Range-Wide Chloroplast and Mitochondrial DNA Imprints Reveal Multiple Lineages and Complex Biogeographic History for Douglas-Fir

Tree Genetics & Genomes (2011) 7:1025–1040 DOI 10.1007/s11295-011-0392-4

ORIGINAL PAPER

Range-wide chloroplast and mitochondrial DNA imprints reveal multiple lineages and complex biogeographic history for Douglas-fir

Xiao-Xin Wei & Jean Beaulieu & Damase P. Khasa & Jesús Vargas-Hernández & Javier López-Upton & Barry Jaquish & Jean Bousquet

Received: 6 December 2010 /Revised: 15 March 2011 /Accepted: 29 March 2011 /Published online: 21 April 2011 # Springer-Verlag 2011

Abstract The contemporary genetic structure of species cpDNA lineages corresponding to the Pacific Coast, the offers key imprints of how organisms responded to past Rocky Mountains, and Mexico were observed. The split geological and climatic events, which have played a crucial time of the two lineages from the Rockies lineage was role in shaping the current geographical distribution of dated back to 8.5 million years (Ma). The most recent north-temperate organisms.Inthisstudy,range-wide common ancestors of Mexican and coastal populations patterns of genetic variation were examined in Douglas-fir were estimated at 3.2 and 4.8 Ma, respectively. The (Pseudotsuga menziesii), a dominant forest tree species northern populations of once glaciated regions were distributed from Mexico to British Columbia in western characterized by a high level of genetic diversity, indicating North America. Two organelle DNA markers with con- a large zone of contact between ancestral lineages. A trasting modes of inheritance were genotyped for 613 possible northern refugium was also inferred. The Mexican individuals from 44 populations. Two mitotypes and 42 lineage, which appeared established by southward migration chlorotypes were recovered in this survey. Both genomes from the Rockies lineage, was characterized by the lowest showed significant population subdivision, indicative of genetic diversity but highest population differentiation. These limited gene flow through seeds and pollen. Three distinct results suggest that the effects of Quaternary climatic

Communicated by S. Aitken Electronic supplementary material The online version of this article (doi:10.1007/s11295-011-0392-4) contains supplementary material, which is available to authorized users. : X.-X. Wei (*) J. Vargas-Hernández J. López-Upton State Key Laboratory of Systematic and Evolutionary of Botany, Programa Forestal–Campus Montecillo, Colegio de Institute of Botany, Chinese Academy of Sciences, Postgraduados, km 36.5 Carretera México-Texcoco, 20 Nanxincun, Xiangshan, Texcoco, México C.P. 56230, Mexico Beijing 100093, China e-mail: [email protected]

X.-X. Wei : J. Beaulieu : D. P. Khasa : J. Bousquet Canada Research Chair in Forest and Environmental Genomics, Forest Research Centre and Institute for Systems and Integrative Biology, 1030 avenue de la Médecine, Université Laval, Québec, Québec G1V 0A6, Canada

