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Resolving the order of speciation events that occurred in rapid succession is inherently hard and typically requires a phylogenomic approach. A case in point concerns the previously unresolved phylogeny of the three species of banded newt (genus Ommatotriton). We obtain c. 7k nuclear DNA markers using target enrichment by sequence capture and analyze the dataset using maximum likelihood inference of concatenated data with RAxML, summary multi-species coalescent analysis with ASTRAL and Bayesian species tree inference using a diffusion model with SNAPPER, and use TreeMix and PhyloNet to test for interspecific gene flow. All analyses recover three distinct species with no evidence of interspecific gene flow. All analyses retrieved the topology (O. nesterovi, (O. ophryticus, O. vittatus)), with high support. SNAPPER did show the tendency to get stuck in a local optimum, resulting in a different but still highly supported topology. Furthermore, we notice that fewer SNAPPER runs get stuck in a local optimum when we include an outgroup. Therefore, we recommend the exploration of multiple independent runs and the use of an outgroup with this approach. The banded newt radiation illustrates the use of genome-wide data to tackle formerly unresolved phylogenies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The world's largest butterfly genus Delias, commonly known as Jezebels, comprises ca. 251 species found throughout Asia, Australia, and Melanesia. Most species are endemic to islands in the Indo-Australian Archipelago or to New Guinea and nearby islands in Melanesia, and many species are restricted to montane habitats over 1200 m. We inferred an extensively sampled and well-supported molecular phylogeny of the group to better understand the spatial and temporal dimensions of its diversification. The remarkable diversity of Delias evolved in just ca. 15-16 Myr (crown age). The most recent common ancestor of a clade with most of the species dispersed out of New Guinea ca. 14 Mya, but at least six subsequently diverging lineages dispersed back to the island. Diversification was associated with frequent dispersal of lineages among the islands of the Indo-Australian Archipelago, and the divergence of sister taxa on a single landmass was rare and occurred only on the largest islands, most notably on New Guinea. We conclude that frequent inter-island dispersal during the Neogene-likely facilitated by frequent sea level change-sparked much diversification during that period. Many extant New Guinea lineages started diversifying 5 Mya, suggesting that orogeny facilitated their diversification. Our results largely agree with the most recently proposed species group classification system, and we use our large taxon sample to extend this system to all described species. Finally, we summarize recent insights to speculate how wing pattern evolution, mimicry, and sexual selection might also contribute to these butterflies' rapid speciation and diversification., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Local adaptation is a key evolutionary process that enhances the growth of plants in their native habitat compared to non-native habitats, resulting in patterns of adaptive genetic variation across the entire geographic range of the species. The study of population adaptation to local environments and predicting their response to future climate change is important because of climate change., Results: Here, we explored the genetic diversity of candidate genes associated with bud burst in pedunculate oak individuals sampled from 6 populations in Poland. Single nucleotide polymorphism (SNP) diversity was assessed in 720 candidate genes using the sequence capture technique, yielding 18,799 SNPs. Using landscape genomic approaches, we identified 8 F ST outliers and 781 unique SNPs in 389 genes associated with geography, climate, and phenotypic variables (individual/family spring and autumn phenology, family diameter at breast height (DBH), height, and survival) that are potentially involved in local adaptation. Then, using a nonlinear multivariate model, Gradient Forests, we identified vulnerable areas of the pedunculate oak distribution in Poland that are at risk from climate change., Conclusions: The model revealed that pedunculate oak populations in the eastern part of the analyzed geographical region are the most sensitive to climate change. Our results might offer an initial evaluation of a potential management strategy for preserving the genetic diversity of pedunculate oak., (© 2024. The Author(s).)

Germline DNA testing using the next-gene-ration sequencing (NGS) technology has become the analytical standard for the diagnostics of hereditary diseases, including cancer. Its increasing use places high demands on correct sample identification, independent confirmation of prioritized variants, and their functional and clinical interpretation. To streamline these processes, we introduced parallel DNA and RNA capture-based NGS using identical capture panel CZECANCA, which is routinely used for DNA analysis of hereditary cancer predisposition. Here, we present the analytical workflow for RNA sample processing and its analytical and diagnostic performance. Parallel DNA/RNA analysis allowed credible sample identification by calculating the kinship coefficient. The RNA capture-based approach enriched transcriptional targets for the majority of clinically relevant cancer predisposition genes to a degree that allowed analysis of the effect of identified DNA variants on mRNA processing. By comparing the panel and whole-exome RNA enrichment, we demonstrated that the tissue-specific gene expression pattern is independent of the capture panel. Moreover, technical replicates confirmed high reproducibility of the tested RNA analysis. We concluded that parallel DNA/RNA NGS using the identical gene panel is a robust and cost-effective diagnostic strategy. In our setting, it allows routine analysis of 48 DNA/RNA pairs using NextSeq 500/550 Mid Output Kit v2.5 (150 cycles) in a single run with sufficient coverage to analyse 226 cancer predisposition and candidate ge-nes. This approach can replace laborious Sanger confirmatory sequencing, increase testing turnaround, reduce analysis costs, and improve interpretation of the impact of variants by analysing their effect on mRNA processing.

Despite the economic, ecological, and scientific importance of the genera Salix L. (willows) and Populus L. (poplars, cottonwoods, and aspens) Salicaceae, we know little about the sources of differences in species diversity between the genera and of the phylogenetic conflict that often confounds estimating phylogenetic trees. Salix subgenera and sections, in particular, have been difficult to classify, with one recent attempt termed a "spectacular failure" due to a speculated radiation of the subgenera Vetrix and Chamaetia. Here, we use targeted sequence capture to understand the evolutionary history of this portion of the Salicaceae plant family. Our phylogenetic hypothesis was based on 787 gene regions and identified extensive phylogenetic conflict among genes. Our analysis supported some previously described subgeneric relationships and confirmed the polyphyly of others. Using an fbranch analysis, we identified several cases of hybridization in deep branches of the phylogeny, which likely contributed to discordance among gene trees. In addition, we identified a rapid increase in diversification rate near the origination of the Vetrix-Chamaetia clade in Salix. This region of the tree coincided with several nodes that lacked strong statistical support, indicating a possible increase in incomplete lineage sorting due to rapid diversification. The extraordinary level of both recent and ancient hybridization in both Salix and Populus have played important roles in the diversification and diversity in these two genera., (© The Author(s) 2023. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

As whole-genome sequencing has become pervasive, some have suggested that reduced genomic representation approaches, for example, sequence capture, are becoming obsolete. In the present study, we argue that these techniques still provide excellent tools in terms of price and quality of data as well as in their ability to provide markers with specific features, as required, for example, in phylogenomics. A potential drawback of the wide-scale application of reduced representation approaches could be their drop in efficiency with increasing phylogenetic distance from the reference species. While some studies have focused on the degree and performance of reduced representation techniques in such situations, to our knowledge, none of them evaluated their applicability to inter-specific hybrids and polyploids. This highlights a significant gap in current knowledge since there is increasing evidence for the frequent occurrence of natural hybrids and polyploids, as well as for the major importance of both phenomena in evolution. The main aim of the present study was to carry out a thorough validation of SEQcap applicability to (1) a set of non-model taxa with a wide range of phylogenetic relatedness and (2) inter-specific hybrids of various ploidies and genomic compositions. Considering the latter point, we especially focused on mechanisms causing allelic bias and consequent allelic dropout, as these could have confounding effects with respect to the evolutionary genomic dynamics of hybrids, especially in asexuals, which virtually reproduce as a frozen F1 generation., (© 2023 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Intra- and interspecific communication is crucial to fitness via its role in facilitating mating, territoriality and defence. Yet, the evolution of animal communication systems is puzzling-how do they originate and change over time? Studying stridulatory morphology provides a tractable opportunity to deduce the origin and diversification of a communication mechanism. Stridulation occurs when two sclerotized structures rub together to produce vibratory and acoustic (vibroacoustic) signals, such as a cricket 'chirp'. We investigated the evolution of stridulatory mechanisms in the superfamily Coreoidea (Hemiptera: Heteroptera), a group of insects known for elaborate male fighting behaviours and enlarged hindlegs. We surveyed a large sampling of taxa and used a phylogenomic dataset to investigate the evolution of stridulatory mechanisms. We identified four mechanisms, with at least five evolutionary gains. One mechanism, occurring only in male Harmostini (Rhopalidae), is described for the first time. Some stridulatory mechanisms appear to be non-homoplastic apomorphies within Rhopalidae, while others are homoplastic or potentially homoplastic within Coreidae and Alydidae, respectively. We detected no losses of these mechanisms once evolved, suggesting they are adaptive. Our work sets the stage for further behavioural, evolutionary and ecological studies to better understand the context in which these traits evolve and change., Competing Interests: We have no competing interests., (© 2023 The Authors.)

