Morphological and Molecular Identification of New Puccinia Species from Qinghai, China
1
College of Agriculture and Animal Husbandry, Qinghai University, Xining 810016, China
2
Datong County Forestry Station, Xining 810199, China
3
Academy of Agriculture and Forestry Sciences, Qinghai University, Xining 810016, China
4
Huzhu County Agriculture, Rural Affairs, and Science & Technology Bureau, Haidong 810599, China
5
Hualong County Forestry and Grassland Bureau, Haidong 810900, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Diversity 2026, 18(8), 487; https://doi.org/10.3390/d18080487
Submission received: 7 July 2026
/
Revised: 8 August 2026
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Accepted: 12 August 2026
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Published: 14 August 2026
(This article belongs to the Special Issue Fungal Diversity—2nd Edition)
Abstract
Rust fungi of the genus Puccinia are important plant pathogens responsible for substantial economic losses in agriculture and forestry. During field surveys conducted in Qinghai Province, China, a novel rust species, Puccinia ligulari-przewalskii, was discovered parasitizing Ligularia przewalskii. Morphological characteristics of its teliospores were examined using light microscopy (LM) and scanning electron microscopy (SEM). Molecular phylogenetic analysis based on the concatenated internal transcribed spacer (ITS) and large subunit (LSU) ribosomal DNA sequences supported its taxonomic distinction as a new species within the genus Puccinia. Phylogenetic reconstructions inferred from maximum likelihood and Bayesian inference showed that the new species forms a distinct, well-supported lineage, clustering as sister to a clade comprising Puccinia argentata and Puccinia komarovii. P. ligulari-przewalskii is characterized by two-celled, spindle-shaped teliospores exhibiting sparse verrucose ornamentation on the spore wall. Only the telial stage was observed across multi-year surveys, and the complete life cycle remains to be determined. This study contributes to the taxonomic knowledge of rust fungi in China and underscores the ecological and phytopathological significance of rust fungi in the Qinghai–Tibet Plateau region.
1. Introduction
Approximately 8400 species of rust fungi have been identified worldwide to date, classified into 24 families and 160 genera. Many of these species cause significant economic losses to agricultural or forestry crops [1,2,3,4]. More than half of these species belong to the genus Puccinia, which is characterized by two-celled teliospores [2].
Rust fungi exhibit highly diverse life cycle patterns. The macrocyclic life cycle produces all five spore stages—spermogonia (0), aecia (I), uredinia (II), telia (III), and basidia (IV)—and can be completed on a single host (autoecious) or on two phylogenetically unrelated hosts (heteroecious) [2]. Derived, reduced life cycles include the demicyclic type (uredinial stage omitted), the hemicyclic type (spermogonial and aecial stages absent), and the microcyclic type, in which the life cycle is reduced to the telial and basidial stages, with or without spermogonia [2,5,6]. Microcyclic rusts do not produce uredinia or aecia and have no known alternate hosts [7,8]. A significantly higher proportion of microcyclic species occurs in arcto-alpine and high-elevation environments compared with temperate regions—a pattern attributed to abbreviated growing seasons that favor the elimination of spore stages requiring two hosts or multiple infection cycles [6,9].
The identification of Puccinia species is primarily based on morphological characteristics, such as teliospore shape, size, surface ornamentation, germ pore position, presence or absence of pore caps, cell wall thickness, pedicel length, and the germ pore position of urediniospores, combined with information on host occurrence and host relationships [2,10,11,12,13,14,15,16]. In recent years, DNA-based phylogenetic approaches, particularly those employing the internal transcribed spacer (ITS) and large subunit (LSU) ribosomal DNA regions, have become essential for precise species delimitation in Puccinia [4,17,18,19].
Globally, more than 5000 species of the genus Puccinia have been documented, with approximately 520 species and varieties reported in China [4,12,13,20,21,22,23,24,25,26,27]. Puccinia species parasitize plants extensively from the families Asteraceae, Cyperaceae, Liliaceae, and Poaceae in China [12,13], causing diseases that significantly affect plant growth and development. Such diseases reduce the yields of economically important crops and have profound impacts on forest ecosystem functionality, warranting increased attention and research. Approximately eleven rust taxa have previously been recorded on Ligularia (Asteraceae) worldwide, including Aecidium nikkense, Aecidium ligulariae, Coleosporium ligulariae, Coleosporium saussureae, Coleosporium senecionis, Puccinia cnici-oleracei, Puccinia eriophori, Puccinia expansa, Puccinia glomerata, Puccinia ligulariae, and Uromyces ligulariae [3,13,15,28,29,30,31]. Among these, only P. cnici-oleracei has been characterized by molecular phylogenetic data. P. ligulariae is listed only by name and host association in the Chinese taxonomic literature, without accompanying morphological descriptions [15,30]. P. eriophori is the teleomorph of A. ligulariae [32,33]; however, its uredinial and telial stages have never been recorded in China, despite the wide distribution of its reported telial host, Eriophorum (Cyperaceae), in the northern part of the country [32]. None of the previously recorded rust taxa on Ligularia have been supported by both morphological and molecular phylogenetic evidence in combination.
During a survey of rust diseases in Qinghai Province, a novel species of Puccinia parasitizing Ligularia przewalskii (Maxim.) Diels was identified and initially recorded in a floristic checklist of the Sanjiangyuan region [34]. Prior to this study, no Puccinia species on Ligularia had been formally described with supporting molecular phylogenetic data. In this study, its morphological characteristics and molecular data were analyzed, its phylogenetic relationships with related Puccinia species were evaluated, and its taxonomic novelty was confirmed. This paper provides a detailed description and comprehensive analysis of this newly discovered rust fungus.
