MycoKeys 95: 33- | 00 (2023) er-reviewed open-access journal oes ADS doi: 10.3897/mycokeys.95.98986 RESEARCH ARTICLE , 03 MycoKkeys https://mycokeys.pensoft. net Launched to accelerate biodiversity research 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) Martin Kukwa!', Pamela Rodriguez-Flakus*, André Aptroot?, Adam Flakus* | Department of Plant Taxonomy and Nature Conservation, Faculty of Biology, University of Gdansk, Wita Stwosza 59, PL-80-308 Gdansk, Poland2. W. Szafer Institute of Botany, Polish Academy of Sciences, Lubicz 46, PL-31-512 Krakéw, Poland 3 Laboratorio de Botinica / Liquenologia, Instituto de Biociéncias, Universidade Federal de Mato Grosso do Sul, Avenida Costa e Silva s/n, Bairro Universitdrio, CEP 79070-900, Campo Grande, Mato Grosso do Sul, Brazil Corresponding author: Pamela Rodriguez-Flakus (p.rodriguez@botany. pl) Academic editor: T. Lumbsch | Received 19 December 2022 | Accepted 18 January 2023 | Published 8 February 2023 Citation: Kukwa M, Rodriguez-Flakus P, Aptroot A, Flakus A (2023) 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). MycoKeys 95: 83-100. https://doi.org/10.3897/mycokeys.95.98986 Abstract Two new species of Astrothelium are described from the Yungas forest in Bolivian Andes. Astrothelium chulumanense 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. Astrothelium isidiatum is known only ina 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 fam- ily Trypetheliaceae for the first time. Keywords lichens, lichenised fungi, Neotropics, South America, taxonomy Copyright Martin Kukwa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 84 Martin Kukwa et al. / MycoKeys 95: 83-100 (2023) Introduction Trypetheliaceae Zenker is the core family of the order Trypetheliales Liicking, Aptroot & Sipman and comprises about 500 species and 19 genera (Liicking 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 (Aptroot et al. 2016a). 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 (Aptroot et al. 2016a; Mendonca et al. 2020). Species of Trypetheliaceae grow in various, mostly tropical and subtropical ecosys- tems in Africa, America, Asia and Australia and are important and common elements in the rain and dry forests and savannahs (Aptroot et al. 2016a). 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 (Liick- ing et al. 2016a; Hongsanan et al. 2020). This resulted in the recognition of several new species (e.g. Aptroot and Caceres (2016); Aptroot and Liicking (2016); Aptroot et al. (2016b, 2019, 2022); Flakus et al. (2016); Liicking et al. (2016b); Caceres 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 (Liicking et al. 2017; Wijayawardene et al. 2022). It is characterised by the following features: corticate thallus, ascomata which can be sim- ple, 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 Liicking 2016). Astrothelium, as presently circumscribed, is paraphyletic and consists of two clades. However, as the relationships between those two clades and the Apérootia Licking & 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 (Liicking et al. 2016a). 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 spe- cies 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 corrobo- rated by molecular analyses. Two new species of Astrothelium from Bolivia 85 Materials and methods 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 solu- tion (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 us- ing transmitted differential interference contrast (DIC) microscopy. All measurements were made in distilled water. Lichen substances were investigated by thin-layer chro- matography (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,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 ITS1E LROR, LR3 and LR5 (Vilgalys and Hester 1990; Rehner and Samuels 1994). Polymerase chain reactions (PCR) were performed in a volume of 25 pl comprising 1 pl of DNA template, 0.2 ul of AmpliTaq 360 DNA polymerase (Applied Biosystems, California, USA), 2.5 wl of 10x AmpliTaq 360 PCR Buffer, 2.5 ul 25mM MgCl, 1 pl of each primer (10 uM), 2 wl GeneAmp dNTPs (10 mM; Applied Biosystems, California, USA), 0.2 ul 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 ul of the PCR product on 1% agarose gels. PCR amplicons were purified using the ExoSAP method (EURx, Gdansk, 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. 86 Martin Kukwa et al. / MycoKeys 95: 83-100 (2023) 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 (Liicking et al. 2016a) (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). GUIDANCE? 