1
Molecular Ecology In press.
2 3
Interspecific phylogenetic analysis enhances intraspecific phylogeographic inference: A
4
case study in Pinus lambertiana
5
AARON LISTON*, MARIAH PARKER-DEFENIKS*, JOHN V. SYRING*,‡, ANN
6
WILLYARD*,§ , and RICHARD CRONN†
7 8
*
9
USA, †Pacific Northwest Research Station, USDA Forest Service, 3200 SW Jefferson Way,
10
Corvallis, Oregon 97331,USA, ‡Current Address: Department of Biological and Physical
11
Sciences, Montana State University – Billings, Billings, Montana 59101, USA, §Current
12
Address:Department of Biology, University of South Dakota, Vermillion, SD 57069 USA
Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331,
13 14 15
Keywords: chloroplast introgression, Cronartium ribicola, Pinus lambertiana,Pinus subsect.
16
Strobus, phylogeography, white pine blister rust resistance
17 18 19
Correspondence: A. Liston. Department of Botany and Plant Pathology, 2082 Cordley Hall,
20
Oregon State University, Corvallis, Oregon 97331, USA. Fax: 1 541 737 3573. E-mail:
21
[email protected]
22 23 24
Running title: Phylogeny and phylogeography
1
25
Abstract
26
Pinus lambertiana (sugar pine) is an economically and ecologically important conifer with a
27
1600 km latitudinal range extending from Oregon, USA to northern Baja California, Mexico.
28
Like all North American white pines (subsect. Strobus), sugar pine is highly susceptible to white
29
pine blister rust, a disease caused by the fungus Cronartium ribicola. We conducted a chloroplast
30
DNA survey of Pinus subsect. Strobus with comprehensive geographic sampling of P.
31
lambertiana. Sequence analysis of 12 sugar pine individuals revealed strong geographic
32
differentiation for two chloroplast haplotypes. A diagnostic restriction site survey of an
33
additional 72 individuals demarcated a narrow 150 km contact zone in northeastern California. In
34
the contact zone, maternal (megagametophtye) and paternal (embryo) haplotypes were identified
35
in 31 single seeds, demonstrating bidirectional pollen flow extending beyond the range of
36
maternal haplotypes. The frequencies of the Cr1 allele for white pine blister rust major gene
37
resistance, previously determined for 41 seed zones, differ significantly among seed zones that
38
are fixed for the alternate haplotypes, or contain a mixture of both haplotypes. Interspecific
39
phylogenetic analysis reveals that the northern sugar pine haplotype belongs to a clade that
40
includes P. albicaulis (whitebark pine) and all of the East Asian white pines. Furthermore, there
41
is little cpDNA divergence between northern sugar pine and whitebark pine (dS = 0.00058).
42
These results are consistent with a Pleistocene migration of whitebark pine into North America
43
and subsequent chloroplast introgression from whitebark pine to sugar pine. This study
44
demonstrates the importance of placing phylogeographic results in a broader phylogentic
45
context.
2
46
Introduction
47 48
Pinus has been described as “the most economically and ecologically significant tree genus in
49
the world” (Richardson and Rundel 1998). Support for this claim is found in the large number of
50
population genetic studies conducted in pine species. Ledig (1998) summarized genic diversity
51
statistics (primarily from isozymes) for 51 of the ca. 110 species of pine. Over the last 15 years,
52
at least 26 species of pine have been evaluated for among population DNA variation (tabulated
53
in Petit et al. 2005, see also Chiang et al. 2006; Navascués et al. 2006). The focus of many of
54
these studies is phylogeographic inference using chloroplast DNA. Despite the prevalence of
55
interspecific hybridization in pines (Ledig 1998) few of these studies sample other related
56
species, and thus cannot place the within species genetic variation in a broader phylogenetic
57
context. This study of Pinus lambertiana, sugar pine, demonstrates how resolution of
58
interspecific phylogeny can have a profound impact on the interpretation of intraspecific
59
phylogeographic results. Just as studies can be enhanced by “putting the geography into
60
phylogeography” (Kidd and Ritchie 2006) it is imperative to incorporate a broad phylogenetic
61
perspective as well.
62 63
Pinus lambertiana is one of the ca. 20 species of subsection Strobus (Gernandt et al. 2005;
64
Syring et al. 2007). This clade is known by the common name “white pines” and is distributed
65
discontinuously throughout the Northern Hemisphere (Table 1). The monophyly of subsect.
66
Strobus is strongly supported by chloroplast sequences (Gernandt et al. 2005; Eckert and Hall
67
2006) and nuclear ribosomal DNA (Liston et al. 1999) and moderately supported by a low copy
68
nuclear locus (Syring et al. 2007). The 20 species share a similar vegetative morphology (five
69
relatively narrow and strongly amphistomatic needles per fascicle) but differ dramatically in
70
ovulate cone size (from 5 cm in P. pumila to 60 cm in P. lambertiana) and shape. The five
71
species (P. albicaulis, P. cembra, P. koraiensis, P. pumila, P. sibirica) with indehiscent “closed”
72
cones adapted for bird dispersal were traditionally treated as “subsect. Cembrae”, or stone pines.
73 74
Like all North American members of subsect. Strobus, sugar pine is highly susceptible to white
75
pine blister rust, a disease caused by the heterocyclic rust fungus Cronartium ribicola. This
76
pathogen is native to Asia (Kinloch 2003) and was accidentally introduced to North America in
3
77
the eastern U.S. and British Columbia in the early 20th century (Mielke 1943; Scharpf 1993). It
78
has subsequently spread to all species of subsect. Strobus that occur in the United States and
79
Canada (Scharpf 1993; Kinloch 2003). The disease results in cankers that girdle the main stem
80
and kill infected seedlings and trees (Kinloch and Scheuner 2004). While a mapped locus (Cr1)
81
that confers qualitative (major gene) resistance has been identified in sugar pine (Kinloch 1992,
82
2003; Devey et al. 1995), its frequency in populations is typically low. Cr1 frequency varies
83
from less than 10% in the southern part of the range of P. lambertiana to near absence in the
84
north (Kinloch 1992, summarized in our Table 2) . Attempts to increase the frequency of white
85
pine blister rust resistance have prompted federal (USDA Forest Service, US Bureau of Land
86
Management) and private agencies to make extensive seed collections for this species, and to
87
initiate large-scale screening programs.