J. Beaulieu B. Jaquish Natural Resources Canada, Canadian Forest Service, Canadian British Columbia Ministry of Forests and Range, Research and Wood Fibre Centre, 1055 du P.E.P.S., Knowledge Management Branch, Kalamalka Forestry Centre, P.O. Box 10380, Stn. Sainte-Foy, 3410 Reservoir Road, Québec, QC G1V 47C, Canada Vernon, BC V1B 2C7, Canada 1026 Tree Genetics & Genomes (2011) 7:1025–1040 oscillations on the population dynamics and genetic diversity America, a more limited number of studies have focused of Douglas-fir varied substantially across the latitudinal on the southern part of North America including Mexico, section. The results emphasize the pressing need for the (reviewed in Jaramillo-Correa et al. 2009), a region conservation of Mexican Douglas-fir. characterized by unusually high species diversity as well as geographical and geological complexity. This complexity Keywords Genetic diversity. Population structure . has been shown to promote vicariance, isolation, and Biogeographic history . Molecular clock . Cytoplasmic genetic drift, given that many tree species have retreated DNA . Douglas-fir to high altitudes with the Holocene warming (Jaramillo- Correa et al. 2006). These processes and their genetic consequences are quite different than those seen in the Introduction north, leading to drastic differences in the patterns of genetic diversity and population genetic structure in Global climate changes, in particular the climatic oscillations various parts of the continent (Jaramillo-Correa et al. of the Quaternary period, have been proposed as critical 2009). Hence, it would be of interest to investigate more factors in inducing speciation and shaping the contemporary species, in particular those with wide distributions encom- distribution of biological diversity in the northern hemisphere passing several regions and potential vicariance factors, to get (Comes and Kadereit 1998;Hewitt1996, 2000;Klickaand a broader picture of how the genetic architecture of wide- Zink 1997; Lemmon et al. 2007;Opgenoorthetal.2010; range species has been affected by past climate changes. Such Perron et al. 2000). In response to Quaternary expansions knowledge will contribute to our understanding of the and contractions of the glacial ice sheets, most surviving contemporary distribution of genetic diversity and assist in organisms either shifted their ranges to southern refugia designing more efficient conservation and sustainable during glacial cycles and recolonized northward following management plans at national and international levels. the receding ice sheets during interglacial cycles or they Douglas-fir (Pseudotsuga menziesii) is an ecologically experienced altitudinal shifts and range fragmentation and economically important coniferous tree species with a (Hewitt 2000; Jaramillo-Correa et al. 2009; Petit et al. broad latitudinal distribution in western North America. It 2003, 2008;Roweetal.2004). As a consequence of occupies a wide variety of climatic conditions from the successive founder events during postglacial colonization, maritime climate of the Pacific Northwest to the mild low genetic diversity in previously northern glaciated areas continental climates of the Rocky Mountains and the warm, and high diversity in the southern refugia are expected. dry climate of central Mexico. Two varieties are well Indeed, numerous genetic surveys in Europe and North recognized: coastal (P. menziesii var. menziesii (Mirb.) America revealed this pattern (e.g., Comes and Kadereit Franco) and interior, or Rocky Mountains, Douglas-fir (P. 1998;Hewitt1996, 1999, 2000; Ibrahim et al. 1996;Soltiset menziesii var. glauca (Beissn.) Franco). The native range of al. 1997; Taberlet et al. 1998). the coastal variety extends from central British Columbia However, this general pattern predicted under simple (55° N) south along the Pacific Coast Range into central models of colonization, i.e., a gradual decrease in diversity California to a latitude of 34°55′ N, whereas the interior away from the source populations (Hewitt 1996, 2000), is variety extends from central British Columbia (55° N) now increasingly challenged by genetic evidences in both south along the Rocky Mountains to a latitude of 17° N in Europe and North America, especially from tree species. In the mountains of Oaxaca in Southern Mexico where it has a Europe, it was found that the genetically most diverse patchier distribution (Debreczy and Racz 1995; Hermann populations of tree and shrub species were located in the and Lavender 1999; Wright et al. 1971). north rather than in the south of the main mountain ranges Li and Adams (1989) contributed the first range-wide (Petit et al. 2003). Such contrasting patterns of diversity molecular investigation of population structure in Douglas-fir were also observed in North America, where zones of based on allozymes using an intensive sampling of distinct or higher diversity in the north were inferred as populations from Canada and the USA and from two footprints of cryptic refugia at high latitudes (Anderson et populations from Mexico. Their study revealed a large al. 2006; Gérardi et al. 2010; Godbout et al. 2008, 2010; genetic differentiation between the coastal and interior Jaramillo-Correa et al. 2004; Provan and Bennett 2008), or varieties but provided less information on the evolution secondary contact on once glaciated regions between of Mexican Douglas-fir. Recently, based on chloroplast genetically distinct glacial lineages (Godbout et al. 2005, and mitochondrial DNA markers, Gugger et al. (2010a) 2008; Jaramillo-Correa et al. 2004, 2009; Naydenov et al. investigated the geographic structure of Douglas-fir 2007; Walter and Epperson 2005). distributed in the USA and Canada. Following this study, In addition, contrary to the intensive studies of the they investigated the phylogeographic history of Mexican postglacial history of plant species in northern North Douglas-fir separately (Gugger et al. 2010b). The divergence Tree Genetics & Genomes (2011) 7:1025–1040 1027 times between major groups were estimated and a number of two species in Asia (Farjon 1990)) were used as outgroups in refugia were inferred in their studies. Given the genetic the cpDNA haplotype network and Bayesian phylogenetic complexity of the species, the present study aimed at analyses (see below), respectively, because of their sister investigating the genetic diversity of Douglas-fir and its relationships with P. menziesii and with North American geographical structure throughout its entire natural range species, respectively (Wei et al. 2010). Eight individuals of from a mixed population genetics and phylogenetic perspec- P. macrocarpa from California (USA) and two individuals of tive. Such an approach should allow us to identify the P. sinensis from Sichuan (China) were used for the analyses. origin of the species, infer the different historical events The locations and numbers of individuals of each Douglas- and geographical factors that have modeled the present fir population are shown in Table 1. population structure of the species, and test the hypothesis of different responses of the species to past events DNA extraction, PCR, and sequencing across the entire latitudinal gradient. In this study, newly identified molecular markers from Needles from adult trees (for Canadian and most of the both the chloroplast and mitochondrial genomes were US populations) and whole seeds (for cpDNA) or employed. As a member of the pine family (Pinaceae), the megagametophytes (for mtDNA; for Mexican popula- cytoplasmic DNA of Pseudotsuga follows contrasting tions and three US populations) were used for DNA modes of inheritance, chloroplast DNA (cpDNA) being extraction. Many of these samples were collected in a paternally inherited and mitochondrial DNA (mtDNA) 30-year-old range-wide provenance test growing near being maternally inherited (Mogensen 1996). Since colo- Vernon, British Columbia (BC Ministry of Forests). Total nization of new habitats occurs through seeds, mtDNA DNA was extracted using the NucleoSpin® 96 Plant II markers provide information on past changes in species (MACHEREY-NAGEL) and DNeasy Plant Mini Kit distribution that is essentially unaffected by subsequent (QIAGEN) for needles and seeds, respectively. DNA pollen movements (Petit et al. 1993). Thus, in the concentration was measured with a GeneSpec spectropho- Pinaceae, the effects of the differential levels of gene flow tometer (MiraiBio). Primers nad7-1 F and nad7-1R between pollen and seed can be directly compared, which (Jaramillo-Correa et al. 2004)andtrnfM and trnS(Shaw should enhance the inference of phylogeographic structure et al. 2005) were used to amplify the first intron of the and identify the underlying causes and evolutionary mitochondrial gene nad7 and the chloroplast DNA trnfM– processes. trnS intergenic spacer, respectively. Polymerase chain reaction (PCR) amplifications were carried out in a volume of 25 μL, containing 10 ng of genomic DNA, 6.25 μMof

Materials and methods each primer, 0.2 mM of each dNTP, 1.5 mM MgCl2,and 0.75 U of Platinum Taq polymerase (Invitrogen). PCR Population sampling amplification was performed in an Eppendorf Mastercycler with the following program: 36 cycles of 20 S at 94°C, 20 S A total of 613 individuals from 44 populations were at 52°C (cpDNA) or 56°C (mtDNA) and 1 min 30 S at 72°C sampled along a latitudinal gradient covering the entire followed by a final extension step of 6 min at 72°C. PCR natural range of Douglas-fir. Except for eight populations, products were separated by 1.5% agarose gel electrophore- which were represented by six to 13 individuals, 15 sis. Direct sequencing of both strands was conducted with an individuals were analyzed for each population (Table 1). automated 3730xl DNA sequencer (Applied Biosystems) Considering the high geographical heterogeneity and com- using the amplification primers and BigDye Terminator kit plexity of North America as well as the genetic disconti- (Applied Biosystems). nuities reported in Douglas-fir (Li and Adams 1989)and A discovery panel composed of 40 individuals from other species in western North America (e.g., Godbout et al. eight populations which represented the major regions of 2008; Jaramillo-Correa et al. 2009; Soltis et al. 1997), we Douglas-fir was initially used for the screening of divided the entire distribution of Douglas-fir into six molecular markers. For the nad7intron1,onlytwo geographical regions (Fig. 1a, b), including two regions for mitotypes were found in the discovery panel. We then the coastal variety (Coastal-north and Coastal-south) and sequenced 225 individuals from 15 other populations and four for the interior variety (Rockies-north, Rockies- confirmed the polymorphisms detected in the discovery transition, Rockies-south, and Mexico). Pseudotsuga macro- panel. Because the two mitotypes could be revealed easily carpa (Vasey) Mayr, one of the only two species of the by DNA restriction with the endonuclease Bsl I(Table2), genus in North America and with a limited distribution in the rest of the individuals were determined by restriction southern California (Flora of North America at efloras.org), enzyme digestion using Bsl I. All sequences were and the Asian species Pseudotsuga sinensis Dode (one of the deposited in Genbank (see “Results” section). 1028 Tree Genetics & Genomes (2011) 7:1025–1040

