Taxonomy and phylogeny of the Pleurotus djamor complex with descriptions of a new species from China

119 Taxonomy and phylogeny of the Pleurotus djamor complex with descriptions of a new species from China Long Zeng1, Yi-Hua Xu1, Le-Le Wan1, Yi-Fei Sun1, Bao-Kai Cui1 1 StateKeyLaboratoryofEfficientProductionofForestResources,SchoolofEcologyandNatureConservation,BeijingForestryUniversity,Beijing100083,China Correspondingauthors:Yi-FeiSun([email protected]);Bao-KaiCui([email protected]) Copyright: © Long Zeng et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract The Pleurotus djamor complex is widely distributed in tropical and subtropical regions and is known for its more or less reddish pileus. Phylogenetic analyses of the P. djamor complex were carried out using multiple loci, including the internal transcribed spacer regions (ITS), the translation elongation factor 1-alpha gene (tef1α), and the second largest subunit of RNA polymerase II (rpb2). In this study, a new species of Pleurotus, P. sinensis, is described based on morphological characters and molecular evidence. Pleurotus sinensis is characterized by a pileus that is white to pinkish buff or flesh-pink, flabelliform to petaloid, with inflexed and sometimes wavy margins. An illustrated description of the novel species is provided. Key words: Basidiomycota, macrofungi, molecular phylogeny, morphology, new species Introduction The genus Pleurotus (Fr.) P. Kumm. has high species diversity and a wide distribution around the world, and most species in this genus have notable edibility and medicinal value (Singer 1949; Guzmán 2000; Ma et al. 2015; Corrêa et al. 2016). However, the utilization and cultivation of Pleurotus have been constrained by misidentification and confused nomenclature (Vilgalys and Sun 1994; Zervakis et al. 2001). Pleurotus djamor (Rumph. ex Fr.) Boedijn was first described by Rumphius (1750) as Boletus fecundus arboreus, and it was described as having an imbricate, circular-oblong to liver-shaped pileus displaying reddish coloration when young, fading to white, dull yellow, or yellowish tan at maturity, with a stipe that is short or sometimes lacking. Boedijn (1959) classified this species into the genus Pleurotus, and this classification has been followed ever since. Corner (1981) examined specimens of P. djamor in Malaysia based on morphological characteristics and habits and proposed six varieties, but these varieties were regarded as synonyms of P. djamor. Nevertheless, due to phenotypic plasticity and interfertility among species, morphology and sexual compatibility alone are insufficient for accurate species delimitation within the P. djamor complex (Nicholl and Petersen 2000). Specimens of P. djamor distributed in Mexico (Huerta et al. 2010), Brazil (Menolli Jr et al. 2014), and Kenya (Otieno et al. 2015) were clarified using ITS sequences. Zervakis et al. (2019) revealed high ITS heterogeneity among specimens of the P. djamor complex, and some materials labeled as Academic editor: Alfredo Vizzini Received: 19 June 2025 Accepted: 29 November 2025 Published: 10 December 2025 Citation: Zeng L, Xu Y-H, Wan L-L, Sun Y-F, Cui B-K (2025) Taxonomy and phylogeny of the Pleurotus djamor complex with descriptions of a new species from China. MycoKeys 126: 119–133. https://doi.org/10.3897/ mycokeys.126.162530 MycoKeys 126: 119–133 (2025) DOI: 10.3897/mycokeys.126.162530 120 MycoKeys 126: 119–133 (2025), DOI: 