Biology subjects

Crawford, A. J.

Publications and source records attributed to Crawford, A. J..

At least 19 recordsLinked to original sources

bioRxiv2026

The Vertebrate Genomes Project Phase I: A global reference genome resource

The Vertebrate Genomes Project (VGP) aims to produce complete and near-error-free reference genomes for all [~]70,000 extant vertebrate species1. Organized in four phases, it progressively targets all vertebrate orders, families, genera, and eventually all species. Here we present the completion of VGP Phase I, delivering reference genomes for [~]95% of vertebrate orders, along with additional lineages within those orders, totaling 816 species and 1.6 trillion base pairs of main haplotype sequence. These genomes were assembled and annotated over an 8-year period (2018-2026) of rapid advances in genome sequencing, assembly, and annotation methods2-4, alongside the growth of associated consortium initiatives and international collaborations5-9. They represent some of the highest-quality vertebrate genomes currently available, and most have become the primary reference for their respective species in public databases. Comparative analyses across a subset of 579 species when we reached a threshold of 85% of orders allowed us to reconstruct the genome of the last common ancestor of all vertebrates 500 million years ago, identify diverse modes of sex chromosome evolution, reveal clade-specific three-dimensional genome architecture, discover methylated epigenetic landscapes across vertebrates, and provide a framework for studying gene and pseudogene evolution, immune loci, cancer-associated genes, and other trait-associated loci. Approximately a quarter of this subset are listed as Vulnerable to Critically Endangered by the IUCN Red List of Threatened Species, and have enabled more advanced genomic investigations of extinction risk. VGP Phase I delivers a reference backbone for vertebrate genomics, enabling discoveries that would otherwise remain out of reach across evolution, conservation, and medicine. Talking pointsO_LIThe flagship paper of VGP Phase I C_LIO_LIThe highest quality collection of genomes within the eukaryotic domain of life C_LIO_LIEvolution of genome sequencing technology quality throughout VGP Phase I C_LIO_LIA driver project that has been a model for multiple large-scale, high-quality reference genome projects C_LIO_LIReleases all currently unpublished genomes in Phase I from scientific study embargoes C_LIO_LIMultiple biological discoveries across the vertebrate tree of life C_LI

genomics↗

bioRxiv2026

Rapid speciation in Harlequin Toads (Anura: Bufonidae) endemic to the Sierra Nevada de Santa Marta

The Neotropical genus Atelopus has experienced a drastic population decline in recent decades. Despite this, a knowledge gap remains regarding the conservation genetic status, phylogeography, and demographic history of most of its species, especially those endemic to areas with limited access like the Sierra Nevada de Santa Marta in Colombia. In this genomic study, we inferred phylogenetic relationships, demographic history, and gene flow among four endemic Atelopus morphospecies in the Sierra Nevada de Santa Marta-SNSM. Additionally, we compared the effective population size (Ne) estimates with the available population census data. NextRAD was used to obtain genomic data from 95 individuals collected at five sites in the SNSM and two in the Colombian Pacific (outgroup). The morphospecies recently diverged in a scenario without gene flow and were recovered as monophyletic. Their phylogenetic relationships were discordant, which is attributed to the presence of incomplete lineage sorting-ILS, which would also explain their shared ancestry among them. The lack of gene flow as well as the recent divergence times given by demography suggests a recent and rapid speciation. However, the reproductive isolation mechanisms that promote or maintain the species boundaries in this group remain unknown and require further investigation. We suggest that this process may have been influenced by the complex topography of the SNSM, traits such as high philopatry, low dispersal ability, and behavioral factors such as habitat preference or to factors related to genetic architecture that influenced the rapid formation of reproductive barriers among populations. Additionally, a pattern of population decline was observed around 200.000 years ago, with recent increases in three morphospecies. Despite the reduction in effective population size, no signs of inbreeding were detected. However, for A. laetissimus, the only species surveyed, the estimated value of Ne and its implications should be interpreted with caution. Ultimately, our findings reveal an evolutionary history shaped by a burst of diversification and abrupt reproductive isolation, highlighting how the resulting endemism and restricted genetic connectivity shape the unique evolutionary trajectory and vulnerability of this threatened montane species. Significance StatementThe mechanisms driving rapid speciation in montane ecosystems remain a central question in evolutionary biology. This study provides crucial genomic insights into the diversification of four endemic Atelopus species in an isolated Neotropical massif. We reveal a compelling evolutionary scenario where species diverge rapidly in absence of gene flow. This rapid speciation was likely facilitated by complex topography, environmental heterogeneity, and ecological and behavioral differences among species. Nevertheless, the evolutionary processes that limited gene flow among SNSM species have not yet been identified.

