Comparative population genetics confirms range-specific lineages of alpine stoneflies Authors: Scott Hotaling1, Lusha M. Tronstad2, J. Joseph Giersch3, Clint C. Muhlfeld3, Debra S. Finn4, and David W. Weisrock1 Affiliations: 1 Department of Biology, University of Kentucky, Lexington, KY 40506 USA 2 Wyoming Natural Diversity Database, University of Wyoming, WY 82071, USA 3 U.S. Geological Survey, Northern Rocky Mountain Science Center, Glacier National Park, West Glacier, MT 59936, USA 4 Department of Biology, Missouri State University, Springfield, MO 65897, USA Correspondence: Scott Hotaling, Department of Biology, University of Kentucky, Lexington, KY 40506; Fax: (859) 257-1717; Phone: (828) 507-9950; Email: [email protected] Keywords: population genomics, Zapada glacier, Lednia, and Rocky Mountains Abstract: Zapada glacier and Lednia tumana are stoneflies (family Nemouridae) that live in cold, alpine streams draining from glaciers, snowfields, or permanent ice. Both species were petitioned and warranted protection under the Endangered Species Act at least partially because of declining habitat (i.e., melting glaciers) as a result of climate change. Zapada glacier was previously only known from Glacier National Park (GNP) but this stonefly was recently discovered in the Absaroka-Beartooth Wilderness (ABW) and Teton Range. The goal of this study was to estimate the degree to which Z. glacier is genetically differentiated across these three mountain ranges. For comparison, we also included L. tumana (only known from the vicinity of GNP) and L. tetonica (only known from the Teton Range). We combined sequence data for all three species from both the mitochondrial (cytochrome oxidase I barcoding) and nuclear (restriction-site associated DNA sequencing) genomes. These data were analyzed in parallel and results were compared both within and across taxa. Taken together, this study represents the most robust systematic assessment of genetic boundaries in Z. glacier, L. tumana, and L. tetonica to date. Multiple analyses of the nuclear data robustly support three independent lineages of Z. glacier that correspond with the three mountain ranges. However, results from mitochondrial DNA are less emphatic, but do show Z. glacier in the Teton Range to be isolated from GNP and ABW. Both nuclear and mitochondrial evidence clearly supported the existing descriptions of L. tumana and L. tetonica as robust, species-level entities. However, additional analyses (both genetic and morphological) should be performed before range-specific lineages of Z. glacier are considered for new species designations. 1 Introduction: Many factors, both evolutionary and environmental, dictate the contemporary distributions of species. While the criteria for defining a species has been the subject of extensive debate (De Queiroz, 2007), at their core, species concepts aim to use varied criteria to identify independently evolving evolutionary lineages (Carstens et al., 2013). From an applied standpoint, the most important aspect of any systematic efforts is to identify underlying lineage independence; whether or not the evidence rises to the level of a new named species has little biological implication. However, when conservation is considered, species definitions, particularly for those taxa that are either already protected under federal statutes, such as the U.S. Endangered Species Act (ESA) or may be considered for ESA protection, are crucial. Because of these overarching policy indications, researchers are faced with a two-fold challenge: (1) To establish biologically accurate delimitations of independently evolving evolutionary lineages from all available data; and (2) To decide if the results cross the threshold of separate species, and, if so, to describe those species accordingly. Every individual in a population has its own evolutionary history, and together, individuals combine into populations, metapopulations, and species. Underpinning these groupings is a shared genetic history that provides a vital link between past and present (Hewitt, 2000; Whiteman et al., 2007). To this end, comparative population genetic studies are particularly powerful, because they highlight the degree to which species have responded to various influences, including past geological processes (e.g., glacial oscillations) and/or variance in dispersal (Lourie et al., 2005). Exemplar studies have shown that both shared geographic distributions and overlapping ecological requirements can be important predictors of similar shared evolutionary trajectories (Lapointe & Rissler, 2005; Whiteman et al., 2007; Satler & Carstens, 2017). Moreover, comparisons between well-resolved species can provide valuable reference points for estimating where similar species may fall on a