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OPEN New lineages of photobionts in Bolivian expand our knowledge on habitat preferences and distribution of Asterochloris algae Magdalena Kosecka1*, Beata Guzow‑Krzemińska1, Ivana Černajová2, Pavel Škaloud2, Agnieszka Jabłońska1 & Martin Kukwa1

We studied the biodiversity of Asterochloris photobionts found in Bolivian lichens to better understand their global spatial distribution and adaptation strategies in the context of a worldwide phylogeny of the . Based on nuclear ITS rDNA, the chloroplast rbcL gene and the actin type I gene we reconstructed a phylogenetic tree that recovered nine new Asterochloris lineages, while 32 Bolivian photobiont samples were assigned to 12 previously recognized Asterochloris lineages. We also show that some previously discovered Asterochloris photobiont species and lineages may occur in a broader spectrum of climatic conditions, and mycobiont species and photobionts may show diferent preferences along an altitude gradient. To reveal general patterns of of mycobiont specifcity towards the photobiont in Asterochloris, we tested the infuence of climate, altitude, geographical distance and efects of symbiotic partner (mycobiont) at the species level of three genera of forming fungi: , Cladonia and Lepraria. Further, we compared the specifcity of mycobionts towards Asterochloris photobionts in cosmopolitan, Neotropical, and Pantropical lichen forming fungi. Interestingly, cosmopolitan species showed the lowest specifcity to their photobionts, but also the lowest haplotype diversity. Neotropical and Paleotropical mycobionts, however, were more specifc.

Lichens are a marvelous example of ubiquitous, symbiotic fungi. Teir thalli contain eukaryotic green algae and/ or , which represent the photosynthetic partners and are called photobionts; however, numerous bacteria and fungi also occur in the lichen symbiosis­ 1–3. Many lichens are widely distributed, but it seems that photobionts in lichen symbioses may show their own habitat preferences independent of the lichenized ­itself4–6. Since the organisms’ environmental preferences may be closely related to their distribution, the geo- graphical patterns of the photobionts may be diferent from their fungal ­partners5. Some phylogenetic lineages, species or OTUs (operational taxonomic units), of photobionts are globally distributed, but many photobionts have only been recorded in a specifc region or habitat­ 5,7–9. In many cases, due to the uneven sampling of the lichen symbionts tested, it is too early to precisely defne biogeographic patterns for lichenized algae; this espe- cially applies to photobionts from the tropics­ 5,7. Asterochloris is one of the most common photobionts in lichen symbioses, but it is mostly restricted to cer- tain phylogenetic lichen groups, of which undoubtedly the best sampled and studied are these associated with members of Cladonia, Lepraria and Stereocaulon4,5,9–18. So far, eighteen species have been identifed within Astero- chloris13,14,19–22. Nevertheless, recent research­ 5,9,14,22,23 demonstrates there are many phylogenetic Asterochloris lineages that represent still unrecognized species, which, apart from diferences in the molecular markers, also difer in climate and substrate preferences. Moreover, Škaloud et al.14 indicated that genetic diversity, ecology, biogeography and specifcity to mycobiont partners should all be taken into account in species delimitation. In addition, it has been revealed that some lichens usually associated with Asterochloris spp. may have additional photobionts in their thalli, e.g., Chloroidium9 or Vulcanochloris9,16.

1Department of Plant and Nature Conservation, Faculty of Biology, University of Gdańsk, Wita Stwosza 59, 80308 Gdańsk, Poland. 2Faculty of Science, Department of Botany, Charles University, Benatska 2, 12801 Praha 2, Czech Republic. *email: [email protected]

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To date, many analyses have been carried out to determine the distribution patterns of lichenized algae, and many authors name climate as the most important factor shaping their distribution­ 4,5,24–26. In addition, photobiont lineages specifc to a particular geographic region have been ­reported24,25, but the exact factors driving photo- biont selection remain unexplored. Some studies have indicated that the mycobiont can be highly specifc to its ­photobiont7,23,27–31; however, mycobiont generalists (sensu Yahr et al.32, i.e. with low photobiont specifcity) are also common, especially in lichens that have wide geographical and ecological ranges­ 33–35. In general, photobiont selection is driven by phylogenetic specialization, mycobiont reproductive strategy, availability of motile and airborne photobiont cells, as well as key ecological factors for both ­symbionts32,33,36–40. Despite extensive research eforts, there are many OTUs that are still unnamed. Few studies have been per- formed using the material from the Neotropics­ 23,41, and from this region most Asterochloris photobionts are of unknown taxonomic afliation­ 9,12,23. In the present study we focused on Asterochloris biodiversity in lichen symbioses in Bolivian Andean vegetation to better understand their global spatial distribution. Bolivia represents the geographical and geological synthesis of South America its geology is a cross-section of all geological eras­ 42. Te eastern Andean foothills have been identifed as the most biologically diverse region of the country. Humidity, rainfall and temperature are important for many biological processes that led to a high speciation degree and endemism. Te Yungas forests are considered the center of diversity for plants dependent on the humid seasonal climate­ 42; this is potentially true for lichens as well. Tis makes Yungas forests the most important center of endemism in Bolivia­ 42,43. Te main aim of this work is to examine the genetic diversity of Asterochloris photobionts in lichens from Bolivia and to reconstruct their phylogenetic relationships. Additionally, to better understand global spatial distribution and adaptation strategies of Asterochloris algae, we studied the role of various factors: mycobiont host, altitude, climatic conditions, and location. For some lineages, we also assessed the level of selectivity of bionts. Additionally, we present the frst record of Vulcanochloris from the Neotropics. Results Phylogenetic analyses. In the present study we generated 54 new ITS rDNA sequences, 29 sequences of the chloroplast rbcL gene fragment, and 14 of the actin type I of Asterochloris algae. In addition, we obtained one ITS rDNA sequence of genus Vulcanochloris (Table S1). Te Bayesian phylogenetic tree inferred the genus Aster- ochloris to be divided into two major clades (Fig. 1), similarly to the results of Vančurová et al.17. Te frst major clade (PP = 0.94, ML = –/84) consists of four poorly supported subclades and a single lineage of A. excentrica, although in Vančurová et al. only two subclades were found­ 17. Te second clade (PP = 0.94, ML = –/84) consists of two subclades. Among them, fve lineages represent previously described species: Asterochloris erici, A. glom- erata, A. irregularis, A. magna and A. pseudoirregularis; an additional four undescribed lineages also occur in this clade. Altogether we recovered 61 lineages (Fig. 1), 52 of which have been previously ­identifed4,5,9,11–13,15,16,22,23, and for those we applied the same nomenclature as in previous papers. 32 Asterochloris samples obtained from Bolivian specimens grouped within 12 of these previously recognized clades, of which only two lineages were ascribed to the species ­level14,20 (Fig. 1). Te remaining nine photobiont lineages (marked as Bol 1–9) consisted only of Bolivian samples and formed new lineages of Asterochloris, probably representing undescribed species, as shown in the ABGD analysis. Furthermore, in 12 localities from Bolivia presented in this study and two previously reported additional Bolivian ­localities23, we found diverse Asterochloris lineages. In locality Chuquisaca 2 (N = 7; for locality codes see Table S1), we found six diferent Asterochloris photobionts; two were detected for the frst time. Additionally, each species of Cladonia and Lepraria from this locality was found to be associated with a diferent Asterochloris lineage. In locality Cochabamba 1 (N = 7), we identifed seven photobionts lineages: A. mediterranea, A. friedlii, Asterochloris spp. StA1, clades 12, P2, Bol2 and Bol3, each associated with a diferent lichen species. Furthermore, we identifed the same ITS rDNA haplotype belonging to Asterochloris sp. clade Bol1 in Lepraria impossibilis in a range of altitudes from 1900 to 2149 m a.s.l. (Table S14). Similarly, photobionts belonging to clade Bol2 from L. congesta and L. af. congesta were collected from 3780 to 4780 m a.s.l. Asterochloris sp. clade MN082 was recorded in localities Chuquisaca 2, La Paz 1, Santa Cruz 1, Tarija 1 and 2, within thalli of Neotropical (Cladonia af. ahtii, C. ceratophylla) or Pantropical (Lepraria sipmaniana) lichens in a range of altitudes from 980 to 2750 m a.s.l., while Asterochloris sp. clade Bol1 associates with Lepraria impossibilis from localities Tarija 1 and 2, and with L. harrisiana from locality Chuquisaca 2. Asterochloris sp. clade P2 was found in Pilophorus cf. cereolus from Costa Rica, and also in eight Bolivian localities in altitudes between 2237 and 3860 m a.s.l. in Cladonia spp., Cladia aggregata and Stereocaulon myriocarpum (Fig. 1; Tables S1, S2). We also observed dissimilarities in photobionts composition within four habitat types: lower montane cloud forest, upper montane cloud forest section 1, upper montane cloud forest section 2 and open high Andean vegeta- tion (Figs. S6, S7). For example, in lower montane forest (N = 12), Asterochloris spp. clades Bol1, Bol3, Bol4, A14, 9 and MN082 were present, while in the frst section of upper montane cloud forest (N = 26), Asterochloris spp. clades 9, A14, L54, P2, S1, MN082 and Bol1, Bol5, Bol6, Bol8 and Bol9, A. mediterranea and A. friedlii occurred. In addition, in the second section of upper montane cloud forest (N = 28), Asterochloris spp. clades 9, P2, S1, 12, A14, StA1, Bol2, A. medirerranea, A. friedlii and Vulcanochloris sp. were detected, while in the open high Andean vegetation (N = 11), Asterochloris spp. clades 8, StA1, StA5, A6, Bol2 and Bol7 were present. Tis shows that diverse photobiont types may occur within a steep gradient of altitude. With increasing altitude above sea level, the average temperature and amount of precipitation diminish (Fig. S7). Tis means that photobionts in the open high Andean vegetation should be resistant to low temperatures and periods of drought, and species of photobionts in the lower montane cloud forest should be resistant to higher temperatures and its seasonal changes. Humidity (as seasonality of rainfall) in Stereocaulon has already been indicated as a determining factor for Asterochloris, Vulcanochloris and Chloroidium9.

