Geographic origins and patterns of radiation of Mertensia (Boraginaceae).

American Journal of Botany 101(1): 104–118. 2014.

GEOGRAPHIC ORIGINS AND PATTERNS OF RADIATION OF MERTENSIA (BORAGINACEAE)1 MARE NAZAIRE2,4, XIAO-QUAN WANG3, AND LARRY HUFFORD2 2School 3State

of Biological Sciences, Washington State University, Pullman, Washington 99164-4236 USA; and Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China

• Premise of the study: Numerous molecular phylogenetic studies have used new biogeographic tools to explain species distributions. However, questions remain about origins, timing, direction of movement, and relationships between range expansion and diversification. We investigated geographic origins and temporal and spatial diversification of Mertensia, giving particular attention to divergence between Asian and North American lineages and radiation of western North American clades. • Methods: Divergence time estimation and biogeographic analyses were based on phylogeny reconstruction inferred from nuclear ribosomal ITS and 12 plastid DNA sequence regions and a broad sampling of Mertensia, Boraginaceae, and core eudicots. • Key results: Mertensia split from Asperugo in the late Oligocene to mid Miocene (26.83–12.22 million years ago [Ma]), followed by the first divergence in the crown group in the late Miocene (10.36–5.19 Ma). The ancestral area is inferred to have been Asia or a widespread distribution across Asia, Beringia, and circumboreal locales. Initial range expansion of North American Mertensia occurred in Beringia and the Pacific Northwest (7.70–4.22 Ma), followed by diversification of three clades (Pacific Northwest, southern Rocky Mountains, central Rocky Mountains). • Conclusions: The crown divergence of extant Mertensia coincides with the onset of extreme cooling and fragmentation of a once extensive mixed mesophytic forest that was subsequently replaced by a boreal coniferous forest. Early diversification likely occurred when Beringia was connected and available for floristic exchange. The north–south orientation of the Rocky Mountain Range and Pleistocene glacial–interglacial cycles appear to have been important in the North American diversification of Mertensia. Key words: ancestral area reconstruction; Beringia; Boraginaceae; divergence time analyses; Mertensia; North America; species richness.

Since the travels of Alexander von Humboldt (1889), scientists have been challenged to explain the geographic patterns of species distributions. In recent years, an increasing number of molecular phylogenetic studies have used new biogeographic tools to help elucidate geographic patterning; however, questions remain about geographic origin, timing, direction of movement, and relationships between range expansion and diversification.

Two striking examples of geographic patterning that have long fascinated biologists are intercontinental disjunctions between Asia and North America (e.g., Tiffney, 1985a; Donoghue et al., 2001; Ricklefs et al., 2004) and geographic radiations associated with montane regions (e.g., McCain, 2007; Drummond, 2008; Kozak and Wiens, 2010). In the northern hemisphere, the striking floristic similarities and patterns of disjunction between Asia and North America have been recognized for more than 150 yr (e.g., Gray, 1840, 1859; Li, 1952; Graham, 1972). The similarities of extant floras in Asia and North America date to the early Tertiary when the boreotropical forest first appeared at mid-latitudes of the northern hemisphere (Wolfe, 1975; Tiffney, 1985a). Over time, floristic exchange between Asia and North America was greatly influenced by continental movements, the availability of land bridges, and climatic oscillations. For example, many authors have hypothesized that arctic, alpine, and boreal taxa originating in the montane regions of central Asia or western North America migrated between continents via the Bering Land Bridge (e.g., Hultén, 1937, 1958; Weber, 1965). Beringia became the primary migration route for biotic exchange following the breakup of the North Atlantic Land Bridge in the Early Eocene (e.g., McKenna, 1983; Tiffney, 1985a, b; Tiffney and Manchester, 2001). The viability of the Bering Land Bridge for biotic exchange was intermittent, however, as physical connectivity between Asia and North America was greatly impacted by climatic oscillations during the Pliocene and Pleistocene (Pielou, 1979; Sanmartín et al., 2001).

1 Manuscript received 2 September 2013; revision accepted 17 November 2013. The authors thank E. Anderson, B. Bennett, J. Collins, G. E. Crow, K. Marr, K. Metter, O. Skaarpas, J. D. Springer, and D.-Y. Tan for assistance in the field and providing material and to E. Roalson, J. J. Schenk, and D. Tank for helpful discussions and technical assistance. The authors thank the staff at the following herbaria for specimen loans and the use of material in analyses: ALA, ARIZ, ASC, BM, BRY, CAS, COLO, CS, HUH, JEPS, MO, MT, NMC, NY, OSC, PE, PH, RM, UNM, UC, US, UTC, WS, WTU, XJA, and XJBI. They also thank two anonymous reviewers for helpful suggestions on a previous version of the manuscript. This project has been supported through an NSF DDIG (DEB-1110484), an NSF East Asia and Pacific Summer Institute Fellowship, China, the Betty W. Higinbotham Trust in Botany through Washington State University, the John W. Marr Fund through the Colorado Native Plant Society, the Margaret Williams Research Grant of the Nevada Native Plant Society, the Native Plant Society of New Mexico Research Grant, and the Washington Native Plant Society Grant to M.N. 4 Author for correspondence (email: [email protected]); present address: Rancho Santa Ana Botanic Garden, Claremont, California 91711, USA

doi:10.3732/ajb.1300320

American Journal of Botany 101(1): 104–118, 2014; http://www.amjbot.org/ © 2014 Botanical Society of America

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NAZAIRE ET AL.—ORIGINS AND RADIATION OF MERTENSIA

Temperate Asia and North America have figured prominently in biogeographic investigations, not only because they share disjunct clades, but also because of their differences in species richness. High species diversity in Asia relative to North America has been extensively documented for temperate plant species (e.g., Latham and Ricklefs, 1993; Qian and Ricklefs, 1999, 2000; Harrison et al., 2001), birds (Mönkkönen and Viro, 1997), and mammals (Mönkkönen and Viro, 1997; Sanmartín et al., 2001). Three main explanations have been proposed for the greater floristic diversity in Asia relative to North America: (1) the complex topography and greater habitat diversity in Asia offered increased opportunities for speciation (Qian and Ricklefs, 1999, 2000; Harrison et al., 2001; Ricklefs et al., 2004); (2) origins of many clades in Asia allowed greater time for speciation and the accumulation of species before they migrated to North America (Latham and Ricklefs, 1993; Qian and Ricklefs, 1999); and (3) a limited impact of Quaternary glaciations in Asia lowered extinction rates relative to those in North America (Qian and Ricklefs, 1999; Wen, 1999). Montane regions play important roles in the diversification of continental floras and have been implicated in shaping distribution patterns and regional diversity in Asia (López-Pujol et al., 2011; Barres et al., 2013) and in North America (Billings, 1974, 1978; Cronquist, 1978; Elias, 1996). Species richness and high levels of endemism in montane regions have often been ascribed to topographic complexity, edaphic diversity, and habitat heterogeneity, which contribute to population subdivision, geographic isolation, and increased opportunities for diversification (e.g., Knowles, 2001; DeChaine and Martin, 2004, 2005a, b; Hughes and Eastwood, 2006). Moreover, longand short-term climatic shifts associated with continental glaciations, especially in montane regions of high- and mid-latitudes, have greatly influenced elevational changes in community structure, large-scale geographic range shifts, and patterns of rapid diversification (e.g., Billings, 1974; Knowles, 2000; Rovito, 2010). North America is characterized by four main mountain systems, with the Appalachian Range in eastern North America and the Rocky Mountain Range, Great Basin ranges, and the Cascades-Sierra Nevada ranges dominating western North America. The ages of the western mountain systems decrease from east to west, with the development of the Rocky Mountain system in the Laramide orogeny of the Late Cretaceous to Middle Eocene (Billings, 1974; Graham, 1993, 1999). The uplift of western North American mountain systems created local and regional climatic changes, such as orographic rainfall and rainshadow effects, which in turn had a significant effect on the evolution of plant diversity and shaping geographic patterns of species richness (Graham, 1999). Most of the mountain ranges in western North America trend in a north–south direction over a broad latitudinal range, and because of their orientation, they are thought to serve as important migration corridors for alpine and montane plants (Billings, 1974). Moreover, these north– south-running mountain blocks have been suggested as important for the diversification of many western North American clades (Axelrod and Raven, 1985; Hewitt and Ibrahim, 2001). The genus Mertensia Roth (Boraginaceae) provides an excellent opportunity to examine origins, timing, and geographic patterns of diversification between Asia and North America and within western North America. Mertensia comprises 62 species and seven varieties of perennial herbs that inhabit primarily alpine, montane, and boreal habitats of both Asia and North America, including Beringia (Williams, 1937; Popov, 1953a, b;

