Rediscovery of Five Rinodina Species Originally Described from Southwest China and One New Species

: Rinodina is a lichenized fungal genus belonging to the Physciaceae, with c. 300 species worldwide. Nearly a century ago, Zahlbruckner described ﬁve species of the genus Rinodina from Southwest China. The type collections were the only records for these species. In the present study, new records for four of these species: Rinodina cornutula , R. globulans , R. handelii


Introduction
Among lichenized fungi with a Holarctic distribution, Rinodina (Ach.) Gray is one of the better understood genera. It has been studied in the Iberian Peninsula [1], Scandinavia [2], South Europe [3,4], the Old World [5], North America [6,7], and Northeast Asia [8]. However, molecular data have shown that Rinodina is a polyphyletic genus [9][10][11][12]. Species-level concepts within Rinodina are becoming better established, with clear outlines of species groups emerging in conjunction with a refined understanding of crustose microlichen transitions [8,12]. However, there remains a substantial knowledge gap for East Asia, especially for Southwest China, a region that has played an important role in the evolution of higher plants [8,[13][14][15][16]. The inclusion of specimens and molecular data from East Asia is vital for the generation of testable hypotheses regarding lichen biogeography [8].
A total of 51 Rinodina species have been reported from China [5,. In the early 20th century, five of these species were described from Southwest China by Zahlbruckner [19]. The chemistry of their lichen substances was not recorded. More recently, Aptroot and Sparrius described a new species, R. pluriloculata Aptroot and Sparrius, from the same area, including the chemistry of its lichen substances but not molecular data [29]. Since the initial publication of these species, no further studies have been conducted. Inter-specific comparisons of chemical compounds are lacking, and the phylogenetic position of these species remains unclear.
During the Second Tibetan Plateau Scientific Expedition and Research Program (STEP), we conducted a thorough survey of the lichen diversity of western China, with a particular focus on the Qinghai-Tibetan Plateau. With the addition of specimens that we previously collected in Yunnan Province, over 150 specimens that were confirmed as Rinodina species

Phylogenetic Analysis
The DNA sequences were aligned using MAFFT v. 7.107 in GENEIOUS v. 8.0.2 with the following parameters: algorithm = auto; scoring matrix = 200 PAM/k = 2; gap open penalty = 1.53; offset value = 0.123 [48]. A 3-locus (nrITS, mtSSU, and nrLSU) concatenated matrix was generated using GENEIOUS v. 8.0.2. This matrix contained both Rinodina species and species belonging to related genera in the Physciaceae and Caliciaceae. In addition to our newly generated sequences, other related sequences were downloaded from GenBank and added to the matrix (Table 1). Subsequently, a maximum likelihood (ML) tree and a Bayesian inference (BI) tree were constructed. Based on the lowest Bayesian information criterion (BIC), the partitioned analysis was selected using ModelFinder in IQ-TREE v.1.6.12 for the subsequent ML and BI analyses [49,50]: In ML analysis, GTR + F + I + G4 is for ITS1 and ITS2, TNe + I + G4 for 5.8S rRNA and nrLSU, and TPM3u + F + I + G4 for mtSSU; in BI analysis, GTR + F + I + G4 is for ITS1 and ITS2, SYM + I + G4 for 5.8S rRNA and nrLSU, and HKY + F + I + G4 for mtSSU. The ML analyses were performed by the IQ-TREE web server [51][52][53]. The BI analyses were performed by MrBayes v. 3.2.7 [54], using three Markov chains running for 10 million generations for the concatenated dataset. The trees were sampled every 100 generations, and the first 25% of the trees were discarded as burn-ins. Posterior probabilities (PPs) were obtained from