88 89
We used DNA sequences of two chloroplast loci to conduct a phylogenetic analysis of North
90
American and Eurasian members of Pinus subsect. Strobus, in concert with a phylogeographic
91
survey of P. lambertiana. Our study documents significant cpDNA divergence between two
92
major haplotypes in P. lambertiana. The distribution of these haplotypes is concordant with the
93
geographic distribution of white pine blister rust major gene resistance. A narrow contact zone
94
between haplotypes, and limited divergence within each haplotype, strongly suggests that
95
secondary contact between two cytoplasmically divergent groups has occurred in the
96
(evolutionarily) recent past. Our phylogenetic analysis provides evidence that the nothern
97
populations of Pinus lambertiana may have obtained their chloroplast via introgression from P.
98
albicaulis, whitebark pine. The integration of phylogenetic and phylogeographic approaches
99
allowed us to recover this unexpected evolutionary history.
100 101 102
Materials and Methods
103
Organismal sampling and DNA genotyping
104
Pinus lambertiana Douglas is an economically and ecologically important conifer with a 1600
105
km latitudinal range extending from Oregon, USA to northern Baja California, Mexico. Eighty-
106
four individuals representing the geographic range of this species were included in this analysis.
107
Nineteen additional species from Pinus subsect. Strobus were sampled for the phylogenetic
4
108
analysis (Table 1), and Pinus gerardiana from subsect. Gerardianae was used as the outgroup.
109
DNA was extracted from the haploid megagametophyte of individual seeds as described in
110
Syring et al. (2007). PCR amplification followed Gernandt et al. (2005). Approximately 90% of
111
the chloroplast matK open reading frame (1404 bp) and ca. 150 bp of the 3' trnK (UUU) intron
112
(see Hausner et al. 2005 for a recent review) were amplified using primers matK1F (Wang et al.
113
1999) and ORF515-900F (Gadek et al. 2000). The chloroplast trnG (UCC) intron (ca. 780 bp)
114
was amplified using the primers 3' trnG and 5' trnG2G (Shaw et al. 2005). For divergence time
115
estimates between P. albicaulis and P. lambertiana (described below), three additional loci were
116
added to the cpDNA data set for these species. These include new sequences for the trnL-trnF
117
intergenic region (including trnL exon 1 and its intron) and the rpl16 intron (Shaw et al. 2005),
118
as well as previously published rbcL sequences (Gernandt et al. 2005). Predicted amplicon
119
lengths were based on the Pinus koraiensis chloroplast genome (Noh et al. unpublished,
120
AY228468). Uncloned PCR products were submitted to High-Throughput Sequencing Solutions
121
(University of Washington) for ExoSAP purification and automated capillary sequencing.
122
Electropherograms were examined and aligned with BioEdit 7.0.5.2 (Hall 1999). All new
123
sequences are deposited in GenBank under the accessions EF546699-EF546759.
124 125
Chloroplast matK and trnG sequences from 12 individuals of P. lambertiana identified two
126
divergent haplotypes (see results) abbreviated N (for North) and S (for South). Using methods
127
described in Liston (1992), we screened 72 individuals for an AluI restriction site that
128
differentiates these two haplotypes. This assay is diagnostic for a C/T polymorphism at position
129
1352 in matK. Since chloroplasts are paternally inherited in Pinus (Neale and Sederoff 1989), the
130
AluI polymorphism was also used to determine maternal versus pollen cpDNA haplotype by
131
extracting DNA from megagametophyte (maternal) and embryo (paternal) tissue in 31 individual
132
seeds.
133 134
Phylogenetic, phylogeographic and statistical analyses
135
Phylogenetic analyses and constraint tests were conducted with PAUP* 4.0b10 (Swofford 2002)
136
following the procedures outlined in Syring et al. (2005). Indels were added to the parsimony
137
data matrix as binary characters. Parsimony analysis was conducted with a heuristic search
138
strategy of 1000 random addition sequences and TBR swapping. Branch support was assessed
5
139
using 2000 bootstrap replicates. Indels and duplicate sequences were excluded, and the most
140
appropriate likelihood model was selected using the method of Posada and Crandall (1998) and
141
AIC scores at the FindModel website (http://hcv.lanl.gov/content/hcv-
142
db/findmodel/findmodel.html). To evaluate whether sequences diverged at clock-like rates,
143
maximum likelihood trees were estimated using the GTR+ γ model as implemented in PAUP*
144
4.0b10 (Swofford 2002). Likelihood scores obtained with and without a molecular clock
145
constraint were evaluated using the likelihood ratio test (LRT) of Muse and Weir (1992). Under
146
assumptions of a molecular clock, the divergence time(Tdiv) between two groups of sequences is
147
approximately Tdiv = dS / 2μ where dS is the average pairwise distance among sequences at
148
presumably neutral (synonymous and non-coding) sites and μ is the neutral mutation rate.
149
Estimates of dS were calculated with DnaSP 4.10.9 (Rozas et al. 2003, note that the program
150
uses the abbreviation Ks). The estimate of μ for Pinus cpDNA (0.22 ×10-9 silent substitutions
151
site-1 year-1; standard error = 0.55 ×10-10) is based on an 85 million years ago (mya) divergence
152
time between the two subgenera of Pinus; see Willyard et al. (2007) for details. Divergence
153
times estimated here are reported with ± one standard error.
154 155
To investigate whether the white pine blister rust major gene resistance allele (Cr1) frequencies
156
were different among chloroplast haplotype classes, 41 tree seed zones used in blister rust
157
screening (Table 2; Kinloch 1992) were classified as either fixed for the N haplotype, fixed for
158
the S haplotype, or polymorphic for the two haplotypes. One-way ANOVA was used to examine
159
variance partitioning and to test the hypothesis that means were equivalent among these three
160
groups. Given the large number of zeros present in estimated Cr1 allele frequencies (especially
161
in the northern part of the range), we also estimated means and confidence intervals for allele
162
frequencies in the N, S, and mixed haplotype groups using 10,000 nonparametric bootstrap
163
resamplings. Statistical and nonparametric bootstraps were performed using PopTools version
164
2.7 (Hood 2006). For mapping, the program TRS2LL (Wefald 2001) was used to convert
165
township/range/section localities to latitude and longitude.
