Global Diversity and Updated Phylogeny of Auricularia (Auriculariales, Basidiomycota)

Auricularia has a worldwide distribution and is very important due to its edibility and medicinal properties. Morphological examinations and multi-gene phylogenetic analyses of 277 samples from 35 countries in Asia, Europe, North and South America, Africa, and Oceania were carried out. Phylogenetic analyses were based on ITS, nLSU, rpb1, and rpb2 sequences using methods of Maximum Likelihood and Bayesian Inference analyses. According to the morphological and/or molecular characters, 37 Auricularia species were identified. Ten new species, A. camposii and A. novozealandica in the A. cornea complex, A. australiana, A. conferta, A. lateralis, A. pilosa and A. sinodelicata in the A. delicata complex, A. africana, A. srilankensis, and A. submesenterica in the A. mesenterica complex, are described. The two known species A. pusio and A. tremellosa, respectively belonging to the A. mesenterica complex and the A. delicata complex, are redefined, while A. angiospermarum, belonging to the A. auricula-judae complex, is validated. The morphological characters, photos, ecological traits, hosts and geographical distributions of those 37 species are outlined and discussed. Morphological differences and phylogenetic relations of species in five Auricularia morphological complexes (the A. auricula-judae, the A. cornea, the A. delicata, the A. fuscosuccinea and the A. mesenterica complexes) are elaborated. Synopsis data on comparisons of species in the five complexes are provided. An identification key for the accepted 37 species is proposed.


Introduction
Members of Auricularia Bull. (Auriculariaceae, Auriculariales), typified by A. mesenterica (Dicks.) Pers. [1], are widely distributed and are recognized for their ecological and economic values and medicinal properties. Most species play an important role in degradation in forest ecosystems, especially in tropical forests, usually inhabiting angiosperm wood, such as dead trees, stumps, fallen trunks and branches, and rotten wood, with a few growing on gymnosperm wood [2,3]. Several species are widely used as important edible and medicinal mushrooms in China and other East Asian countries, e.g., A. heimuer F. Wu & al., and A. cornea Ehrenb. [4,5]. Auricularia heimuer was considered as a delicacy of the emperor in the Eastern Zhou Dynasty 2000 years ago [6], and it has been cultivated for over 1400 years [7]. In China in 2019, 7.1 billion kg (fresh weight) of Auricularia heimuer, valued at more than ¥36.5 billion RMB (≈$5.6 billion USD). The species is widely cultivated and is the second most important edible species, after Lentinula edodes (Berk.) Pegler, in China. Besides being a food, A. heimuer has properties of lowering blood sugar and fat levels, antitumor, antioxidant, and immunity enhancement [8,9].
Morphologically, Auricularia is characterized by gelatinous, resupinate to substipitate basidiomata with hairs on the upper surface, cylindrical to clavate and transversely threeseptate basidia with oil guttules and hyaline, thin-walled and allantoid basidiospores [10][11][12][13][14]. Species identification, for a long time, has been based on macro-morphological characters, such as colour and size of basidiomata and length of hairs as introduced by Barrett and Kobayasi [15,16]. Lowy introduced the hyphal structure of internal stratification of basidiomata as a species differentiating character [10], and subsequently, this method of identification was accepted by Kobayasi [12], Lowy [17], and Li [18]. However, the macromorphological characters of Auricularia species present plasticity, resulting in inaccurate identification of similar species.
The introduction of molecular methods revealed several misidentifications. Scientific names of some important species were revised, and some new species were described. A Chinese Auricularia species named as "Maomuer", widely cultivated in southern China, was identified as A. polytricha (Mont.) Sacc. for over 100 years [4,19]. However, "Maomuer" is actually A. cornea, and A. polytricha, originally described from Jamaica, is a synonymy of A. nigricans (Sw.) Birkebak et al. [20]. Another Chinese species named as "Heimuer", the most important cultivated species of Auricularia in China, has been mistakenly named A. auricula-judae (Bull.) Quél., a species originally described from Europe [21], but it is a new species, A. heimuer [5]. An additional ten new species, including A. scissa  [22][23][24][25][26]. In addition, the phylogenetic analysis based on nLSU sequences showed that Auricularia was polyphyletic [27][28][29], but the genus was shown to be monophyletic in recent multi-gene phylogenetic analyses based on ITS and nLSU sequences [30,31].
Although afore-mentioned studies on phylogeny and taxonomy of Auricularia were carried out [20,[22][23][24][25][26], they are either based on limited samples or focus on one species complex. Thus, the aim of this study is to improve the current knowledge of the phylogeny and species diversity of Auricularia worldwide by analyzing 277 samples (including 27 type specimens) from 35 countries in Asia, Europe, North and South America, Africa and Oceania. According to morphological examinations and phylogenetic analyses, 37 species of Auricularia are recognized, and morphological differences and phylogenetic relations of species in five Auricularia complexes, viz., A. auricula-judae, A. cornea, A. delicata, A. fuscosuccinea, and A. mesenterica, are elaborated.  [23] and Lowy [10,17]. Colour terms follow Petersen [32]. Macro-morphological description of thirteen species based on 31 herbarium specimens are described after their whole basidiomata were rehydrated. Cross-sections of dried basidiomata were mounted in 5% potassium hydroxide (KOH) and 1% Congo Red and examined at 1000× magnification using a Nikon Eclipse 80i microscope. Microscopic structures were photographed using a Nikon Digital Sight DS-Fi1 camera. Basidiospores were measured in Cotton Blue (CB) and to test for cyanophily. Melzer's reagent (IKI) was used to note any chemical reaction of spores. When presenting spore size variation, 5% of measurements from each end of the range were excluded and indicated in parentheses. The following abbreviations were used: IKI + = amyloid or dextrinoid reaction, IKI-= neither amyloid or dextrinoid, CB-= acyanophilous, L = mean spore length (arithmetic average of all spores), W = mean spore width (arithmetic average of all spores), Q = L/W ratio for each specimen studied, n (a/b) = number of spores (a) measured from given number of specimens (b).

