New Species and New Records of Otidea from China Based on Molecular and Morphological Data

Species of genus Otidea previously reported in China are mainly distributed in the northeast, northwest and southwest regions of China, but the species diversity of Otidea in north China is not very clear. In this study, newly collected Otidea specimens from northern China and some herbarium specimens deposited in three important Chinese fungus herbaria (HMAS, HKAS, HMJAU) were studied using morphological and phylogenetic methods. The internal transcribed spacers of the nrDNA (ITS), the nrRNA 28S subunit (nrLSU), the translation elongation factor 1-alpha (tef1-α), and the second largest subunit of RNA polymerase II (rpb2), were employed to elucidate the phylogenetic relationships between Otidea species. Results identified 16 species of Otidea, of which seven new species are described, namely O. aspera, O. cupulata, O. filiformis, O. khakicolorata, O. parvula, O. plicara and O. purpureobrunnea. Otidea bicolor and O. pruinosa are synonymized as O. subpurpurea. Two species, O. mirabilis and O. nannfeldtii, are being reported for the first time in China. The occurrence of O. bufonia, O. leporina and O. onotica are confirmed by molecular data in China.

China has a huge temperate area in the northern hemisphere and likely has diverse Otidea species. However, only a few taxonomic works focus on this genus, and about a quarter of Chinese Otidea species are not supported by molecular evidence [6,8,[16][17][18][23][24][25][26][27][28][29]. Currently, a total of 24 Otidea species are reported in China, including 17 native species and seven known species originally described from Europe and/or North America.
During our investigation of fungal resources in northern China since 2017, many apothecia of the genus Otidea were collected. On the bases of these new collections and some of herbarium specimens deposited in three important Chinese fungus herbaria (HMAS, HKAS, HMJAU), we recognized seven species and two records new to China based on both morphological examination and molecular analysis. Also, both O. bicolor W.Y. Zhuang & Zhu L. Yang, and O. pruinosa Ekanayaka, Q. Zhao & K.D. Hyde were conspecific with O. subpurpurea W.Y. Zhuang. Our aim in this paper is to describe and illustrate these new species and new records and to synonymize O. bicolor and O. pruinosa as O. subpurpurea. Molecular data for some known species existing in China are additionally provided.

Morphological Studies
Fresh specimens were collected and photographed in the field from the Shanxi and Hebi provinces, as well as Beijing, China. The specimens were dried and deposited in the BJTC (Herbarium, Biology Department, Capital Normal University) and the HSA (Herbarium Institute of Edible Fungi, Shanxi Academy of Agricultural Science, Taiyuan, China). Other specimens were studied from the HMAS (Herbarium Mycologicum Academiae Sinicae, Institute of Microbiology, Chinese Academy of Sciences), HKAS (Herbarium of Cryptogams at the Kunming Institute of Botany, Chinese Academy of Sciences) and HMJAU (Herbarium of Mycology, Jilin Agricultural University). Macroscopic characteristics were recorded from fresh specimens. Standardised color values matching the described colour were taken from ColorHexa (http://www.colorhexa.com/, accessed on 30 January 2022). Microscopic characteristics were observed in thin sections of dry specimens mounted in 5% KOH and Melzer's reagent [30]. The dimensions for ascospores are given using notation of the form (a-)b-c(-d).
The range b-c contains a minimum of 90% of the measured values. The extreme values, i.e., a and d, are given in the parentheses. L m and W m indicate the average ascospore length and width for the measured ascospores, respectively. Q is used to represent length/width ratio of a ascospore in side view and Q m represents average Q of all specimens. The number of populations that the statistics are based on is indicated by n.

