The first complete mitochondrional genome of Anopheles gibbinsi using a skimming sequencing approach.

Mosquitoes belonging to the genus Anopheles are the only vectors of human malaria. Anopheles gibbinsi has been linked to malaria transmission in Kenya, with recent collections in Zambia reporting the mosquito species exhibiting zoophilic and exophilic behavioral patterns with occasional contact with humans. Given the paucity of genetic data, and challenges to identification and molecular taxonomy of the mosquitoes belonging to the Anopheles genus; we report the first complete mitochondrial genome of An. gibbinsi using a genome skimming approach. An Illumina Novaseq 6000 platform was used for sequencing, the length of the mitochondrial genome was 15401 bp, with 78.5% AT content comprised of 37 genes. Phylogenetic analysis by maximum likelihood using concatenation of the 13 protein coding genes demonstrated that An. marshallii was the closest relative based on existing sequence data. This study demonstrates that the skimming approach is an inexpensive and efficient approach for mosquito species identification and concurrent taxonomic rectification, which may be a useful alternative for generating reference sequence data for evolutionary studies among the Culicidae.

Ameripathidae, a new family of antipatharian corals (Cnidaria, Anthozoa, Hexacorallia, Antipatharia).

A new family of antipatharian corals, Ameripathidae (Cnidaria: Anthozoa: Antipatharia), is established for Ameripathespseudomyriophylla Opresko & Horowitz, gen. et sp. nov. The new family resembles Myriopathidae and Stylopathidae in terms of the morphology of the polyps and tentacles and the pinnulate branching of the corallum. Phylogenetic analysis using a genomic data set of 741 conserved element loci indicates that the new family is sister to a clade containing the Myriopathidae, Stylopathidae, Antipathidae, and Aphanipathidae.

Corrigendum: LocoGSE, a sequence-based genome size estimator for plants.

[This corrects the article DOI: 10.3389/fpls.2024.1328966.].

Identification of the 18 World War II executed citizens of Adele, Rethymnon, Crete using an ancient DNA approach and low coverage genomes.

In the Battle of Crete during the World War II occupation of Greece, the German forces faced substantial civilian resistance. To retribute the numerous German losses, a series of mass executions took place in numerous places in Crete; a common practice reported from Greece and elsewhere. In Adele, a village in the regional unit of Rethymnon, 18 male civilians were executed and buried in a burial pit at the Sarakina site. In this study, the first one conducted for a conflict that occurred in Greece, we identified for humanitarian purposes the 18 skulls of the Sarakina victims, following a request from the local community of Adele. The molecular identification of historical human remains via ancient DNA approaches and low coverage whole genome sequencing has only recently been introduced. Here, we performed genome skimming on the living relatives of the victims, as well as high throughput historical DNA analysis on the skulls to infer the kinship degrees among the victims via genetic relatedness analyses. We also conducted targeted anthropological analysis to successfully complete the identification of all Sarakina victims. We demonstrate that our methodological approach constitutes a potentially highly informative forensic tool to identify war victims. It can hence be applied to analogous studies on degraded DNA, thus, paving the path for systematic war victim identification in Greece and beyond.

Nuclear Magnetic Resonance Fingerprints and Mini DNA Markers for the Authentication of Cinnamon Species Ingredients Used in Food and Natural Health Products.

Cinnamomum verum (syn C. zeylanicum) is considered 'true' cinnamon. However, it is reported that less expensive sources of cinnamon from C. cassia (syn C. aromaticum), C. loureiroi, and C. burmannii (toxic coumarin) may be used in the place of C. verum. We lack the quality assurance tools that are required to differentiate C. verum from other cinnamon species when verifying that the correct species is sourced from ingredient suppliers. The current research on cinnamon species authentication using DNA tools is limited to a few species and the use of high-quality DNA extracted from raw leaf materials. The cinnamon bark traded in the supply chain contains much less DNA and poorer-quality DNA than leaves. Our research advances DNA methods to authenticate cinnamon, as we utilized full-length chloroplast genomes via a genome skimming approach for C. burmannii and C. cassia to facilitate the design of optimal mini DNA markers. Furthermore, we developed and validated the use of NMR fingerprints for several commercial cinnamon species, including the quantification of 16 molecules. NMR fingerprints provided additional data that were useful for quality assessment in cinnamon extract powders and product consistency. Both the new mini DNA markers and NMR fingerprints were tested on commercial cinnamon products.

