nrDNA Sequences Research Articles

Phylogenetic inference and secondary structure predictions of Turkish genotypes of Coriandrum sativum (L.) based on ITS4 and ITS5 nrDNA sequences

Phylogenetic inference and secondary structure predictions of Turkish genotypes of Coriandrum sativum (L.) based on ITS4 and ITS5 nrDNA sequences

Analysis of the complete plastomes and nuclear ribosomal DNAs from Euonymus hamiltonianus and its relatives sheds light on their diversity and evolution.

Euonymus hamiltonianus and its relatives (Celastraceae family) are used for ornamental and medicinal purposes. However, species identification in Euonymus is difficult due to their morphological diversity. Using plastid genome (plastome) data, we attempt to reveal phylogenetic relationship among Euonymus species and develop useful markers for molecular identification. We assembled the plastome and nuclear ribosomal DNA (nrDNA) sequences from five Euonymus lines collected from South Korea: three Euonymus hamiltonianus accessions, E. europaeus, and E. japonicus. We conducted an in-depth comparative analysis using ten plastomes, including other publicly available plastome data for this genus. The genome structures, gene contents, and gene orders were similar in all Euonymus plastomes in this study. Analysis of nucleotide diversity revealed six divergence hotspots in their plastomes. We identified 339 single nucleotide polymorphisms and 293 insertion or deletions among the four E. hamiltonianus plastomes, pointing to abundant diversity even within the same species. Among 77 commonly shared genes, 9 and 33 were identified as conserved genes in the genus Euonymus and E. hamiltonianus, respectively. Phylogenetic analysis based on plastome and nrDNA sequences revealed the overall consensus and relationships between plastomes and nrDNAs. Finally, we developed six barcoding markers and successfully applied them to 31 E. hamiltonianus lines collected from South Korea. Our findings provide the molecular basis for the classification and molecular taxonomic criteria for the genus Euonymus (at least in Korea), which should aid in more objective classification within this genus. Moreover, the newly developed markers will be useful for understanding the species delimitation of E. hamiltonianus and closely related species.

The determination of the taxonomic position of Peucedanum arenarium subsp. neumayeri and subsp. urbani in Turkey

In this study, the taxonomic status of two subspecies of Peucedanum arenarium grown in Turkey, subsp urbani and subsp. neumayeri were investigated. There are no ripe fruits of this subspecies in the flora of Turkey. In fact, P. arenarium subsp. neumayeri has been misdiagnosed in the flora of Turkey because it is not a ripe fruit. When we looked at the ripe fruit of the plant we collected from the type locality, it was determined that this species was actually Johneria tortusa. P. arenarium subsp. urbani plant samples were collected by us for the first time and morphological, anatomical, DNA sequence analysis and SEM studies were carried out. Molecular (ITS nrDNA sequences), morphological and especially microcarpological data were determined. As a result, in Turkey, P. arenarium subsp. neumayeri is absent. It is true that the P. arenarium subsp urbani species is transferred as Taeniopetalum urbani.

Additions to the Knowledge of the Genus Pezicula (Dermateaceae, Helotiales, Ascomycota) in China.

Simple SummarySpecies of the genus Pezicula are endophytes, phytopathogens, or saprobes, and some of them have biocontrol potential, promoting plant growth and resistance to environmental stress. The species diversity of Pezicula in China was reviewed. Two new species (P. ellipsoids and P. fusispora) and two new Chinese records (P. acericola and P. carpinea) were discovered using morphological and molecular approaches. Descriptions and illustrations of macroscopic and microscopic features were provided for the new and newly found taxa. Pezicula aurantiaca was excluded from the fungal catalogue of China. Together with the previously reported taxa (P. cinnamomea, P. ericae, P. heterochroma, P. magnispora, P. melanigena, P. neosporulosa, P. ocellata, P. rhizophila, P. cf. rubi, and P. subcarnea), 14 Pezicula species are currently known from China. The results provide updated information and improve our understanding of the genus.We describe two new species of Pezicula (Dermateaceae, Ascomycota), P. ellipsoides and P. fusispora, which are discovered in China. Pezicula ellipsoides sp. nov. is distinct in producing 2–3 stipitate apothecia on a basal stroma with a light yellow hymenium, broadly ellipsoid 0–1(–2)-septate ascospores, and divergent DNA sequence data. Pezicula fusispora sp. nov. is characterized by sessile apothecia, 0.3–0.8 mm in diam, a yellowish hymenium, J+ asci 135–170 × 15–21 μm, multiseptate ascospores, 33–48 × 7–10.3 μm, and growing on rotten bamboo. In addition, Pezicula acericola and P. carpinea are reported for the first time from China, and 11 Pezicula species previously reported from China are reviewed and briefly noted, of which one was a misidentified species. Phylogenetic analyses inferred from ITS nrDNA sequences confirm the placement of P. ellipsoides and P. cinnamomea in the genus Pezicula.

