Fungal Diversity (2014) 64:165–179 DOI 10.1007/s13225-013-0267-0 Lepidostromatales , a new order of lichenized fungi (Basidiomycota , Agaricomycetes ), with two new genera, Ertzia and Sulzbacheromyces , and one new species, Lepidostroma winklerianum Brendan P. Hodkinson & Bibiana Moncada & Robert Lücking Received: 4 March 2013 / Accepted: 14 October 2013 / Published online: 16 November 2013 # Mushroom Research Foundation 2013 Abstract We present a revised molecular phylogeny of higher Basidiomycota focusing on Lepidostromataceae based on the large subunit (28S) of the nuclear ribosomal rDNA (nuLSU), with additionl data from the translation elongation factor 1 alpha 1 (TEF1) and the RNA polymerase II second largest subunit (RPB2) genes. Our results suggest that Lepidostromataceae is best recognized in a separate order, Lepidostromatales ordo novum, within subclass Agaricomycetidae. Furthermore, the internal topology of Lepidostromataceae, correlating with thallus features, indicates that three genera, instead of a single genus, should be recognized. We therefore introduce the genera Ertzia genus novum and Sulzbacheromyces genus novum for Lepidostroma akagerae and L. caatingae, respectively. In addition, the new species L. winklerianum spec. nova is described for Mexican material previously identified as L. calocerum . The photobionts of Sulzbacheromyces and Lepidostroma were identified using molecular data of the large subunit of the ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcL) gene, revealing a possibly undescribed genus in B. P. Hodkinson (*) Perelman School of Medicine, University of Pennsylvania, BRB 1046A, 421 Curie Blvd, Philadelphia, PA 19104, USA e-mail: brendan.hodkinson@gmail.com B. Moncada Licenciatura en Biología, Universidad Distrital Francisco José de Caldas, Cra. 4 No. 26B-54, Torre de Laboratorios, Herbario, Bogotá, Colombia e-mail: lbmoncada@udistrital.edu.co R. Lücking Science & Education, Integrative Research Center & Collections Center, The Field Museum, 1400 South Lake Shore Drive, Chicago, IL 60605, USA e-mail: rlucking@fieldmuseum.org Trebouxiophyceae and the first report of lichenization for the genus Bracteacoccus in Chlorophyceae. Keywords Agaricales . Amylocorticiales . Atheliales . Auriculariales . BLAST . Boletales . Dictyonema . Diplosphaera . Gloeophyllales . Lichenomphalia . Marchandiomphalina . Multiclavula Introduction Lichenization is one of the most important mutualistic lifestyles in Fungi, both in terms of the number of described species and the degree to which the symbiotic structures have evolved unique morphologies (Ahmadjian 1993; Alexopoulos et al. 1996; Mueller et al. 2004; Cannon and Kirk 2007; Feuerer and Hawksworth 2007; Kirk et al. 2008; Lücking et al. 2009; Nelsen et al. 2009; Schoch et al. 2009; Kües and Fischer 2010; McLaughlin et al. 2001). Lichenization is unequally distributed across the Fungi, with over 99 % of lichenized species found in the Ascomycota and less than 1 % in the Basidiomycota (Feuerer and Hawksworth 2007; Lawrey et al. 2009; Lücking et al. 2009). The number of independently lichenized clades is, however, comparable in both phyla, with at least five separate clades in the Basidiomycota: Multiclavula , Marchandiomphalina , Lichenomphalia , Dictyonema s.lat., and Lepidostroma (Nelsen et al. 2009; Schoch et al. 2009; Hodkinson 2012; Lücking et al. 2013). Lepidostroma closely resembles Multiclavula in the clavarioid basidiomata and was for a long time included in that genus (Petersen 1967; Oberwinkler 1970), before it was placed in a separate genus on account of its squamulose thallus (Mägdefrau and Winkler 1967; Oberwinkler 1984, 2001, 2012). With the addition of two