DNA Extraction, PCR and Sequencing
CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd., Beijing, China) was used to obtain DNA from 277 dried specimens, according to the manufacturer's instructions with some modifications. 2× EasyTaq PCR SuperMix (Trans-Gen biotech, Beijing, China) was used for reaction to obtain PCR products. ITS region was amplified with primer pairs ITS5 (GGA AGT AAA AGT CGT AAC AAG G) and ITS4 (TCC TCC GCT TAT TGA TAT GC) [33]. Nuclear LSU region was amplified with primer pair LR0R (ACC CGC TGA ACT TAA GC) and LR7 (TAC TAC CAC CAA GAT CT) (https://sites.duke.edu/vilgalyslab/rdna_primers_for_fungi/, accessed on 3 September 2021), the rpb1 with primers Af (GAR TGY CCD GGD CAY TTY GG) and Cr (CCN GCD ATN TCR TTR TCC ATR TA) [34], and the rpb2 with primers b6F (TGG GGK WTG GTY TGY CCT GC) and b7.1R (CCC ATR GCT TGY TTR CCC AT) [35]. PCR conditions were as follows: for ITS and rpb2, initial denaturation at 95 • C for 3 min, followed by 34 cycles at 94 • C for 40 s, 54 • C for 45 s, 72 • C for 1 min; for nLSU, initial denaturation at 94 • C for 1 min, followed by 34 cycles at 94 • C for 30 s, 50 • C for 1 min, 72 • C for 1.5 min, for rpb1 initial denaturation at 95 • C for 3 min, followed by 9 cycles at 94 • C for 40 s, 60 • C for 40 s and 72 • C for 2 min, then followed by 37 cycles at 94 • C for 45 s, 55 • C for 1.5 min and 72 • C for 2 min, and a final extension of 72 • C for 10 min. DNA sequencing was performed at Beijing Genomics Institute, China, with the same primers. Sequences of nLSU, rpb1 and rpb2 were generated in both directions.

Phylogenetic Analyses
All newly generated sequences and additional related sequences downloaded from GenBank are listed in Table 1 and were aligned using MAFFT 7.0 online service with the Q-INS-i strategy using the default parameters [36]. Multiple sequences were concatenated in Mesquite v. 3.10 [37]. Sequence alignments were deposited in TreeBase (submission ID 28649).
To define the high-rank phylogenetic relations within the Auriculariales, we used a dataset composed of concatenated ITS+nLSU sequences resulting in a final alignment comprising 156 different specimens. Heteroradulum deglubens (Berk. & Broome) Spirin & Malysheva was used as the outgroup. The concatenated ITS+nLSU dataset has an aligned length of 1953 characters, of which 600 characters are from locus ITS, and 1353 characters are from locus nLSU. We used another dataset composed of concatenated ITS+nLSU+rpb1+rpb2 sequences resulting in a final alignment comprising 106 different specimens to define phylogenetic relations within Auricularia, especially within closely related Auricularia species. Elmerina efibulata (Y.C. Dai & Y.L. Wei) Y.C. Dai & L.W. Zhou was used as the outgroup to root trees because Elmerina Bres. is closer to Auricularia than other genera of Auriculariales in phylogeny [30]. The concatenated ITS+nLSU+rpb1+rpb2 dataset has an aligned length of 3817 characters, of which 594 characters are from locus ITS, 1353 characters are from locus nLSU, 1336 characters are from locus rpb1, and 534 characters are from locus rpb2. All characters were equally weighted and gaps were treated as missing data.   Maximum Likelihood (ML) and Bayesian Inference (BI) methods were used for each dataset to enhance the reliability of phylogenetic analyses. The optimal substitution models suitable for both datasets were determined using the Akaike information criterion (AIC) implemented in MrModeltest 2.3 [38]. The ML tree was constructed using raxmlGUI 1.2 [39,40], and the BI tree was calculated with MrBayes3.2.5 [41]. The SYM+I+G and GTR+I+G models were selected as the best models for the ITS alignment and the nLSU alignment of the concatenated ITS+nLSU dataset respectively. The GTR+I+G model was selected as the best model for each alignment of the concatenated ITS+nLSU+rpb1+rpb2 dataset. Therefore, the ML analysis based on each dataset was calculated under the model GTRGAMMA. The BI analysis based on the concatenated ITS+nLSU dataset was calculated under the SYM+I+G model and the GTR+I+G model in two alignments, and based on the concatenated ITS+nLSU+rpb1+rpb2 dataset under the GTR+I+G model in four alignments. Four Markov chains were run for two runs from random starting trees for 1 million generations for the concatenated ITS+nLSU dataset, 1 million generations for the concatenated ITS+nLSU+ rpb1+rpb2 dataset, and trees were sampled every 100 generations. BI analysis stopped after effective sample sizes (ESSs) reached more than 200 and the potential scale reduction factors (PSRFs) were close to 1.000 for all parameters. The first one-fourth generations were discarded as burn-in. A majority rule consensus tree of all remaining trees was calculated. Branches that received bootstrap support for Maximum Likelihood (BS) and Bayesian Posterior Probabilities (BPP) greater than or equal to 50% (BS) and 0.90 (BPP) were considered as significantly supported, respectively.

Phylogenetic Analyses
The ML analysis based on the concatenated ITS+nLSU dataset resulted in a similar topology as Bayesian Inference analysis, so only the ML tree is presented (Figure 1). The phylogeny demonstrated that 149 Auricularia specimens formed one large clade with high support, which confirmed the monophyletism of Auricularia. Most of the 31 Auricularia species formed monophyletic lineages with high support, and several species including A. heimuer and A. submesenterica didn't form monophyletic lineages, however, these two species formed two distinct lineages with high support in the phylogeny based on the concatenated ITS+nLSU+rpb1+rpb2 dataset ( Figure 2). The 31 Auricularia species formed three major clades, Clade A-C. Clade A includes 16 species in the A. cornea, A. delicata and A. fuscosuccinea complexes. Clade B includes seven species belonging to the A. auricula-judae complex, and Clade C includes eight species belonging to the A. mesenterica complex. Although species in the A. auricula-judae complex and the A. mesenterica complex clustered into two monophyletic clades, Clade B and Clade C respectively, species in the other three morphological complexes were scattered in small different clades in Clade A. The analyses showed that the morphological complexes do not fully correspond to the phylogenetic clades.
