Floral traits and their connection with Pollinators and Climate.

BACKGROUND AND AIMS: Floral characteristics vary significantly among plant species, and multiple underlying factors govern this diversity. Although it is widely known that spatial variation in pollinator groups can exert selection on floral traits, the relative contribution of pollinators and climate to the variation of floral traits across large geographic areas remains a little-studied area. Besides furthering our conceptual understanding of these processes, gaining insight into the topic is also of conservation relevance: understanding how climate may drive floral traits variation can serve to protect plant-pollinator interactions under global change conditions. METHODS: We used Rhododendron as a model system and collected floral traits (corolla length, nectar volume and concentrations), floral visitors, and climatic data on 21 Rhododendron species across two continents (North America-Appalachians and Asia-Himalaya). Based on this we quantified the influence of climate and pollinators to floral traits using phylogeny-informed analyses. KEY RESULTS: Our results indicate that there is substantial variation in pollinators and morphological traits across Rhododendron species and continents. We came across four pollinator groups: birds, bees, butterflies, and flies. Asian species were commonly visited by birds, bees, and flies, while bees and butterflies were the most common visitors of North American species. The visitor identity explained nectar trait variation, with flowers visited by birds presenting higher volumes of dilute nectar and those visited by insects producing concentrated nectar. Nectar concentration and corolla length exhibited a strong phylogenetic signal across the analysed set of species. We also found that nectar trait variation in the Himalaya could also be explained by climate, which presented significant interactions with pollinator identity. CONCLUSIONS: Our results indicate that both pollinators and climate contribute and interact to drive nectar trait variation, suggesting that both can affect pollination interactions and floral (and plant) evolution individually and interacting with each other.

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.

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.

Patterns of floral morphology in relation to climate and floral visitors.

BACKGROUND AND AIMS: The diversity of floral morphology among plant species has long captured the interest of biologists and led to the development of a number of explanatory theories. Floral morphology varies substantially within species, and the mechanisms maintaining this diversity are diverse. One possibility is that spatial variation in the pollinator fauna drives the evolution of spatially divergent floral ecotypes adapted to the local suite of pollinators. Another possibility is that geographic variation in the abiotic environment and local climatic conditions favours different floral morphologies in different regions. Although both possibilities have been shown to explain floral variation in some cases, they have rarely been competed against one another using data collected from large spatial scales. In this study, we assess floral variation in relation to climate and floral visitors in four oil-reward-specialized pollination interactions. METHODS: We used a combination of large-scale plant and pollinator samplings, morphological measures and climatic data. We analysed the data using spatial approaches, as well as traditional multivariate and structural equation modelling approaches. KEY RESULTS: Our results indicate that the four species have different levels of specialization, and that this can be explained by their climatic niche breadth. In addition, our results show that, at least for some species, floral morphology can be explained by the identity of floral visitors, with climate having only an indirect effect. CONCLUSIONS: Our results demonstrate that, even in very specialized interactions, both biotic and abiotic variables can explain a substantial amount of intraspecific variation in floral morphology.

Integrating life history traits into predictive phylogeography.

Predictive phylogeography seeks to aggregate genetic, environmental and taxonomic data from multiple species in order to make predictions about unsampled taxa using machine-learning techniques such as Random Forests. To date, organismal trait data have infrequently been incorporated into predictive frameworks due to difficulties inherent to the scoring of trait data across a taxonomically broad set of taxa. We refine predictive frameworks from two North American systems, the inland temperate rainforests of the Pacific Northwest and the Southwestern Arid Lands (SWAL), by incorporating a number of organismal trait variables. Our results indicate that incorporating life history traits as predictor variables improves the performance of the supervised machine-learning approach to predictive phylogeography, especially for the SWAL system, in which predictions made from only taxonomic and climate variables meets only moderate success. In particular, traits related to reproduction (e.g., reproductive mode; clutch size) and trophic level appear to be particularly informative to the predictive framework. Predictive frameworks offer an important mechanism for integration of organismal trait, environmental data, and genetic data in phylogeographic studies.

Combining allele frequency and tree-based approaches improves phylogeographic inference from natural history collections.

