Interspecific Phylogenetic Analysis Enhances Intraspecific Phylogeographical Inference

Molecular Ecology (2007) 16, 3926–3937 doi: 10.1111/j.1365-294X.2007.03461.x

BlackwellInterspecific Publishing Ltd phylogenetic analysis enhances intraspecific phylogeographical inference: a case study in Pinus lambertiana

AARON LISTON,* MARIAH PARKER-DEFENIKS,* JOHN V. SYRING,*‡ ANN WILLYARD,*§ and RICHARD CRONN † *Department of Botany and Plant Pathology, 2082 Cordley Hall, Oregon State University, Corvallis, Oregon 97331, USA, †Pacific Northwest Research Station, USDA Forest Service, 3200 SW Jefferson Way, Corvallis, Oregon 97331, USA

Abstract Pinus lambertiana (sugar pine) is an economically and ecologically important conifer with a 1600-km latitudinal range extending from Oregon, USA, to northern Baja California, Mexico. Like all North American white pines (subsect. Strobus), sugar pine is highly susceptible to white pine blister rust, a disease caused by the fungus Cronartium ribicola. We conducted a chloroplast DNA (cpDNA) survey of Pinus subsect. Strobus with com prehensive geographical sampling of P. lambertiana. Sequence analysis of 12 sugar pine individuals revealed strong geographical differentiation for two chloroplast haplotypes. A diagnostic restriction site survey of an additional 72 individuals demarcated a narrow 150-km contact zone in northeastern California. In the contact zone, maternal (megagame tophtye) and paternal (embryo) haplotypes were identified in 31 single seeds, demonstrating bidirectional pollen flow extending beyond the range of maternal haplotypes. The frequencies of the Cr1 allele for white pine blister rust major gene resistance, previously determined for 41 seed zones, differ significantly among seed zones that are fixed for the alternate haplotypes, or contain a mixture of both haplotypes. Interspecific phylogenetic analysis reveals that the northern sugar pine haplotype belongs to a clade that includes Pinus albicaulis (whitebark pine) and all of the East Asian white pines. Furthermore, there is little cpDNA divergence between northern sugar pine and whitebark pine (dS = 0.00058). These results are consistent with a Pleistocene migration of whitebark pine into North America and subsequent chloroplast introgression from whitebark pine to sugar pine. This study demonstrates the importance of placing phylogeographical results in a broader phylogenetic context. Keywords: chloroplast introgression, Cronartium ribicola, phylogeography, Pinus lambertiana, Pinus subsect. Strobus, white pine blister rust resistance Received 13 January 2007; revision received 17 April 2007; accepted 11 June 2007

number of population genetic studies conducted in pine Introduction species. Ledig (1998) summarized genic diversity statistics Pinus has been described as ‘the most economically and (primarily from isozymes) for 51 of the c. 110 species of ecologically significant tree genus in the world’ (Richardson pine. Over the last 15 years, at least 26 species of pine have & Rundel 1998). Support for this claim is found in the large been evaluated for among-population DNA variation (tabulated in Petit et al. 2005; see also Chiang et al. 2006; Navascues et al. 2006). The focus of many of these studies Correspondence: A. Liston, Fax: 1 541 737 3573. is phylogeographical inference using chloroplast DNA E-mail: [email protected] (cpDNA). Despite the prevalence of interspecific §Present address: Department of Biology, University of South Dakota, Vermillion, SD 57069, USA hybridization in pines (Ledig 1998), few of these studies ‡Present address: Department of Biological and Physical Sciences, sample other related species, and thus cannot place the Montana State University–Billings, Billings, MT 59101, USA within-species genetic variation in a broader phylogenetic

