Hidden Species Diversity was Explored in Two Genera of Catapyrenioid Lichens (Verrucariaceae, Ascomycota) from the Deserts of China

Verrucariaceae is the third-largest lichen family with high species diversity. However, this diversity has not been well-explored in China. We carried out a wide-scale field investigation in the arid and semi-arid regions of Northwest China from 2017 to 2021. A large number of lichen groups, especially those commonly distributed in deserts, were collected. Based on molecular phylogeny using ITS and nuLSU sequences by Bayesian and maximum likelihood analyses, combining morphological characters, seven taxa of catapyrenioid lichens in Verricariaceae were found in this study, including one genus (Clavascidium) and one species (Clavascidium lacinulatum) new to China; one genus (Placidium) new to the mainland of China; and four species (Clavascidium sinense, Placidium nitidulum, Placidium nigrum, and Placidium varium) new to science. It enriched our understanding of the high species diversity in Verrucariaceae and the lichen flora of Chinese arid and semi-arid deserts.


Introduction
The lichen family Verrucariaceae Eschw. is affiliated with Verrucariales, Eurotiomycetes, and Ascomycota, including 43 genera and 943 species [1]. Members of this family can colonize on various substrates, such as rock, soil, wood or bark, moss, and even other lichens [2]. Many species can tolerate harsh environments and participate in forming biological soil crusts (BSCs) in arid and semi-arid regions, such as catapyrenioid lichens [3].

Phylogenetic Analysis
A total of 61 sequences newly obtained in this study were used in the single-locus phylogenetic tree construction; among which, 30 sequences of the ITS and nuLSU generated from 15 specimens representing 5 species (7 Clavascidium specimens and 8 Placidium specimens) were combined for constructing the concatenated tree ( Figure 2). The concatenated matrix included 1204 variable positions (448 ITS and 756 nuLSU) after excluding ambiguous regions. The aligned matrix contained 1204 nucleotide position characteristics for the complete data set of 67 members. The concatenated BI phylogenetic tree and two single-gene-locus RAxML trees including 44 and 16 samples are shown in Figures S1-S3. Placidium, Clavascidium, and Heteroplacidium all formed monophyletic branches. Both the ML and BI phylogenetic trees produced similar topologies. There are four new species: Clavascidium sinense sp. nov., Placidium nitidulum sp. nov., Placidium nigrum sp. nov., and Placidium varium sp. nov. are well-supported. Figure 1. Collection sites. The detailed collection areas are marked in a solid red circle, and the corresponding four provinces involved are marked with varying degrees of a gray color.

Figure 2.
The RAxML tree is based on the concatenated ITS + nuLSU data set representing both ML and BI trees. The number in each node represents bootstrap support (BS) and posterior probability Figure 2. The RAxML tree is based on the concatenated ITS + nuLSU data set representing both ML and BI trees. The number in each node represents bootstrap support (BS) and posterior probability (PP) values. BS values ≥ 75 and PP values ≥ 0.95 were plotted on the branches. The taxa in bold indicate that these sequences were newly generated for this study. Green and red clades show Clavascidium and Placidium, respectively. The character status of 1 ascospores arrangement, 2 pycnidia position, and 3 rhizines are listed at the right of the tree, corresponding to each sample of Clavascidium and Placidium. Scale in 0.04 substitution per site.

