Premise of the study: Using sequence data generated via target enrichment for phylogenetics requires reassembly of high‐throughput sequence reads into loci, presenting a number of bioinformatics challenges. We developed HybPiper as a user‐friendly platform for assembly of gene regions, extraction of exon and intron sequences, and identification of paralogous gene copies. We test HybPiper using baits designed to target 333 phylogenetic markers and 125 genes of functional significance in Artocarpus (Moraceae).Methods and Results: HybPiper implements parallel execution of sequence assembly in three phases: read mapping, contig assembly, and target sequence extraction. The pipeline was able to recover nearly complete gene sequences for all genes in 22 species of Artocarpus. HybPiper also recovered more than 500 bp of nontargeted intron sequence in over half of the phylogenetic markers and identified paralogous gene copies in Artocarpus.Conclusions: HybPiper was designed for Linux and Mac OS X and is freely available at https://github.com/mossmatters/HybPiper.

Premise of the Study: Untapped information about allele diversity within populations and individuals (i.e., heterozygosity) could improve phylogenetic resolution and accuracy. Many phylogenetic reconstructions ignore heterozygosity because it is difficult to assemble allele sequences and combine allele data across unlinked loci, and it is unclear how reconstruction methods accommodate variable sequences. We review the common methods of including heterozygosity in phylogenetic studies and present a novel method for assembling allele sequences from target‐enriched Illumina sequencing libraries.Methods: We performed supermatrix phylogeny reconstruction and species tree estimation of Artocarpus based on three methods of accounting for heterozygous sequences: a consensus method based on de novo sequence assembly, the use of ambiguity characters, and a novel method for incorporating read information to phase alleles. We characterize the extent to which highly heterozygous sequences impeded phylogeny reconstruction and determine whether the use of allele sequences improves phylogenetic resolution or decreases topological uncertainty.Key Results: We show here that it is possible to infer phased alleles from target‐enriched Illumina libraries. We find that highly heterozygous sequences do not contribute disproportionately to poor phylogenetic resolution and that the use of allele sequences for phylogeny reconstruction does not have a clear effect on phylogenetic resolution or topological consistency.Conclusions: We provide a framework for inferring phased alleles from target enrichment data and for assessing the contribution of allelic diversity to phylogenetic reconstruction. In our data set, the impact of allele phasing on phylogeny is minimal compared to the impact of using phylogenetic reconstruction methods that account for gene tree incongruence.

We present a 517-gene phylogenetic framework for the breadfruit genus Artocarpus (ca. 70 spp., Moraceae), making use of silica-dried leaves from recent fieldwork and herbarium specimens (some up to 106 years old) to achieve 96% taxon sampling. We explore issues relating to assembly, paralogous loci, partitions, and analysis method to reconstruct a phylogeny that is robust to variation in data and available tools. Although codon partitioning did not result in any substantial topological differences, the inclusion of flanking noncoding sequence in analyses significantly increased the resolution of gene trees. We also found that increasing the size of data sets increased convergence between analysis methods but did not reduce gene-tree conflict. We optimized the HybPiper targeted-enrichment sequence assembly pipeline for short sequences derived from degraded DNA extracted from museum specimens. Although the subgenera of Artocarpus were monophyletic, revision is required at finer scales, particularly with respect to widespread species. We expect our results to provide a basis for further studies in Artocarpus and provide guidelines for future analyses of data sets based on target enrichment data, particularly those using sequences from both fresh and museum material, counseling careful attention to the potential of off-target sequences to improve resolution. [Artocarpus; Moraceae; noncoding sequences; phylogenomics; target enrichment.]