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Nazaire and Hufford, in press). The genus, which has greater species richness in North America than in Asia, is divided into two sections (sensu Nazaire and Hufford, in press): section Stenhammaria (Rchb.) A. Gray (12 species), which occurs in Asia from western China and Russia to northeastern Russia, Beringia, and coastal circumboreal locales of Asia, North America, and Europe; and section Mertensia A. Gray (50 species, seven varieties), which is almost entirely North American and has its geographic center of species diversity in the Rocky Mountains of western United States. One species in section Mertensia, M. rivularis (Turcz.) DC., occurs in western Beringia (northeastern Russia) and is sister to the North American taxa. The North American Mertensia fall primarily into five major clade “complexes” (following Nazaire and Hufford, in press), each of which has numerous species associated with particular geographic regions, although the complexes are not exclusive to those geographic regions. We used molecular phylogenetics, divergence time estimation, and biogeographic analyses to examine the evolutionary origins and broad-scale patterns of geographic diversification between Asia and North America and within western North America in Mertensia. Our investigation applies a broad sampling of taxa that includes Mertensia, other Boraginaceae, and core eudicots to address the following: (1) the geographic area and timing of early radiation in Mertensia; (2) whether extant species richness is greatest in its ancestral area; (3) migration pathways and direction of movement that may have been important in the diversification of the genus; and (4) historical events associated with the geographic diversification of Mertensia.

MATERIALS AND METHODS Taxon sampling—To investigate divergence times and biogeographic patterns in Mertensia, we conducted initial phylogenetic and divergence time analyses based on a broad sampling of Boraginaceae and outgroups. Subsequent biogeographic analyses that centered only on Mertensia used a pruned, dated tree for the genus. The initial broad phylogenetic data set had 234 accessions, including 73 accessions from Mertensia, 106 accessions from other Boraginaceae s.l., and 55 outgroups. Outgroup choice was based on inferred relationships of Boraginaceae in previous phylogenetic analyses (Savolainen et al., 2000; Bremer et al., 2002, 2004; Martínez-Millán, 2010). Outgroups included 51 asterids, representing 10 orders and several families from asterids I and asterids II, and four members from the Saxifragales, which forms a clade in the rosids (APG II, 2003, 2009; Appendix S1, see Supplemental Data with the online version of this article). Taxon selection in Mertensia was guided by our test of lineage composition in the genus (Nazaire and Hufford, in press). Our sampling included exemplars from each of the major clades of Mertensia, encompassing 55 of the currently recognized 62 species (and seven varieties among the sampled species; Appendix S2; see online Supplemental Data). Species of Mertensia that were not sampled were either unavailable to us or we were unable to obtain DNA of sufficient quality for sequencing. Our phylogenetic study of Mertensia (Nazaire and Hufford, in press) indicated selected species were not exclusive lineages, and they were represented by multiple accessions in this study. For example, in our previous phylogenetic reconstructions of Mertensia (Nazaire and Hufford, in press), we recovered M. cana Rydb. in two separate clades; in the present study, we have included both accessions as representing potential independent lineages. Our taxon selection in Boraginaceae was guided by our earlier study that tested the relationships of Mertensia to other Boraginaceae (Nazaire and Hufford, 2012). Each of the six subfamilies of Boraginaceae (Boraginoideae, Cordioideae, Ehretioideae, Heliotropioideae, Hydrophylloideae, and Lennooideae; sensu APG II, 2003, 2009) and the tribes of subfamily Boraginoideae were represented in our analysis. Excluding our accessions of Mertensia, Boraginaceae were represented by 80 genera and 105 species (Appendix S1).

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n/a 3996.4457 4248.2761 5462.9275 4003.0731 5823.4085 3608.5406

5065.1218 3143.4613

14005 614 1260

931

765

1030 1098 753

877

234 73 70 12

67

72

74 23 36

No. of accessions 142 87 171 98 194 sampled (of 234) No. of characters 559 1757 2086 1361 914 (after alignment) AIC score 36095.6422 59628.8231 51209.8711 24099.8716 33557.7698 (GTR + G)

ndhF-rpl32 ndhJ-trnF psbD-trnT psbJ-petA rpl32-trnL trnK-rps16 trnQ-rps16 ycf6-psbM Combined trnL-trnF rbcL ndhF matK

Divergence time estimation—Divergence-time analyses were conducted using the Bayesian MCMC method implemented in the program BEAST ver. 1.7.4 (Drummond et al., 2006; Drummond and Rambaut, 2007). BEAST takes into account phylogenetic and calibration uncertainty in estimating divergence times (Drummond et al., 2006; Drummond and Rambaut, 2007). We assigned the best fitting models of molecular evolution (GTR + G) selected by jModeltest (Guindon and Gascuel, 2003; Posada, 2008; Table 1). Each region was treated as a separate partition. As in the RAxML analysis, trnL-trnF intergenic spacer and trnL intron in the concatenated data set were treated as one partition. An uncorrelated relaxed-clock (UCLN) model was inferred for each partition, and a birth–death speciation process was assumed for the tree prior. We selected the UCLN model because it does not require rates to be heritable (Smith, 2009) and because its accuracy is comparable to an uncorrelated exponential model, although with narrower 95% highest posterior density (HPD) intervals (Drummond et al., 2006). We specified our best ML tree generated from the RAxML analysis as the starting tree after making it ultrametric using the program TreeEdit ver. 10. (Rambaut, 2002). Because there are no known fossils for Mertensia, we used external fossil calibrations for other Boraginaceae and outgroup taxa to estimate divergence times among lineages of Mertensia. To calibrate the Boraginaceae phylogeny, including all exemplar species of Mertensia, we identified 20 calibration points based on fossil data obtained from the literature (online Appendix S3). This approach of using a broad phylogeny to infer divergence times for an ingroup that lacks internal calibration points has been shown to be successful in other studies for estimating divergence times (e.g., Bremer et al., 2004; Martínez-Millán, 2010; Schenk and Hufford, 2010). We assigned log-normally distributed priors (Appendix 1; Ho and Phillips, 2009) to the minimum age estimates of the 20 calibration points, including the eudicot fossils at the root node of the rosid-asterid clade. Although the appearance of tricolpate pollen in the Late Barremian-Early Aptian (~125 Ma) has been frequently used as a maximum age for the origin of eudicots (e.g., Bell and

ITS

Alignment and phylogenetic analysis—Initial sequence alignments for each gene region were prepared using the program MUSCLE ver. 3.8.31 (Edgar, 2004) under the default settings. Following the approach of Simmons (2004), subsequent alignments were manually adjusted using the similarity criterion and performed in the program Se-Al ver. 2.0a11 (Rambaut, 1996). For the ITS region, taxa were initially divided into groups based on sequence similarity, and separate alignments were made for each group. Subsequently, these groups were iteratively aligned with one another using the profile-to-profile alignment method implemented in MUSCLE ver. 3.8.31 (Edgar, 2004). This approach of aligning sequences has been shown to be successful in other largescale phylogenetic studies (e.g., Smith and Donoghue, 2008; Tank and Donoghue, 2010; Smith et al., 2011). For each molecular marker, regions that had ambiguous alignments were excluded from phylogenetic analyses. Concatenation of aligned gene regions and conversion of file types (e.g., FASTA to PHYLIP) were performed in the program Phyutility ver. 2.2 (Smith and Dunn, 2008). Alignments generated in this study were deposited in the database TreeBASE (http://treebase.org, study number S14684). Maximum likelihood (ML; Felsenstein, 1973) analysis was conducted on the concatenated nuclear and plastid data set consisting of all 13 markers using RAxML ver. 7.0.4 (Stamatakis, 2006; Stamatakis et al., 2008). Models of molecular evolution that best fit the data (GTR + G) were selected using the program jModeltest (Guindon and Gascuel, 2003; Posada, 2008), according to the Akaike information criterion (AIC; Table 1). Each region was treated as a separate partition. The trnL-trnF intergenic spacer and trnL intron in the concatenated data set were treated as one partition. The ML analysis consisted of 1000 rapid bootstrap replicates. Every bootstrap tree generated by the rapid bootstrap analyses was used as a starting tree for full ML searches. Trees with the highest ML scores were selected. Bootstrap values (Felsenstein, 1985) were summarized with Phyutility (Smith and Dunn, 2008). Our best phylogenetic tree generated from the maximum likehood analysis was used as a starting tree for divergence time estimation.