Analyses of Nuclear Reads Obtained Using Genome Skimming.

In this protocol paper, we review a set of methods developed in recent years for analyzing nuclear reads obtained from genome skimming. As the cost of sequencing drops, genome skimming (low-coverage shotgun sequencing of a sample) becomes increasingly a cost-effective method of measuring biodiversity at high resolution. While most practitioners only use assembled over-represented organelle reads from a genome skim, the vast majority of the reads are nuclear. Using assembly-free and alignment-free methods described in this protocol, we can compare samples to each other and reference genomes to compute distances, characterize underlying genomes, and infer evolutionary relationships.

Complete mitochondrial genomes of the black corals Alternatipathesmirabilis Opresko & Molodtsova, 2021 and Parantipatheslarix (Esper, 1788) (Cnidaria, Anthozoa, Hexacorallia, Antipatharia, Schizopathidae).

We describe the complete mitogenomes of the black corals Alternatipathesmirabilis Opresko & Molodtsova, 2021 and Parantipatheslarix (Esper, 1790) (Cnidaria, Anthozoa, Hexacorallia, Antipatharia, Schizopathidae). The analysed specimens include the holotype of Alternatipathesmirabilis, collected from Derickson Seamount (North Pacific Ocean; Gulf of Alaska) at 4,685 m depth and a potential topotype of Parantipatheslarix, collected from Secca dei Candelieri (Mediterranean Sea; Tyrrhenian Sea; Salerno Gulf; Italy) at 131 m depth. We also assemble, annotate and make available nine additional black coral mitogenomes that were included in a recent phylogeny (Quattrini et al. 2023b), but not made easily accessible on GenBank. This is the first study to present and compare two mitogenomes from the same species of black coral (Stauropathesarctica (Lütken, 1871)) and, thus, place minimum boundaries on the expected level of intraspecific variation at the mitogenome level. We also compare interspecific variation at the mitogenome-level across five different specimens of Parantipathes Brook, 1889 (representing at least two different species) from the NE Atlantic and Mediterranean Sea.

Insights into the nuclear and mitochondrial genome of the Lemon shark Negaprion brevirostris using low-coverage sequencing: Genome size, repetitive elements, mitochondrial genome, and phylogenetic placement.

The Lemon shark Negaprion brevirostris is an important species experiencing conservation issues that is in need of genomic resources. Herein, we conducted a genome survey sequencing in N. brevirostris and determined genome size, explored repetitive elements, assembled and annotated the 45S rRNA DNA operon, and assembled and described in detail the mitochondrial genome. Lastly, the phylogenetic position of N. brevirostris in the family Carcharhinidae was examined using translated protein coding genes. The estimated haploid genome size ranged between 2.29 and 2.58 Gbp using a k-mer analysis, which is slightly below the genome size estimated for other sharks belonging to the family Carcharhinidae. Using a k-mer analysis, approx. 64-71 % of the genome of N. brevirostris was composed of repetitive elements. A relatively large proportion of the 'repeatome' could not be annotated. Taking into account only annotated repetitive elements, Class I - Long Interspersed Nuclear Element (LINE) were the most abundant repetitive elements followed by Class I - Penelope and Satellite DNA. The nuclear ribosomal operon was fully assembled. The AT-rich complete mitochondrial genome was 16,703 bp long and encoded 13 protein coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. Negaprion brevirostris is closely related to the genera Carcharhinus, Glyphis and Lamiopsis in the family Carcharinidae. This new genomic resources will aid with the development of conservation plans for this large coastal shark.

