﻿Two new species of Astrothelium from Sud Yungas in Bolivia and the first discovery of vegetative propagules in the family Trypetheliaceae (lichen-forming Dothideomycetes, Ascomycota)

﻿Abstract Two new species of Astrothelium are described from the Yungas forest in Bolivian Andes. Astrotheliumchulumanense is characterised by pseudostromata concolorous with the thallus, perithecia immersed for the most part, with the upper portion elevated above the thallus and covered, except the tops, with orange pigment, apical and fused ostioles, the absence of lichexanthone (but thallus UV+ orange-yellow), clear hamathecium, 8-spored asci and amyloid, large, muriform ascospores with median septa. Astrotheliumisidiatum is known only in a sterile state and produces isidia that develop in groups on areoles, but easily break off to reveal a medulla that resembles soralia. Both species, according to the two-locus phylogeny, belong to Astrothelium s.str. The production of isidia is reported from the genus Astrothelium and the family Trypetheliaceae for the first time.


Introduction
Trypetheliaceae Zenker is the core family of the order Trypetheliales Lücking, Aptroot & Sipman and comprises about 500 species and 19 genera (Lücking et al. 2017;Wijayawardene et al. 2022); however, according to Aptroot et al. (2016a), the species diversity is higher. It is predicted that the total number of species is close to 800, with the majority of unrecognised taxa to be found in the Neotropics . Nevertheless, with about 500 species already known, Trypetheliaceae is one of the three, together with Graphidaceae Dumort. and Pyrenulaceae Rabenh., most speciose families of tropical crustose lichens Mendonça et al. 2020).
Species of Trypetheliaceae grow in various, mostly tropical and subtropical ecosystems in Africa, America, Asia and Australia and are important and common elements in the rain and dry forests and savannahs . Despite that, only recently, the generic concept within the family has been revised and the importance of morphological and chemical characters evaluated using molecular approaches Hongsanan et al. 2020). This resulted in the recognition of several new species (e.g. Aptroot and Cáceres (2016); Aptroot and Lücking (2016); Aptroot et al. (2016bAptroot et al. ( , 2019Aptroot et al. ( , 2022; Flakus et al. (2016); Lücking et al. (2016b); Cáceres and Aptroot (2017); Aptroot and Weerakoon (2018); Hongsanan et al. (2020); Jiang et al. (2022)).
Within Trypetheliaceae, the genus Astrothelium Eschw. is the most speciose and comprises about 275 species (Lücking et al. 2017;Wijayawardene et al. 2022). It is characterised by the following features: corticate thallus, ascomata which can be simple, aggregated or forming pseudostromata (often differing in structure and colour) and are immersed to prominent, with apical or eccentric and simple or fused ostioles, hyphal and usually carbonised ascomatal wall (textura intricata), clear or inspersed with oil droplets hamathecium and distoseptate, hyaline, transversely septate or muriform ascospores (Aptroot and Lücking 2016). Astrothelium, as presently circumscribed, is paraphyletic and consists of two clades. However, as the relationships between those two clades and the Aptrootia Lücking & Sipman and Architrypethelium Aptroot, are not fully resolved and supported, the conservative solution was adopted here, with Aptrootia and Architrypethelium treated as separate genera and all other species retained in the large genus Astrothelium .
In Bolivia, 35 species of Astrothelium are known so far, of which 12 have been recently described (Flakus et al. 2016). In this paper, we describe two further species from a mountain forest in Sud Yungas in Bolivia, including the peculiar, sterile species with isidia. This is the first time that vegetative lichenised propagules have been reported from the genus and the family Trypetheliaceae. Both species are characterised morphologically, anatomically and chemically. Additionally, a comparison with similar species is provided. The placement of both novel species in Astrothelium was corroborated by molecular analyses.

