Contrasting demographic history and mutational load in three threatened whitebark pines (Pinus subsect. Gerardianae): implications for conservation.

Demographic history and mutational load are of paramount importance for the adaptation of the endangered species. However, the effects of population evolutionary history and genetic load on the adaptive potential in endangered conifers remain unclear. Here, using population transcriptome sequencing, whole chloroplast genomes and mitochondrial DNA markers, combined with niche analysis, we determined the demographic history and mutational load for three threatened whitebark pines having different endangered statuses, Pinus bungeana, P. gerardiana and P. squamata. Demographic inference indicated that severe bottlenecks occurred in all three pines at different times, coinciding with periods of major climate and geological changes; in contrast, while P. bungeana experienced a recent population expansion, P. gerardiana and P. squamata maintained small population sizes after bottlenecking. Abundant homozygous-derived variants accumulated in the three pines, particularly in P. squamata, while the species with most heterozygous variants was P. gerardiana. Abundant moderately and few highly deleterious variants accumulated in the pine species that have experienced the most severe demographic bottlenecks (P. gerardiana and P. squamata), most likely because of purging effects. Finally, niche modeling showed that the distribution of P. bungeana might experience a significant expansion in the future, and the species' identified genetic clusters are also supported by differences in the ecological niche. The integration of genomic, demographic and niche data has allowed us to prove that the three threatened pines have contrasting patterns of demographic history and mutational load, which may have important implications in their adaptive potential and thus are also key for informing conservation planning.

Demographic dynamics and molecular evolution of the rare and endangered subsect. Gerardianae of Pinus: insights from chloroplast genomes and mitochondrial DNA markers.

MAIN CONCLUSION: The divergence of subsect. Gerardianae was likely triggered by the uplift of the Qinghai-Tibetan Plateau and adjacent mountains. Pinus bungeana might have probably experienced expansion since Last Interglacial period. Historical geological and climatic oscillations have profoundly affected patterns of nucleotide variability, evolutionary history, and species divergence in numerous plants of the Northern Hemisphere. However, how long-lived conifers responded to geological and climatic fluctuations in East Asia remain poorly understood. Here, based on paternally inherited chloroplast genomes and maternally inherited mitochondrial DNA markers, we investigated the population demographic history and molecular evolution of subsect. Gerardianae (only including three species, Pinus bungeana, P. gerardiana, and P. squamata) of Pinus. A low level of nucleotide diversity was found in P. bungeana (π was 0.00016 in chloroplast DNA sequences, and 0.00304 in mitochondrial DNAs). The haplotype-based phylogenetic topology and unimodal distributions of demographic analysis suggested that P. bungeana probably originated in the southern Qinling Mountains and experienced rapid population expansion since Last Interglacial period. Phylogenetic analysis revealed that P. gerardiana and P. squamata had closer genetic relationship. The species divergence of subsect. Gerardianae occurred about 27.18 million years ago (Mya) during the middle to late Oligocene, which was significantly associated with the uplift of the Qinghai-Tibetan Plateau and adjacent mountains from the Eocene to the mid-Pliocene. The molecular evolutionary analysis showed that two chloroplast genes (psaI and ycf1) were under positive selection, the genetic lineages of P. bungeana exhibited higher transition and nonsynonymous mutations, which were involved with the strongly environmental adaptation. These findings shed light on the population evolutionary history of white pine species and provide striking insights for comprehension of their species divergence and molecular evolution.

The hybridization origin of the Chinese endemic herb genus Notopterygium (Apiaceae): Evidence from population genomics and ecological niche analysis.

