Maintenance and expansion of genetic and trait variation following domestication in a clonal crop.

Clonal propagation enables favourable crop genotypes to be rapidly selected and multiplied. However, the absence of sexual propagation can lead to low genetic diversity and accumulation of deleterious mutations, which may eventually render crops less resilient to pathogens or environmental change. To better understand this trade-off, we characterize the domestication and contemporary genetic diversity of Enset (Ensete ventricosum), an indigenous African relative of bananas (Musa) and a principal starch staple for 20 million Ethiopians. Wild enset reproduction occurs strictly by sexual outcrossing, but for cultivation, it is propagated clonally and associated with diversification and specialization into hundreds of named landraces. We applied tGBS sequencing to generate genome-wide genotypes for 192 accessions from across enset's cultivated distribution, and surveyed 1340 farmers on enset agronomic traits. Overall, reduced heterozygosity in the domesticated lineage was consistent with a domestication bottleneck that retained 37% of wild diversity. However, an excess of putatively deleterious missense mutations at low frequency present as heterozygotes suggested an accumulation of mutational load in clonal domesticated lineages. Our evidence indicates that the major domesticated lineages initially arose through historic sexual recombination associated with a domestication bottleneck, followed by the amplification of favourable genotypes through an extended period of clonal propagation. Among domesticated lineages, we found a significant phylogenetic signal for multiple farmer-identified food, nutrition and disease resistance traits and little evidence of contemporary recombination. The development of future-climate adapted genotypes may require crop breeding, but outcrossing risks exposing deleterious alleles as homozygotes. This trade-off may partly explain the ubiquity and persistence of clonal propagation over recent centuries of comparative climate stability.

A New Chromosome-Assigned Mongolian Gerbil Genome Allows Characterization of Complete Centromeres and a Fully Heterochromatic Chromosome.

Chromosome-scale genome assemblies based on ultralong-read sequencing technologies are able to illuminate previously intractable aspects of genome biology such as fine-scale centromere structure and large-scale variation in genome features such as heterochromatin, GC content, recombination rate, and gene content. We present here a new chromosome-scale genome of the Mongolian gerbil (Meriones unguiculatus), which includes the complete sequence of all centromeres. Gerbils are thus the one of the first vertebrates to have their centromeres completely sequenced. Gerbil centromeres are composed of four different repeats of length 6, 37, 127, or 1,747 bp, which occur in simple alternating arrays and span 1-6 Mb. Gerbil genomes have both an extensive set of GC-rich genes and chromosomes strikingly enriched for constitutive heterochromatin. We sought to determine if there was a link between these two phenomena and found that the two heterochromatic chromosomes of the Mongolian gerbil have distinct underpinnings: Chromosome 5 has a large block of intraarm heterochromatin as the result of a massive expansion of centromeric repeats, while chromosome 13 is comprised of extremely large (>150 kb) repeated sequences. In addition to characterizing centromeres, our results demonstrate the importance of including karyotypic features such as chromosome number and the locations of centromeres in the interpretation of genome sequence data and highlight novel patterns involved in the evolution of chromosomes.

Impact of Phylogenetic Tree Completeness and Mis-specification of Sampling Fractions on Trait Dependent Diversification Models.

Understanding the origins of diversity and the factors that drive some clades to be more diverse than others are important issues in evolutionary biology. Sophisticated SSE (state-dependent speciation and extinction) models provide insights into the association between diversification rates and the evolution of a trait. The empirical data used in SSE models and other methods is normally imperfect, yet little is known about how this can affect these models. Here, we evaluate the impact of common phylogenetic issues on inferences drawn from SSE models. Using simulated phylogenetic trees and trait information, we fitted SSE models to determine the effects of sampling fraction (phylogenetic tree completeness) and sampling fraction mis-specification on model selection and parameter estimation (speciation, extinction, and transition rates) under two sampling regimes (random and taxonomically biased). As expected, we found that both model selection and parameter estimate accuracies are reduced at lower sampling fractions (i.e., low tree completeness). Furthermore, when sampling of the tree is imbalanced across sub-clades and tree completeness is ≤ 60%, rates of false positives increase and parameter estimates are less accurate, compared to when sampling is random. Thus, when applying SSE methods to empirical datasets, there are increased risks of false inferences of trait dependent diversification when some sub-clades are heavily under-sampled. Mis-specifying the sampling fraction severely affected the accuracy of parameter estimates: parameter values were over-estimated when the sampling fraction was specified as lower than its true value, and under-estimated when the sampling fraction was specified as higher than its true value. Our results suggest that it is better to cautiously under-estimate sampling efforts, as false positives increased when the sampling fraction was over-estimated. We encourage SSE studies where the sampling fraction can be reasonably estimated and provide recommended best practices for SSE modeling. [Trait dependent diversification; SSE models; phylogenetic tree completeness; sampling fraction.].