ML analysis based on the concatenated ITS+nLSU+rpb1+rpb2 dataset resulted in a similar topology as Bayesian analysis, so only the ML tree was presented ( Figure 2). The phylogeny demonstrated a similar topology in Auricularia as the phylogeny based on the concatenated ITS+nLSU dataset (Figure 1), and also showed three major clades, Clade A-C. In Clade A, species of A. delicata, A. cornea and A. fuscosuccinea complexes did not form their own subclades. Species of A. auricula-judae and A. mesenterica complexes formed their own two clades, Clade B and Clade C.    Etymology-Africana (Lat.): refers to the distribution of the species in Africa. Basidiomata-Gelatinous when rehydrated, greyish brown to fuscous, caespitose, resupinate to effused-reflexed; pileus sometimes observed, free lobed, margin undulate, projecting up to 1 cm, 1.5-2 mm thick, 0.2-0.4 mm thick when dry; upper surface tomentose, concentrically zoned with canescent zones and dark bands, becoming yellowish brown to orange-brown upon drying; hymenophore surface venose with folds, becoming greyish brown to fuscous upon drying.
Spores-Basidiospores allantoid, hyaline, thin-walled, smooth, usually with one to three large guttules, IKI-, CB-, (12 Distribution-USA. Notes-Auricularia angiospermarum was described as a new species by Wu et al. [23], but the name was invalid because the registration identifier cited in the protologue, 'My-coBank no.: MB 812851 , was not issued for the name published. Therefore, the name is validated here. The species was segregated from A. americana. Morphologically, there is no distinct differences between the two species and they were considered as a single species by Looney et al. [20]. However, these two species are clustered in two distinct lineages with strong support in the phylogenies (Figures 1 and 2), and A. angiospermarum grows on angiosperm rather than on gymnosperm wood. Auricularia angiospermarum has so far been reported in North America only, while A. americana occurs both in North America and north Asia. The description taken from Wu et al. [23]. Basidiomata-Gelatinous when fresh, fawn to greyish brown or buff to cream, caespitose, resupinate to effused-reflexed; pileus free lobed, margin undulate, projecting up to 5 cm, 1-3 mm thick, 0.12-0.16 mm thick when dry; upper surface villose, distinctly and concentrically zoned with wide whitish zones and thin black bands in the central pileal surface, becoming clay pink upon drying; hymenophore surface venose with folds, becoming dark greyish blue upon drying.
Spores-Basidiospores allantoid, hyaline, thin-walled, smooth, usually with one or two large guttules, IKI-, CB-, (11-)11.2-12.  Notes-Auricularia asiatica was recently described from Asia by Bandara et al. [26]. It belongs to the A. mesenterica complex and is very similar to A. brasiliana Y.C. Dai & F. Wu by sharing resupinate to effused-reflexed, basidiomata with a free lobed pileus and similar-sized basidiospores, but the latter is distinctly and concentrically zoned with wide whitish zones and thin black bands throughout the pileal surface [25]. Phylogenetically, the species is distant from A. brasiliana and closely related to a new species, A. srilankensis (Figures 1 and 2). Differences between A. asiatica and A. srilankensis are discussed in the notes on A. srilankensis.    Basidiomata-Gelatinous when fresh, reddish brown to fuscous or white, solitary, sometimes caespitose, sessile or substipitate; pileus cupulate or auriculate, sometimes with lobed margin, projecting up to 9 cm, 1-3 mm thick, 0.3-0.48 mm thick when dry; upper sur-face pilose, becoming yellowish brown or pinkish buff upon drying; hymenophore surface usually smooth, without folds, becoming fuscous to black or cinnamon buff upon drying.
Spores-Basidiospores allantoid, hyaline, thin-walled, smooth, usually with one to two large guttules, IKI-, CB-, ( (Figures 1 and 2), but it differs from A. delicata and A. sinodelicata by its wider hairs (5-8 µm in diam in A. delicata and 6-9 µm in diam in A. sinodelicata), and from A. lateralis by shorter hairs (95-250 µm long in A. lateralis). The species is easily confused with A. conferta also described from Australia, but A. conferta has dense and thick folds on the hymenophore surface and wider hairs (45-95 × 8-15 µm in A. conferta) usually with a wide and regular septate lumen.
Distribution-Brazil. Notes-Auricularia brasiliana was described from Brazil [25], and is macro-morphologically similar to A. asiatica, which, however, lacks whitish zones and thin black bands at margin of pileal surface [26]. These two species form two distinct lineages with strong support in our phylogenies (Figures 1 and 2). The morphological description of Auricularia brasiliana taken from Wu et al. [25].   Basidiomata-Gelatinous when rehydrated, orange-brown to reddish brown, solitary or caespitose, sessile or substipitate; pileus discoid or auriculate, margin entire, projecting up to 5 cm, 1-3 mm thick, 0.2-0.45 mm thick when dry; upper surface densely tomentose, becoming greyish brown upon drying; hymenophore surface usually smooth, without folds, becoming bluish grey to greyish blue upon drying.
Spores-Basidiospores reniform, hyaline, thin-walled, 12.5-15 × 5-6 µm. Distribution-Brazil. Notes-Auricularia camposii is characterized by a densely tomentose upper surface, with an obvious medulla in the middle of the cross-section. It is similar to A. cornea in macro-morphology, but has slightly shorter abhymenial hairs (120-250 µm vs. 180-425 µm). In the phylogeny, A. camposii is very distantly related to A. cornea and samples of these two species form two lineages (Figures 1 and 2). Auricularia camposii is relatively related to A. nigricans (Figures 1 and 2) which, however, has distinctly longer abhymenial hairs (300-600 × 7-9 µm vs. 120-250 × 6-7 µm). The type and paratype are good enough and ITS and nLSU sequences are available from them. Therefore, we venture to publish a description for A. camposii even though we know it is inadvisable to do so based on a few basidiospores.
Distribution-Australia. Notes-Auricularia conferta is macro-morphologically similar to A. sinodelicata because A. sinodelicata sometimes also has dense and thick folds on the hymenophore surface.   Basidiomata-Gelatinous when fresh, fawn to reddish brown or buff to white, solitary or caespitose, sessile or substipitate; pileus discoid or auriculate, sometimes with lobed margin, projecting up to 9 cm, 1-2 mm thick, 0.2-0.48 mm thick when dry; upper surface densely pilose, becoming light vinaceous grey or cream upon drying; hymenophore surface usually smooth, without folds, becoming mouse-grey to black or buff-yellow upon drying.