Results and Discussion
By re-collecting these species from their holotype localities and conducting morphological and phylogenetic analyses, we confirmed the identities of Rinodina cornutula, R. globulans, R. handelii, Buellia setschwana (≡ R. setschwana), and R. pluriloculata. Detailed figures of morphology and spores, together with information regarding chemical compounds, have been provided. Furthermore, the phylogenetic positions of these species have been confirmed. An additional species has been described as new to science: R. hengduanensis, for which morphological descriptions and molecular data have been provided. The sequence alignment comprised 105 terminals, of which 25 were newly generated sequences from this study (Table 1). Phylogenetic trees that were based on datasets generated from either ML or BI analyses had almost identical topologies, with minimal differences in the level of statistical support. The phylogenetic analysis showed that the families Physciaceae and Caliciaceae both formed monophyletic clades. Rinodina cornutula, R. globulans, R. handelii, and R. pluriloculata belong to the Physciaceae, while Buellia setschwana (syn. R. setschwana) belongs to the Caliciaceae.
Our results indicate that R. pluriloculata is phylogenetically close to R. conradii. Both species have 3-8-septate ascospores, but R. pluriloculata can be distinguished by its pale to olive green thallus, punctiform, discrete, orbicular, convex to flattened soralia, bluish, granular soredia, and submuriform-type ascospores. R. cornutula is phylogenetically closely related to R. oxydata. The morphological characters of these two species are largely similar: both are characterized by Mischoblastia-type spores and a thallus with atranorin in the cortex. They can be separated by their thallus color and habitat. The molecular data available for R. oxydata showed that this species is not monophyletic, so further research is required based on samples from its type locality. R. handelii appears close to R. orientalis and R. zeorina in the phylogenetic tree, but the three species can be separated by their ascospore morphology and their chemistry. R. handelii lacks secondary metabolites, and it has Milvinato Mischoblastia-type ascospores (up to 27 µm), which are longer than the spores of the other two species (both less than 20 µm). R. orientalis has Physcia-type spores, whereas R. zeorina contains zeorin and has Dirinaria-type ascospores. R. globulans forms a well-supported clade (100/100/1.00), which is also supported by its morphological, chemical, and geographic characteristics. It differs from other species by its round, capitate, and scattered thallus areoles covered with soralia. Interestingly, Buellia setschwana (syn. R. setschwana) forms a monotypic lineage that is close to B. polita. These two species can be distinguished by the characters of their apothecia: B. polita has a reddish brown hypothecium and immersed lecideine apothecia, whereas B. setschwana has a hyaline hypothecium and cryptolecanorine apothecia. The apothecia characters of B. setschwana are identical to those of other species within Rinodina, which was probably the basis for its previous placement within the genus Rinodina.
Ecology and distribution: This species is distributed across subtropical to temperate regions, growing on siliceous rocks, rhyolite, or basalt, at elevations between 950 and 2500 m. Endemic to China, previously reported in Sichuan and Yunnan [19,36].
Ecology and distribution: This species is saxicolous and often grows on sandstone at elevations between 2000 and 3600 m. It is endemic to China, previously reported in Sichuan province [19,36]. This is a new record for Yunnan Province, China.
Notes: R. globulans is a unique species with numerous, scattered, globular, pale green soralia and a prominent black prothallus, which can be reliably distinguished from other Rinodina species in China. Apothecia are rare in this species, and the ascospores are Mischoblastia-Pachysporaria-type. A recently published species, Rinodina punctosorediata Aptroot and Sparrius, is highly similar to this species, with identical external and anatomic morphology [29]. However, R. punctosorediata grows both on trees (Castanopsis sp.) and rocks, whereas the holotype of R. globulans and all our collections grew on rocks. Furthermore, R. punctosorediata has smaller ascospores (<18 µm long) than the Milvina-type. R. globulans would be keyed out at couplet 2 in Lee and Hur's paper because it has scattered thallus areoles covered with abundant, globular, pale green soralia [76].  Chemistry: Spot tests were all negative; no metabolites were detected by TLC. Ecology and distribution: This species is corticolous, growing on bark or branc of Pinus, Quercus, Rhododendron, etc., in coniferous forest or mixed coniferous bro leaved forest, at elevations between 2700 and 3600 m. It has only previously been repor from Yunnan province, China [19,36]. This is a new record for Sichuan province.
Chemistry: Spot tests were all negative; no metabolites were detected by TLC. Ecology and distribution: This species is corticolous, growing on bark or branches of Pinus, Quercus, Rhododendron, etc., in coniferous forest or mixed coniferous broad-leaved forest, at elevations between 2700 and 3600 m. It has only previously been reported from Yunnan province, China [19,36]. This is a new record for Sichuan province.