evolutionary biology↗

bioRxiv2026

Imminent invasion of the chytrid fungus threatens the last naive amphibian biodiversity hotspots

While the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) is driving catastrophic biodiversity loss worldwide, some amphibian communities persist seemingly unaffected despite occurring in climates conducive to pathogen establishment. These amphibian communities may remain epidemiologically naive. As mitigation of Bd is rarely successful after establishment, identifying remaining Bd-free refuges is imperative. Presently, the only known large-scale Bd-free refuge is the island of New Guinea (NG), safeguarding Australasias amphibian phylogenetic diversity otherwise devastated by Bd. Following extensive multi-year disease surveillance, we here uncover a second large-scale Bd-free refuge in the Sierra Nevada de Santa Marta (SNSM), a Neotropical biodiversity hotspot in northern Colombia. We detected no evidence of Bd in SNSM-wide screening, while we uncovered the presence of hypervirulent Bd-GPL in adjacent areas of the tropical Andes. Population genomic analyses in an SNSM-endemic anuran found no evidence for demographic bottlenecks indicative of cryptic epizootic decline. Niche modelling highlights the high risk for Bd establishment and Bd-induced declines in both the SNSM and NG, and the important role of lowland environmental barriers in restricting Bd invasion. Infection trials using three SNSM-endemic amphibians reveal varying disease susceptibility. Together, these data identify the SNSM as an epidemiologically naive refuge likely facing imminent Bd invasion, which could result in the loss of at least 25 endemic amphibian species. We highlight the urgent need for proactive conservation action and strict implementation of biosecurity to safeguard the unique and vast amphibian diversity of the worlds last major Bd-free refuges. Significance StatementAmphibian chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) has driven unprecedented global biodiversity loss. The Neotropics and Australasia comprise epicenters of declines. Identifying remaining Bd-free refuges is crucial to curb further amphibian extinctions, but so far contemporary absence of Bd has only been demonstrated for New Guinea. Here we identify the last known major Bd-free biodiversity hotspot in the Neotropics: the Sierra Nevada de Santa Marta (SNSM) in Colombia. Our results show that amphibian communities in this hotspot are immunologically naive despite occurrence of hypervirulent Bd lineages nearby and climatic conditions within the SNSM conducive to Bd-induced declines. This creates an imminent risk for Bd-driven declines and highlights the urgent need for preventive actions to avert another wave of biodiversity loss.

ecology↗

bioRxiv2025

A field-deployable eDNA metabarcoding workflow including de novo reference assembly for characterizing understudied biodiversity hotspots

Field-deployable DNA metabarcoding offers a transformative approach to biodiversity research and monitoring, yet its application remains limited due to technical constraints and a lack of reference data in poorly studied ecosystems. Combining isothermal Recombinase Polymerase Amplification (RPA) and Oxford Nanopore sequencing, we introduce a two-step approach that uses non-invasive species barcoding to directly generate reference sequences for use in environmental DNA (eDNA) metabarcoding, and enable real-time, PCR-free, and cost-effective molecular assessment of ecological communities in the field. Using an endemic and understudied tropical amphibian assemblage as a model, we demonstrate the practicality and versatility of this novel workflow. De novo generation of a reference sequence library significantly improved the accuracy and taxonomic resolution of sequence assignments from eDNA samples, particularly on the species level, in turn allowing a characterization of fine-scale patterns in community composition. Beyond generating new RPA-compatible amphibian metabarcoding primers, our results show that combining field-based eDNA metabarcoding with the offline assembly of a local reference database can bridge data gaps in molecular biodiversity monitoring, providing a scalable solution for real-time biodiversity assessments in data-deficient ecosystems. This workflow paves the way for broader deployment of molecular tools in global biodiversity hotspots - particularly in remote and resource-limited tropical regions - to directly contribute critical baseline data, and support conservation efforts in regions where they are most urgently needed.

