First Report of powdery mildew of Quercus guyavifolia (Fagaceae) Caused by Erysiphe quercicola.

Oaks are the most abundant trees in naturally regenerated forests in China, play a crucial role in preventing soil erosion and maintaining ecological stability (Du et al. 2022). Quercus guyavifolia H. Léveillé (Fagaceae family, Subgenus Cerris, section Ilex), is endemic in China, distributed in the southeastern boundary of the Qinghai-Tibet Plateau, with elevations from 2, 000 - 4, 500 m a.s.l. (Denk et al. 2018; Sun et al. 2016). Powdery mildew is a prevalent disease of oaks with up to 60% of foliage infection, which can induce leaf necrosis or deformation and might contribute to oak decline (Marçais and Desprez-Loustau 2014). In September 2023, we found leaves of Q. guyavifolia near Yunnan Baima Snow Mountain covered with white fungal colonies. Diseased Q. guyavifolia plants were transplanted into a greenhouse at Yunnan University for pathogenicity tests. Conidia from diseased plants were blown into twenty healthy Q. guyavifolia seedlings by cold air blower and five non-inoculated healthy seedlings were used as control. The inoculated seedlings developed powdery mildew symptoms within ten days on both sides of the leaves. Trypan blue staining was used to identify the pathogen that infects Q. guyavifolia (Xiao et al. 2017). Microscopic examination revealed abundant conidia and extensive branched hyphae on leaves, similar to the characteristics of powdery mildew fungi. The mean length and width of conidia were 29.06 ± 3.96 × 9.52 ± 1.36 µm (n = 50). We collected fungi (YNBAIMAXS01) and extracted genomic DNA from five diseased plants (from the same location) using the CTAB method. We amplified and sequenced the ITS (Gardes and Bruns, 1993), MS294, and MS447 (two nuclear protein-encoding genes; Feau et al. 2011; GenBank numbers: PP079015, PP083693, PP083694). BLAST analysis revealed 100% identity of above three sequences with the ITS of Erysiphe quercicola isolate DACA010 (GenBank accession MT569439), MS294 of E. quercicola isolate GEM09_11_FRTB1 (GenBank accession KY348509), and MS447 of E. quercicola isolate A1I1.5 (GenBank accession KY466619). Therefore, the isolate YNBAIMAXS01 was identified as E. quercicola based on its morphological and molecular characteristics. Sequences from the above three regions for YNBAIMAXS01 and five Erysiphe species were used to construct a Maximum likelihood (ML) tree. In addition, we constructed a ML tree using only the ITS region of YNBAIMAXS01 and eight Erysiphe species from GenBank to better distinguish E. quercicola from these species. Both trees were constructed using MEGA X with K2 + G as best model. The ML trees confirmed the powdery mildew fungi isolated from Q. guyavifolia is closely related to E. alphitoides. To date, thirty-four powdery mildew species belonging to genus Erysiphe have been found affecting Quercus and nine oak species can be infected by E. quercicola (https://fungi.ars.usda.gov/). To our knowledge, this is the first report of powdery mildew caused by E. quercicola on Q. guyavifolia, thus the development of control strategies and disease management is urgently needed.

Species identification through deep learning and geometrical morphology in oaks (Quercus spp.): Pros and cons.

Plant phenotypic characteristics, especially leaf morphology of leaves, are an important indicator for species identification. However, leaf shape can be extraordinarily complex in some species, such as oaks. The great variation in leaf morphology and difficulty of species identification in oaks have attracted the attention of scientists since Charles Darwin. Recent advances in discrimination technology have provided opportunities to understand leaf morphology variation in oaks. Here, we aimed to compare the accuracy and efficiency of species identification in two closely related deciduous oaks by geometric morphometric method (GMM) and deep learning using preliminary identification of simple sequence repeats (nSSRs) as a prior. A total of 538 Asian deciduous oak trees, 16 Q. aliena and 23 Q. dentata populations, were firstly assigned by nSSRs Bayesian clustering analysis to one of the two species or admixture and this grouping served as a priori identification of these trees. Then we analyzed the shapes of 2328 leaves from the 538 trees in terms of 13 characters (landmarks) by GMM. Finally, we trained and classified 2221 leaf-scanned images with Xception architecture using deep learning. The two species can be identified by GMM and deep learning using genetic analysis as a priori. Deep learning is the most cost-efficient method in terms of time-consuming, while GMM can confirm the admixture individuals' leaf shape. These various methods provide high classification accuracy, highlight the application in plant classification research, and are ready to be applied to other morphology analysis.

