Needle morphological evidence of the homoploid hybrid origin of Pinus densata based on analysis of artificial hybrids and the putative parents,Pinus tabuliformis and Pinus yunnanensis

Genetic analyses indicate that Pinus densata is a natural homoploid hybrid originating from Pinus tabuliformis and Pinus yunnanensis. Needle morphological and anatomical features show relative species stability and can be used to identify coniferous species. Comparative analyses of these needle characteristics and phenotypic differences between the artificial hybrids, P. densata, and parental species can be used to determine the genetic and phenotypic evolutionary consequences of natural hybridization. Twelve artificial hybrid families, the two parental species, and P. densata were seeded in a high‐altitude habitat in Linzhi, Tibet. The needles of artificial hybrids and the three pine species were collected, and 24 needle morphological and anatomical traits were analyzed. Based on these results, variations in 10 needle traits among artificial hybrid families and 22 traits among species and artificial hybrids were predicted and found to be under moderate genetic control. Nineteen needle traits in artificial hybrids were similar to those in P. densata and between the two parental species, P. tabuliformis and P. yunnanensis. The ratio of plants with three needle clusters in artificial hybrids was 22.92%, which was very similar to P. densata. The eight needle traits (needle length, the mean number of stomata in sections 2 mm in length of the convex and flat sides of the needle, mean stomatal density, mesophyll/vascular bundle area ratio, mesophyll/resin canal area ratio, mesophyll/(resin canals and vascular bundles) area ratio, vascular bundle/resin canal area ratio) relative to physiological adaptability were similar to the artificial hybrids and P. densata. The similar needle features between the artificial hybrids and P. densata could be used to verify the homoploid hybrid origin of P. densata and helps to better understand of the hybridization roles in adaptation and speciation in plants.     

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.     

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.     

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.     

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。     

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.     

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.     

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.     

Directional mitochondrial introgression and character displacement due to reproductive interference in two closely related Pterostichus ground beetle species

Reproductive interference due to interspecific hybridization can lead to character displacement among related species with overlapping ranges. However, no studies have examined which reproductive traits are most important in reducing reproductive interference. We conducted molecular analyses of two nuclear genes (28S and Wingless) and a mitochondrial gene (COI) from two closely related ground beetle species, Pterostichus thunbergi and Pterostichus habui (Coleoptera: Carabidae), with overlapping distributions. In addition, we examined four reproductive traits (body size, organ morphologies of intromittent and non‐intromittent male genital organs, and female reproductive period) in sympatric and allopatric habitats. We compared male genital morphology using geometric morphometric analysis. The species determined by morphology were classified into separate groups based on the phylogenetic tree constructed by the nuclear gene (Wingless). However, according to the mitochondrial genes examined, P. thunbergi was not monophyletic, whereas at the sympatric sites, these species formed a monophyletic clade. This incongruence suggests that interspecific hybridization and subsequent mitochondrial introgression from P. habui to P. thunbergi have occurred. Concerning genital morphology, both of the intromittent and nonintromittent organs of P. thunbergi differed more from P. habui at the sympatric sites than between allopatric sites, suggesting reproductive character displacement. Pterostichus thunbergi, which likely arrived in P. habui habitat in small numbers, would have experienced stronger selection pressures than P. habui.     

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.     

Trait-dependent resemblance of the flowering phenology and floral morphology of the allopolyploid Cardamine flexuosa to those of the parental diploids in natural habitats

Allopolyploids possess complete sets of genomes derived from different parental species and exhibit a range of variation in various traits. Reproductive traits may play a key role in the reproductive isolation between allopolyploids and their parental species, thus affecting the thriving of allopolyploids. However, empirical data, especially in natural habitats, comparing reproductive trait variation between allopolyploids and their parental species remain rare. Here, we documented the flowering phenology and floral morphology of the allopolyploid wild plant Cardamine flexuosa and its diploid parents C. amara and C. hirsuta in their native range in Switzerland. The flowering of C. flexuosa started at an intermediate time compared with those of the parents and the flowering period of C. flexuosa overlapped with those of the parents. Cardamine flexuosa resembled C. hirsuta in the size of flowers and petals and the length/width ratio of petals, while it resembled C. amara in the length/width ratio of flowers. These results provide empirical evidence of the trait-dependent variation of allopolyploid phenotypes in natural habitats at the local scale. They also suggest that the variation in some reproductive traits in C. flexuosa is associated with self-fertilization. Therefore, it is helpful to consider the mating system in furthering the understanding of the processes that may have shaped trait variation in polyploids in nature.

     

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.     

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.     

