Distinct niche divergence characterizes the homoploid hybrid speciation of Pinus densata on the Tibetan plateau

Ecological divergence and selection for novel adaptations to new habitats have been theoretically proposed to play important roles in promoting homoploid hybrid speciation (HHS). The successful establishment of Pinus densata on the Tibetan Plateau is one of the few known examples of HHS. In this study, we carried out extensive field expeditions to obtain representative coverage of occurrence sites of P. densata and its two putative parents. We then applied a series of geographic information system-based analyses to define the patterns of environmental variation within and among the three pine species, to remove potentially confounding effects of spatial autocorrelation in the environmental data due to allopatric ranges, and to build species distribution models. All results consistently indicated that the ecological preferences of P. densata and its parental species have diverged, and they identified candidate ecological factors associated with habitat-specific adaptation. Projections from niche modeling indicated that P. densata could extend across a vast range along the parallel valley systems of the southeastern Tibetan Plateau. Our findings provide evidence of a distinct niche shift in P. densata and support the hypothesis that local adaptation and geographic isolation help maintain and reinforce between-species differences and reproductive isolation in the species complex.     

Genetic composition and diploid hybrid speciation of a high mountain pine, Pinus densata, native to the Tibetan plateau

Pinus densata has been suggested to have originated from hybridization events involving P. tabulaeformis and P. yunnanensis. In this study, allozyme differentiation at 12 loci was studied in 14 populations of P. tabulaeformis, P. densata, and P. yunnanensis from China. The observed genetic composition of P. densata supported the hybrid hypothesis and showed varying degrees of contribution from P. yunnanensis and P. tabulaeformis among its populations. These data, together with previous chloroplast DNA results, indicated different evolutionary histories among P. densata populations. To examine the possibility of ongoing hybridization among the three species, we analyzed patterns of linkage disequilibria between allozyme loci in ovule, pollen, and zygote pools. None of these tests suggested that there is significant ongoing gene exchange, implying that populations of P. densata have a stabilized hybrid nature. The normal fertility and high fecundity of P. densata indicate that this hybrid is maintained through sexual reproduction. P. densata represents an example of diploid hybrid speciation in an extreme ecological habitat that is both spatially and ecologically separated from that of its parents.     

Molecular genetic and quantitative trait divergence associated with recent homoploid hybrid speciation: a study of Senecio squalidus (Asteraceae)

Hybridization is increasingly seen as a trigger for rapid evolution and speciation. To quantify and qualify divergence associated with recent homoploid hybrid speciation, we compared quantitative trait (QT) and molecular genetic variation between the homoploid hybrid species Senecio squalidus and its parental species, S. aethnensis and S. chrysanthemifolius, and also their naturally occurring Sicilian hybrids. S. squalidus originated and became invasive in the United Kingdom following the introduction of hybrid plants from Mount Etna, Sicily, about 300 years ago. We recorded considerable molecular genetic differentiation between S. squalidus and its parents and their Sicilian hybrids in terms of both reduced genetic diversity and altered allele frequencies, potentially due to the genetic bottleneck associated with introduction to the United Kingdom. S. squalidus is also distinct from its parents and Sicilian hybrids for QTs, but less so than for molecular genetic markers. We suggest that this is due to resilience of polygenic QTs to changes in allele frequency or lack of selection for hybrid niche divergence in geographic isolation. While S. squalidus is intermediate or parental-like for most QTs, some trangressively distinct traits were observed, which might indicate emerging local adaptation in its invasive range. This study emphasizes the important contribution of founder events and geographic isolation to successful homoploid hybrid speciation.     