Phylogenetic Review of Acaulospora (Diversisporales, Glomeromycota) and the Homoplasic Nature of Its Ornamentations.

The genus Acaulospora has undergone many updates since it was first described; however, there are some missing pieces in the phylogenetic relationships among Acaulospora species. The present review aimed to: (i) understand the evolutionary meaning of their different spore wall ornamentations; (ii) define the best molecular marker for phylogenetic inferences, (iii) address some specific issues concerning the polyphyletic nature of Acaulospora lacunosa and Acaulospora scrobiculata, and the inclusion of Kuklospora species; and (iv) update the global geographical distribution of Acaulospora species. As such, the wall ornamentation of previously described Acaulospora species was reviewed and phylogenetic analyses were carried out based on ITS and SSU-ITS-LSU (nrDNA). Moreover, the already available type material of A. sporocarpia was inspected. According to the data obtained, temperate and tropical zones are the richest in Acaulospora species. We also confirmed that A. sporocarpia does not belong to Acaulospora. Furthermore, our phylogeny supported the monophyly of Acaulospora genus, including the Kuklospora species, K. colombiana and K. kentinensis. The nrDNA phylogeny presented the best resolution and revealed the homoplasic nature of many ornamentations in Acaulospora species, pointing out their unfeasible phylogenetic signal. This review reinforces the urgency of more molecular markers, in addition to the nrDNA sequences, for the definition of a multi-locus phylogeny.

Lecaimmeria pakistanica, a new lichen from Azad Jammu and Kashmir, Pakistan

A new lichen species Lecaimmeria pakistanica K.Habib, R.Zulfiqar & Khalid sp. nov. is described and illustrated from rocks in the temperate forests of the Himalaya of Azad Jammu and Kashmir, Pakistan. This species is characterized by its yellow-brown to brown thallus having areoles 0.4 to 1.5 mm across, branched and anastomosing paraphyses, a tall hymenium, large ascospores 20–32 × 10–16 μm, and no substance detected by thin layer chromatography. All other species of the genus have ascospore dimensions in the range of 14–22 × 5–14 μm. A phylogenetic analysis is provided based on ITS nrDNA sequences, and supports the separation of the novel species. Photographs and a comparative analysis with related species of Lecaimmeria are provided to confirm the status of the species.

New combination in the genus Hohenbuehelia (Pleurotaceae)

We studied the type specimens of Pleurotus komarnitzkyi to clarify its taxonomic status. Unlike other species in this genus which grow on wood, P. komarnitzkyi is ecologically distinct as it grows at the base of woody stems of desert herbaceous plants. In addition, its fruiting bodies are small in size, which is not typical for the Pleurotus species. Substrate preferences and morphology of fruiting bodies of P. komarnitzkyi resemble those of Hohenbuehelia species. Based on an analysis of ITS and LSU nrDNA sequences, we propose a new combination in the genus Hohenbuehelia.

Beyond Nuclear Ribosomal DNA Sequences: Evolution, Taxonomy, and Closest Known Saprobic Relatives of Powdery Mildew Fungi (Erysiphaceae) Inferred From Their First Comprehensive Genome-Scale Phylogenetic Analyses.

