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As an indispensable part of insects, intestinal symbiotic bacteria play a vital role in the growth and development of insects and their adaptability. Rhoptroceros cyatheae, the main pest of the relict plant Alsophila spinulosa, poses a serious threat to the development of the A. spinulosa population. In the present study, 16S rDNA and internal transcribed spacer high-throughput sequencing techniques were used to analyze the structure of intestinal microbes and the diversity of the insect feeding on two different plants, as well as the similarities between the intestinal microorganisms of R. cyatheae. The dominant bacteria of leaf endophytes were also compared based on the sequencing data. The results showed that Proteobacteria, Firmicutes, and Actinobacteria were the dominant phyla of intestinal bacteria, and Ascomycota was the dominant phylum of intestinal fungi. Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Methylobacterium-Methylorubrum, and Enterococcus were the dominant genera in the intestine of R. cyatheae feeding on two plants, and the relative abundance was significantly different between the two groups. Candida was the common dominant genus of intestinal fungi in the two groups, and no significant difference was observed in its abundance between the two groups. This showed that compared with the intestinal fungi of R. cyatheae, the abundance of the intestinal bacteria was greatly affected by food. The common core microbiota between the microorganisms in A. spinulosa leaves and the insect gut indicated the presence of a microbial exchange between the two. The network correlation diagram showed that the gut microbes of R. cyatheae feeding on Gymnosphaera metteniana were more closely related to each other, which could help the host to better cope with the adverse external environment. This study provides a theoretical basis for the adaptation mechanism of R. cyatheae and a new direction for the effective prevention and control of R. cyatheae.

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High-mountain and arctic plants are considered especially sensitive to climate change because of their close adaptation to the cold environment. Kalmia procumbens, a typical arctic-alpine species, reaches southernmost European localities in the Pyrenees and Carpathians. The aim of this study was the assessment and comparison of the current potential niche areas of K. procumbens in the Pyrenees and Carpathians and their possible reduction due to climate change, depending on the scenario. The realized niches of K. procumbens in the Pyrenees are compact, while those in the Carpathians are dispersed. In both mountain chains, the species occurs in the alpine and subalpine vegetation belts, going down to elevations of about 1500-1600 m, while the most elevated localities in the Pyrenees are at ca. 3000 m, about 500 m higher than those in the Carpathians. The localities of K. procumbens in the Carpathians have a more continental climate than those in the Pyrenees, with lower precipitation and temperatures but higher seasonality of temperature and precipitation. The species covered a larger area of geographic range during the Last Glacial Maximum, but its geographic range was reduced during the mid-Holocene. Due to climate warming, a reduction in the potential area of occurrence could be expected in 2100; this reduction is expected to be strong in the Carpathians and moderate in the Pyrenees.

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The semi-arid region of the Western Ordos plateau in Inner Mongolia, China, is home to a critically endangered shrub species, Potaninia mongolica, which originates from ancient Mediterranean regions. Root-associated microbiomes play important roles in plant nutrition, productivity, and resistance to environmental stress particularly in the harsh desert environment; however, the succession of root-associated fungi during the growth stages of P. mongolica is still unclear. This study aimed to examine root-associated fungal communities of this relict plant species across three seasons (spring, summer and autumn) using root sampling and Illumina Miseq sequencing of internal transcribed spacer 2 (ITS 2) region to target fungi. The analysis detected 698 fungal OTUs in association with P. mongolica roots, and the fungal richness increased significantly from spring to summer and autumn. Eurotiales, Hypocreales, Chaetothyriales, Pleosporales, Helotiales, Agaricales and Xylariales were the dominant fungal orders. Fungal community composition was significantly different between the three seasons, and the fungal taxa at various levels showed biased distribution and preferences. Stochastic processes predominantly drove community assembly of fungi in spring while deterministic processes acted more in the later seasons. The findings revealed the temporal dynamics of root-associated fungal communities of P. mongolica, which may enhance our understanding of biodiversity and changes along with seasonal alteration in the desert, and predict the response of fungal community to future global changes.

