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Syst. Biol. 56(2):1–19, 2007
c
Society of Systematic Biologists
Copyright ©
ISSN: 1063-5157 print / 1076-836X online
DOI: 10.1080/10635150701258787
Widespread Genealogical Nonmonophyly in Species of Pinus Subgenus Strobus
J OHN S YRING ,1 K ATHLEEN FARRELL,2 R OMAN B USINSK Ý,3 R ICHARD CRONN,4 AND AARON LISTON2
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Department of Biological and Physical Sciences, Montana State University–Billings, Billings, Montana 59101, USA
2
Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331, USA;
E-mail: listona@science.oregonstate.edu (A.L.)
3
Silva Tarouca Research Institute for Landscape and Ornamental Gardening, Průhonice, Czech Republic
4
Pacific Northwest Research Station, USDA Forest Service, 3200 SW Jefferson Way, Corvallis, Oregon 97331, USA
Abstract.—Phylogenetic relationships among Pinus species from subgenus Strobus remain unresolved despite combined
efforts based on nrITS and cpDNA. To provide greater resolution among these taxa, a 900-bp intron from a Late Embryo­
genesis Abundant (LEA)-like gene (IFG8612) was sequenced from 39 pine species, with two or more alleles representing
33 species. Nineteen of 33 species exhibited allelic nonmonphyly in the strict consensus tree, and 10 deviated significantly
from allelic monophyly based on topology incongruence tests. Intraspecific nucleotide diversity ranged from 0.0 to 0.0211,
and analysis of variance shows that nucleotide diversity was strongly associated (P < 0.0001) with the degree of species
monophyly. Although species nonmonophyly complicates phylogenetic interpretations, this nuclear locus offers greater
topological support than previously observed for cpDNA or nrITS. Lacking evidence for hybridization, recombination, or
imperfect taxonomy, we feel that incomplete lineage sorting remains the best explanation for the polymorphisms shared
among species. Depending on the species, coalescent expectations indicate that reciprocal monophyly will be more likely
than paraphyly in 1.71 to 24.0 × 106 years, and that complete genome wide coalescence in these species may require up
to 76.3 × 106 years. The absence of allelic coalescence is a severe constraint in the application of phylogenetic methods in
Pinus, and taxa sharing similar life history traits with Pinus are likely to show species nonmonophyly using nuclear markers.
[Lineage sorting; monophyly; nonmonophyly; nuclear genes; pinaceae; Pinus; phylogeny.]
Whenever a phylogenetic study uses a single individ­
ual to represent a species, an implicit assumption is made
that the species is monophyletic (Shaw and Small, 2005;
Funk and Omland, 2003). To the extent that complicat­
ing factors (e.g., reticulation) are rare in the divergence
history of terminal taxa, this assumption offers a con­
venient simplification for sampling. However, sampling
a single individual per species provides no opportunity
to test the hypothesis of allelic monophyly (coalescence)
within species. In molecular phylogenetics, this issue can
become acute, because gene trees are used to infer or­
ganismal phylogenies. These gene trees are subject to
processes that can result in the nonmonophyly of se­
quences sampled from a single species (lineage sorting,
reticulate evolution; Nei, 1987; Wendel and Doyle, 1998).
Errors in phylogenetic estimation can occur when gene
tree/species tree incongruence exists, but species sam­
pling is insufficient to detect the responsible phenomena.
Many species remain poorly known, and the presence of
cryptic taxa or an inadequate taxonomic treatment can
be recognized when nonmonophyletic species are dis­
covered in a phylogenetic analysis.
Awareness of the existence and complications aris­
ing from intraspecific polymorphism is growing. This is
illustrated by recently published plant molecular phy­
logenetic studies (Appendix 1) that increasingly in­
clude sampling to evaluate species level monophyly.
Not surprisingly, varying levels of nonmonophyly are
encountered as more intensive population-level sam­
pling is included in phylogenetic analyses (Appendix 1).
Despite the prevalence of nonmonophyly, many recent
studies in plants do not include multiple samples per
species, even in the species rich genera Rhododendron (86
included species; Goetsch et al., 2005), Aconitum (54 in­
cluded species; Luo et al., 2005), Utricularia (31 included
species; Müller and Borsch, 2005), Solanum (14 included
species; Levin et al., 2005), Viburnum (41 included species;
Winkworth and Donoghue, 2005), Silene (16 included
species; Popp and Oxelman, 2004), and Dioscorea (67 in­
cluded species; Wilkin et al., 2005).
Although many examples of species nonmonophyly
are the direct result of inadequate phylogenetic sig­
nal (Cross et al., 2002; Roalson and Friar, 2004; Levin
and Miller, 2005; Shaw and Small, 2005), recent pub­
lications across the plant kingdom have demonstrated
that species-level paraphyly and polyphyly can be well
supported (Roalson and Friar, 2004; Álvarez et al., 2005;
Church and Taylor, 2005; Kamiya et al., 2005; Oh and
Potter, 2005; Yuan et al., 2005). Causative factors respon­
sible for species nonmonophyly are often difficult to es­
tablish. Factors commonly cited include introgressive
hybridization (Roalson and Friar, 2004; Kamiya et al.,
2005; Mason-Gamer, 2005; Shaw and Small, 2005), in­
complete lineage sorting (Chiang et al., 2004; Bouillé and
Bousquet, 2005; Kamiya et al., 2005), unrecognized am­
plification of a paralogous locus (Roalson and Friar, 2004;
Álvarez et al., 2005), recombination among divergent al­
leles (Schierup and Hein, 2000), and imperfect taxonomy
including the occurrence of cryptic species (Goodwillie
and Stiller, 2001; Treutlein et al., 2003; Roalson and
Friar, 2004; Shaw and Small, 2005). Further, paraphyletic
species may be the direct result of certain evolutionary
processes, as suggested for recent progenitor-derivative
speciation (Rieseberg and Brouillet, 1994; Rosenberg,
2003).
Funk and Omland (2003) reported a common trend
of species-level coalescence failure from mitochondrial
DNA studies in animals. Their survey of 584 stud­
ies and 2319 species found that 23.1% of the studies
showed species-level paraphyly or polyphyly. Bouillé
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90
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and Bousquet (2005) recently demonstrated a striking
case of trans-species allelic polymorphism in three lowcopy nuclear genes in different species of spruce (Picea).
Allelic coalescence times between randomly selected al­
leles from these spruce species were estimated at 10 to
18 million years ago, values that overlapped with esti­
mated divergence times (13 to 20 million years ago) for
the species studied. Because spruces share many life his­
tory traits with other temperate zone gymnosperm and
angiosperm trees (e.g., highly outcrossing, long-lived
perennials with large effective population sizes), Bouillé
and Bousquet (2005) suggest that the incomplete lineage
sorting phenomenon detected in Picea could hinder the
utility of the nuclear markers in phylogenetic analyses
of conifers and other trees.
The similarities in life history traits between Picea and
Pinus (both genera of Pinaceae) suggest that incomplete
lineage sorting could be a common feature of the 100+
species in this genus, thus creating a potential obstacle to
phylogenetic analyses based on low-copy nuclear genes.
Pinus is a diverse and relatively ancient genus with ori­
gins that date to the late Cretaceous (145 to 125 million
years ago; Alvin, 1960). Paleontological and molecular
data suggest that the first major divergence event sep­
arating extant lineages occurred perhaps 85 to 45 mil­
lion years ago (Miller, 1973; Meijer, 2000; Magallon and
Sanderson, 2002; Willyard et al., 2006), giving rise to
two distinct lineages recognized today as subg. Pinus
and subg. Strobus, the “hard” and “soft” pines, respec­
tively (Liston et al., 1999; Gernandt et al., 2005; Syring
et al., 2005). Although there is extensive morphological
variation in the genus, most character states exhibit ho­
moplasy across subgenera and sections (Gernandt et al.,
2005). The number of fibrovascular bundles per needle is
the only diagnostic character that is nonhomoplastic for
the two subgenera (one for subg. Strobus, two for subg.
Pinus). Recent classifications of subg. Strobus recognize
36 to 40 species (Farjon, 2005; Gernandt et al., 2005), with
complete agreement on 34 species and alternative treat­
ments for the remaining taxa. Specific treatments for re­
gions with high endemism, e.g., Mexico (Perry, 1991) and
East Asia (Businský, 1999, 2004), distinguish narrower
taxonomic limits and thus recognize several additional
species. Within subg. Strobus the recent classification of
Gernandt el al. (2005) recognizes two sections, Quin­
quefoliae and Parrya, each containing three subsections
(Table 1).
Despite the relative antiquity of the subgenus Strobus–
subgenus Pinus split, molecular evidence suggests that
extant species within sections from these subgenera
show a high degree of genetic similarity. For example, av­
erage pairwise nucleotide divergence (1) of species from
subg. Strobus ranges from 0.84% for two plastid genes
(Gernandt et al., 2005) and 5.6% for 11 nuclear genes
(Willyard et al., 2006). By integrating this information
with fossil calibrations, Willyard et al. (2006) showed that
the lineages harboring the greatest number of soft pine
species (Subsects. Strobus [21 species] and Cembroides [11
species]) arose between 10 and 20 million years ago.
The combination of recent divergence and generally very
large effective population sizes (Ne ; Ledig, 1998), make
it likely that mutations have spread and become fixed
slowly across a species’ range. This presents the poten­
tial for long-lived allelic diversity that spans one or more
speciation events.
Pinus subg. Strobus has a long history of systematic
inquiry (reviewed in Critchfield 1986; Price et al., 1998;
Wang et al., 1999; Gernandt et al., 2005; Syring et al.,
2005). To date, however, relationships among the terminal taxa remain nearly unresolved (reviewed in Sy­
ring et al., 2005). In pines, low-copy nuclear genes are
an untapped resource for clarifying these terminal re­
lationships, especially when genetic variation is inter­
preted within a framework where species monophyly
can be assessed, and where the impact of incomplete lin­
eage sorting can be determined. Data from multiple lowcopy nuclear loci in pines (Syring et al., 2005) provide
initial evidence that intraspecific diversity is confined
within Pinus subsections, although the frequency of noncoalescence at the species level has not been previously
addressed.
In this paper, we present a phylogenetic analysis of
subg. Strobus using the most informative nuclear locus
identified in a recent survey (Syring et al., 2005), a ca. 900bp intron localized within a Late Embryogenesis Abun­
dant (LEA)-like gene. The goal of this study is to in­
tensively sample the remaining species of subg. Strobus,
particularly the species-rich subsects. Strobus and Cem­
broides, and to place them within the phylogenetic framework developed in prior studies (Gernandt et al., 2005;
Syring et al., 2005) with multiple markers. In addition,
we seek to examine the impact of intraspecific variation
and patterns of allelic coalescence on the accuracy of the
derived phylogeny. To achieve this goal, we sequenced
a minimum of two alleles for 33 of the 39 species used
in this analysis. This sampling strategy allows us to ad­
dress three important questions: (1) How frequently do
species show complete allelic coalescence at this locus?
(2) In the absence of species monophyly, at what taxonomic rank do the alleles coalesce? and (3) If specieslevel non-coalescence is common, what insights can this
provide regarding the nature of pine species, the process
of speciation in Pinus, or operational species definitions
within this group?
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165
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175
180
185
190
195
M ATERIALS AND M ETHODS
Plant Materials
Thirty-nine species of Pinus subgenus Strobus were
sampled (Table 1). Haploid megagametophyte tissue
was used as the DNA source for most amplifications and 200
extracted using the FastPrep DNA isolation kit (Qbio­
gene, Carlsbad, California, USA). In the select cases
where needle tissue was used, direct sequencing iden­
tified homozygotes and heterozygotes. Homozygous
sequences were directly incorporated into the align- 205
ment, whereas heterozygous sequences were cloned into
pGem-T Easy (Promega, Madison, Wisconsin, USA).
From each of the 39 sampled species, an effort was made
to obtain a minimum of two unique alleles. Depending
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3
TABLE 1. Sampled Pinus subg. Strobus representatives. When two alleles are listed for the same accession then either both alleles came from
the same individual, or the accession is a bulk collection of multiple individuals and is marked as such with a subscript. Clade information refers
to Figures 1 and 2.