高山松及其亲本种群在油松生境下的苗期性状

高山松(Pinus densata)是油松(P. tabuliformis)和云南松(P. yunnanensis)的天然二倍体杂交种, 是为数不多的与亲本种没有严格生殖隔离的同倍体杂交种。为了检测3个种在油松生境下的苗期适应性和生长发育特点, 为高山松成种机制提供数据, 选取了代表高山松及其亲本种遗传多样性的25个种群, 在油松生境下对比分析了苗期11个适应性指标和3个生长发育指标。结果表明, 大部分适应性指标和生长发育指标在种间和种内群体间差异显著, 主要变异存在于种间及种内群体间; 出苗率、2011年11月和2012年10月的封顶率、二年生苗在2012年10月的保存率等指标的种间方差分量较大, 种内变异较小, 是体现种间苗期适应性和生长发育状况的重要指标。油松在大部分适应性指标和3个生长发育指标上均表现最好, 总体适合度高于云南松和高山松; 云南松在封顶率和保存率上都居于最低值, 大部分一年生苗木到11月底仍未出现封顶现象且黄苗比例最高, 二年生苗的存活率为0, 表明云南松在油松生境下适合度最低; 高山松除了在紫苗比例上表现出超亲优势外, 大部分性状居于亲本种之间。此外, 位于青藏高原东北部的高山松祖先种群在适应性上表现较好, 具有在油松生境下发展的潜力; 而位于青藏高原西部的高山松种群及东南部的康定种群对油松生境的适应性较差。研究揭示出生态选择在高山松的同倍性杂交物种形成中起到了关键作用。     

Colonization of the Tibetan Plateau by the homoploid hybrid pine Pinus densata

Pinus densata is an intriguingly successful homoploid hybrid species that occupies vast areas of the southeastern Tibetan Plateau in which neither of its parental species are present, but the colonization processes involved are poorly understood. To shed light on how this species colonized and became established on the plateau, we surveyed paternally inherited chloroplast (cp) and maternally inherited mitochondrial (mt) DNA variation within and among 54 populations of P. densata and its putative parental species throughout their respective ranges. Strong spatial genetic structure of both cp and mtDNA were detected in P. densata populations. Mitotypes specific to P. densata were likely generated by complex recombination events. A putative ancestral hybrid zone in the northeastern periphery of P. densata was identified, and we propose that the species then colonized the plateau by migrating westwards. Along the colonization route, consecutive bottlenecks and surfing of rare alleles caused a significant reduction in genetic diversity and strong population differentiation. The direction and intensity of introgression from parental species varied among geographic regions. In western parts of its range, the species seems to have been isolated from seed and pollen flow from its parent species for a long time. The observed spatial distribution of genetic diversity in P. densata also appears to reflect the persistence of this species on the plateau during the last glaciation. Our results indicate that both ancient and contemporary population dynamics have contributed to the spatial distribution of genetic diversity in P. densata, which accordingly reflects its evolutionary history.     

CASE STUDIES AND MATHEMATICAL MODELS OF ECOLOGICAL SPECIATION. 4. HYBRID SPECIATION IN BUTTERFLIES IN A JUNGLE

We build a spatial individual-based multilocus model of homoploid hybrid speciation tailored for a tentative case of hybrid origin of Heliconius heurippa from H. melpomene and H. cydno in South America. Our model attempts to account for empirical patterns and data on genetic incompatibility, mating preferences and selection by predation (both based on coloration patterns), habitat preference, and local adaptation for all three Heliconius species. Using this model, we study the likelihood of recombinational speciation and identify the effects of various ecological and genetic parameters on the dynamics, patterns, and consequences of hybrid ecological speciation. Overall, our model supports the possibility of hybrid origin of H. heurippa under certain conditions. The most plausible scenario would include hybridization between H. melpomene and H. cydno in an area geographically isolated from the rest of both parental species with subsequent long-lasting geographic isolation of the new hybrid species, followed by changes in the species ranges, the secondary contact, and disappearance of H. melpomene -type ecomorph in the hybrid species. However, much more work (both empirical and theoretical) is necessary to be able to make more definite conclusions on the importance of homoploid hybrid speciation in animals.     

Maternal lineages of Pinus densata,a diploid hybrid

Previous morphological, allozyme and chloroplast DNA data have suggested that Pinus densata originated through hybridization between P. tabuliformis and P. yunnanensis. In the present study, sequence and restriction site analyses of maternally inherited mitochondrial nad1 intron were used to detect variation patterns in 19 populations of P. tabuliformis, P. yunnanensis and P. densata. A total of three mitotypes (A, B, C) were detected. All but one of the populations of P. yunnanensis possessed mitotype B while all populations of P. tabuliformis had mitotype A. Pinus densata populations, on the other hand, harboured both mitotypes A and B, which are characteristic of P. tabuliformis and P. yunnanensis, respectively. This result gives strong additional evidence supporting the hybrid origin of this diploid pine. The distribution of mitotypes indicated very different mating compositions and evolutionary history among P. densata populations. It seems that local founder populations and backcrosses may have played important roles in the early establishment of P. densata populations. The uplift of the Tibetan Plateau had a significant impact on the distribution of maternal lineages of P. densata populations.     

