Introduction
The lichenized fungal genus Placopsis (Nyl.) Linds., with c. 64 accepted species (Index Fungorum Partnership 2023; www.indexfungorum.org), is mainly distributed in highly oceanic temperate or alpine habitats of the Southern Hemisphere, with only a small number of taxa reaching the Northern Hemisphere (Galloway Reference Galloway2013). Placopsis species typically grow on compacted alpine soils or siliceous rocks, often in periglacial areas. It is a remarkable crustose genus with frequent conspicuous cephalodia, structures that enclose cyanobacteria as secondary photobionts that can fix carbon and nitrogen from the atmosphere (Hitch & Stewart Reference Hitch and Stewart1973; Haselkorn Reference Haselkorn, Broughton and Pühler1986; Rai Reference Rai and Rai1990). This allows them to be pioneer colonizers in oligotrophic habitats such as rocks exposed after glacier melting, where they are sometimes a major component of the lichen flora.
With at least 22 species reported in South America (Galloway Reference Galloway2010) and a minimum of 39 in Oceania (Lumbsch et al. Reference Lumbsch, Kashiwadani and Streimann1992; Galloway Reference Galloway2013), it might be expected that Africa, with nearly half of the continent situated in the Southern Hemisphere, could harbour a significant proportion of Placopsis species. However, only three species have been reported so far from Africa (Placopsis gelida (L.) Linds., P. lambii Hertel & V. Wirth, P. parellina (Nyl.) I. M. Lamb) and all three are also present in other continents (Lambinon & Sérusiaux Reference Lambinon and Sérusiaux1983; Moberg & Carlin Reference Moberg and Carlin1999). This reduced number may be partly due to the cold and humid environment preferred by Placopsis, which is not common in Africa. However, such conditions do occur in some regions in the east of the continent, especially around the highlands of Ethiopia, Kenya, the Democratic Republic of Congo and Tanzania. Since the number of collections and knowledge of African lichen diversity is lower than for South America and Oceania, the small number of Placopsis species known from Africa can be explained by sparse sampling, suggesting that the collection of African Placopsis may result in new species for science.
During a field trip conducted in February 2016 to Mount Kilimanjaro and Mount Meru in Tanzania, some lichen specimens were collected. Examination showed new records for the African continent, as well as new lichen species. Among them, two collections of Placopsis could not be identified as any known taxa, and we propose their description here as a new species based on morphological and genetic data.
Materials and Methods
Sampling, morphology and chemistry
Two specimens growing 2 m apart were collected on Mount Meru, Tanzania, at the base of the Little Meru peak (3°12ʹ58.1ʺS, 36°46ʹ27.5ʺE), 3608 m above sea level (Boluda 17841 and Boluda 17842). They were growing on a compacted siliceous soil among small mosses and other crustose lichens in a sparse alpine scrubland. Specimens were examined morphologically using a Leica MZ75 stereomicroscope (up to ×50), and hand-cut sections were studied with a Leica DM2000 microscope. The morphology of these two specimens was compared to all published species and subspecific taxa of the genus Placopsis, using either species descriptions (Lamb Reference Lamb1947; Lambinon & Sérusiaux Reference Lambinon and Sérusiaux1983; Lumbsch et al. Reference Lumbsch, Kashiwadani and Streimann1992; Moberg & Carlin Reference Moberg and Carlin1999; Galloway et al. Reference Galloway, Lewis-Smith and Quilhot2005; Galloway Reference Galloway2010, Reference Galloway2013; Awasthi & Agarwal Reference Awasthi and Agarwal2011) or herbarium specimens deposited in G, the general collection of the Geneva herbarium (Placopsis bicolor (Tuck.) B. de Lesd., P. chilena I. M. Lamb, P. contortuplicata I. M. Lamb, P. gelida, P. parellina, P. perrugosa (Nyl.) Nyl., P. rhodocarpa (Nyl.) Nyl., P. rhodophthalma (Müll. Arg.) Räsänen, P. subcribellans (I. M. Lamb) D. J. Galloway, and Placopsis sp.).
Spot tests reactions were observed using solutions of 10% potassium hydroxide (K), 8% sodium hypochlorite (C) and paraphenylendiamine (P) according to Orange et al. (Reference Orange, James and White2010). Thin-layer chromatography (TLC) was carried out following Orange et al. (Reference Orange, James and White2010). Solvent systems A (toluene:1,4-dioxane:acetic acid, 180:45:5), B (hexane:methyl tert-butylether:formic acid, 140:72:18) and C (toluene:acetic acid, 170:30) were used according to Culberson & Ammann (Reference Culberson and Ammann1979), with solvent B modified according to Culberson & Johnson (Reference Culberson and Johnson1982).