DNA Extraction, PCR Amplification, Sequencing
Herbarium specimens were crushed by shaking for 30 s at 30 Hz 2-4 times (Mixer Mill MM 301, Retsch, Haan, Germany) in a 1.5 mL tube together with one 3 mm diameter tungsten carbide ball, and total genomic DNA was extracted using the modified CTAB method [31]. The following primers were used for PCR amplification and sequencing: ITS1f/ITS4 [31,32] were used for the internal transcribed spacers of the nrDNA (ITS1-5.8S-ITS2 = ITS), LR0R/LR5 [33] for the nrDNA 28S subunit (nrLSU), EF1-983F/EF1-2218R [34] for the translation elongation factor 1-alpha (tef1-α), and RPB2-Otidea6F/RPB2-Otidea7R and fRPB2-7cF/fRPB2-11aR [3] for the RNA polymerase II second largest subunit (rpb2), respectively. PCRs were performed in 50 µL reactions containing 4 µL DNA template, 2 µL of per primer (10 µM), 25 µL 2× Master Mix (Tiangen Biotech Co., Beijing, China), and 17 µL ddH 2 O. PCR reactions were performed as follows: for the ITS gene: initial denaturation at 94 • C for 3 min, followed by 35 cycles at 94 • C for 30 s, 56 • C for 45 s, 72 • C for 1 min, and a final extension at 72 • C for 10 min; for the nrLSU gene: initial denaturation at 94 • C for 4 min, followed by 35 cycles at 94 • C for 30 s, 55 • C for 45 s, 72 • C for 1 min, and a final extension at 72 • C for 10 min; for the tef1-α gene: initial denaturation at 94 • C for 3 min, followed by 35 cycles at 94 • C for 30 s, 60 • C for 45s, 72 • C for 1min, and a final extension at 72 • C for 10 min; for the rpb2 gene: initial denaturation at 94 • C for 3 min, followed by 10 cycles (including denaturation) at 94 • C for 30 s, annealing temperature started at 62 • C (decreased by 1 • C per cycle, until to 52 • C) for 45 s and extension at 72 • C for 1 min, then followed by 30 cycles at 94 • C for 35 s, 55 • C for 45 s, 72 • C for 1 min, and a final extension at 72 • C for 10 min. The PCR products were sent to Beijing Zhongkexilin Biotechnology Co., Ltd. (Beijing, China) for purification, sequencing, and editing. The newly generated sequences were assembled and edited using SeqMan (DNA STAR package, DNAStar Inc., Madison, WI, USA) with generic-level identities for sequences confirmed via BLAST queries of GenBank.

Sequence Alignment and Phylogenetic Analyses
A total of 730 sequences from 283 collections of Otidea were used in the molecular phylogenetic analyses. The detail information about them is provided in Supplementary Table S1, including the geographic origin and accession numbers. Sequences of all DNA regions generated in this study were deposited in GenBank. The sequences obtained from GenBank are based on published literature or selected by using BLASTn search to find similar matches with taxa in Otidea. Two datasets were assembled for this study. Dataset I (ITS/nrLSU) and datasets II (ITS/nrLSU/tef1-α/rpb2) contained the backbone species and all phyloclades of Otidea, which were used to infer the phylogenetic status of Chinese Otidea species of the genus Otidea. The taxa Monascella botryosa Guarro & Arx and Warcupia terrestris Paden & J.V. Cameron were selected as outgroups. The ITS, nrLSU, tef1-α and rpb2 sequences were respectively aligned using the MAFFT v.7.110 online program under the default parameters [35], and manually adjusted to allow for maximum sequence similarity in Se-Al version.2.03a [36]. Ambiguously aligned regions of each sequence were detected and excluded using Gblocks 0.91b [37] before the phylogenetic analyses. Unsampled gene regions were coded as missing data and all introns of tef1-α and rpb2 were excluded because of the alignment difficulty. To examine the conflict among topologies with maximum likelihood (ML), separate single-gene analyses were conducted. Alignments were concatenated using SequenceMatrix v1.7.8 [38] and are provided in Supplementary Files S2 and S3. We conducted maximum likelihood (ML) and Bayesian inference (BI) analyses on the two datasets.
Maximum likelihood (ML) analyses of the two datasets were carried out using RAxML 8.0.14 [39] with all parameters kept to the default settings using a GTRGAMMAI model. The ML bootstrap replicates (1000) were computed in RAxML using a rapid bootstrap analysis searching for the best scoring ML tree. Bayesian inference (BI) analyses were performed with MrBayes v3.1.2 [40] based on the best substitution models for each gene region as determined by MrModeltest 2.3 [41]. The GTR + I+G model was the best model for ITS, nrLSU and rpb2, whereas the best model for tef1-α was the SYM + I+G model. Two independent executions of four chains were conducted: 3,485,000 for ITS/nrLSU and 765,000 for the ITS/nrLSU/tef1-α/rpb2 datasets. Markov chain Monte Carlo generations were conducted using the default settings and sampled every 100 generations. The temperature value was lowered to 0.20, burn-in was set to 0.25, and the program was automatically stopped as soon as the average standard deviation of split frequencies reached below 0.01. A 50% majority-rule consensus tree was constructed. Clades with a bootstrap support (BS) ≥ 70% and a Bayesian posterior probability (PP) ≥ 0.95 were considered as significantly supported [42,43]. All phylogenetic trees were viewed with TreeView32 [44].