PHYLOGENY AND PHYLOGEOGRAPHY 3927 context. This study of Pinus lambertiana, sugar pine, divergence within each haplotype, strongly suggests that demonstrates how resolution of interspecific phylogeny can secondary contact between two cytoplasmically divergent have a profound impact on the interpretation of intraspecific groups has occurred in the (evolutionarily) recent past. phylogeographical results. Just as studies can be enhanced Our phylogenetic analysis provides evidence that the by ‘putting the geography into phylogeography’ (Kidd & northern populations of P. lambertiana may have obtained Ritchie 2006), it is imperative to incorporate a broad their chloroplast via introgression from P. albicaulis, phylogenetic perspective as well. whitebark pine. The integration of phylogenetic and phyl Pinus lambertiana is one of the c. 20 species of subsection ogeographical approaches allowed us to recover this unex Strobus (Gernandt et al. 2005; Syring et al. 2007). This clade is pected evolutionary history. known by the common name ‘white pines’ and is distributed discontinuously throughout the Northern Hemisphere Materials and methods (Table 1). The monophyly of subsect. Strobus is strongly supported by chloroplast sequences (Gernandt et al. 2005; Organismal sampling and DNA genotyping Eckert & Hall 2006) and nuclear ribosomal DNA (Liston et al. 1999) and moderately supported by a low copy Pinus lambertiana Douglas is an economically and eco nuclear locus (Syring et al. 2007). The 20 species share a logically important conifer with a 1600-km latitudinal similar vegetative morphology (five relatively narrow and range extending from Oregon, USA to northern Baja strongly amphistomatic needles per fascicle) but differ California, Mexico. Eighty-four individuals representing dramatically in ovulate cone size (from 5 cm in Pinus pumila the geographical range of this species were included in this to 60 cm in P. lambertiana) and shape. The five Pinus species analysis. Nineteen additional species from Pinus subsect. (P. albicaulis, P. cembra, P. koraiensis, P. pumila, P. sibirica) with Strobus were sampled for the phylogenetic analysis indehiscent ‘closed’ cones adapted for bird dispersal were (Table 1), and Pinus gerardiana from subsect. Gerardianae traditionally treated as ‘subsect. Cembrae’, or stone pines. was used as the outgroup. DNA was extracted from the Like all North American members of subsect. Strobus, haploid megagametophyte of individual seeds as described sugar pine is highly susceptible to white pine blister rust, a in Syring et al. (2007). Polymerase chain reaction (PCR) disease caused by the heterocyclic rust fungus Cronartium amplification followed Gernandt et al. (2005). Approximately ribicola. This pathogen is native to Asia (Kinloch 2003) and 90% of the chloroplast matK open reading frame (1404 bp) was accidentally introduced to North America in the eastern and c. 150 bp of the 3′ trnK (UUU) intron (see Hausner et al. United States and British Columbia in the early 20th 2006 for a recent review) were amplified using primers century (Mielke 1943; Scharpf 1993). It has subsequently matK1F (Wang et al. 1999) and ORF515–900F (Gadek et al. spread to all species of subsect. Strobus that occur in the 2000). The chloroplast trnG (UCC) intron (c. 780 bp) was United States and Canada (Scharpf 1993; Kinloch 2003). amplified using the primers 3′ trnG and 5′ trnG2G (Shaw The disease results in cankers that girdle the main stem et al. 2005). For divergence time estimates between Pinus and kill infected seedlings and trees (Kinloch & Scheuner albicaulis and P. lambertiana (described below), three 2004). While a mapped locus (Cr1) that confers qualitative additional loci were added to the cpDNA data set for these (major gene) resistance has been identified in sugar pine species. These include new sequences for the trnL-trnF (Kinloch 1992, 2003; Devey et al. 1995), its frequency in intergenic region (including trnL exon 1 and its intron) and populations is typically low. Cr1 frequency varies from less the rpl16 intron (Shaw et al. 2005), as well as previously than 10% in the southern part of the range of P. lambertiana published rbcL sequences (Gernandt et al. 2005). Predicted to near absence in the north (Kinloch 1992; summarized in amplicon lengths were based on the Pinus koraiensis our Table 2). Attempts to increase the frequency of white chloroplast genome (EW Noh, JS Lee, YI Choi, MS Han, YS pine blister rust resistance have prompted federal (USDA Yi, and SU Han, unpublished, AY228468). Uncloned PCR Forest Service, US Bureau of Land Management) and private products were submitted to High-Throughput Sequencing agencies to make extensive seed collections for this species, Solutions (University of Washington) for ExoSAP purification and to initiate large-scale screening programmes. and automated capillary sequencing. Electropherograms We used DNA sequences of two chloroplast loci to were examined and aligned with bioedit 7.0.5.2 (Hall conduct a phylogenetic analysis of North American and 1999). All new sequences are deposited in GenBank under Eurasian members of Pinus subsect. Strobus, in concert the Accessions nos EF546699–EF546759. with a phylogeographical survey of P. lambertiana. Our Chloroplast matK and trnG sequences from 12 individuals study documents significant cpDNA divergence between of P. lambertiana identified two divergent haplotypes two major haplotypes in P. lambertiana. The distribution of (see results) abbreviated N (for North) and S (for South). these haplotypes is concordant with the geographical Using methods described in Liston (1992), we screened 72 distribution of white pine blister rust major gene resistance. individuals for an AluI restriction site that differentiates A narrow contact zone between haplotypes, and limited these two haplotypes. This assay is diagnostic for a C/T