squamulose species 166 discovered in tropical Africa, this separation was not accepted and Multiclavula was again emended to include species with squamulose thalli (Fischer et al. 2007). However, molecular phylogenetic analyses eventually confirmed Lepidostroma as a distinct lineage, Lepidostromataceae , unrelated to Multiclavula (Cantharellales) and instead close to Atheliales in subclass Agaricomycetidae (Ertz et al. 2008). With the recent discovery of two further species (Hodkinson et al. 2012a; Sulzbacher et al. 2012), Lepidostroma currently includes five species. The most remarkable of these, in terms of thallus morphology, is L. caatingae, because its thallus is not distinctly squamulose like the other species, but crustosegranulose as in Multiclavula (Sulzbacher et al. 2012) and certain species of Lichenomphalia and Semiomphalina (Oberwinkler 1970, 1984, 2001, 2012). The morphological distinction between Lepidostroma and Multiclavula has therefore become difficult and, to correctly identify species (especially if they have yellow-orange basidiomata), sequence data are indispensable. The photobiont of Lepidostroma is chlorococcoid instead of coccomyxoid, as in Multiclavula (Oberwinkler 1984, 2012; Sulzbacher et al. 2012), but this is not an easily discernible character, which makes it difficult to safely distinguish species of both genera with otherwise similar morphology. Phylogenetic studies that accompanied these recent species descriptions (Hodkinson et al. 2012a; Sulzbacher et al. 2012) also suggested that Lepidostromataceae forms a unique lineage separate from other orders currently recognized in Agaricomycetidae and that the genus might represent several separate lineages correlated with thallus morphology. In this paper, we present an updated phylogeny of Lepidostromataceae which shows that the lineage is best recognized in a separate order, Lepidostromatales. In addition, we describe two new genera (Ertzia and Sulzbacheromyces, for Lepidostroma akagerae and L. caatingae, respectively) and one new species (Lepidostroma winklerianum , for material previously identified as Lepidostroma calocerum from Mexico). The photobionts of two of the three genera were also identified using molecular phylogenetic methods. Material and methods We assembled an alignment of nuLSU sequences downloaded from Genbank (Table 1) from a broad range of higher Basidiomycota, using a set of three species of Dacrymycetes as outgroup and 101 species of Agaricomycetes as the ingroup (Hibbett et al. 2007), including sequences of all known Lepidostroma species (Ertz et al. 2008; Hodkinson et al. 2012a; Sulzbacher et al. 2012). In addition, we assembled a second, 3-gene dataset of a smaller number of taxa, including the translation elongation factor 1 alpha 1 (TEF1) and the RNA polymerase II second largest subunit (RPB2) genes. For this purpose, BM generated new sequences for TEF1 Fungal Diversity (2014) 64:165–179 and RPB2 of four samples of Lepidostroma calocerum from Colombia (Table 2) and assembled a selection of 23 samples (including Lepidostroma) representing the orders Russulales (outgroup), Agaricales , Amylocorticiales , Atheliales , and Boletales, for which nuLSU, TEF1, and RPB2 were available for the same sample (Table 1). To identify the photobionts, BM generated sequences of the algal rbc L gene for two samples of Lepidostroma calocerum from Colombia and one sample of Sulzbacheromyces caatingae from Brazil and aligned these with 64 samples of Chlorophyta from Genbank (Table 3). PCR and sequencing methods, as well as primers, followed Matheny et al. (2007) and Nelsen et al. (2009). The dataset comprising the 104 nuLSU sequences was assembled into a multiple