1

Molecular Ecology In press. 1

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Interspecific phylogenetic analysis enhances intraspecific phylogeographic inference: A 3

case study in Pinus lambertiana 4

AARON LISTON*, MARIAH PARKER-DEFENIKS*, JOHN V. SYRING*,‡, ANN 5

WILLYARD*,§ , and RICHARD CRONN† 6

7 *Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331, 8

USA, †Pacific Northwest Research Station, USDA Forest Service, 3200 SW Jefferson Way, 9

Corvallis, Oregon 97331,USA, ‡Current Address: Department of Biological and Physical 10

Sciences, Montana State University – Billings, Billings, Montana 59101, USA, §Current 11

Address:Department of Biology, University of South Dakota, Vermillion, SD 57069 USA 12

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Keywords: chloroplast introgression, Cronartium ribicola, Pinus lambertiana,Pinus subsect. 15

Strobus, phylogeography, white pine blister rust resistance 16

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Correspondence: A. Liston. Department of Botany and Plant Pathology, 2082 Cordley Hall, 19

Oregon State University, Corvallis, Oregon 97331, USA. Fax: 1 541 737 3573. E-mail: 20

listona@science.oregonstate.edu 21

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Running title: Phylogeny and phylogeography 24

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Abstract 25

Pinus lambertiana (sugar pine) is an economically and ecologically important conifer with a 26

1600 km latitudinal range extending from Oregon, USA to northern Baja California, Mexico. 27

Like all North American white pines (subsect. Strobus), sugar pine is highly susceptible to white 28

pine blister rust, a disease caused by the fungus Cronartium ribicola. We conducted a chloroplast 29

DNA survey of Pinus subsect. Strobus with comprehensive geographic sampling of P. 30

lambertiana. Sequence analysis of 12 sugar pine individuals revealed strong geographic 31

differentiation for two chloroplast haplotypes. A diagnostic restriction site survey of an 32

additional 72 individuals demarcated a narrow 150 km contact zone in northeastern California. In 33

the contact zone, maternal (megagametophtye) and paternal (embryo) haplotypes were identified 34

in 31 single seeds, demonstrating bidirectional pollen flow extending beyond the range of 35

maternal haplotypes. The frequencies of the Cr1 allele for white pine blister rust major gene 36

resistance, previously determined for 41 seed zones, differ significantly among seed zones that 37

are fixed for the alternate haplotypes, or contain a mixture of both haplotypes. Interspecific 38

phylogenetic analysis reveals that the northern sugar pine haplotype belongs to a clade that 39

includes P. albicaulis (whitebark pine) and all of the East Asian white pines. Furthermore, there 40

is little cpDNA divergence between northern sugar pine and whitebark pine (dS = 0.00058). 41

These results are consistent with a Pleistocene migration of whitebark pine into North America 42

and subsequent chloroplast introgression from whitebark pine to sugar pine. This study 43

demonstrates the importance of placing phylogeographic results in a broader phylogentic 44

context. 45

3

Introduction 46

47

Pinus has been described as “the most economically and ecologically significant tree genus in 48

the world” (Richardson and Rundel 1998). Support for this claim is found in the large number of 49

population genetic studies conducted in pine species. Ledig (1998) summarized genic diversity 50

statistics (primarily from isozymes) for 51 of the ca. 110 species of pine. Over the last 15 years, 51

at least 26 species of pine have been evaluated for among population DNA variation (tabulated 52

in Petit et al. 2005, see also Chiang et al. 2006; Navascués et al. 2006). The focus of many of 53

these studies is phylogeographic inference using chloroplast DNA. Despite the prevalence of 54

interspecific hybridization in pines (Ledig 1998) few of these studies sample other related 55

species, and thus cannot place the within species genetic variation in a broader phylogenetic 56

context. This study of Pinus lambertiana, sugar pine, demonstrates how resolution of 57

interspecific phylogeny can have a profound impact on the interpretation of intraspecific 58

phylogeographic results. Just as studies can be enhanced by “putting the geography into 59

phylogeography” (Kidd and Ritchie 2006) it is imperative to incorporate a broad phylogenetic 60

perspective as well. 61

62

Pinus lambertiana is one of the ca. 20 species of subsection Strobus (Gernandt et al. 2005; 63

Syring et al. 2007). This clade is known by the common name “white pines” and is distributed 64

discontinuously throughout the Northern Hemisphere (Table 1). The monophyly of subsect. 65

Strobus is strongly supported by chloroplast sequences (Gernandt et al. 2005; Eckert and Hall 66

2006) and nuclear ribosomal DNA (Liston et al. 1999) and moderately supported by a low copy 67

nuclear locus (Syring et al. 2007). The 20 species share a similar vegetative morphology (five 68

relatively narrow and strongly amphistomatic needles per fascicle) but differ dramatically in 69

ovulate cone size (from 5 cm in P. pumila to 60 cm in P. lambertiana) and shape. The five 70

species (P. albicaulis, P. cembra, P. koraiensis, P. pumila, P. sibirica) with indehiscent “closed” 71

cones adapted for bird dispersal were traditionally treated as “subsect. Cembrae”, or stone pines. 72

73

Like all North American members of subsect. Strobus, sugar pine is highly susceptible to white 74

pine blister rust, a disease caused by the heterocyclic rust fungus Cronartium ribicola. This 75

pathogen is native to Asia (Kinloch 2003) and was accidentally introduced to North America in 76

