The genomes of Darwin's primroses reveal chromosome-scale adaptive introgression and differential permeability of species boundaries.

Introgression is an important source of genetic variation that can determine species adaptation to environmental conditions. Yet, definitive evidence of the genomic and adaptive implications of introgression in nature remains scarce. The widespread hybrid zones of Darwin's primroses (Primula elatior, Primula veris, and Primula vulgaris) provide a unique natural laboratory for studying introgression in flowering plants and the varying permeability of species boundaries. Through analysis of 650 genomes, we provide evidence of an introgressed genomic region likely to confer adaptive advantage in conditions of soil toxicity. We also document unequivocal evidence of chloroplast introgression, an important precursor to species-wide chloroplast capture. Finally, we provide the first evidence that the S-locus supergene, which controls heterostyly in primroses, does not introgress in this clade. Our results contribute novel insights into the adaptive role of introgression and demonstrate the importance of extensive genomic and geographical sampling for illuminating the complex nature of species boundaries.

Parallel evolution of morphological and genomic selfing syndromes accompany the breakdown of heterostyly.

Evolutionary transitions from outcrossing to selfing in flowering plants have convergent morphological and genomic signatures and can involve parallel evolution within related lineages. Adaptive evolution of morphological traits is often assumed to evolve faster than nonadaptive features of the genomic selfing syndrome. We investigated phenotypic and genomic changes associated with transitions from distyly to homostyly in the Primula oreodoxa complex. We determined whether the transition to selfing occurred more than once and investigated stages in the evolution of morphological and genomic selfing syndromes using 22 floral traits and both nuclear and plastid genomic data from 25 populations. Two independent transitions were detected representing an earlier and a more recently derived selfing lineage. The older lineage exhibited classic features of the morphological and genomic selfing syndrome. Although features of both selfing syndromes were less developed in the younger selfing lineage, they exhibited parallel development with the older selfing lineage. This finding contrasts with the prediction that some genomic changes should lag behind adaptive changes to morphological traits. Our findings highlight the value of comparative studies on the timing and extent of transitions from outcrossing to selfing between related lineages for investigating the tempo of morphological and molecular evolution.

Comparative Genomics Elucidates the Origin of a Supergene Controlling Floral Heteromorphism.

Supergenes are nonrecombining genomic regions ensuring the coinheritance of multiple, coadapted genes. Despite the importance of supergenes in adaptation, little is known on how they originate. A classic example of supergene is the S locus controlling heterostyly, a floral heteromorphism occurring in 28 angiosperm families. In Primula, heterostyly is characterized by the cooccurrence of two complementary, self-incompatible floral morphs and is controlled by five genes clustered in the hemizygous, ca. 300-kb S locus. Here, we present the first chromosome-scale genome assembly of any heterostylous species, that of Primula veris (cowslip). By leveraging the high contiguity of the P. veris assembly and comparative genomic analyses, we demonstrated that the S-locus evolved via multiple, asynchronous gene duplications and independent gene translocations. Furthermore, we discovered a new whole-genome duplication in Ericales that is specific to the Primula lineage. We also propose a mechanism for the origin of S-locus hemizygosity via nonhomologous recombination involving the newly discovered two pairs of CFB genes flanking the S locus. Finally, we detected only weak signatures of degeneration in the S locus, as predicted for hemizygous supergenes. The present study provides a useful resource for future research addressing key questions on the evolution of supergenes in general and the S locus in particular: How do supergenes arise? What is the role of genome architecture in the evolution of complex adaptations? Is the molecular architecture of heterostyly supergenes across angiosperms similar to that of Primula?

Whole-genome analyses disentangle reticulate evolution of primroses in a biodiversity hotspot.

