Introduction

Rusts are obligate fungal parasites capable of colonizing fresh plant tissues and producing up to five distinct spore types: basidiospores, spermatia, aeciospores, urediniospores and teliospores, exhibiting either heteroecious or autoecious life cycles [12, 22, 50, 57]. There are almost 8000 accepted species of rust fungi, in which the genus Melampsora Castagne represents foliar rust encompassing approximately 212 species worldwide, including over 65 species in China [10, 26, 48]. Traditionally, Melampsora has been considered the sole genus in Melampsoraceae family in the last few decades. However, recent studies have suggested the possible inclusion of Ceropsora weirii, a species from another genus, in the Melampsoraceae family [2, 64]. Melampsora species were primarily found on Populus and Salix (Salicaceae) trees in the northern hemisphere, mainly due to the prevalence of these host genera. Theire uredinial stages are characterized by colorless, capitate paraphyses and echinulate urediospores, while the telial stages are characterized by one-celled and sessile teliospores [7, 15]. Besides, Melampsora species can also infect various other plants species such as Abies Mill. (Pinaceae), Apocynum L. (Apocynaceae), Chelidonium L. (Papaveraceae), Chosenia (Salicaceae), Corydalis DC. (Papaveraceae), Empetrum L. (Ericaceae), Euonymus L. (Celastraceae), Euphorbia L. (Euphorbiaceae), Flueggea (Phyllanthaceae), Hypericum L. (Hypericaceae), Larix Mill. (Pinaceae), Mercurialis L. (Euphorbiaceae), Moneses Salisb. ex Gray (Ericaceae), Pinus L. (Pinaceae), Pseudotsuga Carrière (Pinaceae), Ribes L. (Grossulariaceae), Ricinus L. (Euphorbiaceae), Tsuga (Endl.) Carrière (Pinaceae), and others [9, 44, 47, 71].

The plant genus Hypericum ranks among the 100 largest genera in angiosperms with nearly 500 species, displaying a global distribution and a fossil record dating back to the Early Cenozoic period [36]. Until now, only three non-host-alternating (autoecious) Melampsora species have been reported infecting Hypericum species, including M. hypericorum on H. androsaemum L., H. attenuatum Fisch. ex Choisy, H. calycinum L., H. oblongifolium Choisy (= H. cernuum Roxb. ex D.Don), H. hirsutum L., H. maculatum Crantz, H. patulum Thunb., H. perforatum L., H. pulchrum L. and H. tetrapterum Fr. [9, 28,29,30, 59], M. hyperici-sampsonii on H. sampsonii Hance [71], and M. kusanoi on H. ascyron L., H. gramineum G. Forst. and H. przewalskii Maxim [961].

Among the three previously mentioned Melampsora rust species, M. hypericorum has been identified globally, with a predominant in Europe, showcasing aecial and telial stages. M. hyperici-sampsonii has only been documented in China with uredinial and telial stages, while the uredinial and telial stages of M. kusanoi have been observed in China, Australia, New Zealand, and Europe [9, 28, 5961, 71]. Initially, M. hypericorum (= Mesopsora hypericorum) was not classified under Melampsora due to its “catenulate urediniospores”; however, subsequent findings revealed these spore stages to be secondary aecia and further confirmed its phylogenetic placement within Melampsora [16, 17, 32]. Distinguishing between these species based solely on morphological characteristics can be challenging owning to subjective interpretations, variations in measurement methods among researchers, the absence or incomprehension of certain spore stage, and morphological concepts leading to a wide species concept [5, 7, 39, 59]. Fortunately, the application of ribosomal DNA-based phylogenetic analysis has significantly enhanced the accurate classification of rust fungi at both generic and species levels [31, 4551, 6366, 6772].