10.3897/mycokeys.126.162530 Long Zeng et al.: Taxonomy and phylogeny of the Pleurotus djamor complex P. flabellatus Sacc., P. ostreatoroseus Singer, and P. salmoneostramineus Lj.N. Vassiljeva also clustered into the same phylogenetic clade as the P. djamor complex. However, a comprehensive phylogenetic revision incorporating both morphological and multi-locus evidence remains lacking. To resolve these taxonomic ambiguities, we conducted multi-locus analyses based on DNA sequences from the ITS, tef1α, and rpb2 loci. In combination with morphological characteristics, these analyses led to the discovery of a new species within the P. djamor complex. Materials and methods Morphological studies Specimens examined in this study were deposited at the herbarium of the Institute of Microbiology, Beijing Forestry University, China (BJFC). The morphological observation followed the protocols used in Lechner et al. (2004) and Camacho et al. (2012). Macro-morphological characteristics and habitats were documented based on field notes and laboratory observations. Special color terms followed Petersen (1996). Examination of the sections was conducted using a Nikon E80i microscope with phase-contrast illumination, capable of magnifications up to 1000× and manufactured by the Nikon Corporation in Tokyo, Japan. The observations, measurements, and illustrations were derived from slide preparations of dried material stained with Cotton Blue, 2% phloxine B and Melzer's reagent (Sun et al. 2022). Basidiospore measurements were taken from at least 30 spores per specimen, excluding 5% of extreme values, which are given in parentheses. The abbreviations used in this paper are as follows: IKI = Melzer’s reagent, IKI− = the absence of dextrinoid or amyloid properties, KOH = 5% solution of potassium hydroxide, CB = Cotton Blue, CB+ = the cyanophilous reaction, CB− = the acyanophilous reaction, L = the mean length of spores (calculated as the arithmetic average), W = the mean width of spores (calculated as the arithmetic average), Q = the variability in the L/W ratio among the samples, and n (a/b) = the total number of spores (specified quantity/number of specimens). DNA extraction, PCR amplification, and sequencing A CTAB plant genome rapid extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd.) was employed for DNA extraction from dried specimens. The polymerase chain reaction (PCR) was conducted following the manufacturer’s guidelines with some modifications (Li et al. 2017; Zervakis et al. 2019). The sequences of three gene loci were derived from PCR amplification. The internal transcribed spacer (ITS) regions were amplified with primer pairs ITS5 and ITS4 (White et al. 1990). The translation elongation factor 1-alpha gene (tef1α) was amplified with primer pairs EF1-983F and EF1-1567R (Rehner 2001). The second largest subunit of RNA polymerase II (rpb2) was amplified with primer pairs RPB2-3.1F and RPB2-6R2 (Liu and Hall 1999). The final PCR volume was 30 µL, consisting of 1 µL of each primer, 1 µL of extracted DNA, 12 µL of ddH2O, and 15 µL of 2× EasyTaq PCR Supermix (TransGen Biotech Co., Ltd., Beijing, China). PCR amplification was performed on an S1000™ Thermal Cycler (Bio-Rad Laboratories, CA, USA). The PCR procedure for ITS was as follows: initial denaturation 121 MycoKeys 126: 119–133 (2025), DOI: 10.3897/mycokeys.126.162530 Long Zeng et al.: Taxonomy and phylogeny of the Pleurotus djamor complex at 94 