Biodiversity hotspots, such as the Caucasus mountains, provide unprecedented opportunities for understanding the evolutionary processes that shape species diversity and richness. Therefore, we investigated the evolution of Primula sect. Primula, a clade with a high degree of endemism in the Caucasus. We performed phylogenetic and network analyses of whole-genome resequencing data from the entire nuclear genome, the entire chloroplast genome, and the entire heterostyly supergene. The different characteristics of the genomic partitions and the resulting phylogenetic incongruences enabled us to disentangle evolutionary histories resulting from tokogenetic vs cladogenetic processes. We provide the first phylogeny inferred from the heterostyly supergene that includes all species of Primula sect. Primula. Our results identified recurrent admixture at deep nodes between lineages in the Caucasus as the cause of non-monophyly in Primula. Biogeographic analyses support the 'out-of-the-Caucasus' hypothesis, emphasizing the importance of this hotspot as a cradle for biodiversity. Our findings provide novel insights into causal processes of phylogenetic discordance, demonstrating that genome-wide analyses from partitions with contrasting genetic characteristics and broad geographic sampling are crucial for disentangling the diversification of species-rich clades in biodiversity hotspots.

Different molecular changes underlie the same phenotypic transition: Origins and consequences of independent shifts to homostyly within species.

The repeated transition from outcrossing to selfing is a key topic in evolutionary biology. However, the molecular basis of such shifts has been rarely examined due to lack of knowledge of the genes controlling these transitions. A classic example of mating system transition is the repeated shift from heterostyly to homostyly. Occurring in 28 angiosperm families, heterostyly is characterized by the reciprocal position of male and female sexual organs in two (or three) distinct, usually self-incompatible floral morphs. Conversely, homostyly is characterized by a single, self-compatible floral morph with reduced separation of male and female organs, facilitating selfing. Here, we investigate the origins of homostyly in Primula vulgaris and its microevolutionary consequences by integrating surveys of the frequency of homostyles in natural populations, DNA sequence analyses of the gene controlling the position of female sexual organs (CYPᵀ), and microsatellite genotyping of both progeny arrays and natural populations characterized by varying frequencies of homostyles. As expected, we found that homostyles displace short-styled individuals, but long-style morphs are maintained at low frequencies within populations. We also demonstrated that homostyles repeatedly evolved from short-styled individuals in association with different types of loss-of-function mutations in CYPᵀ. Additionally, homostyly triggers a shift to selfing, promoting increased inbreeding within and genetic differentiation among populations. Our results elucidate the causes and consequences of repeated transitions to homostyly within species, and the putative mechanisms precluding its fixation in P. vulgaris. This study represents a benchmark for future analyses of losses of heterostyly in other angiosperms.

Supergene evolution via stepwise duplications and neofunctionalization of a floral-organ identity gene.

Heterostyly represents a fascinating adaptation to promote outbreeding in plants that evolved multiple times independently. While l-morph individuals form flowers with long styles, short anthers, and small pollen grains, S-morph individuals have flowers with short styles, long anthers, and large pollen grains. The difference between the morphs is controlled by an S-locus "supergene" consisting of several distinct genes that determine different traits of the syndrome and are held together, because recombination between them is suppressed. In Primula, the S locus is a roughly 300-kb hemizygous region containing five predicted genes. However, with one exception, their roles remain unclear, as does the evolutionary buildup of the S locus. Here we demonstrate that the MADS-box GLOBOSA2 (GLO2) gene at the S locus determines anther position. In Primula forbesii S-morph plants, GLO2 promotes growth by cell expansion in the fused tube of petals and stamen filaments beneath the anther insertion point; by contrast, neither pollen size nor male incompatibility is affected by GLO2 activity. The paralogue GLO1, from which GLO2 arose by duplication, has maintained the ancestral B-class function in specifying petal and stamen identity, indicating that GLO2 underwent neofunctionalization, likely at the level of the encoded protein. Genetic mapping and phylogenetic analysis indicate that the duplications giving rise to the style-length-determining gene CYP734A50 and to GLO2 occurred sequentially, with the CYP734A50 duplication likely the first. Together these results provide the most detailed insight into the assembly of a plant supergene yet and have important implications for the evolution of heterostyly.