alignment using BIOEDIT 7.09 (Hall 1999) and automatically aligned with MAFFT using the--auto option (Katoh and Toh 2005). The unaligned dataset was also subjected to analysis of ambiguously aligned regions using the GUIDANCE webserver (Penn et al. 2010a, 2010b) and introns and regions aligned with low confidence were removed. This resulted in an alignment length of 1302 sites. The alignment was subjected to maximum likelihood (ML) search using RAxML 7.2.6 (Stamatakis et al. 2005; Stamatakis 2006), with parametric bootstrapping using 500 replicates under the GTRGAMMA model. The dataset was also analyzed under a Bayesian framework using MrBAYES 3.1.2 (Huelsenbeck and Ronquist 2001), with two independent runs and four chains per run, a chain length of 10 million generations, resampling every 1000 trees and generating a majority rule consensus tree from the tree sample after discarding 25 % burnin to obtain posterior probability estimates. Bayesian analyses were performed on the CIPRES Science Gateway webserver (Miller et al. 2010). The dataset comprising the 23 OTUs with nuLSU, TEF1, and RPB2 sequences was first analyzed separately to test for conflict and, since no supported conflict was found, combined and analyzed using maximum likelihood (ML) search using RAxML 7.2.6 (Stamatakis et al. 2005; Stamatakis 2006), with parametric bootstrapping using 500 replicates under the GTRGAMMA model, employing seven partitions for the nuLSU and each of the codon positions of the two proteincoding genes. The combined alignment had a length of 4010 bases. The photobiont dataset comprised 67 OTUs and had a length of 1251 bases; it was also analyzed using maximum likelihood under the same parameters as outlined above. We used the SH test implemented in RAxML 7.2.6 to test hypotheses about putative monophyly of accepted orders in Agaricomycetidae with inclusion of Lepidostromataceae : Agaricales, Amylocorticiales, Atheliales, and Boletales. We also performed BLAST searches with each Lepidostroma nuLSU sequence individually against the NCBI nr/nt database and recorded the best BLAST hits in order to evaluate how well a standard BLAST-based approach would predict phylogenetic placement. Fungal Diversity (2014) 64:165–179 Table 1 Genbank accession numbers for the single-gene nuLSU dataset used in this study 167 Taxon Accession Taxon Accession Acantholichen pannarioides Agaricus bisporus Albatrellus skamanius Amphinema byssoides Amphinema byssoides Amphinema diadema Amylocorticium subsulphureum Arrhenia auriscalpia Athelia arachnoidea Athelia arachnoidea Athelia decipiens Athelia epiphylla Athelia singularis Athelidium aurantiacum Athelopsis subinconspicua Auricularia auriculajudae Boletellus projectellus Bondarcevomyces taxi Byssocorticium efibulatum Byssocorticium pulchrum Byssocorticium pulchrum Byssoporia terrestris Calocera cornea Clavulina cristata Columnocystis abietina Cristinia rhenana Dacrymyces sp. Dendrocorticium roseocarneum Dictyonema glabratum Echinodontium tinctorium Eonema pyriforme Ertzia akagerae Ertzia akagerae Exidia uvapassa Exidiopsis calcea Fibulomyces mutabilis Fibulorhizoctonia sp. Fibulorhizoctonia sp. Fibulorhizoctonia sp. Fibulorhizoctonia sp. Fomitiporia mediterranea Galerina marginata Ganoderma applanatum Gautieria otthii Gloeocystidiellum porosum Gloeophyllum sepiarium Gomphidius roseus Guepiniopsis buccina Hydnellum caeruleum Hydnum albidum Hygrophoropsis aurantiaca Irpicodon pendulus EU825953 AY635775 AF393044 AY586626 GQ162810 GQ162811 GU187562 DQ071732 AF518601 GU187557 AY586632 AY586633 GQ162813 EU118606 AY586634 AF291289 AY684158 DQ534672 GQ162815 AY586639 GQ162816 DQ389664 AY701526 DQ284901 EU118619 GU187663 AY691892 AF393053 DQ917660 