© 2007 Society of Systematic Biologists
Widespread Genealogical Nonmonophyly in Species of Pinus Subgenus Strobus
Edited by Vincent Savolainen: Associate Editor
3
1 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{at}science.oregonstate.edu
3 Silva Tarouca Research Institute for Landscape and Ornamental Gardening Pr
honice, Czech Republic
4 Pacific Northwest Research Station, USDA Forest Service 3200 SW Jefferson Way, Corvallis, Oregon 97331, USA
* Corresponding author: John Syring. E-mail: jsyring{at}msubillings.edu
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
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 embryogenesis 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 x 106 years, and that complete genome-wide coalescence in these species may require up to 76.3 x 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.
Keywords: Lineage sorting; monophyly; nonmonophyly; nuclear genes; pinaceae; Pinus; phylogeny
Received February 14, 2006; Revised May 8, 2006; Accepted September 5, 2006
Whenever a phylogenetic study uses a single individual to represent a species, an implicit assumption is made that the species is monophyletic (Funk and Omland, 2003; Shaw and Small, 2005). To the extent that complicating factors (e.g., reticulation)are rare in the divergence history of terminal taxa, this assumption offers a convenient 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 organismal phylogenies. These gene trees are subject to processes that can result in the nonmonophyly of sequences 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 sampling 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 discovered in a phylogenetic analysis.
Awareness of the existence and complications arising from intraspecific polymorphism is growing. This is illustrated by recently published plant molecular phylogenetic studies (Appendix 1)that increasingly include sampling to evaluate species level monophyly. Not surprisingly, varying levels of nonmonophyly are encountered as more intensive population-level sampling 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 included 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 included species; Wilkin et al., 2005).
Although many examples of species nonmonophyly are the direct result of inadequate phylogenetic signal (Cross et al., 2002; Roalson and Friar, 2004; Levin and Miller, 2005; Shaw and Small, 2005), recent publications 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 responsible for species nonmonophyly are often difficult to establish. Factors commonly cited include introgressive hybridization (Roalson and Friar, 2004; Kamiya et al., 2005; Mason-Gamer, 2005; Shaw and Small, 2005), incomplete lineage sorting (Chiang et al., 2004; Bouillé and Bousquet, 2005; Kamiya et al., 2005), unrecognized amplification of a paralogous locus (Roalson and Friar, 2004; Álvarez et al., 2005), recombination among divergent alleles (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 studies and 2319 species found that 23.1% of the studies showed species-level paraphyly or polyphyly. Bouillé and Bousquet (2005) recently demonstrated a striking case of trans-species allelic polymorphism in three low-copy nuclear genes in different species of spruce (Picea). Allelic coalescence times between randomly selected alleles from these spruce species were estimated at 10 to 18 million years ago, values that overlapped with estimated divergence times (13 to 20 million years ago)for the species studied. Because spruces share many life history 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 origins that date to the early Cretaceous (145 to 125 million years ago; Alvin, 1960). Paleontological and molecular data suggest that the first major divergence event separating extant lineages occurred perhaps 85 to 45 million years ago (Miller, 1973; Meijer, 2000; Magallon and Sanderson, 2002; Willyard et al., 2007), giving rise to two distinct lineages recognized today as subg. Pinus and subg. Strobus, the "hard" and "soft" pines, respectively (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 homoplasy 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 treatments for the remaining taxa. Specific treatments for regions with high endemism, e.g., Mexico (Perry, 1991)and East Asia (Businsk, 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, Quinquefoliae 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, average pairwise nucleotide divergence (
)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., 2007). By integrating this information with fossil calibrations, Willyard et al. (2007) 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)makes it likely that mutations have spread and become fixed slowly across a species' range. This presents the potential 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 Syring et al., 2005). In pines, low-copy nuclear genes are an untapped resource for clarifying these terminal relationships, especially when genetic variation is interpreted within a framework where species monophyly can be assessed, and where the impact of incomplete lineage sorting can be determined. Data from multiple low-copy 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. 900-bp intron localized within a Late Embryogenesis Abundant (LEA)-like gene. The goal of this study is to intensively sample the remaining species of subg. Strobus, particularly the species-rich subsects. Strobus and Cembroides, 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 address 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 species-level 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?
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
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 extracted using the FastPrep DNA isolation kit (Qbiogene, Carlsbad, California, USA). In the select cases where needle tissue was used, direct sequencing identified homozygotes and heterozygotes. Homozygous sequences were directly incorporated into the alignment, whereas heterozygous sequences were cloned into pGem-T Easy (Promega, Madison, Wisconsin). From each of the 39 sampled species, an effort was made to obtain a minimum of two unique alleles. Depending on availability, individuals were selected that spanned the geographic range of the species. In cases of narrow endemism or where collections were limited, two alleles were sequenced from the same individual. Estimates of species distribution area were based on published 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 subsection Strobus, three alleles were chosen from a more extensive data set that will be used to evaluate population-level variation (four to seven alleles per species; Syring et al., unpublished data). The three alleles selected represent the most divergent sequences based on calculated p-distances.
Choice of Outgroup
As described in Syring et al. (2005), introns between pine subgenera are frequently unalignable due to numerous 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 evidence 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 nelsonii (Sect. Parrya, subsect. Nelsoniae)is exceptional. Evidence 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 rooting is employed. Because of this uncertainty, we chose to include P. nelsonii as a member of the outgroup in analyzing 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 outgroup 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 investigate 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 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
[GenBank]
, DQ018380
[GenBank]
, AY634346
[GenBank]
, AY634347
[GenBank]
)are incorporated in this study (Table 1). The alignment is available at TreeBase (S1538). Statistics calculated from the alignment include the average number of characters, number of variable and parsimony 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 
2 method)and topological (difference in sum of squares; DSS)approaches. First, the substitution-based "maximum 2" method was chosen 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 adjacent regions of aligned sequences. For all possible sequence 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 x 2 2 was calculated as an expression of the difference in the number of variable sites on each side of the partition for pairs of sequences. Putative recombination points were identified by plotting 2 values along the length of the alignment. An alternative 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 regions of an alignment. For all sequences, a 150-bp window 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 using least squares, and sum of squares for each partition 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 segments were identified by peaks in DSS values. Significance for both methods was determined by 1000 permutations (parametric bootstrapping)using 
= 0.01 as a threshold of significance and the program RDP2 (Martin et al., 2005). Analyses were conducted on data sets where shared indels were discarded to prevent false positives.