Fig. 1 Geographical distribution of mitotypes and chlorotypes in Douglas-fir populations. The green shade indicates the current distribution of Douglas- fir. a Mitotype distribution; white circles indicate mitotype D and blue circles indicate mitotype I. Population names are showed in the circles in reference to Table 1. The Roman numbers indicate the six geographical subdivisions delineated. I Coastal-north, II Coastal-south, III Rockies-north, IV Rockies- transition, V Rockies-south, VI Mexico. b Chlorotype distribution; the distribution of populations and the geographical subdivisions correspond to Fig. 1a

Data analysis were further investigated by Bayesian phylogenetic analysis using P. sinensis as outgroup. The analysis was conducted Sequence alignments were made with CLUSTAL X with MrBayes 3.1 (Ronquist and Huelsenbeck 2003)using (Thompson et al. 1997) and refined manually. Haplotype the K81uf model of nucleotide substitution, which was diversity (Hd) and nucleotide diversity (π) were estimated determined by the AIC test with Modeltest 3.07 (Posada and using DnaSp5.10 (Librado and Rozas 2009). The program Crandall 1998). One cold and three incrementally heated PERMUTE (available at http://www.pierroton.inra.fr/ Markov Chain Monte Carlo (MCMC) chains were run for genetics/labo/software/; Pons and Petit 1996) was applied 1,000,000 cycles. Trees were sampled every 100 genera- to calculate the average gene diversity within populations tions. The 50% majority rule consensus tree was obtained

(HS), total gene diversity (HT), as well as average gene based on the trees sampled after generation 30,000. diversity among-population differentiation (GST) and the A Bayesian approach was used to estimate the diver- degree of population subdivision at the nucleotide level gence time of each major group (node) in the cpDNA

(NST). The occurrence of significant phylogeographical phylogeny using the program BEAST v1.5.3 (Drummond structure was inferred by testing if GST and NST were and Rambaut 2007). The dataset was analyzed using a significantly different using PERMUT with 1,000 permuta- constant size tree prior under the HKY nucleotide tions. Genetic structure was further examined by hierarchical substitution model and gamma-distributed rate heterogeneity. analysis of molecular variance (AMOVA) with pairwise The oldest Pseudotsuga fossil record so far found in the early differences and haplotype frequencies using ARLEQUIN Oligocene lowland Willamette and Rujada paleofloras of 3.11 (Excoffier et al. 2005). Estimates of genetic differenti- west central Oregon (Lakhanpal 1958) could be dated back ation among populations and regions were calculated and the to ∼32 million years (Ma) ago (Schorn 1994). Therefore, the significance was tested using 10,000 permutations. A age of the most recent common ancestor (MRCA) of cpDNA haplotype network was constructed with NET- Pseudotsuga was fixed at 32 Ma (node E in Fig. 3). A WORK 4.516 (http://www.fluxus-engineering.com)bythe random local clock model was used for the estimation. median-joining network algorithm (Bandelt et al. 1999) MCMC chains were run for 10×106 generations, sampling using P. macrocarpa as outgroup. The relationships among every 1,000th generation. BEAST output was analyzed using the cpDNA haplotypes of Douglas-fir and P. macrocarpa TRACER 1.5 (http://beast.bio.ed.ac.uk/Tracer). Tree Genetics & Genomes (2011) 7:1025–1040 1029

Table 1 Population geographical subdivisions and locations of Douglas-fir sampled in this study and mitotype and chlorotype distributions

Region Population Locationa Latitude (oN) Longitude (oW) Altitude (m) Sample size Number of Types and numbers mitotypes of chlorotypes

Coastal- 1142 Lonesome Lake, BC 52.22 125.70 475 15 0 15 H1:6, H5:6, H11:1, north H15:1, H19:1 1115 D’arcy, BC 50.55 122.50 270 15 0 15 H1:9, H5:3, H11:1, H18:2 1101 Alexander, BC 49.70 121.40 170 15 0 15 H1:1, H2:1, H3:1, H4:2, H5:6, H11:2, H15:1, H21:1 161 Twisp, WA 48.38 120.40 795 15 0 15 H1:1, H5:1, H11:1, H12:1, H24:6, H29:5 160 Skykomish, WA 47.70 121.33 305 15 0 15 H1:3, H5:7, H11:4, H15:1 185 Rimrock, WA 46.67 121.03 760 15 0 15 H1:5, H5:5, H11:1, H14:1, H18:1, H21:1, H24:1 Coastal- 229 Oakridge, OR 43.90 122.37 885 15 3 12 H1:3, H5:4, H11:1, H15:2, south H18:1, H20:1, H27:2, H29:1 228 Ashland, OR 42.08 122.82 1,495 15 0 15 H1:7, H5:4, H11:3, H21:1 197 Dunsmuir, CA 41.20 122.30 1,005 15 0 15 H1:2, H5:8, H6:1, H11:1, H15:2, H16:1 200 Burney, CA 41.08 121.65 1,020 15 0 15 H2:1, H5:9, H7:1, H8:1, H11:1, H17:1, H21:1 212 Wildwood,CA 40.38 123.00 1,190 15 0 15 H5:8, H9:1, H10:1, H11:3, H21:2 Rockies- 1191 Takla Lake,BC 55.33 125.83 730 15 0 15 H5:2, H11:4, H27:2, north H29:6, H33:1 194 Mcleod’s Lake, BC 54.83 122.83 925 15 15 0 H11:7, H24:1, H27:1, H29:6 191 Ft. St. James, BC 54.50 124.25 960 15 15 0 H1:5, H24:2, H27:2, H29:3, H30:1, H33:2 193 Tabor Mtn., BC 53.95 122.47 840 15 15 0 H1:1, H20:1, H24:7, H27:1, H29:3, H33:2 192 West Lake, BC 53.67 122.98 840 13 13 0 H1:5, H11:2, H15:1, H24:4, H27:1 1138 Kinney Lake, BC 53.07 119.17 1,070 15 6 9 H1:2, H5:1, H11:1, H24:4, H27:6, H29:1 172 Clearwater, BC 51.65 120.00 460 15 12 3 H1:1, H18:1, H24:6, H27:2, H29:4, H33:1 1141 Lac Des Roches, BC 51.47 120.55 1,130 15 0 15 H1:4, H5:1, H11:1, H24:1, H27:1, H29:5, H30:1, H33:1 189 Golden,BC 51.23 117.00 870 15 15 0 H5:1, H24:1, H27:4, H29:8, H33:1 152 Revelstoke, BC 51.00 118.22 610 11 11 0 H5:1, H20:1, H24:4, H27:1, H29:3, H33:1 157 Monte Ck, BC 50.62 119.90 640 15 0 15 H1:2, H11:1, H24:8, H27:3, H29:1 1104 Arrow lake, BC 50.20 117.77 490 15 15 0 H1:1, H11:1, H24:9, H29:4 1130 Jaffray, BC 49.38 115.33 870 15 15 0 H22:1, H24:5, H27:1, H29:8 147 Republic, WA 48.60 118.73 730 15 15 0 H24:6, H27:5, H29:4 8 Bonner, ID 48.50 116.00 15 12 3 H1:1, H5:1, H15:3, H24:3, H29:5, H30:1, H32:1 Rockies- 43 Horse Creek, ID 43.53 114.41 2,400 15 15 0 H22:9, H24:1, H27:1, transition H29:3, H31:1 44 Pryor Mtns, OR 45.17 108.27 2,280 15 15 0 H22:3, H24:9, H29:3 45 Pinus Creek, MT 44.45 118.20 1,600 15 15 0 H1:6, H13:1, H23:1, H24:3, H27:1, H29:3 Rockies- 4 Jefferson, CO 39.55 105.25 2,290 8 8 0 H24:1, H27:6, H28:1 south 251 Coaldale, CO 38.33 103.83 2,285 9 9 0 H22:1, H24:2, H26:1, H27:5 266 Pagosa, CO 37.25 106.87 2,440 15 15 0 H22:2, H24:9, H27:4 250 Penasco, NM 36.07 105.63 2,930 15 15 0 H22:1, H24:8, H25:1, H27:5 1030 Tree Genetics & Genomes (2011) 7:1025–1040

