Five New Species of the Lichen-Forming Fungal Genus Peltula from China

The genus Peltula is an important cyanobacterial lichen group. We performed a taxonomic study on the Peltula from China using phylogenetic analysis based on three gene loci (ITS, nuSSU, nuLSU) together with additional species delimitation analyses by ABGD, bPTP and GMYC approaches and the phenotypic characteristics. Five new species (Peltula lobulata, P. polycarpa, P. polyphylla, P. pseudoboletiformis and P. submarginata) were found and described. Peltula lobulata is diagnostic in its small thallus with plenty of lobules, rolled down and irregularly lobed margins, and uneven cracked surfaces. Peltula polycarpa has convex and rolled down lobes and numerous apothecia with a thalloid rim covering the whole lobe, and it can be distinguished from fertile P. farinosa (southern Switzerland) by a bright olive-green and epruinose surface, and the absence of isidia. Peltula polyphylla is differentiated from any other known Peltula species by a very small polyphyllous thallus composed of abundant olive-brown to olive-black small lobes growing tightly and sometimes anastomosing and attaching to the substrate by a large and strong umbilical cluster. Peltula submarginata is similar to P. marginata but differs in the presence of encircled epinecral and algae layers, and the absence of a lower cortex. Peltula pseudoboletiformis is different from the similar species P. boletiformis in greener lobes, more yellow–green umbilici and certain phylogenetic differences. Moreover, a key to the species of Peltula in China is also provided here.


Introduction
The lichen family Peltulaceae, affiliated to Lichinales, Lichinomycetes, Ascomycota, has been reported to have more than 50 species all over the world thus far and 14 known in China [1]. Peltula Nyl. is the only genus in this family and is symbiosed with the cyanobacterial photobiont known as Chroococcidiopsis, Myxosarcina, Gloeocapsa [2][3][4][5] or Chroococcales [6][7][8]. Through isolation and culture of lichen specimens and the construction of phylogenetic trees, Jung et al. [9] identified cyanobionts of Peltula as Aliterella and Compactococcus gen. nov., Pseudocyanosarcina gen. nov., and an unknown species, but did not find Chroococcidiopsis, which is the only known unicellular terrestrial genus and could be considered robust support to delimit the genus Peltula.
The genus Peltula is comprised of species with an olive-green peltate, squamulose to subfruticose thalli attached to the substratum by umbilicus or rhizines, immersed apothecia, a gelatinous sheath on the ascus and numerous spores per ascus [10,11]. It is currently subdivided into six growth forms of thallus, viz. peltate-umbilicate, squamulose-semifruticose, squamulose-compound, subfoliose-compound, crustose-areolate and crustose based on the revision of Peltulaceae [12]. The most diagnostic characters within Peltula refer to anatomical structures. The upper cortex is generally absent in Peltula species, with the epinecral layer existing instead, except for P. impressula, which has a translucid upper cortex, as well as P. farinosa [5] and P. sonorensis [4,13] with a primitive upper cortex; the medulla is often composed of interwoven or cellular hyphae with a large air space existing in the

Taxon Sampling
Fifteen specimens for this study were collected from Beijing, capital of China (see Figure 1). The voucher specimens were deposited in the lichen section of fungarium of Institute of Microbiology, Chinese Academy of Sciences (HMAS-L). A LEICA M125 and LEICA DFC450 dissecting microscopes were used for the morphological studies. The internal morphology of the lichen thallus and ascomata was studied using free-hand sections. A Zeiss Axioscope2 compound microscope with a Zeiss Axio Imager A2 was used for the anatomical studies, and a Zeiss AxioCam MRc5 camera was used for taking photographs. Spot tests were performed using K (10% aqueous solution of potassium hydroxide) and IKI (1% aqueous iodine solution with 10% aqueous potassium hydroxide). Lichen substances were examined using standardized thin layer chromatography (TLC, solvent C) [22]. medulla is often composed of interwoven or cellular hyphae with a large air space existin in the subfruticose thallus; and the lower cortex in most species clearly consists of 3layers of large cells. Most of the species in Peltula have immersed apothecia, while P. au riculata and P. lobata are characterized by adnate type and P. imbricata with a sessile typ [6,7,14]. In chemistry, only two closely related yellow pigments, myeloconone D1 and D have been detected from Peltula langei Büdel ex Elix [15].
Previous reports of Peltula in China mainly focused on Hong Kong [16,17], Taiwa [18], Gansu [19,20] and Inner Mongolia [21]. This study is based on an additional surve of Peltula on the national scale, including both the typical arid and semi-arid areas such a Inner Mongolia, Gansu, Ningxia and Qinghai, and some humid and semi-humid area such as Anhui, Beijing, Hebei, etc. Surprisingly, Beijing is found to be abundant in Peltul species diversity for the first time, and all the five new species with umbilici, rhizines o stalks attaching to substrates, such as soil or rock surfaces, are described from here.

