Museum skins enable identification of introgression associated with cytonuclear discordance.

Increased sampling of genomes and populations across closely related species has revealed that levels of genetic exchange during and after speciation are higher than previously thought. One obvious manifestation of such exchange is strong cytonuclear discordance, where the divergence in mitochondrial DNA (mtDNA) differs from that for nuclear genes more (or less) than expected from differences between mtDNA and nuclear DNA (nDNA) in population size and mutation rate. Given genome-scale datasets and coalescent modelling, we can now confidently identify cases of strong discordance and test specifically for historical or recent introgression as the cause. Using population sampling, combining exon capture data from historical museum specimens and recently collected tissues we showcase how genomic tools can resolve complex evolutionary histories in the brachyotis group of rock-wallabies (Petrogale). In particular, applying population and phylogenomic approaches we can assess the role of demographic processes in driving complex evolutionary patterns and assess a role of ancient introgression and hybridisation. We find that described species are well supported as monophyletic taxa for nDNA genes, but not for mtDNA, with cytonuclear discordance involving at least four operational taxonomic units (OTUs) across four species which diverged 183-278 kya. ABC modelling of nDNA gene trees supports introgression during or after speciation for some taxon pairs with cytonuclear discordance. Given substantial differences in body size between the species involved, this evidence for gene flow is surprising. Heterogenous patterns of introgression were identified but do not appear to be associated with chromosome differences between species. These and previous results suggest that dynamic past climates across the monsoonal tropics could have promoted reticulation among related species.

Limited Introgression between Rock-Wallabies with Extensive Chromosomal Rearrangements.

Chromosome rearrangements can result in the rapid evolution of hybrid incompatibilities. Robertsonian fusions, particularly those with monobrachial homology, can drive reproductive isolation amongst recently diverged taxa. The recent radiation of rock-wallabies (genus Petrogale) is an important model to explore the role of Robertsonian fusions in speciation. Here, we pursue that goal using an extensive sampling of populations and genomes of Petrogale from north-eastern Australia. In contrast to previous assessments using mitochondrial DNA or nuclear microsatellite loci, genomic data are able to separate the most closely related species and to resolve their divergence histories. Both phylogenetic and population genetic analyses indicate introgression between two species that differ by a single Robertsonian fusion. Based on the available data, there is also evidence for introgression between two species which share complex chromosomal rearrangements. However, the remaining results show no consistent signature of introgression amongst species pairs and where evident, indicate generally low introgression overall. X-linked loci have elevated divergence compared with autosomal loci indicating a potential role for genic evolution to produce reproductive isolation in concert with chromosome change. Our results highlight the value of genome scale data in evaluating the role of Robertsonian fusions and structural variation in divergence, speciation, and patterns of molecular evolution.

Analyzing trait-climate relationships within and among taxa using machine learning and herbarium specimens.

PREMISE: Continental-scale leaf trait studies can help explain how plants survive in different environments, but large data sets are costly to assemble at this scale. Automating the measurement of digitized herbarium collections could rapidly expand the data available to such studies. We used machine learning to identify and measure leaves from existing, digitized herbarium specimens. The process was developed, validated, and applied to analyses of relationships between leaf size and climate within and among species for two genera: Syzygium (Myrtaceae) and Ficus (Moraceae). METHODS: Convolutional neural network (CNN) models were used to detect and measure complete leaves in images. Predictions of a model trained with a set of 35 randomly selected images and a second model trained with 35 user-selected images were compared using a set of 50 labeled validation images. The validated models were then applied to 1227 Syzygium and 2595 Ficus specimens digitized by the National Herbarium of New South Wales, Australia. Leaf area measurements were made for each genus and used to examine links between leaf size and climate. RESULTS: The user-selected training method for Syzygium found more leaves (9347 vs. 8423) using fewer training masks (218 vs. 225), and found leaves with a greater range of sizes than the random image training method. Within each genus, leaf size was positively associated with temperature and rainfall, consistent with previous observations. However, within species, the associations between leaf size and environmental variables were weaker. CONCLUSIONS: CNNs detected and measured leaves with levels of accuracy useful for trait extraction and analysis and illustrate the potential for machine learning of herbarium specimens to massively increase global leaf trait data sets. Within-species relationships were weak, suggesting that population history and gene flow have a strong effect at this level. Herbarium specimens and machine learning could expand sampling of trait data within many species, offering new insights into trait evolution.