Taxon sampling and morphological studies
Our study was based on specimens freshly collected by the authors and deposited at KRAM, LPB and UGDA. Morphology and anatomy were examined using stereo-and compound microscopes (Nikon SMZ 800, Nikon Eclipse 80i DIC; Tokyo, Japan). Sections were prepared manually using a razor blade. Sections and squash mounts were examined in tap water, 10% potassium hydroxide (KOH) (K) or lactophenol cotton blue (LPCB; Sigma-Aldrich, catalogue no. 61335-100ML; St. Louis, Missouri, USA) and amyloid reactions of anatomical structures were tested using Lugol's solution (I) (Fluka no. 62650-1L-F) or with Lugol's solution preceded by a 10% KOH treatment (K/I). All photomicrographs showing anatomical characters were made using transmitted differential interference contrast (DIC) microscopy. All measurements were made in distilled water. Lichen substances were investigated by thin-layer chromatography (TLC) following the methods by Culberson and Kristinsson (1970) and Orange et al. (2001).

DNA extraction, PCR amplification and DNA sequencing
Freshly collected hymenia or thallus fragments were removed from the specimens and carefully cleaned in double-distilled water (ddH 2 O) on a microscope slide under sterile conditions to remove any visible impurities using ultra-thin tweezers and a razor blade. Genomic DNA was extracted from a few ascomata or thallus pieces using the QIAamp DNA Investigator Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. We amplified both the mtDNA small subunit DNA (mtSSU) using primers pair mrSSU1 and mrSSU3R (Zoller et al. 1999) and nuc rDNA large subunit (nuLSU) with primers ITS1F, LROR, LR3 and LR5 (Vilgalys and Hester 1990;Rehner and Samuels 1994). Polymerase chain reactions (PCR) were performed in a volume of 25 μl comprising 1 μl of DNA template, 0.2 μl of AmpliTaq 360 DNA polymerase (Applied Biosystems, California, USA), 2.5 μl of 10× AmpliTaq 360 PCR Buffer, 2.5 μl 25mM MgCl 2 , 1 μl of each primer (10 μM), 2 μl GeneAmp dNTPs (10 mM; Applied Biosystems, California, USA), 0.2 μl bovine serum albumin (BSA; New England Biolabs, Massachusetts, USA) and sterile distilled water was added to attain the final volume. PCR amplifications were performed using the thermocycling conditions of Rodriguez- Flakus and Printzen (2014). PCR products were visualised by running 3 μl of the PCR product on 1% agarose gels. PCR amplicons were purified using the ExoSAP method (EURx, Gdańsk, Poland) and sequenced by Macrogen (Amsterdam, the Netherlands). The newly-generated mtSSU and nuLSU sequences were checked, assembled and edited manually using Geneious Pro 8.0. (Biomatters, Auckland, New Zealand) and deposited in GenBank.

Phylogenetic analyses and taxon selection
All sequences generated were checked by BLAST (Altschul et al. 1990) to verify potential contaminations by an unrelated fungus. BLAST searches of both mtSSU and nuLSU rDNA sequences from both species revealed the highest similarity with members of Astrothelium (Trypetheliaceae, Dothideomycetes). Therefore, we aligned our sequences with the available sequences of the members of Astrothelium   (Table 1). Alignments were generated for each region using MAFFT (Katoh et al. 2005) as implemented on the GUIDANCE2 Web server (Penn et al. 2010). GUIDANCE2 assigns a confidence score to each ambiguous nucleotide site in the alignment and later removes regions of uncertain columns. We used the default cut-off score of 0.93 in all single gene alignments. The following analyses were performed in the CIPRES Scientific Gateway (Miller et al. 2010). Maximum Likelihood (ML) analyses were carried out in each single-locus alignment using IQ-TREE version 2.1.2 (Nguyen et al. 2015;Chernomor et al. 2016) to detect potential conflicts. We performed 1000 ultrafast bootstrap replicates to estimate branch support amongst the two loci which later were concatenated to a single alignment. The concatenated dataset was used as an input file for analysing the ML in our studies. In which, we performed 5000 replicates under the best-fitting substitution model determined by the ModelFinder Plus (MFP) as implemented in IQ-TREE (Kalyaanamoorthy et al. 2017). The selected model was GTR+F+I+G2 according to AICc in our partitioned per each locus dataset (gene partitioned -s and -m + MFP + MERGE). Bayesian Inference (BI) of the phylogenetic relationships was calculated using the Markov Chain Monte Carlo (MCMC) approach as implemented in MrBayes 3.2.6 on XSEDE (Ronquist et al. 2012) using the partitions and substitution models obtained. Two independent parallel runs were started each with four incrementally heated (0.15) chains. This MCMC was allowed to run for 40 million generations, sampling every 1000 th tree and discarding the first 50% of the sampled tree as a burn-in factor. The resulting ML and BI phylogenetic trees were visualised in TreeView (Page 1996). The tree was rooted by using Architrypethelium and Aptrootia species as the outgroups.