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the eastern U.S. and British Columbia in the early 20th century (Mielke 1943; Scharpf 1993). It 77

has subsequently spread to all species of subsect. Strobus that occur in the United States and 78

Canada (Scharpf 1993; Kinloch 2003). The disease results in cankers that girdle the main stem 79

and kill infected seedlings and trees (Kinloch and Scheuner 2004). While a mapped locus (Cr1) 80

that confers qualitative (major gene) resistance has been identified in sugar pine (Kinloch 1992, 81

2003; Devey et al. 1995), its frequency in populations is typically low. Cr1 frequency varies 82

from less than 10% in the southern part of the range of P. lambertiana to near absence in the 83

north (Kinloch 1992, summarized in our Table 2) . Attempts to increase the frequency of white 84

pine blister rust resistance have prompted federal (USDA Forest Service, US Bureau of Land 85

Management) and private agencies to make extensive seed collections for this species, and to 86

initiate large-scale screening programs. 87

88

We used DNA sequences of two chloroplast loci to conduct a phylogenetic analysis of North 89

American and Eurasian members of Pinus subsect. Strobus, in concert with a phylogeographic 90

survey of P. lambertiana. Our study documents significant cpDNA divergence between two 91

major haplotypes in P. lambertiana. The distribution of these haplotypes is concordant with the 92

geographic distribution of white pine blister rust major gene resistance. A narrow contact zone 93

between haplotypes, and limited divergence within each haplotype, strongly suggests that 94

secondary contact between two cytoplasmically divergent groups has occurred in the 95

(evolutionarily) recent past. Our phylogenetic analysis provides evidence that the nothern 96

populations of Pinus lambertiana may have obtained their chloroplast via introgression from P. 97

albicaulis, whitebark pine. The integration of phylogenetic and phylogeographic approaches 98

allowed us to recover this unexpected evolutionary history. 99

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101

Materials and Methods 102

Organismal sampling and DNA genotyping 103

Pinus lambertiana Douglas is an economically and ecologically important conifer with a 1600 104

km latitudinal range extending from Oregon, USA to northern Baja California, Mexico. Eighty-105

four individuals representing the geographic range of this species were included in this analysis. 106

Nineteen additional species from Pinus subsect. Strobus were sampled for the phylogenetic 107

5

analysis (Table 1), and Pinus gerardiana from subsect. Gerardianae was used as the outgroup. 108

DNA was extracted from the haploid megagametophyte of individual seeds as described in 109

Syring et al. (2007). PCR amplification followed Gernandt et al. (2005). Approximately 90% of 110

the chloroplast matK open reading frame (1404 bp) and ca. 150 bp of the 3' trnK (UUU) intron 111

(see Hausner et al. 2005 for a recent review) were amplified using primers matK1F (Wang et al. 112

1999) and ORF515-900F (Gadek et al. 2000). The chloroplast trnG (UCC) intron (ca. 780 bp) 113

was amplified using the primers 3' trnG and 5' trnG2G (Shaw et al. 2005). For divergence time 114

estimates between P. albicaulis and P. lambertiana (described below), three additional loci were 115

added to the cpDNA data set for these species. These include new sequences for the trnL-trnF 116

intergenic region (including trnL exon 1 and its intron) and the rpl16 intron (Shaw et al. 2005), 117

as well as previously published rbcL sequences (Gernandt et al. 2005). Predicted amplicon 118

lengths were based on the Pinus koraiensis chloroplast genome (Noh et al. unpublished, 119

AY228468). Uncloned PCR products were submitted to High-Throughput Sequencing Solutions 120

(University of Washington) for ExoSAP purification and automated capillary sequencing. 121

Electropherograms were examined and aligned with BioEdit 7.0.5.2 (Hall 1999). All new 122

sequences are deposited in GenBank under the accessions EF546699-EF546759. 123

124

Chloroplast matK and trnG sequences from 12 individuals of P. lambertiana identified two 125

divergent haplotypes (see results) abbreviated N (for North) and S (for South). Using methods 126

described in Liston (1992), we screened 72 individuals for an AluI restriction site that 127

differentiates these two haplotypes. This assay is diagnostic for a C/T polymorphism at position 128

1352 in matK. Since chloroplasts are paternally inherited in Pinus (Neale and Sederoff 1989), the 129

AluI polymorphism was also used to determine maternal versus pollen cpDNA haplotype by 130

extracting DNA from megagametophyte (maternal) and embryo (paternal) tissue in 31 individual 131

seeds. 132

133

Phylogenetic, phylogeographic and statistical analyses 134

Phylogenetic analyses and constraint tests were conducted with PAUP* 4.0b10 (Swofford 2002) 135

following the procedures outlined in Syring et al. (2005). Indels were added to the parsimony 136

data matrix as binary characters. Parsimony analysis was conducted with a heuristic search 137

strategy of 1000 random addition sequences and TBR swapping. Branch support was assessed 138

6

using 2000 bootstrap replicates. Indels and duplicate sequences were excluded, and the most 139

appropriate likelihood model was selected using the method of Posada and Crandall (1998) and 140

AIC scores at the FindModel website (http://hcv.lanl.gov/content/hcv-141

db/findmodel/findmodel.html). To evaluate whether sequences diverged at clock-like rates, 142

maximum likelihood trees were estimated using the GTR+ γ model as implemented in PAUP* 143