Taxona
Section Quinquefoliae
Subsection Gerardianae
P. bungeana Zuccarini ex Endlicher
P. gerardiana Wallich ex D. Don
P. squamata X.W. Li
Subsection Krempfianae
P. krempfii Lecomte
Subsection Strobus
P. albicaulis Engelmann
P. armandii Franchet
P. armandii Franchet subsp. mastersiana
(Hayata) Businský
P. ayacahuite Ehrenberg ex Schlechtendal
var. veitchii (Roezl) Shaw
P. ayacahuite Ehrenberg ex Schlechtendal
var. veitchii (Roezl) Shaw
P. ayacahuite Ehrenberg ex Schlechtendal
P. bhutanica Grierson, Long & Page
P. cembra Linnaeus
P. chiapensis (Martinez) Andresen
P. dalatensis Ferré subsp. procera
Businský
P. dalatensis Ferré subsp. procera
Businský
P. kwangtungensis Chun ex Tsiang
P. flexilis James
P. koraiensis Siebold & Zuccarini
P. lambertiana Douglas
P. monticola Douglas ex D. Don
P. morrisonicola Hayata
P. parviflora Siebold & Zuccarini subsp.
parviflora
P. parviflora Siebold & Zuccarini
P. parviflora Siebold & Zuccarini subsp.
pentaphylla (Mayr) Businský
P. parviflora Siebold & Zuccarini subsp.
pentaphylla (Mayr) Businský
P. peuce Grisebach
Allele
GenBank
A1,
A2
A1
A2
A1
A1
DQ642500
DQ642499
DQ018379
DQ642498
DQ642501
A1
DQ018380
A1
A1
A2
DQ642447
DQ642448
DQ642449
A1
A2
Clade
Collection information
Collector or source (voucherb )
Shanxi Province, Chinae
USDAFS Institute of Forest Genetics (OSC)
Gilgit, Pakistan
Gilgit, Pakistan
Yunnan, China
Yunnan, China
Businský 41105 (RILOGc )
Businský 41123 (RILOGc )
Businský 46118 (RILOGc )
Businský 46120 (RILOGc )
Lam Dong, Vietnam
First Darwin Expedition 242 (RBGE)
A
A
A
California, USA
Montana, USA
Oregon, USA
DQ642471
DQ642472
E
E
Anhui, China
Kaohsiung, Taiwan
Rancho Santa Ana Botanic Garden (OSC)
USDAFS Coeur d’Alene Nursery (OSC)
USDAFS Dorena Genetic Resource Center
(OSC)
Businský 46136 (RILOGc )
Businský 32163 (RILOGc )
A1
DQ642450
B
Mexico, Mexico
USDAFS Institute of Forest Genetics (OSC)
A2
DQ642451
B
Michoacan, Mexico
USDAFS Institute of Forest Genetics (OSC)
A3
A1
A2
A1
DQ642452
DQ642474
DQ642473
DQ642475
B
D
E
D
USDAFS Institute of Forest Genetics (OSC)
Businský 57112 (RILOGc )
Businský 57121 (RILOGc )
Blada (OSC)
A2
DQ642476
D
La Paz, Honduras
Arunachal Pradesh, India
West Kameng, India
North Carpathians,
Romania
Austria
A1
A1
A2
A1
DQ642453
DQ642454
DQ642455
DQ642477
C
C
C
E
A2
DQ642478
E
A1
A1
A2
A3
A1d ,
A2d
A3
A1,
A2
A3
DQ642479
DQ642458
DQ642456
DQ642457
DQ642480
DQ642481
DQ642482
DQ642459
DQ642460
DQ642461
E
B
B
B
A,
A
A
A,
B
D
A1
DQ642464
A
California, USA
A2
DQ642462
B
Oregon, USA
A3
DQ642463
B
California, USA
A1, DQ642483
E,
Taiwane
A2 DQ642484
A1d DQ642485
E
D
Shikoku, Japan
Businský 45155 (RILOGc )
A2
A2
D
D
Japan
Hokkaido, Japan
Iseli Nursery (OSC)
Forest Tree Breeding Center, Japan (OSC)
D
Hokkaido, Japan
Forest Tree Breeding Center, Japan (OSC)
D
Bulgaria
USDAFS Dorena Genetic Resource Center
(OSC)
(Continued on next page)
DQ642486
A2
A1
DQ642487
USDAFS Dorena Genetic Resource Center
(OSC)
Dvorak, CAMCORE (no voucher)
USDAFS Institute of Forest Genetics (OSC)
Hernandez (OSC)
Businský 44116b (RILOGc )
Chiapas, Mexico
Guatemala
Veracruz, Mexico
Kon Tum Province,
Vietnam
Kon Tum Province,
Vietnam
China
Alberta, Cananda
California, USA
Colorado, USA
Khabarovsky, Russia
Washington Park Arboretum 1943-40 (OSC)
Natural Resources Canada (OSC)
USDAFS Institute of Forest Genetics (OSC)
Roelof (OSC)
Krutovsky (no voucher)
Saitama, Japan
California, USAe
Forest Tree Breeding Center, Japan (OSC)
USDAFS Institute of Forest Genetics (OSC)
California, USA
USDAFS Central Zone Genetic Resource
Program (OSC)
USDAFS Central Zone Genetic Resource
Program (OSC)
USDAFS Dorena Genetic Resource Center
(OSC)
USDAFS Central Zone Genetic Resource
Program (OSC)
USDAFS Dorena Genetic Resource Center
(OSC)
Businský 44114 (RILOGc )
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TABLE 1. Sampled Pinus subg. Strobus representatives. When two alleles are listed for the same accession then either both alleles came from
the same individual, or the accession is a bulk collection of multiple individuals and is marked as such with a subscript. Clade information refers
to Figures 1 and 2. (Continuied)
Taxona
P. pumila (Pallas) Regel
P. sibirica Du Tour
P. strobiformis Engelmann
P. strobus Linnaeus
P. wallichiana A. B. Jackson
Section Parrya
Subsection Balfourianae
P. aristata Engelmann
P. balfouriana Balfour
P. longaeva Bailey
Subsection Cembroides
P. cembroides Zuccarini
P. culminicola Andresen & Beaman
P. discolor Bailey & Hawksworth
P. edulis Engelmann
P. johannis Robert-Passini
P. maximartinezii Rzedowski
P. monophylla Torrey & Fremont
P. pinceana Gordon
P. quadrifolia Parlatore ex
Sudworth
P. remota (Little) Bailey &
Hawksworth
P. rzedowskii Madrigal & Caballero
Subsection Nelsoniae
P. nelsonii Shaw
a
Allele
GenBank
Clade
Collection information
Collector or source (voucherb )
A1
A1
A1d
A2
A3
A1
A2
A1
A2
A3
A1
DQ642488
DQ642489
DQ642490
DQ642491
DQ642492
DQ642494
DQ642493
DQ642467
DQ642465
DQ642466
DQ642468
D
D
A
A
A
D
D
A
B
B
A
Macedonia, Yugoslavia
Macedonia, Yugoslavia
Byelorussia, Russia
Hokkaido, Japan
Hokkaido, Japan
Kemerovo District, Russia
Krasnoyarsk Krai, Russia
Texas, USA
Coahuila, Mexico
Nuevo Leon, Mexico
Wisconsin, USA
A2d
A3
A1
A2
A3
DQ642470
DQ642469
DQ642497
DQ642495
DQ642496
A
A
D
E
E
New Jersey, USA
Minnesota, USA
Hutu, Rara, Nepal
Gilgit, Pakistan
Himalayas
USDAFS Institute of Forest Genetics (OSC)
USDAFS Institute of Forest Genetics (OSC)
Krutovsky (no voucher)
Watano (OSC)
Watano (OSC)
Natural Resources Canada (OSC)
Buck (OSC)
Gernandt DSG599 (OSC)
USDAFS Institute of Forest Genetics (OSC)
USDAFS Institute of Forest Genetics (OSC)
USDAFS Dorena Genetic Resource Center
(OSC)
Gernandt DSG00500 (OSC)
USDAFS Oconto River Seed Orchard (OSC)
Natural Resources Canada (OSC)
Businský 41101 (RILOGc )
Natural Resources Canada (OSC)
A1
A2
A1
A2
DQ642503
DQ642504
DQ642505
DQ642506
Colorado, USA
Arizona, USA
S. California, USA
N. California, USA
A1d
A2
AY634346
DQ642507
California, USA
Utah, USA
USDAFS Institute of Forest Genetics (OSC)
Farrell 36 (OSC)
Wisura (OSC)
USDAFS Central Zone Genetic Resource
Program (OSC)
Gernandt 03099 (OSC)
McArthur (OSC)
A1
A2
A3
A1,
A2
A1d ,
A3d
A2
A1
A2
A3
A1
A2
A1
A1
A1
A2
A3
A1
A2
A1
DQ642508
DQ642510
DQ642509
DQ642511
DQ642512
DQ642513
DQ642514
DQ642515
DQ642518
DQ642517
DQ642516
DQ642519
DQ642520
DQ642521
San Luis Potosi, Mexico
Texas, USA
Hidalgo, Mexico
Nuevo Leon, Mexico
USDAFS Institute of Forest Genetics (OSC)
Gernandt DSG593 (OSC)
Hernandez (OSC)
Velazquez (OSC)
San Luis Potosi, Mexico
Gernandt 2101 (MEXU)
DQ642522
DQ642523
DQ642524
DQ642525
DQ642526
DQ642527
F
F
F
F,
G
F,
F
F
F
G
G
G
I
I
I
G
G
G
I
I
H
Arizona, USA
Utah, USA
Utah, USA
New Mexico, USA
Nuevo Leon, Mexico
San Luis Potosi, Mexico
Zacatecas, Mexico
Zacatecas, Mexico
California, USA
Nevada, USA
Utah, USA
Queretaro, Mexico
Coahuila, Mexico
California, USA
Hammond (OSC)
Gernandt DSG489 (OSC)
Gernandt DSG488 (OSC)
USDAFS Institute of Forest Genetics (OSC)
Frankis, M.P. 179 (E)
Gernandt DSG501 (MEXU)
USDAFS Institute of Forest Genetics (OSC)
USDAFS Institute of Forest Genetics (OSC)
Rancho Santa Ana Botanic Garden (OSC)
Halse 6668 (OSC)
Gernandt DSG480 (OSC)
USDAFS Institute of Forest Genetics (OSC)
USDAFS Institute of Forest Genetics (OSC)
Winter (OSC)
A2
A1
DQ642528
DQ642531
H
F
California, USA
Texas, USA
Winter (OSC)
Gernandt DSG588 (OSC)
A2
A3d
A1
DQ642530
DQ642529
DQ642532
F
G
I
Texas, USA
Coahuila, Mexico
Michoacán, Mexico
Zech 382-4 (OSC)
Gernandt DSG 0601 (MEXU)
Businský 47131 (RILOGc )
A1
A2d
DQ642502
AY634347
Nuevo Leon, Mexico
Nuevo Leon, Mexico
Gernandt & Ortiz DSG10398 (OSC, MEXU)
Gernandt & Ortiz DSG11298 (OSC, MEXU)
Taxonomy follows Gernandt et al. (2005).
Herbarium acronyms follow Index Herbariorum: http://sciweb.nybg.org/science2/IndexHerbariorum.asp.
RILOG = Silva Tarouca Research Institute for Landscape and Ornamental Gardening, 252 43 Průhonice, Czech Republic.
d
Cloned product, see Methods for details.
b
c
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on availability, individuals were selected that spanned
the geographic range of the species. In cases of nar­
row endemism or where collections were limited, two
alleles were sequenced from the same individual. Esti­
mates of species distribution area were based on pub­
215 lished maps (Critchfield and Little, 1966; Malusa, 1992;
Farjon and Styles, 1997; Delgado et al., 1999, Ledig et al.,
1999).
For the eight representatives of North American sub­
section Strobus, three alleles were chosen from a more ex­
220 tensive data set that will be used to evaluate populationlevel variation (four to seven alleles per species; Syring
et al., unpublished data). The three alleles selected rep­
resent the most divergent sequences based on calculated
p-distances.
210
225
230
235
240
245
250
Choice of Outgroup
As described in Syring et al. (2005), introns between
pine subgenera are frequently unalignable due to nu­
merous and overlapping indels, repeated motifs, and
uncertain sequence homology. This precludes the use of
members from subgenus Pinus or more distantly related
genera as outgroups in this analysis. Independent evi­
dence from five nuclear genes and cpDNA (Gernandt
et al., 2005; Syring et al., 2005) shows that the sections of
subg. Strobus are monophyletic and sister to each other;
for this reason, members of one section were used as the
outgroup for analyzing the alternative section. Pinus nel­
sonii (Sect. Parrya, subsect. Nelsoniae) is exceptional. Evi­
dence from three nuclear genes (Syring et al., 2005) and
cpDNA (Gernandt et al., 2005) resolve P. nelsonii as the
sister lineage to the remaining members of sect. Parrya.
In contrast, the LEA-like locus used in this study places
P. nelsonii in a unique, moderately supported (71% BS)
position sister to sect. Quinquefoliae when midpoint root­
ing is employed. Because of this uncertainty, we chose
to include P. nelsonii as a member of the outgroup in an­
alyzing both sects. Quinquefoliae and Parrya. Therefore,
P. monticola, P. krempfii, P. gerardiana, and P. nelsonii were
used as outgroup taxa for analyzing sect. Parrya, and P.
aristata, P. monophylla, and P. nelsonii were used as out­
group taxa for analyzing sect. Quinquefoliae. Although
the LEA-like locus is too labile to provide insight into
the placement of P. nelsonii, future studies using more
evolutionarily constrained molecules will be used to in­
vestigate the position of this lineage.
Locus Amplification, Sequencing, Alignment, and
Recombination Analysis
Description of the LEA-like locus and the protocols
for amplification, sequencing, and alignment are given
in Syring et al. (2005). Gaps were coded as phylogenetic
260 characters using the method of Simmons and Ochoterena
(2000) and the online program Gap Recoder (R. Ree, http:
//maen.huh.harvard.edu:8080/services/gap recoder);
all coded gaps were verified manually. Four previously
published sequences (DQ018379, DQ018380, AY634346,
265 AY634347) are incorporated in this study (Table 1). The
alignment is available at TreeBase (S1538). Statistics
255
5
calculated from the alignment include the average
number of characters, number of variable and parsi­
mony informative characters, average within-group
p-distance, and average base composition (determined
using MEGA 2.1; Kumar et al., 2001).
Evidence for recombination was assessed using both
sequence-based (maximum x 2 method) and topological
(difference in sum of squares; DSS) approaches. First,
the substitution-based ‘maximum x 2 ’ method was cho­
sen because of its high power and low false-positive
rate (Posada, 2002). This method (Smith, 1992; Posada
and Crandall, 2001) identifies recombinant segments by
testing for significant differences among proportions of
variable and nonvariable polymorphic positions in ad­
jacent regions of aligned sequences. For all possible se­
quence pairs, a sliding window containing 10% of the
variable positions was divided into two equal partitions
and moved in 1-bp increments along the alignment. At
each increment, a 2 × 2 x 2 was calculated as an expres­
sion of the difference in the number of variable sites on
each side of the partition for pairs of sequences. Puta­
tive recombination points were identified by plotting
x 2 values along the length of the alignment. An alter­
native phylogeny-based test, the difference in sum of
squares method (DSS; McGuire et al., 1997; Milne et al.,
2004) was also used to identify putative recombinants
using phylogenetic trees constructed from adjacent re­
gions of an alignment. For all sequences, a 150-bp win­
dow was divided into two partitions and moved in 20-bp
increments. For each increment, a distance matrix (Fitch
84) was calculated and a least squares tree constructed
for each partition. Branch lengths were estimated us­
ing least squares, and sum of squares for each parti­
tion were recorded. Topologies for partitions were then
swapped, branch lengths for the forced topology were
determined, and sum of squares recorded. DSS values
were plotted along alignments, and recombinant seg­
ments were identified by peaks in DSS values. Signifi­
cance for both methods was determined by 1000 permu­
tations (parametric bootstrapping) using a = 0.01 as a
threshold of significance and the program RDP2 (Mar­
tin et al., 2005). Analyses were conducted on data sets
where shared indels were discarded to prevent false
positives.
