Biology subjects

Faircloth, B. C.

Publications and source records attributed to Faircloth, B. C..

At least 19 recordsLinked to original sources

bioRxiv2026

Distinct molecular mechanisms shape thermal tolerance and its plasticity in a splash pool copepod

Understanding how organisms respond to changes in temperature is becoming increasingly important in our rapidly changing world. While species differ substantially in their thermal tolerance, the exact molecular mechanisms underpinning this trait variation remain largely unknown. Species with broad geographical distribution provide a unique opportunity to examine variable responses to temperature, as populations experience a diverse range of thermal regimes that differ in the intensity, frequency, and duration of thermal stress. We used a splash pool copepod (Tigriopus californicus) with a large geographic range across the North American coast (Baja California to Alaska) to examine variation in thermotolerance and the plasticity of thermotolerance in five populations sampled across 12 degrees of latitude. We found that populations with higher heat tolerance (southern) showed lower plasticity (measured as increased survival at higher temperatures after a prior exposure to sub-lethal temperature). Our comparative transcriptomic analyses revealed that higher heat tolerance was associated with maintaining elevated expression of genes coding for peptidases even in absence of heat shock and higher plasticity of heat tolerance was associated with an increase in gene expression plasticity of genes coding for chitin and extracellular matrix (ECM). Our results highlight ontology-specific patterns associated with changes in the trait means and plasticity of heat tolerance across populations. These findings improve our mechanistic knowledge of thermotolerance as a trait and our ability to predict which populations are most vulnerable to extinction based on the trade-off between fixed thermal limits and plastic responses.

evolutionary biology↗

bioRxiv2026

Geographic structuring of genetic variation differs across two contact zones in the Diglossa carbonaria superspecies

Rapid radiations, where species quickly diversify with little to no change in the genetic composition of the taxa, provide unique opportunities to understand the processes underlying lineage divergence, especially when the newly formed species come into contact. This study examines three parapatrically distributed taxa of the Andean Diglossa carbonaria superspecies that differ strikingly in plumage and have been classified as an example of rapid lineage divergence. Using RADSeq data, we characterized the geographic structuring of genetic variation and investigated the possibility of introgressive hybridization at the contact zones between D. humeralis atterima and D. b. brunneiventris in northern Peru and between D. b. brunneiventris and D. carbonaria in Bolivia. We found weak genetic differentiation between D. humeralis atterima and D. b. brunneiventris and low, but diagnosable, differentiation between D. b. brunneiventris and D. carbonaria. At the contact zone between D. humeralis atterima and D. b. brunneiventris, the lack of genetic differentiation did not allow us to determine if hybridization was occurring between the taxa. In contrast, clustering analyses, diagnostic allele identification, and PCA analyses showed geographic patterns consistent with introgressive hybridization at the contact zone between D. b. brunneiventris and D. carbonaria, a geographic region where birds that are intermediate in plumage have been observed. Finally, we found a genetic break within the distribution of D. b. brunneiventris. The genetic divergence across this [~]450 km wide break is greater than that found between D. humeralis atterima and D. b. brunneiventris. Our results add to previous work documenting weak genetic differentiation between taxa that have striking plumage differences, and in showing that plumage color may be a poor phylogenetic marker. Lay SummaryO_LIWe studied three closely related avian taxa in Peru and Bolivia which have different plumages and adjacent ranges to examine how genetically distinct they are. C_LIO_LITo do this, we used RADseq data from 72 flowerpiercers sampled across their ranges to examine their population structure and to test whether the three taxa hybridize where their ranges meet. C_LIO_LIDespite their striking differences in plumage color, the three species showed few genetic differences. In one area where the ranges are adjacent, the groups were so similar that interbreeding could not be confirmed. In another, there was evidence of admixture, supported by observations of birds with intermediate plumage. C_LIO_LIFor these taxa, feather color may not reflect evolutionary relationships, and more extensive genetic sampling is needed to understand how these plumage differences evolved. C_LI