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0.76/-/90.76/-/98 Pino-Bodas CL281CL281 AsterAsterochloris ssp.p. clade 9 from CladoniaCladonia cocomprmpressa, Bolivia, 2186 m missing data 3501-400 0 2001-250 0 4001-450 0 1001-150 0 2501-300 0 3001-350 0 4501-500 0 1501-200 0

UGDA-LUGDA-L 1901190155 AAsterochloristerochloriss sp. ffrroomm Cladonia dactdactylotylotaa, Santa Cruz 11,, 2250 m 501-1000 AstAstererochloris ClaCladoniadonia ppraetermissaraetermissa ŘŘídkáídká IHIH11 ssp.p. clade 9 frofromm , IndiIndia,a, 1665 m 0-50 0 0.86/-/-0.86/-/- Pino-Bodas CL140CL140 Asterroochlorischloris sp. clade 9 fromfrom CCladonialadonia subrasubradiatdiata, USUSA,FloridA,Florida ŘŘídkáídká IH1IH144 AAstesterroochlorischloris sp. clade 9 from CCladonialadonia coniocraea, Nepal, 1800 m 0.98/-/8/822 ŘŘídkáídká IH20 AstAstererochloris ssp.p. clade 9 from CCladonialadonia scabriuscula, IndiIndia,a, 1410 m PPino-Bodasino-Bodas CCL271L271 AstAstererochloris ssp.p. clade 9 from ClaCladoniadonia conconfusafusa, BoliviBolivia,a, 2946 m 0.0.80/-/880/-/86 PPino-Bodasino-Bodas CCL273L273 AstAstererochloris sp. clade 9 form ClaCladoniadonia calycantcalycanthaha, BoliviaBolivia,, 2186 m Kukwa 1162056205 AAsterochloristerochloriss sp. ffrrom Cladonia granulosa, ChChuquisacauquisaca 2, 2740 m 13 1 3 8 5 5 1 0 0044 UGUGDA-LDA-L 1900190099 AAsterochlorissterochloris sp.p ffrroomm CCladonialadonia arcuata, Santa Cruz 22,, 2330 m Clade 9 Kukwa 1414890890 AAsterochlorissterochloris sp. ffrroomm CCladonialadonia ccf.f. raramulosamulosa, La Paz 22,, 3060 m 00.85/-/-.85/-/- VVaannčurováčurová A5A50404 AstAstererochloris sp. cladcladee A14 frfromom SStertereocaulon ccf.f. obesum, PanamaPanama,, 1776 m Kukwa 1682916829 AsterochlorisAsterochloris sp. ffrroomm LLeprariaepraria cf.cf. crcryptovouauxiyptovouauxii, TTaarijarija 2, 1943 m Kukwa 1690716907 AsterochloriAsterochloriss sp. frfromom LLeprariaepraria pallidpallida, ChChuquisacauquisaca 2, 2600 m Clade A14 0 0 1 2 0 1 1 0 001 Kukwa 1658616586 AsterochlorisAsterochloris sp. frfromom DiploschisteDiploschistess cf.cf. cinereocesiucinereocesiuss, TaTarijarija 1, 1900 m Kukwa 1151355135 AsterochlorisAsterochloris sp. ffrrom CladoniaCladonia subradiata, Cochabamba 1, 2059 m Clade Bol 3 0 0 0 1 1 1 0 0 000 UGDA-L 18555 Asterochloris sp.p from Stereocaulon microcarpup m,,, La Paz 3, 1240 m Clade Bol 4 0 0 1 0 0 0 0 0 000 Pino-Bodas CCL188L188 Asterroochlorischloris sp.p from Cladonia ceratophyllapyy ,,, France, Reunion Pino-Bodas CCL176L176 AstAstererochloris sp. drom ClCladoniaadonia ddidymaidyma, USA, Hawaii PPino-Bodasino-Bodas CCL808L808 Asterroochlorischloris ssp.p. from CCladonialadonia subradiasubradiatata, PortPortugal,ugal, AAzoreszores Kukwa 1184708470 AAsterochloristerochloriss sp. ffrroomm Cladonia ceratophceratophyllaylla, Santa Cruz 11,, 2635 m 1/99/100/100 NNormoreormore 150 AstAstererochloris ssp.p. from ClCladoniaadonia ddidymaidyma, USUSA,A, AlAlabamabama Kukwa 1478147855 AAsterochlorissterochloris sp. ffrrom ClCladoniaadonia ceratoceratophyllaphylla, La Paz 11,, 22750750 m Kukwa 1169096909 AAsterochloristerochloriss sp. ffrroomm CCladoniladonia afaff.f. aahtiihtii, ChChuquisacauquisaca 2, 2600 m Kukwa 1161686168 AAsterochloristerochloriss sp.p form CCladoniladonia afaff.f. aahtiihtii, Chuquisaca 2, 2740 m Kukwa 116915b6915b Asterochloris sp. ffrroomm Lepraria sipmaniana, TTaarijarija 1, 980 m Kukwa 1169416941 AAsterochloristerochloriss sp. ffrrom LLeprariaepraria sisipmanianapmaniana, TTaarijarija 2, 1750 m Clade MN082 0 1 0 1 0 4 0 0 007 Kukwa 16553 Asterochloris sp.p from Cladonia ffurcata, Tarijaj, 1, 2460 m Clade Bol 5 0 0 0 0 1 0 0 0 000 0.79/-/- 1/1/-/-/- Kukwa 1414886886 AAsterochlorissterochloris sp. ffrroomm Cladonia calcalycanthaycantha, La Paz 22,, 3060 m Kukwa 1150445044 AAsterochloristerochloriss sp.p ffrroomm CCladonialadonia ffurcaturcata, Cochabamba 1,1, 3283 m UGUGDA-LDA-L 1861186133 AAsterochloristerochloriss sp. ffrroomm Stereocaulon myriocarpum, La Paz 44,, 3860 m Kukwa 116916911 AAsterochlorissterochloris sp. ffrroomm Cladia aaggregataggregata, ChChuquisacauquisaca 2, 2600 m 1/86/1/86/9292 UGUGDA-LDA-L 1902190211 Asterochloris sp. ffrrom CCladonialadonia arbuscula subssubsp.p. boliviana, Santa Cruz 1, 2950 m DQDQ229895229895 AAstersterochloris ssp.p. clade P2 from PiloPilophorusphorus ccf.f. cerreeoluoluss, Costa RicaRica,, 1542 m UGUGDA-LDA-L 1900190044 AAsterochloristerochloriss sp.p ffrroomm Cladonia dididymadyma, La Paz 44,, 2450 m 1/76/89 Kukwa 1414880a880a AAsterochlorissterochloris sp. ffrroomm Cladonia cf subcariosa, La Paz 2, 3060 m 1/98/99 UGUGDA-LDA-L 1898189877 AAsterochloristerochloriss sp.p ffrroomm Cladonia melanomelanopodapoda,,, La Paz 44,, 3820 m Clade P2 0 0 0 1 2 3 3 3 000 Pino-Bodas CLCL111188 Asterroochlorischloris ssp.p. from Cladonia dacdactylotatylota, BoliviaBolivia,, 2852 m 00.95/-/-.95/-/- UGUGDA-LDA-L 1899189999 AAsterochlorissterochloris sp. ffrroomm Cladonia dididymadyma, La Paz 22,, 3520 m -/-/86 1/89/100/89/100 UGUGDA-LDA-L 2517251733 AAsterochloristerochloriss sp. ffrroomm SStereocaulontereocaulon tomentostomentosumum, Santa Cruz 11,, 2700 m Nelsen 21812181bb Asterroochlorischloris sp. clade S1 from SStertereocaulon sp., Costa Rica, 1542 m Clade S1 0 0 0 1 0 0 2 0 003 1/98/10/100 EEU0087U00871111 AstAstererochloris ssp.p. clade L54 from LLeprariaepraria ssp.,p., Costa Rica, 2071 m Kukwa 1946194688 Asterochloris sp. ffrrom LLeprariaepraria aff. hodkinsoniana, Santa Cruz 1, 2270 m Clade L54 0 0 0 0 2 0 0 0 000 Pino-Bodas CL79 Asterochloris sp.p from Cladonia didymay , Indonesia CL79 0 0 0 0 0 0 0 0 001 0.98/79/-.98/79/- VVaannčurováčurová AA337337 AAstersterochloris woessiae frfromom SStertereocaulon azazororeum, PortPortugal,ugal, Madeira, 671 m 1/99/1099/100 AstAstererochloris woessiae SStertereocaulon azorreeuumm VVaannčurováčurová AA359359 frfromom , PortPortugal,ugal, Madeira, 1250 m 9 0 0 0 0 009 1/84/92 BayerovaBayerova 3401 AsteAsterorochlorischloris woessiae fromfrom LeprariaLepraria borborealis, Bulgaria,Bulgaria, 494 m A. woessiae 7 5 0 Talbot KIS 1873 Asterochloris sp.p clade S3 from Stereocaulon saxatile,,, USA, 138 m Clade S3 1 0 0 0 0 0 0 0 000 0.95/-/80 EU008710EU008710 AAstesterorochlorischloris sp. ccladelade AA66 ffromrom Lepraria nigrnigrocincta, Costa RRicaica KP3186KP31867878 Asterroochlorischloris sp.p clade AA66 frfromom Leprariap crassissima, Czech Republic 1/97/99 EEU008714U008714 AAstersterochloris sp. clade A6 from LLeprariaepraria sp., Costa Rica 0.87/-/86 Kukwa 1414848a848a AAsterochlorissterochloris sp.p ffrroomm LLeprariaepypraria crcryptovouauxiypptovouauxii,,, La Paz 22,, 4370 m Clade A6 0 0 0 0 0 0 0 0 106 00.82/-/-.82/-/- Pino-Bodac CCL822L822 AAstesterroochlorischloris sp.p clade StA3 from CCladonialadonia intermedielintermediellala, TTaanzaninzania 00.94/89/-.94/89/- VVaannčurováčurová OO9898 AstAstererochloris sp. clade StA3 from Cladonia mamacrcrophylla, Denmark 0.83/-/- 1 0 0 0 0 0 0 0 105 0.91/-/98 ŘŘídkáídká TT88 AstAstererochloris ssp.p. clade StA3 from SStertereocaulon vesuvianum, Tanzania, 4430 m Clade StA3 ŘŘídká T20 Asterochloris sp.p from Stereocaulon vesuvianum, Tanzania,, 4430 m Řídká T20 0 0 0 0 0 0 0 0 100 Kukwa 1168586858 AAsterochlorissterochloris sp. ffrroomm LLeprariaepraria imimpossibilipossibilis, TTaarijarija 2, 2149 m Kukwa 1162046204 AAsterochloristerochloriss sp. ffrroomm LLeprariaepraria harrisiana, ChChuquisacauquisaca 2, 2740 m 0.93/-/98 11/98/10/98/100 Kukwa 16828 Asterochloris sp. ffrrom LLeprariaepraria imimpossibilispossibilis, TTaarijarija 2, 1943 m Kukwa 1165846584 AAsterochlorissterochloris sp. ffrroomm LLeprariaepraria impossibilis, TTaarijarija 1, 1900 m Kukwa 1168316831 AAsterochlorissterochloris sp.p ffrroomm LLeprariaepppraria imimpossibilipossibilis, TTaarijarijaj, 2, 1943 m Clade Bol 1 0 0 0 3 1 1 0 0 000 1/100/10000 KGKGS007AS007A AstAstererochloris sejongensis ffromrom Cladonia ppyxidatyxidata, AAntarcticntarctica AAstersterochloris