Plastid Loci Research Articles

Phylogenetics, Taxonomy, and Chromosome Number Analysis of Sanvitalia (Asteraceae-Heliantheae-Zinniinae)

Abstract— Sanvitalia (Asteraceae), a New World endemic, is poorly understood from a phylogenetic perspective. While chromosome number variation is well documented for the genus, previous studies lack a phylogenetic context for chromosome number evolution, with one species, S. fruticosa, lacking a published chromosome count. We collected molecular sequence data from the nuclear ribosomal ITS region and three plastid loci from multiple individuals of each species of Sanvitalia and additional members of the Zinniinae to create phylogenetic trees and study the evolutionary history of the clade. A complete taxonomic treatment was produced as well as a dichotomous key to species. The first chromosome count for S. fruticosa was rendered (2n = 16) and a chromosome number analysis was completed using maximum likelihood methods that account for different modes of chromosome number change. Support for two separate whole genome duplication events in the genus is presented here. This study resulted in deeper understanding of the relationships within Sanvitalia and closely related genera, chromosome number evolution and genome duplication in Zinniinae, and the closest relative (S. ocymoides) of the enigmatic and amphiequatorial disjunct S. versicolor. We also provide support for the prior transfer of S. tenuis to Zinnia, and two new sections within Sanvitalia.

Persistence Over Millennia Through Extreme Clonal Longevity: Phylogenomic Insight Into History of One of the World's Rarest Plant Species

ABSTRACTAimThe evolutionary history of European alpine plant species with medium to large geographical ranges is relatively well explored. Here, we investigate the genetic structure and diversity of an extremely narrow endemic and one of the world's rarest plants.LocationEastern Carpathians, Romania.TaxonAndryala laevitomentosa (Asteraceae), an evolutionarily isolated herb species with a worldwide range limited to five micropopulations distributed along a 1.8 km long mountain ridge.MethodsWe used three plastid loci, nuclear ribosomal ITS and genome‐wide, mostly nuclear 26,272 Single‐Nucleotide Polymorphism (SNPs) obtained from RAD‐seq data. We assessed haplotype and genotype diversity, dated the resulting phylogeographic structure, quantified seed production and inferred vegetative propagation.ResultsMaternally inherited plastid markers and nuclear genomic data revealed a concordant pattern: (i) limited genetic diversity, with seven cpDNA haplotypes and 11 RAD‐seq multilocus genotypes; (ii) a strong geographic structure corresponding to spatially isolated genets (clones). The species is likely of early Pleistocene origin (c. 2 Mya), and the estimated age of individual clones varied from c. 24 to 64 Kya. The average seed set assessed over 3 years was only 0.4%. However, the species reproduces veg by axillary and adventitious rosettes formed on rhizomes and roots, respectively.Main ConclusionsThe strong trade‐off between sexual and vegetative reproduction explains not only a deep and ancient phylogeographic structure but also the rarity of the species. Its survival depends almost entirely on vegetative reproduction. The genets of A. laevitomentosa are amongst the oldest clones ever documented in angiosperms. The persistence of these clones in situ for tens of thousands of years suggests an exceptional ability of this species to adapt to major climatic oscillations throughout the Pleistocene and Holocene and challenge our perception of the extent of resilience in plants.

Plastid phylogenomics clarifies broad-level relationships in Bulbophyllum (Orchidaceae) and provides insights into range evolution of Australasian section Adelopetalum