ecology↗

bioRxiv2024

High-motility pro-tumorigenic monocytes drive macrophage enrichment in the tumor microenvironment

Enrichment of tumor-associated macrophages (TAM{Phi}s) in the tumor microenvironment correlates with worse clinical outcomes in triple-negative breast cancer (TNBC) patients, prompting the development of therapies to inhibit TAM{Phi} infiltration. However, the lackluster efficacy of CCL2-based chemotaxis blockade in clinical trials suggests that a new understanding of monocyte/macrophage infiltration may be necessary. Here we demonstrate that random migration, and not only chemotaxis, drives macrophage tumor infiltration. We identified tumor- associated monocytes (TAMos) that display a dramatically enhanced migration capability, induced rapidly by the tumor microenvironment, that drives effective tumor infiltration, in contrast to low-motility differentiated macrophages. TAMo, not TAM{Phi}, promotes cancer cell proliferation through activation of the MAPK pathway. IL-6 secreted both by cancer cells and TAMo themselves enhances TAMo migration by increasing dendritic protrusion dynamics and myosin- based contractility via the JAK2/STAT3 signaling pathway. Independent from CCL2 mediated chemotaxis, IL-6 driven enhanced migration and pro-proliferative effect of TAMo were validated in a syngeneic TNBC mouse model. Depletion of IL-6 in cancer cells significantly attenuated monocyte infiltration and reversed TAMo-induced cancer cell proliferation. This work reveals the critical role random migration plays in monocyte driven TAM{Phi} enrichment in a tumor and pinpoints IL-6 as a potential therapeutic target in combination with CCL2 to ameliorate current strategies against TAM{Phi} infiltration.

immunology↗

bioRxiv2024

The Amphibian Genomics Consortium: advancing genomic and genetic resources for amphibian research and conservation

Amphibians represent a diverse group of tetrapods, marked by deep divergence times between their three systematic orders and families. Studying amphibian biology through the genomics lens increases our understanding of the features of this animal class and that of other terrestrial vertebrates. The need for amphibian genomic resources is more urgent than ever due to the increasing threats to this group. Amphibians are one of the most imperiled taxonomic groups, with approximately 41% of species threatened with extinction due to habitat loss, changes in land use patterns, disease, climate change, and their synergistic effects. Amphibian genomic resources have provided a better understanding of ontogenetic diversity, tissue regeneration, diverse life history and reproductive modes, anti-predator strategies, and resilience and adaptive responses. They also serve as essential models for studying broad genomic traits, such as evolutionary genome expansions and contractions, as they exhibit the widest range of genome sizes among all animal taxa and possess multiple mechanisms of genetic sex determination. Despite these features, genome sequencing of amphibians has significantly lagged behind that of other vertebrates, primarily due to the challenges of assembling their large, repeat-rich genomes and the relative lack of societal support. The emergence of long-read sequencing technologies, combined with advanced molecular and computational techniques that improve scaffolding and reduce computational workloads, is now making it possible to address some of these challenges. To promote and accelerate the production and use of amphibian genomics research through international coordination and collaboration, we launched the Amphibian Genomics Consortium (AGC, https://mvs.unimelb.edu.au/amphibian-genomics-consortium) in early 2023. This burgeoning community already has more than 282 members from 41 countries. The AGC aims to leverage the diverse capabilities of its members to advance genomic resources for amphibians and bridge the implementation gap between biologists, bioinformaticians, and conservation practitioners. Here we evaluate the state of the field of amphibian genomics, highlight previous studies, present challenges to overcome, and call on the research and conservation communities to unite as part of the AGC to enable amphibian genomics research to "leap" to the next level.