Hybridization is recognized as a major force in species evolution and biodiversity formation, generally leading to the origin and differentiation of new species. Multiple hybridization events cannot easily be reconstructed, yet they offer the potential to study a number of evolutionary processes. Here, we used nuclear expressed sequence tag-simple sequence repeat and large-scale single nucleotide polymorphism variation data, combined with niche analysis, to investigate the putative independent hybridization events in Notopterygium, a group of perennial herb plants endemic to China. Population genomic analysis indicated that the four studied species are genetically well-delimited and that N. forrestii and N. oviforme have originated by hybridization. According to Approximate Bayesian Computation, the best-fit model involved the formation of N. forrestii from the crossing of N. franchetii and N. incisum, with N. forrestii further backcrossing to N. franchetii to form N. oviforme. The niche analyses indicated that niche divergence [likely triggered by the regional climate changes, particularly the intensification of East Asian winter monsoon, and tectonic movements (affecting both Qinghai-Tibetan Plateau and Qinling Mountains)] may have promoted and maintained the reproductive isolation among hybrid species. N. forrestii shows ecological specialization with respect to their parental species, whereas N. oviforme has completely shifted its niche. These results suggested that the climate and environmental factors together triggered the two-step hybridization of the East Asia herb plants. Our study also emphasizes the power of genome-wide SNPs for investigating suspected cases of hybridization, particularly unravelling old hybridization events.

Phylogenetic relationships and molecular evolution of woody forest tree family Aceraceae based on plastid phylogenomics and nuclear gene variations.

The forest tree family Aceraceae is widespread in the northern hemisphere and it has ecological and economic importance. However, the phylogenetic relationships and classifications within the family are still controversial due to transitional intraspecific morphological characteristics and introgression hybridization among species. In this study, we determined the evolutionary relationships and molecular evolution of Aceraceae based on plastid phylogenomics and two nuclear gene variations. Phylogenetic analysis based on the plastid genomes suggested that Aceraceae species can be divided into two larger sub-clades corresponding to the two genera Acer and Dipteronia. Conjoint analysis of the plastid and nuclear gene sequences supported the classification with two genera in the family. Molecular dating showed that the two genera diverged 60.2 million years ago, which is generally consistently with previously reported results. Divergence hotspots and positively selected genes identified in the plastid genomes could be useful genetic resources in Aceraceae.

Evolution and structural variations in chloroplast tRNAs in gymnosperms.

BACKGROUND: Chloroplast transfer RNAs (tRNAs) can participate in various vital processes. Gymnosperms have important ecological and economic value, and they are the dominant species in forest ecosystems in the Northern Hemisphere. However, the evolution and structural changes in chloroplast tRNAs in gymnosperms remain largely unclear. RESULTS: In this study, we determined the nucleotide evolution, phylogenetic relationships, and structural variations in 1779 chloroplast tRNAs in gymnosperms. The numbers and types of tRNA genes present in the chloroplast genomes of different gymnosperms did not differ greatly, where the average number of tRNAs was 33 and the frequencies of occurrence for various types of tRNAs were generally consistent. Nearly half of the anticodons were absent. Molecular sequence variation analysis identified the conserved secondary structures of tRNAs. About a quarter of the tRNA genes were found to contain precoded 3' CCA tails. A few tRNAs have undergone novel structural changes that are closely related to their minimum free energy, and these structural changes affect the stability of the tRNAs. Phylogenetic analysis showed that tRNAs have evolved from multiple common ancestors. The transition rate was higher than the transversion rate in gymnosperm chloroplast tRNAs. More loss events than duplication events have occurred in gymnosperm chloroplast tRNAs during their evolutionary process. CONCLUSIONS: These findings provide novel insights into the molecular evolution and biological characteristics of chloroplast tRNAs in gymnosperms.

Evolutionary characteristics and phylogeny of cotton chloroplast tRNAs.

