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.

Hundreds of nuclear and plastid loci yield novel insights into orchid relationships.

PREMISE: The inference of evolutionary relationships in the species-rich family Orchidaceae has hitherto relied heavily on plastid DNA sequences and limited taxon sampling. Previous studies have provided a robust plastid phylogenetic framework, which was used to classify orchids and investigate the drivers of orchid diversification. However, the extent to which phylogenetic inference based on the plastid genome is congruent with the nuclear genome has been only poorly assessed. METHODS: We inferred higher-level phylogenetic relationships of orchids based on likelihood and ASTRAL analyses of 294 low-copy nuclear genes sequenced using the Angiosperms353 universal probe set for 75 species (representing 69 genera, 16 tribes, 24 subtribes) and a concatenated analysis of 78 plastid genes for 264 species (117 genera, 18 tribes, 28 subtribes). We compared phylogenetic informativeness and support for the nuclear and plastid phylogenetic hypotheses. RESULTS: Phylogenetic inference using nuclear data sets provides well-supported orchid relationships that are highly congruent between analyses. Comparisons of nuclear gene trees and a plastid supermatrix tree showed that the trees are mostly congruent, but revealed instances of strongly supported phylogenetic incongruence in both shallow and deep time. The phylogenetic informativeness of individual Angiosperms353 genes is in general better than that of most plastid genes. CONCLUSIONS: Our study provides the first robust nuclear phylogenomic framework for Orchidaceae and an assessment of intragenomic nuclear discordance, plastid-nuclear tree incongruence, and phylogenetic informativeness across the family. Our results also demonstrate what has long been known but rarely thoroughly documented: nuclear and plastid phylogenetic trees can contain strongly supported discordances, and this incongruence must be reconciled prior to interpretation in evolutionary studies, such as taxonomy, biogeography, and character evolution.

Plastid phylogenomics resolves ambiguous relationships within the orchid family and provides a solid timeframe for biogeography and macroevolution.

Recent phylogenomic analyses based on the maternally inherited plastid organelle have enlightened evolutionary relationships between the subfamilies of Orchidaceae and most of the tribes. However, uncertainty remains within several subtribes and genera for which phylogenetic relationships have not ever been tested in a phylogenomic context. To address these knowledge-gaps, we here provide the most extensively sampled analysis of the orchid family to date, based on 78 plastid coding genes representing 264 species, 117 genera, 18 tribes and 28 subtribes. Divergence times are also provided as inferred from strict and relaxed molecular clocks and birth-death tree models. Our taxon sampling includes 51 newly sequenced plastid genomes produced by a genome skimming approach. We focus our sampling efforts on previously unplaced clades within tribes Cymbidieae and Epidendreae. Our results confirmed phylogenetic relationships in Orchidaceae as recovered in previous studies, most of which were recovered with maximum support (209 of the 262 tree branches). We provide for the first time a clear phylogenetic placement for Codonorchideae within subfamily Orchidoideae, and Podochilieae and Collabieae within subfamily Epidendroideae. We also identify relationships that have been persistently problematic across multiple studies, regardless of the different details of sampling and genomic datasets used for phylogenetic reconstructions. Our study provides an expanded, robust temporal phylogenomic framework of the Orchidaceae that paves the way for biogeographical and macroevolutionary studies.

A Guide to Carrying Out a Phylogenomic Target Sequence Capture Project.

High-throughput DNA sequencing techniques enable time- and cost-effective sequencing of large portions of the genome. Instead of sequencing and annotating whole genomes, many phylogenetic studies focus sequencing effort on large sets of pre-selected loci, which further reduces costs and bioinformatic challenges while increasing coverage. One common approach that enriches loci before sequencing is often referred to as target sequence capture. This technique has been shown to be applicable to phylogenetic studies of greatly varying evolutionary depth. Moreover, it has proven to produce powerful, large multi-locus DNA sequence datasets suitable for phylogenetic analyses. However, target capture requires careful considerations, which may greatly affect the success of experiments. Here we provide a simple flowchart for designing phylogenomic target capture experiments. We discuss necessary decisions from the identification of target loci to the final bioinformatic processing of sequence data. We outline challenges and solutions related to the taxonomic scope, sample quality, and available genomic resources of target capture projects. We hope this review will serve as a useful roadmap for designing and carrying out successful phylogenetic target capture studies.

Frugivory in Canopy Plants in a Western Amazonian Forest: Dispersal Systems, Phylogenetic Ensembles and Keystone Plants.

Frugivory is a widespread mutualistic interaction in which frugivores obtain nutritional resources while favoring plant recruitment through their seed dispersal services. Nonetheless, how these complex interactions are organized in diverse communities, such as tropical forests, is not fully understood. In this study we evaluated the existence of plant-frugivore sub-assemblages and their phylogenetic organization in an undisturbed western Amazonian forest in Colombia. We also explored for potential keystone plants, based on network analyses and an estimate of the amount of fruit going from plants to frugivores. We carried out diurnal observations on 73 canopy plant species during a period of two years. During focal tree sampling, we recorded frugivore identity, the duration of each individual visit, and feeding rates. We did not find support for the existence of sub assemblages, such as specialized vs. generalized dispersal systems. Visitation rates on the vast majority of canopy species were associated with the relative abundance of frugivores, in which ateline monkeys (i.e. Lagothrix and Ateles) played the most important roles. All fruiting plants were visited by a variety of frugivores and the phylogenetic assemblage was random in more than 67% of the cases. In cases of aggregation, the plant species were consumed by only primates or only birds, and filters were associated with fruit protection and likely chemical content. Plants suggested as keystone species based on the amount of pulp going from plants to frugivores differ from those suggested based on network approaches. Our results suggest that in tropical forests most tree-frugivore interactions are generalized, and abundance should be taken into account when assessing the most important plants for frugivores.