2. Materials and Methods
2.1. Specimens
The specimens analyzed in this study were collected in 2023 from forested areas along the Mako River, Guoluo Tibetan Autonomous Prefecture, Qinghai Province, China (Table 1). These specimens are deposited in the Plant Pathology Herbarium of Qinghai University under voucher numbers QHU2023090 and QHU2023092, respectively, and were used for subsequent morphological observation and phylogenetic analysis. Two additional collections from the same locality (QHU2021116, QHU2022139) were previously examined under a light microscope; only telia were observed in these collections, and teliospore morphology was consistent with that of the holotype.
2.2. Morphological Observations
The morphological classification system used in this study follows the 14-family classification framework outlined in the 10th edition of the Dictionary of Fungi [35]. Morphological characteristics were primarily described with reference to the Fungi Identification Manual [36] and Chinese Fungi Flora [12,13,14,15,16], along with other relevant books and literature [20,35,37]. The morphological features of teliospores, including type, shape, attachment position, and color of spore clusters, were examined using a Nikon SMZ800N stereomicroscope (DM; Nikon Corporation, Tokyo, Japan), an optical microscope (LM; Olympus, Tokyo, Japan), and a scanning electron microscope (SEM; Hitachi, Tokyo, Japan). Fifty teliospores were randomly selected for measurements of their length, width, and wall thickness. The surface ornamentation and ultrastructure of teliospores were observed using a Hitachi S-4800 scanning electron microscope (Hitachi, Tokyo, Japan) operating at 15 kV. The measurements obtained were compared with the descriptions of related species in published literature.
2.3. DNA Extraction, PCR Amplification, and Sequencing
DNA extraction was performed following the methods of Tian et al. [38] and Yang [39]. Approximately 100–200 rust fungus spores were placed between two slides, and a few drops of DNA extraction buffer were added. The spores were then thoroughly ground to disrupt the cell walls. The presence of a slurry, observed under an optical microscope, indicated successful cell lysis. The slides were then carefully separated, and DNA was extracted. The primer pairs ITS5-u (5′-CAAGGTTTCTGTAGGTG-3′) [40] and ITS4rust (5′-CAGATTACAAATTTGGGCT-3′) [41] and NL1(5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4(5′-GGTCCGTGTTTCAAGACGG-3′) [42], were used to amplify the ITS and LSU regions, respectively. Polymerase chain reaction (PCR) amplifications were performed in a total volume of 25 µL. The PCR protocol for ITS was as follows: initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 48 °C for 40 s, extension at 72 °C for 60 s, and a final extension at 72 °C for 10 min. The PCR protocol for LSU was as follows: initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 40 s, extension at 72 °C for 60 s, and a final extension at 72 °C for 10 min. Amplification products were separated by electrophoresis on a 1.2% agarose gel in 1× TAE buffer at 120 V for 40–50 min, and visualized under UV light after staining with ethidium bromide or GelRed. The PCR products with bright and distinct single bands were sequenced by Sangon Biotech (Shanghai, China) Co., Ltd.
2.4. Phylogenetic Analyses
The newly generated sequences were subjected to BLASTn searches (https://blast.ncbi.nlm.nih.gov (accessed on 11 March 2024 and 25 June 2026)) against the NCBI database. Sequences of closely related taxa were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/genbank) (Table 2). The newly generated sequences were assigned corresponding GenBank accession numbers. The final combined dataset contained a total of 102 taxa, including 100 ingroup strains of Puccinia and two strains of Austropuccinia psidii designated as outgroup taxa. Alignments were constructed separately for each gene fragment using Multiple Alignment using Fast Fourier Transform (MAFFT) version 7 [43], and manually refined and trimmed in BioEdit version 7.2.5 [44]. The two aligned locus matrices were concatenated into a combined dataset using PhyloSuite v2 [45]. The best-fit nucleotide substitution models for each partition were selected by the built-in ModelFinder under the Bayesian Information Criterion (BIC). Phylogenetic trees were reconstructed using both maximum likelihood (ML) and Bayesian inference (BI). ML analysis was performed in IQ-TREE v3.0.1 [46,47], with branch support assessed via 1000 ultrafast bootstrap replicates. BI analysis was conducted with MrBayes v3.2.7 [48] using two Markov chain Monte Carlo (MCMC) runs of 2 million generations each, sampling every 200 generations, with the first 25% discarded as burn-in. Taxon names, GenBank accession numbers, and collection localities are indicated on the tree. Branch support values are presented as ML ultrafast bootstrap support (BS) followed by Bayesian posterior probability (PP) on the final phylogenetic tree.
2.5. Results
Molecular Phylogeny
The combined sequence data of LSU and ITS comprised 104 strains of Puccinia and two strains of A. psidii as outgroup taxa (Figure 1). A total of 1540 characters, including gaps, were obtained for the phylogenetic analysis. The IQ-TREE analysis of the combined dataset yielded a best-scoring tree with a final ML optimization likelihood value of −8803.859. The matrix had 720 distinct alignment patterns, containing 1041 constant sites, 285 parsimony-informative sites and 214 singleton variable sites; 3 sites contained only gaps or ambiguous characters. The estimated base frequencies were as follows: A = 0.328, C = 0.151, G = 0.232, T = 0.289; substitution rates AC = 1.52670, AG = 3.32150, AT = 1.20335, CG = 0.77246, CT = 5.02495, GT = 1.00000; the proportion of invariable sites was 0.352, and the gamma distribution shape parameter α was 0.528. The topologies recovered by ML and Bayesian inference were highly congruent. Our new isolates of Puccinia ligulari-przewalskii (QHU2023090T and QHU2023092) formed a distinct monophyletic clade with 100% ML and 1.00 BYPP values, and showed a close phylogenetic affinity with Puccinia argentata (strain GC 4X) and Puccinia komarovii (strains Pk23DPN1 and Pk23DPN11) (Figure 1).