During our investigation of rust fungi in Sichuan Province, China, we have identified and meticulously described two novel Melampsora species infecting Hypericum pseudohenryi N. Robson in Pinus armandi Franch. forest. This article provides detailed descriptions and illustrations based on a detailed morphological study. Additionally, phylogenetic analyses were carried out utilizing a combined dataset of internal transcribed spacer regions (ITS) and the large subunit (LSU) sequences to determine their independent phylogenetic placements within the Melampsora genus. As a result of our research, we have proposed two new species: M. danbaensis sp. nov. and M. hyperici-pseudohenryi sp. nov.

Materials and Methods

Sample Collections

Specimens of H. pseudohenryi (GenBank accession no.: PP907815) infected by Melampsora were collected in 2024 from Jiaju Tibetan Village, Danba County, Ganzi Tibetan Autonomous Prefecture, Sichuan Province, China. Voucher specimens were dried and preserved in the Herbarium Mycologicum Academiae Sinicae (nos. HMAS353132 − 353134) at the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. They were also deposited at the Mycological Herbarium of the Forestry College, Northwest A & F University (nos. HMNWFC-SC058 − SC060), Shaanxi Province, China.

Morphological Studies

The macro photographs of specimens were obtained using focus stacking photography techniques. This involved assembling Sony Alpha 7R III camera, Raynox DCR-150 MacroScan Conversion lens, M42 to NEX adapter, Mitutoyo M Plan Apo L 10 × /0.28 ∞/0 f = 200 lenses, Nikon BD Plan 40 × /0.5 ELWD 210/0 lenses, Wemacro Rail®, and Helicon® Focus 7 software, according to our previous study [40]. The rust sori produced on the leaves were examined under a dissecting microscope (Olympus SZX12, Tokyo, Japan). Microphotographs of infection sections and spores were captured using a microscope (Olympus CX41RF, Tokyo, Japan) equipped with a SC600 CCD (Nanjing Lookout Photoelectric Co., Ltd., Nanjing, China) and Micro-Image Analysis System Software. Approximately 50 morphological characteristics were randomly selected for measurements of teliospores, urediniospores, and paraphyses, while about 30 morphological characteristics were randomly selected for measurements of basidia and basidiospores. Images of the surface structures were produced with a field emission scanning electron microscope (SEM) (Nova™ NanoSEM 450, FEI, USA) operated at 5 kV. The various spore stages of rust are designated by the following Roman numerals: spermogonia/spermatia (0), aecia/aeciospores (I), uredinia/urediniospores (II), telia/teliospores (III), and basidia/basidiospore (IV), referring to Cummins and Hiratsuka [13, 14].

DNA Extraction, PCR Amplification, and DNA Sequencing

Rust genomic DNA was extracted using Chelex-100 (Macklin Biochemical Technology Co., Ltd., Shanghai, China) [6, 24]. Rust sori were isolated with a sterile needle under a dissecting microscope (Olympus SZX12, Tokyo, Japan) and then placed into 50 μL 5% Chelex-100 TE Buffer in a sterilized 1.5 mL centrifuge tube. After a 20 min incubation at 56°C, a 1 min centrifugation at 5000 rpm, and then an 8 min incubation at 100°C, the tube was vigorously vortexed and finally centrifuged for 1 min at 15,000 rpm to obtain the supernatant as the template DNA.

The internal transcribed spacer regions and 5.8S nuclear ribosomal RNA gene (ITS) and the large subunit (LSU) rDNA region were amplified using primer pairs ITS1F/ITS4B [21, 58] and NL1/NL4 [37]. Polymerase chain reaction (PCR) was conducted in a 20 μL reaction volume containing 10 μL 2 × Specific™ Taq Master Mix (Novoprotein Scientific Inc., Suzhou, China), 6 μL ddH2O, 2 μL DNA template, and 1 μL of each primer (10 μM). PCR amplification procedures were performed using a GeneAmp PCR TC-96 (Bioer Technology, Hangzhou, China) as follows: 94°C for 90 s; 35 cycles at 94°C for 20 s, 55°C for 20 s, 72°C for 1 min; and a final extension at 72°C for 5 min. The PCR amplification products were examined by electrophoresis in 1% (w/v) agarose gels, and then sequenced by Shanghai Sangon Biotech Co., Ltd.

