Epigenetic architecture of Pseudotaxus chienii: Revealing the synergistic effects of climate and soil variables.

Whether conifers can withstand environmental changes especially temperature fluctuations has been controversial. Epigenetic analysis may provide new perspectives for solving the issue. Pseudotaxus chienii is an endangered gymnosperm species endemic to China. In this study, we have examined the genetic and epigenetic variations in its natural populations aiming to disentangle the synergistic effects of climate and soil on its population (epi)genetic differentiation by using amplified fragment length polymorphism (AFLP) and methylation-sensitive AFLP (MSAP) techniques. We identified 23 AFLP and 26, 7, and 5 MSAP outliers in P. chienii. Twenty-one of the putative adaptive AFLP loci were found associated with climate and/or soil variables including precipitation, temperature, K, Fe, Zn, and Cu, whereas 21, 7, and 4 MSAP outliers were significantly related to precipitation of wettest month (Bio13), precipitation driest of month (Bio14), percent tree cover (PTC), and soil Fe, Mn, and Cu compositions. Total precipitation and precipitation in the driest seasons were the most influential factors for genetic and epigenetic variation, respectively. In addition, a high full-methylation level and a strong correlation between genetic and epigenetic variation were detected in P. chienii. Climate is found of greater importance than soil in shaping adaptive (epi)genetic differentiation, and the synergistic effects of climate and climate-soil variables were also observed. The identified climate and soil variables should be considered when applying ex situ conservation.

EST-SSR-based landscape genetics of Pseudotaxus chienii, a tertiary relict conifer endemic to China.

Pseudotaxus chienii, belonging to the monotypic genus Pseudotaxus (Taxaceae), is a relict conifer endemic to China. Its populations are usually small and patchily distributed, having a low capacity of natural regeneration. To gain a clearer understanding of how landscape variables affect the local adaptation of P. chienii, we applied EST-SSR markers in conjunction with landscape genetics methods: (a) to examine the population genetic pattern and spatial genetic structure; (b) to perform genome scan and selection scan to identify outlier loci and the associated landscape variables; and (c) to model the ecological niche under climate change. As a result, P. chienii was found to have a moderate level of genetic variation and a high level of genetic differentiation. Its populations displayed a significant positive relationship between the genetic and geographical distance (i.e., "isolation by distance" pattern) and a strong fine-scale spatial genetic structure within 2 km. A putatively adaptive locus EMS6 (functionally annotated to cellulose synthase A catalytic subunit 7) was identified, which was found significantly associated with soil Cu, K, and Pb content and the combined effects of temperature and precipitation. Moreover, P. chienii was predicted to experience significant range contractions in future climate change scenarios. Our results highlight the potential of specific soil metal content and climate variables as the driving force of adaptive genetic differentiation in P. chienii. The data would also be useful to develop a conservation action plan for P. chienii.

Population transcriptomic sequencing reveals allopatric divergence and local adaptation in Pseudotaxus chienii (Taxaceae).

BACKGROUND: Elucidating the effects of geography and selection on genetic variation is critical for understanding the relative importance of adaptation in driving differentiation and identifying the environmental factors underlying its occurrence. Adaptive genetic variation is common in tree species, especially widely distributed long-lived species. Pseudotaxus chienii can occupy diverse habitats with environmental heterogeneity and thus provides an ideal material for investigating the process of population adaptive evolution. Here, we characterize genetic and expression variation patterns and investigate adaptive genetic variation in P. chienii populations. RESULTS: We generated population transcriptome data and identified 13,545 single nucleotide polymorphisms (SNPs) in 5037 unigenes across 108 individuals from 10 populations. We observed lower nucleotide diversity (π = 0.000701) among the 10 populations than observed in other gymnosperms. Significant negative correlations between expression diversity and nucleotide diversity in eight populations suggest that when the species adapts to the surrounding environment, gene expression and nucleotide diversity have a reciprocal relationship. Genetic structure analyses indicated that each distribution region contains a distinct genetic group, with high genetic differentiation among them due to geographical isolation and local adaptation. We used FST outlier, redundancy analysis, and latent factor mixed model methods to detect molecular signatures of local adaptation. We identified 244 associations between 164 outlier SNPs and 17 environmental variables. The mean temperature of the coldest quarter, soil Fe and Cu contents, precipitation of the driest month, and altitude were identified as the most important determinants of adaptive genetic variation. Most candidate unigenes with outlier signatures were related to abiotic and biotic stress responses, and the monoterpenoid biosynthesis and ubiquitin-mediated proteolysis KEGG pathways were significantly enriched in certain populations and deserve further attention in other long-lived trees. CONCLUSIONS: Despite the strong population structure in P. chienii, genomic data revealed signatures of divergent selection associated with environmental variables. Our research provides SNPs, candidate unigenes, and biological pathways related to environmental variables to facilitate elucidation of the genetic variation in P. chienii in relation to environmental adaptation. Our study provides a promising tool for population genomic analyses and insights into the molecular basis of local adaptation.