2.6. Taxonomy
Puccinia ligulari-przewalskii L.C. Bai & Q. Xu, sp. nov.
Holotype: CHINA. QINGHAI PROVINCE: Makehe District, Guoluo Tibetan Autonomous Prefecture (100°57′33″ E, 32°40′54″ N; 3368.5 ma.s.l.), on Ligularia przewalskii, 25 Aug 2023, Q. Xu, QHU2023090. GenBank: ITS = PZ575731; LSU = PP469520. Other spore types, except for the teliospore type, have not been found.
Etymology: The epithet ligulari-przewalskii refers to the host plant Ligularia przewalskii, from which the rust fungus was isolated.
Description: Spermogonia, aecia and uredinia: Unknown.
Telia: Present on the underside of leaves, aggregated, round to elliptical, 0.1–0.5 mm in diameter, chestnut-brown, initially covered by the host plant’s epidermis but later rupturing and exposed.
Teliospores: Elliptical, spindle-shaped, or polygonal, two-celled, slightly constricted at the septum, measuring 30.7–44.8 × 17.4–25.3 µm, chestnut-brown, with lateral walls 1.4–2.3 µm thick; papillae 1.6–4.4 µm long, yellow, with apical germ pores; spore surface relatively smooth with sparse warts. (Figure 2).
Ecology and distribution: III (teliospores) on L. przewalskii, known only from Qinghai in China.
Notes: L. przewalskii is a perennial herbaceous plant belonging to the genus Ligularia in the Asteraceae family. It is mainly distributed in the eastern part and marginal areas of the Qinghai–Tibet Plateau. In traditional Tibetan medicine, the entire herb of L. przewalskii can be used as medicine, which has the effects of clearing away heat and toxic substances, dispersing blood stasis and reducing swelling. It is commonly used to treat sore throat, carbuncles and sores, bruises and other diseases. Mashing its fresh product for external application has a significant relieving effect on the redness, swelling, heat and pain in the early stage of sores.
3. Discussion
A new rust species, P. ligulari-przewalskii sp. nov., is described in this study based on both morphological and molecular phylogenetic evidence. It represents the first record of a Puccinia species occurring on Ligularia (Asteraceae) in China to be characterized by both morphological and molecular phylogenetic data. Prior to this discovery, eleven rust taxa had been documented on Ligularia spp.: A. nikkense (on Ligularia stenocephala in China and Japan) [3,28], A. ligulariae (reported from Finland and China without specified host species) [29,31], C. ligulariae (infecting Ligularia jaluensis, Ligularia przewalskii and other species in China, Finland and Romania) [3,13,30], C. saussureae (occurring on Ligularia dentata, L. stenocephala and other hosts, with distribution reflecting host range) [3,13], and C. senecionis (found on Ligularia gigantea and Ligularia macrophylla across multiple regions) [3], P. cnici-oleracei (recorded on Ligularia tsangchanensis and other Asteraceae) [13,15], P. eriophori (reported on Ligularia speciosa and other Ligularia spp. in China) [15,30], P. expansa (recorded on Ligularia sibirica and Ligularia spp. in China) [15,30], P. glomerata (occurring on L. achyrotricha and other Ligularia spp.) [3,15], P. ligulariae (recorded on Ligularia spp. in China) [30], and U. ligulariae (on Ligularia tussilaginea var. formosana in Taiwan) [3].
A. nikkense was originally described from Japan on Ligularia stenocephala var. comosa [28] and has since been recorded on L. stenocephala in Hubei (Shennongjia), China, and on Farfugium japonicum in Japan [3,32]. The Flora Fungorum Sinicorum Vol. 69 treats it as an independent anamorphic species known only from its aecial stage; no telial connection has been established, and no ITS or LSU sequence data are available [32]. It may be noted that the Catalogue of Life China (2023) lists A. nikkense as a synonym of Uromyces veratri (DC.) J. Schröt. [49]; however, the Flora Fungorum Sinicorum Vol. 25 records Aecidium cacaliae Thüm., not A. nikkense, as the aecial stage of U. veratri [14]. The accuracy of this synonymy therefore remains to be verified.
A. ligulariae was originally described from Finland [29]. Tranzschel [50] identified this fungus as the aecial stage of P. eriophori Thüm., and this treatment was subsequently adopted by Tai [30] and Zhuang [33]. Zhuang (1988) listed A. ligulariae as the aecial synonym of P. eriophori and recorded the uredinial and telial stages on Eriophorum (Cyperaceae) [33]. The aeciospores of A. ligulariae recorded in the Chinese rust flora (18–25 × 15–20 µm, with densely and finely verrucose walls) [32] are morphologically consistent with those of Puccinia eriophori var. eriophori described from North America by Savile (1972) (16–24.5 × 12.5–21 µm, with similarly densely and finely verrucose walls) [51]. The species has been recorded on multiple Ligularia species across northern China, including Qinghai Province [32]. Although Eriophorum spp. (Cyperaceae)—the host of P. eriophori recorded—are widely distributed in northern China, the uredinial and telial stages have never been observed on these plants in the country [32]. Savile (1972) described the telial stage of P. eriophori var. eriophori in detail based on North American and European material on Eriophorum [51]: the teliospores are smooth, light yellow-brown to light chestnut, (34–)37–71(–83) × (14–)16.5–28(–29.5) µm, with a rounded or conic apical cap concolorous or paler, and pedicels moderately firm, 48–75 µm long. By contrast, the teliospores of P. ligulari-przewalskii are sparsely verrucose, distinctly smaller (30.7–44.8 × 17.4–25.3 µm), bear a prominent yellow apical papilla 1.6–4.4 µm, and have pedicels that are readily detached (Table 3).