sejongensisjg Cladonia pypyxidatxidata A. sajognensis 4 0 0 0 0 0 0 0 000 0.90/-/92 KKGS064BGS064B ffromrom , AAntarcticntarcticaa MTMT036576036576 AAstesterroochlorischloris antaantarrcticaa from ClCladoniadonia spsp.,., AAntarcticantarctica MT0365MT03657373 AstAstererochloris antarrcctictica from CCladonialadonia ssp.,p., AAntarcticantarctica 1/1/96/96/- MT0365MT03657474 AAstersterochloris antaantarrcticaa from CCladoniladonia spsp.,., AntarcticAntarcticaa MTMT036575036575 AAstersterochloris antaantarrcticaa from CCladonialadonia sp.sp.,p , AntarcticAntarcticaa AstAstererochloris antarrccticatica SStertereocaulon alpinum 0.98/-/- VVaannčurováčurová AA378378 ffromrom , Iceland, 20 m 7 0 0 0 0 0 0 0 001 VVaannčurováčurová AA392392 AAstersterochloris anantatarrccticatica from SStertereocaulon saxatile, Iceland, 43 m A. antarctica/Clade StA2 Pino-Bodas CL100 Asterochloris sp. from Cladonia confusa, South Africa CL100 0 0 0 0 0 0 0 0 001 Asterochloris Lepraria impossibilis Kukwa 19499 sp. from , Santa Cruz 1, 2270 m Clade Bol 6 0 0 0 0 1 0 0 0 000 Pino-Bodas CCL8L81111 AAstersterochloris mediterranea from Cladonia ffoliaceaoliacea, PortPortugal,ugal, AAzoreszores PPino-Bodasino-Bodas SSP3P3 AAstersterochloris mediterranea from CCladonialadonia corsicacorsicanana, PortugaPortugal,l, AAlgarvlgarvee KKP257351P257351 AstAstererochloris mediterranea from Cladonia convoluta/convoluta/foliaceafoliacea, SSpain,pain, 518 m 0.98/81/9/81/98 UGUGDA-LDA-L 1856185666 AsterochloriAsterochloriss sp. ffrrom Stereocaulon pachpachycephaluycephalum, La Paz 4, 3820 m UGUGDA-LDA-L 1896189611 AsterochloriAsterochloriss sp. ffrroomm Cladonia calcalycanthaycantha, La Paz 33,, 22560560 m -/-/95 UGUGDA-LDA-L 25175 AAsterochloristerochloris sp. ffrrom SStereocaulontereocaulon tomentostomentosumum, CCochabambaochabamba 1 2950 m PPino-Bodasino-Bodas CCL799L799 AAstersterochloris mediterranea from ClCladoniaadonia ffoliaceaoliacea, PortPortugal,ugal, AAzoreszores VVaannčurováčurová KKO25.2O25.2 AAstersterochloris mediterranea from SStertereocaulon vesuvianum, Spain, CanarCanaryy Island, 1850 m A. mediterranea 1440 3 1 0 3 0 0 0046 L60 Asterochlorihl s sp.p URa14 ffrom Leprariap sp.,p, China,hi , 2058 m URa14 0 0 0 0 4 0 0 0 000 1/99/971/99/97 Pino-Bodas 33CONISCONIST AAstesterroochlorischloris sp. ffromrom CCladonialadonia conista, UUSA,SA, New Jersey 0.98/-/80.98/-/855 Pino-BodasPino-Bodas 2CONIS2CONIST AAstesterroochlorischloris sp. from CCladonialadonia conistconistaa, USA, ConnecticConnecticutut 0.82/-/80.82/-/822 OOksanenksanen 505 AstAstererochloris sp. from Cladonia furrccataata, USUSA,A, VViirrggininiaia 0.99/-/90.99/-/955 Pino-Bodas CCL133L133 AstAstererochloris sp. from CCladonialadonia santensisantensiss, USA, New Jersey 0.99/-/90.99/-/955 Pino-Bodas 1313HUMIHUMIL AsterAsterochloris ssp.p. ffromrom CCladonialadonia conisconistata, FinlaFinlandnd Pino-Bodas 11CONISTCONIST AAstersterochloris sp. from CCladonialadonia conisconistata, Russia 1/94/101/94/100 AstAstererochloris CCladonialadonia rreeii Peksa 778787 sp.sp. clade 8 from , SlovakiaSlovakia,, 500 m 4 1 0 0 0 0 0 0122 PeksaPeksa 796796 AsteAsterorochlorischloris sp.sp. clade 8 from Cladonia ffimbriatimbriata,,, SlovakiSlovakia,a, 780 m Clade 8 3 Kukwa 19459 AsterochloriAsterochloriss sp. ffrrom LLeprariaepraria finkfinkiiii, Santa Cruz 1, 2270 m 0.77/-/70.77/-/788 Kukwa 1180698069 AAsterochloristerochloriss sp. ffrroomm LLeprariaepraria achariana, Cochabamba 11,, 3120 m EEU008706U008706 AstAstererochloris friedliii frfromom LLeprariaepraria loblobificansificans, USA 1/100/101/100/100 EEU008696U008696 AAstersterochloris friedliii from LLeprariaepraria caesioalcaesioalbaba, USA AAstersterochloris friedliii LLeprariaepraria ffinkiiinkii Nelsen 33974974 from , USA, 349 m 0 0 3 0 1 0029 0.90/-/84 PPeksaeksa 232355 AstAstererochloris ffriedliii ffromrom LLeprariaepraria caesioalba,,, SlovakiaSlovakia,, 998 m A. friedlii 1 3 0 AstAstererochloris Cladonia ranrangiferinagiferina 1/98/1001/98/100 ŘŘídkáídká I6 sp. clade I1 from , IndiaIndia,, 2253 m 0 0 0 0 0 2 0 0 000 ŘŘídkáídká IH31 AstAstererochloris ssp.p. clade I1 from Cladonia corcorymbescensymbescens,,, IndiaIndia,, 2743 m Clade I1 0.92/-/-0.92/-/- VVaannčurováčurová OO1010 AstAstererochloris sp.p clade 12B from SStertereocaulon ssp.,p., Mexico, 1945 m 0.94/-/80.94/-/86 Kukwa 1150405040 Asterochloris sp. ffrroomm Cladonia chlorophaea, Cochabamba 1, 3283 m 1/93/961/93/96 Pino-Bodas CCL125L125 Asterroochlorischloris sp. clade 12B from Cladonia bacilliformis, Russia 1/93/1001/93/100 AstAstererochloris Stereocaulon ppaschale 9 5 0 1 0 0 2 0 005 0.95/-/98 TTaalbotlbot 101011 sp.p clade 12B from , UUSSA Alaska, 55111 m PPeksaeksa 992121 Asterroochlorischloris sp. clade 1212AA from Cladonia rreeii, Czech Republic, 250 m Clade 12 0 0 0 0 0 0 0 0 010 Kukwa 14827 Asterochloris sp. from , La Paz 1, 4780 m Clade Bol 7 Pino-Bodas CL8 Asterochloris sp. from Cladonia pyxidata, New Zealand CL8 0 0 0 0 0 0 0 0 001 UGDA-L 18556 Asterochloris sp. from Lepraria achariana, Santa Cruz 1, 2445 m Clade Bol 8 0 0 0 0 1 0 0 0 000 Kukwa 16907a Asterochloris sp.p from Leprariapp impossibilis, Chuquisacaq 2, 2600 m Clade Bol 9 0 0 0 0 0 1 0 0 000 1/1/97/9997/99 VVaannčurováčurová AA1010 AstAstererochloris italianaa from SStertereocaulon alpinump , AArrggentina,entina, 14 m 11/77/10/77/100 AstAstererochloris italianaa Xanthoria parietina AAM906030M906030 from , IItaltalyy 3 2 4 0 0 0 0 0 0077 1/76/99 Pino-Bodas CCL353L353 AstAstererochloris italianaa from CCladonialadonia aarrcuata, Costa RiRicaca A. italiana 0.99/-/90 Vančurová O70 Asterochloris aff. italiana from Stereocaulon azoreum, Portugal, Madeira, 900 m A. aff. italiana 2 1 1 0 0 0 0 0 007 AAstersterochloris Cladonia corcorymbescensymbescens 0.87/-/95 11/100/10/100/100 Pino-Bodas CCL166L166 ssp.p. from , New Caledonia 0 0 0 0 0 0 0 0 002 Pino-Bodas CCL171L171 AstAstererochloris ssp.p. from Cladonia conconfusafusa, New CaedonCaedoniaia P-B OTU 14 Vančurová A14 Asterochloris sp. from Stereocaulon sp., Argentina, 1062 m Vančurová A14 0 0 1 0 0 0 0 0 000 Kukwa 1414695695 AsterochloriAsterochloriss sp. ffrrom LeLepraripraria aff. congesta, La Paz 11,, 3780 m 1/93/951/93/95 Kukwa 1518151866 AAsterochloristerochloriss sp. ffrrom LLeprariaepraria concongestgesta, Cochabamba 1, 4649 m Kukwa 1414827a827a AAsterochloristerochloriss sp. ffrroomm LLeprariaepraria concongestgesta, La Paz 11,, 4780 m 0.88/-/80.88/-/88 1/90/871/90/87 Kukwa 1159875987 Asterochloris sp. ffrrom hypoleucus, ChChuquisacauquisaca 1, 4150 m Kukwa 1598159888 AAsterochloristerochloriss sp. ffrrom Diploschistes hypoleucus, Chuquisaca 1, 4150 m Kukwa 1414831831 AAsterochloristerochloriss sp.p ffrroomm LLeprariaeppgraria concongestgesta, La Paz 11,, 4780 m Clade Bol 2 0 0 0 0 0 4 0 1 230 0.88/-/-.88/-/- PiPino-Bodasno-Bodas 2RE2REII AstAstererochloris lobophorp a frfromom CCladonialadonia rreeii, CanadCanadaa PPino-Bodasino-Bodas 4RE4REII AstAstererochloris lobophora ffromrom Cladonia rreeii, NNorwayorway 0/99/-/88/-/88 PePeksaksa 161666 AstAstererochloris lobophora frfromom LLeprariaepraria caesioalba, Czech ReRepublic,public, 746 m 1/78/966 PPeksaeksa 868666 AstAstererochloris lobophora frfromom LLeprariaepraria bboorreealisalis, Czech Republic, 850 m VVaannčurováčurová DDS3.1S3.1 Asterroochlorischloris lobophora frfromom SStertereocaulon sp., Russia, 586 m A. lobophora 22 7 0 0 0 0 3 0 0028 0.99/-/84 Nelsen 33950950 AstAstererochloris lobophora fromfrom LeprariaLepraria finkiifinkii, UUSA,SA, 485 m VaVannčurováčurová LL958958 AstAstererochloris sp. clade StA5 from SStertereocaulon alpinum, AAustria,ustria, 2941 m 1/77/98 1/78/101/78/100 VVaannčurováčurová OO5050 AAstersterochloris sp. clade StA5 from SStertereocaulon ccf.f. alpinum, Canada, 1168 m UGUGDA-LDA-L 1896189633 AAsterochlorissterochloris sp.p ffrrom Cladonia ppocillumocillum,,, La Paz 55,, 42425050 m Clade StA5 0 0 4 0 0 6 1 0 101 0.99/-/- SAGSAG 26.81 