Natural hybridization,introgression breeding,and cultivar improvement in the genus Actinidia

China is the original home of kiwifruit which are derived from the species complex Actinidia chinensis. A short domestication history of a little more than 100 years characterizes kiwifruit as a unique fruit crop whose cultivar improvement is heavily dependent on exploitation and selection of wild resources. Actinidia species are widespread geographically; their distributions often overlap, and hybridization between taxa is common naturally. Actinidia species that vary in ploidy and complex mixtures of cytotypes are prevalent in natural populations. Here, we review the recent emerging knowledge of natural distribution, biogeography, and population genetics in Actinidia with a particular focus on the pattern of overlapping distribution and natural hybridization among Actinidia species. Based on the comparison of the original geographical localities where most kiwifruit commercial cultivars were selected and the whole geographical range of the species complex A. chinensis, we propose that introgression breeding for new selections and cultivars of kiwifruit should utilize targeted exploration in natural hybrid zones as well as the genomic tools and related genetic resources that are becoming available to an unprecedented extent.     

The First Complete Chloroplast Genome Sequences in Actinidiaceae: Genome Structure and Comparative Analysis

Actinidia chinensis is an important economic plant belonging to the basal lineage of the asterids. Availability of a complete Actinidia chloroplast genome sequence is crucial to understanding phylogenetic relationships among major lineages of angiosperms and facilitates kiwifruit genetic improvement. We report here the complete nucleotide sequences of the chloroplast genomes for Actinidia chinensis and A. chinensis var deliciosa obtained through de novo assembly of Illumina paired-end reads produced by total DNA sequencing. The total genome size ranges from 155,446 to 157,557 bp, with an inverted repeat (IR) of 24,013 to 24,391 bp, a large single copy region (LSC) of 87,984 to 88,337 bp and a small single copy region (SSC) of 20,332 to 20,336 bp. The genome encodes 113 different genes, including 79 unique protein-coding genes, 30 tRNA genes and 4 ribosomal RNA genes, with 16 duplicated in the inverted repeats, and a tRNA gene (trnfM-CAU) duplicated once in the LSC region. Comparisons of IR boundaries among four asterid species showed that IR/LSC borders were extended into the 5’ portion of the psbA gene and IR contraction occurred in Actinidia. The clap gene has been lost from the chloroplast genome in Actinidia, and may have been transferred to the nucleus during chloroplast evolution. Twenty-seven polymorphic simple sequence repeat (SSR) loci were identified in the Actinidia chloroplast genome. Maximum parsimony analyses of a 72-gene, 16 taxa angiosperm dataset strongly support the placement of Actinidiaceae in Ericales within the basal asterids.     

Fraxin and esculin: two coumarins specific to Actinidia chinensis and A. deliciosa (kiwifruit)

Fraxin and esculin were characterized in stems and fruits of A. deliciosa (kiwifruit) and A. chinensis. These two coumarins were not present in several other Actinidia species belonging to the four sections of the genus Actinidia such as A. callosa, A. eriantha, A. hemsleyana, A. arguta, A. kolomikta, A. melanandra, A. polygama. Our results support the opinion that A. deliciosa and A. chinensis are closely related and likely belonging to the same species.     

Microsatellite DNA in Actinidia chinensis: isolation, characterisation, and homology in related species

 We have isolated and sequenced 263 microsatellite-containing clones from two small insert libraries of Actinidia chinensis enriched for (AC/GT) and (AG/CT) repeats, respectively. Primer pairs were designed for 203 microsatellite loci and successfully amplified from both plasmid and A. chinensis genomic DNA. In this paper we report the sequences of 40 primer pairs for which we have demonstrated Mendelian segregation in the progeny from controlled crosses. The polymorphism of ten microsatellites of each type was evaluated in four diploid and six tetraploid genotypes of A. chinensis. All microsatellites proved to be polymorphic, the number of alleles per locus detected in polyacrylamide sequencing gels ranging from 9 to 17. The high degree of polymorphism in Actinidia renders these markers useful either for mapping in A. chinensis or for fingerprinting cultivars of both domesticated kiwifruit species (A. chinensis and A. deliciosa). While most primer pairs produced single amplification products, about 20% generated banding patterns consistent with the amplification of two different loci. This supports the hypothesis that diploid species of Actinidia (2n=2x=58) are polyploid in origin with a basic chromosome number x=14/15 and that chromosome duplication may have occurred during the evolution of the genus. Finally, we have assayed the cross-species transportability of primer pairs designed from A. chinensis sequences and have found extensive cross-species amplification within the genus Actinidia; 75% of primer pairs gave successful amplification in the eight species assayed (A. arguta, A. rufa, A. polygama, A. chrysantha, A. callosa, A. hemsleyana, A. eriantha, and A. deliciosa), which are representative of the four sections into which the genus is currently split. Received: 14 February 1998 / Accepted: 26 May 1998     

