RESEARCH ARTICLE INVITED SPECIAL ARTICLE For the Special Issue: Using and Navigating the Plant Tree of Life Next- generation lineage discovery: A case study of tuberous Claytonia L. Thomas R. Stoughton1,4, Ricardo Kriebel2, Diana D. Jolles1, and Robin L. O’Quinn3 Manuscript received 14 June 2017; revision accepted 18 December PREMISE OF THE STUDY: Species formation is an intuitive endpoint of reproductive isolation, 2017. but circumscribing taxa that arise during speciation can be difcult because of gene fow, 1 Center for the Environment and Department of Biological morphological continuity, hybridization or polyploidization, and low sequence variation Sciences, Plymouth State University, 17 High Street, Plymouth, among newly diverged lineages. Nonetheless, species complexes are ubiquitous, and their NH 03264-1594, USA classifcation is essential for understanding how diversity infuences ecosystem function. 2 Department of Botany, University of Wisconsin-Madison, Madison, WI 53706, USA METHODS: We used modern sequencing technology to identify lineages of perennial Claytonia 3 Department of Biology, Eastern Washington University, 258 L. and assessed correspondence between genetic lineages and morphological/ecological Science Building, 526 5th Street, Cheney, WA 99004-2440, USA variation. Subsets of 18 taxa from 63 populations were used for (a) lineage discovery using 4 Author for correspondence (e-mail: thomas.stoughton@gmail. network and coalescent analyses, (b) leaf shape analyses using elliptical Fourier analysis and com) ordination, and (c) ecological analyses (soil chemistry, climate) using ANOVA and ordination. Citation: Stoughton, T. R., R. Kriebel, D. D. Jolles, and R. L. O’Quinn. 2018. Next- generation lineage discovery: A case study of KEY RESULTS: Samples mainly aggregated into groups representing each of the previously tuberous Claytonia L. American Journal of Botany 105(3): 536–548. recognized species in each of the genetic data sets. Compared to the double- digest doi:10.1002/ajb2.1061 restriction- site- associated DNA sequencing data set, genome skimming data provided more resolution and further opportunity to probe into patterns of nuclear and chloroplast genome diversity. Morphological and ecological associations are signifcantly diferent (albeit intergrading) among the taxa investigated. A new species, Claytonia crawfordii, is described based on morphological data presented here. CONCLUSIONS: Genetic data presented in this study provide some of the frst insights into phylogenetic relationships among recently diverged perennial Claytonia and are suggestive of past hybridization among caudicose and tuberous species. Given prior difculties in understanding species boundaries among newly diverged plant lineages, this case study demonstrates the revolutionary breakthrough for systematics research that high throughput sequencing represents. KEY WORDS ddRAD; edaphic; elliptical Fourier analysis; genome skimming; Montiaceae; new species; next-generation sequencing; reticulation; statistical ordination; SVD Quartets. Taxonomic circumscription at and below the species bound- and introgression can contribute to complex patterns of gene fow ary can be as difcult technically (Hamilton and Reichard, 1992; and phylogenetic diversifcation, morphological continuity, and Baum and Donoghue, 1995; Spaniel et al., 2012) as it is controver- low sequence variation among newly diverged lineages (Funk and sial (Hey, 2006; Pigliucci, 2003; Stace, 2013; Walsh, 2015; Zachos, Omland, 2003). Evidence of recent gene fow, however infrequent, 2015). Polyploidization or admixture resulting from hybridization violates the rule of reproductive isolation in the strict sense and American Journal of Botany 105(3): 536–548, 2018; http://www.wileyonlinelibrary.com/journal/AJB © 2018 The Authors. American Journal of Botany is published by Wiley Periodicals, Inc. on behalf of the Botanical Society of America. This is an open access article under the terms of the Creative Commons Attribution License, 536 • which permits use, distribution and reproduction in any medium, provided the original work is properly cited. March 2018, Volume 105 • Stoughton et al.