Distribution-Africa, North and South America, Asia and Europe. Notes-Auricularia cornea is characterized macroscopically by the variability in color of fresh basidiomata, dense hairs on the upper surface, and microscopically by the presence of an obvious medulla. It has a wide distribution, being recorded almost all over the world, and is very common in both natural and managed forests of subtropical and tropical areas. It is cultivated in China as "Maomuer" and was first reported as A. polytricha by Patouillard and Olivier [19], a name used in almost all Chinese reports until 2015 and in other Asian and Pacific areas [12,17,[42][43][44][45][46][47][48][49][50][51]. However, A. polytricha is a synonym of A. nigricans, which is distributed throughout America. Zhao & Wang described A. polytricha var. argentea D.Z. Zhao & Chao J. Wang based on its white basidiomata [52]. According to our study, A. polytricha var. argentea [basidia measuring 50-65 × 4-5.5 µm; basidiospores measuring (12.7-)13-15(-15.2) × (4.7-)4.9-5.4(-5.6) µm, L = 13.91 µm, W = 5.02 µm, Q = 2.77 (n = 30/1), from type material] is a synonym of A. cornea. In fact, we found that A. cornea produced both brown and white basidiomata in nature (Figure 15h). In our phylogenies (Figures 1 and 2), all samples of A. cornea, wild A. polytricha var. argentea (Dai 14876) and cultivated A. polytricha var. argentea (Wu 07) form a lineage although with some variations. The white basidiomata of A. cornea is called "Yumuer" and is now cultivated in China (Figure 15g).
In addition, A. reticulata L.J. Li was reported as a new species from China due to its reticulate sterile surface [53]. However, A. cornea sometimes has a reticulate sterile surface, too, e.g., Cui 7517 ( Figure 15f). We examined the type of A. reticulata, and found it has an obvious medulla and the abhymenial hairs are almost the same as those of A. cornea. We did not find basidiospores from the type, but the spores were reported as 14.5 × 5-6 µm in the original description [53], which fit the dimensions of those of A. cornea. For these reasons, we treat A. reticulata as a synonym of A. cornea. Another species, A. leucochroma, was also described from Asia [16], and treated as a synonym of A. nigricans [20]. We rejected this opinion because we examined the type material of A. leucochroma, and treated it as a synonym of A. cornea. Because of the variation of morphology Figure 15a-h) and of the molecular data (Figures 1 and 2), in the present study all these samples with variable morphology are treated as A. cornea for the time being.  Basidiomata-Gelatinous when rehydrated, dark brownish to vinaceous brown, solitary or caespitose, sessile or substipitate; pileus orbricular, sometimes with lobed margin, projecting up to 8 cm, 1.5-2.5 mm thick, 0.18-0.28 mm thick and dark reddish brown to fuscous when dry; upper surface pilose, sometimes with several folds; hymenophore surface conspicuously porose-reticulate.
Distribution-Western Africa. Notes-Auricularia delicata was originally described from Equatorial Guinea in West Africa and it was reported throughout tropical America, Africa, Asia, Australia and South Pacific [12,17]. Auricularia delicata was found to be a species complex, and two new species belonging to the complex, A. scissa and A. subglabra, were described because they have schizomedulla, which is distinctly different from the original description of the medulla of A. delicata by Looney et al. [20]. In addition, samples from Australia and Mexico form two other lineages in the phylogeny, which were treated, respectively, as A. delicata clade I and A. delicata clade II by Looney et al. [20], because of the shortage of samples from the type locality of A. delicata.
In the present study, one collection from Cameroon (K 43873) was morphologically examined and phylogenetically analyzed, and its morphology fits the description of A. delicata. In addition, Cameroon is very close to Equatorial Guinea, so we consider the specimen (K 43873) represents the real A. delicata, and K 43873 is treated as epitype of A. delicata. The A. delicata clade I and A. delicata clade II are, respectively, treated as A. australiana and A. tremellosa (Fr.) Pat. in our study (Figure 1). Auricularia tremellosa was originally described from Mexico [53], and more samples were morphological and phylogenetically studied. These samples form a distinct lineage (Figures 1 and 2) and their morphology fits the description of A. tremellosa.
Distribution-China. Notes-Auricularia eburnea is characterized by cream to honey-yellow fresh basidiomata. According to our observations of the type, the species is macro-morphologically similar to A. cornea, but A. eburnea has obviously larger basidiospores than those in A. cornea (15.6-18 × 5.5-7.5 µm vs 13.8-16.5 × 4.5-6 µm). We failed to extract DNA, because the type material is in poor condition. For the time being we accept A. eburnea as an independent species based on its original description.
Distribution-Africa. Notes-Auricularia eminii is characterized by the longest hairs on the upper surface in the genus and is distributed in Africa only. Lowy indicated that A. squamosa was a synonym of A. eminii [17]. In the present study, we confirm this synonym because the morphological differences between the two type specimens are too small to justify the segregation of these two species.
Spores-Basidiospores allantoid, hyaline, thin-walled, smooth, usually with one or two large guttules, IKI-, CB-, (10.  Notes-Auricularia fibrillifera was originally described from Papua New Guinea [55], and the type specimen (F 234519) was studied. Auricularia thailandica Bandara & K.D. Hyde was recently described from Thailand by Bandara et al. [24], and it is very close to A. fibrillifera both in morphology and phylogeny. Both species occur in Southeast Asia and it is very difficult to distinguish them by morphology, especially the dry basidiomata. However, these two species clustered into two distinct lineages in our phylogeny based on the concatenated ITS+nLSU dataset ( Figure 1); furthermore, the fresh basidiomata of A. fibrillifera are softer than those of A. thailandica. Therefore, we treat A. fibrillifera and A. thailandica as two independent species in the present study.   Basidiomata-Gelatinous when fresh, fawn to cinnamon brown, solitary or caespitose, sessile or substipitate; pileus discoid or auriculate, margin entire, projecting up to 6.5 cm, 1-3 mm thick, 0.15-0.3 mm thick and cinnamon brown to vinaceous brown when dry; upper surface pilose, sometimes with folds; hymenophore surface smooth, sometimes with folds.
