Phylogenetic Analysis Reveals Four New Species of Otidea from China

Simple Summary Otidea is a remarkable genus of ascomycetes. Members of the genus are diverse in ascomatal form, with split to entire, sessile to stipitate, cupulate to ear-shaped epigeous apothecia, as well as closed, solid ascomata in hypogeous taxa. They are distributed in the temperate to arctic-alpine regions of the northern hemisphere. China, located in the northern hemisphere, has a vast temperate zone, and a lot of new species of Otidea have been proposed recently. In this study, some specimens deposited in Chinese fungus herbaria and one newly collected Otidea specimen from northern China were studied using morphological and phylogenetic methods. The results indicate the presence of nine phylogenetic species of Otidea within the sample, four of them are described as new species, namely O. bomiensis, O. gongnaisiensis, O. hanzhongensis, and O. shennongjiana. Recognizing these new species will increase knowledge of species resources of Otidea in China. Abstract The emergence of molecular systematics has greatly helped researchers to identify fungal species. China has abundant Otidea species resources, and a number of new species of Otidea have been recently proposed. However, many old specimens in herbaria are mainly identified by morphology rather than molecular methods. In this study, 11 specimens deposited in Chinese herbaria and one newly collected Otidea species from northern China were identified based on morphological and phylogenetic analyses. Four gene fragments (ITS, LSU, rpb2, and tef1-α) were used to elucidate the phylogenetic relationships of species within Otidea. A total of nine phylogenetic species are recognized, of which four are described as new species, namely O. bomiensis, O. gongnaisiensis, O. hanzhongensis, and O. shennongjiana. Among the known species were O. aspera and O. sinensis.


Introduction
The genus Otidea (Pers.) Bonord. (Pyronemataceae, Pezizales) was established by Bonorden, who elevated Peziza subgenus Otidea to generic rank, and typified it by Otidea onotica (Pers.) Fuckel [1][2][3]. Otidea is a monophyletic but morphologically diverse genus. Members of the genus exhibit a wide range of ascomatal forms, with split to entire, sessile to stipitate, cupulate to ear-shaped epigeous apothecia, as well as closed, solid ascomata in hypogeous taxa, and are mainly distributed in temperate zones of Europe, North America, and Asia in the northern hemisphere, with a few from the arctic-alpine regions. Species of Otidea are considered to be ectomycorrhizal and thus play important roles in forest ecosystems [4][5][6][7][8][9].
Hansen and Olariaga [7] used multilocus phylogenies (LSU, rpb2, tef1-α) to elucidate species relationships within Otidea. Olariaga et al. [8] simultaneously published a monograph of the genus based on relatively extensive taxon sampling and introduced new morphological and histochemical features for species identification. These works laid the Biology 2022, 11, 866 2 of 18 foundation for subsequent research of Otidea. In recent years, a number of new species have been discovered both in Europe and Asia [9][10][11][12][13][14].
During a previous investigation on fungal resources of northern China, we collected many fresh apothecia of the genus Otidea and identified seven new species that were described and illustrated [15]. More recently, following the examination of 11 herbarium specimens (HMAS and HMUABO), four additional Otidea species were discovered that appeared to be undescribed. In this study, we report on the detailed analyses of these Chinese specimens using morphological and phylogenetic methods. Our goals are (i) to evaluate the species identity of herbarium specimens and one freshly collected specimen, (ii) to infer the phylogenetic position of species within Otidea based on multigene sequences (ITS, LSU, tef1-α, rpb2), and (iii) to describe and illustrate the four new species.

Morphological Studies
Specimens were studied from HMAS (Herbarium Mycologicum Academiae Sinicae, Institute of Microbiology, Chinese Academy of Sciences) and HMUABO (Fungal Herbarium of Northwest A & F University, Yangling, China). Macroscopic characters were recorded from fresh specimens. Microscopic characters were observed in 10% KOH, Congo Red, and Melzer's reagent [16]. The form '(a-)b-c(-d)' is used to represent the dimensions of ascospores. The range b-c contains a minimum of 90% of the measured values. The extreme values, i.e., a and d, are given in parentheses. L m and W m are used to represent the average ascospore length and width. Q means length/width ratio; Q m means the average Q. 'n' means the number of populations.

