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Systematics and Phytogeography |
School of Biological Sciences, Washington State University, Pullman, Washington 99164-4236 USA
Received for publication 2 August 2007. Accepted for publication 7 January 2007.
ABSTRACT
We examined the phylogeography of Synthyris sect. Dissecta (Plantaginaceae), which is restricted to the Olympic (S. lanuginosa) and Northern Rocky Mountains (S. dissecta and S. canbyi), to infer effects of sky islands and modes of speciation. Sequences of cpDNA trnT-trnL and psbA-trnH intergenic spacers resolved 22 haplotypes among 185 individuals sampled from 16 populations of the three species. Gene flow in the ancestral lineage and random capture of haplotypes in species lineages of sect. Dissecta have resulted in haplotype clades that are not exclusive to species. Nested clade analysis (NCA) indicates that allopatric fragmentation separated Olympic and Northern Rocky Mountain populations, giving rise to the Olympic endemic S. lanuginosa, which is characterized by unique haplotypes consistent with long temporal isolation. Low haplotype and nucleotide diversity in S. canbyi are consistent with newly founded populations experiencing a bottleneck. Furthermore, we infer S. canbyi evolved as a peripheral isolate of S. dissecta. NCA indicated limited migration in S. dissecta with possible isolation by distance. Both isolation on interglacial sky islands and valley glaciers during at least the last glaciation limited gene flow among populations of S. dissecta in different ranges of the Northern Rocky Mountains.
Key Words: disjunction • haplotypes • phylogeography • Plantaginaceae • sky islands • speciation • Synthyris
Sky islands are landscapes, such as mountain peaks, that have sharp ecological differentiation associated with elevation. Ecological dissimilarities across elevations of sky islands are analogous to the differences between land and water environments in oceanic archipelagos in the limitations they present for the migration of some organisms. Sky islands, because they are situated in continental environments, can differ from oceanic islands when global climate changes periodically shift the elevational associations of ecological communities (VanDyke et al., 2004
). Continental glaciations, associated with long-term Milankovitch cycles (Hays et al., 1976) as well as other shorter-term global cycles (Hewitt, 1996), have shifted the elevational associations of various communities and organisms, especially at middle to high northern latitudes, resulting in the lowering of high elevation communities such as alpine tundra to elevations where they could expand across broader contiguous landscapes (Billings, 1974; VanDyke et al., 2004). The range expansions of alpine organisms during glaciations may have had various consequences, including opportunities to found populations in different mountain ranges (DeChaine and Martin, 2004) and for gene flow following secondary contact with other formerly isolated populations (Arbogast et al., 2001). Conversely, interglacial climate warming may drive alpine organisms into high elevation refugia of sky islands (Hewitt, 1996; DeChaine and Martin, 2004, 2005a, b), where, depending on the dispersability and/or pollen flow of the organism, populations may be reproductively isolated. Sky islands thus differ typically from oceanic islands not only in the ease with which the area of the island can shift with climatic fluctuations, but also in the incorporation of the sky islands with a continent's biotic landscape (VanDyke et al., 2004). Thus, migration opportunities for organisms such as plants in a sky island landscape may vary temporally depending on regional climatic conditions.
The Northern Rocky Mountains of the United States are largely south of the maxima of Pleistocene continental glaciations (Dyke et al., 2002) but were subject to local mountain glaciers (Hollin and Schilling, 1981). Many ranges in the Northern Rocky Mountains have sharp elevational differentiation, creating sky islands that were often separated during interglacials by vast regions of montane forest and meadows that reflect ecological conditions unsuitable for alpine organisms. In recent studies on alpine organisms, including grasshoppers (Knowles, 2001; VanDyke et al., 2004; Knowles and Richards, 2005), butterflies (DeChaine and Martin, 2004, 2005a), and plants (DeChaine and Martin, 2005b), evidence has been reported that Pleistocene climate fluctuations played an important role in isolating alpine populations on Northern Rocky Mountain sky islands and in shaping current organismal distributions and spatial patterns of genetic diversity and may also have played an important role in speciation in many clades (Green et al., 1996; Barber, 1999; Nielson et al., 2001; Demboski and Sullivan, 2003). Global warming is considered to present a particularly strong threat to organisms currently limited to refugial sky islands because of the likelihood they will become even more geographically restricted (Hewitt, 2000) and, consequently, may face greater threat of extinction (Thuiller et al., 2005).