Characterization and Development of Microsatellite Markers in Pseudotaxus chienii (Taxaceae) Based on Transcriptome Sequencing.

Pseudotaxus chienii (Taxaceae) is an endangered conifer species endemic to China. However, a lack of suitable molecular markers hinders the genomic and genetic studies on this species. Here, we characterized and developed the microsatellite markers from a newly sequenced P. chienii transcriptome. A total of 21,835 microsatellite loci were detected from 161,131 non-redundant unigene sequences, and the frequency of SSRs was 13.55%, with an average of one SSR loci per 9.18 kb. Mono-nucleotide, di-nucleotide, and tri-nucleotide were the dominant repeat types, accounting for 50.06, 13.49, and 29.39% of the total SSRs, respectively. In terms of distribution location, the coding regions (CDS) with few microsatellites and mainly consisted of tri-nucleotides. There were significant differences in the length of microsatellite among genic regions and motif types. Functional annotation showed that the unigenes containing microsatellites had a wide range of biological functions, most of which were related to basic metabolism, and a few might be involved in expression regulation of gene and response to environmental stress. In addition, 375 primer pairs were randomly selected and synthesized for the amplification and validation of microsatellite markers. Seventy-seven primer pairs were successfully amplified and 40 primer pairs were found to be polymorphic. Finally, 20 pairs of primers with high polymorphism were selected to assess the genetic diversity in four P. chienii populations. In addition, the newly developed microsatellite markers exhibited high transferability (70%) in Amentotaxus argotaenia. Our study could enable further genetic diversity analysis and functional gene mining on Taxaceae.

Characterization and Analysis of the Full-Length Transcriptomes of Multiple Organs in Pseudotaxus chienii (W.C.Cheng) W.C.Cheng.

Pseudotaxus chienii, a rare tertiary relict species with economic and ecological value, is a representative of the monotypic genus Pseudotaxus that is endemic to China. P. chienii can adapt well to habitat isolation and ecological heterogeneity under a variety of climate and soil conditions, and is able to survive in harsh environments. However, little is known about the molecular and genetic resources of this long-lived conifer. Herein, we sequenced the transcriptomes of four organs of P. chienii using the PacBio Isoform Sequencing and Illumina RNA Sequencing platforms. Based on the PacBio Iso-Seq data, we obtained 44,896, 58,082, 50,485, and 67,638 full-length unigenes from the root, stem, leaf, and strobilus, respectively, with a mean length of 2692 bp, and a mean N50 length of 3010.75 bp. We then comprehensively annotated these unigenes. The number of organ-specific expressed unigenes ranged from 4393 in leaf to 9124 in strobilus, suggesting their special roles in physiological processes, organ development, and adaptability in the different four organs. A total of 16,562 differentially expressed genes (DEGs) were identified among the four organs and clustered into six subclusters. The gene families related to biotic/abiotic factors, including the TPS, CYP450, and HSP families, were characterized. The expression levels of most DEGs in the phenylpropanoid biosynthesis pathway and plant-pathogen interactions were higher in the root than in the three other organs, suggesting that root constitutes the main organ of defensive compound synthesis and accumulation and has a stronger ability to respond to stress. The sequences were analyzed to predict transcription factors, long non-coding RNAs, and alternative splicing events. The expression levels of most DEGs of C2H2, C3H, bHLH, and bZIP families in the root and stem were higher than those in the leaf and strobilus, indicating that these TFs may play a crucial role in the survival of the root and stem. These results comprise the first comprehensive gene expression profiles obtained for different organs of P. chienii. Our findings will facilitate further studies on the functional genomics, adaptive evolution, and phylogeny of P. chienii, and lay the foundation for the development of conservation strategies for this endangered conifer.

Evolutionary history of a relict conifer, Pseudotaxus chienii (Taxaceae), in south-east China during the late Neogene: old lineage, young populations.

