During periods of reduced O2 supply, the most profound changes in gene expression are mediated by hypoxia-inducible factor (HIF) transcription factors that play a key role in cellular responses to low-O2 tension. Using target-enrichment sequencing, we tested whether variation in 26 genes in the HIF signaling pathway was associated with high altitude and therefore corresponding O2 availability in three duck species that colonized the Andes from ancestral low-altitude habitats in South America. We found strong support for convergent evolution in the case of two of the three duck species with the same genes (EGLN1, EPAS1), and even the same exons (exon 12, EPAS1), exhibiting extreme outliers with a high probability of directional selection in the high-altitude populations. These results mirror patterns of adaptation seen in human populations, which showed mutations in EPAS1, and transcriptional regulation differences in EGLN1, causing changes in downstream target transactivation, associated with a blunted hypoxic response.

Sequencing of target enriched libraries is an efficient method for obtaining DNA sequence data for phylogenetic reconstruction.

Arid environments provide ideal ground for investigating the mechanisms of adaptive evolution. High temperatures and low water availability are relentless stressors for many endotherms, including birds; yet birds persist in deserts. While physiological adaptation likely involves metabolic phenotypes, the underlying mechanisms (plasticity, genetics) are largely uncharacterized. To explore this, we took an intra-specific approach that focused on a species that is resident over a mesic to arid gradient, the Karoo scrub-robin (Cercotrichas coryphaeus). Specifically, we integrated environmental (climatic and primary productivity), physiological (metabolic rates: a measure of energy expenditure), genotypic (genetic variation underlying the machinery of energy production) and microbiome (involved in processing food from where energy is retrieved) data, to infer the mechanism of physiological adaptation. We that found the variation in energetic physiology phenotypes and gut microbiome composition are associated with environmental features as well as with variation in genes underlying energy metabolic pathways. Specifically, we identified a small list of candidate adaptive genes, some of them with known ties to relevant physiology phenotypes. Together our results suggest that selective pressures on energetic physiology mediated by genes related to energy homeostasis and possibly microbiota composition may facilitate adaptation to local conditions and provide an explanation to the high avian intra-specific divergence observed in harsh environments. This article is protected by copyright. All rights reserved.

Premise Hybrid capture with high-throughput sequencing (Hyb-Seq) is a powerful tool for evolutionary studies. The applicability of an Asteraceae family-specific Hyb-Seq probe set and the outcomes of different phylogenetic analyses are investigated here. Methods Hyb-Seq data from 112 Asteraceae samples were organized into groups at different taxonomic levels (tribe, genus, and species). For each group, data sets of non-paralogous loci were built and proportions of parsimony informative characters estimated. The impacts of analyzing alternative data sets, removing long branches, and type of analysis on tree resolution and inferred topologies were investigated in tribe Cichorieae. Results Alignments of the Asteraceae family-wide Hyb-Seq locus set were parsimony informative at all taxonomic levels. Levels of resolution and topologies inferred at shallower nodes differed depending on the locus data set and the type of analysis, and were affected by the presence of long branches. Discussion The approach used to build a Hyb-Seq locus data set influenced resolution and topologies inferred in phylogenetic analyses. Removal of long branches improved the reliability of topological inferences in maximum likelihood analyses. The Astereaceae Hyb-Seq probe set is applicable at multiple taxonomic depths, which demonstrates that probe sets do not necessarily need to be lineage-specific.