Where to draw the boundaries? Using landscape genomics to disentangle the scribbly gum species complex.

PREMISE: Species delimitation is an integral part of evolution and ecology and is vital in conservation science. However, in some groups, species delimitation is difficult, especially where ancestral relationships inferred from morphological or genetic characters are discordant, possibly due to a complicated demographic history (e.g., recent divergences between lineages). Modern genetic techniques can take into account complex histories to distinguish species at a reasonable cost and are increasingly used in numerous applications. We focus on the scribbly gums, a group of up to five closely related and morphologically similar "species" within the eucalypts. METHODS: Multiple populations of each recognized scribbly gum species were sampled over a wide region across climates, and genomewide scans were used to resolve species boundaries. RESULTS: None of the taxa were completely divergent, and there were two genetically distinct entities: the inland distributed Eucalyptus rossii and a coastal conglomerate consisting of four species forming three discernible, but highly admixed groups. Divergence among taxa was likely driven by temporal vicariant processes resulting in partial separation across biogeographic barriers. High interspecific gene flow indicated separated taxa reconnected at different points in time, blurring species boundaries. CONCLUSIONS: Our results highlight the need for genetic screening when dealing with closely related taxonomic entities, particularly those with modest morphological differences. We show that high-throughput sequencing can be effective at identifying species groupings and processes driving divergence, even in the most taxonomically complex groups, and be used as a standard practice for disentangling species complexes.

A decision framework for evidence-based climate adaptation interventions.

Climate change is outpacing the ability of species and populations to naturally adapt warranting active interventions to boost climate resilience. In their review paper, Gaitán-Espitia and Hobday discuss how such interventions may, however, counter natural evolutionary processes and adaptive capacity if not underpinned by background knowledge from genes through to ecosystems. They present a robust decision framework to guide implementation of climate adaptation interventions to avoid unintended evolutionary outcomes.

Habitat preference modulates trans-oceanic dispersal in a terrestrial vertebrate.

The importance of long-distance dispersal (LDD) in shaping geographical distributions has been debated since the nineteenth century. In terrestrial vertebrates, LDD events across large water bodies are considered highly improbable, but organismal traits affecting dispersal capacity are generally not taken into account. Here, we focus on a recent lizard radiation and combine a summary-coalescent species tree based on 1225 exons with a probabilistic model that links dispersal capacity to an evolving trait, to investigate whether ecological specialization has influenced the probability of trans-oceanic dispersal. Cryptoblepharus species that occur in coastal habitats have on average dispersed 13 to 14 times more frequently than non-coastal species and coastal specialization has, therefore, led to an extraordinarily widespread distribution that includes multiple continents and distant island archipelagoes. Furthermore, their presence across the Pacific substantially predates the age of human colonization and we can explicitly reject the possibility that these patterns are solely shaped by human-mediated dispersal. Overall, by combining new analytical methods with a comprehensive phylogenomic dataset, we use a quantitative framework to show how coastal specialization can influence dispersal capacity and eventually shape geographical distributions at a macroevolutionary scale.

Analysis of Phylogenomic Tree Space Resolves Relationships Among Marsupial Families.

A fundamental challenge in resolving evolutionary relationships across the tree of life is to account for heterogeneity in the evolutionary signal across loci. Studies of marsupial mammals have demonstrated that this heterogeneity can be substantial, leaving considerable uncertainty in the evolutionary timescale and relationships within the group. Using simulations and a new phylogenomic data set comprising nucleotide sequences of 1550 loci from 18 of the 22 extant marsupial families, we demonstrate the power of a method for identifying clusters of loci that support different phylogenetic trees. We find two distinct clusters of loci, each providing an estimate of the species tree that matches previously proposed resolutions of the marsupial phylogeny. We also identify a well-supported placement for the enigmatic marsupial moles (Notoryctes) that contradicts previous molecular estimates but is consistent with morphological evidence. The pattern of gene-tree variation across tree-space is characterized by changes in information content, GC content, substitution-model adequacy, and signatures of purifying selection in the data. In a simulation study, we show that incomplete lineage sorting can explain the division of loci into the two tree-topology clusters, as found in our phylogenomic analysis of marsupials. We also demonstrate the potential benefits of minimizing uncertainty from phylogenetic conflict for molecular dating. Our analyses reveal that Australasian marsupials appeared in the early Paleocene, whereas the diversification of present-day families occurred primarily during the late Eocene and early Oligocene. Our methods provide an intuitive framework for improving the accuracy and precision of phylogenetic inference and molecular dating using genome-scale data.