The hyperdiverse orchid genus Bulbophyllum is the second largest genus of flowering plants and exhibits a pantropical distribution with a center of diversity in tropical Asia. The only Bulbophyllum section with a center of diversity in Australasia is sect. Adelopetalum. However, the phylogenetic placement, interspecific relationships, and spatio-temporal evolution of this section remain largely unclear. To infer broad-level relationships within Bulbophyllum, and interspecific relationships within sect. Adelopetalum, a genome skimming dataset was generated for 89 samples, which yielded 70 plastid coding regions and a nuclear ribosomal DNA cistron. For 18 additional samples, Sanger data from two plastid loci (matK and ycf1) and nuclear ITS were added using a supermatrix approach. The study provided new insights into broad-level relationships in Bulbophyllum, including phylogenetic evidence for the non-monophyly of sections Beccariana, Brachyantha, Brachypus, Cirrhopetaloides, Cirrhopetalum, Desmosanthes, Minutissima, Oxysepala, Polymeres, and Sestochilos. Section Adelopetalum and sect. Minutissima s.s. formed a highly supported clade that was resolved as a sister group to the remainder of the genus. Divergence time estimations based on a relaxed molecular clock model placed the origin of Bulbophyllum in the Early Oligocene (ca. 33.2 Ma) and sect. Adelopetalum in the Late Oligocene (ca. 23.6 Ma). Ancestral range estimations based on a BAYAREALIKE model identified the Australian continent as the ancestral area of the sect. Adelopetalum. The section underwent crown diversification from the mid-Miocene to the late Pleistocene, predominantly in continental Australia. At least two independent long-distance dispersal events were inferred eastward from the Australian continent to New Zealand and to New Caledonia from the early Pliocene onwards, likely mediated by predominantly westerly winds of the Southern hemisphere. Retraction and fragmentation of the eastern Australian rainforests from the early Miocene onwards are likely drivers of lineage divergence within sect. Adelopetalum facilitating allopatric speciation.

Molecular Identification and Phylogenetic Analysis of Cymbidium Species (Orchidaceae) Based on the Potential DNA Barcodes matK, rbcL, psbA-trnH, and Internal Transcribed Spacer

Numerous Cymbidium species have significant commercial value globally due to their exotic ornamental flowers. Identifying Cymbidium species is challenging due to their similar shapes, which hinders their rational use and the conservation of germplasm resources. In the present study, firstly, four plastid loci (matK, rbcL, psbA-trnH, and atpF-atpH) and a nuclear locus (internal transcribed spacer, ITS) were initially examined to identify Cymbidium species. Secondly, we inferred the interspecific phylogeny of Cymbidium species using ITS sequences. All of these DNA regions, with the exception of atpF-atpH, could be readily amplified from Cymbidium, and the corresponding DNA sequences can be successfully obtained by sequencing. Our research demonstrated that ITS exhibited the highest intra- and interspecific divergences, the greatest barcoding gap, and the highest proportion of species identification. The phylogenetic analysis of Cymbidium species based on the ITS regions primarily corroborated the results obtained using traditional morphological methods. A comparative analysis of candidate DNA barcodes has shown that the ITS can be used not only for barcoding Cymbidium species but also for the phylogenetic analysis of Cymbidium.

One more word on Patagonian Ulota macrodontia: Ulota brachypoda sp. nov. (Orthotrichaceae, Bryophyta)

Abstract The bryophyte genus Ulota has its main diversification area in Patagonia, with 18 currently accepted species, all of which are endemic to the area. Despite this richness, ongoing studies indicate the existence of hitherto unknown species in this territory. However, the strong resemblance between species often complicates the discrimination of new taxa, making the use of integrative taxonomy approaches compulsory. During surveys conducted in past decades, a morphotype of a Ulota, closely resembling Ulota macrodontia, was discovered coexisting with this species. Ulota macrodontia is characterized by unique peristomial features within the genus, and the variant morphotype shares these characteristics while displaying some distinct gametophytic and sporophytic features. Biometric analyses demonstrate that the differences are stable, and without intermediate states. The results obtained from the analyses of one nuclear and four plastid loci reveal a close relationship between both morphotypes, with the samples of U. macrodontia forming a well-supported monophyletic group. We propose to treat the new morphotype at species level (Ulota brachypoda sp. nov.), discussing why this is the most appropriate taxonomic rank, and we provide a detailed and illustrated description. Additionally, a distribution map of the two endemics is included.

Phylogenomics and topological conflicts in the tribe Anthospermeae (Rubiaceae).