MAIN CONCLUSION: The novel structural variations were identified in cotton chloroplast tRNAs and gene loss events were more obvious than duplications in chloroplast tRNAs. Transfer RNAs (tRNA) have long been believed an evolutionary-conserved molecular family, which play the key roles in the process of protein biosynthesis in plant life activities. In this study, we detected the evolutionary characteristics and phylogeny of chloroplast tRNAs in cotton plants, an economic and fibered important taxon in the world. We firstly annotated the chloroplast tRNAs of 27 Gossypium species to analyze their genetic composition, structural characteristics and evolution. Compared with the traditional view of evolutionary conservation of tRNA, some novel tRNA structural variations were identified in cotton plants. I.g., tRNAVal-UAC and tRNAIle-GAU only contained one intron in the anti-condon loop region of tRNA secondary structure, respectively. In the variable region, some tRNAs contained a circle structure with a few nucleotides. Interestingly, the calculation result of free energy indicated that the variation of novel tRNAs contributed to the stability of tRNA structure. Phylogenetic analysis suggested that chloroplast tRNAs have evolved from multiple common ancestors, and the tRNAMet seemed to be an ancestral tRNA, which can be duplicated and diversified to produce other tRNAs. The chloroplast tRNAs contained a group I intron in cotton plants, and the evolutionary analysis of introns indicated that group I intron of chloroplast tRNA originated from cyanobacteria. Analysis of gene duplication and loss events showed that gene loss events were more obvious than duplications in Gossypium chloroplast tRNAs. Additionally, we found that the rate of transition was higher than the ones of transversion in cotton chloroplast tRNAs. This study provided new insights into the structural characteristics and evolution of chloroplast tRNAs in cotton plants.

Using demographic model selection to untangle allopatric divergence and diversification mechanisms in the Rheum palmatum complex in the Eastern Asiatic Region.

Allopatric divergence is often initiated by geological uplift and restriction to sky-islands, climate oscillations, or river capture. However, it can be difficult to establish which mechanism was the most likely to generate the current phylogeographical structure of a species. Recently, genomic data in conjunction with a model testing framework have been applied to address this issue in animals. To test whether such an approach is also likely to be successful in plants, we used population genomic data of the Rheum palmatum complex from the Eastern Asiatic Region, in conjunction with biogeographical reconstruction and demographic model selection, to identify the potential mechanism(s) which have led to the current level of divergence. Our results indicate that the R. palmatum complex originated in the central Hengduan Mts and possibly in regions further east, and then dispersed westward and eastward resulting in genetically distinct lineages. Populations are likely to have diverged in refugia during climate oscillations followed by subsequent expansion and secondary contact. However, model simulations within the western lineage of the R. palmatum complex cannot reject a restriction to sky-islands as a possible mechanism of diversification due to the genetically ambiguous position of one population. This highlights that genetically mixed populations might introduce ambiguity regarding the best diversification model in some cases. Although it might be possible to resolve this ambiguity using other data, sometimes this could prove to be difficult in complex biogeographical areas.

Gene transfer and nucleotide sequence evolution by Gossypium cytoplasmic genomes indicates novel evolutionary characteristics.

KEY MESSAGE: The DNA fragments transferred among cotton cytoplasmic genomes are highly differentiated. The wild D group cotton species have undergone much greater evolution compared with cultivated AD group. Cotton (Gossypium spp.) is one of the most economically important fiber crops worldwide. Gene transfer, nucleotide evolution, and the codon usage preferences in cytoplasmic genomes are important evolutionary characteristics of high plants. In this study, we analyzed the nucleotide sequence evolution, codon usage, and transfer of cytoplasmic DNA fragments in Gossypium chloroplast (cp) and mitochondrial (mt) genomes, including the A genome group, wild D group, and cultivated AD group of cotton species. Our analyses indicated that the differences in the length of transferred cytoplasmic DNA fragments were not significant in mitochondrial and chloroplast sequences. Analysis of the transfer of tRNAs found that trnQ and nine other tRNA genes were commonly transferred between two different cytoplasmic genomes. The Codon Adaptation Index values showed that Gossypium cp genomes prefer A/T-ending codons. Codon preference selection was higher in the D group than the other two groups. Nucleotide sequence evolution analysis showed that intergenic spacer sequences were more variable than coding regions and nonsynonymous mutations were clearly more common in cp genomes than mt genomes. Evolutionary analysis showed that the substitution rate was much higher in cp genomes than mt genomes. Interestingly, the D group cotton species have undergone much faster evolution compared with cultivated AD groups, possibly due to the selection and domestication of diverse cotton species. Our results demonstrate that gene transfer and differential nucleotide sequence evolution have occurred frequently in cotton cytoplasmic genomes.

