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Abstract Background Acer is a taxonomically intractable and speciose genus that contains over 150 species. It is challenging to distinguish Acer species only by morphological method due to their abundant variations. Plastome and nuclear ribosomal DNA (nrDNA) sequences are recommended as powerful next-generation DNA barcodes for species discrimination. However, their efficacies were still poorly studied. The current study will evaluate the application of plastome and nrDNA in species identification and perform phylogenetic analyses for Acer. Result Based on a collection of 83 individuals representing 55 species (c. 55% of Chinese species) from 13 sections, our barcoding analyses demonstrated that plastomes exhibited the highest (90.47%) species discriminatory power among all plastid DNA markers, such as the standard plastid barcodes matK + rbcL + trnH-psbA (61.90%) and ycf1 (76.19%). And the nrDNA (80.95%) revealed higher species resolution than ITS (71.43%). Acer plastomes show abundant interspecific variations, however, species identification failure may be due to the incomplete lineage sorting (ILS) and chloroplast capture resulting from hybridization. We found that the usage of nrDNA contributed to identifying those species that were unidentified by plastomes, implying its capability to some extent to mitigate the impact of hybridization and ILS on species discrimination. However, combining plastome and nrDNA is not recommended given the cytonuclear conflict caused by potential hybridization. Our phylogenetic analysis covering 19 sections (95% sections of Acer) and 128 species (over 80% species of this genus) revealed pervasive inter- and intra-section cytonuclear discordances, hinting that hybridization has played an important role in the evolution of Acer. Conclusion Plastomes and nrDNA can significantly improve the species resolution in Acer. Our phylogenetic analysis uncovered the scope and depth of cytonuclear conflict in Acer, providing important insights into its evolution.

Abstract Background Recent research suggests that periodontitis can increase the risk of chronic obstructive pulmonary disease (COPD). In this study, we performed two-sample Mendelian randomization (MR) and investigated the causal effect of periodontitis (PD) on the genetic prediction of COPD. The study aimed to estimate how exposures affected outcomes. Methods Published data from the Gene-Lifestyle Interaction in the Dental Endpoints (GLIDE) Consortium’s genome-wide association studies (GWAS) for periodontitis (17,353 cases and 28,210 controls) and COPD (16,488 cases and 169,688 controls) from European ancestry were utilized. This study employed a two-sample MR analysis approach and applied several complementary methods, including weighted median, inverse variance weighted (IVW), and MR-Egger regression. Multivariable Mendelian randomization (MVMR) analysis was further conducted to mitigate the influence of smoking on COPD. Results We chose five single-nucleotide polymorphisms (SNPs) as instrumental variables for periodontitis. A strong genetically predicted causal link between periodontitis and COPD, that is, periodontitis as an independent risk factor for COPD was detected. PD (OR = 1.102951, 95% CI: 1.005–1.211, p = 0.039) MR-Egger regression and weighted median analysis results were coincident with those of the IVW method. According to the sensitivity analysis, horizontal pleiotropy’s effect on causal estimations seemed unlikely. However, reverse MR analysis revealed no significant genetic causal association between COPD and periodontitis. IVW (OR = 1.048 > 1, 95%CI: 0.973–1.128, p = 0.2082) MR Egger (OR = 0.826, 95%CI:0.658–1.037, p = 0.1104) and weighted median (OR = 1.043, 95%CI: 0.941–1.156, p = 0.4239). The results of multivariable Mendelian randomization (MVMR) analysis, after adjusting for the confounding effect of smoking, suggest a potential causal relationship between periodontitis and COPD (P = 0.035). Conclusion In this study, periodontitis was found to be independent of COPD and a significant risk factor, providing new insights into periodontitis-mediated mechanisms underlying COPD development.

Abstract Background The ephemeral flora of northern Xinjiang, China, plays an important role in the desert ecosystems. However, the evolutionary history of this flora remains unclear. To gain new insights into its origin and evolutionary dynamics, we comprehensively sampled ephemeral plants of Brassicaceae, one of the essential plant groups of the ephemeral flora. Results We reconstructed a phylogenetic tree using plastid genomes and estimated their divergence times. Our results indicate that ephemeral species began to colonize the arid areas in north Xinjiang during the Early Miocene and there was a greater dispersal of ephemeral species from the surrounding areas into the ephemeral community of north Xinjiang during the Middle and Late Miocene, in contrast to the Early Miocene or Pliocene periods. Conclusions Our findings, together with previous studies, suggest that the ephemeral flora originated in the Early Miocene, and species assembly became rapid from the Middle Miocene onwards, possibly attributable to global climate changes and regional geological events.

Abstract The application of DNA barcoding has been significantly limited by the scarcity of reliable specimens and inadequate coverage and replication across all species. The deficiency of DNA barcode reference coverage is particularly striking for highly biodiverse subtropical and tropical regions. In this study, we present a comprehensive barcode library for woody plants in tropical and subtropical China. Our dataset includes a standard barcode library comprising the four most widely used barcodes (rbcL, matK, ITS, and ITS2) for 2,520 species from 4,654 samples across 49 orders, 144 families, and 693 genera, along with 79 samples identified at the genus level. This dataset also provides a super-barcode library consisting of 1,239 samples from 1,139 species, 411 genera, 113 families, and 40 orders. This newly developed library will serve as a valuable resource for DNA barcoding research in tropical and subtropical China and bordering countries, enable more accurate species identification, and contribute to the conservation and management of tropical and subtropical forests.