Phylogenetic Analyses

The PCR sequencing results were bidirectionally assembled using BioEdit version 7.0.5.3, and the consensus ITS and LSU sequences were deposited in GenBank (refer to Table 1). Reference sequences used for phylogenetic analyses were obtained from GenBank based on published literatures (refer to Table 1), and Chrysomyxa empetri (Pers.) J. Schröt. and Rossmanomyces monesis (Ziller) Aime & McTaggart were used as the outgroups [42, 60, 61, 71]. The ITS and LSU sequences of the reference taxa were aligned individually using the MUSCLE algorithm in MEGA version X and then combined in raxmlGUI version 2.0 [19, 27]. Aligned datasets were converted from FASTA to PHYLIP format using SequenceMatrix version 1.8 software. The best-fitting substitution model for each gene was determined using PAUP version 4.0b10 and MrModeltest version 2.3 [46]. Maximum likelihood (ML) analysis and Bayesian inference (BI) analysis based on a combined ITS and LUS dataset were conducted using raxmlGUI version 2.0 and MrBayes version 3.1.2 with the optimal GTR + G + I site substitution model [41]. Phylogenetic trees were visualized using FigTree version 1.4.3 and manually annotated using Adobe Illustrator 2022 software. Clades with ML bootstrap support (MLBS) of ≥ 70% and Bayesian posterior probability (BPP) of ≥ 0.95 were considered significantly supported [3, 23].

Table 1 Taxa used in the phylogenetic analyses, along with their GenBank accession numbers for ITS and LSU sequence data. “—” indicates the absence of corresponding data. Taxa generated in this study are denoted in boldface

Results

Phylogenetic Analyses

The phylogenetic analyses incorporating ITS and LSU sequences from 62 Melampsora taxa, along with two outgroup taxa, yielded consistent topologies in both ML and BI analyses, as illustrated in Fig. 1. The phylogenetic tree depicted a clustering of all four specimens collected in Sichuan Province within the Melampsora genus. Two specimens (HMAS353133 and HMNWFC-SC059) formed a monophyletic clade with robust support (MLBS = 100%, BPP = 1) and were closely associated with M. kusanoi and M. hyperici-sampsonii. Similarly, the other four specimens (HMAS353132, 353134, and HMNWFC-SC058, SC060) also clustered together with strong support (MLBS = 100%, BPP = 1) and formed a distinct clade, grouped with M. laricis-pentandrae.

Fig. 1
figure 1

Phylogenetic relationships of Melampsora species based on rDNA ITS and LSU sequence data. The best trees generated from maximum likelihood (ML) analyses is shown. The ML bootstrap support (MLBS) values ≥ 50% and Bayesian posterior probabilities (BPP) ≥ 0.75 are indicated at nodes. The clades with orange background are the two novel species, M. danbaensis sp. nov. and M. hyperici-pseudohenryi sp. nov

Taxonomy

Melampsora danbaensis Z.J. Peng, and Z.D. Yu, sp. nov. (Fig. 2).

Fig. 2
figure 2

Melampsora danbaensis Z.J. Peng, and Z.D. Yu, sp. nov. on the leaves of Hypericum pseudohenryi. A: Aecial symptoms on the abaxial leaf; B: Telial symptoms on the abaxial leaf; C: Pulverulent aecia on the abaxial leaf; D: Aecia occurred on the stem; E: Section of aecium; F: Aeciospores; G: Flaps of host epidermal tissue and aeciospores observed under SEM; H: Aeciospores observed under SEM; I: Telia on the abaxial leaf; J: Germinating telia; K: Section of telium; L: Teliospores; M − N: Developing basidia and basidiospores; O: Basidia observed under SEM; P − Q: Basidia and basidiospores observed under SEM

MycoBank number: MB854150.

Etymology: Epithet refers to Danba County, the locality where the type specimen was collected.

Holotype: HMAS353134.