AsterAsterochloris phycobionticay a from Anzina carneonivea, Italy,y, 1777 m A. phycobiontica 0 1 0 2 1 0 0 0 002 0.76/-/-0.76/-/- VaVannčurováčurová L107L10744 AsterAstA erochlorishl i sp.sp. cladeclldade SStA4SAtA44f ffromrom SterStereocaulonl sp.,sp., USA AlAlaska,ask4ka, 40406066 m 0.93/-/8/82 TaTalbotlbot 400400 AstAstererochloris ssp.p. clade StA4 from SStertereocaulon paschale, UUSSA AlaskaAlaska,, 49 m SSteinovateinova CCL383L383 Asterroochlorischloris ssp.p. clade StA4 from Cladonia coccicocciferafera, Czech ReRepublicpublic Clade StA4 5 1 0 0 0 0 0 0 001 1/100/100 AstAstererochloris echinataa LLeprariaepraria caesioalba 1/100/100 PePeksaksa 555511 from , CzeCzechch RRepublic,epublic, 398 m 2 0 3 0 0 0 000 Peksa 181866 AstAstererochloris echinataa from LLeprariaepraria ririgidulgidula, Czech ReRepublic,public, 398 m A. echinata 0 1 0.94/-/84 Peksa 495 Asterochloris sp.p clade A4 from Diploschistesp muscorum,p, Czech Republic,, 383 m Clade A4 2 2 0 0 0 0 0 0 000 VVaannčurováčurová A3 AstAstererochloris ststerereocaulonicolaa from SStertereocaulon vesuvianum, AArrggentina,entina, 990 m 0.97/98/90.97/98/999 MTMT036569036569 AAstersterochloris ststerereocaulonicola from SStertereocaulon alpinum, AntarcticAntarcticaa MTMT036570036570 AstAstererochloris ststerereocaulonicola from SStertereocaulon alpinum, AntarcticAntarcticaa MT0365MT03657171 AstAstererochloris ststerereocaulonicola from SStertereocaulon alpinup mm,, AAntarcticantarctica 1/100/10100/100 PPeksaeksa 101111 AstAstererochloris ststerereocaulonicolaa from SStertereocaulon nanodes, SlovakiSlovakia,a, 860 m 1/-/1/-/- VVaannčurováčurová A386 AstAstererochloris ststerereocaulonicolaa from SStertereocaulon ccf.f. alpinum, Iceland, 238 m 1/98/99 PPeksaeksa 868600 Asterroochlorischloris ststerereocaulonicolaa from Lepraria alpinap , Spain, 1450 m 7 6 3 0 0 0 0 0 003 AstAsterichloriserichloris ststerereocaulonicolaa SStertereocaulon vesuvianum A. stereocaulonicola/Clade A9 0.94/-/82 VVaannčurováčurová AA111 from cf. , AArrggentina,entina, 750 m 0 2 0 0 0 0 0 0 000 Vančurová A1 Asterochloris sp. clade StA6 from Stereocaulon cf. alpinum, Argentina, 990 m Clade StA6 0.97/81/- Pino-Bodas CL183 Asterochloris sp. from Cladonia rappii, Australia CL183 0 0 0 0 0 0 0 0 001 Asterochoris Cladonia ramulosa Pino-Bodas CL345 sp.p from , Denmark CL345 0 0 0 0 0 0 0 0 001 1/95/101/95/100 VVaannčurováčurová A1A133 AAstesterroochlorischloris sp.p clade StA7 from SStertereocaulon ccf.f. vesuvianum, AArrgentina, 14 m 0.87/-/91 Hammer 77000000 AstAstererochloris sp. clade StA7 from Cladia agaggrgregata, AuAustraliastralia Clade StA7 1 0 0 0 0 0 0 0 001 0.97/82/797/82/799 VVaannčurováčurová AA319319 AstAstererochloris sp,p, clade StA8 from SStertereocaulon myriocarpumypy , Costa Rica,, 3042 VVaannčurováčurová AA498498 AAstesterroochlorischloris sp. clade StA8 from StSterereocaulon myriocarpum, Venezuela, 3876 m 1/79/-/91 0.98/88/98/999 VVaannčurováčurová AA496496 AstAstererochloris sp. clade StA8 from SStertereocaulon myriocarpum, Venezuela, 3209 m VVaannčurováčurová A5A50202 AstAstererochloris ssp.p. clade StA8 from SStertereocaulon myriocarpum, Venezuela, 4308 m Clade StA8 0 0 0 0 0 0 3 0 100 PPeksaeksa 181833 AstAstererochloris leprariii ffromrom LLeprariaepraria neneglectaglecta, Czech ReRepublic,public, 1058 m A. leprarii 0 0 1 0 0 0 0 0 004 Asterroochlorischloris LLeprariaepraria 1/100/101/100/100 PPeksaeksa 887070 sp. cladcladee AA111 ffromrom sp., UUSSA California, 13 m 2 2 1 0 0 0 0 0 000 Peksa 887373 Asterroochlorischloris ssp.p. cladcladee AA111 frfromom LLeprariaepraria caesioalba, UUSSA California,, 460 m Clade A11 -/-/83 1/100/10100/100 Peksa 990000 AstAstererochloris gaertnerii from Lepraria rigidula, Czech Republic, 850 m Peksa 232366 AstAstererochloris gaertneri from LLeprariaepraria rigidula, Czech Republic, 11114466 m A. gaertneri 1 2 1 0 0 0 0 0 002 Pino-Bodas CL190 Asterochloris sp. from Cladonia pachyclados, France, Reunion CL190 0 0 0 0 0 0 0 0 001 Asterochloris excentrica Stereocaulon dactylophylluypy y m A. excentrica UTEX 1714 from , USA Vermont,, 265 m 0 0 00 MT036564MT036564 AstAstererochloris ppseudoirpseudoirrregularisg frfromom Cladonia spsp.,p., AAntarcticntarctica 1 0 0 0 0 0 1 MT036565MT036565 AsteAsterroochlorischloris pseudoirpseudoirrregularis from Cladonia gracilis, Antarctica 00.85/93/9.85/93/944 MT036566MT036566 AsterAsterochloris pseudoirrpseudoirregularis fromfrom CladoniCladonia sp.,sp., Antarctica 0.81/85/981/85/93 MT036567MT036567 AsterAsterochloris pseudoirrpseudoirregularis fromfrom CladoniCladonia sp.,sp., AntarcticAntarcticaa MT036568MT036568 AsterAsterochloris pseudoirrpseudoirregularis fromfrom CladoniCladonia sp.,sp., AntarcticAntarctica VaVannčurováčurová LL988988 AsterAsterochloris pseudoirrpseudoirregularis from SterStereocaulon paschale, USUSA Alaska,Alaska, 732 m 0.960.96/94/9/94/944 TaTalbotlbot 281281 AsterAsterochloris ppseudoirrseudoirregularis fromfrom SStertereocauloeocaulonn vesuvianum, UUSASA AAlaska,laska, 112 m A. pseudoirregularis 31 3 0 1 0 0 0 0 0011 VVaannčurováčurová LL992992 AstAstererochloris irirrregularis frfromom SStertereocaulon vesuvianum, AustriAustria,a, 1883 m 0.94/100/9400/94 UUTEXTEX 22322366 AstAstererochloris irirrregularig s ffromrom SStertereocaulon sp.,p Iceland Peksa 999999 Asterroochlorischloris irirrregularig s from SStertereocaulon pileatum, Czech Republic, 356 m 4420 3 4 0 1 0 0 0032 1/100/100 AArmaleormaleo s.n. AAstersterochloris sp. from Cladonia grayi A. irregularis VVaannčurováčurová OO7575 AstAstererochloris gglomerata frfromom SStertereocaulon ppileatum, Portugal,gg, Madeira, 900 m 0.79/81/80.79/81/82 PPeksaeksa 449898 AstAstererochloris glomerata ffromrom Diploschistes muscorum, Czech Republic, 401 m AstAstererochloris gglomerata SStertereocaulon evolutoides 0.91/85/90.91/85/911 UUTEXTEX 895 ffromrom , USUSAA Massachusetts, 387 m 0.87/-/76 UUTEXTEX 11712712 AAstersterochloris glomerata from ClaCladoniadonia squamosquamosasa, UUSSA Massachusetts UUTEXTEX 894 AstAstererochloris glomerata ffromrom SStertereocaulon evolutoides, UUSSA Massachusetts, 387 m A. glomerata 4510 0 1 0 0 0 0 0068 -/-/9-/-/977 UUTEXTEX 910 AstAstererochloris ericii from CCladonialadonia cristatellcristatellaa, UUSSA Massachusetts, 98 m 1/98/9898/98 AstAstererochloris erici SStertereocaulon dactylophylluypy m Nomore 337575 ffromrom , CanadaCanada,, 11116 m 11 0 0 0 0 0 0 0 001 0.95/-/82 UUTEXTEX 99111 AstAstererochloris ericii from CCladonialadonia cristatelcristatellala, UUSSA Massachusetts, 98 m A. erici UTEX 902 Asterochloris magng a from Pilophorusp aciculare, USA Washington,g 1405 m A. magna 11 0 1 0 0 0 0 0 004 1/95/101/95/100 Kukwa 1166216621 AsterochloriAsterochloriss sp. ffrrom Cladonia coconfusnfusa, TTaarijarija 1, 3158 m 0.94/-/84 Kukwa 1414684684 AAsterochloristerochloriss sp. ffrroomm CCladonialadonia andesita, La Paz 11,, 3780 m PPino-Bodasino-Bodas CCL266L266 Asttrreeochloriochloriss ssp.p. clade StA1 from Cladonia leleprprocephala, BolivBoliviaia -/98/--/98/- VVaannčurováčurová OO7676 AAstesterroochlorischloris sp. clade StA1 from SStertereocaulon vesuvianum, DenmarkDenmark,, 143 m 1/100/1001/100/100 Kukwa 115030b5030b Asterochloris sp. ffrrom Cladonia confusa, Cochabamba 11,, 3283 m UGUGDA-LDA-L 1899189922 AAsterochlorissterochloris sp. ffrroomm CCladonialadonia isabellina, La Paz 44,, 33820820 m AstAstererochoris Cladonia melanomelanopodapoda 19 0 0 0 0 1 3 4 201 1/-/87 PPino-Bodasino-Bodas CCL288L288 ssp.p. from , Bolivia Clade StA1 0.92/-/-0.92/-/- ŘŘídkáídká IH23 AAstesterroochlorischloris sp.p clade I2 from ClCladoniaadonia ffuurrccataata, India, 2607 m 0 0 0 0 0 1 2 0 001 0.95/-/- AAstersterochloris ClaCladoniadonia furrccataata ŘŘídkáídká IH2IH266 ssp.p. clade I2 from , India Clade I2 0 0 0 0 0 0 0 0 001 0.99/-/- Kotelko 885 Asterochloris sp. from Cladonia pocillum Kotelko 885 Pino-Bodas CL249 Asterochloris sp. from Cladonia scotteri, Russia CL249 0 0 0 0 0 0 0 0 001