Dipterocarpoidae genomics reveal their demography and adaptations to Asian rainforests.

Dipterocarpoideae species form the emergent layer of Asian rainforests. They are the indicator species for Asian rainforest distribution, but they are severely threatened. Here, to understand their adaptation and population decline, we assemble high-quality genomes of seven Dipterocarpoideae species including two autotetraploid species. We estimate the divergence time between Dipterocarpoideae and Malvaceae and within Dipterocarpoideae to be 108.2 (97.8‒118.2) and 88.4 (77.7‒102.9) million years ago, and we identify a whole genome duplication event preceding dipterocarp lineage diversification. We find several genes that showed a signature of selection, likely associated with the adaptation to Asian rainforests. By resequencing of two endangered species, we detect an expansion of effective population size after the last glacial period and a recent sharp decline coinciding with the history of local human activities. Our findings contribute to understanding the diversification and adaptation of dipterocarps and highlight anthropogenic disturbances as a major factor in their endangered status.

Population and Landscape Genetics Provide Insights Into Species Conservation of Two Evergreen Oaks in Qinghai-Tibet Plateau and Adjacent Regions.

The combination of population and landscape genetics can facilitate the understanding of conservation strategy under the changing climate. Here, we focused on the two most diverse and ecologically important evergreen oaks: Quercus aquifolioides and Quercus spinosa in Qinghai-Tibetan Plateau (QTP), which is considered as world's biodiversity hotspot. We genotyped 1,657 individuals of 106 populations at 15 nuclear microsatellite loci throughout the species distribution range. Spatial patterns of genetic diversity were identified by mapping the allelic richness (AR) and locally common alleles (LCA) according to the circular neighborhood methodology. Migration routes from QTP were detected by historical gene flow estimation. The response pattern of genetic variation to environmental gradient was assessed by the genotype-environment association (GEA) analysis. The overall genetic structure showed a high level of intra-species genetic divergence of a strong west-east pattern. The West-to-East migration route indicated the complex demographic history of two oak species. We found evidence of isolation by the environment in Q. aqu-East and Q. spi-West lineage but not in Q. aqu-West and Q. spi-East lineage. Furthermore, priority for conservation should be given to populations that retain higher spatial genetic diversity or isolated at the edge of the distribution range. Our findings indicate that knowledge of spatial diversity and migration route can provide valuable information for the conservation of existing populations. This study provides an important guide for species conservation for two oak species by the integration of population and landscape genetic methods.

Asymmetric character displacement in mixed oak stands.

Ecological character displacement (ECD) refers to a pattern of increased divergence at sites where species ranges overlap caused by competition for resources. Although ECD is believed to be common, there are few in-depth studies that clearly establish its existence, especially in plants. Thus, we have compared leaf traits in allopatric and sympatric populations of two East Asian deciduous oaks: Quercus dentata and Quercus aliena. In contrast to previous studies, we define sympatry and allopatry at a local scale, thereby comparing populations that can or cannot directly interact. Using genetic markers, we found greater genetic divergence between the two oak species growing in mixed stands and inferred that long-term gene flow has predominantly occurred asymmetrically from the cold-tolerant species (Q. dentata) to the warm-demanding later colonizing species (Q. aliena). Analysis of leaf traits revealed greater divergence in mixed than in pure oak stands. This was mostly due to the later colonizing species being characterized by more resource-conservative traits in the presence of the other species. Controlling for relevant environmental differences did not alter these conclusions. These results suggest that asymmetric trait divergence can take place where species coexist, possibly due to the imbalance in demographic history of species resulting in asymmetric inter-specific selection pressures.

Corrigendum: Landscape Genomics in Tree Conservation Under a Changing Environment.

[This corrects the article DOI: 10.3389/fpls.2022.822217.].

Landscape Genomics in Tree Conservation Under a Changing Environment.

