pipesnake: generalized software for the assembly and analysis of phylogenomic datasets from conserved genomic loci.

MOTIVATION: Phylogenetics has moved into the era of genomics, incorporating enormous volumes of data to study questions at both shallow and deep scales. With this increase in information, phylogeneticists need new tools and skills to manipulate and analyze these data. To facilitate these tasks and encourage reproducibility, the community is increasingly moving toward automated workflows. RESULTS: Here we present pipesnake, a phylogenomics pipeline written in Nextflow for the processing, assembly, and phylogenetic estimation of genomic data from short-read sequences. pipesnake is an easy to use and efficient software package designed for this next era in phylogenetics. AVAILABILITY AND IMPLEMENTATION: pipesnake is publicly available on GitHub at https://github.com/AusARG/pipesnake and accompanied by documentation and a wiki/tutorial.

No link between population isolation and speciation rate in squamate reptiles.

Rates of species formation vary widely across the tree of life and contribute to massive disparities in species richness among clades. This variation can emerge from differences in metapopulation-level processes that affect the rates at which lineages diverge, persist, and evolve reproductive barriers and ecological differentiation. For example, populations that evolve reproductive barriers quickly should form new species at faster rates than populations that acquire reproductive barriers more slowly. This expectation implicitly links microevolutionary processes (the evolution of populations) and macroevolutionary patterns (the profound disparity in speciation rate across taxa). Here, leveraging extensive field sampling from the Neotropical Cerrado biome in a biogeographically controlled natural experiment, we test the role of an important microevolutionary process-the propensity for population isolation-as a control on speciation rate in lizards and snakes. By quantifying population genomic structure across a set of codistributed taxa with extensive and phylogenetically independent variation in speciation rate, we show that broad-scale patterns of species formation are decoupled from demographic and genetic processes that promote the formation of population isolates. Population isolation is likely a critical stage of speciation for many taxa, but our results suggest that interspecific variability in the propensity for isolation has little influence on speciation rates. These results suggest that other stages of speciation-including the rate at which reproductive barriers evolve and the extent to which newly formed populations persist-are likely to play a larger role than population isolation in controlling speciation rate variation in squamates.

A return-on-investment approach for prioritization of rigorous taxonomic research needed to inform responses to the biodiversity crisis.

Global biodiversity loss is a profound consequence of human activity. Disturbingly, biodiversity loss is greater than realized because of the unknown number of undocumented species. Conservation fundamentally relies on taxonomic recognition of species, but only a fraction of biodiversity is described. Here, we provide a new quantitative approach for prioritizing rigorous taxonomic research for conservation. We implement this approach in a highly diverse vertebrate group-Australian lizards and snakes. Of 870 species assessed, we identified 282 (32.4%) with taxonomic uncertainty, of which 17.6% likely comprise undescribed species of conservation concern. We identify 24 species in need of immediate taxonomic attention to facilitate conservation. Using a broadly applicable return-on-investment framework, we demonstrate the importance of prioritizing the fundamental work of identifying species before they are lost.

Repeated patterns of reptile diversification in Western North America supported by the Northern Alligator Lizard (Elgaria coerulea).

Understanding the processes that shape genetic diversity by either promoting or preventing population divergence can help identify geographic areas that either facilitate or limit gene flow. Furthermore, broadly distributed species allow us to understand how biogeographic and ecogeographic transitions affect gene flow. We investigated these processes using genomic data in the Northern Alligator Lizard (Elgaria coerulea), which is widely distributed in Western North America across diverse ecoregions (California Floristic Province and Pacific Northwest) and mountain ranges (Sierra Nevada, Coastal Ranges, and Cascades). We collected single-nucleotide polymorphism data from 120 samples of E. coerulea. Biogeographic analyses of squamate reptiles with similar distributions have identified several shared diversification patterns that provide testable predictions for E. coerulea, including deep genetic divisions in the Sierra Nevada, demographic stability of southern populations, and recent post-Pleistocene expansion into the Pacific Northwest. We use genomic data to test these predictions by estimating the structure, connectivity, and phylogenetic history of populations. At least 10 distinct populations are supported, with mixed-ancestry individuals situated at most population boundaries. A species tree analysis provides strong support for the early divergence of populations in the Sierra Nevada Mountains and recent diversification into the Pacific Northwest. Admixture and migration analyses detect gene flow among populations in the Lower Cascades and Northern California, and a spatial analysis of gene flow identified significant barriers to gene flow across both the Sierra Nevada and Coast Ranges. The distribution of genetic diversity in E. coerulea is uneven, patchy, and interconnected at population boundaries. The biogeographic patterns seen in E. coerulea are consistent with predictions from co-distributed species.