0.02

Figure 1. Majority-rule consensus tree from Bayesian analysis of Asterochloris based on ITS rDNA, rbcL and actin type I locus data set with posterior probabilities and bootstrap support values from RaxML and IQ-TREE analysis presented near the branches. For each record GenBank accession no. or voucher no. (for newly sequenced samples) are followed with photobiont name (if known), their mycobiont host name and the origin of specimen together with altitude (if known). Newly sequenced photobionts from Bolivia are marked in bold. Species or phylogenetic lineages are marked in boxes with appropriate names. Based on altitude of the samples, which belongs to particular phylogenetic lineage, we added summary of abundance for each lineage. Samples from 0 to 500 m a.s.l. are in frst box, from 501 to 1000 are in second box, from 1001 to 1500 are in third box, from 1501 to 2000 are in fourth box, from 2001 to 2500 are in ffh box, from 2501 to 3000 are in sixth box, from 3001 to 3500 are in seventh box, from 3501 to 4000 are in eighth box, from 4001 to 4500 are in ninth box, from 4501 to 5000 are in tenth box and samples with missing data of altitude are in eleventh box, marked in grey.

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Based on the observed diferences in composition of photobionts of four habitat types in Bolivia, we con- cluded that some previously known Asterochloris lineages may occur in a broader spectrum of climatic condi- tions (data summarized in Table S9 and in Fig. 1 in reference to altitude). Tus, A. mediterranea only known from the Mediterranean, temperate Europe and islands of the North Atlantic and Indian Oceans can also occur in the Neotropics. Annual precipitation in areas in which this species occurs ranges between 18 and 974 mm, with precipitation in the driest quarter between 2 and 95 mm (Fig. 2). Asterochloris sp. OTU25­ 23 was found on islands of the North Atlantic, Indian and Pacifc Oceans. We also identifed this particular lineage in Bolivia in Cladonia ceratophylla, C. af. ahtii and Lepraria sipmaniana. Our phylogenetic analysis revealed that this lineage is similar to a photobiont from Cladonia didyma (clade MN082) from USA­ 31 and we conclude it may possess tolerance to a wide range of climatic condition. We do not have complete data for all specimens, but our rough estimate for annual precipitation for this lineage ranges between 591 and 4930 mm, with precipitation of the driest quarter between 11 and 1009 mm. Asterochloris friedlii was previously known from temperate Europe and USA as well as tropical areas of China and Korea­ 5,12, and we found it in a Bolivian sample of the widespread Lepraria fnkii. A similar situation was observed for Asterochloris sp. clade StA5, which to date has been found to associate with Stereocaulon spp.9. Likewise, Asterochloris sp. clade 9 found in South America (this study), India and Southern North America is associated only with Cladonia spp.5,18,23,32,41; based on our analysis, this lineage shows tolerance for a broad annual mean temperature range (− 5.2–24.9 °C) and is characterized by drought resistance and rehydration (annual precipitation: 591–4930 mm, precipitation of driest quarter: 8–1009 mm, precipitation of wettest quarter: 143–3968 mm). Te widespread lineage Asterochloris sp. clade 12 (also found in this study) associates with Cladonia spp. and Stereocaulon spp.5,9,11,13,23 and is characterized by the widest annual temperature range we have seen: − 16.9–18.1 °C and high drought resistance (precipitation of driest quarter: 10–165 mm). Asterochloris sp. clade P2 was found to associate with diferent lichen species and genera (Cladia, Cladonia, Pilophorus and Stereocaulon) [this ­study11,23]. Tis lineage, together with closely related Asterochloris spp. clades L54 and S1, were all found only in the Neotropics [this ­study11,23]. So far, Asterochloris spp. clades L54 and A6 have been found to form a symbiosis exclusively with Lepraria spp. (Fig. 1) [this ­study4,11].