Understanding the genetic basis of how species respond to changing environments is essential to the conservation of species. However, the molecular mechanisms of adaptation remain largely unknown for long-lived tree species which always have large population sizes, long generation time, and extensive gene flow. Recent advances in landscape genomics can reveal the signals of adaptive selection linking genetic variations and landscape characteristics and therefore have created novel insights into tree conservation strategies. In this review article, we first summarized the methods of landscape genomics used in tree conservation and elucidated the advantages and disadvantages of these methods. We then highlighted the newly developed method "Risk of Non-adaptedness," which can predict the genetic offset or genomic vulnerability of species via allele frequency change under multiple scenarios of climate change. Finally, we provided prospects concerning how our introduced approaches of landscape genomics can assist policymaking and improve the existing conservation strategies for tree species under the ongoing global changes.

Contrasted patterns of local adaptation to climate change across the range of an evergreen oak, Quercus aquifolioides.

Long-lived tree species are genetically differentiated and locally adapted with respect to fitness-related traits, but the genetic basis of local adaptation remains largely unresolved. Recent advances in population genetics and landscape genomic analyses enable identification of putative adaptive loci and specific selective pressures acting on local adaptation. Here, we sampled 60 evergreen oak (Quercus aquifolioides) populations throughout the species' range and pool-sequenced 587 individuals at drought-stress candidate genes. We analyzed patterns of genetic diversity and differentiation for 381 single nucleotide polymorphisms (SNPs) from 65 candidate genes and eight microsatellites. Outlier loci were identified by genetic differentiation analysis and genome-environment associations. The response pattern of genetic variation to environmental gradient was assessed by linear isolation-by-distance/environment tests, redundancy analysis, and nonlinear methods. SNPs and microsatellites revealed two genetic lineages: Tibet and Hengduan Mountains-Western Sichuan Plateau (HDM-WSP), with reduced genetic diversity in Tibet lineage. More outlier loci were detected in HDM-WSP lineage than Tibet lineage. Among these, three SNPs in two genes responded to dry season precipitation in the HDM-WSP lineage but not in Tibet. By contrast, genetic variation in the Tibet lineage was related to geographic distance instead of the environment. Furthermore, risk of nonadaptedness (RONA) analyses suggested HDM-WSP lineage will have a better capacity to adapt in the predicted future climate compared with the Tibet lineage. We detected genetic imprints consistent with natural selection and molecular adaptation to drought on the Qinghai-Tibet Plateau (QTP) over a range of long-lived and widely distributed oak species in a changing environment. Our results suggest that different within-species adaptation processes occur in species occurring in heterogeneous environments.

The complete chloroplast genome of Quercus fabri (Fagaceae) from China.

The complete chloroplast (cp) genome sequence of Quercus fabri Hance has been characterized in this study. The length of cp genome was 161,227 bp, containing a large single-copy region (LSC) of 90,539 bp and a small single-copy region (SSC) of 19,048 bp, which were separated by a pair of 25,820 bp inverted repeat regions (IRs). The genome contained 134 genes, including 86 protein-coding genes, 40 tRNA genes, and 8 rRNA genes. The overall GC content is 36.82%. Further, phylogenetic analysis suggested that Q. fabri is clustered to section Quercus.

Comparative Genomic Analysis Reveals the Mechanism Driving the Diversification of Plastomic Structure in Taxaceae Species.

Inverted repeat (IR) regions in the plastomes from land plants induce homologous recombination, generating isomeric plastomes. While the plastomes of Taxaceae species often lose one of the IR regions, considerable isomeric plastomes were created in Taxaceae species with a hitherto unclarified mechanism. To investigate the detailed mechanism underpinning the IR-independent genesis of plastomic diversity, we sequenced four Taxaceae plastomes, including Taxus cuspidata Siebold & Zuccarini, Taxus fauna Nan Li & R. R. Mill, and two individuals of Taxus wallichiana Zuccarini. Then we compared these structures with those of previously reported Taxaceae plastomes. Our analysis identified four distinct plastome forms that originated from the rearrangements of two IR-flanking inverted fragments. The presence of isomeric plastomes was then verified in T. cuspidata individuals. Both rearrangement analyses and phylogenetic results indicated that Taxaceae were separated into two clades, one including Taxus and Pseudotaxus and another formed by Amentotaxus and Torreya. Our reconstructed scenario suggests that the minimum number of inversion events required for the transformation of the plastome of Cephalotaxus oliveri Masters into the diversified Taxaceae plastomes ranged from three to six. To sum up, our study reveals a distinct pattern and the mechanism driving the structural diversification of Taxaceae plastomes, which will advance our understanding of the maintenance of plastomic diversity and complexity in conifers.