Effective management of protected species requires information on appropriate evolutionary and geographic population boundaries and knowledge of how the physical environment and life-history traits combine to shape the population structure and connectivity. Saltwater crocodiles ( Crocodylus porosus ) are the largest and most widely distributed of living crocodilians, extending from Sri Lanka to Southeast Asia and down to northern Australia. Given the long-distance movement capabilities reported for C. porosus , management units are hypothesised to be highly connected by migration. However, the magnitude, scale, and consistency of connection across managed populations are not fully understood. Here we used an efficient genotyping method that combines DArTseq and sequence capture to survey ≈ 3000 high-quality genome-wide single nucleotide polymorphisms from 1176 C. porosus sampled across nearly the entire range of the species in Queensland, Australia. We investigated historical and present-day connectivity patterns using fixation and diversity indices coupled with clustering methods and the spatial distribution of kin pairs. We inferred kinship using forward simulation coupled with a kinship estimation method that is robust to unspecified population structure. The results demonstrated that the C. porosus population has substantial genetic structure with six broad populations correlated with geographical location. The rate of gene flow was highly correlated with spatial distance, with greater differentiation along the east coast compared to the west. Kinship analyses revealed evidence of reproductive philopatry and limited dispersal, with approximately 90% of reported first and second-degree relatives showing a pairwise distance of <50 km between sampling locations. Given the limited dispersal, lack of suitable habitat, low densities of crocodiles and the high proportion of immature animals in the population, future management and conservation interventions should be considered at regional and state-wide scales., (© 2023 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

Sapindales is an angiosperm order of high economic and ecological value comprising nine families, c. 479 genera, and c. 6570 species. However, family and subfamily relationships in Sapindales remain unclear, making reconstruction of the order's spatio-temporal and morphological evolution difficult. In this study, we used Angiosperms353 target capture data to generate the most densely sampled phylogenetic trees of Sapindales to date, with 448 samples and c. 85% of genera represented. The percentage of paralogous loci and allele divergence was characterized across the phylogeny, which was time-calibrated using 29 rigorously assessed fossil calibrations. All families were supported as monophyletic. Two core family clades subdivide the order, the first comprising Kirkiaceae, Burseraceae, and Anacardiaceae, the second comprising Simaroubaceae, Meliaceae, and Rutaceae. Kirkiaceae is sister to Burseraceae and Anacardiaceae, and, contrary to current understanding, Simaroubaceae is sister to Meliaceae and Rutaceae. Sapindaceae is placed with Nitrariaceae and Biebersteiniaceae as sister to the core Sapindales families, but the relationships between these families remain unclear, likely due to their rapid and ancient diversification. Sapindales families emerged in rapid succession, coincident with the climatic change of the Mid-Cretaceous Hothouse event. Subfamily and tribal relationships within the major families need revision, particularly in Sapindaceae, Rutaceae and Meliaceae. Much of the difficulty in reconstructing relationships at this level may be caused by the prevalence of paralogous loci, particularly in Meliaceae and Rutaceae, that are likely indicative of ancient gene duplication events such as hybridization and polyploidization playing a role in the evolutionary history of these families. This study provides key insights into factors that may affect phylogenetic reconstructions in Sapindales across multiple scales, and provides a state-of-the-art phylogenetic framework for further research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Yanã Campos Rizzieri, School of Integrative Plant Sciences, Plant Biology Section, Cornell University, in collaboration with reviewer JO., (Copyright © 2023 Joyce, Appelhans, Buerki, Cheek, de Vos, Pirani, Zuntini, Bachelier, Bayly, Callmander, Devecchi, Pell, Groppo, Lowry, Mitchell, Siniscalchi, Munzinger, Orel, Pannell, Nauheimer, Sauquet, Weeks, Muellner-Riehl, Leitch, Maurin, Forest, Nargar, Thiele, Baker and Crayn.)

The plant family Bignoniaceae is a conspicuous and charismatic element of the tropical flora. The family has a complex taxonomic history, with substantial changes in the classification of the group during the past two centuries. Recent re-classifications at the tribal and generic levels have been largely possible by the availability of molecular phylogenies reconstructed using Sanger sequencing data. However, our complete understanding of the systematics, evolution, and biogeography of the family remains incomplete, especially due to the low resolution and support of different portions of the Bignoniaceae phylogeny. To overcome these limitations and increase the amount of molecular data available for phylogeny reconstruction within this plant family, we developed a bait kit targeting 762 nuclear genes, including 329 genes selected specifically for the Bignoniaceae; 348 genes obtained from the Angiosperms353 with baits designed specifically for the family; and, 85 low-copy genes of known function. On average, 77.4% of the reads mapped to the targets, and 755 genes were obtained per species. After removing genes with putative paralogs, 677 loci were used for phylogenetic analyses. On-target genes were compared and combined in the Exon-Only dataset, and on-target + off-target regions were combined in the Supercontig dataset. We tested the performance of the bait kit at different taxonomic levels, from family to species-level, using 38 specimens of 36 different species of Bignoniaceae, representing: 1) six (out of eight) tribal level-clades (e.g., Bignonieae, Oroxyleae, Tabebuia Alliance, Paleotropical Clade, Tecomeae, and Jacarandeae), only Tourrettieae and Catalpeae were not sampled; 2) all 20 genera of Bignonieae; 3) seven (out of nine) species of Dolichandra (e.g., D. chodatii , D. cynanchoides , D. dentata , D. hispida , D. quadrivalvis , D. uncata , and D. uniguis-cati ), only D. steyermarkii and D. unguiculata were not sampled; and 4) three individuals of Dolichandra unguis-cati . Our data reconstructed a well-supported phylogeny of the Bignoniaceae at different taxonomic scales, opening new perspectives for a comprehensive phylogenetic framework for the family as a whole., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fonseca, Carlsen, Fine and Lohmann.)

Whole genome duplications (WGD) are frequent in many plant lineages; however, ploidy level variation is unknown in most species. The most widely used methods to estimate ploidy levels in plants are chromosome counts, which require living specimens, and flow cytometry estimates, which necessitate living or relatively recently collected samples. Newly described bioinformatic methods have been developed to estimate ploidy levels using high-throughput sequencing data, and these have been optimized in plants by calculating allelic ratio values from target capture data. This method relies on the maintenance of allelic ratios from the genome to the sequence data. For example, diploid organisms will generate allelic data in a 1:1 proportion, with an increasing number of possible allelic ratio combinations occurring in individuals with higher ploidy levels. In this chapter, we explain step-by-step this bioinformatic approach for the estimation of ploidy level., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Although recent molecular phylogenetic analyses of Lepidothrix manakins (family Pipridae) have helped clarify their evolutionary relationships, the placement of several lineages remains in question because of low or conflicting branch support. In particular, the relationship of L. coronata to other members of the genus and relationships within the L. nattereri + L. vilasboasi + L. iris clade have been difficult to resolve. We used RADcap to collect restriction site-associated DNA sequence data and estimate the first subspecies-level phylogeny of the genus Lepidothrix (17 of 18 currently recognized subspecies), and we included extensive geographic representation of the widespread and phenotypically variable L. coronata. We found strong support for the phylogenetic position and monophyly of L. coronata, and we resolved two clades separated by the Andes that, along with previous divergence time estimates and our assessment of morphological and vocal evidence, suggest the presence of two biological species: Velvety Manakin (L. velutina) west of the Andes and Blue-capped Manakin (L. coronata) east of the Andes. Species-level relationships within the L. nattereri + L. vilasboasi + L. iris clade remained poorly resolved in concatenated and coalescent-based analyses, with SNAPP analyses suggesting that the lack of reciprocal monophyly is due to extensive allele sharing among these taxa. Finally, we confirmed a previously documented hybrid between L. coronata and L. suavissima as an F1 individual, consistent with the view that hybridization between these two species is a rare event and that postmating reproductive barriers prevent successful backcrossing., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Schistosomatidae Stiles and Hassall 1898 is a medically significant family of digenetic trematodes (Trematoda: Digenea), members of which infect mammals or birds as definitive hosts and aquatic or amphibious gastropods as intermediate hosts. Currently, there are 17 named genera, for many of which evolutionary interrelationships remain unresolved. The lack of a resolved phylogeny has encumbered our understanding of schistosomatid evolution, specifically patterns of host-use and the role of host-switching in diversification. Here, we used targeted sequence capture of ultra-conserved elements (UCEs) from representatives of 13 of the 17 named genera and 11 undescribed lineages that are presumed to represent either novel genera or species to generate a phylogenomic dataset for the estimation of schistosomatid interrelationships. This study represents the largest phylogenetic effort within the Schistosomatidae in both the number of loci and breadth of taxon sampling. We present a near-comprehensive family-level phylogeny providing resolution to several clades of long-standing uncertainty within Schistosomatidae, including resolution for the placement of the North American mammalian schistosomes, implying a second separate capture of mammalian hosts. Additionally, we present evidence for the placement of Macrobilharzia at the base of the Schistosoma + Bivitellobilharzia radiation. Patterns of definitive and intermediate host use and a strong role for intermediate host-switching are discussed relative to schistosomatid diversification.