Genome skimming (shallow whole-genome sequencing) offers time- and cost-efficient production of large amounts of DNA data that can be used to address unsolved evolutionary questions. Here we address phylogenetic relationships and topological incongruence in the tribe Anthospermeae (Rubiaceae), using phylogenomic data from the mitochondrion, the nuclear ribosomal cistron, and the plastome. All three genomic compartments resolve relationships in the Anthospermeae; the tribe is monophyletic and consists of three major subclades. Carpacoce Sond. is sister to the remaining clade, which comprises an African subclade and a Pacific subclade. Most results, from all three genomic compartments, are statistically well supported; however, not fully consistent. Intergenomic topological incongruence is most notable in the Pacific subclade but present also in the African subclade. Hybridization and introgression followed by organelle capture may explain these conflicts but other processes, such as incomplete lineage sorting (ILS), can yield similar patterns and cannot be ruled out based on the results. Whereas the null hypothesis of congruence among all sequenced loci in the individual genomes could not be rejected for nuclear and mitochondrial data, it was rejected for plastid data. Phylogenetic analyses of three subsets of plastid loci identified using the hierarchical likelihood ratio test demonstrated statistically supported intragenomic topological incongruence. Given that plastid genes are thought to be fully linked, this result is surprising and may suggest modeling or sampling error. However, biological processes such as biparental inheritance and inter-plastome recombination have been reported and may be responsible for the observed intragenomic incongruence. Mitochondrial insertions into the plastome are rarely documented in angiosperms. Our results indicate that a mitochondrial insertion event in the plastid trnS GGA - rps4 IGS region occurred in the common ancestor of the Pacific clade of Anthospermeae. Exclusion/inclusion of this locus in phylogenetic analyses had a strong impact on topological results in the Pacific clade.

A New Endemic Smilax (Smilacaceae) from Tafelberg, Suriname, Supported by Morphometric and Phylogenetic Evidence

Abstract— Taxonomy of Smilax in the Neotropics is poorly known with many undescribed species, unresolved synonymy, and regional treatments with conflicting taxon concepts. This issue is amplified in the Guiana Shield, which has arguably received the least amount of taxonomic research in Smilax. Here, we investigate multiple specimens from Tafelberg, Suriname that we hypothesized to represent an undescribed species. We used univariate and multivariate morphometrics to analyze linear measurements and leaf outlines to test if and how Tafelberg specimens differ from those of morphologically similar species in the Guiana Shield, Smilax lasseriana and S. maypurensis. We used a genome skimming approach to sequence and assemble plastomes, which also allowed us to extract plastid loci previously employed in Sanger-based studies and place the putative new species in a phylogeny with expanded taxon sampling. We recovered the Smilax from Tafelberg as more closely related to species with ranges centered in eastern Brazil than to its morphologically hypothesized congeners, S. lasseriana and S. maypurensis. We describe this new species as Smilax tafelbergensis with detailed morphological comparisons to similar taxa, a distribution map, and illustration.

Development of CAPS-markers for studying plastid loci polymorphism in Rubus L. subgenus Idaeobathus Focke

Genus Rubus L. including subgenus Idaeobathus Focke (raspberry) has difficult structure. Studying this subgenus researchers have special interest to West Siberia and especially Altai region because two species R. idaeus L. and R. sachalinensis H. Lev. grow together on this territory. Usually in phylogenetic research chloroplast DNA loci are studied. This article describes the attempt to develop relevant plastid CAPS-markers for studying raspberry genetic diversity. Nine markers that revealed polymorphism among raspberry cultivars have been developed. However, the sample of R. idaeus and R. sachalinensis herbarium specimens from Altai Krai and Republic of Altai showed that all of them are monomorphic aside from two combinations “primers/restrictase” indicated intraspecies polymorphism within R. sachalinensis. In addition, two potential markers able to differentiate R. idaeus and R. sachalinensis without using restrictases are detected. Further research devoted to creating markers for revealing plastid loci polymorphism will continue.

A target enrichment probe set for resolving phylogenetic relationships in the coffee family, Rubiaceae.