Effects of 24-Epibrassinolide, melatonin and their combined effect on cadmium tolerance in Primula forbesii Franch.

This study aimed to investigate the interaction between 24-Epibrassinolide (EBR) and melatonin (MT) and their effects on cadmium (Cd)-stressed Primula forbesii Franch. P. forbesii seedlings were hydroponically acclimatized at 6-7 weeks, then treated with Cd (200 µmol L-1), 24-EBR (0.1 µmol L-1), and MT (100 µmol L-1) after two weeks. Cd stress significantly reduced crown width, shoot, root length, shoot fresh weight, and fresh and dry root weights. Herein, 24-EBR, MT, and 24-EBR+MT treatments attenuated the growth inhibition caused by Cd stress and improved the morphology, growth indexes, and ornamental characteristics of P. forbesii under Cd stress. 24-EBR had the best effect by effectively alleviating Cd stress and promoting plant growth and development. 24-EBR significantly increased all growth parameters compared to Cd treatment. In addition, 24-EBR significantly improved the gas exchange parameters, activities of antioxidant enzymes, and the cycle efficiency of AsA-GSH. Furthermore, 24-EBR increased the activities of ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase (MDHAR) by 127.29%, 61.31%, 61.22%, and 51.04%, respectively, compared with the Cd treatment. Therefore, 24-EBR removed the reactive oxygen species produced by stress, thus protecting plants against stress damage. These results indicate that 24-EBR can effectively enhance the tolerance of P. forbesii to Cd stress.

Transcriptome Analyses Reveal the Aroma Terpeniods Biosynthesis Pathways of Primula forbesii Franch. and the Functional Characterization of the PfDXS2 Gene.

Primula forbesii Franch. is a unique biennial herb with a strong floral fragrance, making it an excellent material for studying the aroma characteristics of the genus Primula. The floral scent is an important ornamental trait that facilitates fertilization. However, the molecular mechanism regulating the floral scent in Primula is unknown. In order to better understand the biological mechanisms of floral scents in this species, this study used RNA sequencing analysis to discuss the first transcriptome sequence of four flowering stages of P. forbesii, which generated 12 P. forbesii cDNA libraries with 79.64 Gb of clean data that formed 51,849 unigenes. Moreover, 53.26% of the unigenes were annotated using public databases. P. forbesii contained 44 candidate genes covering all known enzymatic steps for the biosynthesis of volatile terpenes, the major contributor to the flower's scent. Finally, 1-deoxy-d-xylulose 5-phosphate synthase gene of P. forbesii (PfDXS2, MK370094), the first key enzyme gene in the 2-c-methyl-d-erythritol 4-phosphate (MEP) pathway of terpenoids, was cloned and functionally verified using virus-induced gene silencing (VIGs). The results showed that PfDXS2-silencing significantly reduced the relative concentrations of main volatile terpenes. This report is the first to present molecular data related to aroma metabolites biosynthesis pathways and the functional characterization of any P. forbesii gene. The data on RNA sequencing provide comprehensive information for further analysis of other plants of the genus Primula.

The ameliorative effect of Primula vulgaris on cisplatin-induced nephrotoxicity in rats and quantification of its phenolic components using LC-ESI-MS/MS.