166 167
Results
168
Phylogeographic haplotype variation in sugar pine
6
169
Sequences of cpDNA matK and trnG intron in 12 Pinus lambertiana individuals revealed two
170
predominant haplotypes with fixed differences at 10 sites (seven in matK, including three amino
171
acid replacements; three in the trnG intron). One additional haplotype was observed in the Baja
172
California individual (an autapomorphic matK replacement substitution). The sequence results
173
combined with AluI restriction digest assays for 72 individuals demonstrated an abrupt transition
174
in the distribution of the two predominant haplotypes (Fig. 1A). Plants from Oregon and
175
northwestern California (Klamath mountains and North Coast range) were fixed for a common
176
haplotype “N” (thymine at position 1352 of matK), while plants from the Sierra Nevada and
177
Transverse and Peninsular ranges in California were fixed for the alternate haplotype “S”
178
(cytosine at position 1352). The contact zone between the N and S haplotypes occurs near
179
latitude 40˚ 30' N in northeastern California, and the zone of polymorphism is remarkably well-
180
defined, spanning less than 150 km of the ca. 1600 km latitudinal range of this species (Figs. 1,
181
2). Megagametophyte and embryo comparisons in 31 individuals from the contact zone revealed
182
that 12 (39%) seeds contained different maternal and paternal haplotypes, indicating that
183
seedlings are frequently sired by pollen parents with different haplotypes than the ovulate parent.
184
Embryos with the S haplotype were found in seeds from eight N haplotype maternal trees
185
distributed in seed zones 522, 523, 732 and 771 (Fig. 2). The converse pattern was also observed
186
as four S haplotype maternal plants from seed zones 522 and 731 produced seed containing N
187
haplotype embryos (Fig. 2).
188 189
At least one sugar pine individual was assayed for the S vs. N haplotype in 35 of the 41 seed
190
zones sampled by Kinloch (1992) in his survey of Cr1 allele frequencies (the factor conferring
191
major gene resistance to white pine blister rust; Table 2), with intensive sampling in the contact
192
zone (Fig. 2). The haplotype for six unassayed seed zones (one in northwestern California and
193
five in Oregon) was inferred to be N based on results from adjacent seed zones. The Baja
194
California haplotype S' (differing from S by one substitution, Fig. 3) was grouped with S for this
195
analysis. The N, S, and polymorphic seed zones showed significantly different Cr1 frequencies
196
by one-way ANOVA (F = 32.3, P = 7.6e-9). The N haplotype seed zones had the lowest Cr1
197
allele frequency (0.0032 + 0.0038; n = 23), mixed haplotype seed zones had intermediate Cr1
198
frequencies (0.0380 + 0.0185; n = 3), and S haplotype seed zones had the highest Cr1
7
199
frequencies (0.0484 + 0.0260; n = 15). Nonparametric bootstrapping resulted in the same mean
200
allele frequencies and confidence intervals.
201 202
Phylogenetic relationships and variation in sugar pine and other white pine species. Alignment
203
of matK and trnG intron sequences required one 6 bp indel in the matK ORF and four indels (1,
204
4, 10 and 15 bp) in the trnG intron. Three of the indels were confined to P. parviflora, the 15 bp
205
trnG intron indel was shared by P. ayacahuite, P. flexilis, P. strobiformis and P. peuce, and the 1
206
bp indel was autapomorphic in P. armandii ‘b’. Combined phylogenetic analysis of matK (1545
207
bp), trnG intron (746 bp) and the five indels resulted in two most parsimonious trees of length 57
208
with a consistency index of 0.91 (Fig. 3). The two trees differ in the resolution of P. monticola as
209
a grade (shown) or clade (not shown). All Asian species of subsect. Strobus form a strongly
210
supported clade that includes the North American P. albicaulis and the P. lambertiana N
211
haplotype. At these two loci, no sequence divergence was found among northern P. lambertiana,
212
P. albicaulis and representatives of six Asian species (P. dalatensis, P. koraiensis, P. pumila, P.
213
sibirica, P. wallichiana, P. kwangtungensis) and P. cembra from Romania. The P. lambertiana S
214
haplotypes and the remaining North American white pine species form a grade, with P.
215
monticola in a strongly supported sister position to the Eurasian clade. Constraining P.
216
lambertiana samples to monophyly results in trees of 66 steps, which is significantly longer
217
based on both Templeton (p = 0.0039) and Kishino-Hasegawa tests (p = 0.0027).
218 219
Maximum likelihood trees obtained with and without a molecular clock were topologically
220
identical to one of the most parsimonious trees and were not significantly different from each
221
other based on the LRT (ΔlnL = 10.44013, χ2 = 20.88, d.f. = 19, p = 0.34). This suggests that the
222
sequences are diverging at equivalent rates, and a simple molecular clock calculation can be
223
applied. The mean dS between P. monticola and the Asian clade is 0.00232, resulting in an
224
estimated divergence of 5.3 ± 1.3 mya (Late Miocene – Early Pliocene). Sequences of 5799 bp
225
of cpDNA (adding rbcL, rpl16 and trnL-F to the matK and trnG used in the phylogenetic
226
analysis) revealed a single substitution in trnL-F between a P. albicaulis accession (Washington
227
state) and an N haplotype P. lambertiana (dS = 0.00058), resulting in an estimated divergence of
228
0.6 ± 0.15 mya (Pleistocene). Sequences of trnL-F from an additional five accessions of P.
229
albicaulis and three of N haplotype P. lambertiana (Table 1) confirmed that this is a fixed
8
230
difference between these two taxa. The estimated divergence time between the two P.
231
lambertiana chloroplast haplotypes is 15.5 ± 3.9 mya (dS = 0.00681) and between the S
232
haplotype of P. lambertiana and the other North American white pines is 9.0 ± 2.25 mya (dS =
233
0.00396).
234 235
Discussion
236
The pattern of genetic subdivision in sugar pine
237
Previous accounts of sugar pine have described neither morphological nor ecological differences
238
that can be associated with the phylogeographic pattern observed here. In fact, Mirov (1967)
239
described Pinus lambertiana as “rather stable morphologically” (p. 142) and he considered it to
240
be a prime example of a “good species” that is “clearly delimited [and] can be identified without
241
difficulty” (p. 531). The southern limit of the contact zone (Fig. 1A) does coincide with the
242
interface of the Cascade and Sierra Nevada mountain ranges, characterized by relatively recent
243
volcanics and predominantly metamorphics (with granitic intrusions and volcanics), respectively
244
(Hickman 1993). Although this geological transition is used to demarcate two floristic
245
subregions, there is no apparent vegetational break between the forests of the Cascades and
246
northern Sierra Nevada (Hickman 1993).