Taxon Sampling
Fifteen specimens for this study were collected from Beijing, capital of China (se Figure 1). The voucher specimens were deposited in the lichen section of fungarium o Institute of Microbiology, Chinese Academy of Sciences (HMAS-L). A LEICA M125 an LEICA DFC450 dissecting microscopes were used for the morphological studies. The in ternal morphology of the lichen thallus and ascomata was studied using free-hand sec tions. A Zeiss Axioscope2 compound microscope with a Zeiss Axio Imager A2 was use for the anatomical studies, and a Zeiss AxioCam MRc5 camera was used for taking pho tographs. Spot tests were performed using K (10% aqueous solution of potassium hydrox ide) and IKI (1% aqueous iodine solution with 10% aqueous potassium hydroxide). Liche substances were examined using standardized thin layer chromatography (TLC, solven C) [22].

DNA Extraction, PCR and Sequencing
Fourteen fresh specimens were chosen for DNA extraction in this study. The extrac tion procedure followed a modified CTAB method [23]. PCR was performed to amplif three gene loci: nuclear ribosomal DNA internal transcribed spacer (ITS), small subun (nuSSU) and large subunit (nuLSU). The PCR primers and procedures of nuSSU an

DNA Extraction, PCR and Sequencing
Fourteen fresh specimens were chosen for DNA extraction in this study. The extraction procedure followed a modified CTAB method [23]. PCR was performed to amplify three gene loci: nuclear ribosomal DNA internal transcribed spacer (ITS), small subunit (nuSSU) and large subunit (nuLSU). The PCR primers and procedures of nuSSU and nuLSU followed Kauff et al. [12]. ITS was amplified using the primers ITS4 and ITS5 [24] in 25 µL reactions containing 12.5 µL 2 × Taq PCR MasterMix®(Beijing Jiangchen Biotechnology Co., Ltd., Beijing, China), 1 µL each primer solution (10 µM), 9 µL ddH 2 O and 1.5 µL genomic DNA, and the PCR cycling conditions comprised of an 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. The target PCR products were checked by electrophoresis on 1% agarose gels and then sequenced in Ruibio BioTech Co., Ltd. (Beijing, China) and BioSune (Shanghai) (Beijing, China). The new sequences generated for this study were deposited in GenBank. A total of 162 DNA sequences including 37 new sequences (14 ITS, 14 nuLSU and 9 nuSSU) generated for this study under accession numbers MT499282-MT499304 and MT499313-MT499326 were used in this study (Table 1). Lichinella iodopulchra (Lichinales) was chosen as the outgroup. All sequences of three loci (ITS, nuSSU, nuLSU) were aligned separately using ClustalW Multiple Alignment [25] in BioEdit v7.0.5 [26]. The program Gblocks v0.91b [27,28] was used to remove regions of alignment uncertainty, using options for a "less stringent" selection on the Gblocks web server (http://molevol. cmima.csic.es/castresana/Gblocks_server.html, accessed on 1 May 2021).  Notes: Newly generated sequences are in bold font. "-" indicates that the corresponding sequence is absent.

Congruence among Loci
To test the level of congruence among loci, highly supported clades (equal to or more than 75% bootstrap) from single-gene trees were compared and assessed [29,30]. When there was no conflict using a 75% bootstrap value threshold, in situations where a monophyletic group was supported with bootstrap values ≥75% at one locus and the same group of taxa was supported (≤75%) as nonmonophyletic with another locus, then the group was assumed to be congruent, and the data set was concatenated [30]. Each locus was subjected to a randomized accelerated maximum likelihood (RAxML) analysis, involving 1000 pseudoreplicates with RAxML-HPC BlackBox 8.2.6 [31] on the Cipres Science Gateway (http://www.phylo.org, accessed on 1 May 2021), and all three single-locus RAxML trees were compared. We removed the conflicting gene sequences based on the significant topological differences and repeated the test until no further conflicts could be detected. The best model for the three single genes used in the phylogenetic analysis was identified using PartitionFinder 2 [32]. The results were visualized with FigTree 1.4.2.