zoology↗

bioRxiv2026

The curious case of a Chilean copepod (Tigriopus aff. angulatus) genome assembly

Tigriopus copepods are found in splash pools on all seven continents from the equator to Arctic and Antarctic regions. Given their geographic distribution, frequent exposure to extreme environmental conditions, and strong signatures of local adaptation, these copepods have become models for exploring patterns of adaptation to stressful environments. However, most studies focus on a small subset of Tigriopus species, and there are few genome resources representing the diversity of Tigriopus species and populations. Here, we combine long-read, Pacific Biosciences HiFi data with short-read, Illumina HiC and RNA-seq data to assemble and annotate a genome representing a Tigriopus population from the coast of central Chile. Based on the level of divergence that we observed in mitochondrial genes, we also performed a comparison of morphological characteristics between this population and members of the T. angulatus complex. The assembly that we generated (qhTigAngs1.1.pri) includes 12 major scaffolds (N50 19Mbp, L50 7), equivalent to the number of chromosomes in other Tigriopus species. BUSCO and k-mer analyses of the assembly and BUSCO analyses of gene models are relatively complete (89-99%) with respect to gene or k-mer content. The level of divergence that we observed, morphological differences we recorded, and the spatial distance of this population from the type locality suggest that this Chilean population of Tigriopus may represent a novel species that we call Tigriopus aff. angulatus. These genomic resources will help us understand the diversity and structure of Tigriopus species and populations as well as facilitate future comparisons of adaptation across parallel environmental gradients.

genomics↗

bioRxiv2025

Integrative taxonomy using traits and genomic data for Species Delimitation with Deep learning

Recognizing species boundaries in complex speciation scenarios, including those involving gene flow and demographic fluctuations, remains a challenge, particularly given the diversity of existing species concepts. Promising recent approaches adopt an integrative taxonomy that combines multiple sources of evidence (e.g., genetic, morphology, geographic distributions), reflecting different properties associated with the dynamics of the speciation continuum. The use of statistical inference methods for model comparison, such as approximate Bayesian computation, approximate likelihood approaches, and machine learning, has improved the better assessment of species boundaries in such contexts. However, most existing approaches involve analyzing genetic and phenotypic/geographical information separately, followed by visual/qualitative comparison. Methods that integrate genetic information with other sources of evidence remain limited to simple evolutionary models and are typically unable to analyze more than a few hundred loci across a maximum of a few tens of samples. Here, we present a deep learning approach (DeepID) that combines two convolutional neural networks to integrate genomic data (thousands of loci or single nucleotide polymorphisms, SNPs) and trait information into a unified framework. Using both simulated and empirical data sets, we evaluate the power and accuracy of this approach for discriminating among competing divergence speciation scenarios (with minimal ongoing gene flow) across a varying number of SNPs and traits, as well as different levels of missing data. Analyses based on genomic or trait data alone yielded a slight lower accuracy, whereas integrating genomic and trait data resulted in improved performance. When we violated the speciation model by including extensive migration, approaches incorporating trait data were less affected than those relying solely on genomic information. Together, these results suggest that combining genomic and trait data may capture complementary signals associated with different stages of the speciation process. Moreover, our approach successfully recovered the expected delimitation scenarios in empirical data sets from a plant (Euphorbia balsamifera) and a fish (Lepomis megalotis) species complex. We argue that our method is a flexible and promising approach, allowing for complex scenario comparison and the use of multiple types of data. Combining genomic and trait data likely captures complementary signals associated with different stages of the speciation process, reflecting the fact that speciation is a continuum in which genetic and phenotypic divergence may proceed at different rates.