AF393056 EU118605 FJ171733 FJ171734 AY645056 AY293180 GQ162817 AY635779 FJ232044 FJ232045 FJ232046 AY684157 DQ457669 EU232274 AF336249 AF310100 HM536061 DQ534669 AY745711 EU522835 AY293186 AY684156 DQ144619 Junghuhnia subundata Kjeldsenia aureispora Kuehneromyces rostratus Laetisaria fuciformis Lepidostroma calocerum Lepidostroma rugaramae Lepidostroma rugaramae Lepidostroma vilgalysii Lepidostroma winklerianum Leptosporomyces galzinii Leptosporomyces raunkiaeri Lichenomphalia hudsoniana Lycoperdon perlatum Macrolepiota procera Marchandiomphalina foliacea Multiclavula corynoides Multiclavula mucida Multiclavula vernalis Paxillus vernalis Peniophora nuda Phallus impudicus Phellinus punctatus Piloderma byssinum Piloderma byssinum Piloderma byssinum Piloderma byssinum Piloderma byssinum Piloderma byssinum Piloderma lanatum Piloderma lanatum Piloderma olivaceum Piloderma olivaceum Piloderma olivaceum Plicaturopsis crispa Polyporoletus sublividus Polyporus squamosus Porphyrellus porphyrosporus Pterula echo Russula nigricans Sarcodon imbricatus Schizopora paradoxa Serpula himantioides Sistotrema eximum Stephanospora caroticolor Sulzbacheromyces caatingae Sulzbacheromyces caatingae Thelephora sp. Tricholoma vaccinum Tylospora asterophora Tylospora asterophora Veluticeps fimbriata Xerocomus chrysenteron AF518625 DQ218637 DQ457684 AY293192 FJ171737 FJ171735 FJ171736 JN698908 FJ171738 EU118642 GU187588 U66446 AF518630 U85275 AY542864 U66440 AY885163 U66439 AY645059 AF287880 AY152404 EF429230 AY586699 DQ469279 DQ469280 DQ469281 DQ469282 DQ469283 AY586700 DQ469288 DQ469289 DQ469290 DQ469291 AY293203 AF393066 AF393069 DQ534643 AY629315 JF834479 AF518646 AY059067 AJ440943 AF393076 AF518652 KC170318 KC170319 AF287890 AY207307 AF325323 AY463480 HM536083 AF071537 168 Table 2 Genbank accession numbers for the nuLSU, TEF1, and RPB2 3-gene dataset used in this study Fungal Diversity (2014) 64:165–179 Taxon Accession nuLSU Accession TEF1 Accession RPB2 Agrocybe praecox Amanita brunnescens Amylocorticium cebennense Amylocorticium subsulphureum Athelia arachnoidea Athelia epiphylla Aureoboletus thibetanus Boletellus projectellus Calostoma cinnabarinum Cantharocybe gruberi AY646101 AY631902 GU187561 GU187562 GU187557 GU187558 AY700189 AY684158 AY645054 DQ234540 DQ061276 AY881021 — GU187680 GU187672 GU187676 DQ029199 AY879116 AY879117 DQ059045 DQ385876 AY780936 GU187770 GU187773 GU187769 GU187771 DQ366279 AY787218 AY780939 DQ385879 Collybia tuberosa Coprinus comatus Entoloma prunuloides Lactarius deceptivus Lepidostroma calocerum(1) Lepidostroma calocerum(1) Lepidostroma calocerum(2) Lepidostroma calocerum(3) Lepidostroma calocerum(4) Lepista irina Leptosporomyces raunkiaeri Mythicomyces corneipes Rhodocollybia maculata Serpula himantioides AY639884 AY635772 AY700180 AY631899 FJ171737 — — — — DQ234538 GU187588 AY745707 AY639880 — AY881025 AY881026 DQ457633 AY885158 — KF774182 KF774183 KF774184 KF774185 DQ028591 GU187719 DQ029197 DQ061279 DQ059046 AY787219 AY780934 DQ385883 AY803749 — KF774186 KF774187 KF774188 KF774189 DQ385885 GU187791 DQ408110 AY787220 DQ366283 Results Maximum likelihood and Bayesian analyses showed no significant topological differences for the nuLSU dataset. The Bayesian majority rule consensus tree (with maximum likelihood bootstrap support values indicated) recovered the following major clades within Agaricomycetes with mostly good to strong support (Fig. 1): Auriculariales as sister to the remaining Agaricomycetes ; Cantharellales , including the lichenized genus Multiclavula , as sister (unsupported) to the remaining Agaricomycetes ; a number of order-level clades without backbone support comprising Phallales , Corticiales , including the lichenized