A Species-Wide Inventory of NLR Genes and Alleles in Arabidopsis thaliana.

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.

Signatures of local adaptation to current and future climate in phenology-related genes in natural populations of Quercus robur.

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 FST 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.

Phylogenomics and morphology of Celmisiinae (Asteraceae: Astereae): Taxonomic and evolutionary implications.

The tribe Astereae (Asteraceae) includes 36 subtribes and 252 genera, and is distributed worldwide in temperate and tropical regions. One of the subtribes, Celmisiinae Saldivia, has been recently circumscribed to include six genera and ca. 160 species, and is restricted to eastern Australia, New Zealand, and New Guinea. The species show an impressive range of growth habit, from small herbs and ericoid subshrubs to medium-sized trees. They live in a wide range of habitats and are often dominant in subalpine and alpine vegetation. Despite the well-supported circumscription of Celmisiinae, uncertainties have remained about their internal relationships and classification at genus and species levels. This study exploited recent advances in high-throughput sequencing to build a robust multi-gene phylogeny for the subtribe Celmisiinae. The target enrichment Angiosperms353 bait set and the hybpiper-nf and paragone-nf pipelines were used to retrieve, infer, and assemble orthologous loci from 75 taxa representing all the main putative clades within the subtribe. Because of the diploidised ploidy level in Celmisiinae, as well as missing data in the assemblies, uncertainty remains surrounding the inference of orthology detection. However, based on a variety of gene-family sets, coalescent and concatenation-based phylogenetic reconstructions recovered similar topologies. Paralogy and missing data in the gene-families caused some problems, but the estimated phylogenies were well-supported and well-resolved. The phylogenomic evidence supported Celmisiinae and three main clades: the Pleurophyllum clade (Pleurophyllum, Macrolearia and Damnamenia), mostly in the New Zealand Subantarctic Islands, Celmisia of mainland New Zealand and Australia, and Shawia (including 'Olearia pro parte' and Pachystegia) of New Zealand, Australia and New Guinea. The results presented here add to the accumulating support for the Angiosperms353 bait set as an efficient method for documenting plant diversity.

To and fro in the archipelago: Repeated inter-island dispersal and New Guinea's orogeny affect diversification of Delias, the world's largest butterfly genus.

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.

Phylogenomics reveals patterns of ancient hybridization and differential diversification that contribute to phylogenetic conflict in willows, poplars, and close relatives.

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.

Phylogeny, biogeography and ecological diversification of New Caledonian palms (Arecaceae).

BACKGROUND AND AIMS: The geographical origin and evolutionary mechanisms underpinning the rich and distinctive New Caledonian flora remain poorly understood. This is attributable to the complex geological past of the island and to the scarcity of well-resolved species-level phylogenies. Here, we infer phylogenetic relationships and divergence times of New Caledonian palms, which comprise 40 species. We use this framework to elucidate the biogeography of New Caledonian palm lineages and to explore how extant species might have formed. METHODS: A phylogenetic tree including 37 New Caledonian palm species and 77 relatives from tribe Areceae was inferred from 151 nuclear genes obtained by targeted sequencing. Fossil-calibrated divergence times were estimated and ancestral ranges inferred. Ancestral and extant ecological preferences in terms of elevation, precipitation and substrate were compared between New Caledonian sister species to explore their possible roles as drivers of speciation. KEY RESULTS: New Caledonian palms form four well-supported clades, inside which relationships are well resolved. Our results support the current classification but suggest that Veillonia and Campecarpus should be resurrected and fail to clarify whether Rhopalostylidinae is sister to or nested in Basseliniinae. New Caledonian palm lineages are derived from New Guinean and Australian ancestors, which reached the island through at least three independent dispersal events between the Eocene and Miocene. Palms then dispersed out of New Caledonia at least five times, mainly towards Pacific islands. Geographical and ecological transitions associated with speciation events differed across time and genera. Substrate transitions were more frequently associated with older events than with younger ones. CONCLUSIONS: Neighbouring areas and a mosaic of local habitats shaped the palm flora of New Caledonia, and the island played a significant role in generating palm diversity across the Pacific region. This new spatio-temporal framework will enable population-level ecological and genetic studies to unpick the mechanisms underpinning New Caledonian palm endemism.

Parallel DNA/RNA NGS Using an Identical Target Enrichment Panel in the Analysis of Hereditary Cancer Predisposition.

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.

Systematics of Lepidothrix manakins (Aves: Passeriformes: Pipridae) using RADcap markers.

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.

Independent evolutionary transitions to pueriparity across multiple timescales in the viviparous genus Salamandra.

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.

Phylogenomic relationships and historical biogeography in the South American vegetable ivory palms (Phytelepheae).

The palm tribe Phytelepheae form a clade of three genera and eight species whose phylogenetic relationships and historical biogeography are not fully understood. Based on morphological similarities and phylogenetic relatedness, it has been suggested that Phytelephas seemannii and Phytelephas schottii are synonyms of Phytelephas macrocarpa, implying the existence of only six species within the Phytelepheae. In addition, uncertainty in their phylogenetic relationships in turn results in blurred biogeographic history. We inferred the phylogenomic relationships in the Phytelepheae by target-capturing 176 nuclear genes and estimated divergence times by using four fossils for time calibration. We lastly explored the biogeographic history of the tribe by inferring its ancestral range evolution. Our phylogenomic trees showed that P. seemannii and P. schottii are not closely related with P. macrocarpa, and therefore, support the existence of eight species in the Phytelepheae. The ancestor of the tribe was widely-distributed in the Chocó, Magdalena, and Amazonia during the Miocene at 19.25 Ma. Early diversification in Phytelephas at 5.27 Ma could have occurred by trans-Andean vicariance after the western Andes uplifted rapidly at âˆ¼ 10 Ma. Our results show the utility of phylogenomic approaches to shed light on species relationships and their biogeographic history.