Rubiaceae is among the most species-rich plant families, as well as one of the most morphologically and geographically diverse. Currently available phylogenies have mostly relied on few genomic and plastid loci, as opposed to large-scale genomic data. Target enrichment provides the ability to generate sequence data for hundreds to thousands of phylogenetically informative, single-copy loci, which often leads to improved phylogenetic resolution at both shallow and deep taxonomic scales; however, a publicly accessible Rubiaceae-specific probe set that allows for comparable phylogenetic inference across clades is lacking. Here, we use publicly accessible genomic resources to identify putatively single-copy nuclear loci for target enrichment in two Rubiaceae groups: tribe Hillieae (Cinchonoideae) and tribal complex Palicoureeae+Psychotrieae (Rubioideae). We sequenced 2270 exonic regions corresponding to 1059 loci in our target clades and generated in silico target enrichment sequences for other Rubiaceae taxa using our designed probe set. To test the utility of our probe set for phylogenetic inference across Rubiaceae, we performed a coalescent-aware phylogenetic analysis using a subset of 27 Rubiaceae taxa from 10 different tribes and three subfamilies, and one outgroup in Apocynaceae. We recovered an average of 75% and 84% of targeted exons and loci, respectively, per Rubiaceae sample. Probes designed using genomic resources from a particular subfamily were most efficient at targeting sequences from taxa in that subfamily. The number of paralogs recovered during assembly varied for each clade. Phylogenetic inference of Rubiaceae with our target regions resolves relationships at various scales. Relationships are largely consistent with previous studies of relationships in the family with high support (≥0.98 local posterior probability) at nearly all nodes and evidence of gene tree discordance. Our probe set, which we call Rubiaceae2270x, was effective for targeting loci in species across and even outside of Rubiaceae. This probe set will facilitate phylogenomic studies in Rubiaceae and advance systematics and macroevolutionary studies in the family.

Dissecting the continuum and unravelling the phylogeographic knot of plastid DNA in European white oaks (Quercus sect. Quercus): ancient signatures and multiple diversity reservoirs

Available knowledge on the European white oaks (Quercus, sect. Quercus) plastome still exhibits large gaps, mostly in respect of detailed phylogeny and information from southern Europe and adjacent Near East. We investigated DNA sequence polymorphism at two plastid loci on 270 individuals representing 13 white oak species/taxa and retrieved several hundred sequences of the same two DNA regions from GenBank to fill the above gaps, and contribute to a better understanding of white oak diversity and evolution. The modern European sect. Quercus species exhibits a poorly differentiated plastid DNA, despite its current highly diversified taxonomy. Twenty-nine haplotypes were identified across the Euro-Mediterranean and Near East regions, including two ancestral variants that are westerly and south-easterly partitioned. Other high- and low-frequency haplotypes also showed a well-structured geographic distribution consistent with the current phytogeographic framework of the European continent. The climatic and geological events that characterized the end of the Tertiary and Quaternary periods played a crucial role in triggering haplotype mixing, isolation, and in offering niche opportunities for more recent diversification. Euro-Mediterranean southern territories host a high and as yet poorly studied genetic variation; the role of the Italian Peninsula as a crossroad and threshold for haplotype diversity and distribution clearly emerges from this study.

DNA barcodes, ISSR, RAPD and SCAR markers as potential quality control tools for molecular authentication of black and white mulberry

Morus species, especially black mulberry (Morus nigra L.), are well known for their nutritive and economic values. Black mulberry is often a subject for deliberate and undeliberate substitution with the morphologically similar white mulberry (Morus alba L.). In the current study, the genetic diversity of Morus nigra L. and Morus alba L. was assessed using different genetic profiling and fingerprinting techniques namely namely DNA barcoding, Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR). DNA barcoding was implemented using 2 plastid loci (matK, and rbcL-1) and 2 nuclear spacers (ITS and ITS2) aiming at accurate authentication of the studied species. DNA barcoding, targeting the selected loci succeeded to identify and authenticate the studies Morus species but failed to differentiate them, owing to their close genetic resemblance. Out of 52 screened RAPD primers, 27 were able to produce reproducible and clear profiles with 115 amplified fragments of which 37 were polymorphic accounting for 32.17% polymorphism. The number of amplified bands varied from 2 to 7 ranging in size from 100 to 700 bp. Moreover, 55 ISSR primers were evaluated, 108 fragments were produced, using 20 primers, of which 28 were polymorphic accounting for 25.92% polymorphism with size range of 200–800 bp. Sequence Characterized Amplified Region (SCAR) marker was developed based on reproducible banding pattern obtained by RAPD primer (OPG-03), for the purpose of accelerating the initial screening of Morus batches and enabling the preventative rejection of adulterated samples. The polymorphic band obtained was sequenced and specific primers were designed to develop a SCAR marker of approximately 558 bp exclusively for M. nigra, allowing detection of down to 1 ng, of M. nigra. The SCAR marker showed 100% success in differentiating authentic M. nigra and M. alba samples. In addition, the application of the developed SCAR marker was extended to distinguish M. nigra from its four common adulterants namely Ficus carica L., Fragaria vesca L., Ficus racemosa L. and Vitis vinifera L.. The proposed protocol was successfully implemented for the evaluation of 47 commercial M. nigra samples collected from different Egyptian locations.