LocoGSE, a sequence-based genome size estimator for plants.

Extensive research has focused on exploring the range of genome sizes in eukaryotes, with a particular emphasis on land plants, where significant variability has been observed. Accurate estimation of genome size is essential for various research purposes, but existing sequence-based methods have limitations, particularly for low-coverage datasets. In this study, we introduce LocoGSE, a novel genome size estimator designed specifically for low-coverage datasets generated by genome skimming approaches. LocoGSE relies on mapping the reads on single copy consensus proteins without the need for a reference genome assembly. We calibrated LocoGSE using 430 low-coverage Angiosperm genome skimming datasets and compared its performance against other estimators. Our results demonstrate that LocoGSE accurately predicts monoploid genome size even at very low depth of coverage (<1X) and on highly heterozygous samples. Additionally, LocoGSE provides stable estimates across individuals with varying ploidy levels. LocoGSE fills a gap in sequence-based plant genome size estimation by offering a user-friendly and reliable tool that does not rely on high coverage or reference assemblies. We anticipate that LocoGSE will facilitate plant genome size analysis and contribute to evolutionary and ecological studies in the field. Furthermore, at the cost of an initial calibration, LocoGSE can be used in other lineages.

Giants among Cnidaria: Large Nuclear Genomes and Rearranged Mitochondrial Genomes in Siphonophores.

Siphonophores (Cnidaria: Hydrozoa) are abundant predators found throughout the ocean and are important constituents of the global zooplankton community. They range in length from a few centimeters to tens of meters. They are gelatinous, fragile, and difficult to collect, so many aspects of the biology of these roughly 200 species remain poorly understood. To survey siphonophore genome diversity, we performed Illumina sequencing of 32 species sampled broadly across the phylogeny. Sequencing depth was sufficient to estimate nuclear genome size from k-mer spectra in six specimens, ranging from 0.7 to 2.3 Gb, with heterozygosity estimates between 0.69% and 2.32%. Incremental k-mer counting indicates k-mer peaks can be absent with nearly 20× read coverage, suggesting minimum genome sizes range from 1.4 to 5.6 Gb in the 25 samples without peaks in the k-mer spectra. This work confirms most siphonophore nuclear genomes are large relative to the genomes of other cnidarians, but also identifies several with reduced size that are tractable targets for future siphonophore nuclear genome assembly projects. We also assembled complete mitochondrial genomes for 33 specimens from these new data, indicating a conserved gene order shared among nonsiphonophore hydrozoans, Cystonectae, and some Physonectae, revealing the ancestral mitochondrial gene order of siphonophores. Our results also suggest extensive rearrangement of mitochondrial genomes within other Physonectae and in Calycophorae. Though siphonophores comprise a small fraction of cnidarian species, this survey greatly expands our understanding of cnidarian genome diversity. This study further illustrates both the importance of deep phylogenetic sampling and the utility of k-mer-based genome skimming in understanding the genomic diversity of a clade.

Complete Mitochondrial Genome and Phylogenetic Position of Nurudea zhengii Ren (Insecta, Hemiptera, Aphididae).

Nurudea zhengii Ren was identified by aphid morphological characteristics as well as the gall shape and host plant species, and placed in the tribe Fordini (Hemiptera, Aphididae, Eriosomatinae). Here, its whole genome was firstly sequenced by a genome-skimming method and its mitochondrial genome (mitogenome) was assembled to examine its genetic variation and phylogenetic position. The complete mitogenome of Nurudea zhengii is 15,392 bp in length, and consists of 13 protein-coding genes, 22 tRNAs, two rRNAs and one D-loop region. The gene order follows the mitogenomes of the other Rhus gall aphids, and similarly has an AT bias with the content of 83.9%. The majority strand is A-skewed and C-skewed, and shows opposite skewness for G-skewed in the minority strands. The ratios of nonsynonymous to synonymous substitution rates of protein-coding genes are lower than one except for ATP8, which indicated that ATP8 was undergoing positive selection. Phylogenetic analysis among the Rhus gall aphids based on 13 protein-coding genes and two rRNA genes showed that N. zhengii was sister to N. shiraii, and then clustered with N. yanoniella as a group with high support value. The two species, N. shiraii and N. yanoniella, share the same host plant Rhus chinensis, while the host of N. zhengii is R. hypoleuca. However, the phylogenetic relationship indicated that the taxa sharing the same host plant were not absolutely clustered as the closest taxa at least at species level.