Phylogenetic Analyses
No topological incongruence was detected when the four genes were analyzed individually. Dataset I (ITS/nrLSU) contained 528 sequences from 51 species, including 93 novel sequences the two genes from Chinese collections, and four from the outgroups (Monascella botryosa and Warcupia terrestris). The dataset had an aligned length of 1363 characters (551 bp from ITS and 812 bp from nrLSU), of which 647 were constant, 716 were variable, and 609 of these variable sites were informative. ML and BI analyses yielded similar tree topologies. Only the tree inferred from the ML analyses is shown (Figure 1). The species of Otidea formed a monophyletic clade with high support values (BS = 100%, PP = 1.00). A total of 10 clades were recognized in the two-gene phylogram, which is consistent with Olariaga et al. [4] and Hansen and Olariaga [3].  .84% similarity in the ITS region, which implied they may be conspecific, although they have some difference in apothecial color. Fortunately, we borrowed the type specimens of these three species for observation and research. We thought that they should be conspecific and formally synonymized O. bicolor and O. pruinosa in this study.   contained 501 sequences from 49 species, including 73 novel sequences these four genes from Chinese collections, and 8 from the outgroups (M. botryosa and W. terrestris). The dataset had an aligned length of 4400 characters (551 bp from ITS, 812 bp from nrLSU, 1383bp from tef1-α and 1654 bp from rpb2), of which 2456 were constant, 1854 were variable, and 1753 of these variable sites were informative. ML and BI analyses yielded similar tree topologies. Only the tree inferred from the ML analysis is shown ( Figure 2).  purpureobrunnea and O. cupulata in the O. bufonia-onotica clade), which may be due to the fact that these species have a lower support value between them.

