Accepted Manuscript SPECIES BOUNDARIES IN THE ASTRAGALUS CUSICKII COMPLEX DE- LIMITED USING MOLECULAR PHYLOGENETIC TECHNIQUES J.C. Zimmers, M. Thomas, L. Yang, A. Bombarely, M.M. Mancuso, M.F. Wojciechowski, J.F. Smith PII: S1055-7903(17)30039-8 DOI: http://dx.doi.org/10.1016/j.ympev.2017.06.004 Reference: YMPEV 5845 To appear in: Molecular Phylogenetics and Evolution Received Date: 11 January 2017 Revised Date: 1 May 2017 Accepted Date: 7 June 2017 Please cite this article as: Zimmers, J.C., Thomas, M., Yang, L., Bombarely, A., Mancuso, M.M., Wojciechowski, M.F., Smith, J.F., SPECIES BOUNDARIES IN THE ASTRAGALUS CUSICKII COMPLEX DELIMITED USING MOLECULAR PHYLOGENETIC TECHNIQUES, Molecular Phylogenetics and Evolution (2017), doi: http:// dx.doi.org/10.1016/j.ympev.2017.06.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. SPECIES BOUNDARIES IN THE ASTRAGALUS CUSICKII COMPLEX DELIMITED USING MOLECULAR PHYLOGENETIC TECHNIQUES J. C. Zimmers1, M. Thomas2, L. Yang2, A. Bombarely3, M. M. Mancuso1, M. F. Wojciechowski4, and J. F. Smith1 1Department of Biological Sciences, Boise State University, 1910 University Drive, Boise, Idaho 83725 U. S. A. 2Department of Biological Sciences, Idaho State University, 921 South 8th Avenue Pocatello, Idaho, 83209-8007 U.S.A. 3Department of Horticulture, Virginia Tech, Latham Hall 216, 220 Ag Quad Lane, Blacksburg, Virginia 24060 U.S.A. 4School of Life Sciences, Arizona State University, Tempe, Arizona 85287-4501, USA Abstract— Understanding the source of phenotypic variability is a challenge in the biological sciences. Variation in phenotypes is the result of variation in the genetics and environment the organism experiences, but elucidating the relative contribution of these two parameters can pose problems, especially in the field of systematics. Systematists are challenged to classify biological diversity into groups that share common ancestry. Phenotypic variation can be useful to demonstrate common ancestry, but only when the primary contributor to the variation is under strong genetic control, and thus heritable. Cusick’s milkvetch (Astragalus cusickii) is a perennial forb endemic to the northwestern intermountain region of the United States. The species currently comprises four varieties based on subtle morphological dissimilarities, such as leaf size and density, and the size and shape of the seed pods. The taxonomic organization of the varieties of A. cusickii and related species of Astragalus were reexamined through phylogenetic analysis of low copy nuclear, nuclear-ribosomal, and chloroplast gene regions. Maximum parsimony, maximum likelihood, Bayesian inference, the genealogical sorting index, and an approximately unbiased test were used to determine appropriate species boundaries under the phylogenetic species concept. The results support reclassification of A. cusickii var. packardiae and A. cusickii var. sterilis as separate species. Additionally, evidence suggests a chloroplast capture event may have occurred in one population of A. cusickii var. 2 packardiae. Keywords — coalescence; reciprocal monophyly; species boundaries; varieties 1. Introduction Distinct populations under different environmental conditions can have markedly different phenotypes. Such differences are the basis for recognizing distinct taxa, but can be a challenge if the phenotype is the result of the environmental conditions on a common genotype. Such phenotypic plasticity, where environmental factors alter the phenotype despite identical genetic backgrounds, erodes the reliability of phenotypic variation as the sole method of diagnosing species boundaries (Mayr, 1969). The impact of phenotypic plasticity is especially critical in plants that lack the ability to directly move in the landscape and therefore must contend with the environment where they germinate. Consequently, plants may exhibit large- scale morphological and physiological responses to variations in environmental factors such as soil nutrient content, temperature, and water availability which can have profound effects on their phenotypes (Sultan, 2000; Gurevitch et al., 2002; Valladares et al., 2007). These challenges have been a source of confusion for hundreds of years and are being resolved with more analyses that examine the genotypic variation directly (Belton et al., 2014; Muggia et al., 2014; DeBiasse and Heilberg, 2015). The advent of molecular systematics has revolutionized our classification of biodiversity from the most inclusive (Adl et al., 2005; Soltis et al., 2005; Hibbett et al., 2007) to the least inclusive