Attribute

Phylogenetic markers—For Mertensia, we applied 11 plastid markers (matK, ndhF, ndhF-rpl32, ndhJ-trnF, psbD-trnT, psbJ-petA, rpl32-trnL, trnK-rps16, trnL-trnF, trnQ-rps16, ycf6-psbM) and also included the nuclear ribosomal 5.8S subunit and internal transcribed spacers (= ITS region) for six accessions that were used in Nazaire and Hufford (2012; Appendix S2). For Boraginaceae (other than Mertensia) and outgroups, we applied DNA sequences obtained from GenBank for the ITS region and plastid matK, ndhF, rbcL, and trnL-trnF. These markers were chosen for phylogeny reconstructions because they currently have the broadest and most complete sampling across Boraginaceae and our selected outgroups.

Summary of phylogenetic data sets and AIC scores for models of molecular evolution that best fit the data (GTR + G). AIC score was not generated for the combined data set (n/a).

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TABLE 1.

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Donoghue, 2005; Magallón and Sanderson, 2005; Schenk and Hufford, 2010), recent investigations suggest that this fixed calibration may underestimate the origin of eudicots (Smith et al., 2010). We used 125 Ma as a minimum age for the origin of eudicots and conservatively placed the log-normal distribution at a maximum age of 135 Ma. All stratigraphic ages in this study were based on the 2012 Geological Time Scale from the Geological Society of America (Geological Society of America, 2012). BEAST analyses were conducted on the concatenated plastid and nuclear data set on teragrid in the Cipres Portal (Miller et al., 2010). We initiated three independent Markov chain Monte Carlo (MCMC) runs, each consisting of 100 million generations, sampling every 5000 steps. Convergence on the same posterior distribution of each run was examined using the program Tracer ver. 1.6 (Rambaut and Drummond, 2007). Effective sample sizes for all relevant estimated parameters and node ages were well above 200. The first 10 000 000 trees were discarded as burn-in from each run. The remaining trees for each replicate were combined using the program LogCombiner ver. 1.7.4 (Drummond et al., 2006; Drummond and Rambaut, 2007). Trees were summarized with TreeAnnotator ver. 1.7.4 (Drummond et al., 2006; Drummond and Rambaut, 2007) and represent the maximum clade credibility tree. We also ran an analysis without the data to assess their informativeness. This analysis indicated that the effective priors were similar to the original priors and that the posteriors obtained after adding the data departed from both. Posterior probabilities (PP) ≥ 0.95/bootstrap proportions (BS) ≥ 85% were considered as strong support, while 0.85–0.94 PP/75–84% BS were considered as moderate support, and values of 0.70–0.84 PP/50–74% BS were considered as low or weak support. Biogeographic analyses—To test hypotheses of ancestral areas and broadscale patterns of diversification between Asia and North America, we conducted maximum likelihood-based analyses using Lagrange (Ree et al., 2005; Ree and Smith, 2007, 2008). Under a specified biogeographic model, Lagrange identifies the biogeographic history that maximizes the likelihood of realizing the observed geographic distributions of extant taxa. Because Lagrange requires an ultrametric tree for ancestral area reconstructions, we used the maximum clade-credibility (MCC) tree created from the posterior distribution of trees generated from the divergence time analyses. Phyutility ver. 2.2 (Smith and Dunn, 2008) was used to prune this tree to include only accessions of Mertensia. Our sampling for biogeographic reconstructions represents all geographic areas inhabited by Mertensia, including North America, Asia, Beringia, and circumboreal locales (Appendix S2). Geographic areas were based on Takhtajan’s (1986) definition of floristic regions. We explored three separate coding schemes to represent the geographic areas encompassed by the current distribution of Mertensia. We first implemented a simple coding scheme to include three geographic areas: North America (N), Asia (A), and circumboreal (C; for circumboreal coastal regions of North America, Asia, and Europe). Our second coding scheme, with five geographic areas, accounted for taxa restricted to Beringia and to eastern North America and were coded as the following: western North America (W), eastern North America (E), Asia (A), Beringia (B), and circumboreal (C; for circumboreal coastal regions of North America, Asia, and Europe). Last, our third coding scheme included six geographic areas to account for taxa that had distributions restricted to either western Beringia or to eastern Beringia and were coded as the following: western North America (W), eastern North America (E), Asia (A), western Beringia (R; for the Beringian region in Russia), eastern Beringia (B; for the Beringian region in North America), and circumboreal (C; for circumboreal coastal regions of North America, Asia, and Europe). Each of the three coding schemes was first run as an unconstrained analysis. In the second run, we implemented dispersal constraints for each of the three coding schemes to reflect two separate periods when the Bering Land Bridge was connected and thus likely to facilitate dispersal between Asia and North America: (1) the middle– late Miocene (14–10 Ma) to Pliocene (3.5 Ma; Hopkins, 1967; Pielou, 1979); and (2) the Illinoian and Wisconsin glaciations of the Pleistocene (1.5–0.01 Ma; Pielou, 1979; Sanmartín et al., 2001). Following the approach of Buerki et al. (2012) and Giribet et al. (2012), we set dispersal constraints to 1.0 (if landmasses were connected) or 0.1 (if landmasses were disjunct). We also examined geographic patterns of diversification within North America using Lagrange. As in the ancestral area reconstructions noted above, we used the MCC tree generated from the posterior distribution of trees from our divergence time analyses and pruned this tree using Phyutility ver. 2.2 (Smith and Dunn, 2008) to include accessions of North American Mertensia and the Beringian taxon, M. rivularis (section Mertensia, sensu Nazaire and Hufford, in press). Based on Cronquist’s (1982), Takhtajan’s (1986), and McLaughlin’s (1992) definitions of North American floristic regions, we specified nine geographic areas encompassing the current distribution of North American Mertensia:

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Appalachian (A), Prairies (P), Canadian (C), Pacific Northwest (N), Beringia (B), Great Basin-Columbia Plateau (G), central Rocky Mountains (R), southern Rocky Mountains-Mogollon (S), and Colorado Plateau (O). For monophyletic species, we coded those accessions as representing the geographic range of that species. For cases in which species were not exclusive lineages, we treated independent lineages as cryptic “species” and coded these segregate species according to their respective geographic range. For example, our previous phylogenetic analysis of Mertensia (Nazaire and Hufford, in press) recovered two separate clades of M. ciliata (James ex Torr.) G. Don, including one lineage of accessions from the Pacific Northwest and central Rocky Mountains and a second lineage of accessions from the central Rocky Mountains and Colorado Plateau; here, we treat these two lineages as independent cryptic species coded separately for these geographic regions.