In summary, A. nikkense remains an anamorphic name whose telial connection is uncertain and disputed across authoritative sources. A. ligulariae is recognized as the aecial stage of P. eriophori, a rust for which Eriophorum (Cyperaceae) is recorded as the host in the Chinese rust flora; however, the uredinial and telial stages of P. eriophori on Eriophorum (Cyperaceae) have never been recorded in China. The teliospores of P. ligulari-przewalskii are morphologically distinct from those of P. eriophori described by Savile (1972) [51] (Table 3). The available evidence does not support a connection between the uredinial or telial stages of A. nikkense or A. ligulariae and the telial material on L. przewalskii described here. P. ligulari-przewalskii is therefore proposed as a new species on the basis of the morphological and molecular data presented. The ITS and LSU sequences, voucher specimens, and morphological documentation from this study are available for comparison should the teleomorph of A. nikkense or A. ligulariae be identified.
The three Coleosporium species recorded on Ligularia—C. ligulariae, C. saussureae, and C. senecionis—differ fundamentally from Puccinia in their teliospore morphology: Coleosporium teliospores are one-celled, cylindrical, thin-walled, colorless, and formed in a single-layered crust, germinating without dormancy by producing an external basidium [3,13]. In contrast, Puccinia teliospores are two-celled, thick-walled, pigmented, pedicellate, and germinate via apical germ pores [2]. The three Coleosporium species and U. ligulariae are readily distinguished from Puccinia by their one-celled teliospores [3,13], in contrast to the two-celled teliospores of P. ligulari-przewalskii. Unlike the aforementioned Uromyces and Coleosporium species having one-celled teliospores, P. ligulari-przewalskii is assigned to Puccinia, characterized by its two-celled teliospores.
Phylogenetic analysis based on the concatenated ITS and LSU sequences places P. ligulari-przewalskii within the genus Puccinia, clustering as a sister lineage to P. argentata and P. komarovii, further corroborating its generic placement. The teliospores of P. argentata are smooth or very finely obscurely verruculose, ellipsoid or oblong, measuring 25–40 × 14–21 µm, with a distinct hyaline pore cap 3–5 µm and an apex that is not thickened [13]. P. ligulari-przewalskii produces sparsely verrucose teliospores that are ellipsoid, fusiform or polygonal, 30.7–44.8 × 17.4–25.3 µm, with a yellow apical papilla 1.6–4.4 µm (Figure 2; Table 3). The teliospores of P. komarovii are smooth or nearly smooth, subrectangular or obovoid, 25–40 × 16–23 µm, with a uniform wall thickness of approximately 2.0 µm and a distinct hyaline pore cap [13]. P. ligulari-przewalskii differs in its sparsely verrucose ornamentation, ellipsoid to fusiform shape, and the presence of a yellow apical papilla. Furthermore, P. argentata and P. komarovii both occur on Impatiens (Balsaminaceae) [13], whereas the new species parasitizes Ligularia (Asteraceae).
Among the morphologically compared species, Puccinia gentianae merits particular attention. It is widely distributed in the North Temperate Zone, including the Qinghai–Tibet Plateau region, where it parasitizes various Gentiana species (Gentianaceae) [13]. In the Sanjiangyuan region, P. gentianae has been recorded on Gentiana straminea [34], indicating that it co-occurs with P. ligulari-przewalskii in the same geographic area. P. gentianae has a macrocyclic life cycle, producing spermogonia, aecia, uredinia, and telia [13]. The teliospores of P. gentianae are smooth and broadly ellipsoid to oblong, measuring 28–43 × 20–30 µm, with uniformly thickened lateral walls (2.0–3.0 µm) and a colorless small pore cap at the apical germ pore [13]. In contrast, P. ligulari-przewalskii has sparsely verrucose, ellipsoid to fusiform teliospores (30.7–44.8 × 17.4–25.3 µm), with thinner lateral walls (1.4–2.3 µm) and a distinct yellow apical papilla (1.6–4.4 µm) (Figure 2; Table 3). The host genera Gentiana (Gentianaceae) and Ligularia (Asteraceae) are phylogenetically distant, further corroborating the distinction between these two species.
Among the Puccinia species previously recorded on Ligularia, Puccinia glomerata is the most comparable taxon [3,15]. Its teliospores are smooth, ellipsoid or irregularly obovoid, 33–53 × 15–28 µm, with a hyaline pore cap 3–5 µm [13]. The new species produces smaller, sparsely verrucose teliospores with a yellow papilla. Moreover, P. glomerata produces two teliospore morphs, small and large, according to Cummins [52], whereas P. ligulari-przewalskii produces a single teliospore type. P. cnici-oleracei, recorded on L. tsangchanensis [15], has smooth, clavate or oblong-ellipsoid teliospores measuring 33–58 × 13–20 µm, with a considerably thickened apical wall of 3–13 µm [13], clearly distinct from the sparsely verrucose, papilla-bearing spores of the new species. P. expansa, also recorded on Ligularia [15,30], is morphologically similar to P. glomerata and has been treated as distinct by several authors [53]; however, both P. expansa and P. glomerata lack molecular characterization and differ from the new species in teliospore ornamentation and pore structure.
P. ligulari-przewalskii is characterized by a unique combination of sparsely verrucose teliospores, a yellow apical papilla 1.6–4.4 µm, and the absence of a hyaline pore cap. This combination distinguishes it from all other Puccinia species compared in this study (Table 3). Among these, Puccinia cremanthodii has reticulate-rugose spores; Puccinia hieracii, although finely verrucose and similarly lacking a pore cap, has more broadly ellipsoid spores with the lower cell germ pore positioned away from the septum; Puccinia minussensis has verrucose spores with a distinct hyaline pore cap; Puccinia violae is densely verrucose and occurs on Violaceae; P. senecionis and Puccinia artemisiae-keiskeanae both have smooth teliospores; and Puccinia ferruginosa, shown to be polyphyletic by Engkhaninun et al. [54], has smooth, elongate or clavate spores with a thickened apex [55].