Phylogeny of the Genus Peltula
Phylogenetic analyses of Peltula were performed using the concatenated data set using RAxML-HPC v. 8.2.6 [31] and MrBayes v.3.2.6 [33,34] on the Cipres Science Gateway (http://www.phylo.org, accessed on 1 May 2021). 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 (http://tree.bio.ed.ac. uk/software/figtree/, accessed on 1 May 2021). The multilocus data alignment file was submitted to TreeBASE (submission ID 26385).

Species Delimitation Analyses
Three species delimitation methods, Automatic Barcode Gap Discovery (ABGD) [35], a Bayesian implementation of the Poisson tree process model (bPTP) [36] and the General Mixed Yule Coalescent (GMYC) approach [37,38], were used to circumscribe species boundaries in the genus Peltula from the ITS and nuLSU sequence alignments.
ABGD analysis was performed using the ABGD webserver (http://wwwabi.snv. jussieu.fr/public/abgd/abgdweb.html, accessed on 20 October 2021). We used default parameters except for using a Pmax at 0.01 and a relative gap width of 1.5 with the Jukes-Cantor model (JC69). The PTP model is intended for delimiting species in three gene-locus (ITS + nuSSU + nuLSU) molecular phylogenies and provides an objective approach for delimiting putative species boundaries that are consistent with the phylogenetic species criteria. We used the bPTP web server (http://species.h-its.org, accessed on 20 October 2021) [36] to delimit putative species groups using the concatenated topology as the input tree and implementing default settings. The GMYC method aims to detect shifts in branching rates between intra-and inter-specific relationships. Within a likelihood framework, it uses chronograms to compare a null model under which the whole sample belongs to a single species and hence follows a coalescent process and an alternative general mixed Yule coalescent (GMYC) model. The latter combines equations describing branching patterns within and among lineages. A likelihood ratio test (LRT) was used to evaluate whether the null model can be significantly rejected. If the GMYC model fits the data significantly better than the null model, the threshold T allows for the estimation of the number of species present in the data set. First, ultrametric trees were estimated using the program BEAST v2.6.3 [39]. We ran two independent Markov Chain Monte Carlo (MCMC) chains for 15 million generations under the coalescent model with a constant population size and a constant clock as the tree prior. Default values were used for the remaining priors. The outputs were diagnosed for convergence using TRACER v.1.7.2 [40] after removing 10% of the samples as a burn-in. The effective sample size (ESS) values greater than 200 were considered a good indicator. Tree files from two independent runs were combined using LogCombiner [39]. A consensus tree was generated using TreeAnnotator 1.8.2 [39] after discarding the first 3000 trees. The GMYC analysis was performed on an ultrametric consensus tree under the single-threshold model using the SPLITS package [38] available for R 4.0.5 [41].

Phylogenetic Analysis
The aligned matrix contained 3380 unambiguous nucleotide position characteristics for the full data set of 54 members. BI and ML phylogenetic trees of Peltula were constructed, and they had similar topological structures. The RAxML tree is shown in Figure 2 with both bootstrap support (BS) and posterior probability (PP) values of BI analysis. In the tree, all the Peltula species clustered into a monophyletic clade and obviously separated from the outgroup (PP = 1), within which five well-supported (BS = 100, PP = 1.00) branches corresponding to the five new species were included. The multilocus phylogenetic analysis highly supports (BS100/PP1.00) that Peltula farinosa, P. polycarpa and P. lobulate clustered closely but also distinctly (see Figure 2), and they can be easily distinguished in morphology. The BI phylogenetic tree and three single-genelocus RAxML trees are shown in Figures S1-S4.
The results of species delimitation analyses are shown in Figure 2. Different colour patches correspond to different species, and the same colour patches refer to the same species. The ABGD analysis, based on the concatenated data set, provided evidence supporting the species delimitation scenario similar to the phylogenetic results, for example, Peltula farinosa, P. polycarpa and P. lobulata are delimited into three separate species, but the same species Peltula euploca and P. eupcola ssp. sorediosa are delimited into two different ones (Table S1). The tree-based bPTP analysis obtained the same results as ABGD analysis (Table S2), and the GMYC analysis provided further and stronger support (most of the posterior probability = 1.0) for delimiting all the species (Table S3).
species. The ABGD analysis, based on the concatenated data set, provided evidence supporting the species delimitation scenario similar to the phylogenetic results, for example, Peltula farinosa, P. polycarpa and P. lobulata are delimited into three separate species, but the same species Peltula euploca and P. eupcola ssp. sorediosa are delimited into two different ones (Table S1). The tree-based bPTP analysis obtained the same results as ABGD analysis (Table S2), and the GMYC analysis provided further and stronger support (most of the posterior probability = 1.0) for delimiting all the species (Table S3).