Our phylogeographic study of Synthyris section Dissecta (Plantaginaceae) examines evolutionary processes related to disruptions of gene flow in the Northern Rocky Mountains. Section Dissecta is a clade of three species (Hufford and McMahon, 2004) and is restricted to alpine environments of the Northern Rocky Mountains (2600–3400 m a.s.l.) and the Olympic Mountains (1500–2000 m a.s.l.). These plants are rhizomatous, herbaceous perennials that have capsular fruits and small discoid seeds that can be distributed short distances by wind (Schaack, 1983; Hufford, 1992b; Hufford and McMahon, 2004). Although taxon circumscriptions in sect. Dissecta have been controversial, we follow Hufford and McMahon's (2004) recognition of the three species S. canbyi Pennell, S. dissecta Rydb. (= S. dissecta var. dissecta sensu Schaack [1983]), and S. lanuginosa (Piper) Pennell & J. W. Thomps. (= S. pinnatifida subsp. lanuginosa sensu Piper [1906] and S. dissecta var. lanuginosa sensu Schaack [1983]). These species, which are differentiated by leaf and capsule forms (Hufford, 1992a, b), have allopatric distributions (Fig. 1) and are currently not known to hybridize. Synthyris lanuginosa, an Olympic Mountains endemic, is geographically disjunct from the rest of the section and is characterized by distinctive, densely tomentose shoots. Synthyris canbyi is restricted to the Mission, Swan, and Jocko Mountains of western Montana. Both S. canbyi and S. lanuginosa are conservation concerns because of their small population sizes. Synthyris dissecta is the most widespread species of the section, consisting of populations in southwestern Montana and adjacent Idaho. Following Schaack (1983), we provisionally recognize S. dissecta as including populations in Idaho that Pennell (1933) described as S. hendersonii Pennell and S. cymopteroides Pennell, although these taxonomic differences may indicate possible cryptic species. Tomentose pubescence on the shoots of variant Idaho populations has been used to call attention to their possible genetic differentiation from other populations of S. dissecta (Pennell, 1933). This tomentose pubescence is, however, similar to that found in S. lanuginosa, and this raises the possibility that populations of S. dissecta in Idaho are the closest relatives of the disjunct populations in the Olympic Mountains of northwestern Washington.
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; Buckingham et al., 1995; Demboski and Cook, 2001; Good and Sullivan, 2001; Nielson et al., 2001; Wilke and Duncan, 2004; Carstens et al., 2005; Brunsfeld and Sullivan, 2006). The Northern Rocky Mountains–Cascade/Olympic disjunction has been inferred to result especially from aridification of the Inland Northwest in association with the uplift of the Cascade Mountains (Glew, 1994; Buckingham et al., 1995; Brunsfeld et al., 2001; Demboski and Sullivan, 2003). We tested whether genetic signatures are consistent with a hypothesis of vicariance rather than dispersal as the source of this disjunction in sect. Dissecta. Under the ancient vicariance hypothesis of Brunsfeld et al. (2001), we would expect long-separated coastal and Northern Rocky Mountain lineages to be genetically divergent, whereas a similar disjunction resulting from a relatively recent dispersal could lead to expectations of shared haplotypes between the two regions (e.g., McDaniel and Shaw, 2003; Carstens et al., 2005; Waters et al., 2005). Although the disjunction between S. canbyi and S. dissecta is no greater than among populations of the latter more widespread species, we test similarly for signatures of relatively deep vicariance vs. fragmentation following a possibly recent northward range expansion in the origin of S. canbyi, which we hypothesize evolved as a peripheral isolate of S. dissecta or a common ancestor of these two species. We test also for similar processes in the possible peripheral isolation of S. dissecta populations in the southwest part of its range, where Pennell had recognized S. hendersonii and S. cymopteroides.