Genetic assimilation of ancestral plasticity during parallel adaptation to zinc contamination in Silene uniflora.

Phenotypic plasticity in ancestral populations is hypothesized to facilitate adaptation, but evidence is piecemeal and often contradictory. Further, whether ancestral plasticity increases the probability of parallel adaptive changes has not been explored. The most general finding is that ancestral responses to a new environment are reversed following adaptation (known as reversion). We investigated the contribution of ancestral plasticity to adaptive evolution of gene expression in two independently evolved lineages of zinc-tolerant Silene uniflora. We found that the general pattern of reversion is driven by the absence of a widespread stress response in zinc-adapted plants compared with zinc-sensitive plants. We show that ancestral plasticity that moves expression closer to the optimum value in the new environment influences the evolution of gene expression among genes that are likely to be involved in adaptation and increases the chance that genes are recruited repeatedly during adaptation. However, despite convergence in gene expression levels between independently adapted lineages, ancestral plasticity does not influence how similar expression values of adaptive genes become. Surprisingly, we also observed that ancestral plasticity that increases fitness often becomes genetically determined and fixed, that is, genetically assimilated. These results emphasize the important role of ancestral plasticity in parallel adaptation.

Mapping the root systems of individual trees in a natural community using genotyping-by-sequencing.

The architecture of root systems is an important driver of plant fitness, competition and ecosystem processes. However, the methodological difficulty of mapping roots hampers the study of these processes. Existing approaches to match individual plants to belowground samples are low throughput and species specific. Here, we developed a scalable sequencing-based method to map the root systems of individual trees across multiple species. We successfully applied it to a tropical dry forest community in the Brazilian Caatinga containing 14 species. We sequenced all 42 individual shrubs and trees in a 14 × 14 m plot using double-digest restriction site-associated sequencing (ddRADseq). We identified species-specific markers and individual-specific haplotypes from the data. We matched these markers to the ddRADseq data from 100 mixed root samples from across the centre (10 × 10 m) of the plot at four different depths using a newly developed R package. We identified individual root samples for all species and all but one individual. There was a strong significant correlation between belowground and aboveground size measurements, and we also detected significant species-level root-depth preference for two species. The method is more scalable and less labour intensive than the current techniques and is broadly applicable to ecology, forestry and agricultural biology.

Madagascar's extraordinary biodiversity: Evolution, distribution, and use.

Madagascar's biota is hyperdiverse and includes exceptional levels of endemicity. We review the current state of knowledge on Madagascar's past and current terrestrial and freshwater biodiversity by compiling and presenting comprehensive data on species diversity, endemism, and rates of species description and human uses, in addition to presenting an updated and simplified map of vegetation types. We report a substantial increase of records and species new to science in recent years; however, the diversity and evolution of many groups remain practically unknown (e.g., fungi and most invertebrates). Digitization efforts are increasing the resolution of species richness patterns and we highlight the crucial role of field- and collections-based research for advancing biodiversity knowledge and identifying gaps in our understanding, particularly as species richness corresponds closely to collection effort. Phylogenetic diversity patterns mirror that of species richness and endemism in most of the analyzed groups. We highlight humid forests as centers of diversity and endemism because of their role as refugia and centers of recent and rapid radiations. However, the distinct endemism of other areas, such as the grassland-woodland mosaic of the Central Highlands and the spiny forest of the southwest, is also biologically important despite lower species richness. The documented uses of Malagasy biodiversity are manifold, with much potential for the uncovering of new useful traits for food, medicine, and climate mitigation. The data presented here showcase Madagascar as a unique "living laboratory" for our understanding of evolution and the complex interactions between people and nature. The gathering and analysis of biodiversity data must continue and accelerate if we are to fully understand and safeguard this unique subset of Earth's biodiversity.

