Information Dropout Patterns in Restriction Site Associated DNA Phylogenomics and a Comparison with Multilocus Sanger Data in a Species-Rich Moth Genus

Abstract

A rapid shift from traditional Sanger sequencing-based molecular methods to the phylogenomic approach with large numbers of loci is underway. Among phylogenomic methods, restriction site associated DNA (RAD) sequencing approaches have gained much attention as they enable rapid generation of up to thousands of loci randomly scattered across the genome and are suitable for nonmodel species. RAD data sets however suffer from large amounts of missing data and rapid locus dropout along with decreasing relatedness among taxa. The relationship between locus dropout and the amount of phylogenetic information retained in the data has remained largely uninvestigated. Similarly, phylogenetic hypotheses based on RAD have rarely been compared with phylogenetic hypotheses based on multilocus Sanger sequencing, even less so using exactly the same species and specimens. We compared the Sanger-based phylogenetic hypothesis (8 loci; 6172 bp) of 32 species of the diverse moth genus Eupithecia (Lepidoptera, Geometridae) to that based on double-digest RAD sequencing (3256 loci; 726,658 bp). We observed that topologies were largely congruent, with some notable exceptions that we discuss. The locus dropout effect was strong. We demonstrate that number of loci is not a precise measure of phylogenetic information since the number of single-nucleotide polymorphisms (SNPs) may remain low at very shallow phylogenetic levels despite large numbers of loci. As we hypothesize, the number of SNPs and parsimony informative SNPs (PIS) is low at shallow phylogenetic levels, peaks at intermediate levels and, thereafter, declines again at the deepest levels as a result of decay of available loci. Similarly, we demonstrate with empirical data that the locus dropout affects the type of loci retained, the loci found in many species tending to show lower interspecific distances than those shared among fewer species. We also examine the effects of the numbers of loci, SNPs, and PIS on nodal bootstrap support, but could not demonstrate with our data our expectation of a positive correlation between them. We conclude that RAD methods provide a powerful tool for phylogenomics at an intermediate phylogenetic level as indicated by its broad congruence with an eight-gene Sanger data set in a genus of moths. When assessing the quality of the data for phylogenetic inference, the focus should be on the distribution and number of SNPs and PIS rather than on loci.

Double-digest RAD-sequencing: do wet and dry protocol parameters impact biological results?

“…A potential “false” signal due to genotyping inconsistencies at some loci seems negligible compared to the abundance of “true” signal provided by most of the RAD loci. Similar observations have also been made for another species model, i.e.. Galapagos sea lion ( Zalophus wollebaeki ) in Shafer et al (2017), and similarly, several studies have demonstrated that clustering parameters have no impact on phylogenetic reconstruction (Herrera et al, 2015; Hou et al, 2015; Lee et al, 2018).…”

“…Among these, double-digest RADseq, or ddRADseq (Peterson et al, 2012), is highly customizable as regards the final number of loci, depending on the choice of enzymes and range of fragment size selected. The ddRADseq approach has been applied with success to many purposes including population genetic studies (Kjeldsen et al, 2016; Black, Seears, Hollenbeck, & Samollow, 2017; Sherpa, Rioux, Goindin, et al, 2018), phylogenetic reconstructions (DaCosta & Sorenson, 2016; Vargas, Ortiz, & Simpson, 2017; Boubli et al, 2018; Lee et al, 2018; Sherpa, Rioux, Pougnet-Lagarde, & Després, 2018), demographic inferences (Capblancq, Després, Rioux, & Mavárez, 2015; Nunziata, Lance, Scott, Lemmon, & Weisrock, 2017; Settepani et al, 2017; Elleouet & Aitken, 2018) and landscape genetic analyses (Saenz-Agudelo et al, 2015; Johnson, Gaddis, Cairns, Konganti, & Krutovsky, 2017). Despite the recognized advantages of the ddRADseq technique, several limitations and weaknesses arose in the literature (Davey et al, 2013; K. R. Andrews et al, 2016; Lowry et al, 2017).…”

Single nucleotide polymorphism‐based species phylogeny of greater fritillary butterflies (Lepidoptera: Nymphalidae: Speyeria) demonstrates widespread mitonuclear discordance

“…During parameter testing and final data processing in stacks , we output all SNPs for each locus. Some studies have advocated the inclusion of invariant flanking sequence in phylogenetic analysis of SNPs (Wagner et al ., ; Leaché et al ., ); however, given the evolutionary distance in this study, we expected to retain fewer loci in our dataset relative to other studies at shallower evolutionary scales (Arnold et al ., ; Lee et al ., ), as well as to find an overall increase in per‐locus polymorphism (Paris et al ., ). Although parameter testing with the r80 rule reduces the number of spurious or paralogous loci being built in stacks , it does not control removal of potentially erroneous SNPs within a locus; instead, those SNPs are typically filtered by specifying a minimum genotype quality score and/or minor allele frequency.…”

Using genomic information for management planning of an endangered perennial, Viola uliginosa

“…The quantity of genomic DNA (gDNA) was determined with the PicoGreen Kit (Molecular Probes, Eugene, OR, USA). The ddRADseq library was implemented following protocols described in Peterson, Weber, Kay, Fisher, and Hoekstra (2012) and Lee et al (2018) with the following modifications. Briefly, gDNA was digested at 37°C for 3 hr using the restriction enzymes PstI and Ranta et al (2016) with slight modifications according to Matulevičiūté (2015)…”