Statistical analyses. To identify the impact of selected factors on the distribution of Asterochloris pho- tobionts, we performed variation partitioning analyses (Tables S7, S8) that showed that our selected variables explained 46–47% of the variation for Bolivian samples. 25–26% of the variation was explained by the species of the mycobiont (Table S7), which shows low correlation between photobiont distribution and mycobiont hosts. 22–23% was explained by environmental factors. 5% of the variability was shared between mycobiont host, climate and altitude or habitat type, while, 6% of the variability was explained by climate as independent factor. In the case of Stereocaulon, 39% of the variation was explained by our selected variables. Te largest part of the variation in diversity of photobionts was explained by the mycobiont hosts (12%, Table S7). As independent factors, altitude explained 8% of the variation, while geographical distance—explained 4%. For Cladonia spp., 58% of the variability was explained by our selected variables (Table S7). 31% was explained by mycobiont spe- cies, whereas climate and geographical distance explained 5% and 3%, respectively. However, ecological factors shared 11% of the variability. In the case of Lepraria spp., altitude appeared to be insignifcant (Table S6). Results of PCoA analyses for Lepraria spp. show moderate correlation between photobiont distribution and mycobiont host (35% of the variation explained by mycobiont). Te remaining factors did not exert signifcant infuence. In addition, we selected 12 cosmopolitan, 17 Neotropical and ten Pantropical species from our dataset, and supplemented by data available in GenBank (Table S5). In the case of cosmopolitan species, variation partitioning analyses revealed that 0–13% of the variability was explained by mycobiont hosts (Table S8), climate accounted 15% of the variability; altitude and substrate appeared to be insignifcant (dbRDA analyses; Table S6). Overall, 59% of the variability of Asterochloris distribution in Neotropical and Pantropical lichens can be explained by the mycobiont hosts. We performed PCA analyses to identify which photobionts were represented by selected groups of lichens, as a function of climate (Fig. S2). PCA ordination of all analyzed species resulted in 72.7% cumula- tive variance explained on the frst 2 axes (frst—47.8%, second—24.9%). Lichen groups and their photobionts, respectively, showed indiference relative to climatic parameters: i.e., they are scattered across the hyperdimen- sional climatic space (Fig. S2). However, cosmopolitan, Neotropical and Pantropical species as groups represent diferent range of climatic conditions. In the case of cosmopolitan species, the total explained variation in the biplot was 68.4% by component 1 and 2 (Fig. S3). PCA ordination of Neotropical species, on the other hand resulted in 84.6% (Fig. S4) cumulative variance explained on the frst 2 axes; and for Pantropical species it was 78.7% (Fig. S5). However, in cosmopolitan, Neotropical and Pantropical groups of lichens (and also between them), diferent genera show indiferences, dependent on diverse climatic conditions (potentially climatic preferences). Tropi- cal species prefer higher temperatures in warm and wet seasons, while the cosmopolitan species are adapted to extreme temperatures (BIO4, 7). Due to the dissimilarities we observed in the photobionts composition of the four habitat types in Bolivia, we performed PCA to visualize variability of photobionts of those habitat types in the context of climatic factors (Fig. S6). Tis shows that diferent conditions prevail in each habitat, however, the ranges of photobionts from diferent habitats may overlap.

Haplotypes. Due to diferences in the impact of the mycobiont species on the distribution of photobionts in selected genera of lichens, as well as diferences in tolerance for climatic conditions, we performed ITS rDNA haplotype analyses to study the associations of mycobiont hosts and photobionts in selected groups of lichens. In the case of Asterochloris photobionts from 200 Stereocaulon samples (Table S10), we found low haplotype diversity (0.28) and detected 55 photobiont ITS rDNA haplotypes (Tables S10, S16), of which 24 were identifed

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Annual Mean Temperature (°C) Temerature Annual Range (°C)

70

20 60

50

10

40

0

30

20 −10

10 2 2 a a iedlii iedlii A, fr A, fr , Clade 9 , Clade 9 , Clade 12 , Clade 12 , Clade P2 , Clade P2 , Clade Sta 5 , Clade Sta 5 , Clade StA1 , Clade StA1 A, sp A, sp , Clade MN08 , Clade MN08 A, sp A, sp A, sp A, sp A, mediterrane A, mediterrane A, sp A, sp A, sp A, sp A, sp A, sp

Annual Precipitation (mm) Precipitation of Driest Quarter (mm)

5000 1000

4000 800

3000 600

400 2000

200 1000

0

0 i 2 2 iedli iedlii A. fr A. fr . Clade 9 . Clade 9 . Clade 12 . Clade 12 . Clade P2 . Clade P2 . Clade Sta 5 . Clade Sta 5 . Clade StA1 . Clade StA1 A. sp A. sp . Clade MN08 . Clade MN08 A. sp A. sp A. sp A. sp A. mediterranea A. mediterranea A. sp A. sp A. sp A. sp . sp A. sp A

Figure 2. Box-plot diagram representing diferences in climatic preferences for selected species and lineages of Asterochloris photobionts. Climatic data were obtained from the Global Climate Data—WorldClim. BIO1 = Annual Mean Temperature (°C), BIO7 = Temperature Annual Range (°C), BIO12 = Annual Precipitation (mm), BIO17 = Precipitation of Driest Quarter (mm).

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in samples from tropics, 36 in samples from the temperate zone, and 5 from both regions. However, haplotype diversity for tropical samples appeared to be higher (0.52) than that from temperate areas (0.24) (Table S17). Asterochloris irregularis appeared to be the most common photobiont associated with Stereocaulon spp. in tem- perate climates (62 samples). In Stereocaulon samples from Bolivia, we found fve distinct haplotypes represent- ing A. mediterranea, Asterochloris spp. clades P2, S1, and Bol4 and Bol7. Moreover, some species showed a high fexibility in their photobiont choice, and furthermore, it appears their photobiont pools may difer depending on the climate, e.g. in Stereocaulon vesuvianum we found haplotypes belonging to Asterochloris glomerata, A. irregularis, A. pseudoirregularis, Asterochloris spp. clades A9, StA1 and StA7 in temperate areas, while Asterochlo- ris spp. clades StA3, StA5, T20 and A. mediterranea (Table S10) in tropical regions. In the case of Asterochloris associated with Cladonia spp., we found 268 ITS rDNA haplotypes (haplotype diversity = 0.42) based on 630 samples (Tables S11, S16). 132 originated from tropical regions, 143 from temper- ate areas and only 10 Asterochloris haplotypes were noted in both regions. Moreover, we observed diferences in photobiont haplotype diversity within the tropical and temperate Cladonia samples (tropical—0.63, temper- ate—0.35). Te most common photobiont in Cladonia spp. from tropical areas seems to be Asterochloris sp. clade 9 (71 samples); this potentially new species is represented by 54 haplotypes in our dataset. Moreover, samples of this clade were also found in 13 Cladonia specimens from temperate regions. Bolivian specimens of Cladonia spp. were found to associate with 34 haplotypes belonging to A. mediterranea, Asterochloris spp. clades 9, 12, A14, S1, StA1, StA5, P2, MN082, Bol3 and Bol5. Photobionts from Lepraria spp. (Tables S12, S16), represented by 44 haplotypes of Asterochloris, showed high haplotype diversity (0.54). In tropical climates 14 haplotypes were found, while in temperate areas we found 30 haplotypes. Te most common photobiont in Lepraria spp. is a haplotype of A. friedlii (14 sequences from both regions). Moreover, haplotype diversity was higher in tropical (0.70) than temperate (0.49) regions (Table S17). In the case of lichens representing cosmopolitan distribution patterns, we observed 62 Asterochloris haplo- types (Table S13) with high haplotype diversity (0.53) (Table S16). Tese haplotypes belong to 30 Asterochlo- ris lineages. Samples from tropical climates were found to associate with 14 lineages, while specimens from temperate climates associated with 21. Only one was found in both regions (haplotype 28 in Table S13). Some lichen species appear to adopt diferent Asterochloris haplotypes in diferent climatic regions, e.g. Cladonia fur- cata associates with Asterochloris spp. clades 12 or 8 in temperate areas, while other haplotypes were found in tropical areas (clades I1 and I2 in India, and clades P2 and Bol5 in Bolivia). Remarkably, Stereocaulon alpinum appears to associate with at least 11 diferent Asterochloris lineages, of which clade Bol7 and A. lobophora have only been found in tropical climates. Interestingly, Asterochloris sp. clade StA5 was found both in tropical and temperate regions: Bolivia, Austria (one haplotype) and in Canada (a diferent haplotype). Additionally, Ste- reocaulon alpinum collected in temperate regions was also found to associate with A. irregularis, A. italiana, A. phycobiontica, A. woessiae and Asterochloris spp. clades StA2, StA4 and A9 (Table S10). In the case of Cladonia pyxidata, it was found to associate with haplotypes of A. glomerata, A. lobophora, and Asterochloris sp. clade12 in temperate regions; with A. sejongensis in Antarctica; and with Asterochloris spp. clades 12 and I1 in tropical regions. Indeed, Asterochloris sp. clade 12 may associate with this species in both regions (Table S11). Seventeen Neotropical lichen species were analyzed; among them we identifed 29 haplotypes of Asterochloris belonging to 16 lineages (Table S14) with high haplotype diversity (0.83) (Table S16). In the case of the Pantropical group, ten lichen species associated with 32 Asterochloris haplotypes belonging to 13 lineages (Table S15) and showed high haplotype diversity (0.80) (Table S16). Within Neotropical and Pantropical lichens, we found multiple Asterochloris haplotypes either restricted to a single species or with potentially wide selectivity (i.e., occurring in diferent species in similar or diverse localities). Discussion Little is still known about the nature of the associations between mycobionts and photobionts. Only several works have explored the diversity of Asterochloris photobionts in the tropics­ 5,9,11,12,18,31,32,41. In this study, we recovered nine new Asterochloris lineages, while 29 Bolivian photobiont samples were assigned to 12 previously recognized Asterochloris lineages, from which only two have been so far formally described (Fig. 1). In this study we also showed that some previously known Asterochloris photobiont lineages may occur in a broader spectrum of climatic conditions, e.g. A. mediterranea is here reported for the frst time from Neo- tropics. On the other hand, alpine and psychrophilic Asterochloris sp. clade StA5­ 9 may occur in open high Andean vegetation and may show higher drought resistance (Precipitation of Driest Quarter for sample UGDA-L 18963 = 30 mm) than previously thought (80–341 mm) (Fig. 2). Asterochloris spp. clades P2, A14 and L54 are considered Neotropical lineages, as well as clades Bol1 and Bol2 recovered in this study, neither of which is closely related to previously described lineages. Preferences of mycobionts for certain types of photobionts have been repeatedly recognized for Trebouxia8,29,44, Asterochloris9,14, or ­Trentepohliaceae7. Te photobiont’s ecological specialization probably determines the mycobi- ont’s selection for its symbiont­ 4,44,45. Furthermore, horizontal transmission has probably increased the taxonomic range of compatible photobiont ­partners15; for example Vančurová et al.9 showed that several algal species or lineages show specifcity towards a single mycobiont species. On the other hand, they also recovered lineages that were not specifc towards a single mycobiont. Our data shows that previously described Asterochloris lineages can associate with broader ranges of mycobionts. For example, Asterochloris spp. clades StA1 and StA5, were previously found only within Stereocaulon spp., but we found them associated with diferent Cladonia species, suggesting a lower level of selectivity and specifcity (sensu Beck et al.10). Interestingly, Asterochloris sp. clade MN082 was found in samples of L. sipmaniana, Cladonia af. ahtii, and C. ceratophylla from diferent localities ranging in altitude from 980 to 2750 m a.s.l; these results are preliminary, however, further analyses are necessary to determine the exact selectivity level of these algae.