247 248
In her PhD thesis, Martinson (1997) conducted an analysis of allozyme data collected by Conkle
249
(unpublished; 1996). Forty populations and 400 individuals of P. lambertiana were assayed at 30
250
allozyme loci. Average heterozygosity was 0.22, and no geographic region showed reduced
251
genetic diversity. Clustering of genetic distances separated populations from Oregon and
252
northwestern California from populations in the Sierra Nevada. Unfortunately, no populations
253
were sampled in the chloroplast haplotype contact zone in northeastern California. Thus,
254
although the reported allozyme differentiation may correspond to the chloroplast haplotype
255
distribution, it will require allozyme sampling in the contact zone to confirm this. The allozyme
256
data also separated the Sierra Nevada populations from those sampled in southern California and
257
Baja California. This division is not apparent in our data set. However, a unique substitution was
258
found in the matK sequence of the Baja California individual. This evidence for genetic isolation
259
is consistent with the geographic isolation of this disjunct population (Fig. 1A, B).
260
9
261
The narrow transition zone between the N and S haplotypes of P. lambertiana suggests that these
262
populations have only recently come into contact. This would be consistent with a Holocene
263
range expansion from separate northern and southern refugia. There is abundant evidence from
264
the pollen record for post-glacial movement of pines (Mohr et al. 2000; Thompson and Anderson
265
2000). Unfortunately, individual Pinus species cannot be identified from pollen. Narrow
266
haplotype (chloroplast or mitochondrial) transition zones observed in other western North
267
American plants (Soltis et al. 1997; Aagaard et al. 1998; Latta and Mitton 1999; Johansen and
268
Latta 2003) have also been attributed to post-glacial contact of previously separated populations.
269 270
The examination of maternal and paternal haplotypes offers insight into the dynamics of
271
dispersal at the contact zone (Fig. 2). The two P. lambertiana trees sampled in the southern part
272
of seed zone 731 represent a population that is isolated on Happy Camp mountain, Modoc
273
County. These trees have the S haplotype, and are presumed to have colonized this location by
274
long distance seed dispersal. The closest sampled potential source is ca. 90 km away. The large
275
(228 ± 40 mg) and “flimsy” winged seeds of P. lambertiana are “seldom dispersed far by wind”,
276
but caching by Steller’s jays (Thayer and Vander Wall 2005) and Clark’s nutcrackers (D.
277
Tomback, pers. comm.) can potentially lead to long-distance dispersal. No other example of a
278
disjunct haplotype was observed. Embryos possessing the S haplotype occur up to 25 km north
279
of the northernmost potential source trees, indicating that pollen flow advances ahead of seed
280
dispersal. The two Happy Camp mountain trees whose megagametophytes carry the S haplotype
281
have apparently been pollinated by trees of the N haplotype. Comparison of chloroplast and
282
mitochondrial haplotypes in a Pinus ponderosa contact zone in western Montana has found a
283
similar pattern of more extensive pollen flow and rare long distance seed dispersal (Latta and
284
Mitton 1993; Johansen and Latta 2003).
285 286
One of the most surprising results of our study is the concordance between the distribution of the
287
two cpDNA haplotypes and the relative frequency of a white pine blister rust major gene
288
resistance locus (Cr1) in sugar pine. Kinloch (1992) described the pattern of Cr1 frequency as a
289
cline. In contrast, the significant differences in Cr1 frequency observed among the three
290
haplotype groups (N, S, and mixed) suggests that the gene frequency does not change in a
291
gradual manner, but rather shows the same abrupt transition as observed in the chloroplast. There
10
292
is no evidence for a causal link between the cpDNA haplotype and Cr1 distribution patterns. It is
293
well-established that resistance shows nuclear inheritance (Kinloch 1992), and the highest
294
frequencies of Cr1 (4 – 9% per seed zone) are far lower than the frequency of the S haplotype.
295
Furthermore, examination of three resistant trees (heterozygous for the dominant Cr1 allele; J.
296
Gleason, unpublished data) in the contact zone found both N and S haplotypes (Fig. 2). Note that
297
all other genotyped individuals in the contact zone are non-resistant (J. Gleason, unpublished
298
data). We predict that the concordance between the Cr1 and chloroplast haplotype frequencies
299
reflects a common history of genetic isolation, followed by recent migration and contact (see
300
below).
301 302
Evidence for chloroplast introgression
303
The chloroplast haplotypes of P. lambertiana resolve in two different clades in the phylogenetic
304
analysis of Pinus subsect. Strobus, one comprised of five other North American species and the
305
other encompassing 12 Eurasian species and the North American P. albicaulis (whitebark pine).
306
Two biological processes could explain these results: incomplete lineage sorting of an ancestral
307
polymorphism, or chloroplast introgression. Incomplete lineage sorting has been determined to
308
be the most probable source of widespread allelic non-monophyly at nuclear loci in species of
309
Pinus subgenus Strobus (Syring et al. 2007). It has also been considered a potential cause of
310
similar patterns observed in chloroplast studies in other plant species (Tsitrone et al. 2003).
311
However, the stochastic process of incomplete lineage sorting is not expected to show the strong
312
geographic partitioning observed for the two chloroplast haplotypes. On the other hand, if the
313
two subgroups of sugar pine have been separated since the Miocene (15.1 ± 3.8 mya) and each
314
retained a different haplotype, one might expect to find morphological divergence between (and
315
sequence variation within) the two groups, particularly since this same interval has apparently
316
been accompanied by multiple speciation events in these lineages. Although some sequence
317
divergence was found in the S haplotype clade (in the geographically isolated Baja California
318
population), none was found in the N haplotype clade. The amount of sequence divergence
319
between the S haplotype of P. lambertiana and the other North American white pines is
320
consistent with genetic isolation since the Miocene. In contrast, the high sequence similarity
321
between the N haplotype of sugar pine and the Asian clade is suggestive of a much more recent
322
shared plastid ancestry.
11
323 324
To account for this unexpected genetic similarity, we suggest that the N haplotype of P.
325
lambertiana may have its origin in a chloroplast introgression event involving P. albicaulis.
326
Introgression-mediated chloroplast transfer has been named “chloroplast capture” (Rieseberg and
327
Soltis 1991; Tsitrone et al. 2003) and has been offered as an explanation for similar patterns of
328
cytonuclear incongruence observed in many plant genera (reviewed in Rieseberg et al. 1996;
329
Wendel and Doyle 1998). Tsitrone et al. (2003) have modeled the process under the assumption
330
of maternal chloroplast inheritance and they determined conditions likely to promote its
331
occurence. A key aspect of their model is the observation that cytonuclear incompatibility often
332
results in full or partial male steritily and thus can increase maternal fitness through enhanced
333
seed production. Although they do not explicitly model paternal inheritance (the situation in
334
Pinaceae), they suggest that chloroplast introgression should be less common here, since
335
cytonuclear interactions typically reduce male fitness. Theoretically, chloroplast substitution
336
could result in an advantage in male function, but this situation has apparently not been
337
documented (Tsitrone et al. 2003). However, a pattern consistent with chloroplast introgression
338
has been observed in other species of Pinaceae, e.g. Pinus montezumae (Matos and Schaal 2000),
339
Pinus muricata (Hong et al. 2003) and Larix sibirica (Wei and Wang 2003).