genomics↗

bioRxiv2024

A 3D in vitro assay to study combined immune cell infiltration and cytotoxicity

Immune cell-mediated killing of cancer cells in a solid tumor is prefaced by a multi-step infiltration cascade of invasion, directed migration, and cytotoxic activities. In particular, immune cells must invade and migrate through a series of different extracellular matrix (ECM) boundaries and domains before reaching and killing their target tumor cells. These infiltration events are a central challenge to the clinical success of CAR T cells against solid tumors. The current standard in vitro cell killing assays measure cell cytotoxicity in an obstacle-free, two-dimensional (2D) microenvironment, which precludes the study of 3D immune cell-ECM interactions. Here, we present a 3D combined infiltration/cytotoxicity assay based on an oil-in-water microtechnology. This assay measures stromal invasion following extravasation, migration through the stromal matrix, and invasion of the solid tumor in addition to cell killing. We compare this 3D cytotoxicity assay to the benchmark 2D assay through tumor assembloid cocultures with immune cells and engineered immune cells. This assay is amenable to an array of imaging techniques, which allows direct observation and quantification of each stage of infiltration in different immune and oncological contexts. We establish the 3D infiltration/cytotoxicity assay as an important tool for the mechanistic study of immune cell interactions with the tumor microenvironment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/586980v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@6a161dorg.highwire.dtl.DTLVardef@d1e5d3org.highwire.dtl.DTLVardef@47b87corg.highwire.dtl.DTLVardef@a0e051_HPS_FORMAT_FIGEXP M_FIG The 3D combined infiltration/cytotoxicity assay captures three important steps of immune cell infiltration into the solid tumor microenvironment: (1) circulating immune cells extravasate and invade the stromal matrix, (2) immune cells migrate through the stromal matrix to reach the tumor core, and (3) immune cells that successfully navigate the stroma must cross a basement membrane boundary secreted by the cancer cells to contact and kill the cancer cells within a solid tumor. C_FIG

cancer biology↗

bioRxiv2024

Honoring the Afro-Colombian musical culture with the naming of Epipedobates sp. nov. (Anura: Dendrobatidae), a frog from the Pacific rainforests

The number of amphibian species described yearly shows no signs of slowing down, especially in tropical regions, implying that the biodiversity of amphibians remains woefully underestimated. We describe a new species of poison frog from the Pacific lowlands of southwestern Colombia: Epipedobates currulao sp. nov., named for the Pacific music and dance genre known as bambuco viejo or currulao. This species inhabits lowland forests from 0-260 m a.s.l. This taxon differs from congeners by having a combination of bright yellow blotches in the dorsal anterior region of the thigh and upper arm, homogenous dark-brown dorsal coloration, and advertisement calls of long duration and many pulses. We also describe the courtship call of E. currulao, which is lower in frequency and shorter in duration than its advertisement call. Molecular phylogenetic analyses confirm the monophyly of the populations sampled and its position as the sister species of Epipedobates narinensis, which occurs in southwestern Colombia. Among species of Epipedobates, the new species has been previously confused with E. boulengeri, but we find that the two species are allopatric and represent two divergent clades (1.77% divergent for 12S-16S and 5.39% for CYTB). These species can be distinguished by the presence of a bright yellow blotch on the dorsal anterior region of the thigh and on the upper arm of E. currulao, blotches that are either more white than yellow or absent in E. boulengeri. In addition, the advertisement calls are distinct, with E. currulao having a single but long call in each call series while E. boulengeri has 2-6 calls in a series with each call being much shorter in length. Epipedobates currulao is the most northern species of Epipedobates, which extends southwards along the western edge of the Andes. Known as the Choco, this biogeographic region has been largely converted to agriculture in Ecuador and is experiencing widespread transformation in Colombia, which may endanger E. currulao and biodiversity in the region. A Spanish translation of the main text is available in the Supplementary Materials. ResumenEl numero de especies de anfibios descritas cada ano continua aumentando, especialmente en las regiones tropicales, lo que implica que la biodiversidad de anfibios sigue siendo subestimada. Describimos una nueva especie de rana venenosa de las tierras bajas del Pacifico del suroccidente de Colombia: Epipedobates currulao sp. nov., nombrada asi por el genero de musica y danza del Pacifico conocido como bambuco viejo o currulao. Las ranas de esta especie habitan en bosques de tierras bajas desde el nivel del mar hasta los 260 m. Este taxon se diferencia de sus congeneres por tener una combinacion de manchas amarillas brillantes en la region dorsal anterior del muslo y los brazos, una coloracion dorsal homogenea marron oscuro y cantos de advertencia mas largos y en consecuencia con mayor numero de pulsos. Tambien describimos la llamada de cortejo de E. currulao, con menor frecuencia pico y duracion que la llamada de advertencia. Los analisis filogeneticos confirman la monofilia de la especie y su posicion como hermana de Epipedobates narinensis, la cual se distribuye en el suroccidente de Colombia. Entre las especies de Epipedobates, la nueva especie ha sido previamente asignada a E. boulengeri, pero las dos especies son alopatricas y representan dos clados filogeneticamente divergentes (1.77% divergentes para 12S-16S y 5.39% para CYTB). Estas especies se pueden distinguir fenotipicamente por la presencia de una mancha amarilla brillante en la region dorsal anterior del muslo y en la parte superior del brazo en E. currulao, que son mas blancas que amarillas o estan ausentes en E. boulengeri. Ademas, los cantos de advertencia son distintos, E. currulao tiene una unica y larga llamada en una serie de llamadas, mientras que E. boulengeri tiene de 2 a 6 llamadas por serie, siendo cada llamada mucho mas corta. Epipedobates currulao es la especie distribuida mas al norte del genero Epipedobates, el cual se extiende hacia el sur a lo largo del flanco occidental de la cordillera de los Andes. Esta region conocida como el Choco biogeografico, ha sido fuertemente transformada por agricultura en Ecuador y esta experimentando una transformacion generalizada de sus bosques en Colombia, lo cual pone en peligro a E. currulao y toda su biodiversidad en un futuro cercano. Una traduccion al espanol del texto principal esta disponible en el material suplementario.