Genetic divergence, range expansion and possible homoploid hybrid speciation among pine species in Northeast China

Although homoploid hybrid speciation in plants is probably more common than previously realized, there are few well-documented cases of homoploid hybrid origin in conifers. We examined genetic divergence between two currently widespread pines in Northeast China, Pinus sylvestris var. mongolica and Pinus densiflora, and also whether two narrowly distributed pines in the same region, Pinus funebris and Pinus takahasii, might have originated from the two widespread species by homoploid hybrid speciation. Our results, based on population genetic analysis of chloroplast (cp), mitochondrial (mt) DNA, and nuclear gene sequence variation, showed that the two widespread species were divergent for both cp- and mtDNA variation, and also for haplotype variation at two of eight nuclear gene loci surveyed. Our analysis further indicated that P. sylvestris var. mongolica and P. densiflora remained allopatric during the most severe Quaternary glacial period that occurred in Northeast China, but subsequently exhibited rapid range expansions. P. funebris and P. takahasii, were found to contain a mixture of chlorotypes and nuclear haplotypes that distinguish P. sylvestris var. mongolica and P. densiflora, in support of the hypothesis that they possibly originated via homoploid hybrid speciation following secondary contact and hybridization between P. sylvestris var. mongolica and P. densiflora.     

Hybrid speciation through sorting of parental incompatibilities in Italian sparrows

Speciation by hybridization is emerging as a significant contributor to biological diversification. Yet, little is known about the relative contributions of (i) evolutionary novelty and (ii) sorting of pre‐existing parental incompatibilities to the build‐up of reproductive isolation under this mode of speciation. Few studies have addressed empirically whether hybrid animal taxa are intrinsically isolated from their parents, and no study has so far investigated by which of the two aforementioned routes intrinsic barriers evolve. Here, we show that sorting of pre‐existing parental incompatibilities contributes to intrinsic isolation of a hybrid animal taxon. Using a genomic cline framework, we demonstrate that the sex‐linked and mitonuclear incompatibilities isolating the homoploid hybrid Italian sparrow at its two geographically separated hybrid–parent boundaries represent a subset of those contributing to reproductive isolation between its parent species, house and Spanish sparrows. Should such a sorting mechanism prove to be pervasive, the circumstances promoting homoploid hybrid speciation may be broader than currently thought, and indeed, there may be many cryptic hybrid taxa separated from their parent species by sorted, inherited incompatibilities.     

Demography and speciation history of the homoploid hybrid pine Pinus densata on the Tibetan Plateau

Pinus densata is an ecologically successful homoploid hybrid that inhabits vast areas of heterogeneous terrain on the south‐eastern Tibetan Plateau as a result of multiple waves of colonization. Its region of origin, route of colonization onto the plateau and the directions of introgression with its parental species have previously been defined, but little is known about the isolation and divergence history of its populations. In this study, we surveyed nucleotide polymorphism over eight nuclear loci in 19 representative populations of P. densata and its parental species. Using this information and coalescence simulations, we assessed the historical changes in its population size, gene flow and divergence in time and space. The results indicate a late Miocene origin for P. densata associated with the recent uplift of south‐eastern Tibet. The subsequent differentiation between geographical regions of this species began in the late Pliocene and was induced by regional topographical changes and Pleistocene glaciations. The ancestral P. densata population had a large effective population size but the central and western populations were established by limited founders, suggesting that there were severe bottlenecks during the westward migration out of the ancestral hybrid zone. After separating from their ancestral populations, population expansion occurred in all geographical regions especially in the western range. Gene flow in P. densata was restricted to geographically neighbouring populations, resulting in significant differentiation between regional groups. The new information on the divergence and demographic history of P. densata reported herein enhances our understanding of its speciation process on the Tibetan Plateau.     