Recent Fragmentation May Not Alter Genetic Patterns in Endangered Long-Lived Species: Evidence From Taxus cuspidata.

Forestland fragmentation caused by overexploitation of forest resources can in principle reduce genetic diversity, limit gene flow and eventually lead to species developing strong genetic structure. However, the genetic consequences of recent anthropogenic fragmentation of tree species remain unclear. Taxus cuspidata, which has extremely small populations distributed mainly in Changbai Mt. in Northeast (NE) China, has recently endured severe habitat fragmentation. Here, we investigate the pattern of genetic diversity and structure, identify risk factors, predict the future distribution and finally provide guidelines for the conservation and management of this species. We used three chloroplast and two mitochondrial DNA fragments, which are both paternally inherited in yews but differ in mutation rates, to genotype a total of 265 individuals from 26 populations covering the distribution of the species in China. Both chloroplast and mitochondrial data showed high degrees of genetic diversity, extensive gene flow over the entire geographical range and historical stability of both effective population size and distribution of the species. However, ecological niche modeling suggests a decrease in suitable areas for this species by the years 2050 and 2070. The maintenance of high genetic diversity and the existence of sufficient gene flow suggest that recent fragmentation has not affected the genetic composition of the long-lived tree T. cuspidata. However, severe impacts of anthropogenic activities are already threatening the species. Conservation and management strategies should be implemented in order to protect the remnant populations.

Species Boundaries Between Three Sympatric Oak Species: Quercus aliena, Q. dentata, and Q. variabilis at the Northern Edge of Their Distribution in China.

Oaks are important timber trees with wide distributions in China, but few genetic studies have been conducted on a fine scale. In this study, we seek to investigate the genetic diversity and differentiation of three sympatric oak species (Quercus aliena Blume, Quercus dentata Thunb. ex Murray, and Quercus variabilis Blume) in their northern distribution in China using 17 bi-parentally inherited nSSRs markers and five maternally inherited chloroplast DNA (cpDNA) fragments. Both the cpDNA and the nSSRs show a high level of genetic differentiation between different oak sections. The chloroplast haplotypes are clustered into two lineages. Clear species boundaries are detected between Q. variabilis and either Q. aliena or Q. dentata. The sharing of chloroplast haplotype H1 between Q. aliena and Q. dentata suggests very recent speciation and incomplete lineage sorting or introgression of H1 from one species to another. The nSSRs data indicate a complete fixation of variation within sites for all three oak species, and that extensive gene flow occurs within species whereas only limited gene flow is detected between Q. aliena and Q. dentata and nearly no gene flow can be detected between Q. aliena and Q. variabilis and between Q. dentata and Q. variabilis. Prezygotic isolation may have contributed to the species boundaries of these three sympatric oak species.

Different Natural Selection Pressures on the atpF Gene in Evergreen Sclerophyllous and Deciduous Oak Species: Evidence from Comparative Analysis of the Complete Chloroplast Genome of Quercus aquifolioides with Other Oak Species.