Species delimitation and interspecific relationships of the endangered herb genus Notopterygium inferred from multilocus variations.

Species identification and discrimination is the basis of biodiversity research. In general, it is considered that numerous nucleotide variations (e.g., whole chloroplast genomes) can identify species with higher resolution than a few loci, e.g., partial chloroplast or nuclear gene fragments. In this study, we tested this hypothesis by sampling population genetics samples of the endangered herb genus Notopterygium. We sequenced the complete plastomes, five nuclear gene regions, three chloroplast DNA fragments, and a nuclear internal transcribed spacer (nrITS) region for 18 populations sampled throughout most of the geographic ranges of all six Notopterygium species. Species identification analysis showed that four DNA barcodes (matK, rbcL, trnS-trnG, and nrITS) and/or combinations of these markers achieved Notopterygium species discrimination at higher resolution than the general plastomes and nuclear gene sequences. In particular, nrITS had the highest discriminatory power among all of the individual markers. Molecular data sets and morphological evidence indicated that all six Notopterygium species could be reclassified unambiguously to four putative species clades. N. oviforme and N. franchetii had the closest relationship. Molecular dating showed that the origin and divergence of Notopterygium species was significantly associated with geological and climatic fluctuations during the middle of the Pliocene. In conclusion, our results suggest that a few nucleotide variations can achieve species discrimination with higher resolution than numerous plastomes and general nuclear gene fragments when discerning related Notopterygium species.

Complex population evolutionary history of four cold-tolerant Notopterygium herb species in the Qinghai-Tibetan Plateau and adjacent areas.

Historical geological and climatic events are the most important drivers of population expansions/contractions, range shifts, and interspecific divergence in plants. However, the species divergence and spatiotemporal population dynamics of alpine cold-tolerant herbal plants in the high-altitude Qinghai-Tibetan Plateau (QTP) and adjacent areas remain poorly understood. In this study, we investigated population evolutionary history of four endangered Notopterygium herb species in the QTP and adjacent regions. We sequenced 10 nuclear loci, 2 mitochondrial DNA regions, and 4 chloroplast DNA regions in a total of 72 natural populations from the 4 species, and tested the hypothesis that the population history of these alpine herbs was markedly affected by the Miocene-Pliocene QTP uplifts and Quaternary climatic oscillations. We found that the four Notopterygium species had generally low levels of nucleotide variability within populations. Molecular dating and isolation-with-migration analyses suggested that Notopterygium species diverged ~1.74-7.82 million years ago and their differentiation was significantly associated with recent uplifts of the eastern margin of the QTP. In addition, ecological niche modeling and population history analysis showed that N. incisum and N. franchetii underwent considerable demographic expansions during the last glacial period of the Pleistocene, whereas a demographic contraction and a expansion occurred for N. forrestii and N. oviforme during the antepenultimate interglacial period and penultimate glacial period, respectively. These findings highlight the importance of geological and climatic changes during the Miocene-Pliocene and Pleistocene as causes of species divergence and changes in population structure within cold-tolerant herbs in the QTP biodiversity hotspot.

Evolutionary Analysis of Plastid Genomes of Seven Lonicera L. Species: Implications for Sequence Divergence and Phylogenetic Relationships.