Abstract Musa acuminata is a main wild contributor to banana cultivars. Here, we reported a haplotype-resolved and telomere-to-telomere reference genome of M. acuminata by incorporating PacBio HiFi reads, Nanopore ultra-long reads, and Hi-C data. The genome size of the two haploid assemblies was estimated to be 469.83 Mb and 470.21 Mb, respectively. Multiple assessments confirmed the contiguity (contig N50: 16.53 Mb and 18.58 Mb; LAI: 20.18 and 19.48), completeness (BUSCOs: 98.57% and 98.57%), and correctness (QV: 45.97 and 46.12) of the genome. The repetitive sequences accounted for about half of the genome size. In total, 40,889 and 38,269 protein-coding genes were annotated in the two haploid assemblies, respectively, of which 9.56% and 3.37% were newly predicted. Genome comparison identified a large reciprocal translocation involving 3 Mb and 10 Mb from chromosomes 01 and 04 within M. acuminata. This reference genome of M. acuminata provides a valuable resource for further understanding of subgenome evolution of Musa species, and precise genetic improvement of banana.

Abstract Background Lysimachia L., the second largest genus within the subfamily Myrsinoideae of Primulaceae, comprises approximately 250 species worldwide. China is the species diversity center of Lysimachia, containing approximately 150 species. Despite advances in the backbone phylogeny of Lysimachia, species-level relationships remain poorly understood due to limited genomic information. This study analyzed 50 complete plastomes for 46 Lysimachia species. We aimed to identify the plastome structure features and hypervariable loci of Lysimachia. Additionally, the phylogenetic relationships and phylogenetic conflict signals in Lysimachia were examined. Results These fifty plastomes within Lysimachia had the typical quadripartite structure, with lengths varying from 152,691 to 155,784 bp. Plastome size was positively correlated with IR and intron length. Thirteen highly variable regions in Lysimachia plastomes were identified. Additionally, ndhB, petB and ycf2 were found to be under positive selection. Plastid ML trees and species tree strongly supported that L. maritima as sister to subg. Palladia + subg. Lysimachia (Christinae clade), while the nrDNA ML tree clearly placed L. maritima and subg. Palladia as a sister group. Conclusions The structures of these plastomes of Lysimachia were generally conserved, but potential plastid markers and signatures of positive selection were detected. These genomic data provided new insights into the interspecific relationships of Lysimachia, including the cytonuclear discordance of the position of L. maritima, which may be the result of ghost introgression in the past. Our findings have established a basis for further exploration of the taxonomy, phylogeny and evolutionary history within Lysimachia.

Abstract Introduction Previous observational studies have associated periodontitis (PD) with migraine; however, the results are inconclusive and the causality of the association between PD and migraine remains unclear. This two-sample Mendelian randomization (MR) study was performed to explore the bi-directional causal relationship between PD and migraine. Methods To investigate the relationship between PD (17,353 cases; 28,210 controls) and migraine (1072 cases; 360,122 controls), we used genetic tools from the largest available genome-wide association study of European descent. Inverse variance-weighted (IVW) and a series of sensitivity analyses were used to explore the association between migraine and PD. We performed an MR study using seven SNPs (single nucleotide polymorphisms) as instrumental variables for PD to investigate the causal relationship between migraine and PD. Results We found no significant causal relationship between PD and migraine (odds ratio, OR = 1.000; 95% confidence interval, CI = 0.99–1.00; p = 0.65). Similarly, no evidence supported a causal relationship between migraine and PD (OR = 0.07; CI = 2.04 × 10–9–2.65 × 106; p = 0.77). A sensitivity analysis revealed that no potential polymorphic effect (p = 0.356) and heterogeneity (p = 0.652) exists for the variants used in constructing the genetic instrument. Conclusions Based on the results of our MR study, there is no causal relationship between PD and migraines or migraines and PD.

Abstract Lomas formations or “fog oases” are islands of vegetation in the desert belt of the west coast of South America, with a unique vegetation composition among the world’s deserts. However, plant diversity and conservation studies have long been neglected, and there exists a severe gap in plant DNA sequence information. To address the lack of DNA information, we conducted field collections and laboratory DNA sequencing to establish a DNA barcode reference library of Lomas plants from Peru. This database provides 1,207 plant specimens and 3,129 DNA barcodes data corresponding with collections from 16 Lomas locations in Peru, during 2017 and 2018. This database will facilitate both rapid species identification and basic studies on plant diversity, thereby enhancing our understanding of Lomas flora’s composition and temporal variation, and providing valuable resources for conserving plant diversity and maintaining the stability of the fragile Lomas ecosystems.

Abstract Background Over the past decade, phylogenomics has greatly advanced our knowledge of angiosperm evolution. However, phylogenomic studies of large angiosperm families with complete species or genus-level sampling are still lacking. The palms, Arecaceae, are a large family with ca. 181 genera and 2600 species and are important components of tropical rainforests bearing great cultural and economic significance. Taxonomy and phylogeny of the family have been extensively investigated by a series of molecular phylogenetic studies in the last two decades. Nevertheless, some phylogenetic relationships within the family are not yet well-resolved, especially at the tribal and generic levels, with consequent impacts for downstream research. Results Plastomes of 182 palm species representing 111 genera were newly sequenced. Combining these with previously published plastid DNA data, we were able to sample 98% of palm genera and conduct a plastid phylogenomic investigation of the family. Maximum likelihood analyses yielded a robustly supported phylogenetic hypothesis. Phylogenetic relationships among all five palm subfamilies and 28 tribes were well-resolved, and most inter-generic phylogenetic relationships were also resolved with strong support. Conclusions The inclusion of nearly complete generic-level sampling coupled with nearly complete plastid genomes strengthened our understanding of plastid-based relationships of the palms. This comprehensive plastid genome dataset complements a growing body of nuclear genomic data. Together, these datasets form a novel phylogenomic baseline for the palms and an increasingly robust framework for future comparative biological studies of this exceptionally important plant family.