270
275
280
285
290
295
300
305
310
Phylogenetic and Statistical Analysis
Phylogenetic analyses were performed by taxonomic
section using maximum parsimony (MP; PAUP* ver­
sion 4.0b10; Swofford, 2003). Most parsimonious trees
were found from branch-and-bound searches, with all 315
characters weighted equally and treated as unordered.
Branch support was evaluated using the nonparamet­
ric bootstrap (Felsenstein, 1985), with 1000 replicates
and TBR branch swapping. Alternative phylogenetic
hypotheses and statistical strength for species nonmono­ 320
phyly were analyzed using constraints on tree topolo­
gies in PAUP*. The Wilcoxon signed-rank test (WSR;
Templeton, 1983) was employed to test for significant dif­
ferences among topologies. For this test, up to 1000 of the
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most-parsimonious trees were used as constraint topolo­
gies. The range of P values across all topologies is re­
ported for every test.
Statistical associations between intraspecific nu­
cleotide diversity, geographic range (a proxy for species
abundance and census population size), and extent of
monophyly were explored using analysis of variance
(ANOVA). For these tests, species were categorized into
two “PHYLY” classes, either strongly nonmonophyletic
(i.e., intraspecific allelic coalescence resulted in statis­
tically significant topological distortion as shown by
the WSR constraint test) or weakly nonmonophyletic to
monophyletic (i.e., WSR constraint tests were insignif­
icant). Nucleotide diversity was analyzed directly, and
geographic ranges (km2 ; Critchfield and Little, 1966)
were log10-transformed to minimize skewness and kur­
tosis. Differences in univariate measures between PHYLY
classes were tested using one-factor ANOVA. Statistical
analyses were performed using SAS (SAS Institute, 1999).
Coalescent Expectations of Genic and Genomic Monophyly
According to Rosenberg (2003) reciprocal monophyly
is predicted to be more likely than paraphyly under
conditions of neutrality at ∼1.67 × 2Ne diploid gener­
ations, and complete genome-wide coalescence may re­
quire ∼5.30 × 2Ne diploid generations. In order to make
these calculations for species of Pinus, Watterson’s esti­
mate of Theta (8) was determined for select species using
DnaSP v.4.00.6 (Rozas et al., 2004). Effective population
sizes were estimated using the formula Ne = 8/(4 ×j G ),
where j G is the absolute silent mutation rate per nu­
cleotide, adjusted for generation time. The absolute silent
mutation rate for Pinus was recently estimated from 11
nuclear genes (Willyard et al., 2006) to average 7.0 × 10−10
substitutions/site/year, assuming an 85 million year di­
vergence of pine subgenera. Generation times for indi­
vidual species were taken as the average for the range of
years to seed bearing age cited in Krugman and Jenkin­
son (Woody Plant Seed Manual, 2nd edition [R. G. Nis­
ley, ed.], http://www.nsl.fs.fed.us/wpsm/). Although
estimating the generation time of species with uneven­
aged populations and overlapping generations can be
contentious, the range of variability in this parameter is
not great enough to affect the order of magnitude for the
calculations of Ne .
In this study we test the extent of monophyly for
LEA-like allele lineages within species. For simplicity,
we abbreviate this as ‘species monophyly’ or ‘species
nonmonophyly,’ but want to emphasize that we are re­
ferring to the coalescence of genealogies of the LEA-like
gene and not the extent of monophyly for actual species.
375
R ESULTS
380
Sequences, Alignment Characteristics, Allelic Diversity, and
Recombination
We acquired 86 LEA-like alleles from 39 species of Pinus subgenus Strobus (Table 1). From 14 of the 39 species
we sequenced three unique alleles, and from 19 species
VOL. 56
we sequenced two unique alleles. From the remaining
six species we obtained a single allele, although in three
of these cases multiple sequences from different indi­
viduals returned an identical allele (see Table 1); these
include P. peuce (N = 3 different seeds), P. maximartinezii
(N = 2), P. squamata (N = 2), and P. kwangtungensis, P.
krempfii, and P. rzedowskii (N = 1 template each). In Pinus krempfii alone, the locus was amplified directly from
diploid needle tissue; direct sequencing indicated this in­
dividual was homozygous. For three species having two
unique alleles, further seed sampling from other trees
recovered one of the known alleles: albicaulisA1 (allele
A1, N = 2), chiapensisA1 (allele A1; N = 2), and parvi­
floraA2 (allele A2; N = 3). For P. bungeana, P. culminicola,
and P. morrisonicola both alleles were sequenced from the
same individual; for P. discolor (A1 and A3), P. koraien­
sis (A1 and A2), and P. lambertiana (A1 and A2) two of
the three alleles were from the same individual. Ten se­
quences included in this data set were cloned: koraien­
sisA1, koraiensisA2, longaevaA1, nelsoniiA2, parvifloraA1,
pumilaA1, remotaA3, and strobusA2. Based on this sam­
ple, our phylogenetic estimates of allelic monophyly can
be assessed in 33 of 39 included species.
Our aligned sequence for the LEA-like locus was 1554
bp in length, with individual sequences averaging 898.5
bp from sect. Quinquefoliae (range = 821–971 bp) and
987.3 bp from sect. Parrya (range = 937–1132 bp). The
aligned sequence includes a partial exon on the 31 end
with 44 complete and two partial codons (135 bp). The
remaining sequence is intron and has an aligned length
of 1419 bp. The exon has 19 variable positions within
subg. Strobus (12 in sect. Quinquefoliae, 8 in sect. Parrya),
of which 3 were localized in first codon positions, 4 in
second positions, and 12 in third positions. Within subg.
Strobus, inferred amino acid replacements occur at 7 of
44 sites. The intron segment included 249 variable sites
and 136 parsimony informative (PI) sites within subg.
Strobus. This included 157 variable sites (88 PI) in sect.
Quinquefoliae, and 110 variable sites (41 PI) in sect. Parrya.
Nucleotide frequencies were relatively AT-rich (25.9% A,
33.5% T, 20.2% G, 20.6% C). Complex and simple indels
are frequent across the length of the intron and range in
length from a single nucleotide to a 110-bp deletion in
armandiiA1. In total, 74 gaps were scored and appended
to the alignment.
Average interspecific 1 for subg. Strobus at the LEAlike locus is 0.0330 ±0.0029. Estimates of intraspecific 1
show that soft pine species display a wide range of ge­
netic diversity and differentiation. Across the 33 species
with nonidentical alleles, intraspecific nucleotide diversity ranged from 0.0 in P. albicaulis (alleles differ by one
indel) to 0.0211 in P. strobiformis, and averaged 0.0085
(Table 2). Other species showing high nucleotide diver­
sity in our sample include P. lambertiana (1 = 0.0196), P. jo­
hannis (0.0186), P. bhutanica (0.0185), P. monticola (0.0155),
P. edulis (0.0158), and P. aristata (0.0149). Members of sect.
Parrya showed a trend toward higher intraspecific nu­
cleotide diversity relative to sect. Quinquefoliae, with 10
of 13 species (76.9%) versus 11 of 20 species (55.0%) show­
ing 1 ≥ 0.005, respectively (Table 2).
385
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395
400
405
410
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420
425
430
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440
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TABLE 2. Interspecific nucleotide diversity (1), pproximate geo­
graphic ranges, and monophyletic status of the species included in this
study. N = number of unique alleles upon which 1 was based. 1 was
determined using p-distances; SD = standard deviation of the 1 Cal­
culations. Approximate ranges for each species were determined from
several sources and represent best estimates (see Methods).
Species
N
1 (SD)
Approx.
range
(km2 )
P. strobiformis
P. lambertiana
P. johannis
P. bhutanica
P. edulis
P. monticola
P. aristata
P. longaeva
P. discolor
P. remota
P. wallichiana
P. balfouriana
P. cembra
P. pumila
P. sibirica
P. strobus
P. cembroides
P. culminicola
P. dalatensis
P. monophylla
P. armandii
P. pinceana
P. nelsonii
P. koraiensis
P. gerardiana
P. chiapensis
P. ayacahuite
P. parviflora
P. flexilis
P. albicaulis
P. bungeana
P. morrisonicola
P. quadrifolia
P. kwangtungensis
P. krempfii
P. maximartinezii
P. peuce
P. rzedowskii
P. squamata
3
3
2
2
3
3
2
2
3
3
3
2
2
3
2
3
3
2
2
3
2
2
2
3
2
2
3
2
3
2
2
2
2
1
1
1
1
1
1
0.0211 (0.0041)a
0.0196 (0.0038)
0.0186 (0.0040)
0.0185 (0.0045)
0.0158 (0.0033)
0.0155 (0.0033)
0.0149 (0.0040)
0.0128 (0.0038)
0.0112 (0.0026)
0.0112 (0.0026)
0.0112 (0.0028)
0.0107 (0.0034)
0.0100 (0.0032)
0.0092 (0.0025)
0.0088 (0.0033)
0.0078 (0.0024)
0.0076 (0.0023)
0.0075 (0.0022)
0.0077 (0.0028)
0.0058 (0.0021)
0.0051 (0.0027)
0.0047 (0.0023)
0.0044 (0.0022)
0.0038 (0.0018)
0.0035 (0.0019)
0.0031 (0.0017)
0.0022 (0.0012)
0.0022 (0.0015)
0.0014 (0.0010)
0.0000 (0.0000)
0.0012 (0.0012)
0.0011 (0.0011)
0.0010 (0.0011)
n/a
n/a
n/a
n/a
n/a
n/a
160,000
110,000
18,000
8000
260,000
370,000
15,000
10,000
27,000
27,000
150,000
2500
80,000
6,000,000
5,000,000
1,800,000
190,000
50
50
75,000
600,000
25,000
6000
600,000
25,000
5000
50,000
140,000
250,000
400,000
50,000
5000
5000
500
300
4
2000
100
0.05
Monophyletic
in the Strict
Consensus Tree?
N
N
N
N
N
N
Y
Y
N
N
N
N
N
Y
N
Y
N
N
N
N
N
Y
Y
Y
Y
Y
N
Y
N
Y
Y
Y
Y
n/a
n/a
n/a
n/a
n/a
n/a
a
Value excludes the region where tests indicated potential recombination be­
tween sites 388 and 399, see text for details.
Only one allele was shared between two species,
namely ayacahuiteA1 and flexilisA1. This observation
was striking, because these samples came from wild
collections separated by ∼3600 km (P. ayacahuite orig­
445 inated from the state of Mexico, Mexico and P. flex­
ilis from Alberta, Canada). This allele has also been
sequenced for P. strobiformis from both New Mexico
and Texas (J. Syring, unpublished data). All other com­
parisons between P. ayacahuite and P. flexilis alleles
450 show pairwise distances between 0.0011 and 0.0043,
suggesting a close genetic affinity. Although not con­
taining identical alleles, seven interspecific comparisons
yielded highly similar alleles with p-distances ≤ 0.0011:
ayacahuiteA1/flexilisA3, ayacahuiteA2/flexilisA1, ayac­
455 ahuiteA3/strobiformisA2, cembraA1/parvifloraA2, bhutan­
7
icaA2/wallichianaA2, bhutanicaA2/wallichianaA3, and
culminicolaA2/remotaA3.
Of the 86 LEA-like sequences used in this analysis,
only one allele showed evidence of recombination, de­
spite a relatively lax threshold for significance (a = 0.01).
The maximum x 2 method identified one recombinant re­
gion from strobiformisA1 between positions 388 and 399.
Close inspection shows that strobiformisA1 contains an
11-nucleotide motif (CTTTTGTAGCC) with 37% identity
to the same region (e.g., TTCYACAGGA) from all other
species of sect. Quinquefoliae. If this segment is recom­
binant, it likely arose via xenologous recombination or
the PCR process because it lacks similarity to other ho­
mologous alleles. The topology-based DSS method pro­
vides no evidence for recombination in strobiformisA1 or
other sequences. Because the single putatively recom­
binant segment from strobiformisA1 only adds autapo­
morphic steps to this lineage and does not distort the
topology (data not shown), we included this sequence in
all analyses (the potentially recombinant 12 bases were
omitted from estimates of 1).
460
465
470
475
Phylogenetic Analyses
Section Quinquefoliae.—From the branch and bound
search of the sect. Quinquefoliae data set, twenty most par­
simonious trees were recovered (Fig. 1). Trees were 350
steps in length, had a consistency index (CI) of 0.8229,
and a retention index (RI) of 0.8835. Subsection Krempfi­
anae is sister to a clade of subsects. Strobus and Gerardianae
(67% bootstrap support, BS). Support for the topology
within subsect. Gerardianae is relatively high with 81%
BS for the monophyly of the subsection, and 90% BS for
the resolution of the rare endemic P. squamata as sister to
P. gerardiana and P. bungeana.
Alleles from species of subsect. Strobus resolved into
five major groups (Fig. 1), all of which were present in
the strict consensus tree. Bootstrap support ranged from
lacking (<50%; clades A and D), or moderate (74%; clade
E), to very strong (98%; clades B and C). Clade A includes
alleles from five North America species (P. strobiformis, P.
albicaulis, P. strobus, P. lambertiana, P. monticola) and two
East Asian species (P. koraiensis, P. pumila). Noteworthy is
the 100% BS uniting alleles lambertianaA1 and monticola
A1. Despite the sister relationship among these alleles,
a sister relationship among these species is contradicted
by the lack of allelic coalescence within these two species
(described below). Sister to clade A are two strongly sup­
ported and exclusively North American groups, one of
which includes alleles from five species (clade B) and the
second of which includes only P. chiapensis alleles (clade
C). Clade C is the only group where species do not share
alleles with another clade. Relationships between clades
A, B, and C are essentially unresolved, and the node
supporting clade C as sister to clades A/B collapses in
the strict consensus tree. The remaining alleles from P.
monticola (A2, A3) both resolved within clade B, as did
one allele from P. lambertiana (A2). Clade D is composed
entirely of alleles sampled from European (P. cembra, P.
peuce) and Asian (P. bhutanica, P. wallichiana, P. sibirica, P.