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 AlCc 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" 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. Two new species of Astrothelium from Bolivia 87 Table |. Voucher data and GenBank accession numbers for the sequences included in this study. Newly- generated sequences are shown in bold. Taxon Origin Collector | Voucher Herbarium _Isolate GenBank accession numbers - mtSSU- ss nu LSU Aptrootia elatior New Zealand Knight 061815 OTA MPNS560B ~=KM453821 KM453754 Aptrootia robusta Australia Lumbsch 20012 F MPN235B KM453822 KM453755 Aptrootia terricola Costa Rica Liicking 17211 F DNAI1501 DQ328995 KM453756 Architrypethelium lauropaluanum Peru Nelsen Cit1P F MPN48 KX215566 = KX215605 Architrypethelium nitens Panama Liicking 27038 F MPN257_—-KM453823. = KM453757 Architrypethelium uberinum Brazil Nelsen s.n. F MPN489 - KM453758 Astrothelium aenascens I Thailand Luangsuphabool 27887. RAMK HRK93 LC128018 LC127403 Astrothelium aenascens 2 Thailand Luangsuphabool 27888 © RAMK HRK98 LC128019 LC127404 Astrothelium aeneum Panama Liicking 27056 F MPN302 - KX215606 Astrothelium bicolor USA Nelsen 4002a F MPN139_ = _GU327706 = =GU327728 Astrothelium carassense Brazil Liicking 31004 F MPN438 =KM453849_ £.KM453784 Astrothelium cecidiogenum Costa Rica Liicking s.n. F N/A DQ328991 = Astrothelium chulumanense Bolivia Flakus 29985 KRAM 14-31 OQ275191 OQ281430 Astrothelium cinereorosellum 2 Philippines RivasPlata 2106 F MPN199C - KX215610 Astrothelium cinereorosellum I Philippines RivasPlata 2110 F MPN191 KM453873 =§.KM453809 Astrothelium cinnamomeum Costa Rica Liicking 15322b DUKE AFTOLI10 AY584632 AY584652 Astrothelium crassum Peru Nelsen $.n. F MPN98 GU327685 GU327710 Astrothelium aff. crassum Brazil Caceres 6011 F MPN335 KM453827. ~KM453761 Astrothelium croceum Peru Nelsen 211D F MPN55 KX215567 KX215611 Astrothelium degenerans 1 Costa Rica Liicking 17502b CR DNA1496_ _DQ328987 = Astrothelium degenerans 2 Panama Liicking 27109 F MPN267 =9.KM453835 =». KM453770 Astrothelium diplocarpum 2 Nicaragua Liicking 28529 F MPN210 KM453846 KM453781 Astrothelium diplocarpum 1 USA Nelsen s.n. F MPN134 KX215568 - Astrothelium endochryseum Brazil Liicking 31088 F MPN436 ~=—=KM453837. = KM453772 Astrothelium erubescens Peru Nelsen AnaG F MPN96 KX215569 KX215614 Astrothelium euthelium 1 Thailand Liicking 24075 F MPN226 - KX215615 Astrothelium euthelium 2 Philippines RivasPlata 1194B F MPN22B KX215616 Astrothelium flavocoronatum 1 Thailand Luangsuphabool 27890 RAMK KY859 LC128014 LC127398 Astrothelium flavocoronatum 2 Thailand Luangsuphabool 27889 §RAMK TSL63 AB759874 LC127397 Astrothelium floridanum 1 USA Nelsen 4008 F MPN132. GU327705 =GU327727 Astrothelium floridanum 2 Panama Licking 27131a F MPN304 KM453876 KM453811 Astrothelium gigantosporum Panama Liicking 33037 F MPN590 = KM453851 = KM453786 Astrothelium grossum 2 Panama Liicking 27045 F MPN259 KM453834 = KM453769 Astrothelium grossum I Peru Nelsen 4000a F MPN47 GU327689 =_GU327713 Astrothelium inspersoaeneum Peru Nelsen Citlk F MPN45 KX215571 7 Astrothelium isidiatum Bolivia Flakus 30000 KRAM 14-8 0Q275190 OQ281431 Astrothelium kunzei 1 Salvador Liicking 28120 F MPN201B - KX215624 Astrothelium kunzei 2 Salvador Liicking 28137 F MPN203B 7 KX215625 Astrothelium laevigatum Brazil Liicking 31010 F MPN430 KX215572 - Astrothelium laevithallinum Brazil Liicking 31061 F MPN442 KM453836 KM453771 Astrothelium leucoconicum Peru Nelsen 4000c F MPN42 KM453830 KM453764 Astrothelium leucosessile 1 Panama Liicking 27059 F MPN258 KM453828 KM453762 Astrothelium leucosessile 2 Brazil Caceres 11201 F MPN713 KM453869 KM453805 Astrothelium macrocarpum I Panama Liicking 27077 F MPN260 KM453829 =.KM453763 Astrothelium macrocarpum 2 Thailand n/a 27892 RAMK UBN37 LC128015 LC127400 Astrothelium macrocarpum 3 Thailand n/a 27894 RAMK UBN43 LC128016 LC127399 Astrothelium macrostiolatum Thailand Luangsuphabool 27895 RAMK PHL84 LC128022 LC127407 Astrothelium megaspermum 2 Gabon Ertz 9725 BR AFTOL2094 GU561847 FJ267702 88 Taxon Astrothelium megaspermum 3 Astrothelium megaspermum I Astrothelium meristosporum 2 Astrothelium meristosporum I Astrothelium neglectum I Astrothelium neglectum 2 Astrothelium neglectum 3 Astrothelium neogalbineum 1 Astrothelium neogalbineum 2 Astrothelium neoinspersum 2 Astrothelium neoinspersum I Astrothelium neovariolosum I Astrothelium neovariolosum 2 Astrothelium nicaraguense I Astrothelium nicaraguense 2 Astrothelium nitidiusculum 2 Astrothelium nitidiusculum 1 Astrothelium norisianum Astrothelium aff. norisianum Astrothelium aff. obscurum Astrothelium obtectum Astrothelium perspersum Astrothelium phlyctaena I Astrothelium phlyctaena 2 Astrothelium pulcherrimum Astrothelium pupula Astrothelium purpurascens Astrothelium robustum 1 Astrothelium robustum 2 Astrothelium robustum 3 Astrothelium rufescens 1 Astrothelium rufescens 2 Astrothelium sanguinarium I Astrothelium sanguinarium 2 Astrothelium sanguinarium 3 Astrothelium scoria Astrothelium scorizum Astrothelium aff. sepultum 2 Astrothelium aff. sepultum 1 Astrothelium siamense I Astrothelium siamense 2 Astrothelium subcatervarium Astrothelium subendochryseum Astrothelium subinterjectum Astrothelium subscoria 1 Astrothelium subscoria 2 Astrothelium tuberculosum Astrothelium variolosum I Astrothelium variolosum 2 Martin Kukwa et al. / MycoKeys 95: 83-100 (2023) Origin USA Thailand Philippines Philippines ‘Thailand ‘Thailand Thailand Brazil Peru Peru Peru Thailand Thailand Nicaragua Nicaragua Fiji Brazil Peru Peru Philippines Brazil Gabon USA USA Panama Colombia Peru Costa Rica Nicaragua Nicaragua Brazil Argentina Brazil Brazil Brazil Panama