Testing plastomes and nuclear ribosomal DNA sequences as the next-generation DNA barcodes for species identification and phylogenetic analysis in Acer.

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.

Plastid genome and its phylogenetic implications of Asiatic Spiraea (Rosaceae).

BACKGROUND: Spiraea L. is a genus comprising approximately 90 species that are distributed throughout the northern temperate regions. China is recognized as the center of species diversity for this genus, hosting more than 70 species, including 47 endemic species. While Spiraea is well-known for its ornamental value, its taxonomic and phylogenetic studies have been insufficient. RESULTS: In this study, we conducted sequencing and assembly of the plastid genomes (plastomes) of 34 Asiatic Spiraea accessions (representing 27 Asiatic Spiraea species) from China and neighboring regions. The Spiraea plastid genome exhibits typical quadripartite structures and encodes 113-114 genes, including 78-79 protein-coding genes (PCGs), 30 tRNA genes, and 4 rRNA genes. Linear regression analysis revealed a significant correlation between genome size and the length of the SC region. By the sliding windows method, we identified several hypervariable hotspots within the Spiraea plastome, all of which were localized in the SC regions. Our phylogenomic analysis successfully established a robust phylogenetic framework for Spiraea, but it did not support the current defined section boundaries. Additionally, we discovered that the genus underwent diversification after the Early Oligocene (~ 30 Ma), followed by a rapid speciation process during the Pliocene and Pleistocene periods. CONCLUSIONS: The plastomes of Spiraea provided us invaluable insights into its phylogenetic relationships and evolutionary history. In conjunction with plastome data, further investigations utilizing other genomes, such as the nuclear genome, are urgently needed to enhance our understanding of the evolutionary history of this genus.

Plastome evolution and phylogenomic insights into the evolution of Lysimachia (Primulaceae: Myrsinoideae).

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.

Flora diversity survey and establishment of a plant DNA barcode database of Lomas ecosystems in Peru.

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.

Lysimachiafenghwaiana (Primulaceae), a new species from Hunan Province, China.

A new species, Lysimachiafenghwaiana G.Hao & H.F.Yan (Primulaceae), from Hunan Province, China, is described and illustrated. This new species belongs to Lysimachiasubgen.Lysimachiasect.Nummularia and is morphologically similar to L.crista-galli and L.carinata, but is distinctive in its leaf shape and arrangement of flowers. It can be further distinguished from L.crista-galli by the absence of calyx lobule spur, and from L.carinata by the black glandular striates in the corolla lobes, rather than punctate.

Plant DNA barcode library for native flowering plants in the arid region of northwestern China.

DNA barcoding is a well-established tool for rapid species identification and biodiversity monitoring. A reliable and traceable DNA barcode reference library with extensive coverage is necessary but unavailable for many geographical regions. The arid region in northwestern China, a vast area of about 2.5 million km2 , is ecologically fragile and often overlooked in biodiversity studies. In particular, DNA barcode data from the arid region in China are lacking. We develop and evaluate the efficacy of an extensive DNA barcode library for native flowering plants in the arid region of northwestern China. Plant specimens were collected, identified and vouchered for this purpose. The database utilized four DNA barcode markers, namely rbcL, matK, ITS and ITS2, for 1816 accessions (representing 890 species from 385 genera and 72 families), and consisted of 5196 barcode sequences. Individual barcodes varied in resolution rates: species- and genus-level rates for rbcL, matK, ITS and ITS2 were 79.9%-51.1%/76.1%, 79.9%-67.2%/88.9%, 85.0%-72.0%/88.2% and 81.0%-67.4%/84.9%, respectively. The three-barcode combination of rbcL + matK + ITS (RMI) revealed a higher species- and genus-level resolution (75.5%/92.1%, respectively). A total of 110 plastomes were newly generated as super-barcodes to increase species resolution for seven species-rich genera, namely Astragalus, Caragana, Lactuca, Lappula, Lepidium, Silene and Zygophyllum. Plastomes revealed higher species resolution compared to standard DNA barcodes and their combination. We suggest future databases include super-barcodes, especially for species-rich and complex genera. The plant DNA barcode library in the current study provides a valuable resource for future biological investigations in the arid regions of China.