RESULTS Phylogenetic reconstructions— The combined matrix consisted of 14 005 characters, including 559 nuclear and 13 446 plastid characters (Table 1). The ML analysis with 234 accessions resulted in one most likely tree (−ln L score = 120 095.731185). The ML topology based on a broad set of outgroups and selected Boraginaceae recovered a strongly supported monophyletic Boraginaceae, with six moderate to strongly supported (79–100% BS) clades that correspond to the six subfamilies recognized in recent phylogenetic studies: Boraginoideae, Cordioideae, Ehretioideae, Heliotropioideae, Hydrophylloideae, and Lennooideae (online Appendix S4). We recovered a monophyletic subfamily Boraginoideae, with six moderately to strongly supported (75–100% BS) clades that correspond to tribal designations in Boraginoideae: Boragineae, Cynoglosseae, Codoneae, Echiochileae, Lithospermeae, and Wellstedieae. Mertensia accessions were monophyletic and sister to the monotypic genus Asperugo. They were strongly supported in the tribe Cynoglosseae (Appendix S4). In Mertensia we recovered three major clades in two sections: section Stenhammaria consisted primarily of Asian lineages; section Mertensia consisted of the Beringian taxon M. rivularis placed sister the monophyletic North American species (Appendix S4). Phylogeny reconstructions also indicated that Asian diversity is paraphyletic (Appendix S4), including both Beringian and circumboreal clades nested among Asian species of section Stenhammaria, and the Asian M. rivularis placed sister to the North American clade. Divergence time estimation— Our divergence time results indicated that Boraginaceae split from its sister clade consisting of Gentianales, Solanales, Lamiales, and Vahliaceae at 102.10– 73.29 Ma (online Appendix S5; mean divergence times, 95% highest probability density [HPD], and posterior probabilities (PP) are listed in Table 2). The crown of Boraginaceae originated 87.69–54.34 Ma (95% HPD; Appendix S5), the crown of subfamily Boraginoideae originated 76.89–46.49 Ma (95% HPD; Fig. 2B), and the crown group of tribe Cynoglosseae within subfamily Boraginoideae originated 38.27–23.25 Ma (95% HPD; Appendix S5). The inferred divergence time for the split between Mertensia and its sister clade, Asperugo, was dated to the early to mid Miocene at 26.83–12.22 Ma (95% HPD; Appendix S5). Divergence time estimates indicate a late Miocene origin for the Mertensia crown group at 10.36–5.19 Ma (95% HPD), with crown groups of sections Stenhammaria and Mertensia (sensu Nazaire and Hufford, in press) originating 9.33–4.52 Ma (95% HPD) and 8.23–4.37 Ma (95% HPD), respectively (Table 2;

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TABLE 2.

Divergence time estimates for Boraginaceae, including major clades of the family, and the major lineages of Mertensia that are the focus of this study. Crown group ages (Ma)

Clade

Mean

95% HPD

PP

Boraginaceae + Gentianales, Solanales, Lamiales, Vahliaceae Boraginaceae Subfamily Boraginoideae Tribe Cynoglosseae Mertensia + Asperugo Mertensia Section Stenhammaria (Clade 1) Section Mertensia (including M. rivularis) Asia 1 clade Asia 2 clade Circumboreal clade Asia 3 clade Beringia clade North American Mertensia (Clade 3) Pacific Northwest clade (paniculata complex) Southern Rocky Mountain clade arizonica complex Central Rocky Mountain clade lanceolata complex ciliata complex oblongifolia complex

89.08

102.10–73.29

1.0

72.63 63.52 31.06 18.95 7.94 7.11 6.34

87.69–54.34 76.89–46.49 38.27–23.25 26.83–12.22 10.36–5.19 9.33–4.52 8.23–4.37

1.0 1.0 1.0 0.69 0.72 0.92 0.79

6.72 6.72 5.79 3.62 3.79 5.93 5.42

9.27–4.03 9.27–4.03 8.09–3.70 5.64–1.80 5.99–1.67 7.70–4.22 7.27–3.68

0.31 0.31 0.9 0.98 0.98 0.73 1.0

5.21 4.45 4.18 2.55 2.03 1.79

6.69–3.68 5.85–2.99 5.54–2.77 3.55–1.52 2.86–1.23 2.49–1.10

0.3 0.88 0.92 0.54 0.99 0.98

Appendix S5). The four major lineages of section Stenhammaria, encompassing Asian, Beringian, and circumboreal locales, diverged during the late Miocene to the Pleistocene (Table 2; Appendix S5). Within North American Mertensia, the divergence times of five major complexes (paniculata complex, arizonica complex, lanceolata complex, ciliata complex, and oblongifolia complex) fall between the late Miocene and Pleistocene (Table 2; Appendix S5). Biogeographic analyses— The three separate coding schemes applied to ancestral-area reconstructions between Asia and North America had similar patterns (Fig. 1; online Appendix S6). Notably, increasing the number of geographic areas (from three to six) had the effect of refining the ancestral-range reconstructions at nodes, but also increased conflict in the level of relative probabilities for the same ancestral areas in our range reconstructions. For example, under the three-region coding scheme, Lagrange reconstructed two possible scenarios for the ancestral area of Mertensia; the scenario with the highest relative probability reconstructed the ancestral area as Asia with a small probability of North American ancestry (A|AN with 0.83 relative probability at node 1; Fig. 1B). In contrast, the ancestral area with the highest relative probability in reconstructions based on a five-region coding scheme reconstructed

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Asia–Beringia–circumboreal as the ancestral area (ABC|B with 0.56 relative probability at node 1; Fig. 1A); however, three other scenarios, albeit with lower relative probabilities were also reconstructed for the same node (Appendix S6). Under the six-region coding scheme, the ancestral area was recovered as western Beringia–Asia–circumboreal (ARC|R with 0.57 relative probability at node 1; Fig. 1C), with two additional reconstructed scenarios. Constraining the model to optimize times when the Bering Land Bridge was available between Asia and North America improved log likelihood scores and relative probability on some nodes in each of the coding schemes, but generally these were not significant differences. Ancestral area reconstructions generated from the five- and six-region coding schemes identified two dispersal events in the lanceolata complex of the North American clade that were not evident from results obtained from reconstruction based on coding three regions. Both the five- and six-region coding schemes, which coded for eastern North America and Beringia, showed a dispersal event from western North America into eastern North America (five-region coding scheme: E|WE with 0.91 relative probability at node 47; Fig. 1A), as well as a migration from western North America northward to Beringia (five-region coding scheme: B|W with 1.0 relative probability at node 49; Fig. 1A). Lagrange reconstruction of geographic diversification of Mertensia within North America indicated the ancestral area of section Mertensia (which included the Beringian taxon M. rivularis, sensu Nazaire and Hufford, in press) as Beringia–Pacific Northwest (B|N with 0.37 relative probability at node 1; Fig. 2), but five additional scenarios with significant relative probabilities (>0.05) were also reconstructed for this node (online Appendix S7). Similar to ancestral area reconstructions that focused on the Asia–North America pattern (as discussed already), we noted that certain nodes showed evidence of conflict in the level of relative probabilities for the same ancestral areas, and this appeared to occur on nodes that were either coded for multiple regions (e.g., node 5; Appendix S7) or nodes that had low phylogenetic support values (e.g., node 60; Appendix S7). Our analyses recovered three major clades within North America, which we designate as the Pacific Northwest clade, the southern Rocky Mountain clade, and the central Rocky Mountain clade (Fig. 2). We infer that each of these clades had an ancestral area that was Beringia–Pacific Northwest. The Pacific Northwest clade shows a range expansion from the Pacific Northwest (N|N with 0.64 relative probability at node 3; Fig. 2) eastward into Canadian regions and prairies. In the southern Rocky Mountain clade, one subclade remained largely in the Pacific Northwest (N|NS with 0.23 relative probability at node 11; Fig. 2). A second subclade, which includes the arizonica complex, dispersed from the Pacific Northwest southward into the southern Rocky Mountains (S|S with 0.34 relative probability at node 15; Fig. 2), with subsequent range expansion into the Colorado Plateau and the Great Basin-Columbia Plateau regions (G|GO with 0.43 relative probability at node 25; Fig. 2). →

Fig. 1. Ancestral area reconstructions for Mertensia inferred by Lagrange analysis, based on models constrained to reflect likely periods of dispersal between Asia and North America. Colored squares at terminals indicate ranges occupied by sampled species. Colored squares on nodes indicate ranges reconstructed for hypothetical ancestors. Numbers on nodes refer to nodes in online Appendix S6 and correspond to reconstructed splits, log likelihoods, and relative probabilities for each node. (A) Ancestral area reconstruction based on a five-region coding scheme with corresponding map. (B) Ancestral area reconstruction based on a three-region coding scheme. (C) Ancestral area reconstruction based on a six-region coding scheme. Gray box in (A) references portion of tree illustrated in ancestral area reconstructions for (B) and (C). Sections Stenhammaria and Mertensia of Mertensia identified by gray solid lines. Major lineages (complexes) in North American Mertensia indicated by white bars.

