Fungal Diversity (2020) 102:205–224 https://doi.org/10.1007/s13225-020-00451-9 Evolution of non‑lichenized, saprotrophic species of Arthonia (Ascomycota, Arthoniales) and resurrection of Naevia, with notes on Mycoporum Vinodhini Thiyagaraja1,2,3,4 · Robert Lücking5 · Damien Ertz6,7 · Dhanushka N. Wanasinghe1,4 · Samantha C. Karunarathna1,4 · Erio Camporesi8,9,10 · Kevin D. Hyde1,2,4 Received: 20 January 2020 / Accepted: 2 June 2020 / Published online: 3 July 2020 © MUSHROOM RESEARCH FOUNDATION 2020 Abstract Fungi that are barely lichenized or non-lichenized and closely related to lichenized taxa, the so-called borderline fungi, are an important element in reconstructing the evolutionary history of lichenized lineages. Arthoniaceae is a prime example includ- ing non-lichenized, saprotrophic lineages which potentially were precursors to lichenized taxa. In this study, we focused on saprotrophic species of Arthonia sensu lato, including new sequence data for Arthonia pinastri. We obtained fresh material of this taxon from a living branch of Fraxinus ornus in Italy to assess its taxonomic status and to elucidate its phylogenetic relationships within Arthonia. Thin sections of the thallus and ascomata of A. pinastri confrmed the absence of a photobiont. Maximum likelihood and Bayesian analyses of combined mtSSU, nuLSU and RPB2 sequence data placed the species close to A. dispersa (barely lichenized or non-lichenized) and A. punctiformis (non-lichenized) in a clade closely related to Arthonia sensu stricto, and the A. pinastri clade is here resurrected under the name Naevia. Ancestral character state analysis within a broader context of Arthoniales does not support the saprotrophic lifestyle to be a plesiomorphic feature, but suggests loss of lichenization in Naevia, as well as loss and possible regain in a second clade containing saprotrophic species and including taxa resembling Mycoporum, underlining the evolutionary plasticity of Arthoniales. These two clades constitute model taxa to further investigate the evolution of alternative biological lifestyles within the context of chiefy lichenized taxa. Keywords Arthonia susa · Arthothelium · Evolution · Lichenization · Mycarthonia · Pseudoarthonia · Saprobes Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1322 5-020-00451 -9) contains supplementary material, which is available to authorized users. * Kevin D. Hyde 6 Research Department, Meise Botanic Garden, Nieuwelaan [email protected] 38, 1860 Meise, Belgium 7 Service Général de l’Enseignement Supérieur et de la 1 CAS Key Laboratory for Plant Biodiversity Recherche Scientifque, Fédération Wallonie-Bruxelles, Rue and Biogeography of East Asia, Kunming Institute A. Lavallée 1, 1080 Brussels, Belgium of Botany, Chinese Academy of Science, Kunming 650201, Yunnan, People’s Republic of China 8 A.M.B.G. Gruppo Micologico Forlivese “Antonio Cicognani”, Via Roma 18, Forli, Italy 2 Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand 9 A.M.B, Circolo Micologico “Giovanni carini”, C.P. 314, Brescia, Italy 3 Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50002, 10 Societa per gli Studi Naturalistici della Romagna, C.P. 144, Thailand Bagnacavallo, RA, Italy 4 World Agro Forestry Centre East and Central Asia, Kunming 650201, Yunnan, People’s Republic of China 5 Botanischer Garten und Botanisches Museum, Freie Universität Berlin, Königin-Luise-Str. 6–8, 14195 Berlin, Germany Vol.:(0123456789)1 3 206 Fungal Diversity (2020) 102:205–224 Introduction et al. 2014, 2015; Ertz et al. 2018; Van den Broeck et al. 2018). Lichenization has evolved several times independently Due to its versatile biology and the fact that Arthoniales within the Phylum Ascomycota, but its exact origins and forms a separate order and class of chiefy lichenized fungi, the relationships between lichenized and non-lichenized Arthonia sensu lato is an ideal group to investigate the role lineages is often unclear (Gargas et al. 1995; Lutzoni et al. of so-called borderline lichenized fungi in the evolution of 2001, 2004; Nelsen et al. 2009, 2011; Lücking and Nelsen lichenization. Borderline lichenized fungi are characterized 2018). One example is the order Arthoniales, which is by forming autonomous thalli on bark, i.e. not lichenicolous, predominantly lichenized, but contains a number of non- but growing intermingled with other lichens; the presence of lichenized, lichenicolous or saprotrophic taxa, particular a photobiont is ambiguous or a photobiont is absent. Some of in the family Arthoniaceae, which comprises more than these lineages are optionally lichenized or saprotrophic, such 700 species in about 20 genera (Grube 1998; Frisch et al. as in the genera Schizoxylon and Stictis in