Only the telial stage was observed in the specimens of P. ligulari-przewalskii examined in this study. Specimens were collected from L. przewalskii at the type locality on multiple occasions between 2021 and 2023, with collection efforts concentrated in July through September, corresponding to the flowering and fruiting period of the host [56]. No spermogonia, aecia, or uredinia were detected in any collection. However, because the surveys did not span the entire growing season—particularly the early season (May–June) when leaf emergence and the development of spermogonial and aecial stages of rust fungi typically occur—the possibility that additional spore stages are present during unsampled periods cannot be excluded.
Several lines of evidence suggest that P. ligulari-przewalskii may not be microcyclic. First, its closest phylogenetic relatives, P. argentata and P. komarovii, are both non-microcyclic: P. argentata is macrocyclic and heteroecious, producing spermogonia and aecia on Adoxa moschatellina and uredinia and telia on Impatiens species [13], while P. komarovii produces uredinia and telia on Impatiens [13]. The derivation of a microcyclic species from within this non-microcyclic lineage would represent an evolutionary exception, though not an impossibility. Second, microcyclic Puccinia species are rare on Asteraceae. Among the more than 1000 rust species reported on this family, the vast majority are macrocyclic or demicyclic [6], and only a handful of microcyclic taxa have been documented, e.g., P. cremanthodii on Cremanthodium at elevations above 4000 m on the Qinghai–Tibet Plateau [13]. P. glomerata, previously considered microcyclic on Ligularia [3,15], has recently been shown to occasionally produce urediniospores [13]. Third, collections were made during the flowering and fruiting phase of the host [56], which is suboptimal for detecting spermogonial and aecial stages that typically develop on newly emerged leaves in early summer.
Nevertheless, the type locality lies in a high-elevation alpine environment (3370 m) on the Qinghai–Tibet Plateau, where abbreviated growing seasons are known to favor life cycle reduction. Microcyclic rusts are disproportionately represented in arcto-alpine floras, a pattern attributed to the selective advantage of completing the life cycle within a short thermal window without reliance on an alternate host [6,9]. Whether P. ligulari-przewalskii is microcyclic, demicyclic, or macrocyclic remains an open question. Full-season phenological monitoring from leaf emergence (June) through senescence (September), combined with basidiospore inoculation experiments on healthy L. przewalskii seedlings, would be required to definitively establish the life cycle type.
Collectively, both morphological and molecular evidence support the recognition of P. ligulari-przewalskii as a species new to science, and it represents the first Puccinia species occurring on Ligularia (Asteraceae) in China to be characterized by both morphological and molecular phylogenetic data.
Author Contributions
Conceptualization, L.B. and Y.Z.; Methodology, Q.X. and L.Q.; Sample collection, L.B., Q.X. and W.Z.; Writing—Original Draft Preparation, Q.X. and L.Q.; Writing—Review & Editing, L.B. and Y.C. All authors have read and agreed to the published version of the manuscript.
Funding
This paper was supported by Qinghai Province Key Research and Development and Transformation Plan (2023-SF-119).
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.
Acknowledgments
We thank the Qinghai Provincial Department of Science & Technology for technical guidance and Sanjiangyuan National Administration for ecological stewardship.
Conflicts of Interest
The authors declare no competing interests.
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Figure 1.
Phylogram generated from ML analysis based on the combined LSU and ITS of Puccinia species. Bootstrap support values for ML equal to or higher than 65% and BYPP equal to or greater than 0.90 are shown above the nodes. The new species are highlighted in red font. The tree is rooted with A. psidii (APG1 and PUCPY02). The scale bar represents 0.02 nucleotide substitutions per site.
Figure 1.
Phylogram generated from ML analysis based on the combined LSU and ITS of Puccinia species. Bootstrap support values for ML equal to or higher than 65% and BYPP equal to or greater than 0.90 are shown above the nodes. The new species are highlighted in red font. The tree is rooted with A. psidii (APG1 and PUCPY02). The scale bar represents 0.02 nucleotide substitutions per site.
Figure 2.
Morphological characteristics of P. ligulari-przewalskii on L. przewalskii. (A,B) Host plant habitat. (C) Telia. (D) Teliospores (LM). (E) Teliospores (SEM).
Figure 2.
Morphological characteristics of P. ligulari-przewalskii on L. przewalskii. (A,B) Host plant habitat. (C) Telia. (D) Teliospores (LM). (E) Teliospores (SEM).
Table 1.
Herbarium specimens used for morphological examination and molecular phylogenetic analyses.
Table 1.
Herbarium specimens used for morphological examination and molecular phylogenetic analyses.
| Host Plants | Locality | Time | Elevation/m | Longitude & Latitude | Specimen No. | Mycobank No. |
|---|---|---|---|---|---|---|
| Ligularia przewalskii | Makehe | 25 August 2023 | 3368.5 | 100°57′33″ E 32°40′54″ N | QHU2023090 (Holotype) QHU2023092 (Isotype) | 860240 |
Table 2.