Powdery mildew fungi (Erysiphaceae), common obligate biotrophic pathogens of many plants, including important agricultural and horticultural crops, represent a monophyletic lineage within the Ascomycota. Within the Erysiphaceae, molecular phylogenetic relationships and DNA-based species and genera delimitations were up to now mostly based on nuclear ribosomal DNA (nrDNA) phylogenies. This is the first comprehensive genome-scale phylogenetic analysis of this group using 751 single-copy orthologous sequences extracted from 24 selected powdery mildew genomes and 14 additional genomes from Helotiales, the fungal order that includes the Erysiphaceae. Representative genomes of all powdery mildew species with publicly available whole-genome sequencing (WGS) data that were of sufficient quality were included in the analyses. The 24 powdery mildew genomes included in the analysis represented 17 species belonging to eight out of 19 genera recognized within the Erysiphaceae. The epiphytic genera, all but one represented by multiple genomes, belonged each to distinct, well-supported lineages. Three hemiendophytic genera, each represented by a single genome, together formed the hemiendophytic lineage. Out of the 14 other taxa from the Helotiales, Arachnopeziza araneosa, a saprobic species, was the only taxon that grouped together with the 24 genome-sequenced powdery mildew fungi in a monophyletic clade. The close phylogenetic relationship between the Erysiphaceae and Arachnopeziza was revealed earlier by a phylogenomic study of the Leotiomycetes. Further analyses of powdery mildew and Arachnopeziza genomes may discover signatures of the evolutionary processes that have led to obligate biotrophy from a saprobic way of life. A separate phylogeny was produced using the 18S, 5.8S, and 28S nrDNA sequences of the same set of powdery mildew specimens and compared to the genome-scale phylogeny. The nrDNA phylogeny was largely congruent to the phylogeny produced using 751 orthologs. This part of the study has revealed multiple contamination and other quality issues in some powdery mildew genomes. We recommend that the presence of 28S, internal transcribed spacer (ITS), and 18S nrDNA sequences in powdery mildew WGS datasets that are identical to those determined by Sanger sequencing should be used to assess the quality of assemblies, in addition to the commonly used Benchmarking Universal Single-Copy Orthologs (BUSCO) values.

Taxonomy and ecology of Physisporinus forming synnema-like structures in freshwater environments

ABSTRACT Since 2011, we have collected fungi that form synnema-like structures (SSs) bearing many acanthophyses at the apex on water-splashed wood in streams in various regions of Japan. A provisional phylogenetic analysis of strains isolated from SSs based on their nrDNA sequences implied affinity with Physisporinus (Polyporales, Basidiomycota). However, it has not been reported that this genus forms SSs in freshwater habitats. We found a fungus forming not only SSs on the water-boundary part of wood but also resupinate basidiocarps with poroid hymenophores on nonsubmerged parts, and the morphological characteristics of the basidiocarps matched those of Physisporinus. Therefore, we investigated the relationship between SS-forming fungi and their sexual states by taxonomic approaches. Phylogenetic analyses based on nrDNA internal transcribed spacer (ITS) and large subunit (LSU) sequences indicated that SS-forming fungi diverged into five clades in Physisporinus. Each clade was discriminated by the color of SSs, morphology of acanthophyses, and cultural characteristics. Of the five clades, Clade 1, which consisted only of sequences of strains isolated from SSs and basidiocarps produced on rhizomorphs, was closely related to P. eminens and P. undatus, but the morphology of basidiocarps and the manner of basidiocarp development differed. Clade 5 was closely related to P. castanopsidis, P. crocatus, P. pouzarii, P. sanguinolentus, P. subcrocatus, P. tibeticus, and P. vitreus, but the basidiocarp morphology differed. Therefore, Clades 1 and 5 were described as two new species. Regarding Clades 2, 3, and 4, further taxonomic studies with additional specimens are required. SS and acanthophysis formation in wet habitats in streams and in culture could be recognized as new taxonomic and ecological characters of Physisporinus.

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

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

Taxonomic synopsis of Berberis (Berberidaceae) from the northern Hengduan mountains region in China, with descriptions of seven new species

Though Berberis (Berberidaceae) is widely distributed across the Eurasian landmass it is most diverse in the Himalaya–Hengduan Mountain (HHM) region. There are more than 200 species in China where it is one of the most common mountain shrubs. The study on the taxonomy and evolution of Berberis in this region can thus provide an important insight into the origin and diversification of its flora. A prerequisite to this is mapping and describing the various species of Berberis in the region – a task that despite recent progress is by no means complete. It is clear that in China there may be a significant number of species still to be described and that even with published species much about their distribution remains to be discovered. As a contribution to the first of these tasks seven new species from the northern Hengduan Mountain of N. Sichuan and S. Qinghai: Berberis chinduensis, Berberis degexianensis, Berberis jiajinshanensis, Berberis jinwu, Berberis litangensis, Berberis longquensis and Berberis riparia, are described here. Differences in overall morphology and especially in floral structures with each other and with similar species of Berberis in the same region are presented. The report is the result of phylogenetic analyses based on plastome and partial nrDNA sequences of both the seven proposed new species and a significant number of similar species already published. Provisional conclusions as to the insights provides on the history of the genetic divergence are discussed.