Brazil Costa Rica Peru Thailand Thailand Peru Salvador Brazil Nicaragua Bolivia Costa Rica Peru Peru Collector Nelsen Nelsen RivasPlata RivasPlata Luangsuphabool Luangsuphabool Luangsuphabool Caceres Nelsen Nelsen Nelsen Luangsuphabool Luangsuphabool Liicking Liicking Lumbsch Caceres Nelsen Nelsen RivasPlata Liicking Ertz Nelsen Nelsen Liicking Liicking Nelsen Mercado Liicking Liicking Nelsen Liicking Cafiez Cafiez Cafiez Liicking Liicking Liicking Nelsen Luangsuphabool Luangsuphabool Nelsen Liicking Nelsen Liicking Liicking Liicking Nelsen Nelsen Voucher Herbarium s.n. s.n. 2128 2108 27898 27896 27897 11100 Cit1T AnaJ s.n. 27899 27900 28503 28551 20547i 11297 4000d Cit1B 2175 31242 9716 4167 4149 27046 26305 s.n. 586 28519 28547 Bl 30511 3133 3135 3137a 27181 29814 21027 4001la 27901 27902 4009a 28121 B15 28640 29010 16306a s.n. Cit1F Ena Ene -Fr. “ry RR BR Added, Isolate MPN 138 MPN32B MPN198 MPN189 TAK8 TAK12 TAKI17 MPN711 MPNS51 MPNGI1C MPN62 KY777 KY848 MPN205 MPN213 MPN768 MPN704 MPN52C MPN23B MPN194 MPN422 AFTOL2099 MPN373 MPN386 MPN313 MPN224 MPN53C MPN754 MPN209 MPN212 MPN143 MPN346 MPN765 MPN766 MPN767 MPN310 MPN336 MPN229 MPN63C KRB105 KRB139 MPN97 MPN202B MPN157 MPN217 MPN325 DNA1504 MPN43 MPN41 GenBank accession numbers mtSSU nuLSU KX215574 KX215632 KX215576 - = KX215634 KM453850 KM453785 LC128025 LC127410 LC128026 LC127411 LC128027 LC127412 KM453877. =. KM453812 KX215577 KX215635 = KX215636 KM453866 KM453802 LC128023 LC127408 LC128024 LC127409 = KX215637 = KX215639 = KX215640 KM453868 KM453804 KM453848 KM453783 KX215578 KX215607 2B: KX215608 KM453832 KM453767 GU561848 FJ267701 = KX215641 = KX215644 KM453879 KM453814 KM453880 KM453815 KM453847. =.KM453782 KM453826 KM453760 = KX215645 = KX215646 = KX215650 _ KX215652 KM453853 ~=9KM453788 KX215579 KX215653 KX215580 KX215654 _ KX215655 KM453872 KM453808 = KX215609 GU327690 = GU327714 LC128020 LC127405 LC128021 LC127406 GU327707. = =£.GU327729 i KX215659 KX215583 KX215660 KM453878 KM453813 KX215584 KX215661 DQ329008 — KM453833 KM453768 KX215585 KX215662 Two new species of Astrothelium from Bolivia 89 The phylogenetic reconstruction shows that all Astrothelium species form a well- supported clade divided into two subclades, of which the smaller and well-supported (six species) refers to the clade labelled as Astothelium s.lat. by Licking 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 Liicking et al. (2016a) as all species of Astrothe- lium, although still divided into two groups, form one clade, with Aptrootia and Ar- chitrypethelium 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 Liicking et al. (2016a) as Astrothelium s.str. Astrothelium chulumanense forms a strongly-supported clade together with A. robustum Mill. Arg.; however, the relation- ship 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 Mill. 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; Ekman and Tonsberg 2002; Kukwa and Pérez-Ortega 2010; Hodkinson and Lendemer 2013; Len- demer and Hodkinson 2013; Guzow-Krzemiriska et al. 2019). 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 Ameri- can 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), which may suggest their restricted distribution. This situation can be similar to the genus Sticta 90 99/1 40/4 87/1 72/4 Martin Kukwa et al. / MycoKeys 95: 83-100 (2023) Architrypethelium lauropaluanum Architrypethelium nitens Architrypethelium uberinum Aptrootia robusta Aptrootia terricola Aptrootia elatior Astrothelium scorizum 75/- 4100/1 66/1 86 /- Astrothelium laevithallinum Astrothelium endochryseum Astrothelium subendochryseum Astrothelium obtectum Astrothelium subinterjectum Astrothelium cinnamomeum Astrothelium grossum | Astrothelium grossum 2 Astrothelium aff. norisianum Astrothelium purpurascens Astrothelium sanguinarium 2 Astrothelium sanguinarium | Astrothelium sanguinarium 3 Astrothelium carassense Astrothelium norisianum ww Astrothelium aff. sepultum 1 Astrothelium aff. sepultum 2 Astrothelium neglectum 3 Astrothelium neglectum | Astrothelium neglectum 2 Astrothelium erubescens Astrothelium subcatervarium Astrothelium leucosessile 1 Astrothelium leucosessile 2 Astrothelium nitidiusculum | 99/1 99/1 98/1 88/1 87). 4100/4 4100/1 400/1 100/- 4100/1 40/1 Astrothelium aeneum Astrothelium croceum Astrothelium neogalbineum | Astrothelium neogalbineum 2 Astrothelium inspersoaeneum Astrothelium neoinspersum | Astrothelium neoinspersum 2 4100/1 Zi. 30/1 94/- 1400/1 Astrothelium scoria Astrothelium rufescens | Astrothelium rufescens 2 86 /- Astrothelium bicolor Astrothelium nitidiusculum 2 4100/1 Astrothelium degenerans | 2 Astrothelium degenerans 2 Astrothelium degenerans 3 Astrothelium kunzei | Astrothelium kunzei 2 79). 4100/1 86/- Astrothelium pulcherrimum 4100/1 26/. Astrothelium phlyctaena 1 Astrothelium phlyctaena 2 Astrothelium aff. crassum Astrothelium euthelium 1 Astrothelium euthelium 2 Astrothelium megaspermum | Astrothelium megaspermum 2 Astrothelium megaspermum 3 Astrothelium neovariolosum 1 Astrothelium neovariolosum 2 Astrothelium perspersum Astrothelium macrostiolatum Astrothelium meristosporum | Astrothelium meristosporum 2 toy Astrothelium aenascens 1 Astrothelium aenascens 2 z.