speciation timeline. In the North American Rockies, two alpine stoneflies, Zapada glacier and Lednia tumana have been recommended for listing under the U.S. Endangered Species Act (ESA) due to climate change-induced habitat loss (U.S. Fish and Wildlife Service, 2016). A third stonefly endemic to the Teton Range – Lednia tetonica – was previously only known from a single stream fed by permanent subterranean ice. In GNP, L. tumana and Z. glacier co-occur and in the Teton Range, L. tetonica and Z. glacier co-occur. All three species are highly similar in many ways: they belong to the same order and family (Plecoptera: Nemouridae), are phytophagous with short (< 30 days) winged adult stages, and are tightly linked to the hydrologic conditions associated with melting glaciers, ice, and snow (Baumann, 1975; Muhlfeld et al., 2011; Baumann & Call, 2012; Giersch et al., 2015; Giersch et al., 2016). Previous mitochondrial DNA (mtDNA) evidence has indicated genetic distinctiveness among populations of Z. glacier generally corresponding with mountain ranges, suggesting that Z. glacier may actually represent one or more mountain range- specific species, similar to the morphology-based descriptions of L. tumana and L. tetonica (Baumann & Call, 2012; Giersch et al., 2015; Giersch et al., 2016). Moreover, addressing these population genetic questions holds significant implications for conservation in the region, as the alpine streams that Z. glacier, L. tumana, and L. tetonica inhabit, as well as those worldwide, are under significant threat as rapid warming drives substantial glacier recession (Hall & Fagre, 2003; Hansen et al., 2005; Pederson et al., 2010; Roe et al., 2016). Linked to this decline in the alpine cryosphere is the potential for loss of an entire community of meltwater-dependent alpine organisms (Muhlfeld et al., 2011; Giersch et al., 2016; Hotaling et al., 2017b; Hotaling et al., 2017a). 2 A lack of agreement can exist between mtDNA and nuclear genomes of the same species (Gompert et al., 2008). This “mito-nuclear discordance” is typically a result of unique evolutionary characteristics of the mtDNA genome (e.g., matrilineal inheritance, interspecific introgression; Boratyński et al., 2011). This discordance can obscure signals of differentiation as lineages that are distinct according to the nuclear genome may actually appear to be closely- related according to mtDNA data. Thus, it is important for researchers to incorporate sequence data from both the mtDNA and nuclear genomes in any analyses of species histories. However, when a disconnect arises, the two sources of information should not be considered equal – rather, multiple independent nuclear loci, and the story they collectively tell, should be given greater credence. In this study, we combined mtDNA and nuclear sequence data for Z. glacier, L. tumana, and L. tetonica to assess (1) whether range-specific lineage boundaries within Z. glacier as inferred from mtDNA are supported by genome-scale nuclear data and (2) to assess the degree to which patterns of Z. glacier differentiation correspond with those from co-occurring Lednia species. Given that our study species were described based solely upon the presence (L. tumana and L. tetonica; Baumann & Kondratieff 2010; Baumann & Call 2012) or absence (Z. glacier; Baumann 1975) of distinguishing morphological characters, and no comparative genetic perspectives have been presented, we hypothesized that both comparisons (Z. glacier by mountain range and L. tumana vs. L. tetonica) would reveal similar levels of differentiation and gene flow, and that results would largely align between mtDNA and nuclear data. From a conservation perspective, our study sheds light on a specific, applied question: is the currently described Z. glacier species comprised of several mountain range-specific species? Materials and Methods: Study species and field sampling Zapada glacier (Plecoptera: Nemouridae; Figure 1A) is known to occur in three mountainous regions: GNP in northwestern Montana, the Absaroka-Beartooth Wilderness (ABW) in southern Montana, and the Teton Range in northwestern Wyoming (Figure 2; Giersch et al., 2016). Conversely, both Lednia (Plecoptera: Nemouridae) species are endemic to a single mountain range: L. tumana (GNP; Figure 1B) and L. tetonica (Teton Range; Figures 1, 1C), can co-occur with Z. glacier in their respective ranges. Beyond Z. glacier, the Zapada genus is widely distributed, with seven recognized species in the western United States (Baumann, 1975; Baumann et al., 1977) whereas Lednia contains only two other species, both of which are also mountain range endemics: L. borealis of the Cascades, and L. sierra of the Sierra Nevada (Baumann & Kondratieff, 2010). While no Lednia species overlap geographically,