Phylogenetic and Statistical Analysis
Phylogenetic analyses were performed by taxonomic section using maximum parsimony (MP; PAUP* version 4.0b10; Swofford, 2003). Most parsimonious trees were found from branch-and-bound searches, with all characters weighted equally and treated as unordered. Branch support was evaluated using the nonparametric bootstrap (Felsenstein, 1985), with 1000 replicates and TBR branch swapping. Alternative phylogenetic hypotheses and statistical strength for species nonmonophyly were analyzed using constraints on tree topologies in PAUP*. The Wilcoxon signed-rank test (WSR; Templeton, 1983)was employed to test for significant differences among topologies. For this test, up to 1000 of the most-parsimonious trees were used as constraint topologies. The range of P values across all topologies is reported for every test.
Statistical associations between intraspecific nucleotide 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 statistically significant topological distortion as shown by the WSR constraint test)or weakly nonmonophyletic to monophyletic (i.e., WSR constraint tests were insignificant). Nucleotide diversity was analyzed directly, and geographic ranges (km2; Critchfield and Little, 1966) were log10-transformed to minimize skewness and kurtosis. 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 x 2Ne diploid generations, and complete genome-wide coalescence may require 5.30 x 2Ne diploid generations. In order to make these calculations for species of Pinus, Watterson's estimate of Theta (
)was determined for select species using DnaSP v.4.00.6 (Rozas et al., 2004). Effective population sizes were estimated using the formula Ne = /(4 x µG), where µ G is the absolute silent mutation rate per nucleotide, adjusted for generation time. The absolute silent mutation rate for Pinus was recently estimated from 11 nuclear genes (Willyard et al., 2007)to average 7.0 x 10– 10 substitutions/site/year, assuming an 85 million year divergence of pine subgenera. Generation times for individual species were taken as the average for the range of years to seed bearing age cited in Krugman and Jenkinson (Woody Plant Seed Manual, 2nd edition [R. G. Nisley, 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 referring to the coalescence of genealogies of the LEA-like gene and not the extent of monophyly for actual species.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
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 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 individuals 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 individual 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 parvifloraA2 (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. koraiensis (A1 and A2), and P. lambertiana (A1 and A2), two of the three alleles were from the same individual. Ten sequences included in this data set were cloned: discolor A1, discolor A3, koraiensisA1, koraiensisA2, longaevaA1, nelsoniiA2, parvifloraA1, pumilaA1, remota A3, and strobusA2. Based on this sample, 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 = 821–983 bp)and 987.3 bp from sect. Parrya (range = 937–1132 bp). The aligned sequence includes a partial exon on the 3' 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 20 variable positions within subg. Strobus (12 in sect. Quinquefoliae, 8 in sect. Parrya), of which 3 were localized in first codon positions, 5 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 267 variable sites and 152 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 for subg. Strobus at the LEA-like locus is 0.0330 ± 0.0029. Estimates of intraspecific show that soft pine species display a wide range of genetic 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 diversity in our sample include P. lambertiana ( = 0.0196), P. johannis (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 nucleotide diversity relative to sect. Quinquefoliae, with 10 of 13 species (76.9%)versus 11 of 20 species (55.0%)showing 
0.005, respectively (Table 2).
|
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 originated from the state of Mexico, Mexico and P. flexilis from Alberta, Canada). This allele has also been sequenced for P. strobiformis from both New Mexico and Texas (J. Syring, unpublished data). All other comparisons between P. ayacahuite and P. flexilis alleles show pairwise distances between 0.0011 and 0.0043, suggesting a close genetic affinity. Although not containing identical alleles, seven interspecific comparisons yielded highly similar alleles with p-distances 
0.0011: ayacahuiteA1/flexilisA3, ayacahuiteA2/flexilisA1, ayac-ahuiteA3/strobiformisA2, cembraA1/parvifloraA2, bhuta-nicaA2/wallichianaA2, bhutanicaA2/wallichianaA3, and culminicolaA2/remotaA3.
Of the 86 LEA-like sequences used in this analysis, only one allele showed evidence of recombination, despite a relatively lax threshold for significance ( = 0.01). The maximum 2 method identified one recombinant region 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 recombinant, it likely arose via xenologous recombination or the PCR process because it lacks similarity to other homologous alleles. The topology-based DSS method provides no evidence for recombination in strobiformisA1 or other sequences. Because the single putatively recombinant segment from strobiformisA1 only adds autapomorphic 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 ; Table 2).
Phylogenetic Analyses
Section Quinquefoliae
From the branch-and-bound search of the sect. Quinquefoliae data set, twenty most parsimonious 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 Krempfianae 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 American 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 supported 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. 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 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 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 I; Fig. 2), all of which were present in the strict consensus tree with the exception of the culminicolaA1 and discolorA3 alleles (these collapse out of clade F in the strict consensus tree). With the exclusion of these two poorly supported alleles and johannisA2 from clade I, each of the four major clades within subsect. Cembroides have moderate to strong support (78% to 90% BS), though relationships among and within the clades remain largely unresolved.