genus Marchandiomphalina , Thelephorales, Gloeophyllales , Polyporales, Hymenochaetales, and Russulales, with several genera previously placed in Atheliales or Polyporales; and finally subclass Agaricomycetidae , including the orders Atheliales, Boletales, Amylocorticiales, and Agaricales s.lat. (unsupported), with the lichenized Lichenomphalia and Dictyonema s.lat. in a single, supported clade, as well as Lepidostromataceae, which falls outside of any of the other orders in Agaricomycetidae. The 3-gene dataset focusing on Voucher (for new sequences) Costa Rica, Lücking R05 Colombia, Lücking 35836a Colombia, Lücking 35836a Colombia, Tisnes 1a Colombia, Tisnes 1b Agaricales , Atheliales , Amylocorticiales , Boletales , and Lepidostromatales, recovered each order except for Agaricales with support (Fig. 2). However, overall support was lower compared to the single-gene nuLSU dataset, and backbone support was absent. Hence, the nuLSU dataset provided better resolution and support than the 3-gene dataset. The SH test on the nuLSU dataset showed that inclusion of Lepidostromataceae in either Agaricales, Amylocorticiales, Atheliales, or Boletales, cannot be rejected at either the 5 %, 2 %, or 1 % level (Table 4), therefore not providing conclusive evidence whether the family should be included in any of the accepted orders in subclass Agaricomycetidae . The same applied to the 3-gene dataset (not shown). However, the nuLSU topology demonstrates that Lepidostromataceae are outside a clade including Agaricales, Amylocorticiales, and Boletales, with support. The strong support for Atheliales as a separate clade in basal position does not allow for inclusion of Lepidostromataceae in that order either. Lepidostromataceae itself forms three strongly supported clades on long branches: the Lepidostroma akagerae group, the L. caatingae group, and Lepidostroma s.str. with the type species, L. calocerum , and the species L. vilgalysii and L. Fungal Diversity (2014) 64:165–179 Table 3 Genbank accession numbers for the rbcL photobiont dataset used in this study 169 Taxon Accession Taxon Accession Actinastrum hantzschii EF113405 KF754418 Acutodesmus obliquus Ankistrodesmus falcatus Ankyra starrii Aphanochaete magna Asterochloris phycobiontica Atractomorpha echinata Auxenochlorella protothecoides Bracteacoccus aerius Bracteacoccus cohaerens Bracteacoccus giganteus Bracteacoccus grandis Bracteacoccus medionucleatus Bracteacoccus minor Bracteacoccus pseudominor Bracteacoccus ruber KC810305 JQ394814 EF113409 EF113410 JF502538 EF113412 EU038285 JQ259860 JF717390 HQ246366 GQ985396 GQ985400 JQ259886 HQ246365 JQ259863 Unknown (L. calocerum, Colombia, Tisnes 1b) Heterochlorella luteoviridis Hydrodictyon reticulatum Kalinella bambusicola Leptochlorella corticola Meyerella planktonica Microthamnion kuetzingianum Monactinus simplex Monoraphidium circinale Muriella zofingiensis Myrmecia biatorellae Oedogonium nodulosum Oocystis apiculata Ourococcus multisporus Parachlorella kessleri Parapediastrum biradiatum HE984580 EF078307 HE984581 HE984583 AY543047 EF589152 EF078333 AB175934 HQ902940 AF499685 DQ481204 EF113459 JQ717306 AB260912 EF078303 Bulbochaete rectangularis var. hiloensis Chaetopeltis orbicularis Chaetophora lobata Characiopodium hindakii Chlamydomonas reinhardtii Chlorella vulgaris EF113415 EU380527 JQ394810 EF113418 AB511845 EF589154 Pediastrum integrum Prasiola crispa Pseudomuriella aurantiaca Pseudopediastrum boryanum Raphidonema nivale Rosenvingiella radicans EF078387 JQ669722 HM852438 EF078342 EF589151 AY694201 Chloromonas reticulata Closteriopsis acicularis Coccobotrys verrucariae Desmodesmus pseudoserratus Dictyococcus schumacherensis Diplosphaera mucosa Dunaliella