Abstract Background Although knowledge of the sizes, contents, and forms of plant mitochondrial genomes (mitogenomes) is increasing, little is known about the mechanisms underlying their structural diversity. Evolutionary information on the mitogenomes of Primula, an important ornamental taxon, is more limited than the information on their nuclear and plastid counterparts, which has hindered the comprehensive understanding of Primula mitogenomic diversity and evolution. The present study reported and compared three Primula mitogenomes and discussed the size expansion of mitogenomes in Ericales. Results Mitogenome master circles were sequenced and successfully assembled for three Primula taxa and were compared with publicly available Ericales mitogenomes. The three mitogenomes contained similar gene contents and varied primarily in their structures. The Primula mitogenomes possessed relatively high nucleotide diversity among all examined plant lineages. In addition, high nucleotide diversity was found among Primula species between the Mediterranean and Himalaya-Hengduan Mountains. Most predicted RNA editing sites appeared in the second amino acid codon, increasing the hydrophobic character of the protein. An early stop in atp6 caused by RNA editing was conserved across all examined Ericales species. The interfamilial relationships within Ericales and interspecific relationships within Primula could be well resolved based on mitochondrial data. Transfer of the two longest mitochondrial plastid sequences (MTPTs) occurred before the divergence of Primula and its close relatives, and multiple independent transfers could also occur in a single MTPT sequence. Foreign sequence [MTPTs and mitochondrial nuclear DNA sequences (NUMTs)] uptake and repeats were to some extent associated with changes in Ericales mitogenome size, although none of these relationships were significant overall. Conclusions The present study revealed relatively conserved gene contents, gene clusters, RNA editing, and MTPTs but considerable structural variation in Primula mitogenomes. Relatively high nucleotide diversity was found in the Primula mitogenomes. In addition, mitogenomic genes, collinear gene clusters, and locally collinear blocks (LCBs) all showed phylogenetic signals. The evolutionary history of MTPTs in Primula was complicated, even in a single MTPT sequence. Various reasons for the size variation observed in Ericales mitogenomes were found.

Abstract Background Tribe Cinnamomeae is a species-rich and ecologically important group in tropical and subtropical forests. Previous studies explored its phylogenetic relationships and historical biogeography using limited loci, which might result in biased molecular dating due to insufficient parsimony-informative sites. Thus, 15 plastomes were newly sequenced and combined with published plastomes to study plastome structural variations, gene evolution, phylogenetic relationships, and divergence times of this tribe. Results Among the 15 newly generated plastomes, 14 ranged from 152,551 bp to 152,847 bp, and the remaining one (Cinnamomum chartophyllum XTBGLQM0164) was 158,657 bp. The inverted repeat (IR) regions of XTBGLQM0164 contained complete ycf2, trnI CAU , rpl32, and rpl2. Four hypervariable plastid loci (ycf1, ycf2, ndhF-rpl32-trnL UAG , and petA-psbJ) were identified as candidate DNA barcodes. Divergence times based on a few loci were primarily determined by prior age constraints rather than by DNA data. In contrast, molecular dating using complete plastid protein-coding genes (PCGs) was determined by DNA data rather than by prior age constraints. Dating analyses using PCGs showed that Cinnamomum sect. Camphora diverged from C. sect. Cinnamomum in the late Oligocene (27.47 Ma). Conclusions This study reports the first case of drastic IR expansion in tribe Cinnamomeae, and indicates that plastomes have sufficient parsimony-informative sites for molecular dating. Besides, the dating analyses provide preliminary insights into the divergence time within tribe Cinnamomeae and can facilitate future studies on its historical biogeography.

Abstract Background Musaceae is an economically important family consisting of 70-80 species. Elucidation of the interspecific relationships of this family is essential for a more efficient conservation and utilization of genetic resources for banana improvement. However, the scarcity of herbarium specimens and quality molecular markers have limited our understanding of the phylogenetic relationships in wild species of Musaceae. Aiming at improving the phylogenetic resolution of Musaceae, we analyzed a comprehensive set of 49 plastomes for 48 species/subspecies representing all three genera of this family. Results Musaceae plastomes have a relatively well-conserved genomic size and gene content, with a full length ranging from 166,782 bp to 172,514 bp. Variations in the IR borders were found to show phylogenetic signals to a certain extent in Musa. Codon usage bias analysis showed different preferences for the same codon between species and three genera and a common preference for A/T-ending codons. Among the two genes detected under positive selection (dN/dS > 1), ycf2 was indicated under an intensive positive selection. The divergent hotspot analysis allowed the identification of four regions (ndhF-trnL, ndhF, matK-rps16, and accD) as specific DNA barcodes for Musaceae species. Bayesian and maximum likelihood phylogenetic analyses using full plastome resulted in nearly identical tree topologies with highly supported relationships between species. The monospecies genus Musella is sister to Ensete, and the genus Musa was divided into two large clades, which corresponded well to the basic number of n = x = 11 and n = x =10/9/7, respectively. Four subclades were divided within the genus Musa. A dating analysis covering the whole Zingiberales indicated that the divergence of Musaceae family originated in the Palaeocene (59.19 Ma), and the genus Musa diverged into two clades in the Eocene (50.70 Ma) and then started to diversify from the late Oligocene (29.92 Ma) to the late Miocene. Two lineages (Rhodochlamys and Australimusa) radiated recently in the Pliocene /Pleistocene periods. Conclusions The plastome sequences performed well in resolving the phylogenetic relationships of Musaceae and generated new insights into its evolution. Plastome sequences provided valuable resources for population genetics and phylogenetics at lower taxon.