Madagascar's extraordinary biodiversity: Threats and opportunities.

Madagascar's unique biota is heavily affected by human activity and is under intense threat. Here, we review the current state of knowledge on the conservation status of Madagascar's terrestrial and freshwater biodiversity by presenting data and analyses on documented and predicted species-level conservation statuses, the most prevalent and relevant threats, ex situ collections and programs, and the coverage and comprehensiveness of protected areas. The existing terrestrial protected area network in Madagascar covers 10.4% of its land area and includes at least part of the range of the majority of described native species of vertebrates with known distributions (97.1% of freshwater fishes, amphibians, reptiles, birds, and mammals combined) and plants (67.7%). The overall figures are higher for threatened species (97.7% of threatened vertebrates and 79.6% of threatened plants occurring within at least one protected area). International Union for Conservation of Nature (IUCN) Red List assessments and Bayesian neural network analyses for plants identify overexploitation of biological resources and unsustainable agriculture as the most prominent threats to biodiversity. We highlight five opportunities for action at multiple levels to ensure that conservation and ecological restoration objectives, programs, and activities take account of complex underlying and interacting factors and produce tangible benefits for the biodiversity and people of Madagascar.

Hybridisation and chloroplast capture between distinct Themeda triandra lineages in Australia.

Ecotypes are distinct populations within a species that are adapted to specific environmental conditions. Understanding how these ecotypes become established, and how they interact when reunited, is fundamental to elucidating how ecological adaptations are maintained. This study focuses on Themeda triandra, a dominant grassland species across Asia, Africa and Australia. It is the most widespread plant in Australia, where it has distinct ecotypes that are usually restricted to either wetter and cooler coastal regions or the drier and hotter interior. We generate a reference genome for T. triandra and use whole genome sequencing for over 80 Themeda accessions to reconstruct the evolutionary history of T. triandra and related taxa. Organelle phylogenies confirm that Australia was colonized by T. triandra twice, with the division between ecotypes predating their arrival in Australia. The nuclear genome provides evidence of differences in the dominant ploidal level and gene-flow among the ecotypes. In northern Queensland there appears to be a hybrid zone between ecotypes with admixed nuclear genomes and shared chloroplast haplotypes. Conversely, in the cracking claypans of Western Australia, there is cytonuclear discordance with individuals possessing the coastal chloroplast and interior clade nuclear genome. This chloroplast capture is potentially a result of adaptive introgression, with selection detected in the rpoC2 gene which is associated with water use efficiency. The reason that T. triandra is the most widespread plant in Australia appears to be a result of distinct ecotypic genetic variation and genome duplication, with the importance of each depending on the geographic scale considered.

The demographic history of Madagascan micro-endemics: have rare species always been rare?

Extinction has increased as human activities impact ecosystems, yet relatively few species have conservation assessments. Novel approaches are needed to highlight threatened species that are currently data-deficient. Many Madagascan plant species have extremely narrow ranges, but this may not have always been the case-it is unclear how the island's diverse flora evolved. To assess this, we generated restriction-site associated DNA sequence data for 10 Madagascan plant species, estimated effective population size (Ne) for each species and compared this to census (Nc) sizes. In each case, Ne was an order of magnitude larger than Nc-signifying rapid, recent population decline. We then estimated species' demographic history, tracking changes in Ne over time. We show that it is possible to predict extinction risk, particularly in the most threatened species. Furthermore, simulations showed that our approach has the power to detect population decline during the Anthropocene. Our analyses reveal that Madagascar's micro-endemics were not always rare, having experienced a rapid decline in their recent history. This casts further uncertainty over the processes that generated Madagascar's exceptional biodiversity. Our approach targets data-deficient species in need of conservation assessment, particularly in regions where human modification of the environment has been rapid.