Results and discussion
Two new sequences of each marker (mtSSU and nuLSU) from two new species of Astrothelium were generated for this study (Table 1). The final DNA alignment consisted of sequences obtained from 98 specimens and two markers with a total of 1128 characters, 487 distinct patterns, 288 parsimony-informative, 102 singleton sites and 738 constant sites. The ML phylogenetic tree is presented in Fig. 1. The phylogenetic reconstruction shows that all Astrothelium species form a wellsupported clade divided into two subclades, of which the smaller and well-supported (six species) refers to the clade labelled as Astothelium s.lat. by Lücking et al. (2016a) and the larger one refers to Astrothelium s.str., but is poorly supported (Fig. 1). Our results differ from those received by Lücking et al. (2016a) as all species of Astrothelium, although still divided into two groups, form one clade, with Aptrootia and Architrypethelium forming the sister clade. However, our analyses were restricted only to Astrothelium and two related genera, Aptrootia and Architrypethelium.
Astrothelium chulumanense and A. isidiatum are placed in the larger clade defined by Lücking et al. (2016a) as Astrothelium s.str. Astrothelium chulumanense forms a strongly-supported clade together with A. robustum Müll. Arg.; however, the relationship of this two-species clade with other species within Astrothelium s.str. is not well resolved (Fig. 1). Astrothelium isidiatum is grouped with A. laevigatum Müll. Arg., but the support is weak (Fig. 1). In addition, the relationships of this two-species clade within Astrothelium s.str. are not supported.
The most surprising finding is the presence of isidia in one of the new species, Astrothelium isidiatum. This is the first case when vegetative lichenised diaspores are reported in Trypetheliaceae. Moreover, the new species is sterile and lichen taxa being sterile, but reproducing by isidia or other similar propagules consisting of mycobiont and photobiont, are known in several other groups of lichenised fungi. In extreme cases even entire lineages evolved into permanently asexually reproducing genera, like Botryolepraria Canals et al., Lepraria Ach. and others (Canals et al. 1997 In some genera, sterile taxa producing vegetative diaspores prevail, like in Herpothallon Tobler (Aptroot et al. 2009), but in others, they are rarer, for example, in Ochrolechia A. Massal. (Kukwa 2011). It seems that, in groups of perithecioid lichens, they are much rarer than in apothecioid lichens (e.g. Diederich and Ertz (2020); Orange and Chhetri (2022)). Astrothelium isidiatum is the first species of the Trypetheliaceae, as mentioned above, reproducing by lichenised propagules. However, it is highly possible that more such taxa can be discovered in poorly-explored areas, like Bolivian and other South American ecosystems, but such sterile lichens cause difficulties in placing them properly in higher taxa without molecular approaches; therefore, they can be easily omitted in taxonomic revisions. Additionally, they may have more inconspicuous thalli compared to fertile species (thallus areoles of A. isidiatum were found dispersed amongst other lichens) and can be easily overlooked.