Revised Taxonomy of theArctotisAnnual Clade (Arctotideae, Asteraceae) from Southern Africa: Integration of Molecular Phylogenetic and Morphological Evidence

Abstract—Previous phylogenetic analysis of ITS nrDNA sequence data for Arctotidinae species resolved a strongly supported clade containing all but one of the showy annualArctotisspecies (informally designated theArctotisAnnual Clade). In the present study, phylogenetic relationships in theArctotisAnnual Clade were investigated by Bayesian inference and maximum parsimony analyses of cpDNA (trnT-trnL-trnFandtrnH-psbA) and nrDNA (ITS) sequence data. The cpDNA and nrDNA phylogenies were notably incongruent.Arctotis venustaand a putative unnamed species (A.‘sp. B’) were strongly supported as monophyletic by both data sets. The monophyly ofA. leiocarpawas strongly supported by the ITS data set, whereas the remaining accessions formed a poorly resolved complex (the ‘A. fastuosacomplex’). Within theA. fastuosacomplex,A. hirsutawas monophyletic with strong support in the ITS phylogeny. A statistical parsimony-derived cpDNA haplotype network resolved five broad groups of haplotypes and showed no consistent geographical structure, but species-specific haplotype lineages forA. venustaandA.sp. B were resolved.Arctotis fastuosaaccessions were distributed among four haplotype groups. Incongruence between the data sets and poor resolution within theA. fastuosacomplex may reflect reticulate evolution, ancestral polymorphism, and incomplete lineage sorting, in tandem with the low information content of the data sets. The greatest phenotypic diversification in the clade is in cypsela morphology. Comparison of cypsela morphology with the phylogenies suggests a general trend for reduction in the sizes of the cypsela, abaxial wings, pappus scales, and loss of pubescence during diversification. A revised taxonomy, integrating currently available evidence, accompanied by full descriptive accounts and a key to the taxa are presented. Eight species are recognized, including the nomenclatural noveltiesArctotis chrysantha(sp. nov.) andArctotis namibiensis(sp. nov.). The namesArctotis karasmontana,Venidium fugax, andVenidium macrocephalumare lectotypified.

Increasing Taxa Sampling Provides New Insights on the Phylogenetic Relationship Between Eriobotrya and Rhaphiolepis.

Eriobotrya (Rosaceae) is an economically important genus with around 30 species. It is widely distributed in tropical and warm temperate regions of Asia, with most of its species in China, Myanmar, and Vietnam. However, Eriobotrya is often confused with the smaller genus Rhaphiolepis, and the phylogenetic relationships between the two genera are controversial. Here we present phylogenetic analyses of 38 newly generated Eriobotrya and Rhaphiolepis nrDNA together with 16 sequences of nrDNA and 28 sequences of ITS obtained from GenBank, representing 28 species of Eriobotrya and 12 species of Rhaphiolepis, in order to reconstruct highly supported relationships for the two genera. Contrary to previous research based on limited sampling, our results highlight the monophyly of Eriobotrya as well as Rhaphiolepis. The topology recovered here is consistent with key morphological synapomorphies such as the persistent sepals in Eriobotrya. Our findings show that increased sampling of taxa can provide a more robust phylogeny through reducing phylogenetic error and increasing overall phylogenetic accuracy.

Plastome phylogenomics of Allaeanthus, Broussonetia and Malaisia (Dorstenieae, Moraceae) and the origin of B.× kazinoki.