Figure 2.
The RAxML tree of Peltula species based on the concatenated ITS + nuSSU + nuLSU data set. The numbers in each node represent bootstrap support (BS) and posterior probability (PP) values. BS values ≥ 75 and PP values ≥ 0.95 were plotted on the branches of the tree. The clades corresponding to the new species are in bold, which indicates that these sequences were newly generated for this study. Scale in 0.08 substitution per site. Three species delimitation analysis results are listed on the right side of the tree, among which different colour patches correspond to the different species recognized by the software, and the same colour patches refer to the same species.

Taxonomy
The genus Peltula is a cyanobacterial lichen group. It should be recognized that the cyanobiont morphology described here refers to the lichenized state in thallus not the freeliving form in nature [42]. A key to the species of Peltula is listed in Table 2.
Peltula lobulata Q.X. Yang & X.L. Wei, sp. nov. (Figure 3). MycoBank: MB 840880. The clades corresponding to the new species are in bold, which indicates that these sequences were newly generated for this study. Scale in 0.08 substitution per site. Three species delimitation analysis results are listed on the right side of the tree, among which different colour patches correspond to the different species recognized by the software, and the same colour patches refer to the same species.

Taxonomy
The genus Peltula is a cyanobacterial lichen group. It should be recognized that the cyanobiont morphology described here refers to the lichenized state in thallus not the free-living form in nature [42]. A key to the species of Peltula is listed in Table 2.
Chemistry: No substances detected by TLC.
Habitat and distribution: The species sample was found on sun-exposed red shale and gray sandstone along the road on the low altitude mountain. It is dispersed and not associated with other species and known only in China up to now.
Additional Notes: This new species is easily overlooked in the field because the thallus is very small and the colour is very similar to the surrounding environment. It is rather difficult to be identified only based on the external morphology but needs additional anatomical structures and DNA sequences. The thallus of this species is irregular ( Figure 3A,B), with plenty of lobules on each squamule as the most diagnostic feature, which is more obvious in section ( Figure 3C Diagnosis: It is characterized by numerous apothecia with thalloid rims and large discs (to 0.4 mm) and strongly curved thalli.
Description: Thallus terricolous, polyphyllous, up to 2.5 mm in diameter, to 0.9 mm in diameter, concave, margins slightly to deeply lobed, undulate and upper surface olive-brown to olive-black, epruinose; lower surface smooth, pale y brown, attached to the substrate by a large and strong umbilicus cluster compose bilicus of each lobe; isidia and soredia absent. Thallus 550 µm thick, lobes 130 thick, upper cortex not developed but with a yellowish epinecral layer, 5 µm th Notes: This new species is very distinctive and can be readily identifiable in the field by its numerous and bright red apothecia with thalloid rims, especially at maturity, its strongly revolute lobe tips and the light colour of its young lobes. The lobes of most species in Peltula have been known to be attached the substrate by rhizines or central umbilici; however, the umbilicus of this species is in the lateral position, which is more prominent in a polyphyllous thallus. Phylogenetic analyses confirm that this species is closely related to P. farinosa. Indeed, this new species looks very similar to fertile P. farinosa (southern Switzerland) because they both have large and broad squamules and numerous apothecia with thalloid rims when mature. However, these two species can still be distinguished quickly in morphology. Fertile Peltula farinosa has simple gray squamules without lobules, with a pruinose upper surface and sorediate undulated margins [5,43], while P. polycarpa is initially pale-yellow and formed by tongue-shaped squamules, later turns into bright olive-green and polyphyllous thalli, characterized by several lobes growing around a same umbilicus or different umbilici aggregating together with strongly convex thalli, occasionally superficial lobules, and an absence of isidia, soredia and pruina. Although the systematic positions of the two species are close, the morphological characteristics and species delimitation analyses support that they are two independent species.