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The central Rocky Mountain clade, which includes the lanceolata, ciliata, and oblongifolia complexes, shows a migration from the Pacific Northwest south into the central Rocky Mountains (R|R with 0.62 relative probability at node 31; Fig. 2). Three subsequent range expansions are noted in the central Rocky Mountain clade: (1) dispersal eastward into the Appalachian and Prairies regions (A|AP with 0.96 relative probability at node 38; Fig. 2); (2) migration northward to Beringia (B|P with 0.81 relative probability at node 15; Fig. 2); and (3) movement westward into the Great Basin Columbia Plateau region, which largely includes the oblongifolia complex (G|GR with 0.33 relative probability at node 15; Fig. 2). DISCUSSION Phylogenetic relationships—Our phylogenetic results are consistent with recent molecular phylogenetic studies of Boraginaceae (Nazaire and Hufford, 2012) and of the six subfamilies recognized in recent phylogenetic studies: Boraginoideae, Cordioideae, Ehretioideae, Heliotropioideae, Hydrophylloideae, and Lennooideae (Långström and Chase, 2002; Nazaire and Hufford, 2012; Appendix S4). Consistent with recent phylogenetic studies of Boraginaceae (Gottschling et al., 2001; Nazaire and Hufford, 2012), we recovered a monophyletic subfamily Boraginoideae, with six moderately to strongly supported clades that correspond to tribal designations: Boragineae, Cynoglosseae, Codoneae, Echiochileae, Lithospermeae, and Wellstedieae. We note the addition of Wellstedia dinteri Pilg. in the present analysis, a taxon previously not included in Nazaire and Hufford (2012). Wellstedia Balf. f. was strongly supported as sister to tribes Boragineae, Cynoglosseae, Echiochileae, and Lithospermeae. Our phylogenetic results are also consistent with the broader sampling of Mertensia in Nazaire and Hufford (in press), with the exception of a few minor differences in taxon placement in weakly supported clades. Divergence times for Boraginaceae— Topologies recovered from both the ML analysis and the BEAST divergence time analyses were congruent and placed Boraginaceae as sister to a clade consisting of Gentianales, Solanales, Lamiales, and Vahliaceae, with divergence time estimates indicating a Late Cretaceous–early Tertiary origin (87.69–54.34 Ma) for Boraginaceae (Appendix S5). Previous investigations of borage diversification have found somewhat earlier divergence times, with a Late Cretaceous origin for the family (e.g., Wikström et al., 2001; Bremer et al., 2004; Magallón and Castillo, 2009). Our divergence time estimates are also notably younger than those yielded in Gottschling et al. (2004). They estimated divergence times for the primarily woody members of the family (Cordioideae, Ehretioideae, Heliotropioideae) as originating during the midCretaceous (~90 Ma), suggestive of an earlier divergence time estimate for the family. The Gottschling et al. (2004) divergence time estimates were based primarily on the placement of a well-dated fossilized fruit that shares features with the extant

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Ehretia II subclade of Ehretioideae, at ~50 Ma (Gottschling et al., 2002, 2004) and thus implies that Ehretia P. Browne and Ehretioideae were likely present at an earlier time period. However, in our divergence time analysis, we could not place this fossil appropriately to constrain the Ehretia II clade, because the node in our phylogeny occurred in a more derived position compared with the corresponding node in which this fossil was placed by Gottschling et al. (2004). Based on our other fossil placements, BEAST showed this fossil to be incongruent in our divergence time analysis. Similarly, Moore and Jansen (2006) encountered difficulty in placing the Ehretia fossil to constrain the Ehretia II clade for their divergence time estimates of Tiquilia Pers., but addressed the issue by placing the fossil to constrain Ehretioideae. Consistent with previous studies of Boraginaceae radiations (Moore and Jansen, 2006; Mansion et al., 2009), our divergence time estimates show a relatively rapid radiation of the major lineages of Boraginaceae, which occurred shortly after the end of the Eocene (~33.5 Ma). If our divergence time estimates are correct, it appears that much of the diversification within the family, and notably, diversification of herbaceous lineages, coincides with the development of widespread aridification and cooling of the Earth, which began during the end of the Eocene (Wolfe, 1992, 1997; Graham, 1999). The Eocene–Oligocene boundary marks the transition from boreotropical forests to a mixed mesophytic forest, in which cool-adapted deciduous and herbaceous plant families originated (Wolfe, 1997). Given that many borage lineages are herbaceous, particularly in subfamily Boraginoideae, it seems plausible that the climatic conditions during this time period could have provided new opportunities for geographic expansion and diversification of these lineages. Ancestral area and timing of early geographic radiation in Mertensia— Our divergence time estimates indicate that Mertensia is a relatively young lineage. The stem lineage of Mertensia is inferred to have split from its nearest extant relative, Asperugo, in the late Oligocene to mid Miocene (26.83– 12.22 Ma), with the first divergence in the crown group in the late Miocene (10.36–5.19 Ma). Ancestral area reconstructions based on three regions infer that the ancestral area for Mertensia was Asia, with a small probability of North American ancestry (Fig. 1B). Reconstructions based on five and six regions suggest that a widespread ancestor of Mertensia was distributed across northern latitudes encompassing Asia, Beringia, and circumboreal locales. Early diversification of stem and crown lineages of Mertensia occurred during a period of time when climatic changes were dramatically influencing plant distribution patterns. With the onset of extreme cooling in the northern hemisphere during the middle to late Miocene, a belt of coniferous forests connected northeastern Asia and northwestern North America across Beringia, dominating northern latitudes and alpine to subalpine zones in more southerly locations (Hopkins, 1967; Wen, 1999; Sanmartín et al., 2001). Ecological conditions and floristic elements of the coniferous forests present during that time (e.g., Picea A. Dietr. and Abies Mill.; Baker, →

Fig. 2. Biogeographic reconstructions for North American Mertensia inferred by Lagrange analysis, based on nine coded regions. Colored squares at terminals indicate ranges occupied by sampled species. Colored squares on nodes indicate ranges reconstructed for hypothetical ancestors. Numbers on nodes refer to nodes in online Appendix S7 and correspond to reconstructed splits, log likelihoods, and relative probabilities for each node. Reconstructed nodes with highest relative probability are shown. Numbered nodes in pink indicate other likely ranges were reconstructed with high relative probabilities and are referred to in online Appendix S7. Major geographic clades in North American Mertensia identified by gray solid lines. Major lineages (complexes) indicated by white bars.