Abstract Background The East Asian subtropical evergreen broad-leaved forests (EBLFs) harbor remarkable biodiversity. However, their historical assembly remains unclear. To gain new insights into the assembly of this biome, we generated a molecular phylogeny of one of its essential plant groups, the tribe Perseeae (Lauraceae). Results Our plastid tree topologies were robust to analyses based on different plastid regions and different strategies for data partitioning, nucleotide substitution saturation, and gap handling. We found that tribe Perseeae comprised six major clades and began to colonize the subtropical EBLFs of East Asia in the early Miocene. The diversification rates of tribe Perseeae accelerated twice in the late Miocene. Conclusions Our findings suggest that the intensified precipitation in East Asia in the early Miocene may have facilitated range expansions of the subtropical EBLFs and establishment of tribe Perseeae within this biome. By the late Miocene, species assembly and diversification within the EBLFs had become rapid.

Abstract The Andean plant endemic Puya is a striking example of recent and rapid diversification from central Chile to the northern Andes, tracking mountain uplift. This study generated 12 complete plastomes representing nine Puya species and compared them to five published plastomes for their features, genomic evolution, and phylogeny. The total size of the Puya plastomes ranged from 159,542 to 159,839 bp with 37.3%–37.4% GC content. The Puya plastomes were highly conserved in organization and structure with a typical quadripartite genome structure. Each of the 17 consensus plastomes harbored 133 genes, including 87 protein‐coding genes, 38 tRNA (transfer RNA) genes, and eight rRNA (ribosomal RNA) genes; we found 69–78 tandem repeats, 45–60 SSRs (simple sequence repeats), and 8–22 repeat structures among 13 species. Four protein‐coding genes were identified under positive site‐specific selection in Puya. The complete plastomes and hypervariable regions collectively provided pronounced species discrimination in Puya and a practical tool for future phylogenetic studies. The reconstructed phylogeny and estimated divergence time for the lineage suggest that the diversification of Puya is related to Andean orogeny and Pleistocene climatic oscillations. This study provides plastome resources for species delimitation and novel phylogenetic and biogeographic studies.

The complete chloroplast genome of Isatis minima, a typical ephemeral plant of Brassicaceae in the Central Asia desert, was sequenced and characterized in this study. The genome 153,642 bp in size, contains a typical quadripartite genome organization including LSC and SSC regions of 83,423 bp and 17,709 bp, and two copies of the IR regions of 26,255 bp. It has 113 unique genes, including 79 protein-coding, 30 tRNA, and four rRNA genes. Phylogenetic analysis fully resolved I. minima in a monophyletic clade with I. tinctoria. This bioinformatic data contributes to the phylogenetics systematics and evolutionary history of Brassicaceae.

Abstract The strength of biodiversity–biomass production relationships increases with increasing environmental stress and time. However, we know little about the effects of abiotic (e.g., climate) and biotic (e.g., species pool and community composition) factors on this trend. Whether variation in biomass production is best explained by phylogenetic diversity metrics or traditional measures of species richness also remains elusive. We compiled estimates of community composition and biomass production for tree species in 111 permanent quadrats spanning three natural forests (tropical, subtropical, and temperate) in China. Based on ~10 years of data, we compared temperature, rainfall, species pool size, and community composition in each forest each year. We estimated species richness and phylogenetic diversity in each quadrat each year; the latter metric was based on the sum of branch lengths of a phylogeny that connects species in each quadrat each year. Using generalized linear mixed‐effect models, we found that top‐ranked models included the interaction between forest and biodiversity and the interaction between forest and year for both biodiversity metrics. Variation in biomass production was best explained by phylogenetic diversity; biomass production generally increased with phylogenetic diversity, and the relationship was stronger in subtropical and temperate forests. Increasing species pool size, temperature, and rainfall and decreasing inter‐quadrat dissimilarity range shifted the relationship between biomass production and phylogenetic diversity from positive to neutral. When considered alone, species pool size had the strongest influence on biomass production, while species pool size, rainfall, and their interaction with phylogenetic diversity constituted the top‐ranked model. Our study highlights the importance of species pool size and rainfall on the relationship between phylogenetic diversity and biomass production in natural forest ecosystems.

Abstract Background Calligonum (Polygonaceae) is distributed from southern Europe through northern Africa to central Asia, and is typically found in arid, desert regions. Previous studies have revealed that standard DNA barcodes fail to discriminate Calligonum species. In this study, the complete plastid genomes (plastome) for 32 accessions of 21 Calligonum species is sequenced to not only generate the first complete plastome sequence for the genus Calligonum but to also 1) Assess the ability of the complete plastome sequence to discern species within the group, and 2) screen the plastome sequence for a cost-effective DNA barcode that can be used in future studies to resolve taxonomic relationships within the group. Results The whole plastomes of Calligonum species possess a typical quadripartite structure. The size of the Calligonum plastome is approximately 161 kilobase pairs (kbp), and encodes 113 genes, including 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. Based on ML phylogenetic tree analyses, the complete plastome has higher species identification (78%) than combinations of standard DNA barcodes (rbcL + matK + nrITS, 56%). Five newly screened gene regions (ndhF, trnS-G, trnC-petN, ndhF-rpl32, rpl32-trnL) had high species resolution, where ndhF and trnS-G were able to distinguish the highest proportion of Calligonum species (56%). Conclusions The entire plastid genome was the most effective barcode for the genus Calligonum, although other gene regions showed great potential as taxon-specific barcodes for species identification in Calligonum.

