With the increasing availability of high-throughput sequencing, phylogenetic analyses are no longer constrained by the limited availability of a few loci. Here, we describe a sequence capture methodology, which we used to collect data for analyses of diversification within Sabal (Arecaceae), a palm genus native to the south-eastern USA, Caribbean, Bermuda and Central America. RNA probes were developed and used to enrich DNA samples for putatively low copy nuclear genes and the plastomes for all Sabal species and two outgroup species. Sequence data were generated on an Illumina MiSeq sequencer and target sequences were assembled using custom workflows. Both coalescence and supermatrix analyses of 133 nuclear genes were used to estimate species trees relationships. Plastid genomes were also analysed, yielding generally poor resolution with regard to species relationships. Species relationships described in both nuclear gene and plastome sequences largely reflect the biogeography of the group and, to a lesser extent, previous morphology-based hypotheses. Beyond the biological implications, this research validates a high-throughput methodology for generating a large number of genes for coalescence-based phylogenetic analyses in plant lineages.

Museums hold most of the world’s most valuable biological specimens and tissues collected, including type material that is often decades or even centuries old. Unfortunately, traditional museum collection and storage methods were not designed to preserve the nucleic acids held within the material, often reducing its potential viability and value for many genetic applications. High-throughput sequencing technologies and associated applications offer new opportunities for obtaining sequence data from museum samples. In particular, target sequence capture offers a promising approach for recovering large numbers of orthologous loci from relatively small amounts of starting material. In the present study, we test the utility of target sequence capture for obtaining data from museum-held material from a speciose mammalian genus: the horseshoe bats (Rhinolophidae: Chiroptera). We designed a ‘bait’ for capturing > 3600 genes and applied this to 10 species of horseshoe bat that had been collected between 93 and 7 years ago and preserved using a range of methods. We found that the mean recovery rate per species was approximately 89% of target genes with partial sequence coverage, ranging from 3024 to 3186 genes recovered. On average, we recovered 1206 genes with ≥ 90% sequence coverage, per species. Our findings provide good support for the application of large-scale bait capture across congeneric species spanning approximately 15 Myr of evolution. On the other hand, we observed no clear association between the success of capture and the phylogenetic distance from the bait model, although sample sizes precluded a formal test.

The genus Cucurbita (squashes, pumpkins, gourds) contains numerous domesticated lineages with ancient New World origins. It was broadly distributed in the past but has declined to the point that several of the crops’ progenitor species are scarce or unknown in the wild. We hypothesize that Holocene ecological shifts and megafaunal extinctions severely impacted wild Cucurbita, whereas their domestic counterparts adapted to changing conditions via symbiosis with human cultivators. First, we used high-throughput sequencing to analyze complete plastid genomes of 91 total Cucurbita samples, comprising ancient (n = 19), modern wild (n = 30), and modern domestic (n = 42) taxa. This analysis demonstrates independent domestication in eastern North America, evidence of a previously unknown pathway to domestication in northeastern Mexico, and broad archaeological distributions of taxa currently unknown in the wild. Further, sequence similarity between distant wild populations suggests recent fragmentation. Collectively, these results point to wild-type declines coinciding with widespread domestication. Second, we hypothesize that the disappearance of large herbivores struck a critical ecological blow against wild Cucurbita, and we take initial steps to consider this hypothesis through cross-mammal analyses of bitter taste receptor gene repertoires. Directly, megafauna consumed Cucurbita fruits and dispersed their seeds; wild Cucurbita were likely left without mutualistic dispersal partners in the Holocene because they are unpalatable to smaller surviving mammals with more bitter taste receptor genes. Indirectly, megafauna maintained mosaic-like landscapes ideal for Cucurbita, and vegetative changes following the megafaunal extinctions likely crowded out their disturbed-ground niche. Thus, anthropogenic landscapes provided favorable growth habitats and willing dispersal partners in the wake of ecological upheaval.

The formation of allopolyploid cotton precipitated a rapid diversification and colonization of dry coastal American tropical and subtropical regions. Previous phylogenetic analyses, combined with molecular divergence analyses, have offered a temporal framework for this radiation, but provide only weak support for some of the resolved branches. Moreover, these earlier analyses did not include the recently recognized sixth polyploid species, G. ekmanianum Wittmack. Here we use targeted sequence capture of multiple loci in conjunction with both concatenated and Bayesian concordance analyses to reevaluate the phylogeny of allopolyploid cotton species. Although phylogenetic resolution afforded by individual genes is often low, sufficient signal was attained both through the concatenated and concordance analyses to provide robust support for the Gossypium polyploid clade, which is reported here.

Targeted Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach to identify novel sequence variation in genomes, with the aims of investigating gene function and/or developing useful alleles for breeding. Despite recent advances in wheat genomics, most current TILLING methods are low to medium in throughput, being based on PCR amplification of the target genes. We performed a pilot-scale evaluation of TILLING in wheat by next-generation sequencing through exon capture. An oligonucleotide-based enrichment array covering ~2 Mbp of wheat coding sequence was used to carry out exon capture and sequencing on three mutagenised lines of wheat containing previously-identified mutations in the TaGA20ox1 homoeologous genes. After testing different mapping algorithms and settings, candidate SNPs were identified by mapping to the IWGSC wheat Chromosome Survey Sequences. Where sequence data for all three homoeologues were found in the reference, mutant calls were unambiguous; however, where the reference lacked one or two of the homoeologues, captured reads from these genes were mis-mapped to other homoeologues, resulting either in dilution of the variant allele frequency or assignment of mutations to the wrong homoeologue. Competitive PCR assays were used to validate the putative SNPs and estimate cut-off levels for SNP filtering. At least 464 high-confidence SNPs were detected across the three mutagenized lines, including the three known alleles in TaGA20ox1, indicating a mutation rate of ~35 SNPs per Mb, similar to that estimated by PCR-based TILLING. This demonstrates the feasibility of using exon capture for genome re-sequencing as a method of mutation detection in polyploid wheat, but accurate mutation calling will require an improved genomic reference with more comprehensive coverage of homoeologues.