Plant plastomes play crucial roles in species evolution and phylogenetic reconstruction studies due to being maternally inherited and due to the moderate evolutionary rate of genomes. However, patterns of sequence divergence and molecular evolution of the plastid genomes in the horticulturally- and economically-important Lonicera L. species are poorly understood. In this study, we collected the complete plastomes of seven Lonicera species and determined the various repeat sequence variations and protein sequence evolution by comparative genomic analysis. A total of 498 repeats were identified in plastid genomes, which included tandem (130), dispersed (277), and palindromic (91) types of repeat variations. Simple sequence repeat (SSR) elements analysis indicated the enriched SSRs in seven genomes to be mononucleotides, followed by tetra-nucleotides, dinucleotides, tri-nucleotides, hex-nucleotides, and penta-nucleotides. We identified 18 divergence hotspot regions (rps15, rps16, rps18, rpl23, psaJ, infA, ycf1, trnN-GUU-ndhF, rpoC2-rpoC1, rbcL-psaI, trnI-CAU-ycf2, psbZ-trnG-UCC, trnK-UUU-rps16, infA-rps8, rpl14-rpl16, trnV-GAC-rrn16, trnL-UAA intron, and rps12-clpP) that could be used as the potential molecular genetic markers for the further study of population genetics and phylogenetic evolution of Lonicera species. We found that a large number of repeat sequences were distributed in the divergence hotspots of plastid genomes. Interestingly, 16 genes were determined under positive selection, which included four genes for the subunits of ribosome proteins (rps7, rpl2, rpl16, and rpl22), three genes for the subunits of photosystem proteins (psaJ, psbC, and ycf4), three NADH oxidoreductase genes (ndhB, ndhH, and ndhK), two subunits of ATP genes (atpA and atpB), and four other genes (infA, rbcL, ycf1, and ycf2). Phylogenetic analysis based on the whole plastome demonstrated that the seven Lonicera species form a highly-supported monophyletic clade. The availability of these plastid genomes provides important genetic information for further species identification and biological research on Lonicera.

Molecular Evolution of Chloroplast Genomes of Orchid Species: Insights into Phylogenetic Relationship and Adaptive Evolution.

Orchidaceae is the 3rd largest family of angiosperms, an evolved young branch of monocotyledons. This family contains a number of economically-important horticulture and flowering plants. However, the limited availability of genomic information largely hindered the study of molecular evolution and phylogeny of Orchidaceae. In this study, we determined the evolutionary characteristics of whole chloroplast (cp) genomes and the phylogenetic relationships of the family Orchidaceae. We firstly characterized the cp genomes of four orchid species: Cremastra appendiculata, Calanthe davidii, Epipactis mairei, and Platanthera japonica. The size of the chloroplast genome ranged from 153,629 bp (C. davidi) to 160,427 bp (E. mairei). The gene order, GC content, and gene compositions are similar to those of other previously-reported angiosperms. We identified that the genes of ndhC, ndhI, and ndhK were lost in C. appendiculata, in that the ndh I gene was lost in P. japonica and E. mairei. In addition, the four types of repeats (forward, palindromic, reverse, and complement repeats) were examined in orchid species. E. mairei had the highest number of repeats (81), while C. davidii had the lowest number (57). The total number of Simple Sequence Repeats is at least 50 in C. davidii, and, at most, 78 in P. japonica. Interestingly, we identified 16 genes with positive selection sites (the psbH, petD, petL, rpl22, rpl32, rpoC1, rpoC2, rps12, rps15, rps16, accD, ccsA, rbcL, ycf1, ycf2, and ycf4 genes), which might play an important role in the orchid species' adaptation to diverse environments. Additionally, 11 mutational hotspot regions were determined, including five non-coding regions (ndhB intron, ccsA-ndhD, rpl33-rps18, ndhE-ndhG, and ndhF-rpl32) and six coding regions (rps16, ndhC, rpl32, ndhI, ndhK, and ndhF). The phylogenetic analysis based on whole cp genomes showed that C. appendiculata was closely related to C. striata var. vreelandii, while C. davidii and C. triplicate formed a small monophyletic evolutionary clade with a high bootstrap support. In addition, five subfamilies of Orchidaceae, Apostasioideae, Cypripedioideae, Epidendroideae, Orchidoideae, and Vanilloideae, formed a nested evolutionary relationship in the phylogenetic tree. These results provide important insights into the adaptive evolution and phylogeny of Orchidaceae.

Comparative Transcriptome Analysis Reveals Adaptive Evolution of Notopterygium incisum and Notopterygium franchetii, Two High-Alpine Herbal Species Endemic to China.

