Genome-wide SNPs resolve phylogenetic relationships in the North American spruce budworm (Choristoneura fumiferana) species complex

Highlights

• Genotyping-by-sequencing has resolved relationships in the spruce budworm complex.

• Multiple analyses agreed on an unexpectedly basal placement for Choristoneura pinus.

• Relationships remain ambiguous for a clade of western species.

• Speciation has likely been driven by a combination of ecological factors.

Abstract

High throughput sequencing technologies have revolutionized the potential to reconcile incongruence between gene and species trees, and numerous approaches have been developed to take advantage of these advances. Genotyping-by-sequencing is becoming a regular tool for gathering phylogenetic data, yet comprehensive evaluations of phylogenetic methods using these data are sparse. Here we use multiple phylogenetic and population genetic methods for genotyping-by-sequencing data to assess species relationships in a group of forest insect pests, the spruce budworm (Choristoneura fumiferana) species complex. With few exceptions, all methods agree on the same relationships, most notably placing C. pinus as basal to the remainder of the group, rather than C. fumiferana as previously suggested. We found strong support for the monophyly of C. pinus, C. fumiferana, and C. retiniana, but more ambiguous relationships and signatures of introgression in a clade of western lineages, including C. carnana, C. lambertiana, C. occidentalis occidentalis, C. occidentalis biennis, and C. orae. This represents the most taxonomically comprehensive genomic treatment of the spruce budworm species group, which is further supported by the broad agreement among multiple methodologies.

Introduction

Incongruence between gene and species trees has been a long-recognized concept in molecular systematics and phylogenetics (e.g. Fitch, 1970, Avise et al., 1983, Maddison, 1997). Conceptually, this incongruence can be accommodated by considering the modal gene phylogeny (the most frequently occurring gene phylogeny) as the species phylogeny (Pamilo and Nei, 1988, Maddison, 1997, Sperling, 2003). Ten to twenty years ago, the main limitation of this conceptual solution was methodological: the prohibitive cost of gathering sufficient data to estimate a modal gene phylogeny, especially for many taxa. This particular limitation has been substantially decreased by the immense growth of high-throughput next-generation sequencing (NGS) (Metzker, 2009). However, systematists now face another suite of methodological challenges regarding how to appropriately use these genomic data to construct phylogenies. Genotyping-by-sequencing (GBS: Elshire et al., 2011, Poland et al., 2012) has garnered particular interest for phylogenetic reconstruction (Miller et al., 2007, Baird et al., 2008, Davey et al., 2011), particularly for relatively shallow phylogenetic context (see Rubin et al., 2012) (note: although we focus on GBS here, the same methodological considerations apply to other restriction-site-associated DNA sequencing (RAD-seq) techniques; for simplicity in referring to methodological considerations we use “GBS” exclusively). Flexible requirements for a priori genomic resources and cost-effectiveness at the scales of loci and individuals (Peterson et al., 2012) make GBS ideal for systematics in the current era, where the distinction between traditional phylogenetics and population genetics is fading (Edwards, 2009). Although a growing number of studies have illustrated the utility of GBS for reconstructing phylogenies (e.g. Rubin et al., 2012, Wagner et al., 2012, Eaton and Ree, 2013, Jones et al., 2013, Nadeau et al., 2013, Cruaud et al., 2014, Hipp et al., 2014, Gohli et al., 2015, DaCosta and Sorenson, 2016, Díaz-Arce et al., 2016, Stervander et al., 2016, Rivers et al., 2016), critical and comprehensive evaluations of the phylogenetic methods used for these datasets are still relatively rare (e.g. DaCosta and Sorenson, 2016).

Most empirical studies using GBS for phylogenomics have relied on concatenation (or supermatrix) methods, under the assumption that the volume of phylogenetic signal in thousands of loci outweighs any potential gene tree/species tree discordance (e.g. Nadeau et al., 2013, Wagner et al., 2012, Cruaud et al., 2014). Concatenation methods simplify model selection for genomic data and have been shown to be robust for estimating species trees from GBS data (Rivers et al., 2016). However, many studies have also shown that such treatment of phylogenomic data can be misleading for both determination of species relationships and evaluation of tree support (e.g. strong bootstrap support for incorrect relationships) (Kubatko and Degnan, 2007, Weisrock et al., 2012, McVay and Carstens, 2013, Wielstra et al., 2014, Giarla and Esselstyn, 2015). Alternatively, coalescent methods (such as BEAST: Drummond and Rambaut, 2007), which may be more appropriate when genealogical discordance exists, present computational challenges for GBS data, and may become unfeasible with large datasets (e.g. Zimmerman et al., 2014). GBS loci are also relatively short (generally <100 bp) and have few variable sites per locus, which can lead to unresolved relationships when analyzed individually to construct “gene” trees (following “traditional” methods to determine species trees: Knowles and Kubatko, 2010). Likewise, emerging methods that use single nucleotide polymorphisms (SNPs) rather than sequence-based data (e.g. SNAPP: Bryant et al., 2012) can also be computationally demanding with large datasets. Given the methodological uncertainty surrounding GBS data and phylogenomics, and apparent impracticality of some approaches, critical evaluation of the leading methods is vital to establishing best practices for its application in phylogenetic contexts.

Here, we assessed phylogenetic relationships among lineages of the spruce budworm (Choristoneura fumiferana (Clemens, 1865)) species complex in North America using GBS data. Species in this group (most notably C. fumiferana, considered North America’s most destructive insect defoliator of living conifers: Volney and Fleming, 2007) exhibit wide population oscillations, with outbreak densities causing high tree mortality (Gray and MacKinnon, 2006) and serious economic losses. The complex is composed of eight or nine species (Lumley and Sperling, 2011a, Gilligan et al., 2014, Brunet et al., 2016) that differ primarily on the basis of larval host preferences (Stehr, 1967, Harvey, 1985), female sex-pheromone chemistry (Sanders et al., 1977), diapause characteristics (Harvey, 1967) and geography (Stehr, 1967). However, phylogenetic relationships in the complex remain uncertain, especially the placement of the eastern pine feeding species, Choristoneura pinus Freeman 1953 (Fig. 1). While most marker systems place C. fumiferana as the sister taxon to the rest of the complex, a recent analysis employing a genome-wide set of SNPs has cast doubt on this hypothesis by placing C. pinus basal to the group (Bird, 2013). However, these findings were confounded by the omission of several vital taxa within the group, including other pine feeders (e.g. C. lambertiana (Busck 1915)), and limited by the sole use of concatenation methods for phylogeny reconstruction.

In this study, we include all consistently recognized species in the complex and assess phylogenetic relationships of the group using multiple methods and datasets. First, we implement traditional phylogenetic methods with a concatenated (supermatrix) dataset. Given the common use of GBS datasets for population genomics, we compare the results of concatenated phylogenetic analyses to individual-based Bayesian clustering and ordinations using SNPs. Finally, we implement several approaches to assess gene tree/species tree incongruence, with and without a coalescent framework using both SNPs and sequence data. We find strong support for a single set of species-level relationships, and use this phylogeny to interpret mechanisms for speciation in the complex. This study represents the most taxonomically comprehensive phylogenomic evaluation of the spruce budworm species complex to date.