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 40 s, annealing at 52 °C for 40 s, and extension at 72 °C for 1 min, with a final extension at 72 °C for 7 min. The PCR process for rpb2 and tef1α was as follows: initial denaturation at 94 °C for 4 min, followed by 35 cycles at 94 °C for 40 s, 55 °C for 1 min, and 72 °C for 90 s, with a final extension at 72 °C for 10 min. The PCR products were subsequently purified and sequenced at the Beijing Genomics Institute (BGI, China) using the same primers. The newly generated and downloaded sequences in this study were deposited at GenBank and are listed in Table 1. Phylogenetic analyses In this study, 87 sequences derived from 59 fungal samples representing nine species were used to reconstruct the phylogenetic trees, including 59 ITS sequences, 14 tef1α sequences, and 14 rpb2 sequences. Among them, 30 sequences were newly generated, comprising 10 ITS sequences, 10 tef1α sequences, and 10 rpb2 sequences. The phylogenetic relationships within the P. djamor complex were inferred using ITS and the combined three-gene (ITS + tef1α + rpb2) datasets. The ITS dataset was used to reconstruct the general phylogenetic framework of the complex, whereas the combined three-gene datasets were used to further determine the phylogenetic differences between P. sinensis and P. djamor. Pleurotus abieticola R.H. Petersen & K.W. Hughes was selected as the outgroup. The datasets were aligned in MAFFT 7 (Katoh and Standley 2013; https://mafft.cbrc.jp/alignment/ server/) and manually adjusted in BioEdit (Hall 1999). Alignment statistics, including the numbers of constant, parsimony-uninformative, and parsimony-informative sites, were calculated in PAUP version 4.0b10 (Swofford 2002) to evaluate the phylogenetic information content of the datasets. Alignments were spliced in Mesquite v. 3.2 (Maddison and Maddison 2017). Phylogenetic analyses were performed using the maximum likelihood (ML) and Bayesian inference (BI) methods. ML analyses were performed using RAxML-HPC v. 8.2.3 (Stamatakis 2014) with 1000 ML searches under the GTRGAMMA model, and only the maximum likelihood best tree from all searches was kept. In addition, 1000 rapid bootstrap replicates were run with the GTRCAT model to assess ML bootstrap values. BI analyses were performed using MrBayes v. 3.2 (Ronquist and Huelsenbeck 2003) with two simultaneous independent chains for datasets, performing 10 million generations until the split deviation frequency value was < 0.01 and sampled every 100 generations. The first 25% of sampled trees were discarded as burn-in, while the remaining ones were used to calculate Bayesian posterior probabilities (BPP) of the clades. The ML bootstrap (ML-BS) ≥ 70% and Bayesian posterior probabilities (BPP) ≥ 0.90 were presented on topologies from the ML analyses. The trees were viewed in FigTree v. 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/). Results Phylogenetic analyses The ITS dataset included sequences from 59 fungal samples representing nine taxa. The dataset had an aligned length of 647 characters, including 467 constant characters, three parsimony-uninformative variables, and 177 122 MycoKeys 126: 119–133 (2025), DOI: 10.3897/mycokeys.126.162530 Long Zeng et al.: Taxonomy and phylogeny of the Pleurotus djamor complex Table 1. Specimen information and GenBank accession numbers for the sequences used in this study. Species name Geographic Origin Voucher GenBank accession