Description: Spermogonia and uredinia not found. Aecia Uredo-type, surrounded by flaps of host epidermal tissue, hypophyllous, on leaves and stem, subepidermal, erumpent, pulverulent, globose to ellipsoidal, gregarious or grouped, bright yellow to pale orange, 0.39 − 1.33 mm, shallowly pulvinate (Fig. 2A, C − E). Aeciospores globose to ellipsoidal, strongly verrucose, with pale orange contents, 18.2 − 33.7 × 15.9 − 30.1 µm, uneven in size, wall 1.4–3.5 µm thick, hyaline (Fig. 2F − H). Telia hypophyllous, on leaves and stem, appeared like crusts of one spore deep, subepidermal, gelatinous, from round to elongated or irregular, seldom scattered, always aggregated in patches covering whole leaves, orange when fresh and orangish-brown to brown when dry, 0.14–0.36 mm (Fig. 2B, I − K). Teliospores sessile, clavate to cylindrical, yellowish, 32.5–65.8 × 7.2–14.8 µm, lateral wall 0.6–2.8 µm thick, sometimes with thickened apical wall up to 5 µm (Fig. 2K − L). Basidia arose from teliospore, external, septate, yellowish, 22.3–37.2 × 6.1–8.0 µm (Fig. 2M–O). Basidiospores globose, yellowish, 5.1–7.3 × 4.9–7.0 µm (Fig. 2N, P, Q).

Materials examined: China, Sichuan Province, Ganzi Tibetan Autonomous Prefecture, Danba County, I, III, IV on H. pseudohenryi, 15 May 2024, H.C. Tong, HMAS353134 (holotype), ibid. HMNWFC-SC060; China, Sichuan Province, Ganzi Tibetan Autonomous Prefecture, Danba County, III, IV on H. pseudohenryi, 24 April 2024, Z.J. Peng & Z.M. Xu, HMAS353132, ibid. HMNWFC-SC058.

Notes: M. danbaensis is phylogenetically closely related to M. laricis-pentandrae (Fig. 1), which has been documented on Salix pentandra L., S. fragilis × pentandra, and Larix spp. [43]. Morphologically, M. danbaensis can be distinguished from M. laricis-pentandrae by its longer teliospores ranging from 32.5 to 65.8 µm, whereas M. laricis-pentandrae typically features smaller teliospores of up to 48 µm. By now, M. hypericorum, M. kusanoi and M. hyperici-sampsonii have been known to infect Hypericum species [28, 61, 71]. Notably, M. danbaensis stands out with the longest teliospores, reaching up to 65.8 µm, in contrasting to the shorter teliospores found in M. hypericorum, M. kusanoi and M. hyperici-sampsonii, which range up to 30 µm, 39 µm, and 32 µm respectively [28, 61, 71]. Furthermore, the uredinial stage of both M. hypericorum and M. danbaensis remains unreported. M. danbaensis features Uredo-type aecia with aeciospores up to 33.7 µm, distinguishing it from M. hypericorum, which exhibits Caeoma-type aecia with aeciospores formed in short chains and reaching up to 25 µm [9, 28].

Melampsora hyperici-pseudohenryi Z.J. Peng, and Z.D. Yu, sp. nov. (Fig. 3).

Fig. 3
figure 3

Melampsora hyperici-pseudohenryi Z.J. Peng, and Z.D. Yu, sp. nov. on leaves of Hypericum pseudohenryi. A: Uredinial symptoms on the abaxial leaf; B − D: Uredinia on the abaxia leaf; E: Section of uredinium; F: Flaps of host epidermal tissue; G: Urediniospores and paraphyses; H: Uredinium observed under SEM; I: Urediniospores, paraphyses and flaps of host epidermal tissue observed under SEM; J: Urediniospores and paraphyses observed under SEM

MycoBank number: MB854151.

Etymology: Name reflects the host plant species Hypericum pseudohenryi, from which the type specimen was collected.

Holotype: HMAS353133.