340 341
Petit et al. (2003) offer “pollen swamping” as an alternative explanation for the lack of
342
cytoplasmic (cpDNA and mtDNA) differentiation between Quercus petraea and Q. robur, two
343
sympatric oaks that are consistently differentiated at nuclear markers. In their scenario, Q. robur
344
seed disperses into new habitats which are subsequently colonized by Q. petraea via pollen flow,
345
resulting in F1 hybrids. Asymmetric introgression and strong selection for the Q. petraea
346
phenotype results in mixed populations that share a single cytoplasm. Petit et al. invoke the fact
347
that the seeds of Q. robur are better adapted to bird-dispersal than Q. petraea, and thus are more
348
likely to establish through long-distance dispersal. A similar relationships exists between P.
349
albicaulis (dispersed mainly by the pine seed specialist, Clark’s nutcracker, Tomback 2005) and
350
P. lambertiana (dispersed to a limited extent by wind and primarily by generalist Steller’s jays
351
and yellow pine chipmunks, Thayer and Vander Wall 2005). In both cases, the better disperser is
352
thought to have contributed its chloroplast to the other species. An important caveat is that the
12
353
cytoplasm is maternally inherited in oaks, and thus carried by the seed, and not the pollen as in
354
pines.
355 356 357
If chloroplast introgression is responsible for this pattern, how does one account for the
358
otherwise “Eurasian” haplotype in two species of North American subsect. Strobus? The
359
cpDNA-based phylogeny (Fig. 3) requires two dispersal events between western North America
360
and Asia. The disjunction between P. monticola and the Eurasian clade can be dated to the late
361
Miocene or early Pliocene. This timing is consistent with estimates of 2.6 – 16.7 mya from
362
eleven other eastern Asian / western North American plant disjunctions (Zhu et al. 2006; Zhang
363
et al. 2007). It is noteworthy that well-preserved Pliocene and Pleistocene fossils of P. monticola
364
have been collected in northeastern Siberia and Alaska (reviewed in Bingham et al. 1974).
365
Following diversification of the Eurasian white pines, and origin of the “closed cone”
366
morphology characteristic of stone pines, we propose that the ancestor of P. albicaulis dispersed
367
from Asia to North America via Beringia, presumably during the Pleistocene. Beringia was
368
largely unglaciated during the Pleistocene, and is known to have served as a refugium for trees
369
and shrubs, including Pinus, through the late glacial maximum (Brubaker et al. 2005).
370
Krutovskii et al. (1995) proposed a similar scenario based on allozyme and chloroplast
371
restriction fragment analysis of the “subsect. Cembrae” pines, but placed the migration at an
372
earlier period (Pliocene).
373 374
Evidence from mtDNA haplotypes has been used to infer the existence of three late Pleistocene
375
refugia for P. albicaulis (Richardson et al. 2002): western Wyoming, western Idaho and the
376
southern Cascades of Oregon. The southern Cascades currently support large populations of P.
377
lambertiana, and we propose that this region could also have served as a northern glacial
378
refugium for sugar pine, or possibly further west in the Klamath/Siskiyou Mts. (a region with
379
several paleo-endemic conifers, e.g. Picea breweriana and Chamaecyparis lawsoniana). Sugar
380
pine is common here, but whitebark pine has only recently been discovered in a small population
381
on Mt. Ashland (nine individuals; Murray 2005). Regardless of their current geographic
382
distributions, sympatry in a glacial refugium could have provided the opportunity for the
383
northern populations of sugar pine to acquire the chloroplast of whitebark pine. Although P.
13
384
albicaulis generally occurs at higher elevations than P. lambertiana, the two are partly sympatric
385
in northern California and southern Oregon (Fig. 1B). There is also evidence that P. lambertiana
386
formerly occurred at higher elevations than its current distribution (May 1974; Millar et al.
387
2006).
388 389
Two factors are required for chloroplast introgression to occur; sympatry and reproductive
390
compatibility. While many interspecific crosses have been conducted in subsect. Strobus, there is
391
no record of attempts to cross P. lambertiana and P. albicaulis (Critchfield 1986, R. Sniezko,
392
pers. comm.). Critchfield and Kinloch (1986) do, however, document interspecific hybridization
393
between P. lambertiana and Asian members of subsect. Strobus, namely P. armandii and P.
394
koraiensis. Seed set in these artificial crosses averaged 2.1% and 0.2% viable seed per cone,
395
respectively. In contrast, P. lambertiana is apparently intersterile with all other North American
396
white pines (Critchfield 1986; Fernando et al. 2005). No naturally occurring hybrids of P.
397
lambertiana have been recorded (Mirov 1967; Critchfield 1986; R. Sniezko, pers. comm.).
398 399
Our results explain why Pinus lambertiana was resolved in conflicting positions in recent
400
chloroplast sequence-based phylogenetic analyses of pines. Gernandt et al. (2005) sampled P.
401
lambertiana in Oregon (a region fixed for the N haplotype), while Eckert and Hall (2006)
402
sampled an individual from southern California (a region fixed for the S haplotype). Each study
403
placed P. lambertiana in a position that is consistent with the resolution of the respective
404
haplotypes in our study (Fig. 3). This demonstrates the importance of sampling multiple
405
individuals per species in phylogenetic analyses of closely related species (see also Syring et al.
406
2007).
407 408
The results reported here provide the first phylogeographic hypothesis for an ecologically and
409
economically important conifer, Pinus lambertiana. By placing the intraspecific results within a
410
broader phylogenetic context, further insights were gained into the evolutionary history of this
411
species. The novel hypotheses of two Pleistocene refugia for P. lambertiana and chloroplast
412
introgression with P. albicaulis can be tested with additional molecular markers, in particular
413
nuclear and mitochondrial loci. The observation that the two chloroplast haplotypes demarcate
414
population groups that differ in their vulnerability to white pine blister rust is also a significant
14
415
result that merits further attention. Beyond sugar pine, this study demonstrates the value of
416
including an interspecific phylogenetic component in phylogeographic research. Without this
417
broader perspective, the antiquity of the haplotype groups would remain unknown, as would the
418
unexpected, and potentially reticulate, history of these species.
