Molecular Phylogenetics and Evolution 93 (2015) 234–248 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Comparison of five methods for delimitating species in Ophion Fabricius, a diverse genus of parasitoid wasps (Hymenoptera, Ichneumonidae) q ⇑ Marla D. Schwarzfeld , Felix A.H. Sperling Department of Biological Sciences, CW 405 Biological Sciences Building, University of Alberta, Edmonton, Alberta T6G 2E9, Canada article info abstract Article history: DNA taxonomy has been proposed as a method to quickly assess diversity and species limits in highly Received 6 January 2015 diverse, understudied taxa. Here we use five methods for species delimitation and two genetic markers Revised 31 July 2015 (COI and ITS2) to assess species diversity within the parasitoid genus, Ophion. We searched for compen- Accepted 4 August 2015 satory base changes (CBC’s) in ITS2, and determined that they are too rare to be of practical use in delim- Available online 8 August 2015 iting species in this genus. The other four methods used both COI and ITS2, and included distance-based (threshold analysis and ABGD) and tree-based (GMYC and PTP) models. We compared the results of these Keywords: analyses to each other under various parameters and tested their performance with respect to 11 ABGD Nearctic species/morphospecies and 15 described Palearctic species. We also computed barcode accumu- Compensatory base change DNA barcoding lation curves of COI sequences to assess the completeness of sampling. The species count was highly vari- GMYC able depending on the method and parameters used, ranging from 47 to 168 species, with more ITS2 conservative estimates of 89–121 species. Despite this range, many of the Nearctic test species were fairly Poisson tree processes robust with respect to method. We concluded that while there was often good congruence between Species delimitation methods, GMYC and PTP were less reliant on arbitrary parameters than the other two methods and more easily applied to genetic markers other than COI. However, PTP was less successful at delimiting test spe- cies than was GMYC. All methods, as well as the barcode accumulation curves, indicate that several Palearctic species remain undescribed and that we have scarcely begun to appreciate the Nearctic diver- sity within this genus. Ó 2015 Elsevier Inc. All rights reserved. 1. Introduction Many studies using DNA taxonomy, particularly of the COI gene, have delimited species with distance-based clustering methods. Species are a basic unit in studies of ecology, biogeography, and For example, species have been defined as terminal monophyletic evolution, and are essential to applications in conservation or bio- clusters on neighbor-joining trees (Hajibabaei et al., 2006), by a logical control (Claridge et al., 1997; Sites and Marshall, 2004). But standard interspecific threshold of 1–3% (Hebert et al., 2003a,b, most of Earth’s species remain undescribed, while taxonomists are 2004; Smith et al., 2009, 2013; Stalhut et al., 2013; too few and traditional taxonomy is too slow to deal fully with this Strutzenberger et al., 2011; Tang et al., 2012), or by an interspecific unknown diversity (Brooks and Hoberg, 2000; Godfray, 2002; distance of 10X the intraspecific distance (Hebert et al., 2004). Wheeler, 2004). With the advent of rapid DNA sequencing, molec- While it is widely acknowledged that a single threshold divergence ular taxonomy has been proposed for quickly assessing species will not apply to all taxa (Cognato, 2006; Hendrich et al., 2010; diversity in diverse, little-known taxa (Blaxter, 2004; Pons et al., Monaghan et al., 2009), pre-defined thresholds have nonetheless 2006; Tautz et al., 2003). In particular, the Barcode of Life project been implicitly or explicitly used as criteria for assessing species has popularized use of half of the cytochrome oxidase I (COI) gene diversity in a number of studies (Barrett and Hebert, 2005; of mitochondrial DNA as a standardized ‘‘barcode” for species Hebert et al., 2003a; Smith et al., 2009, 2013; Strutzenberger identification and discovery (Hebert et al., 2003a,b; Miller, 2007). et al., 2011). As well, the universality of barcoding gaps is contro- versial, with several studies finding significant overlap between intra- and interspecific divergences (Meyer and Paulay, 2005; q Wiemers and Fiedler, 2007). The challenge of objectively determin- This paper was edited by the Associate Editor S.L. Cameron. ing this gap led to the development of the automated barcode gap ⇑ Corresponding author at: Canadian National Collection of Insects, Arachnids and Nematodes, 960 Carling Ave., Ottawa, Ontario K1A 0C6, Canada. discovery (ABGD) algorithm, which infers confidence intervals for E-mail address: [email protected] (M.D. Schwarzfeld). intraspecific divergences, and recursively partitions the sequences http://dx.doi.org/10.1016/j.ympev.2015.08.003 1055-7903/Ó 2015 Elsevier Inc. All rights reserved. M.D. Schwarzfeld, F.A.H. Sperling / Molecular Phylogenetics and Evolution 93 (2015) 234–248 235 according to the empirically observed divergences (Puillandre of conspecificity, as closely related species may not have any CBC’s et al., 2012a). (Müller et al., 2007; Wiemers et al., 2009). CBC’s therefore only Distance-based methods have been criticized for being purely indicate a minimum number of species among the specimens phenetic, rather than incorporating evolutionary theory (DeSalle, examined. 