Grasslands are widespread and economically relevant ecosystems at the basis of sustainable roughage production. Plant genetic diversity (PGD; i.e., within-species diversity) is related to many beneficial effects on the ecosystem functioning of grasslands. The monitoring of PGD in temperate grasslands is complicated by the multiplicity of species present and by a shortage of methods for large-scale assessments. However, the continuous advancement of high-throughput DNA sequencing approaches has improved the prospects of broad, multispecies PGD monitoring. Among them, amplicon sequencing stands out as a robust and cost-effective method. Here, we report a set of 12 multispecies primer pairs that can be used for high-throughput PGD assessments in multiple grassland plant species. The target loci were selected and tested in two phases: a "discovery phase" based on a sequence capture assay (611 nuclear loci assessed in 16 grassland plant species), which resulted in the selection of 11 loci; and a "validation phase", in which the selected loci were targeted and sequenced using multispecies primers in test populations of Dactylis glomerata L., Lolium perenne L., Festuca pratensis Huds., Trifolium pratense L. and T. repens L. The multispecies amplicons had nucleotide diversities per species from 5.19 × 10 -3 to 1.29 × 10 -2 , which is in the range of flowering-related genes but slightly lower than pathogen resistance genes. We conclude that the methodology, the DNA sequence resources, and the primer pairs reported in this study provide the basis for large-scale, multispecies PGD monitoring in grassland plants., (© 2021 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Fungal dye-decolorizing peroxidases (DyPs) have found applications in the treatment of dye-contaminated industrial wastes or to improve biomass digestibility. Their roles in fungal biology are uncertain, although it has been repeatedly suggested that they could participate in lignin degradation and/or modification. Using a comprehensive set of 162 fully sequenced fungal species, we defined seven distinct fungal DyP clades on basis of a sequence similarity network. Sequences from one of these clades clearly diverged from all others, having on average the lower isoelectric points and hydropathy indices, the highest number of N-glycosylation sites, and N-terminal sequence peptides for secretion. Putative proteins from this clade are absent from brown-rot and ectomycorrhizal species that have lost the capability of degrading lignin enzymatically. They are almost exclusively present in white-rot and other saprotrophic Basidiomycota that digest lignin enzymatically, thus lending support for a specific role of DyPs from this clade in biochemical lignin modification. Additional nearly full-length fungal DyP genes were isolated from the environment by sequence capture by hybridization; they all belonged to the clade of the presumably secreted DyPs and to another related clade. We suggest focusing our attention on the presumably intracellular DyPs from the other clades, which have not been characterized thus far and could represent enzyme proteins with novel catalytic properties. KEY POINTS: • A fungal DyP phylogeny delineates seven main sequence clades. • Putative extracellular DyPs form a single clade of Basidiomycota sequences. • Extracellular DyPs are associated to white-rot fungi., (© 2022. The Author(s).)

Background: Madin-Darby canine kidney (MDCK) cells are widely used for vaccine production, however, the safety of MDCK cells needs to be considered seriously because of high tumorigenicity. Micro RNAs (miRNAs) that are involved in the tumorigenicity of MDCK cells have been never been reported., Objective: To reveal the role of miRNA in the tumorigenic phenotype of MDCK cell line., Methods: The miRNA expression profiles of two monoclonal MDCK cells (M09CL and M35CL) with low tumorigenicity and one MDCK cell line (M73P) with high tumorigenicity were characterized and investigated by using small RNA-seq technology., Results: A total of 5 known miRNAs and 5 novel miRNAs were highly expressed in M73P. In addition, 4 known miRNAs and 4 novel miRNAs were highly expressed in M09CL and M35CL. The target genes of the differentially expressed miRNAs were significantly enriched in several biological processes, and the majority of these genes were involved in pathways in cancer and the MAPK signaling pathway. Through interaction analysis, 4 up-regulated miRNAs (cfa-miR-452, cfa-miR-8826, cfa-miR-224, and cfa-miR-2387) and their crucial target genes related to the tumor regulation network were identified. Results indicated these 4 miRNAs might play crucial roles in the tumorigenesis of MDCK cells., Conclusion: Our findings, which were based on the functional prediction of miRNAs and target genes, suggested that miRNAs might influence the tumorigenicity of MDCK cells by regulating target genes. Moreover, the results provided important data for understanding the miRNA-mediated regulatory networks that control the tumorigenicities of MDCK cells., (© 2021. The Author(s) under exclusive licence to The Genetics Society of Korea.)

The ability to bear live offspring, viviparity, has evolved multiple times across the tree of life and is a remarkable adaptation with profound life-history and ecological implications. Within amphibians the ancestral reproductive mode is oviparity followed by a larval life stage, but viviparity has evolved independently in all three amphibian orders. Two types of viviparous reproduction can be distinguished in amphibians; larviparity and pueriparity. Larviparous amphibians deliver larvae into nearby ponds and streams, while pueriparous amphibians deliver fully developed juveniles and thus do not require waterbodies for reproduction. Among amphibians, the salamander genus Salamandra is remarkable as it exhibits both inter- and intraspecific variation in the occurrence of larviparity and pueriparity. While the evolutionary relationships among Salamandra lineages have been the focus of several recent studies, our understanding of how often and when transitions between modes occurred is still incomplete. Furthermore, in species with intraspecific variation, the reproductive mode of a given population can only be confirmed by direct observation of births and thus the prevalence of pueriparous populations is also incompletely documented. We used sequence capture to obtain 1,326 loci from 94 individuals from across the geographic range of the genus, focusing on potential reproductive mode transition zones. We also report additional direct observations of pueriparous births for 20 new locations and multiple lineages. We identify at least five independent transitions from the ancestral mode of larviparity to pueriparity among and within species, occurring at different evolutionary timescales ranging from the Pliocene to the Holocene. Four of these transitions occurred within species. Based on a distinct set of markers and analyses, we also confirm previous findings of introgression between species and the need for taxonomic revisions in the genus. We discuss the implications of our findings with respect to the evolution of this complex trait, and the potential of using five independent convergent transitions for further studies on the ecological context in which pueriparity evolves and the genetic architecture of this specialized reproductive mode., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The diversity of avian visual phenotypes provides a framework for studying mechanisms of trait diversification generally, and the evolution of vertebrate vision, specifically. Previous research has focused on opsins, but to fully understand visual adaptation, we must study the complete phototransduction cascade (PTC). Here, we developed a probe set that captures exonic regions of 46 genes representing the PTC and other light responses. For a subset of species, we directly compared gene capture between our probe set and low-coverage whole genome sequencing (WGS), and we discuss considerations for choosing between these methods. Finally, we developed a unique strategy to avoid chimeric assembly by using "decoy" reference sequences. We successfully captured an average of 64% of our targeted exome in 46 species across 14 orders using the probe set and had similar recovery using the WGS data. Compared to WGS or transcriptomes, our probe set: (1) reduces sequencing requirements by efficiently capturing vision genes, (2) employs a simpler bioinformatic pipeline by limiting required assembly and negating annotation, and (3) eliminates the need for fresh tissues, enabling researchers to leverage existing museum collections. We then utilized our vision exome data to identify positively selected genes in two evolutionary scenarios-evolution of night vision in nocturnal birds and evolution of high-speed vision specific to manakins (Pipridae). We found parallel positive selection of SLC24A1 in both scenarios, implicating the alteration of rod response kinetics, which could improve color discrimination in dim light conditions and/or facilitate higher temporal resolution., (© 2021 John Wiley & Sons Ltd. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Technological advances in genome sequencing have improved our ability to catalog genomic variation and have led to an expansion of the scope and scale of genetic studies over the past decade. Yet, for agronomically important plant pathogens such as the downy mildews ( Peronosporaceae ), the scale of genetic studies remains limited. This is, in part, due to the difficulties associated with maintaining obligate pathogens and the logistical constraints involved in the genotyping of these species (e.g., obtaining DNA of sufficient quantity and quality). To gain an evolutionary and ecological perspective of downy mildews, adaptable methods for the genotyping of their populations are required. Here, we describe a targeted enrichment (TE) protocol to genotype isolates from two Pseudoperonospora species ( P. cubensis and P. humuli ), using less than 50 ng of mixed pathogen and plant DNA for library preparation. We were able to enrich 830 target genes across 128 samples and identified 2,514 high-quality single nucleotide polymorphism (SNP) variants. Using these SNPs, we detected significant genetic differentiation (analysis of molecular variance [AMOVA], P = 0.01) between P. cubensis subpopulations from Cucurbita moschata (clade I) and Cucumis sativus (clade II) in the state of Michigan. No evidence of location-based differentiation was detected within the P. cubensis (clade II) subpopulation in Michigan. However, a significant effect of location on the genetic variation of the P. humuli subpopulation was detected in the state (AMOVA, P = 0.01). Mantel tests found evidence that the genetic distance among P. humuli samples was associated with the physical distance of the hop yards from which the samples were collected ( P = 0.005). The differences in the distribution of genetic variation of the Michigan P. humuli and P. cubensis subpopulations suggest differences in the dispersal of these two species. The TE protocol described here provides an additional tool for genotyping obligate biotrophic plant pathogens and the execution of new genetic studies.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The sustainability of many crops is hindered by the lack of genomic resources and a poor understanding of natural genetic diversity. Particularly, application of modern breeding requires high-density linkage maps that are integrated into a highly contiguous reference genome. Here, we present a rapid method for deriving haplotypes and developing linkage maps, and its application to Mentha suaveolens, one of the diploid progenitors of cultivated mints. Using sequence-capture via DNA hybridization to target single nucleotide polymorphisms (SNPs), we successfully genotyped ∼5000 SNPs within the genome of >400 individuals derived from a self cross. After stringent quality control, and identification of nonredundant SNPs, 1919 informative SNPs were retained for linkage map construction. The resulting linkage map defined a total genetic space of 942.17 cM divided among 12 linkage groups, ranging from 56.32 to 122.61 cM in length. The linkage map is in good agreement with pseudomolecules from our preliminary genome assembly, proving this resource effective for the correction and validation of the reference genome. We discuss the advantages of this method for the rapid creation of linkage maps., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