evolutionary biology↗

bioRxiv2025

Efficient Inference of Macrophylogenies: Insights from the Avian Tree of Life

The exponential growth of molecular sequence data over the past decade has enabled the construction of numerous clade-specific phylogenies encompassing hundreds or thousands of taxa. These independent studies often include overlapping data, presenting a unique opportunity to build macrophylogenies (phylogenies sampling > 1,000 taxa) for entire classes across the Tree of Life. However, the inference of large trees remains constrained by logistical, computational, and methodological challenges. The Avian Tree of Life provides an ideal model for evaluating strategies to robustly infer macrophylogenies from intersecting datasets derived from smaller studies. In this study, we leveraged a comprehensive resource of sequence capture datasets to evaluate the phylogenetic accuracy and computational costs of four methodological approaches: (1) supermatrix approaches using concatenation, including the "fast" maximum likelihood (ML) methods, (2) filtering datasets to reduce heterogeneity, (3) supertree estimation based on published phylogenomic trees, and (4) a "divide-and-conquer" strategy, wherein smaller ML trees were estimated and subsequently combined using a supertree approach. Additionally, we examined the impact of these methods on divergence time estimation using a dataset that includes newly vetted fossil calibrations for the Avian Tree of Life. Our findings highlight that recently developed fast tree search approaches offer a reasonable compromise between computational efficiency and phylogenetic accuracy, facilitating inference of macrophylogenies.

evolutionary biology↗

bioRxiv2024

Testing the mettle of METAL: A comparison of phylogenomic methods using a challenging but well-resolved phylogeny

The evolutionary histories of different genomic regions typically differ from each other and from the underlying species phylogeny. This makes species tree estimation challenging. Here, we examine the performance of phylogenomic methods using a well-resolved phylogeny that nevertheless contains many difficult nodes, the species tree of living birds. We compared trees generated by maximum likelihood (ML) analysis of concatenated data, gene tree summary methods, and SVDquartets. We also conduct the first empirical test of a "new" method called METAL (Metric algorithm for Estimation of Trees based on Aggregation of Loci), which is based on evolutionary distances calculated using concatenated data. We conducted this test using a novel dataset comprising more than 4,000 ultraconserved element (UCE) loci from almost all bird families and two existing UCE and intron datasets sampled from almost all avian orders. We identified "reliable clades" very likely to be present in the true avian species tree and used them to assess method performance. ML analyses of concatenated data recovered almost all reliable clades with less data and greater robustness to missing data than other methods. METAL recovered many reliable clades, but only performed well with the largest datasets. Gene tree summary methods (weighted ASTRAL and weighted ASTRID) performed well; they required less data than METAL but more data than ML concatenation. SVDquartets exhibited the worst performance of the methods tested. In addition to the methodological insights, this study provides a novel estimate of avian phylogeny with almost 99% of the currently recognized avian families. Only one of the 181 reliable clades we examined was consistently resolved differently by ML concatenation versus other methods, suggesting that it may be possible to achieve consensus on the deep phylogeny of extant birds.

evolutionary biology↗

bioRxiv2023

The AEGEAN-169 clade of bacterioplankton is synonymous with SAR11 subclade V (HIMB59) and metabolically distinct

Bacterioplankton of the SAR11 clade are the most abundant marine microorganisms and consist of numerous subclades spanning Order level divergence (Pelagibacterales). The assignment of the earliest diverging subclade V (a.k.a. HIMB59) to the Pelagibacterales is highly controversial, with multiple recent phylogenetic studies placing them completely separate from SAR11. Other than through phylogenomics, subclade V has not received detailed examination due to limited genomes from this group. Here, we assessed the ecogenomic characteristics of subclade V to better understand the role of this group in comparison to the Pelagibacterales. We used a new isolate genome, recently released single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs), and previously established SAR11 genomes to perform a comprehensive comparative genomics analysis. We paired this analysis with recruitment of metagenomes spanning open ocean, coastal, and brackish systems. Phylogenomics, average amino acid identity, and 16S rRNA gene phylogeny indicate that SAR11 subclade V is synonymous with the ubiquitous AEGEAN-169 clade, and support the contention that this group represents a taxonomic Family. AEGEAN-169 shared many bulk genome qualities with SAR11, such as streamlining and low GC content, but genomes were generally larger. AEGEAN-169 had overlapping distributions with SAR11 but was metabolically distinct from SAR11 in its potential to transport and utilize a broader range of sugars as well as in transport of trace metals and thiamin. Thus, regardless of the ultimate phylogenetic placement of AEGEAN-169, these organisms have distinct metabolic capacities that likely allow them to differentiate their niche from canonical SAR11 taxa. IMPORTANCEOne goal of marine microbiologists is to uncover the roles various microorganisms are playing in biogeochemical cycles. Success in this endeavor relies on differentiating groups of microbes and circumscribing their relationships. An early-diverging group (subclade V) of the most abundant bacterioplankton, SAR11, has recently been proposed as a separate lineage that does not share a most recent common ancestor. But beyond phylogenetics, little has been done to evaluate how these organisms compare with SAR11. Our work leverages dozens of new genomes to demonstrate the similarities and differences between subclade V and SAR11. In our analysis, we also establish that subclade V is synonymous with a group of bacteria established from 16S rRNA gene sequences, AEGEAN-169. Subclade V/AEGEAN-169 has clear metabolic distinctions from SAR11 and their shared traits point to remarkable convergent evolution if they do not share a most recent common ancestor.