4.0b10 (Swofford 2002). Likelihood scores obtained with and without a molecular clock 144

constraint were evaluated using the likelihood ratio test (LRT) of Muse and Weir (1992). Under 145

assumptions of a molecular clock, the divergence time(Tdiv) between two groups of sequences is 146

approximately Tdiv = dS / 2μ where dS is the average pairwise distance among sequences at 147

presumably neutral (synonymous and non-coding) sites and μ is the neutral mutation rate. 148

Estimates of dS were calculated with DnaSP 4.10.9 (Rozas et al. 2003, note that the program 149

uses the abbreviation Ks). The estimate of μ for Pinus cpDNA (0.22 ×10-9 silent substitutions 150

site-1 year-1; standard error = 0.55 ×10-10) is based on an 85 million years ago (mya) divergence 151

time between the two subgenera of Pinus; see Willyard et al. (2007) for details. Divergence 152

times estimated here are reported with ± one standard error. 153

154

To investigate whether the white pine blister rust major gene resistance allele (Cr1) frequencies 155

were different among chloroplast haplotype classes, 41 tree seed zones used in blister rust 156

screening (Table 2; Kinloch 1992) were classified as either fixed for the N haplotype, fixed for 157

the S haplotype, or polymorphic for the two haplotypes. One-way ANOVA was used to examine 158

variance partitioning and to test the hypothesis that means were equivalent among these three 159

groups. Given the large number of zeros present in estimated Cr1 allele frequencies (especially 160

in the northern part of the range), we also estimated means and confidence intervals for allele 161

frequencies in the N, S, and mixed haplotype groups using 10,000 nonparametric bootstrap 162

resamplings. Statistical and nonparametric bootstraps were performed using PopTools version 163

2.7 (Hood 2006). For mapping, the program TRS2LL (Wefald 2001) was used to convert 164

township/range/section localities to latitude and longitude. 165

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Results 167

Phylogeographic haplotype variation in sugar pine 168

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Sequences of cpDNA matK and trnG intron in 12 Pinus lambertiana individuals revealed two 169

predominant haplotypes with fixed differences at 10 sites (seven in matK, including three amino 170

acid replacements; three in the trnG intron). One additional haplotype was observed in the Baja 171

California individual (an autapomorphic matK replacement substitution). The sequence results 172

combined with AluI restriction digest assays for 72 individuals demonstrated an abrupt transition 173

in the distribution of the two predominant haplotypes (Fig. 1A). Plants from Oregon and 174

northwestern California (Klamath mountains and North Coast range) were fixed for a common 175

haplotype “N” (thymine at position 1352 of matK), while plants from the Sierra Nevada and 176

Transverse and Peninsular ranges in California were fixed for the alternate haplotype “S” 177

(cytosine at position 1352). The contact zone between the N and S haplotypes occurs near 178

latitude 40˚ 30' N in northeastern California, and the zone of polymorphism is remarkably well-179

defined, spanning less than 150 km of the ca. 1600 km latitudinal range of this species (Figs. 1, 180

2). Megagametophyte and embryo comparisons in 31 individuals from the contact zone revealed 181

that 12 (39%) seeds contained different maternal and paternal haplotypes, indicating that 182

seedlings are frequently sired by pollen parents with different haplotypes than the ovulate parent. 183

Embryos with the S haplotype were found in seeds from eight N haplotype maternal trees 184

distributed in seed zones 522, 523, 732 and 771 (Fig. 2). The converse pattern was also observed 185

as four S haplotype maternal plants from seed zones 522 and 731 produced seed containing N 186

haplotype embryos (Fig. 2). 187

188

At least one sugar pine individual was assayed for the S vs. N haplotype in 35 of the 41 seed 189

zones sampled by Kinloch (1992) in his survey of Cr1 allele frequencies (the factor conferring 190

major gene resistance to white pine blister rust; Table 2), with intensive sampling in the contact 191

zone (Fig. 2). The haplotype for six unassayed seed zones (one in northwestern California and 192

five in Oregon) was inferred to be N based on results from adjacent seed zones. The Baja 193

California haplotype S' (differing from S by one substitution, Fig. 3) was grouped with S for this 194

analysis. The N, S, and polymorphic seed zones showed significantly different Cr1 frequencies 195

by one-way ANOVA (F = 32.3, P = 7.6e-9). The N haplotype seed zones had the lowest Cr1 196

allele frequency (0.0032 + 0.0038; n = 23), mixed haplotype seed zones had intermediate Cr1 197

frequencies (0.0380 + 0.0185; n = 3), and S haplotype seed zones had the highest Cr1 198

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frequencies (0.0484 + 0.0260; n = 15). Nonparametric bootstrapping resulted in the same mean 199

allele frequencies and confidence intervals. 200

201

Phylogenetic relationships and variation in sugar pine and other white pine species. Alignment 202

of matK and trnG intron sequences required one 6 bp indel in the matK ORF and four indels (1, 203

