Empirical assessment of the reproductive fitness components of the hybrid pine Pinus densata on the Tibetan Plateau

Pinus densata is distributed on the Tibetan Plateau, where it forms extensive forests at high elevations. Genetic studies have provided evidence that P. densata originated through hybridization between P. yunnanensis and P. tabuliformis. To clarify the relationships among these pines, and assess their reproductive fitness in their respective habitats, we conducted a comparative analysis of eight cone and seed morphometric traits and six reproductive traits in them. Among the eight morphometric traits examined, six appeared to be intermediate in P. densata between those of P. yunnanensis and P. tabuliformis. There were significant differences among the three pines in all of the morphometric traits, and P. densata showed greater variability in these traits than the other two pines. In contrast to the morphometric traits, the reproductive traits (including the proportions of filled and empty seeds, ovule abortion rate, seed efficiency, meiotic abnormalities during microsporogenesis and pollen viability) differed little among the three pines, indicating that they have similar overall rates of effective pollination and fertilization in their respective natural environments. Despite their location on the high plateau, natural populations of P. densata appeared to have normal levels of reproductive success, comparable to those of the two parental species in their natural habitats. This study provides empirical data characterizing the reproductive success and adaptation of a stabilized homoploid hybrid in a novel habitat that is ecologically and spatially inaccessible to its parental species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Physiological responses to gradual drought stress in the diploid hybrid Pinus densata and its two parental species

Pinus densata is a homoploid hybrid species, originating from P. tabuliformis × P. yunnanensis. The physiological fitness of this natural hybrid compared to its two parental species remains unknown. In this study, we investigated physiological responses of the three species by exposing artificially breed seedlings of each to drought stress lasting 28 days. Our results suggest that, in all three species, drought affected the contents of the plants’ chlorophyll, stomatal conductance, TBARS, hydrogen peroxide, and free proline and increased the activities of antioxidant enzymes, including superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), and peroxidase (POD; EC 1.11.1.7). The drought stress also induced significant changes in the activity of ascorbate peroxidase (APX; EC 1.11.1.11), monodehydroascorbate reductase (MDHAR; EC 1.6.5.4), dehydroascorbate reductase (DHAR; EC 1.8.5.1), glutathione reductase (GR; EC 1.6.4.2), and levels of ascorbate and glutathione in the ascorbate–glutathione cycle. The hybrid species P. densata appeared to achieve greater drought tolerance and exhibit hybrid superiority in antioxidant processes and other related physiological traits compared to the two parental species, although a few of the hybrid’s measured variables were similar to those of P. tabuliformis. However, P. yunnanensis was more sensitive to drought and appeared to have the lowest resistance to such stress. These physiological differences are largely consistent with the species’ habitat preferences, which may reflect their early genetic divergences and niche differentiation. These findings provide important information for management and forest restoration efforts of these species in the future.     

Adaptive Differentiation in Seedling Traits in a Hybrid Pine Species Complex,Pinus densata and Its Parental Species,on the Tibetan Plateau

Evidence from molecular genetics demonstrates that Pinus densata is a natural homoploid hybrid originating from the parent species Pinus tabuliformis and Pinus yunnanensis, and ecological selection may have played a role in the speciation of P. densata. However, data on differentiation in adaptive traits in the species complex are scarce. In this study, we performed a common garden test on 16 seedling traits to examine the differences between P. densata and its parental species in a high altitude environment. We found that among the 16 analyzed traits, 15 were significantly different among the species. Pinus tabuliformis had much earlier bud set and a relatively higher bud set ratio but poorer seedling growth, and P. yunnanensis had opposite responses for the same traits. P. densata had the greatest fitness with higher viability and growth rates than the parents. The relatively high genetic contribution of seedling traits among populations suggested that within each species the evolutionary background is complex. The correlations between the seedling traits of a population within a species and the environmental factors indicated different impacts of the environment on species evolution. The winter temperature is among the most important climate factors that affected the fitness of the three pine species. Our investigation provides empirical evidence on adaptive differentiation among this pine species complex at seedling stages.     