Notes from the taxonomic disaster zone: Evolutionary drivers of intractable species boundaries in an Australian lizard clade (Scincidae: Ctenotus).

Genomic-scale datasets, sophisticated analytical techniques, and conceptual advances have disproportionately failed to resolve species boundaries in some groups relative to others. To understand the processes that underlie taxonomic intractability, we dissect the speciation history of an Australian lizard clade that arguably represents a "worst-case" scenario for species delimitation within vertebrates: the Ctenotus inornatus species group, a clade beset with decoupled genetic and phenotypic breaks, uncertain geographic ranges, and parallelism in purportedly diagnostic morphological characters. We sampled hundreds of localities to generate a genomic perspective on population divergence, structure, and admixture. Our results revealed rampant paraphyly of nominate taxa in the group, with lineages that are either morphologically cryptic or polytypic. Isolation-by-distance patterns reflect spatially continuous differentiation among certain pairs of putative species, yet genetic and geographic distances are decoupled in other pairs. Comparisons of mitochondrial and nuclear gene trees, tests of nuclear introgression, and historical demographic modelling identified gene flow between divergent candidate species. Levels of admixture are decoupled from phylogenetic relatedness; gene flow is often higher between sympatric species than between parapatric populations of the same species. Such idiosyncratic patterns of introgression contribute to species boundaries that are fuzzy while also varying in fuzziness. Our results suggest that "taxonomic disaster zones" like the C. inornatus species group result from spatial variation in the porosity of species boundaries and the resulting patterns of genetic and phenotypic variation. This study raises questions about the origin and persistence of hybridizing species and highlights the unique insights provided by taxa that have long eluded straightforward taxonomic categorization.

Natural selection maintains species despite frequent hybridization in the desert shrub Encelia.

Natural selection is an important driver of genetic and phenotypic differentiation between species. For species in which potential gene flow is high but realized gene flow is low, adaptation via natural selection may be a particularly important force maintaining species. For a recent radiation of New World desert shrubs (Encelia: Asteraceae), we use fine-scale geographic sampling and population genomics to determine patterns of gene flow across two hybrid zones formed between two independent pairs of species with parapatric distributions. After finding evidence for extremely strong selection at both hybrid zones, we use a combination of field experiments, high-resolution imaging, and physiological measurements to determine the ecological basis for selection at one of the hybrid zones. Our results identify multiple ecological mechanisms of selection (drought, salinity, herbivory, and burial) that together are sufficient to maintain species boundaries despite high rates of hybridization. Given that multiple pairs of Encelia species hybridize at ecologically divergent parapatric boundaries, such mechanisms may maintain species boundaries throughout Encelia.

Genetic and Ecogeographic Controls on Species Cohesion in Australia's Most Diverse Lizard Radiation.