numbers Reference ITS tef1 rpb2 P. abieticola China HKAS91342 KX836361 KX840302 KX870442 Li et al. (2017) P. abieticola China HKAS45720 KP771696 KX885093 KX885220 Liu et al. (2015); Li et al. (2017) P. agave Mexico CP-194 GU722262 – – Huerta et al. (2010) P. agave Mexico ECS-0165 GU722264 – – Huerta et al. (2010) P. agave (Submitted to NCBI under the name P. opuntiae) Mexico ET3313 AY450339 – – From NCBI P. agave (Submitted to NCBI under the name P. opuntiae) Mexico TENN52368 AY450340 – – From NCBI P. calyptratus Austria TENN57451 AY450338 – – From NCBI P. calyptratus Slovakia CBS 325.85 EU424283 – – From NCBI P. calyptratus Russia 1935 KF932720 – – Shnyreva and Shnyreva (2015) P. djamor Cuba CBS596.96 FJ040176 – – From NCBI P. djamor Mexico ECS-0123 GU722265 – – Huerta et al. (2010) P. djamor Mexico ECS-0128 GU722266 – – Huerta et al. (2010) P. djamor Mexico ECS-0130 GU722267 – – Huerta et al. (2010) P. djamor Mexico ECS-0150 GU722268 – – Huerta et al. (2010) P. djamor Mexico ECS-0151 GU722269 – – Huerta et al. (2010) P. djamor Mexico ECS-0159 GU722273 – – Huerta et al. (2010) P. djamor Mexico CP-170 GU722271 – – Huerta et al. (2010) P. djamor Brazil SP445682 KF280324 – – Menolli Jr et al. (2014) P. djamor Brazil SP445798 KF280326 – – Menolli Jr et al. (2014) P. djamor Dominican Republic TENN F-59778 KP026246 – – From NCBI P. djamor Mexico CC050 KX573921 – – Aguilar Doroteo et al. 2018 P. djamor Mexico CC051 KX573922 – – Aguilar Doroteo et al. 2018 P. djamor Mexico CC053 KX573924 – – Aguilar Doroteo et al. 2018 P. djamor Mexico CC056 KX573927 – – Aguilar Doroteo et al. 2018 P. djamor Sri Lanka HKAS94069 KX061789 KX840308 –Li et al. (2017) P. djamor (Submitted to NCBI under the name P. ostreatoroseus) unknown P94 MG282434 – – From NCBI P. djamor (Submitted to NCBI under the name P. ostreatoroseus) Peru ITA-308 ON426447 – – From NCBI P. djamor Puerto Rico Cui 16861 PV771009 PX608114 PX608104 This study P. djamor Puerto Rico Cui 16862 PV771010 PX608115 PX608105 This study P. djamor Puerto Rico Cui 16890 PV771011 PX608116 PX608106 This study P. djamor Puerto Rico Cui 16902 PV771012 PX608117 PX608107 This study P. djamor var. fuscopruinosus (Submitted to NCBI under the name P. salmoneostramineus) unknown ACCC50836 EU424302 – – From NCBI P. djamor var. fuscopruinosus (Submitted to NCBI under the name P. djamor var. roseus) unknown ABM1049204 KC582640 – – From NCBI P. djamor var. fuscopruinosus (Submitted to NCBI under the name P. flabellatus) Malaysia ATCC38137 AY265827 – – From NCBI P. djamor var. fuscopruinosus (Submitted to NCBI under the name P. flabellatus) unknown ATCC38140 AY368660 – – From NCBI P. djamor var. fuscopruinosus (Submitted to NCBI under the name P. djamor) Malaysia FUM-093 KY951475 – – Avin et al. (2017) 123 MycoKeys 126: 119–133 (2025), DOI: 10.3897/mycokeys.126.162530 Long Zeng et al.: Taxonomy and phylogeny of the Pleurotus djamor complex Species name Geographic Origin Voucher GenBank accession numbers Reference ITS tef1 rpb2 P. djamor var. fuscopruinosus Thailand MFLUCC24-0062 PP192013 – – Phonemany et al. (2025) Pleurotus sp. 1 (Submitted to NCBI under the name P. opuntiae) New Zealand ICMP 11566 MH395961 – – From NCBI Pleurotus sp. 1 (Submitted to NCBI under the name P. opuntiae) New Zealand ICMP 11670 MH395966 – – From NCBI Pleurotus sp. 1 (Submitted to NCBI under the name P. opuntiae) New Zealand ICMP 11671 MH395967 – – From NCBI Pleurotus sp. 1 (Submitted to NCBI under