-— Astrothelium cinereorosellum | Astrothelium cinereorosellum 2 Astrothelium siamense 1 Astrothelium siamense 2 87/- 66/1 92k 82/1 4100/4 99). aa 4100/4 96/1 oT 96/1 88/- Astrothelium laevigatum Astrothelium pupula Astrothelium diplocarpum 2 Astrothelium diplocarpum 1 Astrothelium gigantosporum 4100/4 84). 80h 98/41 Astrothelium subscoria 1 Astrothelium subscoria 2 Astrothelium isidiatum Astrothelium nicaraguense | Astrothelium nicaraguense 2 Astrothelium cecidiogenum Astrothelium tuberculosum 4100/1 Astrothelium macrocarpum | Astrothelium macrocarpum 2 Astrothelium macrocarpum 3 Astrothelium crassum Astrothelium variolosum 1 Astrothelium variolosum 2 Astrothelium chulumanense Astrothelium robustum 3 Astrothelium robustum | Astrothelium robustum 2 Astrothelium leucoconicum Astrothelium aff. obscurum Astrothelium flavocoronatum | 16-4 Astrothelium flavocoronatum 2 4100/4 4100/1 80/- 94/- 99). 73)- 1400/1 Astrothelium floridanum Astrothelium floridanum 2 Figure |. Phylogenetic placement of the two new species of Astrothelium within Trypetheliaceae inferred from ML analyses of combined mtSSU and nuLSU rDNA dataset. Aptrootia and Architrypethelium spe- cies were used as the outgroups. Bold branches represent either bootstrap values > 70 and/or Bayesian posterior probabilities > 0.95. Two new species of Astrothelium from Bolivia 91 (Schreb.) Ach. in which several species are confined only to some regions (Moncada et al. 2014, 2018, 2020; Dal Forno et al. 2018; Simon et al. 2018; Mercado-Diaz et al. 2020; Ossowska et al. 2022). Taxonomy Astrothelium chulumanense Flakus, Kukwa & Aptroot, sp. nov. MycoBank No: 847215 Fig. 2 Diagnosis. Characterised by pseudostromata not differing in colour from the thal- lus, perithecia immersed for the most part in thallus, with the upper part elevated above the thallus and covered, except the tops, with orange pigment, apical and fused ostioles, the absence of lichexanthone, clear hamathecium, 8-spored asci and amyloid, large (125-167 x 27-35 um), muriform ascospores with a thickened median septum. Type. Botivia. Dept. La Paz; Prov. Sud Yungas, Pataloa, near estacién bioldgica Santiago de Chirca, near Chulumani, 16°23'57.16"S, 67°34'33.96"W, elev. 2271 m, Yungas montane forest, corticolous, 22 Jan 2020, A. Flakus 29985 & P. Rodriguez- Flakus (holotype KRAM-L 73244, isotypes LPB, UGDA). Description. Thallus corticate, with corticiform layer 10—20 um 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., emerg- ing 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 um wide in the upper part and thinner, up to ca. 20 um wide, near the base. Ostioles apical, fused, black. Hamathecium clear, composed of thin and anastomosing paraphysoids, 1.5—2.5 um wide. Asci 8-spored, 350-470 x 56-60 um. Ascospores distoseptate, hya- line, I+ violet, densely muriform, with a gelatinous layer in younger stages, with a dis- tinct thickened median septum, sometimes breaking into two parts in the septa, nar- rowly ellipsoid, 125-167 x 27-35 um, 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-, vis- ible part of perithecia K+ red. Trace of unidentified substance detected in the thallus by thin layer chromatography; pigment on the top of perithecia. 92 Martin Kukwa et al. / MycoKeys 95: 83-100 (2023) | i. | ‘ . “a | \ | 9 \ —_~ 7 XN Figure 2 2. Astrothelium chulumanense (holotype) A, B thallus and ascomata C vertical cross section through pseudostromata D horizontal cross section through pseudostromata E asci (violet ascospores in Lugol’s solution) F ascospores (violet in Lugol’s solution). Scale bars: 1000 um (A, B); 500 um (C, D); 50 um (E); 10 um (F). 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. Two new species of Astrothelium from Bolivia ve} Notes. Astrothelium chulumanense can be distinguished by pseudostromata not dif- fering 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 im- mersed 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. Ihe 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 Licking 2016; Aptroot 2021). Only four Astrothelium species have clear hamathecium, 8-spored asci and large, mu- riform ascospores, which react I+ violet. Astrothelium amylosporum Flakus & Aptroot has pseudostromata not covered by thallus and lacks pigments, whereas A. palaeoexostemmatis Sipman & Aptroot lacks pigments, has smaller ascospores (85-100 x 20-24 pm) and ascomata are almost completely covered by the thallus and do not form distinct pseudostromata. Astrothelium sanguinarium (Malme) Aptroot & Liicking differs in the shape of pseudostromata, the pigment is red (isohypocrellin), reacts K+ yellow- green and is present internally within pseudostromata. Astrothelium sanguineoxanthum Aptroot has smaller (up to 86 um long) ascospores, whitish pseudostromata and pro- duces lichexanthone and isohypocrellin (internal in pseudostromata) (Aptroot and Liicking 2016; Aptroot et al. 2016b, 2019; Flakus et al. 2016; Aptroot 2021). Several other species of the genus have pseudostromata or aggregated ascomata often with fused