microbiology↗

bioRxiv2022

Widespread sympatry in a species-rich clade of marine fishes (Carangoidei)

The patterns of speciation in marine fishes are largely unknown, in part due to the deficiency of species-level phylogenies and information on species distributions, and partly due to conflicting relationships between species dispersal, range size, and patterns of co- occurrence. Most research on global patterns of marine fish speciation has focused on coral reef or pelagic species. Carangoidei is a clade of marine fishes including the trevallies, remoras, and dolphinfishes that utilize both coral reef and pelagic environments, spanning the ecologies of coral reef obligate and open-ocean species. We used sequence capture of 1314 ultraconserved elements (UCEs) from 154 taxa to generate a phylogeny of Carangoidei and its parent clade, Carangiformes. Age-range correlation analyses of the geographic distributions and divergence times of sister species pairs reveal widespread sympatry, with 73% of sister species pairs exhibiting a sympatric geographic distribution, regardless of node age, and most species pairs co-existing across large portions of their ranges. We also observe greater disparity in body size and water column depth utilization between sympatric than allopatric sister species. These and other ecological or behavioral attributes likely facilitate sympatry among the most closely related carangoid species, which exhibit sympatry at a larger taxonomic scale than has previously been described in marine fishes.

evolutionary biology↗

bioRxiv2022

An island "endemic" born out of hybridization between introduced lineages

Humans have profoundly impacted the distribution of plant and animal species over thousands of years. The most direct example of these effects is human-mediated movement of individuals, either through translocation of individuals within their range or the introduction of species to new habitats. While human involvement may be suspected in species with obvious range disjunctions, it can be difficult to detect natural versus human-mediated dispersal events for populations at the edge of a species range, and this uncertainty muddles how we understand the evolutionary history of populations and broad biogeographic patterns. Studies combining genetic data with archeological, linguistic, and historical evidence have confirmed prehistoric examples of human-mediated dispersal; however, it is unclear whether these methods can disentangle recent dispersal events, such as species translocated by European colonizers during the past 500 years. We use genomic DNA from historical specimens and historical records to evaluate three hypotheses regarding the timing and origin of Northern Bobwhites (Colinus virginianus) in Cuba, whose status as an endemic or introduced population has long been debated. We discovered that bobwhites from southern Mexico arrived in Cuba between the 12th and 16th centuries, followed by the subsequent introduction of bobwhites from the southeastern USA to Cuba between the 18th and 20th centuries. These dates suggest the introduction of bobwhites to Cuba was human-mediated and concomitant with Spanish colonial shipping routes between Veracruz, Mexico and Havana, Cuba during this period. Our results identify endemic Cuban bobwhites as a genetically distinct population born of hybridization between divergent, introduced lineages.