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500
505
510
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VOL. 56
FIGURE 1. One of 20 most-parsimonious trees for section Quinquefoliae. Trees are rooted with P. aristata, P. edulis, and P. nelsonii. Bootstrap
values from 1000 replicates and TBR branch swapping are shown near nodes. Number of characters = 1554; length of trees = 358; consistency
index = 0.827; retention index = 0.889. Asterisks (∗ ) indicate nodes that collapse in the strict consensus tree. Numbers in parentheses following
alleles refer to the number of additional times the same allele was sequenced. The letters “L” and “M” are placed on the node of the most recent
common ancestors for all alleles of P. lambertiana and P. monticola, respectively.
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9
FIGURE 2. One of 4495 most-parsimonious trees for section Parrya. Trees are rooted with P. gerardiana, P. krempfii, P. monticola, and P. nelsonii.
Bootstrap values from 1000 replicates and TBR branch swapping are shown near nodes. Number of characters = 1554; length of trees = 300;
consistency index = 0.843; retention index = 0.883. Asterisks (∗ ) indicate nodes that collapse in the strict consensus tree. Numbers in parentheses
following alleles refer to the number of additional times the same allele was sequenced.
515
parviflora) species, with the exception of lambertianaA3,
which is in an unsupported position as the sister to the
remaining members of this clade. Pinus bhutanica and P.
wallichiana have alleles in both clade D and clade E. The
latter clade is composed strictly of alleles from species
distributed in southeastern Asia.
Section Parrya.—Branch and bound searches on the 520
sect. Parrya data recovered 4495 most parsimonious
trees that were 292 steps in length (Fig. 2; CI = 0.843,
RI = 0.883). Subsections Balfourianae and Cembroides were
monophyletic sister lineages, with 97% BS each. Support
for relationships within subsect. Balfourianae is high, with 525
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P. longaeva resolving as sister to the clade of P. aristata/P.
balfouriana (97% BS, although alleles of P. balfouriana are
nonmonophyletic in the strict consensus tree). Species of
subsect. Cembroides resolved into four clades (F through
530 I; Fig. 2), all of which were present in the strict consen­
sus tree with the exception of the culminicolaA1 and dis­
colorA3 alleles (these collapse out of clade F in the strict
consensus tree). With the exclusion of these two poorly
supported alleles andjohannisA2 from clade I, each of the
535 four major clades within subsect. Cembroides have mod­
erate to strong support (78% to 90% BS), though rela­
tionships among and within the clades remain largely
unresolved.
Clade F contains all alleles from P. cembroides and P.
540 discolor, along with some of the alleles for P. culminicola,
P. edulis, and P. remota, the remainder of which are found
in clade G. Clade G contains all three alleles of P. mono­
phylla and one of two P. johannis alleles. Support for the
monophyly and relationships among the three alleles of
545 P. monophylla is weak (57% and 55% BS, respectively),
and all remaining support within clade G unites alleles
from differing species. Clade H contains only the two
alleles of P. quadrifolia (90% BS), and is unique in be­
ing the only clade from this section where species do
550 not share alleles with another clade. Clade I contains
both alleles of P. pinceana and the singleton alleles of
P. maximartinezii and P. rzedowskii. The support for the
node uniting these three species is strong (89% BS), but
the node supporting the sister relationship of P. maxi­
555 martinezii and P. pinceana is only weakly supported (68%
BS). Sister to these three species is the poorly supported
johannisA2 (62% BS), whose other allele is found in a
strongly supported position within clade G.
Species Monophyly and Topological Conflict Arising from
Genealogical Nonmonophyly
Section Quinquefoliae.—The strict consensus tree indi­
cates that of the 20 species for which multiple unique al­
leles were sequenced, 9 species (45%) were monophyletic
(Table 2). For species exhibiting allelic monophyly, seven
565 showed moderate to very high support (82% to 100%
BS). For species exhibiting paraphyly, eight had at least
one well-supported allele (83% to 100% BS), ensuring
nonmonophyly. Most severe in this regard were the five
species that possess alleles in two or more of the major
570 clades: these include P. bhutanica (clades D, E), P. lamber­
tiana (clades A, B, D), P. monticola (clades A, B), P. strobi­
formis (clades A, B), and P. wallichiana (clades D, E). For
species including three alleles, the proportion of monophyletic species was nearly identical to the broader sam­
575 ple (50%; 6 of 12 species), suggesting that small sample
sizes generally capture enough intraspecfic heterogene­
ity to be indicative of allelic monophyly in this section.
Species-level monophyly in the strict consensus tree
is not necessarily dependent on having low nucleotide
580 diversity (Table 2). Although intraspecific mean pdistances are low for P. flexilis (0.001) and P. armandii
(0.005), these species are not monophyletic. In contrast, P.
strobus has a relatively high mean intraspecific p-distance
560
VOL. 56
(0.008) and is well supported as monophyletic (88% BS).
However, of the seven species having mean intraspecific
p-distances >0.008, only P. pumila was monophyletic in
the strict consensus tree, and none had bootstrap support
>50%.
Enforcing topological constraints for species mono­
phyly shows that 6 of the 11 nonmonophyletic members of sect. Quinquefoliae return significant results in
the WSR test (Table 3), indicating statistical support for
their nonmonophyly (a = 0.05). For example, constrain­
ing topologies for the monophyly of P. flexilis or P. ay­
acahuite led to trees with lengths 351 and 352, respectively (one step and two steps longer than unconstrained
topologies). These topologies returned insignificant WSR
results (P = 0.3173–0.6547 for P. flexilis and 0.1573–0.4142
for P. ayacahuite), suggesting that the nonmonophyly
of these species is not strongly supported. In contrast,
results from P. lambertiana (369 steps, P = <0.0001–
0.0003), P. monticola (368 steps, P = <0.0001–0.0001), and
P. bhutanica (367 steps, P = <0.0001–0.0002) strongly sup­
port the polyphyly of alleles from these species.
Section Parrya.—The strict consensus tree indicates
that of the 13 species for which multiple alleles were se­
quenced, only 5 species (38.5%) were monophyletic (Ta­
ble 2). The remaining eight species (61.5%) were either
paraphyletic or polyphyletic. For those species showing
allelic monophyly, four were strongly supported (85% to
100% BS; the monophyly of P. longaeva has 70% BS). For
those species showing paraphyly or polyphyly, five had
at least one allele in a moderately or strongly supported
position (79% to 90% BS) ensuring nonmonophyly. Alle­
les from four species (P. culmicola, P. edulis,P. johannis, P.
remota) are spread across two or more of the major clades.
None of the five species represented with three sequences
were monophyletic in the strict consensus tree, suggest­
ing that, in contrast to sect. Quinquefoliae, additional sam­
pling may reduce the number of monophyletic species
in sect. Parrya.
Following the trend in sect. Quinquefoliae, allelic mono­
phyly is not a simple function of intraspecific genetic di­
versity (Table 2). Although nucleotide diversity is low
for the three alleles of P. monophylla (1 = 0.006), this
species is not monophyletic in the strict consensus tree.
In contrast, P. aristata (1 = 0.015) and P. longaeva (1 =
0.013), once considered to be a single species (see Bailey,
1970), show high nucleotide diversity yet are each monophyletic.
Enforcing topological constraints for species mono­
phyly shows that four of the eight nonmonophyletic
members of sect. Parrya return significant results in the
WSR test (Table 3), and 335 of the 951 trees (35.2%) indi­
cate that the nonmonophyly of P. discolor is significant. It
is noteworthy that in both sects. Quinquefoliae and Parrya,
all species whose alleles fall into two or more of the major
clades return significant WSR results, thereby providing
further confidence in the distinctiveness of these clades.
Subgenus summary.—Across 33 species from subgenus
Strobus, we found that 14 were monophyletic (42.4%) and
19 (57.6%) were nonmonphyletic at the LEA-like locus
in the strict consensus tree (Table 2). Ten of 33 species
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TABLE 3. Wilcoxon signed rank (WSR) tests of species nonmonophyly. All species from the strict consensus tree that were either para- or
polyphyletic were constrained to monophyly and the resulting trees were tested for topological incongruence against the unconstrained trees.
Up to 1000 most parsimonious trees were saved in the constraint analysis. Significant results at the a = 0.05 level are marked with an asterisk (∗ ).
Species
sect. Quinquefoliae Figure 1
P. armandii
P. ayacahuite
P. bhutanica
P. cembra
P. dalatensis
P. flexilis
P. lambertiana
P. monticola
P. siberica
P. strobiformis
P. wallichiana
sect. Parrya Figure 2
P. balfouriana
P. cembroides
P. culminicola
P. discolor
P. edulis
P. johannis
P. monophylla
P. remota
No. of trees saved
from constraint
analysis
Length of
Treesb
Range of P
values
E-2
B-3
D-1, E-1
D-2
E-2
B-3
A-1, B-1, D-1
A-1, B-2
D-2
A-1, B-2
D-1, E-2
114
150
1000
397
61
487
1000
831
35
154
70
351
352
367
359
351
351
369
368
353
364
364
0.3173–0.6547
0.1573–0.4142
<0.0001–0.0002∗
0.0027–0.0126∗
0.3173–0.6547
0.3173–0.6547
<0.0001–0.0003∗
<0.0001–0.0001∗
0.2568–0.3173
0.0005–0.0010∗
0.0005–0.0017∗
sect. Balfourianae
F-3
F-1, G-1
F-3
F-1, G-2
G-1, I-1
G-3
F-2, G-1
1000
1000
1000
951
594
1000
1000
1000
292
294
302
298
304
302
292
308
1.0000
0.1573–0.5637
0.0016–0.0352∗
0.0339∗ –0.1336c
0.0047–0.0073∗
0.0016–0.0124∗
1.0000
0.0001–0.0036∗
Clades in which
alleles appeara
a
Letters refer to clades in Figures 1 and 2, numbers following letters indicate the number of sequences in each clade.
Length of most parsimonious trees without constraints is 350 steps for sect. Quinquefoliae and 292 steps for sect. Parrya
35% of the topologies for P. discolor returned significant results in the WSR test.
b
c
645
650
655
660
665
670
675
(30.3%) were supported as nonmonophyletic both in the
strict consensus tree and in the WSR constraint anal­
yses (referred to as cases of “strong” nonmonophyly;
Table 3). Data for the remaining nine species that were
nonmonophyletic in the strict consensus tree, but did
not return significant results in the WSR tests are consid­
ered ambiguous (including P. discolor; referred to as cases
of “weak” nonmonophyly). Excluding cases of weak
nonmonophyly, 6 of 15 species from sect. Quinquefoliae
(40.0%), and 4 of 9 species (44.4%) from sect. Parrya show
strong allelic nonmonophyly.
Across both pine sections, the PHYLY of a sample of
alleles from a species (e.g., strongly nonmonophyletic
versus weakly nonmonophyletic or monophyletic) was
evaluated for statistical associations with two depen­
dent variables, 1 and log10[geographic range]. These
two variables are uncorrelated with each other (Pearson’s
R = 0.0554; P = 0.759). Individually, PHYLY shows a
statistically significant association with nucleotide di­
versity. Average nucleotide diversities in species show­
ing strong deviation from monophyly are significantly
higher (1 = 0.0149) that comparable values from monophyletic and weakly nonmonophyletic species com­
bined (1 = 0.0056; one-way ANOVA, P < 0.0000; Table
4). Monophyletic and weakly nonmonophyletic species
are not significantly different (1 = 0.0049 and 0.0067
respectively; one-way ANOVA, P = 0.339; results not
shown). A relationship between PHYLY and geographic
range of a species, in contrast, was not detected. Aver­
age geographic ranges for these two classes are essen­
tially indistinguishable (one-way ANOVA, P < 0.8007;
Table 4), with the average geographic range for strongly
nonmonophyletic species at 40,615 km2 , and the aver­
age range for weakly nonmonophyletic-to-monophyletic
species at 52,516 km2 . In combination, these analyses
indicate that the most important determinant of allelic
monophyly is intraspecific variation (and possibly ap­ 680
portionment of genetic variation across species), rather
than the geographic range of a species or their census
population size.
Estimating Ne , the Time to Reciprocal Monophyly,
and Genome-wide Coalesence
Estimates of Ne , the number of years for reciprocal
monophyly to be more likely than paraphyly, and the
number of years for complete genome-wide coalescence
were calculated for three species of pines (Table 5). These
species were chosen because they represent a range of
nucleotide diversities (Table 2), as well as the full phylo­
genetic spectrum from essentially monophyletic (P. flex­
ilis; 1 = 0.0014), to weakly non-monophyletic (P. discolor;
1 = 0.0112), to strongly nonmonophyletic (P. lamber­
tiana; 1 = 0.0196). Estimates of 8 are not significantly
different than values of 1 (based on Tajima’s D statis­
tic; data not shown), so our estimates of 8 are unlikely to
reflect effects from natural selection. Estimated values for
Ne range from ca. 1.7 × 104 for P. discolor to 12 × 104 for P.
lambertiana (Table 5). Based on these estimates, the time
for reciprocal monophyly to become more likely than
paraphyly at this locus is substantially different across
species, ranging from 1.7 million years for the nearly
monophyletic species P. flexilis, to 24 million years for the
strongly nonmonophyletic P. lambertiana. The estimated
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VOL. 56
SYSTEMATIC BIOLOGY
TABLE 4. Nucleotide diversity and geographic range of strongly nonmonophyletic versus monophyletic to weakly nonmonophyletic species
of pines. Significant differences between phyly categories were tested using one-way ANOVA.
Dependent variable
Phyly
Nucleotide diversity
Log10(geographic range)
time for complete genome-wide coalescence ranges from
5.4 million years to 76 million years.
710
715
720
725
730
735
N
Strongly nonmonophyletic
10
Monophyletic to weakly nonmonophyletic
23
F = 32.02; df = 1 between groups, 31 within groups; P< 0.0000
Strongly nonmonophyletic
10
Monophyletic to weakly nonmonophyletic
23
F = 0.06; df = 1 between groups, 31 within groups; P = 0.8007
D ISCUSSION
Considering that widespread allelic nonmonophyly
was observed and the fact that this is a single-locus
estimate of relationships, phylogenetic conclusions re­
garding relationships among the terminal taxa remain
premature, even in those cases where species monophyly
is clear and support values are high. However, the phy­
logeny presented in this paper is in strong agreement
with both cpDNA (Gernandt et al., 2005) and nrDNA ITS
(Liston et al., 1999) in resolving the same monophyletic
subsections, thereby further increasing our confidence
in these intrageneric ranks. Although this study has de­
tected nine cases of species having alleles in two or more
of the major clades in subsects. Strobus and Cembroides,
no intraspecific variability crossed the taxonomic subsec­
tions defined by Gernandt et al. (2005). Lack of species
monophyly appears to be a greater complication within
the species-rich subsects. Strobus and Cembroides than in
the species-poor subsects. Gerardianae and Balfourianae—
11 of 28 species tested for allelic monophyly in subsects.