2007; Rubinoff et al., 2006; Will and Rubinoff, 2004; Will et al., Ophion is a genus of large-sized (10–20 mm) nocturnal para- 2005). Some explicitly evolutionary approaches have been devel- sitoid wasps in the subfamily Ophioninae of the family oped to address this concern, such as coalescence or tree-based Ichneumonidae. Unlike the majority of ophionine genera, Ophion methods, and these may be used to delimit large numbers of spe- are most diverse in temperate regions, with few species in tropical cies (Carstens and Dewey, 2010; Fujita et al., 2012; Leaché and habitats (Gauld, 1980, 1988). Ophion are large, abundant, and come Fujita, 2010; O’Meara, 2010; Yang and Rannala, 2010). One of these readily to lights; however, Nearctic species have received almost methods, the generalized mixed Yule coalescent (GMYC) model, no taxonomic attention despite being highly apparent and easily determines the point at which coalescent branching patterns collected. Seventeen Nearctic species are currently described (within species) transition to Yule patterns (between species) (Schwarzfeld and Sperling, 2014; Yu et al., 2012), yet it has been (Fontaneto et al., 2007; Pons et al., 2006). This method has shown estimated that the fauna consists of approximately 50 species promise for identifying species boundaries in diverse, little known (Gauld, 1985). In the Palearctic region, Ophion are better known, taxa (Astrin et al., 2012; Ceccarelli et al., 2012; Monaghan et al., with 79 described species (Yu et al., 2012), but there have been 2009); however, the results depend on the choice of parameters no species revisions that span the Palearctic. The fauna has been used to construct the ultrametric tree (Ceccarelli et al., 2012). most comprehensively examined in the United Kingdom, with a The Poisson Tree Processes model (PTP) is another tree-based number of revisions (Gauld, 1973, 1976, 1978) culminating in a method that uses a non-ultrametric phylogenetic tree as input, comprehensive revision of 16 known species (Brock, 1982). thus avoiding the challenges of constructing an accurate ultramet- Ophion are notoriously difficult to identify as they are morpho- ric tree (Zhang et al., 2013). The method delimits species under the logically homogenous among species and yet variable within spe- assumption that the number of substitutions within a species is cies (Brock, 1982; Gauld, 1980). They are thus well-suited to the significantly lower than the number of substitutions between application of molecular taxonomic methods as a first step toward species. species delimitation. Here we estimate Ophion species diversity Regardless of the delimitation method used, reliance on a single using tree-based methods (GMYC, PTP), distance-based methods genetic marker may give misleading results (Dupuis et al., 2012). (ABGD, threshold analysis), and by assessing compensatory base While COI has many advantages for studies of species delimitation, changes of ITS2. The study is mainly based on Canadian specimens, such as its ease of amplification and relatively rapid rate of muta- with the addition of specimens from the United States and repre- tion (Li et al., 2010; Monti et al., 2005; Williams et al., 2012), it may sentatives of all species, except one, that have been described from not accurately delimit species due to factors such as introgression, the United Kingdom. retained ancestral polymorphisms, or coamplification of nuclear pseudogenes (numts) (Cognato, 2006; Dupuis et al., 2012; Funk 2. Methods and Omland, 2003; Meier et al., 2006; Schmidt and Sperling, 2008). It is therefore important to use additional data, such as from 2.1. Specimen collection other genes or morphology, in an integrative taxonomic analysis to more accurately delimit species (Dayrat, 2005; Roe and Sperling, A total of 674 specimens of Ophion were included in this study, 2007; Schlick-Steiner et al., 2010; Schwarzfeld and Sperling, 2014). including 572 from Canada, 81 from Europe, 19 from the United The internal transcribed spacer 2 (ITS2) of ribosomal