Table 1 (continued)

Region Population Locationa Latitude (oN) Longitude (oW) Altitude (m) Sample size Number of Types and numbers mitotypes of chlorotypes

261 Honeymoon, AZ 33.30 109.02 2,745 15 15 0 H22:1, H24:8, H27:6 260 Cloudcroft, NM 32.92 105.50 2,440 15 15 0 H24:12, H27:3 Mexico 1 Mohinora, Chih 25.96 107.04 3,000 15 15 0 H24:4, H34:9, H36:2 4 Coahuital,Dgo 23.46 104.80 2,700 10 10 0 H34:4, H37:1, H42:5 6 Santa Anita, Coah 25.45 100.57 2,520 15 15 0 H27:1, H34:9, H36:4, H42:1 12 Peña Nevada, NL 23.82 99.86 2,890 15 15 0 H34:1, H36:12, H41:2 13 Pinal de Amoles, Qro 21.16 99.69 3,000 7 7 0 H34:1, H38:1, H39:4, H40:1 16 Presa Jaramillo, Hgo 20.17 98.73 2,765 15 15 0 H34:3, H36:12 17 Tlaxco,Tlax 19.65 98.05 2,960 15 15 0 H35:11, H36:4 21 Apizaquito, Puebla 19.20 97.31 3,100 6 6 0 H42:6 22 Ixtepeji Peña Prieta, Oax 17.16 96.64 2,700 9 9 0 H36:9 a List of abbreviations in alphabetical order: AZ Arizona, BC British Columbia, CA California, Chih Chihuahua, CO Colorado, Coah Coahuila, Dgo Durango, Hgo Hidalgo, ID Idaho, MT Montana, NL Nuevo León, NM New Mexico, Oax Oaxaca, OR Oregon, Qro Querétaro, Tlax Tlaxcala, WA Washington Results 1138, 172, and 8 (Table 1, Fig. 1a). All eight individuals of P. macrocarpa, the sister species of Douglas-fir in North Mitotype distribution America, had an identical sequence to that of mitotype D in the same region of the nad7 intron 1 (GenBank accession The sequences of nad7 intron 1 in Douglas-fir ranged from number: HM030968). The sequence of an Asian species, P. 796 to 810 bp in size. One 15-bp and three 1-bp indels, and sinensis, was also highly similar to this mitotype (GenBank five substitutions (single-nucleotide polymorphisms) were accession number: HM030969). Comparison with the P. found in the aligned sequences. All these polymorphisms macrocarpa and P. sinensis sequences indicated that were located in the region between nucleotides 576–622 mitotype D was likely the ancestral type and mitotype I (Table 2). The three 1-bp indels and all substitution was a derivative, given its 15-bp apomorphic insertion that polymorphisms were completely linked with the 15-bp was unique to Douglas-fir and restricted to certain regions. indel and therefore, only two mitotypes (mitotype I and mitotype D, GenBank accession numbers: HM030966 and Chlorotype distribution and genealogy HM030967) were identified throughout all the individuals analyzed (Table 1). With the exception of three individuals In Douglas-fir, 14 indels and 13 substitutions were detected from population 229 which possessed mitotype I, all coastal from the cpDNA trnfM–trnS intergenic spacer (956– variety populations were fixed for mitotype D, whereas all 963 bp), which resulted in 42 chlorotypes (GenBank interior variety populations from Mexico, Rockies-south accession numbers: HM030924–HM030965, Table 1, and Rockies-transition zones harbored mitotype I (Table 1, Fig. 1b). Two substitutions at the nucleotide positions 39 Fig. 1a). The mitotype distribution in the Rockies-north was and 52 delineated three cpDNA groups (Table 3), which more complex. Nine out of 15 populations were fixed for corresponded to three major lineages (coastal, Rockies, and mitotype I, along with mitotype D in population 1191, Mexican) shown in Fig. 2. The coastal lineage was 1141, and 157. Both mitotypes were present in population composed by chlorotypes 1–21, which harbored an A

Table 2 Variable sites along the sequence of the mtDNA nad7 intron 1 for Douglas-fir (Pseudotsuga menziesii), P. macrocarpa, and P. sinensis

The sequence underlined contains the recognition sites for Bsl I Tree Genetics & Genomes (2011) 7:1025–1040 1031

Table 3 Variable nucleotide sites of the cpDNA trnfM-trnS intergenic spacer in Douglas-fir and Pseudotsuga macrocarpa

a A dot indicates that the character states are the same as in haplotype 1 and a dash indicates an alignment gap 1032 Tree Genetics & Genomes (2011) 7:1025–1040