Clade F contains all alleles from P. cembroides and P. 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. monophylla and one of two P. johannis alleles. Support for the monophyly and relationships among the three alleles of 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 being the only clade from this section where species do 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. maximartinezii 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 indicates that of the 20 species for which multiple unique alleles were sequenced, 9 species (45%)were monophyletic (Table 2). For species exhibiting allelic monophyly, seven 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 clades: these include P. bhutanica (clades D, E), P. lambertiana (clades A, B, D), P. monticola (clades A, B), P. strobiformis (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 sample (50%; 6 of 12 species), suggesting that small sample sizes generally capture enough intraspecfic heterogeneity 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 diversity (Table 2). Although intraspecific mean p-distances 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 (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 monophyly 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 ( = 0.05). For example, constraining topologies for the monophyly of P. flexilis or P. ayacahuite 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 support the polyphyly of alleles from these species.
|
Section Parrya
The strict consensus tree indicates that of the 13 species for which multiple alleles were sequenced, only 5 species (38.5%)were monophyletic (Table 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. Alleles 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, suggesting that, in contrast to sect. Quinquefoliae, additional sampling may reduce the number of monophyletic species in sect. Parrya.
Following the trend in sect. Quinquefoliae, allelic monophyly is not a simple function of intraspecific genetic diversity (Table 2). Although nucleotide diversity is low for the three alleles of P. monophylla ( = 0.006), this species is not monophyletic in the strict consensus tree. In contrast, P. aristata ( = 0.015)and P. longaeva ( = 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 monophyly 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%)indicate 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 (30.3%)were supported as nonmonophyletic both in the strict consensus tree and in the WSR constraint analyses (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 considered 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 dependent variables, 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 diversity. Average nucleotide diversities in species showing strong deviation from monophyly are significantly higher ( = 0.0149)than comparable values from monophyletic and weakly nonmonophyletic species combined ( = 0.0056; one-way ANOVA, P < 0.0000; Table 4). Monophyletic and weakly nonmonophyletic species are not significantly different ( = 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. Average geographic ranges for these two classes are essentially indistinguishable (one-way ANOVA, P < 0.8007; Table 4), with the average geographic range for strongly nonmonophyletic species at 40,615 km2, and the average 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 apportionment 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 phylogenetic spectrum from essentially monophyletic (P. flexilis;
= 0.0014), to weakly non-monophyletic (P. discolor; = 0.0112), to strongly nonmonophyletic (P. lambertiana; = 0.0196). Estimates of are not significantly different than values of (based on Tajima's D statistic; data not shown), so our estimates of are unlikely to reflect effects from natural selection. Estimated values for Ne range from ca. 1.7 x 104 for P. discolor to 12 x 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 time for complete genome-wide coalescence ranges from 5.4 million years to 76 million years.
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Considering that widespread allelic nonmonophyly was observed and the fact that this is a single-locus estimate of relationships, phylogenetic conclusions regarding relationships among the terminal taxa remain premature, even in those cases where species monophyly is clear and support values are high. However, the phylogeny 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 detected 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 subsections 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 relationships 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 range from 0.0131 (subsect. Cembroides)to 0.0177 (subsect. Strobus), which are 4.5 times (subsects. Strobus, Cembroides)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), orthological 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, further 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 potential 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; Hudson and Coyne, 2002; Rosenberg and Nordborg, 2002; Funk and Omland, 2003; Rosenberg, 2003; Sites and Marshall, 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 nonmonophyly in subg. Strobus. As Funk and Omland (2003) point out, definitive causes for species polyphyly are difficult to prove, however, from the observed topological patterns causal inferences can be made.
Inadequate phylogenetic signal.— The signature of insufficient phylogenetic signal is apparent, but not universal, 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. balfouriana and P. monophylla, which have no alleles separated by supported nodes in the strict consensus tree, and show insignificant nonmonophyly in WSR tests (Table 3). However, inadequate phylogenetic information cannot 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 position 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 nonmonophyletic, show strong nodal support, and have significant WSR results provides compelling evidence that the species level polyphyly uncovered in this study is the product of true biological signal and not simply inadequate 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; Businsk, 2004; Gernandt et al., 2005). Although issues of taxonomic uncertainty 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 synonymizing 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. cembroidessensu lato due to the position of johannis A2 in clade I (Fig. 2).
The situation in sect. Quinquefoliae is similar to that of sect. Parrya. Pinusbhutanica was recently recognized as a subspecies of P. wallichiana (Businsk, 1999, 2004). Although 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 allele lineage. Pinus kwangtungensis, recently considered to be conspecific with P. wangii Hu & W. C. Cheng and related to P. parviflora (Businsk, 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 (Martínez, 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. ayacahuite to the south. Neither a broadly circumscribed P. ayacahuite nor P. flexilis would restore species monophyly 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 conditions (P. monophylla x P. edulis: Lanner 1974a; Lanner and Phillips, 1992; P. parvifloraxP. 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 local 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 explain 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, hybridization between P. pumilax 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 analysis. 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 x P. monophylla is widely recognized (Lanner, 1974a). In this case, the introgression of alleles in regions of sympatry 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. juarazensis" Lanner from Baja California and P. monophylla from southern California.
Pinus lambertiana serves to illustrate the potential for allelic 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 divergence 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 x P. monticola crosses traces to prefertilization barriers (Fernando et al., 2005). It is noteworthy that lambertianaA3 is found associated with clade D, an otherwise strictly Eurasian clade. There remains the possibility 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 suggest that the observed pattern of species nonmonophyly is the result of paralogous gene amplification. On the contrary, evidence suggests that the sequences amplified 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 (Krutovsky et al., 2004). The locus was amplified from haploid (megagametophyte)tissue, and this provides a powerful screen for evaluating PCR pool heterogeneity. If duplicate 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 target 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. Incomplete lineage sorting, by contrast, is the persistence and retention of ancestral polymorphisms through multiple speciation events. Incomplete lineage sorting can potentially impact any single-locus gene tree in any taxon. The theoretical impact of incomplete lineage sorting on gene trees and inferred species trees has been known for some time (Nei, 1987; Doyle, 1992). Incomplete lineage sorting can be promoted by biological conditions that encourage the retention of genetic variability in a species, e.g., long life spans, large Ne, and outcrossing. The retention of shared ancestral polymorphisms is also affected by natural selection (Broughton and Harrison, 2003), in that balancing selection works to oppose directional selection and maintain genetic diversity. 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 between 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 animals (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 balancing 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 subsections. According to the molecular clock divergence estimates of Willyard et al. (2007), the species-rich subsections Cembroides and Strobus diverged from their sister lineages (subsects. Balfourianae and Gerardianae, respectively)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 sorting rarely approached allelic fixation within daughter lineages.