WEAK CROSSABILITY BARRIER BUT STRONG JUVENILE SELECTION SUPPORTS ECOLOGICAL SPECIATION OF THE HYBRID PINE PINUS DENSATA ON THE TIBETAN PLATEAU

Determining how a new hybrid lineage can achieve reproductive isolation is a key to understanding the process and mechanisms of homoploid hybrid speciation. Here, we evaluated the degree and nature of reproductive isolation between the ecologically successful hybrid species Pinus densata and its parental species P. tabuliformis and P. yunnanensis. We performed interspecific crosses among the three species to assess their crossability. We then conducted reciprocal transplantation experiments to evaluate their fitness differentiation, and to examine how natural populations representing different directions of introgression differ in adaptation. The crossing experiments revealed weak genetic barriers among the species. The transplantation trials showed manifest evidence of local adaptation as the three species all performed best in their native habitats. Pinus densata populations from the western edge of its distribution have evolved a strong local adaptation to the specific habitat in that range; populations representing different directions of introgressants with the two parental species all showed fitness disadvantages in this P. densata habitat. These observations illustrate that premating isolation through selection against immigrants from other habitat types or postzygotic isolation through selection against backcrosses between the three species is strong. Thus, ecological selection in combination with endogenous components and geographic isolation has likely played a significant role in the speciation of P. densata.     

Morphological evaluation of the Pinus kesiya complex (Pinaceae)

The main aim of this study is to compare all taxa from the Pinus kesiya complex and related P. tabuliformis using statistical, ordination and discrimination techniques focusing on ten most discriminating morphological traits. Special emphasis was placed on the recently described taxa Pinus densata subsp. tibetica and P. × naxiorum (=P. yunnanensis × P. densata). Population comparisons of the particular main geographic distribution areas of Pinus kesiya, P. densata subsp. tibetica and P. yunnanensis were also conducted separately within each mentioned taxon using the same data. Pinus densata subsp. tibetica proved to be sufficiently morphologically differentiated from subsp. densata as well as from other species of the P. kesiya complex and P. tabuliformis. The recently originating hybrid P. × naxiorum appears to be intermediate between its parents. The unique shrubby taxon P. densata var. pygmaea is clearly closer to P. densata than to P. yunnanensis, to which it has been classified in Chinese floras. Populations of four distant Pinus kesiya geographic distribution areas lack substantial differences that would support the recognition of infraspecific taxa such as subsp. insularis or var. langbianensis. Pinus yunnanensis is very similar to P. kesiya, with only one trait, leaf length, being significantly different.     

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.     

HYBRIDIZATION AND CHLOROPLAST DNA VARIATION IN A PINUS SPECIES COMPLEX FROM ASIA

Heterologous hybridization of chloroplast DNA (cpDNA) involving 30 endonucleaseprobe combinations was used to analyze cpDNA variation in multiple individuals and populations of Pinus tabulaeformis (Carr.), Pinus yunnanensis (Franchèt) and Pinus massoniana (Lamb.). Restriction fragment patterns detected by several combinations distinguished among the three species. The obtained cpDNA markers were subsequently used to examine cpDNA variation of Pinus densata (Masters), a putative tertiary hybrid between P. tabulaeformis and P. yunnanensis. The analysis demonstrated that P. densata populations harbor three different haplotypes. Two of these haplotypes are characteristic of P. tabulaeformis and P. yunnanensis. However, the third haplotype found in P. densata appears to be absent in other extant Asian Pinus species. It is suggested that the observed cpDNA composition of P. densata populations is a result of past hybridization involving P. tabulaeformis, P. yunnanensis, and a third unknown or extinct taxon. Chloroplast DNA polymorphism in P. densata was much greater than that for nuclear allozyme markers in this and the other Pinus species. Population differentiation was also substantial in P. densata and exceeded that for allozyme markers. In contrast, no cpDNA polymorphism was detected in populations of P. tabulaeformis, P. yunnanensis, and P. massoniana. The study suggests that interspecific gene exchange may lead to the creation of stable cpDNA polymorphism in conifer hybrids.     

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.     