4, 10 and 15 bp) in the trnG intron. Three of the indels were confined to P. parviflora, the 15 bp 204

trnG intron indel was shared by P. ayacahuite, P. flexilis, P. strobiformis and P. peuce, and the 1 205

bp indel was autapomorphic in P. armandii ‘b’. Combined phylogenetic analysis of matK (1545 206

bp), trnG intron (746 bp) and the five indels resulted in two most parsimonious trees of length 57 207

with a consistency index of 0.91 (Fig. 3). The two trees differ in the resolution of P. monticola as 208

a grade (shown) or clade (not shown). All Asian species of subsect. Strobus form a strongly 209

supported clade that includes the North American P. albicaulis and the P. lambertiana N 210

haplotype. At these two loci, no sequence divergence was found among northern P. lambertiana, 211

P. albicaulis and representatives of six Asian species (P. dalatensis, P. koraiensis, P. pumila, P. 212

sibirica, P. wallichiana, P. kwangtungensis) and P. cembra from Romania. The P. lambertiana S 213

haplotypes and the remaining North American white pine species form a grade, with P. 214

monticola in a strongly supported sister position to the Eurasian clade. Constraining P. 215

lambertiana samples to monophyly results in trees of 66 steps, which is significantly longer 216

based on both Templeton (p = 0.0039) and Kishino-Hasegawa tests (p = 0.0027). 217

218

Maximum likelihood trees obtained with and without a molecular clock were topologically 219

identical to one of the most parsimonious trees and were not significantly different from each 220

other based on the LRT (ΔlnL = 10.44013, χ2 = 20.88, d.f. = 19, p = 0.34). This suggests that the 221

sequences are diverging at equivalent rates, and a simple molecular clock calculation can be 222

applied. The mean dS between P. monticola and the Asian clade is 0.00232, resulting in an 223

estimated divergence of 5.3 ± 1.3 mya (Late Miocene – Early Pliocene). Sequences of 5799 bp 224

of cpDNA (adding rbcL, rpl16 and trnL-F to the matK and trnG used in the phylogenetic 225

analysis) revealed a single substitution in trnL-F between a P. albicaulis accession (Washington 226

state) and an N haplotype P. lambertiana (dS = 0.00058), resulting in an estimated divergence of 227

0.6 ± 0.15 mya (Pleistocene). Sequences of trnL-F from an additional five accessions of P. 228

albicaulis and three of N haplotype P. lambertiana (Table 1) confirmed that this is a fixed 229

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difference between these two taxa. The estimated divergence time between the two P. 230

lambertiana chloroplast haplotypes is 15.5 ± 3.9 mya (dS = 0.00681) and between the S 231

haplotype of P. lambertiana and the other North American white pines is 9.0 ± 2.25 mya (dS = 232

0.00396). 233

234

Discussion 235

The pattern of genetic subdivision in sugar pine 236

Previous accounts of sugar pine have described neither morphological nor ecological differences 237

that can be associated with the phylogeographic pattern observed here. In fact, Mirov (1967) 238

described Pinus lambertiana as “rather stable morphologically” (p. 142) and he considered it to 239

be a prime example of a “good species” that is “clearly delimited [and] can be identified without 240

difficulty” (p. 531). The southern limit of the contact zone (Fig. 1A) does coincide with the 241

interface of the Cascade and Sierra Nevada mountain ranges, characterized by relatively recent 242

volcanics and predominantly metamorphics (with granitic intrusions and volcanics), respectively 243

(Hickman 1993). Although this geological transition is used to demarcate two floristic 244

subregions, there is no apparent vegetational break between the forests of the Cascades and 245

northern Sierra Nevada (Hickman 1993). 246

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In her PhD thesis, Martinson (1997) conducted an analysis of allozyme data collected by Conkle 248

(unpublished; 1996). Forty populations and 400 individuals of P. lambertiana were assayed at 30 249

allozyme loci. Average heterozygosity was 0.22, and no geographic region showed reduced 250

genetic diversity. Clustering of genetic distances separated populations from Oregon and 251

northwestern California from populations in the Sierra Nevada. Unfortunately, no populations 252

were sampled in the chloroplast haplotype contact zone in northeastern California. Thus, 253

although the reported allozyme differentiation may correspond to the chloroplast haplotype 254

distribution, it will require allozyme sampling in the contact zone to confirm this. The allozyme 255

data also separated the Sierra Nevada populations from those sampled in southern California and 256

Baja California. This division is not apparent in our data set. However, a unique substitution was 257

found in the matK sequence of the Baja California individual. This evidence for genetic isolation 258

is consistent with the geographic isolation of this disjunct population (Fig. 1A, B). 259

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The narrow transition zone between the N and S haplotypes of P. lambertiana suggests that these 261

populations have only recently come into contact. This would be consistent with a Holocene 262

range expansion from separate northern and southern refugia. There is abundant evidence from 263

the pollen record for post-glacial movement of pines (Mohr et al. 2000; Thompson and Anderson 264

2000). Unfortunately, individual Pinus species cannot be identified from pollen. Narrow 265

haplotype (chloroplast or mitochondrial) transition zones observed in other western North 266

American plants (Soltis et al. 1997; Aagaard et al. 1998; Latta and Mitton 1999; Johansen and 267

Latta 2003) have also been attributed to post-glacial contact of previously separated populations. 268

269

The examination of maternal and paternal haplotypes offers insight into the dynamics of 270

dispersal at the contact zone (Fig. 2). The two P. lambertiana trees sampled in the southern part 271

of seed zone 731 represent a population that is isolated on Happy Camp mountain, Modoc 272