Extensive chromosomal repatterning and the evolution of sterility barriers in hybrid sunflower species

New species may arise via hybridization and without a change in ploidy. This process, termed homoploid hybrid speciation, is theoretically difficult because it requires the development of reproductive barriers in sympatry or parapatry. Theory suggests that isolation may arise through rapid karyotypic evolution and/or ecological divergence of hybrid neospecies. Here, we investigate the role of karyotypic change in homoploid hybrid speciation by generating detailed genetic linkage maps for three hybrid sunflower species, Helianthus anomalus, H. deserticola, and H. paradoxus, and comparing these maps to those previously generated for the parental species, H. annuus and H. petiolaris. We also conduct a quantitative trait locus (QTL) analysis of pollen fertility in a BC2 population between the parental species and assess levels of pollen and seed fertility in all cross-combinations of the hybrid and parental species. The three hybrid species are massively divergent from their parental species in karyotype; gene order differences were observed for between 9 and 11 linkage groups (of 17 total), depending on the comparison. About one-third of the karyoypic differences arose through the sorting of chromosomal rearrangements that differentiate the parental species, but the remainder appear to have arisen de novo (six breakages/six fusions in H. anomalus, four breakages/three fusions in H. deserticola, and five breakages/five fusions in H. paradoxus). QTL analyses indicate that the karyotypic differences contribute to reproductive isolation. Nine of 11 pollen viability QTL occur on rearranged chromosomes and all but one map close to a rearrangement breakpoint. Finally, pollen and seed fertility estimates for F1's between the hybrid and parental species fall below 11%, which is sufficient for evolutionary independence of the hybrid neospecies.     

同倍杂交种高山松与亲本种云南松的地理隔离研究

为了探讨横断山区同倍杂交种高山松与其亲本种云南松的地理隔离机制,采用实际的路线踏查方法,调查了高山松和云南松群体的地理分布情况和开花物候特征,并基于7个气象因子进行了聚类分析。调查研究表明高山松和云南松群体分布在不同的地貌类型中,以云南中甸为临界区,沿着金沙江河谷向北分布高山松,向南分布云南松。云南松的开花物候日期明显早于高山松,聚类分析表明两个物种分别聚为一支。在地质历史过程中,高大山系的形成以及海拔的升高将高山松和云南松从地理上隔离开来,两物种的适生区的气象因子的差异造成花期不遇,从而阻碍了两个物种问的基因交流,进而隔离开来。     

Impacts of climate warming on hybrid zone movement: Geographically diffuse and biologically porous “species borders”

Abstract The ecology and evolutionary biology of insect–plant associations has realized extensive attention, especially during the past 60 years. The classifications (categorical designations) of continuous variation in biodiversity, ranging from global patterns (e.g., latitudinal gradients in species richness/diversity and degree of herbivore feeding specialization) to localized insect–plant associations that span the biospectrum from polyphenisms, polymorphisms, biotypes, demes, host races, to cryptic species, remain academically contentious. Semantic and biosystematic (taxonomical) disagreements sometimes detract from more important ecological and evolutionary processes that drive diversification, the dynamics of gene flow and local extinctions. This review addresses several aspects of insect specialization, host‐associated divergence and ecological (including “hybrid”) speciation, with special reference to the climate warming impacts on species borders of hybridizing swallowtail butterflies (Papilionidae). Interspecific hybrid introgression may result in collapse of multi‐species communities or increase species numbers via homoploid hybrid speciation. We may see diverging, merging, or emerging genotypes across hybrid zones, all part of the ongoing processes of evolution. Molecular analyses of genetic mosaics and genomic dynamics with “divergence hitchhiking”, combined with ecological, ethological and physiological studies of “species porosity”, have already begun to unveil some answers for some important ecological/evolutionary questions. (i) How rapidly can host‐associated divergence lead to new species (and why doesn't it always do so, e.g., resulting in “incomplete” speciation)? (ii) How might “speciation genes” function, and how/where would we find them? (iii) Can oscillations from specialists to generalists and back to specialists help explain global diversity in herbivorous insects? (iv) How could recombinant interspecific hybridization lead to divergence and speciation? From ancient phytochemically defined angiosperm affiliations to recent and very local geographical mosaics, the Papilionidae (swallowtail butterflies) have provided a model for enhanced understanding of ecological patterns and evolutionary processes, including host‐associated genetic divergence, genomic mosaics, genetic hitchhiking and sex‐linked speciation genes. Apparent homoploid hybrid speciation in Papilio appears to have been catalyzed by climate warming‐induced interspecific introgression of some, but not all, species diagnostic traits, reflecting strong divergent selection (discordant), especially on the Z (= X) chromosome. Reproductive isolation of these novel recombinant hybrid genotypes appears to be accomplished via a delayed post‐diapause emergence or temporal isolation, and is perhaps aided by the thermal landscape. Changing thermal landscapes appear to have created (and may destroy) novel recombinant hybrid genotypes and hybrid species.     