Distribution-Tropical and subtropical America. Notes-Auricularia fuscosuccinea is characterized by macroscopically cinnamon brown basidiomata when dry and microscopically by a medulla distinctly present near the abhymenium. It is similar to A. fibrillifera and A. thailandica but differs by having slightly thicker basidiomata and hyphae with simple septa. In the phylogeny, A. fuscosuccinea is distantly related to A. fibrillifera and A. thailandica (Figures 1 and 2). The species was previously reported in China [44]. Vouchers of Chinese materials were studied, and they are actually A. fibrillifera or A. thailandica. Because the three species share similar morphology, A. fuscosuccinea is temporarily considered as a species complex even if the phylogenetic relations are distant (Figures 1 and 2). Basidiomata-Gelatinous when rehydrated, fuscous to vinaceous brown, solitary, sessile or substipitate; pileus auriculate, margin entire, projecting up to 3.5 cm, 2-3.1 mm thick, 0.6-0.7 mm thick and mouse-grey to black when dry; upper surface scantly pilose, sometimes with folds; hymenophore surface with obvious folds.
Distribution-China. Notes-Auricularia hainanensis is characterized by very thick basidiomata when dried and very small basidiospores. According to the original description and our observation of the type, the species is morphologically similar to A. minor Kobayasi, but that species has thinner basidiomata (0.1-0.2 mm vs. 0.6-0.7 mm) and hymenophore surface without distinct folds. Because the type materials of both A. hainanensis and A. minor are in poor condition, and DNA extraction from the types failed, their phylogenetic relations remain uncertain.  Basidiomata-Gelatinous when fresh, fawn to reddish brown, solitary or caespitose, sessile or substipitate; pileus auriculate or petaloid, margin entire, projecting up to 12 cm, 0.8-1.5 mm thick, 0.04-0.24 mm thick and greyish brown to vinaceous brown when dry; upper surface pilose, sometimes with folds; hymenophore surface smooth, sometimes with shallow folds.
Distribution-Temperate Asia. Notes-Morphologically A. heimuer is similar to A. villosula Malysheva, but the latter has larger basidiospores (13-15.5 × 5-6.1 µm). Auricularia heimuer has a wide distribution in temperate Asia and grows mostly on Quercus, but basidiomata from the wild are uncommon. Previously the Asian A. heimuer was considered the same species as the European A. auricula-judae [56,57]. The cultivated and the wild A. heimuer (Figure 5e,f) were different from A. auricula-judae in both morphology (refer to the notes on A. auricula-judae) and phylogeny, in which they clustered into two different lineages, each one with high support (Figure 2). The morphological description of A. heimuer taken fromWu et al. [5].
Distribution-China. Notes-Auricularia lateralis is characterized macroscopically by a conspicuously porosereticulate hymenophore, and microscopically by wide abhymenial hairs with slightly swollen center. The species belongs to the A. delicata complex and it is morphologically close to A. pilosa Y.C. Dai & F. Wu sharing abhymenial hairs > 100 µm long, but A. pilosa has shorter basidia (35-45 × 4-5.5 µm) and is distributed in Africa. Phylogenetically, the species is distantly related to A. pilosa and forms a distinct lineage with high support (Figures 1 and 2).  Basidiomata-Gelatinous when fresh, greyish brown to fuscous or buff to white, caespitose, resupinate to effused-reflexed; pileus free lobed, margin undulate, projecting up to 7 cm, 1.5-3 mm thick, 0.2-0.3 mm thick when dry; upper surface hispid, distinctly and concentrically zoned with canescent zones and dark bands, becoming olivaceous buff upon drying; hymenophore surface venose with obvious folds, becoming fawn to reddish brown upon drying.
Spores-Basidiospores allantoid, hyaline, thin-walled, smooth, usually with one or two large guttules, IKI-, CB-, (13.8-)14-17(-17.6) × (4.5-)4.7-5.  Notes-Auricularia mesenterica is a common species in Europe and grows on species of different angiosperm wood usually in summer to autumn. The species was originally described from Europe, but is also reported from the Americas and Asia [14,20,43]. However, A. mesenterica was reported as a species complex with three species (A. asiatica, A. brasiliana and A. orientalis Y.C. Dai & F. Wu) [25,26]. In the present study, the other two new species, A. africana and A. submesenterica Y.C. Dai & F. Wu, are described. In addition, A. pusio, originally described from Australia [58], is re-studied and phylogenetically analyzed. We confirmed that it belongs to the A. mesenterica complex and is an independent species.
Morphologically, the A. mesenterica complex is distinct from other species in the genus by its resupinate to effused-reflexed basidiomata, the upper surface usually with distinct and concentric zones and a venose hymenophore surface with obvious folds. Auricularia mesenterica has the largest basidiospores among the species in the complex. Phylogenetically, A. mesenterica forms a single lineage with strong support and is closely related to A. orientalis and A. submesenterica (Figures 1 and 2).  Basidiomata-Gelatinous when rehydrated, greyish brown to fawn, solitary, sessile; pileus discoid or auriculate, margin entire, projecting up to 6.5 cm, 0.65-0.8 mm thick, 0.1-0.2 mm thick when dry; upper surface scantly pilose, becoming mouse-grey upon drying; hymenophore surface smooth, sometimes with indistinct folds, becoming black upon drying.
Notes-Auricularia minor was originally described from tropical Asia [12], and is characterized by its solitary and very small basidiomata. We failed to extract DNA because the type material and other samples are in poor condition, so its phylogenetic relations with other species are uncertain. Morphologically, A. minor is very similar to A. hainanensis described from tropical Asia, but the abhymenial hairs in A. minor (90-120 × 6-10 µm) are longer than those in A. hainanensis (30-  Basidiomata-Gelatinous when fresh, vinaceous grey to fuscous or clay buff to pinkish buff, solitary or caespitose, sessile; pileus discoid or auriculate, sometimes with lobed margin, projecting up to 4 cm, 0.5-2 mm thick, 0.04-0.2 mm thick when dry; upper surface pilose, sometimes with folds, becoming vinaceous grey upon drying; hymenophore surface smooth or with obvious folds, becoming fuscous or black upon drying. Internal features-Medulla present in the middle of the cross-section; crystals present, usually scattered in the hymenium; abhymenial hairs with a slightly swollen base, hyaline, thick-walled, with a narrow lumen, sometimes septate, apical tips acute or obtuse, single or tufted, 50-85(-105) × 5-6 µm; hyphae with clamp connections, 1-3.5 µm in diam in KOH; basidia clavate, transversely 3-septate, with oil guttules, 46-67 × 5-8 µm, sterigmata occasionally observed; cystidioles absent.
