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Research Article
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)
expand article infoMartin Kukwa‡, Pamela Rodriguez-Flakus§, André Aptroot|, Adam Flakus§
‡ University of Gdańsk, Gdańsk, Poland
§ W. Szafer Institute of Botany, Polish Academy of Sciences, Krakow, Poland
| Universidade Federal de Mato Grosso do Sul, Mato Grosso do Sul, Brazil

Corresponding author: Pamela Rodriguez-Flakus ( p.rodriguez@botany.pl )

Academic editor: Thorsten Lumbsch
© 2023 Martin Kukwa, Pamela Rodriguez-Flakus, André Aptroot, Adam Flakus.
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.
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
Open Access

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 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.

Keywords

lichens, lichenised fungi, Neotropics, South America, taxonomy

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 (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; 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 (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 (Lücking et al. 2016a; 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. (2016b, 2019, 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 (Lücking 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 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.

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 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 (ddH2O) 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 MgCl2, 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 (Lücking 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). 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 1000th 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.

Table 1.
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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 nuLSU
Aptrootia elatior New Zealand Knight O61815 OTA MPN560B KM453821 KM453754
Aptrootia robusta Australia Lumbsch 20012 F MPN235B KM453822 KM453755
Aptrootia terricola Costa Rica Lücking 17211 F DNA1501 DQ328995 KM453756
Architrypethelium lauropaluanum Peru Nelsen Cit1P F MPN48 KX215566 KX215605
Architrypethelium nitens Panama Lücking 27038 F MPN257 KM453823 KM453757
Architrypethelium uberinum Brazil Nelsen s.n. F MPN489 – KM453758
Astrothelium aenascens 1 Thailand Luangsuphabool 27887 RAMK HRK93 LC128018 LC127403
Astrothelium aenascens 2 Thailand Luangsuphabool 27888 RAMK HRK98 LC128019 LC127404
Astrothelium aeneum Panama Lücking 27056 F MPN302 – KX215606
Astrothelium bicolor USA Nelsen 4002a F MPN139 GU327706 GU327728
Astrothelium carassense Brazil Lücking 31004 F MPN438 KM453849 KM453784
Astrothelium cecidiogenum Costa Rica Lücking 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