3928 A. LISTON ET AL.

Table 1 Geographical origin and GenBank Accessions for samples sequenced for the cpDNA matK and trnG intron loci

Pinus species Haplotype Country Administrative unit matK accession trnG accession albicaulis* USA California: Mono Co. EF546699 EF546730 albicaulis* USA California: Siskiyou Co. ″ ″ albicaulis* USA Montana ″ ″ albicaulis* USA Oregon ″ ″ albicaulis*‡ USA Washington ″ ″ albicaulis* USA 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 USA California EF546709 EF546737 flexilis b USA Colorado EF546710 ″ flexilis c Canada Alberta EF546711 ″ koraiensis Russia EF546712 EF546738 koraiensis Japan ″ ″ koraiensis South Korea ″ ″ kwangtungensis China EF546713 EF546739 lambertiana* N USA California: seed zone 091 EF546715 EF546741 lambertiana N USA California: seed zone 372 ″ ″ lambertiana* N USA California: seed zone 516 ″ ″ lambertiana*‡ N USA California: seed zone 732 ″ ″ lambertiana* N USA Oregon: seed zone 472 ″ ″ lambertiana N USA Oregon: seed zone 731 ″ ″ lambertiana S USA California: seed zone 120 EF546714 EF546740 lambertiana S USA California: seed zone 526 ″ ″ lambertiana*‡ S USA California: seed zone 731 ″ ″ lambertiana S USA California: seed zone 992 ″ ″ lambertiana S USA California: seed zone 994 ″ ″ lambertiana S′ Mexico Baja California EF546716 ″ monticola a USA Oregon EF546717 EF546742 monticola b Canada British Columbia EF546718 EF546743 monticola b USA 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 USA Texas ″ ″ strobus USA Minnesota EF546727 EF546750 strobus Canada Newfoundland ″ ″ strobus USA North Carolina ″ ″ wallichiana† a Pakistan Punjab EF546728 EF546751 wallichiana b Nepal Karnali EF546729 ″ gerardiana† Pakistan Gilgit AY115801 EF546752

Haplotypes N, S and S’ for P. lambertiana are described in the text. In other species, the letters a, b and c were applied as needed for multiple haplotypes within a species. Voucher specimens are deposited at Oregon State University (OSC), unless indicated otherwise; *sequenced for trnL-F, GenBank Accession nos EF546753–EF546756; †Voucher deposited at the Silva Tarouca Research Institute for Landscape and Ornamental Gardening, Prùhonice, Czech Republic (RILOG); ‡ sequenced for rpl16, GenBank Accession nos EF546757–EF546759.

Table 2 The frequency of the white pine blister rust resistance gene Cr1 in sugar pine seed zones, compared to the number of southern and northern haplotypes. Data in columns 2 and 3 are from the 41 seed zones sampled by Kinloch (1992)

Trees/seeds Frequency of major Haplotype Frequency of Seed zone sampled gene resistance group N haplotype S haplotype 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 0.00 322 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 1.00 994 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

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd 3930 A. LISTON ET AL. polymorphism at position 1352 in matK. Since chloroplasts nonparametric bootstraps were performed using poptools are paternally inherited in Pinus (Neale & Sederoff 1989), version 2.7 (Hood 2006). For mapping, the program trs2ll the AluI polymorphism was also used to determine mater (Wefald 2001) was used to convert township/range/section nal vs. pollen cpDNA haplotype by extracting DNA from localities to latitude and longitude. megagametophyte (maternal) and embryo (paternal) tissue in 31 individual seeds. Results