Phylogenetic reconstruction
DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Barcelona, Spain) with a slight modification to the manufacturer's instructions (Crespo et al. Reference Crespo, Blanco and Hawksworth2001; Divakar et al. Reference Divakar, Del-Prado, Lumbsch, Wedin, Esslinger, Leavitt and Crespo2012). The ITS locus (internal transcribed spacers of the nuclear ribosomal DNA including the 5.8S region and partial sequences of the 18S and 28S) was amplified using the primers ITS1FKYO2 (5ʹ-TAG AGG AAG TAA AAG TCG TAA-3ʹ) and ITS4KYO2 (5ʹ-RBT TTC TTT TCC TCC GCT-3ʹ; Toju et al. Reference Toju, Tanabe, Yamamoto and Sato2012). For PCR amplification, a reaction mixture of 25 μl was used containing 18 μl sterile water, 2.5 μl 10× buffer with 2 mM MgCl2, 0.5 μl dNTPs (10 mM of each base), 1.25 μl of each primer at 10 μM, 0.625 μl of DNA polymerase (1U μl−1), and 1 μl DNA template. Amplifications were run in a thermocycler (XP Cycler, Bioer, Hangzhou, China) using the following parameters: initial denaturation of 5 min at 95 °C, then 35 cycles of 1 min at 95 °C, 1 min at 56 °C, 1 min 30 s at 72 °C, and a final extension of 10 min at 72 °C. Polymerase chain reaction (PCR) products were cleaned using IllustraTM ExoProStar (GE Healthcare, Little Chalfont, UK), according to the manufacturer's instructions. Sequencing was performed by the Unidad de Genómica (Parque Científico de Madrid).
BLAST (Altschul et al. Reference Altschul, Gish, Miller, Myers and Lipman1990) was used to search for the most similar ITS sequences available on GenBank. Those sequences, as well as others representing all the known Placopsis species available in the database, were downloaded (Fig. 1). Sequence alignment was performed using MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server/; Katoh & Standley Reference Katoh and Standley2013) with the G-INS-i alignment algorithm, a ‘200PAM/k = 2’ scoring matrix, with an offset value of 0.1, and the remaining parameters set to default values. Gblocks v. 0.91b (Talavera & Castresana Reference Talavera and Castresana2007) was used to remove ambiguously aligned positions. The final DNA alignment contained 486 base pairs. PartitionFinder (Lanfear et al. Reference Lanfear, Calcott, Ho and Guindon2012) was used to detect possible intra-locus substitution model variability, resulting in the splitting of the ITS region into ITS1, 5.8S and ITS2. DNA substitution models for each locus partition were selected with jModelTest v. 2.0 (Darriba et al. Reference Darriba, Taboada, Doallo and Posada2012), using the Akaike information criterion (AIC; Akaike Reference Akaike1974). The best-fit models of evolution obtained were: TVM + G for ITS1, TrNef + I for 5.8S, and TIM3 + I + G for ITS2.
Phylogenetic reconstruction of 85 Placopsis specimens representing at least 26 species, based on the nuITS matrix with 486 base pairs. Tree topology depicts the results of the Bayesian Markov chain Monte Carlo (B/MCMC). Posterior probabilities and maximum likelihood bootstrap values, when congruent with the Bayesian tree, are given on each branch. The nuITS GenBank Accession numbers are provided for each specimen.
Datasets were analyzed using maximum likelihood (ML) and Bayesian (B/MCMCMC) approaches. For ML tree reconstruction, we used RAxML v. 8.2.10 (Stamatakis Reference Stamatakis2006) implemented on the CIPRES Science Gateway (https://www.phylo.org/; Miller et al. Reference Miller, Pfeiffer and Schwartz2010) with the GTRGAMMA model (Stamatakis Reference Stamatakis2006, Reference Stamatakis2014; Stamatakis et al. Reference Stamatakis, Hoover and Rougemont2008). Support values were assessed using the ‘rapid bootstrapping’ option with 1000 replicates. For the Bayesian reconstruction, MrBayes v. 3.2.1 (Ronquist & Huelsenbeck Reference Ronquist and Huelsenbeck2003) was used. Two simultaneous runs with 2 million generations each, starting with a random tree and employing 12 simultaneous chains, were executed. Every 100th tree was saved to a file. To correct for putative overestimation of branch lengths, we used the uniform compound Dirichlet prior ‘brlenspr = unconstrained:gammadir (1, 1, 1, 1)’ (Zamora et al. Reference Zamora, Calonge and Martín2015). We plotted the log-likelihood scores of sample points against generations using Tracer v. 1.5 (Rambaut et al. Reference Rambaut, Suchard, Xie, Baele and Suchard2014) and determined that stationarity had been achieved when the log-likelihood values of the sample points reached an equilibrium and effective sampling size (ESS) values exceeded 200 (Huelsenbeck & Ronquist Reference Huelsenbeck and Ronquist2001). Posterior probabilities (PPs) were obtained from the 50% majority-rule consensus of sampled trees after excluding the initial 25% as burn-in. The phylogenetic tree was drawn with FigTree v. 1.4 (Rambaut Reference Rambaut2009) and edited with CorelDRAW v. 11.