Astrotheliumcinereorosellum 1 Philippines RivasPlata 2110 F MPN191 KM453873 KM453809
Astrothelium cinnamomeum Costa Rica Lücking 15322b DUKE AFTOL110 AY584632 AY584652
Astrothelium crassum Peru Nelsen s.n. F MPN98 GU327685 GU327710
Astrothelium aff. crassum Brazil Cáceres 6011 F MPN335 KM453827 KM453761
Astrothelium croceum Peru Nelsen 211D F MPN55 KX215567 KX215611
Astrothelium degenerans 1 Costa Rica Lücking 17502b CR DNA1496 DQ328987 –
Astrothelium degenerans 2 Panama Lücking 27109 F MPN267 KM453835 KM453770
Astrothelium diplocarpum 2 Nicaragua Lücking 28529 F MPN210 KM453846 KM453781
Astrothelium diplocarpum 1 USA Nelsen s.n. F MPN134 KX215568 –
Astrothelium endochryseum Brazil Lücking 31088 F MPN436 KM453837 KM453772
Astrothelium erubescens Peru Nelsen AnaG F MPN96 KX215569 KX215614
Astrothelium euthelium 1 Thailand Lücking 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 Lücking 27131a F MPN304 KM453876 KM453811
Astrothelium gigantosporum Panama Lücking 33037 F MPN590 KM453851 KM453786
Astrothelium grossum 2 Panama Lücking 27045 F MPN259 KM453834 KM453769
Astrothelium grossum 1 Peru Nelsen 4000a F MPN47 GU327689 GU327713
Astrothelium inspersoaeneum Peru Nelsen Cit1K F MPN45 KX215571 –
Astrothelium isidiatum Bolivia Flakus 30000 KRAM 14-8 OQ275190 OQ281431
Astrothelium kunzei 1 Salvador Lücking 28120 F MPN201B – KX215624
Astrotheliumkunzei 2 Salvador Lücking 28137 F MPN203B – KX215625
Astrothelium laevigatum Brazil Lücking 31010 F MPN430 KX215572 –
Astrothelium laevithallinum Brazil Lücking 31061 F MPN442 KM453836 KM453771
Astrothelium leucoconicum Peru Nelsen 4000c F MPN42 KM453830 KM453764
Astrothelium leucosessile 1 Panama Lücking 27059 F MPN258 KM453828 KM453762
Astrothelium leucosessile 2 Brazil Cáceres 11201 F MPN713 KM453869 KM453805
Astrothelium macrocarpum 1 Panama Lücking 27077 F MPN260 KM453829 KM453763
Astrothelium macrocarpum 2 Thailand n/a 27892 RAMK UBN37 LC128015 LC127400
Astrotheliummacrocarpum 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
Astrothelium megaspermum 3 USA Nelsen s.n. F MPN138 KX215574 KX215632
Astrothelium megaspermum 1 Thailand Nelsen s.n. F MPN32B KX215576 –
Astrothelium meristosporum 2 Philippines RivasPlata 2128 F MPN198 – KX215634
Astrothelium meristosporum 1 Philippines RivasPlata 2108 F MPN189 KM453850 KM453785
Astrothelium neglectum 1 Thailand Luangsuphabool 27898 RAMK TAK8 LC128025 LC127410
Astrothelium neglectum 2 Thailand Luangsuphabool 27896 RAMK TAK12 LC128026 LC127411
Astrothelium neglectum 3 Thailand Luangsuphabool 27897 RAMK TAK17 LC128027 LC127412
Astrothelium neogalbineum 1 Brazil Cáceres 11100 F MPN711 KM453877 KM453812
Astrothelium neogalbineum 2 Peru Nelsen Cit1T F MPN51 KX215577 KX215635
Astrothelium neoinspersum 2 Peru Nelsen AnaJ F MPN61C – KX215636