Species of Broussonetia have been essential in the development of papermaking technology. In Japan and Korea, a hybrid between B. monoica and B. papyrifera (= B. × kazinoki) known as kōzo and daknamu is still the major source of raw materials for making traditional paper washi and hanji, respectively. Despite their cultural and practical significance, however, the origin and taxonomy of kōzo and daknamu remain controversial. Additionally, the long-held generic concept of Broussonetia s.l., which included Sect. Allaeanthus and Sect. Broussonetia, was challenged as phylogenetic analyses showed Malaisia is sister to the latter section. To re-examine the taxonomic proposition that recognizes Allaeanthus, Broussonetia, and Malaisia (i.e., Broussonetia alliance), plastome and nuclear ribosomal DNA (nrDNA) sequences of six species of the alliance were assembled. Characterized by the canonical quadripartite structure, genome alignments and contents of the six plastomes (160,121-162,594bp) are highly conserved, except for the pseudogenization and/or loss of the rpl22 gene. Relationships of the Broussonetia alliance are identical between plastome and nrDNA trees, supporting the maintenance of Malaisia and the resurrection of Allaeanthus. The phylogenomic relationships also indicate that the monoecy in B. monoica is a derived state, possibly resulting from hybridization between the dioecious B. kaempferi (♀) and B. papyrifera (♂). Based on the hypervariable ndhF-rpl32 intergenic spacer selected by sliding window analysis, phylogeographic analysis indicates that B. monoica is the sole maternal parent of B. × kazinoki and that daknamu carries multiple haplotypes, while only one haplotype was detected in kōzo. Because hybridizations between B. monoica and B. papyrifera are unidirectional and have occurred rarely in nature, our data suggest that daknamu might have originated via deliberate hybrid breeding selected for making hanji in Korea. On the contrary, kōzo appears to have a single origin and the possibility of a Korean origin cannot be ruled out.

Limacella subtropicana (Amanitaceae, Agaricales), a new species from Pakistan based on morphological and molecular phylogenetic evidences

Basidiomata of a hitherto undescribed Limacella species were collected from the grasslands of Punjab, Pakistan. We describe it as Limacella subtropicana, which is characterized by its viscid, white to creamy pileus, white to the creamy stipe, changing to brown when bruised, subglobose basidiospores measuring 4.0?6.3 × 3.7?5.5 ?m, an ixotrichodermal pileipellis with subcylindric to clavate terminal elements and the common presence of clamp connections in all tissues. Maximum likelihood and Bayesian inference analyses of ITS sequences and LSU sequences of nrDNA confirmed its placement in the genus Limacella. A comprehensive description and a comparison of the new taxon with other Limacella species are provided.

Bergera unifolia (Rutaceae), A New Species from Guangxi (China) Based on Morphological and Molecular Data

A new species, Bergera unifolia C.L. Deng & F.J. Mou (Rutaceae, Aurantioideae, Clauseneae) is described and illustrated from Dahua County, Guangxi Province, China, based on morphological characters as well as on nrDNA (ITS) and cpDNA (trnL-F, atpB-rbcL, rbcL, matK, psbH-petB and psbA-trnH) sequences. It is characterised by unifoliate leaves similar to those of Murraya stenocarpa (Murraya sect. Bergera, or Chalcas sect. Bergera) but differs from it by having smaller and thinner subcoriaceous leaflets with irregularly toothed margins. The leaflets resemble those of M. kwangsiensis (Murraya sect. Bergera) in having hairs under the leaflets, oil glands and similar odour. Based on molecular data, B. unifolia is closely related to M. stenocarpa and M. kwangsiensis and thus it is important for resolving the classification of those species within Murraya sect. Bergera that should be formally transferred to Bergera.

Endophytes from blueberry roots and their antifungal activity and plant growth enhancement effects

Root endophytic fungi that live with plants in a mutualistic fashion could be promising biological agents to improve biotic resistance and plant growth. In this study, 71 endophytic fungi isolates were obtained from blueberry roots, and they were grouped into 2 phyla, 6 classes, 8 orders, and 16 genera based on characterization of morphological features and variances of ITS1, ITS2, and 5.8s nrDNA sequences. Diaporthe and Fusarium were the most abundant genera. Among them, 16 strains exhibited various levels of antagonicstic abilities against five blueberry pathogens including Botrytis cinerea, Phomopsis sp. , Botryosphaeria dothidea, Pestalotiopsis sp., and Fusarium oxysporum . The isolate FLR13, which was classified as Pezicula sp., exhibited the strongest level of antifungal effect (>60%) against these five pathogens and displayed potential biological control effect against Botrytis cinerea in blueberry seedlings. In addition, FLR13 could form typical microsclerotia of dark septate endophyte (DSE) in blueberry root cells to evidently enhance blueberry growth, resulting in the significant increases of 24.13% in height, 88.10% in leaf size, 43.32% in stem diameter, and 54.19% in fresh plant weight as compared with non-FLR13 colonized seedlings. Those results suggest that FLR13 should have potential application in disease control and plant growth enhancement.