Pleistocene climatic fluctuations are hypothesized to have significantly impacted the ranges of alpine organisms (Billings, 1974). We test for possible effects of Pleistocene glaciations within species of sect. Dissecta. For example, if glaciations resulted in range expansions that allowed populations to expand out of mountain ranges across intermountain valleys, then we would expect relatively little genetic geographic structure within species. In contrast, if populations have been restricted largely to mountain ranges through recent glacial cycles, then we would expect stronger signatures of geographic isolation and greater geographic structure within species. Alpine species can also remain in mountaintop refugia (nunataks) during glaciations (Brubaker and McLachlan, 1996; DeChaine and Martin, 2005b; Schönswetter et al., 2005), and this can be associated with population crashes and genetic drift (Knowles and Richards, 2005). Nunataks may have been important in the Olympic Mountains where all but one endemic species—including S. lanuginosa—are found above the maximum height of the continental ice sheets and survived on nunataks exposed above the ice (Peterson et al., 1997). We test especially for genetic signatures of nunatak survival in S. lanuginosa by comparing genetic variation within and among populations. We expect populations that survived on different nunataks will have low genetic variation and strong differentiation among populations on different nunataks (Stehlik, 2003). We use cpDNA haplotype data based on DNA sequences of trnT-trnL and psbA-trnH intergenic spacers to examine the geographic structure of genetic diversity in this clade. These genetic regions have been found to be variable within and among populations of Synthyris (Streit, 2004; Brustkern, 2006; Marlowe, 2007).
MATERIALS AND METHODS
Taxon sampling
We collected from populations across the distributional and elevational ranges of the species of Synthyris sect. Dissecta, including 10 populations of S. dissecta, two of S. canbyi, and four of S. lanuginosa (see Appendix). We sampled 12 individuals per population except for the Central Bitterroot population for which 13 were sampled and the Swan Range population where fewer individuals were present (Table 1). Two additional individuals of S. canbyi were sampled using herbarium specimens, for a total of 185 individuals. Because Synthyris have short subterranean rhizomes that permit asexual reproduction, individuals sampled within each population were at least 6 m apart. We attempted to collect over the range of the population unless it was geographically very widespread. To represent the diversity of leaf pubescence in S. dissecta, our sampling included one population (White Cloud Mountains; Fig. 1) with tomentose shoots that was treated as S. cymopteroides by Pennell (1933). All populations sampled are vouchered by collections deposited in the Marion Ownbey Herbarium (WS; leaf and inflorescence/infructescence samples only for Olympic National Park collections).
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. The trnT-trnL intergenic spacer regions were amplified using primers a and b (Taberlet et al., 1991), and the psbA-trnH intergenic spacer regions were amplified using primers psbA and trnH (GUG; Hamilton, 1999). Each 25-µL sample for PCR amplification contained 10.8 µL double-distilled (dd) H2O, 2.5 µL 10x reaction buffer (Promega, Madison, Wisconsin, USA), 2.5 µL 5' 5 µM primer, 2.5 µL 3' 5 µM primer, 3.0 µL MgCl2, 1.5 µL dNTP, 0.2 µL Taq polymerase (Promega), and 2.0 µL diluted DNA template (1.5 µL water + 0.5 µL 
20 ng DNA concentration). PCR conditions in a Biometra thermocycler (Whatman, Göttingen, Germany) included initial denaturation at 94°C for 5 min, followed by 30 cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min, with a final extension at 72°C for 7 min. PCR products were visualized by 1.5% agarose gel electrophoresis and purified using 20% polyethylene glycol (PEG) 8000 in 2.5 M NaCl. The 10-µL cycle sequencing reaction volumes contained between 3.67 and 6.67 µL ddH2O, 0.33 µL 10 µM primer, 1.0 µL 5x sequencing buffer (Applied Biosystems, Foster City, California, USA), 1.0 µL BigDye Terminator v3.1 (Applied Biosystems), and between 1.0 and 4.0 µL clean PCR product. Cycle sequence reactions included 25 cycles of 96°C for 10 s and 50°C for 5 s, with a final extension at 60°C for 4 min. Cycle sequence products were purified using 75% isopropanol precipitation, and DNA sequences were obtained on a 48-capillary 3730 DNA Analyzer (Applied Biosystems). Contigs were assembled and edited using Sequencher version 4.2.2 (Gene Codes Corp., Ann Arbor, Michigan, USA). Manual sequence alignment was unambiguous in Se-Al version 2.0a11 (Rambaut, 1996–2002) using the similarity criterion (Simmons, 2004). Sequences for all haplotypes generated in this study have been deposited with NCBI GenBank (see Appendix). The combined trnT-trnL and psbA-trnH data matrix and tree were deposited in TreeBASE (http://www.treebase.org; M3557, S1931).