Controlling and monitoring the still ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic regarding geographical distribution, evolution, and emergence of new mutations of the SARS-CoV-2 virus is only possible due to continuous next-generation sequencing (NGS) and sharing sequence data worldwide. Efficient sequencing strategies enable the retrieval of increasing numbers of high-quality, full-length genomes and are, hence, indispensable. Two opposed enrichment methods, tiling multiplex PCR and sequence hybridization by bait capture, have been established for SARS-CoV-2 sequencing and are both frequently used, depending on the quality of the patient sample and the question at hand. Here, we focused on the evaluation of the sequence hybridization method by studying five commercially available sequence capture bait panels with regard to sensitivity and capture efficiency. We discovered the SARS-CoV-2-specific panel of Twist Bioscience to be the most efficient panel, followed by two respiratory panels from Twist Bioscience and Illumina, respectively. Our results provide on the one hand a decision basis for the sequencing community including a computation for using the full capacity of the flow cell and on the other hand potential improvements for the manufacturers. IMPORTANCE Sequencing the genomes of the circulating SARS-CoV-2 strains is the only way to monitor the viral spread and evolution of the virus. Two different approaches, namely, tiling multiplex PCR and sequence hybridization by bait capture, are commonly used to fulfill this task. This study describes for the first time a combined approach of droplet digital PCR (ddPCR) and NGS to evaluate five commercially available sequence capture panels targeting SARS-CoV-2. In doing so, we were able to determine the most sensitive and efficient capture panel, distinguish the mode of action of the various bait panels, and compute the number of read pairs needed to recover a high-quality full-length genome. By calculating the minimum number of read pairs needed, we are providing optimized flow cell loading conditions for all sequencing laboratories worldwide that are striving for maximizing sequencing output and simultaneously minimizing time, costs, and sequencing resources.

An overlooked aspect of current microbiome studies is the role of viruses in human health. Compared to bacterial studies, laboratory and analytical methods to study the entirety of viral communities in clinical samples are rudimentary and need further refinement. In order to address this need, we developed Virobiome-Seq, a sequence capture method and an accompanying bioinformatics analysis pipeline, that identifies viral reads in human samples. Virobiome-Seq is able to enrich for and detect multiple types of viruses in human samples, including novel subtypes that diverge at the sequence level. In addition, Virobiome-Seq is able to detect RNA transcripts from DNA viruses and may provide a sensitive method for detecting viral activity in vivo. Since Virobiome-Seq also yields the viral sequence, it makes it possible to investigate associations between viral genotype and psychiatric illness. In this proof of concept study, we detected HIV1, Torque Teno, Pegi, Herpes and Papilloma virus sequences in Peripheral Blood Mononuclear Cells, plasma and stool samples collected from individuals with psychiatric disorders. We also detected the presence of numerous novel circular RNA viruses but were unable to determine whether these viruses originate from the sample or represent contaminants. Despite this challenge, we demonstrate that our knowledge of viral diversity is incomplete and opportunities for novel virus discovery exist. Virobiome-Seq will enable a more sophisticated analysis of the virome and has the potential of uncovering complex interactions between viral activity and psychiatric disease., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Diversity among phenology-related genes is predicted to be a contributing factor in local adaptations seen in widely distributed plant species that grow in climatically variable geographic areas, such as forest trees. European beech (Fagus sylvatica L.) is widespread, and is one of the most important broadleaved tree species in Europe; however, its potential for adaptation to climate change is a matter of uncertainty, and little is known about the molecular basis of climate change-relevant traits like bud burst., Results: We explored single nucleotide polymorphisms (SNP) at candidate genes related to bud burst in beech individuals sampled across 47 populations from Europe. SNP diversity was monitored for 380 candidate genes using a sequence capture approach, providing 2909 unlinked SNP loci. We used two complementary analytical methods to find loci significantly associated with geographic variables, climatic variables (expressed as principal components), or phenotypic variables (spring and autumn phenology, height, survival). Redundancy analysis (RDA) was used to detect candidate markers across two spatial scales (entire study area and within subregions). We revealed 201 candidate SNPs at the broadest scale, 53.2% of which were associated with phenotypic variables. Additive polygenic scores, which provide a measure of the cumulative signal across significant candidate SNPs, were correlated with a climate variable (first principal component, PC1) related to temperature and precipitation availability, and spring phenology. However, different genotype-environment associations were identified within Southeastern Europe as compared to the entire geographic range of European beech., Conclusions: Environmental conditions play important roles as drivers of genetic diversity of phenology-related genes that could influence local adaptation in European beech. Selection in beech favors genotypes with earlier bud burst under warmer and wetter habitats within its range; however, selection pressures may differ across spatial scales., (© 2021. The Author(s).)

Cis-regulatory sequences play a crucial role in regulating gene expression and are evolutionary hot spots that drive phenotypic divergence among organisms. Sequencing some cis-regulatory regions of interest in many different species is common in comparative genetic studies. For nonmodel organisms lacking genomic data, genome walking is often the preferred method for this type of application. However, applying genome walking will be laborious and time-consuming when the number of cis-regulatory regions and species to be analyzed is large. In this study, we propose a novel method called amplicon genome fishing (AGF), which can isolate and sequence cis-regulatory regions of interest for any organism. The main idea of the AGF method is to use fragments amplified from the target cis-regulatory regions as enrichment baits to capture and sequence the whole target cis-regulatory regions from genomic library pools. Unlike genome walking, the AGF method is based on hybridization capture and high-throughput sequencing, which makes this method rapid and efficient for projects where some cis-regulatory regions have to be sequenced for many species. We used human amplicons as capture baits and successfully sequenced five target enhancer regions of Homo sapiens, Mus musculus, Gallus gallus, and Xenopus tropicalis, proving the feasibility and repeatability of AGF. To show the utility of the AGF method in real studies, we used it to sequence the ZRS enhancer, a cis-regulatory region associated with the limb loss of snakes, for twenty-three vertebrate species (includes many limbless species never sequenced before). The newly obtained ZRS sequences provide new perspectives into the relationship between the ZRS enhancer's evolution and limb loss in major tetrapod lineages.

