Mitchell et al. IMA Fungus (2021) 12:6 https://doi.org/10.1186/s43008-021-00056-0 IMA Fungus RESEARCH Open Access Sareomycetes: more diverse than meets the eye James K. Mitchell1,2* , Isaac Garrido-Benavent3 , Luis Quijada1,4 and Donald H. Pfister1,4 Abstract Since its resurrection, the resinicolous discomycete genus Sarea has been accepted as containing two species, one with black apothecia and pycnidia, and one with orange. We investigate this hypothesis using three ribosomal (nuITS, nuLSU, mtSSU) regions from and morphological examination of 70 specimens collected primarily in Europe and North America. The results of our analyses support separation of the traditional Sarea difformis s.lat. and Sarea resinae s.lat. into two distinct genera, Sarea and Zythia. Sarea as circumscribed is shown to conservatively comprise three phylospecies, with one corresponding to Sarea difformis s.str. and two, morphologically indistinguishable, corresponding to the newly combined Sarea coeloplata. Zythia is provisionally maintained as monotypic, containing only a genetically and morphologically variable Z. resinae. The new genus Atrozythia is erected for the new species A. klamathica. Arthrographis lignicola is placed in this genus on molecular grounds, expanding the concept of Sareomycetes by inclusion of a previously unknown type of asexual morph. Dating analyses using additional marker regions indicate the emergence of the Sareomycetes was roughly concurrent with the diversification of the genus Pinus, suggesting that this group of fungi emerged to exploit the newly-available resinous ecological niche supplied by Pinus or another, extinct group of conifers. Our phylogeographic studies also permitted us to study the introductions of these fungi to areas where they are not native, including Antarctica, Cape Verde, and New Zealand and are consistent with historical hypotheses of introduction. Keywords: Atrozythia, Cryptic diversity, Integrative taxonomy, Resinicolous fungi, Sarea, Zythia, New taxa INTRODUCTION physical barrier to penetration by fungal hyphae (Verrall Conifers, particularly in the families Araucariaceae, 1938; Shain 1971; Rishbeth 1972; Prior 1976). When Pinaceae, and Cupressaceae, produce resins in their tis- soft, resin can flow, trapping fungal hyphae and spores; sues (Langenheim 2003) as part of a complex defence when hard, the resin is difficult to penetrate. Further- system to protect against herbivores (Smith 1961; more, the components of the resin can inhibit the Rudinsky 1966; van Buijtenen and Santamour 1972), growth of fungi, acting as a chemical barrier (Cobb Jr pathogenic fungi (Whitney and Denyer 1969; Gibbs et al. 1968; Hintikka 1970; De Groot 1972; Fries 1973; 1972; Hart et al. 1975; Yamada 2001), protists (Krupa Väisälä 1974; Chou and Zabkiewicz 1976; Bridges 1987; and Nylund 1972; Bunny and Tippett 1988), and bacteria Yamamoto et al. 1997). Despite this apparently inhospit- (Hemingway and Greaves 1973; Hartmann et al. 1981). able environment, a number of so-called "resinicolous" To protect against fungi, resins have the potential to act fungi have evolved to exploit this niche (Cappelletti in several different manners. First, they present a 1924; Selva and Tuovila 2016). The study of fungi growing on conifer resins has a * Correspondence: [email protected] long history, dating back to the fathers of mycology (Per- 1Farlow Reference Library and Herbarium of Cryptogamic Botany, Harvard soon 1801; Fries 1815, 1822). The first species described University, 22 Divinity Avenue, Cambridge, MA 02138, USA was Helotium aureum, described in 1801 by Christiaan 2Department of Physics, Harvard University, 17 Oxford Street, Cambridge, MA 02138, USA Persoon, though he made no mention of the resinicolous Full list of author information is available at the end of the article habit (Seifert and Carpenter 1987). Thus, the first author © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Mitchell et al. IMA Fungus (2021) 12:6 Page 2 of 36 to describe fungi dwelling on resin was Elias Fries, who phylogenetic relationships and to provide further in- described three such fungi in 1815. Sphaeria resinae and sights on the evolutionary history of Sareomycetes on a Lecidea resinae were described as sharing the same habi- temporal and spatial scale. tat and easily confused; these were later determined to represent