Taxonomy
Based on our phylogenies and morphological data, a total of seven new species, a known species, and two new records of Otidea from China were described and illustrated here.
Notes: Otidea aspera is diagnosed by the combination of the stipitate, broadly ear shaped to cup-shaped, greyish yellow to light brown hymenium, pale-yellow to yellowishbrown receptacle surface, ellipsoid to slightly subfusoid ascospores and straight to slightly curved paraphyses. Otidea aspera and O. parvispora have comparable apothecia color, however O. parvispora differs from O. aspera by the smaller ascospores ((11.0-) 11.5-13.0 × 5.0-6.5 µm) and shorter asci. DNA analysis showed that O. aspera shared less than 93.39% ITS sequence similarity with other Otidea species. Phylogenetic analyses revealed that the sequences of O. aspera were grouped into an independent clade with a strong support value (Figures 1 and 2). These supported the erection of the new species.
Other in the ectal excipulum turning amber and brown in KOH. Otidea purpureobrunnea is distinguished by its grayish-purple-brown to dark-purple-brown receptacle surface and mostly smooth basal mycelium. Otidea simithii differs in having typically narrower, ear-shaped apothecia, and resinous exudates of the ectal excipulum that does not turn bright yellow in KOH. Otidea subpurpurea in having smaller ascospores (9-12 × 4.5-6 µm) and lilac to purplish receptacle surface.
Other Phylogenetic analyses revealed that the sequences of O. filiformis were grouped into an independent clade with a strong support value (Figures 1 and 2). DNA analysis showed that O. filiformis shared less than 95.88% ITS sequence similarity with other Otidea species. These supported the erection of the new species. One Finnish collection (MCVE 29372) identified as O. bufonia by Carbone et al. [22] is clustered into the O. filiformis clade with strong support values in our phylogenetic trees (Figures 1 and 2), and it shared more than 98% ITS similarity with our O. filiformis and less than 94.6% with O. bufonia. This evidence showed MCVE 29372 is more closely related to O. filiformis, and whether it is conspecific with O. filiformis need further morphological identification.
Notes: Otidea khakicolorata is characterized by khaki to pale-ochre, long, narrowly earshaped apothecia, small ascospores and resinous exudates on the ectal excipulum turning reddish brown in KOH. Otidea nannfeldtii and O. khakicolorata share similar apothecia shape and the reaction of the resinous exudate in the ectal excipulum and basal mycelium in MLZ and KOH, but O. nannfeldtii can be distinguished by an ochre to orangish-ochre hymenium surface with pink tones, higher warts (45-85 µm) on the apothecial outer surface, medullary excipulum of textura intricata differentiated into two parts, and relatively bigger ascospores ((9-) 9.5-10.5 (-11.5) × 5.5-6.5 (-7) µm). Otidea khakicolorata is phylogenetically close to O. nannfeldtii; however, they are separated by a low support value (Figures 1 and 2). DNA analyses showed that O. khakicolorata shared less than 91% similarity in ITS sequence with O. nannfeldtii.
Other O. purpurea: 8-10 × 4.5-6 µm). Otidea purpureogrisea is distinguished by the purple-gray tone of the receptacle surface near the base and resinous exudates of the ectal excipulum turning amber in MLZ and turning brown in KOH. Otidea smithii is distinguished by typically narrower, ear-shaped apothecia, relatively shorter ascospores (12-14 × 6-7.5 µm) with a lower Q m value (1.9-2), and resinous exudates of the ectal excipulum not turning bright yellow in KOH. For a comparison with O. cupulata see under that species below.
Phylogenetic analyses revealed that O. purpureobrunnea and O. filiformis are grouped together with a low support value (Figure 2), but O. filiformis is easy to distinguish from O. purpureobrunnea by its apothecia without purple tones, fusoid ascospores, same width and narrow paraphyses (≤3 µm), as well as its basal mycelium with abundant spheroid, pale brown, resinous exudates. DNA analysis showed that O. purpureobrunnea shared less than 94.53% similarity in its ITS sequence with O. filiformis. These indicate that they are two different species.
Otidea  [22,25]; however, due to the unavailability of specimens, this issue has not been formally addressed. In this study, we examined the type specimens and obtained multiple loci sequences from them. DNA analyses revealed that O. pruinosa, O. bicolor, and O. subpurpurea share high sequence similarity (ITS: >98.87%; nrLSU: >99.53%; tef1-α: >99.72%; rpb2: >99.45%). We performed morphological observation on these type specimens and found that there was no obvious difference in microscopic features. The reaction of the resinous exudate in the ectal excipulum and basal mycelium in MLZ and KOH are also the same. Although the receptacle surface of O. bicolor and O. subpurpurea is purplish in tint when fresh, the receptacle surface of O. pruinosa is without a purplish tint [23,28,29], but that may be influenced by its habitat. Otidea pruinosa is proposed as a new species because of receptacle surface with pruinose, but we found a similar granulate on the surface of dry specimens of O. bicolor and O. subpurpurea. In addition, phylogenetic analyses based on the two-gene and four-gene datasets also confirmed that they represent the same species, so here we formally treat O. bicolor and O. pruinosa as synonyms of O. subpurpurea. The sequence from ZMU124 (label as O. bufonia) from Guizhou province of China grouped with O. subpurpurea with a high support value (Figure 1), indicating that O. subpurpurea seems widely distributed in southwest China. Similarly, the sequence from JS150904-08 from Korea named O. bufonia [45] was also grouped into this clade (Figure 1). We checked the original morphological description by Jin et al. [45] and found that its ascospores size does not conform to O. bufonia, but instead to O. subpurpurea. This indicates that O. subpurpurea also occurs in Korea.
Notes: The occurrence of O. mirabilis is confirmed in China based on morphological and DNA evidence in this study. Olariaga et al. [4] showed already that O. mirabilis occur in China using nrLSU sequences from two Chinese collections in GenBank (identified as O. leporina by Zhuang [17] and Liu and Zhuang [18], but by morphology it has not been previously confirmed, as Olariaga et al. did not study those two collections morphologically. It is interesting that two distinct clades were revealed, one comprising Chinese specimens, and another comprising specimens from Europe. The Chinese specimens shared 98.46-99.84% ITS sequence similarity and the European ones had 99.54-99.85% similarity, while the similarities between the two proveniences were 97-98.5%. However, we found no significant morphological differences between the Chinese and European specimens, which probably resulted from the geographic distance. Notes: The occurrence of O. nannfeldtii in China is first confirmed based on molecular and morphological evidence. Otidea nannfeldtii is originally described in Europe, and also reported from North America [4]. Before this study, there are no DNA data that support the existence of this species in China.

Discussion
Temperate China is surely rich in Otidea species. Nine species are added to this genus by this study. A total of 31 species is thus recorded in this huge country currently. Of these species, 27 species are supported by morphological and molecular data, but four species

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The sequencing data were submitted to GenBank.