Phylogenetic analysis
A phylogenetic analysis was used to infer evolutionary relationships among haplotypes. Redundant haplotypes were removed and models of molecular evolution were evaluated using DT-ModSel (Minin et al., 2003), which uses decision theory to incorporate estimates of branch length error along with a Bayesian information criterion and includes a penalty for over-parameterization. The selected model was chosen based on the data set in which the indels were not coded as single evolutionary events. The specified parameters of a K81uf model (six substitution rates: rAC = 1.00 rAG = 1.48, rAT = 0.19, rCG = 0.92, rCT = 1.48, rGT = 1.00; base frequencies: A = 0.3882, C = 0.1176, G = 0.1499, T = 0.3443) were applied in maximum likelihood (ML; Felsenstein, 1973) searches of the combined data set. ML analyses were conducted in PAUP* version 4.0b10 (Swofford, 2002) using heuristic ML searches (1000 replicates), including random taxon addition and branch-swapping by tree bisection-reconnection, saving one tree per replicate. Bootstrap analysis (Felsenstein, 1985) was used to assess support for branches recovered by ML using PAUP*. Maximum likelihood bootstrap analyses used the same model of evolution and a single random addition per replicate of haplotypes as the heuristic search for 1000 replicates.
Phylogeographic analyses
Nested clade analysis (NCA; Templeton et al., 1995; Templeton, 1998) was used to assess geographic associations among haplotypes. Haplotype networks were constructed using TCS version 1.21 (Clement et al., 2000) under the criterion of a parsimony network in which connections have a probability of at least 95%. Four indels (ranging from three to 10 nucleotides) resulting from a duplication of adjacent sequences were coded as single evolutionary events following Simmons and Ochoterena (2000). Coding these areas as "missing data" would lead to branch length inflation, and if excluded, would possibly lead to loss of resolution (Cox and Chase, 1995). One region of 35 nucleotides was polymorphic, having two possible sequences among the sampled haplotypes. This region of nucleotides was coded as a single base to avoid inflating the number of nucleotide changes and to allow haplotypes to be combined into one network. Single gaps that were not the result of a multinucleotide indel were treated as a fifth character state to be informative in TCS. Closed loops in the network were resolved using procedures from Templeton et al. (1992) and Templeton and Sing (1993) in combination with the ML topology. On the ML topology that includes branch lengths (not shown), haplotype D is topologically closer to haplotype B than to haplotype C; haplotype D is topologically closer to haplotypes E, I, and K than to haplotypes T and U; and haplotype U is topologically closer to haplotypes K, M, and N than to haplotype P. The guidelines of Crandall (1996), Templeton et al. (1987, 1992), and Templeton and Sing (1993) were used to infer nested clades based on the TCS network. NCA was performed using GeoDis version 2.5 (Posada et al., 2000). Significance in the NCA was determined using an alpha = 0.05 for clade distances. Posada and Templeton's (2005) inference key was used to infer geographic processes associated with statistically significant patterns determined by NCA. The haplotype network reconstruction and NCA were performed using the three species to identify possible evolutionary processes that led to speciation within the group, as well as differentiation within species.
Evidence for range expansion was tested under the expansion model of Rogers and Harpending (1992) by examining pairwise mismatch distributions using Arlequin version 3.0 (Excoffier and Schneider, 2005). The mismatch distribution of a population that has been constant in size for a long time is expected to be ragged and erratic, whereas a population that has undergone recent expansion is expected to generate a smooth distribution with a peak (Harpending, 1994). Mismatch distributions were calculated (1000 replicates) using Arlequin for samples in each of the clades identified by the NCA. The Harpending (1994) raggedness index was used to evaluate deviation from a model of population expansion.
Because of its geographic breadth and numerous populations, S. dissecta was analyzed in greater detail than S. canbyi and S. lanuginosa. A Mantel test (Sokal and Rohlf, 1995) was performed on S. dissecta populations using Arlequin to look for evidence of isolation by distance. A distribution was created from 1000 random pairwise permutations, and statistical significance was determined by comparing the observed r-value to this distribution. Estimated pairwise FST values were plotted against corresponding geographical distance to illustrate the distribution of pairwise comparisons.