ostioles, clear hymenium, large (at least some over 80 um long) and muriform, but I negative ascospores and 8-spored asci. They differ significantly in other characters (for the key to all species, see Aptroot (2021)). In A. alboverrucum (Makhija & Patw.) Aptroot & Liicking, ascomata are solitary to diffusely pseudostro- matic, prominent, with whitish surrounding the black ostiolar area (Aptroot and Liick- ing 2016). Astrothelium carassense Licking, M. P. Nelsen & Marcelli differs in perithe- cia completely immersed in pseudostromata, which are covered with orange pigment (Liicking et al. 2016b). Astrothelium chapadense (Malme) Aptroot & Liicking differs in dark brown pseudostromata, up to 100 um long ascospores and the lack of sec- ondary metabolites (Aptroot and Lticking 2016). Astrothelium confluens (Mill. Arg.) Aptroot & Licking has ascomata completely covered by the thallus and ascospores measuring ca. 130 x 20 um (Aptroot and Liicking 2016). Astrothelium defossum (Mill. Arg.) Aptroot & Liicking has joined ascomata, which are dispersed to confluent or diffusely pseudostromatic with lichexanthone on the surface (Aptroot and Liicking 2016). Astrothelium elixii Flakus & Aptroot develops white pruinose pseudostromata and produces lichexanthone and isohypocrellin (internal in pseudostromata) (Flakus et al. 2016). Astrothelium flavoduplex Aptroot & M. Caceres differs from the new species by the presence of lichexanthone, oval to irregular or reticulate in outline pseudostro- mata, which are yellow to brownish and contain up to 50 ascomata with no fused osti- oles (Aptroot and Caceres 2016). Astrothelium flavomurisporum Aptroot & M. Caceres has aggregated ascomata (but without pseudostroma) covered with the thallus, lumina of ascospores with yellow oil and lacks secondary metabolites (Aptroot and Caceres 94 Martin Kukwa et al. / MycoKeys 95: 83-100 (2023) 2016). Astrothelium megeustomum Aptroot & Fraga Jr produces ascomata mostly im- mersed in the bark tissue below pseudostromata, up to 125 um long ascospores and lichexanthone around ostiolar region (Aptroot et al. 2016b). Astrothelium mesoduplex Aptroot & M. Caceres has ascomata immersed in superficially yellow to orange, pale yellow inside pseudostromata and shorter, up to 100 um long ascospores (Aptroot and Caceres 2016). Astrothelium octosporoides Aptroot & Liicking differs in solitary or a few grouped ascomata covered by the thallus and the lack of secondary metabolites (Aptroot and Liicking 2016). Astrothelium purpurascens (Mill. Arg.) Aptroot & Liick- ing develops ascomata with fused ostioles covered with the thallus, produces isohypo- crellin and has mostly shorter ascospores (100-130 pm) (Aptroot and Liicking 2016). Astrothelium variabile Flakus & Aptroot has aggregated ascomata in well-delimited and white pseudostromata, not fused ostioles, lacks pigments and produces lichexanthone (Flakus et al. 2016). Astrothelium xanthosuperbum Aptroot & M. Caceres differs in black, raised above the thallus pseudostromata, which are usually in lines, the lack of pigments and the production of lichexanthone (Aptroot and Caceres 2016). Astrothelium isidiatum Kukwa, Flakus & Rodr. Flakus, sp. nov. MycoBank No: 847216 Fig. 3 Diagnosis. ‘The new species differs from all known species of the genus by develop- ing groups of isidia on the surface of areoles, which break off to reveal a medulla that resembles soralia. Type. Bottvia. Dept. La Paz; Prov. Sud Yungas, near Reserva Ecolégica de Apa Apa, Sanani near Chulumani, 16°20'39.70"S, 67°29'54.32"W, elev. 2423 m, Yungas montane forest, corticolous, 23 Jan 2020, A. Flakus 30000 & P. Rodriguez-Flakus (KRAM-L 73245 holotype; LPB, UGDA isotypes). Description. Thallus endosubstratal to episubstratal and then grey-green, shiny, folded in non-areolate parts, with areoles, isidiate. Areoles tuberculate, sometimes with cylindrical outgrowth developing at the lateral parts of areoles (Fig. 3C), constricted at the base (especially when young) or not, rounded to elongate and up to 1.2 mm wide. Isidia mostly cylindrical, globose when young, simple, rarely branched, con- stricted at the base or not, developing on areoles, up to 0.5 mm long and 0.2 mm wide, often shed from areoles and then exposing the yellow medulla of areoles, which then resemble soralia; sometimes elongated isidia-like outgrowth developing directly from the endosubstratal thallus present (Fig. 3D). Cortex up to 30-50 um in width, of two layers, lower part prosoplectenchymatous and visible mostly in young areoles and upper part gelatinous. Photobiont layer up to 35 um wide. Medulla whitish (only in young areoles) to yellow, densely filled with rhomboid or irregular crystals (crystals not dissolving in K), crystals 4-35 x 3-12 um. The upper layer of areoles with shed isidia pseudoparenchymatous. Ascomata and pycnidia unknown. Two new species of Astrothelium from Bolivia iP) —_ —e 2 Weava rf - — Figure 3. Astrothelium isidiatum (type collection) A-D thallus morphology A, B isidia developing in groups on areoles which are partly shed exposing the medulla of the areoles C isidia-like outgrows de- veloping on lateral parts of areoles D isidia-like outgrowths developing directly from the endosubstratal parts of the thallus E, F a vertical cross-section through thallus with crystals present in the medulla (E) (in LPCB) G, H vertical cross-section through cortical layer (in LPCB). Scale bars: 1000 um (A, B); 500 um (C, D); 50 um (E, F); 10 um (G, H). 