Quercus is an economically important and phylogenetically complex genus in the family Fagaceae. Due to extensive hybridization and introgression, it is considered to be one of the most challenging plant taxa, both taxonomically and phylogenetically. Quercus aquifolioides is an evergreen sclerophyllous oak species that is endemic to, but widely distributed across, the Hengduanshan Biodiversity Hotspot in the Eastern Himalayas. Here, we compared the fully assembled chloroplast (cp) genome of Q. aquifolioides with those of three closely related species. The analysis revealed a cp genome ranging in size from 160,415 to 161,304 bp and with a typical quadripartite structure, composed of two inverted repeats (IRs) separated by a small single copy (SSC) and a large single copy (LSC) region. The genome organization, gene number, gene order, and GC content of these four Quercus cp genomes are similar to those of many angiosperm cp genomes. We also analyzed the Q. aquifolioides repeats and microsatellites. Investigating the effects of selection events on shared protein-coding genes using the Ka/Ks ratio showed that significant positive selection had acted on the atpF gene of Q. aquifolioides compared to two deciduous oak species, and that there had been significant purifying selection on the atpF gene in the chloroplast of evergreen sclerophyllous oak trees. In addition, site-specific selection analysis identified positively selected sites in 12 genes. Phylogenetic analysis based on shared protein-coding genes from 14 species defined Q. aquifolioides as belonging to sect. Heterobalanus and being closely related to Q. rubra and Q. aliena. Our findings provide valuable genetic information for use in accurately identifying species, resolving taxonomy, and reconstructing the phylogeny of the genus Quercus.

Development of microsatellite markers for Fargesia denudata (Poaceae), the staple-food bamboo of the giant panda.

PREMISE OF THE STUDY: There is a need for microsatellite primers to analyze genetic parameters of Fargesia denudata (Poaceae), the staple-food bamboo of the giant panda (Ailuropoda melanoleuca). METHODS AND RESULTS: Using next-generation sequencing technology, we obtained a 75-Mb assembled sequence of F. denudata and identified 182 microsatellites. Primer pairs for 70 candidate microsatellite markers were selected and validated in four individuals, and 42 primer pairs generated reliable amplicons. Fourteen of 16 tested markers were found to be polymorphic in 72 individuals from four F. denudata populations. The number of alleles ranged from two to 19 per locus; the observed and expected heterozygosities ranged from 0 to 1 and from 0 to 0.87, respectively. The transferability of these 16 novel microsatellite markers was validated in five related species. CONCLUSIONS: These markers will be useful for examining the genetic diversity, genetic structure, and cloning of F. denudata, the staple-food bamboo of the giant panda, and related bamboo species.

The complete chloroplast genome of Cathay Poplar: Poplus cathayana Rehder.

The complete chloroplast genome of Populus cathayana was determined in this study. The total length of the chloroplast genome size is 155 449 bp, with 36.95% GC content. A pair of inverted repeats of 27 525 bp are separated by a large single-copy region (LSC, 83 911 bp) and a small single-copy region (SSC, 16 488 bp). About 104 unique genes were annotated, including 76 protein coding genes, 24 tRNA genes and 4 rRNA genes.

Complete chloroplast genome of the Oriental white oak: Quercus aliena Blume.

The complete chloroplast (cp) genome sequence of the Oriental white oak: Quercus aliena Blume, the first sequenced member of the section Quercus, is reported in this study. The length of cp genome size is 160,921 bp, with 36.9% GC content. A pair of 25,841 bp inverted repeat regions (IRs) is separated by a 90,258 bp large single copy region (LSC) and an 18,980 bp small single copy region (SSC). This genome contains 115 unique genes, including 89 coding genes, four rRNA genes, and 39 tRNA genes. Border analysis of cp genome of Quercus aliena and other 10 most closely related cp genomes revealed that most Fagaceae species have high similarity either in structure or distribution of these genes except for Trigonobalanus doichangensis.

Protein domain analysis of genomic sequence data reveals regulation of LRR related domains in plant transpiration in Ficus.

Predicting protein domains is essential for understanding a protein's function at the molecular level. However, up till now, there has been no direct and straightforward method for predicting protein domains in species without a reference genome sequence. In this study, we developed a functionality with a set of programs that can predict protein domains directly from genomic sequence data without a reference genome. Using whole genome sequence data, the programming functionality mainly comprised DNA assembly in combination with next-generation sequencing (NGS) assembly methods and traditional methods, peptide prediction and protein domain prediction. The proposed new functionality avoids problems associated with de novo assembly due to micro reads and small single repeats. Furthermore, we applied our functionality for the prediction of leucine rich repeat (LRR) domains in four species of Ficus with no reference genome, based on NGS genomic data. We found that the LRRNT_2 and LRR_8 domains are related to plant transpiration efficiency, as indicated by the stomata index, in the four species of Ficus. The programming functionality established in this study provides new insights for protein domain prediction, which is particularly timely in the current age of NGS data expansion.