The Microgeographical Patterns of Morphological and Molecular Variation of a Mixed Ploidy Population in the Species Complex Actinidia chinensis

Polyploidy and hybridization are thought to have significant impacts on both the evolution and diversification of the genus Actinidia, but the structure and patterns of morphology and molecular diversity relating to ploidy variation of wild Actinidia plants remain much less understood. Here, we examine the distribution of morphological variation and ploidy levels along geographic and environmental variables of a large mixed-ploidy population of the A. chinensis species complex. We then characterize the extent of both genetic and epigenetic diversity and differentiation exhibited between individuals of different ploidy levels. Our results showed that while there are three ploidy levels in this population, hexaploids were constituted the majority (70.3%). Individuals with different ploidy levels were microgeographically structured in relation to elevation and extent of niche disturbance. The morphological characters examined revealed clear difference between diploids and hexaploids, however tetraploids exhibited intermediate forms. Both genetic and epigenetic diversity were high but the differentiation among cytotypes was weak, suggesting extensive gene flow and/or shared ancestral variation occurred in this population even across ploidy levels. Epigenetic variation was clearly correlated with changes in altitudes, a trend of continuous genetic variation and gradual increase of epigenomic heterogeneities of individuals was also observed. Our results show that complex interactions between the locally microgeographical environment, ploidy and gene flow impact A. chinensis genetic and epigenetic variation. We posit that an increase in ploidy does not broaden the species habitat range, but rather permits A. chinensis adaptation to specific niches.     

Development of sex-linked PCR markers for gender identification in Actinidia

 Two sex-linked random amplified polymorphic DNA (RAPD) markers identified from Actinidia chinensis were converted into sequence-characterised amplified regions (SCARs) for the large-scale screening of Actinidia breeding populations. Initial SCAR primers converted one RAPD (SmX) into a dominant marker, but the other (SmY), which was potentially more useful because of its linkage to the male determining ‘Y’ locus, failed to retain polymorphism. This difficulty was overcome by cloning and sequencing the alternate ‘allele’ from female plants, and then designing ‘allele’-specific primers that utilised nucleotide differences between the sexes. Using a quick squash-blot method of DNA extraction, the SCAR primers were tested in 120 A. chinensis plants to determine their gender. The system is now in use for large-scale screening of seedling populations in the Actinidia breeding programme. The sex-linked SCAR primers also functioned with plants from some other geographically separate accessions of A. chinensis and with plants in the closely related polyploid species A. deliciosa, but did not amplify a sex-linked band in more distantly related species of Actinidia. Received: 27 December 1997 / Accepted: 5 March 1998     

Cryopreservation of shoot tips,evaluations of vegetative growth,and assessments of genetic and epigenetic changes in cryo-derived plants of Actinidia spp.

A droplet-vitrification protocol was described for cryopreservation of shoot tips of kiwifruit ‘Yuxiang’ (Actinidia chinensis var. deliciosa). No significant differences were found in root formation and shoot growth between the in vitro-derived shoots (the control) and cryo-derived ones when cultured in vitro. No significant differences were detected in survival and vegetative growth between the in vitro-derived plants (the control) and cryo-derived ones after re-establishment in greenhouse conditions. Inter-simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) did not detect any polymorphic bands in the cryo-derived shoots when cultured in vitro and the cryo-derived plants after re-establishment in greenhouse conditions. These data indicate rooting ability, vegetative growth and genetic stability are maintained in the cryo-derived kiwifruit plants recovered from the droplet-vitrification cryopreservation. Methylation sensitive amplification polymorphism (MSAP) detected 12.8% and 1.6% DNA methylation in the cryo-derived shoots when cultured in vitro and the cryo-derived plants after re-established in greenhouse conditions, respectively. This droplet-vitrification was applied to five cultivars and three rootstocks belonging to A. chinensis var. deliciosa, A. chinensis var. chinensis, A. macrosperma, A. polygama and A. valvata. The highest (68.3%) and lowest (22.5%) shoot regrowth were obtained in A. macrosperma and A. chinensis var. chinensis ‘Jinmi’, respectively, with an average of 46.4% shoot regrowth obtained across the eight genotypes. The droplet-vitrification protocol described here can be considered the most applicable cryopreservation method so far reported for the genus Actinidia. Results reported here provide theoretical and technical supports for setting up cryo-banks of genetic resources of Actinidia spp.     