Model selection approaches in phylogeography have allowed researchers to evaluate the support for competing demographic histories, which provides a mode of inference and a measure of uncertainty in understanding climatic and spatial influences on intraspecific diversity. Here, to rank all models in the comparison set and determine what proportion of the total support the top-ranked model garners, we conduct model selection using two analytical approaches-allele frequency-based, implemented in fastsimcoal2, and gene tree-based, implemented in phrapl. We then expand this model selection framework by including an assessment of absolute fit of the models to the data. For this, we utilize DNA isolated from existing natural history collections that span the distribution of red alder (Alnus rubra) in the Pacific Northwest of North America to generate genomic data for the evaluation of 13 demographic scenarios. The quality of DNA recovered from herbarium specimen leaf tissue was assessed for its utility and effectiveness in demographic model selection, specifically in the two approaches mentioned. We present strong support for the use of herbarium tissue in the generation of genomic DNA, albeit with the inclusion of additional quality control checks prior to library preparation and analyses with multiple approaches that incorporate various data. Analyses with allele frequency spectra and gene trees predominantly support A. rubra having experienced an ancient vicariance event with intermittent and frequent gene flow between the disjunct populations. Additionally, the data consistently fit the most frequently selected model, corroborating the model selection techniques. Finally, these results suggest that the A. rubra disjunct populations do not represent separate species.

Ecological intensification to mitigate impacts of conventional intensive land use on pollinators and pollination.

Worldwide, human appropriation of ecosystems is disrupting plant-pollinator communities and pollination function through habitat conversion and landscape homogenisation. Conversion to agriculture is destroying and degrading semi-natural ecosystems while conventional land-use intensification (e.g. industrial management of large-scale monocultures with high chemical inputs) homogenises landscape structure and quality. Together, these anthropogenic processes reduce the connectivity of populations and erode floral and nesting resources to undermine pollinator abundance and diversity, and ultimately pollination services. Ecological intensification of agriculture represents a strategic alternative to ameliorate these drivers of pollinator decline while supporting sustainable food production, by promoting biodiversity beneficial to agricultural production through management practices such as intercropping, crop rotations, farm-level diversification and reduced agrochemical use. We critically evaluate its potential to address and reverse the land use and management trends currently degrading pollinator communities and potentially causing widespread pollination deficits. We find that many of the practices that constitute ecological intensification can contribute to mitigating the drivers of pollinator decline. Our findings support ecological intensification as a solution to pollinator declines, and we discuss ways to promote it in agricultural policy and practice.

Demographic model selection using random forests and the site frequency spectrum.

Phylogeographic data sets have grown from tens to thousands of loci in recent years, but extant statistical methods do not take full advantage of these large data sets. For example, approximate Bayesian computation (ABC) is a commonly used method for the explicit comparison of alternate demographic histories, but it is limited by the "curse of dimensionality" and issues related to the simulation and summarization of data when applied to next-generation sequencing (NGS) data sets. We implement here several improvements to overcome these difficulties. We use a Random Forest (RF) classifier for model selection to circumvent the curse of dimensionality and apply a binned representation of the multidimensional site frequency spectrum (mSFS) to address issues related to the simulation and summarization of large SNP data sets. We evaluate the performance of these improvements using simulation and find low overall error rates (~7%). We then apply the approach to data from Haplotrema vancouverense, a land snail endemic to the Pacific Northwest of North America. Fifteen demographic models were compared, and our results support a model of recent dispersal from coastal to inland rainforests. Our results demonstrate that binning is an effective strategy for the construction of a mSFS and imply that the statistical power of RF when applied to demographic model selection is at least comparable to traditional ABC algorithms. Importantly, by combining these strategies, large sets of models with differing numbers of populations can be evaluated.

Assessing the potential of RAD-sequencing to resolve phylogenetic relationships within species radiations: The fly genus Chiastocheta (Diptera: Anthomyiidae) as a case study.