AbstractSpecies vary extensively in geographic range size and climatic niche breadth. If range limits are primarily determined by climatic factors, species with broad climatic tolerances and those that track geographically widespread climates should have large ranges. However, large ranges might increase the probability of population fragmentation and adaptive divergence, potentially decoupling climatic niche breadth and range size. Conversely, ecological generalism in large-ranged species might lead to higher gene flow across climatic transitions, increasing species' cohesion and thus decreasing genetic isolation by distance (IBD). Focusing on Australia's iconic Ctenotus lizard radiation, we ask whether species range size scales with climatic niche breadth and the degree of population isolation. To this end, we infer independently evolving operational taxonomic units (OTUs), their geographic and climatic ranges, and the strength of IBD within OTUs based on genome-wide loci from 722 individuals spanning 75 taxa. Large-ranged OTUs were common and had broader climatic niches than small-ranged OTUs; thus, large ranges do not appear to simply result from passive tracking of widespread climatic zones. OTUs with larger ranges and broader climatic niches showed relatively weaker IBD, suggesting that large-ranged species might possess intrinsic attributes that facilitate genetic cohesion across large distances and varied climates. By influencing population divergence and persistence, traits that affect species cohesion may play a central role in large-scale patterns of diversification and species richness.

A lizard with two tales: What diversification within Sceloporus occidentalis teaches us about species formation.

In 1859, Charles Darwin proposed that species are not fundamentally different from subspecies or the varieties from which they evolve. A century later, Dobzhansky (1958) suggested that many such lineages are ephemeral and are likely to reverse differentiation through introgression (Figure 1a); only a few evolve complete reproductive isolation and persist in sympatry. In this issue of Molecular Ecology, Bouzid et al. (2021) showed how new analytical methods, when applied to genomic data, allow us to more precisely determine whether or not species formation follows the paths outlined by Darwin and Dobzhansky (Figure 1b). The authors studied the diversification of the lizard Sceloporus occidentalis, finding a continuum of genetic interactions between the preservation of genetic identity to genetic merger, analogous to what is exemplified by ring species. In doing so, they teach us two tales about species formation: that lineages are fractal byproducts of evolutionary processes such as genetic drift and selection, and that lineages are often ephemeral and do not always progress into fully reproductively isolated taxa. Studying ephemeral lineages like those in S. occidentalis allows us to capture divergence at its earliest stages, and potentially to determine the factors that allow lineages to remain distinct despite pervasive gene flow. These lineages thus serve as a natural laboratory to address long standing hypotheses about species formation.

Genetic variability and the ecology of geographic range: A test of the central-marginal hypothesis in Australian scincid lizards.

For many species, both local abundance and regional occupancy are highest near the centre of their geographic distributions. One hypothesis for this pattern is that niche suitability declines with increasing distance from a species geographic centre, such that populations near range margins are characterized by reduced density and increased patchiness. In these smaller edge populations, genetic drift is more powerful, leading to the loss of genetic diversity. This simple verbal model has been formalized as the central-marginal hypothesis, which predicts that core populations should have greater genetic diversity than edge populations. Here, we tested the central-marginal hypothesis using a genomic data set of 25 species-level taxa of Australian scincid lizards in the genera Ctenotus and Lerista. A majority of taxa in our data set showed range-wide patterns of genetic variation consistent with central-marginal hypothesis, and eight of 25 taxa showed significantly greater genetic diversity in the centre of their range. We then explored biological, historical, and methodological factors that might predict which taxa support the central-marginal hypothesis. We found that taxa with the strongest evidence for range expansion were the least likely to follow predictions of the central-marginal hypothesis. The majority of these taxa had range expansions that originated at the range edge, which led to a gradient of decreasing genetic diversity from the range edge to the core, contrary to the central-marginal hypothesis.

Congruence and Conflict in the Higher-Level Phylogenetics of Squamate Reptiles: An Expanded Phylogenomic Perspective.