Yang & W.C. Wang 20,191,645 (HMAS-L 145475). Notes: The thallus of this new species is very small and grows together with other species of Peltula, such as P. euploca and P. placodizans, so it is not easy to be noticed in the field. However, when being observed under a dissecting microscope, this species is readily distinguishable due to its polyphyllous thallus composed of many small lobes. The small lobes grow tightly, sometimes connecting with each other and anastomosing ( Figure  5C). The thallus attaches to the substrate by a large and strong umbilical cluster ( Figure  5B).
Peltula polyphylla is related to P. auriculata, but they can be easily distinguished. Thallus of P. auriculata attached to the substrate by umbilicus and its lobes have ear-shaped appendices while P. polyphylla attached to the substrate by a large and strong umbilical cluster with peltate lobes [6]. Phylogenetic analysis indicated that P. polyphylla and P. auriculata clustered into the same clade, which supported that they are closely related but different. The additional species delimitation analyses also defined P. polyphylla and P. auriculata into separate species. Besides, this new species is similar to P. imbricata due to having polyphyllous thalli, but the thallus of P. imbricata is made up of small imbricate lobes, sometimes with pruinose upper surface, tightly appressed to the substrate, forming small colonies or a continuous crust to 10 mm in diam. While P. polyphyllais consists of many peltate and epruinose lobes, attached to the substrate by a large and strong umbilical cluster, and the colonies up to 2.5 mm in diameter [14]. Diagnosis: The new species is characterized by a polyphyllous thallus composed of abundant olive-brown to olive-black small lobes.
Chemistry: No substances detected by TLC.
Habitat and distribution: This species sample was found on sun-exposed rocks covered with a thin layer of soil. Peltula euploca and P. placodizans are on the bare rocks nearby. Notes: The thallus of this new species is very small and grows together with other species of Peltula, such as P. euploca and P. placodizans, so it is not easy to be noticed in the field. However, when being observed under a dissecting microscope, this species is readily distinguishable due to its polyphyllous thallus composed of many small lobes. The small lobes grow tightly, sometimes connecting with each other and anastomosing ( Figure 5C). The thallus attaches to the substrate by a large and strong umbilical cluster ( Figure 5B).
Peltula polyphylla is related to P. auriculata, but they can be easily distinguished. Thallus of P. auriculata attached to the substrate by umbilicus and its lobes have ear-shaped appendices while P. polyphylla attached to the substrate by a large and strong umbilical cluster with peltate lobes [6]. Phylogenetic analysis indicated that P. polyphylla and P. auriculata clustered into the same clade, which supported that they are closely related but different. The additional species delimitation analyses also defined P. polyphylla and P. auriculata into separate species. Besides, this new species is similar to P. imbricata due to having polyphyllous thalli, but the thallus of P. imbricata is made up of small imbricate lobes, sometimes with pruinose upper surface, tightly appressed to the substrate, forming small colonies or a continuous crust to 10 mm in diam. While P. polyphylla is consists of many peltate and epruinose lobes, attached to the substrate by a large and strong umbilical cluster, and the colonies up to 2.5 mm in diameter [14].
Peltula pseudoboletiformis Q.X. Yang & X.L. Wei, sp. nov. (Figure 6). MycoBank: MB 840883. Etymology: The epithet 'pseudoboletiformis' refers to the thallus morphology similar to that of the mushroom Boletus, which is why this new species is similar to Peltula boletiformis, so the epithet 'pseudoboletiformis' is chosen to define this new species. Diagnosis: This new species is different from the similar species P. boletiformis in more yellow-green umbilici and larger conidia.