—Next- generation Claytonia lineage discovery • 537 relative reproductive isolation (resulting from introgression) is dif- Tere is a trade- of with NGS between sampling breadth (meas- fcult to quantify in a way that directly translates to taxonomic rank ured both in number of loci and samples) and depth of coverage (Hamilton and Reichard, 1992; Cronin, 2006). Regardless of these when it comes to sequencing: maximizing breadth and sacrifcing challenges, groups of actively diverging or recently diverged lineages depth (our metric for statistical confdence in a consensus sequence (i.e., species complexes) likely make up a considerable portion of the for any particular locus) would result in a less accurate phylogenetic standing diversity in plant communities across the globe, undoubt- estimate. As noted above, the list of sampling possibilities, alternative edly have important ecological roles in ecosystems, and should be methods, and scenarios worth considering for starting a NGS project, recognized at some taxonomic level for the purposes of conservation including explanation of terms, have been discussed fairly thoroughly and management. Distinctions among species, metapopulations, by other authors (Godden et al., 2012; Lemmon and Lemmon, 2013; and populations (Cronin, 2006) are essential for understanding Edwards et al., 2015; Zimmer and Wen, 2015; Andrews et al., 2016), whether sufcient isolation has occurred, and will be maintained, so we focus instead on high- throughput sequencing methodologies as to warrant species rank. More and more workers (Baum, 2009; de they are applied to species delimitation, a topic that has not yet been Querioz, 2007) seem to favor the idea that speciation, rather than broadly addressed with regard to efectiveness and feasibility. Eforts being a strictly punctuated event, is the gradual establishment of by plant systematists to sample and sequence greater taxonomic diferent types of isolation (e.g., phenological, ecological, gametic). breadth across the plant tree of life (Palmer et al., 2004; Matasci et al., Species delimitation for recently diverged lineages based solely 2014; Wickett et al., 2014) has yielded precious resources: genomic on morphological criteria lost favor among many taxonomists when reference sequences for a variety of model and nonmodel plant spe- PCR and Sanger sequencing in biological studies became more avail- cies that can be used to explore microevolutionary patterns and pro- able and afordable. Before the advent of high- throughput sequencing cesses. Delineating taxa at the specifc and infraspecifc level will be technology, genetic data were sparse and therefore most ofen used increasingly possible, allowing conservation botanists to better un- simply to corroborate or contradict morphology- based phylogenetic derstand, identify, and manage taxa. hypotheses. As genetic data have become even cheaper and easier to Te genus Claytonia L. (Montiaceae, order Caryophyllales) is one obtain, molecular phylogenies are used as best approximations of the of many plant lineages that underwent a recent radiation during or “true tree”, and morphological data are increasingly used in the con- afer the Miocene (crown age ca. 10–20 million years; Jefers, 2015), text of molecular- based hypotheses as post- hoc assessments of char- and this diversifcation is likely associated with allopolyploidization acter evolution (e.g., adaptation). Morphology alone can be difcult (Miller and Chambers, 2006). Other examples of hybridization- and to interpret and today may seem like scant support for species given polyploidy- driven radiations in plants include the Hawaiian silver- the relative ease of obtaining molecular data and using modern com- swords (Argyroxiphium D.C.; Barrier et al., 1999), North American putational phylogenetic methods to support or refute hypotheses. Silene L. (Popp and Oxelman, 2007), Penstemon Schmidel (Wolfe High- throughput next- generation sequencing (NGS) methods are et al., 2006), and the mustard family (Brassicaceae Burnett; Marhold also still relatively unapproachable for many researchers who identify and Lihová, 2006). Accurate estimates of standing biodiversity for as feld biologists rather than molecular biologists. these complex groups and others are needed because species are es- Beyond the formidable task of learning a specialized lexicon, sential metrics in ecological studies that either use biodiversity as an NGS methods are not a “one size fts all” for molecular systemat- experimental variable or aim to explain complex species interactions. ics, especially in the case of species complexes. When considering In addition, many analytical methods for phylogenetic estimation a project, proximity to species boundaries and questions related to require thoughtful sampling of taxa to estimate diversity parame- the biological integrity of these boundaries tend to dictate which ters such as fxation indices with high accuracy. Tese phylogenetic approach is best applied in a given situation (Godden et al., 2012; analyses will be greatly improved by more precise taxonomic classif- Lemmon and Lemmon, 2013; Edwards et al., 2015; Zimmer and cations, which will in turn enable accurate biodiversity assessments. Wen, 2015; Andrews et al., 2016). A restriction enzyme- based ap- Last, recognition of discrete lineages will advance our understand-