Lithocarpus hancei (Benth.) Rehd is a widely distributed evergreen tree with broad-leaves that dominates the lower stories of the forest in China. Here, we sequenced and assembled the complete chloroplast genome of L. hancei. The genome is 161,304 bp with one large single copy (LSC: 90,585 bp), one small single copy (SSC: 18,959 bp), and two inverted repeat (IR) regions (IRa and IRb, each 25,880 bp). It contains 117 genes, including 80 protein-coding genes, 33 tRNA genes, and four rRNA genes. Phylogenetic analysis of 21 representative cp genomes of the Fagaceae suggests Lithocarpus is monophyletic with strong bootstrap support and also that L. hancei is closely related to L. polystachyus. The cp genome is important for constructing a robust phylogeny of Lithocarpus and Fagaceae for future study.

Background Gene tree discordance is common in phylogenetic analyses. Many phylogenetic studies have excluded non-coding regions of the plastome without evaluating their impact on tree topology. In general, plastid loci have often been treated as a single unit, and tree discordance among these loci has seldom been examined. Using samples of Laureae (Lauraceae) plastomes, we explored plastome variation among the tribe, examined the influence of non-coding regions on tree topology, and quantified intra-plastome conflict. Results We found that the plastomes of Laureae have low inter-specific variation and are highly similar in structure, size, and gene content. Laureae was divided into three groups, subclades I, II and III. The inclusion of non-coding regions changed the phylogenetic relationship among the three subclades. Topologies based on coding and non-coding regions were largely congruent except for the relationship among subclades I, II and III. By measuring the distribution of phylogenetic signal across loci that supported different topologies, we found that nine loci (two coding regions, two introns and five intergenic spacers) played a critical role at the contentious node. Conclusions Our results suggest that subclade III and subclade II are successively sister to subclade I. Conflicting phylogenetic signals exist between coding and non-coding regions of Laureae plastomes. Our study highlights the importance of evaluating the influence of non-coding regions on tree topology and emphasizes the necessity of examining discordance among different plastid loci in phylogenetic studies.

The palm family (Arecaceae) is of high ecological and economic value, yet identification in the family remains a challenge for both taxonomists and horticulturalists. The family consists of approximately 2600 species across 181 genera and DNA barcoding may be a useful tool for species identification within the group. However, there have been few systematic evaluations of DNA barcodes for the palm family. In the present study, five DNA barcodes (rbcL, matK, trnH-psbA, ITS, ITS2) were evaluated for species identification ability across 669 samples representing 314 species and 100 genera in the Arecaceae, employing four analytical methods. The ITS gene region was found to not be a suitable barcode for the palm family, due in part, to low recovery rates and paralogous gene copies. Among the four analyses used, species resolution for ITS2 was much higher than that achieved with the plastid barcodes alone (rbcL, matK, trnH-psbA), and the barcode combination ITS2 + matK + rbcL gave the highest resolution among all single barcodes and their combinations, followed by ITS2 + matK. Among 669 palm samples analyzed, 110 samples (16.3%) were found to be misidentified. The 2992 DNA barcode sequences generated in this study greatly enriches the existing identification toolbox available to plant taxonomists that are interested in researching genetic relationships among palm taxa as well as for horticulturalists that need to confirm palm collections for botanical garden curation and horticultural applications. Our results indicate that the use of the ITS2 DNA barcode gene region provides a useful and cost-effective tool to confirm the identity of taxa in the Palm family.

Abstract Background Species delimitation is a challenging but essential task in conservation biology. Morphologically similar species are sometimes difficult to recognize even after examination by experienced taxonomists. With the advent of molecular approaches in species delimitation, this hidden diversity has received much recent attention. In addition to DNA barcoding approaches, analytical tools based on the multi-species coalescence model (MSC) have been developed for species delimitation. Musa itinerans is widely distributed in subtropical Asia, and at least six varieties have been documented. However, the number of evolutionarily distinct lineages remains unknown. Results Using genome resequencing data of five populations (making up four varieties), we examined genome-wide variation and found four varieties that were evolutionary significant units. A Bayesian Phylogenetics and Phylogeography (BP&P) analysis using 123 single copy nuclear genes support three speciation events of M. itinerans varieties with robust posterior speciation probabilities; However, a Bayes factor delimitation of species with genomic data (BFD*) analysis using 1201 unlinked single nucleotide polymorphisms gave decisive support for a five-lineage model. When reconciling divergence time estimates with a speciation time scale, a modified three-lineage model was consistent with that of BP&P, in which the speciation time of two varieties (M. itinerans var. itinerans and M. itinerans var. lechangensis) were dated to 26.2 kya and 10.7 kya, respectively. In contrast, other two varieties (M. itinerans var. chinensis and M. itinerans var. guangdongensis) diverged only 3.8 kya in the Anthropocene; this may be a consequence of genetic drift rather than a speciation event. Conclusion Our results showed that the M. itinerans species complex harbours high cryptic species diversity. We recommend that M. itinerans var. itinerans and M. itinerans var. lechangensis be elevated to subspecies status, and the extremely rare latter subspecies be given priority for conservation. We also recommend that the very recently diverged M. itinerans var. chinensis and M. itinerans var. guangdongensis should be merged under the subspecies M. itinerans var. chinensis. Finally, we speculate that species delimitation of recently diverged lineages may be more effective using genome-wide bi-allelic SNP markers with BFD* than by using unlinked loci and BP&P.