Phylogenetic, phylogeographical and statistical analyses Phylogeographical haplotype variation in sugar pine Phylogenetic analyses and constraint tests were conducted Sequences of cpDNA matK and trnG intron in 12 Pinus with paup* 4.0b10 (Swofford 2002) following the procedures lambertiana individuals revealed two predominant outlined in Syring et al. (2005). Indels were added to the haplotypes with fixed differences at 10 sites (seven in matK, parsimony data matrix as binary characters. Parsimony including three amino acid replacements; three in the trnG analysis was conducted with a heuristic search strategy intron). One additional haplotype was observed in the Baja of 1000 random addition sequences and tree-bisection– California individual (an autapomorphic matK replacement reconnection swapping. Branch support was assessed substitution). The sequence results combined with AluI using 2000 bootstrap replicates. Indels and duplicate restriction digest assays for 72 individuals demonstrated sequences were excluded, and the most appropriate an abrupt transition in the distribution of the two likelihood model was selected using the method of predominant haplotypes (Fig. 1A). Plants from Oregon Posada & Crandall (1998) and AIC scores at the ﬁndmodel and northwestern California (Klamath mountains and website (http://hcv.lanl.gov/content/hcv-db/findmodel/ North Coast range) were fixed for a common haplotype ‘N’ findmodel.html). To evaluate whether sequences diverged (thymine at position 1352 of matK), while plants from at clock-like rates, maximum-likelihood trees were estimated the Sierra Nevada and Transverse and Peninsular ranges using the GTR + γ model as implemented in paup* 4.0b10 in California were fixed for the alternate haplotype ‘S’ (Swofford 2002). Likelihood scores obtained with and (cytosine at position 1352). The contact zone between the without a molecular clock constraint were evaluated using N and S haplotypes occurs near latitude 40°30′N in the likelihood ratio test (LRT) of Muse & Weir (1992). northeastern California, and the zone of polymorphism is Under assumptions of a molecular clock, the divergence remarkably well-defined, spanning less than 150 km of the time (Tdiv) between two groups of sequences is c. 1600-km latitudinal range of this species (Figs 1 and 2). µ approximately Tdiv = dS/2 , where dS is the average Megagametophyte and embryo comparisons in 31 pairwise distance among sequences at presumably neutral individuals from the contact zone revealed that 12 (39%) (synonymous and noncoding) sites and µ is the neutral seeds contained different maternal and paternal haplotypes, mutation rate. Estimates of dS were calculated with dnasp indicating that seedlings are frequently sired by pollen 4.10.9 (Rozas et al. 2003; note that the program uses the parents with different haplotypes than the ovulate parent. abbreviation Ks). The estimate of µ for Pinus cpDNA Embryos with the S haplotype were found in seeds from (0.22 × 10–9 silent substitutions site–1 years–1; standard eight N haplotype maternal trees distributed in seed zones error = 0.55 × 10–10) is based on a divergence time of 85 522, 523, 732 and 771 (Fig. 2). The converse pattern was also million years between the two subgenera of Pinus; see observed as four S haplotype maternal plants from seed Willyard et al. (2007) for details. Divergence times estimated zones 522 and 731 produced seed containing N haplotype here are reported with ± one standard error. embryos (Fig. 2). To investigate whether the white pine blister rust major At least one sugar pine individual was assayed for the S gene resistance allele (Cr1) frequencies were different vs. N haplotype in 35 of the 41 seed zones sampled by among chloroplast haplotype classes, 41 tree seed zones Kinloch (1992) in his survey of Cr1 allele frequencies (the used in blister rust screening (Table 2; Kinloch 1992) were factor conferring major gene resistance to white pine classified as either fixed for the N haplotype, fixed for the blister rust; Table 2), with intensive sampling in the contact S haplotype, or polymorphic for the two haplotypes. One- zone (Fig. 2). The haplotype for six unassayed seed zones way analysis of variance (anova) was used to examine (one in northwestern California and five in Oregon) was variance partitioning and to test the hypothesis that means inferred to be N based on results from adjacent seed zones. were equivalent among these three groups. Given the large The Baja California haplotype S′ (differing from S by one number of zeros present in estimated Cr1 allele frequencies substitution, Fig. 3) was grouped with S for this analysis. (especially in the northern part of the range), we also esti The N, S, and polymorphic seed zones showed significantly mated means and confidence intervals for allele frequencies different Cr1 frequencies by one-way anova (F = 32.3, in the N, S, and mixed haplotype groups using 10 000 P = 7.6 × 10– 9). The N haplotype seed zones had the lowest nonparametric bootstrap resamplings. Statistical and Cr1 allele frequency (0.0032 ± 0.0038; n = 23), mixed

Fig. 1 (A) Sampled individuals of Pinus lambertiana in western North America. Yellow squares represent the S haplotype and blue squares represent the N haplotype. Forest tree seed zones sampled by Kinloch (1992) and/or this study are shaded according to the frequency of the Cr1 allele (Table 2), ranging from 0% (no shading) to 8.9% (dark grey, seed zone 120). Seed zones outlined in red were not sampled for Cr1. The haplotype contact zone (green rectangle) is shown in more detail in Fig. 2. (B) Approximate geographical distribution of P. lambertiana (dark grey) and P. albicaulis (light grey) from Critchfield & Little (1966). Red triangles represent P. albicaulis samples used in this study.