Meiotic chromosome pairing in Actinidia chinensis var. deliciosa

Polyploids are defined as either autopolyploids or allopolyploids, depending on their mode of origin and/or chromosome pairing behaviour. Autopolyploids have chromosome sets that are the result of the duplication or combination of related genomes (e.g., AAAA), while allopolyploids result from the combination of sets of chromosomes from two or more different taxa (e.g., AABB, AABBCC). Allopolyploids are expected to show preferential pairing of homologous chromosomes from within each parental sub-genome, leading to disomic inheritance. In contrast, autopolyploids are expected to show random pairing of chromosomes (non-preferential pairing), potentially leading to polysomic inheritance. The two main cultivated taxa of Actinidia (kiwifruit) are A. chinensis (2x and 4x) and A. chinensis var. deliciosa (6x). There is debate whether A. chinensis var. deliciosa is an autopolyploid derived solely from A. chinensis or whether it is an allopolyploid derived from A. chinensis and one or two other Actinidia taxa. To investigate whether preferential or non-preferential chromosome pairing occurs in A. chinensis var. deliciosa, the inheritance of microsatellite alleles was analysed in the tetraploid progeny of a cross between A. chinensis var. deliciosa and the distantly related Actinidia eriantha Benth. (2x). The frequencies of inherited microsatellite allelic combinations in the hybrids suggested that non-preferential chromosome pairing had occurred in the A. chinensis var. deliciosa parent. Meiotic chromosome analysis showed predominantly bivalent formation in A. chinensis var. deliciosa, but a low frequency of quadrivalent chromosome formations was observed (1 observed in 20 pollen mother cells).     

Paternal inheritance of plastids in interspecific hybrids of the genus Actinidia revealed by PCR-amplification of chloroplast DNA fragments

RFLPs (restriction fragment length polymorphisms) of PCR (polymerase chain reaction) -amplified fragments were used to trace the pattern of plastid DNA inheritance in the genus Actinidia. A total of 51 progeny originating from interspecific crosses between three A. arguta cultivars and A. deliciosa, the kiwifruit, and 12 progeny originating from the cross between A. kolomikta and A. chinensis were analysed together with their parents. No reciprocal crosses could be tested since they all failed to set viable seeds. Attempts to rescue immature embryos failed in all cases as well. The A. arguta×A. deliciosa crosses were checked for the RFLP patterns of a sequence encoding part of the Rubisco large subunit (rbcL), using either AluI or MseI, and for a sequence encoding part of the photosystem II D1 protein (psbA), using HinfI. The A. kolomikta×A. chinensis cross was checked for the RFLP patterns of sequences encoding the spacers between trnT and the 5′-trnL exon (a-b spacer DNA) and the trnL 3′ exon and trnF (e-f spacer DNA), respectively. The first spacer revealed a natural polymorphism between the two parent species due to a large deletion occurring in A. kolomikta detectable without further restriction enzyme treatment. The e-f spacer DNA was digested with HinfI. The comparison of the RFLP patterns in the parents and their progeny showed a strictly paternal inheritance of chloroplast DNA in Actinidia, with no exception found in any of the crosses examined. As the reciprocal crosses were not available, we do not know whether paternal inheritance of plastids is restricted to the crosses we analysed or if this is the general rule for plastid inheritance in the genus Actinidia. Actinidia is dioecious and is the first purely outbreeding species for which a paternal plastid inheritance has so far been documented.     