evolutionary biology↗

bioRxiv2022

Ecophysiology and genomics of the brackish water adapted SAR11 subclade IIIa

The Order Pelagibacterales (SAR11) is the most abundant group of heterotrophic bacterioplankton in global oceans and comprises multiple subclades with unique spatiotemporal distributions. Subclade IIIa is the primary SAR11 group in brackish waters and shares a common ancestor with the dominant freshwater IIIb (LD12) subclade. Despite its dominance in brackish environments, subclade IIIa lacks systematic genomic or ecological studies. Here, we combine closed genomes from new IIIa isolates, new IIIa MAGS from San Francisco Bay (SFB), and 466 high-quality publicly available SAR11 genomes for the most comprehensive pangenomic study of subclade IIIa to date. Subclade IIIa represents a taxonomic family containing three genera (denoted as subgroups IIIa.1, IIIa.2, and IIIa.3) that had distinct ecological distributions related to salinity. The expansion of taxon selection within subclade IIIa also established previously noted metabolic differentiation in subclade IIIa compared to other SAR11 subclades such as glycine/serine prototrophy, mosaic glyoxylate shunt presence, and polyhydroxyalkanoate synthesis potential. Our analysis further shows metabolic flexibility among subgroups within IIIa. Additionally, we find that subclade IIIa.3 bridges the marine and freshwater clades based on its potential for compatible solute transport, iron utilization, and bicarbonate management potential. Pure culture experimentation validated differential salinity ranges in IIIa.1 and IIIa.3 and provided the first IIIa cell size and volume data. This study is an important step forward for understanding the genomic, ecological, and physiological differentiation of subclade IIIa and the overall evolutionary history of SAR11.

microbiology↗

bioRxiv2021

Historical specimens and the limits of subspecies phylogenomics in the New World quails (Odontophoridae)

As phylogenomics focuses on comprehensive taxon sampling at the species and population/subspecies levels, incorporating genomic data from historical specimens has become increasingly common. While historical samples can fill critical gaps in our understanding of the evolutionary history of diverse groups, they also introduce additional sources of phylogenomic uncertainty, making it difficult to discern novel evolutionary relationships from artifacts caused by sample quality issues. These problems highlight the need for improved strategies to disentangle artifactual patterns from true biological signal as historical specimens become more prevalent in phylogenomic datasets. Here, we tested the limits of historical specimen-driven phylogenomics to resolve subspecies-level relationships within a highly polytypic family, the New World quails (Odontophoridae), using thousands of ultraconserved elements (UCEs). We found that relationships at and above the species-level were well-resolved and highly supported across all analyses, with the exception of discordant relationships within the two most polytypic genera which included many historical specimens. We examined the causes of discordance and found that inferring phylogenies from subsets of taxa resolved the disagreements, suggesting that analyzing subclades can help remove artifactual causes of discordance in datasets that include historical samples. At the subspecies-level, we found well-resolved geographic structure within the two most polytypic genera, including the most polytypic species in this family, Northern Bobwhites (Colinus virginianus), demonstrating that variable sites within UCEs are capable of resolving phylogenetic structure below the species level. Our results highlight the importance of complete taxonomic sampling for resolving relationships among polytypic species, often through the inclusion of historical specimens, and we propose an integrative strategy for understanding and addressing the uncertainty that historical samples sometimes introduce to phylogenetic analyses.

evolutionary biology↗

bioRxiv2021

Prolonged morphological expansion of spiny-rayed fishes following the end-Cretaceous

Spiny-rayed fishes (Acanthomorpha) dominate modern marine habitats and comprise more than a quarter of all living vertebrate species1-3. It is believed that this dominance resulted from explosive lineage and phenotypic diversification coincident with the Cretaceous-Paleogene (K-Pg) mass-extinction event4. It remains unclear, however, if living acanthomorph diversity is the result of a punctuated burst or gradual accumulation of diversity following the K-Pg. We assess these hypotheses with a time-calibrated phylogeny inferred using ultraconserved elements from a sampling of species that represent over 91% of all acanthomorph families, as well as an extensive body shape dataset of extant species. Our results indicate that several million years after the end-Cretaceous, acanthomorphs underwent a prolonged and significant expansion of morphological disparity primarily driven by changes in body elongation, and that acanthomorph lineages containing the bulk of the living species diversity originated throughout the Cenozoic. These acanthomorph lineages radiated into distinct regions of morphospace and retained their iconic phenotypes, including a large group of laterally compressed reef fishes, fast-swimming open-ocean predators, bottom-dwelling flatfishes, seahorses, and pufferfishes. The evolutionary success of spiny-rayed fishes is the culmination of a post K-Pg adaptive radiation in which rates of lineage diversification were decoupled from periods of high phenotypic disparity.