419 420
Acknowledgements
421
We are indebted to John Gleason (USFS, Placerville Nursery and Disease Resistance Program),
422
Jerry Berdeen and Richard Sniezko (USFS, Dorena Genetic Resource Center) and David
423
Johnson (USFS, Institute of Forest Genetics) for supplying sugar pine seeds and Roman
424
Businský for providing his collections of Asian species. We thank David Gernandt, Bohun
425
Kinloch, Todd Ott, Paul Severns, Richard Sniezko, and Diana Tomback for constructive
426
comments on the manuscript. This research was funded by National Science Foundation grants
427
DEB 0317103 and ATOL 0629508, an NSF Research Experience for Undergraduates
428
suppplement, and the Pacific Northwest Research Station, USDA Forest Service.
429 430
References
431
Aagaard JE, Krutovskii KV, Strauss SH (1998) RAPD markers of mitochondrial origin exhibit
432
lower population diversity and higher differentiation than RAPDs of nuclear origin in
433
Douglas fir. Molecular Ecology, 7, 801-812.
434 435 436
Bingham RT, Hoff RJ, Steinhoff RJ (1972) Genetics of Western White Pine, USDA Forest Service Research Paper WO-12, USDA Forest Service, Washington, DC. Brubaker LB, Anderson PM, Edwards ME, Lozhkin AV (2005) Beringia as a glacial refugium
437
for boreal trees and shrubs: new perspectives from mapped pollen data. Journal of
438
Biogeography, 32, 833-848.
439
Chiang YC, Hung KH, Schaal BA et al. (2006) Contrasting phylogeographical patterns between
440
mainland and island taxa of the Pinus luchuensis complex. Molecular Ecology, 15, 765-
441
779.
442
Conkle MT (1996) Patterns of variation in isozymes of sugar pine. In: Sugar Pine: Status,
443
Values, and Roles in Ecosystems: Proceedings of a Symposium presented by the
444
California Sugar Pine Management Committee (eds. Kinloch BB, Marosy M, Huddleston
15
445
ME), p. 99. University of California Division of Agriculture and Natural Resources,
446
Davis, CA.
447 448
Critchfield WB (1986) Hybridization and classification of the white pines (Pinus section Strobus). Taxon, 35, 647-656.
449
Critchfield WB, Kinloch BB (1986) Sugar pine and its hybrids. Silvae Genetica, 35, 138-145.
450
Critchfield WB, Little EL, Jr. (1966) Geographic Distribution of the Pines of the World, Forest
451 452
Service Misc. Publication 991, USDA Forest Service, Washington, DC. Devey ME, Delfino-Mix A, Kinloch BB, Neale DB (1995) Random amplified polymorphic
453
DNA markers tightly linked to a gene for resistance to white pine blister rust in sugar
454
pine. Proceedings of the National Academy of Sciences, 92, 2066-2070.
455
Eckert AJ, Hall BD (2006) Phylogeny, historical biogeography, and patterns of diversification
456
for Pinus (Pinaceae): phylogenetic tests of fossil-based hypotheses. Molecular
457
Phylogenetics and Evolution, 40, 166-182.
458
Fernando DD, Long SM, Sniezko RA (2005) Sexual reproduction and crossing barriers in white
459
pines: the case between Pinus lambertiana (sugar pine) and P. monticola (western white
460
pine). Tree Genetics & Genomes, 1, 143-150.
461
Gadek PA, Alpers DL, Heslewood MM, Quinn CJ (2000) Relationships within Cupressaceae
462
sensu lato: a combined morphological and molecular approach. American Journal of
463
Botany, 87, 1044-1057.
464 465 466 467
Gernandt DS, Gaeda López G, Ortiz García S, Liston A (2005) Phylogeny and classification of Pinus. Taxon, 54, 29-42. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95-98.
468
Hausner G, Olson R, Simon D, et al. (2006) Origin and evolution of the chloroplast trnK (matK)
469
intron: A model for evolution of group II intron RNA structures. Molecular Biology and
470
Evolution, 23, 380-391.
471
Hickman JC (1993) Geographic subdivisions of California. In: The Jepson Manual: Higher
472
Plants of California (ed. Hickman JC), pp. 37-48. University of California, Berkeley,
473
CA.
16
474
Hong Y-P, Krupkin AB, Strauss SH (1993) Chloroplast DNA transgresses species boundaries
475
and evolves at variable rates in the California closed-cone pines (P. radiata, P. muricata,
476
and P. attenuata). Molecular Phylogenetics and Evolution, 2, 322-329.
477 478 479 480 481 482 483 484
Hood GM (2006) PopTools version 2.7.5. CSIRO, Canberra, Australia, http://www.cse.csiro.au/poptools/. Johansen AD, Latta RG (2003) Mitochondrial haplotype distribution, seed dispersal and patterns of postglacial expansion of ponderosa pine. Molecular Ecology, 12, 293-298. Kidd DM, Ritchie MG (2006) Phylogeographic information systems: putting the geography into phylogeography. Journal of Biogeography, 33, 1851-1865. Kinloch BB (2003) White pine blister rust in North America: past and prognosis. Phytopathology, 93, 1044-1047.
485
Kinloch BB (1992) Distribution and frequency of a gene for resistance to white pine blister rust
486
in natural populations of sugar pine. Canadian Journal of Botany, 70, 1319-1323.
487
Kinloch BB, Scheuner WH (2004) Pinus lambertiana Dougl., Sugar Pine, USDA Forest Service,
488
Washington, DC.
489
http://na.fs.fed.us/spfo/pubs/silvics_manual/Volume_1/vol1_Table_of_contents.htm.
490
Krutovskii KV, Politov DV, Altukhov YP (1995) Isozyme study of population genetic structure,
491
mating system and phylogenetic relationships of the five stone pine species (subsection
492
Cembrae, section Strobi, subgenus Strobus). In: Population genetics and genetic
493
conservation of forest trees (eds. Baradat P, Adams WT, Müller-Starck G), pp. 279-304.
494
Academic Publishing, Amsterdam.
495 496
Latta RG, Mitton JB (1999) Historical separation and present gene flow through a zone of secondary contact in ponderosa pine. Evolution, 53, 769-776.
497
Ledig FT (1998) Genetic variation in Pinus. In: Ecology and biogeography of Pinus (ed.
498
Richardson DM), pp. 251-280. Cambridge University Press, Cambridge.
499
Liston A (1992) Variation in the chloroplast genes rpoC1 and rpoC2 of the genus Astragalus
500
(Fabaceae): Evidence from restriction site mapping of a PCR-amplified fragment.
501
American Journal of Botany, 79, 953-961.