Plastid DNA Barcoding and RtActin cDNA Fragment Isolation of Reutealis Trisperma: A Promising Bioresource for Biodiesel Production.

Reutealis trisperma belonging to the family Euphorbiaceae is currently used for biodiesel production, and rapid development in plant-based biofuel production has led to its increasing demand. However, massive utilization of bio-industrial plants has led to conservation issues. Moreover, genetic information on R trisperma is still limited, which is crucial for developmental, physiological, and molecular studies. Studying gene expression is essential to explain plant physiological processes. Nonetheless, this technique requires sensitive and precise measurement of messenger RNA (mRNA). In addition, the presence of internal control genes is important to avoid bias. Therefore, collecting and preserving genetic data for R trisperma is indispensable. In this study, we aimed to evaluate the application of plastid loci, rbcL, and matK, to the DNA barcode of R trisperma for use in conservation programs. In addition, we isolated and cloned the RtActin (RtACT) gene fragment for use in gene expression studies. Sequence information was analyzed in silico by comparison with other Euphorbiaceae plants. For actin fragment isolation, reverse-transcription polymerase chain reaction was used. Molecular cloning of RtActin was performed using the pTA2 plasmid before sequencing. We successfully isolated and cloned 592 and 840 bp of RtrbcL and RtmatK fragment genes, respectively. The RtrbcL barcoding marker, rather than the RtmatK plastidial marker, provided discriminative molecular phylogenetic data for R Trisperma. We also isolated 986 bp of RtACT gene fragments. Our phylogenetic analysis demonstrated that R trisperma is closely related to the Vernicia fordii Actin gene (97% identity). Our results suggest that RtrbcL could be further developed and used as a barcoding marker for R trisperma. Moreover, the RtACT gene could be further investigated for use in gene expression studies of plant.

Phylogeny and systematics of the tribe Sonerileae (Melastomataceae) in Africa: A revised taxonomic classification

AbstractThe tribe Sonerileae in tropical Africa and Madagascar is a morphologically diverse lineage that consists of 239 species in 10 genera. In this study, we present the first in‐depth phylogenetic analysis of African Sonerileae to test monophyly of the currently recognized genera. Phylogenetic analyses were performed using sequence data from two nuclear (nrITS and nrETS) and three plastid loci (accD‐psaI, ndhF and psbK‐psbL). Sampling consisted of 140 accessions including 64 African, 27 Malagasy, 46 Asian, and three neotropical Sonerileae together with a broad outgroup sampling (105 spp.). Phylogenetic relationships were inferred using maximum likelihood and Bayesian inference approaches, and a careful reassessment of morphological characters was carried out. Our results neither support the monophyly of the Old World nor African Sonerileae. The monospecific African genus Benna is partially supported as sister to Phainantha, one of the basal neotropical lineages, while African and Malagasy Medinilla are nested among the SE Asian genera. Gravesia (116 spp.), the most species‐rich and morphologically diverse genus in Madagascar, is recovered as monophyletic. The African genera of Sonerileae Calvoa, Dicellandra, and Preussiella form well‐supported clades. In contrast, Amphiblemma (including Amphiblemma molle) and Cincinnobotrys s.l. (including Cincinnobotrys felicis) are not monophyletic. To accommodate the caulescent C. felicis we propose reinstatement of the monospecific genus Bourdaria. For the distinctive A. molle a new genus Mendelia is described. Calvoa hirsuta is designated here as the type of genus Calvoa, lectotypes are designated for Medinilla engleri and Veprecella lutea, and a neotype is designated for Preussiella kamerunensis.