Recombination
The effect of recombination on reconstructing genealogies has been well documented (reviewed in Posada et al., 2002). Because recombination produces sequence segments that have different genealogical histories, organismal history cannot be accurately depicted by a single phylogenetic "tree," but rather a set of correlated trees across recombinant segments in the alignment. Under scenarios of ancient recombination, 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 (Schierup and Hein, 2000; Posada and Crandall, 2002). In contrast, recombination between divergent sequences or high recombination rates can produce inaccurate phylogenies that show artifactually long terminal branches, apparent trans-specific polymorphism (as shown here and the study of Bouillé and Bousquet, 2005), and phylogenies that are significantly different from the true histories underlying the data (Posada and Crandall, 2002).
The locus examined in this study has certainly experienced recombination during the divergence of sections of subg. Strobus, but different methods employed (maximum 2; DSS)fail to detect recombination in our data. We chose these methods because they use different approaches to infer recombination and because the maximum 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 ( = 0.02 for Sect. Quinquefoliae and 0.03 for Sect. Parrya)may be the primary obstacle in detecting recombination because 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 sequences 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 between parental alleles and yield more "star-like" phylogenies than would be obtained with nonrecombined sequences. Clearly, the presence of divergent allelic polymorphism is primarily responsible for the pattern of nonmonophyly in pines; recombination simply adds complexity and uncertainty to the pattern.
How Do Pine Species Attain Monophyly?
Rieseberg and Brouillet (1994) suggest that species concepts based on monophyly are inadequate because paraphyletic species should be expected from progenitor-derivative speciation. Under that mode of speciation, paraphyly should be expected as a direct result 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)as long as balancing selection 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 postspeciation mutation. The situation in Pinus is more complex 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 allelic 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 are based on a small sample, the values in Table 5 are instructive. For example, the estimated time for reciprocal 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 calculated at 19 million years (Willyard et al., 2007). Topological analyses suggest that P. discolor is weakly nonmonophyletic (35% of WSR tests were significant; Table 3)at the LEA-like locus, a finding that shows surprisingly good agreement with the approximations of allele coalescence and molecular evolutionary age of the Cembriodes lineage. Nevertheless, the estimate for genome-wide 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 , 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-wide coalescence would extend far beyond the divergence of the two sections from subg. Strobus. Clearly, the accuracy of these estimates depends upon many assumptions; nevertheless, they highlight the fallacy of assuming ‘species monophyly’ in groups characterized by large population sizes, and the complexity of resolving phylogenetic relationships among pine species.
In light of this information, the historic effective population 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 current geographic ranges (a proxy for census population sizes and global abundance)are uncorrelated with nucleotide diversity, and show no association with the extent 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 geographic 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. 5,000 km2)shows far greater nucleotide diversity than P. albicaulis ( = 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 reflect nonequilibrium processes of recent expansion (e.g., species with low genetic diversity and large geographic ranges like P. koraiensis and P. strobus), recent range contraction (e.g., species with high genetic diversity and small ranges like P. bhutanica and P. johannis), and possibly geographic or ecological isolation coupled with long-distance dispersal events (e.g., P. chiapensis and P. albicaulis). This observation has implications for molecular phylogenetic studies focusing on recently diverged taxa (e.g., 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).
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 during speciation, and continue to the present. Processes responsible for genic nonmonophyly in species include hybridization with subsequent introgression, incomplete lineage sorting, and recombination. These processes may become superimposed, and their present-day genealogical patterns may reflect ancient and recent events. Sequences 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 monophyly is less than 0.1. In the absence of complicating factors (e.g., organellar introgression, nuclear recombination), 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 theory 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 inappropriate for pines and other species sharing similar life history traits. Distinct morphological and ecological differences are readily apparent between most of the species of subgenus Strobus included in this study, yet species-level 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 related species well after speciation has occurred (Rosenberg, 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 harbor ancestral polymorphisms at a large fraction of their genome.
The demonstration of widespread species-level nonmonophyly 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 histories among closely related species. Although we generally agree with this conclusion for our work in deciphering 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 organellar 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 diversity 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 (Liston 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 detectable by sampling multiple individuals per species, and perhaps multiple loci. In Pinus, large Ne, long generation 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., Quercus (400 species), Salix (450 species), Ficus (750 species), and Eucalyptus (680 species), should be prone to similar levels of nonmonophyly if examined by nuclear markers. 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 recommend that researchers explicitly test the monophyly of species and lower level taxa by including multiple individuals across the range of a species, especially prior to proposing new classifications or delimiting species. In the absence of such sampling,2 and working under the assumption of species monophyly, it seems likely that many more cases of species nonmonophyly will remain undetected.
|
|
Acknowledgements |
|---|
We thank Martin Gardner, Fiona Inches, and Philip Thomas (Royal 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), 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 Breeding Center (Japan), Sanderson McArthur (USDAFS Rocky Mt. Research Station), Paul Halladin (Iseli Nursery, Oregon), Dennis Ringnes (USDAFS Central Zone Genetic Resource Program, California), Carrie 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 Management 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.
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
-
Álvarez I., Cronn R., Wendel J. F. Phylogeny of the New World diploid cottons (Gossypium L. Malvaceae) based on sequences of three low-copy nuclear genes. Plant Syst. Evol. (2005) 252:199–214.[CrossRef]
Alvin K. Further conifers of the Pinaceae from the Wealden Formation of Belgium. Mem. Inst. Roy. Sci. Nat. Belg. (1960) 146:1–39.
Andresen J. W. A multivariate analysis of the Pinus chiapensis–monticola–strobus phylad. Rhodora (1966) 68:1–24.