BACKGROUND AND AIMS: Many monotypic gymnosperm lineages in south-east China paradoxically remain in relict status despite long evolutionary histories and ample opportunities for allopatric speciation, but this paradox has received little attention and has yet to be resolved. Here, we address this issue by investigating the evolutionary history of a relict conifer, Pseudotaxus chienii (Taxaceae). METHODS: DNA sequences from two chloroplast regions and 14 nuclear loci were obtained for 134 samples. The demographic history was inferred and the contribution of isolation by environment (IBE) in patterning genetic divergence was compared with that of isolation by distance (IBD). KEY RESULTS: Three genetic clusters were identified. Approximate Bayesian computation analyses showed that the three clusters diverged in the late Pliocene (~3.68 Ma) and two admixture events were detected. Asymmetric gene flow and similar population divergence times (~ 3.74 Ma) were characterized using the isolation with migration model. Neither IBD nor IBE contributed significantly to genetic divergence, and the contribution of IBE was much smaller than that of IBD. CONCLUSIONS: These results suggest that several monotypic relict gymnosperm lineages like P. chienii in south-east China did not remain in situ and undiversified for millions of years. On the contrary, they have been evolving and the extant populations have become established more recently, having insufficient time to speciate. Our findings provide a new perspective for understanding the formation and evolution of the relict gymnosperm flora of China as well as of the Sino-Japanese Flora.

Methylation data from Pseudotaxus chienii obtained using methylation-dependent restriction-site associated DNA sequencing.

Pseudotaxus chienii is an endangered coniferous plant that is endemic to China. Because P. chienii is sessile and has a long life cycle, its options for responding to drastic or rapid changes in climate are limited. To survive locally, P. chienii must be able to adapt, and the species shows variations in leaf size along an environmental gradient from east to west. It is important to determine whether this phenotypic variation is driven by DNA methylation. Therefore, we performed a preliminarily survey using methylation-dependent restriction-site associated DNA sequencing (MethylRAD) to investigate the methylation status of three P. chienii individuals from heterogeneous ecological niches. In total, 372,611 CCGG tags and 726,332 CCHGG tags were obtained. The rate of high quality methylation tags for a specific site in the genome varied from 42.31% (Gxdms3-4) to 50.01% (Jxbj3-4) and 50.18% (Zjdxg3-6). The level of CCHGG methylation (16.63%) was higher than that of CCGG (13.60%), which may be why P. chienii has low levels of phenotypic variation. The methylation data can be accessed using the Sequence Read Archive (SRA) database (SRP128155).

Chloroplast microsatellite markers for Pseudotaxus chienii developed from the whole chloroplast genome of Taxus chinensis var. mairei (Taxaceae).

PREMISE OF THE STUDY: Pseudotaxus chienii (Taxaceae) is an old rare species endemic to China that has adapted well to ecological heterogeneity with high genetic diversity in its nuclear genome. However, the genetic variation in its chloroplast genome is unknown. METHODS AND RESULTS: Eighteen chloroplast microsatellite markers (cpSSRs) were developed from the whole chloroplast genome of Taxus chinensis var. mairei and successfully amplified in four P. chienii populations and one T. chinensis var. mairei population. Of these loci, 10 were polymorphic in P. chienii, whereas six were polymorphic in T. chinensis var. mairei. The unbiased haploid diversity per locus ranged from 0.000 to 0.641 and 0.000 to 0.545 for P. chienii and T. chinensis var. mairei, respectively. CONCLUSIONS: The 18 cpSSRs will be used to further investigate the chloroplast genetic structure and adaptive evolution in P. chienii populations.

Microsatellite loci for an old rare species, Pseudotaxus chienii, and transferability in Taxus wallichiana var. mairei (Taxaceae).

PREMISE OF THE STUDY: Microsatellite loci were developed for Pseudotaxus chienii, an old rare species endemic to China, and which provided a useful tool for investigating the patterns of population genetic structure, phylogeography, evolutionary history, and adaptive potential. Transferability was assayed in the related species, Taxus wallichiana var. mairei. • METHODS AND RESULTS: A total of 15 microsatellite loci were targeted in P. chienii using the Fast Isolation by AFLP of Sequences COntaining Repeats (FIASCO) protocol. Polymorphism was evaluated in five populations of P. chienii and five populations of T. wallichiana var. mairei. Of these loci, 13 were polymorphic in P. chienii, whereas 15 were polymorphic in T. wallichiana var. mairei. • CONCLUSIONS: The 15 microsatellite loci developed lay a solid foundation for further studies on population genetic variability and investigations of local adaptation. Additionally, cross-species amplification in T. wallichiana var. mairei showed that these loci may also have potential utility in other genera of Taxaceae.