taxonomic levels (Stepansky et al., 1999; 3 Bronikowski and Arnold, 2001; Clark et al., 2003; Abbasi et al., 2005; Tomasello et al., 2014), and has been a major means of improving our overall concept of biological diversity. One group of plants that has a complicated taxonomic history in terms of interpreting phenotypic diversity and its ability to resolve taxonomic boundaries is the genus Astragalus L. (Polhill, 1981; Wojciechowski, 2005). Astragalus (Fabaceae) is a diverse group of approximately 2,500 species in the Old and New Worlds, containing more recognized species than any other genus of angiosperms (Frodin, 2004; Lock and Schrire, 2005; Mabberley, 2008). Until recently many systematicists regarded Astragalus as a ‘wastebasket’ genus, likely to be paraphyletic (Polhill, 1981; Wojciechowski, 2005). However, while the monophyly of Astragalus sensu stricto has been well-supported (Sanderson, 1991; Sanderson and Doyle, 1993; Wojciechowski et al., 1993, 1999), many species-level relationships within the genus remain poorly resolved. Species of Astragalus can be found on every continent except Australia and Antarctica (Lewis et al., 2005). The nearly cosmopolitan distribution and extreme morphological diversity of Astragalus make it a difficult genus for systematic studies (Sanderson and Doyle, 1993; Scherson et al., 2008). Mating systems have been studied in fewer than 1% of the species within Astragalus (Watrous and Cane, 2011) and most species in the Old World had not been revised since the late 19th century (Bunge, 1868, 1869; Taubert, 1894) until only recently (Podlech and Zarre, 2013). Astragalus exhibits rich diversity in four geographic areas (southwest and south-central Asia, the Sino-Himalayan region, the Mediterranean Basin, and western North America; in addition the Andes in South America have at least 100 species). The most diverse of these areas, home to approximately 2000 species, and assumed to be the place of origin of the genus, 4 are the steppes and mountains of southwest and south-central Asia and the Himalayan plateau (Wojciechowski, 2005). Second to Eurasia in terms of species diversity is the New World, with approximately 400-450 species. The Intermountain Region of western North America (Barneby, 1989) is especially diverse, and an estimated 70 species of Astragalus can be found in Idaho alone, including several endemic taxa (Mancuso, 1999). Among the species of Astragalus in the North American Intermountain Region is Astragalus cusickii. First described by Gray (1878), A. cusickii is a sparsely leafy, multi- stemmed, perennial forb found in western Idaho, eastern Oregon, and the extreme southeast corner of Washington (Fig. 1). The species overall has small to moderately sized flowers and conspicuous papery inflated pods (Fig. 2). It is found primarily on relatively sparsely vegetated to canyon grassland sites, often steep hillsides, ash soils, and talus slopes (Barneby, 1989; Mancuso, 1999). The species currently comprises four infraspecific taxa, varieties cusickii, flexilipes, sterilis, and packardiae. The inclusion of these four taxa into a single species was made on the basis of morphological similarity. However, it is possible that edaphic and microclimatic conditions where A. cusickii var. sterilis and A. cusickii var. packardiae occur may alter the morphologies of these varieties and as such, they may not merit taxonomic distinction. Herein we resolve the taxonomic boundaries for the four varieties of A. cusickii using molecular phylogenetic techniques. Species status is evaluated using the criteria of the phylogenetic species concept, monophyly with diagnosable differences within a larger understanding of species as separately evolving meta-population lineages, as described in the unified species concept (de Queiroz, 2003). This concept has been selected because 1) monophyly can be assessed, 2) morphological differences are known and presumably the result 5 of inheritance from a common ancestor if monophyly is found, and 3) there are few studies to directly address the breeding system of these plants. Recent studies of species in Astragalus have also employed monophyly with diagnosable differences between populations as the criteria for recognizing species (Scherson et al., 2008; Riahi et al., 2011). 2. Materials and Methods 2.1 Study Species Astragalus cusickii var. cusickii (Fig. 2) has the widest geographic distribution of the four varieties. It is found in western Idaho, eastern Oregon, and southeast Washington, although with a concentration in the Hells Canyon area (Fig.