Bailey D. K. Phytogeography and taxonomy of Pinus subsection Balfourianae. Ann. Missouri Bot. Gard. (1970) 57:210–249.[CrossRef][Web of Science]
Baum D. A., Donoghue M. J. Choosing among alternative "phylogenetic" species concepts. Syst. Bot. (1995) 20:560–573.[CrossRef]
Berry P. E., Hipp A. L., Wurdack K. J., Van Ee B., Riina R. Molecular phylogenetics of the giant genus Croton and tribe Crotoneae (Euphorbiaceae sensu stricto) using ITS and trnL-trnF DNA sequence data. Am. J. Bot. (2005) 92:1520–1534.
Bouillé M., Bousquet J. Trans-species shared polymorphisms at orthologous nuclear gene loci among distant species in the conifer Picea (Pinaceae): Implications for the long-term maintenance of genetic diversity in trees. Am. J. Bot. (2005) 92:63–73.
Broughton R. E., Harrison R. G. Nuclear gene genealogies reveal historical, demographic and selective factors associated with speciation in field crickets. Genetics (2003) 163:1389–1401.
Businsk R. Taxonomic revision of Eurasian pines (genus Pinus L.)—Survey of species and infraspecific taxa according to latest knowledge. Acta Pruhoniciana (1999) 68:7–86.
Businsk R. A revision of the Asian Pinus subsection Strobus (Pinaceae). Willdenowia (2004) 34:209–257.[CrossRef]
Chase M. W., Salamin N., Wilkinson M., Dunwell J. M., Kesanakurthi R. P., Haidar N., Savolainen V. Land plants and DNA barcodes: Short-term and long-term goals. Phil. Trans. R. Soc. Lond. B (2005) 360:1889–1895.
Chiang T. Y., Hung K. H., Hsu T. W., Wu W. L. Lineage sorting and phylogeography in Lithocarpus formosanus L. dodonaeifolius (Fagaceae) from Taiwan. Ann. Missouri Bot. Gard. (2004) 91:207–222.[Web of Science]
Church S. A., Taylor D. R. Speciation and hybridization among Houstonia (Rubiaceae) species: The influence of polyploidy on reticulate evolution. Am. J. Bot. (2005) 92:1372–1380.
Comes H. P., Abbott R. J. Molecular phylogeography, reticulation, and lineage sorting in Mediterranean Senecio sect. Senecio (Asteraceae). Evolution (2001) 55:1943–1962.[Web of Science][Medline]
Crisp M. D., Chandler G. T. Paraphyletic species. Telopea (1996) 6:813–844.
Critchfield W. B. Interspecific hybridization in Pinus: A summary review. Fowler D. P., Yeatman C. W., eds. (1975) Proceedings 14th Canadian Tree Improvement Association, Part 2, Symposium on interspecific hybridization in forest trees, NB. Ottawa, Ontario: Canadian Forestry Service. 99–105.
Critchfield W. B. Hybridization and classification of the white pines (Pinus section Strobus). Taxon (1986) 35:647–656.[CrossRef][Web of Science]
Critchfield W. B., Little E. L. Jr. Geographic distribution of the pines of the world (1966) Washington, DC. USDA Forest Service Miscellaneous Publication 991.
Cross E. W., Quinn C. J., Wagstaff S. J. Molecular evidence for the polyphyly of Olearia (Astereae: Asteraceae). Plant. Syst. Evol. (2002) 235:99–120.[CrossRef]
de Queiroz K., Donoghue M. J. Phylogenetic systematics and the species problem. Cladistics (1988) 4:317–338.[CrossRef][Web of Science]
Delgado P., Piñero D., Chaos A., Pérez-Nasser N., Alvarez-Buylla E. R. High population differentiation and genetic variation in the endangered Mexican pine Pinus rzedowskii (Pinaceae). Am. J. Bot. (1999) 86:669–676.
Doyle J. J. Trees within trees: Genes and species, molecules and morphology. Syst. Biol. (1997) 46:537–553.
Doyle J. J. Gene trees and species trees: Molecular systematics as one-character taxonomy. Syst. Bot. (1992) 17:144–163.[CrossRef]
Farjon A. World checklist and bibliography of conifers (1998) Richmond, UK: Royal Botanical Gardens at Kew.
Farjon A. Drawings and descriptions of the genus Pinus (2005) 2nd edition. Leiden, Netherlands: E. J. Brill and W. Backhuys.
Farjon A., Styles B. T. Pinus (Pinaceae) (1997) New York: The New York Botanical Garden. Flora Neotropica Monograph 75.
Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution (1985) 39:783–791.[CrossRef][Web of Science]
Feng Y., Oh S. H., Manos P. S. Phylogeny and historical biogeography of the genus Platanus as inferred from nuclear and chloroplast DNA. Syst. Bot. (2005) 30:786–799.[CrossRef]
Fernando D. D., Long S. M., Sniezko R. A. Sexual reproduction and crossing barriers in white pines: The case between Pinus lambertiana (sugar pine) and P. monticola (western white pine). Tree Genet. Genomes (2005) 1:143–150.
Funk D. J., Omland K. E. Species-level paraphyly and polyphyly: Frequency, causes, and consequences, with insights from animal mitochondrial DNA. Annu. Rev. Ecol. Evol. Syst. (2003) 34:397–423.[CrossRef]
Garrett P. W. Species hybridization in the genus Pinus (1979) Northeast Forest Experiment Station: USDA Forest Service. Station Research Paper 436.
Gernandt D. S., Liston A., Piñero D. Variation in the nrDNA ITS of Pinussubsection Cembroides: Implications for molecular systematic studies of pine species complexes. Mol. Phylogenet. Evol. (2001) 21:449–467.[CrossRef][Web of Science][Medline]
Gernandt D. S., Liston A., Piñero D. Phylogenetics of Pinus subsections Cembroides Nelsoniae inferred from cpDNA sequences. Syst. Bot. (2003) 4:657–673.