502
Liston A, Robinson WA, Piñero D, Alvarez-Buylla ER (1999) Phylogenetics of Pinus (Pinaceae)
503
based on nuclear ribosomal DNA internal transcribed spacer sequences. Molecular
504
Phylogenetics and Evolution, 11, 95-109.
17
505
Martinson SR (1997) Association among geographic, allozyme, and growth variables for sugar
506
pine (Pinus lambertiana Dougl.) in southwest Oregon and throughout the species range.
507
PhD thesis, North Carolina State University.
508
Matos JA, Schaal BA (2000) Chloroplast evolution in the Pinus montezumae complex: A
509
coalescent approach to hybridization. Evolution 54, 1218-1233.
510
May RH (1974) An observation of some sugar pine relicts. Madroño, 22, 276.
511
Mielke JL (1943) White Pine Blister Rust in Western North America, Bulletin No. 52, Yale
512 513
University School of Forestry, New Haven. Millar CI, King JC, Westfall RD, Alden HA, Delany DL (2006) Late Holocene forest dynamics,
514
volcanism, and climate change at Whitewing Mountain and San Joaquin Ridge, Mono
515
County, Sierra Nevada, CA, USA. Quaternary Research, 66, 273-287.
516
Mirov NT (1967) The genus Pinus, The Ronald Press Company, New York.
517
Mohr JA, Whitlock C, Skinner CN (2000) Postglacial vegetation and fire history, eastern
518 519 520
Klamath mountains, California, USA. The Holocene, 10, 587-601. Murray M (2005) Our threatened timberlines: the plight of whitebark pine ecosystems. Kalmiopsis, 12, 25-29.
521
Muse SV, Weir BS (1992) Testing for equality of evolutionary rates. Genetics, 132, 269-276.
522
Navascues M, Vaxevanidou Z, Gonzalez-Martinez SC, et al. (2006) Chloroplast microsatellites
523
reveal colonization and metapopulation dynamics in the Canary Island pine. Molecular
524
Ecology, 15, 2691-2698.
525 526 527
Neale DB, Sederoff RR (1989) Paternal inheritance of chloroplast DNA and maternal inheritance of mitochondrial DNA in loblolly pine. Theoretical and Applied Genetics, 77, 212-216. Petit RJ, Duminil J, Fineschi S, et al. (2005) Comparative organization of chloroplast,
528
mitochondrial and nuclear diversity in plant populations. Molecular Ecology, 14, 689-
529
701.
530 531 532 533
Petit RJ, Bodénès C, Ducousso A, Roussel G, Kremer A (2003) Hybridization as a mechanism of invasion in oaks. New Phytologist 161, 151-164. Posada D, Crandall KA (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics, 14, 817-818.
18
534
Richardson BA, Brunsfeld SJ, Klopfenstein NB (2002) DNA from bird-dispersed seed and wind-
535
disseminated pollen provides insights into postglacial colonization and population genetic
536
structure of whitebark pine (Pinus albicaulis). Molecular Ecology, 11, 215-227.
537
Richardson DM, Rundel PW (1998) Ecology and biogeography of Pinus: an introduction. In:
538
Ecology and biogeography of Pinus (ed. Richardson DM), pp. 3-46. Cambridge
539
University Press, Cambridge.
540 541 542 543
Rieseberg LH, Soltis DE (1991) Phylogenetic consequences of cytoplasmic gene flow in plants. Evolutionary Trends in Plants, 5, 65-84. Rieseberg LH, Whitton J, Linder CR (1996) Molecular marker incongruence in plants hybrid zones and phylogenetic trees. Acta Botanica Neerlandica, 45, 243-262.
544
Rozas J, Sánchez-DelBarrio JC, Messeguer X, Rozas R (2003) DnaSP, DNA polymorphism
545
analyses by the coalescent and other methods. Bioinformatics, 19, 2496-2497.
546
Scharpf RF (1993) Diseases of Pacific Coast Conifers, Handbook 521, USDA Forest Service,
547 548
Washington D.C. Shaw J, Lickey EB, Beck JT, et al. (2005) The tortoise and the hare II: relative utility of 21
549
noncoding chloroplast DNA sequences for phylogenetic analysis. American Journal of
550
Botany, 92, 142-166.
551
Soltis DE, Gitzendanner MA, Strenge DD, Soltis PS (1997) Chloroplast DNA intraspecific
552
phylogeography of plants from the Pacific Northwest of North America. Plant
553
Systematics and Evolution, 206, 353-373.
554 555 556 557 558
Swofford DL (2002) PAUP*, Phylogenetic Analysis Using Parsimony (*and Other Methods), Version 4, Sinauer Associates, Sunderland, Massachusetts. Syring J, Farrell K, Businský R, Cronn R, Liston A (2007) Widespread genealogical nonmonophyly in species of Pinus subgenus Strobus. Systematic Biology, 56, 163-181. Syring J, Willyard A, Cronn R, Liston A (2005) Evolutionary relationships among Pinus
559
(Pinaceae) subsections inferred from multiple low-copy nuclear loci. American Journal
560
of Botany, 92, 2086-2100.
561
Thayer TC, Vander Wall SB (2005) Interactions between Steller’s jays and yellow pine
562
chipmunks over scatter-hoarded sugar pine seeds. Journal of Animal Ecology, 74, 364-
563
375.
19
564
Thompson RS, Anderson KH (2000) Biomes of western North America at 18,000, 6000 and 0
565
14
566
27, 555-584.
567
C yr bp reconstructed from pollen and packrat midden data. Journal of Biogeography,
Tomback DF (2005) The impact of seed dispersal by Clark's nutcracker on whitebark pine:
568
multi-scale perspective on a high mountain mutualism. In: Mountain ecosystems: studies
569
in treeline ecology (eds. Broll G, Keplin B), pp. 181-201. Springer, Berlin.
570 571
Tsitrone A, Kirkpatrick M, Levin DA (2003) A model for chloroplast capture. Evolution, 57, 1776-1782.
572
Wang XR, Tsumura Y, Yoshimaru H, Nagasaka K, Szmidt AE (1999) Phylogenetic relationships
573
of Eurasian pines (Pinus, Pinaceae) based on chloroplast rbcL, matK, rpl20-rps18 spacer,
574
and trnV intron sequences. American Journal of Botany, 86, 1742-1753.
575
Wefald M (2001) TRS2LL, http://www.geocities.com/jeremiahobrien/trs2ll.html.