evolutionary biology↗

bioRxiv2019

Adapterama II: Universal amplicon sequencing on Illumina platforms (TaggiMatrix)

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. Most NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5 end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (eight forward primers + 12 reverse primers yield 8 x 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

genomics↗

bioRxiv2018

A target enrichment bait set for studying relationships among ostariophysan fishes

O_LITarget enrichment of conserved nuclear loci has helped reconstruct evolutionary relationships among a wide variety of species. While there are preexisting bait sets to enrich a few hundred loci across all fishes or a thousand loci from acanthomorph fishes, no bait set exists to enrich large numbers (>1000 loci) of ultraconserved nuclear loci from ostariophysans, the second largest actinopterygian superorder.\nC_LIO_LIIn this manuscript, we describe how we designed a bait set to enrich 2,708 ultraconserved nuclear loci from ostariophysan fishes by combining an existing genome assembly with low coverage sequence data collected from two ostariophysan lineages.\nC_LIO_LIWe perform a series of enrichment experiments using this bait set across the ostariophysan Tree of Life, from the deepest splits among the major groups (>150 MYA) to more recent divergence events that have occured during the last 50 million years.\nC_LIO_LIOur results demonstrate that the bait set we designed is useful for addressing phylogenetic questions from the origin of crown ostariophysans to more recent divergence events, and our in silico results suggest that this bait set may be useful for addressing evolutionary questions in closely related groups of fishes, like Clupeiformes.\nC_LI

evolutionary biology↗

bioRxiv2018

Allele Phasing Greatly Improves the Phylogenetic Utility of Ultraconserved Elements

Advances in high-throughput sequencing techniques now allow relatively easy and affordable sequencing of large portions of the genome, even for non-model organisms. Many phylogenetic studies reduce costs by focusing their sequencing efforts on a selected set of targeted loci, commonly enriched using sequence capture. The advantage of this approach is that it recovers a consistent set of loci, each with high sequencing depth, which leads to more confidence in the assembly of target sequences. High sequencing depth can also be used to identify phylogenetically informative allelic variation within sequenced individuals, but allele sequences are infrequently assembled in phylogenetic studies.\n\nInstead, many scientists perform their phylogenetic analyses using contig sequences which result from the de novo assembly of sequencing reads into contigs containing only canonical nucleobases, and this may reduce both statistical power and phylogenetic accuracy. Here, we develop an easy-to-use pipeline to recover allele sequences from sequence capture data, and we use simulated and empirical data to demonstrate the utility of integrating these allele sequences to analyses performed under the Multispecies Coalescent (MSC) model. Our empirical analyses of Ultraconserved Element (UCE) locus data collected from the South American hummingbird genus Topaza demonstrate that phased allele sequences carry sufficient phylogenetic information to infer the genetic structure, lineage divergence, and biogeographic history of a genus that diversified during the last three million years. The phylogenetic results support the recognition of two species, and suggest a high rate of gene flow across large distances of rainforest habitats but rare admixture across the Amazon River. Our simulations provide evidence that analyzing allele sequences leads to more accurate estimates of tree topology and divergence times than the more common approach of using contig sequences.

evolutionary biology↗

bioRxiv2017

Phylogenomics of montane frogs of the Brazilian Atlantic Forest supports a scenario of isolation in sky islands followed by relative climatic stability

Despite encompassing a relatively small geographical area, montane regions harbor disproportionately high levels of species diversity and endemism. Yet, relatively little is known about the evolutionary mechanisms ultimately leading to montane diversity. In this study, we use target capture of ultraconserved elements to investigate the phylogenetic relationships and diversification patterns of Melanophryniscus (Bufonidae) and Brachycephalus (Brachycephalidae), two frog genera that occur in sky islands of the southern Atlantic Forest of Brazil. Specifically, we test whether diversification of montane species in these genera can be explained by a single climatic shift leading to isolation in sky islands, followed by relative climatic stability that maintained populations in allopatry. In both genera, the topologies inferred using concatenation and coalescent-based methods were concordant and had strong nodal support, except for a few recent splits. These recent splits tended to be supported by more informative loci (those with higher average bootstrap support), suggesting that, while individual trees may be well resolved, the relationships they recover are being obscured by non-informative data. Divergence dating of a combined dataset using both genera is consistent with concordant timing of their diversification. These results support the scenario of diversification-by-isolation in sky islands, and suggest that allopatry due to climatic gradients in montane regions are an important mechanism for generating species diversity and endemism in these regions.