haplotype seed zones had intermediate Cr1 frequencies (shown) or clade (not shown). All Asian species of subsect. (0.0380 ± 0.0185; n = 3), and S haplotype seed zones had Strobus form a strongly supported clade that includes the the highest Cr1 frequencies (0.0484 ± 0.0260; n = 15). North American Pinus albicaulis and the P. lambertiana Nonparametric bootstrapping resulted in the same mean N haplotype. At these two loci, no sequence divergence allele frequencies and confidence intervals. was found among northern P. lambertiana, P. albicaulis and representatives of six Asian Pinus species (P. dalatensis, Phylogenetic relationships and variation in sugar pine and other P. koraiensis, P. pumila, P. sibirica, P. wallichiana, P. kwan white pine species. Alignment of matK and trnG intron gtungensis) and P. cembra from Romania. The P. lambertiana sequences required one 6-bp indel in the matK ORF and S haplotypes and the remaining North American white four indels (1, 4, 10 and 15 bp) in the trnG intron. Three of pine species form a grade, with P. monticola in a strongly the indels were confined to Pinus parviflora, the 15-bp supported sister position to the Eurasian clade. trnG intron indel was shared by P. ayacahuite, P. flexilis, Constraining P. lambertiana samples to monophyly results P. strobiformis and P. peuce, and the 1-bp indel was in trees of 66 steps, which is significantly longer based on autapomorphic in P. armandii ‘b’. Combined phylogenetic both Templeton (P = 0.0039) and Kishino–Hasegawa tests analysis of matK (1545 bp), trnG intron (746 bp) and the five (P = 0.0027). indels resulted in two most parsimonious trees of length 57 Maximum-likelihood trees obtained with and without with a consistency index of 0.91 (Fig. 3). The two trees a molecular clock were topologically identical to one of differ in the resolution of Pinus monticola as a grade the most parsimonious trees and were not significantly

Fig. 2 Detail of the contact zone between the S (yellow) and N (blue) cpDNA haplotypes of Pinus lambertiana. Squares represent the maternal (megagametophyte) haplotype and circles represent the pater nal (embryo) haplotype. Asterisks denote white pine blister rust resistant individuals.

different from each other based on the LRT (∆ln L = haplotypes is 15.5 ± 3.9 Ma (dS = 0.00681) and between the 10.44013, χ2 = 20.88, d.f. = 19, P = 0.34). This suggests that the S haplotype of P. lambertiana and the other North American sequences are diverging at equivalent rates, and a simple white pines is 9.0 ± 2.25 Ma (dS = 0.00396). molecular clock calculation can be applied. The mean dS between P. monticola and the Asian clade is 0.00232, resulting Discussion in an estimated divergence of 5.3 ± 1.3 million years ago (Ma; Late Miocene–Early Pliocene). Sequences of 5799 bp The pattern of genetic subdivision in sugar pine of cpDNA (adding rbcL, rpl16 and trnL-F to the matK and trnG used in the phylogenetic analysis) revealed a single Previous accounts of sugar pine have described neither substitution in trnL-F between a P. albicaulis accession morphological nor ecological differences that can be (Washington state) and an N haplotype P. lambertiana associated with the phylogeographical pattern observed (dS = 0.00058), resulting in an estimated divergence of here. In fact, Mirov (1967) described Pinus lambertiana as 0.6 ± 0.15 Ma (Pleistocene). Sequences of trnL-F from an ‘rather stable morphologically’ (p. 142) and he considered additional five accessions of P. albicaulis and three of N it to be a prime example of a ‘good species’ that is ‘clearly haplotype P. lambertiana (Table 1) confirmed that this is a delimited [and] can be identified without difficulty’ fixed difference between these two taxa. The estimated (p. 531). The southern limit of the contact zone (Fig. 1A) divergence time between the two P. lambertiana chloroplast does coincide with the interface of the Cascade and Sierra