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1983; Spaulding et al., 1983) are similar to those in which many species of Mertensia grow today (Hitchcock et al., 1959; Pelton, 1961; Cronquist et al., 1984). Hypotheses to explain geographic patterns of diversity in the northern hemisphere (e.g., Hopkins, 1967; Murray, 1981, 1995; DeChaine, 2008) often emphasize origins of clades in Asia. Many arctic, alpine, and boreal taxa are thought to have originated in the high montane regions of central Asia (and to a lesser extent western North America) and migrated via the Bering Land Bridge (Hultén, 1958; Hedberg, 1992; Murray, 1995). Asian Mertensia, such as those included in our analyses [e.g., M. davurica (Sims) G. Don, M. sibirica (L.) G. Don] are restricted primarily to alpine and subalpine environments in central to western Russia and China (Popov, 1953a, b; Ge-ling et al., 1995). Origins of Mertensia in Asia, including western Beringia, are consistent with the geographic regions reconstructed at the base of our phylogeny and our ancestral area reconstructions. Early diversification of Mertensia may have involved migrations from low latitude mountain systems to high latitudes and/or elevational movements into alpine and subalpine habitats, thus leading to fragmentation and isolation of populations and potential opportunities for diversification. Because many extant species of Mertensia occupy either alpine or subalpine habitats [e.g., M. alpina (Torr.) G. Don, M. davurica], northern latitude boreal and arctic habitats [e.g., M. paniculata (Ait.) G. Don, M. maritima (L.) Gray], or cool, moist, lower elevation sites (e.g., M. virginica L. Pers. ex Link), it is very likely that the most recent common ancestor of Mertensia was also adapted to cool climatic conditions. Beringia and boreal Mertensia— Based on our divergence time results, early diversification of Mertensia may have occurred during a period when Beringia was connected and available for floristic exchange. There has been some diversification of Mertensia in Beringia, as some species are endemic to that area. Interestingly, some species are restricted to western Beringia [e.g., M. kamczatica (Turcz.) DC., M. pterocarpa (Turcz.) Tatew. & Ohwi, M. rivularis] and some to eastern Beringia (e.g., M. eastwoodiae J.F. Macbr., M. paniculata). Mertensia may be consistent with Hultén’s (1937) postulate that many arctic and boreal taxa initially radiated east and west from Beringia during the late Tertiary and persisted in refugia during the glacial events of the Pleistocene. Extensive fossil (Sher et al., 2005) and molecular evidence (e.g., Abbott et al., 2000; Geml et al., 2006; DeChaine, 2008) support Hultén’s hypothesis. A similar hypothesis proposed by Popov (1953a) suggested that Beringia served as the “primary native land” for Mertensia during the Pliocene, from which both Asian and North American clades diverged and underwent range expansion during the Pleistocene. With origins in Beringia, we would expect to find a grade of Beringian taxa at the base of our phylogeny. However, our phylogenetic results and ancestral area reconstructions are not congruent with this hypothesis (Fig. 3; Appendix S2). Rather, we find Beringian taxa nested in Asian and North American clades. Beringia may have been included as part of the ancestral area, but is unlikely to be solely the ancestral area. Although uncommon for Boraginaceae, the present transBeringian distribution of Mertensia suggests that Beringia likely played an important role in the early geographic radiation of the genus, as inferred from our ancestral area reconstructions (Fig. 1; Appendix S6). Our phylogenetic results recovered three independent clades of Beringian taxa. One clade (M. kamczatica,

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M. pterocarpa) is closely related to Asian and circumboreal members of the genus (section Stenhammaria [sensu Nazaire and Hufford, in press]) and is inferred to have diverged from an Asian ancestor (Fig. 1). A second clade, which includes the Beringian taxon M. rivularis, is sister to North American Mertensia. A third clade, which includes the narrow endemic, M. drummondii (Lehm.) G. Don, is restricted to eastern Beringia. Results from our biogeographic reconstructions (Fig. 1A, 1B) are consistent with a scenario of range expansion from Beringia into North America. Hultén (1937) considered dispersal from coastal refugia following interglacial periods to be important for the geographic radiation of arctic and boreal taxa. He suggested that M. maritima, a species of coastal habitats, survived in arctic coastal refugia and achieved its widespread circumpolar distribution along Pacific and Atlantic tracks. Furthermore, Hultén speculated that European populations of M. maritima were derived from North American ancestors via Atlantic movements. Our ancestral area reconstructions imply that a circumboreal coastal ancestry may have been important in the early geographic radiation of Mertensia (Fig. 1). Similar scenarios that suggest the importance of arctic refugia in explaining current circumpolar distributions have been inferred for diverse plant groups (Abbott et al., 2000; Eidesen et al., 2007; Schönswetter et al., 2008). Our sampling, which includes two accessions of M. maritima, represents a subset of a broader sampling of M. maritima in our previous phylogenetic analyses of Mertensia (Nazaire and Hufford, in press). In that study, accessions representing the current geographic range of M. maritima were recovered in two clades that correspond with Hultén’s (1937) Pacific and Atlantic migrations. Atlantic and European coastal accessions of M. maritima form a clade; however, our results were equivocal about whether European populations were derived from a North American ancestor. Dispersal via ocean currents may perhaps be the most reasonable explanation for the current circumboreal pattern of M. maritima. Extant species richness— In Mertensia, the greater species richness in North America relative to Asia is anomalous for groups inferred to have an Asian origin. Although our ancestral area reconstructions cannot determine the exact place of origin for Mertensia, it is unlikely that North America was its ancestral area; we infer Asia or more widely Asia, Beringia, and circumboreal locales as the ancestral area. The geography of species richness in Mertensia is inconsistent with the hypothesis of a “time-for-speciation effect” (Latham and Ricklefs, 1993; Qian and Ricklefs, 1999; Stephens and Wiens, 2003; Kozak and Wiens, 2010), which would predict regions that were occupied the longest had more time for speciation and the accumulation of species. In contrast, our divergence time estimates indicate that lineages in North America are relatively younger than those of Asian clades. High species diversity in Asia relative to North America has frequently been attributed to low extinction rates resulting from lesser impacts of Quaternary glaciations in Asia relative to North America (Qian and Ricklefs, 1999; Wen, 1999). However, glaciations have also been associated with increased rates of speciation in mammals (George, 1988; Talbot and Shields, 1996), birds (Voelker, 1999; Milá et al., 2007), and grasshoppers (Knowles, 2001). Our divergence time analysis shows that origins of several lineages of Mertensia in North America coincide with events of the Pleistocene, from which we infer that

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Fig. 3. Postulated migration pathways for the three major clades in North American Mertensia. Colored regions correspond to nine regions used in Lagrange analysis, referenced in Fig. 2. Large arrows indicate major routes, while smaller arrows indicate smaller or postulated routes. (A) Pacific Northwest clade. (B) southern Rocky Mountain clade. (C) central Rocky Mountain clade.

glaciation may have influenced rates of diversification in the North American clades. High species diversity in Asia has traditionally been explained as a consequence of a complex topography and diverse habitats that create opportunities for speciation (Qian and Ricklefs, 1999, 2000; Harrison et al., 2001; Ricklefs et al., 2004). In western North America, landscapes are also marked by topographic, edaphic, and habitat diversity and are notably characterized by three major north–south-running mountain blocks. In North America, Mertensia occupies a range of habitats and elevations, including alpine (e.g., M. alpina), montane (e.g., M. ciliata), and boreal (e.g., M. paniculata) communities, low mesic forests (e.g., M. virginica), prairies [e.g., M. lanceolata (Pursh) DC. ex A. DC.], and sagebrush slopes [e.g., M. oblongifolia (Nutt.) G. Don], but Asian Mertensia are largely restricted to alpine and subalpine habitats (Popov, 1953a, b; Ge-ling et al., 1995). While niches of Asian Mertensia are largely conservative, diversification of North American Mertensia likely involved ecological niche shifts as populations expanded over the mountainous American West. Diversification of Mertensia in North America— From our biogeographic reconstructions, we infer that North American Mertensia diversified from a widespread ancestral lineage of boreal, arctic, and alpine habitats in Beringia and the Pacific Northwest (together, these regions are regarded here as northwestern North America) during the late Miocene to early Pliocene (7.70–4.22 Ma). Early diversification of North American Mertensia would have coincided with onset of increased topographic diversity and climatic cooling during this period. The Rocky Mountains were largely elevated to their present heights, many of the Great Basin ranges were undergoing rapid uplift, and the Cascade Range had attained significant heights by the mid Pliocene (McKee, 1972; Billings, 1978; Axelrod and Raven, 1985; Graham, 1999). Further, the north–south orientation of western North American ranges has been suggested as important for the migration of arctic, alpine, and boreal plants (Billings, 1978; Axelrod and Raven, 1985; DeChaine and Martin, 2005a). Both the onset of physical changes in western North America and the north–south-running mountain blocks provided new opportunities for migration, expansion, and diversification of North American Mertensia. Our results indicated that early northwestern North American Mertensia differentiated in that region into three deep clades, including the Pacific