The rate of genome stabilization in homoploid hybrid species

Homoploid hybrid speciation has been recognized for its potential rapid completion, an idea that has received support from experimental and modeling studies. Following initial hybridization, the genomes of parental species recombine and junctions between chromosomal blocks of different parental origin leave a record of recombination and the time period before homogenization of the derived genome. We use detailed genetic maps of three hybrid species of sunflowers and models to estimate the time required for the stabilization of the new hybrid genome. In contrast to previous estimates of 60 or fewer generations, we find that the genomes of three hybrid sunflower species were not stabilized for hundreds of generations. These results are reconciled with previous research by recognizing that the stabilization of a hybrid species' genome is not synonymous with hybrid speciation. Segregating factors that contribute to initial ecological or intrinsic genetic isolation may become stabilized quickly. The remainder of the genome likely becomes stabilized over a longer time interval, with recombination and drift dictating the contributions of the parental genomes. Our modeling of genome stabilization provides an upper bound for the time interval for reproductive isolation to be established and confirms the rapid nature of homoploid hybrid speciation.     

Genetic divergence and hybrid speciation

Although the evolutionary importance of natural hybridization has been debated for decades, it has become increasingly clear that hybridization plays a fundamental role in the evolution of many plant and animal taxa, sometimes resulting in the formation of entirely new species. Although some hybrid species retain the base chromosome number of their parents, others combine the full chromosomal complements of their progenitors. Hybrid speciation can thus produce two fundamentally different types of evolutionary lineages, yet relatively little is known about the factors influencing ploidy level in hybrid neospecies. We estimated genetic divergence between species pairs that have given rise to homoploid and polyploid hybrid species and found that divergence is significantly greater for the parents of polyploids, even after controlling for potentially confounding factors. Our data thus provide the first direct evidence in support of the notion that the extent of genomic divergence between hybridizing species influences the likelihood of diploid versus polyploid hybrid speciation.     

Homoploid hybrid speciation in a rare endemic Castilleja from Idaho (Castilleja christii,Orobanchaceae)

• Premise of the study: Hybridization is an important evolutionary force in the history of angiosperms; however, there are few examples of stabilized species derived through homoploid hybrid speciation. Homoploid hybrid species are generally detected via the presence of genetic additivity of parental markers, novel ecological and spatial distinctions, and novel morphological traits, all of which may aid in the successful establishment of hybrid species from parental types. Speciation and diversification within the genus Castilleja (Orobanchaceae) has been attributed to high levels of hybridization and polyploidy, though currently there are no examples of homoploid hybrid speciation within the genus. We employed multiple lines of evidence to examine a putative hybrid origin in C. christii, a rare endemic, known only from 80 hectares at the summit of Mt. Harrison (Cassia Co., Idaho). • Methods: We used granule-bound starch synthase II (waxy) sequences and 26 morphological characters to address hybridization between C. christii and widespread congeners C. miniata and/or C. linariifolia in an area of sympatry. Chromosomes of C. christii were also counted for the first time. • Key results: All 230 direct-sequenced C. christii individuals had the additive genomes of both C. miniata and C. linariifolia. Castilleja christii shares traits with both parents but also has floral characters that are unique and transgressive. Cytological counts indicated that all three taxa are diploid. • Conclusions: We conclude that C. christii is a stabilized homoploid hybrid derivative of C. linariifolia and C. miniata and is likely following an independent evolutionary trajectory from its progenitors.     

种间杂种染色体配对所揭示的披碱草属植物StY基因组分化及其进化意义

在同倍体(homoploid)植物杂交-分化的物种形成(speciation)过程中,杂交后代与亲本之间的有效生殖隔离是新物种形成的关键。杂种与亲本在时间、空间、生态环境和基因水平上的隔离,保证了杂种后代的分化和稳定,并逐渐形成新物种。为了研究披碱草属(Elymus)含StY基因组四倍体物种的系统演化关系,本文对来自亚洲不同地理分布区的26种披碱草属植物进行了大规模的种间杂交和杂种F1减数分裂染色体配对行为的分析。结果表明各物种之间有不同程度的杂交亲合力,杂交结实率在各杂交组合之间有较大的变异(在4．8％-100％之间);但各物种之间的杂种F1完全不育。证明各物种之间形成了明显的生殖隔离。种间杂种F1减数分裂中期-I染色体配对分析的结果进一步表明,StY基因组随各披碱草属物种地理分布的不同而有不同程度的分化。来自同一分布区(如东亚或西亚分布区之内)物种的StY基因组分化程度较低,但来自不同分布区(如东亚和西亚)物种之间相同的StY基因组具有显著的分化。表明地理隔离对含StY基因组物种的分化起到了十分重要的作用。通过对种间和种内杂种F1的减数分裂异常现象和染色体配对频率变化规律的分析,作者认为细胞学水平的变化,如基因组同源性的分化和染色体结构的变异等都在杂种后代与亲本之间产生生殖隔离并逐渐形成新物种的进化过程中起到了积极的作用。     

