Genomic convergence underlying high-altitude adaptation in alpine plants

Evolutionary convergence is one of the most striking examples of adaptation driven by natural selection.However, genomic evidence for convergent adaptation to extreme environments remains scarce.Here, we assembled reference genomes of two alpine plants, Saussurea obvallata(Asteraceae)and Rheum alexandrae(Polygonaceae), with 37,938 and 61,463 annotated protein-coding genes. By integrating an additional five alpine genomes,we elucidated genomic convergence underlying high-altitude adaptation in alpine plants. Our results detected convergent contractions of diseaseresistance genes in alpine genomes, which might be an energy-saving strategy for surviving in hostile environments with only a few pathogens present.We identified signatures of positive selection on a set of genes involved in reproduction and respiration(e.g., MMD1, NBS1, and HPR), and revealed signatures of molecular convergence on genes involved in self-incompatibility, cell wall modification,DNA repair and stress resistance, which may underlie adaptation to extreme cold, high ultraviolet radiation and hypoxia environments. Incorporating transcriptomic data, we further demonstrated that genes associated with cuticular wax and flavonoid biosynthetic pathways exhibit higher expression levels in leafy bracts, shedding light on the genetic mechanisms of the adaptive “greenhouse” morphology. Our integrative data provide novel insights into convergent evolution at a high-taxonomic level,aiding in a deep understanding of genetic adaptation to complex environments.

Changes in leaf vein traits among vein types of alpine grassland plants on the Tibetan Plateau

Vein traits influence photosynthesis and drought resistance and are sensitive to climate change.It is unclear whether vein traits,similar to other leaf traits,have obvious regional characteristics and covariance with other leaf traits,especially in Tibetan Plateau grasslands.We collected 66 species from 37 sites in late July of 2012 and early August of 2013 to investigate leaf vein traits and their relationships with other leaf traits in comparison with the available global database data and to elucidate vein investment of plants with different vein types.The average vein length per area(VLA)of plants in the Tibetan Plateau was within the range of the global dataset,and the relationships among vein traits and other leaf traits of alpine plants were consistent with the global models.The VLAs of parallel-veined grasses and pinnateveined forbs were significantly lower and higher than the global mean value,respectively.The leaf mass perarea and total nitrogen content of parallel-veined grasses were significantly lower and higher,respectively,than the global mean values;the opposite was observed in pinnate-veined forbs.The parallel-veined grasses exhibited almost a four-fold higher vein biomass investment(i.e.,vein mass per leaf mass)than pinnate-veined forbs in the same region.The average VLA and its relationships with other leaf traits of the alpine grassland plants on the Tibetan Plateau had no regional characteristics,suggesting a convergence in plant leaf functioning.Plants with different leaf types differ in their adaptation strategies to plateau environments,and this may relate to biomass investment into leaf veins.Our study fills the gap with regard to vein density in alpine grassland species and provides a new perspective for understanding plant physiology and ecology by calculating and comparing the proportion of vein investment among different vein types.

Alpine tundra species phenology is mostly driven by climate-related variables rather than by photoperiod

The study of plant phenology has frequently been used to link phenological events to various factors,such as temperature or photoperiod.In the high-alpine environment,proper timing of the phenological cycle has always been crucial to overcome harsh conditions and potential extreme events(i.e.spring frosts)but little is known about the response dynamics of the vegetation,which could shape the alpine landscape in a future of changing climate.Alpine tundra vegetation is composed by an array of species belonging to different phytosociological optima and with various survival strategies,and snowbed communities are a relevant expression of such an extreme-climate adapted flora.We set eight permanent plots with each one in a snowbed located on the Cimalegna plateau in Northwestern Italy and then we selected 10 most recurring species among our plots,all typical of the alpine tundra environment and classified in 3different pools:snowbed specialists,grassland species and rocky debris species.For 3 years we registered the phenophases of each species during the whole growing season using an adaptation of the BBCH scale.We later focused on the three most biologically relevant phenophases,i.e.,flower buds visible,full flowering,and beginning of seed dispersion.Three important season-related variables were chosen to investigate their relationship with the phenological cycle of the studied species:(i)the Day Of Year(DOY),the progressive number of days starting from the 1 st of January,used as a proxy of photoperiod,(ii)Days From Snow Melt(DFSM),selected to include the relevance of the snow dynamics,and(iii)Growing Degree Days(GDD),computed as a thermal sum.Our analysis highlighted that phenological development correlated better with DFSM and GDD than with DOY.Indeed,models showed that DOY was always a worse predictor since it failed to overcome interannual variations,while DFSM and marginally GDD were better suited to predict the phenological development of most of the species,despite differences intemperature and snowmelt date among the three years.Even if the response pattern to the three variables was mainly consistent for all the species,the timing of their phenological response was different.Indeed,species such as Salix herbacea and Ranunculus glacialis were always earlier in the achievement of the phenophases,while Agrostis rupestris and Euphrasia minima developed later and the remaining species showed an intermediate behavior.However,we did not detect significant differences among the three functional pools of species.