Description: Thallus saxicolous, squamulose-subfruticose, loosely or tightly clustered, up to 1.5 mm in diameter, 2 mm high, stalk 0.9 mm long in average, twisted, orange to tan; lobes circular, elliptical to angular and irregular, top almost flattened, margin entire; upper surface dark olive-green, smooth, epruinose; attached the substrate by umbilicus, isidia and soredia absent. Lobes 350-600 µm thick, upper cortex not developed, with a thin yellowish epinecral layer, 5-8 µm thick; the algal layer of immature thalli 57.5-67.3 µm thick, medulla with interwoven hyphae and small air spaces and lower cortex not clear, made up of several layers of circular cells; later algal layer encircled, 119-240 µm thick, algae in clumps of 1-4 cells, cells up to 13 × 15 µm in diameter, medulla with rare and loose hyphae and big hollow areas, hyphae 2.5-3.3 µm wide. Apothecia 1 or 2 per squamule, immersed and punctiform. Epihymenium yellow; hymenium 95-215 µm tall, paraphyses 1.5-2 µm wide, I+ wine red, K-; subhymenium up to 100 µm tall, IKI+ blue after K pretreatment; asci clavate to obclavate, 70-95 × 18-20.5 µm, with a lacerate gelatinous sheath, more than 100 spores, ascus wall IKI+ blue after K pretreatment; ascospores hyaline, ellipsoid, simple, 4.5-5.5 × 3-3.8 µm. Pycnidia immersed, cerebriform. Conidia ellipsoid, hyaline, 3.5-4.1(4.5) × 1.6-2.3 µm.  Notes: In the early stage, the thallus of this new species is squamulose attached to the substrate by the umbilicus. Gradually, the umbilicus elongated and thickened into a stalk. In the process of growing, the internal structure of thalli also changed. The boundary between the algal layer and the medulla becomes distinct, the medulla tends to be surrounded by the algal layer and the medullary cavities correspondingly become larger, varying from the previous small air spaces to the later large medullary cavities ( Figure 6D,E). Peltula pseudoboletiformis is similar to P. boletiformis; the only external minor difference is the colour of thalli. Peltula pseudoboletiformis has a dark olive-green lobe and yellow-green umbilicus of mature thalli, while the lobe of P. boletiformis is black to blackbrown, and the umbilicus is olive-brown. However, the genetic distance in the phylogenetic tree and the additional species delimitation analysis support them to be separate species. Therefore, the distinction between P. pseudoboletiformis and P. boletiformis mainly depends on the DNA sequences. Diagnosis: This species is similar to P. marginata but differs in the epinecral layer covering the whole surface and the underlying algal layer surrounding the whole perimeter of squamules, larger conidia, and absence of lower cortex.
Chemistry: No substances detected by TLC.
Habitat and distribution: This species sample was found on sun-exposed sandstone, and it occupies most of the rock face where no other lichens are nearby. It is known only in China up to now. deed, the new species P. submarginata is also similar to the new sp in growth types. However, more regular squamules, brighter up tiform apothecia and rounded algae layer are observed in P. su irregular and dark olive-green squamules, rare apothecia, and cle surrounded algae layer in P. pseudoboletiformis.  Notes: This new species is easily confused with P. marginata due to their similarity in general morphology, such as the colour of thalli, the shape of lobes and the size and number of apothecia. However, they clearly differ in presence of surrounding epinecral and algae layers and the absence of an algal layer in the lower cortex. Büdel [11] mentioned that the algal layer of P. marginata is obviously stratified, while the epinecral layer of P. submarginata covers the whole surface and the underlying algal layer surrounds the whole perimeter of squamules ( Figure 7C,D). The epinecral and lower cortex layers are not distinguished but forming a whole. This feature can be also seen in Peltula cylindrica, but the latter species has cylindrical lobes [10]. In the phylogenetic tree, P. submarginata and P. marginata are in relatively close clades, supporting them as the most similar species. Indeed, the new species P. submarginata is also similar to the new species P. pseudoboletiformis in growth types. However, more regular squamules, brighter upper surface, black punctiform apothecia and rounded algae layer are observed in P. submarginata compared to irregular and dark olive-green squamules, rare apothecia, and clearly stratified or slightly surrounded algae layer in P. pseudoboletiformis.