Fig. 2 Evolutionary relationships among chlorotypes of the cpDNA intergenic spacer trnfM–trnS of Douglas-fir. The size of circle is proportional to the relative frequency of the chlorotype instead of a C in the chlorotypes 22–42 at the nucleotide and six, respectively) and both had three private chlorotypes. position 52 (Table 3). The Rockies lineage was composed by Mexican populations showed a moderate level of chlorotype chlorotypes 22–33, which could be distinguished from the variation but nine out of 11 chlorotypes were endemic to this Mexican lineage (chlorotypes 34–42) by an A instead of a T region. Additionally, in Mexico, the northern populations (1, at position 39 (Table 3). The coastal lineage occurred 4, 6, 12, and 13) harbored three or four chlorotypes. By predominantly in the coastal variety populations, whereas comparison, only two chlorotypes occurred in southern the Rockies lineage was predominant in the interior pop- populations 16 and 17 and one chlorotype in the southernmost ulations from the Rocky Mountains (Fig. 1b). All Mexican populations 21 and 22. Chlorotype 36 was the most populations were fixed for the chlorotypes found unique to widespread, occurring in six out of nine populations from the Mexican lineage with the exception of four individuals north to south in Mexico. from population 1 carrying chlorotype 24 and one individual The Bayesian phylogenetic analysis of the 42 cpDNA from population 6 carrying chlorotype 27, both of these sequences generated a topology that included two mono- chlorotypes being representative of the Rockies lineage. phyletic groups corresponding to the coastal and Mexican ThefivepredominantchlorotypesinCanadaandtheUSA, lineages (Fig. 3). The Rockies lineage was paraphyletic and i.e., chlorotypes 1, 5, 24, 27, and 29, had the highest frequency consisted of two subgroups. Chlorotypes 29–33 were throughout all populations and each of these chlorotypes placed at the basal position and chlorotypes 22–28 diverged occurred in three or more geographic regions (Table 1). The next (Fig. 3). The basal placement of the Rockies lineage in populations from the Coastal-south region harbored 17 both the chlorotype network and the Bayesian cpDNA tree chlorotypes, of which seven were endemic. The Coastal- indicated that this lineage is ancestral to the others. On the north and Rockies-north regions came up next, containing 14 contrary, the mtDNA data showed that the coastal lineage and 13 chlorotypes, of which five and three were endemic to was most likely ancestral since mitotype D, which was their region, respectively. The Rockies-transition and Rockies- shared with the outgroup P. macrocarpa,washighly south regions had the smallest numbers of chlorotypes (eight prevalent in the coastal variety. Tree Genetics & Genomes (2011) 7:1025–1040 1033

Molecular dating with BEAST based on the cpDNA population differentiation was observed, as indicated by the phylogeny indicated that the MRCA for each of the coastal values of GST=0.289 and NST=0.452 (Table 4). The and Mexican lineages was dated at 4.8 Ma (95% highest permutation test showed that GST and NST were significantly posterior density (HPD) interval, 9.7–1.2 Ma; Fig. 3, node different from each other (NST>GST, P<0.01), indicating A) and 3.2 Ma (95% HPD interval, 7.0–0.5 Ma; Fig. 3, significant phylogeographic structure. By comparison, at node B), respectively. The most recent common ancestor of the mitotype level over all populations, the total gene all Douglas-fir was dated back to 9.0 Ma (95% HPD diversity and within-population gene diversity were interval, 17.5–2.8 Ma; Fig. 3, node D), and the split time of relatively low (0.480 and 0.038, respectively) but a high the coastal and Mexican lineages from the Rocky lineage GST value (0.922) was estimated. When estimating chlorotype was estimated at 8.5 Ma (95% HPD interval, 16.5–2.4 Ma; variation based on the six previously defined large Fig. 3, node C). The estimated nucleotide substitution rate regions, the Rockies-north and Rockies-south regions −9 for the cpDNA trnfM-trnS intergenic region was 4.6×10 showed the highest and lowest π and Hd, respectively per site per year. (Table 4). For the Coastal-north region, the inclusion or exclusion of population 161 in the calculation caused

Genetic diversity and population structure quite different results. All regional values of HT, HS, π, and Hd were slightly lower, and indices of population The HT estimated at the chlorotype level over all populations, differentiation decreased markedly when population 161 was 0.908 and the average HS was 0.646. Sizeable was excluded. This was caused by the high frequency of

Fig. 3 Bayesian 50% consensus tree for chlorotypes of the cpDNA intergenic spacer trnfM–trnS in Douglas-fir. Numbers above branches indicate Bayesian posterior probabilities. Bold letters under branches are node names. The molecular clock time estimates are denoted by italic numbers above branches. The colors of chlorotype names correspond to Fig. 1b (asterisk indicates the 95% highest posterior density (HPD) interval: A 9.7–1.2 Ma, B 7.0–0.5 Ma, C 16.5–2.4 Ma, D is 17.5–2.8 Ma, E fixed age of 32 Ma based on the fossil record) 1034 Tree Genetics & Genomes (2011) 7:1025–1040

Table 4 Estimates of average gene diversity within populations (HS), mean±SE in parentheses) as well as nucleotide diversity per site (π) total gene diversity (HT), among-population differentiation (GST), and and haplotype diversity (Hd) based on the cpDNA intergenic spacer the degree of population subdivision at the nucleotide level (NST; trnfM-trnS sequence in Douglas-fir

Region HS HT GST NST π Hd

Coastal-north 0.743 (0.0321) 0.822 (0.0473) 0.097 (0.0490) 0.193 (0.0743)* 0.00168 0.810 Coastal-northa 0.739 (0.0390) 0.763 (0.0337) 0.031 (0.0386) −0.013 (0.0294) 0.00113 0.758 Coastal-south 0.735 (0.0413) 0.776 (0.0414) 0.053 (0.0246) 0.035 (NC) 0.00111 0.768 Rockies-north 0.750 (0.0213) 0.815 (0.0185) 0.079 (0.0193) 0.076 (0.0257) 0.00181 0.810 Rockies-transition 0.676 (0.0625) 0.821 (0.0638) 0.214 (NC) 0.280 (NC) 0.00147 0.802 Rockies-south 0.553 (0.0523) 0.620 (0.0318) 0.107 (0.0831) 0.107 (0.1064) 0.00011 0.591 Mexico 0.408 (0.0881) 0.826 (0.0633) 0.506 (0.1005) 0.528 (0.0876) 0.00088 0.755 North Americab 0.646 (0.0295) 0.908 (0.0116) 0.289 (0.0363) 0.452 (0.0463)* 0.00232 0.897