Taxonomy
A key to the species of Clavascidium and Placidium is listed in Table 1. The genus Clavascidium is characterized by squamulose thallus, the presence of rhizines, and clavate to (sub) cylindrical asci containing biseriate ascospores. Gueidan [2,9] introduced Clavascidium as a sister genus of Placidium based on morphological characters and a phylogenetic analysis, between which Clavascidium has clavate asci with biseriate ascospores, whereas Placidium has cylindrical asci with uniseriate ascospores. However, uniseriate or both biseriate and uniseriate ascospores were also reported [30]. Description: Thallus squamulose, terricolous, 300 µm thick; lobes 1-4 mm wide, roundish to deeply lobed, contiguous, rarely overlapping; upper surface dark brown, dull; lower surface pale ± rhizines; epinecral layer transparent, up to 15 µm thick; upper cortex paraplectenchymous, 50-70 µm thick, cells 4 × 10 µm in diam.; uppmost layer brown, 25-30 µm thick; photobiont layer 50-100 µm thick, algal cells 6-10 µm in diam; medullar tissue paraplectenchymous; lower cortex not delimited from the medulla, 40 µm thick, paraplectenchymous, composed of irregularly arranged roundish cells up to 7.5 µm in diam. Rhizohyphae hyaline, 4-6 µm wide. Pycnidia laminal, Dermatocarpon-type, immersed, subglobose, light brown, conidia oblong-ellipsoid to bacilliform, 1-1.3 × 3-3.7 µm in size. Perithecia immersed, broadly pyriform (150 × 180 µm) to subglobose (up to 230 µm) wide; perithecia wall bright, 30-35 µm thick; hymenium bright, 35-45 µm thick; involucrellum brown, 42-44 µm thick; pyriphyses 35-40 × 2.5-3 µm; asci cylindrical to clavate, 14-18 × 40-52 µm, ascospores 8 per ascus, uniseriate to biseriate, ellipsoid to fusiform to ovoid, 5-7 × 10-12 µm. Chemistry: All the spot tests were negative, and no substances were detected by TLC. Habitat and distribution: These species grow on the surface of sandy soil in the semi-arid and arid region of Northwest China, located in the open areas with sun exposure. The surrounding environment is characterized by interlace of meadows, fixed undulating sand dunes under shrubs, and the Gobi Desert, with the elevations greatly varying from 923 to 3175 m. It distributes worldwide [30] and is new to China. Notes: This species is widespread in the desert regions of China as a crust ( Figure 1).Our specimens are well-clustered with Cl. lacinulatum in the phylogeny [4] (Figures 2 and S1-S3) and, also, with a high consistency of the phenotype described by Nash et al. [30]. Although this species is variable in the external morphology of thallus and squamules, several important taxonomic characters such as laminal pycnidia, oblong-ellipsoidal to subcylindrical conidia, and rhizines are constantly present. Breuss described uniseriate ascospores in this species [31]; however, we found this character is variable ( Figure 3).    