zoology↗

bioRxiv2023

Precision-engineered biomimetics: the human fallopian tube

The fallopian tube has an essential role in several physiological and pathological processes from pregnancy to ovarian cancer. However, there are no biologically relevant models to study its pathophysiology. The state-of-the-art organoid model has been compared to two-dimensional tissue sections and molecularly assessed providing only cursory analyses of the models accuracy. We developed a novel multi-compartment organoid model of the human fallopian tube that was meticulously tuned to reflect the compartmentalization and heterogeneity of the tissues composition. We validated this organoids molecular expression patterns, cilia-driven transport function, and structural accuracy through a highly iterative platform wherein organoids are compared to a three-dimensional, single-cell resolution reference map of a healthy, transplantation-quality human fallopian tube. This organoid model was precision-engineered to match the human microanatomy. One sentence summaryTunable organoid modeling and CODA architectural quantification in tandem help design a tissue-validated organoid model.

bioengineering↗

bioRxiv2023

Serine synthesis pathway upregulated by E-cadherin is essential for the proliferation and metastasis of breast cancers

The loss of E-cadherin (E-cad), an epithelial cell adhesion molecule, has been implicated in the epithelial-mesenchymal transition (EMT), promoting invasion and migration of cancer cells and, consequently, metastasis. However, recent studies have demonstrated that E-cad supports the survival and proliferation of metastatic cancer cells, suggesting that our understanding of E-cad in metastasis is far from comprehensive. Here, we report that E-cad upregulates the de novo serine synthesis pathway (SSP) in breast cancer cells. The SSP provides metabolic precursors for biosynthesis and resistance to oxidative stress, critically beneficial for E-cad-positive breast cancer cells to achieve faster tumor growth and more metastases. Inhibition of PHGDH, a rate-limiting enzyme in the SSP, significantly and specifically hampered the proliferation of E-cad-positive breast cancer cells and rendered them vulnerable to oxidative stress, inhibiting their metastatic potential. Our findings reveal that E-cad adhesion molecule significantly reprograms cellular metabolism, promoting tumor growth and metastasis of breast cancers.