Phylogenomic analyses sheds new light on the phylogeny and diversification of Corydalis DC. in Himalaya-Hengduan Mountains and adjacent regions.

The Himalaya-Hengduan Mountains (HHM), a renowned biodiversity hotspot of the world, harbors the most extensive habitats for alpine plants with extraordinary high levels of endemism. Although the general evolution pattern has been elucidated, the underlying processes driving spectacular radiations in many species-rich groups remain elusive. Corydalis DC. is widely distributed throughout the Northern Hemisphere containing more than 500 species, with high diversity in HHM and adjacent regions. Using 95 plastid genes, 3,258,640 nuclear single nucleotide polymorphisms (SNPs) and eight single-copy nuclear genes (SCNs) generated from genome skimming data, we reconstructed a robust time-calibrated phylogeny of Corydalis comprising more than 100 species that represented all subgenera and most sections. Molecular dating indicated that all main clades of Corydalis began to diverge in the Eocene, with the majority of extant species in HHM emerged from a diversification burst after the middle Miocene. Global pattern of mean divergence times indicated that species distributed in HHM were considerably younger than those in other regions, particularly for the two most species-rich clades (V and VI) of Corydalis. The early divergence and the recent diversification of Corydalis were most likely promoted by the continuous orogenesis and climate change associated with the uplift of the Qinghai-Tibetan Plateau (QTP). Our study demonstrates the effectivity of phylogenomic analyses with genome skimming data on the phylogeny of species-rich taxa, and sheds lights on how the uplift of QTP has triggered the evolutionary radiations of large plant genera in HHM and adjacent regions.

Insights into the genome of the 'Loco' Concholepas concholepas (Gastropoda: Muricidae) from low-coverage short-read sequencing: genome size, ploidy, transposable elements, nuclear RNA gene operon, mitochondrial genome, and phylogenetic placement in the family Muricidae.

BACKGROUND: The Peruvian 'chanque' or Chilean 'loco' Concholepas concholepas is an economically, ecologically, and culturally important muricid gastropod heavily exploited by artisanal fisheries in the temperate southeastern Pacific Ocean. In this study, we have profited from a set of bioinformatics tools to recover important biological information of C. concholepas from low-coverage short-read NGS datasets. Specifically, we calculated the size of the nuclear genome, ploidy, and estimated transposable elements content using an in silico k-mer approach, we discovered, annotated, and quantified those transposable elements, we assembled and annotated the 45S rDNA RNA operon and mitochondrial genome, and we confirmed the phylogenetic position of C. concholepas within the muricid subfamily Rapaninae based on translated protein coding genes. RESULTS: Using a k-mer approach, the haploid genome size estimated for the predicted diploid genome of C. concholepas varied between 1.83 Gbp (with kmer = 24) and 2.32 Gbp (with kmer = 36). Between half and two thirds of the nuclear genome of C. concholepas was composed of transposable elements. The most common transposable elements were classified as Long Interspersed Nuclear Elements and Short Interspersed Nuclear Elements, which were more abundant than DNA transposons, simple repeats, and Long Terminal Repeats. Less abundant repeat elements included Helitron mobile elements, 45S rRNA DNA, and Satellite DNA, among a few others.The 45S rRNA DNA operon of C. concholepas that encodes for the ssrRNA, 5.8S rRNA, and lsrRNA genes was assembled into a single contig 8,090 bp long. The assembled mitochondrial genome of C. concholepas is 15,449 bp long and encodes 13 protein coding genes, two ribosomal genes, and 22 transfer RNAs. CONCLUSION: The information gained by this study will inform the assembly of a high quality nuclear genome for C. concholepas and will support bioprospecting and biomonitoring using environmental DNA to advance development of conservation and management plans in this overexploited marine snail.