Gernandt D. S., Lopez G. G., García S. O., Liston A. Phylogeny and classification of Pinus. Taxon (2005) 54:29–42.[Web of Science]
Gilbert C., Dempcy J., Ganong C., Patterson R., Spicer G. S. Phylogenetic relationships within Phacelia subgenus Phacelia (Hydrophyllaceae) inferred from nuclear rDNA ITS sequence data. Syst. Bot. (2005) 30:627–634.[CrossRef]
Goetsch L., Eckert A. J., Hall B. D. The molecular systematics of Rhododendron (Ericaceae): A phylogeny based upon RPB2 gene sequences. Syst. Bot. (2005) 30:616–626.[CrossRef]
Goodwillie C., Stiller J. W. Evidence for polyphyly in a species of Linanthus (Polemoniaceae): Convergent evolution in self-fertilizing taxa. Syst. Bot. (2001) 26:273–282.
Hare M. P., Cipriano F., Palumbi S. R. Genetic evidence on the demography of speciation in allopatric dolphin species. Evolution (2002) 56:804–816.[CrossRef][Web of Science][Medline]
Hudson R. R., Coyne J. A. Mathematical consequences of the genealogical species concept. Evolution (2002) 56:1557–1565.[CrossRef][Web of Science][Medline]
Ioerger T. R., Clark A. G., Kao T. H. Polymorphism at the self-incompatibility locus in Solanaceae predates speciation. Proc. Natl. Acad. Sci. USA (1990) 87:9732–9735.
Kamiya K., Harada K., Tachida H., Ashton P. S. Phylogeny of PgiC gene in Shorea and its closely related genera (Dipterocarpaceae), the dominant trees in southeast Asian tropical rain forests. Am. J. Bot. (2005) 92:775–788.
Kral R. Pinus. In: Flora of North America (North of Mexico)—Flora of North America Editorial Committee, ed. (1993) volume 2. New York: Oxford University Press. 373–398.
Kress W. J., Wurdack K. J., Zimmer E. A., Weigt L. A., Janzen D. H. Use of DNA barcodes to identify flowering plants. Proc. Natl. Acad. Sci. USA (2005) 102:8369–8374.
Krutovsky K. V., Troggio M., Brown G. R., Jermstad K. D., Neale D. B. Comparative mapping in the Pinaceae. Genetics (2004) 168:447–461.
Kumar S., Tamura K., Jakobsen I. B., Nei M. MEGA2: Molecular evolutionary genetics analysis software. Bioinformatics (2001) 17:1244–1245.
Lanner R. M. Natural hybridization between Pinus edulis and Pinus monophylla in the American Southwest. Silvae Genet. (1974a) 23:108–116.
Lanner R. M. A new pine from Baja California and the hybrid origin of Pinus quadrifolia. Southwest. Nat. (1974b) 19:75–95.[CrossRef]
Lanner R. M., Phillips A. M. III. Natural hybridization and introgression of pinyon pines in northwestern Arizona. Int. J. Plant Sci. (1992) 153:250–257.[CrossRef][Web of Science]
Ledig F. T. Genetic variation in Pinus. In: Ecology and biogeography of Pinus—Richardson D. M., ed. (1998) Cambridge, UK: Cambridge University Press. 251–280.
Ledig F. T., Conkle M. T., Bermejo-Velazaquez B., Eguiluz-Piedra T., Hodgskiss P. D., Johnson D. R., Dvorak W. S. Evidence for an extreme bottleneck in a rare Mexican pinyon: Genetic diversity, disequilibrium, and the mating system in Pinus maximartinezii. Evolution (1999) 53:91–99.[CrossRef][Web of Science]
Lee C., Kim S. C., Lundy K., Santos-Guerra A. Chloroplast DNA phylogeny of the woody Sonchus alliance (Asteraceae: Sonchinae) in the Macaronesian Islands. Am. J. Bot. (2005) 92:2072–2085.
Levin R. A., Miller J. S. Relationships within tribe Lycieae (Solanaceae): Paraphyly of Lycium and multiple origins of gender dimorphism. Am. J. Bot. (2005) 92:2044–2053.
Levin R. A., Watson K., Bohs L. A four-gene study of evolutionary relationships in Solanum section Acanthophora. Am. J. Bot. (2005) 92:603–612.
Liston A., Robinson W. A., Piñero D., Alvarez-Buylla E. R. Phylogenetics of Pinus (Pinaceae) based on nuclear ribosomal DNA internal transcribed spacer region sequences. Mol. Phylogenet. Evol. (1999) 11:95–109.[CrossRef][Web of Science][Medline]
Little E. L. Jr. Two new pinyon varieties from Arizona. Phytologia (1968) 17:329–342.
Little E. L. Jr., Critchfield W. B. Subdivisions of the genus Pinus (1969) Washington, DC. USDA Forest Service Miscellaneous Publication 1144.
Little E. L. Jr., Righter F. I. Botanical descriptions of forty artificial pine hybrids. USDA Forest Service Technical Bulletin (1965) 1345:1–47. Washington, DC.
Luo Y., Zhang F., Yang Q. E. Phylogeny of Aconitumsubgenus Aconitum(Ranunculaceae) inferred from ITS sequences. Plant Syst. Evol. (2005) 252:11–25.[CrossRef]
MacDonald G. M., Cwynar L. C., Whitlock C. The late Quaternary dynamics of pines in northern North America. In: Ecology and biogeography of Pinus—Richardson D. M., ed. (1998) Cambridge, UK: Cambridge University Press. 122–149.
Magallön S., Sanderson M. J. Relationships among seed plants inferred from highly conserved genes: Sorting conflicting phylogenetic signals among ancient lineages. Am. J. Bot. (2002) 89:1991–2006.
Malusa J. Phylogeny and biogeography of the pinyon pines (Pinus subsect. Cembroides). Syst. Bot. (1992) 17:42–66.[CrossRef]
Martin D. P., Williamson C., Posada D. RDP2: Recombination detection and analysis from sequence alignments. Bioinformatics (2005) 21:260–262.