Sequence data of Puccinia species from GenBank and used for phylogenetic analyses.
| Taxon Name | Strain Number | ITS | LSU | Host | Country |
|---|---|---|---|---|---|
| Austropuccinia psidii | PUCPY02 | MH513948 | MH513948 | Eucalyptus benthamii | Paraguay |
| Austropuccinia psidii | APG1 | MN238767 | MN238767 | Guava | Brazil |
| Puccinia achnatheri-sibirici | HMJAU8663 | MW404809 | MW404969 | Deyeuxia purpurea | China |
| Puccinia acroptili | U-733 | JN204196 | JN204196 | Rhaponticum repens | Kazakhstan |
| Puccinia adenocauli | HMJAU8276 | MW447796 | MW414403 | Adenocaulon himalaicum | China |
| Puccinia adenocauli | HMJAU8628 | MK785270 | MK785295 | Adenocaulon himalaicum | China |
| Puccinia aff. verbesinae | DAOM 115974 | MW009569 | MW009569 | Verbesina sp. | Mexico |
| Puccinia albispora | TSH R11044 | MT560796 | MT560813 | South Africa | |
| Puccinia allii | HSZ0340 | AF511087 | AF511087 | Allium | USA |
| Puccinia amari | BPI 064435T | KX190836 | KX190836 | Panicum amarum | USA |
| Puccinia ampliaticoronata | HMJAU8736T | MW404765 | MW404925 | Melica grandiflora | China |
| Puccinia andropogonis | HSZ0264 | DQ344519 | DQ344519 | Andropogon spp. | USA |
| Puccinia andropogonis var. onobrychidis | Ba aecia | EF583818 | EF583818 | Baptisia australis | USA |
| Puccinia argentata | GC-4X | PP273009 | PP266821 | China | |
| Puccinia artemisiae-keiskeanae | TSH-R4183 | AB188133 | AB190893 | Artemisia keiskeana | Japan |
| Puccinia atrofusca | DAOM:151011 | MW009474 | MW009474 | Carex rossii | Canada |
| Puccinia bartholomaei | Av aecia | EF583820 | EF583820 | Asclepias viridis | USA |
| Puccinia brachypodii | DAOM:984862 | MT965646 | MT965646 | Brachypodium sylvaticum | UK |
| Puccinia calcitrapae var. centaureae | U-209 | JN204183 | JN204183 | Centaurea calcitrapa | Turkey |
| Puccinia canaliculata | U-677 | HQ412647 | HQ412647 | Cyperus rotundus | Oman |
| Puccinia canaliculata var. tenuis | TA430 | OL437018 | OL437033 | Cyperus esculentus | Benin |
| Puccinia caricina | PDD:98313 | KX985750 | KX985750 | Carex geminata | New Zealand |
| Puccinia caricis-hebeiensis | HMJAU8895T | MW447818 | MW414427 | Carex lanceolata | China |
| Puccinia caricis-jilinensis | HMJAU8842T | MW447898 | MW414367 | Carex siderosticta | China |
| Puccinia caricis-lactucae | HMJAU8899T | MW447778 | NG_228873 | Lactuca sp. | China |
| Puccinia caricis-pediformis | HMJAU8836T | MW447855 | MW414323 | Carex pediformis | China |
| Puccinia caricis-rafaensis | HMJAU8798T | MW447892 | MW414360 | Carex sp. | China |
| Puccinia caricis-ribicola | HMJAU8871 | MW447805 | MW414413 | Carex dispalata | China |
| Puccinia caricis-shepherdiae | DAOM:181319 | MW009426 | MW009426 | Shepherdia canadensis | Canada |
| Puccinia caricis-stipatae | 10451 | AB188137 | AB190887 | Carex stipata | Japan |
| Puccinia caricis-tenuiformis | HMJAU8851T | MW447858 | MW414326 | Carex sp. | China |
| Puccinia carthami | DAOM:134964 | MW009544 | MW009544 | Carthamus tinctorius | Canada |
| Puccinia caulophylli | HMJAU8621 | MK785279 | MK785304 | Milium effusum | China |
| Puccinia cerinthes-agropyrina | HMJAU8687 | MW404828 | MW404988 | Clematis terniflora | China |
| Puccinia cf. allii | BRIP 61618 | KU296870 | KU296870 | Allium sativum | Australia |
| Puccinia chrysanthemi | CA1 | EU816926 | EU816926 | Chrysanthemum | USA |
| Puccinia clavata | PDD:74903 | KX985761 | KX985761 | Clematis foetida | New Zealand |
| Puccinia cnici-oleracei | BPI 879277 | GU058017 | GU058017 | Aster sp. | Mexico |
| Puccinia coleataeniae | BPI 006819T | KX190843 | KX190843 | Coleataenia anceps | USA |
| Puccinia coronata f. sp. avenae | 93MN437 | AY114290 | AY114290 | Avenae | USA |
| Puccinia coronata var. coronata | QHU2023068 | PP471872.1 | PP469628.1 | Clematis sp. | China |
| Puccinia coronati-hordei | HMJAU8724 | MW404779 | MW404939 | Poa sp. | China |
| Puccinia crotonopsidis | BPI 006810T | KX190844 | KX190844 | Croton michauxii | USA |
| Puccinia cumminsii | DAOM 114236T | KX190845 | KX190845 | Panicum sp. | USA |
| Puccinia cyperi | BRIP 60997 | KU296885 | KU296885 | Cyperus iria | Australia |
| Puccinia digitaticoronata | HMJAU8782T | MW404711 | MW404871 | Poa sp. | China |
| Puccinia dioicae | BPI 879279 | GU058019 | GU058019 | Oenothera fruticosa | USA |
| Puccinia dioicae var. micropuncta | TSH:R16540 | AB188129 | LC626796 | Carex breviculmis | Japan |
| Puccinia dulichii | DAOM:107660 | MW009481 | MW009481 | Dulichium arundinaceum | USA |
| Puccinia eleganticoronata | HMJAU8792T | MW404723 | MW404883 | Poa sphondylodes | China |
| Puccinia elymi | HMJAU8694 | MW404827 | MW404987 | Poaceae | China |
| Puccinia elymi-albispora | HMJAU8678T | MW404819 | MW404979 | Elymus ciliaris | China |