Premise Microsatellite primers were developed for Puya raimondii (Bromeliaceae), an endangered species distributed in the Andean Mountains of Bolivia and Peru. Methods and Results Genome skimming of P. raimondii, P. macrura, and P. hutchisonii resulted in the selection of 46 pairs of cross‐species microsatellite markers. Of these, 12 microsatellite primer pairs produced clear and polymorphic bands in P. raimondii. These primer sets were then used for the detection of potential polymorphisms in 84 P. raimondii individuals collected from four populations in Peru. The number of alleles per locus ranged from one to six, and the observed and expected levels of heterozygosity ranged from 0.000 to 0.8929 and from 0.000 to 0.7662, respectively. Conclusions The microsatellite markers developed in this study will be useful for future population genetic analyses and breeding system studies in P. raimondii.

Pogostemon Desf., the largest genus of the tribe Pogostemoneae (Lamiaceae), consists of ca. 80 species distributed mainly from South and Southeast Asia to China. The genus contains many patchouli plants, which are of great economic importance but taxonomically difficult. Therefore, it is necessary to characterize more chloroplast (cp) genomes for infrageneric phylogeny analyses and species identification of Pogostemon, especially for patchouli plants. In this study, we newly generated four cp genomes for three patchouli plants (i.e., Pogostemon plectranthoides Desf., P. septentrionalis C. Y. Wu et Y. C. Huang, and two cultivars of P. cablin (Blanoco) Benth.). Comparison of all samples (including online available cp genomes of P. yatabeanus (Makino) Press and P. stellatus (Lour.) Kuntze) suggested that Pogostemon cp genomes are highly conserved in terms of genome size and gene content, with a typical quadripartite circle structure. Interspecific divergence of cp genomes has been maintained at a relatively low level, though seven divergence hotspot regions were identified by stepwise window analysis. The nucleotide diversity (Pi) value was correlated significantly with gap proportion (indels), but significantly negative with GC content. Our phylogenetic analyses based on 80 protein-coding genes yielded high-resolution backbone topologies for the Lamiaceae and Pogostemon. For the overall mean substitution rates, the synonymous (dS) and nonsynonymous (dN) substitution rate values of protein-coding genes varied approximately threefold, while the dN values among different functional gene groups showed a wider variation range. Overall, the cp genomes of Pogostemon will be useful for phylogenetic reconstruction, species delimitation and identification in the future.

Species-rich genus Primula L. is a typical plant group with which to understand genetic variance between species in different levels of relationships. Chloroplast genome sequences are used to be the information resource for quantifying this difference and reconstructing evolutionary history. In this study, we reported the complete chloroplast genome sequence of Primula sinensis and compared it with other related species. This genome of chloroplast showed a typical circular quadripartite structure with 150,859 bp in sequence length consisting of 37.2% GC base. Two inverted repeated regions (25,535 bp) were separated by a large single-copy region (82,064 bp) and a small single-copy region (17,725 bp). The genome consists of 112 genes, including 78 protein-coding genes, 30 tRNA genes and four rRNA genes. Among them, seven coding genes, seven tRNA genes and four rRNA genes have two copies due to their locations in the IR regions. The accD and infA genes lacking intact open reading frames (ORF) were identified as pseudogenes. SSR and sequence variation analyses were also performed on the plastome of Primula sinensis, comparing with another available plastome of P. poissonii. The four most variable regions, rpl36–rps8, rps16–trnQ, trnH–psbA and ndhC–trnV, were identified. Phylogenetic relationship estimates using three sub-datasets extracted from a matrix of 57 protein-coding gene sequences showed the identical result that was consistent with previous studies. A transcript found from P. sinensis transcriptome showed a high similarity to plastid accD functional region and was identified as a putative plastid transit peptide at the N-terminal region. The result strongly suggested that plastid accD has been functionally transferred to the nucleus in P. sinensis.

DNA barcoding of plants poses particular challenges, especially in differentiating, recently diverged taxa. The genus Gentiana (Gentianaceae) is a species-rich plant group which rapidly radiated in the Himalaya-Hengduan Mountains in China. In this study, we tested the core plant barcode (rbcL + matK) and three promising complementary barcodes (trnH-psbA, ITS and ITS2) in 30 Gentiana species across 6 sections using three methods (the genetic distance-based method, Best Close Match and tree-based method). rbcL had the highest PCR efficiency and sequencing success (100%), while the lowest sequence recoverability was from ITS (68.35%). The presence of indels and inversions in trnH-psbA in Gentiana led to difficulties in sequence alignment. When using a single region for analysis, ITS exhibited the highest discriminatory power (60%-74.42%). Of the combinations, matK + ITS provided the highest discrimination success (71.43%-88.24%) and is recommended as the DNA barcode for the genus Gentiana. DNA barcoding proved effective in assigning most species to sections, though it performed poorly in some closely related species in sect. Cruciata because of hybridization events. Our analysis suggests that the status of G. pseudosquarrosa needs to be studied further. The utility of DNA barcoding was also verified in authenticating 'Qin-Jiao' Gentiana medicinal plants (G. macrophylla, G. crassicaulis, G. straminea, and G. dahurica), which can help ensure safe and correct usage of these well-known Chinese traditional medicinal herbs.