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Sassafras tzumu (Hemsl.) Hemsl., as an Arctic Tertiary relict woody species, is an ecologically and economically important deciduous tree endemic to southern China. Nonetheless, the genetic resources and backgrounds of S. tzumu are still lacking and remain largely unclear. Here, we predicted 16,215 candidate polymorphic nuclear microsatellite (nSSR) loci from the assembled nucleus databases of six geographic-distant individuals of S. tzumu via CandiSSR. Among these nSSRs, the di- (75.53%) and tri-nucleotide (19.75%) repeats were the most abundant, and 27 new polymorphic SSRs were developed and characterized in 136 individuals from six natural populations of S. tzumu. The majority of the above 27 SSRs (24 loci, 88.89%) presented moderate polymorphism (mean PIC = 0.356), and the transferability of these markers in other Sassafras species was high (85.19%). A moderately low level of genetic diversity and a high variation (FST = 0.286) of six wild populations of S. tzumu were illuminated by 16 selected polymorphic nSSRs, with the average expected heterozygosity (HE) of 0.430 at the species level and HE ranging from 0.195 to 0.387 at the population level. Meanwhile, a bottleneck effect was shown in two populations. Consistent with the results of the principal coordinate analysis (PCoA) and phylogenetic trees, structure analysis optimally divided these six S. tzumu populations into two clusters, and the further strong population subdivision appeared from K = 2 to K = 5, which corresponded to two evolutionarily significant units (ESUs). Moreover, the significant correlation between genetic and geographic distance was tested by the Mantel test (r = 0.742, p = 0.006), clarifying the effect about isolation by distance (IBD), which could be possibly explained by the low gene flow (Nm = 0.625), a relatively high degree of inbreeding (FIS = 0.166), a relatively large distribution, and mountainous barriers. Above all, our research not only enlarged the useful genetic resources for future studies of population genetics, molecular breeding, and germplasm management of S. tzumu and its siblings but also contributed to proposing scientific conservation strategies and schemes for the better preservation of S. tzumu and other Sassafras (Lauraceae) species.

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Lotus (Nelumbo Adans.), a relict plant, is the testimony of long-term sustained ecological success, but the underlying genetic changes related to its survival strategy remains unclear. Here, we assembled the high-quality lotus genome, investigated genome variation of lotus mutation accumulation (MA) lines and reconstructed the demographic history of wild Asian lotus, respectively. We identified and validated 43 base substitutions fixed in MA lines, implying a spontaneous mutation rate of 1.4 × 10-9 base/generation in lotus shoot stem cells. The past history of lotus revealed that the ancestors of lotus in eastern and southern Asia could be traced back ~20 million years ago (Mya) and experienced twice significant bottlenecks and population splits. We further identified the selected genes among three lotus groups in different habitats, suggesting that 453 genes between tropical and temperate group and 410 genes between two subgroups from Northeastern China and the Yangtze River - Yellow River Basin might play important roles in natural selection in lotus's adaptation and resilience. Our findings not only improve an understanding of the lotus evolutionary history and the genetic basis of its survival advantages, but also provide valuable data for addressing various questions in evolution and protection for the relict plants.

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Salixlapponum is a cold-tolerant relict species in Europe that occurs in several sites, probably reflecting previous migration routes of S.lapponum during the Pleistocene. However, only a few data are available on the genetic structures of populations of S.lapponum. In this study, we use PCR-ISSR markers to investigate genetic variation in 19 European populations of S.lapponum L. AMOVA analysis shows that most of the variation (55.8%) occurs within populations; variability among groups accounts for 19.7%. An AMOVA analysis based on four groups determined by STRUCTURE analysis shows similar results: variability of 54.1% within the population and variability of 18.9% between the four population groups, based on geographic regions. Within individual geographic groups, which are characterised by the studied populations, the lowest variability (as well as the highest homogeneity) was found in populations located in Belarus. The obtained results are consistent with our expectations that the European Lowland could be a significant geographic barrier for gene flow over large geographic distances for S.lapponum. Both the Scandinavian and Belarusian populations, as well as those coming from NE Poland, are characterised by significant genetic distinctiveness. However, some populations from NE Poland and the Sudetes show similarities with populations from other geographic regions, indicating existing genetic relationships between them. Moreover, the results suggest a fairly clear division of the population into 4 emerging geographic regions, although separated by a geographical barrier: the Polish lowland, which forms part of the larger geographic unit known as the European Lowland.