The extreme conditions (e.g., cold, low oxygen, and strong ultraviolet radiation) of the high mountains provide an ideal natural laboratory for studies on speciation and the adaptive evolution of organisms. Up to now, few genome/transcriptome-based studies have been carried out on how plants adapt to conditions at extremely high altitudes. Notopterygium incisum and Notopterygium franchetii (Notopterygium, Apiaceae) are two endangered high-alpine herbal plants endemic to China. To explore the molecular genetic mechanisms of adaptation to high altitudes, we performed high-throughput RNA sequencing (RNA-seq) to characterize the transcriptomes of the two species. In total, more than 130 million sequence reads, 81,446 and 63,153 unigenes with total lengths of 86,924,837 and 62,615,693 bp, were generated for the two herbal species, respectively. OrthoMCL analysis identified 6375 single-copy orthologous genes between N. incisum and N. franchetii. In total, 381 positively-selected candidate genes were identified for both plants by using estimations of the non-synonymous to synonymous substitution rate. At least 18 of these genes potentially participate in RNA splicing, DNA repair, glutathione metabolism and the plant-pathogen interaction pathway, which were further enriched in various functional gene categories possibly responsible for environment adaptation in high mountains. Meanwhile, we detected various transcription factors that regulated the material and energy metabolism in N. incisum and N. franchetii, which probably play vital roles in the tolerance to stress in surroundings. In addition, 60 primer pairs based on orthologous microsatellite-containing sequences between the both Notopterygium species were determined. Finally, 17 polymorphic microsatellite markers (SSR) were successfully characterized for the two endangered species. Based on these candidate orthologous and SSR markers, we detected that the adaptive evolution and species divergence of N. incisum and N. franchetii were significantly associated with the extremely heterogeneous environments and climatic oscillations in high-altitude areas. This work provides important insights into the molecular mechanisms of adaptation to high-altitudes in alpine herbal plants.

Transcriptome Sequencing and Development of Genic SSR Markers of an Endangered Chinese Endemic Genus Dipteronia Oliver (Aceraceae).

Dipteronia Oliver (Aceraceae) is an endangered Chinese endemic genus consisting of two living species, Dipteronia sinensis and Dipteronia dyeriana. However, studies on the population genetics and evolutionary analyses of Dipteronia have been hindered by limited genomic resources and genetic markers. Here, the generation, de novo assembly and annotation of transcriptome datasets, and a large set of microsatellite or simple sequence repeat (SSR) markers derived from Dipteronia have been described. After Illumina pair-end sequencing, approximately 93.2 million reads were generated and assembled to yield a total of 99,358 unigenes. A majority of these unigenes (53%, 52,789) had at least one blast hit against the public protein databases. Further, 12,377 SSR loci were detected and 4179 primer pairs were designed for experimental validation. Of these 4179 primer pairs, 435 primer pairs were randomly selected to test polymorphism. Our results show that products from 132 primer pairs were polymorphic, in which 97 polymorphic SSR markers were further selected to analyze the genetic diversity of 10 natural populations of Dipteronia. The identification of SSR markers during our research will provide the much valuable data for population genetic analyses and evolutionary studies in Dipteronia.

Characterization of Global Transcriptome Using Illumina Paired-End Sequencing and Development of EST-SSR Markers in Two Species of Gynostemma (Cucurbitaceae).

Gynostemma pentaphyllum is an important medicinal herb of the Cucurbitaceae family, but limited genomic data have hindered genetic studies. In this study, transcriptomes of two closely-related Gynostemma species, Gynostemma cardiospermum and G. pentaphyllum, were sequenced using Illumina paired-end sequencing technology. A total of 71,607 nonredundant unigenes were assembled. Of these unigenes, 60.45% (43,288) were annotated based on sequence similarity search with known proteins. A total of 11,059 unigenes were identified in the Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG) database. A total of 3891 simple sequence repeats (SSRs) were detected in 3526 nonredundant unigenes, 2596 primer pairs were designed and 360 of them were randomly selected for validation. Of these, 268 primer pairs yielded clear products among six G. pentaphyllum samples. Thirty polymorphic SSR markers were used to test polymorphism and transferability in Gynostemma. Finally, 15 SSR makers that amplified in all 12 Gynostemma species were used to assess genetic diversity. Our results generated a comprehensive sequence resource for Gynostemma research.