Cisplatin (CDDP) is an important chemotherapeutic agent, accumulation of which in kidney tissue causes nephrotoxicity and renal failure. The aim of this study was to evaluate, for the first time in the literature, the protective effect of dimethyl sulfoxide (DMSO) extract of Primula vulgaris leaf (PVE) against CDDP-induced nephrotoxicity in rats. The PVE content was characterized using liquid chromatography-mass spectrometry. Nephrotoxicity was induced with a single dose of CDDP (7.5 mg/kg). Thirty female Wistar-Albino rats were divided into five groups (control, DMSO, CDDP (7.5 mg/kg), CDDP + PVE (25 mg/kg), and CDDP + PVE (50 mg/kg)). Biochemical and histopathological analyses were then performed. Rutin, gallic acid, p-coumaric acid and protocatechuic acid were identified as major components of PVE. Total antioxidant status and glutathione (GSH) values increased significantly in the serum samples from the treatment group compared to the CDDP group, while blood urea nitrogen, creatinine, oxidative stress index, malondialdehyde (MDA), tumor necrosis factor-alpha (TNF-α), total oxidant status, and 8-hydroxy-2'-deoxyguanosine (8-OHdG) values decreased significantly. GSH levels increased significantly in the treatment group compared to the CDDP group, while TNF-α, caspase-3, 8-OHdG, MDA levels and damage scores decreased significantly. In conclusion, PVE exhibited strong protective effects through its anti-apoptotic, antioxidant, and anti-inflammatory activities against nephrotoxicity and oxidative damage caused by CDDP in rats.

The Genomic Selfing Syndrome Accompanies the Evolutionary Breakdown of Heterostyly.

The evolutionary transition from outcrossing to selfing can have important genomic consequences. Decreased effective population size and the reduced efficacy of selection are predicted to play an important role in the molecular evolution of the genomes of selfing species. We investigated evidence for molecular signatures of the genomic selfing syndrome using 66 species of Primula including distylous (outcrossing) and derived homostylous (selfing) taxa. We complemented our comparative analysis with a microevolutionary study of P. chungensis, which is polymorphic for mating system and consists of both distylous and homostylous populations. We generated chloroplast and nuclear genomic data sets for distylous, homostylous, and distylous-homostylous species and identified patterns of nonsynonymous to synonymous divergence (dN/dS) and polymorphism (πN/πS) in species or lineages with contrasting mating systems. Our analysis of coding sequence divergence and polymorphism detected strongly reduced genetic diversity and heterozygosity, decreased efficacy of purifying selection, purging of large-effect deleterious mutations, and lower rates of adaptive evolution in samples from homostylous compared with distylous populations, consistent with theoretical expectations of the genomic selfing syndrome. Our results demonstrate that self-fertilization is a major driver of molecular evolutionary processes with genomic signatures of selfing evident in both old and relatively young homostylous populations.

Asymmetries of reproductive isolation are reflected in directionalities of hybridization: integrative evidence on the complexity of species boundaries.

The complex nature of species boundaries has been a central topic in evolutionary biology ever since Darwin. Despite numerous separate studies on reproductive isolation and hybridization, their relationship remains underinvestigated. Are the strengths and asymmetries of reproductive barriers reflected in the extent and directionalities of interspecific genetic exchange? We combined field, experimental, and molecular data to quantify strengths and asymmetries of sympatric reproductive barriers and hybridization between florally heteromorphic primroses. We also assessed whether generalist pollinators discriminate between different floral cues and contribute to reproductive isolation, a long-debated topic. Sympatric reproductive isolation is high but incomplete, and most phenotypic intermediates are genetic F1 hybrids, whereas backcrosses are rare, revealing low interspecific gene flow. Species integrity rests on multiple barriers, but ethological isolation is among the strongest, demonstrating that even generalist pollinators crucially contribute to the maintenance of species boundaries. Furthermore, reproductive barriers are weaker for Primula veris and short-styled plants, results corroborated by molecular data. Thus, in florally heteromorphic systems, both species- and morph-dependent asymmetries affect permeability of species boundaries. Our study illustrates how the interactions between complex floral syndromes and pollinators shape species boundaries in unique, previously undescribed ways.

Very-Long-Chain Wax Constituents from Primula veris and P. acaulis: Does the Paradigm of Non-Branched vs. Branched Chain Dominance Universally Hold in all Plant Taxa?