Characterisation of microsatellites from Actinidia chinensis

We have identified a set of informative microsatellite markers for genome analysis in kiwifruit and related Actinidia species. A small-insert genomic library was constructed from Actinidia chinensis DNA, and screened for microsatellites. About 1.2% of the total colonies hybridised to a (GA)₈ probe, 0.4% to (GT)₈, and 0.1% to a mixture of three different trinucleotide repeat probes, (CAA)₅, (GAA)₅ and (CTA)₅. From the DNA sequences of 35 hybridising clones, 18 primer pairs were designed, and used to amplify genomic DNA from 38 individual plants, representing 30 different accessions of ten Actinidia species. The banding patterns for most of the dinucleotide repeats showed a high degree of polymorphism in the diploid and tetraploid A. chinensis, and in the hexaploid A. deliciosa (kiwifruit). Heterozygosity levels of up to 100% were found among eight diploid accessions of A. chinensis examined, and the number of different-sized bands among all the species varied from 3 to 36 for each microsatellite. One simple CT microsatellite gave 21 bands with sizes suggesting that the number of repeats ranged from 9 to 37. The highest number of bands (36) and the largest size variation (>100 bp) were observed with a complex microsatellite harbouring four different repeat motifs. The majority of primer pairs amplified bands from most of the ten Actinidia species tested. The most polymorphic primer pairs were used successfully to fingerprint a range of closely related varieties of kiwifruit (A. deliciosa).Abbreviations PCR polymerase chain reaction - RFLP restriction fragment length polymorphism - VNTR variable number of tandem repeats     

Genetic variation and natural hybridization among sympatric <Emphasis Type="Italic">Actinidia</Emphasis> species and the implications for introgression breeding of kiwifruit

The overlapping ranges of closely related species provide a natural setting for the investigation of reticulate hybridization and other evolutionary processes. In the present study, we examined the pattern of genetic variation and interspecific gene flow in seven Actinidia species across ten localities in which sympatry among at least two species occurs. Our results showed that 48.7% of the alleles across the nine nuclear microsatellite loci examined were shared among the seven Actinidia species. Moreover, at the species level, Actinidia chinensis and Actinidia deliciosa exhibited the highest genetic similarity, with a large percentage of shared alleles (P _s = 81.3%) and a significant consistency between the distribution frequency of their allele sizes (r = 0.859, P = 0.045). Yet, the genetic distinctions between species are obvious except for the species pair A. chinensis and A. deliciosa. Interspecific introgression was detected among the two main species pairs (Actinidia latifolia–Actinidia eriantha and A. chinensis–A. deliciosa), but more apparent in the latter in which 30% of the A. chinensis individuals and 49% of the A. deliciosa individuals showed genetic admixture in the STRUCTURE analysis. Possibly active hybrid zones relating to the two main species pairs were discussed at last, which are expected to pave the way for the introgression breeding of kiwifruit from natural sympatric populations.     

Restriction-site variation of PCR-amplified chloroplast DNA regions and its implication for the evolution and taxonomy of Actinidia

 Twenty six restriction sites from five PCR-amplified chloroplast DNA sequences (rbcL, psbA, rpoB, and two spacers flanking the trnL gene) were mapped and analysed in 20 Actinidia taxa, encompassing all four sections into which the genus is divided. At least three species out of the 20 examined have been found to have originated through natural interspecific hybridisation on the basis of the discrepancy between morphological and biochemical traits and the cpDNA profiles of pairs of species. A widely reticulate evolution has therefore been postulated in Actinidia. Wagner and weighted parsimony analysis produced consensus trees that did not match the traditional taxonomy based on morphological characters. The molecular data clearly showed that some taxa, such as A. rufa and A. kolomikta, occupy a wrong position and most, if not all, of the traditional groups represented by sections and series are weakly supported, since they appear as polyphyletic. A. chinensis and A. deliciosa were confirmed to be very closely related. Since chloroplast DNA is paternally inherited in Actinidia, A. chinensis is a paternal progenitor, if not the only one, of A. deliciosa, the domesticated kiwifruit. Received: 18 August 1997 / Accepted: 6 October 1997     

Cross-species amplification of microsatellite loci within the dioecious, polyploid genus Actinidia (Actinidiaceae)

Microsatellite marker transfer across species in the dioecious genus Actinidia (kiwifruit) could offer an efficient and time-effective technique for use during trait transfer for vine and fruit improvement in breeding programmes. We evaluated the cross-species amplification of 20 EST-derived microsatellite markers that were fully informative in an Actinidia chinensis mapping family. We tested all 20 markers on 120 genotypes belonging to 21 species, 5 with varieties and/or chromosome races. These 26 taxa included 16 diploids, 7 tetraploids, 2 hexaploids and 1 octaploid, and represented all four taxonomic sections in the genus. All 20 markers showed some level of cross-species amplification. The most successful marker amplified in all genotypes from all species from all sections of the genus, the least successful amplified fragments only in A. chinensis and A. deliciosa. One species, A. glaucophylla, failed to amplify with all but 2 markers. PIC (Polymorphism information content) values were high, with 14 of 17 markers recording values of 0.90 and above. Sequence data demonstrated the presence of the microsatellite in all the amplified products. Sequence homology was less 5′ of the microsatellite and increased toward the start codon of the translated region of the EST from which the marker was derived. The data confirm that EST-derived microsatellite markers from Actinidia species show cross-species amplification with high levels of polymorphism which could make them useful markers in breeding programmes.     