Phenotypic plasticity of floral traits and pollination adaption in an alpine plant Pedicularis siphonantha D. Don when transplanted from higher to lower elevation in Eastern Himalaya

Phenotypic plasticity and/or pollinatormediated selection may be responsible for the changes in floral traits of plants when they are forced to live in new conditions. Although the two events could be independent, we hypothesized that phenotypic plasticity in floral traits might help to coordinate plant-pollinator interactions and enhance plant reproductive success in changing habitats. To test this hypothesis, we investigated floral traits and pollination on three natural populations of a lousewort(Pedicularis siphonantha) ranging at different elevations, as well as two downward transplanted populations in Shangeri-La County and Deqin County, northwest Yunnan, China. The results indicated that floral traits, i.e. phenology, longevity,display size, corolla tube length and pollen production differed significantly among populations. Moreover,or the two transplanted populations, floral traits diverged from their original populations, but converged to their host populations. Although the phenotypic plasticity in floral traits might be a rapid response to abiotic factor such as warmer environment, the changes in floral traits were found to be well adapted to pollination environment of the host population. Compared with plants of their original habitats in higher elevation, the transplanted individuals advanced flowering time, shortened flower longevity, reduced floral display size and pollen production, received higher visiting frequency and yielded more seeds. These findings suggested that phenotypic plasticity of floral traits might help plants adjust their resource allocation strategy between preand post-pollination stages in response to harsh or temperate conditions, which might correspondingly meet a pollinator-poor or hyphen rich environment.This would be beneficial for the widely-distributed species to adapt to various environmental changes.

Comparison of Photosynthetic Adaptability Between Kobresia humilis and Polygonum viviparum on Qinghai Plateau

The chlorophyll fluorescence parameters of Kobresia humilis Serg. and Polygonum viviparum L. grown at two different altitudes (3?200 m, 3?980 m) were measured and the ultrastructure of chloroplasts were observed for studying the photosynthetic adaptability of plants to the influences of stress conditions in alpine environment. Rfd _values, the vitality index, in leaves of K. humilis and P.viviparum grown at 3?980 m were higher than those at 3?200 m. The higher ratio of F v/F o and F v/F m in leaves of K. humilis and P.viviparum indicated that the rate of photosynthetic conversion of light energy increased at higher altitude. Ratios of F v/F o and F v/F m and Rfd _values in K.humilis were higher than that in P.viviparum grown at the same altitude. There were more irregular chloroplasts in leaves of both species grown at higher altitude. Many irregular chloroplasts such as swollen thylakoid, deformed chloroplast envelope, were observed in P.viviparum grown at 3?980 m, but few in K. humilis . These results were discussed in relation to the photosynthetic adaptability of alpine plants and the different adaptive competence between K.humilis and P.viviparum .

Effects of environment and genotype-by-environment interaction on phenotype of Rorippa elata(Brassicaceae),an endemic alpine plant in the Hengduan mountains

Global climate change poses a severe threat to mountain biodiversity.Phenotypic plasticity and local adaptation are two common strategies for alpine plant to cope with such change.They may facilitate organismal adaptation to contrasting environments,depending on the influences of the environment or genotype or their interacted effects.In this study,we use an endemic alpine plant(Rorippa elata)in the Hengduan mountains(HDM)to unravel its phenotypic basis of adaptation strategy and evaluate the relative contributions of environment and genotype to its phenotype.We transplanted 37 genotypes of R.elata into two common gardens across low and high elevations(2800 vs.3800 m)during 2021-2022.Nine fitness-related traits were measured,including flowering probability and glucosinolates(GS)content.We estimated the environmental or genotypic contributions to the phenotype and identified the main environmental components.Our results revealed that both environment and genotype-by-environment interactions contributed to the phenotypes of R.elata.Latitudinal heterogeneity was identified as a key factor that explained 24%of the total phenotypic variation.In particular,genotypes of the northern HDM showed significantly higher plasticity in flowering probability than those of the southern HDM.Furthermore,within the southern HDM,GS content indicated local adaptation to herbivory stresses for R.elata genotypes along elevations.In conclusion,our results suggest that R.elata may have adapted to the alpine environment through species-level plasticity or regional-level local adaptation.These processes were shaped by either complex topography or interactions between genotype and mountain environments.Our study provides empirical evidence on the adaptation of alpine plants.