Genome-scale data have the potential to clarify phylogenetic relationships across the tree of life but have also revealed extensive gene tree conflict. This seeming paradox, whereby larger data sets both increase statistical confidence and uncover significant discordance, suggests that understanding sources of conflict is important for accurate reconstruction of evolutionary history. We explore this paradox in squamate reptiles, the vertebrate clade comprising lizards, snakes, and amphisbaenians. We collected an average of 5103 loci for 91 species of squamates that span higher-level diversity within the clade, which we augmented with publicly available sequences for an additional 17 taxa. Using a locus-by-locus approach, we evaluated support for alternative topologies at 17 contentious nodes in the phylogeny. We identified shared properties of conflicting loci, finding that rate and compositional heterogeneity drives discordance between gene trees and species tree and that conflicting loci rarely overlap across contentious nodes. Finally, by comparing our tests of nodal conflict to previous phylogenomic studies, we confidently resolve 9 of the 17 problematic nodes. We suggest this locus-by-locus and node-by-node approach can build consensus on which topological resolutions remain uncertain in phylogenomic studies of other contentious groups. [Anchored hybrid enrichment (AHE); gene tree conflict; molecular evolution; phylogenomic concordance; target capture; ultraconserved elements (UCE).].

Temporal genomic contrasts reveal rapid evolutionary responses in an alpine mammal during recent climate change.

Many species have experienced dramatic changes in their abundance and distribution during recent climate change, but it is often unclear whether such ecological responses are accompanied by evolutionary change. We used targeted exon sequencing of 294 museum specimens (160 historic, 134 modern) to generate independent temporal genomic contrasts spanning a century of climate change (1911-2012) for two co-distributed chipmunk species: an endemic alpine specialist (Tamias alpinus) undergoing severe range contraction and a stable mid-elevation species (T. speciosus). Using a novel analytical approach, we reconstructed the demographic histories of these populations and tested for evidence of recent positive directional selection. Only the retracting species showed substantial population genetic fragmentation through time and this was coupled with positive selection and substantial shifts in allele frequencies at a gene, Alox15, involved in regulation of inflammation and response to hypoxia. However, these rapid population and gene-level responses were not detected in an analogous temporal contrast from another area where T. alpinus has also undergone severe range contraction. Collectively, these results highlight that evolutionary responses may be variable and context dependent across populations, even when they show seemingly synchronous ecological shifts. Our results demonstrate that temporal genomic contrasts can be used to detect very recent evolutionary responses within and among contemporary populations, even in the face of complex demographic changes. Given the wealth of specimens archived in natural history museums, comparative analyses of temporal population genomic data have the potential to improve our understanding of recent and ongoing evolutionary responses to rapidly changing environments.

2-(Anthracen-9-yl)benzothiazole-modified graphene oxide-nickel ferrite nanocomposite for anodic stripping voltammetric detection of heavy metal ions.

A novel electrochemical sensor, 2-(anthracen-9-yl)benzothiazole (ABT)-modified nickel ferrite reduced graphene oxide (NF@rGO) has been designed for the individual and simultaneous detection of Cd2+, Cu2+, and Hg2+ ions. Herein, NF@rGO nanocomposite, synthesized by a simple hydrothermal methodology, was hooked to ABT under easy and simple stirring conditions. Chelation of active functional groups of ABT with metal ions was augmented with higher adsorption and conductivity provided by NF@rGO. The created synergy resulted in analytical signals via selective oxidation of the ions within a potential ranging from - 1.2 to + 1.2 V vs sat. KCl. The proposed protocol exhibited a wide linear range from 0.05 to 1250 nM with excellent detection limit of 123, 54.1, and 86.6 pM via anodic stripping voltammetry for the simultaneous determination of Cd2+, Cu2+, and Hg2+ ions, respectively. Simple cost-effective synthetic approach, improved sensitivity with high selectivity, noteworthy repeatability (RSD less than 3%), and reproducibility (RSD less than 7%) equipped with successful real time monitoring (apparent recovery more than 90%) bring about a spiffing sensing platform for the detection of hazardous metal ions.

Diversification, disparification and hybridization in the desert shrubs Encelia.