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Tetracentron sinense and Trochodendron aralioides are two Tertiary relict species of large trees in the family Trochodendraceae with narrow distributions on the mainland and islands of eastern Asia. They belong to the order Trochodendrales, which is one of the four early-diverged eudicot lineages. These two relict species provide a good system in which to examine genomic changes that occurred as they survived during repeated climatic oscillations in the Quaternary. We sequenced the genome of Te. sinense and compared it with that of Tr. aralioides. We found that Te. sinense has a smaller genome size (986.3 Mb) than that of Tr. aralioides (1610 Mb). Repetitive elements made the major contribution to the contrasting genome sizes in the two species, with most bursts of repeats occurring within the past four million years when the climate oscillated greatly. These species share two rounds of whole-genome duplications. The mainland species Te. sinense had a larger effective population size than the island species Tr. aralioides after the largest glaciation during the Quaternary climatic oscillation. However, soon after this recovery stage, the effective population sizes of both species continued to decrease, although the current effective population size of Te. sinense is still larger than that of Tr. aralioides. We recovered three distinctly diverged clades through resequencing the genomes of 50 individuals across the distributional range of Te. sinense in China. Our results provide an important genomic resource with which to examine early trait evolution in the core eudicots and assist efforts to conserve this relict tree species.

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MAIN CONCLUSION: The current state-of-the-art creates a sound basis for the preservation of unique species Cupressus dupreziana provided that targeted effort and care is devoted to the accomplishment of multiplication protocols. This review is to summarize known data on Cupressus dupreziana-specific characteristics, including abiotic stress resistance, and natural reproduction, and estimates the possibilities of ex situ conservation with an emphasis on the cultivation in vitro of this endangered species. As there is only limited information about the cultivation of C. dupreziana in vitro, we have included relevant data on the related species Cupressus sempervirens, where micropropagation techniques are well established, along with other information on species with similar fates and life strategies-Saharan olive Olea europaea subsp. laperrinei and myrtle Myrthus nivellei. The aim of this work is to enhance general understanding and to promote an interest in this relict plant species to contribute to more wide-ranging studies and to increase its chance of preservation. Besides others, reducing the number of species threatened with extinction is an essential and immediate task as high genetic variability of ecosystems is crucial for their stability under changing climatic conditions.

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The Chinese water pine Glyptostrobus pensilis is the sole surviving species of the genus Glyptostrobus. It is endemic to southern China, central Vietnam, and eastern Laos, and today it is nearly extinct in the wild. Forest community characteristics and population structure of G. pensilis in China have remained unknown up to now. We investigated six swamp forest stands and analyzed their forest community characteristics (i.e. vertical stratification, species composition, and diversity) and population structure, including the frequency distribution of DBH (diameter at breast height) and age-classes as found in Fujian Province, southeastern China. The vertical stratifications of all the forest stands were rather simple. The remaining wild specimens ranged from roughly 15 to some 357 years for an average of ca. 85 years, with only a few individuals less than 20 years old. Compared with the stands and populations of G. pensilis in Vietnam, the taxonomic compositions of the stands in the two regions were different, except for the dominant species-G. pensilis. The Shannon-Wiener index showed the overstory of each stand had much lower diversity (0.26 on average) in Fujian Province than that (1.97 on average) in Vietnam, whereas the diversity indices were about the same (around 2.41) for the understories in the two regions. Furthermore, we discovered 18 G. pensilis seedlings at the study sites in Fujian Province. This discovery demonstrates that G. pensilis regeneration is extremely poor and its populations are declining, although these populations are relatively healthier than those in Vietnam.

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The population structure, regeneration ways and spatial relationships of six relict deci-duous broadleaved trees were examined based on community investigation on Mount Tianmu, East China. The results showed that relict deciduous broadleaved trees had a strong ability of vegetative reproduction and their population structure was generally belonged to intermittent type. Some relict species such as Cyclocarya paliurus and Liquidamb aracalycina regenerated discontinuously at appropriate sites via long-distance diffusion. Some species such as Emmenoptery henryi and Euptelea pleiospermum regenerated discontinuously around mature trees due to habitat limitation and frequent disturbance. After occupying forest gaps by seedling regeneration, C. paliurus, L. acalycina, Nyssasinensis and Liquidambar formosana could recruit and form multi-stemmed individuals by their inherent sprouting ability at relatively stable sites such as crest slope and side slope. At riverbed, valley slope and head hollow, recognized as unstable habitats, Emmenopterys henryi, E. pleiospermum, and L. formosana could also recruit by strong sprouting ability due to sparse seedlings and individual loss incurred by disturbance. Therefore, the relict deciduous broadleaved trees could be classified into two ecological species groups (repairment species for forest gap and supplement species for special habitat) based on spatial distribution and strategy of regeneration and competition. We suggested that the protection of special habitats of their communities should be strengthened.