Strobus and Cembroides were strongly nonmonophyletic,
whereas none of the six members of subsects. Gerardianae
and Balfourianae were strongly nonmonophyletic.
The LEA-like intron offers greater support for the rela­
tionships among and within subsections than previously
observed for cpDNA (Wang et al., 1999; Gernandt et al.,
2005) and nrITS (Liston et al., 1999). For example, average
p-distances at the LEA-like locus across four subsections
TABLE 5. Population and genetic parameters for select Pinus species
(see text for details on calculations).
Species
ga
b
8(sil)
Ne
Years for
Ymonophyly
to be more
likely than
paraphyly
P. lambertiana
P. discolor
P. flexilis
60
50
30
0.02016
0.01124
0.00143
12.0 × 104
8.03 × 104
1.70 × 104
24.0 × 106
13.4 × 106
1.71 × 106
Years to
reach
genomewide
coalescence
76.3 × 106
42.6 × 106
5.41 × 106
a
Generation time in years, taken as the average number of years to seed bear­
ing (Krugman and Jenkinson, online Woody Plant Seed Manual). Estimates for
P. discolor are not available, so we used values from P. edulis, which is highly
similar.
b
Estimates of theta are calculated using Watterson’s approximation (Rozas
et al., 2004) based on silent positions.
Mean
SD
0.01494
0.00566
0.00471
0.00416
4.6087
4.7203
1.1613
1.1554
range from 0.0131 (subsect. Cembroides) to 0.0177 (subsect. Strobus), which are 4.5 times (subsects. Strobus, Cem­
broides) to 6.1 times (subsect. Balfourianae) greater than
comparable values for cpDNA (Gernandt et al., 2005).
Despite greater divergence for nrITS (Liston et al., 1999)
relative to the LEA-like locus in the range of 1.6- (subsect.
Cembroides) to 2.9-fold (subsect. Gerardianae), orthologi­
cal complexity in rDNA precludes its use for assessing
species-level monophyly.
Our results suggest that the allele pool for any given
pine species may be very large, and it is likely that our
small sample sizes failed to capture all of the major allele
lineages within any given species. For example, genetic
differences partitioned by geographical subdivision may
not have been sampled in many species, particularly in
those cases where monophyly was assessed with two
alleles from a single individual (Table 1) or where the
range of a species was extensive (Table 2). Therefore, fur­
ther allele sampling may increase the number of species
exhibiting allelic nonmonophyly.
Possible Factors Leading to Species-Level Nonmonophyly
in Pinus
A number of factors have been suggested as poten­
tial mechanisms for apparent species nonmonophyly
based on cytoplasmic and nuclear markers (Pamilo and
Nei, 1988; Rieseberg and Broulliet, 1994; Moore, 1995;
Crisp and Chandler, 1996; Doyle, 1997; Shaw, 2001; Hud­
son and Coyne, 2002; Rosenberg and Nordborg, 2002;
Funk and Omland, 2003; Rosenberg, 2003; Sites and Mar­
shall, 2003; Bouillé and Bosquet, 2005). Here, we review
the main factors in reference to the LEA-like topologies
(Figs. 1 and 2), and consider how these factors may have
played a role in species level monophyly and nonmono­
phyly in subg. Strobus. As Funk and Omland (2003) point
out, definitive causes for species polyphyly are difficult
to prove, however, from the observed topological pat­
terns causal inferences can be made.
Inadequate phylogenetic signal.—The signature of in­
sufficient phylogenetic signal is apparent, but not uni­
versal, in this data set. Inadequate information almost
certainly plays a role in the nine cases of weak allelic
nonmonophyly across the subgenus where insufficient
data were available to determine the status of species
monophyly. Two of the clearest examples are P. balfouri­
ana and P. monophylla, which have no alleles separated
by supported nodes in the strict consensus tree, and
show insignificant nonmonophyly in WSR tests (Table
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SYRING ET AL.—WIDESPREAD NONMONOPHYLY IN PINUS SUBGENUS STROBUS
3). However, inadequate phylogenetic information can­
not explain many of the cases of nonmonophyly. There
were 14 species across the subgenus that had one or more
alleles in a supported (79% to 100% BS) topological posi­
tion that ensured allelic nonmonophyly. Further, of these
14 species, constrained topologies resulted in 10 species
being significantly nonmonophyletic (WSR tests; Table
3). The fact that 10 species are resolved as nonmono­
phyletic, show strong nodal support, and have signifi­
cant WSR results provides compelling evidence that the
species level polyphyly uncovered in this study is the
product of true biological signal and not simply inade­
quate phylogenetic information.
Imperfect taxonomy.—In pines, overreliance on labile
characters, such as number of needles per fascicle, or the
misinterpretation of intraspecific variation has caused
imperfect taxonomic circumscriptions in subg. Strobus
(reviewed in Farjon and Styles 1997; Businský, 2004;
Gernandt et al., 2005). Although issues of taxonomic un­
certainty remain, particularly in the Asiatic members of
subsect. Strobus and the Mexican members of subsect.
Cembroides, it appears that imperfect taxonomy plays
only a minor role in explaining the observed species
nonmonophyly. For taxa in subsect. Cembroides, broader
species circumscription (e.g., synonymizing P. cembroides
and P. remota [Farjon and Styles, 1997], synonymizing P.
cembroides, P. discolor, and P. remota [Kral, 1993], or syn­
onymizing P. cembroides, P. discolor, and P. johannis [Farjon
and Styles, 1997]) does not result in monophyly due to
allele sharing across the major clades or well-supported
nodes within one of the major clades. Even the broadest
species concept, which treats all taxa of clades F, G, and
H as varieties of P. cembroides (Shaw, 1914, with the caveat
that several of these taxa were described subsequent to
his publication), would fail to yield a monophyletic P.
cembroides sensu lato due to the position of johannisA2 in
clade I (Fig. 2).
The situation in sect. Quinquefoliae is similar to that of
sect. Parrya. Pinus bhutanica was recently recognized as
a subspecies of P. wallichiana (Businský, 1999, 2004). Al­
though their alleles are closely related, they appear in
two separate clades (D and E), and thus even a broader
species concept does not result in a monophyletic al­
lele lineage. Pinus kwangtungensis, recently considered
to be conspecific with P. wangii Hu & W. C. Cheng and
related to P. parviflora (Businský, 2004), is found in the
well-supported (98% BS) clade E, several steps removed
from P. parviflora. Pinus chiapensis, first described as a
variety of P. strobus (Martı́nez, 1940), was very strongly
supported (100% BS) as monophyletic in a monotypic
clade C, and with no allele sharing between this species
and P. strobus. Further, in the 20 recovered trees for sect.
Quinquefoliae, the alternative placement for P. chiapensis
(clade C) was sister to clade B, which contains two of
three alleles from P. monticola, but none of the diversity
of P. strobus. In a final example, Critchfield and Little
(1966) recognize P. strobiformis as a morphological and
geographical link between P. flexilis to the north and P. ay­
acahuite to the south. Neither a broadly circumscribed P.
ayacahuite nor P. flexilis would restore species monophyly
13
in this taxonomic complex due to the diversity within
P. strobiformis.
Hybridization and introgression.—The ability for pine
species to interbreed under artificial (Little and Righter,
1965; Garrett, 1979; Critchfield, 1986) and natural con­
ditions (P. monophylla × P. edulis: Lanner 1974a; Lanner
and Phillips, 1992; P. parviflora × P. pumila: Watano et al.,
2004) has been well documented. In addition, there are at
least three cases where hypothesized hybridization has
given rise to a named taxon in subg. Strobus, namely P.
hakkodensis (Farjon, 1998), P. quadrifolia (Lanner, 1974b),
and P. edulis var. fallax (Little, 1968). However, none of
these cases have been supported with molecular evidence, in contrast to the well-documented hybrid origin
of P. densata in subg. Pinus (Wang et al., 2001; Song et al.,
2003). In Pinus, natural hybridization is usually of lo­
cal occurrence in areas of sympatry, and often associated
with disturbance (Ledig, 1998). The two subgenera, Pinus
and Strobus, are completely isolated, and crossing among
subsections is very rare (Little and Critchfield, 1969).
In order to determine whether hybridization could ex­
plain the distribution of alleles in the nonmonophyletic
species, we looked specifically at the groups where hybridization has been documented either in the wild or
under artificial conditions. In subsect. Strobus, hybridiza­
tion between P. pumila × P. parviflora (Watano et al., 2004)
is not reflected in Figure 1, where both species are monophyletic and found in unique clades (A and D, respectively). Gernandt et al. (2005) resolve Pinus parviflora in a
clade of North American species based on a cpDNA anal­
ysis. The discrepancy between cpDNA and the LEA-like
locus may be a result of cpDNA introgression. In subsect.
Parrya, the documented hybridization between P. edulis
× P. monophylla is widely recognized (Lanner, 1974a). In
this case, the introgression of alleles in regions of sympa­
try could be observed, but we uncovered no cases of allele
sharing among these species (Fig. 2). The hybrid origin
of P. quadrifolia could not be evaluated, because we did
not sample populations of putative parents “P. juarazen­
sis” Lanner from Baja California and P. monophylla from
southern California.
Pinus lambertiana serves to illustrate the potential for al­
lelic nonmonophyly in the absence of hybridization. This
species is easily distinguished from all other members of
subg. Strobus, and it is reproductively isolated from all
North American pines (Critchfield, 1986; Fernando et al.,
2005). In our study, P. lambertiana is polyphyletic, with
three alleles appearing in as many clades in the strict
consensus tree. The most recent common ancestor for
all P. lambertiana alleles is at the node supporting the di­
vergence of clade D from clades A/B/C (Fig. 1, marked
with an “L”). The allele lambertianaA1 (clade A) is sister
to monticolaA1 with 100% BS, and could be interpreted as
a potentially introgressant allele between these species.
However, Critchfield (1975, 1986) demonstrated that P.
lambertiana can hybridize only with Eurasian members
of subsect. Strobus (Critchfield, 1986), and the failure of
P. lambertiana × P. monticola crosses traces to prefertilization barriers (Fernando et al., 2005). It is noteworthy
that lambertianaA3 is found associated with clade D, an
845
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SYSTEMATIC BIOLOGY
otherwise strictly Eurasian clade. There remains the pos­
sibility that allele A3 represents an ancient hybridization
event, but this seems unlikely given the geographic and
reproductive isolation of P. lambertiana.
Paralogy versus orthology.—We have no evidence to sug­
gest that the observed pattern of species nonmonophyly
is the result of paralogous gene amplification. On the
contrary, evidence suggests that the sequences ampli­
fied from across subg. Strobus were orthologous. First,
the LEA-like locus has been shown to have a low copy
number and is not a member of a large gene family (Kru­
tovsky et al., 2004). The locus was amplified from haploid
(megagametophyte) tissue, and this provides a power­
ful screen for evaluating PCR pool heterogeneity. If du­
plicate paralogs were amplified from the genome, this
would produce a heterogeneous PCR pool that would
be easily identified by direct DNA sequencing. This was
not observed, so we infer that only a single LEA-like tar­
get was amplified and seqeunced.
Lineage sorting.—Lineage sorting is the process by
which ancestral polymorphism is ‘sorted’ and later fixed
in daughter lineages, either by drift or by selection. In­
complete lineage sorting, by contrast, is the persistence
and retention of ancestral polymorphisms through mul­
tiple speciation events. Incomplete lineage sorting can
potentially impact any single-locus gene tree in any
taxon. The theoretical impact of incomplete lineage sort­
ing on gene trees and inferred species trees has been
known for some time (Nei, 1987; Doyle, 1992). Incom­
plete lineage sorting can be promoted by biological con­
ditions that encourage the retention of genetic variabil­
ity in a species, e.g., long life spans, large Ne , and out­
crossing. The retention of shared ancestral polymor­
phisms is also affected by natural selection (Broughton
and Harrison, 2003), since balancing selection works to
oppose directional selection and maintain genetic diver­
sity. The timing of speciation events is another critical
factor impacting the process of lineage sorting, because
rapid radiations or multiple speciation events in quick
succession reduce the chances for lineage sorting to reach
completion before cladogenesis. If the time intervals be­
tween species divergence events are short relative to
the time intervals between lineage-branching events in
each species, ancestral polymorphisms may be carried
through successive rounds of divergence.
Recent studies implicate incomplete lineage sorting
as a major factor in the retention of polymorphism in
plants (Ioerger et al., 1990; Comes and Abbott, 2001;
Chiang et al., 2004; Bouillé and Bousquet, 2005) and ani­
mals (Nagl et al., 1998; Hare et al., 2002; Broughton and
Harrison, 2003; citations in Funk and Omland, 2003).
Estimates for the retention of ancestral polymorphisms
range from 27 to 36 million years for SI alleles under bal­
ancing selection in the Solanaceae (Ioerger et al., 1990),
to 10 to 18 million years for genes of unknown function
in three species of Picea (Bouillé and Bousquet, 2005). In
Pinus, ancestral retention on this order could explain all
cases of species nonmonophyly within each of the sub­
sections. According to the molecular clock divergence
VOL. 56
estimates of Willyard et al. (2006), the species-rich sub­
sections Cembroides and Strobus diverged from their sister
lineages (subsects. Balfourianae and Gerardianae, respec­
tively) between 20 and 10 million years ago, depending upon the calibration date used. Given the rapidity
with which lineages radiated in these subsections (e.g.,
clades A to E in Fig. 1, clades F to I in Fig. 2), divergence
events may have occurred so rapidly that lineage sort­
ing rarely approached allelic fixation within daughter
lineages.