County. These trees have the S haplotype, and are presumed to have colonized this location by 273

long distance seed dispersal. The closest sampled potential source is ca. 90 km away. The large 274

(228 ± 40 mg) and “flimsy” winged seeds of P. lambertiana are “seldom dispersed far by wind”, 275

but caching by Steller’s jays (Thayer and Vander Wall 2005) and Clark’s nutcrackers (D. 276

Tomback, pers. comm.) can potentially lead to long-distance dispersal. No other example of a 277

disjunct haplotype was observed. Embryos possessing the S haplotype occur up to 25 km north 278

of the northernmost potential source trees, indicating that pollen flow advances ahead of seed 279

dispersal. The two Happy Camp mountain trees whose megagametophytes carry the S haplotype 280

have apparently been pollinated by trees of the N haplotype. Comparison of chloroplast and 281

mitochondrial haplotypes in a Pinus ponderosa contact zone in western Montana has found a 282

similar pattern of more extensive pollen flow and rare long distance seed dispersal (Latta and 283

Mitton 1993; Johansen and Latta 2003). 284

285

One of the most surprising results of our study is the concordance between the distribution of the 286

two cpDNA haplotypes and the relative frequency of a white pine blister rust major gene 287

resistance locus (Cr1) in sugar pine. Kinloch (1992) described the pattern of Cr1 frequency as a 288

cline. In contrast, the significant differences in Cr1 frequency observed among the three 289

haplotype groups (N, S, and mixed) suggests that the gene frequency does not change in a 290

gradual manner, but rather shows the same abrupt transition as observed in the chloroplast. There 291

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is no evidence for a causal link between the cpDNA haplotype and Cr1 distribution patterns. It is 292

well-established that resistance shows nuclear inheritance (Kinloch 1992), and the highest 293

frequencies of Cr1 (4 – 9% per seed zone) are far lower than the frequency of the S haplotype. 294

Furthermore, examination of three resistant trees (heterozygous for the dominant Cr1 allele; J. 295

Gleason, unpublished data) in the contact zone found both N and S haplotypes (Fig. 2). Note that 296

all other genotyped individuals in the contact zone are non-resistant (J. Gleason, unpublished 297

data). We predict that the concordance between the Cr1 and chloroplast haplotype frequencies 298

reflects a common history of genetic isolation, followed by recent migration and contact (see 299

below). 300

301

Evidence for chloroplast introgression 302

The chloroplast haplotypes of P. lambertiana resolve in two different clades in the phylogenetic 303

analysis of Pinus subsect. Strobus, one comprised of five other North American species and the 304

other encompassing 12 Eurasian species and the North American P. albicaulis (whitebark pine). 305

Two biological processes could explain these results: incomplete lineage sorting of an ancestral 306

polymorphism, or chloroplast introgression. Incomplete lineage sorting has been determined to 307

be the most probable source of widespread allelic non-monophyly at nuclear loci in species of 308

Pinus subgenus Strobus (Syring et al. 2007). It has also been considered a potential cause of 309

similar patterns observed in chloroplast studies in other plant species (Tsitrone et al. 2003). 310

However, the stochastic process of incomplete lineage sorting is not expected to show the strong 311

geographic partitioning observed for the two chloroplast haplotypes. On the other hand, if the 312

two subgroups of sugar pine have been separated since the Miocene (15.1 ± 3.8 mya) and each 313

retained a different haplotype, one might expect to find morphological divergence between (and 314

sequence variation within) the two groups, particularly since this same interval has apparently 315

been accompanied by multiple speciation events in these lineages. Although some sequence 316

divergence was found in the S haplotype clade (in the geographically isolated Baja California 317

population), none was found in the N haplotype clade. The amount of sequence divergence 318

between the S haplotype of P. lambertiana and the other North American white pines is 319

consistent with genetic isolation since the Miocene. In contrast, the high sequence similarity 320

between the N haplotype of sugar pine and the Asian clade is suggestive of a much more recent 321

shared plastid ancestry. 322

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323

To account for this unexpected genetic similarity, we suggest that the N haplotype of P. 324

lambertiana may have its origin in a chloroplast introgression event involving P. albicaulis. 325

Introgression-mediated chloroplast transfer has been named “chloroplast capture” (Rieseberg and 326

Soltis 1991; Tsitrone et al. 2003) and has been offered as an explanation for similar patterns of 327

cytonuclear incongruence observed in many plant genera (reviewed in Rieseberg et al. 1996; 328

Wendel and Doyle 1998). Tsitrone et al. (2003) have modeled the process under the assumption 329

of maternal chloroplast inheritance and they determined conditions likely to promote its 330

occurence. A key aspect of their model is the observation that cytonuclear incompatibility often 331

results in full or partial male steritily and thus can increase maternal fitness through enhanced 332

seed production. Although they do not explicitly model paternal inheritance (the situation in 333

Pinaceae), they suggest that chloroplast introgression should be less common here, since 334

cytonuclear interactions typically reduce male fitness. Theoretically, chloroplast substitution 335

could result in an advantage in male function, but this situation has apparently not been 336

documented (Tsitrone et al. 2003). However, a pattern consistent with chloroplast introgression 337

has been observed in other species of Pinaceae, e.g. Pinus montezumae (Matos and Schaal 2000), 338