Extreme changes to gene expression associated with homoploid hybrid speciation

Hybridization is an important cause of abrupt speciation. Hybrid speciation without a change in ploidy (homoploid hybrid speciation) is well-established in plants but has also been reported in animals and fungi. A notable example of recent homoploid hybrid speciation is Senecio squalidus (Oxford ragwort), which originated in the UK in the 18th Century following introduction of hybrid material from a hybrid zone between S. chrysanthemifolius and S. aethnensis on Mount Etna, Sicily. To investigate genetic divergence between these taxa, we used complementary DNA microarrays to compare patterns of floral gene expression. These analyses revealed major differences in gene expression between the parent species and wild and resynthesized S. squalidus . Comparisons of gene expression between S. aethnensis , S. chrysanthemifolius and natural S. squalidus identified genes potentially involved in local environmental adaptation. The analysis also revealed non-additive patterns of gene expression in the hybrid relative to its progenitors. These expression changes were more dramatic and widespread in resynthesized hybrids than in natural S. squalidus , suggesting that a unique expression pattern may have been fixed during the allopatric divergence of British S. squalidus . We speculate that hybridization-induced gene-expression change may provide an immediate source of novel phenotypic variation upon which selection can act to facilitate homoploid hybrid speciation in plants.     

The role of parental and hybrid species in multiple introgression events: evidence of homoploid hybrid speciation in Centaurea (Cardueae,Asteraceae)

Molecular approaches have greatly increased the number of confirmed homoploid hybrids, which suggests that the frequency of this phenomenon was underestimated in the past because it was much more difficult to detect than allopolyploidy. Centaurea is a suitable model group for studying homoploid speciation, as hybridization events have been commonly reported for this genus. Based on this, here we study Centaurea × forsythiana, a naturally occurring homoploid hybrid between two Sardinian endemics, C. horrida and C. filiformis, using a molecular approach involving nuclear and plastid markers, to understand the underlying population dynamics between homoploid hybrids and their parents. Our results confirm that C. × forsythiana is a hybrid between the above‐mentioned species and define the roles of the parents. Plastid markers point towards C. horrida as the maternal progenitor, and nuclear markers reveal that the other parental species, C. filiformis, is itself an old, stabilized homoploid hybrid related to the C. paniculata complex from the Italian mainland. Homoploid hybrid speciation is discussed and C. × forsythiana and C. filiformis are compared with other similar examples. The study confirms the importance of introgression between parental species mediated by hybrids and its potential implications in conservation. Furthermore, it shows how hybridization studies become even more complex when the parents are themselves of probable hybrid origin. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 175 , 453–467.     

Intragenomic and interspecific 5S rDNA sequence variation in five Asian pines

Patterns of intragenomic and interspecific variation of 5S rDNA in Pinus (Pinaceae) were studied by cloning and sequencing multiple 5S rDNA repeats from individual trees. Five pines, from both subgenera, Pinus and Strobus, were selected. The 5S rDNA repeat in pines has a conserved 120-base pair (bp) transcribed region and an intergenic spacer region of variable length (382-608 bp). The evolutionary rate in the spacer region is three- to sevenfold higher than in the genic region. We found substantial sequence divergence between the two subgenera. Intragenomic sequence heterogeneity was high for all species, and more than 86% of the clones within each individual were unique. The 5S gene tree revealed that different 5S repeats within individuals are polyphyletic, indicating that their ancestral divergence preceded the speciation events. The degrees of interspecific and intragenomic divergence among diploxylon pines are similar. The observed sequence patterns suggest that concerted evolution has been acting after the diversification of the two subgenera but very weak after the speciation of the four diploxylon pines. Sequence patterns in P. densata are consistent with hybrid origin. It had higher intragenomic diversity and maintained polymorphic copies of the parental types in addition to new and recombinant types unique to the hybrid.