Exploiting the transcriptome of Euphrates Poplar, Populus euphratica (Salicaceae) to develop and characterize new EST-SSR markers and construct an EST-SSR database.

BACKGROUND: Microsatellite markers or Simple Sequence Repeats (SSRs) are the most popular markers in population/conservation genetics. However, the development of novel microsatellite markers has been impeded by high costs, a lack of available sequence data and technical difficulties. New species-specific microsatellite markers were required to investigate the evolutionary history of the Euphratica tree, Populus euphratica, the only tree species found in the desert regions of Western China and adjacent Central Asian countries. METHODOLOGY/PRINCIPAL FINDINGS: A total of 94,090 non-redundant Expressed Sequence Tags (ESTs) from P. euphratica comprising around 63 Mb of sequence data were searched for SSRs. 4,202 SSRs were found in 3,839 ESTs, with 311 ESTs containing multiple SSRs. The most common motif types were trinucleotides (37%) and hexanucleotides (33%) repeats. We developed primer pairs for all of the identified EST-SSRs (eSSRs) and selected 673 of these pairs at random for further validation. 575 pairs (85%) gave successful amplification, of which, 464 (80.7%) were polymorphic in six to 24 individuals from natural populations across Northern China. We also tested the transferability of the polymorphic eSSRs to nine other Populus species. In addition, to facilitate the use of these new eSSR markers by other researchers, we mapped them onto Populus trichocarpa scaffolds in silico and compiled our data into a web-based database (http://202.205.131.253:8080/poplar/resources/static_page/index.html). CONCLUSIONS: The large set of validated eSSRs identified in this work will have many potential applications in studies on P. euphratica and other poplar species, in fields such as population genetics, comparative genomics, linkage mapping, QTL, and marker-assisted breeding. Their use will be facilitated by their incorporation into a user-friendly web-based database.

Two highly validated SSR multiplexes (8-plex) for Euphrates' poplar, Populus euphratica (Salicaceae).

Multiplex PCR amplification of microsatellites has significantly increased the throughput and decreased the costs of genotyping. We have developed two highly polymorphic microsatellite multiplexes for Populus euphratica, the only tree species found in desert regions of Western China and adjacent Central Asian countries. The first of these multiplex kits comprises an eight-Plex of genomic SSRs (gSSRs) obtained from published databases. The second comprises an eight-plex of newly designed EST-SSRs (eSSRs) based on expressed sequence tags for P. euphratica. Both kits were tested on a sample of 170 individuals from four populations. The gSSRs exhibited slightly more polymorphism than the eSSRs. The new multiplex protocols yielded consistent results in the hands of multiple researchers, demonstrating their robustness. The 16 loci used in the kits exhibited a high transferability rate (82.0%) in eight other poplar species belonging to five different sections of the genus. Both kits should therefore be useful for further investigations of population genetics in P. euphratica and related species. Our results indicate that it is essential to follow recently established recommendations when developing microsatellite markers, including verifying the amplification efficiency, detecting null alleles and carefully measuring error rates.

Direction and extent of organelle DNA introgression between two spruce species in the Qinghai-Tibetan Plateau.

A recent model has shown that, during range expansion of one species in a territory already occupied by a related species, introgression should take place preferentially from the resident species towards the invading species and genome components experiencing low rates of gene flow should introgress more readily than those experiencing high rates of gene flow. Here, we use molecular markers from two organelle genomes with contrasted rates of gene flow to test these predictions by examining genetic exchanges between two morphologically distinct spruce Picea species growing in the Qinghai-Tibetan Plateau. The haplotypes from both mitochondrial (mt) DNA and chloroplast (cp) DNA cluster into two distinct lineages that differentiate allopatric populations of the two species. By contrast, in sympatry, the species share the same haplotypes, suggesting interspecific genetic exchanges. As predicted by the neutral model, all sympatric populations of the expanding species had received their maternally inherited mtDNA from the resident species, whereas for paternally inherited cpDNA introgression is more limited and not strictly unidirectional. Our results underscore cryptic introgressions of organelle DNAs in plants and the importance of considering rates of gene flow and range shifts to predict direction and extent of interspecific genetic exchanges.