| Puccinia emaculata | BPI 851570T | KX190848 | KX190848 | Panicum capillare | USA |
| Puccinia epigejos | HMJAU8738 | MW404769 | MW404929 | Calamagrostis epigeios | China |
| Puccinia esclavensis | BPI 893096 | KX190866 | KX190866 | Mirabilis melanotricha | Mexico |
| Puccinia ferruginosa | IBA7553 | AB193001 | AB190901 | Artemisia freyniana | Japan |
| Puccinia festucae | HMJAU8706 | MW404786 | MW404946 | Festuca extremiorientalis | China |
| Puccinia fumosa | BPI 910240 | KY764123 | KY764123 | Loeselia mexicana | USA |
| Puccinia fumosa | BPI 910239 | KY764122 | KY764122 | Loeselia mexicana | USA |
| Puccinia graminicola | BPI 006909T | KX190868 | KX190868 | Panicum virgatum | USA |
| Puccinia graminis | PG1 | AY874153 | MK952782 | Triticum aestivum | USA |
| Puccinia haemodori | BRIP:57767 | KF690675 | KF690693 | Anigozanthos sp. | Australia |
| Puccinia heeringiana | POLL_0060000615 | OR568547 | OR568547 | Tanacetum parthenium | Germany |
| Puccinia helianthin | BPI 880747 | KY764127 | KY764127 | Helianthus annuus | USA |
| Puccinia hemerocallidis | BRIP:53476 | KM249855 | KM249855 | Hemerocallis sp. | Australia |
| Puccinia hieracii | ZP-R501 | MK518930 | MK518610 | China | |
| Puccinia hordei | BPI 910245 | KY764129 | KY764129 | Ornithogalum sp. | USA |
| Puccinia horiana | BPI 910316 | KY798398 | KY798398 | Chrysanthemum x morifolium | USA |
| Puccinia jaceae | OL 33481 | KX468974 | KX468974 | Centaurea macrocephala | Czech Republic |
| Puccinia junci | PDD:99243 | KX985745 | KX985745 | Juncus tenuis | New Zealand |
| Puccinia klugkistiana | HMJAU8649 | MW406957 | MW397084 | Cleistogenes hackelii | China |
| Puccinia komarovii | Pk23DPN11 | OR475557 | OR475550 | Impatiens parviflora | Poland |
| Puccinia komarovii | Pk23DPN1 | OR475551 | OR473652 | Impatiens parviflora | Poland |
| Puccinia lagenophorae | KSAN | FJ655869 | FJ669236 | Senecio arenarius | South Africa |
| Puccinia latimamma | HGUP21188 | OR470000 | OR548163 | Pleuropterus multiflorus | China |
| Puccinia latimamma | HGUP21186 | OR469998 | OR548161 | Pleuropterus multiflorus | China |
| Puccinia ligulari-przewalskii | QHU2023090T | PZ575731 | PP469520 | Ligularia przewalskii | China |
| Puccinia ligulari-przewalskii | QHU2023092 | PZ575735 | PZ575738 | Ligularia przewalskii | China |
| Puccinia magnusiana | HMJAU8376 | MW406953 | MW397080 | Clematis terniflora | China |
| Puccinia menthae | BPI 910256 | KY764142 | KY764142 | Mentha sp. | USA |
| Puccinia menthae | BPI 910255 | KY764141 | KY764141 | Mentha sp. | USA |
| Puccinia mixta | HMUT 8510 | MW429286 | MW429286 | Allium platyspathum | China |
| Puccinia moiwensis | HMJAU8807 | MW447888 | MW414356 | Carex sp. | China |
| Puccinia moiwensis | HMJAU8800 | MW447882 | MW414350 | Urtica angustifolia | China |
| Puccinia mysuruensis | HSZ2119 | KC847089 | KC847089 | Psychotria nervosa | USA |
| Puccinia novopanici | BPI 747673T | KX190874 | KX190874 | Panicum virgatum | USA |
| Puccinia obscura | KR14322 | FJ655874 | FJ669234 | Luzula sylvatica | Germany |
| Puccinia oncospora | HMJAU8688 | MW404825 | MW404985 | Trillium kamtschaticum | China |
| Puccinia polygoni-amphibii | ZP-R716 | MK519001 | MK518706 | China | |
| Puccinia rhei-undulati | PDD:101523 | KX985739 | KX985739 | Rheum rhabarbarum | New Zealand |
| Puccinia rhei-undulati | PDD:104489 | KX985743 | KX985743 | Rheum rhabarbarum | New Zealand |
| Puccinia rupestris | HMJAU8600 | PP937689 | PP930982 | Flueggea suffruticosa | China |
| Puccinia sp. YG-2024B | GY44AT | PQ435764 | PQ435766 | Poaceae | China |
| Puccinia suaveolens | DAOM:195480 | MW009554 | MW009554 | Cirsium arvense | Canada |
| Puccinia suaveolens | XJ1 | OR600240 | OR598614 | China | |
| Puccinia striiformis f. sp. tritici | HSZ1847 | GU382671 | GU382671 | Triticum aestivum | USA |
| Puccinia tiritea | PDD:107783 | KX985736 | KX985736 | Muehlenbeckia australis | New Zealand |
| Puccinia tiritea | PDD:97495 | KX985742 | KX985742 | Muehlenbeckia australis | New Zealand |
| Puccinia triticina | HMJAU8693 | MW404826 | MW404986 | China | |
| Puccinia triticina | DAOM:240974 | MT965572 | MT965572 | Triticum sp. | Canada |
| Puccinia triticina | DAOM:984848 | MT965605 | MT965605 | Triticum sp. | Canada |
| Puccinia urticae-inflatae | HMJAU8824 | MW447878 | MW414346 | Carex sp. | China |
| Puccinia urticae-inflatae | HMJAU8946 | MW447879 | MW414347 | Carex sp. | China |
| Puccinia urticae-inflatae | HMJAU8947 | MW447880 | MW414348 | Carex sp. | China |
| Puccinia violae | HSZ0478 | KM096424 | KM096424 | Viola sp. | India |
| Puccinia violae | DAOM 240967 | HQ317513 | HQ317513 | Viola sp. | Canada |
Note: Sequences from type collections are indicated with (T). The new taxa are in bold.