Ostropales (Wedin 2014; Lücking et al. 2017; Ertz et al. 2018; Wijayawardene et al. 2004; Muggia et al. 2011). Borderline lichenized fungi et al. 2020). Most of the non-lichenized taxa are concen- can be deeply nested within lichenized clades (e.g. Ostro- trated in the collective genus Arthonia sensu lato, one of pales), or they may represent early diverging clades, such as the largest genera of crustose lichens comprising ca. 500 in Trypetheliales (Nelsen et al. 2011; Lücking et al. 2017; species (Acharius 1806; Sundin and Tehler 1998; Sundin Miranda-González et al. 2020). A potential placement as 1999; Lücking et al. 2017). According to Index Fungorum early diverging lineages is of special interest in reconstruct- (2020), more than 1560 names are linked to this genus. ing the evolution of lichenization. Phylogenetically, Arthonia sensu lato forms a largely para- In cases of borderline lichenized fungi currently classifed phyletic clade comprising various distinct lineages (Grube in Arthonia sensu lato, it is unclear whether they are precur- et al. 1995; Grube 2001; Ertz et al. 2009, 2018; Frisch sors to lichenized lineages or are secondarily delichenized. et al. 2014; Van den Broeck et al. 2018). To elucidate their evolution, it is necessary to analyze their Arthonia in its broad sense, including various recent preferred life strategies in a molecular phylogenetic frame- segregates, exhibits a broad biological amplitude: while work (Divakar et al. 2013; Ismail et al. 2016). To this end, comprising mostly lichenized species, about one quarter we obtained fresh material of the borderline lichenized, pre- of the genus (139 species) represents lichenicolous taxa sumably saprotrophic taxon Arthonia pinastri from a living (Grube et al. 1995; Kantvilas and Wedin 2015; Dieder- branch of Fraxinus ornus in Italy. We generated morpho- ich et al. 2018) and some species are non-lichenized and anatomical, microchemical and molecular data in order to saprotrophic on bark (Sundin 1999; Grube 2007; Smith (1) infer the placement of this species using maximum like- et al. 2009). In historical classifcations, saprotrophic lihood and Bayesian analyses of combined sequence data species have variously been separated in genera such within a broad phylogenetic framework of Arthoniaceae; as Celidiopsis, Celidium, Conida, Conidella, Lecideop- (2) document morpho-anatomical and chemical characteris- sis, Mycarthonia, and Naevia (Fries 1824, 1825; Reinke tics of this taxon, particularly its biological lifestyle; and (3) 1895; Schneider 1897; Vainio 1901; Engler 1903; Ser- perform ancestral character analysis to reconstruct whether nander 1907; Zahlbruckner 1907; Petrak 1953; von Arx the species is primarily or secondarily non-lichenized. By 1954; Sundin and Tehler 1998; Grube 2007). Lichenized including a broad set of Arthoniaceae comprising other pre- species are associated chiefy with trentepohlioid algae, sumably saprotrophic lineages, we also expected to clarify with the exception of the Bryostigma clade and Arthonia lifestyle shifts in this family at a more general level. mediella, which feature chlorococcoid algae (Frisch et al. 2014). However, the phylogenetic position of several taxa with chlorococcoid algae and obviously unrelated to these Materials and methods clades such as Arthonia phlyctiformis (Gerstmans and Ertz 2016) still needs to be determined. Notably, lichenicol- Fresh material of Arthonia pinastri was collected from Passo ous species are also often associated with hosts having del Carnaio Bagno di Romangna, Forli-Cesena Province, chlorococcoid algae (Sundin and Tehler 1998; Wedin and Italy, in September 2017. Samples were examined using a Hafellner 1998; Frisch and Holien 2018; Diederich et al. Motic SMZ 168 Series dissecting microscope. Hand sec- 2018). Species of Arthonia sensu lato occupy a wide range tions of the ascomata were mounted on water, 10% KOH and of habitats, occurring in dry to wet environments, from lit- Lugol’s solution. Sections of ascomata and other micro-mor- toral to alpine regions (Sundin 1999; Grube 2007; Smith phological characteristics were photographed using a Nikon et al. 2009). Even after the removal of various recent seg- ECLIPSE 80i compound microscope ftted with a Canon regates, the genus remains highly heterogenous (Frisch 550D digital camera. Microscopic measurements were made with Tarosoft Image Frame Work 0.9.0.7 and images 1 3 Fungal Diversity (2020) 102:205–224 207 used for fgures were processed with Adobe Photoshop CS6 Phylogenetic trees were reconstructed based on mtSSU, Extended 10.0 (Adobe Systems, USA). The material was nuLSU and RPB2 sequence data of a broad set of Artho- deposited in the Mae Fah Luang University