cancer biology↗

bioRxiv2023

Comparative analysis of amphibian genomes: an emerging resource for basic and applied research

Amphibians are the most threatened group of vertebrates and are in dire need of conservation intervention to ensure their continued survival. They exhibit unique features including a high diversity of reproductive strategies, permeable and specialized skin capable of producing toxins and antimicrobial compounds, multiple genetic mechanisms of sex determination, and in some lineages, the ability to regenerate limbs and organs. Although genomics approaches would shed light on these unique traits and aid conservation, sequencing and assembly of amphibian genomes has lagged behind other taxa due to their comparatively large genome sizes. Fortunately, the development of long-read sequencing technologies and initiatives has led to a recent burst of new amphibian genome assemblies. Although growing, the field of amphibian genomics suffers from the lack of annotation resources, tools for working with challenging genomes, and lack of high-quality assemblies in multiple clades of amphibians. Here we analyze 51 publicly available amphibian genomes to evaluate their usefulness for functional genomics research. We report considerable variation in genome assembly quality and completeness, and report some of the highest transposable element and repeat contents of any vertebrate. Additionally, we detected an association between transposable element content and climatic variables. Our analysis provides evidence of conserved genome synteny despite the long divergence times of this group, but we also highlight inconsistencies in chromosome naming and orientation across genome assemblies. We discuss sequencing gaps in the phylogeny and suggest key targets for future sequencing endeavors. Finally, we propose increased investment in amphibian genomics research to promote their conservation.

genomics↗

bioRxiv2022

Tumor proliferation and invasion are coupled through cell-extracellular matrix friction

Cell proliferation and invasion are two key drivers of tumor progression and are traditionally considered two independent cellular processes regulated by distinct pathways. Through in vitro and in silico methods, we provide evidence that these two processes are intrinsically coupled through matrix-adhesion friction. Using novel tumor spheroids, we show that both tumor cell proliferation and invasion are limited by a volumetric carrying capacity of the system, i.e. maximum spatial cell concentration supported by the systems total cell count, nutrient consumption rate, and collagen gel mechanical properties. To manipulate these phenotypes in breast cancer cells, we modulate the expression of E-cadherin and its associated role in adhesion, invasion, and proliferation. We integrate these results into a mixed-constitutive formulation to computationally delineate the contributions of cellular and extracellular adhesion, stiffness, and mechanical properties of the extracellular matrix (ECM) to the proliferative and invasive fates of breast cancer tumor spheroids. Both approaches conclude that the dominant drivers of tumor fate are system properties modulating cell-ECM friction, such as E-cadherin dependent cell-ECM adhesion and matrix pore size.

biophysics↗

bioRxiv2022

Epistatic effects between amino acid insertions and substitutions mediate toxin-resistance of vertebrate Na+,K+-ATPases

The recurrent evolution of resistance to cardiotonic steroids (CTS) across diverse animals most frequently involves convergent amino-acid substitutions to the H1-H2 extracellular loop of Na+,K+-ATPase (NKA). Previous work established that hystricognath rodents (e.g. chinchilla) and pterocliform birds (sandgrouse) have convergently evolved amino-acid insertions in the H1-H2 loop, but their functional significance is not known. Using protein engineering, we show that these insertions have distinct effects on CTS resistance of NKA in the two lineages that strongly depend on intramolecular interactions with other residues. Removing the insertion in the chinchilla lineage unexpectedly increases CTS resistance and decreases NKA activity. In the sandgrouse lineage, the insertion works in concert with the substitution Q111R to increase CTS resistance while maintaining wild-type ATPase activity levels. Molecular docking simulations provide additional insight into the biophysical mechanisms responsible for the context-specific CTS insensitivity of the enzyme. Our results highlight the diversity of genetic substrates that underlie CTS insensitivity in vertebrate NKA and reveal how amino-acid insertions can alter the phenotypic effects of point mutations at key sites in the same protein domain.

evolutionary biology↗

bioRxiv2021

Epistasis is not a strong constraint on the recurrent evolution of toxin-resistant Na+,K+-ATPases among tetrapods