There are multiple hypotheses for the spectacular plant diversity found in deserts. We explore how different factors, including the roles of ecological opportunity and selection, promote diversification and disparification in Encelia, a lineage of woody plants in the deserts of the Americas. Using a nearly complete species-level phylogeny based on double-digest restriction-aided sequencing along with a broad set of phenotypic traits, we estimate divergence times and diversification rates, identify instances of hybridization, quantify trait disparity and assess phenotypic divergence across environmental gradients. We show that Encelia originated and diversified recently (mid-Pleistocene) and rapidly, with rates comparable to notable adaptive radiations in plants. Encelia probably originated in the hot deserts of North America, with subsequent diversification across steep environmental gradients. We uncover multiple instances of gene flow between species. The radiation of Encelia is characterized by fast rates of phenotypic evolution, trait lability and extreme disparity across environments and between species pairs with overlapping geographic ranges. Encelia exemplifies how interspecific gene flow in combination with high trait lability can enable exceptionally fast diversification and disparification across steep environmental gradients.

Dispersal Predicts Hybrid Zone Widths across Animal Diversity: Implications for Species Borders under Incomplete Reproductive Isolation.

Hybrid zones occur as range boundaries for many animal taxa. One model for how hybrid zones form and stabilize is the tension zone model, a version of which predicts that hybrid zone widths are determined by a balance between random dispersal into hybrid zones and selection against hybrids. Here, we examine whether random dispersal and proxies for selection against hybrids (genetic distances between hybridizing pairs) can explain variation in hybrid zone widths across 131 hybridizing pairs of animals. We show that these factors alone can explain ∼40% of the variation in zone width among animal hybrid zones, with dispersal explaining far more of the variation than genetic distances. Patterns within clades were idiosyncratic. Genetic distances predicted hybrid zone widths particularly well for reptiles, while this relationship was opposite tension zone predictions in birds. Last, the data suggest that dispersal and molecular divergence set lower bounds on hybrid zone widths in animals, indicating that there are geographic restrictions on hybrid zone formation. Overall, our analyses reinforce the fundamental importance of dispersal in hybrid zone formation and more generally in the ecology of range boundaries.

Predicting speciation probability from replicated population histories.

In this issue of Molecular Ecology, Yamasaki et al. (2020) use genetic data from extensive sampling of Rhinogobius goby fish across the Ryukyu Archipelago in Japan to demonstrate the parallel speciation of a freshwater form from an ancestral amphidromous form. They then show that ecosystem size strongly predicts the probability of speciation between the two forms across islands. In doing so, this study connects population-level processes (microevolution) to broad-scale biodiversity patterns (macroevolution), an important but understudied link in evolutionary biology. Moving forward, we can build on this research to (a) more directly determine how geographic, ecological and historical factors influence the different stages of the speciation process, and (b) understand whether mechanisms inferred from insular radiations extend to those on continents, where both demographic histories and environmental regimes are likely more complex.

Glutathione Modified Fluorescent CdS QDs Synthesized Using Environmentally Benign Pathway for Detection of Mercury Ions in Aqueous Phase.

An adept, rapid and novel water-soluble glutathione functionalized CdS quantum dots (GSH@CdS QDs) were fabricated using green pathway for sensing of heavy metal contamination prevalent in industrial wastewater. GSH@CdS QDs were facilely synthesized in an aqueous phase reaction and were effectively characterized using FT-IR, XRD, FESEM, HRTEM and EDX techniques. The distinct fluorescence characteristics of GSH@CdS QDs were explored and the QDs showed selective sensitivity towards mercury ions with a low limit of detection of 0.54 nM under optimal conditions. The detailed interaction between GSH@CdS QDs and Hg2+ and the probable fluorescence quenching mechanism were established in this study. In comparison to already reported fluorescent probes, GSH@CdS QDs showed high sensitivity, biocompatibility, long fluorescence stability and convenient removal of mercury ions. Graphical Abstract Facile green route for the fabrication of glutathione capped CdS quantum dots for fluorescence-based detection of toxic Hg2+ ions.

Is genomic diversity a useful proxy for census population size? Evidence from a species-rich community of desert lizards.