Distribution-China and Russia. Notes-Auricularia minutissima belongs to the A. auricula-judae complex and is characterized by its small basidiomata. The species is similar to A. heimuer and A. minor, but the latter two species have smaller basidiospores (11-13 × 4-5 µm in A. heimuer and 7-8 × 3-4 µm in A. minor). A. minutissima has two kinds of basidiomata (Figure 5h,i) that are different in macro-morphology, but have the similar basidia, basidiospores and DNA data. In the phylogeny, A. minutissima is closely related to A. heimuer, but it forms a single lineage with strong support (Figures 1 and 2). The morphological description of A. minutissima taken from Wu et al. [23].   Internal features-Medulla obviously present near the abhymenium; crystals present, usually scattered in the hymenium; abhymenial hairs with a slightly swollen base, hyaline, thick-walled, with a narrow lumen, apical tips acute or obtuse, tufted, 300-600 × 7-9 µm; hyphae with clamp connections and simple septa, 0.5-4 µm in diam in KOH; basidia clavate, transversely 3-septate, with oil guttules, 50-60 × 4-6 µm, sterigmata rarely observed; cystidioles absent.
Distribution-North America. Notes-Auricularia polytricha is a name that has been applied to this species for a long time [12,17,51], but Looney et al. recently found that Exidia polytricha Mont. and Peziza nigricans represent the same species [20]. Since P. nigricans was an earlier name and thus had the priority, they proposed the combination A. nigricans (Sw.) Birkebak & al., and A. polytricha is treated as a synonym of A. nigricans.
Auricularia nigricans is characterized by the densely hispid upper surface and obvious medulla near the abhymenium. It is easily confused with A. cornea because of the variable morphology of A. cornea, but A. nigricans usually has a more hispid upper surface and is distributed in North America only. Phylogenetically, both species are distantly related (Figures 1 and 2). Etymology-Novozealandica (Lat.): refers to the species from New Zealand. Basidiomata-Gelatinous when rehydrated, reddish brown to fuscous, solitary or caespitose, sessile or substipitate; pileus cupulate or auriculate, sometimes with lobed margin, projecting up to 15 cm, 0.8-1.5 mm thick, 0.3-0.4 mm thick when dry; upper surface densely pilose, becoming greyish brown to clay buff upon drying; hymenophore surface usually smooth, without folds, becoming vinaceous grey upon drying.
Distribution-China. Notes-Auricularia orientalis was described as a new species in the A. mesenterica complex [25]. Compared to other species in this complex, only A. orientalis and A. mesenterica have cystidioles, but A. orientalis has smaller basidiospores than those of A. mesenterica (12.5-14.2 × 5-6 µm vs. 14-17 × 4.7-5.2 µm). Phylogenetically, samples of A. orientalis clustered in a single lineage with robust support (Figures 1 and 2). The morphological description of A. orientalis taken from Wu et al. [25].
Distribution-Ethiopia and Tanzania. Notes-Auricularia pilosa is characterized by the pilose upper surface, porose-reticulate hymenophore, and wide abhymenial hairs with slightly swollen center. The species belongs to the A. delicata complex and is easily confused with A. lateralis because of the distinctly pilose abhymenial surface, but it differs by the slightly shorter hairs and basidia (in A. lateralis 95-250 µm long and 50-70 µm long, respectively) and is distributed in Africa. Phylogenetically, A. pilosa forms a single lineage distant from other species in the A. delicata complex (Figures 1 and 2).
Distribution-Australia and Zambia. Notes-Auricularia pusio was originally described from Queensland (Australia) and was considered a synonym of A. mesenterica in the light of morphological characters (http://www.indexfungorum.org/Names/NamesRecord.asp?RecordID=156648, accessed on 3 September 2021), but A. mesenterica is a species complex [25]. Basidiospores of A. pusio are distinctly shorter and wider than those in A. mesenterica sensu stricto (14-17 × 4.7-5.2 µm). Additionally, our phylogenies show that the two species form two distinct and distantly related lineages (Figures 1 and 2). Therefore, we accept A. pusio as an independent species in the A. mesenterica complex.
Distribution-Dominican Republic and USA. Notes-Auricularia scissa was recently described from the Dominican Republic by Looney et al. [20], and is characterized macroscopically by conspicuously porose-reticulate hymenophores and microscopically by the presence of schizomedulla. It is like A. subglabra as both have porose-reticulate hymenophores and a schizomedulla, but the latter species has an almost smooth abhymenium and very short hairs (up to 45 µm). Phylogenetically, A. scissa forms a single lineage with high support (Figures 1 and 2) and is not closely related to A. subglabra.
Distribution-Tropical North and South America. Notes-Auricularia subglabra was described from Costa Rica by Looney et al. [20], and is characterized by slightly porose-reticulate hymenophores and the presence of schizomedulla. The species is similar to A. scissa, which, however, has a pilose upper surface and longer abhymenial hairs (40-100 µm). Phylogenetically, both species nest in two distinct lineages with high support (Figures 1 and 2).  Etymology-Submesenterica (Lat.): refers to the species being similar to Auricularia mesenterica. Basidiomata-Gelatinous when fresh, greyish brown to fuscous, caespitose, resupinate to effused-reflexed; pileus free lobed, margin undulate, projecting up to 7 cm, 1-2 mm thick, 0.12-0.2 mm thick when dry; upper surface hispid, distinctly and concentrically zoned with canescent zones and dark bands, becoming greyish brown to deep olive upon drying; hymenophore surface venose with obvious folds, becoming dark bluish grey to dark grey upon drying.

Distribution-China.
Notes-Auricularia submesenterica is characterized by caespitose, resupinate to effusedreflexed basidiomata with a lobed pileus, distinctly and concentrically zoned with canescent zones and dark bands on the upper surface, absence of medulla and cystidioles, and small basidia and basidiospores. Auricularia submesenterica definitely belongs to the A. mesenterica complex, but A. mesenterica sensu stricto differs from A. submesenterica by longer abhymenial hairs (1000-2000 µm), presence of cystidioles and larger basidiospores (14-17 × 4.7-5.2 µm). Auricularia orientalis is another member in the A. mesenterica complex and was recently described from China [25]; it resembles A. submesenterica by sharing similar abhymenial hairs and basidiospores, but A. orientalis usually has resupinate basidiomata and cystidioles in the hymenium. In addition, A. submesenterica, A. orientalis and A. mesenterica form three different lineages in our phylogenies (Figures 1 and 2).