Astrothelium neoinspersum 1 Peru Nelsen s.n. F MPN62 KM453866 KM453802
Astrothelium neovariolosum 1 Thailand Luangsuphabool 27899 RAMK KY777 LC128023 LC127408
Astrothelium neovariolosum 2 Thailand Luangsuphabool 27900 RAMK KY848 LC128024 LC127409
Astrothelium nicaraguense 1 Nicaragua Lücking 28503 F MPN205 – KX215637
Astrothelium nicaraguense 2 Nicaragua Lücking 28551 F MPN213 – KX215639
Astrothelium nitidiusculum 2 Fiji Lumbsch 20547i F MPN768 – KX215640
Astrothelium nitidiusculum 1 Brazil Cáceres 11297 F MPN704 KM453868 KM453804
Astrothelium norisianum Peru Nelsen 4000d F MPN52C KM453848 KM453783
Astrothelium aff. norisianum Peru Nelsen Cit1B F MPN23B KX215578 KX215607
Astrothelium aff. obscurum Philippines RivasPlata 2175 F MPN194 – KX215608
Astrothelium obtectum Brazil Lücking 31242 F MPN422 KM453832 KM453767
Astrothelium perspersum Gabon Ertz 9716 BR AFTOL2099 GU561848 FJ267701
Astrothelium phlyctaena 1 USA Nelsen 4167 F MPN373 – KX215641
Astrothelium phlyctaena 2 USA Nelsen 4149 F MPN386 – KX215644
Astrothelium pulcherrimum Panama Lücking 27046 F MPN313 KM453879 KM453814
Astrothelium pupula Colombia Lücking 26305 F MPN224 KM453880 KM453815
Astrothelium purpurascens Peru Nelsen s.n. F MPN53C KM453847 KM453782
Astrothelium robustum 1 Costa Rica Mercado 586 F MPN754 KM453826 KM453760
Astrothelium robustum 2 Nicaragua Lücking 28519 F MPN209 – KX215645
Astrothelium robustum 3 Nicaragua Lücking 28547 F MPN212 – KX215646
Astrothelium rufescens 1 Brazil Nelsen B1 F MPN143 – KX215650
Astrothelium rufescens 2 Argentina Lücking 30511 CTES MPN346 – KX215652
Astrothelium sanguinarium 1 Brazil Cañez 3133 CGMS MPN765 KM453853 KM453788
Astrothelium sanguinarium 2 Brazil Cañez 3135 CGMS MPN766 KX215579 KX215653
Astrothelium sanguinarium 3 Brazil Cañez 3137a CGMS MPN767 KX215580 KX215654
Astrothelium scoria Panama Lücking 27181 F MPN310 – KX215655
Astrothelium scorizum Brazil Lücking 29814 F MPN336 KM453872 KM453808
Astrothelium aff. sepultum 2 Costa Rica Lücking 21027 F MPN229 – KX215609
Astrotheliumaff. sepultum 1 Peru Nelsen 4001a F MPN63C GU327690 GU327714
Astrothelium siamense 1 Thailand Luangsuphabool 27901 RAMK KRB105 LC128020 LC127405
Astrothelium siamense 2 Thailand Luangsuphabool 27902 RAMK KRB139 LC128021 LC127406
Astrothelium subcatervarium Peru Nelsen 4009a F MPN97 GU327707 GU327729
Astrothelium subendochryseum Salvador Lücking 28121 F MPN202B – KX215659
Astrothelium subinterjectum Brazil Nelsen B15 F MPN157 KX215583 KX215660
Astrothelium subscoria 1 Nicaragua Lücking 28640 F MPN217 KM453878 KM453813
Astrothelium subscoria 2 Bolivia Lücking 29010 F MPN325 KX215584 KX215661
Astrothelium tuberculosum Costa Rica Lücking 16306a F DNA1504 DQ329008 –
Astrothelium variolosum 1 Peru Nelsen s.n. F MPN43 KM453833 KM453768
Astrothelium variolosum 2 Peru Nelsen Cit1F F MPN41 KX215585 KX215662