Allozyme Diversity and Population Genetic Structure of Pinus densata Master in Northwestern Yunnan, China

We investigated the levels and patterns of genetic diversity of Pinus densata Master in Yunnan. Horizontal starch-gel electrophoresis was performed on macrogametophytes collected from nine populations in northwestern Yunnan, China. Compared with other gymnosperm species, P. densata has higher mean values for all measures of genetic diversity. Allozyme polymorphism (0.99 criterion) was 97.0% and 71.4% at the species and population levels, respectively. The average number of alleles per locus was 3.1 and 2.0 at the species and population levels. Mean expected heterozygosity was substantially higher in P. densata than average values investigated for other gymnosperms both at the population (H _ep = 0.174±0.031) and at the species (H _es = 0.190) levels. Of the total genetic variation, less than 12% was partitioned among populations (G _ST = 0.112). Our allozyme survey supports the suggestion that the observed higher diversity in P. densata may be attributed partly to its hybrid origin between two genetically distinct species, P. yunnanensis and P. tabulaeformis. In addition, we suggest that introgression would give rise to the increase in genetic diversity occurring in P. densata.     

Evolutionary analysis of Pinus densata (Masters), a putative Tertiary hybrid.

Summary Restriction fragment analysis and heterologous hybridization of chloroplast (cp) DNA was used to develop species-specific markers for P. tabulaeformis, P. yunnanensis and P. massoniana. Fragment patterns created by the BclI and DraI restriction enzymes and hybridization patterns to the psbC and psbD probes were distinctive among the three species. No intraspecific variation was detected with respect to any of the cpDNA markers developed in this study. The cpDNA markers obtained were subsequently used to examine the parentage of P. densata, a putative Tertiary hybrid between P. tabulaeformis and P. yunnanensis. The analysis demonstrated for the first time that P. densata populations accommodate chloroplast genomes of P. tabulaeformis and P. yunnanensis, which strongly supports earlier suggestions of the hybrid origin of this species. It appears that P. densata represents a stabilized natural hybrid that has become adapted to high mountain environments where neither of the parental species can normally grow.     

The alpine homoploid hybrid Pinus densata has greater cold photosynthesis tolerance than its progenitors

The diploid hybrid tree species Pinus densata inhabits high-altitude regions, usually at higher altitudes than its parental species, P. tabulaeformis and P. yunnanensis. Its success in alpine habitat has been hypothetically attributed to enhanced cold tolerance. We tested this hypothesis by subjecting all three species to cold stress for 33 days (12/5 °C, day/night) and measuring changes in their photosynthetic function. Significant decreases in net photosynthetic rate (P_n), stomatal conductance (g_s) and transpiration rate (E) were observed in all three species in response to cold stress. The depression of P_n was primarily attributable to stomatal closure in P. densata, but to both stomatal and non-stomatal limitations in its progenitors, as the parental species had increased intercellular CO₂ concentrations (C_i) under the stress. Significant decreases in chlorophyll fluorescence parameters were also observed in both parental species, but they varied little throughout the experiment in P. densata. Furthermore, the chlorophyll and carotenoid concentrations decreased significantly under the cold stress, while anthocyanin concentrations increased, and the anthocyanin concentration in P. densata was significantly higher than in either parental species at the end of the exposure. Finally, P. densata recovered more quickly than its parental species when the cold treatment ended. These results show that P. densata maintains a high photosynthetic rate and exhibits elevated PSII activity and anthocyanin levels under cold stress. These adaptations suggest that P. densata is more cold-tolerant than its parental species and may have been important for its colonization of high alpine habitats.     

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。     

Systematic position of Pinus henryi (Pinaceae) as revealed by multiple evidence

The systematic position of Henry's pine (Pinus henryi Mast.), a rare species endemic to China, is still ambiguous. In this study, the phylogenetic relationships of P. henryi and its allies (P. hwangshanensis, P. tabuliformis, P. yunnanensis and P. massoniana) were revealed by multiple markers, including chemical components, DNA sequences, chloroplast microsatellites and amplified fragment length polymorphisms. The results refuted the hypothesis that P. henryi is a subspecies of P. tabuliformis or P. massoniana and supported that it might be a distinct species close to P. hwangshanensis. Pinus tabuliformis and P. yunnanensis are sister taxa. Incomplete genomic sorting, hybridization and recent divergence may explain the controversial relationships among them. Their diversification may be ascribed to habitat adaptation followed by geographic and climate changes during the Pliocene period.     