Gesneriaceae (ca. 3400 species) is a pantropical plant family with a wide range of growth form and floral morphology that are associated with repeated adaptations to different environments and pollinators. Although Gesneriaceae systematics has been largely improved by the use of Sanger sequencing data, our understanding of the evolutionary history of the group is still far from complete due to the limited number of informative characters provided by this type of data. To overcome this limitation, we developed here a Gesneriaceae-specific gene capture kit targeting 830 single-copy loci (776,754 bp in total), including 279 genes from the Universal Angiosperms-353 kit. With an average of 557,600 reads and 87.8% gene recovery, our target capture was successful across the family Gesneriaceae and also in other families of Lamiales. From our bait set, we selected the most informative 418 loci to resolve phylogenetic relationships across the entire Gesneriaceae family using maximum likelihood and coalescent-based methods. Upon testing the phylogenetic performance of our baits on 78 taxa representing 20 out of 24 subtribes within the family, we showed that our data provided high support for the phylogenetic relationships among the major lineages, and were able to provide high resolution within more recent radiations. Overall, the molecular resources we developed here open new perspectives for the study of Gesneriaceae phylogeny at different taxonomical levels and the identification of the factors underlying the diversification of this plant group., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Premise: During plant domestication, traits can be subject to a variety of types of selection, ranging from strong directional selection for traits such as seed or fruit size to diversifying selection for traits like color or flavor. These types of selection interact with other evolutionary processes including genetic bottlenecks and interspecific gene flow to generate different levels of genetic diversity across the genome and at target genes in domesticated lineages, but little is known about the impacts of these processes in perennial fruit crops., Methods: We used sequence capture by hybridization to examine patterns of diversity at a suite of candidate domestication and anonymous background genes in domesticated apple (Malus ×domestica) in comparison to its wild relatives Malus sieversii and Malus orientalis., Results: We found no change in average diversity at these candidate domestication genes across the three species. However, a subset of the genes did exhibit patterns of very high or very low diversity in M. ×domestica compared to its progenitor, M. sieversii. Of the genes with characterized function, the low-diversity genes mainly contributed to fruit quality traits like color and flavor, predicted to be under conscious, directional selection relatively late in the domestication process, while the high-diversity genes included a variety of functions., Conclusions: Overall, these results are consistent with predictions based on the likely timing and nature of selection during domestication and open new avenues for understanding genes with high diversity in a perennial crop compared to its wild relatives., (© 2021 Botanical Society of America.)

Many wheat varieties have the potential to develop unacceptably high levels of α-amylase in the grains if exposed to a cool temperature shock or simply cool temperature during the early to middle stages of grain filling. This phenomenon is referred to as late maturity α-amylase (LMA). The enzyme persists in the grain until harvest and may result in wheat with a low Falling Number that does not meet receival and export specifications. Resistance to LMA is therefore a valuable target for wheat breeders and wheat industries in general. Genetic evidence implicating a locus on the long arm of chromosome 7B in variation in LMA phenotype was confirmed in this investigation. Through intensive fine-mapping an ent -copalyl diphosphate synthase (CPS), hitherto named LMA-1 , was identified as the likely candidate gene associated with variation in LMA phenotype. Single Nucleotide Polymorphisms (SNPs) within the LMA-1 coding sequence of Chinese Spring, Maringa and Halberd result in either prematurely terminated or functionally altered proteins that are associated with useful levels of resistance to LMA. LMA-1 transcripts detected in de-embryonated grain tissue from around 15 days after anthesis, several days before the synthesis of α-amylase, were low in the resistant varieties Chinese Spring and Maringa compared with LMA susceptible genotype Spica. This was associated with a dramatic reduction in the concentrations of intermediates in the gibberellin biosynthesis pathway such as GA 19 , evidence that LMA-1 was functioning as CPS in the gibberellin biosynthesis pathway. A survey of a large collection of Australian and international wheat varieties distinguished 9 major haplotypes at the LMA-1 locus. Generally, within classes, there was notable variation for LMA phenotype and evidence for genotypes whose resistance is presumed to be due to genetic loci located elsewhere on the wheat genome. Further investigation is required to characterize the sequence of steps between LMA-1 and α-amylase synthesis as well as to gain a better understanding of the role and potential impact of other genetic loci. Diagnostic markers for sources of resistance and SNP variation reported in this study should assist breeders to deploy resistance associated with LMA-1 variants in breeding programs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Derkx, Baumann, Cheong, Mrva, Sharma, Pallotta and Mares.)

Transposable elements (TEs) are repetitive DNA sequences that have the ability to mobilize in the genome and create major effect mutations. Despite the importance of transposition as a source of genetic novelty, we still know little about the rate, landscape, and consequences of TE mobilization. This situation stems in large part from the repetitive nature of TEs, which complicates their analysis. Moreover, TE mobilization is typically rare and therefore new TE (i.e., non-reference) insertions tend to be missed in small-scale population studies. This chapter describes a TE-sequence capture approach designed to identify transposition events for most of the TE families that are potentially active in Arabidopsis thaliana. We show that our TE-sequence capture design provides an efficient means to detect with high sensitivity and specificity insertions that are present at a frequency as low as 1/1000 within a DNA sample.

Lack of knowledge of taxonomic biodiversity and reliable genetic markers in Trypanosomatidae limit our understanding of their phylogenetic relationships. Ultraconserved elements (UCEs) have improved phylogenetic analyses and inferences in many vertebrate and invertebrate taxa. However, it is unknown whether protozoans have these markers, their abundance, and if these could be reliably used for phylogenetics. In this study I design a target enrichment bait set for UCE loci for this group. In silico testing showed good loci recovery rates across 63 taxa and produced consistent, highly supported phylogenetic trees. This bait set adds a new resource of useful genetic markers for Trypanosomatidae phylogenetics., (Copyright © 2020 Elsevier Inc. All rights reserved.)

With recent advances in sequencing technology, genomic data are changing how important conservation management decisions are made. Applications such as Close-Kin Mark-Recapture demand large amounts of data to estimate population size and structure, and their full potential can only be realised through ongoing improvements in genotyping strategies. Here we introduce DArTcap, a cost-efficient method that combines DArTseq and sequence capture, and illustrate its use in a high resolution population analysis of Glyphis garricki, a rare, poorly known and threatened euryhaline shark. Clustering analyses and spatial distribution of kin pairs from four different regions across northern Australia and one in Papua New Guinea, representing its entire known range, revealed that each region hosts at least one distinct population. Further structuring is likely within Van Diemen Gulf, the region that included the most rivers sampled, suggesting additional population structuring would be found if other rivers were sampled. Coalescent analyses and spatially explicit modelling suggest that G. garricki experienced a recent range expansion during the opening of the Gulf of Carpentaria following the conclusion of the Last Glacial Maximum. The low migration rates between neighbouring populations of a species that is found only in restricted coastal and riverine habitats show the importance of managing each population separately, including careful monitoring of local and remote anthropogenic activities that may affect their environments. Overall we demonstrated how a carefully chosen SNP panel combined with DArTcap can provide highly accurate kinship inference and also support population structure and historical demography analyses, therefore maximising cost-effectiveness., (© 2020 John Wiley & Sons Ltd.)