Reticulation, divergence, and the phylogeography-phylogenetics continuum.

Phylogeography, and its extensions into comparative phylogeography, have their roots in the layering of gene trees across geography, a paradigm that was greatly facilitated by the nonrecombining, fast evolution provided by animal mtDNA. As phylogeography moves into the era of next-generation sequencing, the specter of reticulation at several levels-within loci and genomes in the form of recombination and across populations and species in the form of introgression-has raised its head with a prominence even greater than glimpsed during the nuclear gene PCR era. Here we explore the theme of reticulation in comparative phylogeography, speciation analysis, and phylogenomics, and ask how the centrality of gene trees has fared in the next-generation era. To frame these issues, we first provide a snapshot of multilocus phylogeographic studies across the Carpentarian Barrier, a prominent biogeographic barrier dividing faunas spanning the monsoon tropics in northern Australia. We find that divergence across this barrier is evident in most species, but is heterogeneous in time and demographic history, often reflecting the taxonomic distinctness of lineages spanning it. We then discuss a variety of forces generating reticulate patterns in phylogeography, including introgression, contact zones, and the potential selection-driven outliers on next-generation molecular markers. We emphasize the continued need for demographic models incorporating reticulation at the level of genomes and populations, and conclude that gene trees, whether explicit or implicit, should continue to play a role in the future of phylogeography.

Phylogenomics of a rapid radiation: the Australian rainbow skinks.

BACKGROUND: The application of target capture with next-generation sequencing now enables phylogenomic analyses of rapidly radiating clades of species. But such analyses are complicated by extensive incomplete lineage sorting, demanding the use of methods that consider this process explicitly, such as the multispecies coalescent (MSC) model. However, the MSC makes strong assumptions about divergence history and population structure, and when using the full Bayesian implementation, current computational limits mean that relatively few loci and samples can be analysed for even modest sized radiations. We explore these issues through analyses of an extensive (> 1000 loci) dataset for the Australian rainbow skinks. This group consists of 3 genera and 41 described species, which likely diversified rapidly in Australia during the mid-late Miocene to occupy rainforest, woodland, and rocky habitats with corresponding diversity of morphology and breeding colouration. Previous phylogenetic analyses of this group have revealed short inter-nodes and high discordance among loci, limiting the resolution of inferred trees. A further complication is that many species have deep phylogeographic structure - this poses the question of how to sample individuals within species for analyses using the MSC. RESULTS: Phylogenies obtained using concatenation and summary coalescent species tree approaches to the full dataset are well resolved with generally consistent topology, including for previously intractable relationships near the base of the clade. As expected, branch lengths at the tips are substantially overestimated using concatenation. Comparisons of different strategies for sampling haplotypes for full Bayesian MSC analyses (for one clade and using smaller sets of loci) revealed, unexpectedly, that combining haplotypes across divergent phylogeographic lineages yielded consistent species trees. CONCLUSIONS: This study of more than 1000 loci provides a strongly-supported estimate of the phylogeny of the Australian rainbow skinks, which will inform future research on the evolution and taxonomy of this group. Our analyses suggest that species tree estimation with the MSC can be quite robust to violation of the assumption that the individuals representing a taxon are sampled from a panmictic population.

Pleistocene climatic changes drive diversification across a tropical savanna.

Spatial responses of species to past climate change depend on both intrinsic traits (climatic niche breadth, dispersal rates) and the scale of climatic fluctuations across the landscape. New capabilities in generating and analysing population genomic data, along with spatial modelling, have unleashed our capacity to infer how past climate changes have shaped populations, and by extension, complex communities. Combining these approaches, we uncover lineage diversity across four codistributed lizards from the Australian Monsoonal Tropics and explore how varying climatic tolerances interact with regional climate history to generate common vs. disparate responses to late Pleistocene change. We find more divergent spatial structuring and temporal demographic responses in the drier Kimberley region compared to the more mesic and consistently suitable Top End. We hypothesize that, in general, the effects of species' traits on sensitivity to climate fluctuation will be more evident in climatically marginal regions. If true, this points to the need in climatically marginal areas to craft more species-(or trait)-specific strategies for persistence under future climate change.