Hybridization and genome duplication for early evolutionary success in the Asian Palmate group of Araliaceae

AbstractThe phenomenal advances in sequencing techniques and analytical development during the last decade have provided a unique opportunity to unravel the evolutionary history of lineages under complex patterns of evolution. This is the case of the largest clade of the ginseng family (Araliaceae), the Asian Palmate group (AsPG), where the large internal polytomies and genome incongruences detected in previous studies pointed to a scenario of radiation with hybridization events between genera for the early evolution of the group. In this study, we aim to obtain well‐resolved nuclear and plastid phylogenies of the AsPG using Hyb‐Seq to evaluate the radiation hypothesis and assess the role of hybridization in the early evolution of the group. We performed concatenated‐ and coalescent‐based phylogenetic analyses from the 936 targeted nuclear loci and 261 plastid loci obtained for 72 species representing 20 genera of the AsPG and the main clades of Araliaceae. The impact of hybridization and incomplete lineage sorting (ILS) was assessed with SNaQ, and genome duplications were evaluated with ChromEvol. Our nuclear and plastid phylogenies are compatible with a scenario of early radiation in the AsPG. Also, the identification of extensive signals of hybridization and ILS behind the genome incongruences supports hybridization as a major driving force during the early radiation. We hypothesize a whole‐genome duplication event at the origin of the AsPG, followed by a radiation that led to extensive ILS, which, alongside the early inter‐genera hybridization, is obscuring the phylogenetic signal in the early evolution of this major clade.

Plastome structure, phylogenomics, and divergence times of tribe Cinnamomeae (Lauraceae)

BackgroundTribe 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.ResultsAmong 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, trnICAU, rpl32, and rpl2. Four hypervariable plastid loci (ycf1, ycf2, ndhF-rpl32-trnLUAG, 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).ConclusionsThis 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.

Plastome sequences fail to resolve shallow level relationships within the rapidly radiated genus Isodon (Lamiaceae).

As one of the largest genera of Lamiaceae and of great medicinal importance, Isodon is also phylogenetically and taxonomically recalcitrant largely ascribed to its recent rapid radiation in the Hengduan Mountains. Previous molecular phylogenetic studies using limited loci have only successfully resolved the backbone topology of the genus, but the interspecific relationships suffered from low resolution, especially within the largest clade (Clade IV) which comprises over 80% species. In this study, we attempted to further elucidate the phylogenetic relationships within Isodon especially Clade IV using plastome sequences with a broad taxon sampling of ca. 80% species of the genus. To reduce systematic errors, twelve different plastome data sets (coding and non-coding regions with ambiguously aligned regions and saturated loci removed or not) were employed to reconstruct phylogeny using maximum likelihood and Bayesian inference. Our results revealed largely congruent topologies of the 12 data sets and recovered major lineages of Isodon consistent with previous studies, but several incongruences are also found among these data sets and among single plastid loci. Most of the shallow nodes within Clade IV were resolved with high support but extremely short branch lengths in plastid trees, and showed tremendous conflicts with the nrDNA tree, morphology and geographic distribution. These incongruences may largely result from stochasticity (due to insufficient phylogenetic signal) and hybridization and plastid capture. Therefore, the uniparental-inherited plastome sequences are insufficient to disentangle relationships within a genus which has undergone recent rapid diversification. Our findings highlight a need for additional data from nuclear genome to resolve the relationships within Clade IV and more focused studies to assess the influences of multiple processes in the evolutionary history of Isodon. Nevertheless, the morphology of the shape and surface sculpture/indumentum of nutlets is of systematic importance that they can distinguish the four major clades of Isodon.

Reinstatement of Ticanto (Leguminosae-Caesalpinioideae) - the final piece in the Caesalpinia group puzzle.

A recent molecular phylogenetic analysis of the Caesalpinia group demonstrated that it comprises 26 genera, but the recognition of a putative 27th genus, Ticanto, remained in doubt. This study presents a phylogenetic analysis of ITS and five plastid loci revealing a robustly supported monophyletic group representing the Ticanto clade, sister to the morphologically distinct genus Pterolobium. Based upon this evidence, along with a morphological evaluation, the genus Ticanto is here reinstated. Descriptions are provided for all nine species of Ticanto, together with a key to the species, maps, and colour photographs. Nine new combinations are made: Ticantocaesia (Hand.-Mazz.) R. Clark & Gagnon, T.crista (L.) R. Clark & Gagnon, T.elliptifolia (S. J. Li, Z. Y. Chen & D. X. Zhang) R. Clark & Gagnon, T.magnifoliolata (Metcalf) R. Clark & Gagnon, T.rhombifolia R. Clark & Gagnon, T.sinensis (Hemsl.) R. Clark & Gagnon, T.szechuenensis (Craib) R. Clark & Gagnon, T.vernalis (Champion ex Benth.) R. Clark & Gagnon and T.yunnanensis (S. J. Li, D. X. Zhang & Z.Y. Chen) R. Clark & Gagnon. The final major question in the delimitation of segregate genera from within Caesalpinia sensu lato and the Caesalpinia group is thus resolved.