the name P. parsonsiae) New Zealand ICMP 18169 MH395975 – – From NCBI Pleurotus sp. 2 (Submitted to NCBI under the name P. djamor) Nigeria ELEB27 KT273359 – – Adedokun et al. (2016) Pleurotus sp. 2 (Submitted to NCBI under the name P. djamor) Kenya KKF8428 KJ754106 – – Otieno et al. (2015) Pleurotus sp. 2 (Submitted to NCBI under the name P. djamor) Kenya KKF2710 KJ754108 – – Otieno et al. (2015) Pleurotus sp. 2 (Submitted to NCBI under the name P. djamor) Kenya KKF0121 KJ754109 – – Otieno et al. (2015) P. opuntiae Italy SAF 250 MH620770 – – Zervakis et al. (2019) P. opuntiae Italy SAF 251 MH620771 – – Zervakis et al. (2019) P. opuntiae Italy SAF 252 MH620772 – – Zervakis et al. (2019) P. sinensis China HKAS90179 KX836373 KX840306 KX870451 Li et al. (2017) P. sinensis China HKAS90178 KX836374 –KX870452 Li et al. (2017) P. sinensis (Submitted to NCBI under the name P. salmoneostramineus) unknown ASI 2172 AY265845 – – From NCBI P. sinensis China Dai 16572 PX612360 PX608118 PX608108 This study P. sinensis China Cui 23262 PV771017 PX608119 PX608109 This study P. sinensis China Cui 23263 PV771018 PX608120 PX608110 This study P. sinensis China Cui 23999 PX612361 PX608123 PX608113 This study P. sinensis China Cui 23439 PV771021 PX608121 PX608111 This study P. sinensis China Cui 23450 PV771022 PX608122 PX608112 This study parsimony-informative characters. Bayesian and ML analyses resulted in the same topology. Only the ML tree is provided in Fig. 1, and the ML bootstrap (MLBS) and Bayesian posterior probabilities (BPP) are shown at the nodes. The phylogenetic tree showed that the species originally labeled as Pleurotus djamor were divided into five distinct monophyletic clades (Fig. 1): P. djamor from Sri Lanka and the Americas (95% ML-BS, 0.99 BPP), P. djamor var. fuscopruinosus from Thailand and Malaysia (100% ML-BS, 0.93 BPP), Pleurotus sinensis sp. nov. from China (99% ML-BS, 0.90 BPP), Pleurotus sp. 1 from New Zealand (99% ML-BS, 0.99 BPP), and Pleurotus sp. 2 from Africa (100% ML-BS, 1.00 BPP). The combined three-gene (ITS + tef1α + rpb2) dataset included sequences from 39 fungal samples representing nine taxa. The dataset had an aligned length of 1989 bp, including 1474 constant characters, 34 parsimony-uninformative variables, and 481 parsimony-informative characters. Bayesian and ML analyses resulted in the same topology. Only the ML tree is provided in Fig. 2, and the ML bootstrap (ML-BS) and Bayesian posterior probabilities (BPP) are shown at the nodes. The phylogenetic tree showed that P. djamor and P. sinensis clustered into two distinct groups with high support (95% ML-BS, 1.00 BPP for P. djamor; 98% ML-BS, 1.00 BPP for P. sinensis; Fig. 2). 124 MycoKeys 126: 119–133 (2025), DOI: 10.3897/mycokeys.126.162530 Long Zeng et al.: Taxonomy and phylogeny of the Pleurotus djamor complex Taxonomy Pleurotus sinensis L. Zeng, Y.F. Sun & B.K. Cui, sp. nov. MycoBank No: 861390 Figs 3, 4 Diagnosis. Pleurotus sinensis is characterized by a pileus that is white to pinkish buff or flesh-pink pileus when young, turning yellow at maturity, flabelliform, spatulate, or petaloid in shape, with inflexed and occasionally wavy margins with age. Holotype. China • Liaoning Province, Shenyang, Dadong District, 3 July 2024, Cui 23439 (BJFC). Etymology. “sinensis” (Lat.) refers to specimens derived from China. Description. Pileus 30–70 × 35–90 mm, flabelliform, spatulate, or petaloid, white to pinkish buff (5A3) or flesh-pink (8A3/9A4) when young, becoming yellow at maturity; with inflexed and wavy margin with age; glabrous, smooth to touch; margin entire (Fig. 4). Lamellae decurrent, margin entire, l.5–3 mm in dry state, white when young becoming pinkish buff (5A3) when old. Stipe 5–15 mm long × 5–10 mm diam, laterally stipitate or sessile. Context 1–1.5 mm thick when dry. Figure 1. Phylogenetic analysis of the Pleurotus djamor complex based on the ITS sequence dataset. 