Population genomics of the Isoetes appalachiana (Isoetaceae) complex supports a 'diploids-first' approach to conservation.

BACKGROUND AND AIMS: Allopolyploidy is an important driver of diversification and a key contributor to genetic novelty across the tree of life. However, many studies have questioned the importance of extant polyploid lineages, suggesting that the vast majority may constitute evolutionary 'dead ends'. This has important implications for conservation efforts where polyploids and diploid progenitors often compete for wildlife management resources. Isoetes appalachiana is an allotetraploid that is broadly distributed throughout the eastern USA alongside its diploid progenitors, I. valida and I. engelmannii. As such, this species complex provides an excellent opportunity to investigate the processes that underpin the formation and survival of allopolyploid lineages. METHODS: Here we utilized RADseq and whole-chloroplast sequencing to unravel the demographic and evolutionary history of hybridization in this widespread species complex. We developed a modified protocol for phasing RADseq loci from an allopolyploid in order to examine each progenitor's genetic contribution independently in a phylogenetic context. Additionally, we conducted population-level analyses to examine genetic diversity and evidence of gene flow within species. KEY RESULTS: Isoetes appalachiana is the product of multiple phylogenetic origins, suggesting that formation and establishment of allopolyploids are common in this group. Hybridization appears to be unidirectional, with I. engelmannii consistently being the maternal progenitor. Additionally, we find that polyploid lineages are genetically isolated, rarely if ever experiencing gene flow between geographically distinct populations. CONCLUSIONS: Allopolyploid lineages of I. appalachiana appear to form frequently and experience a high degree of genetic isolation following formation. Thus, our results appear to corroborate the hypothesis that the vast majority of recently formed polyploids may represent evolutionary dead ends. However, this does not necessarily lessen the evolutionary importance or ecological impact of polyploidy per se. Accordingly, we propose a conservation strategy that prioritizes diploid taxa, thus preserving downstream processes that recurrently generate allopolyploid diversity.

Patchwork: Alignment-Based Retrieval and Concatenation of Phylogenetic Markers from Genomic Data.

Low-coverage whole-genome sequencing (also known as "genome skimming") is becoming an increasingly affordable approach to large-scale phylogenetic analyses. While already routinely used to recover organellar genomes, genome skimming is rather rarely utilized for recovering single-copy nuclear markers. One reason might be that only few tools exist to work with this data type within a phylogenomic context, especially to deal with fragmented genome assemblies. We here present a new software tool called Patchwork for mining phylogenetic markers from highly fragmented short-read assemblies as well as directly from sequence reads. Patchwork is an alignment-based tool that utilizes the sequence aligner DIAMOND and is written in the programming language Julia. Homologous regions are obtained via a sequence similarity search, followed by a "hit stitching" phase, in which adjacent or overlapping regions are merged into a single unit. The novel sliding window algorithm trims away any noncoding regions from the resulting sequence. We demonstrate the utility of Patchwork by recovering near-universal single-copy orthologs within a benchmarking study, and we additionally assess the performance of Patchwork in comparison with other programs. We find that Patchwork allows for accurate retrieval of (putatively) single-copy genes from genome skimming data sets at different sequencing depths with high computational speed, outperforming existing software targeting similar tasks. Patchwork is released under the GNU General Public License version 3. Installation instructions, additional documentation, and the source code itself are all available via GitHub at https://github.com/fethalen/Patchwork.

Shallow Whole-Genome Sequencing of Aedes japonicus and Aedes koreicus from Italy and an Updated Picture of Their Evolution Based on Mitogenomics and Barcoding.