NC not calculated a Excluding population 161 (see “Results” section) b Conducted on overall populations *P<0.05, significant chlorotype 24 (40%) and chlorotype 29 (33%) in this that 49.8% and 31.5% of the total chlorotype variance population, which were the most frequent ones in the were assigned between the two varieties and within populations from the Rocky Mountains. populations, respectively (Table 5). Mitotype variation The non-hierarchical AMOVA showed that the chlorotype mostly occurred between the coastal and interior varieties diversity among populations was only slightly higher (55.2%) (76.4% of the total variance), and diversity within popula- than that within populations (44.8%; Table 5). By tions accounted for only 4.3% of the total variance (Table 5). comparison, most of the mitotype diversity (92.1%) was A similar distribution pattern of variation was detected found to reside among populations, and the within- when the range of Douglas-fir was further partitioned into population component only accounted for 7.9% of the six regions (Table 5). The non-hierarchical AMOVA analysis mitotype variation. The hierarchical AMOVA revealed of each region showed that more than 71% of cpDNA

Table 5 Non-hierarchical and hierarchical analysis of molecular variance (AMOVA) of populations and regions of Douglas-fir based on chlorotype frequencies for the cpDNA trnfM-trnS intergenic spacer and mitotype frequencies for the mtDNA nad7 intron 1

Source of variation Decrees of Sum of squares Variance Percent of Fixation index freedom components variation

cpDNA mtDNA cpDNA mtDNA cpDNA mtDNA cpDNA mtDNA cpDNA mtDNA

North America

Among populations 43 43 397.7 665.8 0.628 1.105 55.2 92.1 FST=0.552 FST=0.921 Within populations 569 569 289.5 54.0 0.509 0.095 44.8 7.9 Total 612 612 687.2 719.8 1.137 1.200 Divided into two groups to the distribution of varieties (i.e., coastal and interior)

Among varieties 1 1 199.5 413.2 0.806 1.687 49.8 76.4 FCT=0.498 FCT=0.764

Among populations 42 42 198.2 252.6 0.303 0.426 18.7 19.3 FST=0.686 FST=0.957 within varieties Within populations 569 569 289.5 54.0 0.509 0.095 31.5 4.3 FSC=0.373 FSC=0.8l8 Total 612 612 687.2 719.8 1.618 2.207 Divided into six region (Coastal-north and -south, Rockies-north, -south, and -transition, Mexico)

Among regions 5 5 306.9 469.1 0.6 13 0.920 48.7 66.6 FCT=0.487 FCT=0.666

Among populations 38 38 90.8 196.7 0.136 0.367 10.8 26.5 FST=0.595 FST=0.931 within regions Within populations 569 569 289.5 54.0 0.509 0.095 40.5 6.9 FSC=0.210 FSC=0.794 Total 612 612 687.2 719.8 1.257 1.381 Tree Genetics & Genomes (2011) 7:1025–1040 1035 variation resided within populations in all subdivisions reductions in genetic variability in the recolonized areas except Mexico, where only 39.6% of the total variation given that the leading edge colonizing population may have resided within populations (Table S1). gone through a series of bottlenecks (Hewitt 1996; Soltis et al. 1997). However, in Douglas-fir, the genetic diversity

indices (HS, HT, Hd,andπ) suggested that northern Discussion populations of the coastal variety (Coastal-north region) had similar levels of chlorotype variation as southern Population genetic structure populations (Coastal-south region), whereas the genetic diversity of the northern populations (Rockies-north In the present study, moderate to high levels of chlorotype region) of the interior variety was even significantly and mitotype differentiation were observed in Douglas-fir, higher than that of the populations from the Rockies- as recently reported by Gugger et al. (2010a, b) using a southregion(Fig.1,Table4). No significant loss of different set of markers and populations. The level of diversity with latitude was also reported by Gugger et al. mitotype differentiation among all populations was very (2010a). This pattern might be interpreted as the result of high (92.1%), with much of the differentiation accounted for secondary contact between differentiated glacial lineages by varieties (76.4%). These trends indicate very limited gene migrating from the south and the subsequent admixture of flow by seeds between varieties. In contrast, the level of the two divergent cpDNA lineages, i.e., coastal and chlorotype differentiation among populations was lower than Rockies lineages. that found for mtDNA, as 55.2% of chlorotype variation was The presumed recolonization routes to the north can be detected among all populations. However, this value is still inferred by the geographical distribution of the two nad7-1 notably high for pollen-dispersed cpDNA in the Pinaceae mitotypes (Fig. 1a), which are representative of seed (Petit et al. 2005). It is likely inflated given the pronounced dispersal only. The first lineage carrying mitotype D would varietal structure of Douglas-fir. In fact, AMOVA revealed originate from a coastal refugium, whereas the other lineage that only 18.7% of chlorotype variation occurred among carrying mitotype I would derive from a refugium in the populations within varieties. This value is more similar to the Rocky Mountains. The hypothesis of subsequent admixture average of 16.5% among-population variation reported for of these distinct migration routes is concordant with the cpDNA in conifers (Petit et al. 2005). Coupled with the high distribution pattern of cpDNA trnfM-trnShaplotypes level of chlorotype variation seen among populations at the (Figs. 1b and 2). Chlorotypes 1, 5, and 11, which were range-wide scale, the significantly higher value of NST over prevalent in populations from the coastal variety, also GST noted for cpDNA at the range level further reflected a occurred at a high frequency of 32%, 10%, and 47%, strong phylogeographic structure. However, less or no respectively, in populations from the Rockies-north region, phylogeographic structure was detected within regions. in particular in some populations along the Cascade ranges These various trends indicate that partial reproductive and in the interior of British Columbia where the two isolation between the two varieties likely contributed much varieties appear to have come into contact. Such large to the high level of among-population differentiation and zones of contact on once glaciated lands between phylogeographic structure of cpDNA at the range-wide genetically distinct glacial lineages have been reported scale. Indeed, when considering a grouping that takes into for other conifers (e.g., Picea mariana, Pinus banksiana, account only the varietal structure (coastal and interior), an Pinus sylvestris) with large natural longitudinal distributions

