Two New Species and Two New Records of the Lichen-Forming Fungal Genus Peltula (Ascomycota: Peltulaceae) from China

Simple Summary This paper reports on two new species and two new records of the lichen-forming fungal genus Peltula Nyl., highlighting progress in our investigation of Chinese Peltula spp. The external morphology, anatomy, and molecular systematics were comprehensively analyzed to support the species delimitation. The results contribute to the knowledge of species diversity and geographical distributions of Peltula species in China. Abstract In this study, two new species, Peltula confusa Q.X. Yang & X.L. Wei, sp. nov., growing in a dry microenvironment within a semi-humid area, and Peltula subpatellata Q.X. Yang & X.L. Wei, sp. nov., occurring in arid and semi-arid regions, are described. In addition, two species, P. polyspora (Tuck.) Wetmore and P. obscuratula (Nyl.) Poelt ex Egea, are recorded for the first time in China. All four species are described based on morphological, anatomical and molecular data. Peltula confusa is characterized by a lighter, brighter, and more yellowish upper surface than other species of this genus, with discs concentrated in the central part of squamules, and a thick lower cortex. Peltula subpatellata can be distinguished from P. patellata (Bagl.) Swinscow & Krog by its non-thickened and sometimes darkened margins and sometimes the presence of peltate squamules. Phylogenetic analysis based on DNA sequences of four loci (ITS, nrSSU, nrLSU, and RPB2) demonstrates the placement of these species within Peltula, and supports current species delimitations. We suggest that the growth substrate should be considered as an additional characteristic for species delimitation.


Introduction
The lichen genus Peltula (Lichinales, Lichinomycetes, Ascomycota) has grown to comprise more than 60 species since the type species, Peltula radicata Nyl., was described by Nylander in 1853. After being established, this genus name was not used further for a long period of time and many species that are now accommodated in the genus Peltula were described as part of the genus Heppia Naeg. It was not until 1935 that Peltula was mentioned again and began to be used as a separate genus [1]. Based on the development of ascocarps, the unique structure and function of the ascus, and numerous ascospores in the ascus, Büdel considered Peltula to be a monophyletic genus and established a new family, Peltulaceae, for it, including four growth types: leaf-like, squamulose, crustose and fruticose [2]. Büdel and Kalb added the genera Neoheppia Zahlbr. and Phyllopeltula Kalb to the family, respectively [3,4]. Kauff et al. confirmed that Peltulaceae was a monophyletic family using phylogenetic analysis, and combined the two above-mentioned genera, i.e., Neoheppia and Phyllopeltula, into Peltula [5]. Based on the newest revision of Peltulaceae, Peltula is characterized by five growth forms of the thallus, viz. peltate, squamulose, subfruticose, subfoliose, and crustose. About 20 species of Peltula from China have been reported. Previously, P. euploca (Ach.) Poelt, P. impressula (H. Magn.) N.S. Golubk, P. minuta (H. Magn.) N.S. Golubk, P. placodizans (Zahlbr.) Wetmore and P. tortuosa (Nees) Wetmore were known to be present in arid and semi-arid desert regions [6][7][8]; P. applanata (Zahlbr.) J.C. Wei was described as a new species from Guizhou province, which is a humid region [9]; P. bolanderi (Tuck.) Wetmore, P. clavata (Kremp.) Wetmore, P. coriacea Büdel, Henssen & Wessels, P. corticola Büdel & R. Sant., P. euploca, and P. placodizans were identified in Hong Kong and Taiwan [10][11][12]; and P. cylindrica Wetmore, P. euploca, P. lobulate Q.X. Yang & X.L. Wei, P. polycarpa Q.X. Yang & X.L. Wei, P. polyphylla Q.X. Yang & X.L. Wei, P. pseudoboletiformis Q.X. Yang & X.L. Wei, and P. submarginata Q.X. Yang & X.L. Wei were reported in humid and semi-humid areas [13,14]. Such a high diversity of species indicates that China is an important reservoir of Peltula, and it is likely that more species may be found.
We conducted an investigation into lichens in several provinces across China and collected numerous specimens, among which Peltula species were identified. The Peltula specimens collected from Beijing, Inner Mongolia, Gansu, and Ningxia are mainly squamulose in growth type. Among these localities, Beijing is rich in natural forest resources and has distinct wet and dry seasons, while the other three regions are (semi) arid regions.