Verification of CRISPR editing and finding transgenic inserts by Xdrop indirect sequence capture followed by short- and long-read sequencing.

Validation of CRISPR-Cas9 editing typically explores the immediate vicinity of the gene editing site and distal off-target sequences, which has led to the conclusion that CRISPR-Cas9 editing is very specific. However, an increasing number of studies suggest that on-target unintended editing events like deletions and insertions are relatively frequent but unfortunately often missed in the validation of CRISPR-Cas9 editing. The deletions may be several kilobases-long and only affect one allele. The gold standard in molecular validation of gene editing is direct sequencing of relatively short PCR amplicons. This approach allows the detection of small editing events but fails in detecting large rearrangements, in particular when only one allele is affected. Detection of large rearrangements requires that an extended region is analyzed and the characterization of events may benefit from long-read sequencing. Here we implemented Xdrop™, a new microfluidic technology that allows targeted enrichment of long regions (~100 kb) using just a single standard PCR primer set. Sequencing of the enriched CRISPR-Cas9 gene-edited region in four cell lines on long- and short-read sequencing platforms unravelled unknown and unintended genome editing events. The analysis revealed accidental kilobases-large insertions in three of the cell lines, which remained undetected using standard procedures. We also applied the targeted enrichment approach to identify the integration site of a transgene in a mouse line. The results demonstrate the potential of this technology in gene editing validation as well as in more classic transgenics.

Fungal dye-decolorizing peroxidase diversity: roles in either intra- or extracellular processes.

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.

Insertional mutagenesis of Brachypodium distachyon using the Tnt1 retrotransposable element.

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.

Application of Target Enrichment Sequencing for Population Genetic Analyses of the Obligate Plant Pathogens Pseudoperonospora cubensis and P. humuli in Michigan.

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.

Genomic signatures of natural selection at phenology-related genes in a widely distributed tree species Fagus sylvatica L.

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.

Optimizing Phylogenomics with Rapidly Evolving Long Exons: Comparison with Anchored Hybrid Enrichment and Ultraconserved Elements.

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.

Amplicon genome fishing (AGF): a rapid and efficient method for sequencing target cis-regulatory regions in nonmodel organisms.

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.

A bioinformatic platform to integrate target capture and whole genome sequences of various read depths for phylogenomics.

The increasing availability of short-read whole genome sequencing (WGS) provides unprecedented opportunities to study ecological and evolutionary processes. Although loci of interest can be extracted from WGS data and combined with target sequence data, this requires suitable bioinformatic workflows. Here, we test different assembly and locus extraction strategies and implement them into secapr, a pipeline that processes short-read data into multilocus alignments for phylogenetics and molecular ecology analyses. We integrate the processing of data from low-coverage WGS (<30×) and target sequence capture into a flexible framework, while optimizing de novo contig assembly and loci extraction. Specifically, we test different assembly strategies by contrasting their ability to recover loci from targeted butterfly protein-coding genes, using four data sets: a WGS data set across different average coverages (10×, 5× and 2×) and a data set for which these loci were enriched prior to sequencing via target sequence capture. Using the resulting de novo contigs, we account for potential errors within contigs and infer phylogenetic trees to evaluate the ability of each assembly strategy to recover species relationships. We demonstrate that choosing multiple sizes of kmer simultaneously for assembly results in the highest yield of extracted loci from de novo assembled contigs, while data sets derived from sequencing read depths as low as 5× recovers the expected species relationships in phylogenetic trees. By making the tested assembly approaches available in the secapr pipeline, we hope to inspire future studies to incorporate complementary data and make an informed choice on the optimal assembly strategy.

Comparison of sequence-capture and ddRAD approaches in resolving species and populations in hexacorallian anthozoans.

Genome-level sequencing is the next step in understanding species-level relationships within Anthozoa (soft corals, anemones, stony corals, and their kin) as morphological and PCR-directed (single-locus) sequencing methods often fall short of differentiating species. The sea anemone genus Metridium is a common northern temperate sea anemone whose species are difficult to differentiate using morphology alone. Here we use Metridium as a case study to confirm the low level of information available in six loci for species differentiation commonly sequenced for Actiniaria and explore and compare the efficacy of ddRAD and sequence-capture methods in species-level systematics and biogeographic studies. We produce phylogenetic trees from concatenated datasets and perform DAPC and STRUCTURE analyses using SNP data. The six conventional loci are not able to consistently differentiate species within Metridium. The sequence-capture dataset resulted in high support and resolution for both current species and relationships between geographic areas. The ddRAD datasets displayed ambiguity among species, and support between major geographic groupings was not as high as the sequence-capture datasets. The level of resolution and support resulting from the sequence-capture data, combined with the ability to add additional individuals and expand beyond the genus Metridium over time, emphasizes the utility of sequence-capture methods for both systematics and future biogeographic studies within anthozoans. We discuss the strengths and weaknesses of the genomic approaches in light of our findings and suggest potential implications for the biogeography of Metridium based on our sampling.

Phylogenomics of Gesneriaceae using targeted capture of nuclear genes.

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.