1 Molecular systematics and character evolution in the lichen family Ramalinaceae (Lecanorales, 2 Ascomycota) 3 4 Sonja Kistenich1*, Einar Timdal1, Mika Bendiksby1,2 & Stefan Ekman3 5 6 1 Natural History Museum, University of Oslo, P.O. Box 1172 Blindern, 0318 Oslo, Norway 7 2 NTNU University Museum, Norwegian University of Science and Technology, Elvegata 17, 7012 8 Trondheim, Norway 9 3 Museum of Evolution, Uppsala University, Norbyvägen 16, 75236 Uppsala, Sweden 10 11 * Corresponding author: [email protected] 12 13 14 15 16 17 1 18 Abstract The Ramalinaceae is the fourth largest family of lichenized ascomycetes with 42 genera 19 and 913 species exhibiting considerable morphological variation. Historically, generic boundaries in the 20 Ramalinaceae were primarily based on morphological characters. However, molecular systematic 21 investigations of subgroups revealed that current taxonomy is at odds with evolutionary relationships. 22 Tropical members of the family remain particularly understudied, including the large genus Phyllopsora. 23 We have generated and collected multilocus sequence data (mtSSU, nrITS, nrLSU, RPB1, RPB2) for 149 24 species associated with the Ramalinaceae and present the first comprehensive molecular phylogeny of the 25 family. We used ancestral state reconstructions on our molecular family phylogeny to trace the evolution 26 of character states. Our results indicate that the Ramalinaceae have arisen from an ancestor with long, 27 multiseptate ascospores living in humid temperate forests, and that the phyllopsoroid growth form has 28 evolved multiple times within the family. Based on our results using integrative taxonomy, we discuss 29 sister-relations and taxon-delimitation within five well-supported clades: The Bacidia-, Biatora-, 30 Ramalina-, Rolfidium-, and Toninia-groups. We reduce six genera into synonymy and make 49 new 31 nomenclatural combinations. The genera Bacidia, Phyllopsora, Physcidia and Toninia are polyphyletic 32 and herein split into segregates. We describe the two genera Bellicidia and Parallopsora and resurrect the 33 genera Bibbya, Kiliasia, Thalloidima and Sporacestra. According to our new circumscription, the family 34 Ramalinaceae now comprises 38 genera. 35 36 Keywords Ancestral state reconstruction, integrative taxonomy, multilocus phylogeny, 37 Phyllopsora, Toninia, tropical lichens 38 39 Supplementary Material The Electronic Supplement (Tables S1–S2; Figs. S1–S2) is available in 40 the Supplementary Data section of the online version of this article (xx). 41 2 42 INTRODUCTION 43 44 The Ramalinaceae C. Agardh is the fourth largest family of lichen-forming ascomycetes, with 42 45 genera and 913 species (Lücking & al. 2017a, b). This family contains members with a chlorococcoid 46 photobiont, mostly biatorine or lecideine apothecia (when present) and hyaline and often transversely 47 septate ascospores. Historically, generic boundaries were primarily based on growth form, ascospore 48 septation and even ecological preferences. Growth form varies considerably among species. The majority 49 of species form crustose or squamulose thalli, but fruticose or foliose species occur in a couple of genera. 50 Members of the family inhabit a wide spectrum of habitats, including lichenicolous life forms, but 51 corticolous and saxicolous species are the most numerous. Being globally distributed, the Ramalinaceae 52 span all climatic zones from arctic to temperate and tropical, and occur in humid as well as dry habitats. 53 The Ramalinaceae, as currently circumscribed (sensu Lücking & al. 2017a, b), has been variously 54 delimited in the past. Originally, it was described by Agardh in 1821 as ‘Ramalineae’ and included the 55 eight genera Sphaerophoron (now Sphaerophorus, Sphaerophoraceae), Roccella (Roccellaceae), Evernia 56 (Parmeliaceae), Dufourea (Teloschistaceae), Alectoria (Parmeliaceae), Ramalina (Ramalinaceae), 57 Cornicularia (Parmeliaceae) and Usnea (Parmeliaceae; Agardh 1821). More than 100 years later, Watson 58 (1929) excluded all other genera besides Ramalina and was the first to use the correct family name, 59 Ramalinaceae. Zahlbruckner (1921–1940) assigned some foliose and fruticose genera to this family, 60 while crustose species now included here were placed by him in the family Lecideaceae Chevall. He 61 divided the genus Ramalina into three sections: Ecorticatae, Desmaziera and Euramalina (Zahlbruckner 62 1921–1940). Keuck (1979) was the first to investigate morphological and anatomical features of the 63 family’s – at that point – five genera: Cenozosia, Niebla, Ramalina, Ramalinopsis and Trichoramalina. 64 Crustose genera were at the time still included in other families, for example Bacidiaceae Walt. Watson, 65 Biatoraceae A. Massal. ex Stizenb., Lecaniaceae Walt. Watson, Lecideaceae, and Phyllopsoraceae Zahlbr. 