Estimates of genetic diversity
Arlequin was used for analyses of molecular variance (AMOVA), based on pairwise distances, to assess the partitioning of genetic variation within and among populations, within and among recognized species, and within and among regions (Rocky Mountains vs. Olympic Peninsula). Nei's (1987) haplotypic diversity (the mean number of differences between all pairs of haplotypes in the sample) and nucleotide diversity (the probability that two randomly chosen homologous nucleotides are different; Nei and Tajima, 1981) were calculated also using Arlequin. In addition, AMOVA was performed separately for S. dissecta haplotypes to assess genetic variation partitioning solely within this widespread species.
RESULTS
Haplotypes
The aligned DNA sequences for the trnT-trnL and psbA-trnH data partitions were 687 and 312 bp, respectively (before indel coding; after indel coding, 679 and 271), 28 of which were variable and nine parsimony-informative. We recovered 22 haplotypes based on the concatenated trnT-trnL and psbA-trnH sequences (Table 1). Three haplotypes (A, B, and M) were recovered from the four populations of S. lanuginosa and were exclusive to this species; haplotype A, the most common haplotype, was shared by 45 individuals among the four S. lanuginosa populations. Haplotypes C, E–J, L, and N–V were exclusive to and shared among S. dissecta populations; haplotype D was found in only one S. dissecta population (Marlowe 79). The two populations of S. canbyi (Marlowe 84/104, Marlowe 85/Gilbert 3) were represented by only one haplotype (K), which was shared with two populations of S. dissecta (Marlowe 74, Thomson 110). Haplotype C was exclusive to the tomentose Idaho S. dissecta (White Cloud Mountains) population; haplotype L of this population was also found in individuals from the Tobacco Root population. To illustrate the geographic spread of genetic diversity, haplotypes were placed onto the distribution map (Fig. 2). The ML tree (Fig. 3; –ln 1480.8501) has nonexclusive haplotype phylogenies of the three species. The species are intermixed throughout the phylogeny.
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). The mismatch distributions failed to reject a model of population expansion for all clades except 2–2 (P = 0.0090; 
= 0.05) and 2–3 (P = 0.0430).
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DISCUSSION
Genetic variation
Chloroplast DNA haplotypes are shared broadly among the three currently recognized species of sect. Dissecta (Figs. 2–4), which could result from gene flow or incomplete lineage sorting. These species are not known to hybridize, and no intermediate forms have been identified that would be indicative of hybridization. The absence of sympatric and even closely adjacent populations is likely to prevent interspecific gene flow. Chloroplast DNA haplotype polymorphism among species is common throughout Synthyris (e.g., Streit, 2004; L. Hufford, unpublished data). Based on the factors that limit gene flow in this clade and the extent of haplotype polymorphism among species of Synthyris, we contend that the sharing of haplotypes among sect. Dissecta species can be attributed to the ancient gene flow in early panmictic ancestors prior to the cladogenesis that resulted in extant species and that persistence of ancestral polymorphism is a consequence of incomplete lineage sorting.
Our AMOVA found that a high portion (70.48%; Table 5) of genetic variation could be explained by differences among populations. Such partitioning of molecular variance is typical for glacial relict populations that were subjected to geographic isolation and, potentially, genetic drift in populations restricted to refugia (Reisch et al., 2003). We would expect plants that have undergone recent dispersal to show instead low levels of differentiation between populations. The strong geographical differentiation of sect. Dissecta is therefore most likely due to genetic drift in isolated populations associated with glaciation, especially warm interglacials. The high level of differentiation among sect. Dissecta populations is not associated with a strong loss of genetic diversity within populations (Table 5). Life-history traits of Synthyris, such as long life span, asexual propagation, and the ability to both self-fertilize and outcross (McKone et al., 1995), may contribute to this genetic diversity within populations.