Accounting for Uncertainty in Gene Tree Estimation: Summary-Coalescent Species Tree Inference in a Challenging Radiation of Australian Lizards.

Accurate gene tree inference is an important aspect of species tree estimation in a summary-coalescent framework. Yet, in empirical studies, inferred gene trees differ in accuracy due to stochastic variation in phylogenetic signal between targeted loci. Empiricists should, therefore, examine the consistency of species tree inference, while accounting for the observed heterogeneity in gene tree resolution of phylogenomic data sets. Here, we assess the impact of gene tree estimation error on summary-coalescent species tree inference by screening ${\sim}2000$ exonic loci based on gene tree resolution prior to phylogenetic inference. We focus on a phylogenetically challenging radiation of Australian lizards (genus Cryptoblepharus, Scincidae) and explore effects on topology and support. We identify a well-supported topology based on all loci and find that a relatively small number of high-resolution gene trees can be sufficient to converge on the same topology. Adding gene trees with decreasing resolution produced a generally consistent topology, and increased confidence for specific bipartitions that were poorly supported when using a small number of informative loci. This corroborates coalescent-based simulation studies that have highlighted the need for a large number of loci to confidently resolve challenging relationships and refutes the notion that low-resolution gene trees introduce phylogenetic noise. Further, our study also highlights the value of quantifying changes in nodal support across locus subsets of increasing size (but decreasing gene tree resolution). Such detailed analyses can reveal anomalous fluctuations in support at some nodes, suggesting the possibility of model violation. By characterizing the heterogeneity in phylogenetic signal among loci, we can account for uncertainty in gene tree estimation and assess its effect on the consistency of the species tree estimate. We suggest that the evaluation of gene tree resolution should be incorporated in the analysis of empirical phylogenomic data sets. This will ultimately increase our confidence in species tree estimation using summary-coalescent methods and enable us to exploit genomic data for phylogenetic inference. [Coalescence; concatenation; Cryptoblepharus; exon capture; gene tree; phylogenomics; species tree.].

Speciation in the presence of gene flow: population genomics of closely related and diverging Eucalyptus species.

Speciation is a complex process that is fundamental to the origins of biological diversity. While there has been considerable progress in our understanding of speciation, there are still many unanswered questions, especially regarding barriers to gene flow in diverging populations. Eucalyptus is an appropriate system for investigating speciation mechanisms since it comprises species that are rapidly evolving across heterogeneous environments. We examined patterns of genetic variation within and among six closely related Eucalyptus species in subgenus Eucalyptus section Eucalyptus in south-eastern Australia (commonly known as the "green ashes"). We used reduced representation genome sequencing to genotype samples from populations across altitudinal and latitudinal gradients. We found one species, Eucalyptus cunninghamii, to be highly genetically differentiated from the others, and a population of mallees from Mount Banks to be genetically distinct and therefore likely to be a new undescribed species. Only modest levels of differentiation were found between all other species in the study. There was population structure within some species (e.g., E. obstans) corresponding to geographical factors, indicating that vicariance may have played a role in the evolution of the group. Overall, we found that lineages within the green ashes are differentiated to varying extents, from strongly diverged to much earlier stages of the speciation continuum. Furthermore, our results suggest the green ashes represent a group where a range of mechanisms (e.g., reticulate evolution and vicariance) have been operating in concert. These findings not only offer insights into recent speciation mechanisms in Eucalyptus, but also other species complexes.

Tropical specialist vs. climate generalist: Diversification and demographic history of sister species of Carlia skinks from northwestern Australia.