Table 3.
Morphological comparison of teliospores among P. ligulari-przewalskii and related species.
| Species | Size (µm) | Shape | Wall | Thickness (µm) | Germ Pore & Pore Cap | Host |
|---|---|---|---|---|---|---|
| P. ligulari-przewalskii | 30.7–44.8 × 17.4–25.3 | Ellipsoid, spindle-shaped or polygonal | Sparsely verruculose | Lateral wall 1.4–2.3 | Apical germ pore; yellow papilla 1.6–4.4 µm long | L. przewalskii |
| P. eriophori var. eriophori | (34–)37–71(–83) × (14–)16.5–28(–29.5) | Ellipsoid to oblong, slightly to moderately constricted | Smooth | Lateral (0.7–)1.0–1.8(–2.3); apical 4.5–14(–19) | Upper cell apical, slightly to moderately eccentric; lower cell near septum; rounded or conic cap, concolorous or paler | Eriophorum spp. (Cyperaceae) |
| P. argentata | 25–40 × 14–21 | Ellipsoid or oblong | Smooth or very finely obscurely verruculose | 1.5–2.0; apex not thickened | Upper cell apical, lower cell near septum; distinct hyaline pore cap 3–5 µm | Impatiens spp. |
| P. komarovii | 25–40 × 16–23 | Subrectangular or obovoid | Smooth or nearly smooth | ~2.0, uniform | Upper cell apical, lower cell near septum; distinct hyaline pore cap | Impatiens spp. |
| P. gentianae | 28–43 × 20–30 | Broadly ellipsoid or oblong | Smooth | 2–3 | Upper cell apical, lower cell near septum or slightly below; colorless small pore cap | Gentiana spp. |
| P. cnici-oleracei | 33–58(–73) × 13–20(–25) | Clavate or oblong-ellipsoid | Smooth | Lateral wall 1–2.5; apical wall 3–13(–18) | Upper cell apical; lower cell near septum | Asteraceae (L. tsangchanensis) |
| P. ferruginosa | (38–)45–77 × 14–30 | Elongate or clavate | Smooth | Apex thickened up to 8 | Apical; apex thickened | Artemisia spp. |
| P. senecionis | 28–38 × 14–23 | Broadly ellipsoid or obovoid | Smooth | 1.5–2.0 (apex + cap 2–3) | Upper cell apical, lower cell near septum or slightly below; small hyaline pore cap | Senecio spp. |
| P. hieracii | (25–)29–40(–45) × (18–)21–25(–29) | Oblong, ellipsoid or broadly ellipsoid | Finely verrucose | 1.5–2.5; apex not thickened | Upper cell apical or slightly depressed; lower cell away from septum, median; no pore cap | Taraxacum spp., Crepis spp., Hieracium spp. |
| P. artemisiae-keiskeanae | 35–55 × 20–25 | Ellipsoid or oblong | Smooth | Lateral wall 1.5–3.0; apical wall 4–13(–18) | Upper cell apical, lower cell near septum | Artemisia spp. |
| P. violae | (20–)30–45 × 17–23(–28) | Ellipsoid or obovoid | Densely verrucose | 1.5–2.5 | Upper cell apical, lower cell near septum; pale hyaline pore cap | Viola spp. |
| P. glomerata | 33–53 × 15–28 | Ellipsoid or irregularly obovoid | Smooth | 1.5–2 | Upper cell apical, lower cell near septum; hyaline pore cap 3–5 µm thick | Ligularia spp., Cacalia spp. |
| P. cremanthodii | (32–)35–45(–48) × (19–)22–28(–32) | Ellipsoid or subobovoid | Reticulate-rugose | 2.0–3.5; apex + pore cap up to 5 | Upper cell apical, lower cell near septum | Cremanthodium spp. |
| P. minussensis | (27–)30–38(–45) × (16–)19–25(–28) | Ellipsoid or obovoid | Verrucose | 1.5–2.5; apex not thickened | Upper cell apical, lower cell median or below; distinct hyaline pore cap | Ixeris spp., Lactuca spp. |
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Qi, L.; Xu, Q.; Zhang, Y.; Bai, L.; Cao, Y.; Zhang, W. Morphological and Molecular Identification of New Puccinia Species from Qinghai, China. Diversity 2026, 18, 487. https://doi.org/10.3390/d18080487
AMA Style
Qi L, Xu Q, Zhang Y, Bai L, Cao Y, Zhang W. Morphological and Molecular Identification of New Puccinia Species from Qinghai, China. Diversity. 2026; 18(8):487. https://doi.org/10.3390/d18080487
Chicago/Turabian StyleQi, Lin, Qi Xu, Yang Zhang, Luchao Bai, Yingtai Cao, and Weidong Zhang. 2026. "Morphological and Molecular Identification of New Puccinia Species from Qinghai, China" Diversity 18, no. 8: 487. https://doi.org/10.3390/d18080487
APA StyleQi, L., Xu, Q., Zhang, Y., Bai, L., Cao, Y., & Zhang, W. (2026). Morphological and Molecular Identification of New Puccinia Species from Qinghai, China. Diversity, 18(8), 487. https://doi.org/10.3390/d18080487
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