125 MycoKeys 126: 119–133 (2025), DOI: 10.3897/mycokeys.126.162530 Long Zeng et al.: Taxonomy and phylogeny of the Pleurotus djamor complex Figure 2. Phylogenetic analysis of the Pleurotus djamor complex based on the combined ITS + tef1α + rpb2 dataset. Basidiospores (6.2–) 6.8–9.8 (–10.2) × (3.3–) 3.5–5.5 (–5.7) μm, L = 8.08 μm, W = 4.18 μm, Q = 1.78–2.11 (n = 60/2), cylindrical-oblong, hyaline, thin-walled, smooth, IKI–, CB–. Basidia 23.6–28.3 × 5.2–6.8 μm, clavate, 4–spored, hyaline, thin-walled. Basidioles 15.8–24.2 × 4.9–5.2 μm, in shape similar to basidia. Cheilocystidia hyaline, thin-walled, clavate with mucronate, 22–41.7 × 2.6–3.4 μm. Pleurocystidia absent. Hymenophoral trama dimitic, with clamped generative hyphae 3.6–6.8 μm diam and skeletal hyphae 2.5–4.9 μm diam. Pileus trama dimitic, with generative hyphae 3.4–7.7 μm diam and skeletal 126 MycoKeys 126: 119–133 (2025), DOI: 10.3897/mycokeys.126.162530 Long Zeng et al.: Taxonomy and phylogeny of the Pleurotus djamor complex Figure 3. Basidiomata of Pleurotus sinensis in the field. A. (Cui 23439, holotype); B. (Cui 23450, paratype); C. (Cui 23262, paratype); D. (Cui 23263, paratype). Scale bars: 1 cm. Figure 4. Microscopic structures of Pleurotus sinensis (Cui 23439, holotype). A. Basidiospores; B. Hyphae of the hymenium; C. Basidia and basidioles; D. Lamella section; E, F. Cheilocystidia. Scale bars: 10 µm (A–F). 127 MycoKeys 126: 119–133 (2025), DOI: 10.3897/mycokeys.126.162530 Long Zeng et al.: Taxonomy and phylogeny of the Pleurotus djamor complex hyphae 2.5–4.8 μm diam. Stem context also dimitic, with generative hyphae 4.5–6.8 μm diam and skeletal 2.6–5.2 μm diam. Habitat and distribution. Solitary, gregarious to imbricate, on angiosperm trees or on dead and decaying wood in the subtropical and temperate zones of China. Additional specimens examined (paratypes). China • Guizhou Province, Guiyang, Guizhou Academy of Agricultural Sciences, 17 June 2016, Dai 16572 (BJFC); • Guangdong Province, Shenzhen, Futian District, Futian Mangrove Ecological Park, 20 April 2024, Cui 23262 (BJFC), Cui 23263 (BJFC); • Sichuan Province, Chengdu, 1 August 2024, Cui 23999 (BJFC); • Liaoning Province, Fushun, 4 July 2024, Cui 23450 (BJFC). Pleurotus djamor (Rumph. ex Fr.) Boedijn, Rumphius Mem. Vol. 292, 1959. MycoBank No: 355683 Figs 5, 6 Diagnosis. Pleurotus djamor is characterized by a pileus that is clay-pink to salmon or white when young, becoming thicker and turning brown at maturity. Description. Pileus 20–70 × 20–100 mm, flabelliform, clay-buff to salmon (6B/C4–6A4) or white when young, brown at maturity; surface dry, glabrous; margin entire. Lamellae decurrent, margin entire, l–7.5 mm in dry state, white when young becoming pinkish buff (5A3) when old. Stipe 5–10 mm long, 4–8 mm in diam., laterally stipitate or sessile. Context 1–3 mm thick when dry. Figure 5. Basidiomata of Pleurotus djamor in the field. A. (Cui 16861); B. (Cui 16862); C. (Cui 16902); D. (Cui 16890). Scale bars: 1 cm.