Species abundance data are critical for testing ecological theory, but obtaining accurate empirical estimates for many taxa is challenging. Proxies for species abundance can help researchers circumvent time and cost constraints that are prohibitive for long-term sampling. Under simple demographic models, genetic diversity is expected to correlate with census size, such that genome-wide heterozygosity may provide a surrogate measure of species abundance. We tested whether nucleotide diversity is correlated with long-term estimates of abundance, occupancy and degree of ecological specialization in a diverse lizard community from arid Australia. Using targeted sequence capture, we obtained estimates of genomic diversity from 30 species of lizards, recovering an average of 5,066 loci covering 3.6 Mb of DNA sequence per individual. We compared measures of individual heterozygosity to a metric of habitat specialization to investigate whether ecological preference exerts a measurable effect on genetic diversity. We find that heterozygosity is significantly correlated with species abundance and occupancy, but not habitat specialization. Demonstrating the power of genomic sampling, the correlation between heterozygosity and abundance/occupancy emerged from considering just one or two individuals per species. However, genetic diversity does no better at predicting abundance than a single day of traditional sampling in this community. We conclude that genetic diversity is a useful proxy for regional-scale species abundance and occupancy, but a large amount of unexplained variation in heterozygosity suggests additional constraints or a failure of ecological sampling to adequately capture variation in true population size.

A Framework for Resolving Cryptic Species: A Case Study from the Lizards of the Australian Wet Tropics.

As we collect range-wide genetic data for morphologically-defined species, we increasingly unearth evidence for cryptic diversity. Delimiting this cryptic diversity is challenging, both because the divergences span a continuum and because the lack of overt morphological differentiation suggests divergence has proceeded heterogeneously. Herein, we address these challenges as we diagnose and describe species in three co-occurring species groups of Australian lizards. By integrating genomic and morphological data with data on hybridization and introgression from contact zones, we explore several approaches-and their relative benefits and weaknesses-for testing the validity of cryptic lineages. More generally, we advocate that genetic delimitations of cryptic diversity must consider whether these lineages are likely to be durable and persistent through evolutionary time.

Does Population Structure Predict the Rate of Speciation? A Comparative Test across Australia's Most Diverse Vertebrate Radiation.

Population divergence is the first step in allopatric speciation, as has long been recognized in both theoretical models of speciation and empirical explorations of natural systems. All else being equal, lineages with substantial population differentiation should form new species more quickly than lineages that maintain range-wide genetic cohesion through high levels of gene flow. However, there have been few direct tests of the extent to which population differentiation predicts speciation rates as measured on phylogenetic trees. Here, we explicitly test the links between organismal traits, population-level processes, and phylogenetic speciation rates across a diverse clade of Australian lizards that shows remarkable variation in speciation rate. Using genome-wide double digest restriction site-associated DNA data from 892 individuals, we generated a comparative data set on isolation by distance and population differentiation across 104 putative species-level lineages (operational taxonomic units). We find that species show substantial variation in the extent of population differentiation, and this variation is predicted by organismal traits that are thought to be proxies for dispersal and deme size. However, variation in population structure does not predict variation in speciation rate. Our results suggest that population differentiation is not the rate-limiting step in species formation and that other ecological and historical factors are primary determinants of speciation rates at macroevolutionary scales.

Genetic diversity is largely unpredictable but scales with museum occurrences in a species-rich clade of Australian lizards.

Genetic diversity is a fundamental characteristic of species and is affected by many factors, including mutation rate, population size, life history and demography. To better understand the processes that influence levels of genetic diversity across taxa, we collected genome-wide restriction-associated DNA data from more than 500 individuals spanning 76 nominal species of Australian scincid lizards in the genus Ctenotus To avoid potential biases associated with variation in taxonomic practice across the group, we used coalescent-based species delimitation to delineate 83 species-level lineages within the genus for downstream analyses. We then used these genetic data to infer levels of within-population genetic diversity. Using a phylogenetically informed approach, we tested whether variation in genetic diversity could be explained by population size, environmental heterogeneity or historical demography. We find that the strongest predictor of genetic diversity is a novel proxy for census population size: the number of vouchered occurrences in museum databases. However, museum occurrences only explain a limited proportion of the variance in genetic diversity, suggesting that genetic diversity might be difficult to predict at shallower phylogenetic scales.