Accumulation of livestock manure-derived heavy metals in the Hexi Corridor oasis agricultural alkaline soil and bioavailability to Chinese cabbage (Brassica pekinensis L.) after 4-year continuous application.

Hexi Corridor is one of the most important base of vegetable producing areas in China. Livestock manure (LM) applied to agricultural field could lead to soil heavy metal (HM) pollution. Previous studies have focused on HM pollution following LM application in acidic polluted soils; however, fewer studies have been conducted in alkaline unpolluted soils. A 4-year field vegetable production experiment was conducted using pig manure (PM) and chicken manure (CM) at five application rates (0, 15, 30, 45, and 60 t ha-1) to elucidate potential risks of HMs in an alkaline unpolluted soil in the Hexi Corridor oasis agricultural area and HM uptake by Chinese cabbage. The results showed that LM application caused a significant build-up of Cu, Zn, Pb, Cd, and Ni content in topsoil by 30.6-99.7%, 11.4-51.7%, 1.4-31.3%, 5.6-44.9%, 14%-40.8%, respectively. The Cd, Cu, Zn could potentially exceed the soil threshold in next 8-65 years after 15-60 t ha-1 LM application. Under LM treatment, the soil DTPA-extractable Cu, Zn, Fe, the acid-extractable fraction of Cu, Zn, Fe, Cd, Ni, and the Oxidable fraction of Cu, Zn, Fe, Mn, Cd, Ni significantly increased, but the DTPA-extractable Pb, Cd, the acid-extractable fraction of Pb, and the reducible fraction of Cd significantly decreased. Cu and Zn could migrate to the deeper soil and relatively increase in DTPA-extracted Cu, Zn were found in 20-40 cm soil depth after LM application. The pH and SOM could influence the bioavailability of HMs in soil. The bioaccumulation factor and transfer factor (TF) values were <1 except Mn (TF > 1). HMs in leaf did not approach the threshold for HM toxicity due to the "dilution effect". Recommend the type of manure was the PM and the annual PM application rate was 30 t ha-1 to ensure a 20-year period of clean production in alkaline unpolluted Fluvo-aqiuc vegetable soils.

Testing complete plastomes and nuclear ribosomal DNA sequences for species identification in a taxonomically difficult bamboo genus Fargesia.

Fargesia, the largest genus within the temperate bamboo tribe Arundinarieae, has more than 90 species mainly distributed in the mountains of Southwest China. The Fargesia bamboos are important components of the subalpine forest ecosystems that provide food and habitat for many endangered animals, including the giant panda. However, species-level identification of Fargesia is difficult. Moreover, the rapid radiation and slow molecular evolutionary rate of Fargesia pose a significant challenge to using DNA barcoding with standard plant barcodes (rbcL, matK, and ITS) in bamboos. With progress in the sequencing technologies, complete plastid genomes (plastomes) and nuclear ribosomal DNA (nrDNA) sequences have been proposed as organelle barcodes for species identification; however, these have not been tested in bamboos. We collected 196 individuals representing 62 species of Fargesia to comprehensively evaluate the discriminatory power of plastomes and nrDNA sequences compared to standard barcodes. Our analysis indicates that complete plastomes have substantially higher discriminatory power (28.6%) than standard barcodes (5.7%), whereas nrDNA sequences show a moderate improvement (65.4%) compared to ITS (47.2%). We also found that nuclear markers performed better than plastid markers, and ITS alone had higher discriminatory power than complete plastomes. The study also demonstrated that plastomes and nrDNA sequences can contribute to intrageneric phylogenetic resolution in Fargesia. However, neither of these sequences were able to discriminate all the sampled species, and therefore, more nuclear markers need to be identified.

Clinicopathological Characteristics, Prognosis, and Correlated Tumor Cell Function of Tropomodulin-3 in Pancreatic Adenocarcinoma.