Anatomical changes with needle length are correlated with leaf structural and physiological traits across five Pinus species

The genus Pinus has wide geographical range and includes species that are the most economically valued among forest trees worldwide. Pine needle length varies greatly among species, but the effects of needle length on anatomy, function, and coordination and trade‐offs among traits are poorly understood. We examined variation in leaf morphological, anatomical, mechanical, chemical, and physiological characteristics among five southern pine species: Pinus echinata, Pinus elliottii, Pinus palustris, Pinus taeda, and Pinus virginiana. We found that increasing needle length contributed to a trade‐off between the relative fractions of support versus photosynthetic tissue (mesophyll) across species. From the shortest (7 cm) to the longest (36 cm) needles, mechanical tissue fraction increased by 50%, whereas needle dry density decreased by 21%, revealing multiple adjustments to a greater need for mechanical support in longer needles. We also found a fourfold increase in leaf hydraulic conductance over the range of needle length across species, associated with weaker upward trends in stomatal conductance and photosynthetic capacity. Our results suggest that the leaf size strongly influences their anatomical traits, which, in turn, are reflected in leaf mechanical support and physiological capacity.     

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。     

Evolution of a hybrid zone of two willow species (Salix L.) in the European Alps analyzed by RAD-seq and morphometrics

Natural hybridization of plants can result in many outcomes with several evolutionary consequences, such as hybrid speciation and introgression. Natural hybrid zones can arise in mountain systems as a result of fluctuating climate during the exchange of glacial and interglacial periods, where species retract and expand their territories, resulting in secondary contacts. Willows are a large genus of woody plants with an immense capability of interspecific crossing. In this study, the sympatric area of two diploid sister species, S. foetida and S. waldsteiniana in the eastern European Alps, was investigated to study the genomic structure of populations within and outside their contact zone and to analyze congruence of morphological phenotypes with genetic data. Eleven populations of the two species were sampled across the Alps and examined using phylogenetic network and population genetic structure analyses of RAD Seq data and morphometric analyses of leaves. The results showed that a homoploid hybrid zone between the two species was established within their sympatric area. Patterns of genetic admixture in homoploid hybrids indicated introgression with asymmetric backcrossing to not only one of the parental species but also one hybrid population forming a separate lineage. The lack of F1 hybrids indicated a long-term persistence of the hybrid populations. Insignificant isolation by distance suggests that gene flow can act over large geographical scales. Morphometric characteristics of hybrids supported the molecular data and clearly separated populations of the parental species, but showed intermediacy in the hybrid zone populations with a bias toward S. waldsteiniana. The homoploid hybrid zone might have been established via secondary contact hybridization, and its establishment was fostered by the low genetic divergence of parental species and a lack of strong intrinsic crossing barriers. Incomplete ecological separation and the ability of long-distance dispersal of willows could have contributed to the spatial expansion of the hybrid zone.     

Genetic structure and evolutionary history of a diploid hybrid pine Pinus densata inferred from the nucleotide variation at seven gene loci

Although homoploid hybridization is increasingly recognized as an important phenomenon in plant evolution, its evolutionary genetic mechanisms are poorly documented and understood. Pinus densata, a pine native to the Tibetan Plateau, represents a good example of a homoploid hybrid speciation facilitated by adaptation to extreme environment and ecological isolation from the parents. Its ecologically and reproductively stabilized nature offers excellent opportunity for studying genetic processes associated with hybrid speciation. In this study, we investigated the levels and patterns of nucleotide variation in P. densata and its putative parents. Haplotype composition, gene genealogies, and the levels and patterns of nucleotide variation gave further support to the hybrid nature of P. densata. Allelic history, as revealed by our data, suggests the ancient nature of the hybrid preceding elevation of the Tibetan Plateau. We detected more deviations from neutrality in P. densata than in the parental species. Thus, at least some of the evolutionary forces that have shaped the genetic variation in P. densata are likely to be different from those acting upon parental species. We speculate that when populations of P. densata invaded new territories, they had elevated rates of response to selection in order to develop traits that help them to survive and adapt in the new environments.     