66 In his extensive work, Hafellner (1984) reclassified the large families Lecanoraceae and Lecideaceae and 67 erected the following additional families for some of the genera now placed in Ramalinaceae: 68 Catillariaceae Hafellner, Catinariaceae Hale ex Hafellner, Crocyniaceae M. Choisy ex Hafellner, 69 Megalariaceae Hafellner, Mycobilimbiaceae Hafellner, and Schadoniaceae Hafellner. Later, most of those 70 families were reduced into synonymy with Bacidiaceae (see Ekman 2001 for a thorough historical 71 overview). Ekman (2001) investigated taxa belonging to both Bacidiaceae and Ramalinaceae in a broad 72 molecular phylogeny and pointed out the striking morphological similarities in the ascus structure 73 between the two families. The inclusion of the Bacidiaceae, Megalariaceae (Ekman 2001), and later 74 Crocyniaceae (Ekman & al. 2008) resulted in 42 component genera (Lücking & al. 2017a, b). Even so, 75 the synonymy of Bacidiaceae with Ramalinaceae does not seem to have been broadly accepted, as the 76 Bacidiaceae as a separate family is still being used in some modern literature (e.g., Sérusiaux & al. 2012). 77 The first molecular studies of the Ramalinaceae that included more than five genera (e.g., Ekman 78 2001; Reese Næsborg & al. 2007; Schmull & al. 2011; Miadlikowska & al. 2014) rendered the family 79 circumscription non-monophyletic as several genera and species were nested in other lichen families. A 80 few Ramalinaceae genera have undergone further molecular phylogenetic investigations, for example 81 Bacidia De Not. (Ekman 2001), Biatora Fr. (Printzen 2014), and Lecania A. Massal. (Reese Næsborg & 82 al. 2007). None of these, however, included a family-wide taxonomic sampling, which left most generic 83 boundaries largely unexamined, while at the same time rendering these genera polyphyletic. The recent 84 compilation by Lücking & al. (2017a, b) reported that only 55% of the 42 Ramalinaceae genera are 85 represented by DNA sequence data in GenBank, and less than half of those genera have ten or more 86 sequence records. Despite recent molecular efforts to delimit the family within Lecanorales 87 (Miadlikowska & al. 2014), comprehensive phylogenetic investigations of the family with an exhaustive 88 genus-level sampling have so far not been conducted. Genera lacking molecular sequences to verify their 89 placement in the Ramalinaceae include the enigmatic Heppsora D.D. Awasthi & Kr.P. Singh, Physcidia 90 Tuck., Pseudohepatica P.M. Jørg., and Tasmidella Kantvilas, Hafellner & Elix, among others (Lücking & 91 al. 2017a, b: Table S1). 92 While many morphological studies and monographs have been published on temperate species of 93 the Ramalinaceae in North America and Europe (Howe 1913a, 1913b; Mayrhofer 1988; Timdal 1992; 94 Printzen 1995; Ekman 1996a; Printzen and Tønsberg 1999), tropical members of the family remain 95 understudied. A set of genera occurring almost uniquely in the tropics include Bacidiopsora Kalb, 96 Crocynia (Ach.) A. Massal., Eschatogonia Trevis., Krogia Timdal, Phyllopsora Müll. Arg. and Physcidia 97 Tuck. Except for Crocynia, all are characterized by largely squamulose thalli, with the areoles or 98 squamules often overgrowing a thick prothallus (Fig. 1). This morphology is here termed ‘phyllopsoroid’. 99 Even though Crocynia has a more felt-like thallus, we consider this genus also associated with the 3 100 phyllopsoroid growthform due to the distinct prothallus. Phyllopsora is the largest of these genera and has 101 been placed in various families, for example Phyllopsoraceae (Hafellner 1984; Elix 2009), Bacidiaceae 102 (Brako 1989, 1991), and Ramalinaceae (Timdal 2008, 2011). The circumscription of the Phyllopsoraceae 103 was thereby mainly based on the phyllopsoroid growth form, which may have evolved independently in 104 the family as a response to similar ecological conditions (shaded tree trunks in tropical rainforests). 105 Furthermore, many of these species form vegetative dispersal units such as phyllidia or lacinules that are 106 rarely found in other temperate genera of the Ramalinaceae. Apart from Crocynia and Phyllopsora, there 107 are no published molecular studies or sequences in GenBank for the abovementioned tropical genera. 108 Hence, the evolution of the phyllopsoroid growth form has so far not been explored in a phylogenetic 109 context, nor has the inclusion of all these genera in the Ramalinaceae been confirmed. 110 The aim of this study was to improve the knowledge about phylogenetic relationships and 111 character evolution in the family Ramalinaceae and to update its classification accordingly. To achieve 112 this aim, we conducted phylogenetic analyses with multilocus DNA sequence data