Taxon Sampling
Specimens for this study were collected from Beijing, Gansu, Inner Mongolia, and Ningxia. The voucher specimens were deposited in the Fungarium of the Institute of Microbiology, Chinese Academy of Sciences (HMAS-L). Leica M125 (Leica Microsystems, Wetzlar, Germany) and DFC450 (Leica Microsystems, Wetzlar, Germany) dissecting microscopes were used for the morphological studies. Free-hand sections were used to study the internal morphology of the lichen thallus and ascomata. A Zeiss Axioscope2 compound microscope (Zeiss Corporation, Göttingen, Germany) with a Zeiss Axio Imager A2 (Zeiss Corporation, Göttingen, Germany) was used for the anatomical studies, and an AxioCam MRc5 camera (Zeiss Corporation, Göttingen, Germany) was used to take photographs. Spot tests were performed using K (10% aqueous solution of potassium hydroxide) and IKI (1% aqueous iodine solution with 10% aqueous potassium hydroxide, Sinopharm Chemical Reagent Co., Ltd, Beijing, China).

DNA Extraction, Amplification, and Sequencing
Thirty-four fresh specimens were chosen for DNA extraction. The modified CTAB method was used as the extraction procedure [15]. In general, PCR amplifications and primers used followed the work by Kauff et al. [5]. The ITS amplification primers were ITS1 / ITS4, and ITS4 / ITS5 [16], and the ITS cycling parameters consisted of the initial denaturation at 94 • C for 2 min; 33 denaturation cycles at 94 • C for 30 s, annealing at 55 • C for 30 s, extension at 72 • C for 1 min 10 s; and a final extension at 72 • C for 2 min. Reactions were carried out in a volume of 25 µL containing 12.5 µL of 2 × Taq PCR MasterMix®(Beijing Jiangchen Biotechnology Co., Ltd, Beijing, China), 1 µL of each primer solution (10 µM), 9 of µL ddH 2 O and 1.5 µL of genomic DNA. The new sequences generated in this study were deposited in GenBank (www.ncbi.nlm.nih.gov, accessed on 27 August 2022) (

Congruence among Loci
Highly supported clades (≥75% bootstrap) from single-locus trees were compared to assess the level of congruence among loci [21,22]. When there was no conflict using a 75% bootstrap value threshold, the data set was concatenated. In situations where a monophyletic group was supported with bootstrap values ≥ 75% at one locus and the same group of taxa was supported by ≤75% as non-monophyletic with another locus, the group was still assumed to be congruent [22]. Each locus was subjected to a randomized accelerated maximum likelihood (RAxML) analysis involving 1000 pseudoreplicates with RAxML-HPC BlackBox 8.2.6 [23] on the Cipres Science Gateway (http://www.phylo.org, accessed on 21 July 2022) and all four single-locus RAxML trees were compared. The conflicting gene sequences were removed based on the significant topological differences and the analysis was repeated until no further conflicts were detected. The four single-locus RAxML trees are shown in Supplementary Figures S1-S4.

Phylogeny of the Genus Peltula
The concatenated data set was subjected to phylogenetic analysis using RAxML-HPC v. 8.2.6 [23] and MrBayes v.3.2.6 [24,25] on the Cipres Science Gateway (http://www.phylo.org, accessed on 21 July 2022). The GTR + I + G model was selected in both ML and Bayesian analyses with 1000 pseudoreplicates. Two parallel Markov chain Monte Carlo (MCMC) runs were performed in MrBayes, each using 8 million generations and sampling every 1000 steps. A 50% majority-rule consensus tree was generated from the combined sampled trees of both runs after discarding the first 25% as burn-in. Tree files were visualized with FigTree v.1.4.2.

Phylogenetic Analysis
A total of 295 DNA sequences, including 104 new sequences (34 ITS, 33 nrLSU, 23 nrSSU and 14 RPB2) generated for this study, were used (Table 1). Bayesian Inference (BI) and Maximum Likelihood (ML) phylogenetic trees of the concatenated data set of the four gene markers were constructed, and they had similar topological structures. The RAxML tree is shown in Figure 1 with both bootstrap support (BS) and posterior probability (PP) values of BI analysis. The resulting tree showed two well-supported (BS = 100, PP = 1.00) branches corresponding to the two new species and almost all species had strong support. Although the new species, Peltula confusa, showed small genetic differences within the species, all specimens were consistent in terms of their morphology. The results also revealed the high genetic diversity of P. impressula. Initially, some specimens of P. impressula were mistakenly considered to be P. radicata, but all the specimens of P. impressula consistently had peltate squamules with small white dots (versus P. radicata with its fissured surface), a thin upper cortex (versus P. radicata without an upper cortex), and root-like rhizoids [6,8,26]. Our results also highly support the distinctness of the new species Peltula subpatellata from P. patellata and it can be easily distinguished by its morphology. The BI phylogenetic tree and four single-gene-locus RAxML trees are shown in Figures S1-S5.