Impacts of ontogenetic and altitudinal changes on morphological traits and biomass allocation patterns of Fritillaria unibracteata

Environmental variations and ontogeny may affect plant morphological traits and biomass allocation patterns that are related to the adjustments of plant ecological strategies. We selected 2-, 3-and 4-year-old Fritillaria unibracteata plants to explore the ontogenetic and altitudinal changes that impact their morphological traits(i.e., plant height, single leaf area,and specific leaf area) and biomass allocations [i.e.,biomass allocations of roots, bulbs, leaves, stems, and flowers] at relatively low altitudinal ranges(3400 m to 3600 m asl) and high altitudinal ranges(3600 m to4000 m asl). Our results indicated that plant height,root biomass allocation, and stem biomass allocation significantly increased during the process of individual growth and development, but single leaf area, specific leaf area, bulb biomass allocation, and leaf biomass allocation showed opposite trends.Furthermore, the impacts of altitudinal changes on morphological traits and biomass allocations had no significant differences at low altitude, except for single leaf area of 2-year-old plants. At high altitude,significantly reduced plant height, single leaf area and leaf biomass allocation for the 2-year-old plants,specific leaf area for the 2-and 4-year-old plants, and stem biomass allocation were found along altitudinal gradients. Significantly increased sexual reproductive allocation and relatively stable single leaf area and leaf biomass allocation were also observed for the 3-and 4-year-old plants. In addition, stable specific leaf area for the 3-year-old plants and root biomass allocation were recorded. These results suggested that the adaptive adjustments of alpine plants, in particular F. unibracteata were simultaneously influenced by altitudinal gradients and ontogeny.

Cold stratification,temperature,light,GA3,and KNO3 effects on seed germination of Primula beesiana from Yunnan,China

Primula beesiana Forr.is an attractive wildflower endemically distributed in the wet habitats of subalpine/alpine regions of southwestern China.This study is an attempt to understand how this plant adapts to wet habitats and high altitudes.Specifically,we examined the effects of cold stratification,light,GA3,KNO3,and temperature on P.beesiana seed germination.KNO3 and GA3 increased germination percentage and germination rate compared to control treatments at 15/5 and 25/15℃.Untreated seeds germinated well(>80%)at higher temperatures(20,25 and 28℃),whereas at lower(5,10 and15℃)and extremely high temperatures(30 and 32℃)germination decreased significantly.However,after cold stratification(4-16 weeks),the germination percentage of P.beesiana seeds at low temperatures(5-15℃)and the germination rate at high temperatures(30℃)increased significantly,suggesting that P.beesiana has type 3 non-deep physiological dormancy.The base temperature and thermal time for germination decreased in seeds that were cold stratified for 16 weeks.Cold-stratified seeds incubated at fluctuating temperatures(especially at 15/5℃)had significantly high germination percentages and germination rates in light,but not in dark,compared to the corresponding constant temperature(10℃).Seeds had a strict light requirement at all temperatures,even after experiencing cold stratification;however,the combinations of cold stratification and fluctuating temperature increased germination when seeds were transferred from dark to light.Such dormancy/germination responses to light and temperature are likely mechanisms that ensure germination occurs only in spring and at/near the soil surface,thus avoiding seedling death by freezing,inundation and/or germination deep in the soil.

Variations in leaf functional traits and physiological characteristics of Abies georgei var.smithii along the altitude gradient in the Southeastern Tibetan Plateau

Variations in leaf functional traits of Abies georgei var. smithii at 3700, 3900, 4100, 4300, and 4390 m altitude were investigated in 15 typical plots in the Southeastern Tibetan Plateau. In each plot, three seedlings were selected, of which functional leaves in current-year sunny branches were chosen for the measurement of morphological, photosynthetic, and physiological and biochemical characteristics, and their variations were analyzed. Results showed that significant variations existed among the leaf functional traits of A. georgei var. smithii along the altitudinal gradient, as well as their physiological adaption indicators. Leaf area decreased, while the mass per area and thickness of leaf increased at an altitude above 4,100 m. The maxima of pigment, total nitrogen concentration, net photosynthesis rate during light-saturated, and when water use efficiency appeared at 4100 m altitude. In addition, A. georgei var. smithii seedlings regulated the activities of superoxide dismutase and ascorbate peroxidase to resist abiotic stress under 4100 m altitude. Meanwhile, malondialdehyde concentration and the dark respiration rate rapidly increased, which indicates that A. georgei var. smithii seedlingssuffered from heavy abiotic stress from 4100 m to 4390 m altitude. Basing on variations in leaf functional traits along the altitude gradient, we inferred that 4100 m altitude was the suitable region for A. georgei var. smithii growth in the Sygera Mountain. Moreover, the harsh environment was the main limiting factor for A. georgei var. smithii population expansion to high altitude.