evolutionary biology↗

bioRxiv2017

Adapterama III: Quadruple-indexed, triple-enzyme RADseq libraries for about $1USD per Sample (3RAD)

Molecular ecologists frequently use genome reduction strategies that rely upon restriction enzyme digestion of genomic DNA to sample consistent portions of the genome from many individuals (e.g., RADseq, GBS). However, researchers often find the existing methods expensive to initiate and/or difficult to implement consistently, especially due to the inability to highly-multiplex samples to fill entire sequencing lanes. Here, we introduce a low-cost and highly robust approach for the construction of dual-digest RADseq libraries that relies on adapters and primers designed in Adapterama I. Major features of our method include: 1) minimizing the number of processing steps; 2) focusing on a single strand of sample DNA for library construction, allowing the use of a non-phosphorylated adapter on one end; 3) ligating adapters in the presence of active restriction enzymes, thereby reducing chimeras; 4) including an optional third restriction enzyme to cut apart adapter-dimers formed by the phosphorylated adapter, thus increasing the efficiency of adapter ligation to sample DNA, which is particularly effective when only low quantity/quality DNA samples are available; 5) interchangeable adapter designs; 6) incorporating variable-length internal indexes within the adapters to increase the scope of sample indexing, facilitate pooling, and increase sequence diversity; 7) maintaining compatibility with universal dual-indexed primers and thus, Illumina sequencing reagents and libraries; and, 8) easy modification for the identification of PCR duplicates. We present eight adapter designs that work with 72 restriction enzyme combinations. We demonstrate the efficiency of our approach by comparing it with existing methods, and we validate its utility through the discovery of many variable loci in a variety of non-model organisms. Our 2RAD/3RAD method is easy to perform, has low startup costs, has increased utility with low-concentration input DNA, and produces libraries that can be highly-multiplexed and pooled with other Illumina libraries.

genomics↗

bioRxiv2017

Phylogenomic Species Delimitation In Microendemic Frogs Of The Brazilian Atlantic Forest

The advent of next-generation sequencing allows researchers to use large-scale datasets for species delimitation analyses, yet one can envision an inflection point where the added accuracy of including more loci does not offset the increased computational burden. One alternative to including all loci could be to prioritize the analysis of loci for which there is an expectation of high informativeness, such as those with higher numbers of parsimony-informative sites. Here, we explore the issue of species delimitation and locus selection with species from two anuran genera: Melanophryniscus (Bufonidae) and Brachycephalus (Brachycephalidae). Montane species in these genera have been isolated in sky islands across the southern Brazilian Atlantic Forest, which led to the formation of a number of microendemic species. To delimit species, we obtained genetic data using target enrichment of ultraconserved elements from 32 populations (13 for Melanophryniscus and 19 for Brachycephalus), and we were able to create datasets that included over 800 loci with no missing data. We ranked loci according to their corresponding number of parsimony-informative sites, and we performed species delimitation analyses using BPP in each genus based on the top 10, 20, 40, 80, 160, 320, and 640 loci. We also conducted several additional analyses using 10 randomly sampled datasets containing the same numbers of loci to discriminate the relative contribution of increasing the number of loci from prioritizing those with higher informativeness. We identified three types of node: nodes with either consistently high or low support regardless of the number of loci or their informativeness, and nodes that were initially poorly supported, but their support became stronger with more data. Adding more loci had a stronger impact on model support than prioritizing loci for their informativeness, but this effect was less apparent in datasets with more than 160 loci. When viewed across all sensitivity analyses, our results suggest that the current species richness in both genera might have been underestimated. In addition, our results provide useful guidelines to the use of different sampling strategies to carry out species delimitation with phylogenomic datasets.

evolutionary biology↗