Maternal inheritance of mitochondrial genomes and complex inheritance of chloroplast genomes in Actinidia Lind.: evidences from interspecific crosses

The inheritance pattern of chloroplast and mitochondria is a critical determinant in studying plant phylogenetics, biogeography and hybridization. To better understand chloroplast and mitochondrial inheritance patterns in Actinidia (traditionally called kiwifruit), we performed 11 artificial interspecific crosses and studied the ploidy levels, morphology, and sequence polymorphisms of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) of parents and progenies. Sequence analysis showed that the mtDNA haplotypes of F1 hybrids entirely matched those of the female parents, indicating strictly maternal inheritance of Actinidia mtDNA. However, the cpDNA haplotypes of F1 hybrids, which were predominantly derived from the male parent (9 crosses), could also originate from the mother (1 cross) or both parents (1 cross), demonstrating paternal, maternal, and biparental inheritance of Actinidia cpDNA. The inheritance patterns of the cpDNA in Actinidia hybrids differed according to the species and genotypes chosen to be the parents, rather than the ploidy levels of the parent selected. The multiple inheritance modes of Actinidia cpDNA contradicted the strictly paternal inheritance patterns observed in previous studies, and provided new insights into the use of cpDNA markers in studies of phylogenetics, biogeography and introgression in Actinidia and other angiosperms.     

Induced polyploidy dramatically increases the size and alters the shape of fruit in Actinidia chinensis

Background and Aims

Some otherwise promising selections of Actinidia chinensis (kiwifruit) have fruit that are too small for successful commercialization. We have therefore made the first detailed study in diploid kiwifruit of the effects of chromosome doubling induced by colchicine on fruit size, shape and crop loading.

Methods

Flow cytometric analysis of young leaves and chromosome analysis of flower buds and root tips was used to confirm the stability of induced autotetraploids. Fruit weight, size and crop load were measured in the third year after planting in the field and for three consecutive years. DNA fingerprinting was used to confirm the origin of the material.

Key Results

There was a very significant increase in fruit size in induced autotetraploids of different genotypes of A. chinensis. With the commercially important diploid cultivar ‘Hort16A’, most regenerants, Type A plants, had fruit which were much the same shape as fruit of the diploid but, at the same fruit load, were much larger and heavier. Some regenerants, Type B plants, produced fruit similar to ‘fasciated’ fruit. Fruit of the autotetraploids induced from three female red-fleshed A. chinensis selections were also 50–60 % larger than fruit of their diploid progenitors. The main increase in fruit dimensions was in their diameters. These improved fruit characteristics were stable over several seasons.

Conclusions

Chromosome doubling has been shown to increase significantly fruit size in autotetraploid A. chinensis, highlighting the considerable potential of this technique to produce new cultivars with fruit of adequate size. Other variants with differently shaped fruit were also produced but the genetic basis of this variation remains to be elucidated. Autoploids of other Actinidia species with commercial potential may also show improved fruit characteristics, opening up many new possibilities for commercial development.     

Exon primed intron-crossing (EPIC) markers reveal natural hybridization and introgression in Actinidia (Actinidiaceae) with sympatric distribution

Interspecific hybridization is widespread among plants. Understanding the phylogenetic relationships among species is necessary for revealing the potential hybridization events. Actinidia, best known as kiwifruit genus found throughout a wide range in eastern Asian from Indonesia to Siberia. In this study, phylogenetic relationships of Actinidia species with sympatric distributions were investigated using three chloroplast introns (trnL-F, atpB-rbcL and rpl32-trnL) and three Exon primed intron-crossing (EPIC) markers. Chloroplast phylogeny supports non-monophyly of the five species studied excluding Actinidia fulvicoma var. lanata. The non-monophyly was also revealed by EPIC markers. Our results showed EPIC markers are more variable and informative for phylogenetic inference than that of chloroplast markers. The incongruences between loci from the plastid and nuclear DNA phylogenic trees may stem from incomplete lineage sorting or historical introgression hybridization. Incomplete lineage sorting may explain the non-monophyly between Actinidia chrysantha (section Maculatae) and other four species (section Stellatae), and introgression hybridization and high level of interspecific gene flow may explain the non-monophyly among the species of sect. Stellatae. Thus, natural hybridization and introgression may be common in Actinidia with sympatric distribution.     