Determining phylogenetic relationships among recently diverged species has long been a challenge in evolutionary biology. Cytoplasmic DNA markers, which have been widely used, notably in the context of molecular barcoding, have not always proved successful in resolving such phylogenies. However, with the advent of next-generation-sequencing technologies and associated techniques of reduced genome representation, phylogenies of closely related species have been resolved at a much higher detail in the last couple of years. Here we examine the potential and limitations of one of such techniques-Restriction-site Associated DNA (RAD) sequencing, a method that produces thousands of (mostly) anonymous nuclear markers, in disentangling the phylogeny of the fly genus Chiastocheta (Diptera: Anthomyiidae). In Europe, this genus encompasses seven species of seed predators, which have been widely studied in the context of their ecological and evolutionary interactions with the plant Trollius europaeus (Ranunculaceae). So far, phylogenetic analyses using mitochondrial markers failed to resolve monophyly of most of the species from this recently diversified genus, suggesting that their taxonomy may need a revision. However, relying on a single, non-recombining marker and ignoring potential incongruences between mitochondrial and nuclear loci may provide an incomplete account of the lineage history. In this study, we applied both classical Sanger sequencing of three mtDNA regions and RAD-sequencing, for reconstructing the phylogeny of the genus. Contrasting with results based on mitochondrial markers, RAD-sequencing analyses retrieved the monophyly of all seven species, in agreement with the morphological species assignment. We found robust nuclear-based species assignment of individual samples, and low levels of estimated contemporary gene flow among them. However, despite recovering species' monophyly, interspecific relationships varied depending on the set of RAD loci considered, producing contradictory topologies. Moreover, coalescence-based phylogenetic analyses revealed low supports for most of the interspecific relationships. Our results indicate that despite the higher performance of RAD-sequencing in terms of species trees resolution compared to cytoplasmic markers, reconstructing inter-specific relationships among recently-diverged lineages may lie beyond the possibilities offered by large sets of RAD-sequencing markers in cases of strong gene tree incongruence.

Identifying cryptic diversity with predictive phylogeography.

Identifying units of biological diversity is a major goal of organismal biology. An increasing literature has focused on the importance of cryptic diversity, defined as the presence of deeply diverged lineages within a single species. While most discoveries of cryptic lineages proceed on a taxon-by-taxon basis, rapid assessments of biodiversity are needed to inform conservation policy and decision-making. Here, we introduce a predictive framework for phylogeography that allows rapidly identifying cryptic diversity. Our approach proceeds by collecting environmental, taxonomic and genetic data from codistributed taxa with known phylogeographic histories. We define these taxa as a reference set, and categorize them as either harbouring or lacking cryptic diversity. We then build a random forest classifier that allows us to predict which other taxa endemic to the same biome are likely to contain cryptic diversity. We apply this framework to data from two sets of disjunct ecosystems known to harbour taxa with cryptic diversity: the mesic temperate forests of the Pacific Northwest of North America and the arid lands of Southwestern North America. The predictive approach presented here is accurate, with prediction accuracies placed between 65% and 98.79% depending of the ecosystem. This seems to indicate that our method can be successfully used to address ecosystem-level questions about cryptic diversity. Further, our application for the prediction of the cryptic/non-cryptic nature of unknown species is easily applicable and provides results that agree with recent discoveries from those systems. Our results demonstrate that the transition of phylogeography from a descriptive to a predictive discipline is possible and effective.

Genetic Structure across Broad Spatial and Temporal Scales: Rocky Mountain Tailed Frogs (Ascaphus montanus; Anura: Ascaphidae) in the Inland Temperate Rainforest.

Contemporary and historical processes interact to structure genetic variation, however discerning between these can be difficult. Here, we analyze range-wide variation at 13 microsatellite loci in 2098 Rocky Mountain tailed frogs, Ascaphus montanus, collected from 117 streams across the species distribution in the Inland Northwest (INW) and interpret that variation in light of historical phylogeography, contemporary landscape genetics, and the reconstructed paleodistribution of the species. Further, we project species distribution models (SDMs) to predict future changes in the range as a function of changing climate. Genetic structure has a strong spatial signature that is precisely congruent with a deep (~1.8 MY) phylogeographic split in mtDNA when we partition populations into 2 clusters (K = 2), and is congruent with refugia areas inferred from our paleorange reconstructions. There is a hierarchical pattern of geographic structure as we permit additional clusters, with populations clustering following mountain ranges. Nevertheless, genetic diversity is the highest in populations at the center of the range and is attenuated in populations closer to the range edges. Similarly, geographic distance is the single best predictor of pairwise genetic differentiation, but connectivity also is an important predictor. At intermediate and local geographic scales, deviations from isolation-by-distance are more apparent, at least in the northern portion of the distribution. These results indicate that both historical and landscape factors are contributing to the genetic structure and diversity of tailed frogs in the Inland Northwest.

Integrated phylogenomic approaches in insect systematics.