Results
The morphological comparison of the specimens Boluda 17841 and Boluda 17842 against all other published Placopsis taxa (101 taxa including species and subspecific categories; www.indexfungorum.org) showed a combination of characters absent in any other taxon. These two Placopsis collections are characterized by their circular not confluent soralia, with a protruding white margin and coarse granular pinkish central soredia that do not form pits after soredia dispersion.
The maximum likelihood and Bayesian phylogenetic trees (Fig. 1) are congruent except for a small number of unsupported branches indicated with the symbol ‘-’. However, they show some unsupported backbone nodes. This may be expected when a single locus is used. With few exceptions, the different species appear highly supported. The species Aspiciliopsis macrophthalma (Hook. f. & Taylor) B. de Lesd. appeared nested within Placopsis, but lacked support. The collections Boluda 17841 and Boluda 17842 appear clustered together with 100/1 bootstrap/posterior probability values, with a branch length similar to other Placopsis species. However, the relationships between the Tanzanian collections and the other species are not supported.
Discussion
The specimens Boluda 17841 and 17842 appear genetically distinct from all sequenced Placopsis species and differ from all the described species in their soralia characteristics. Soralia are not frequent in the genus Placopsis, with 51 of the 69 taxa published at species-rank being esorediate. Following Galloway (Reference Galloway2013), the soralia of Placopsis can be classified into two distinct types: capitate, lacking a characteristic margin (as observed in P. rhodocarpa and P. lateritioides I. M. Lamb), and eroding, with a well-defined and often sharp margin (as seen in most sorediate species, such as P. argillacea (C. Knight) Malcolm & Vězda, P. lambii, P. erosa D. J. Galloway, P. fusciduloides D. J. Galloway or P. microphylla (I. M. Lamb) D. J. Galloway). Occasionally, both types of soralia may be found in the same species, as in Placopsis gelida. Additionally, some taxa may produce soralia that do not align well with these two types, as seen in Placopsis antarctica D. J. Galloway, R. I. L. Sm. & Quilhot, where they form on eroding dactyls (Galloway et al. Reference Galloway, Lewis-Smith and Quilhot2005).
The Mount Meru collections produce soralia of the eroding type, yet these are never confluent as commonly seen in most sorediate species. Furthermore, the soralia margin is noticeably thicker, and more developed and raised compared to any described species. The soredia are pinkish to cream, different from the greenish, greyish, olive or blackened colouring found in other taxa, at least when fresh (Galloway Reference Galloway2010, Reference Galloway2013). Placopsis erosa forms discrete, coarsely pustular, crater-like soralia that generate coarsely granular soredia, which are the most similar to those in the Tanzanian collections. However, after soredia dispersion they form pits, whereas the soralia in the Tanzanian samples remain protruding.
The species Aspiciliopsis macrophthalma was found to be nested within Placopsis in the present study. However, its inclusion in this genus lacks support, since the phylogenetic tree's backbone is unsupported. Aspiciliopsis, along with Ducatina and Orceolina, formed a clade sister to Placopsis in previous phylogenetic studies (Schmitt et al. Reference Schmitt, Lumbsch and Søchting2003; Ertz et al. Reference Ertz, Søchting, Gadea, Charrier and Poulsen2017), including reconstructions that utilized only nuITS. These studies were conducted with fewer specimens than were used in the present study, and including more specimens could potentially reduce the support of the tree's backbone due to the presence of missing data. This could result in long branch attraction phenomena that might misplace Aspiciliopsis within Placopsis, especially if Ducatina and Orceolina are not included.
The combination of morphological, genetic and geographical data indicate that the specimens Boluda 17841 and Boluda 17842 belong to an undescribed species of the genus Placopsis. Consequently, we propose its description below as Placopsis craterifera Boluda sp. nov.
The Species
Placopsis craterifera Boluda sp. nov.