Sequence Alignment and Phylogenetic Analyses
Three datasets were assembled for this study. Dataset I (LSU-tef1-α-rpb2) contained our collections and the backbone species in all phyloclades of Otidea, which was used to infer the phylogenetic status of our collections at the phyloclade level in the genus Otidea. Warcupia terrestris Paden & J.V. Cameron and Monascella botryosa Guarro & Arx were used as outgroups. Datasets II and III (ITS-LSU) corresponded to the Otidea formicarum phyloclade and the O. alutacea phyloclade, respectively, and contained the representative species of their own and all available sequences of Chinese Otidea species (including our collections) separately classified in these two clades. These two datasets were used to evaluate the relationship between the four new species and their close relatives. Otidea cantharella (Fr.) Quél. and O. platyspora Nannf. were selected as outgroups for Datasets II and III, respectively.
The sequences (ITS, LSU, tef1-α, and rpb2) were independently aligned in an online version of MAFFT 7.110 using default parameters [21] and manually modified where necessary in Se-Al 2.03a. [22]. Ambiguous aligned regions for each sequence were detected and excluded through Gblocks 0.91b [23] using default options with default parameters. To check for the degree of congruence among different genes, phylogenetic analyses were first conducted separately for each gene and later for the genes combined. These genes were then concatenated using SequenceMatrix v1.7.8 [24], and the alignment files of the three datasets were provided in Supplementary File S1-S3. Maximum likelihood (ML) and Bayesian inference (BI) were used to infer phylogenetic analyses.
ML analysis was performed in RAxML [25] by running 1000 bootstrap replicates with all parameters at default settings using the GTRGAMMAI model. BI analysis was performed in MrBayes [26] based on the best substitution models (GTR+I+G for ITS, LSU, and rpb2; SYM+I+G for tef1-α) determined by MrModeltest 2.3 [27]. Two independent runs of four chains were conducted: 1,000,000 for the LSU/tef1-α/rpb2 dataset, 395,000 for the O. formicarum dataset, and 615,000 for the O. alutacea dataset. Markov Chain Monte Carlo generations were conducted using the default settings and sampled every 100 generations. The average standard deviations of split frequency (ASDSF) values were far lower than 0.01 at the end of the runs. Trees were sampled every 100 generations after burn-in, and 50% of the majority-rule consensus trees were constructed. Maximum likelihood bootstrap support (BS) ≥ 50% and Bayesian posterior probability (PP) ≥ 0.95 were shown on the nodes [28,29]. Trees were visualized using TreeView [30].

Phylogenetic Analyses
The combined LSU/tef1-α/rpb2 dataset contained 360 sequences from 146 taxa, including 39 newly obtained sequences in this study. The length of the aligned dataset was 3547 bp (802 bp for LSU, 969 bp for tef1-α, 1776 bp for rpb2). The topologies of ML and BI phylogenetic trees were similar, so only the ML tree is shown ( Figure 1). The Otidea species formed a monophyletic lineage with high support values. Our Otidea collections were nested in four clades, i.e., O. bufonia-onotica clade, O. concinna clade, O. formicarum clade, and O. alutacea clade, and a total of nine phylogenetic species were recognized from our Chinese collections. In the O. bufonia-onotica clade, one species was recognized. Although this species contained one collection, HMAS 85660, and probably represented an undescribed species, we could not describe it due to the poor condition of its ascomata (see discussion). Here, we temporally named it Otidea sp.'c'. In the O. concinna clade, two collections were placed in a well-supported clade, represented by a known species, O. sinensis J.Z. Cao & L. Fan. In the O. formicarum clade, two species were recognized. They were described as new species O. gongnaisiensis and O. shennongjiana, respectively. In the O. alutacea clade, five species were recognized. Two of them were described as new species, O. bomiensis and O. hanzhongensis, in this paper. One (HSA 251) is corresponding to the known species O. aspera L. Fan & Y.Y. Xu. The remaining two phylogenetic species (clades 6 and 7) were not treated taxonomically here due to the poor condition of Chinese collections included in the two clades (see discussion). Further, we could not examine the Swedish specimen (C-F-48045).
The O. formicarum dataset contained 42 sequences from 21 taxa, including four newly obtained sequences and four sequences from outgroups (O. cantharella). The length of the aligned dataset was 1416 bp (582 bp for ITS, and 834 bp for LSU). The topologies of ML and BI phylogenetic trees were similar, so only the ML tree is shown ( Figure 2    The O. alutacea dataset contained 81 sequences from 41 taxa, including ten newly obtained sequences and four sequences from outgroups (O. platyspora). The length of the aligned dataset was 1357 bp (572 bp for ITS, and 785 bp for LSU). The topologies of ML and BI phylogenetic trees were similar, so only the ML tree is shown ( Figure 3). Similar to the three-gene phylogram result, the Chinese collections were placed in five well-supported clades. Of them, one clade is represented by a known species (O. aspera), and two are new species (O. bomiensis and O. hanzhongensis). The remaining two clades (clades 6 and 7) are two distinct species, but we are not able to treat these two taxa taxonomically (see discussion). The O. alutacea dataset contained 81 sequences from 41 taxa, including ten newly obtained sequences and four sequences from outgroups (O. platyspora). The length of the aligned dataset was 1357 bp (572 bp for ITS, and 785 bp for LSU). The topologies of ML and BI phylogenetic trees were similar, so only the ML tree is shown ( Figure 3). Similar to the three-gene phylogram result, the Chinese collections were placed in five well-supported clades. Of them, one clade is represented by a known species (O. aspera), and two are new species (O. bomiensis and O. hanzhongensis). The remaining two clades (clades 6 and 7) are two distinct species, but we are not able to treat these two taxa taxonomically (see discussion).