Synthyris lanuginosa
Several taxa, including plants (Buckingham et al., 1995; Brunsfeld and Sullivan, 2006), lichens (Glew, 1994), and animals (Demboski and Cook, 2001; Good and Sullivan, 2001; Wilke and Duncan, 2004; Carstens et al., 2005) are disjunct between the Olympic and/or Cascade Mountains on the western side of the Pacific Northwest and the Northern Rocky Mountain populations. Brunsfeld et al. (2001) identified both ancient vicariance and more recent long-distance dispersal as possible causes for the disjunction. Synthyris lanuginosa, an Olympic Mountains endemic, was found by Hufford and McMahon (2004) to be the sister species of S. dissecta and S. canbyi of the Northern Rocky Mountains of western Montana and adjacent Idaho. Synthyris lanuginosa has relatively limited genetic diversity compared to its sister clade, which could be a consequence of relatively recent dispersal, its relatively fewer populations spread over a smaller geographic area, or population bottlenecks. With a recent dispersal, however, we would expect to find shared haplotypes between the two regions (e.g., McDaniel and Shaw, 2003; Carstens et al., 2005; Waters et al., 2005). The absence of shared haplotypes between the Olympic and the Northern Rocky Mountains points toward a more ancient source of the disjunction. The low number of S. lanuginosa individuals in its limited geographic distribution could be consistent with a population bottleneck. NCA inferred allopatric fragmentation as the evolutionary process that led to the differentiation between S. lanuginosa and its sister clade (clades 1–1 and 1–10; Fig. 4; Table 2), and we hypothesize this occurred in association with vicariance that resulted in the capture of at least two different haplotype lineages in the Olympic Mountains populations (Figs. 2, 4). Our AMOVA, when partitioned by region (Olympic Mountains vs. Northern Rocky Mountains; Table 5), recovered a higher amount of genetic variation associated with differences between regions (42%) than among populations within regions (36%), which is likely due to a relatively deep temporal split between the two regions.
Uplift of the Cascade Range during the Miocene and Pliocene (McKee, 1972; Graham, 1999) resulted in the aridification of the Inland Northwest, where there was substantial biotic change (Daubenmire, 1975), especially after 4.5 mya (Leopold and Denton, 1987). This mountain building and aridification has been associated with disjunction in various taxa in the northwest (Buckingham et al., 1995, Nielson et al., 2001), although it is unclear whether these processes impacted high elevation lineages, such as sect. Dissecta, as much as those at lower elevations in the Columbia Basin. Another causal factor that could have affected the high mountain environments between the Olympics and the Northern Rocky Mountains in Montana was Pleistocene glaciation (Crandell, 1965). The earliest record of alpine glaciation in the region dates to approximately 1.75 mya (Kaufman et al., 2004). Thus, alpine glaciation in the Canadian Rockies and especially high elevation glaciation in the USA–Canada border region represents a possible force in the extirpation of intervening populations of sect. Dissecta and the isolation and disjunction of Olympic S. lanuginosa from its sister clade in the Northern Rocky Mountains.
Glacial refugia have previously been hypothesized for the northeastern portion of the Olympic Peninsula, where S. lanuginosa is distributed (Heusser, 1977; Heusser and Heusser, 1990; Brubaker and McLachlan, 1996). During the last glacial period, a portion of the Buckhorn Mountain area was not covered by ice and is considered a refugium for numerous rare plants (Glew, 1994). Among the species endemic to the Olympic Peninsula, all (including S. lanuginosa) but one are found at elevations exceeding the maximum height of the Cordilleran ice sheet, which has led to inferences that glacial survival of these endemics was in refugia on mountain peaks exposed above the ice (Buckingham et al., 1995; Peterson et al., 1997). Inbreeding resulting from isolation of small high-alpine nunatak populations should lead to relatively low genetic variation within populations and to strong differentiation among populations on different nunataks (Stehlik, 2003). With S. lanuginosa, we found low genetic variation within populations; however, we did not find strong differentiation among populations. This finding may be an indication that isolation of the Olympic populations is relatively recent and may also reflect the close geographic proximity of these populations. Alternatively, the genetic signatures of the Olympic populations could be consistent with expansion out of a bottleneck. The long-term separation of S. lanuginosa from its sister clade in the Northern Rocky Mountains and its persistence in small, isolated refugia are consistent with our data. Persistence of S. lanuginosa in small populations in nunatak refugia, in which genetic drift occurred, could account for both the low genetic diversity of the populations and the origin of the unique haplotypes B and M.