The increased accessibility of genomic and imaging methods, and the improved access to ecological, spatial, and other natural history-related data is allowing for insect systematics to grow and find answers to central evolutionary and taxonomic questions. Today, integrated studies in insect phylogenomics and systematics are combining natural history, behavior, developmental biology, morphology, fossils, geographic range data, and ecological interactions. This integration is contributing to the clarification of evolutionary relationships, and the recognition of the role played by these factors on the evolution of insects. Future work should continue to build on these advances, seeking to further increase open-access databasing and support for natural history research, as well as expand its analytical palettes.

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.

Predicting present and future intra-specific genetic structure through niche hindcasting across 24 millennia.

Paleoclimatic reconstructions coupled with species distribution models and identification of extant spatial genetic structure have the potential to provide insights into the demographic events that shape the distribution of intra-specific genetic variation across time. Using the globeflower Trollius europaeus as a case-study, we combined (1) Amplified Fragment Length Polymorphisms, (2) suites of 1000-years stepwise hindcasted species distributions and (3) a model of diffusion through time over the last 24,000 years, to trace the spatial dynamics that most likely fits the species' current genetic structure. We show that the globeflower comprises four gene pools in Europe which, from the dry period preceding the Last Glacial Maximum, dispersed while tracking the conditions fitting its climatic niche. Among these four gene pools, two are predicted to experience drastic range retraction in the near future. Our interdisciplinary approach, applicable to virtually any taxon, is an advance in inferring how climate change impacts species' genetic structures.

Ecological and historical drivers of diversification in the fly genus Chiastocheta Pokorny.

Coevolution is among the main forces shaping the biodiversity on Earth. In Eurasia, one of the best-known plant-insect interactions showing highly coevolved features involves the fly genus Chiastocheta and its host-plant Trollius. Although this system has been widely studied from an ecological point of view, the phylogenetic relationships and biogeographic history of the flies have remained little investigated. In this integrative study, we aim to test the monophyly of the five Chiastocheta eco-morphological groups, defined by Pellmyr in 1992, by inferring a mitochondrial phylogeny. We further apply a new approach to assess the effect of (i) different molecular substitution rates and (ii) phylogenetic uncertainty on the inference of the spatio-temporal evolution of the group. From a taxonomic point of view, we demonstrate that only two of Pellmyr's groups (rotundiventris and dentifera) are phylogenetically supported, the other species appearing para- or polyphyletic. We also identify the position of C. lophota, which was not included in previous surveys. From a spatio-temporal perspective, we show that the genus arose during the Pliocene in Europe. Our results also indicate that at least four large-scale dispersal events are required to explain the current distribution of Chiastocheta. Moreover, each dispersal to or from Asia is associated with a host-shift and seems to correspond to an increase in speciation rates. Finally, we highlight the correlation between diversification and climatic fluctuations, which indicate that the cycles of global cooling over the last million years had an influence on the radiation of the group.

Comparative phylogeography of two Northern Rocky Mountain endemics: the widespread Anguispira kochi occidentalis and the narrow-range Anguispira nimapuna (Gastropoda: Discidae).

The Northern Rocky Mountain ecosystem supports rich biological diversity with many endemic and rare species. Extant endemics display two biogeographic patterns: widespread species with fragmented populations, and narrow-range endemics. These distributions are shown by the congeneric snails Anguispira kochi occidentalis and Anguispira nimapuna. These two taxa are disjunct from the remaining species of the genus, which achieves its greatest diversity in eastern North America. Given the disjunct nature of A. k. occidentalis and A. nimapuna, we here present a mtDNA phylogeny of the genus that includes both eastern and western species to assess the phylogenetic position of A. k. occidentalis and A. nimapuna. We then reconstruct the demographic history of A. k. occidentalis and A. nimapuna by analysing current patterns of genetic variation and interpreting the results considering the historical biogeography of the region. Both A. k. occidentalis and A. nimapuna represent unique taxa that are genetically and geographically distinct from their congeners. The current distribution and genetic structure of A. k. occidentalis has been shaped by both historical isolation in refugia and more recent northward shifts, whereas A. nimapuna is represented by two populations with shallow divergence in an area of long-term habitat stability.

Self-Compatibility Not Associated with Morphological or Genetic Diversity Reduction in Oil-Rewarding Calceolaria Species.