Herein n-, iso- and anteiso-series of very-long-chained (VLC) alkanes (C21 -C35 ), fatty acid benzyl esters (FABEs; C20 -C32 ), and 2-alkanones (C23 -C35 ) were identified in the wax of Primula veris L. and P. acaulis (L.) L. (Primulaceae). For the very first time in a sample of natural origin, the presence of iso- and anteiso-VLC FABEs and 2-alkanones was unequivocally confirmed by synthetic work, derivatization, and NMR. It should be noted that the studied species produced unusually high amounts of branched wax constituents (e. g., >50 % of 2-alkanones were branched isomers). The domination of iso-isomers, probably biosynthesized from leucine-derived starters, is a unique feature in the Plant Kingdom. The plant organ distribution of these VLC compounds in P. acaulis samples (different habitats and phenological phases) pointed to their possible ecological value. This was supported by a eutectic behavior of binary blends of FABEs and alkanes, as well as by high UV-C absorption by FABEs.

Phylogenomic analysis reveals multiple evolutionary origins of selfing from outcrossing in a lineage of heterostylous plants.

Evolutionary transitions from outcrossing to selfing often occur in heterostylous plants. Selfing homostyles originate within distylous populations and frequently evolve to become reproductively isolated species. We investigated this process in 10 species of Primula section Obconicolisteri using phylogenomic approaches and inferred how often homostyly originated from distyly and its consequences for population genetic diversity and floral trait evolution. We estimated phylogenetic relationships and reconstructed character evolution using the whole plastome comprised of 76 protein-coding genes. To investigate mating patterns and genetic diversity we screened 15 microsatellite loci in 40 populations. We compared floral traits among distylous and homostylous populations to determine how phenotypically differentiated homostyles were from their distylous ancestors. Section Obconicolisteri was monophyletic and we estimated multiple independent transitions from distyly to homostyly. High selfing rates characterised homostylous populations and this was associated with reduced genetic diversity. Flower size and pollen production were reduced in homostylous populations, but pollen size was significantly larger in some homostyles than in distylous morphs. Repeated transitions to selfing in section Obconicolisteri are likely to have been fostered by the complex montane environments that species occupy. Unsatisfactory pollinator service is likely to have promoted reproductive assurance in homostyles leading to subsequent population divergence through isolation.

Fiber-optic refractometer for in vivo sugar concentration measurements of low-nectar-producing flowers.

Sugar concentration in floral nectars is an assessment required in several diverse fields of application. The widely used analysis, consisting of nectar extraction with a microcapillary and sugar concentration measurement with a light refractometer, is not reliable when the nectar is secreted in small quantities, unextractable with a microcapillary. Ancillary methods adopted in such cases are destructive, rather complicated and often provide much less precise and accurate results. The microscopic-size, low cost and biocompatibility of optical fibers were exploited to deliver light directly inside the flower with minimal invasiveness and measure instantaneously the refractometric properties of the nectar without extracting it. After comparing the new and old methods using two known nectariferous species, the new approach was validated on Primula palinuri, whose nectar is unextractable with microcapillaries. The fiber-optic probe was able to measure the nectar refractive index in P. palinuri flowers making it possible to highlight a previously undetected significant trend of the sugar concentration throughout the long anthesis of the single flowers. Changes in nectar concentrations are similar in both longistylous and brevistylous flowers. The fiber-optic refractometer is an advancement of light refractometer analysis. Further customization of the laboratory set-up into portable equipment will boost applications.

Assessment of the Bioactive Compounds in White and Red Wines Enriched with a Primula veris L.