96 Martin Kukwa et al. / MycoKeys 95: 83-100 (2023) Chemistry. Thallus surface UV—, K-, C-, KC-; medulla with yellow pigment, K+ yellow going into solution, C+ yellow-orange; upper parts of areoles with shed isidia with patches of orange pigment reacting K+ purple. Unidentified substances (probably some of them are anthraquinones) in trace to minor amounts detected by thin layer chromatography. 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, cylindri- cal 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. Some species of Trypetheliaceae, for example, Architrypethelium lauropaluanum Liicking, M. P. Nelsen & Marcelli, Astrothelium komposchii Aptroot or A. puigearii (Mill. Arg.) Aptroot & Liicking (Aptroot and Licking 2016; Aptroot et al. 2016c; Liicking et al. 2016b), develop thalli with areoles resembling isidia which somehow are similar to these of A. isidiatum (Fig. 3C, D). However, A. isidiatum differs by developing cylindrical and often constricted at the base isidia which are covering the entire areoles (Fig. 3A, B). The isidia are easily broken and shed from areoles revealing the medulla of areoles that then resemble soralia. We are not aware of any other similar species in other groups, which remind us of the unique taxon described here. Acknowledgements We would like to thank Robert Liicking for his constructive comments on the manu- script, members of Herbario Nacional de Bolivia, Instituto de Ecologia, Universidad Mayor de San Andrés La Paz, for their generous cooperation and, in particular, our friend Silvia C. Gallegos for her invaluable assistance during the fieldwork. This re- search received support from the National Science Centre (project no 2015/17/B/ NZ8/02441: Hidden genetic diversity in sterile crustose lichens in the Neotropical for- ests — an innovative case study in Bolivia, a hotspot of biodiversity) and statutory funds from the W. Szafer Institute of Botany, Polish Academy of Sciences, Krakow, Poland. References Altschul SE, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. Journal of Molecular Biology 215(3): 403-410. https://doi.org/10.1016/S0022- 2836(05)80360-2 Aptroot A (2021) World key to the species of Pyrenulaceae and Trypetheliaceae. Archive For Lichenology 29: 1-91. Two new species of Astrothelium from Bolivia 97 Aptroot A, Caceres MES (2016) New Trypetheliaceae from the Amazon basin in Rondénia (Brazil), the centre of diversity of the genus Astrothelium. Lichenologist 48(6): 693-712. https://doi.org/10.1017/S00242829 15000584 Aptroot A, Liicking R (2016) A revisionary synopsis of the Trypetheliaceae (Ascomycota: Trypetheliales). Lichenologist48 (6):763—982.https://doi.org/10.1017/S00242829 16000487 Aptroot A, Weerakoon G (2018) Three new species and ten new records of Trypetheliaceae (Ascomycota) from Sri Lanka. Cryptogamie. Mycologie 39(3): 373-378. https://doi. org/10.7872/crym/v39.iss3.2018.373 Aptroot A, Thor G, Liicking R, Elix JA, Chaves JL (2009) The lichen genus Herpothallon reinstated. Bibliotheca Lichenologica 99: 19-66. Aptroot A, Caceres MES, Johnston MK, Liicking R (2016a) How diverse is the lichenized fungal family Trypetheliaceae (Ascomycota: Dothideomycetes)? A quantitative predic- tion of global species richness. Lichenologist 48(6): 983-1011. https://doi.org/10.1017/ S00242829 16000463 Aptroot A, Mendonga CO, Andrade DS, Silva JR, Martins SMA, Gumboski E, Fraga CAV, Caceres MES (2016b) New Trypetheliaceae from northern and southern At- lantic rainforests in Brazil. Lichenologist 48(6): 713-725. https://doi.org/10.1017/ S00242829 16000037 Aptroot A, Ertz D, Etayo Salazar J, Gueidan C, Mercado Diaz J, Schumm FE, Weerakoon G (2016c) Forty-six new species of Trypetheliaceae from the tropics. Lichenologist 48(6): 609-638. https://doi.org/10.1017/S002428291600013X Aptroot A, Sipman HJM, Barreto FMO, Nunes AD, Caceres MES (2019) Ten new species and 34 new country records of Trypetheliaceae. Lichenologist 51(1): 27-43. https://doi. org/10.1017/S002428291800052X Aptroot A, de Souza MF, dos Santos LA, Junior IO, Barbosa BMC, Caceres MES (2022) New species of lichenized fungi from Brazil, with a record report of 492 species in a small area of the Amazon Forest. The Bryologist 125(3): 433-465. https://doi.org/10.1639/0007- 2745-125.3.433 Caceres MES, Aptroot A (2017) Lichens from the Brazilian Amazon, with special reference to the genus Astrothelium. The Bryologist 120(2): 165-181. https://doi.org/10.1639/0007- 2745-120.2.165 Canals A, Hernandez-Mariné M, Gomez-Bolea A, Llimona X (1997) Botryolepraria, a new monotypic genus segregated from Lepraria. Lichenologist 29(4): 339-345. https://doi. org/10.1006/lich.1997.0081 Chernomor O, von Haeseler A, Quang Minh B (2016) Terrace Aware Data Structure for Phylogenomic