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Ecological factors may modulate the availability of photobiont species/strains at various sites, thus reducing the number of possible associations in isolated populations. Te infuence of altitude gradient on photobiont population structure was previously described­ 7,46–48. In our study, we found some changes in photobiont popu- lations along altitude gradients; e.g., Asterochloris spp. clades Bol1 and MN082 were present in lower montane cloud forest and upper montane cloud forest section 1; in upper montane cloud forest section 1 and section 2 were Asterochloris spp. clade P2 and S1, as well as A. mediterranea and A. friedlii. In each altitude dependent habitat, unique Asterochloris lineages occur. We need more data to defne exact ecological niche for those lineages. To reveal global patterns of specifcity of the mycobionts towards the photobiont in Asterochloris, we tested the infuence of climate, altitude, geographical distance and efects of the symbiotic partner (mycobiont) at the species level for three genera of lichen forming fungi: Stereocaulon, Cladonia and Lepraria. Stereocaulon species are widespread and have broad ecological requirements­ 9, and associate with many Asterochloris species [­ 14, this study], but also Chloroidium9,10 and Vulcanochloris ­[16, this study]. Te diversity of photobionts in Stereocaulon and the association between their diversity and environmental conditions was previously conducted on a global scale by Vančurová et al.16. Te photobionts distribution in Stereocaulon species found in that study appeared to follow a pattern that was highly infuenced by substrate type. However, in that study this was mostly evident in Chloroidium and Vulcanochloris. Our analyses confrmed the hypothesis that photobiont variability may be dependent on geographical distance­ 32. In the case of Stereocaulon9, 4% of the photobiont variability was explained by a net efect of geographical distance. Furthermore, the distribution of photobiont diversity in Stereocaulon may also be afected by altitude (8%). It has been previously shown that the majority of Stereocaulon mycobiont species appear to be specifc towards ­phycobionts9,17. In our study, Stereocaulon displayed a low level of selectivity toward photobiont lineages (Table S10). Furthermore, we report low haplotype diversity for Asterochloris photobionts of Stereocaulon that can be correlated with oversampled photobionts from temperate regions. Yahr et al.32 demonstrated that geographic position and habitat are the best predictors of algal genotype distri- bution in Cladonia. Steinová et al.15 showed that photobionts are signifcantly structured by climate and geogra- phy, but the mode of reproduction was revealed to have the greatest impact on Cladonia photobiont diversity. In our analyses we found that 36% of the variability was explained by mycobiont at the species level. Furthermore, we report higher haplotype diversity in photobionts from Cladonia spp. than Stereocaulon spp., in which the predominating photobiont species was Asterochloris glomerata (21 haplotypes represented by 104 sequences; the most common haplotype represented by 51 sequences). Nonetheless, Cladonia and Lepraria are known to associate with a wide range of Asterochloris species­ 4,14. With regards to Lepraria, we found that these mycobiont species associated with particular Asterochloris taxa from lineages B and C (sensu Vančurová, et al.9) and (like Cladonia), show moderate selectivity toward their photobionts, with a moderate infuence of mycobiont species and a very low infuence of climate and spatial structure. In addition, species of both genera may show diferent ranges of adaptation strategies. Tese results support a hypothesis that mycobionts can associate with numerous symbiotic partners in the case of ubiquitous lichen species adapted to various ecological conditions­ 8,13,23,24. Te dominant species of Asterochloris found within Lepraria spp. was A. friedlii (found in 22 samples, 27% of tested samples). However, more research is needed to reveal the most common Asterochloris spp. in Lepraria as well as to fully explain the factors afecting photobiont distribution. Lichens exhibit various distribution patterns at the micro- and macro-levels. In the case of lichen-forming fungi, 16 main biogeographic patterns were distinguished (including cosmopolitan, bipolar, paleotropical, Neo- tropical, Pantropical, Mediterranean)49, but the biogeography of lichen photobionts is still poorly known. Prefer- ential associations with locally adapted symbionts have been reported repeatedly­ 8,50,51. It was hypothesized that low specifcity of the host towards its symbiotic partner helps the host to take advantage of the locally adapted symbiotic partners and colonize broader geographic areas­ 8. In the case of higher specifcity, the host is expected to have a narrower ecological niche, and a restricted geographical distribution. In the case of lichen symbioses, the generalist pattern is more ­common8,24,47; however, a specialist pattern has been reported for -associated lichen ­fungi52. We compared the specifcity of mycobionts towards Asterochloris photobionts in three groups of lichens: cosmopolitan, Neotropical, and Pantropical. Interestingly, cosmopolitan species repeatedly showed the lowest specifcity towards photobionts, but also had the lowest haplotype photobiont diversity. More haplotypes were identifed in temperate regions; however, the haplotype diversity of tropical photobiont lineages was higher. Tis may indicate a signifcant under-sampling of tropical regions. In addition, the distribution of the diversity of photobionts within cosmopolitan lichens was infuenced by a moderate impact of climatic conditions (15%), while in the case of Neotropical and Pantropical lichens there was a correlation between the mycobiont species and climatic conditions. Tis indicates the selection of locally adapted photobionts in cosmopolitan lichens, while tropical species mostly demonstrate habitat preferences. However, in the light of the results obtained here and in previous research­ 8,53, we assume that the selection of a symbiotic partner may be based on the search of the best-suited photobiont. To obtain more accurate results, additional data on one species commonly found in the Neotropical and Pantropical regions are needed, with emphasis on the impact of habitat conditions. In conclusion, to further investigate these questions, we suggest that a suitable species, representative of each fungal genus associating with Asterochloris, should be selected for future studies before sampling. Furthermore, to avoid geographical gaps, sampling should be extended to the whole world. In addition, on the basis of the results obtained here, it can be assumed that Bolivia, due to its geological and ecological diversity, may refect the biodi- versity of the entire Andean region in the Neotropics, where the issue of photobionts’ biodiversity still remains unresolved. Indeed, the tropics may be an important, under-explored source of hidden photobionts biodiversity.

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Materials and methods Material. 54 lichens samples representing 4 genera: Cladonia, Diploschistes, Lepraria and Stereocaulon con- taining Asterochloris photobionts from various habitats in Bolivia were selected randomly for studying the bio- diversity of Bolivian Asterochloris photobionts. Lichens were collected from various substrata (rocks, soil, tree bark, wood, and bryophytes). Te majority of samples were obtained from Yungas cloud forest, but also from Tucuman-Bolivian forest and dry inter-Andean forest. Lichen samples were collected in Bolivia with the permission of Ministerio de Media Ambiente y Agua (MMAYA/VMABCC GDF/DGBAP/MEG No 03272/2016) and in cooperation with Herbario Nacional de Bolivia (LPB), who in turn made specimens available to the herbarium of University of Gdańsk. Original samples are deposited in herbarium LPB, with duplicates stored in University of Gdańsk (UGDA). Lichen species were determined using appropriate identifcation keys­ 54–58. Morphology, secondary chemistry, and for Lepraria spp., sequencing of nucITS rDNA of ­mycobiont59 were used for specimen identifcation. Lichen substances were identifed with thin-layer chromatography (TLC) in solvents A and ­C60. Detailed locality data are presented in Supplementary Data Table S1. Te ranges of distribution were defned for lichen species used in this study—Cosmopolitan, Neotropical or Pantropical, based on the division proposed by Galloway­ 49. Te best sampled species were selected, and afer scrutiny of the literature, a database was created for further analysis.