Pinus muricata (Hong et al. 2003) and Larix sibirica (Wei and Wang 2003). 339

340

Petit et al. (2003) offer “pollen swamping” as an alternative explanation for the lack of 341

cytoplasmic (cpDNA and mtDNA) differentiation between Quercus petraea and Q. robur, two 342

sympatric oaks that are consistently differentiated at nuclear markers. In their scenario, Q. robur 343

seed disperses into new habitats which are subsequently colonized by Q. petraea via pollen flow, 344

resulting in F1 hybrids. Asymmetric introgression and strong selection for the Q. petraea 345

phenotype results in mixed populations that share a single cytoplasm. Petit et al. invoke the fact 346

that the seeds of Q. robur are better adapted to bird-dispersal than Q. petraea, and thus are more 347

likely to establish through long-distance dispersal. A similar relationships exists between P. 348

albicaulis (dispersed mainly by the pine seed specialist, Clark’s nutcracker, Tomback 2005) and 349

P. lambertiana (dispersed to a limited extent by wind and primarily by generalist Steller’s jays 350

and yellow pine chipmunks, Thayer and Vander Wall 2005). In both cases, the better disperser is 351

thought to have contributed its chloroplast to the other species. An important caveat is that the 352

13

cytoplasm is maternally inherited in oaks, and thus carried by the seed, and not the pollen as in 353

pines. 354

355

356

If chloroplast introgression is responsible for this pattern, how does one account for the 357

otherwise “Eurasian” haplotype in two species of North American subsect. Strobus? The 358

cpDNA-based phylogeny (Fig. 3) requires two dispersal events between western North America 359

and Asia. The disjunction between P. monticola and the Eurasian clade can be dated to the late 360

Miocene or early Pliocene. This timing is consistent with estimates of 2.6 – 16.7 mya from 361

eleven other eastern Asian / western North American plant disjunctions (Zhu et al. 2006; Zhang 362

et al. 2007). It is noteworthy that well-preserved Pliocene and Pleistocene fossils of P. monticola 363

have been collected in northeastern Siberia and Alaska (reviewed in Bingham et al. 1974). 364

Following diversification of the Eurasian white pines, and origin of the “closed cone” 365

morphology characteristic of stone pines, we propose that the ancestor of P. albicaulis dispersed 366

from Asia to North America via Beringia, presumably during the Pleistocene. Beringia was 367

largely unglaciated during the Pleistocene, and is known to have served as a refugium for trees 368

and shrubs, including Pinus, through the late glacial maximum (Brubaker et al. 2005). 369

Krutovskii et al. (1995) proposed a similar scenario based on allozyme and chloroplast 370

restriction fragment analysis of the “subsect. Cembrae” pines, but placed the migration at an 371

earlier period (Pliocene). 372

373

Evidence from mtDNA haplotypes has been used to infer the existence of three late Pleistocene 374

refugia for P. albicaulis (Richardson et al. 2002): western Wyoming, western Idaho and the 375

southern Cascades of Oregon. The southern Cascades currently support large populations of P. 376

lambertiana, and we propose that this region could also have served as a northern glacial 377

refugium for sugar pine, or possibly further west in the Klamath/Siskiyou Mts. (a region with 378

several paleo-endemic conifers, e.g. Picea breweriana and Chamaecyparis lawsoniana). Sugar 379

pine is common here, but whitebark pine has only recently been discovered in a small population 380

on Mt. Ashland (nine individuals; Murray 2005). Regardless of their current geographic 381

distributions, sympatry in a glacial refugium could have provided the opportunity for the 382

northern populations of sugar pine to acquire the chloroplast of whitebark pine. Although P. 383

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albicaulis generally occurs at higher elevations than P. lambertiana, the two are partly sympatric 384

in northern California and southern Oregon (Fig. 1B). There is also evidence that P. lambertiana 385

formerly occurred at higher elevations than its current distribution (May 1974; Millar et al. 386

2006). 387

388

Two factors are required for chloroplast introgression to occur; sympatry and reproductive 389

compatibility. While many interspecific crosses have been conducted in subsect. Strobus, there is 390

no record of attempts to cross P. lambertiana and P. albicaulis (Critchfield 1986, R. Sniezko, 391

pers. comm.). Critchfield and Kinloch (1986) do, however, document interspecific hybridization 392

between P. lambertiana and Asian members of subsect. Strobus, namely P. armandii and P. 393

koraiensis. Seed set in these artificial crosses averaged 2.1% and 0.2% viable seed per cone, 394

respectively. In contrast, P. lambertiana is apparently intersterile with all other North American 395

white pines (Critchfield 1986; Fernando et al. 2005). No naturally occurring hybrids of P. 396

lambertiana have been recorded (Mirov 1967; Critchfield 1986; R. Sniezko, pers. comm.). 397

398

Our results explain why Pinus lambertiana was resolved in conflicting positions in recent 399

chloroplast sequence-based phylogenetic analyses of pines. Gernandt et al. (2005) sampled P. 400

lambertiana in Oregon (a region fixed for the N haplotype), while Eckert and Hall (2006) 401

sampled an individual from southern California (a region fixed for the S haplotype). Each study 402

placed P. lambertiana in a position that is consistent with the resolution of the respective 403

haplotypes in our study (Fig. 3). This demonstrates the importance of sampling multiple 404

individuals per species in phylogenetic analyses of closely related species (see also Syring et al. 405