Closely related taxa often exhibit mitonuclear discordance attributed to introgression of mitochondrial DNA (mtDNA), yet few studies have considered the underlying causes of mtDNA introgression. Here we test for demographic versus adaptive processes as explanations for mtDNA introgression in three subspecies of the intermediate horseshoe bat (Rhinolophus affinis). We generated sequences of 1692 nuclear genes and 13 mitochondrial protein-coding genes for 48 individuals. Phylogenetic reconstructions based on 320 exon sequences and 2217 single nucleotide polymorphisms (SNPs) both revealed conflicts between the species tree and mtDNA tree. These results, together with geographic patterns of mitonuclear discordance, and shared identical or near-identical mtDNA sequences, suggest extensive introgression of mtDNA between the two parapatric mainland subspecies. Under demographic hypotheses, we would also expect to uncover traces of ncDNA introgression, however, population structure and gene flow analyses revealed little nuclear admixture. Furthermore, we found inconsistent estimates of the timing of population expansion and that of the most recent common ancestor for the clade containing introgressed haplotypes. Without a clear demographic explanation, we also examined whether introgression likely arises from adaptation. We found that five mtDNA genes contained fixed amino acid differences between introgressed and non-introgressed individuals, including putative positive selection found in one codon, although this did not show introgression. While our evidence for rejecting demographic hypotheses is arguably stronger than that for rejecting adaptation, we find no definitive support for either explanation. Future efforts will focus on larger-scale resequencing to decipher the underlying causes of discordant mitonuclear introgression in this system., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The family Epicopeiidae is a small group of day-flying moths, known for mimicking many different groups of butterflies and moths. So far, there still lacks a reliable phylogenetic framework of Epicopeiidae that is necessary to our understanding of the evolutionary process of their mimicry. In this study, we sequenced 94 nuclear protein-coding markers for 56 epicopeiid samples and 11 outgroups, covering all ten genera of Epicopeiidae. We used homemade PCR-generated baits to capture target sequences, which allowed us to utilize old and dried specimens that were difficult to handle by conventional PCR + Sanger sequencing. Maximum likelihood and Bayesian analyses of the newly obtained dataset (86,388 bp) at both DNA and protein levels produced identical phylogenies with strong support. The non-mimicry genus Deuveia is the sister group of other epicopeiid genera. Epicopeia and Nossa are not monophyletic, and these two genera nest together to form a clade. We also estimated divergence times of Epicopeiidae and found that their initial diversification happened in Eocene about 41 million years ago. The ancestral state reconstruction of mimicry type for this family suggested that thelast common ancestor of epicopeiid moths is non-mimetic, and the Riodinidae-mimicry type evolved first. In summary, our work provides a comprehensive and robust time-calibrated phylogeny of Epicopeiidae that provides a sound framework for revising their classification and interpreting character evolution., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Brachypodium distachyon is an annual C3 grass used as a monocot model system in functional genomics research. Insertional mutagenesis is a powerful tool for both forward and reverse genetics studies. In this study, we explored the possibility of using the tobacco retrotransposon Tnt1 to create a transposon-based insertion mutant population in B. distachyon. We developed transgenic B. distachyon plants expressing Tnt1 (R0) and in the subsequent regenerants (R1) we observed that Tnt1 actively transposed during somatic embryogenesis, generating an average of 6.37 insertions per line in a population of 19 independent R1 regenerant plants analyzed. In seed-derived progeny of R1 plants, Tnt1 segregated in a Mendelian ratio of 3:1 and no new Tnt1 transposition was observed. A total of 126 flanking sequence tags (FSTs) were recovered from the analyzed R0 and R1 lines. Analysis of the FSTs showed a uniform pattern of insertion in all the chromosomes (1-5) without any preference for a particular chromosome region. Considering the average length of a gene transcript to be 3.37 kb, we estimated that 29 613 lines are required to achieve a 90% possibility of tagging a given gene in the B. distachyon genome using the Tnt1-based mutagenesis approach. Our results show the possibility of using Tnt1 to achieve near-saturation mutagenesis in B. distachyon, which will aid in functional genomics studies of other C3 grasses., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

For population genetic studies in nonmodel organisms, it is important to use every single source of genomic information. This paper presents EXFI, a Python pipeline that predicts the splice graph and exon sequences using an assembled transcriptome and raw whole-genome sequencing reads. The main algorithm uses Bloom filters to remove reads that are not part of the transcriptome, to predict the intron-exon boundaries, to then proceed to call exons from the assembly, and to generate the underlying splice graph. The results are returned in GFA1 format, which encodes both the predicted exon sequences and how they are connected to form transcripts. EXFI is written in Python, tested on Linux platforms, and the source code is available under the MIT License at https://github.com/jlanga/exfi., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

The plant immune system involves detection of pathogens via both cell-surface and intracellular receptors. Both receptor classes can induce transcriptional reprogramming that elevates disease resistance. To assess differential gene expression during plant immunity, we developed and deployed quantitative sequence capture (CAP-I). We designed and synthesized biotinylated single-strand RNA bait libraries targeted to a subset of defense genes, and generated sequence capture data from 99 RNA-seq libraries. We built a data processing pipeline to quantify the RNA-CAP-I-seq data, and visualize differential gene expression. Sequence capture in combination with quantitative RNA-seq enabled cost-effective assessment of the expression profile of a specified subset of genes. Quantitative sequence capture is not limited to RNA-seq or any specific organism and can potentially be incorporated into automated platforms for high-throughput sequencing., (© 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The torrent frog genus Amolops contains nearly sixty species distributed in swift mountain streams throughout southeast Asia. The taxonomy of this genus has proven complicated due to unstable morphological diagnostic characters. The relationships of Amolops species and species groups were not readily resolved with a small number of molecular markers. Here, we applied the novel AFLP-Capture approach and acquired two large datasets (242 anonymous nuclear sequences and the mitochondrial genome) from 70 Chinese Amolops samples to study their relationships. The phylogenies inferred from the nuclear data and the mitochondrial data were both robust and revealed a primary phylogenetic split between eastern and western Chinese Amolops species. The relationships of the six species groups were clarified. While the three species groups in east China (the A. ricketti, A. daiyunensis and A. hainanensis groups) were monophyletic, the three species groups in the west (the A. mantzorum, A. monticola and A. marmoratus groups) were not monophyletic, suggesting a need for further investigation and revision. The robust phylogenies also provided new insights into species relationships, especially for the A. mantzorum group, which has been difficult to resolve due to multiple speciation events occurring approximately 7-8 million years ago. The divergence times estimated with the nuclear data indicated that the ancestor of the Chinese Amolops appeared in the late Eocene or early Oligocene, and that speciation events in the Chinese Amolops were often related to geological events (e.g. the uprising of mountains and the formation of islands). By including the mitochondrial sequences from GenBank, a more comprehensive Amolops phylogeny was constructed that reflected the origin of the Chinese Amolops. Based on all these results, a dispersal scenario of the torrent frogs was hypothesized. Our research serves as the first example of using AFLP-Capture to obtain a large amount of data for shallow-scale phylogenetic and taxonomic studies, which should be useful for other nonmodel organism groups., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Palms are conspicuous floristic elements across the tropics. In continental Africa, even though there are less than 70 documented species, they are omnipresent across the tropical landscape. The genus Raphia has 20 accepted species in Africa and one species endemic to the Neotropics. It is the most economically important genus of African palms with most of its species producing food and construction material. Raphia is divided into five sections based on inflorescence morphology. Nevertheless, the taxonomy of Raphia is problematic with no intra-generic phylogenetic study available. We present a phylogenetic study of the genus using a targeted exon capture approach sequencing of 56 individuals representing 18 out of the 21 species. Our results recovered five well supported clades within the genus. Three sections correspond to those based on inflorescence morphology. R. regalis is strongly supported as sister to all other Raphia species and is placed into a newly described section: Erectae. Overall, morphological based identifications agreed well with our phylogenetic analyses, with 12 species recovered as monophyletic based on our sampling. Species delimitation analyses recovered 17 or 23 species depending on the confidence level used. Species delimitation is especially problematic in the Raphiate and Temulentae sections. In addition, our clustering analysis using SNP data suggested that individual clusters matched geographic distribution. The Neotropical species R. taedigera is supported as a distinct species, rejecting the hypothesis of a recent introduction into South America. Our analyses support the hypothesis that the Raphia individuals from Madagascar are potentially a distinct species different from the widely distributed R. farinifera . In conclusion, our results support the infra generic classification of Raphia based on inflorescence morphology, which is shown to be phylogenetically useful. Classification and species delimitation within sections remains problematic even with our phylogenomic approach. Certain widely distributed species could potentially contain cryptic species. More in-depth studies should be undertaken using morphometrics, increased sampling, and more variable markers. Our study provides a robust phylogenomic framework that enables further investigation on the biogeographic history, morphological evolution, and other eco-evolutionary aspects of this charismatic, socially, and economically important palm genus.