Synthyris canbyi
Synthyris canbyi is restricted to the Jocko, Mission, and Swan mountain ranges of northwestern Montana (Fig. 1), a distribution continuous with but north of S. dissecta. All sampled individuals of S. canbyi had the same haplotype (K; Table 1), which is also found in the Central Bitterroot Range and Gravelly Range populations of S. dissecta. This haplotype was also recovered in S. plantaginea of the Southern Rocky Mountain clade of Synthyris sect. Besseya (Marlowe, 2007) and, thus, may be one of the more common and ancient haplotypes in Synthyris. Synthyris canbyi, which has scattered populations on inaccessible, rugged terrain, has not been well collected. Our sampling includes 14 individuals from the Mission Range, only two individuals from the Swan Range, and none from the Jocko Mountains populations, which could have affected the diversity of haplotypes recovered. We hypothesize, however, that S. canbyi evolved from a founder population expanding geographically from the margin of an ancestral population of the S. canbyi/S. dissecta lineage. NCA did not find significant evolutionary processes that explain the S. canbyi haplotypes relative to the rest of sect. Dissecta; however, the low haplotype and nucleotide diversity of S. canbyi is consistent with newly founded populations experiencing a genetic bottleneck (Nei et al., 1975). Many high-elevation areas in the Mission and Swan ranges and Jocko Mountains were unaffected by glaciation (Davis, 1920), with glacial reconstructions inferring several nunataks in the Swan and Mission ranges (Locke, 1995), and it is likely that S. canbyi had achieved its present distribution during or somewhat prior to the latest glaciation of the Pleistocene.
Factors other than geographic isolation may also have played a role in the origin of S. canbyi. Raven's (1964) observations that marginal populations often are found on soil types unusual for a lineage as a whole and that populations found on unusual soil types are almost inevitably genetically distinct could help to account for the differentiation of S. canbyi, which is limited to calcareous substrate (Schaack, 1983). The bedrock material beneath the Swan and Mission ranges' populations of S. canbyi is derived from the same calcareous geologic formation (Schaack and Stickney, 1981). Synthyris dissecta is also sometimes found on limestone but is usually found on igneous intrusive material (Schaack, 1983). Polyploidization could also have played a role in the origin of S. canbyi, serving to provide reproductive isolation of its populations from those of S. dissecta. The single chromosome count for the species revealed it was tetraploid in contrast to the diploid counts for S. dissecta and S. lanuginosa (Schaack, 1983).
Synthyris dissecta
The relatively widespread S. dissecta has several populations that consist of haplotypes that are phylogenetically distantly related (e.g., Northern Bitterroot, Tobacco Root, Gravelly Range, and White Cloud Mountains populations; Fig. 1) and some haplotypes (e.g., E, K, and L) that are geographically widespread (Fig. 2). Both of these patterns are consistent with incomplete lineage sorting and random capture of haplotypes in refugia (Neigel and Avise, 1986). Contiguous range expansion was inferred by NCA as the process leading to genetic variation for the total cladogram, in which S. dissecta populations were widespread, as well as for two clades that consisted only of S. dissecta populations (clades 2–2 and 2–3; Fig. 4; Table 2). In contrast to the NCA results, the mismatch distribution rejected a hypothesis of range expansion for S. dissecta. Printzen et al. (2003) observed that persistence of ancestral haplotypes from the prefragmentation era in the fragmented subpopulations can preclude NCA from detecting fragmentation in the shallow genealogies. The signal in the NCA, therefore, may result from relatively recent fragmentation.
Genetic variation in S. dissecta is highly structured with most variation found among populations (Table 5). The populations have high haplotype diversity with populations harboring between two and four haplotypes (only one in the Anaconda Range population; Fig. 2). High haplotype diversity combined with the observed low nucleotide diversity (Table 1) is characteristic of rapid population expansion after a bottleneck (Nei et al., 1975). The phylogeographic pattern for S. dissecta could be explained by the stochastic partitioning of ancestral variation into isolated populations, followed by genetic drift associated with isolation of populations in interglacial refugia.
Population range expansion out of sky islands during glaciations may have been insufficient to permit gene flow among populations of different mountain ranges. This hypothesis is consistent with data from DeChaine and Martin (2004, 2005a) who found little population expansion in alpine butterflies among mountain ranges during glaciations in the Northern Rocky Mountains. Earlier workers (Davis, 1920; Daubenmire, 1952; Pierce, 2004) had also hypothesized the persistence of populations at high elevations in ranges of the Northern Rocky Mountains.