Recombination.—The effect of recombination on recon­
structing genealogies has been well documented (re­
viewed in Posada et al., 2002). Because recombination
produces sequence segments that have different genealogical histories, organismal history cannot be accu­
rately depicted by a single phylogenetic ‘tree,’ but rather
a set of correlated trees across recombinant segments in
the alignment. Under scenarios of ancient recombina­
tion, recombination between highly similar segments,
or low recombination rates, the majority of positions
sampled will accurately reflect phylogenetic history and
the impact of recombination will be limited to alleles
within species or recently diverged species (Posada and
Crandall, 2002; Schierup and Hein, 2000). In contrast,
recombination between divergent sequences or high re­
combination rates can produce inaccurate phylogenies
that show artifactually long terminal branches, appar­
ent trans-specific polymorphism (as shown here and the
study of Bouillé and Bousquet, 2005), and phylogenies
that are significantly different from the true histories un­
derlying the data (Posada and Crandall, 2002).
The locus examined in this study has certainly ex­
perienced recombination during the divergence of sec­
tions of subg. Strobus, but different methods employed
(maximum x 2 ; DSS) fail to detect recombination in our
data. We chose these methods because they use differ­
ent approaches to infer recombination, and because the
maximum x 2 method shows high sensitivity and a low
false-error rate in detecting recombination from empirical data sets (Posada, 2002). The low nucleotide diversity
characteristic of pines from subg. Strobus (1 = 0.02 for
Sect. Quinquefoliae and 0.03 for Sect. Parrya) may be the
primary obstacle in detecting recombination because 1
values of ∼5% are required to obtain statistical power
(Posada and Crandall, 2002). The added combination of
high haplotype diversity and large effective population
sizes for these species makes the detection of parental se­
quences and daughter recombinants unlikely given our
sampling strategy.
For pine species exhibiting monophyly across multiple
LEA-like alleles (e.g., P. albicaulis, P. chiapensis; Figs. 1, 2),
undetected recombination will have mimimal impact on
phylogenetic resolution because recombinants between
‘divergent’ alleles will show congruent phylogenetic signal. For species deviating significantly from monophyly,
recombination among divergent alleles could have a
pronounced impact. Simulations by Schierup and Hein
(2000) show that low levels of recombination can produce
trees that underestimate the amount of true divergence
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SYRING ET AL.—WIDESPREAD NONMONOPHYLY IN PINUS SUBGENUS STROBUS
between parental alleles, and yield more ‘star-like’ phy­
logenies than would be obtained with nonrecombined
sequences. Clearly, the presence of divergent allelic poly­
morphism is primarily responsible for the pattern of non­
1025 monophyly in pines; recombination simply adds com­
plexity and uncertainty to the pattern.
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How Do Pine Species Attain Monophyly?
Rieseberg and Brouillet (1994) suggest that species
concepts based on monophyly are inadequate, be­
cause paraphyletic species should be expected from
progenitor-derivative speciation. Under that mode of
speciation, paraphyly should be expected as a direct re­
sult of incomplete lineage sorting. Given enough time
following a speciation event, drift or directional selection
should theoretically lead to genome-wide monophyly
via the sorting and extinction of lineages (Rieseberg and
Brouillet, 1994; Rosenberg, 2003), so long as balancing se­
lection does not maintain polymorphisms that predate
speciation events. At the point when lineage sorting is
complete, all new mutation will result from the same
lineage, and intraspecific variation will reflect postspe­
ciation mutation. The situation in Pinus is more com­
plex because multiple speciation events appear to have
occurred before lineage sorting was completed in any
single bifurcation event. As a consequence, ancestral
polymorphisms have been retained through multiple
speciation events. One potential outcome of ancestral al­
lelic retention on this order is that allele lineages within
species become polyphyletic.
The occurrence of paraphyletic and polyphyletic
species in both Figures 1 and 2 appears consistent with
our estimates for the number of years until reciprocal
monophyly is expected to be more likely than paraphyly
(Table 5; Rosenberg, 2003). Even though our estimates
of 8 are based on a small sample, the values in Table 5
are instructive. For example, the estimated time for re­
ciprocal monophyly to be more likely than paraphyly is
13.4 million years for P. discolor. This value is bounded
by the estimated age of sect. Cembroides, which is cal­
culated at 19 million years (Willyard et al., 2006). Topo­
logical analyses suggest that P. discolor is weakly non­
monophyletic (35% of WSR tests were significant; Table
3) at the LEA-like locus, a finding that shows surpris­
ingly good agreement with the approximations of allele
coalescence and molecular evolutionary age of the Cem­
briodes lineage. Nevertheless, the estimate for genomewide coalescence in this species is ca. 43 million years,
suggesting that portions of the genome may harbor deep
trans-species polymorphisms, even under neutrality.
For species with large values of 8, such as P. lambertiana,
the phylogenetic implications are striking. A calculated
value of ca. 24 million years until reciprocal monophyly is
expected to be more likely than paraphyly would predate
the divergence of subsects. Strobus and Gerardianae. If this
estimate is correct, it suggests that the potential exists for
trans-species polymorphisms to be shared among pine
species from different subsections. Further, a calculated
value of ca. 76 million years for complete genome-
15
wide coalescence would extend far beyond the diver­
gence of the two sections from subg. Strobus. Clearly, the
accuracy of these estimates depends upon many assump­
tions; nevertheless, they highlight the fallacy of assum­
ing ‘species monophyly’ in groups characterized by large
population sizes, and the complexity of resolving phylo­
genetic relationships among pine species.
In light of this information, the historic effective pop­
ulation size, as reflected by nucleotide diversity within
contemporary species, seems to be the driving factor in
determining whether pine species are genetically unique,
and whether genes can accurately trace a species phylogenetic history. Noteworthy in this regard is that cur­
rent geographic ranges (a proxy for census population
sizes and global abundance) are uncorrelated with nu­
cleotide diversity, and show no association with the ex­
tent of monophyly or nonmonophyly of a species (Table
4). Based on this analysis (Table 4), it seems that effective
population size either has an unpredictable association
with monophyly in pines, or perhaps more likely, that ge­
ographic range (or census count) is a poor predictor of the
effective population size of a species. These results were
not entirely unexpected, because studies of pines have
shown that geographically widespread species can lack
genetic diversity, whereas narrowly distributed pines
can show ample genetic diversity (Ledig, 1998; Ledig
et al., 1999; Delgado et al., 1999). We note, for instance,
that P. chiapensis (a narrow endemic of Mexico, limited
to ca. 15,000 km2 ) shows far greater nucleotide diversity
than P. albicaulis (1 = 0.0031 versus 0.0000), even though
the latter species is dispersed across ca. 400,000 km2 of
western North America.
The multiple cycles of glaciation in North America,
Europe, and Asia have had a pronounced impact on
conifer genetic diversity (MacDonald et al., 1998; Petit
et al., 2003), and it is likely that contemporary ranges re­
flect nonequilibrium processes of recent expansion (e.g.,
species with low genetic diversity and large geographic
ranges like P.koraiensis and P. strobus), recent range con­
traction (e.g., species with high genetic diversity and
small ranges like P. bhutanica and P. johannis), and possi­
bly geographic or ecological isolation coupled with long
distance dispersal events (e.g., P. chiapensis and P. albi­
caulis). This observation has implications for molecular
phylogenetic studies focusing on recently diverged taxa
(e.g., tribes, genera, and species complexes) because it
highlights the importance of considering the magnitude
of intraspecific diversity within the overall pattern of
phyletic divergence. Until accurate estimates of relative
or absolute effective population sizes become available,
the connection between monophyly and Ne can only be
inferred from simulation studies (e.g., Rosenberg, 2003).
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Broader Implications for Phylogenic Studies of Seed Plants
Coalescence of allele lineages is dependent on a suite
of interacting processes that occurred prior to and dur­
ing speciation, and continue to the present. Processes re­
sponsible for genic nonmonophyly in species include hy- 1135
bridization with subsequent introgression, incomplete
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SYSTEMATIC BIOLOGY
lineage sorting, and recombination. These processes may
become superimposed, and their present-day genealog­
ical patterns may reflect ancient and recent events. Se­
quences of this nature track the mutational history of an
allele, but they are unlikely to track the comparatively
simple cladistic history of recently diverged species. It
is noteworthy that these processes are found to varying
degrees in cpDNA and mtDNA, thus the lack of species
monophyly cannot be dismissed as exclusively a nuclear
gene phenomenon. Nonetheless, coalescent simulations
by Rosenberg (2003) show that when three coalescent
units of time have passed for haploid organellar genes,
the probability of reciprocal monophyly exceeds 0.8; this
same length of time equates to 0.75 coalescent units for
nuclear loci, at which time the probability of genic mono­
phyly is less than 0.1. In the absence of complicating fac­
tors (e.g., organellar introgression, nuclear recombina­
tion), the prevalence of noncoalescence is expected to be
much more problematic for nuclear loci than organellar
loci.
Given the long time frame predicted by coalescent the­
ory for species of Pinus to attain monophyly (Table 5),
genealogical-based species concepts (de Queiroz and
Donoghue, 1988; Baum and Donoghue, 1995; Shaw, 1998)
derived from molecular markers may be inappropri­
ate for pines and other species sharing similar life his­
tory traits. Distinct morphological and ecological differ­
ences are readily apparent between most of the species
of subgenus Strobus included in this study, yet specieslevel paraphyly or polyphyly appears in nearly half of
the species examined. Evidence from Pinus is consistent
with the theoretical expectation that a large portion of
the genome frequently remains common to closely re­
lated species well after speciation has occurred (Rosen­
berg, 2003). Wu (2001) outlined a theory of speciation
whereby “differentiation loci” become fixed within a pair
of species whereas regions of “neutral divergence” in
between these fixed sites are free to have unrestricted
gene flow. Under this theory, the fixed regions in both
genomes become larger over time as gene flow is further
restricted. Our data suggest the possibility for retention
of ancestral polymorphisms in these regions of neutral
divergence, regardless of whether gene flow is ongoing.
In other words, even after interspecific mating barriers
become fixed at many loci, pines can be expected to har­
bor ancestral polymorphisms at a large fraction of their
genome.
The demonstration of widespread species-level non­
monophyly appears to be a severe constraint in the
application of nuclear genes in resolving organismal
evolutionary history of pines at low taxonomic ranks.
Similar conclusions were reported by Bouillé and
Bousquet (2005) and Broughton and Harrison (2003), all
of whom have suggested that nuclear gene genealogies
have limited potential to reconstruct evolutionary histo­
ries among closely related species. Although we gener­
ally agree with this conclusion for our work in decipher­
ing relationships among the terminal species of Pinus, it
is difficult to extrapolate these findings to dissimilar taxa
with different life history traits (e.g., small Ne , short life
spans, higher inbreeding rates). Even with the limitation
of noncoalescence, discrete nuclear genes can provide
important insights into the historical, demographic, and
possibly even selective processes that forge new species.
This information offers new perspectives (relative to or­
ganellar DNA or nrITS) as to the biological basis for the
presence (or absence) of phylogenetic patterns.
Given the patterns of nonmonophyly detected in this
study, the use of nuclear genes (including nrITS; see Gernandt et al., 2001) for the identification of species using
DNA barcoding (Chase et al., 2005; Kress et al., 2005)
is probably inappropriate in pines because species are
segregating for polymorphisms that are retained across
multiple and ancient speciation events. In some cases
(e.g., P. lambertiana and P. edulis) intraspecific allelic diver­
sity is located in widely divergent phylogenetic positions
(Figs. 1 and 2). Preliminary data from the matK region
of the chloroplast genome among members of the North
American subsection Quinquefoliae (Syring et al., unpublished data) indicate that although allelic nonmonophyly
occurs, it may not be as widespread as in the nuclear
genome.
Perhaps the most important general finding of this
work is that coalescence failure can lead to significantly
different phylogenetic interpretations that are only de­
tectable by sampling multiple individuals per species,
and perhaps multiple loci. In Pinus, large Ne , long gener­
ation times, and high outcrossing rates combine to make
allelic noncoalescence a readily detected pattern, even
with a small sample size. Given this combination of traits,
it seems reasonable to expect that other species-rich tree
genera with large, widespread populations, e.g., Quer­
cus (400 species), Salix (450 species), Ficus (750 species),
and Eucalyptus (680 species), should be prone to similar
levels of nonmonophyly if examined by nuclear mark­
ers. Less clear is the impact noncoalescence will have
on less widespread, more genetically uniform taxa. In
order to ensure the robustness of future studies, we rec­
ommend that researchers explicitly test the monophyly
of species and lower-level taxa by including multiple in­
dividuals across the range of a species, especially prior
to proposing new classifications or delimiting species. In
the absence of such sampling, and working under the
assumption of species monophyly, it seems likely that
many more cases of species nonmonophyly will remain
undetected.
1200
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ACKNOWLEDGEMENTS
We thank Martin Gardner, Fiona Inches, and Philip Thomas (Royal 1245
Botanic Garden Edinburgh, Scotland), David Gernandt (Universidad
Autónoma de Hidalgo, Mexico), Dave Johnson (USDA Forest Service,
Institute of Forest Genetics, Placerville, CA), Richard Sneizko (USDA
Forest Service, Dorena Genetic Resource Center, Cottage Grove, OR),
Michael Wall (Rancho Santa Ana Botanic Garden, Claremont, CA), 1250
Jesus Vargas Hernandez (Instituto de Recursos Naturales, Mexico),
Randall Hitchin (Washington Park Arboretum, Seattle), Dale Simpson
and Jean Beaulieu (Natural Resources Canada), Steven Roelof (Native
Plant Society of Oregon), Jim Buck (University of Michigan), Richard
Halse (Oregon State University), Bill Dvorak (CAMCORE), Forest Tree 1255
Breeding Center (Japan), Sanderson McArthur (USDAFS Rocky Mt. Re­
search Station), Paul Halladin (Iseli Nursery, Oregon), Dennis Ringnes
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February 27, 2007
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SYRING ET AL.—WIDESPREAD NONMONOPHYLY IN PINUS SUBGENUS STROBUS
(USDAFS Central Zone Genetic Resource Program, California), Car­
rie Sweeney (USDAFS Oconto River Seed Orchard, Minnesota), Joe
Myers (USDAFS Coeur d’Alene Nursery, Idaho), Yasayuki Watano
(Chiba University, Japan) , Ioan Blada (Forest Research and Manage­
ment Institute of Bucharest, Romania), Frank Hammond (U.S. Army,
Fort Huachuca, AZ), Kirsten Winter (Cleveland National Forest, San
Diego, CA), James C. Zech (Sull Ross State University, Alpine, TX),
and Konstantin Krutovsky (Texas A&M University) for the generous
contributions of seed and needle tissue. Funding for this study was
provided by the National Science Foundation grant DEB 0317103 to
Aaron Liston and Richard Cronn, and the USDA Forest Service Pacific
Northwest Research Station.