Discussion
Peltula occurs worldwide in semiarid/arid regions and mainly grows on the fully exposed rock or soil surfaces, on seepage faces, at the border of small rivulets in the spray water region or along rain-water tracks [3][4][5][6][7]14]. Species of Peltula have different preferences to the growth environment. Saxicolous species of Peltula are more distributed in alternating dry and wet areas, while terricolous species are more distributed in desert areas with low rainfall. All the five new species in this study were collected from Beijing area, where has a typical temperate monsoon climate with obviously dry and rainy seasons and is a suitable habitat for saxicolous species, especially for those growing on calcareous rocks. In addition to the five new species, Peltula euploca, P. placodizans and P. obscurans etc. were also found here. Based on our current investigation and analysis, it is speculated that Beijing area may be a distribution hotspot with high species diversity of Peltula in China.
Subfruticose growth type has been considered as the most advanced trait in Peltulaceae [11,12,44]. The factors affecting the distribution of squamulose-subfruticose Peltula submarginata and P. pseudoboletiformis may be same to the other subfruticose species of Peltula, such as water conditions and light. Peltula submarginata and P. pseudoboletiformis are similar to P. marginata and P. boletiformis, respectively, among which the latter two were described to be squamulose species originally by Büdel [11] and were further defined into squamulose-semifruticose growth form by Kuaff et al. [12]. In this study, we defined these two new species as squamulose-subfruticose growth type considering the small to large medullary cavities inside the thalli [12]. They have small lobes and thick stalks, but these traits are not stable. Lobes are generally not developed when young, then the lobes enlarge, and small umbilicus gradually develop into thick and long stalks. The phylogenetic tree also shows that P. submarginata and P. pseudoboletiformis clustered in a moderately supported clade together with P. tortuosa, P. lingulata and P. clavata (Figure 2), which have a subfruticose growth type.
Due to different water conditions in different regions or growth environments, the subfruticose growth type may be an intermediate type between the clavate type and lingulate type or peltate type [11]. Severe ecological environments or different habitats would affect anatomical and morphological characters of lichens, which means that lichens will have a correspondingly adaptive evolution to the different environments [45][46][47][48]. Wessels and Büdel [48] summarized that black subfruticose species of Peltula seem to grow specifically near water sources where they can even be submerged for short periods. Combining drought and water needs, subfruticose species of Peltula possess both drought periods and sufficiently short water supply periods. Instantaneously sufficient water makes the thallus grow higher, while seasonal drought limits the endless growth of the thallus into fully fruticose, and the alternation between sufficient water and rapid drying promotes the generation of bubbles or cavities in the medulla to store water and exchange gas [11,47,48]. Obviously, the subfruticose species of Peltula can be fully drought resistant before sufficient water comes.
In addition to the specific water supply, light may also play a decisive role in the distribution of Peltula [11,49]. Therefore, to obtain a higher ecological niche and get more available light sources, the algae layer of subfruticose species such as Peltula cylindrica, P. tortuosa and P. submarginata might evolve into encircled algae layer with a larger area than the flat algae layer. These adaptive evolutionary traits in morphology and anatomy could help the species of Peltula colonize both arid and humid habitats well.
In China, there is still a large gap in recognizing Peltula and the whole Lichinales. This Peltula study may also be a tip of the iceberg. Therefore, it should be noted that it is necessary to add more molecular data of more species and more fresh specimens to better understand and solve the existing uncertainty within this genus. For example, the results of species delimitation analyses in this study showed that Peltula euploca and P. euploca ssp. sorediosa, which have always been considered to be the same species previously, are two separate ones. Although we cannot immediately deny the previous conclusions based on our results, the question of Peltula eupcola and P. eupcola ssp. sorediosa being conspecific or not, attracted our attention. In addition, we also found that Peltula impressula has great intraspecific differences in the phylogenetic tree compared with what we have understood in the past. The phylogenetic differences may be related to the colour and size of thalli. However, the specimens within the range of phylogenetic differences all have peltate and strongly rhizoid thallus, and dotted upper surface with shallow, K+ reddish violet upper cortex [19]. We consider that these differences tend to be intraspecific more than interspecific. It is believed that molecular data can allow us to better understand the intra-and inter-species differences and distances. Besides, more comprehensive and extensive research in the future will greatly improve the taxonomic status of Peltula and Lichinales.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof8020134/s1, Figure S1: The Bayesian tree of Peltula species based on the concatenated ITS + nuSSU + nuLSU (three-gene) data set; Figure S2: The maximum likelihood tree of Peltula species based on the nuSSU sequences; Figure S3: The maximum likelihood tree of Peltula species based on the ITS sequences; Figure S4: The maximum likelihood tree of Peltula species based on the nuLSU sequences; Table S1: ABGD species delimitation results; Table S2: bPTP species delimitation results based on Maximum Likelihood partition; Table S3: GMYC species delimitation results.
Author Contributions: X.W. conceived and designed the study. Q.Y., X.W., T.Z. and X.C. collected specimens from China. Q.Y. generated the DNA sequence data, Q.Y. and X.W. performed the phenotypic analysis, Q.Y., X.L. and X.W. analyzed the DNA data. Q.Y. and X.W. checked issues related to nomenclatural articles. Q.Y. wrote the manuscript draft. Q.Y., X.L. and X.W. revised the draft and all authors approved the final manuscript. All authors have read and agreed to the published version of the manuscript.