FCT differentiation estimate of 0.498 was obtained using in North America or Eurasia (Godbout et al. 2005;Jaramillo- AMOVA. The amplitude of this cpDNA differentiation Correa et al. 2004, 2009;Naydenovetal.2007), including estimate indicates that gene flow via pollen is much reduced lodgepole pine (Pinus contorta) in the same area as that between the two varieties of Douglas-fir, but substantial detected for Douglas-fir (Godbout et al. 2008). Thus, within varieties and regions. contrary to common agreement, the present results on Douglas-fir indicate that recolonized unglaciated lands can Coastal and Rockies lineages and their contact in the north carry genetically highly diverse populations of tree species (Jaramillo-Correa et al. 2009). The Quaternary glacial and interglacial cycles, especially In addition, all individuals of populations 161, 1141, the Last Glacial Maximum which covered essentially all of 172, and 157 from the contact zone were fixed for mitotype Canada and further south to 40° N, induced great changes D, which is typical of the coastal lineage, with the in the distribution of species in North America: most tree exception of three individuals from population 172 which species from the north were forced to move or survive in harbored mitotype I. This pattern would be indicative of the southern refugia (Jaramillo-Correa et al. 2009). Generally, early colonization of the area by the coastal lineage. Almost range expansions out of refugia are expected to cause half (47% in population 1141) or over half of the 1036 Tree Genetics & Genomes (2011) 7:1025–1040 individuals (73% in population 161, 80% in population of Mexico, all other subdivisions were characterized by 157, 67% in population 172) from these populations higher level of cpDNA variation within populations (>71%, harbored chlorotypes 24, 27, and 29, three predominant Table S1) than among populations. In sharp contrast, the chlorotypes typical of the Rocky Mountains. This inter- Mexican populations were much more differentiated for lineage genetic composition within the same individuals cpDNA, given the higher level of variation among represents new and likely recent organelle genome populations (60.4%, Table S1) than within populations assemblages; it implies active interbreeding between the (39.6%). Substantial population differentiation in Mexico is two ancestral lineages in the contact zone and most also indicated by the strikingly high cpDNA GST value, to likely, the capture of coastal mtDNA by the invading cpDNA which corresponded the lowest level of genetic diversity Rockies lineage. Such new postglacial interlineage genome within populations (Table 4). Population isolation is usually assemblages (mtDNA/cpDNA) at the intraspecific level accompanied by evolutionary forces such as genetic drift have also been reported recently in black spruce (P. and inbreeding, leading to decreased variation within mariana), similarly in a zone of contact between two populations and increased variation among populations genetically distinct glacial lineages in western Canada (Ledig et al. 1997). Indeed, the Mexican natural distribution (Gérardi et al. 2010). The adaptive implications of such of Douglas-fir is spatially restricted; the populations are genomic intermixing are unclear. fragmented and found in heterogeneous environments at high altitudes. Such a pattern would limit the opportunities A possible northern refugium for gene exchange among populations and result in genetic pauperization (Jaramillo-Correa et al. 2006). A key signature of glacial refugia is that they might harbor a In particular, the southern populations of Mexican number of private alleles or haplotypes (e.g., Anderson et al. Douglas-fir have been reported as isolated, with small census 2006; Gérardi et al. 2010; Godbout et al. 2008, 2010). In the size and low tree density in nature (Mapula-Larreta et al. present study, although only chlorotype 33 was found 2007). This observation is corroborated by the trend detected endemic to the zone of contact in central and northern British in the present study of decreasing numbers of chlorotypes Columbia, its unique and broad presence (Table 1)mayimply from north to south in Mexican populations. Given the high the existence of a genetically distinct northern refugium. The genetic differentiation and the unique chlorotypes observed existence of such northwestern refugia, such as in Alaska or in Mexican populations (Table 3), distinct conservation Yukon, has been inferred or suggested for several conifers in strategies should be considered for Mexican Douglas-fir, in the region, such as white spruce (Picea glauca; Anderson et particular for the southern populations which appear al. 2006), lodgepole pine (Godbout et al. 2008; Strong 2010), genetically more depauperated at the cpDNA level. and black spruce (Gérardi et al. 2010). Similar genetic patterns have been reported in other Other rare chlorotypes were observed in British Columbia, Mexican conifers (Jaramillo-Correa et al. 2006;Lediget closer to the coast (e.g., chlorotypes 4 and 19 in populations al. 1997) and more recently for Mexican Douglas-fir 1101 and 1142, respectively; Table 1). However, these populations (Gugger et al. 2010b). As such, we may chlorotypes would not be indicative of genetically distinct conclude that plants with similar life history, demography, refugia. Contrary to the widely distributed chlorotype 33, and reproductive system are likely to have experienced which was found at the tip of a branch in the chlorotype similar genetic consequences. network (Fig. 2), these two chlorotypes were at low frequency with private geographic distribution, and they Inferring the biogeographic history of Douglas-fir were found at intermediate positions in the network (Fig. 2). These features are likely indicative of recent intragenic The present divergence time estimates based on the recombination (Jaramillo-Correa and Bousquet 2005), which present cpDNA trnfM–trnS dataset indicated that modern has also been previously inferred for cpDNA microsatellites Douglas-fir shared a common ancestor at 9.0 Ma (95% in P. contorta (Marshall et al. 2001). Further examination is HPD interval, 17.5–2.8 Ma). This ancestor was likely needed to investigate the likelihood of this phenomenum and typical of the interior Rocky Mountains lineage, which its evolutionary consequences. appears paraphyletic. Almost simultaneous splits of the coastal and Mexican lineages from the Rockies lineage Different population dynamics in Mexico would have occurred soon after around 8.5 Ma (95% HPD interval, 16.5–2.4 Ma; Fig. 3). There was a large interval The AMOVA revealed that the population genetic structure between the split time (8.5 Ma) and the dates for the two differed substantially among the six regions analyzed, most recent common ancestors of the coastal and Mexican Coastal-north, Coastal-south, Rockies-north, Rockies- lineages (4.8 and 3.1 Ma, respectively; Fig. 3). It is likely transition, Rockies-south, and Mexico. With the exception that the diversification of each lineage had occurred Tree Genetics & Genomes (2011) 7:1025–1040 1037 during this period but resulted in evolutionary dead ends. speciation as a major cause for the birth of new species Our estimates are consistent with the hypothesis that the in the Pinaceae (Bouillé et al. 2011;Perronetal.2000). coastal and interior varieties have existed in the Miocene, Thus, the geographically well-defined major intraspecific as proposed by Critchfield (1984) based on paleobotanical lineages and varieties of Douglas-fir likely reflect an evidence. The present estimates are also congruent with intermediate stage of speciation potentially leading towards the Neogene origin of the