Northwest clade, southern Rocky Mountain clade, and central Rocky Mountain clade. Lineages in the Pacific Northwest clade became centered largely in the Pacific Northwest, although some lineages expanded into Canada and prairies (Figs. 2, 3A). Both the southern and central Rocky Mountain clades underwent range expansion from northwestern North America in separate migrations into the Rocky Mountains (Figs. 2, 3B, 3C). Signatures of an ancestral range expansion followed by fragmentation and diversification in western North American clades are often supported by contemporary geographic patterns of endemism (Stebbins and Major, 1965; Harper and Reveal, 1978; Marlowe, 2007). Endemism is characteristic of many speciesrich groups in western North America (e.g., Astragalus L., Eriogonum Michx., Lomatium Raf., Mentzelia L., Penstemon Schmidel, Primula L.) as well as Mertensia (especially alpine and montane species in the southern and central Rocky Mountain clades). In many cases, endemics are restricted to a single mountain range (e.g., M. toyabensis J.F. Macbr. in the Toiyabe Mountains of Nevada, M. humilis Rydb. in the Big Horn Mountains of Wyoming) or geographic region (e.g., M. subcordata Greene in lowland forests west of the Cascades in Oregon). As we discuss below, repeated cycles of range expansion for alpine and montane species across relatively low elevation continuous landscapes during glacial maxima followed by range contraction and fragmentation during interglacials may in part explain contemporary distribution patterns and high endemism of Mertensia in western North America. Pacific Northwest clade—Divergence times for the origin of the crown group of the Pacific Northwest clade were estimated at 7.27–3.68 Ma (Fig. 2, Table 2). Origin and early diversification of the crown group appears to correspond with the uplift of the Cascade Range during the Miocene and Pliocene (McKee, 1972; Graham, 1999). Subsequent range expansions of lineages in the Pacific Northwest clade coincide with the onset and duration of continental glaciations in the Pleistocene, when ice sheets repeatedly covered extensive portions of Canada, Alaska, and the Pacific Northwest, pushing populations and suitable habitats southward and down-slope (e.g., Pielou, 1991; Graham, 1993; Shafer et al., 2010). Postglacial range expansion is thought to be important for many species that occupied refugia south of the ice sheets during glacial maxima (Hewitt, 2000). Some lineages in the Pacific Northwest clade, such as M. paniculata var. paniculata, are widespread in areas that were occupied

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by continental ice sheets, which is indicative of postglacial range expansion (Hewitt, 2000; Shafer et al., 2010). Similar patterns of postglacial range expansion have been noted in other boreal forest elements, including spruce, pine, and quaking aspen (Strong and Hills, 2005), white spruce (Yansa, 2006), and one-sided wintergreen (Beatty and Provan, 2010). Southern Rocky Mountain clade—Our biogeographic reconstructions indicated that the Rocky Mountain Range served as an important migration corridor during two major dispersal events of North American Mertensia as they moved from northwestern North America: (1) migration into the southern Rocky Mountain Range (southern Rocky Mountain clade; Figs. 2, 3B) and (2) migration into the central Rocky Mountain Range (central Rocky Mountain clade; Figs. 2, 3C). Movement of the southern Rocky Mountain clade into the southern Rocky Mountains during the late Miocene to mid Pliocene (6.69–3.68 Ma) is characterized by three geographic patterns: (1) some lineages became centered largely in the Pacific Northwest, with selected species subsequently expanding their ranges into the southern Rocky Mountains and Great Basin-Columbia Plateau; (2) some lineages in the arizonica complex diversified in the southern Rocky Mountains and selected subclades expanded into the Colorado Plateau; and (3) a subsequent migration of arizonica complex lineages westward from the Colorado Plateau into the Great Basin-Columbia Plateau region. Lineages centered largely in the Pacific Northwest (e.g., M. bella Piper, M. campanulata A. Nelson, M. longiflora Greene) are characterized by contemporary ranges that are disjunct, fragmented, or narrowly restricted. These geographic patterns may have originated in response to mountain building and aridification of inland regions in the Pacific Northwest (Graham, 1993, 1999). For example, the disjunction of M. bella between Montana and Idaho mountain populations east of the Cascade Range and Oregon populations west of the Cascades is common among organisms associated with mesic coniferous forests of the Pacific Northwest and is thought to have originated through vicariance that resulted from aridification of Columbia Basin following the Cascadian orogeny (Brunsfeld et al., 2001, 2007). Some lineages in the arizonica complex (e.g., M. cynoglossoides Greene, M. franciscana A. Heller, M. ovata Rydb.) expanded their range from the southern Rocky Mountains into the montane regions of the Colorado Plateau. The floristic similarities shared between the Rocky Mountains and mountain ranges in the Colorado Plateau have led some workers to hypothesize the availability of habitat corridors during the Pleistocene (e.g., Moore, 1965; Billings, 1978; Schaak, 1983; Wells, 1983). Although the southern Rocky Mountains were south of the continental ice sheets, this region was still greatly impacted by the Pleistocene glaciations. During glacial maxima, montanesubalpine coniferous forests expanded their ranges across broad contiguous landscapes at lower elevations, which facilitated migrations through dispersal corridors (e.g., Chabot and Billings, 1972; Billings, 1974, 1978; Schaak, 1983). Hypothesized dispersal corridors are corroborated by recent phylogeographic work in the southern Rocky Mountains (Marlowe, 2007). Subsequent migration from the Colorado Plateau into the Great Basin-Columbia Plateau, as exemplified by the westward movement of a clade (node 25; Fig. 2) in the arizonica complex may have also used similar Pleistocene dispersal corridors. Such patterns are shared with other alpine and montane plants (Chabot and Billings, 1972; Kelso et al., 2009), butterflies (Nice and

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Shapiro, 2001; Schoville et al., 2011), and pikas (Hafner and Sullivan, 1995). Central Rocky Mountain clade—A second episode of dispersal into the Rocky Mountains, involving migration of the central Rocky Mountain clade southward into the central Rocky Mountains from northwestern North America, occurred during the Pliocene to early Pleistocene (5.54–2.77 Ma; Figs. 2, 3C). Three key events characterize central Rocky Mountain clade geography: (1) lineages in the lanceolata complex migrated from the central Rocky Mountains eastward into eastern North America and northward, resulting in a reintroduction to Beringia; (2) lineages of the ciliata complex diversified in the central Rocky Mountains; and (3) lineages in the oblongifolia complex migrated westward from the central Rocky Mountains into the Great Basin-Columbia Plateau region. The lanceolata complex of the central Rocky Mountain clade is characterized by two dispersal events: (1) eastward dispersal from the central Rocky Mountains into the Appalachian region, exemplified by M. virginica and (2) a northward migration to the prairies of central United States (M. lanceolata) and to Beringia (M. drummondii; Figs. 2, 3C). The eastward movement of lineages into eastern North America, from which M. virginica, a species of lowland mesic forests in the Appalachian region, is probably derived, appears to have occurred during the early Pleistocene (Fig. 2), when boreal forests were pushed southward in response to advancing ice sheets (Davis, 1969, 1983; Strong and Hills, 2005) and formed broad contiguous landscapes that may have served as migration corridors. Northward migration of the lanceolata complex may have tracked recently deglaciated terrain, following the retreat of continental ice sheets. For example, the contemporary distribution of M. lanceolata in prairie habitats of north central United States indicates a northwestern movement from eastern United States (Figs. 2, 3C). Placement of the Beringian M. drummondii as sister to M. lanceolata points toward a migration from the prairies of north central United States northward to Beringia. Lineages in the ciliata complex largely remained in the central Rocky Mountains, where they diversified. Several species have relatively narrow geographic ranges in alpine, subalpine, and/or high elevation habitats, often isolated on mountaintops (e.g., M. cana, M. parvifolia (L. O. Williams) Nazaire & L. Hufford, M. perplexa), or may be more broadly distributed along the alpine habitats of the Rocky Mountain axis (e.g., M. alpina). The contemporary distribution of alpine and subalpine plants in the ciliata complex is consistent with a sky-island archipelago model of expansion and contraction of populations as proposed by DeChaine and Martin (2004). Shifting distribution patterns of alpine and subalpine habitats in response to Quaternary glacial–interglacial cycles occurred repeatedly throughout the Quaternary. Phylogeographic studies of alpine butterflies (DeChaine and Martin, 2004; Schoville et al., 2011), grasshoppers (Knowles, 2000), and plants (DeChaine and Martin, 2005b) corroborate the sky-island archipelago model. Biogeographic reconstructions identified a second dispersal event into the Great Basin-Columbia Plateau region, independent of the dispersal of lineages in the southern Rocky Mountain clade discussed above. Range expansion of several lineages in the oblongifolia complex of the central Rocky Mountain clade may have used similar Pleistocene dispersal corridors as lineages in the southern Rocky Mountain clade or different pathways. For example, Billings (1974, 1978) proposed that some movements into the Great Basin-Columbia Plateau from