Prieto. However, Cl. sinense has distinct traits compared with these three species, for example, Cl. antillarum is characterized by dark brown to black lower surface and rhizines [5], while Cl. sinense is characterized by a pale lower surface and rhizines. Cl. imitans and Cl. krylovianum can be distinguished by the morphology of medulla; the former medullary hyphae divide into many short and swollen cells, but the latter medullary hyphae are filamentous [5]; in comparison, Cl. sinense has a mixed-type medulla. The genus Placidium has squamulose thallus, laminal or marginal Dermatocarpon-type pycnidia, cylindrical asci, uniseriate ascospores, and rhizohyphae [8]. Two Placidium species have been reported from Taiwan by Dr. Aptroot [13]. This genus is firstly reported from Mainland China in this study, and we found some additional morphological characters in Placidium such as a glossy upper surface and aggregated pycnidia, which can be used to distinguish some species.   The genus Placidium has squamulose thallus, laminal or marginal Dermatocarpon-type pycnidia, cylindrical asci, uniseriate ascospores, and rhizohyphae [8]. Two Placidium species have been reported from Taiwan by Dr. Aptroot [13]. This genus is firstly reported from Mainland China in this study, and we found some additional morphological characters in Placidium such as a glossy upper surface and aggregated pycnidia, which can be used to distinguish some species.  Notes: This new species can be easily recognized by its glossy appearance of the upper surface and tiny lobes, which is very distinctive and different from all the other known Placidium species. The medulla zone is obscure due to being fully covered by the thick algal layer, so the lower cortex is difficult to delimit from the medulla, similar to Placidium tenellum (Breuss) Breuss in this character. However, the upper surface of Pl. tenellum is matt, the algal layer is thinner ((40) 93 ± 24 (155) µm), and the pycnidia is much broader (up to 500 µm). The distribution is more frequent in coastal areas [8]. Based on the phylogenetic trees ( Figure 2 and Figures S1-S3), the new species is close to Placidium fingens (Breuss) Breuss, Pl. pilosellum, and Pl. tenellum, among which the first two are more intimate than the last one to the new species in phylogeny; however, the lobe widths of Pl. fingens and Pl. pilosellum (up to 6 mm) are nearly three times the new species (0.7-2 mm). Pl. nitidulum is closer to Pl. fingens, both of which have laminal pycnidia, while the species Pl. pilosellum within this subclade with a little far distance has marginal pycnidia. Pl. nitidulum has more slender asci (45-65 × 5-8 µm) than the species Pl. pilosellum (70-90 × 10-15 µm). Additionally, Pl. nitidulum has squamules with a smooth margin, but Pl. pilosellum has squamules with hairy margins [8].    Notes: This species is distinctive by having both uniseriate and (sub) biseriate ascospores arrangement, both laminal and marginal pycnidia, and a surrounding black area due to the aggregation of abundant pycnidia, which well-separate it from all the other Placidium species. Placidium is generally known as the only genus comprising species with laminal or marginal pycnidia [31]. Within this genus, very few species have both laminal and marginal pycnidia such as Pl. velebiticum (Zahlbr. ex Zschacke) Breuss [4,8]; however, Pl. velebiticum only has a uniseriate ascospores arrangement, which is different from Pl. nigrum.  [4,8]; however, Pl. velebiticum only has a uniseriate ascospores arrangement, which is different from Pl. nigrum.