576
Wei XX, Wang XQ (2003) Phylogenetic split of Larix: evidence from paternally inherited
577 578
cpDNA trnT- trnF region. Plant Systematics and Evolution, 239, 67-77. Wendel JF, Doyle JJ (1998) Phylogenetic incongruence: window into genome history and
579
molecular evolution. In: Molecular Systematics of Plants: DNA Sequencing (eds. Soltis
580
DE, Soltis PS, Doyle JJ), pp. 265-296. Kluwer, Boston.
581
Willyard A, Syring J, Gernandt DS, Liston A, Cronn R (2007) Fossil calibration of molecular
582
divergence infers a moderate mutation rate and recent radiations for Pinus. Molecular
583
Biology and Evolution, 24, 90-101.
584
Zhang M-L, Uhink CH, Kadereit JW (2007) Phylogeny and biogeography of
585
Epimedium/Vancouveria (Berberidaceae): Western North American - East Asian
586
disjunctions, the origin of European mountain plant taxa, and East Asian species
587
diversity. Systematic Botany 32, 81-92.
588
Zhu Y-P, Wen J, Zhang Z-Y, Chen Z-D (2006) Evolutionary relationships and diversification of
589
Stachyuraceae based on sequences of four chloroplast markers and the nuclear ribosomal
590
ITS region. Taxon, 55, 931-940.
20
591
Table 1 Geographic origin and Genbank accessions for samples sequenced for the cpDNA matK
592
and trnG intron loci.
593 Species
Haplo-
Country
Administrative Unit
type
matK
trnG
accession
accession
EF546699
EF546730
albicaulis *
U.S.
California: Mono Co.
albicaulis *
U.S.
California: Siskiyou Co.
"
"
*
U.S.
Montana
"
"
albicaulis *
U.S.
Oregon
"
"
albicaulis *, ‡
U.S.
Washington
"
"
albicaulis *
U.S.
Wyoming
"
"
albicaulis
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
21
lambertiana *
N
U.S.
California: seedzone 091
EF546715
EF546741
lambertiana
N
U.S.
California: seedzone 372
"
"
N
U.S.
California: seedzone 516
"
"
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
"
"
EF546719
EF546744
EF546720
EF546745
"
"
EF546721
EF546746
lambertiana * lambertiana
*, ‡
morrisonicola
Taiwan
parviflora
Japan
Hokkaido
parviflora
Japan
Honshu
peuce
Bulgaria
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
"
"
Pakistan
Punjab
EF546728
EF546751
wallichiana †
a
22
wallichiana gerardiana †
b
Nepal
Karnali
EF546729
"
Pakistan
Gilgit
AY115801
EF546752
594 595
Haplotypes N, S and S' for P. lambertiana are described in the text. In other species, the letters a,
596
b and c were applied as needed for multiple haplotypes within a species. Voucher specimens are
597
deposited at Oregon State University (OSC), unless indicated otherwise.
598
*
599
Genbank accessions EF546757 - EF546759.
600
†
601
Gardening, Průhonice, Czech Republic (RILOG).
Sequenced for trnL-F, GenBank accessions EF546753 - EF546756. ‡ Sequenced for rpl16, Voucher deposited at the Silva Tarouca Research Institute for Landscape and Ornamental
602 603
23
604
Table 2 The frequency of the white pine blister rust resistance gene Cr1 in sugar pine seed zones
605
from the region of haplotype contact, compared to the number of southern and northern
606
haplotypes. Data in columns 2 and 3 are from the 41 seed zones sampled by Kinloch (1992).
607 seed
trees / seeds
zone
sampled
frequency of
haplo-
N
S
frequency
major gene
type
haplo-
haplo-
of S
resistance
group
type
type
haplotype
Oregon: Coast ranges and Cascades 090
10 / 74
0.0000
N
452
N
1
0.00
472
N
1
0.00
1
0.00
491
24 / 430
0.0000
N
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
N
1
0.00
703
8 / 157
0.0000
721
N
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
N
1
0.00
303 311
28 / 683
0.0073
N
1
0.00
321
141 / 5197
0.0073
N
1
0.00
24
322
5 / 302
0.00
N
1
0.00
331
3 / 44
0.00
N
1
0.00
N
1
0.00
332 340
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
mixed
1
2
0.67
4
0.33
731 732
7 / 174
0.0632
mixed
8
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
S
1
1.00
California: Central Coast range 120
93 / 2133
0.0886
Southern California: Transverse ranges 992
8 / 644
0.0699
S
1
1.00
993
29 / 463
0.0324
S
1
1.00
25
994
31 / 337
0.0297
S
1
1.00
997
32 / 517
0.0561
S
1
1.00
S
1
1.00
Baja California: Sierra San Pedro Martír 12 / 254
0.00
608 609
26
610
Fig. 1A Sampled individuals of P. lambertiana in western North America. Yellow squares
611
represent the S haplotype and blue squares represent the N haplotype. Forest tree seed zones
612
sampled by Kinloch (1992) and / or this study are shaded according to the frequency of the Cr1
613
allele (Table 2), ranging from 0% (no shading) to 8.9% (dark gray, seed zone 120). Seed zones
614
outlined in red were not sampled for Cr1. The haplotype contact zone (green rectangle) is shown
615
in more detail in Figure 2.
616 617
Fig. 1B Approximate geographic distribution of Pinus lambertiana (dark gray) and P. albicaulis
618
(light gray) from Critchfield and Little (1966). Red triangles represent P. albicaulis samples used
619
in this study.
620 621
Fig. 2 Detail of the contact zone between the S (yellow) and N (blue) cpDNA haplotypes of P.
622
lambertiana. Squares represent the maternal (megagametophyte) haplotype and circles represent
623
the paternal (embryo) haplotype. Asterisks denote white pine blister rust resistant individuals.
624 625
Fig. 3 One of two most parsimonious trees estimated from cpDNA matK and trnG intron
626
sequences. Bootstrap values are shown below the branches. Tree length = 57, consistency index
627
= 0.91, retention index = 0.97. When applicable, haplotypes (see Table 2) and the number of
628
individuals that share a particular sequence and haplotypes follow the species names.
629 630
Author Information
631 632 633 634 635 636 637
Aaron Liston and Rich Cronn collaboratively study the systematics, population genetics, and evolution of pines. John Syring and Ann Willyard are former PhD students of Cronn and Liston. Dr. Syring is now an Assistant Professor of Plant Systematics at Montana State UniversityBillings, and Dr. Willyard is a post-doc at the University of South Dakota. Mariah ParkerdeFeniks is a Sociology major at Oregon State University, pursuing a career in Criminal Justice and Social Research.
27
638
Figure 1
639
28
640
Figure 2
641
29
642
Figure 3
643
30