Inference from Supermatrices. Systematic Biology 65(6): 997-1008. https://doi.org/10.1093/sysbio/syw037 Culberson CE, Kristinsson H (1970) A standardized method for the identification of lichen products. Journal of Chromatography A 46: 85-93. https://doi.org/10.1016/S0021- 9673(00)83967-9 Dal Forno M, Moncada B, Liicking R (2018) Sticta aongstroemii, a newly recognized species in the S. damicornis morphodeme (Lobariaceae) potentially endemic to the Atlantic Forest in Brazil. Lichenologist 50(6): 691-696. https://doi.org/10.1017/S00242829 18000403 98 Martin Kukwa et al. / MycoKeys 95: 83-100 (2023) Diederich P, Ertz D (2020) First checklist of lichens and lichenicolous fungi from Mauritius, with phylogenetic analyses and descriptions of new taxa. Plant and Fungal Systematics 65(1): 13-75. https://doi.org/10.35535/pfsyst-2020-0003 Ekman S, Tonsberg T (2002) Most species of Lepraria and Leproloma form a monophy- letic group closely related to Stereocaulon. Mycological Research 106(11): 1262-1276. https://doi.org/10.1017/S0953756202006718 Flakus A, Kukwa M, Aptroot A (2016) Trypetheliaceae of Bolivia: An updated checklist with descriptions of twenty-four new species. Lichenologist 48(6): 661-692. https://doi. org/10.1017/S0024282915000559 Guzow-Krzeminska B, Jablonska A, Flakus A, Rodriguez-Flakus P, Kosecka M, Kukwa M (2019) Phylogenetic placement of Lepraria cryptovouauxii sp. nov. (Lecanorales, Lecanoromycetes, Ascomycota) with notes on other Lepraria species from South America. MycoKeys 53: 1-22. https://doi.org/10.3897/mycokeys.53.33508 Hodkinson BP, Lendemer JC (2013) Next-generation sequencing reveals sterile crustose lichen phylogeny. Mycosphere 4(6): 1028-1039. https://doi.org/10.5943/mycosphere/4/6/1 Hongsanan S, Hyde KD, Phookamsak R, Wanasinghe DN, McKenzie EHC, Sarma VV, Liick- ing R, Boonmee S, Bhat JD, Liu N-G, Tennakoon DS, Pem D, Karunarathna A, Jiang S-H, Jones GEB, Phillips AJL, Manawasinghe IS, Tibpromma S, Jayasiri SC, Sandamali D, Jayawardena RS, Wijayawardene NN, Ekanayaka AH, Jeewon R, Lu Y-Z, Phukhamsakda C, Dissanayake AJ, Zeng X-Y, Luo Z-L, Tian Q, Thambugala KM, Dai D, Samarakoon MC, Chethana KWT, Ertz D, Doilom M, Liu J-K, Pérez-Ortega S, Suija A, Senwan- na C, Wijesinghe SN, Niranjan M, Zhang S-N, Ariyawansa HA, Jiang H-B, Zhang J-F, Norphanphoun C, de Silva NI, Thiyagaraja V, Zhang H, Bezerra JDP, Miranda-Gonzalez R, Aptroot A, Kashiwadani H, Harishchandra D, Sérusiaux E, Abeywickrama PD, Bao D-F, Devadatha B, Wu H-X, Moon KH, Gueidan C, Schumm F, Bundhun D, Mapook A, Monkai J, Bhunjun CS, Chomnunti P, Suetrong S, Chaiwan N, Dayarathne MC, Yang J, Rathnayaka AR, Xu JC, Zheng J, Liu G, Feng Y, Xie N (2020) Refined families of Dothideomycetes: Orders and families incertae sedis in Dothideomycetes. Fungal Diver- sity 105(1): 17-318. https://doi.org/10.1007/s13225-020-00462-6 Jiang S-H, Zhang C, Xue X-D, Aptroot A, Wei J-C, Wei X-L (2022) Morphological and phy- logenetic characterizations reveal five new species of Astrothelium (Trypetheliales, Ascomy- cota) from China. Journal of Fungi 8(10): 994. https://doi.org/10.3390/jof8 100994 Kalyaanamoorthy S, Minh BQ, Wong TKE, Von Haeseler A, Jermiin LS (2017) ModelFinder: fast model selection for accurate phylogenetic estimates. Nature Methods 14(6): 587-589. https://doi.org/10.1038/nmeth.4285 Katoh K, Kuma K, Toh H, Miyata T (2005) MAFFT version 5: Improvement in accuracy of multiple sequence alignment. Nucleic Acids Research 33(2): 511-518. https://doi. org/10.1093/nar/gkil98 Kukwa M (2011) The lichen genus Ochrolechia in Europe. Fundacja Rozwoju Uniwersytetu Gdaniskiego, Gdarisk, 309 pp. Kukwa M, Pérez-Ortega S (2010) A second species of Botryolepraria from the Neotropics and the phylogenetic placement of the genus within Ascomycota. Mycological Progress 9(3): 345-351. https://doi.org/10.1007/s11557-009-0642-0 Two new species of Astrothelium from Bolivia 99 Lendemer JC, Hodkinson BP (2013) A radical shift in the taxonomy of Lepraria s.|.: Molecular and morphological studies shed new light on the evolution of asexuality and lichen growth form diversification. Mycologia 105(4): 994-1018. https://doi.org/10.3852/12-338 Liicking R, Nelsen MP, Aptroot A, Klee RB, Bawingan PA, Benatti MN, Binh NQ, Bungartz FE Caceres MES, Canez LS, Chaves J-L, Ertz D, Esquivel RE, Ferraro LI, Grijalva A, Gueidan C, Hernandez JE, Knight A, Lumbsch HT, Marcelli MP, Mercado-Diaz JA, Moncada B, Mo- rales EA, Naksuwankul K, Orozco T, Parnmen S, Rivas Plata E, Salazar-Allen N, Spielmann AA, Ventura N (2016a) A phylogenetic framework for reassessing generic concepts and spe- cies delimitation in the lichenized family Trypetheliaceae (Ascomycota: Dothideomycetes). Lichenologist 48(6): 739-762. https://doi.org/10.1017/S00242829 16000505 Liicking R, Nelsen MP, Aptroot A, Benatti MN, Binh NQ, Gueidan C, Gutiérrez MC, Jun- gbluth P, Lumbsch HT, Marcelli MP, Moncada B, Naksuwankul K, Orozco T, Salazar- Allen N, Upreti DK (2016b) A pot-pourri of new species of Trypetheliaceae resulting from molecular phylogenetic studies. Lichenologist 48(6): 639-660. https://doi.org/10.1017/ S00242829 