The allozyme data also separated the Sierra Nevada populations from those sampled in southern California and Baja California. This division is not apparent in our data set. However, a unique substitution was found in the matK sequence of the Baja California individual. This evidence for genetic isolation is consistent with the geographical isolation of this disjunct population (Fig. 1A,B). The narrow transition zone between the N and S haplo types of P. lambertiana suggests that these populations have only recently come into contact. This would be consistent with a Holocene range expansion from separate northern and southern refugia. There is abundant evidence from the pollen record for postglacial movement of pines (Mohr et al. 2000; Thompson & Anderson 2000). Unfortunately, individual Pinus species cannot be identified from pollen. Narrow haplotype (chloroplast or mitochondrial) transition zones observed in other western North American plants (Soltis et al. 1997; Aagaard et al. 1998; Latta & Mitton 1999; Johansen & Latta 2003) have also been attributed to postglacial contact of previously separated populations. The examination of maternal and paternal haplotypes offers insight into the dynamics of dispersal at the contact zone (Fig. 2). The two P. lambertiana trees sampled in the southern part of seed zone 731 represent a population that is isolated on Happy Camp mountain, Modoc County. These trees have the S haplotype, and are presumed to Fig. 3 One of two most parsimonious trees estimated from have colonized this location by long-distance seed dispersal. cpDNA matK and trnG intron sequences. Bootstrap values are The closest sampled potential source is c. 90 km away. shown below the branches. Tree length = 57, consistency The large (228 ± 40 mg) and ‘flimsy’ winged seeds of P. index = 0.91, retention index = 0.97. When applicable, haplotypes lambertiana are ‘seldom dispersed far by wind’, but caching (see Table 2) and the number of individuals that share a particular by Steller’s jays (Thayer & Vander Wall 2005) and Clark’s sequence and haplotypes follow the species names. nutcrackers (D. Tomback, personal communication) can potentially lead to long-distance dispersal. No other example of a disjunct haplotype was observed. Embryos possessing Nevada mountain ranges, characterized by relatively recent the S haplotype occur up to 25 km north of the northern volcanic activities and predominantly metamorphics (with most potential source trees, indicating that pollen flow granitic intrusions and volcanic activities), respectively advances ahead of seed dispersal. The two Happy Camp (Hickman 1993). Although this geological transition is mountain trees whose megagametophytes carry the S used to demarcate two floristic subregions, there is no haplotype have apparently been pollinated by trees of the apparent vegetational break between the forests of the N haplotype. Comparison of chloroplast and mitochondrial Cascades and northern Sierra Nevada (Hickman 1993). haplotypes in a Pinus ponderosa contact zone in western In her PhD thesis, Martinson (1997) conducted an analysis Montana has found a similar pattern of more extensive of allozyme data collected by Conkle (1996, abstract only). pollen flow and rare long-distance seed dispersal (Latta & Forty populations and 400 individuals of P. lambertiana Mitton 1999; Johansen & Latta 2003). were assayed at 30 allozyme loci. Average heterozygosity One of the most surprising results of our study is the was 0.22, and no geographical region showed reduced concordance between the distribution of the two cpDNA genetic diversity. Clustering of genetic distances separated haplotypes and the relative frequency of a white pine blister populations from Oregon and northwestern California rust major gene resistance locus (Cr1) in sugar pine. Kinloch from populations in the Sierra Nevada. Unfortunately, no (1992) described the pattern of Cr1 frequency as a cline. populations were sampled in the chloroplast haplotype In contrast, the significant differences in Cr1 frequency contact zone in northeastern California. Thus, although observed among the three haplotype groups (N, S, and the reported allozyme differentiation may correspond to mixed) suggests that the gene frequency does not change the chloroplast haplotype distribution, it will require in a gradual manner, but rather shows the same abrupt allozyme sampling in the contact zone to confirm this. transition as observed in the chloroplast. There is no