Comparative analyses of three complete Primula mitogenomes with insights into mitogenome size variation in Ericales.

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.

Fingerprints of climatic changes through the late Cenozoic in southern Asian flora: Magnolia section Michelia (Magnoliaceae).

BACKGROUND AND AIMS: Ongoing global warming is a challenge for humankind. A series of drastic climatic changes have been proven to have occurred throughout the Cenozoic based on a variety of geological evidence, which helps to better understand our planet's future climate. Notably, extant biomes have recorded drastic environmental shifts. The climate in southern Asia, which hosts high biodiversity, is deeply impacted by the Asian monsoon. The origins and evolutionary dynamics of biomes occurring between the tropics and sub-tropics in southern Asia have probably been deeply impacted by climatic changes; however, these aspects remain poorly studied. We tested whether the evolutionary dynamics of the above biomes have recorded the drastic, late Cenozoic environmental shifts, by focusing on Magnolia section Michelia of the family Magnoliaceae. METHODS: We established a fine time-calibrated phylogeny of M. section Michelia based on complete plastid genomes and inferred its ancestral ranges. Finally, we estimated the evolutionary dynamics of this section through time, determining its diversification rate and the dispersal events that occurred between tropical and sub-tropical areas. KEY RESULTS: The tropical origin of M. section Michelia was dated to the late Oligocene; however, the diversification of its core group (i.e. M. section Michelia subsection Michelia) has occurred mainly from the late Miocene onward. Two key evolutionary shifts (dated approx. 8 and approx. 3 million years ago, respectively) were identified, each of them probably in response to drastic climatic changes. CONCLUSION: Here, we inferred the underlying evolutionary dynamics of biomes in southern Asia, which probably reflect late Cenozoic climatic changes. The occurrence of modern Asian monsoons was probably fundamental for the origin of M. section Michelia; moreover, the occurrence of asymmetric dispersal events between the tropics and sub-tropics hint at an adaptation strategy of M. section Michelia to global cooling, in agreement with the tropical conservatism hypothesis.

Comparative plastome analysis of Musaceae and new insights into phylogenetic relationships.

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.

Plastid phylogenomics of tribe Perseeae (Lauraceae) yields insights into the evolution of East Asian subtropical evergreen broad-leaved forests.

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.

Lysimachiacoriacea (Primulaceae, Myrsinoideae), a new species from Chongqing, China.

A new species, Lysimachiacoriacea, from Chongqing, China, is described and illustrated. It is assigned to subgen. Lysimachiasect.Nummulariaser.Paridiformes and resembles L.paridiformisvar.stenophylla, but is characterised by smaller leathery leaves with black glandular striations near the margin. It is also similar to L.nanpingensis in its two to three pairs of leaves sub-whorled at the stem apices, but differs by smaller leathery leaves and densely glandular stem, petiole and pedicel, and calyx lobes with sparse black glandular stripes.

Further clarification on Androsacemollis Hand.-Mazz. (Primulaceae), with a description of a new species of Androsace.

The syntypes (H.R.E. Handel-Mazzetti 8896 and H.R.E. Handel-Mazzetti 9280) of Androsacemollis Hand.-Mazz. are identified as two separate taxa based on critical examinations of herbarium specimens and field investigation. While H.R.E. Handel-Mazzetti 8896 has been designated as the lectotype of A.mollis, we describe the other taxon, represented by H.R.E. Handel-Mazzetti 9280, as A.chimingiana Y.Xu & G.Hao sp. nov. The new species is morphologically similar to A.hookeriana Klatt and A.laxa C.M.Hu & Y.C.Yang but can be easily differentiated by its white corolla and obovate bracts.

The complete chloroplast genome of Lithocarpus hancei (Benth.) Rehd (Fagaceae) from Zhejiang, China.

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.

Characterization of the complete plastid genome of Lysimachia christinae Hance (Primulaceae).