The two new species of Astrothelium, as well as some of these recently described taxa within Trypetheliaceae from Bolivia by Flakus et al. (2016), may be potentially endemic to some areas in this country. With tens of thousands of samples collected by our team across all major ecosystems in Bolivia over almost 20 years, single or only very few records of each new species have been found (Flakus et al. 2016  (Schreb.) Ach. in which several species are confined only to some regions (Moncada et al. 2014Dal Forno et al. 2018;Simon et al. 2018;Mercado-Díaz et al. 2020;Ossowska et al. 2022 Description. Thallus corticate, with corticiform layer 10-20 μm thick, uneven, folded to bumpy, somewhat shiny, continuous, ca. 0.1mm thick, greenish, surrounded by a dark prothallus, not inducing swellings of the host bark, covering areas ≤ 8 cm diam. Pseudostromata with a surface similar to the thallus, distinctly raised above the thallus, hemispherical to wart-shaped, ca. 1.5-3 mm in diam. and 0.5-1.5 mm high, the same colour like thallus with black to orange-black apical spot, inside containing bark tissue. Ascomata perithecia, pyriform to hemispherical, aggregated, 0.6-1 mm diam., emerging from beneath the upper periderm layers of the bark and surrounded by bark tissues in outside part, immersed in most parts in regular in outline pseudostromata, upper part elevated above the thallus and covered, except the tops, with orange pigment. Ostioles apical, centrally fused to form a shared channel leading to various chambers. Wall fully carbonised, not differentiated into excipulum and involucrellum, thicker, ≤ ca. 100 μm wide in the upper part and thinner, up to ca. 20 μm wide, near the base. Ostioles apical, fused, black. Hamathecium clear, composed of thin and anastomosing paraphysoids, 1.5-2.5 μm wide. Asci 8-spored, 350-470 × 56-60 μm. Ascospores distoseptate, hyaline, I+ violet, densely muriform, with a gelatinous layer in younger stages, with a distinct thickened median septum, sometimes breaking into two parts in the septa, narrowly ellipsoid, 125-167 × 27-35 μm, ends rounded, lumina diamond-shaped.
Chemistry. Thallus surface UV+ orange-yellow, K-, C-, KC-, thallus medulla K-; pseudostromata surface UV+ orange-yellow, K-, inner part of pseudostromata K-, visible part of perithecia K+ red. Trace of unidentified substance detected in the thallus by thin layer chromatography; pigment on the top of perithecia.
Etymology. The species is named after its locus classicus located near Chulumani town in Bolivia.
Distribution and habitat. So far, the species is known only from the type locality in Yungas forest in Bolivia. Notes. Astrothelium chulumanense can be distinguished by pseudostromata not differing in colour from the thallus, the orange-yellow reaction in UV (perhaps due to the presence of an unknown substance), the absence of lichexanthone, perithecia immersed for the most part in the thallus, but with upper part elevated above the thallus and covered, except the tops, with orange pigment, apical and fused ostioles, clear hamathecium, 8-spored asci and amyloid, large, muriform ascospores with median septa. The new species is phylogenetically related and externally similar to A. robustum. Both species have also ascomata with fused ostioles; however, ascospores in A. robustum are (3-)5-7(-9)-septate and I negative. Furthermore, the species does not produce secondary metabolites (Aptroot and Lücking 2016;Aptroot 2021).
Etymology. The name refers to the production of isidia, which are unique in the genus. Distribution and habitat. So far, the species is known only from the type locality in the Yungas forest in Bolivia.
Notes. This is a very characteristic species with areoles filled with crystals, cylindrical isidia developing on the areoles and usually yellow thallus medulla. The ascomata were not found in the studied material. It differs from all species of Astrothelium and Trypetheliaceae in the presence of isidia.
We are not aware of any other similar species in other groups, which remind us of the unique taxon described here.