Species endemic to the tropical regions are expected to be vulnerable to future climate change due in part to their relatively narrow climatic niches. In addition, these species are more likely to have responded strongly to past climatic change, and this can be explored through phylogeographic analyses. To test the hypothesis that tropical specialists are more sensitive to climate change than climate generalists, we generated and analyse sequence data from mtDNA and ~2500 exons to compare scales of historical persistence and population fluctuation in two sister species of Australian rainbow skinks: the tropical specialist Carlia johnstonei and the climate generalist C. triacantha. We expect the tropical specialist species to have deeper and finer-scale phylogeographic structure and stronger demographic fluctuations relative to the closely related climate generalist species, which should have had more stable populations through periods of harsh climate in the late Quaternary. Within C. johnstonei, we find that some populations from the northern Kimberley islands are highly divergent from mainland populations. In C. triacantha, one major clade occurs across the deserts and into the mesic Top End, and another occurs primarily in the Kimberley with scattered records eastwards. Where their ranges overlap in the Kimberley, both mitochondrial DNA and nuclear DNA suggest stronger phylogeographic structure and range expansion within the tropical specialist, whereas the climate generalist has minimal structuring and no evidence of recent past range expansion. These results are consistent with the hypothesis that tropical specialists are more sensitive to past climatic change.

Phylogenomics at the tips: inferring lineages and their demographic history in a tropical lizard, Carlia amax.

High-throughput sequencing approaches offer opportunities to better understand the evolutionary processes driving diversification, particularly in nonmodel organisms. In particular, the 100-1000's of loci that can now be sequenced are providing unprecedented power in population, speciation and phylogenetic studies. Here, we apply an exon capture approach to generate >99% complete sequence and SNP data across >2000 loci from a tropical skink, Carlia amax, and exploit these data to identify divergent lineages and infer their relationships and demographic histories. This is especially relevant to low-dispersal tropical taxa that often have cryptic diversity and spatially dynamic histories. For C. amax, clustering of nuclear SNPs and coalescent-based species delimitation analyses identify four divergent lineages, one fewer than predicted based on geographically coherent mtDNA clades (>9.4% sequence divergence). Three of these lineages are widespread and parapatric on the mainland, whereas the most divergent is restricted to islands off the northeast Northern Territory. Tests for population expansion reject an equilibrium isolation-by-distance model for two of the three widespread lineages and infer refugial expansion sources in the relatively mesic northeast Top End and northwest Kimberley. The latter is already recognized as a hotspot of endemism, but our results also suggest that a stronger focus on the northeast Top End, and adjacent islands is warranted. More generally, our results show how genome-reduction methods such as exon capture can yield insights into the pattern and dynamics of biodiversity across complex landscapes with as yet poorly understood biogeographic history and how exon data can link between population and phylogenetic questions.

Population and phylogenomic decomposition via genotyping-by-sequencing in Australian Pelargonium.

Species delimitation has seen a paradigm shift as increasing accessibility of genomic-scale data enables separation of lineages with convergent morphological traits and the merging of recently diverged ecotypes that have distinguishing characteristics. We inferred the process of lineage formation among Australian species in the widespread and highly variable genus Pelargonium by combining phylogenomic and population genomic analyses along with breeding system studies and character analysis. Phylogenomic analysis and population genetic clustering supported seven of the eight currently described species but provided little evidence for differences in genetic structure within the most widely distributed group that containing P. australe. In contrast, morphometric analysis detected three deep lineages within Australian Pelargonium; with P. australe consisting of five previously unrecognized entities occupying separate geographic ranges. The genomic approach enabled elucidation of parallel evolution in some traits formerly used to delineate species, as well as identification of ecotypic morphological differentiation within recognized species. Highly variable morphology and trait convergence each contribute to the discordance between phylogenomic relationships and morphological taxonomy. Data suggest that genetic divergence among species within the Australian Pelargonium may result from allopatric speciation while morphological differentiation within and among species may be more strongly driven by environmental differences.

Evaluating the performance of anchored hybrid enrichment at the tips of the tree of life: a phylogenetic analysis of Australian Eugongylus group scincid lizards.