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd 3934 A. LISTON ET AL. evidence for a causal link between the cpDNA haplotype been named ‘chloroplast capture’ (Rieseberg & Soltis 1991; and Cr1 distribution patterns. It is well-established that Tsitrone et al. 2003) and has been offered as an explanation resistance shows nuclear inheritance (Kinloch 1992), and for similar patterns of cytonuclear incongruence observed the highest frequencies of Cr1 (4–9% per seed zone) are far in many plant genera (reviewed in Rieseberg et al. 1996; lower than the frequency of the S haplotype. Furthermore, Wendel & Doyle 1998). Tsitrone et al. (2003) have modelled examination of three resistant trees (heterozygous for the the process under the assumption of maternal chloroplast dominant Cr1 allele; J. Gleason, unpublished data) in inheritance and they determined conditions likely to the contact zone found both N and S haplotypes (Fig. 2). promote its occurrence. A key aspect of their model is the Note that all other genotyped individuals in the contact observation that cytonuclear incompatibility often results zone are nonresistant (J. Gleason, unpublished data). in full or partial male sterility and thus can increase maternal We predict that the concordance between the Cr1 and fitness through enhanced seed production. Although they chloroplast haplotype frequencies reflects a common do not explicitly model paternal inheritance (the situation history of genetic isolation, followed by recent migration in Pinaceae), they suggest that chloroplast introgression and contact (see below). should be less common here, since cytonuclear interactions typically reduce male fitness. Theoretically, chloroplast substitution could result in an advantage in male function, Evidence for chloroplast introgression but this situation has apparently not been documented The chloroplast haplotypes of P. lambertiana resolve in two (Tsitrone et al. 2003). However, a pattern consistent with different clades in the phylogenetic analysis of Pinus chloroplast introgression has been observed in other subsect. Strobus, one comprised of five other North species of Pinaceae, e.g. Pinus montezumae (Matos & Schaal American species and the other encompassing 12 Eurasian 2000), Pinus muricata (Hong et al. 2003) and Larix sibirica species and the North American Pinus albicaulis (whitebark (Wei & Wang 2003). pine). Two biological processes could explain these results: Petit et al. (2003) offer ‘pollen swamping’ as an alternative incomplete lineage sorting of an ancestral polymorphism, explanation for the lack of cytoplasmic (cpDNA and mito or chloroplast introgression. Incomplete lineage sorting chondrial DNA) differentiation between Quercus petraea has been determined to be the most probable source of and Quercus robur, two sympatric oaks that are consistently widespread allelic nonmonophyly at nuclear loci in species differentiated at nuclear markers. In their scenario, Q. robur of Pinus subgenus Strobus (Syring et al. 2007). It has also seed disperses into new habitats which are subsequently been considered a potential cause of similar patterns colonized by Q. petraea via pollen flow, resulting in F1 observed in chloroplast studies in other plant species hybrids. Asymmetric introgression and strong selection (Tsitrone et al. 2003). However, the stochastic process of for the Q. petraea phenotype results in mixed populations incomplete lineage sorting is not expected to show the that share a single cytoplasm. Petit et al. (2003) invoke the strong geographical partitioning observed for the two fact that the seeds of Q. robur are better adapted to bird- chloroplast haplotypes. On the other hand, if the two dispersal than Q. petraea, and thus are more likely to establish subgroups of sugar pine have been separated since the through long-distance dispersal. A similar relationships Miocene (15.1 ± 3.8 Ma) and each retained a different exists between P. albicaulis (dispersed mainly by the pine haplotype, one might expect to find morphological seed specialist, Clark’s nutcracker, Tomback 2005) and divergence between (and sequence variation within) the P. lambertiana (dispersed to a limited extent by wind and two groups, particularly since this same interval has primarily by generalist Steller’s jays and yellow pine apparently been accompanied by multiple speciation events chipmunks, Thayer & Vander Wall 2005). In both cases, in these lineages. Although some sequence divergence was the better disperser is thought to have contributed its found in the S haplotype clade (in the geographically chloroplast to the other species. An important caveat is isolated Baja California population), none was found in the that the cytoplasm is maternally inherited in oaks, and thus N haplotype clade. The amount of sequence divergence carried by the seed and not by the pollen as in pines. between the S haplotype of P. lambertiana and the other If chloroplast introgression is responsible for this pattern, North American white pines is consistent with genetic how does one account for the otherwise ‘Eurasian’ haplotype isolation since the Miocene. In contrast, the high sequence in two species of North American subsect. Strobus? The similarity between the N haplotype of sugar pine and the cpDNA-based phylogeny (Fig. 3) requires two dispersal Asian clade is suggestive of a much more recent shared events between western North America and Asia. The plastid ancestry. disjunction between P. monticola and the Eurasian clade To account for this unexpected genetic similarity, we can be dated to the Late Miocene or Early Pliocene. This suggest that the N haplotype of P. lambertiana may have timing is consistent with estimates of 2.6–16.7 Ma from its origin in a chloroplast introgression event involving P. 11 other eastern Asian/western North American plant albicaulis. Introgression-mediated chloroplast transfer has disjunctions (Zhu et al. 2006; Zhang et al. 2007). It is