With its large proportion of endemic taxa, complex geological past, and location at the confluence of the highly diverse Malesian and Australian floristic regions, Papuasia - the floristic region comprising the Bismarck Archipelago, New Guinea, and the Solomon Islands - represents an ideal natural experiment in plant biogeography. However, scattered knowledge of its flora and limited representation in herbaria have hindered our understanding of the drivers of its diversity. Focusing on the woody angiosperm genus Schefflera (Araliaceae), we ask whether its morphologically defined infrageneric groupings are monophyletic, when these lineages diverged, and where (within Papuasia or elsewhere) they diversified. To address these questions, we use a high-throughput sequencing approach (Hyb-Seq) which combines target capture (with an angiosperm-wide bait kit targeting 353 single-copy nuclear loci) and genome shotgun sequencing (which allows retrieval of regions in high-copy number, e.g., organellar DNA) of historical herbarium collections. To reconstruct the evolutionary history of the genus we reconstruct molecular phylogenies with Bayesian inference, maximum likelihood, and pseudo-coalescent approaches, and co-estimate divergence times and ancestral areas in a Bayesian framework. We find strong support for most infrageneric morphological groupings, as currently circumscribed, and we show the efficacy of the Angiosperms-353 probe kit in resolving both deep and shallow phylogenetic relationships. We infer a sequence of colonization to explain the present-day distribution of Schefflera in Papuasia: from the Sunda Shelf, Schefflera arrived to the Woodlark plate (present-day eastern New Guinea) in the late Oligocene (when most of New Guinea was submerged) and, subsequently (throughout the Miocene), it migrated westwards (to the Maoke and Bird's Head Plates and thereon) and further diversified, in agreement with previous reconstructions., (Copyright © 2020 Shee, Frodin, Cámara-Leret and Pokorny.)

Madagascar's biota is characterized by a high degree of microendemism at different taxonomic levels, but how colonization and in-situ speciation contribute to the assembly of local species communities has rarely been studied on this island. Here we analyze the phylogenetic relationships of riparian frogs of the Mantidactylus ambreensis species complex, which is distributed in the north of Madagascar and was originally described from Montagne d'Ambre, an isolated mountain of volcanic origin, currently protected within Montagne d'Ambre National Park (MANP). Data from mitochondrial DNA, and phylogenomic data from FrogCap, a sequence capture method, independently confirm that this species complex is monophyletic within the subgenus Ochthomantis, and identify two main clades within it. These two clades are separated by 5.6-6.8% pairwise distance in the mitochondrial 16S rRNA gene and co-occur in MANP, with one distributed at high elevations (940-1375 m a.s.l.) and the other at lower elevations (535-1010 m a.s.l.), but show almost no haplotype sharing in the nuclear RAG1 gene. This occurrence in syntopy without admixture confirms them as independent evolutionary lineages that merit recognition as separate species, and we here refer to them as high-elevation (HE) and low-elevation (LE) lineage; they will warrant taxonomic assessment to confidently assign the name ambreensis to one or the other. Populations of the M. ambreensis complex from elsewhere in northern Madagascar all belong to the LE lineage, although they do occur over a larger elevational range than in Montagne d'Ambre (285-1040 m a.s.l.). Within LE there are several phylogroups (LE1-LE4) of moderately deep divergence (1.5-2.8% in 16S), but phylogroup LE4 that occurs in MANP has a deeply nested phylogenetic position, as recovered separately by mitochondrial and sequence capture datasets. This suggests that HE and LE did not diverge by a local fission of lower and upper populations, but instead arose through a more complex biogeographic scenario. The branching pattern of phylogroups LE1-LE4 shows a clear south-to-north phylogeographic pattern. We derive from these results a testable hypothesis of vicariant speciation that restricted the HE lineage to MANP and the LE candidate species to a climatic refugium further south, with subsequent northwards range expansion and secondary colonization of MANP by LE. These results provide an example for complex assembly of local microendemic amphibian faunas on Madagascar., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Marker selection has emerged as an important component of phylogenomic study design due to rising concerns of the effects of gene tree estimation error, model misspecification, and data-type differences. Researchers must balance various trade-offs associated with locus length and evolutionary rate among other factors. The most commonly used reduced representation data sets for phylogenomics are ultraconserved elements (UCEs) and Anchored Hybrid Enrichment (AHE). Here, we introduce Rapidly Evolving Long Exon Capture (RELEC), a new set of loci that targets single exons that are both rapidly evolving (evolutionary rate faster than RAG1) and relatively long in length (>1,500 bp), while at the same time avoiding paralogy issues across amniotes. We compare the RELEC data set to UCEs and AHE in squamate reptiles by aligning and analyzing orthologous sequences from 17 squamate genomes, composed of 10 snakes and 7 lizards. The RELEC data set (179 loci) outperforms AHE and UCEs by maximizing per-locus genetic variation while maintaining presence and orthology across a range of evolutionary scales. RELEC markers show higher phylogenetic informativeness than UCE and AHE loci, and RELEC gene trees show greater similarity to the species tree than AHE or UCE gene trees. Furthermore, with fewer loci, RELEC remains computationally tractable for full Bayesian coalescent species tree analyses. We contrast RELEC to and discuss important aspects of comparable methods, and demonstrate how RELEC may be the most effective set of loci for resolving difficult nodes and rapid radiations. We provide several resources for capturing or extracting RELEC loci from other amniote groups., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The origin of the mammalian order Eulipotyphla has been debated intensively with arguments around whether they began diversifying before or after the Cretaceous-Palaeogene (K-Pg) boundary at 66 Ma. Here, we used an in-solution nucleotide capture method and next generation DNA sequencing to determine the sequence of hundreds of ultra-conserved elements (UCEs), and conducted phylogenomic and molecular dating analyses for the four extant eulipotyphlan lineages-Erinaceidae, Solenodontidae, Soricidae, and Talpidae. Concatenated maximum-likelihood analyses with single or partitioned models and a coalescent species-tree analysis showed that divergences among the four major eulipotyphlan lineages occurred within a short period of evolutionary time, but did not resolve the interrelationships among them. Alternative suboptimal phylogenetic hypotheses received consistently the same amount of support from different UCE loci, and were not significantly different from the maximum likelihood tree topology, suggesting the prevalence of stochastic lineage sorting. Molecular dating analyses that incorporated among-lineage evolutionary rate differences supported a scenario where the four eulipotyphlan families diversified between 57.8 and 63.2 Ma. Given short branch lengths with low support values, traces of rampant genome-wide stochastic lineage sorting, and post K-Pg diversification, we concluded that the crown eulipotyphlan lineages arose through a rapid diversification after the K-Pg boundary when novel niches were created by the mass extinction of species., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Populus trichocarpa is an important forest tree species for the generation of lignocellulosic ethanol. Understanding the genomic basis of biomass production and chemical composition of wood is fundamental in supporting genetic improvement programs. Considerable variation has been observed in this species for complex traits related to growth, phenology, ecophysiology and wood chemistry. Those traits are influenced by both polygenic control and environmental effects, and their genome architecture and regulation are only partially understood. Genome wide association studies (GWAS) represent an approach to advance that aim using thousands of single nucleotide polymorphisms (SNPs). Genotyping using exome capture methodologies represent an efficient approach to identify specific functional regions of genomes underlying phenotypic variation., Results: We identified 813 K SNPs, which were utilized for genotyping 461 P. trichocarpa clones, representing 101 provenances collected from Oregon and Washington, and established in California. A GWAS performed on 20 traits, considering single SNP-marker tests identified a variable number of significant SNPs (p-value < 6.1479E-8) in association with diameter, height, leaf carbon and nitrogen contents, and δ 15 N. The number of significant SNPs ranged from 2 to 220 per trait. Additionally, multiple-marker analyses by sliding-windows tests detected between 6 and 192 significant windows for the analyzed traits. The significant SNPs resided within genes that encode proteins belonging to different functional classes as such protein synthesis, energy/metabolism and DNA/RNA metabolism, among others., Conclusions: SNP-markers within genes associated with traits of importance for biomass production were detected. They contribute to characterize the genomic architecture of P. trichocarpa biomass required to support the development and application of marker breeding technologies.