Molecular Clocks and Archeogenomics of a Late Period Egyptian Date Palm Leaf Reveal Introgression from Wild Relatives and Add Timestamps on the Domestication.

The date palm, Phoenix dactylifera, has been a cornerstone of Middle Eastern and North African agriculture for millennia. It was first domesticated in the Persian Gulf, and its evolution appears to have been influenced by gene flow from two wild relatives, P. theophrasti, currently restricted to Crete and Turkey, and P. sylvestris, widespread from Bangladesh to the West Himalayas. Genomes of ancient date palm seeds show that gene flow from P. theophrasti to P. dactylifera may have occurred by ∼2,200 years ago, but traces of P. sylvestris could not be detected. We here integrate archeogenomics of a ∼2,100-year-old P. dactylifera leaf from Saqqara (Egypt), molecular-clock dating, and coalescence approaches with population genomic tests, to probe the hybridization between the date palm and its two closest relatives and provide minimum and maximum timestamps for its reticulated evolution. The Saqqara date palm shares a close genetic affinity with North African date palm populations, and we find clear genomic admixture from both P. theophrasti, and P. sylvestris, indicating that both had contributed to the date palm genome by 2,100 years ago. Molecular-clocks placed the divergence of P. theophrasti from P. dactylifera/P. sylvestris and that of P. dactylifera from P. sylvestris in the Upper Miocene, but strongly supported, conflicting topologies point to older gene flow between P. theophrasti and P. dactylifera, and P. sylvestris and P. dactylifera. Our work highlights the ancient hybrid origin of the date palms, and prompts the investigation of the functional significance of genetic material introgressed from both close relatives, which in turn could prove useful for modern date palm breeding.

Rapid Parallel Adaptation to Anthropogenic Heavy Metal Pollution.

The impact of human-mediated environmental change on the evolutionary trajectories of wild organisms is poorly understood. In particular, capacity of species to adapt rapidly (in hundreds of generations or less), reproducibly and predictably to extreme environmental change is unclear. Silene uniflora is predominantly a coastal species, but it has also colonized isolated, disused mines with phytotoxic, zinc-contaminated soils. To test whether rapid, parallel adaptation to anthropogenic pollution has taken place, we used reduced representation sequencing (ddRAD) to reconstruct the evolutionary history of geographically proximate mine and coastal population pairs and found largely independent colonization of mines from different coastal sites. Furthermore, our results show that parallel evolution of zinc tolerance has occurred without gene flow spreading adaptive alleles between mine populations. In genomic regions where signatures of selection were detected across multiple mine-coast pairs, we identified genes with functions linked to physiological differences between the putative ecotypes, although genetic differentiation at specific loci is only partially shared between mine populations. Our results are consistent with a complex, polygenic genetic architecture underpinning rapid adaptation. This shows that even under a scenario of strong selection and rapid adaptation, evolutionary responses to human activities (and other environmental challenges) may be idiosyncratic at the genetic level and, therefore, difficult to predict from genomic data.

Inference of natural selection from ancient DNA.

Evolutionary processes, including selection, can be indirectly inferred based on patterns of genomic variation among contemporary populations or species. However, this often requires unrealistic assumptions of ancestral demography and selective regimes. Sequencing ancient DNA from temporally spaced samples can inform about past selection processes, as time series data allow direct quantification of population parameters collected before, during, and after genetic changes driven by selection. In this Comment and Opinion, we advocate for the inclusion of temporal sampling and the generation of paleogenomic datasets in evolutionary biology, and highlight some of the recent advances that have yet to be broadly applied by evolutionary biologists. In doing so, we consider the expected signatures of balancing, purifying, and positive selection in time series data, and detail how this can advance our understanding of the chronology and tempo of genomic change driven by selection. However, we also recognize the limitations of such data, which can suffer from postmortem damage, fragmentation, low coverage, and typically low sample size. We therefore highlight the many assumptions and considerations associated with analyzing paleogenomic data and the assumptions associated with analytical methods.

Strong divergent selection at multiple loci in two closely related species of ragworts adapted to high and low elevations on Mount Etna.