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd PHYLOGENY AND PHYLOGEOGRAPHY 3935 noteworthy that well-preserved Pliocene and Pleistocene phylogenetic analyses of pines. Gernandt et al. (2005) fossils of P. monticola have been collected in northeastern sampled P. lambertiana in Oregon (a region fixed for the N Siberia and Alaska (reviewed in Bingham et al. 1972). haplotype), while Eckert & Hall (2006) sampled an individual Following diversification of the Eurasian white pines, and from southern California (a region fixed for the S haplo origin of the ‘closed cone’ morphology characteristic of type). Each study placed P. lambertiana in a position that is stone pines, we propose that the ancestor of P. albicaulis consistent with the resolution of the respective haplotypes dispersed from Asia to North America via Beringia, in our study (Fig. 3). This demonstrates the importance of presumably during the Pleistocene. Beringia was largely sampling multiple individuals per species in phylogenetic unglaciated during the Pleistocene, and is known to have analyses of closely related species (see also Syring et al. served as a refugium for trees and shrubs, including 2007). Pinus, through the late glacial maximum (Brubaker et al. The results reported here provide the first phylogeo 2005). Krutovskii et al. (1995) proposed a similar scenario graphical hypothesis for an ecologically and economically based on allozyme and chloroplast restriction fragment important conifer, P. lambertiana. By placing the intraspecific analysis of the ‘subsect. Cembrae’ pines, but placed the results within a broader phylogenetic context, further migration at an earlier period (Pliocene). insights were gained into the evolutionary history of this Evidence from mitochondrial DNA haplotypes has been species. The novel hypotheses of two Pleistocene refugia used to infer the existence of three Late Pleistocene refugia for P. lambertiana and chloroplast introgression with P. for P. albicaulis (Richardson et al. 2002): western Wyoming, albicaulis can be tested with additional molecular markers, western Idaho and the southern Cascades of Oregon. The in particular nuclear and mitochondrial loci. The observation southern Cascades currently support large populations of that the two chloroplast haplotypes demarcate population P. lambertiana, and we propose that this region could also groups that differ in their vulnerability to white pine blister have served as a northern glacial refugium for sugar pine, rust is also a significant result that merits further attention. or possibly farther west in the Klamath/Siskiyou Mountains Beyond sugar pine, this study demonstrates the value of (a region with several palaeo-endemic conifers, e.g. Picea including an interspecific phylogenetic component in breweriana and Chamaecyparis lawsoniana). Sugar pine is phylogeographical research. Without this broader per common here, but whitebark pine has only recently been spective, the antiquity of the haplotype groups would discovered in a small population on Mount Ashland (nine remain unknown, as would the unexpected, and potentially individuals; Murray 2005). Regardless of their current reticulate, history of these species. geographical distributions, sympatry in a glacial refugium could have provided the opportunity for the northern Acknowledgements populations of sugar pine to acquire the chloroplast of whitebark pine. Although P. albicaulis generally occurs at We are indebted to John Gleason (USFS, Placerville Nursery and higher altitudes than P. lambertiana, the two are partly Disease Resistance Program), Jerry Berdeen and Richard Sniezko sympatric in northern California and southern Oregon (USFS, Dorena Genetic Resource Center) and David Johnson (USFS, Institute of Forest Genetics) for supplying sugar pine seeds (Fig. 1B). There is also evidence that P. lambertiana formerly and Roman Businsky for providing his collections of Asian occurred at higher altitudes than its current distribution species. We thank David Gernandt, Bohun Kinloch, Todd Ott, (May 1974; Millar et al. 2006). Paul Severns, Richard Sniezko, and Diana Tomback for constructive Two factors are required for chloroplast introgression to comments on the manuscript. This research was funded by occur; sympatry and reproductive compatibility. While National Science Foundation grants DEB 0317103 and ATOL many interspecific crosses have been conducted in subsect. 0629508, an NSF Research Experience for Undergraduates Strobus, there is no record of attempts to cross P. lambertiana supplement, and the Pacific Northwest Research Station, USDA Forest Service. and P. albicaulis (Critchfield 1986; R. Sniezko, personal communication). Critchfield & Kinloch (1986) do, however, document interspecific hybridization between P. lambertiana References and Asian members of subsect. Strobus, namely P. armandii Aagaard JE, Krutovskii KV, Strauss SH (1998) RAPD markers of and P. koraiensis. Seed set in these artificial crosses averaged mitochondrial origin exhibit lower population diversity and 2.1% and 0.2% viable seed per cone, respectively. In higher differentiation than RAPDs of nuclear origin in Douglas contrast, P. lambertiana is apparently intersterile with all fir. Molecular Ecology, 7, 801–812. other North American white pines (Critchfield 1986; Bingham RT, Hoff RJ, Steinhoff RJ (1972) Genetics of Western White Fernando et al. 2005). No naturally occurring hybrids of Pine, USDA Forest Service Research Paper WO-12. USDA Forest Service, Washington, D.C. P. lambertiana have been recorded (Mirov 1967; Critchfield Brubaker LB, Anderson PM, Edwards ME, Lozhkin AV (2005) 1986; R. Sniezko, personal communication). Beringia as a glacial refugium for boreal trees and shrubs: new Our results explain why P. lambertiana was resolved in perspectives from mapped pollen data. Journal of Biogeography, conflicting positions in recent chloroplast sequence-based 32, 833–848.

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