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the Rocky Mountains might have occurred via northward movements along the Rocky Mountain axis followed by a southward migration along the Cascade Range into the Great Basin-Columbia Plateau region. Our biogeographic reconstructions show that contemporary distributions of several species in the oblongifolia complex [e.g., M. longipedunculata (A. Nelson) Nazaire & L. Hufford, M. oblongifolia var. nevadensis (A. Nelson) L. O. Williams, M. oblongifolia var. oblongifolia, M. subpubescens Rydb.] encompass the Great Basin-Columbia Plateau and the Pacific Northwest, a geographic pattern that could be consistent with Billings’s hypothesis. Alternatively, lineages in the oblongifolia complex may have migrated westward from the central Rocky Mountains into the Great Basin-Columbia Plateau region and subsequently expanded their range northward to the Pacific Northwest. In conclusion, results from our molecular dating analysis of Mertensia recover signatures of a relatively young lineage that faced rapid diversification over the last 7–10 million years. From biogeographic reconstructions, we infer the ancestral area to have been Asia or a more widespread distribution encompassing parts of Asia, Beringia, and circumboreal locales. It is unlikely that North America, which for Mertensia has greater taxic diversity than Asia, was the ancestral area for the genus. Mertensia likely first appeared in the mid Miocene as a floristic element of boreal, arctic, and alpine habitats and probably remained largely restricted to cool, edaphically moist sites throughout much of its history. Early diversification likely occurred during a period when Beringia was connected and available for floristic exchange. Initial range expansion of North American Mertensia from Beringia and the Pacific Northwest probably coincided with the onset of extreme cooling and increased topographic diversity during the late Miocene to early Pliocene, followed by the diversification of three deep North American clades. The Rocky Mountain range served an important role in the diversification of the southern and central Rocky Mountain clades as climatic fluctuations associated with glacial and interglacial cycles of the Pleistocene shifted populations over latitudinal and elevational gradients to shape patterns of distribution and regional diversity observed today. LITERATURE CITED ABBOTT, R. J., L. C. SMITH, R. I. MILNE, R. M. M. CRAWFORD, K. WOLFF, AND J. BALFOUR. 2000. Molecular analysis of plant migration and refugia in the Arctic. Science 289: 1343–1346. APG II [ANGIOSPERM PHYLOGENY GROUP II]. 2003. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG II. Botanical Journal of the Linnean Society 141: 399–436. APG III [ANGIOSPERM PHYLOGENY GROUP III]. 2009. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III. Botanical Journal of the Linnean Society 161: 105–121. AXELROD, D. I., AND P. H. RAVEN. 1985. Origins of the Cordilleran flora. Journal of Biogeography 12: 21–47. BAKER, R. G. 1983. Holocene vegetation and history of the western United States. In S. C. Porter [ed.], Late Quaternary environments of the United States, vol. 2, The Holocene, 109–127. University of Minnesota Press, Minneapolis, Minnesota, USA. BARRES, L., I. SANMARTÍN, C. L. ANDERSON, A. SUSANNA, S. BUERKI, M. GALBANY-CASALS, AND R. VILATERSANA. 2013. Reconstructing the evolution and biogeographic history of tribe Cardueae (Compositae). American Journal of Botany 100: 867–882. BEATTY, G. E., AND J. PROVAN. 2010. Refugial persistence and postglacial recolonization of North America by the cold-tolerant herbaceous plant Orthilia secunda. Molecular Ecology 19: 5009–5021.

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APPENDIX 1. Fossils, minimum ages (Min. Age [Ma]), lognormal prior mean

(LNP mean), and standard deviation (SD) used to calibrate divergence times for Boraginaceae and Mertensia: Fossil no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Fossil Core eudicots—tricolpate pollen Archamamelis bivalvis Divisestylus brevistamineus Ribes stevenii Cornus multilocularis Hironia fusiformis Dichroa bornensis Schizophragma polonica Jamesia caplanii, Fendlera coloradensis Parasaurauia allonensis Ilex hercynica Dendropanax eocenensis, Paleopanax oregonensis Campanula paleopyramidalis Donatia novae-zelandiae Eucommia montana Emmenopterys dilcheri Apocynospermum sp. Acanthus rugatus Lithospermum dakotense Cryptantha coroniformis

Min. Age (Ma) LNP Mean SD 125 83.5 90 26.5 49 85.8 28.4 2.6 26.5

0.7 1.0 1.0 0.1 0.2 0.2 0.2 0.2 0.1

0.7 1.0 1.0 0.3 0.65 0.65 0.65 0.5 0.3

70.6 61.7 43

0.5 0.2 0.1

0.75 0.5 0.5

5.3 0.01 48.6 43 40.4 28.4 5.3 2.6

0.1 0.2 0.2 0.1 1.0 0.1 0.2 0.1

0.75 0.5 0.6 0.4 0.5 0.5 0.6 0.5

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Cytomictic anomalous male meiosis and 2n pollen grain formation in Mertensia echioides Benth. (Boraginaceae) from Kashmir Himalaya.

Geographic patterns of immunity and vascular disease.

Radiation in medicine: Origins, risks and aspirations.

Patterns and causes of geographic variation in bat echolocation pulses.

The classification and origins of protein folding patterns.

Genetic characterization of Bombyx mori (Lepidoptera: Bombycidae) breeding and hybrid lines with different geographic origins.

Components and Anti-HepG2 Activity Comparison of Lycopodium Alkaloids from Four Geographic Origins.

Raman Radiation Patterns of Graphene.

The utilization of stable isotope analysis for the estimation of the geographic origins of unidentified cadavers.

Assessment of the geographic origins of pinewood nematode isolates via single nucleotide polymorphism in effector genes.

Genomic Patterns of Geographic Differentiation in Drosophila simulans.

Geographic patterns of breast cancer in the United States.

Multiple evolutionary origins of prochlorophytes within the cyanobacterial radiation.

Geographic patterns of large bowel cancer in the United States.

Molecular Evolution of PvMSP3α Block II in Plasmodium vivax from Diverse Geographic Origins.

PTR-ToF-MS characterisation of roasted coffees (C. arabica) from different geographic origins.

Geographic population structure analysis of worldwide human populations infers their biogeographical origins.

Intensity patterns of solar ultraviolet radiation.

Intraspecific N and P stoichiometry of Phragmites australis: geographic patterns and variation among climatic regions.

Community-Engaged Modeling of Geographic and Demographic Patterns of Multiple Public Health Risk Factors.

Temporal patterns and geographic heterogeneity of Zika virus (ZIKV) outbreaks in French Polynesia and Central America.

The origins and radiation of Australian Coptotermes termites: from rainforest to desert dwellers.

Geographic patterns of chromosomal variation in South American marsh rats, Holochilus brasiliensis and H. vulpinus.

Onosma anatolica, a new species of Boraginaceae from Turkey.