Traditional approaches for interrogating the mitochondrial genome often involve laborious extraction and enrichment protocols followed by Sanger sequencing. Although preparation techniques are still demanding, the advent of next-generation or massively parallel sequencing has made it possible to routinely obtain nucleotide-level data with relative ease. These short-read sequencing platforms offer deep coverage with unparalleled read accuracy in high-complexity genomic regions but encounter numerous difficulties in the low-complexity homopolymeric sequences characteristic of the mitochondrial genome. The inability to discern identical units within monomeric repeats and resolve copy-number variations for heteroplasmy detection results in suboptimal genome assemblies that ultimately complicate downstream data analysis and interpretation of biological significance. Oxford Nanopore Technologies offers the ability to generate long-read sequencing data on a pocket-sized device known as the MinION. Nanopore-based sequencing is scalable, portable, and theoretically capable of sequencing the entire mitochondrial genome in a single contig. Furthermore, the recent development of a nanopore protein with dual reader heads allows for clear identification of nucleotides within homopolymeric stretches, significantly increasing resolution throughout these regions. The unrestricted read lengths, superior homopolymeric resolution, and affordability of the MinION device make it an attractive alternative to the labor-intensive, time-consuming, and costly mainstay deep-sequencing platforms. This article describes three approaches to extract, prepare, and sequence mitochondrial DNA on the Oxford Nanopore MinION device. Two of the workflows include enrichment of mitochondrial DNA prior to sequencing, whereas the other relies on direct sequencing of native genomic DNA to allow for simultaneous assessment of the nuclear and mitochondrial genomes. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Enrichment-free mitochondrial DNA sequencing Alternate Protocol 1: Mitochondrial DNA sequencing following enrichment with polymerase chain reaction (PCR) Alternate Protocol 2: Mitochondrial DNA sequencing following enrichment with PCR-free hybridization capture Support Protocol 1: DNA quantification and quality assessment using the Agilent 4200 TapeStation System Support Protocol 2: AMPure XP bead clean-up Support Protocol 3: Suggested data analysis pipeline

Exon capture across species has been one of the most broadly applied approaches to acquire multi-locus data in phylogenomic studies of non-model organisms. Methods for assembling loci from short-read sequences (eg, Illumina platforms) that rely on mapping reads to a reference genome may not be suitable for studies comprising species across a wide phylogenetic spectrum; thus, de novo assembling methods are more generally applied. Current approaches for assembling targeted exons from short reads are not particularly optimized as they cannot (1) assemble loci with low read depth, (2) handle large files efficiently, and (3) reliably address issues with paralogs. Thus, we present Assexon: a streamlined pipeline that de novo assembles targeted exons and their flanking sequences from raw reads. We tested our method using reads from Lepisosteus osseus (4.37 Gb) and Boleophthalmus pectinirostris (2.43 Gb), which are captured using baits that were designed based on genome sequence of Lepisosteus oculatus and Oreochromis niloticus, respectively. We compared performance of Assexon to PHYLUCE and HybPiper, which are commonly used pipelines to assemble ultra-conserved element (UCE) and Hyb-seq data. A custom exon capture analysis pipeline (CP) developed by Yuan et al was compared as well. Assexon accurately assembled more than 3400 to 3800 (20%-28%) loci than PHYLUCE and more than 1900 to 2300 (8%-14%) loci than HybPiper across different levels of phylogenetic divergence. Assexon ran at least twice as fast as PHYLUCE and HybPiper. Number of loci assembled using CP was comparable with Assexon in both tests, while Assexon ran at least 7 times faster than CP. In addition, some steps of CP require the user’s interaction and are not fully automated, and this user time was not counted in our calculation. Both Assexon and CP retrieved no paralogs in the testing runs, but PHYLUCE and Hybpiper did. In conclusion, Assexon is a tool for accurate and efficient assembling of large read sets from exon capture experiments. Furthermore, Assexon includes scripts to filter poorly aligned coding regions and flanking regions, calculate summary statistics of loci, and select loci with reliable phylogenetic signal. Assexon is available at https://github.com/yhadevol/Assexon.

Abstract. Army ants are a charismatic group of organisms characterized by a suite of morphological and behavioral adaptations that includes obligate collective

Premise The genus Antennaria has a complex evolutionary history due to dioecism, excessive polyploidy, and the evolution of polyploid agamic complexes. We developed microsatellite markers from A. corymbosa to investigate genetic diversity and population genetic structure in Antennaria species. Methods and Results Twenty-four novel microsatellite markers (16 nuclear and eight chloroplast) were developed from A. corymbosa using an enriched genomic library. Ten polymorphic nuclear markers were used to characterize genetic variation in five populations of A. corymbosa. One to four alleles were found per locus, and the expected heterozygosity and fixation index ranged from 0.00 to 0.675 and −0.033 to 0.610, respectively. We were also able to successfully amplify these markers in five additional Antennaria species. Conclusions These markers are promising tools to study the population genetics of sexual Antennaria species and to investigate interspecific gene flow, clonal diversity, and parentage of Antennaria polyploid agamic complexes.