中国西南四种常见松树火适应性状比较

野火对松属植物的进化和分布有重要的影响。在与野火长期抗争中,松属植物形成了一系列性状提高在易火生境中的适合度,维持种群生存与繁衍。西南地区是中国野火高发区,作为这一地区特有松树,云南松、思茅松和高山松具有一些典型的火适应性状,如厚树皮。以这3种松树和东部常见的马尾松为研究对象,比较4种松树的火适应对策。结果表明：4种松树的火适应性状存在一定的差异。与火适应相关的13项性状主成分分析显示,4个种在空间上总体是分离的,也表现出一些重叠。其中云南松火适应生活史对策是火耐受型和火依赖型的中间类型,适应会发生林冠火的生境。高山松、思茅松和马尾松都是火耐受型,通过快速高生长、厚树皮等性状适应生境不同频度的地表火。四种松树火适应对策与分布区火险基本相符。表明野火在这一区域广泛存在,并对植物进化和森林格局有重要影响。野火在西南地区松属分布和种群维持中的生态作用应被给予足够重视。     

Are hybrid species more fit than ancestral parent species in the current hybrid species habitats?

Hybrid speciation is thought to be facilitated by escape of early generation hybrids into new habitats, subsequent environmental selection and adaptation. Here, we ask whether two homoploid hybrid plant species (Helianthus anomalus, H. deserticola) diverged sufficiently from their ancestral parent species (H. annuus, H. petiolaris) during hybrid speciation so that they are more fit than the parent species in hybrid species habitats. Hybrid and parental species were reciprocally transplanted into hybrid and parental habitats. Helianthus anomalus was more fit than parental species in the H. anomalus actively moving desert dune habitat. The abilities to tolerate burial and excavation and to obtain nutrients appear to be important for success in the H. anomalus habitat. In contrast, H. deserticola failed to outperform the parental species in the H. deserticola stabilized desert dune habitat, and several possible explanations are discussed. The home site advantage of H. anomalus is consistent with environmental selection having been a mechanism for adaptive divergence and hybrid speciation and supports the use of H. anomalus as a valuable system for further assessment of environmental selection and adaptive traits.     

Differentiation of F1 hybrids P. nigra J. F. Arnold × P. sylvestris L., P. nigra J. F. Arnold × P. densiflora Siebold et Zucc., P. nigra J. F. Arnold × P. thunbergiana Franco and their parental species by needle volatile composition

The volatile composition of needles from three F₁ hard pine hybrids produced by the controlled hybridization and their parental species were researched with gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) in order to explore the utility of terpenes in hybrid identification (their differentiation from the parental species) as well as confirmation of hybridity. The analysed hybrids were: 1. Pinus nigra J. F. Arnold × Pinus sylvestris L. (= nisy), 2. P. nigra × Pinus densiflora Siebold et Zucc. (= nide) and 3. P. nigra × Pinus thunbergiana Franco (= nith). A total of 55 compounds were identified. All identified compounds were terpenes, except trans-2-hexenal.Three analysed F₁ hybrids showed the same qualitative pattern of the needle volatile composition as their parental species. However, there were quantitative differences in several major terpenes. The volatile composition of the needles from the hybrids nisy were equally similar to both parents, the hybrids nide were more similar to the female parent (P. nigra), whereas the hybrids nith were more similar to the male parent (P. thunbergiana). According to the content of germacrene D, as the specific component of P. nigra (female parent of the three analysed F₁ hybrids), all hybrids were intermediary in relation to the parental species. The content of Δ-3-carene (the specific component of P. sylvestris) in the hybrids nisy was also intermediary. The hybrids nide had a higher content of thunbergol (specific component of P. densiflora) than the other analysed hybrids. In view of the content of β-pinene, the specific component of P. thunbergiana, the hybrids nith were intermediary to the parental species and that content was considerably higher than in the other analysed hybrids. The intermediary quality of F₁ hybrids for these specific components in relation to the parental species confirms their hybrid character.The needle volatile composition analysis as well as the previous morphometric analysis confirm the hybrid character of three F₁ hybrids, whose female parent is P. nigra, and male parents are P. sylvestris, P. densiflora, i.e. P. thunbergiana.