifers.

Biodiversity hotspots are important for understanding how areas of high species richness form, but disentangling the processes that produce them is difficult. We combine geographical ranges, phylogenetic relationships and trait data for 606 conifer species in order to explore the mechanisms underlying richness hotspot formation. We identify eight richness hotspots that overlap known centres of plant endemism and diversity, and find that conifer richness hotspots occur in mountainous areas within broader regions of long-term climate stability. Conifer hotspots are not unique in their species composition, traits or phylogenetic structure; however, a large percentage of their species are not restricted to hotspots and they rarely show either a preponderance of new radiating lineages or old relictual lineages. We suggest that conifer hotspots have primarily formed as a result of lineages accumulating over evolutionary time scales in stable mountainous areas rather than through high origination, preferential retention of relictual lineages or radiation of species with unique traits, although such processes may contribute to nuanced differences among hotspots. Conifers suggest that a simple accumulation of regional diversity can generate high species richness without additional processes and that geography rather than biology may play a primary role in hotspot formation.

Differences in flowering time maintain species boundaries in a continental radiation of Viburnum.

PREMISE: We take an integrative approach in assessing how introgression and Pleistocene climate fluctuations have shaped the diversification of the core Lentago clade of Viburnum, a group of five interfertile species with broad areas of sympatry. We specifically tested whether flowering time plays a role in maintaining species isolation. METHODS: RAD-seq data for 103 individuals were used to infer the species relationships and the genetic structure within each species. Flowering times were compared among species on the basis of historical flowering dates documented by herbarium specimens. RESULTS: Within each species, we found a strong relationship between flowering date and latitude, such that southern populations flower earlier than northern ones. In areas of sympatry, the species flower in sequence rather than simultaneously, with flowering dates offset by ≥9 d for all species pairs. In two cases it appears that the offset in flowering times is an incidental consequence of adaptation to differing climates, but in the recently diverged sister species V. prunifolium and V. rufidulum, we find evidence that reinforcement led to reproductive character displacement. Long-term trends suggest that the two northern-most species are flowering earlier in response to recent climate change. CONCLUSIONS: We argue that speciation in the Lentago clade has primarily occurred through ecological divergence of allopatric populations, but differences in flowering time were essential to maintain separation of incipient species when they came into secondary contact. This combination of factors may underlie diversification in many other plant clades.

Phylogeography of a widespread eastern North American shrub, Viburnum lantanoides.

PREMISE OF THE STUDY: There have been relatively few phylogeographic studies of eastern North American plants, especially of animal-dispersed shrubby species, and this leaves a significant gap in our understanding of how such species were affected by glacial events. Here, we analyzed the phylogeography of the widespread understory shrub Viburnum lantanoides. METHODS: We generated RADseq data and paleoclimatic species distribution models (SDMs) to identify the locations of refugia where V. lantanoides may have survived the Last Glacial Maximum (LGM) and how its range expanded as glaciers receded. KEY RESULTS: Genetic diversity falls off with increasing latitude and longitude, indicating that range expansion likely occurred via serial founder events from southern source populations. Samples from the southern Appalachians form a grade, while those from the north form a clade, suggesting that a single genetic lineage recolonized the north. SDMs indicate that V. lantanoides probably survived the LGM in refugia on the mid-Atlantic Coastal Plain and/or the interior Gulf Coastal Plain. CONCLUSIONS: Our analyses indicate that V. lantanoides survived the LGM in refugia south of the glacier but north of the extensive refugium along the Gulf Coast. Following the LGM, a single population expanded northward along the Appalachian Mountains and eventually into eastern Canada. The patterns observed here suggest that range expansion occurred in a stepwise manner, similar to postglacial dynamics observed in a number of European plant species.