Taxonomy
Peltula confusa Q.X. Yang & X.L. Wei, sp. nov. (Figure 2).  Diagnosis: The new species is characterized by a lighter, brighter and more yellowish upper surface than in other species of this genus, apothecial discs concentrated in the central part of the squamules and a thick lower cortex.
Habitat and distribution: The species was found on sun-exposed sandstone along the road in low-altitude mountains. It was intermixed with other species of this genus and other rock-inhabiting groups. It is presently known only to occur in China.
Additional Notes: This species was only found in Beijing, and it is very abundant in this area. We obtained many specimens of this species, and frequently free-living cyanobacteria are found to be associated with the thallus. Considering the seasonally wet and dry climate, we assumed that the rain washed the cyanobacteria from elsewhere, a phenomenon that is also common in Peltula pseudoboletiformis, another species we collected. Peltula confusa grows with other species of this genus, i.e., P. lobulata and P. placodizans, but the upper surface of the new species is lighter, brighter, and more yellowish, so they can be easily distinguished by their thallus color.
Peltula confusa often does not have prominent features, such as appendage structures or a special thallus outline. It is similar to P. sonorensis Büdel & T.H. Nash, which also has a simple thallus; however, the new species has a matte and tarnished upper surface, the absence of an upper cortex, a thicker lower cortex (45-85 µm, 10 layers of cells), and reddish black apothecial discs concentrated in the central part of the squamules, while P. sonorensis has a smooth upper surface with a primitive upper cortex, a thinner lower cortex (18-30 µm, 4-5 layers of cells), and yellow-brown discs randomly distributed within the squamules [27].
In the phylogenetic tree, Peltula confusa and the several rock-inhabiting squamulose species are clustered into a clade, among which P. polycarpa and P. lobulata are also dis-tributed in the Beijing area, indicating the three species live in a similar habitat and have evolved from a common ancestor [14].
Habitat and distribution: The new species was found on sandy soil in semi-arid and arid regions. It grows on stable or semi-stable soil with other soil-inhabiting species, but not on flowing sand. It is presently known only to occur in China.
Additional Notes: Peltula subpatellata strongly resembles the common and easily recognizable species P. patellata due to its flat to deeply concave squamules with ascending and darker margins and the existence of a tuft of hyphae connecting it to the substrate. The squamule margins of Peltula subpatellata are smooth, shiny, and not thickened, and are sometimes not obvious or darker in very mature peltate squamules; however, the squamule margins of P. patellata are thickened, resulting in a typical patellate form. The molecular data support Peltula subpatellata as a separate species, although it is closely related to P. patellata. Based on the four-locus phylogenetic tree, Peltula subpatellata, P. polyspora, P. psammophila (Nyl.) Egea, P. radicata, P. patellata, and P. richardsii (Herre) Wetmore form a highly supported clade, and they are all soil-inhabiting species found in (semi) arid regions. Although Kauff et al. attempted to group the genus by different types of growth forms, it was finally found that this grouping was unsuitable [5]. Our finding that species sharing the same substrates are phylogenetically related provides a new taxonomic characteristic for the group of this genus.
It should be noted that the ascospores of this new species contain oil drops. Among all the reported species in this genus, this characteristic has only been mentioned for Peltula impressula (reported as P. oleifera (H. Magn.) J.C. Wei) [6,28,29]. Based on our own observations, oil droplets are found in both the hymenium and ascospores of Peltula impressula. As for Peltula patellata, we did not find any information about oil droplets in past reports [30][31][32], but we recently noted their presence in the ascospores of a voucher specimen from the Santa Monica Mountains in Southern California, U.S.A. (J. Hollinger 9078, herb. Noell & Hollinger).

Discussion
In the four-locus phylogenetic analysis, the distance and genetic relationships among species of Peltula are clearly revealed. In particular, in species with unclear morphological boundaries, DNA sequences could resolve their phylogenetic relationships. The specimens of Peltula subpatellata were preliminarily identified as P. patellata, but the phylogenetic analysis and further morphological comparison with Jason Hollinger's specimen (J. Hollinger 9078) revealed that these specimens represent a new species, different from P. patellata. Previously, there have been several reports of P. patellata, but in the absence of DNA sequence analysis, identification of these specimens was mainly based on their morphology alone. For example, Upreti & Budel described P. patellata from India as having a K-epithecium and noted that this species was similar to P. obscurans [32]. In fact, P. patellata has a K+ epithecium and it is not similar to P. obscurans. To reach a more natural taxonomy and species delimitation it is essential that molecular data supplement morphological observations.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biology11101518/s1, Figure S1: The maximum likelihood tree of Peltula species based on the nrSSU sequences; Figure S2: The maximum likelihood tree of Peltula species based on the ITS sequences; Figure S3: The maximum likelihood tree of Peltula species based on the nrLSU sequences; Figure S4: The maximum likelihood tree of Peltula species based on the RPB2 sequences. Figure S5: The Bayesian tree of Peltula species based on the concatenated ITS + nrSSU + nrLSU + RPB2 data set.  Data Availability Statement: All data used in this study are reported in the paper.