cool-to-cold-temperate plants in the formation of new species. Mexico (Graham 1999a). As for the Mexican Douglas-fir lineage, Graham (1995) However, these estimates are quite earlier than the proposed early on that most of the modern tree genera recently reported estimate of 2.11 Ma (4.37 Ma–755 ka) endemic to Mexico migrated from more northerly areas of between the coastal and Rocky Mountains populations North America during the Tertiary or from northern South (Gugger et al. 2010a), and the mid-Pleistocene (958 ka) America during the Quaternary, and experienced recurrent split between the Mexican and Rocky Mountains populations latitudinal and altitudinal shifts since their establishment. (Gugger et al. 2010b). These more recent estimates must be The present estimates indicated that the Mexican and the taken with caution, given their uncertainties and possible coastal lineages diverged from the Rocky lineage around sources of bias, as indicated in Gugger et al. (2010a; e.g., the 8.5 Ma (95% HPD interval, 16.5–2.4 Ma), whereas the large discrepancy of estimates based on mtDNA and MRCA of the lineage leading to current Mexican Douglas-fir cpDNA, likely unfit models employed by the program populations would be much after, around 3.2 Ma (95% IMa for the mtDNA dataset, the possible violation of the HPD interval, 7.0–0.5 Ma). Thus, Douglas-fir would molecular clock assumption between the outgroup P. have migrated southwards to Mexico from the southern macrocarpa and Douglas-fir and thus possibly inaccurate Rocky Mountains during a time when the climate cooled substitution rate used in IMa). We also acknowledge that down, most likely before the Pleistocene and no later our time estimates have large error ranges that overlap than the onset of this period. The genetic imprint of this with those of Gugger et al. (2010a, b), but our estimates process is possibly seen in the distribution pattern of were well before the Pleistocene. chlorotypes 24 and 27, two prevalent haplotypes in the The present estimates suggest that the diversification of southern Rocky Mountains, which were also present at Douglas-fir in its three main phylogenetic lineages and their low frequency in the two northernmost populations 1 and subsequent diversification occurred from the late Miocene 6 of Mexico, respectively (Fig. 1). through the Pliocene (10–2 Ma), a period of general cooling Additionally, the distribution patterns of both mitotypes at the global level (Zachos et al. 2001). This period also and chlorotypes (Fig. 1) indicated a closer affinity between corresponds to a time when western North America populations from Mexico and the Rocky Mountains than experienced a series of complex geological events, such between coastal and Rocky Mountains populations, as the orogeny of the Sierra–Cascades and the subsequent paralleling their common classification into the interior xerification of the Columbian basin, which presumably split variety. Therefore, the idea that Mexican Douglas-fir a formerly continuous coniferous forest (Graham 1993, could be a different species, as suggested by the large 1999b). Both the divergence time estimates and the differentiation observed for one Mexican population in a significant phylogeographical differentiation we found range-wide allozyme analysis in Douglas-fir (Li and between the coastal and interior varieties imply that the Adams 1989), was not supported by our study, neither orogeny of the Cascade/Sierra Chain may have played a the hypothesis of multiple species within Mexico (Flous crucial role in the vicariance initiating the varietal differen- 1934;Martinez1963). However, in line with Gugger et al. tiation of Douglas-fir. The arid areas in southern Idaho and (2010b), our study also provided strong phylogenetic Wyoming, which developed during the late Tertiary evidence that the Mexican populations of Douglas-fir are (Brunsfeld et al. 2007; Li and Adams 1989), may have monophyletic and should be treated as a distinct variety, also acted as a potential physical barrier to gene flow between given the large differentiation observed for cpDNA data the northern (including the transition zone) and southern and quite ancient divergence time from the Rockies interior populations in the Rocky Mountains, as suggested by lineage. the relatively large cpDNA differentiation between these Another interesting finding in the present study is that regions observed in the present study (FCT=0.141). Such the cpDNA and mtDNA datasets indicated a geographically vicariance factors have been shown to be crucial for the different ancestral lineage for Douglas-fir. Both the network diversification of widely distributed North American forest (Fig. 2) and Bayesian phylogenetic tree (Fig. 3), based on conifers in their major subspecific lineages (Gérardi et al. cpDNA sequence variation, indicated that the interior 2010; Godbout et al. 2005, 2008; Jaramillo-Correa et al. variety (the Rockies lineage) was ancestral to the coastal 2009). In addition, this evolutionary trend is in line with the and Mexican lineages, given its closer phylogenetic recognition of vicariance and geographical/allopatric proximity to the outgroup P. macrocarpa. 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Koeltz Scientific Books, Koenigstein, as well as M. Deslauriers and P. Laplante (Natural Resources Canada, Germany Canadian Forest Service) for laboratory assistance. We are grateful to Flous F (1934) Deux espèces nouvelles de Pseudotsuga Américains. S. Gérardi, J. Godbout, and M. Lemieux (Canada Research Chair in Bull Soc Hist Nat Toulouse 66:211–224 Forest and Environmental Genomics) for their help with data analysis Gérardi J, Jaramillo-Correa JP, Beaulieu J, Bousquet J (2010) and for valuable discussions. We also thank the two anonymous From glacial refugia to modern populations: new assemblages reviewers and the associate editor S. Aitken for their insightful of organelle genomes generated by differential cytoplasmic comments and suggestions on this manuscript. Our thanks are also due gene flow in transcontinental black spruce. Mol Ecol to G.E. Rehfeldt (USDA Forest Service in Moscow, Idaho) and F.T. 19:5265–5280 Ledig (UC Davis, California) for providing seeds of the three Godbout J, Jaramillo-Correa JP, Beaulieu J, Bousquet J (2005) A Douglas-fir populations of the Rockies-transition region, and of P. mitochondrial DNA minisatellite reveals the postglacial history macrocarpa, respectively. The collections of samples for Mexican of jack pine (Pinus banksiana), a broad-range North American populations and for P. sinensis were supported by CONACYT (Grant conifer. Mol Ecol 14:3497–3512 33617-B and 2002-C01-6416) and the National Natural Science Godbout J, Fazekas A, Newton CH, Yeh FC, Bousquet J (2008) Foundation of China (Grant No. 30500030), respectively. This study Glacial vicariance in the Pacific Northwest: evidence from a was supported by grants from the Mexico-Québec Program of the lodgepole pine mitochondrial DNA minisatellite for multiple Ministère du Développement économique, de l’innovation et de genetically distinct and widely separated refugia. Mol Ecol l’exportation du Québec, the National Sciences and Engineering 17:2463–2475 Research Council of Canada, and Natural Resources Canada. This Godbout J, Beaulieu J, Bousquet J (2010) The phylogeographic study was part of a task of the Forest Genetic Resources Working structure of jack pine (Pinus banksiana; Pinaceae) supports the Group of the North American Forest Commission/Food and Agricul- existence of a coastal glacial refugium in northeastern North tural Organization of the United Nations. America. Am J Bot 97:1903–1912 Tree Genetics & Genomes (2011) 7:1025–1040 1039

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