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Parrotia subaequalis (Hamamelidaceae) is a Tertiary relic species endemic in eastern China. We used inter-simple sequence repeat (ISSR) markers to access genetic diversity and population genetic structure in natural five populations of P. subaequalis. The levels of genetic diversity were higher at species level (H = 0.2031) but lower at population level (H = 0.1096). The higher genetic diversity at species levels might be attributed to the accumulation of distinctive genotypes which adapted to the different habitats after Quaternary glaciations. Meanwhile, founder effects on the early stage, and subsequent bottleneck of population regeneration due to its biological characteristics, environmental features, and human activities, seemed to explain the low population levels of genetic diversity. The hierarchical AMOVA revealed high levels (42.60%) of among-population genetic differentiation, which was in congruence with the high levels of Nei's genetic differentiation index (G ST = 0.4629) and limited gene flow (N m = 0.5801) among the studied populations. Mantel test showed a significant isolation-by-distance, indicating that geographic isolation has a significant effect on genetic structure in this species. Unweighted pair-group method with arithmetic average clustering, PCoA, and Bayesian analyses uniformly recovered groups that matched the geographical distribution of this species. In particular, our results suggest that Yangtze River has served as a natural barrier to gene flow between populations occurred on both riversides. Concerning the management of P. subaequalis, the high genetic differentiation among populations indicates that preserving all five natural populations in situ and collecting enough individuals from these populations for ex situ conservation are necessary.

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PREMISE OF THE STUDY: Metasequoia glyptostroboides is an endangered relict conifer species endemic to China. In this study, expressed sequence tag-simple sequence repeat (EST-SSR) markers were developed using transcriptome mining for future genetic and functional studies. METHODS AND RESULTS: We collected 97,565 unigene sequences generated by 454 pyrosequencing. A bioinformatics analysis identified 2087 unique and putative microsatellites, from which 96 novel microsatellite markers were developed. Fifty-three of the 96 primer sets successfully amplified clear fragments of the expected sizes; 23 of those loci were polymorphic. The number of alleles per locus ranged from two to eight, with an average of three, and the observed and expected heterozygosity values ranged from 0 to 1.0 and 0.117 to 0.813, respectively. CONCLUSIONS: These microsatellite loci will enrich the genetic resources to develop functional studies and conservation strategies for this endangered relict species.

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Metasequoia glyptostroboides Hu et Cheng is the only species in the genus Metasequoia Miki ex Hu et Cheng, which belongs to the Cupressaceae family. There were around 10 species in the Metasequoia genus, which were widely spread across the Northern Hemisphere during the Cretaceous of the Mesozoic and in the Cenozoic. M. glyptostroboides is the only remaining representative of this genus. Here, we report the complete chloroplast (cp) genome sequence and the cp genomic features of M. glyptostroboides. The M. glyptostroboides cp genome is 131,887 bp in length, with a total of 117 genes comprised of 82 protein-coding genes, 31 tRNA genes and four rRNA genes. In this genome, 11 forward repeats, nine palindromic repeats, and 15 tandem repeats were detected. A total of 188 perfect microsatellites were detected through simple sequence repeat (SSR) analysis and these were distributed unevenly within the cp genome. Comparison of the cp genome structure and gene order to those of several other land plants indicated that a copy of the inverted repeat (IR) region, which was found to be IR region A (IRA), was lost in the M. glyptostroboides cp genome. The five most divergent and five most conserved genes were determined and further phylogenetic analysis was performed among plant species, especially for related species in conifers. Finally, phylogenetic analysis demonstrated that M. glyptostroboides is a sister species to Cryptomeria japonica (L. F.) D. Don and to Taiwania cryptomerioides Hayata. The complete cp genome sequence information of M. glyptostroboides will be great helpful for further investigations of this endemic relict woody plant and for in-depth understanding of the evolutionary history of the coniferous cp genomes, especially for the position of M. glyptostroboides in plant systematics and evolution.