Description: Spermogonia and aecia not found. Uredinia Uredo-type, surrounded by ruptured epidermis, hypophyllous, subepidermal, erumpent, pulverulent, globose to ellipsoidal, scattered or grouped, bright yellow to orange, 0.18 − 0.46 mm, mixed with numerous paraphyses (Fig. 3A–F). Urediniospores globose, ellipsoidal or obovoid, echinulate, with orange contents, 19.5–28.4 × 16.5–23.8 µm, wall 1.1–2.1 µm thick, hyaline, paraphyses clavate to capitate, 38.3–77.6 × 11.9–26.6 µm, wall 1.9–4.8 µm thick, hyaline, without thickened apex (Fig. 3G–J). Telia not found.

Materials examined: China, Sichuan Province, Ganzi Tibetan Autonomous Prefecture, Danba County, II on H. pseudohenryi, 24 April 2024, Z.J. Peng & Z.M. Xu, HMAS353133 (holotype), ibid. HMNWFC-SC059.

Notes: In terms of phylogeny, M. hyperici-pseudohenryi demonstrates a close genetic affinity with M. kusanoi and M. hyperici-sampsonii, both known to infect Hypericum spp., and has a quite far genetic relationship with another two Melampsora rust colonized on Hypericum spp., M. danbaensis and M. hypericorum (Fig. 1). Morphologically, M. hyperici-pseudohenryi can be differentiated from M. danbaensis and M. hypericorum by its production of larger urediniospores, reaching up to 28.4 µm, and paraphyses that extend up to 77.6 µm without a thickened apex. In contrast, M. kusanoi possesses smaller urediniospores (up to 20.01 µm) and smaller paraphyses (up to 70 µm) with a thickened apical wall, while M. hyperici-sampsonii has smaller urediniospores (up to 21 µm) and shorter paraphyses only up to 38 μm [9, 28, 61, 71].

A key to Melampsora species on Hypericum

1. Aecial stage present…………………………………………………………………………2

1. Uredinial stage present………………………………………………………………………3

2. Aecia Caeoma-type; Telia scattered or aggregated…………………………. M. hypericorum

2. Aecia Uredo-type; Telia aggregated in patches covering whole leaves……… M. danbaensis

3. Paraphyses with a thickened apical wall (16–25 μm)…………………………… M. kusanoi

3. Paraphyses with apex evenly thickened……………………………………………………..4

4. Paraphyses 23–38 × 10–15 μm……………………………………….M. hyperici-sampsonii

4. Paraphyses 38.3–77.6 × 11.9–26.6 µm……………………….….. M. hyperici-pseudohenryi.

Discussion

Through detailed observations and DNA barcoding analysis of aecia/aeciospores, uredinia/urediniospores, and telia/teliospores on H. pseudohenryi, we uncovered the presence of two novel Melampsora species from China, namely M. danbaensis and M. hyperici-pseudohenryi. Despite initial assumptions of them being different stages of one rust species due to their presence on the same host plant, the examination of ITS and LSU sequences revealed a notable distinction: aecia/aeciospores and telia/teliospores shared identical genetic information, setting them apart from uredinia/urediniospores. Leveraging a consolidated ITS + LSU dataset, we delineated a phylogenetic tree of Melampsora genus through ML and BI analyses and found both M. danbaensis and M. hyperici-pseudohenryi formed a distinct clade in Melampsora (Fig. 1), respectively. The resulting topology was consistent with previous studies by Zhao et al. [71] and Wu et al. [60]. Based on ITS + LSU phylogeny, Melampsora species colonizing Hypericaceae, Salicaceae and Euphorbiaceae were distributed in several distinct clades (Fig. 1), which suggests the occurrence of host jumps in Melampsora rusts, analogous to the phenomenon driving the diversification of Phakopsora [33], Puccinia/Uromyces [34, 52] and Uromycladium [18]. Host jumps explain how rust fungi have evolved to widespread pathogens infecting a diverse range of plants [35]. However, our data was unable to determine the precise events and numbers of host jumps in Melampsora on Hypericaceae, Salicaceae and Euphorbiaceae due to the low MLBS values/BPPs at most early nodes in the phylogenetic tree, possibly resulting from an insufficient sequence dataset. As suggested by Thines [49], clades that diversified after a large host jump will almost always include pathogens from closely related hosts. To further elucidate the host-Melampsora evolutionary associations, it is imperative to incorporate more specimens and additional molecular markers for rust fungi, such as RNA polymerase II subunits 1 (RPB1) and 2 (RPB2), elongation factor 1-alpha (EF1α), mitochondrial ATPase subunit 6 (ATP6), β-tubulin, cytochrome oxidase subunit 1 (CO1), dehydrogenase subunit 6 (Nad6), and single-copy protein coding genes MS277 and MS208, into the phylogenetic analysis [1525470].