2007). 406

407

The results reported here provide the first phylogeographic hypothesis for an ecologically and 408

economically important conifer, Pinus lambertiana. By placing the intraspecific results within a 409

broader phylogenetic context, further insights were gained into the evolutionary history of this 410

species. The novel hypotheses of two Pleistocene refugia for P. lambertiana and chloroplast 411

introgression with P. albicaulis can be tested with additional molecular markers, in particular 412

nuclear and mitochondrial loci. The observation that the two chloroplast haplotypes demarcate 413

population groups that differ in their vulnerability to white pine blister rust is also a significant 414

15

result that merits further attention. Beyond sugar pine, this study demonstrates the value of 415

including an interspecific phylogenetic component in phylogeographic research. Without this 416

broader perspective, the antiquity of the haplotype groups would remain unknown, as would the 417

unexpected, and potentially reticulate, history of these species. 418

419

Acknowledgements 420

We are indebted to John Gleason (USFS, Placerville Nursery and Disease Resistance Program), 421

Jerry Berdeen and Richard Sniezko (USFS, Dorena Genetic Resource Center) and David 422

Johnson (USFS, Institute of Forest Genetics) for supplying sugar pine seeds and Roman 423

Businský for providing his collections of Asian species. We thank David Gernandt, Bohun 424

Kinloch, Todd Ott, Paul Severns, Richard Sniezko, and Diana Tomback for constructive 425

comments on the manuscript. This research was funded by National Science Foundation grants 426

DEB 0317103 and ATOL 0629508, an NSF Research Experience for Undergraduates 427

suppplement, and the Pacific Northwest Research Station, USDA Forest Service. 428

429

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21

Table 1 Geographic origin and Genbank accessions for samples sequenced for the cpDNA matK 591

and trnG intron loci. 592

593

Species Haplo-

type

Country Administrative Unit matK

accession

trnG

accession

albicaulis * U.S. California: Mono Co. EF546699 EF546730

albicaulis * U.S. California: Siskiyou Co. " "

albicaulis * U.S. Montana " "

albicaulis * U.S. Oregon " "

albicaulis *, ‡ U.S. Washington " "

albicaulis * U.S. Wyoming " "

armandii † a China Anhui EF546700 EF546731

armandii † b Taiwan Kaohsiung EF546701 "

ayacahuite Honduras La Paz EF546702 EF546732

ayacahuite Mexico Mexico " "

ayacahuite Mexico Michoacan " "

bhutanica India West Kameng EF546703 EF546733

cembra a Austria EF546704 EF546734

cembra b Switzerland EF546705 "

cembra c Romania EF546706 "

chiapensis Guatemala EF546707 EF546735

chiapensis Mexico Chiapas " "

chiapensis Mexico Guerrero " "

dalatensis † Vietnam Kon Tum EF546708 EF546736

flexilis a U.S. California EF546709 EF546737

flexilis b U.S. Colorado EF546710 "

flexilis c Canada Alberta EF546711 "

koraiensis Russia EF546712 EF546738

koraiensis Japan " "

koraiensis S. Korea " "

kwangtungensis China EF546713 EF546739

22

lambertiana * N U.S. California: seedzone 091 EF546715 EF546741

lambertiana N U.S. California: seedzone 372 " "

lambertiana * N U.S. California: seedzone 516 " "

lambertiana *, ‡ N U.S. California: seedzone 732 " "

lambertiana * N U.S. Oregon: seedzone 472 " "

lambertiana N U.S. Oregon: seedzone 731 " "

lambertiana S U.S. California: seedzone 120 EF546714 EF546740

lambertiana S U.S. California: seedzone 526 " "

lambertiana *, ‡ S U.S. California: seedzone 731 " "

lambertiana S U.S. California: seedzone 992 " "

lambertiana S U.S. California: seedzone 994 " "

lambertiana S' Mexico Baja California EF546716 "

monticola a U.S. Oregon EF546717 EF546742

monticola b Canada British Columbia EF546718 EF546743

monticola b U.S. California " "

morrisonicola Taiwan EF546719 EF546744

parviflora Japan Hokkaido EF546720 EF546745

parviflora Japan Honshu " "

peuce Bulgaria EF546721 EF546746

pumila Japan Hokkaido EF546722 EF546747

pumila Japan Hokkaido " "

pumila unknown " "

sibirica a Russia Krasnoyarsk Krai EF546723 EF546748

sibirica b Russia Kemorovo EF546724 "

strobiformis a Mexico Coahuila EF546725 EF546749

strobiformis b Mexico Durango EF546726 "

strobiformis b U.S. Texas " "

strobus U.S. Minnesota EF546727 EF546750

strobus Canada Newfoundland " "

strobus U.S. North Carolina " "

wallichiana † a Pakistan Punjab EF546728 EF546751

23

wallichiana b Nepal Karnali EF546729 "

gerardiana † Pakistan Gilgit AY115801 EF546752

594

Haplotypes N, S and S' for P. lambertiana are described in the text. In other species, the letters a, 595

b and c were applied as needed for multiple haplotypes within a species. Voucher specimens are 596

deposited at Oregon State University (OSC), unless indicated otherwise. 597 * Sequenced for trnL-F, GenBank accessions EF546753 - EF546756. ‡ Sequenced for rpl16, 598