In situ hybridization in Actinidia using repeat DNA and genomic probes

 In situ hybridization has been used to probe chromosome spreads of hexaploid Actinidia deliciosa (kiwifruit; 2n=6x=174) and tetraploid A. chinensis (2n=4x=116). When a species-specific repeat sequence, pKIWI516, was used, six hybridization sites were observed in some accessions of tetraploid A. chinensis and all of A. deliciosa. Southern analysis with the pKIWI516 probe revealed that there are two types of tetraploid A. chinensis. Genomic probes from diploid A. chinensis (2n=2x=58) did not differentiate the genomes of hexaploid A. deliciosa and tetraploid A. chinensis, irrespective of the presence or absence of blocking DNA. The results indicate that the genomes of polyploid Actinidia species are similar but not identical. The origin of A. deliciosa is discussed. Received: 29 June 1996 / Accepted: 5 July 1996     

A genome-specific repeat sequence from kiwifruit (Actinidia deliciosa var. deliciosa)

Summary Six members of a family of moderately repetitive DNA sequences from kiwifruit (Actinidia deliciosa var. deliciosa) have been cloned and characterized. The repeat family is composed of elements that have a unit length of 463 bp, are highly methylated, occur in tandem arrays of at least 50 kb in length, and constitute about 0.5% of the kiwifruit genome. Individual elements diverge in nucleotide sequence by up to 5%, which suggests that the repeat sequence is evolving rapidly. Homologous sequences were found in A. deliciosa var. chlorocarpa. The repeat sequence was not found under low stringency hybridization conditions in the diploid A. chinensis, the species most closely related to the hexaploid kiwifruit, or in eight other Actinidia species. However, homologous repeats were detected in a tetraploid species, A. chrysantha. The results provide the first molecular evidence to suggest that kiwifruit may be an allopolyploid species.     

Correlation between population size of pear Psylla (Cacopsylla chinensis) and leaf structure features in different pear cultivars

The feeding preference of pear psylla (Cacopsylla chinensis) varies among pear cultivars. To clarify whether leaf structural factors are related to C. chinensis resistance, eleven pear cultivars of three pear species and their hybrids in China were studied. The population size of C. chinensis in plots of different cultivars was investigated, and leaf paraffin sections of each pear cultivar were observed. Correlation analysis results showed that the thickness of palisade tissue (PT) and the thickness ratio (TR) of PT to leaf (L) were the main factors influencing resistance to C. chinensis. The leaves of cultivar Wujiuxiang and those species from France and the USA exhibited the thickest PT and the highest TR; these three cultivars demonstrated much higher resistance to C. chinensis than did the other cultivars. The cultivar Gold Nijisseiki, with a large C. chinensis population size, showed low PT and TR. The findings of this study may provide valuable information to pear growers, experts and scientists regarding plant protection, cultivation and breeding when selecting suitable varieties to culture from the perspective of pest control.     

The changes in water-use efficiency and stoma density of Leymus chinensis along Northeast China Transect

Leymus chinensis is a keystone species in the temperate zone grassland of China. Along the NECT (Northeast China Transect) in 2001, water-use efficiency of L. chinensis was analyzed with δ¹³C, and changes in the stoma density of its leaves were observed and computed under a microscope. Results showed that the ecological plasticity modulation of the stoma density of L. chinensis and its water-use efficiency were two important mechanisms for its broad ecological adaptability. From east to west along the NECT, the δ¹³C of the species varied from -27.49‰ to -23.57‰, consisting with the reduction of annual precipitation, soil water and annual average temperature, but increased with the increase of the elevation of sampling sites. The stoma density increased from 96.9/mm² to 169.5/mm², indicating that the water-use efficiency for the species was improved along the gradient. The linear coefficient between the two parameters was significant (R² = 0.7338). The results of a stepwise regression analysis showed that the soil water was the first marked factor for determining the stoma density, and the next was the annual precipitation, which suggested that the water factors were the primary ecological factors influencing the stoma density of L. chinensis. The findings in this study showed that the responses of the stoma density and the water-use efficiency for L. chinensis to environmental changes were very complicated. They may be the outcome operated synthetically by all environmental factors in the long-term adaptation to different ecological environments, including human activity, for L. chinensis.