Recently diverged species present particularly informative systems for studying speciation and maintenance of genetic divergence in the face of gene flow. We investigated speciation in two closely related Senecio species, S. aethnensis and S. chrysanthemifolius, which grow at high and low elevations, respectively, on Mount Etna, Sicily and form a hybrid zone at intermediate elevations. We used a newly generated genome-wide single nucleotide polymorphism (SNP) dataset from 192 individuals collected over 18 localities along an elevational gradient to reconstruct the likely history of speciation, identify highly differentiated SNPs, and estimate the strength of divergent selection. We found that speciation in this system involved heterogeneous and bidirectional gene flow along the genome, and species experienced marked population size changes in the past. Furthermore, we identified highly-differentiated SNPs between the species, some of which are located in genes potentially involved in ecological differences between species (such as photosynthesis and UV response). We analysed the shape of these SNPs' allele frequency clines along the elevational gradient. These clines show significantly variable coincidence and concordance, indicative of the presence of multifarious selective forces. Selection against hybrids is estimated to be very strong (0.16-0.78) and one of the highest reported in literature. The combination of strong cumulative selection across the genome and previously identified intrinsic incompatibilities probably work together to maintain the genetic and phenotypic differentiation between these species - pointing to the importance of considering both intrinsic and extrinsic factors when studying divergence and speciation.

Ecological speciation in sympatric palms: 3. Genetic map reveals genomic islands underlying species divergence in Howea.

Although it is now widely accepted that speciation can occur in the face of continuous gene flow, with little or no spatial separation, the mechanisms and genomic architectures that permit such divergence are still debated. Here, we examined speciation in the face of gene flow in the Howea palms of Lord Howe Island, Australia. We built a genetic map using a novel method applicable to long-lived tree species, combining it with double digest restriction site-associated DNA sequencing of multiple individuals. Based upon various metrics, we detected 46 highly differentiated regions throughout the genome, four of which contained genes with functions that are particularly relevant to the speciation scenario for Howea, specifically salt and drought tolerance.

Ecological speciation in sympatric palms: 4. Demographic analyses support speciation of Howea in the face of high gene flow.

The idea that populations must be geographically isolated (allopatric) to evolve into separate species has persisted for a long time. It is now clear that new species can also diverge despite ongoing genetic exchange, but few accepted cases of speciation in sympatry have held up when scrutinized using modern approaches. Here, we examined evidence for speciation of the Howea palms of Lord Howe Island, Australia, in light of new genomic data. We used coalescence-based demographic models combined with double digest restriction site associated DNA sequencing of multiple individuals and provide support for previous claims by Savolainen et al. that speciation in Howea did occur in the face of gene flow.

Evolutionary genetics of immunological supertypes reveals two faces of the Red Queen.

Red Queen host-parasite co-evolution can drive adaptations of immune genes by positive selection that erodes genetic variation (Red Queen arms race) or results in a balanced polymorphism (Red Queen dynamics) and long-term preservation of genetic variation (trans-species polymorphism). These two Red Queen processes are opposite extremes of the co-evolutionary spectrum. Here we show that both Red Queen processes can operate simultaneously by analysing the major histocompatibility complex (MHC) in guppies (Poecilia reticulata and P. obscura) and swamp guppies (Micropoecilia picta). Sub-functionalisation of MHC alleles into 'supertypes' explains how polymorphisms persist during rapid host-parasite co-evolution. Simulations show the maintenance of supertypes as balanced polymorphisms, consistent with Red Queen dynamics, whereas alleles within supertypes are subject to positive selection in a Red Queen arms race. Building on the divergent allele advantage hypothesis, we show that functional aspects of allelic diversity help to elucidate the evolution of polymorphic genes involved in Red Queen co-evolution.

Field-based species identification of closely-related plants using real-time nanopore sequencing.