Distribution-Mexico and tropical South America. Notes-Auricularia tremellosa was originally described from Mexico and then considered as synonym of A. delicata based on morphology by Lowy and Kobayasi [12,17]. However, in the present study, we accept A. tremellosa as an independent species because it is different from A. delicata sensu stricto not only in morphology but also in the phylogenies (Figures 1 and 2). Six Brazilian specimens were studied and their morphological characters fit the description of A. tremellosa. Auricularia tremellosa differs from A. delicata by its poroid hymenophore, smaller basidia (36-42 × 5-5.5 µm vs. 48-65 × 4-6 µm) and distribution in Basidiomata-Gelatinous when fresh, fawn to fuscous or buff yellow to orange yellow, solitary or caespitose, sessile or substipitate; pileus auriculate or petaloid, sometimes with lobed margin, projecting up to 5 cm, 1-2 mm thick, 0.08-0.3 mm thick and greyish brown to vinaceous brown when dry; upper surface pilose, sometimes with folds; hymenophore surface with obvious folds.
Distribution-China. Notes-Auricularia xishaensis has very thin basidiomata when fresh like A. fibrillifera, but it has distinctly larger basidiospores than those of A. fibrillifera (11-14 × 4-5 µm). In addition, A. xishaensis is easily identified by obvious folds and white particles on the hymenophore surface and plenty of crystals scattered throughout the cross-section. We failed to extract DNA because the type is in poor condition, so the placement of A. xishaensis in Auricularia remain uncertain.

Discussion
Currently 37 Auricularia species belonging to five species complexes (A. auriculajudae, A. cornea, A. delicata, A. fuscosuccinea and A. mesenterica) are recognized based on morphological and/or molecular data. DNA sequences of 31 Auricularia species were obtained, while the samples of another six species (A. eburnea, A. eminii, A. hainanensis, A. minor, A. papyracea and A. xishaensis) were poorly dried or contaminated during sampling and DNA extraction failed. The molecular data of these six species will tentatively be accessed when new samples from their type locality are collected in the future.
Auricularia was considered a polyphyletic group based on nLSU sequences [27,29], but it is shown to be monophyletic in our phylogeny based on the concatenated ITS+nLSU dataset (Figure 1), which confirms the results of recent phylogenetic analyses [30,31]. The phylogenetic analyses inferred from both datasets result in similar topology of Auricularia with three clades (Clade A, Clade B and Clade C). Clade A is somewhat complicated and includes the A. cornea, the A. delicata and the A. fuscosuccinea complexes, while the A. auricula-judae and the A. mesenterica complexes are nested in Clade B and Clade C, respectively (Figures 1 and 2). Clade A includes 16 species in both phylogenies. Those taxa cluster in three groups unrelated to the species complexes in the phylogeny based on the concatenated ITS+nLSU dataset. Group I and Group III form two distinct clades with strong support in the phylogenies (Figures 1 and 2). For the convenience of discussion, species in Group II in the phylogeny based on the concatenated ITS+nLSU dataset (Figure 1) are still regarded as one group, although these species do not cluster in one clade in the phylogeny based on the concatenated ITS+nLSU+rpb1+rpb2 dataset (Figure 2).
Group I is divided into two small clades which include two species respectively (A. cornea and A. novozealandica) in the A. cornea complex and five species (A. sinodelicata, A. delicata, A. australiana, A. conferta, and A. lateralis) in the A. delicata complex. Auricularia cornea was originally collected in the Marianna Islands [17], and it has been widely reported from Asian and Pacific areas [12,20,[49][50][51]. Although the lineage of A. cornea is not strongly supported and includes some small lineages in both phylogenies, those small lineages are not stable in both phylogenies (Figures 1 and 2). In addition, samples with mostly similar macro-morphology are distantly related, e.g., Wu 07 ( Figure 15g) and Dai 14876 (Figure 15h), and samples with slightly different morphology are closely related, e.g., Dai 15336 ( Figure 15e) and Cui 7517 (Figure 15f). However, all these samples have similar micro-morphology and share the characteristics of A. cornea [17,20], so they are treated as A. cornea in the present study. These variations in macro-morphology and molecular data may be due to a wide distribution in Africa, North and South America, Asia, and Europe. The new species A. novozealandica is closely related to A. cornea in our phylogenies (Figures 1 and 2) and macro-morphologically similar to a few specimens of A. cornea, but it has distinctly larger basidiospores (Table 2) and distribution restricted to New Zealand so far. The lineage of A. australiana from Australia was defined as A. delicata clade I in Looney et al. [20], and it is not supported in our phylogeny based on the concatenated ITS+nLSU dataset (Figure 1), but it is strongly supported in our phylogeny based on the concatenated ITS+nLSU+rpb1+rpb2 dataset (Figure 2). Auricularia conferta also from Australia has dense thick folds on the hymenophore surface and wider hairs than A. australiana (Table 3) with a wide and regular septate lumen. Auricularia sinodelicata and A. lateralis form two distinct lineages separated from A. delicata in the phylogenies (Figures 1 and 2), and they are different from A. delicata in morphology. Therefore, the four species are considered as new species in the A. delicata complex in the present study. Group II includes seven species: A. fuscosuccinea, A. subglabra, A. scissa, A. pilosa, A. nigricans, A. camposii, and A. tremellosa. The lineages of the species in this group are strongly supported, but their morphologies are not corresponding to their phylogenetic relations. Auricularia fuscosuccinea, originally collected in Cuba [17], resembles A. fibrillifera and A. thailandica because of the red-brown fresh basidiomata and all three species belong to the A. fuscosuccinea complex ( Figure 20). Auricularia subglabra, A. scissa, A. pilosa, and A. tremellosa have more or less fleshy basidiomata, pilose pileal surface and porose-reticulate hymenophore, and they belong to the A. delicata complex ( Figure 10). Auricularia camposii and A. nigricans have more or less leathery basidiomata, tomentose to hispid pileal surface and smooth hymenophore and they belong to the A. cornea complex (Figure 15). The new species, A. pilosa, from Ethiopia is described here based on only one small specimen (LWZ20190421-7), but the specimen together with JMH 45 from Tanzania form one lineage with high support in the phylogeny (Figure 1). So, these two specimens are recognized as the new species. Auricularia camposii is morphologically similar to A. cornea, but it has tomentose upper surface and forms one lineage distantly from A. cornea in the phylogenies (Figures 1 and 2). The lineage of A. tremellosa was defined as the "A. delicata clade II" in Looney et al. [20], and it is recognized as the known species A. tremellosa originally described from Mexico [53].