Linkages among the functional traits,insect visitation rate and seed set of Gentiana taxa on the Tibetan Plateau

Maximum plant height(H_(max)),stem tissue mass density(SD),leaf mass per area(LMA),and relative growth rate(RGR)have all been linked to flowering phenology.However,it is still unknown whether these functional traits varying with flowering phenology are related to other floral traits associated with pollinator preference and reproductive success.We investigated the relationship between vegetative and floral traits,as well as the rates of insect visitation and seed set of fiveGentiana species in eastern Tibetan meadows.Our results showed that flowering onset and flowering offset time were all found to be positively correlated with the H_(max),SD,and LMA,but negatively correlated with the RGR.Flowering onset time was positively correlated with corolla diameter and pollen grain number,whereas was negatively correlated with flower number and flower longevity.The rates of insect visitation were positively related to flowering onset time,pollen grain number,corolla diameter,and seed set,but negatively related to flower number and longevity.Early flowering species have higher RGR but lower SD,LMA andH_(max),as well as smaller flowers,fewer pollen grains,longer flower longevity,and lower insect visitation rates than late-flowering plant species.Our findings indicate that floral traits are related to vegetative traits in Gentiana species.Also these plant trait relationships were associated with pollinator preference,and plant reproductive success of eastern Tibetan meadows.We speculate that these traits relationships are likely adaptive in unpredictable and often pollinator-limited environments in the Tibet alpine meadows.

Phylogeography of Potentilla fruticosa,an alpine shrub on the Qinghai-Tibetan Plateau

Aims Our objectives were(i)to elucidate the phylogeography of chloroplast DNA(cpDNA)in Potentilla fruticosa in relation to Quaternary climate change and postglacial colonization,(ii)to infer historical population range expansion using mismatch distribution analyses and(iii)to locate the refugia of this alpine species on the Qinghai-Tibetan plateau during glacial-interglacial periods.Methods Potentilla fruticosa is a widespread species distributed on the Qinghai-Tibetan Plateau.We sampled leaves of P.fruticosa from 10 locations along a route of;1300 km from the northeastern plateau(Haibei,Qinghai)to the southern plateau(Dangxiong,Tibet).We examined the cpDNA of 15 haplotypes for 87 individuals from the 10 populations based on the sequence data from;1000 base pairs of the trnS-trnG and rpl20-rps12.Phylogenetic relationship of haplotypes was analyzed using the Phylip software package and the program TCS.The diversity of populations indices was obtained using the program ARLEQUIN.Important Findings With the limited samples,we found that(i)higher nucleotide diversity often occurs in high-altitude populations,(ii)the ancestral haplotypes distribute in the populations with higher nucleotide diversity than recent haplotypes,(iii)the expansion time of population in the high altitudes was estimated to be approximately at 52-25 ka BP(1000 years Before Present,where"Present"is AD 1950)and that in the low altitudes to be;5.1-2.5 ka BPand(iv)the source location of P.fruticosa is at the high altitudes,which might provide refugia for the species during the interglacial warm periods.The species expanded from the high-elevated locations on the Tanggula Mountains during the Holocene.

Grazing alters warming effects on leaf photosynthesis and respiration in Gentiana straminea,an alpine forb species