The recognition of Melampsora species has traditionally relied on the ecological species concept, emphasizing aecial or telial host ranges alongside a few morphological traits in uredinial and telial stages [10]. Morphologically, M. danbaensis stands out among the five Melampsora species colonizing Hypericum with its Uredo-type aecia, strongly verrucose aeciospores, gelatinous telia tending to covering whole leaves, and elongated teliospores up to 65.8 µm with thickened apical walls. Generally, five types of aecia, namely peridermioid, roestelioid, aecidioid, caeomoid and uredinoid, have been generally accepted, and Melampsora species have been characterized by Caeoma-type aecia producing verrucose aeciospores in chains without peridia, exemplified by M. hypericorum [7, 10, 11, 14, 25]. However, M. danbaensis deviates from this norm with its distinctive Uredo-type aecia, making it an anomaly among Melampsora species. Both M. danbaensis and M. hypericorum are recognized for possessing the aecial stage while lacking the uredinial stage, enabling their differentiation from the other three Melampsora species on Hypercium [9, 28, 71]. In contrast to M. kusanoi and M. hyperici-sampsonii, M. hyperici-pseudohenryi showcases the largest urediniospores (up to 28.4 µm) and longest paraphyses (up to 77.6 µm) without a thickened apex. Notably, no other Melampsora species share the same host species from Hypericum except M. danbaensis and M. hyperici-pseudohenryi, facilitating the differentiation of these two species from all other Melampsora species on Hypericum based solely on morphological characteristics and host range.

Over the past 50 years, 65 volumes of Flora Fungorum Sinicorum, considered as the most systematic and professional monograph on microbiology in China, have been published by successive generations of Chinese fungal taxonomists [78]. However, it is noteworthy that the genus Melampsora has not been incorporated in any of the five Uredinales volumes of Flora Fungorum Sinicorum [56, 74,75,76,77]. Although 65 Melampsora species were documented in the Catalogue of Life China: 2024 Annual Checklist [48], various new species and newly recorded species published these years have been inadvertently overlooked or omitted, including M. ferrinii Toome & Aime, M. hyperici-sampsonii P. Zhao & L. Cai, M. iranica Damadi, M.H. Pei, J.A. Sm. & M. Abbasi, M. linearis P. Zhao & L. Cai, M. salicis-argyraceae P. Zhao & Yamaoka, M. salicis-bakko P. Zhao, M. salicis-futurae P. Zhao & L. Cai, M. salicis-delavayanae P. Zhao & L. Cai, M. salicis-michelsonii C.M. Tian & L.L. Wang, M. salicis-purpureae P. Zhao & Yamaoka, M. salicis-sinicae P. Zhao, C.M. Tian & Y.J. Yao, and M. salicis-triandrae P. Zhao & L. Cai [42, 55, 65, 67,68,69,70,71]. Together with two novel species we proposed in this study, the total count of Melampsora species in China now approaches nearly 80. Nonetheless, our understanding of the inventory, distribution, life cycles, and host specificity of Melampsora rusts in China remains limited [71], and a systemic list of Melampsora species in China is currently unavailable. Further investigations and specimen collections are imperative to augment our comprehension of Melampsora species in China. By delving deeper into these aspects, researchers can advance our understanding of the diversity, ecological roles, and evolutionary dynamics of Melampsora rust fungi in the Chinese region.