A growing body of theoretical and experimental evidence suggests that intramolecular epistasis is a major determinant of rates and patterns of protein evolution and imposes a substantial constraint on the evolution of novel protein functions. Here, we examine the role of intramolecular epistasis in the recurrent evolution of resistance to cardiotonic steroids (CTS) across tetrapods, which occurs via specific amino acid substitutions to the -subunit family of Na,K-ATPases (ATP1A). After identifying a series of recurrent substitutions at two key sites of ATP1A that are predicted to confer CTS resistance in diverse tetrapods, we then performed protein engineering experiments to test the functional consequences of introducing these substitutions onto divergent species backgrounds. In line with previous results, we find that substitutions at these sites can have substantial background-dependent effects on CTS resistance. Globally, however, these substitutions also have pleiotropic effects that are consistent with additive rather than background-dependent effects. Moreover, the magnitude of a substitutions effect on activity does not depend on the overall extent of ATP1A sequence divergence between species. Our results suggest that epistatic constraints on the evolution of CTS-resistant forms of Na,K-ATPase likely depend on a small number of sites, with little dependence on overall levels of protein divergence. We propose that dependence on a limited number sites may account for the observation of convergent CTS resistance substitutions observed among taxa with highly divergent Na,K-ATPases. Significance StatementIndividual amino acid residues within a protein work in concert to produce a functionally coherent structure that must be maintained even as orthologous proteins in different species diverge over time. Given this dependence, we expect identical mutations to have more similar effects on protein function in more closely related species. We tested this hypothesis by performing protein-engineering experiments on ATP1A, an enzyme mediating target-site insensitivity to cardiotonic steroids (CTS) in diverse animals. These experiments reveal that the phenotypic effects of substitutions can sometimes be background-dependent, but also that the magnitude of these phenotypic effects does not correlate with overall levels of ATP1A sequence divergence. Our results suggest that epistatic constraints are determined by states at a small number of sites, potentially explaining the frequent convergent CTS resistance substitutions among Na,K-ATPases of highly divergent taxa.

evolutionary biology↗

bioRxiv2021

Benchmarking ultra-high molecular weight DNA preservation methods for long-read and long-range sequencing

Studies in vertebrate genomics require sampling from a broad range of tissue types, taxa, and localities. Recent advancements in long-read and long-range genome sequencing have made it possible to produce high-quality chromosome-level genome assemblies for almost any organism. However, adequate tissue preservation for the requisite ultra-high molecular weight DNA (uHMW DNA) remains a major challenge. Here we present a comparative study of preservation methods for field and laboratory tissue sampling, across vertebrate classes and different tissue types. We find that no single method is best for all cases. Instead, the optimal storage and extraction methods vary by taxa, by tissue, and by down-stream application. Therefore, we provide sample preservation guidelines that ensure sufficient DNA integrity and amount required for use with long-read and long-range sequencing technologies across vertebrates. Our best practices generated the uHMW DNA needed for the high-quality reference genomes for Phase 1 of the Vertebrate Genomes Project (VGP), whose ultimate mission is to generate chromosome-level reference genome assemblies of all [~]70,000 extant vertebrate species.

genomics↗

bioRxiv2020

Landscape genetics and species delimitation in the Andean palm rocket frog, Rheobates spp.

The complex topography of the species-rich northern Andes creates heterogeneous environmental landscapes that are hypothesized to have promoted population fragmentation and diversification by vicariance, gradients and/or the adaptation of species. Previous phylogenetic work on the Palm Rocket Frog (Anura: Aromobatidae: Rheobates spp.), endemic to mid-elevation forests of Colombia, suggested valleys were important in promoting divergence between lineages. In this study, we use a spatially, multi-locus population genetic approach of two mitochondrial and four nuclear genes from 25 samples representing the complete geographic range of the genus to delimit species and test for landscape effects on genetic divergence within Rheobates. We tested three landscape genetic models: isolation by distance, isolation by resistance, and isolation by environment. Bayesian species delimitation (BPP) and a Poisson Tree Process (PTP) model both recovered five highly divergent genetic lineages within Rheobates, rather than the three inferred in a previous study. We found that an isolation by environment provided the only variable significantly correlated with genetic distances for both mitochondrial and nuclear genes, suggesting that local adaptation may have a role driving the genetic divergence within this genus of frogs. Thus, genetic divergence in Rheobates may be driven by the local environments where these frogs live, even more so that by the environmental characteristics of the intervening regions among populations (i.e., geographic barriers).