The aim of this paper was to analyze selected physicochemical properties and the pro-health potential of wines produced in southeastern Poland, in the Subcarpathian region, and commercial Carlo Rossi wines enhanced with cowslip (Primula veris L.). This study used ultra-performance reverse-phase liquid chromatography (UPLC)-PDA-MS/MS to perform most of the analysis, including the polyphenolic compounds and saponin content in wines enriched by Primula veris L. The initial anthocyanin content in Subcarpathian (Regional) red wine samples increased four times to the level of 1956.85 mg/L after a 10% addition of Primula veris L. flowers. For white wines, a five-fold increase in flavonol content was found in Subcarpathian (Regional) and wine samples, and an almost 25-fold increase in flavonol content was found in Carlo Rossi (Commercial) wine samples at the lowest (2.5%) Primula veris L. flower addition. Qualitative analysis of the regional white wines with a 10% Primula veris L. flower enhancement demonstrated the highest kaempferol content (197.75 mg/L) and a high quercetin content (31.35 mg/L). Thanks to wine enrichment in triterpenoid saponins and in polyphenolic compounds from Primula veris L. flowers, which are effectively extracted to wine under mild conditions, both white and red wines can constitute a highly pro-health component of diets, which is valuable in preventing chronic heart failure.

Lanostane-Type Saponins from Vitaliana primuliflora.

The phytochemistry of the genera Androsace, Cortusa, Soldanella, and Vitaliana, belonging to the Primulaceae family is not well studied so far. Hence, in this paper, we present the results of UHPLC-MS/MS analysis of several primrose family members as well as isolation and structure determination of two new saponins from Vitaliana primuliflora subsp. praetutiana. These two nor-triterpenoid saponins were characterized as (23S)-17α,23-epoxy-29-hydroxy-3ß-[(O-ß-d-glucopyranosyl-(1→2)-O-α-l-rhamnopyranosyl-(1→2)-O-ß-d-glucopyranosyl-(1→2)-O-α-l-arabinopyranosyl-(1→6)-ß-d-glucopyranosyl)oxy]-27-nor-lanost-8-en-25-one and (23S)-17α,23-epoxy-29-hydroxy-3ß-[(O-α-l-rhamnopyranosyl-(1→2)-O-ß-d-glucopyranosyl-(1→2)-O-α-l-arabinopyranosyl-(1→6)-ß-d-glucopyranosyl)oxy]-27-nor-lanost-8-en-25-one, respectively. Their structures were determined by high resolution mass spectrometry (HRMS), tandem mass spectrometry (MS/MS), one- and two-dimensional nuclear magnetic resonance spectroscopy (1D-, and 2D-NMR) analyses. So far, the 27-nor-lanostane monodesmosides were rarely found in dicotyledon plants. Therefore their presence in Vitaliana and also in Androsace species belonging to the Aretia section is unique and reported here for the first time. Additionally, eleven other saponins were determined by HRMS and MS/MS spectra. The isolated lanostane saponins can be considered as chemotaxonomic markers of the family Primulaceae.

Comparative transcriptome analyses reveal genes related to pigmentation in the petals of red and white Primula vulgaris cultivars.

Primula vulgaris is an important ornamental plant species with various flower color. To explore the molecular mechanism of its color formation, comparative transcriptome analyses of the petals in red and white cultivars was performed. A total of 4451 differentially expressed genes were identified and annotated into 128 metabolic pathways. Candidate genes FLS, F3'H, DFR, ANS and AOMT in the anthocyanin pathway were expressed significantly higher in the red cultivar than the white and may be responsible for the red coloration. In the red petals, a putative transcription factors bHLH (c52273.graph_c0) was up-regulated about 14-fold, while a R2R3-MYB unigene (c36140.graph_c0) was identified as a repressor involved in anthocyanin regulation and was significantly down-regulated. In addition, the anatomy analyses and pigments composition in the red and white petals were also analyzed. The papillae on the adaxial epidermis of the red petals of P. vulgaris display a triangle-shapes, in contrast with a spherical shape for the white petals. Although flavonoids were detected in both cultivars, anthocyanins could only be identified in the red cultivar. Gossypetin and peonidin/rosinin were the most abundant pigments in red petals. This study shed light on the genetic and biochemistry mechanisms underlying the flower coloration in Primula.

Grazers affect selection on inflorescence height both directly and indirectly and effects change over time.