Pennell (1933) recognized populations in Idaho as S. hendersonii or S. cymopteroides, although Schaack (1983) treated these as conspecific with S. dissecta. A population sampled from the White Cloud Mountains had two haplotypes, C and L (Figs. 2, 4). Haplotype C is unique to this population although L is also found in the Tobacco Root population of southwestern Montana. The presence of the unique haplotype C may be indicative of a relatively long period of isolation of the White Cloud Mountains population from other populations of S. dissecta. Shoot systems and inflorescences of the White Cloud Mountains population have more tomentose vestiture than most other populations of S. dissecta, although they are much less tomentose than populations of the densely woolly S. lanuginosa. The haplotype network (Fig. 4) recovers haplotype C as a terminal haplotype differing by only one step from A, which is found only in S. lanuginosa. Three different scenarios could explain the shared tomentose vestiture of S. lanuginosa and the White Cloud Mountains populations of S. dissecta. First, tomentose leaves could have arisen in the Idaho populations of S. dissecta and these served as the founders via long-distance dispersal for S. lanuginosa in the Olympic Mountains; however, we have not detected genetic signatures associated clearly with long-distance dispersal. Second, tomentose leaves could have arisen independently in S. lanuginosa and the Idaho populations of S. dissecta, but such homoplasy is perhaps inconsistent with the phylogenetic relationship of haplotypes A and C. Third, tomentose vestiture could be plesiomorphic for sect. Dissecta and retained only in S. lanuginosa and selected populations of S. dissecta, including those in the White Cloud Mountains. However, if this hypothesis were true, then we might expect the common and likely ancient haplotype K (or other internal haplotypes in the haplotype network derived more directly from K) to be present in the tomentose Idaho populations. Our observations point to considerable variation in vestiture, ranging from glabrous to villous, among other populations of the section. Further studies of vestiture in sect. Dissecta may be warranted to understand the differences in vestiture among populations and how they coordinate with geography, variations within populations, and variation in vestiture over developmental time of individual plants.
Conclusions
Our results recover genetic signatures of temporally relatively deep as well as more shallow demographic and evolutionary events. A widespread, panmictic ancestor is inferred for sect. Dissecta. Similarly our other studies of Synthyris, including a clade of sect. Besseya in the Southern Rocky Mountains (Marlowe, 2007) and a lineage of subgenus Missurica in the Pacific Northwest (Streit, 2004), have also recovered signatures of widespread gene flow prior to the speciations that resulted in extant taxa. Allopatric fragmentation, splitting the Northern Rocky Mountain populations from those of the Olympic Mountains, may have been an early evolutionary event in sect. Dissecta, resulting in the origin of S. lanuginosa. Isolation of populations at the northern periphery of the S. dissecta range in the Northern Rocky Mountains may have led to the origin of S. canbyi. Within all three species, we see genetic signatures of life in sky island environments. Populations of each species appear to have persisted through Pleistocene glaciations in high-elevation nunatak refugia, where population bottlenecks and genetic drift account for much of the genetic novelty. In the relatively widespread S. dissecta, we recovered no signatures of gene flow among populations that could have resulted from east–west migrations between mountain ranges during glaciations. Because of the deep sharing of haplotypes among species, our data may not have provided an adequate test of the hypothesis that the Idaho population of S. dissecta, recognized by Pennell (1933) as S. hendersonii because of its markedly tomentose vestiture, was a distinct species. This population shared a haplotype found in a geographically distant population of S. dissecta and had only a single private haplotype. We provisionally retain the tomentose Idaho populations as part of S. dissecta and recommend further investigation of vestiture variation among populations of S. dissecta, which we observed to be more variable than previously recognized.
Appendix. GenBank accession numbers and locality information for taxa used in this study. Locality is given for the first individual of each population. Voucher specimens are deposited in WS unless otherwise indicated. State abbreviations: ID, Idaho; MT, Montana; WA, Washington.
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FOOTNOTES
1 The authors thank E. Roalson, M. Webster, C. Steele, and M. King for their helpful discussions, technical assistance, and support; P. Stickney, E. Schreiner (National Park Service), U. S. Forest Service, and The Confederated Salish and Kootenai Tribes of the Flathead Nation for locality information and issue of collecting permits; the curator of MONTU for supplying plant material; A. Thomson for assistance with field collections; and M. Simmons and two anonymous reviewers for providing helpful comments on the manuscript. This project was supported by awards from NSF (DEB0608026), American Society of Plant Taxonomists, Northwest Scientific Association, Washington Native Plant Society, and the Rexford Daubenmire Grant for Graduate Education and Betty W. Higinbotham Trust from Washington State University. 
2 Author for correspondence (e-mail: marlowe{at}wsu.edu)
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