BACKGROUND: Pancreatic adenocarcinoma (PAAD) is a frequent malignant tumor with a high mortality rate. Searching for novel biomarkers that can influence its prognosis may help patients. It has been shown that tropomodulin-3 (TMOD3) may influence tumor progression, but its role in pancreatic cancer is not clear. We aimed to explore the expression and prognostic value of TMOD3 in PAAD. METHODS: We used bioinformatics analysis to analyze the relationship between TMOD3 expression and clinicopathological features and prognosis and verified it with clinical data from tissue microarray. We also conducted in vitro cell experiments to explore the effects of TMOD3 on the function of PAAD cells. RESULTS: TMOD3 expression was found to be significantly higher in PAAD tissues than in matched paracancerous tissues (P < 0.05). Meanwhile, high TMOD3 expression was associated with significantly poorer overall survival (P < 0.05). Analysis of relevant clinicopathological characteristics data obtained from TCGA showed that high TMOD3 expression correlated with age, TNM stage, N stage, and M stage (P < 0.05). Analysis of correlation data obtained from tissue microarrays showed that high TMOD3 expression was associated with lymph node invasion, nerve invasion, macrovascular invasion, and TNM stage (P < 0.05). In addition, siRNA knockdown of TMOD3 significantly reduced the migration and invasion of PAAD cells. CONCLUSION: Our study shows that TMOD3 may be associated with the progression of PAAD cells, and that it is an independent risk factor for poor pathological features and prognosis of PAAD. It may be helpful as a prognostic indicator of clinical outcomes in PAAD patients.

Characterization of the complete chloroplast genome of Gypsophila huashanensis Y. W. Tsui & D. Q. Lu, an endemic herb species in China.

Gypsophila huashanensis Y. W. Tsui & D. Q. Lu (Caryophyllaceae) is an endemic herb species to the Qinling Mountains in China. In this study, we characterized its whole plastid genome using the Illumina sequencing platform. The complete plastid genome of G. huashanensis is 152,457 bp in length, including a large single-copy DNA region of 83,476 bp, a small single-copy DNA region of 17,345 bp, and a pair of inverted repeat DNA sequences of 25,818 bp. The genome contains 130 genes comprising 85 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Evolutionary analysis showed that the non-coding regions of Caryophyllaceae exhibit a higher level of divergence than the exon regions. Gene site selection analysis suggested that 11 coding protein genes (accD, atpF, ndhA, ndhB, petB, petD, rpoCl, rpoC2, rps16, ycfl, and ycf2) have some sites under protein sequence evolution. Phylogenetic analysis showed that G. huashanensis is most closely related to the congeneric species G. oldhamiana. These results are very useful for studying phylogenetic evolution and species divergence in the family Caryophyllaceae.

Change of geographic distributions of the genus Morchella species responding to climate oscillations in East Asia.

Morchella is a rare macrofungi taxon with high medicinal and edible values. Influenced by recent climate oscillations and human activities, habitat fragmentation of this genus has been critical, leading to a rapid decline of the resource of Morchella. It is thus urgent to preserve Morchella species. Based on maximum entropy model (MaxEnt), and 102 geographic distribution records of Morchella species with 10 environmental factors, we simulated the changes of potential geographic distributions under the climatic conditions of the last glacial maximum (LGM), last interglacial (LIG), in contemporary period and future (2050, 2070). We further analyzed the potential changes of geographic distributions of Morchella species in East Asia under climate change and formulated the effective conservation strategies for Morchella. The results showed that the dominant environmental factors affecting the geographic distributions of Morchella species were mean temperature of coldest quarter, annual precipitation, elevation and temperature annual range, with the mean temperature of coldest quarter having the greatest contribution. Results of the species distribution models showed that the highly suitable regions for Morchella species were mainly distributed in parts of western China under contemporary period. From the LIG to LGM and then the current to the future period, the total suitable regions of Morchella species showed a trend of firstly decrease and then increase, while the highly suitable regions showed similar change with the total suitable regions. At present, there is an urgent need to conduct in situ conservation for the resources of Morchella species in highly suitable regions in western China, and to carry out ex situ conservation in the marginal ranges of highly suitable regions and moderately suitable regions of Shaanxi, Hebei, Shandong, and other regions in China.