The genus Primula is extremely diverse in the east Himalaya-Hengduan Mountains (HHM) in China as a result of rapid radiation. In order to overcome the difficulty of morphological classification of this genus, we surveyed three plastid regions (rbcL, matK, and trnH-psbA) and two nuclear markers (ITS and ITS2) from 227 accessions representing 66 Primula species across 18 sections, to assess their discriminatory power as barcodes. We found that ITS alone or combined with plastid regions showed the best discrimination across different infrageneric ranks and at species level. We suggest rbcL + matK + ITS as the first choice at present to barcode Primula plants. Although the present barcoding combination performed poorly in many closely related species of Primula, it still provided many new insights into current Primula taxonomy, such as the underlying presence of cryptic species, and several potential improper taxonomic treatments. DNA barcoding is one useful technique in the integrative taxonomy of the genus Primula, but it still requires further efforts to improve its effectiveness in some taxonomically challenging groups.

Advances in high-throughput sequencing have generated a vast amount of transcriptomic data that are being increasingly used in phylogenetic reconstruction. However, processing the vast datasets for a huge number of genes and even identifying optimal analytical methodology are challenging. Through de novo sequenced and retrieved data from public databases, we identified 221 orthologous protein-coding genes to reconstruct the phylogeny of Ericales, an order characterized by rapid ancient radiation. Seven species representing different families in Ericales were used as in-groups. Both concatenation and coalescence methods yielded the same well-supported topology as previous studies, with only two nodes conflicting with previously reported relationships. The results revealed that a partitioning strategy could improve the traditional concatenation methodology. Rapidly evolving genes negatively affected the concatenation analysis, while slowly evolving genes slightly affected the coalescence analysis. The coalescence methods usually accommodated rate heterogeneity better and required fewer genes to yield well-supported topologies than the concatenation methods with both real and simulated data.

Cultivated bananas are large, vegetatively-propagated members of the genus Musa. More than 1,000 cultivars are grown worldwide and they are major economic and food resources in numerous developing countries. It has been suggested that cultivated bananas originated from the islands of Southeast Asia (ISEA) and have been developed through complex geodomestication pathways. However, the maternal and parental donors of most cultivars are unknown, and the pattern of nucleotide diversity in domesticated banana has not been fully resolved.We studied the genetics of 16 cultivated and 18 wild Musa accessions using two single-copy nuclear (granule-bound starch synthase I, GBSS I, also known as Waxy, and alcohol dehydrogenase 1, Adh1) and two chloroplast (maturase K, matK, and the trnL-F gene cluster) genes. The results of phylogenetic analyses showed that all A-genome haplotypes of cultivated bananas were grouped together with those of ISEA subspecies of M. acuminata (A-genome). Similarly, the B- and S-genome haplotypes of cultivated bananas clustered with the wild species M. balbisiana (B-genome) and M. schizocarpa (S-genome), respectively. Notably, it has been shown that distinct haplotypes of each cultivar (A-genome group) were nested together to different ISEA subspecies M. acuminata. Analyses of nucleotide polymorphism in the Waxy and Adh1 genes revealed that, in comparison to the wild relatives, cultivated banana exhibited slightly lower nucleotide diversity both across all sites and specifically at silent sites. However, dramatically reduced nucleotide diversity was found at nonsynonymous sites for cultivated bananas.Our study not only confirmed the origin of cultivated banana as arising from multiple intra- and inter-specific hybridization events, but also showed that cultivated banana may have not suffered a severe genetic bottleneck during the domestication process. Importantly, our findings suggested that multiple maternal origins and a reduction in nucleotide diversity at nonsynonymous sites are general attributes of cultivated bananas.

Primula poissonii (Primulaceae) is a perennial herb, widely distributed in the Hengduan Mountain region of Southwest China. In this study, Roche 454 pyrosequencing was used to isolate microsatellite markers. A total of 4528 unique sequences were identified from 68,070 unique reads. Of these, eighty-seven microsatellite loci were screened for utility using two criteria: successful PCR amplification and variation of these loci within three wild P. poissonii populations. Twenty loci were successfully amplified and exhibited polymorphic alleles. The number of observed alleles ranged from 1 to 9 with an average of 3.5. The observed and expected heterozygosities ranged from 0.087 to 1.000 and from 0.124 to 0.828, respectively. Among these SSR loci, only the P69 locus could not be cross-amplified successfully in two closely related species P. wilsonii and P. anisodora. The microsatellite loci developed in this study will be useful for studying genetic diversity and speciation events between P. poissonii and closely related Primula species.

Amentotaxus, a genus of Taxaceae, is an ancient lineage with six relic and endangered species. Four Amentotaxus species, namely A. argotaenia, A. formosana, A. yunnanensis, and A. poilanei, are considered a species complex because of their morphological similarities. Small populations of these species are allopatrically distributed in Asian forests. However, only a few codominant markers have been developed and applied to study population genetic structure of these endangered species. In this study, we developed and characterized polymorphic expressed sequence tag-simple sequence repeats (EST-SSRs) from the transcriptome of A. formosana. We identified 4955 putative EST-SSRs from 68,281 unigenes as potential molecular markers. Twenty-six EST-SSRs were selected for estimating polymorphism and transferability among Amentotaxus species, of which 23 EST-SSRs were polymorphic within Amentotaxus species. Among these, the number of alleles ranged from 1–4, the polymorphism information content ranged from 0.000–0.692, and the observed and expected heterozygosity were 0.000–1.000 and 0.080–0.740, respectively. Population genetic structure analyses confirmed that A. argotaenia and A. formosana were separate species and A. yunnanensis and A. poilanei were the same species. These novel EST-SSRs can facilitate further population genetic structure research of Amentotaxus species.