Reinstatement of Loxogramme yigongensis (Polypodiaceae), an endemic species from southeastern Xizang, China

Loxogramme yigongensis, so far known only from the type specimens collected in 1965 and treated as a synonym of L. grammitoides, is reinstated. Morphologically, these two species are closely related but L. yigongensis differs in having succulent lamina, becoming rugose when dry, lamina lanceolate with acute apex, widest near middle of lamina, and costa not or slightly raised on lower surface. Molecular analyses based on five plastid loci resolved these two species as sister to each other with distinct divergence. In addition, the ploidy level of the two species are reported for the first time.

The RadOrgMiner pipeline: Automated genotyping of organellar loci from RADseq data

Abstract Different versions of Restriction‐site‐Associated DNA sequencing (RADseq) have become powerful and popular tools in molecular ecology. Although RADseq datasets are generally regarded as representative of the nuclear genome, reduced representation genomic libraries may also sample the organellar (i.e. the mitochondrial and, in the case of plants, the plastid) DNA. This cytoplasmic genetic variance provides a better understanding of evolutionary history by uncovering past hybridisation and identifying maternal or, rarely, paternal lineage due to rapid lineage sorting. We developed a pipeline that is based on existing bioinformatic tools to automatically mine and genotype organellar loci from RADseq libraries. The efficacy of our pipeline is tested on eight publicly available datasets spanning different phylogenetic levels (i.e. from family‐level phylogenies to phylogeography) and RADseq methods (sdRAD, ddRAD, ezRAD, GBS) for genotyping mitochondrial and plastid loci, which were subject to phylogenetic tree reconstruction. In all cases, organellar phylogenies adequately supplemented the original studies by corroborating the large‐scale picture based on RADseq or by bringing additional evidence on past or contemporary hybridisation. RADseq methods designed to achieve larger horizontal coverage (i.e. ddRAD, ezRAD) yielded longer organellar alignments, but sdRAD and GBS still provided valuable polymorphic organellar loci at no additional sequencing effort. Our newly developed pipeline can be run under a Unix‐like operating system and is freely accessible at https://doi.org/10.5281/zenodo.6619190

Plant DNA barcoding and metabolomics for comprehensive discrimination of German Chamomile from its poisonous adulterants for food safety

German Chamomile (Matricaria recutita L.) is one of the most consumed herbal teas in the world. Deliberate and undeliberate adulteration in German Chamomile raw material represents serious concerns for consumers’ safety. Authentication of Matricaria recutita L. samples was performed using an integrative approach of DNA barcoding and chemical profiling. Discrimination of M. recutita from three morphologically related species namely Anthemis cotula L., Senecio desfontainei Druce and Senecio vulgaris L. was accomplished. Three plastid loci (mat K, rbcL and psbA-trnH) and two nuclear spacers (ITS and ITS2) were utilized for the DNA barcoding of M. recutita. The Chemical profiling of M. recutita was achieved using untargeted HPTLC-image analysis. Among the tested barcoding loci, ITS2 revealed the maximum genetic diversity among the studied plants. ITS2 showed adequate polymorphic sites to detect interspecific variation with high amplification and sequencing success. Chemical profiling showed that flavonoids, coumarins, sesquiterpene lactones and phenolic acids are the main secondary metabolite classes of the candidate plants. While, flavonoids and phenolic acids served as quality markers, sesquiterpene lactone (anthecotulide) and pyrrolizidine alkaloid (senecionine) accounted as toxicity markers for the adulterants. Notably, coumarin accumulation pattern could be utilized to discriminate M. recutita from its adulterants due to the absence of hernarin and umbelliferone from Senecio species. The results of this study suggest that, integrating genetic and metabolomic fingerprinting could serve as an effective tool, with 100% success rate, in authenticating M. recutita and discriminating its samples from the morphologically related toxic adulterants. This offers a comprehensive tool for the quality control of M. recutita samples in food industry.