Molecular methods. Well-preserved specimens lacking any visible symptoms of fungal infection were used for DNA isolation (total lichen DNA) following the CTAB ­protocol61. For molecular identifcation of the photobionts (Asterochloris), the nuclear internal transcribed spacer (ITS, ITS1-5.8S-ITS2)11,31,62, the chloroplast rbcL gene and in few cases of unique lineages of Asterochloris63–65, and the actin type I gene were amplifed­ 10. Te PCR condition are presented in Supplementary Data Table S3. Sequencing was performed in Macrogen® in Amsterdam, Netherlands using amplifcation primers. Sequences were compared to the sequences available in GenBank using Megablast ­searchers66 to verify their identity and detect potential contaminants. Te newly obtained sequences of the ITS rDNA, and fragments of the rblcL gene and the actin type I regions were deposited in GenBank (Table S1).

Phylogenetic analyses. Te Asterochloris sequence datasets were analyzed as a concatenated dataset of all loci. In total, 189 sequences with a total of 2618 sites were generated. Te ITS rDNA dataset consisted of 188 sequences, including 54 newly obtained and 134 previously published representative sequences of taxa retrieved from GenBank. Tese representative sequences were selected following the latest review paper by Vančurová et al.9, as well as additional, new Asterochloris spp. described by Kim et al.22 and Pino-Bodas and ­Stenroos23. To avoid confusion, the nomenclature of some mycobiont species associated with Asterochloris has not been updated according to the newest taxonomical works; these are: Lepraria borealis and L. caesiolaba (both now subsumed under L. neglecta), L. lobifcans auct. (now L. fnkii), L. nigrocincta (now L. yunnaniana) and Parmel- inopis minarum (now Hypotrachyna minarum). Te chloroplast rbcL gene dataset consisted of 64 sequences (29 newly obtained sequences). Te actin type I dataset consisted of 132 sequences: 14 newly obtained sequences, and previously published sequences. Sequence alignment was automatically performed using MAFFT—Mul- tiple Alignment using Fast Fourier ­Transform67 as implemented in ­UGENE68. Phylogenetic relationships were inferred with Bayesian Inference (BI) carried out in MrBayes v.3.2.269. In cases of which rbcL or actin sequences were lacking, they were treated as missing data. Two parallel MCMC runs were performed, using four inde- pendent heated chains and 10 million generations, sampling every 1000th tree. Te initial 2500 trees of each run (25%) were discarded as burn-in, and posterior probabilities were estimated by constructing a majority-rule consensus tree of all sampled post-burn-in trees. Maximum likelihood (ML) analyses were performed using RAxML-HPC v.8 with 1000 bootstrap replicates (ML-BS), the GTRGAMMAI model­ 70 and the edge-linked partition model in IQ-TREE71,72 on the CIPRES Science Gateway­ 73. Te best-ft substitution models and block (Table S4) were selected using Akaike Information Criterion (AIC) as implemented in PartitionFinder ­274. Te tree topology obtained by ML method did not contradict the Bayesian tree; therefore, only the Bayesian tree is shown. Te consensus trees were visualized using FigTree v1.4.275. Branches with bootstrap support ≥ 70% and posterior probabilities ≥ 0.95 were considered to be strongly supported. Analogically, an ITS rDNA dataset of Vulcanochloris, including a single new sequence of Vulcanochloris sp. detected in Stereocaulon pityryzans (UGDA-L 18522), was analyzed. It consisted of 16 sequences, of which 15 were previously published sequences were retrieved from GenBank.

OTUs delimitation. To delimit Asterochloris OTUs, the Automatic Barcode Gap Discovery method (ABGD) was used, following Leavitt et al.27. Te analyses were performed using the webserver (https://​bioin​fo.​ mnhn.​fr/​abi/​public/​abgd/​abgdw​eb.​html) separately for the ITS rDNA and actin datasets. Te Jukes and Cantor (JC69) model was applied to calculate the genetic distance. Pmin = 0.001, Pmax = 0.01, step = 10, Nb bins = 20 and X (relative gap width) = 0.5 were used. Next, comparisons of the inferred OTUs were made based on both markers. Afliation to individual species or Asterochloris phylogenetic lineages used in further analyses were estimated based on published data. Tese data are available in Table S2. In total, 61 OTUs were inferred.

Statistical analyses. Te relationship between species richness of Bolivian photobionts and lichen species, as well as the relative efects of climate, altitude, geographical distances, substrate, and habitat type, were ana- lyzed. In the case of data obtained from GenBank, only records with precise geospatial coordinates were used. Te comparative efects of selected variables were analyzed by variation partitioning in redundancy analyses, using the varpart function in the vegan ­package76. Te phylogenetic distances of photobionts were used as a

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response variable, coded as the frst 10 PCoA axes. Climatic data were obtained from the Global Climate Data— WorldClim Version ­277 at a resolution of 2.5 arc minutes. Te 19 environmental variables, altitude, substrate and habitat type were transformed into principal component variables (PCs). Principal coordinates of neigh- bor matrices vectors (PCNM) representing the geographical distances at various spatial ­scales78 were obtained from the transformation of geographical distance values (latitude and longitude). PCNM vectors were calculated based on the pairwise geographical distances obtained by the distGPS function of the BoSSA package­ 79. Dis- tance-based redundancy analyses (dbRDA)80 were used to select statistically signifcant predictors for explaining variation for each of the data-sets used in the variation partitioning analyses. Variation partitioning analyses were carried out separately for all Bolivian data obtained in this study (N = 54) and in Pino-Bodas & ­Stenroos23, and also for Asterochloris from selected lichen genera, i.e. Cladonia (N = 179), Stereocaulon (N = 169) and Lepraria (N = 34) due to better sampling of these genera. We found that the substrate infuence appeared to be statistically insignifcant and did not correlate with our Bolivian datasets (Table S6). Furthermore, due to multiple missing data for this particular variable, we decided to omit this factor in the analyses of the datasets of the three genera. Variation partitioning analyses were also performed for selected group of lichen forming fungi representing diferent distribution patterns, i.e. cosmopolitan (N = 77), Neotropical (N = 30) and Pantropical (N = 23) (patterns of distribution identifed based on the literature­ 54–58; Table S5). Due to limited data for species showing diferent distribution patterns, diferent PCoA analyses schemes were used. A series of analyses were performed using mycobiont host as an explanatory variable constantly together with an other variable, which was replaced in subsequent series (geographical distance, altitude, substrate and climate; Table S8). We took into account only morphospecies; we did not consider the possibility of one morphospecies comprising diferent phylogenetic lineages (i.e.cryptic species) a situation that can potentially be geographically or ecologically more restrictive, as has been demonstrated in the ­Cladoniaceae81. To detect and visualize the diferential ordination (tendency or strategies) of samples in the hyperspace, we performed Principal Component Analyses (PCA) according to climatic factors (BIO1–BIO19) and the grouped results depending on the distribution patterns of lichen species and the genus of mycobiont host. In addition, we performed these analyses for Bolivian samples, grouping results by habitat type. All analyses were performed in R v 3.6.082, using RStudio v.1.2.133583.

Haplotypes. To visualize the precise interaction between mycobionts and photobionts, we inferred hap- lotypes from a pairwise character diference matrix of ITS rDNA sequences using the haplotype function in haplotypes ­package84. Tese analyses were carried out for six data sets (Tables S10–14) consisting of sequences newly obtained for this paper aligned with additional sequences downloaded from GenBank of Asterochloris from genera Cladonia, Stereocaulon and Lepraria. We also ran analyses for lichen-forming fungi representing diferent distribution patterns: cosmopolitan, Neotropical, Pantropical. Lastly, we measured nucleotide diversity, the number of haplotypes, and haplotype diversity as further estimations of diferences between groups. Data availability DNA Sequences are deposited in GenBank: ITS rDNA MW847807-MW847861, actin type I locus MW845956- MW845969, and rbcL gene MW847862- MW847890. DNA matrix used for phylogenetic analyses of Astero- chloris, as a concatenated dataset of all loci is available at TreeBASE version 2 web ­server85,86, http://​purl.​org/​ phylo/​treeb​ase/​phylo​ws/​study/​TB2:​S28062.

Received: 16 November 2020; Accepted: 8 April 2021

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H., Chan, L., Dominus, M. J., Ruan, J., Vos, R. A., and V. Tannen. TreeBASE v. 2: A Database of Phylogenetic Knowledge. In: e-BioSphere (2009) Acknowledgements We greatly appreciate Adam Flakus and Pamela Rodriguez-Flakus (Polish Academy of Sciences, Kraków) for great support in the feld studies. We would like to thank members of Herbario Nacional de Bolivia, Instituto de Ecología, Universidad Mayor de San Andrés La Paz, for the generous cooperation and our colleagues and all protected areas staf (http://sernap.​ gob.​ bo​ ) for their help during the feldwork. Scott LaGreca (Duke University) is acknowledged for language editing and proofreading. Tis 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). Author contributions M.K1. and B.G.-K. designed the study. M.K. conducted feldwork, collected specimens and delimit the species of mycobionts. M.K., I.Č., and A.J. performed laboratory work with contributions from P.Š. M.K1. analyzed the data with contributions from P.Š. and B.G.-K. M.K1., M.K., B.G.-K. and P.Š. wrote the manuscript.

Competing interests Te authors declare no competing interests. Additional information Supplementary Information Te online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​021-​88110-0. Correspondence and requests for materials should be addressed to M.K.

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