Discussion
Nowadays, dry lands cover about 41% of Earth's land surface and influence more than 38% of the global population [33]. Curbing the spread of land desertification in arid and semi-arid areas has become an urgent problem that needs to be focused on. Biological soil crusts (BSCs), as a ubiquitous phenomenon in these regions, play an essential role in soil nutrient cycling, sand stability, and hydrological processes [34]. When the BSCs

Discussion
Nowadays, dry lands cover about 41% of Earth's land surface and influence more than 38% of the global population [33]. Curbing the spread of land desertification in arid and semi-arid areas has become an urgent problem that needs to be focused on. Biological soil crusts (BSCs), as a ubiquitous phenomenon in these regions, play an essential role in soil nutrient cycling, sand stability, and hydrological processes [34]. When the BSCs developed to the lichen crust stage, they would contribute to the greater compressive strength and carbon and nitrogen fixation [35,36]. Lichens existing as the crust in the desert regions are very popular, such as Endocarpon and catapyrenioid lichens, which have started to be explored as a species diversity in China [37,38], but due to the small size of the lichen thallus and diverse, continuously changing, and transitional phenotypes, sometimes there will inevitably bring difficulties in correctly recognizing and choosing some related genera and species in the field survey, which then affects the sampling for the further study in the lab especially when we also should consider small sampling for not destroying too much the BSCs, all of which could lead to the missing findings for new taxa and the hidden species diversity in deserts.
Our phylogenetic analysis (Figure 2) well-supported Clavascidium, Heteroplacidium, and Placidium formed separate monophyletic clades, especially the genus Heteroplacidium situated in the outermost position, consistent with its only-exiting paraplectenchymatous medulla, while variable medulla types exited in Clavascidium and Placidium. Clavascidium and Placidium can be clearly distinguished by presence or absence of rhizines. Within the genus Placidium, it is generally known only uniseriate ascospores arrangement existed [8,9], but sometimes, both uniseriate and biseriate ascospores arrangements co-existed in some species such as Placidium acarosporoides (Zahlbr.) Breuss [30] and two new species Pl. nigrum and Pl. varium, indicating uniseriate and biseriate ascospores arrangements are continuously changing character between Clavascidium and Placidium, and within each of these two genera; nevertheless, a single biseriate ascospores arrangement was not seen in Placidium. As comparison, within the genus Clavascidium, it is generally known only biseriate ascospores arrangement existed [6,8,9], but sometimes, both uniseriate and biseriate ascospores arrangements also coexisted in some species such as Cl. pseudorufescens and Cl. semaforonense [30] and Cl. lacinulatum; however, single a uniseriate ascospores arrangement was not seen in Clavascidium. Laminal pycnidia are almost an exclusive character in most genera of catapyrenioid lichens, except Placidium with laminal or marginal pycnidia; however, marginal pycnidia were also found in Cl. semaforonense [30] and new species Cl. sinensel; further supporting pycnidia position is also a continuously changing character between Clavascidium and Placidium. The only difference in the pycnidia position between Clavascidium and Placidium is both laminal and marginally coexisted in some species of Placidium but not in Clavascidium. Besides, some new characters have been put forward to define species in Placidium, well-supported by the phylogenetic analysis, such as aggregated pycnidia and a glossy upper surface.
This study found that Clavascidium and Placidium are distributed in Northwest China, where are harsh environments such as drought and oligotrophic. The previous studies showed that ecological environments could influence the lichen distribution, similar to Placidium [8,39]; therefore, the relationship between the adaptative characters and distribution should be paid more attention. The finding of the new species Cl. sinense provides a further clue to reconsider the generic relationship between Clavascidium and Placidium. Moreover, the coexistence of uniseriate and biseriate ascospores arrangement may promote the chance of ascospores discharge for better reproduction. Pl. nitidulum only grows at high altitudes above 3000 m alt., and the adaptive phenotype may have produced, for example, tiny lobes and thick algal layer may increase photosynthetic efficiency, and the developed paraphyses in the perithecia would be helpful in squeezing the asci for a better discharge of ascospores through swelling caused by absorbing water. Pl. nigrum has a relatively wide ecological niche, ranging from low altitude to high altitude (400-3500 m alt.), with the climate type from arid to wet (90-523 mm annual precipitation) [40]; we hypothesize it is so abundant, pycnidia could help itself to propagate in the variable environments through its asexual reproduction. Pl. varium grows in the Xinjiang Autonomous Region dry area with annual precipitation of 180-270 mm; small size and macro guttule of the ascospores may decrease their weight and correspondingly increase their propagative velocity for col-onizing a farther and broader living space. However, whether these phenotypic characters are significantly related to the adaptive mechanism need to be further studied.
Therefore, to better recognize the species diversity, understand the adaptive phenotype and other mechanism, and explore more potential species resources that could be applied to restrain sustainable desertification; more taxonomic studies should be continuously carried out on the BSCs-related taxa, such as catapyrenioid lichens, and genomic-scale adaptive evolution studies also need to be explored in the near future.
Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/jof8070729/s1: Table S1: Details of specimens (species name, voucher information, and GenBank numbers) used in this study. Figure S1: The Bayesian tree based on the concatenated ITS + nuLSU (two genes) data set. Figure S2: The maximum-likelihood tree based on the ITS data set. Figure S3: The maximum-likelihood tree based on the nuLSU data set.
Author Contributions: X.W. conceived and designed the study. X.W. and T.Z. collected the specimens from China. T.Z. and X.Z. generated the DNA sequence data, T.Z., Q.Y. and X.W. performed the phenotypic analysis, T.Z. and X.W. analyzed the DNA data. T.Z. and X.W. checked the issues related to the nomenclatural articles. T.Z. wrote the manuscript draft. T.Z. and X.W. revised the draft. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the National Natural Science Foundation of China (32070096).

Institutional Review Board Statement: Not applicable.
Data Availability Statement: Publicly available datasets were analyzed in this study. All newly generated sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/; Table S1, accessed on 4 June 2022). All new taxa were deposited in Fungal names (https://www.fungalinfo.im. ac.cn, accessed on 8 June 2022).