Group III includes two species: A. fibrillifera and A. thailandica. Both species have thin basidiomata when fresh becoming fragile when dry and are distributed in subtropical to tropical areas of Africa and Asia. The Chinese samples were previously considered as "A. fuscosuccinea" because of the similar morphology [42,44]. In the present study, we still list A. fibrillifera and A. thailandica as members of the A. fuscosuccinea complex although A. fuscosuccinea is only distantly related to A. fibrillifera and A. thailandica in the phylogenies (Figures 1 and 2).
The species from the A. cornea complex differ from other species in the genus by the smooth hymenophore surface, densely pilose, tomentose or hispid upper surface, long abhymenium hairs (>150 µm) and the presence of a medulla. Another two species, A. eburnea and A. eminii, also have the afore-mentioned characteristics, and we think they belong to the same complex even if the molecular data is lacking. The main characteristics of the seven species in the A. cornea complex are summarized in Table 2.
The Auricularia delicata complex is characterized by its porose-reticulate hymenophore surface, the absence of crystals, usually short abhymenium hairs (<100 µm) and small basidiospores. A synoptic table of a comparison of species in the complex is provided in Table 3.
The species from the A. fuscosuccinea complex are different from other species in the genus by the thin and fragile basidiomata when dry, pilose upper surface and short abhymenium hairs (<150 µm). The main characteristics of members in the complex are summarized in Table 4. Auricularia papyracea and A. xishaensis have very thin dry basidiomata and are similar to A. fuscosuccinea. We temporarily treat them as members of the A. fuscosuccinea complex although the phylogenetic relations among the three species are unknown. Clade B includes seven species belonging to the Auricularia auricula-judae complex (Figures 1 and 2). Samples of these seven species in the complex are nested in seven lineages with high support in our phylogenies, Intraspecific genetic variations are obviously present among samples of these taxa which is probably the reason why A. auricula-judae has considerable variability in the size of the basidiospores. Auricularia auricula-judae was originally described from Europe and was recognized as a species complex by Looney et al. [20]. They also confirmed that the so-called "A. auricula-judae" in the USA was actually A. americana, but two sublineages, represented by samples, on angiosperm and gymnosperm, respectively, clustered in their phylogeny based on sequences of ITS and rpb2. Later, based on a comprehensive phylogeny, Wu et al. considered that samples on gymnosperm represented A. americana sensu stricto because A. americana was originally described from gymnosperm [23], and samples on angiosperm were described as A. angiospermarum. Subsequently, A. heimuer, A. minutissima, A. tibetica and A. villosula were described based on phylogenetic analyses [5,20,22].
The species from the A. auricula-judae complex are characterized by usually reddish brown basidiomata, a pilose upper surface with short abhymenium hairs (<150 µm), and mainly smooth hymenophores ( Figure 5). Although we do not know the phylogenetic relations of A. hainanensis and A. minor with other members in the A. auricula-judae complex, we list them in this complex because they share these afore-mentioned morphological features. A synoptic table of a comparison on species in the complex is provided in Table 5. Clade C includes eight species belonging to the A. mesenterica complex (Figures 1 and 2). All species in this complex have similar macro-morphology, and were considered A. mesenterica previously [12,20]. Among them, A. asiatica, A. brasiliana and A. orientalis were recently described [25,26], while A. africana, A. srilankensis and A. submesenterica are described as new species in the present study. These eight species form eight independent lineages with high support in the phylogeny based on the concatenated ITS+nLSU dataset except for A. submesenterica (Figure 1), but six samples of A. submesenterica form a single lineage with high support in the phylogeny based on the concatenated ITS+nLSU+rpb1+rpb2 dataset ( Figure 2). The lineage of A. africana from Africa is strongly supported and distant from other species of the complex in both phylogenies. The lineage of A. srilankensis is closely related to A. asiatica, but A. srilankensis morphologically differs from A. asiatica by the usually resupinate basidiomata (Figure 3h), larger basidia and longer basidiospores ( Table 6). The species from the A. mesenterica complex are distinctly different from other species of the genus by resupinate to effused-reflexed basidiomata, a usually concentrically zoned upper surface (Figure 3), the absence of medulla and usually inflated hyphae in KOH. Characteristics of these seven species in the complex are summarized in Table 6.
The morphological characters of the members of the A. auricula-judae and the Auricularia mesenterica complexes totally correspond to molecular data, but not so in the members of the A. cornea, the A. delicata and A. fuscosuccinea complexes. Species in these three complexes may be at a stage of speciation. So far, the most important morphological characters to delimit a species in the genus are the presence or absence of the medulla, the texture, length and diameter of hairs, and the lengths and widths of basidia and basidiospores, which is more or less the same as observed in previous studies [12,17,20]. Morphological features displaying evidence for evolution and phenotypic plasticity are worthy of being studied in the future.
In addition to the molecular data, the present study provides data about host and geographical distribution of 277 samples from 35 countries in Asia, Europe, North and South America, Africa, and Oceania. The two species growing on gymnosperm wood, A. americana and A. tibetica, cluster in the A. auricula-judae complex in the phylogeny, and they form two independent lineages separated from other species growing on angiosperms. Thus, host may also be important in the taxonomy of Auricularia as with some other wood-inhabiting genera [59,60]. Regarding the geographical distribution, most species are distributed in the same continent, while a few species are widely distributed in different continents, e.g., A. cornea (Figure 1), which, however, remains as an unresolved lineage. The species in Group II are mostly distributed in the Americas, while other species are mainly distributed in Asia (Figure 1). Therefore, the host and geographical distribution probably have an influence on speciation of Auricularia.