16000475 Liicking R, Hodkinson BP, Leavitt SD (2017) [(2016)] The 2016 classification of lichenized fungi in the Ascomycota and Basidiomycota — Approaching one thousand genera. The Bryologist 119(4): 361-416. https://doi.org/10.1639/0007-2745-119.4.361 Mendonga CO, Aptroot A, Liicking R, Caceres MES (2020) Global species richness prediction for Pyrenulaceae (Ascomycota: Pyrenulales), the last of the “big three” most speciose tropi- cal microlichen families. Biodiversity and Conservation 29(3): 1059-1079. https://doi. org/10.1007/s10531-019-01925-2 Mercado-Diaz JA, Liicking R, Moncada B, Widhelm TJ, Lumbsch HT (2020) Elucidating spe- cies richness in lichen fungi: The genus Sticta (Ascomycota: Peltigeraceae) in Puerto Rico. Taxon 69(5): 851-891. https://doi.org/10.1002/tax. 12320 Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for infer- ence of large phylogenetic trees. Proceedings of the Gateway Computing Environments Workshop (GCE). 14 Nov. 2010. New Orleans Convention Center, New Orleans, LA, 1-8. https://doi.org/10.1109/GCE.2010.5676129 Moncada B, Liicking R, Suarez A (2014) Molecular phylogeny of the genus Sticta (lichenized Ascomycota: Lobariaceae) in Colombia. Fungal Diversity 64(1): 205-231. https://doi. org/10.1007/s13225-013-0230-0 Moncada B, Mercado-Diaz JA, Liicking R (2018) The identity of Sticta damicornis (Ascomy- cota: Lobariaceae): a presumably widespread taxon is a Caribbean endemic. Lichenologist 50(5): 591-597. https://doi.org/10.1017/S00242829 18000373 Moncada B, Liicking R, Lumbsch HT (2020) Rewriting the evolutionary history of the lichen genus Sticta (Ascomycota: Peltigeraceae subfam. Lobarioideae) in the Hawaiian islands. Plant and Fungal Systematics 65(1): 95-119. https://doi.org/10.35535/pfsyst-2020-0005 Nguyen L-T, Schmidt HA, Von Haeseler A, Minh BQ (2014) IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology and Evolution 32: 268-274. https://doi.org/10.1093/molbev/msu300 Orange A, Chhetri SG (2022) Verrucariaceae from Nepal. Lichenologist 54(3—4): 139-174. https://doi.org/10.1017/S0024282922000160 100 Martin Kukwa et al. / MycoKeys 95: 83-100 (2023) Orange A, James PW, White FJ (2001) Microchemical Methods for the Identification of Lichens. British Lichen Society, London, 101 pp. Ossowska E, Moncada B, Kukwa M, Flakus A, Rodriguez-Flakus P, Olszewska S, Liicking R (2022) New species of Sticta (lichenised Ascomycota, lobarioid Peltigeraceae) from Bo- livia suggest a high level of endemism in the Central Andes. MycoKeys 92: 131-160. https://doi.org/10.3897/mycokeys.92.89960 Page RDM (1996) Tree View: An application to display phylogenetic trees on personal computers. Bioinformatics 12(4): 357-358. https://doi.org/10.1093/bioinformat- ics/12.4.357 Penn O, Privman E, Ashkenazy H, Landan G. Graur D, Pupko T (2010) GUIDANCE: a web server for assessing alignment confidence scores. Nucleic Acids Research 38(Web Server): W23-W 28. https://doi.org/10.1093/nar/gkq443 Rehner SA, Samuels GJ (1994) Taxonomy and phylogeny of Gliocladium analysed from nu- clear large subunit ribosomal DNA sequences. Mycological Research 98(6): 625-634. https://doi.org/10.1016/S0953-7562(09)80409-7 Rodriguez-Flakus P, Printzen C (2014) Palicella, a new genus of lichenized fungi and its phy- logenetic position within Lecanoraceae. The Lichenologist 46(4): 535-552. https://doi. org/10.1017/S00242829 14000127 Ronquist E, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian Phylogenetic Infer- ence and Model Choice Across a Large Model Space. Systematic Biology 61(3): 539-542. https://doi.org/10.1093/sysbio/sys029 Simon A, Goffinet B, Magain N, Sérusiaux E (2018) High diversity, high insular endemism and recent origin in the lichen genus Sticta (lichenized Ascomycota, Peltigerales) in Madagascar and the Mascarenes. Molecular Phylogenetics and Evolution 122: 15-28. https://doi. org/10.1016/j.ympev.2018.01.012 Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically ampli- fied ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172(8): 4238-4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990 Wijayawardene NN, Hyde KD, Dai DQ, Sanchez-Garcia M, Goto BT, Saxena RK, Erdogdu M, Selcuk F, Rajeshkumar KC, Aptroot A, Btaszkowski J, Boonyuen N, da Silva GA, de Souza FA, Dong W, Ertz D, Haelewaters D, Jones EBG, Karunarathna SC, Kirk PM, Kukwa M, Kumla J, Leontyev DV, Lumbsch HT, Maharachchikumbura SSN, Marguno F, Martinez-Rodriguez P, Mesi¢ A, Monteiro JS, Oehl E Pawtowska J, Pem D, Pfliegler WP, Phillips AJL, Posta A, He MQ, Li JX, Raza M, Sruthi OP, Suetrong S, Suwannarach N, Tedersoo L, Thiyagaraja V, Tibpromma S, Tkalcéec Z, Tokarev YS, Wanasinghe DN, Wijesundara DSA, Wimalaseana SDMK, Madrid H, Zhang GQ, Gao Y, Sanchez-Castro I, Tang LZ, Stadler M, Yurkov A, Thines M (2022) Outline of Fungi and fungus-like taxa — 2021. Mycosphere 13(1): 53-453. https://doi.org/10.5943/mycosphere/13/1/2 Zoller S, Scheidegger C, Sperisen C (1999) PCR primers for the amplification of mitochondrial small subunit ribosomal DNA of lichen-forming ascomycetes. Lichenologist 31(5): 511- 516. https://doi.org/10.1006/lich.1999.0220