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.

Figure 1. 

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 species were used as the outgroups. Bold branches represent either bootstrap values ≥ 70 and/or Bayesian posterior probabilities ≥ 0.95.

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 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; Ekman and Tønsberg 2002; Kukwa and Pérez-Ortega 2010; Hodkinson and Lendemer 2013; Lendemer and Hodkinson 2013; Guzow-Krzemińska 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 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), which may suggest their restricted distribution. This situation can be similar to the genus Sticta (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-Díaz et al. 2020; Ossowska et al. 2022).

Taxonomy

Astrothelium chulumanense Flakus, Kukwa & Aptroot, sp. nov.

MycoBank No: MycoBank No: 847215
Fig. 2

Diagnosis

Characterised by pseudostromata not differing in colour from the thallus, 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 × 27–35 μm), muriform ascospores with a thickened median septum.

Figure 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 μm (A, B); 500 μm (C, D); 50 μm (E); 10 μm (F).

Type

Bolivia. Dept. La Paz; Prov. Sud Yungas, Pataloa, near estación biológica 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 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).

Only four Astrothelium species have clear hamathecium, 8-spored asci and large, muriform 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 × 20–24 μm) and ascomata are almost completely covered by the thallus and do not form distinct pseudostromata. Astrothelium sanguinarium (Malme) Aptroot & Lücking 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 μm long) ascospores, whitish pseudostromata and produces lichexanthone and isohypocrellin (internal in pseudostromata) (Aptroot and Lücking 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 μm 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 & Lücking, ascomata are solitary to diffusely pseudostromatic, prominent, with whitish surrounding the black ostiolar area (Aptroot and Lücking 2016). Astrothelium carassense Lücking, M. P. Nelsen & Marcelli differs in perithecia completely immersed in pseudostromata, which are covered with orange pigment (Lücking et al. 2016b). Astrothelium chapadense (Malme) Aptroot & Lücking differs in dark brown pseudostromata, up to 100 μm long ascospores and the lack of secondary metabolites (Aptroot and Lücking 2016). Astrothelium confluens (Müll. Arg.) Aptroot & Lücking has ascomata completely covered by the thallus and ascospores measuring ca. 130 × 20 μm (Aptroot and Lücking 2016). Astrothelium defossum (Müll. Arg.) Aptroot & Lücking has joined ascomata, which are dispersed to confluent or diffusely pseudostromatic with lichexanthone on the surface (Aptroot and Lücking 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. Cáceres differs from the new species by the presence of lichexanthone, oval to irregular or reticulate in outline pseudostromata, which are yellow to brownish and contain up to 50 ascomata with no fused ostioles (Aptroot and Cáceres 2016). Astrothelium flavomurisporum Aptroot & M. Cáceres has aggregated ascomata (but without pseudostroma) covered with the thallus, lumina of ascospores with yellow oil and lacks secondary metabolites (Aptroot and Cáceres 2016). Astrothelium megeustomum Aptroot & Fraga Jr produces ascomata mostly immersed in the bark tissue below pseudostromata, up to 125 μm long ascospores and lichexanthone around ostiolar region (Aptroot et al. 2016b). Astrothelium mesoduplex Aptroot & M. Cáceres has ascomata immersed in superficially yellow to orange, pale yellow inside pseudostromata and shorter, up to 100 μm long ascospores (Aptroot and Cáceres 2016). Astrothelium octosporoides Aptroot & Lücking differs in solitary or a few grouped ascomata covered by the thallus and the lack of secondary metabolites (Aptroot and Lücking 2016). Astrothelium purpurascens (Müll. Arg.) Aptroot & Lücking develops ascomata with fused ostioles covered with the thallus, produces isohypocrellin and has mostly shorter ascospores (100–130 μm) (Aptroot and Lücking 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. Cáceres differs in black, raised above the thallus pseudostromata, which are usually in lines, the lack of pigments and the production of lichexanthone (Aptroot and Cáceres 2016).

Astrothelium isidiatum Kukwa, Flakus & Rodr. Flakus, sp. nov.

MycoBank No: MycoBank No: 847216
Fig. 3

Diagnosis

The new species differs from all known species of the genus by developing groups of isidia on the surface of areoles, which break off to reveal a medulla that resembles soralia.

Type

Bolivia. Dept. La Paz; Prov. Sud Yungas, near Reserva Ecológica 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, constricted 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 µm in width, of two layers, lower part prosoplectenchymatous and visible mostly in young areoles and upper part gelatinous. Photobiont layer up to 35 µm wide. Medulla whitish (only in young areoles) to yellow, densely filled with rhomboid or irregular crystals (crystals not dissolving in K), crystals 4–35 × 3–12 µm. The upper layer of areoles with shed isidia pseudoparenchymatous. Ascomata and pycnidia unknown.

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 developing 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 μm (A, B); 500 μm (C, D); 50 μm (E, F); 10 μm (G, H).

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, 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.

Some species of Trypetheliaceae, for example, Architrypethelium lauropaluanum Lücking, M. P. Nelsen & Marcelli, Astrothelium komposchii Aptroot or A. puiggarii (Müll. Arg.) Aptroot & Lücking (Aptroot and Lücking 2016; Aptroot et al. 2016c; Lücking 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 Lücking for his constructive comments on the manuscript, members of Herbario Nacional de Bolivia, Instituto de Ecología, 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 research received support from the National Science Centre (project no 2015/17/B/NZ8/02441: Hidden genetic diversity in sterile crustose lichens in the Neotropical forests – 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.

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