Genbank accessions EF546757 - EF546759. 599 † Voucher deposited at the Silva Tarouca Research Institute for Landscape and Ornamental 600

Gardening, Průhonice, Czech Republic (RILOG). 601

602

603

24

Table 2 The frequency of the white pine blister rust resistance gene Cr1 in sugar pine seed zones 604

from the region of haplotype contact, compared to the number of southern and northern 605

haplotypes. Data in columns 2 and 3 are from the 41 seed zones sampled by Kinloch (1992). 606

607

seed

zone

trees / seeds

sampled

frequency of

major gene

resistance

haplo-

type

group

N

haplo-

type

S

haplo-

type

frequency

of S

haplotype

Oregon: Coast ranges and Cascades

090 10 / 74 0.0000 N

452 N 1 0.00

472 N 1 0.00

491 24 / 430 0.0000 N 1 0.00

492 7 / 231 0.0000 N

501 8 / 237 0.0127 N

502 18 / 379 0.0053 N

511 12 / 204 0.0049 N 1 0.00

512 9 / 205 0.0000 N 1 0.00

681 N 1 0.00

701 N 1 0.00

702 N 1 0.00

703 8 / 157 0.0000 N

721 N 1 0.00

Northwest California: Coast ranges

081 2 / 15 0.0000 N 2 0.00

091 36 / 1763 0.0085 N 2 0.00

095 8 / 136 0.00 N

301 162 / 4752 0.0061 N 1 0.00

302 5 / 137 0.00 N 1 0.00

303 N 1 0.00

311 28 / 683 0.0073 N 1 0.00

321 141 / 5197 0.0073 N 1 0.00

25

322 5 / 302 0.00 N 1 0.00

331 3 / 44 0.00 N 1 0.00

332 N 1 0.00

340 33 / 2030 0.0030 N 1 0.00

371 4 / 190 0.0053 N 1 0.00

372 101 / 4065 0.0096 N 1 0.00

Northeast California: Cascades

516 8 / 265 0.00 N 1 0.00

521 12 / 779 0.0026 N 3 0.00

522 264 / 15815 0.0192 mixed 4 3 0.43

523 42 / 1869 0.0316 mixed 1 3 0.75

731 mixed 1 2 0.67

732 7 / 174 0.0632 mixed 8 4 0.33

741 9 / 538 0.00 N 2 0.00

742 N 4 0.00

771 mixed 1 2 0.67

Eastern California: Sierra Nevada

524 85 / 3141 0.0274 S 3 1.00

525 116 / 5099 0.0322 S 2 1.00

526 222 / 7714 0.0460 S 2 1.00

531 91 / 2976 0.0339 S 1 1.00

532 16 / 941 0.0659 S 1 1.00

533 16 / 635 0.0819 S 1 1.00

534 124 / 2668 0.0727 S 1 1.00

540 44 / 1077 0.0706 S 1 1.00

772 14 / 222 0.0180 S 3 1.00

California: Central Coast range

120 93 / 2133 0.0886 S 1 1.00

Southern California: Transverse ranges

992 8 / 644 0.0699 S 1 1.00

993 29 / 463 0.0324 S 1 1.00

26

994 31 / 337 0.0297 S 1 1.00

997 32 / 517 0.0561 S 1 1.00

Baja California: Sierra San Pedro Martír

12 / 254 0.00 S 1 1.00

608

609

27

Fig. 1A Sampled individuals of P. lambertiana in western North America. Yellow squares 610

represent the S haplotype and blue squares represent the N haplotype. Forest tree seed zones 611

sampled by Kinloch (1992) and / or this study are shaded according to the frequency of the Cr1 612

allele (Table 2), ranging from 0% (no shading) to 8.9% (dark gray, seed zone 120). Seed zones 613

outlined in red were not sampled for Cr1. The haplotype contact zone (green rectangle) is shown 614

in more detail in Figure 2. 615

616

Fig. 1B Approximate geographic distribution of Pinus lambertiana (dark gray) and P. albicaulis 617

(light gray) from Critchfield and Little (1966). Red triangles represent P. albicaulis samples used 618

in this study. 619

620

Fig. 2 Detail of the contact zone between the S (yellow) and N (blue) cpDNA haplotypes of P. 621

lambertiana. Squares represent the maternal (megagametophyte) haplotype and circles represent 622

the paternal (embryo) haplotype. Asterisks denote white pine blister rust resistant individuals. 623

624

Fig. 3 One of two most parsimonious trees estimated from cpDNA matK and trnG intron 625

sequences. Bootstrap values are shown below the branches. Tree length = 57, consistency index 626

= 0.91, retention index = 0.97. When applicable, haplotypes (see Table 2) and the number of 627

individuals that share a particular sequence and haplotypes follow the species names. 628

629

Author Information 630

Aaron Liston and Rich Cronn collaboratively study the systematics, population genetics, and 631 evolution of pines. John Syring and Ann Willyard are former PhD students of Cronn and Liston. 632 Dr. Syring is now an Assistant Professor of Plant Systematics at Montana State University-633 Billings, and Dr. Willyard is a post-doc at the University of South Dakota. Mariah Parker-634 deFeniks is a Sociology major at Oregon State University, pursuing a career in Criminal Justice 635 and Social Research. 636 637

28

Figure 1 638

639

29

Figure 2 640

641

30

Figure 3 642

643