genetics↗

bioRxiv2020

Concerted evolution reveals co-adapted amino acid substitutions in frogs that prey on toxic toads

Gene duplication is an important source of evolutionary innovation, but the adaptive division-of-labor between duplicates can be opposed by ongoing gene conversion between them. Here we document a tandem duplication of Na+,K+-ATPase subunit 1 (ATP1A1) shared by frogs in the genus Leptodactylus, a group of species that feeds on toxic toads. One ATP1A1 paralog evolved resistance to toad toxins while the other paralog retained ancestral susceptibility. We show that the two Leptodactylus paralogs are distinguished by 12 amino acid substitutions that were maintained by strong selection that counteracted the homogenizing effect of gene conversion. Protein-engineering experiments show that two major-effect substitutions confer toxin resistance, whereas the 10 additional substitutions mitigate deleterious pleiotropic effects on enzyme function. Our results highlight how trans-specific, neofunctionalized gene duplicates can provide unique insights into interactions between adaptive substitutions and the genetic backgrounds on which they arise. One Sentence SummarySelection counteracts gene conversion to maintain an adaptive division-of-labor between tandemly duplicated genes.

evolutionary biology↗

bioRxiv2020

Synthesis of geological and comparative phylogeographic data point to climate, not mountain uplift, as driver of divergence across the Eastern Andean Cordillera

AimTo evaluate the potential role of the orogeny of the Eastern Cordillera (EC) of the Colombian Andes and the Merida Andes (MA) of Venezuela as drivers of vicariance between populations of 37 tetrapod lineages co-distributed on both flanks, through geological reconstruction and comparative phylogeographic analyses. LocationNorthwestern South America MethodsWe first reviewed and synthesized published geological data on the timing of uplift for the EC-MA. We then combined newly generated mitochondrial DNA sequence data with published datasets to create a comparative phylogeographic dataset for 37 independent tetrapod lineages. We reconstructed time-calibrated molecular phylogenies for each lineage under Bayesian inference to estimate divergence times between lineages located East and West of the Andes. We performed a comparative phylogeographic analysis of all lineages within each class of tetrapod using hierarchical approximate Bayesian computation (hABC) to test for synchronous vicariance across the EC-MA. To evaluate the potential role of life history in explaining variation in divergence times among lineages, we evaluated 13 general linear models (GLM) containing up to six variables each (maximum elevation, range size, body length, thermoregulation, type of dispersal, and taxonomic class). ResultsOur synthesis of geological evidence suggested that the EC-MA reached significant heights by 38-33 million years ago (Ma) along most of its length, and we reject the oft-cited date of 2-5 Ma. Based on mtDNA divergence from 37 lineages, however, the median estimated divergence time across the EC-MA was 3.26 Ma (SE = 2.84) in amphibians, 2.58 Ma (SE = 1.81) in birds, 2.99 Ma (SE = 4.68) in reptiles and 1.43 Ma (SE = 1.23) in mammals. Using Bayes Factors, the hypothesis for a single temporal divergence interval containing synchronous divergence events was supported for mammals and but not supported for amphibians, non-avian reptiles, or birds. Among the six life-history variables tested, only thermoregulation successfully explained variation in divergence times (minimum AICc, R2 0.10), with homeotherms showing more recent divergence relative to poikilotherms. Main conclusionsOur results reject the hypothesis of the rise Andean Cordillera as driver of vicariance of lowland population because divergence dates are too recent and too asynchronous. We discuss alternative explanations, including dispersal through mountain passes, and suggest that changes in the climatic conditions during the Pliocene and Pleistocene interacted with tetrapod physiology, promoting older divergences in amphibians and reptiles relative to mammals and birds on an already established orogen.

evolutionary biology↗