Lysimachia christinae Hance is widely distributed in subtropical China at the elevational range from 500-2300 m. The species is an important medicinal herb for treating jaundice, urinary disorders, and the liver. Here, we sequenced and characterized the whole plastid genome of L. christinae. It is 154,810 bp in length, containing two copies of inverted repeat (IR) regions (26,034 bp, each), a large single-copy (LSC) region (84,809 bp), and a small single-copy (SSC) region (17,933 bp). It has 114 genes, of which 80 are protein-coding, 30 are tRNA, and 4 are rRNA genes. The ML tree indicates L. christinae is closely related to Lysimachia congestiflora Hemsl. This genome information can help us better construct a backbone phylogeny of Lysimachia in the future.

Characterization of the complete plastid genome of Chinese medicinal plant Isodon serra (Lamiaceae).

The herb Isodon serra (Maximowicz) Kudô, which is widely distributed in China and its neighbor regions, is a well-known traditional Chinese medicinal plant. In this study, we characterized the complete plastid genome sequence of I. serra using Illumina sequencing data. The plastome is 152,676 bp in length and contains a typical quadripartite structure. The inverted repeat (IR), large-single copy (LSC) and small-single copy (SSC) regions each has 25,716 bp, 83,564 bp, and 17,680 bp. The genome contains 80 protein coding genes (PCGs), 30 transfer RNAs (tRNA), and four ribosomal RNAs (rRNA). The phylogenetic result indicates I. serra together with genera Ocimum and Lavandula formed tribe Ocimeae clade.

Comparative Analyses of the Chloroplast Genomes of Patchouli Plants and Their Relatives in Pogostemon (Lamiaceae).

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.

Conflicting phylogenetic signals in plastomes of the tribe Laureae (Lauraceae).

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.

Liparisnapoensis (Orchidaceae), a new species from Guangxi, China.

Liparisnapoensis, a new orchid species belonging to section Cestichis from Guangxi, China is described and illustrated. It occurs in the karst limestone forest. The new species is morphologically similar to L.viridiflora and L.somae, but can be readily distinguished by having narrowly oblong-falcate petals; flabellate-quadrate lip distinctly concave at base and emarginate at apex; conspicuously arcuate column with a pair of wedge-shaped wings.

What explains high plant richness in East Asia? Time and diversification in the tribe Lysimachieae (Primulaceae).

What causes the disparity in biodiversity among regions is a fundamental question in biogeography, ecology, and evolutionary biology. Evolutionary and biogeographic processes (speciation, extinction, dispersal) directly determine species richness patterns, and can be studied using integrative phylogenetic approaches. However, the strikingly high richness of East Asia relative to other Northern Hemisphere regions remains poorly understood from this perspective. Here, for the first time, we test two general hypotheses (older colonization time, faster diversification rate) to explain this pattern, using the plant tribe Lysimachieae (Primulaceae) as a model system. We generated a new time-calibrated phylogeny for Lysimachieae (13 genes, 126 species), to estimate colonization times and diversification rates for each region and to test the relative importance of these two factors for explaining regional richness patterns. We find that neither time nor diversification rates alone explain richness patterns among regions in Lysimachieae. Instead, a new index that combines both factors explains global richness patterns in the group and their high East Asian biodiversity. Based on our results from Lysimachieae, we suggest that the high richness of plants in East Asia may be explained by a combination of older colonization times and faster diversification rates in this region.

Characterization of polymorphic microsatellite markers for Primula sikkimensis (Primulaceae) using a 454 sequencing approach.

PREMISE OF THE STUDY: Microsatellite markers from Primula sikkimensis (Primulaceae) were developed for testing deep lineage divergence and speciation events. METHODS AND RESULTS: A total of 3112 microsatellites were identified from 61,755 unique reads though 454 pyrosequencing technology. Twenty-nine microsatellite loci were selected for PCR amplification and polymorphic analyses. Among the 29 tested markers, 17 microsatellite loci were further used for genotyping in three wild P. sikkimensis populations. The number of alleles varied from one to eight, and the observed heterozygosity ranged from 0.111 to 1.000. Ten simple sequence repeat loci could be successfully cross-amplified in two Primula species. The transferability values were 76.5% in P. florindae and 58.8% in P. alpicola, respectively. CONCLUSIONS: These microsatellite markers will be valuable for testing the hypothesis of lineage divergence, genetic introgression, and cryptic speciation events between P. sikkimensis and its closely related taxa.