Ecology and distribution: This species grows on rocks at elevations between 1100 m and 2000 m. It has previously been reported from Sichuan Province, China [19]. This is a new record for Yunnan Province.
Notes: Buellia setschwana is characterized by its thin, rimose thallus; black and prominent prothallus; innate cryptolecanorine to lecideine; black apothecia; and containing atranorin and norstictic acid as main compounds. It resembles Buellia polita in its external morphology and chemical compounds but could be distinguished by the hyaline hypothecium and cryptolecanorine apothecia. Both B. setschwana and the globally distributed Buellia halonia contain the secondary metabolite arthothelin. This could lead to confusion between the two species. However, R. halonia has exciples with typical aeruginose pigments (HNO 3 + purple), a reddish brown hypothecium, and adnate apothecia (innate in B. setschwana). Furthermore, R. halonia is always maritime, whereas B. setschwana is continental.
Rinodina setschwana was originally collected in Sichuan Province, Dechang County. It was placed in the genus Rinodina based on its cryptolecanorine apothecia and hyaline hypothecium [19]. After the collection of fresh specimens from the same locality and comparison with the type specimen, we confirmed that our collections were morphologically identical to the holotype. However, phylogenetic analyses demonstrated that this species should be placed within the genus Buellia rather than Rinodina. We consider that the type specimens were previously misassigned as species of Rinodina. Based on the morphology, chemistry, and phylogeny of specimens from the type locality, a new combination is proposed here, Buellia setschwana.  Notes: R. hengduanensis is characterized by subsquamulose, greyish-brown to olivaceous thallus, jigsaw-like areoles, and Dirinaria-type ascospores. Rinodina gennarii Bagl. is a saxicolous species with the same type of ascospores that has been reported from China [26,27,29,78]. It differs from this new species by having a whitish thallus, crowded brown apothecia, and smaller ascospores (length <17.5 µm). Rinodina pycnocarpa H. Magn. was  Characterized by saxicolous, subsquamulose, greyish brown to olivaceous thallus, lecanorine apothecium, and Dirinaria-type ascospores.
Etymology: The epithet 'hengduanensis' refers to its distribution, which is mainly in the Hengduan Mountains region.
Chemistry: Spot tests for cortex were all negative; medulla I− contained traces of gyrophoric acid and skyrin, confirmed by TLC.
Ecology and distribution: This species grows on rocks at approximately 2800 m elevation. It is reported from Sichuan and Yunnan provinces in China.
Notes: R. hengduanensis is characterized by subsquamulose, greyish-brown to olivaceous thallus, jigsaw-like areoles, and Dirinaria-type ascospores. Rinodina gennarii Bagl. is a saxicolous species with the same type of ascospores that has been reported from China [26,27,29,78]. It differs from this new species by having a whitish thallus, crowded brown apothecia, and smaller ascospores (length < 17.5 µm). Rinodina pycnocarpa H. Magn. was originally reported from China. It also has a subsquamulose thallus and saxicolous habit, but it can be differentiated by its Bicincta-type and smaller ascospores (<18 µm). Three other species of Rinodina have similar subsquamulose thalli: R. dolichospora Malme can be differentiated by its Pachysporaria-type I spores and lack of substances; R. intermedia Bagl. can be recognized by its 3-septate to submuriform ascospores with type A development; whereas R. willeyi Sheard and Giralt can be separated by having soredia on the areole margin (sublabriform soralia) and its pannarin and zeorin content. R. hengduanensis would key out at couplet 4 in Lee and Hur's paper because it has a continental distribution and has Dirinaria-type, larger (length > 20 µm) ascospores [76]. margin (sublabriform soralia) and its pannarin and zeorin content. R. hengduanensis wo key out at couplet 4 in Lee and Hur's paper because it has a continental distribution has Dirinaria-type, larger (length >20 µm) ascospores [76].

Conclusions
In the present study, using morphological, chemical, and phylogenetic methods, new records for the five endemic species Rinodina cornutula, R. globulans, R. handelii, R. setschwana, and R. pluriloculata from Southwest China were documented based on specimens collected from the holotype localities. Rinodina setschwana was transferred to the genus Buellia and proposed as Buellia setschwana. One new species was discovered: Rinodina hengduanensis. Photographs, chemical compounds, and sequences support the circumscription of these species. However, such details are lacking for many of the previously proposed species of Rinodina. The absence of genetic material means that no molecular phylogeny can yet be constructed that includes all species currently assigned to the genus. For those species lacking genetic material, further new collections are required from the type localities.
Currently available keys based on morphology and chemistry are either outdated, limited in their geographic scope, or do not include the recently described Rinodina spp. A full revision of Rinodina is required in order to produce a key that should incorporate all described species across its full global distribution.

Data Availability Statement:
Publicly available datasets were analyzed in this study. This data can be found from here: https://www.ncbi.nlm.nih.gov/ (accessed on 24 September 2022).