BackgroundCurrent and historical events have both affected the current distribution patterns and intraspecific divergence of plants. While numerous studies have focused on the Qinghai-Tibetan Plateau (QTP), the impacts of such events on the flora of subtropical China remain poorly understood. Subtropical China is famous for its highly complex topography and the limited impact from glaciation during the Pleistocene; this may have resulted in a different genetic legacy for species in this region compared to fully glaciated areas.Methodology/principal findingsWe used plastid and nuclear DNA sequence data and distribution modeling to analyze the divergence patterns and demographic history of Primula obconica Hance, a widespread herbaceous montane species in subtropical China. The phylogenetic analysis revealed two major lineages (lineage A and lineage B), representing a west-east split into the Yunnan and Eastern groups, and the Sichuan and Central groups, respectively. The Eastern and Central groups comprised relatively new derived haplotypes. Nested Clade Analysis and Bayesian Skyline Plot analyses both indicated that P. obconica mainly experienced a gradual expansion of populations. In addition, the simulated distribution of P. obconica during the Last Glacial Maximum was slightly larger than its present-day distribution.Conclusion/significanceOur results are the first to identify a west-east migration of P. obconica. The gradual expansion pattern and a larger potential distribution range in cold periods detected for P. obconica indicate that the population expansion of this species is consistent with the phalanx model. In addition, the current patterns of genetic differentiation have persisted as a result of the extensive environmental heterogeneity that exists in subtropical China.

Pinus massoniana, an ecologically and economically important conifer, is widespread across central and southern mainland China and Taiwan. In this study, we tested the central-marginal paradigm that predicts that the marginal populations tend to be less polymorphic than the central ones in their genetic composition, and examined a founders' effect in the island population.We examined the phylogeography and population structuring of the P. massoniana based on nucleotide sequences of cpDNA atpB-rbcL intergenic spacer, intron regions of the AdhC2 locus, and microsatellite fingerprints. SAMOVA analysis of nucleotide sequences indicated that most genetic variants resided among geographical regions. High levels of genetic diversity in the marginal populations in the south region, a pattern seemingly contradicting the central-marginal paradigm, and the fixation of private haplotypes in most populations indicate that multiple refugia may have existed over the glacial maxima. STRUCTURE analyses on microsatellites revealed that genetic structure of mainland populations was mediated with recent genetic exchanges mostly via pollen flow, and that the genetic composition in east region was intermixed between south and west regions, a pattern likely shaped by gene introgression and maintenance of ancestral polymorphisms. As expected, the small island population in Taiwan was genetically differentiated from mainland populations.The marginal populations in south region possessed divergent gene pools, suggesting that the past glaciations might have low impacts on these populations at low latitudes. Estimates of ancestral population sizes interestingly reflect a recent expansion in mainland from a rather smaller population, a pattern that seemingly agrees with the pollen record.

Elucidating the ecological mechanisms underlying community assembly in subtropical forests remains a central challenge for ecologists. The assembly of species into communities can be due to interspecific differences in habitat associations, and there is increasing evidence that these associations may have an underlying phylogenetic structure in contemporary terrestrial communities. In other words, by examining the degree to which closely related species prefer similar habitats and the degree to which they co-occur, ecologists are able to infer the mechanisms underlying community assembly. Here we implement this approach in a diverse subtropical tree community in China using a long-term forest dynamics plot and a molecular phylogeny generated from three DNA barcode loci. We find that there is phylogenetic signal in plant-habitat associations (i.e. closely related species tend to prefer similar habitats) and that patterns of co-occurrence within habitats are typically non-random with respect to phylogeny. In particular, we found phylogenetic clustering in valley and low-slope habitats in this forest, indicating a filtering of lineages plays a dominant role in structuring communities in these habitats and we found evidence of phylogenetic overdispersion in high-slope, ridge-top and high-gully habitats, indicating that distantly related species tended to co-occur in these high elevation habitats and that lineage filtering is less important in structuring these communities. Thus we infer that non-neutral niche-based processes acting upon evolutionarily conserved habitat preferences explain the assembly of local scale communities in the forest studied.

Premise of the study: Pinus wangii is an endemic and endangered species in southwestern China, and microsatellite primers were developed to characterize its genetic diversity and population structure. Methods and Results: Using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) protocol, nine sets of microsatellite primers were developed in P. wangii. One population with 26 individuals of P. wangii, as well as 11 individuals each for two congeneric species, P. taiwanensis and P. squamata, were used to test their polymorphism and transferability. The number of alleles per locus ranged from one to seven with an average of 3.7, and the observed heterozygosity and expected heterozygosity ranged from 0 to 0.91 and 0 to 0.75, respectively. Conclusions: We developed nine sets of polymorphic microsatellite loci that are suitable for investigating genetic diversity and population structure of P. wangii, and these markers may be useful for other Pinus species.

Premise of the study: Microsatellite markers were developed to investigate the genetic diversity and population structure of the endangered species Paraisometrum mileense, as well as its evolutionary history. Methods and Results: Using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) protocol, 11 polymorphic primer sets were obtained in P. mileense. The number of alleles per locus ranged from one to nine with an average 3.8, and the observed and expected heterozygosities ranged from 0 to 1 and 0 to 0.89, respectively, for the available three populations. Conclusions: These markers may be useful for further investigation of the conservation genetics of P. mileense.