MycoBank No.: MB 849792
Vaguely resembling the Australasian Placopsis erosa in its soralia, but with a bullate-squamulose thallus instead of angular areolated, and soralia that are pustular to crateriform, 0.7–1.1 mm diam., not forming pits after soredia spreading, versus 0.2–0.4 mm diam. and soon eroding and spreading to form pits.
Type: Tanzania, Reg. Arusha, Mount Meru, base of the Little Meru peak, on compacted siliceous soil in an alpine sparse scrubland, 3608 m, 3°12ʹ58.1ʺS, 36°46ʹ27.5ʺE, 20 February 2016, Boluda 17841 (G 00576113—holotype) (GenBank Accession no.: OR094466).
(Fig. 2)
A–D, Field views of four non-collected thalli from the type locality of Placopsis craterifera sp. nov.; 1 = cephalodia, 2 = soralia, 3 = thallus squamules. Scales: A, C & D = 1 cm; B = 0.5 cm. Images courtesy of Christoph Scheidegger.
Thallus closely attached to the substratum, forming irregular rosettes when young, squamulose when mature, coalescing to form larger colonies, 1–8 cm diam., 0.3–0.8 mm thick, margins shortly lobed or squamulose, without any marginal prothallus. Marginal lobes shallowly to strongly convex, contiguous to imbricate, 0.6–4.2 × 0.4–2.1 mm. Squamules 0.5–1.3(–2.3) mm diam., isodiametric or frequently elongated or lobed, slightly to strongly convex, ±contiguous, sometimes brownish at the edges. Upper surface pale greyish green to greyish brown, matt, sometimes faintly rough, sorediate; isidia, maculae and pseudocyphellae absent. Soralia scattered, solitary, never confluent, at the beginning slightly prominent, circular, brownish, c. 0.3 mm diam., soon becoming very prominent, pustular to crateriform, circular, 0.7–1.1(–1.5) mm diam.; with up to 40 (sometimes more) coarse granular soredia longer than wide, 90–210 × 60–120 μm, more or less guttiform, inserted by the thinnest end, pale pink to pale brown, easily eroding, becoming an apothecium-like structure with a whitish wrinkled-uneven disc with scars originating from the soredia insertions; margin of soralia permanent, 120–300 μm wide, 90–210 μm in height, whitish. Medulla white. Primary photobiont a green chlorococcoid alga, cells rounded, 6–10 μm diam. Cephalodia sessile, spreading slightly or strongly over the thallus surface, orbicular at first, 0.4–0.9 mm diam., smooth or shallowly wrinkled, becoming deeply plicate at maturity, furrowed, with the margin lobulated and sometimes splitting and separating, 1–5 mm diam., from pinkish brown to orange-brown, blackened when old or in older parts, epruinose. Cyanobiont in chains, cells rounded, 3–6 μm diam., somewhat compressed.
Apothecia and conidiomata unknown.
Chemistry
Gyrophoric acid.
Etymology
Named after its crateriform soralia, as well as its habitat in the volcanic crater of Mount Meru.
Distribution and ecology
So far known only from the type locality on the Little Meru peak, a secondary summit of Mount Meru, the African continent's fifth highest mountain, in Arusha, Tanzania. It grows on compacted siliceous soil of volcanic origin on subvertical to horizontal substrata, in exposed areas in sparse alpine scrubland with frequent fogs and warm days with cold nights.
Conservation status
We are reluctant to perform a conservation assessment based only on two collections, which would end in a Critically Endangered status according to the IUCN (2012) criteria. These two collections were situated in a protected area; however, the type locality is affected by frequent fires and human activities, especially damage resulting from trampling by mountain climbers.
Additional specimen examined (paratype)
Tanzania: Reg. Arusha: Mount Meru, base of the Little Meru peak, on compacted siliceous soil in an alpine sparse scrubland, 3608 m, 3°12ʹ58.1ʺS, 36°46ʹ27.5ʺE, 2016, Boluda 17842 (G 00576112) (GenBank Accession no.: OR094467).
Acknowledgements
We thank the Tanzania Wildlife Research Institute (TAWIRI), Arusha National Park (ANAPA) and Tanzania National Park (TANAPA) authorities for their great support and for granting us access to the National Park areas. We also thank Christoph Scheidegger for providing images of the new species, Philippe Clerc for performing the thin-layer chromatography, and Yamama Naciri for checking the English. This study was conducted within the framework of research collaboration between the Department of Forest Biology at Sokoine University of Agriculture (SUA) and the Department of Biodiversity and Conservation Biology at the Swiss Federal Institute for Forest, Snow and Landscape Research (WSL).
Author ORCIDs
Carlos G. Boluda, 0000-0001-7922-8718; Nuru N. Kitara, 0000-0002-5725-4621.
Competing Interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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