R EFERENCES
Álvarez, I., R. Cronn, and J. F. Wendel. 2005. Phylogeny of the New
World diploid cottons (Gossypium L., Malvaceae) based on sequences
of three low-copy nuclear genes. Plant Syst. Evol. 252:199–214.
Alvin, K. 1960. Further conifers of the Pinaceae from the Wealden For­
mation of Belgium. Mem. Inst. Roy. Sci. Nat. Belg. 146:1–39.
Andresen, J. W. 1966. A multivariate analysis of the Pinus chiapensis–
monticola–strobus phylad. Rhodora 68:1–24.
Bailey, D. K. 1970. Phytogeography and taxonomy of Pinus subsection
Balfourianae. Ann. Missouri Bot. Gard. 57:210–249.
Baum, D. A., and M. J. Donoghue. 1995. Choosing among alternative
“phylogenetic” species concepts. Syst. Bot. 20:560–573.
Berry, P. E., A. L. Hipp, K. J. Wurdack, B. Van Ee, and R. Riina. 2005.
Molecular phylogenetics of the giant genus Croton and tribe Cro­
toneae (Euphorbiaceae sensu stricto) using ITS and trnL-trnF DNA
sequence data. Am. J. Bot. 92:1520–1534.
Bouillé, M., and J. Bousquet. 2005. Trans-species shared polymorphisms
at orthologous nuclear gene loci among distant species in the conifer
Picea (Pinaceae): Implications for the long-term maintenance of ge­
netic diversity in trees. Am. J. Bot. 92:63–73.
Broughton, R. E., and R. G. Harrison. 2003. Nuclear gene genealogies
reveal historical, demographic and selective factors associated with
speciation in field crickets. Genetics 163:1389–1401.
Businský, R. 1999. Taxonomic revision of Eurasian pines (genus Pinus L.)—Survey of species and infraspecific taxa according to latest
knowledge. Acta Pruhoniciana 68:7–86.
Businský, R. 2004. A revision of the Asian Pinus subsection Strobus
(Pinaceae). Willdenowia 34:209–257.
Chase, M. W., N. Salamin, M. Wilkinson, J. M. Dunwell, R. P.
Kesanakurthi, N. Haidar, and V. Savolainen. 2005. Land plants and
DNA barcodes: Short-term and long-term goals. Phil. Trans. R. Soc.
Lond. B 360:1889–1895.
Chiang, T.-Y., K.-H. Hung, T.-W. Hsu, and W.-L. Wu. 2004. Lineage
sorting and phylogeography in Lithocarpus formosanus and L. dodon­
aeifolius (Fagaceae) from Taiwan. Ann. Missouri Bot. Gard. 91:207–
222.
Church, S. A., and D. R. Taylor. 2005. Speciation and hybridization
among Houstonia (Rubiaceae) species: The influence of polyploidy
on reticulate evolution. Am. J. Bot. 92:1372–1380.
Comes, H. P., and R. J. Abbott. 2001. Molecular phylogeography, retic­
ulation, and lineage sorting in Mediterranean Senecio sect. Senecio
(Asteraceae). Evolution 55:1943–1962.
Crisp, M. D., and G. T. Chandler. 1996. Paraphyletic species. Telopea
6:813–844.
Critchfield, W. B. 1986. Hybridization and classification of the white
pines (Pinus section Strobus). Taxon 35:647–656.
Critchfield, W. B. 1975. Interspecific hybridization in Pinus: A summary
review. Pages 99–105 in Proceedings 14th Canadian Tree Improve­
ment Association, Part 2, Symposium on interspecific hybridization
in forest trees, NB, 28–30 August 1973 (Fowler, D. P., and C. W.
Yeatman, eds.). Canadian Forestry Service, Ottawa, Ontario.
Critchfield, W. B., and E. L. Little, Jr. 1966. Geographic distribution of the
pines of the world. USDA Forest Service Miscellaneous Publication
991. Washington, DC.
Cross, E. W., C. J. Quinn, and S. J. Wagstaff. 2002. Molecular evidence
for the polyphyly of Olearia (Astereae: Asteraceae). Plant. Syst. Evol.
235:99–120.
de Queiroz, K., and M. J. Donoghue. 1988. Phylogenetic systematics
and the species problem. Cladistics 4:317–338.
17
Delgado, P., D Piñero, A. Chaos, N. Pérez-Nasser, and E. R. AlavarezBuylla. 1999. High population differentiation and genetic variation 1330
in the endangered Mexican pine Pinus rzedowskii (Pinaceae). Am. J.
Bot. 86:669–676.
Doyle, J. J. 1997. Trees within trees: Genes and species, molecules and
morphology. Syst. Biol. 46:537–553.
Doyle, J. J. 1992. Gene trees and species trees: Molecular systematics as 1335
one-character taxonomy. Syst. Bot. 17:144–163.
Farjon, A. 2005. Drawings and descriptions of the genus Pinus, 2nd
edition. E. J. Brill and W. Backhuys, Leiden, Netherlands.
Farjon, A. 1998. World checklist and bibliography of conifers. Royal
Botanical Gardens at Kew, Richmond, UK.
1340
Farjon, A., and B. T. Styles. 1997. Pinus (Pinaceae). Flora Neotropica
Monograph 75. The New York Botanical Garden, New York.
Felsenstein, J. 1985. Confidence limits on phylogenies: An approach
using the bootstrap. Evolution 39:783–791.
Feng, Y., S.-H. Oh, and P. S. Manos. 2005. Phylogeny and historical 1345
biogeography of the genus Platanus as inferred from nuclear and
chloroplast DNA. Syst. Bot. 30:786–799.
Fernando, D. D., S. M. Long, and R. A. Sniezko. 2005. Sexual reproduc­
tion and crossing barriers in white pines: The case between Pinus
lambertiana (sugar pine) and P. monticola (western white pine). Tree 1350
Genet. Genomes 1:143–150.
Funk, D. J., and K. E. Omland. 2003. Species-level paraphyly and
polyphyly: Frequency, causes, and consequences, with insights from
animal mitochondrial DNA. Annu. Rev. Ecol. Evol. Syst. 34:397–
423.
1355
Garrett, P. W. 1979. Species hybridization in the genus Pinus. USDA For­
est Service, Northeast Forest Experiment Station, Station Research
Paper 436.
Gernandt, D. S., A. Liston, and D. Piñero. 2001. Variation in the nrDNA
ITS of Pinus subsection Cembroides: Implications for molecular sys- 1360
tematic studies of pine species complexes. Mol. Phylogenet. Evol.
21:449–467.
Gernandt, D. S., A. Liston, and D. Piñero. 2003. Phylogenetics of Pinus subsections Cembroides and Nelsoniae inferred from cpDNA se­
quences. Syst. Bot. 4:657–673.
1365
Gernandt, D. S., G. G. Lopez, S. O. Garcia, and A. Liston. 2005. Phy­
logeny and classification of Pinus. Taxon 54:29–42.
Gilbert, C., J. Dempcy, C. Ganong, R. Patterson, and G. S. Spicer. 2005.
Phylogenetic relationships within Phacelia subgenus Phacelia (Hy­
drophyllaceae) inferred from nuclear rDNA ITS sequence data. Syst. 1370
Bot. 30:627–634.
Goetsch, L., A. J. Eckert, and B. D. Hall. 2005. The molecular systematics
of Rhododendron (Ericaceae): A phylogeny based upon RPB2 gene
sequences. Syst. Bot. 30:616–626.
Goodwillie, C., and J. W. Stiller. 2001. Evidence for polyphyly in a 1375
species of Linanthus (Polemoniaceae): Convergent evolution in selffertilizing taxa. Syst. Bot. 26:273–282.
Hare, M. P., F. Cipriano, and S. R. Palumbi. 2002. Genetic evidence on the
demography of speciation in allopatric dolphin species. Evolution
56:804–816.
1380
Hudson, R. R., and J. A. Coyne. 2002. Mathematical consequences of
the genealogical species concept. Evolution 56:1557–1565.
Ioerger, T. R., A. G. Clark, and T. H. Kao. 1990. Polymorphism at the
self-incompatibility locus in Solanaceae predates speciation. Proc.
Natl. Acad. Sci. USA 87:9732–9735.
1385
Kamiya, K., K. Harada, H. Tachida, and P. S. Ashton. 2005. Phylogeny of
PgiC gene in Shorea and its closely related genera (Dipterocarpaceae),
the dominant trees in southeast Asian tropical rain forests. Am. J. Bot.
92:775–788.
Kral, R. 1993. Pinus. in Flora of North America (North of Mexico), 1390
volume 2 (Flora of North America Editorial Committee, ed.). Oxford
University Press, New York.
Q3
Kress, W. J., K. J. Wurdack, E. A. Zimmer, L. A. Weigt, and D. H. Janzen.
2005. Use of DNA barcodes to identify flowering plants. Proc. Natl.
1395
Acad. Sci. USA 102:8369–8374.
Krutovsky, K. V., M. Troggio, G. R. Brown, K. D. Jermstad, and D.
B. Neale. 2004. Comparative mapping in the Pinaceae. Genetics
168:447–461.
Kumar, S., K. Tamura, I. B. Jakobsen, and M. Nei. 2001. MEGA2: Molec­
ular evolutionary genetics analysis software. Bioinformatics 17:1244– 1400
1245.
P1: PXN
TF-SYB
USYB˙03˙225790
18
1405
1410
1415
1420
1425
1430
1435
1440
1445
1450
1455
1460
1465
1470
February 27, 2007
14:32
SYSTEMATIC BIOLOGY
Lanner, R. M. 1974a. Natural hybridization between Pinus edulis and
Pinus monophylla in the American Southwest. Silvae Genet. 23:108–
116.
Lanner, R. M. 1974b. A new pine from Baja California and the hybrid
origin of Pinus quadrifolia. Southwest. Nat. 19:75–95.
Lanner, R. M., and A. M. Phillips III. 1992. Natural hybridization and
introgression of pinyon pines in northwestern Arizona. Int. J. Plant
Sci. 153:250–257.
Ledig, F. T. 1998. Genetic variation in Pinus. Pages 251–280 in Ecol­
ogy and biogeography of Pinus (D. M. Richardson, ed.). Cambridge
University Press, Cambridge, UK.
Ledig, F. T., M. T. Conkle, B. Bermejo-Velazaquez, T. Eguiluz-Piedra,
P. D. Hodgskiss, D. R. Johnson, W. S. Dvorak. 1999. Evidence for an
extreme bottleneck in a rare Mexican pinyon: Genetic diversity, dise­
quilibrium, and the mating system in Pinus maximartinezii. Evolution
53:91–99.
Lee, C., S.-C. Kim, K. Lundy, and A. Santos-Guerra. 2005. Chloroplast
DNA phylogeny of the woody Sonchus alliance (Asteraceae: Sonchi­
nae) in the Macaronesian Islands. Am. J. Bot. 92:2072–2085.
Levin, R. A., and J. S. Miller. 2005. Relationships within tribe Lycieae
(Solanaceae): Paraphyly of Lycium and multiple origins of gender
dimorphism. Am. J. Bot. 92:2044–2053.
Levin, R. A., K. Watson, and L. Bohs. 2005. A four-gene study of evo­
lutionary relationships in Solanum section Acanthophora. Am. J. Bot.
92:603–612.
Liston, A., W. A. Robinson, D. Piñero, and E. R. Alvarez-Buylla. 1999.
Phylogenetics of Pinus (Pinaceae) based on nuclear ribosomal DNA
internal transcribed spacer region sequences. Mol. Phylogenet. Evol.
11:95–109.
Little, E. L., Jr. 1968. Two new pinyon varieties from Arizona. Phytologia
17:329–342.
Little, E. L., Jr., and W. B. Critchfield. 1969. Subdivisions of the
genus Pinus. USDA Forest Service Miscellaneous Publication 1144.
Washington, DC.
Little, E. L., Jr., and F. I. Righter. 1965. Botanical descriptions of forty arti­
ficial pine hybrids. USDA Forest Service Technical Bulletin 1345:1–47.
Washington, DC.
Luo, Y., F. Zhang, and Q.-E. Yang. 2005. Phylogeny of Aconitum sub­
genus Aconitum (Ranunculaceae) inferred from ITS sequences. Plant
Syst. Evol. 252:11–25.
MacDonald, G. M., L. C. Cwynar, and C. Whitlock. 1998. The late Qua­
ternary dynamics of pines in northern North America. Pages 122–149
in Ecology and biogeography of Pinus (D. M. Richardson, ed.). Cam­
bridge University Press, Cambridge, UK.
Magallon, S., and M. J. Sanderson. 2002. Relationships among seed
plants inferred from highly conserved genes: Sorting conflicting
phylogenetic signals among ancient lineages. Am. J. Bot. 89:1991–
2006.
Malusa, J. 1992. Phylogeny and biogeography of the pinyon pines (Pinus subsect. Cembroides). Syst. Bot. 17:42–66.
Martin, D. P., C. Williamson, and D. Posada. 2005. RDP2: Recombina­
tion detection and analysis from sequence alignments. Bioinformat­
ics 21:260–262.
Martı́nez, M. 1940. Pinaceas Mexicanas. Anales del Instituto de Biologia
de Mexico 11:57–84.
Mason-Gamer, R. J. 2005. The {beta}-amylase genes of grasses and a
phylogenetic analysis of the Triticeae (Poaceae). Am. J. Bot. 92:1045–
1058.
McGuire, G., F. Wright, and M. J. Prentice. 1997. A graphical method for
detecting recombination in phylogenetic data sets. Mol. Biol. Evol.
14:1125–1131.
McKown, A. D., J.-M. Moncalvo, and N. G. Dengler. 2005. Phylogeny of
Flaveria (Asteraceae) and inference of C4 photosynthesis evolution.
Am. J. Bot. 92:1911–1928.
Meijer, J. 2000. Fossil woods from the late Cretaceous Aachen Forma­
tion. Rev. Palaeobot. Palynol. 112:297–336.