玉龙雪山不同针叶树种树轮宽度年表特征及其与气候因子的关系北大核心CSCD

为揭示滇西北高原树木径向生长与气候关系的规律,该研究分别在玉龙雪山采集高山松(Pinus densata)、云南松(Pinus yunnanensis)和云南铁杉(Tsuga dumosa)年轮样本,建立3个树种树轮宽度差值年表,将年轮指数与气候因子进行响应分析和冗余分析,以明确影响玉龙雪山树木径向生长的主要气候因子,为该区域森林生态系统的管理保护提供依据,并为探讨气候变化背景下滇西北森林动态提供依据。结果表明:(1)所建立的3个树种树轮宽度差值年表具有较少的低频变化,且离散程度较低、逐年变化共性较高,可代表采样点树木的年轮特征;高山松、云南松和云南铁杉差值年表的平均敏感度依次分别为0.21、0.22和0.17,样本总体代表性分别为0.96、0.96和0.94,均高于0.85的阈值,说明年表中信息具有代表性。(2)响应分析结果显示,高山松径向生长与当年2月的降水量呈显著正相关关系;云南松径向生长与当年5月的平均气温和最高温呈显著负相关关系,与当年10月平均气温呈显著正相关关系;云南铁杉径向生长与当年1月和5月的降水、当年10月相对湿度均呈显著正相关关系,与当年5月最高气温呈显著负相关关系。(3)冗余分析结果显示,当年1月降水、2月降水、5月干旱指数对3个树种均具有促进作用,且5月干旱指数的显著相关性最强;当年10月降水的增加有利于高山松和云南铁杉的径向生长,但对云南松径向生长具有抑制作用。研究认为,当年冬季降水、5月湿润条件以及10月降水是影响玉龙雪山3个树种径向生长的重要环境因子;若干暖化趋势加剧,将不利于滇西北高原3个树种的生长,从而影响区域森林生态系统结构和功能。     

Colonization of Pinus halepensis in Mediterranean habitats: consequences of afforestation, grazing and fire

The expansion of P. halepensis from plantations into natural sites of high conservation value is becoming a frequent occurrence across the Mediterranean zone of Israel. We studied how colonization of Pinus halepensis in natural Mediterranean habitats is related to afforestation, cattle grazing and fire. The study was conducted in a Mediterranean garrigue (Shrubland) located in Ramat Hanadiv Nature Park, southern Mt. Carmel region, Israel. The study area (ca. 350 ha) was divided into cells (100 × 100 m) each of which was categorized with respect to distance from planted pines, grazing (grazed since 1990/ungrazed), fire (burned in 1980/unburned), and vegetation structure (garrigue, dense garrigue, dense woodland). The location of colonizing pines, typically three m or more in height, was determined using an aerial photograph. Density of colonizing pines decreased linearly with the distance from planted pines within 300 m from planted pines with a long tail that extended out to ca 1,000 m. Over 90% of the colonizing pines that were found were located within a distance of 300 m (56% of the park area) from planted pines. Colonization was about two times greater under grazing than without grazing. The effects of fire and of the interaction fire × grazing were found insignificant. A separate analysis reveled that colonization was about 2 times larger in patches of sparse woody cover than in those of dense cover. In conclusion, pine colonization was mainly determined by the proximity to seed sources. Additionally, pine colonization was enhanced by cattle grazing probably through reduction of the natural vegetation cover.     

Reproducing under a warming climate: long winter flowering and extended flower longevity in the only Mediterranean and maritime Primula

Under the pressure of global warming, general expectations of species migration and evolution of adaptive traits should always be confirmed with species‐specific studies. Within this framework, some species can be used as study systems to predict possible consequences of global warming also on other relatives. Unlike its mountain congeneric, Primula palinuri Petagn. has endured all the climatic fluctuations since the Pleistocene, while surviving on Mediterranean coastal cliffs. The aim of this work was to investigate the possible evolution of reproductive biological and ecological traits in P. palinuri adaptation to a warmer environment. Data showed that flowering starts in mid‐winter; single flowers remain open for over a month, changing from pendulous to erect. The number of insects visiting flowers of P. palinuri increases during the flowering season, and pollination reduces flower longevity. Overall, the best pollen performances, in terms of viability and germinability, occur at winter temperatures, while pollinator activity prolongs flowering until spring. Moreover, extended longevity of single flowers optimises reproductive success. Both phenotypic plasticity and selective processes might have occurred in P. palinuri. However, we found that reproductive traits of the only Mediterranean Primula remain more associated with cold mountain habitats than warm coastal cliffs. Given the rapid trend of climate warming, migration and new adaptive processes in P. palinuri are unlikely. Response to past climate warming of P. palinuri provides useful indications for future scenarios in other Primula species.     

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.     

Effects of combined ozone and nitrogen deposition on the in situ properties of eleven key plant species of a subalpine pasture

Tropospheric O₃ and deposition of reactive N threaten the composition and function of natural and semi-natural vegetation even in remote regions. However, little is known about effects of these pollutants individually or in combination on plant species in alpine habitats. We analyzed 11 frequent plant species of a subalpine Geo-Montani-Nardetum pasture exposed at 2,000 m a.s.l. in the Swiss Alps during 3 years using a factorial free-air exposure system with three concentrations of O₃ and five rates of N application. The aim was to detect subtle effects on leaf chlorophyll and N concentrations, leaf weight, specific leaf area (SLA), and δ¹⁸O and δ¹³C as proxies for gas exchange. We expected that the species’ responsiveness to O₃ and N would be related to their functional traits and that N-induced changes in these traits would modify the species’ response to O₃ via increased growth and higher leaf conductance (g _s). Most species reacted to N supply with the accumulation of N and chlorophyll, but with no change in SLA, g _s, and growth, except Carex sempervirens which showed increased water use efficiency and leaf weight. Elevated O₃ reduced g _s in most species, but this was not related to a reduction in leaf weight, which was recorded in half of the species. Contrary to our expectation, the magnitude of the response to both O₃ and N was not related to species-specific traits such as SLA or g _s. No pronounced O₃ × N interactions were observed. In conclusion, since for most species neither N nor gas exchange limited growth, their short-term response to O₃ and N and to their combination was small. O₃ × N interactive effects are expected to be more pronounced in habitats where species are more responsive to N due to favorable growth conditions in terms of nutrient availability and temperature. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.