Aims Vast grasslands on the Tibetan Plateau are almost all under livestock grazing.It is unclear,however,what is the role that the grazing will play in carbon cycle of the grassland under future climate warming.We found in our previous study that experimental warming can shift the optimum temperature of saturated photosynthetic rate into higher temperature in alpine plants.In this study,we proposed and tested the hypothesis that livestock grazing would alter the warming effect on photosynthetic and respiration through changing physical environments of grassland plants.Methods Experimental warming was carried by using an infrared heating system to increase the air temperature by 1.2 and 1.7℃ during the day and night,respectively.The warming and ambient temperature treatments were crossed over to the two grazing treatments,grazing and un-grazed treatments,respectively.To assess the effects of grazing and warming,we examined photosynthesis,dark respiration,maximum rates of the photosynthetic electron transport(J_(max)),RuBP carboxylation(V_(cmax))and temperature sensitivity of respiration Q_(10) in Gentiana straminea,an alpine species widely distributed on the Tibetan grassland.Leaf morphological and chemical properties were also examined to understand the physiological responses.Important findings 1)Light-saturated photosynthetic rate(A_(max))of G.straminea showed similar temperature optimum at around 16℃ in plants from all experimental conditions.Experimental warming increased A_(max) at all measuring temperatures from 10 to 25℃,but the positive effect of the warming occurred only in plants grown under the un-grazed conditions.Under the same measuring temperature,A_(max) was significantly higher in plants from the grazed than the un-grazed condition.2)There was significant crossing effect of warming and grazing on the temperature sensitivity(Q_(10))of leaf dark respiration.Under the un-grazed condition,plants from the warming treatment showed lower respiration rate but similar Q_(10) in comparison with plants from the ambient temperature treatment.However,under the grazed condition Q_(10) was significantly lower in plants from the warming than the ambient treatment.3)The results indicate that livestock grazing can alter the warming effects on leaf photosynthesis and temperature sensitivity of leaf dark respiration through changing physical environment of the grassland plants.The study suggests for the first time that grazing effects should be taken into account in predicting global warming effects on photosynthesis and respiration of plants in those grasslands with livestock grazing.

Patterns of plant diversity at high altitudes on the Qinghai-Tibetan Plateau

To describe the biodiversity patterns of plants along an altitudinal gradient on the Qinghai-Tibetan Plateau and to clarify the bias in plant specimen records at high altitude.Methods We conducted a large-scale investigation of vegetation at a wide range of altitudes,focusing on a high-altitudinal range(3200-5200 m)at different locations on the Qinghai-Tibetan Plateau.We then compared the altitudinal distribution of plant species obtained from our field investigation with that in plant specimen records from published sources and an online database.Important Findings Our data provide evidence that altitude plays a large role in regulating species composition on the Qinghai-Tibetan Plateau.We could not,however,detect a clear relationship between altitude and species richness,although a weak monotonically increasing trend of richness was detected with increasing altitude.According to specimen records,most species have been sampled at a wide range of altitudes,and the average range of 145 species is>2000 m.Despite this wide range,more than half of the species we observed were at higher altitudes than the specimen records indicate.High-altitude areas have probably been so poorly sampled that only a small fraction of the resident species has been recorded.This study clearly shows the regional bias of specimen records in the Qinghai-Tibetan Plateau.

Leaf photosynthesis and simulated carbon budget of Gentiana straminea from a decade-long warming experiment

Aims Alpine ecosystems may experience larger temperature increases due to global warming as compared with lowland ecosystems.Information on physiological adjustment of alpine plants to temperature changes can provide insights into our understanding how these plants are responding to current and future warming.We tested the hypothesis that alpine plants would exhibit acclimation in photosynthesis and respiration under long-term elevated temperature,and the acclimation may relatively increase leaf carbon gain under warming conditions.Methods Open-top chambers(OTCs)were set up for a period of 11 years to artificially increase the temperature in an alpine meadow ecosystem.We measured leaf photosynthesis and dark respiration under different light,temperature and ambient CO_(2)concentrations for Gentiana straminea,a species widely distributed on the Tibetan Plateau.Maximum rates of the photosynthetic electron transport(J_(max)),RuBP carboxylation(V_(cmax))and temperature sensitivity of respiration Q10 were obtained from the measurements.We further estimated the leaf carbon budget of G.straminea using the physiological parameters and environmental variables obtained in the study.Important findings 1)The OTCs consistently elevated the daily mean air temperature by;1.6℃and soil temperature by;0.5℃during the growing season.2)Despite the small difference in the temperature environment,there was strong tendency in the temperature acclimation of photosynthesis.The estimated temperature optimum of light-saturated photosynthetic CO_(2)uptake(A_(max))shifted;1℃higher from the plants under the ambient regime to those under the OTCs warming regime,and the Amax was significantly lower in the warming-acclimated leaves than the leaves outside the OTCs.3)Temperature acclimation of respiration was large and significant:the dark respiration rates of leaves developed in the warming regime were significantly lower than leaves from the ambient environments.4)The simulated net leaf carbon gain was significantly lower in the in situ leaves under the OTCs warming regime than under the ambient open regime.However,in comparison with the assumed non-acclimation leaves,the in situ warmingacclimated leaves exhibited significantly higher net leaf carbon gain.5)The results suggest that there was a strong and significant temperature acclimation in physiology of G.straminea in response to long-term warming,and the physiological acclimation can reduce the decrease of leaf carbon gain,i.e.increase relatively leaf carbon gain under the warming condition in the alpine species.