Recently diverged taxa are often characterised by high rates of introgressive hybridization and incomplete lineage sorting, both of which can complicate phylogenetic reconstructions of species histories. Here we use a sequence capture approach to obtain genome-wide data to resolve the evolutionary relationships, and infer the extent and timescale of hybridization and introgression events, among six recently diverged taxa of the horseshoe bat species complexes Rhinolophus sinicus and R. thomasi. We show that two different methods of species tree reconstruction applied to a set of ~1500 nuclear loci all recover species trees with similar topologies, differing from the previous phylogeny based on two nuclear loci. By comparing the tree topology obtained from the nuclear loci with that inferred from the mitochondrial genome, we observed at least three cases of conflict, each of which likely results from past introgression. Of these, the occurrence of a highly similar mitogenome sequence shared by individuals of two taxa in a sympatric region points to very recent mtDNA introgression. The other cases are characterised by greater divergence and strong phylogeographic structure among putative introgressed individuals and their source populations, and thus likely reflect more ancient hybridization events. These results also suggest that two of the subspecies (R. s. septentrionalis and the undescribed taxon R. s. ssp) are likely to represent full species, warranting full taxonomic descriptions. This work adds a growing number of studies showing the potential problems of relying solely on mitochondrial sequences, or a limited number of loci, to infer phylogenetic relationships among recently diverged taxa., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Norway spruce is a boreal forest tree species of significant ecological and economic importance. Hence there is a strong imperative to dissect the genetics underlying important wood quality traits in the species. We performed a functional genome-wide association study (GWAS) of 17 wood traits in Norway spruce using 178 101 single nucleotide polymorphisms (SNPs) generated from exome genotyping of 517 mother trees. The wood traits were defined using functional modelling of wood properties across annual growth rings. We applied a Least Absolute Shrinkage and Selection Operator (LASSO-based) association mapping method using a functional multilocus mapping approach that utilizes latent traits, with a stability selection probability method as the hypothesis testing approach to determine a significant quantitative trait locus. The analysis provided 52 significant SNPs from 39 candidate genes, including genes previously implicated in wood formation and tree growth in spruce and other species. Our study represents a multilocus GWAS for complex wood traits in Norway spruce. The results advance our understanding of the genetics influencing wood traits and identifies candidate genes for future functional studies., (© 2019 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

High-throughput DNA sequencing (HTS) presents great opportunities for plant systematics, yet genomic complexity needs to be reduced for HTS to be effectively applied. We highlight Hyb-Seq as a promising approach, especially in light of the recent development of probes enriching 353 low-copy nuclear genes from any flowering plant taxon., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Infectious disease is both a major force of selection in nature and a prime cause of yield loss in agriculture. In plants, disease resistance is often conferred by nucleotide-binding leucine-rich repeat (NLR) proteins, intracellular immune receptors that recognize pathogen proteins and their effects on the host. Consistent with extensive balancing and positive selection, NLRs are encoded by one of the most variable gene families in plants, but the true extent of intraspecific NLR diversity has been unclear. Here, we define a nearly complete species-wide pan-NLRome in Arabidopsis thaliana based on sequence enrichment and long-read sequencing. The pan-NLRome largely saturates with approximately 40 well-chosen wild strains, with half of the pan-NLRome being present in most accessions. We chart NLR architectural diversity, identify new architectures, and quantify selective forces that act on specific NLRs and NLR domains. Our study provides a blueprint for defining pan-NLRomes., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Whole genome duplication (WGD) events are common in many plant lineages, but the ploidy status and possible occurrence of intraspecific ploidy variation are unknown for most species. Standard methods for ploidy determination are chromosome counting and flow cytometry approaches. While flow cytometry approaches typically use fresh tissue, an increasing number of studies have shown that recently dried specimens can be used to yield ploidy data. Recent studies have started to explore whether high-throughput sequencing (HTS) data can be used to assess ploidy levels by analyzing allelic frequencies from single copy nuclear genes. Here, we compare different approaches using a range of yam ( Dioscorea ) tissues of varying ages, drying methods and quality, including herbarium tissue. Our aims were to: (1) explore the limits of flow cytometry in estimating ploidy level from dried samples, including herbarium vouchers collected between 1831 and 2011, and (2) optimize a HTS-based method to estimate ploidy by considering allelic frequencies from nuclear genes obtained using a target-capture method. We show that, although flow cytometry can be used to estimate ploidy levels from herbarium specimens collected up to fifteen years ago, success rate is low (5.9%). We validated our HTS-based estimates of ploidy using 260 genes by benchmarking with dried samples of species of known ploidy ( Dioscorea alata , D. communis , and D. sylvatica ). Subsequently, we successfully applied the method to the 85 herbarium samples analyzed with flow cytometry, and successfully provided results for 91.7% of them, comprising species across the phylogenetic tree of Dioscorea . We also explored the limits of using this HTS-based approach for identifying high ploidy levels in herbarium material and the effects of heterozygosity and sequence coverage. Overall, we demonstrated that ploidy diversity within and between species may be ascertained from historical collections, allowing the determination of polyploidization events from samples collected up to two centuries ago. This approach has the potential to provide insights into the drivers and dynamics of ploidy level changes during plant evolution and crop domestication.

Objective: Hybrid zones are geographic regions where genetically distinct taxa interbreed, resulting in offspring of mixed ancestry. California Scrub-Jays (Aphelocoma californica) and Woodhouse's Scrub-Jays (A. woodhouseii) come into secondary contact and hybridize in western Nevada. Although previous work investigated divergence and gene flow between these species using a handful of microsatellite markers, the hybrid zone has not been studied using genome-scale markers, providing an opportunity to assess genome-wide introgression, test for a genetic basis for ecomorphological traits, and compare these estimates to those derived from microsatellites., Results: Using variant sites flanking ultraconserved elements (UCEs), we performed population assignment and quantified hybrid ancestry for 16 individuals across the zone of secondary contact. Our study included 2468 SNPs distributed throughout the genome, allowing discrimination of genetic affinities of hybrid individuals that were similar to estimates from microsatellites. We show a relationship between bill and wing length and the genetic composition of individuals that was not found in prior work using microsatellites, suggesting a genetic basis for these traits. Our analyses demonstrate the utility of UCEs for the analysis of hybrid zones and provide a basis for future studies to identify the genomic architecture of speciation and phenotypic differences between these incipient species.

Sequence capture across large phylogenetic scales is not easy because hybridization capture is only effective when the genetic distance between the bait and target is small. Here, we propose a simple but effective strategy to tackle this issue: pooling DNA from a number of selected representative species of different clades to prepare PCR-generated baits to minimize the genetic distance between the bait and target. To demonstrate the utility of this strategy, we newly developed a set of universal nuclear markers (including 94 nuclear protein-coding genes) for Lepidoptera, a superdiverse insect group. We used a DNA pool from six lepidopteran species (representing six superfamilies) to prepare PCR baits for the 94 markers. These homemade PCR baits were used to capture sequence data from 43 species of 17 lepidopteran families, and 94% of the target loci were recovered. We constructed two data sets from the obtained data (one containing ~90 kb target coding sequences and the other containing ~120 kb target + flanking coding sequences). Both data sets yielded highly similar and well-resolved trees with 90% of nodes having >95% bootstrap support. Our capture experiment indicated that using DNA mixtures pooled from different clade-representative species of Lepidoptera to prepare PCR baits can reliably capture a large number of targeted nuclear markers across different Lepidoptera lineages. We hope that this newly developed nuclear marker set will serve as a new phylogenetic tool for Lepidoptera phylogenetics, and the PCR bait preparation strategy can facilitate the application of sequence capture techniques by researchers to accelerate data collection., (© 2019 John Wiley & Sons Ltd.)

Sequencing of target-enriched libraries is an efficient and cost-effective method for obtaining DNA sequence data from hundreds of nuclear loci for phylogeny reconstruction. Much of the cost of developing targeted sequencing approaches is associated with the generation of preliminary data needed for the identification of orthologous loci for probe design. In plants, identifying orthologous loci has proven difficult due to a large number of whole-genome duplication events, especially in the angiosperms (flowering plants). We used multiple sequence alignments from over 600 angiosperms for 353 putatively single-copy protein-coding genes identified by the One Thousand Plant Transcriptomes Initiative to design a set of targeted sequencing probes for phylogenetic studies of any angiosperm group. To maximize the phylogenetic potential of the probes, while minimizing the cost of production, we introduce a k-medoids clustering approach to identify the minimum number of sequences necessary to represent each coding sequence in the final probe set. Using this method, 5-15 representative sequences were selected per orthologous locus, representing the sequence diversity of angiosperms more efficiently than if probes were designed using available sequenced genomes alone. To test our approximately 80,000 probes, we hybridized libraries from 42 species spanning all higher-order groups of angiosperms, with a focus on taxa not present in the sequence alignments used to design the probes. Out of a possible 353 coding sequences, we recovered an average of 283 per species and at least 100 in all species. Differences among taxa in sequence recovery could not be explained by relatedness to the representative taxa selected for probe design, suggesting that there is no phylogenetic bias in the probe set. Our probe set, which targeted 260 kbp of coding sequence, achieved a median recovery of 137 kbp per taxon in coding regions, a maximum recovery of 250 kbp, and an additional median of 212 kbp per taxon in flanking non-coding regions across all species. These results suggest that the Angiosperms353 probe set described here is effective for any group of flowering plants and would be useful for phylogenetic studies from the species level to higher-order groups, including the entire angiosperm clade itself., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Society of Systematic Biologists.)