Miller, C. 1973. Silicified cones and vegetative remains of Pinus from
the Eocene of British Columbia. Cont. Univ. Mich. Museum Paleo.
24:101–118.
Milne, I., F. Wright, G. Rowe, D. F. Marshall, D. Husmeier, and G.
McGuire. 2004. TOPALi: Software for automatic identification of re­
combinant sequences within DNA multiple alignments. Bioinfor­
matics 20:1806–1807.
VOL.
56
Moore, W. S. 1995. Inferring phylogenies from mtDNA variation: 1475
Mitochondrial-gene trees versus nuclear-gene trees. Evolution
49:718–726.
Müller, K., and T. Borsch. 2005. Phylogenetics of Utricularia (Lentibu­
lariaceae) and molecular evolution of the trnK intron in a lineage
with high substitutional rates. Plant Syst. Evol. 250:39–67.
1480
Nagl, S., H. Tichy, W. E. Mayer, N. Takahata, and J. Klein. 1998. Persis­
tence of neutral polymorphisms in Lake Victoria cichlid fish. Proc.
Natl. Acad. Sci. USA 95:14238–14243.
Nei, M. 1987. Molecular evoltionary genetics. Columbia University
1485
Press, New York.
Oh, S.-H., and D. Potter. 2005. Molecular phylogenetic systematics and
biogeography of tribe Neillieae (Rosaceae) using DNA sequences of
cpDNA, rDNA, and LEAFY. Am. J. Bot. 92:179–192.
Oline, D. K., J. B. Mitton, and M. C. Grant. 2000. Population and sub­
specific genetic differentiation in the foxtail pine (Pinus balfouriana). 1490
Evolution 54:1813–1819.
Pamilo, P., and M. Nei. 1988. Relationships between gene trees and
species trees. Mol. Biol. Evol. 5:568–583.
Perez de la Rosa, J., S. A. Harris, and A. Farjon. 1995. Noncoding chloro­
plast DNA variation in Mexican pines. Theor. Appl. Genet. 91:1101– 1495
1106.
Perry, J. P. 1991. The pines of México and Central America. Timber
Press, Portland, Oregon.
Petit, R. J., I. Aguinagalde, J. L. de Beaulieu, C. Bittkau, S. Brewer,
R. Cheddadi, R. Ennos, S. Fineschi, D. Grivet, M. Lascoux, A. Mo- 1500
hanty, G. Müller-Starck, B. Demesure-Musch, A. Palmé, J. P. Martı́n,
S. Rendell, and G. G. Vendramin. 2003. Glacial refugia: Hotspots but
not melting pots of genetic diversity. Science 300:1563–1565.
Popp, M., and B. Oxelman. 2004. Evolution of a RNA polymerase gene
family in Silene (Caryophyllaceae)—incomplete concerted evolution 1505
and topological congruence among paralogues. Syst. Biol. 53:914–
932.
Posada, D. 2002. Evaluation of methods for detecting recombination
from DNA sequences: Empirical data. Mol. Biol. Evol. 19:708–717.
Posada, D., and K. A. Crandall. 2001. Evaluation of methods for de- 1510
tecting recombination from DNA sequences: Computer simulations.
Proc. Natl. Acad. Sci. USA 98:13757–13762.
Posada, D., and K. A. Crandall. 2002. The effect of recombination on
the accuracy of phylogeny estimation. J. Mol. Evol. 54:396–402.
Price, R. A., A. Liston , and S. H. Strauss. 1998. Phylogeny and system- 1515
atics of Pinus. Pages 49–68 in Ecology and biogeography of Pinus (D.
M. Richardson, ed.). Cambridge University Press, Cambridge, UK.
Rieseberg, L. H., and L. Broulliet. 1994. Are many plant species paraphyletic? Taxon 43:21–32.
Roalson, E. H., and E. A. Friar. 2004. Phylogenetic relationships and 1520
biogeographic patterns in North American members of Carex section
Acrocystis (Cyperaceae) using nrDNA ITS and ETS sequence data.
Plant Syst. Evol. 243:175–187.
Robba, L., M. A. Carine, S. J. Russell, and F. M. Raimondo. 2005. The
monophyly and evolution of Cynara L. (Asteraceae) sensu lato: Evi- 1525
dence from the internal transcribed spacer region of nrDNA. Plant
Syst. Evol. 253:53–64.
Rosenberg, N. A. 2003. The shapes of neutral gene genealogies in two
species: Probabilities of monophyly, paraphyly, and polyphyly in a
coalescent model. Evolution 57:1465–1477.
1530
Rosenberg, N. A., and M. Nordborg. 2002. Genealogical trees, coa­
lescent theory and the analysis of genetic polymorphisms. Nature
3:380–390.
Rozas, J., J. Sánchez-DelBarrio, X. Messeguer, and R. Rozas. 2004.
DnaSP: DNA sequence polymorphism, v4.00.6. Bioinformatics 1535
19:2496–2497.
Rzedowski, J., and L. Vela. 1966. Pinus strobus var. chiapensis en la Sierra
Madre del Sur de México. Ciencia 24:211–216.
SAS Institute, Inc. 1999. SAS/STAT user’s guide, version 8, volume 1.
SAS Institute, Cary, North Carolina.
1540
Schierup, M. H., and J. Hein. 2000. Consequences of recombination on
traditional phylogenetic analysis. Genetics 156:879–891.
Schneeweiss, G., P. Schonswetter, S. Kelso, and H. Niklfeld. 2004. Com­
plex biogeographic patterns in Androsace (Primulaceae) and related
genera: Evidence from phylogenetic analyses of nuclear internal 1545
transcribed spacer and plastid trnL-F sequences. Syst. Biol. 53:856–
876.
P1: PXN
TF-SYB
USYB˙03˙225790
February 27, 2007
2007
1550
1555
1560
1565
1570
1575
1580
1585
14:32
19
SYRING ET AL.—WIDESPREAD NONMONOPHYLY IN PINUS SUBGENUS STROBUS
Shaw, G. R. 1914. The genus Pinus. Publications of the Arnold Arbore­
tum No. 5, Riverside Press, Cambridge, Massachusetts.
Shaw, J. 2001. Biogeographic patterns and cryptic speciation in
bryophytes. J. Biogeogr. 28:253–261.
Shaw, J., and R. L. Small. 2005. Chloroplast DNA phylogeny and phylo­
geography of the North American plums (Prunus subgenus Prunus
section Prunocerasus, Rosaceae). Am. J. Bot. 92:2011–2030.
Shaw, K. L. 1998. Species and the diversity of natural groups. Pages
44–56 in Endless forms: Species and speciation (D. J. Howard and S.
J. Berlocher, eds.). Oxford University Press, Oxford, UK.
Simmons, M. P., and H. Ochoterena. 2000. Gaps as characters in
sequence-based phylogenetic analyses. Syst. Biol. 49:369–381.
Sites, J. W., and J. C. Marshall. 2003. Delimiting species: A renaissance
issue in systematic biology. Trends Ecol. Evol. 18:462–470.
Smith, J. M. 1992. Analyzing the mosaic structure of genes. J. Mol. Evol.
34:126–129.
Song, B. H., X. Q. Wang, X. R. Wang, K. Y. Ding, and D. Y. Hong. 2003.
Cytoplasmic composition in Pinus densata and population establish­
ment of the diploid hybrid pine. Mol. Ecol. 12:2995–3001.
Swofford, D. L. 2003. PAUP*4.0b10: Phylogenetic analysis using parsi­
mony (*and other methods). Sinauer Associates, Sunderland, Mas­
sachusetts.
Syring, J., A. Willyard, R. Cronn, and A. Liston. 2005. Evolutionary rela­
tionships among Pinus (Pinaceae) subsections inferred from multiple
low-copy nuclear loci. Am. J. Bot. 92:2086–2100.
Templeton, A. R. 1983. Phylogenetic inference from restriction endonu­
clease cleavage site maps with particular reference to the evolution
of humans and the apes. Evolution 37:221–244.
Treutlein, J., G. F. Smith, B.-E. van Wyk, and M. Wink. 2003. Evidence for
the polyphyly of Haworthia (Asphodelaceae Subfamily Alooideae;
Asparagales) inferred from nucleotide sequences of rbcL, matK, ITS1
and genomic fingerprinting with ISSR-PCR. Plant Biol. 513–521.
Wang, X. R., A. E. Szmidt, and O. Savolainen. 2001. Genetic composition
and diploid hybrid speciation of a high mountain pine, Pinus densata,
native to the Tibetan plateau. Genetics 159:337–346.
Wang, X. R., Y. Tsumura, H. Yoshimaru, K. Nagasaka, and A. E. Szmidt.
1999. Phylogenetic relationships of Eurasian pines (Pinus, Pinaceae)
based on chloroplast rbcL, MATK, RPL20-RPS18 spacer, and TRNV
intron sequences. Am J Bot 86:1742–1753.
Watano, Y., A. Kanai, and N. Tani. 2004. Genetic structure of hy­
brid zones between Pinus pumila and P. parviflora var. pentaphylla
(Pinaceae) revealed by molecular hybrid index analysis. Am. J. Bot.
91:65–72.
Wendel, J. F., and J. J. Doyle. 1998. Phylogenetic incongruence: Window into genome history and molecular evolution. Pages 265–296 in
Molecular systematics of plants II: DNA sequencing (P. S. Soltis, D. E.
Soltis, and J. J. Doyle, eds.). Kluwer Academic Publishers, Dordrecht,
Netherlands.
Wilkin, P., P. Schols, M. W. Chase, K. Chavamarit, C. A. Furness, S.
Huysmans, F. Rakotonasolo, E. Smets, and C. Thapyai. 2005. A plas­
tid gene phylogeny of the yam genus, Dioscorea: Roots, fruits and
Madagascar. Syst. Bot. 30:736–749.
Willyard, A., J. Syring, D. Gernandt, A. Liston, and R.Cronn. 2006.
Molecular evolutionary rates indicate a recent and rapid diversification for modern pine lineages. Mol. Biol. Evol 23:1–12.
Winkworth, R. C., and M. J. Donoghue. 2005. Viburnum phylogeny
based on combined molecular data: Implications for taxonomy and
biogeography. Am. J. Bot. 92:653–666.
Wright, J. A., A. M. Marin V, and W. S. Dvorak. 1996. Conservation
and use of the Pinus chiapensis genetic resource in Columbia. Forest
Ecol.Manag. 88:283–288.
Wu, C.-I. 2001. The genic view of the process of speciation. J. Evol. Biol.
14:851–865.
Yamane, K., and T. Kawahara. 2005. Intra- and interspecific phylogenetic relationships among diploid Triticum-Aegilops species
(Poaceae) based on base-pair substitutions, indels, and microsatel­
lites in chloroplast noncoding sequences. Am. J. Bot. 92:1887–
1898.
Yuan, Y. M., S. Wohlhauser, M. Moller, J. Klackenberg, M. Callmander, and P. Kupfer. 2005. Phylogeny and biogeography of Exacum
(Gentianaceae): A disjunctive distribution in the Indian ocean basin
resulted from long distance dispersal and extensive radiation. Syst.
Biol. 54:21–34.
Authors
Taxonomic group
Loci
ITS + trnL-F
ITS + trnL-F
ITS1
ITS + trnT-L
ITS
trnL-F + trnD-T + psbA-trnK
+ matK-trnK + ITS + ETS
+ leafy
Yamane and Kawahara, Triticum–Aegilops, Poaceae trnC-rpoB + trnF-ndhJ +
2005
ndhF-rpl132 + atpI–atpH +
7 microsatellites
Yuan et al., 2005
Exacum, Gentianaceae
ITS + trnL intron
Lee et al., 2005
Sonchus, Asteraceae
ITS + trnT-L-F + matK
Álvarez et al., 2005
Gossypium, Malvaceae
AdhC
Church and Taylor,
Houstonia, Rubiaceae
trnL intron + trnS-trnG spacer
2005
Kamiya et al., 2005
Shorea, Dipterocarpaceae PgiC
Levin and Miller, 2005 Lycium, Solanaceae
waxy+ trnT-F
McKown et al., 2005
Flaveria, Asteraceae
trnL-F
Roalson and Friar, 2004 Carex, Cyperaceae
ITS + ETS
Shaw and Small, 2005
Prunus, Rosaceae
rpL16
Croton, Euphorbiaceae
Androsace, Primulaceae
Cynara, Asteraceae
Platanus, Platanaceae
Phacelia, Hydrophyllaceae
Tribe Neillieae, Rosaceae
1595
1600
1605
1610
1615
1620
First submitted 14 February 2006; reviews returned 08 May 2006;
final acceptance 05 September 2006
Associate Editor: Vincent Savolainen
APPENDIX 1. Representative phylogenetic studies that highlight the issue of species nonmonophyly. Papers include recent studies from four
prominent journals (Systematic Biology, Systematic Botany, American Journal of Botany, Plant Systematics and Evolution). Only studies that included
multiple samples per species and studies that sampled closely related species within a genus are included. Where possible, determinations of
species monophyly were based on strict consensus trees (or ML trees).
Berry et al., 2005
Schneeweiss et al., 2004
Robba et al., 2005
Feng et al., 2005
Gilbert et al., 2005
Oh and Potter, 2005
1590
Number of species
Number of species
Number of
with multiple accessions that were monophyletic, accessions
(total species in study,
(% of species tested
included per
% of species tested)
that were monophyletic) spp. (range)
7 (78, 9.0%)
15 (47, 31.9%)
5 (8, 62.5%)
4 (5, 80.0%)
17 (53, 32.1%)
12 (16, 75.0%)
7 (100%)
14 (93%)
4 (80%)
4 (80%)
13 (76%)
8 (66%)
2–3
2–4
2–4
2–6
2–4
2–4
13 (13, 100%)
8 (62%)
4–13
5 (30, 16.7%)
12 (27, 44.4%)
6 (14, 42.9%)
14 (17, 82.4%)
3 (60%)
6 (50%)
3 (50%)
6 (43%)
2–4
2–4
2–5
2–13
17 (48, 35.4%)
6 (47, 12.8%)
20 (21, 95.2%)
4 (22, 18.2%)
13 (14, 92.9%)
6 (35%)
2 (33%)
5 (25%)
1 (25%)
0
2–5
2–3
2–7
2–4
3–53