Aedes japonicus and Aedes koreicus are two invasive mosquitoes native to East Asia that are quickly establishing in temperate regions of Europe. Both species are vectors of arboviruses, but we currently lack a clear understanding of their evolution. Here, we present new short-read, shallow genome sequencing of A. japonicus and A. koreicus individuals from northern Italy, which we used for downstream phylogenetic and barcode analyses. We explored associated microbial DNA and found high occurrences of Delftia bacteria in both samples, but neither Asaia nor Wolbachia. We then assembled complete mitogenomes and used these data to infer divergence times estimating the split of A. japonicus from A. koreicus in the Oligocene, which was more recent than that previously reported using mitochondrial markers. We recover a younger age for most other nodes within Aedini and other Culicidae. COI barcoding and phylogenetic analyses indicate that A. japonicus yaeyamensis, A. japonicus amamiensis, and the two A. koreicus sampled from Europe should be considered as separate species within a monophyletic species complex. Our studies further clarify the evolution of A. japonicus and A. koreicus, and indicate the need to obtain whole-genome data from putative species in order to disentangle their complex patterns of evolution.

Phylogenetic Relationships and Next-Generation Barcodes in the Genus Torreya Reveal a High Proportion of Misidentified Cultivated Plants.

Accurate species identification is key to conservation and phylogenetic inference. Living plant collections from botanical gardens/arboretum are important resources for the purpose of scientific research, but the proportion of cultivated plant misidentification are un-tested using DNA barcodes. Here, we assembled the next-generation barcode (complete plastid genome and complete nrDNA cistron) and mitochondrial genes from genome skimming data of Torreya species with multiple accessions for each species to test the species discrimination and the misidentification proportion of cultivated plants used in Torreya studies. A total of 38 accessions were included for analyses, representing all nine recognized species of genus Torreya. The plastid phylogeny showed that all 21 wild samples formed species-specific clades, except T. jiulongshanensis. Disregarding this putative hybrid, seven recognized species sampled here were successfully discriminated by the plastid genome. Only the T. nucifera accessions grouped into two grades. The species identification rate of the nrDNA cistron was 62.5%. The Skmer analysis based on nuclear reads from genome skims showed promise for species identification with seven species discriminated. The proportion of misidentified cultivated plants from arboreta/botanical gardens was relatively high with four accessions (23.5%) representing three species. Interspecific relationships within Torreya were fully resolved with maximum support by plastomes, where Torreya jackii was on the earliest diverging branch, though sister to T. grandis in the nrDNA cistron tree, suggesting that this is likely a hybrid species between T. grandis and an extinct Torreya ancestor lineage. The findings here provide quantitative insights into the usage of cultivated samples for phylogenetic study.

Low coverage sequencing provides insights into the key features of the nuclear and mitochondrial genomes of the Alligator Snapping Turtle Macrochelys temminckii.

The alligator snapping turtle Macrochelys temminckii is a culturally, ecologically, and evolutionary relevant species of conservation concern. In this study, we conducted a genome survey of M. temminckii. Using a low-coverage short read sequencing strategy, this study estimated the genome size, repetitive genome content, annotated and quantified repetitive elements, assembled the 45S rRNA DNA operon, and characterized in detail the mitochondrial genome of M. temminckii. Using a k-mer strategy, the estimated haploid genome size varied between 3.77 and 3.19 Gbp, which is within the range previously reported for other representatives of the family Chelydridae. Repetitive genome content estimates using different k-mers (21 to 51) indicated that more than 75 % of the genome of M. temminckii comprised repetitive elements. Taking into account only annotated repetitive elements, the most common repetitive elements were classified as Class I - Long Interspersed Nuclear Element (LINE) which were more abundant than Class I - Penelope and Class I - Long Terminal Repeat (LTR) Ty3 mobile elements. Less abundant repeat element families in the nuclear genome of M. temminckii included Class I - DIRS mobile elements and Satellite DNA. The nuclear ribosomal operon was partially assembled into three contigs, one encoding the complete ssrRNA gene, a second encoding the complete 5.8S rRNA gene, and a third comprising the full lsrRNA gene. The AT-rich complete mitochondrial genome was 16,570 bp long. These new genomic resources are of utmost importance to aid in the development of conservation plans for this iconic freshwater turtle.