One of the most common evolutionary transitions in angiosperms is the reproductive change from outcrossing to selfing, commonly associated with changes in floral biology and genetic diversity. Here, we aim to test whether self-compatibility leads to a reduction of floral traits and genetic diversity. For this, we experimentally estimate levels of self-compatibility, measure three floral traits and estimate four genetic diversity parameters using nine microsatellites in nine Calceolaria species. Our analysis indicated that four of the study species were self-incompatible. In addition, we found that self-compatible species did not show a reduction in floral traits size, but rather displayed larger corolla and elaiophore areas. Our analyses of genetic diversity identified larger allele number and observed heterozygosity in selfers than in outcrossers, but did not find larger inbreeding in the self-compatible species. Even though our results contradict our expectations, in the case of Calceolaria, their high dependence on only two genera of oil-bees puts the genus in a vulnerable reproductive position, probably facilitating the evolution of reproductive assurance mechanisms in the absence of pollinators. As a result, plants maintain their pollinator attraction traits while evolving the ability to self, possibly in a delayed way.

Risks to pollinators and pollination from invasive alien species.

Invasive alien species modify pollinator biodiversity and the services they provide that underpin ecosystem function and human well-being. Building on the Intergovernmental Science-Policy Platform for Biodiversity and Ecosystem Services (IPBES) global assessment of pollinators and pollination, we synthesize current understanding of invasive alien impacts on pollinators and pollination. Invasive alien species create risks and opportunities for pollinator nutrition, re-organize species interactions to affect native pollination and community stability, and spread and select for virulent diseases. Risks are complex but substantial, and depend greatly on the ecological function and evolutionary history of both the invader and the recipient ecosystem. We highlight evolutionary implications for pollination from invasive alien species, and identify future research directions, key messages and options for decision-making.

Gene flow among wild and domesticated almond species: insights from chloroplast and nuclear markers.

Hybridization has played a central role in the evolutionary history of domesticated plants. Notably, several breeding programs relying on gene introgression from the wild compartment have been performed in fruit tree species within the genus Prunus but few studies investigated spontaneous gene flow among wild and domesticated Prunus species. Consequently, a comprehensive understanding of genetic relationships and levels of gene flow between domesticated and wild Prunus species is needed. Combining nuclear and chloroplastic microsatellites, we investigated the gene flow and hybridization among two key almond tree species, the cultivated Prunus dulcis and one of the most widespread wild relative Prunus orientalis in the Fertile Crescent. We detected high genetic diversity levels in both species along with substantial and symmetric gene flow between the domesticated P. dulcis and the wild P. orientalis. These results were discussed in light of the cultivated species diversity, by outlining the frequent spontaneous genetic contributions of wild species to the domesticated compartment. In addition, crop-to-wild gene flow suggests that ad hoc transgene containment strategies would be required if genetically modified cultivars were introduced in the northwestern Mediterranean.

Comparative phylogeography in a specific and obligate pollination antagonism.

In specific and obligate interactions the nature and abundance of a given species can have important effects on the survival and population dynamics of associated organisms. In a phylogeographic framework, we therefore expect that the fates of organisms interacting specifically are also tightly interrelated. Here we investigate such a scenario by analyzing the genetic structures of species interacting in an obligate plant-insect pollination lure-and-trap antagonism, involving Arum maculatum (Araceae) and its specific psychodid (Diptera) visitors Psychoda phalaenoides and Psycha grisescens. Because the interaction is asymmetric (i.e., only the plant depends on the insect), we expect the genetic structure of the plant to be related with the historical pollinator availability, yielding incongruent phylogeographic patterns between the interacting organisms.Using insect mtDNA sequences and plant AFLP genome fingerprinting, we inferred the large-scale phylogeographies of each species and the distribution of genetic diversities throughout the sampled range, and evaluated the congruence in their respective genetic structures using hierarchical analyses of molecular variances (AMOVA). Because the composition of pollinator species varies in Europe, we also examined its association with the spatial genetic structure of the plant.Our findings indicate that while the plant presents a spatially well-defined genetic structure, this is not the case in the insects. Patterns of genetic diversities also show dissimilar distributions among the three interacting species. Phylogeographic histories of the plant and its pollinating insects are thus not congruent, a result that would indicate that plant and insect lineages do not share the same glacial and postglacial histories. However, the genetic structure of the plant can, at least partially, be explained by the type of pollinators available at a regional scale. Differences in life-history traits of available pollinators might therefore have influenced the genetic structure of the plant, the dependent organism, in this antagonistic interaction.