the asexual and sexual morphs of the same MATERIALS AND METHODS fungus, currently known as Sarea resinae (Ayers 1941; Specimens examined and microscopic examination Hawksworth and Sherwood 1981). The third species, During the course of this study, a number of specimens Racodium resinae, described from Picea resin, is a syn- of Sarea were collected and examined by us. The host nematous hyphomycete now called Sorocybe resinae range and distribution of these specimens was broad, (Seifert et al. 2007). These three Friesian species were with collections from the United States (California, followed by Cytospora resinae, described by Ehrenberg Georgia, Maine, Massachusetts, Minnesota, New Hamp- (1818); this was later determined to be a synonym of shire, Rhode Island, and Vermont) made by J.K.M. and Fries' Sphaeria resinae (Fries 1823; von Thümen 1880). collections from Austria, Cape Verde, Spain, and The last of these early species was described in 1822, Switzerland made by I.G.-B. Further specimens were col- again by Fries, as Peziza difformis, currently known as lected by and lent by Tomás J. Curtis (Ohio), Alden C. Sarea difformis. No additional new resinicolous taxa Dirks (Michigan, Wisconsin), Michael Haldeman (Idaho, were noted until Arnold (1858). Washington), Jason M. Karakehian (Maine, Massachu- The two species assigned to the genus Sarea, S. setts, Newfoundland), Elizabeth Kneiper (Maine, Massa- resinae and S. difformis, are the most commonly col- chusetts), JiříMalíček (Czechia), Rubén Negrín Piñero lected and reported of these resinicolous fungi. A (Canary Islands), Donald H. Pfister (Dominican Repub- search of the Global Biodiversity Information Facility lic), Michaela Schmull (New York), Judi Thomas (Mis- (GBIF) database for S. resinae yielded 1261 records, souri), Per Vetlesen (Norway), and Andrus Voitk and one for "Sarea resinae" on Google Scholar 249 (Newfoundland); these specimens are deposited in FH, results; S. difformis gave 519 records and 196 results, KE, MICH, VAL, and several personal herbaria. Further respectively. In contrast, Sorocybe resinae gives only specimens of Sarea and other critical materials from the 24 records and 56 results (accessed 13 July 2020). In following fungaria were studied: B, CANL, DUKE, FH, addition to frequent reports, the two Sarea species H, K, LD, MICH, NCSLG, NY, TFM, TNS, and TROM. have also been a subject of some interest regarding Microscopic examination of hymenial elements was their systematic placement, which has been unclear conducted using free-hand sections cut under a dissect- (Reeb et al. 2004;Miadlikowskaetal.2014). A recent ing microscope (Wild M5; Leica Geosystems, Heerbrugg, study resolved the uncertainty and has supported the Switzerland) and of the excipulum using sections made erection of a new class in Pezizomycotina, Sareomy- on a freezing microtome. Microtome sections were pre- cetes (Beimforde et al. 2020). This study, as well as a pared by stabilizing water-hydrated apothecia on a freez- recent study that yielded 31 endolichenic isolates of ing stage (Physitemp BFS-MP; Physitemp Instruments, Sarea species (Masumoto and Degawa 2019), have il- Clifton, NJ) with a diluted gum arabic solution and sec- lustrated that both Sarea species are genetically di- tioning with a sliding microtome (Bausch & Lomb Op- verse. This pattern is present in published sequences tical, Rochester, NY) set at approximately 25 μm. The of both Sarea species deposited in public repositories. resulting sections were applied serially to a clean glass Sequence similarity and phylogenetic analyses also slide and allowed to adhere by drying in the remaining suggest that Arthrographis lignicola,thoughmorpho- gum arabic. Slides were prepared under a dissecting logically unlike Sarea species, is a close relative (Gir- microscope (Olympus SZX9; Olympus Corporation, aldo et al. 2014). This, combined with the wide Tokyo, Japan) and studied with a compound microscope distributions of these species, suggest a higher than (Olympus BX40; Olympus Corporation, Tokyo, Japan). known diversity, both obvious and cryptic, in Sareo- Digital images were captured with an Olympus XC50 mycetes. The aim of this study is to assess this USB camera (Olympus Corporation, Tokyo, Japan). diversity. Hand sections were studied with a compound micro- To assess this diversity within Sareomycetes, an inte-