Martínez M. Pinaceas Mexicanas. Anales del Instituto de Biologia de Mexico (1940) 11:57–84.
Mason-Gamer R. J. The β-amylase genes of grasses and a phylogenetic analysis of the Triticeae (Poaceae). Am. J. Bot. (2005) 92:1045–1058.
McGuire G., Wright F., Prentice M. J. A graphical method for detecting recombination in phylogenetic data sets. Mol. Biol. Evol. (1997) 14:1125–1131.[Abstract]
McKown A. D., Moncalvo J. M., Dengler N. G. Phylogeny of Flaveria (Asteraceae) and inference of C4 photosynthesis evolution. Am. J. Bot. (2005) 92:1911–1928.
Meijer J. Fossil woods from the late Cretaceous Aachen Formation. Rev. Palaeobot. Palynol. (2000) 112:297–336.[CrossRef][Web of Science]
Miller C. Silicified cones and vegetative remains of Pinus from the Eocene of British Columbia. Cont. Univ. Mich. Museum Paleo. (1973) 24:101–118.
Milne I., Wright F., Rowe G., Marshall D. F., Husmeier D., McGuire G. TOPALi: Software for automatic identification of recombinant sequences within DNA multiple alignments. Bioinformatics (2004) 20:1806–1807.
Moore W. S. Inferring phylogenies from mtDNA variation: Mitochondrial-gene trees versus nuclear-gene trees. Evolution (1995) 49:718–726.[CrossRef][Web of Science]
Müller K., Borsch T. Phylogenetics of Utricularia (Lentibulariaceae) and molecular evolution of the trnK intron in a lineage with high substitutional rates. Plant Syst. Evol. (2005) 250:39–67.[CrossRef]
Nagl S., Tichy H., Mayer W. E., Takahata N., Klein J. Persistence of neutral polymorphisms in Lake Victoria cichlid fish. Proc. Natl. Acad. Sci. USA (1998) 95:14238–14243.
Nei M. Molecular evoltionary genetics (1987) New York: Columbia University Press.
Oh S. H., Potter D. Molecular phylogenetic systematics and biogeography of tribe Neillieae (Rosaceae) using DNA sequences of cpDNA, rDNA, and LEAFY. Am. J. Bot. (2005) 92:179–192.
Oline D. K., Mitton J. B., Grant M. C. Population and subspecific genetic differentiation in the foxtail pine (Pinus balfouriana). Evolution (2000) 54:1813–1819.[Web of Science][Medline]
Pamilo P., Nei M. Relationships between gene trees and species trees. Mol. Biol. Evol. (1988) 5:568–583.[Abstract]
Perez de la Rosa J., Harris S. A., Farjon A. Noncoding chloroplast DNA variation in Mexican pines. Theor. Appl. Genet. (1995) 91:1101–1106.[Web of Science]
Perry J. P. The pines of México and Central America (1991) Portland, Oregon: Timber Press.
Petit R. J., Aguinagalde I., de Beaulieu J. L., Bittkau C., Brewer S., Cheddadi R., Ennos R., Fineschi S., Grivet D., Lascoux M., Mohanty A., Müller-Starck G., Demesure-Musch B., Palmé A., Martín J. P., Rendell S., Vendramin G. G. Glacial refugia: Hotspots but not melting pots of genetic diversity. Science (2003) 300:1563–1565.
Popp M., Oxelman B. Evolution of a RNA polymerase gene family in Silene (Caryophyllaceae)—Incomplete concerted evolution and topological congruence among paralogues. Syst. Biol. (2004) 53:914–932.
Posada D. Evaluation of methods for detecting recombination from DNA sequences: Empirical data. Mol. Biol. Evol. (2002) 19:708–717.
Posada D., Crandall K. A. Evaluation of methods for detecting recombination from DNA sequences: Computer simulations. Proc. Natl. Acad. Sci. USA (2001) 98:13757–13762.
Posada D., Crandall K. A. The effect of recombination on the accuracy of phylogeny estimation. J. Mol. Evol. (2002) 54:396–402.[Web of Science][Medline]
Price R. A., Liston A., Strauss S. H. Phylogeny and systematics of Pinus. In: Ecology and biogeography of Pinus—Richardson D. M., ed. (1998) Cambridge, UK: Cambridge University Press. 49–68.
Rieseberg L. H., Broulliet L. Are many plant species paraphyletic? Taxon (1994) 43:21–32.[CrossRef][Web of Science]
Roalson E. H., Friar E. A. Phylogenetic relationships and biogeographic patterns in North American members of Carexsection Acrocystis(Cyperaceae) using nrDNA ITS and ETS sequence data. Plant Syst. Evol. (2004) 243:175–187.[CrossRef]
Robba L., Carine M. A., Russell S. J., Raimondo F. M. The monophyly and evolution of Cynara L. (Asteraceae) sensu lato: Evidence from the internal transcribed spacer region of nrDNA. Plant Syst. Evol. (2005) 253:53–64.[CrossRef]
Rosenberg N. A. The shapes of neutral gene genealogies in two species: Probabilities of monophyly, paraphyly, and polyphyly in a coalescent model. Evolution (2003) 57:1465–1477.[CrossRef][Web of Science][Medline]
Rosenberg N. A., Nordborg M. Genealogical trees, coalescent theory and the analysis of genetic polymorphisms. Nature (2002) 3:380–390.
Rozas J., Sánchez-DelBarrio J., Messeguer X., Rozas R. DnaSP: DNA sequence polymorphism, v4.00.6. Bioinformatics (2004) 19:2496–2497.[CrossRef][Web of Science]
Rzedowski J., Vela L. Pinus strobusvar. chiapensis en la Sierra Madre del Sur de México. Ciencia (1966) 24:211–216.[Web of Science]
SAS Institute, Inc. SAS/STAT user's guide, version 8, volume 1 (1999) Cary, North Carolina: SAS Institute.
Schierup M. H., Hein J. Consequences of recombination on traditional phylogenetic analysis. Genetics (2000) 156:879–891.