An updated classification for the hyper-diverse genus Corydalis (Papaveraceae: Fumarioideae) based on phylogenomic and morphological evidence.

The genus Corydalis, with ca. 530 species, has long been considered taxonomically challenging because of its great variability. Previous molecular analyses, based on a few molecular markers and incomplete taxonomic sampling, were clearly inadequate to delimit sections and subgenera. We have performed phylogenetic analyses of Corydalis and related taxa, using 65 shared protein-coding plastid genes from 313 accessions (including 280 samples of ca. 226 species of Corydalis) and 152 universal low-copy nuclear genes from 296 accessions (including 271 samples of Corydalis) covering all 42 previously recognized sections and five independent "series". Phylogenetic trees were inferred using Bayesian Inference and Maximum Likelihood. Eight selected morphological characters were estimated using ancestral state reconstructions. Results include: (i) of the three subgenera of Corydalis, two are fully supported by both the plastid and nuclear data; the third, subg. Cremnocapnos, is weakly supported by plastid DNA only, whereas in the nuclear data the two included sections form successive outgroups to the rest of the genus; (ii) among all 42 sections and five "series", 25 sections and one "series" are resolved as monophyletic in both data sets; (iii) the common ancestor of Corydalis is likely to be a perennial plant with a taproot, yellow flowers with a short saccate spur, linear fruits with recurved fruiting pedicels, and seeds with elaiosomes; (iv) we provide a new classification of Corydalis with four subgenera (of which subg. Bipapillatae is here newly described), 39 sections, 16 of which are consistent with the previous classification, 16 sections have been recircumscribed, one section has been reinstated and six new sections are established. Characters associated with lifespan, underground structures, floral spur, fruit and elaiosomes are important for the recognition of subgenera and sections. These new phylogenetic analyses combined with ancestral character reconstructions uncovered previously unrecognized relationships, and greatly improved our understanding of the evolution of the genus.

Phylogenetic relationship and comparative analysis of the main Bupleuri Radix species in China.

Background: Bupleuri Radix (Chaihu) is a famous traditional Chinese medicine derived from Bupleurum, Apiaceae. The origin of cultivated Chaihu germplasm in China is unclear, which has led to unstable Chaihu quality. In this study, we reconstructed the phylogeny of the main Chaihu germplasm species in China and identified potential molecular markers to authenticate its origin. Methods: Three Bupleurum species (eight individuals), B. bicaule, B. chinense, and B. scorzonerifolium, were selected for genome skimming. Published genomes from B. falcatum and B. marginatum var. stenophyllum were used for comparative analysis. Results: Sequences of the complete plastid genomes were conserved with 113 identical genes ranging from 155,540 to 155,866 bp in length. Phylogenetic reconstruction based on complete plastid genomes resolved intrageneric relationships of the five Bupleurum species with high support. Conflicts between the plastid and nuclear phylogenies were observed, which were mainly ascribed to introgressive hybridization. Comparative analysis showed that noncoding regions of the plastomes had most of the variable sequences. Eight regions (atpF-atpH, petN-psbM, rps16-psbK, petA-psbJ, ndhC-trnV/UAC and ycf1) had high divergence values in Bupleurum species and could be promising DNA barcodes for Chaihu authentication. A total of seven polymorphic cpSSRs and 438 polymorphic nSSRs were detected across the five Chaihu germplasms. Three photosynthesis-related genes were under positive selection, of which accD reflected the adaptation fingerprint of B. chinense to different ecological habitats. Our study provides valuable genetic information for phylogenetic investigation, germplasm authentication, and molecular breeding of Chaihu species.