Advances in DNA sequencing and informatics have revolutionised biology over the past four decades, but technological limitations have left many applications unexplored. Recently, portable, real-time, nanopore sequencing (RTnS) has become available. This offers opportunities to rapidly collect and analyse genomic data anywhere. However, generation of datasets from large, complex genomes has been constrained to laboratories. The portability and long DNA sequences of RTnS offer great potential for field-based species identification, but the feasibility and accuracy of these technologies for this purpose have not been assessed. Here, we show that a field-based RTnS analysis of closely-related plant species (Arabidopsis spp.) has many advantages over laboratory-based high-throughput sequencing (HTS) methods for species level identification and phylogenomics. Samples were collected and sequenced in a single day by RTnS using a portable, "al fresco" laboratory. Our analyses demonstrate that correctly identifying unknown reads from matches to a reference database with RTnS reads enables rapid and confident species identification. Individually annotated RTnS reads can be used to infer the evolutionary relationships of A. thaliana. Furthermore, hybrid genome assembly with RTnS and HTS reads substantially improved upon a genome assembled from HTS reads alone. Field-based RTnS makes real-time, rapid specimen identification and genome wide analyses possible.

Seed size and its rate of evolution correlate with species diversification across angiosperms.

Species diversity varies greatly across the different taxonomic groups that comprise the Tree of Life (ToL). This imbalance is particularly conspicuous within angiosperms, but is largely unexplained. Seed mass is one trait that may help clarify why some lineages diversify more than others because it confers adaptation to different environments, which can subsequently influence speciation and extinction. The rate at which seed mass changes across the angiosperm phylogeny may also be linked to diversification by increasing reproductive isolation and allowing access to novel ecological niches. However, the magnitude and direction of the association between seed mass and diversification has not been assessed across the angiosperm phylogeny. Here, we show that absolute seed size and the rate of change in seed size are both associated with variation in diversification rates. Based on the largest available angiosperm phylogenetic tree, we found that smaller-seeded plants had higher rates of diversification, possibly due to improved colonisation potential. The rate of phenotypic change in seed size was also strongly positively correlated with speciation rates, providing rare, large-scale evidence that rapid morphological change is associated with species divergence. Our study now reveals that variation in morphological traits and, importantly, the rate at which they evolve can contribute to explaining the extremely uneven distribution of diversity across the ToL.

Viviparity stimulates diversification in an order of fish.

Species richness is distributed unevenly across the tree of life and this may be influenced by the evolution of novel phenotypes that promote diversification. Viviparity has originated ∼150 times in vertebrates and is considered to be an adaptation to highly variable environments. Likewise, possessing an annual life cycle is common in plants and insects, where it enables the colonization of seasonal environments, but rare in vertebrates. The extent to which these reproductive life-history traits have enhanced diversification and their relative importance in the process remains unknown. We show that convergent evolution of viviparity causes bursts of diversification in fish. We built a phylogenetic tree for Cyprinodontiformes, an order in which both annualism and viviparity have arisen, and reveal that while both traits have evolved multiple times, only viviparity played a major role in shaping the patterns of diversity. These results demonstrate that changes in reproductive life-history strategy can stimulate diversification.

Demographic history of speciation in a Senecio altitudinal hybrid zone on Mt. Etna.

Hybrid zones typically form as a result of species coming into secondary contact, but can also be established in situ as an ecotonal hybrid zone, a situation which has been reported far less frequently. An altitudinal hybrid zone on Mount Etna between two ragwort species (the low elevation Senecio chrysanthemifolius and high elevation S. aethnensis) could potentially represent either of these possibilities. However, a scenario of secondary contact vs. speciation with gene flow has not been explicitly tested. Here, we test these alternatives and demonstrate that the data do not support secondary contact. Furthermore, we report that the previous analyses of speciation history of these species were based on admixed populations, which has led to inflated estimates of ongoing, interspecific gene flow. Our new analyses, based on 'pure' S. aethnensis and S. chrysanthemifolius populations, reveal gene exchange of less than one effective migrant per generation, a level low enough to allow the species to accumulate neutral, genomewide differences. Overall, our results are consistent with a scenario of speciation with gene flow and a divergence time which coincides with the rise of Mt. Etna to altitudes above 2000 m (~150 KY). Further work to quantify the role of adaptation to contrasting environments of high and low altitudes will be needed to support the scenario of recent ecological speciation in this system.