BACKGROUND: High-throughput sequencing using targeted enrichment and transcriptomic methods enables rapid construction of phylogenomic data sets incorporating hundreds to thousands of loci. These advances have enabled access to an unprecedented amount of nucleotide sequence data, but they also pose new questions. Given that the loci targeted for enrichment are often highly conserved, how informative are they at different taxonomic scales, especially at the intraspecific/phylogeographic scale? We investigate this question using Australian scincid lizards in the Eugongylus group (Squamata: Scincidae). We sequenced 415 anchored hybrid enriched (AHE) loci for 43 individuals and mined 1650 exons (1648 loci) from transcriptomes (transcriptome mining) from 11 individuals, including multiple phylogeographic lineages within several species of Carlia, Lampropholis, and Saproscincus skinks. We assessed the phylogenetic information content of these loci at the intergeneric, interspecific, and phylogeographic scales. As a further test of the utility at the phylogeographic scale, we used the anchor hybrid enriched loci to infer lineage divergence parameters using coalescent models of isolation with migration. RESULTS: Phylogenetic analyses of both data sets inferred very strongly supported trees at all taxonomic levels. Further, AHE loci yielded estimates of divergence times between closely related lineages that were broadly consistent with previous population-level analyses. CONCLUSIONS: Anchored-enriched loci are useful at the deep phylogeny and phylogeographic scales. Although overall phylogenetic support was high throughout the Australian Eugongylus group phylogeny, there were nonetheless some conflicting or unresolved relationships, especially regarding the placement of Pseudemoia, Cryptoblepharus, and the relationships amongst closely-related species of Tasmanian Niveoscincus skinks.

Genomic variation across landscapes: insights and applications.

The distribution of genomic variation across landscapes can provide insights into the complex interactions between the environment and the genome that influence the distribution of species, and mediate phenotypic adaptation to local conditions. High throughput sequencing technologies now offer unprecedented power to explore these interactions, allowing powerful inferences about historical processes of colonization, gene flow and divergence, as well as the identification of loci that mediate local adaptation. These 'landscape genomic' approaches have been validated in model species and are now being applied to nonmodel organisms, including foundation species that have substantial effects on ecosystem processes. Here we review the growing field of landscape genomics from a very broad perspective. In particular, we describe the inferential power that is gained by taking a genome-wide view of genetic variation, strategies for study design to best capture adaptive variation, and how to apply this information to practical challenges, such as restoration.

Incorporating evolutionary processes into population viability models.

We examined how ecological and evolutionary (eco-evo) processes in population dynamics could be better integrated into population viability analysis (PVA). Complementary advances in computation and population genomics can be combined into an eco-evo PVA to offer powerful new approaches to understand the influence of evolutionary processes on population persistence. We developed the mechanistic basis of an eco-evo PVA using individual-based models with individual-level genotype tracking and dynamic genotype-phenotype mapping to model emergent population-level effects, such as local adaptation and genetic rescue. We then outline how genomics can allow or improve parameter estimation for PVA models by providing genotypic information at large numbers of loci for neutral and functional genome regions. As climate change and other threatening processes increase in rate and scale, eco-evo PVAs will become essential research tools to evaluate the effects of adaptive potential, evolutionary rescue, and locally adapted traits on persistence.

Capturing Genetic Diversity in Seed Collections: An Empirical Study of Two Congeners with Contrasting Mating Systems.

Plant mating systems shape patterns of genetic diversity and impact the long-term success of populations. As such, they are relevant to the design of seed collections aiming to maximise genetic diversity (e.g., germplasm conservation, ecological restoration). However, for most species, little is known empirically about how variation in mating systems and genetic diversity is distributed. We investigated the relationship between genetic diversity and mating systems in two functionally similar, co-occurring species of Hakea (Proteaceae), and evaluated the extent to which genetic diversity was captured in seeds. We genotyped hundreds of seedlings and mother plants via DArTseq, and developed novel implementations of two approaches to inferring the mating system from SNP data. A striking contrast in patterns of genetic diversity between H. sericea and H. teretifolia was revealed, consistent with a contrast in their mating systems. While both species had mixed mating systems, H. sericea was found to be habitually selfing, while H. teretifolia more evenly employed both selfing and outcrossing. In both species, seed collection schemes maximised genetic diversity by increasing the number of maternal lines and sites sampled, but twice as many sites were needed for the selfing species to capture equivalent levels of genetic variation at a regional scale.

Protein material costs: single atoms can make an evolutionary difference.

The process of gene expression has material costs caused by the quantities of carbon, nitrogen, sulfur and phosphorus that are needed to make mRNAs and proteins. When any such chemical element is ecologically limiting, mutations increasing these costs can reduce growth. Here, we ask if such mutations are 'visible' to natural selection in the yeast Saccharomyces cerevisiae. We find that mutations causing small increases in expression and even single amino acid replacements can be subject to natural selection on the basis of their material costs.