Two new psathyrelloid species of Coprinopsis (Agaricales, Psathyrellaceae) from China.

In this study, Coprinopsisjilinensis and Coprinopsispusilla were introduced, based on their morphological characteristics, the internal transcribed spacer (ITS) and large subunit ribosomal (LSU) region sequences of nrDNA. These new psathyrelloid species were found in Jilin Province, China. Coprinopsisjilinensis has brown pileus, utriform pleurocystidia, brown, smooth, dextrinoid basidiospores and tiny pore. It mainly grows on humus. Coprinopsispusilla has small basidiomata, greyish-white pileus, thick and distinct veil at edges, subcolourless and verrucose basidiospores. It is poreless and it grows on the decaying wood of broad-leaved trees. Both of them belong to the C.sect.Melanthinae. A supplementary description of C.sect.Melanthinae was given in combination with the newly-discovered taxa and an identification key to the fourteen psathyrelloid species of Coprinopsis is provided. Coprinopsissect.Canocipes and C.sect.Quartoconatae were evaluated and the phylogenetic position of the psathyrelloid species of Coprinopsis was discussed. Psathyrellasubagraria, as a confusing species, was also discussed in this study.

The genus Heteromera (Compositae, Anthemideae)

A comprehensive treatment of the N African genus Heteromera Pomel (Compositae, Anthemideae) is presented. Morphological, anatomical and karyological features as well as distribution maps are reported. The analysis of nrDNA and cpDNA sequence variation reveals the close relationship of the two species, with H. philaenorum exhibiting little variation and being well separated from a more diverse H. fuscata. The chromosome numbers of H. fuscata and H. philaenorum are reported for the first time. Heteromera philaenorum Maire & Weiller, Chrysanthemum fuscatum f. pubescens Trotter and Chrysanthemum fuscatum f. subcanescens Trotter are lectotypified and H. philaenorum is reported as new for the flora of Algeria and Tunisia. Citation: Vogt R., Wagner F. & Oberprieler C. 2021: The genus Heteromera (Compositae, Anthemideae). – Willdenowia 51: 233–249. Version of record first published online on 17 August 2021 ahead of inclusion in August 2021 issue.

Testing genome skimming for species discrimination in the large and taxonomically difficult genus Rhododendron.

Standard plant DNA barcodes based on 2-3 plastid regions, and nrDNA ITS show variable levels of resolution, and fail to discriminate among species in many plant groups. Genome skimming to recover complete plastid genome sequences and nrDNA arrays has been proposed as a solution to address these resolution limitations. However, few studies have empirically tested what gains are achieved in practice. Of particular interest is whether adding substantially more plastid and nrDNA characters will lead to an increase in discriminatory power, or whether the resolution limitations of standard plant barcodes are fundamentally due to plastid genomes and nrDNA not tracking species boundaries. To address this, we used genome skimming to recover near-complete plastid genomes and nuclear ribosomal DNA from Rhododendron species and compared discrimination success with standard plant barcodes. We sampled 218 individuals representing 145species of this species-rich and taxonomically difficult genus, focusing on the global biodiversity hotspots of the Himalaya-Hengduan Mountains. Only 33% of species were distinguished using ITS+matK+rbcL+trnH-psbA. In contrast, 55% of species were distinguished using plastid genome and nrDNA sequences. The vast majority of this increase is due to the additional plastid characters. Thus, despite previous studies showing an asymptote in discrimination success beyond 3-4 plastid regions, these results show that a demonstrable increase in discriminatory power is possible with extensive plastid genome data. However, despite these gains, many species remain unresolved, and these results also reinforce the need to access multiple unlinked nuclear loci to obtain transformative gains in species discrimination in plants.