Selection mediated by one biotic agent will often be modified by the presence of other biotic interactions, and the importance of such indirect effects might change over time. We conducted an 11-yr field experiment to test the prediction that large grazers affect selection on floral display of the dimorphic herb Primula farinosa not only directly through differential grazing damage, but also indirectly by affecting vegetation height and thereby selection mediated by pollinators and seed predators. Exclusion of large grazers increased vegetation height and the strength of pollinator-mediated selection for tall inflorescences and seed-predator-mediated selection for short inflorescences. The direct effect of grazers on selection resulting from differential grazing damage to the two scape morphs showed no temporal trend. By contrast, the increase in vegetation height in exclosures over time was associated with an increase in selection mediated by pollinators and seed predators. In the early years of the experiment, the indirect effects of grazers on selection mediated by pollinators and seed predators were weak, whereas at the end of the experiment, the indirect effects were of similar magnitude as the direct effect due to differential grazing damage. The results demonstrate that the indirect effects of a selective agent can be as strong as its direct effects, and that the relative importance of direct vs. indirect effects on selection can change over time. A full understanding of the ecological processes governing variation in selection thus requires that both direct and indirect effects of biotic interactions are assessed.

Effects of climate warming and prolonged snow cover on phenology of the early life history stages of four alpine herbs on the southeastern Tibetan Plateau.

PREMISE OF THE STUDY: Much research has focused on plant responses to ongoing climate change, but there is relatively little information about how climate change will affect the early plant life history stages. Understanding how global warming and changes in winter snow pattern will affect seed germination and seedling establishment is crucial for predicting future alpine population and vegetation dynamics. METHODS: In a 2-year study, we tested how warming and alteration in the snowmelt regime, both in isolation and combination, influence seedling emergence phenology, first-year growth, biomass allocation, and survival of four native alpine perennial herbs on the southeastern Tibetan Plateau. KEY RESULTS: Warming promoted seedling emergence phenology of all four species and biomass per plant of two species but reduced seedling survival of three species. Prolonged snow cover partly mediated the affects of warming on Primula alpicola (survival and biomass), Pedicularis fletcheri (phenology, biomass, and root:shoot ratio) and Meconopsis integrifolia (survival). For the narrowly distributed species M. racemosa, seedling growth was additively decreased by warming and prolonged snow cover. CONCLUSIONS: Both warming and alteration of the snow cover regime can influence plant recruitment by affecting seedling phenology, growth, and survival, and the effects are largely species-specific. Thus, climate change is likely to affect population dynamics and community structure of the alpine ecosystem. This is the first experimental demonstration of the phenological advancement of seedling emergence in the field by simulated climate warming.

Comparative Plastid Genomes of Primula Species: Sequence Divergence and Phylogenetic Relationships.

Compared to traditional DNA markers, genome-scale datasets can provide mass information to effectively address historically difficult phylogenies. Primula is the largest genus in the family Primulaceae, with members distributed mainly throughout temperate and arctic areas of the Northern Hemisphere. The phylogenetic relationships among Primula taxa still maintain unresolved, mainly due to intra- and interspecific morphological variation, which was caused by frequent hybridization and introgression. In this study, we sequenced and assembled four complete plastid genomes (Primula handeliana, Primula woodwardii, Primula knuthiana, and Androsace laxa) by Illumina paired-end sequencing. A total of 10 Primula species (including 7 published plastid genomes) were analyzed to investigate the plastid genome sequence divergence and their inferences for the phylogeny of Primula. The 10 Primula plastid genomes were similar in terms of their gene content and order, GC content, and codon usage, but slightly different in the number of the repeat. Moderate sequence divergence was observed among Primula plastid genomes. Phylogenetic analysis strongly supported that Primula was monophyletic and more closely related to Androsace in the Primulaceae family. The phylogenetic relationships among the 10 Primula species showed that the placement of P. knuthiana-P. veris clade was uncertain in the phylogenetic tree. This study indicated that plastid genome data were highly effective to investigate the phylogeny.