Schneeweiss G., Schonswetter P., Kelso S., Niklfeld H. Complex biogeographic patterns in Androsace (Primulaceae) and related genera: Evidence from phylogenetic analyses of nuclear internal transcribed spacer and plastid trnL-F sequences. Syst. Biol. (2004) 53:856–876.
Shaw G. R. The genus Pinus (1914) Cambridge, Massachusetts: Riverside Press. Publications of the Arnold Arboretum No. 5.
Shaw J. Biogeographic patterns and cryptic speciation in bryophytes. J. Biogeogr. (2001) 28:253–261.[CrossRef]
Shaw J., Small R. L. Chloroplast DNA phylogeny and phylogeography of the North American plums (Prunus subgenus Prunussection Prunocerasus, Rosaceae). Am. J. Bot. (2005) 92:2011–2030.
Shaw K. L. Species and the diversity of natural groups. In: Endless forms: Species and speciation—Howard D. J., Berlocher S. J., eds. (1998) Oxford, UK: Oxford University Press. 44–56.
Simmons M. P., Ochoterena H. Gaps as characters in sequence-based phylogenetic analyses. Syst. Biol. (2000) 49:369–381.
Sites J. W., Marshall J. C. Delimiting species: A renaissance issue in systematic biology. Trends Ecol. Evol. (2003) 18:462–470.[CrossRef]
Smith J. M. Analyzing the mosaic structure of genes. J. Mol. Evol. (1992) 34:126–129.[Web of Science][Medline]
Song B. H., Wang X. Q., Wang X. R., Ding K. Y., Hong D. Y. Cytoplasmic composition in Pinus densata and population establishment of the diploid hybrid pine. Mol. Ecol. (2003) 12:2995–3001.[CrossRef][Medline]
Swofford D. L. PAUP*4.0b10: Phylogenetic analysis using parsimony (*and other methods) (2003) Sunderland, Massachusetts: Sinauer Associates.
Syring J., Willyard A., Cronn R., Liston A. Evolutionary relationships among Pinus(Pinaceae) subsections inferred from multiple low-copy nuclear loci. Am. J. Bot. (2005) 92:2086–2100.
Templeton A. R. Phylogenetic inference from restriction endonuclease cleavage site maps with particular reference to the evolution of humans and the apes. Evolution (1983) 37:221–244.[CrossRef][Web of Science]
Treutlein J., Smith G. F., van Wyk B. E., Wink M. Evidence for the polyphyly of Haworthia(Asphodelaceae Subfamily Alooideae; Asparagales) inferred from nucleotide sequences of rbc L, matK, ITS1 and genomic fingerprinting with ISSR-PCR. Plant Biol. (2003) 513–521.
Wang X. R., Szmidt A. E., Savolainen O. Genetic composition and diploid hybrid speciation of a high mountain pine, Pinus densata, native to the Tibetan plateau. Genetics (2001) 159:337–346.
Wang X. R., Tsumura Y., Yoshimaru H., Nagasaka K., Szmidt A. E. Phylogenetic relationships of Eurasian pines (Pinus, Pinaceae) based on chloroplast rbcL matK, rpl20-rps18 spacer, and trnV intron sequences. Am J Bot (1999) 86:1742–1753.
Watano Y., Kanai A., Tani N. Genetic structure of hybrid zones between Pinus pumila P. parviflora var. pentaphylla (Pinaceae) revealed by molecular hybrid index analysis. Am. J. Bot. (2004) 91:65–72.
Wendel J. F., Doyle J. J. Phylogenetic incongruence: Window into genome history and molecular evolution. In: Molecular systematics of plants II: DNA sequencing—Soltis P. S., Soltis D. E., Doyle J. J., eds. (1998) Dordrecht, Netherlands: Kluwer Academic Publishers. 265–296.
Wilkin P., Schols P., Chase M. W., Chavamarit K., Furness C. A., Huysmans S., Rakotonasolo F., Smets E., Thapyai C. A plastid gene phylogeny of the yam genus, Dioscorea: Roots, fruits and Madagascar. Syst. Bot. (2005) 30:736–749.[CrossRef]
Willyard A., Syring J., Gernandt D., Liston A., Cronn R. Molecular evolutionary rates indicate a recent and rapid diversification for modern pine lineages. Mol. Biol. Evol (2007) 23:1–12.[CrossRef]
Winkworth R. C., Donoghue M. J. Viburnum phylogeny based on combined molecular data: Implications for taxonomy and biogeography. Am. J. Bot. (2005) 92:653–666.
Wright J. A., Marin A. M. V., Dvorak W. S. Conservation and use of the Pinus chiapensis genetic resource in Columbia. Forest Ecol. Manag (1996) 88:283–288.[CrossRef]
Wu C. I. The genic view of the process of speciation. J. Evol. Biol. (2001) 14:851–865.[CrossRef][Web of Science]
Yamane K., Kawahara T. Intra-and interspecific phylogenetic relationships among diploid Triticum-Aegilopsspecies (Poaceae) based on base-pair substitutions, indels, and microsatellites in chloroplast noncoding sequences. Am. J. Bot. (2005) 92:1887–1898.
Yuan Y. M., Wohlhauser S., Moller M., Klackenberg J., Callmander M., Kupfer P. Phylogeny and biogeography of Exacum(Gentianaceae): A disjunctive distribution in the Indian ocean basin resulted from long distance dispersal and extensive radiation. Syst. Biol. (2005) 54:21–34.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. A. Dragon and D. S. Barrington Systematics of the Carex aquatilis and C. lenticularis lineages: Geographically and ecologically divergent sister clades of Carex section Phacocystis (Cyperaceae) Am. J. Botany, October 1, 2009; 96(10): 1896 - 1906. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. J. Maureira-Butler, B. E. Pfeil, A. Muangprom, T. C. Osborn, and J. J. Doyle The Reticulate History of Medicago (Fabaceae) Syst Biol, June 1, 2008; 57(3): 466 - 482. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||