Taxonomy
Based on our phylogenies and morphological data, four new species and two known species of Otidea from China were described and illustrated here.
Notes: The two specimens (HMAS 268390 and HMAS 268544) grouped together with the type specimen of O. sinensis with strong a support value (BS = 91%, PP = 1.00). We examined the morphology of these two specimens, which are consistent with the original description by Cao et al. [31]. The two specimens were, therefore, confirmed to be O. sinensis. The type specimen of O. sinensis was collected from Heilongjiang Province in northeast China. Our results demonstrate that O. sinensis is also distributed in southwest China.

Discussion of Additional Specimens
In the phylogeny derived from the combined datasets (LSU, tef1-α, and rpb2) (Figure 1), a specimen (HMAS 85660) collected from Heilongjiang Province, China, clustered in the O. bufonia-onotica clade, and formed a sister clade with O. cupulata L. Fan & Y.Y. Xu with high support values. This specimen collected from rotted wood has an ascospore size of (13.5-)14-15.7(-16) × (6.3-)6.6-7.7(-8) µm, Q = 1.9-2.2, which overlaps with O. cupulata. However, the ascospore shape of the two species is different. The ascospores in HMAS 85660 is narrowly fusoid, while ellipsoid to slightly subfusoid in O. cupulata. Furthermore, HMAS 85660 had less than 95.28% ITS similarity with O. cupulata. Therefore, HMAS 85660 may represent a new species, but due to the poor condition of this specimen, its formal taxonomic treatment awaits the discovery of new fruitbodies.
In the O. alutacea clade, Otidea bomiensis forms a sister clade with clade 6 (containing four Chinese collections) with a moderate support value (BS = 73%, PP = 1.00) (Figure 1). The four specimens in clade 6 are of poor quality due to the early collection time, and the macroscopic features can hardly be observed, but we checked their microscopic features ( Figure 8D). The ascospore measurements we obtained were 15-20 × 6-8.5 µm (Q = 2-2.5, Q m = 2.2), which is somewhat longer than that described by Xu et al. [14] (14-17.5 × 6-8 µm, Q m = 2). Therefore, Otidea bomiensis differs from clade 6 by having shorter ascospores and a smaller Q m value. Furthermore, in the phylogenetic tree derived from the O. alutacea dataset (Figure 3), O. bomiensis and clade 6 form two distant clades, indicating that they are two distinct species.
Clade 7 contains two specimens, the Swedish C-F-48045 is without any published morphological data, and the other HMAS 88262 is from the Xinjiang Autonomous Region of China. Otidea hanzhongensis forms a sister clade with clade 7 with good support values (BS = 73%, PP = 1.00) (Figure 1), but HMAS 88262 differs from O. hanzhongensis (ascospore length < 13.5 µm, Q = 1.7-2) in having significantly larger ascospores (16.5-18.5 × 7.4-8.2 µm, Q = 2.1-2.4). The phylogenetic tree derived from the O. alutacea dataset ( Figure 3) also confirmed that they are two different species. Xu et al. [14] treated C-F-48045 as a separate clade (clade 7); our collections (HMAS 88262) fall into this clade, but here we did not treat this taxon taxonomically, as we could not study the Swedish collection, and our collection (HMAS 88262) is in poor condition.
Regarding the species represented by Otidea sp. 'c', clades 6 and 7, we still need to go to the original collection site to collect new specimens for further research. In addition, many sequences of the genus Otidea obtained from ectomycorrhizal, and soil samples have been submitted into public databases without confirmed species identification. These should be considered in future research to extend and improve our knowledge of the biogeography and diversification of Otidea species worldwide.

Conclusions
In this study, 11 Otidea specimens deposited in Chinese herbaria and one newly collected specimen from northern China were examined based on morphological and phylogenetic analyses. The results revealed a total of nine phylogenetic species, of which four were described as new, namely O. bomiensis, O. gongnaisiensis, O. hanzhongensis, and O. shennongjiana. In addition, two known species were identified, namely O. aspera and O. sinensis. The remaining three taxa are putative new species, but more specimens need to be collected for further study before attempting a formal taxonomic treatment. Our findings emphasize that the diversity of the Otidea species in China is extremely high and that more studies are needed to fully appreciate the exact species number.

Informed Consent Statement: Not applicable.
Data Availability Statement: The sequencing data were submitted to GenBank.