Exon probe sets and bioinformatics pipelines for all levels of fish phylogenomics

Abstract

24Exon markers have a long history of use in phylogenetics of ray-finned fishes, the most diverse 25 clade of vertebrates with more than 35,000 species. As the number of published genomes 26 increases, it has become easier to test exons and other genetic markers for signals of ancient 27 duplication events and filter out paralogs that can mislead phylogenetic analysis. We present 28 seven new probe sets for current target-capture phylogenomic protocols that capture 1,104 exons 29 explicitly filtered for paralogs using gene trees. These seven probe sets span the diversity of 30 teleost fishes, including four sets that target five hyper-diverse percomorph clades which 31 together comprise ca. 17,000 species (Carangaria, Ovalentaria, Eupercaria, and Syngnatharia + 32 Pelagiaria combined). We additionally included probes to capture exon markers that have been 33 commonly used in fish phylogenetics, despite some being flagged for paralogs, to facilitate 34 integration of old and new molecular phylogenetic matrices. We tested these probes and merged 35 new exon-capture sequence data into an existing data matrix of 1,105 exons and 300 ray-finned 36 fish species. We provide an optimized bioinformatics pipeline to assemble exon capture data 37 from raw reads to alignments for downstream analysis. We show that loci with known paralogs 38 are at risk of assembling duplicated sequences with target-capture, but we also assembled many 39 useful orthologous sequences. These probe sets are a valuable resource for advancing fish 40 phylogenomics because they can be easily extracted from increasingly available whole genome 41 and transcriptome datasets, and also may be integrated with existing PCR-based exon and 42 mitochondrial datasets. 43 44 Exon markers have played a pivotal role in resolving phylogenetic relationships among ray-46 finned fishes (Li et al. 2007;Near et al. 2012;Betancur-R. et al. 2013;Betancur-R et al. 2017; 47 Hughes et al. 2018;Rabosky et al. 2018). Identification of these exons has typically involved the 48 comparison of a small number of fish model genomes. For example, a suite of 154 single-copy 49 exons conserved enough for PCR amplification was identified by Li et al. (2007). This study 50 used a reciprocal BLAST approach on two genomes, the pufferfish Takifugu rubripes and the 51 zebrafish Danio rerio, with 10 exons initially optimized with PCR primers for sequencing (Li et 52 al. 2007). These exons demonstrated their utility for resolving previously enigmatic relationships 53 among fishes (Li et al. 2008), and were the basis for largescale reappraisals of the ray-finned fish 54

Extracting ‘legacy loci’ from an invertebrate sequence capture data set

“…Although NGS and sequence capture approaches continue to supplant Sanger‐sequencing approaches based on one to a few loci (herein referred to as ‘legacy loci’), there has been recent interest in exploring how legacy loci can complement sequence capture data (e.g. Blaimer et al., 2015; Branstetter et al., 2017, 2021; Derkarabetian et al., 2019; Hughes et al., 2021; Simon et al., 2019; Zhang et al., 2019). Specifically, the generation of more comprehensive data sets may increase resolution power for phylogenetic inference and allow for the inclusion of rare and vital species that may be difficult to sample repeatedly (Branstetter et al., 2017; Derkarabetian et al., 2019; Zhang, Williams, et al., 2019).…”

“…One approach to integrating legacy loci with sequence capture data involves designing capture baits of legacy loci for inclusion in existing sequence capture bait kits, such as optimized UCE and exon‐capture bait sets (Branstetter et al., 2017; Hughes et al., 2021; Simon et al., 2019). However, this approach may increase the cost of generating custom probe kits and may require more baits across more species due to higher rates of substitutions in some legacy loci (particularly mitochondrial DNA [mtDNA]).…”

Evolutionary determinism and convergence associated with water-column transitions in marine fishes

“…High-quality DNA extractions were sent to Arbor Biosciences for target enrichment and sequencing. Our target capture probes are based on a set of 1,104 single-copy exons optimized for rayfinned fish phylogenetics (26,27). We also included 15 legacy exons into the probe set.…”

“…Extended results are reported in the SI Appendix. Using exon capture approaches (26,27), we assembled two main phylogenomic data matrices: 1) an expanded supermatrix that includes all genes and taxa sequenced for this study, with the addition of GenBank sequences aimed at increasing taxonomic coverage for downstream comparative analyses (1,115 exons and 474,132 nucleotide sites for 110 out of ca. 136 species; 37% missing cells), and 2) a reduced (phylogenomic-only) matrix obtained with a matrix reduction algorithm, used to assess the sensitivity of phylogenomic results to missing data (1,047 exons and 448,410 nucleotide sites for 84 species; 16% missing cells).…”

Phylogenomics of piranhas and pacus (Serrasalmidae) uncovers how convergent diets obfuscate traditional morphological taxonomy

“…We used the bioinformatics pipeline optimized by Hughes et al (2020) to obtain sequence alignments for 951 exon markers from an initial set of 1051 (S1 appendix). Raw FASTQ files were trimmed with Trimmomatic v0.36 (Bolger et al 2014), to remove low quality sequences and adapter contamination.…”