bioculata Eudorina elegans Golenkinia minutissima Hariotina reticulata Bracteacoccus sp. (S. caatingae, Brazil, Sulzbacher 1479) Unknown (L. calocerum, Colombia, Lücking 35836c) AB022534 EF113433 AM260447 GU192421 HM852434 AM260444 AB127991 D88804 EF113445 JQ394815 KF754420 Scenedesmus bajacalifornicus Selenastrum capricornutum Sorastrum spinulosum Sphaeroplea robusta Stauridium privum Stichococcus spec Tetraselmis marina Trebouxia arboricola Uronema belkae Volvox aureus Yamagishiella unicocca HQ246355 EF113471 EF078311 EF113472 EF078392 EF589148 U30284 AM158960 EF113481 D63445 AB359065 rugaramae . The two specimens previously classified as Lepidostroma calocerum form a paraphyletic group, with one sample from Mexico and the other from Costa Rica, indicating that the two samples likely represent different species. The genetic difference between each of the three major clades is substantial, with a total of 74 parsimonyinformative sites (Fig. 3) out of 1302 (5.7 %) in the relatively conserved nuLSU locus. Lepidostromataceae nuLSU sequences showed highest BLAST-based similarity to sequences of Piloderma (Atheliales), with 92 % maximum identity (Table 5). Further KF754419 BLAST hits with 91–92 % maximum identity included Boreostereum, Chaetodermella, Columnocystis, Gloeophyllum, Neolentinus, and Veluticeps (all Gloeophyllales), Cortinarius, Pleurotus , Rhodocybe , and Termitomyces (Agaricales), Pseudomerulius and Tapinella (Boletales), Amylostereum and Gloecystidiellum (Russulales), Heliocybe , Laccocephalum , Piloporia , and Tyromyces (Polyporales), and Jaapia (Jaapiales). This is remarkable since Gloeophyllales , Polyporales, and Russulales, including the very sequences showing high BLAST-based similarity to Lepidostroma, fall outside of subclass Agaricomycetidae. Therefore, although 170 Fungal Diversity (2014) 64:165–179 Fig. 1 Maximum-likelihood tree of nuLSU sequences of selected Basidiomycota focusing on Atheliales and Lepidostroma s.lat. Supported branches for major and order-level clades and for genus-level clades within Lepidostromatales are indicated by thick lines and both bootstrap support values (above branches) and Bayesian posterior support (below branches) are given (only values of 70 % bootstrap support and larger and 0.95 posterior probability and larger are given). Order-level clades are highlighted in color to facilitate their distinction. The newly proposed genera for Lepidostromatales are also indicated BLAST search also recovered Agaricales, Atheliales, and Boletales as partial hits, the search results are not wellcorrelated with sequence phylogenies. The photobiont of Sulzbacheromyces caatingae is strongly supported as belonging to the genus Bracteacoccus in class Chlorophyceae (Fig. 4). This is the first report of this Fungal Diversity (2014) 64:165–179 171 Fig. 2 Maximum-likelihood tree of combined nuLSU, TEF1, and RPB2 sequences of selected Basidiomycota focusing on Agaricomycetidae. Supported branches for order-level clades are indicated by thick lines and bootstrap support values are given freshwater genus (Lewis 1997) as lichenized, underlining the uniqueness of the Lepidostromatales and its independent lichenization. According to Leliaert et al. (2012), this genus belongs in Sphaeropleales, but members of that order are here split into at least four lineages, with Sphaeroplea only distantly related to Bracteacoccus. The photobiont of Lepidostroma caatingae is an unidentified, possibly undescribed genus close to the order Prasiolales (Prasiola clade; Leliaert et al. 2012) in class Trebouxiophyceae (Fig. 4). The closest lichenized genus, although not directly related, appears to be Diplosphaera. Discussion Overall, the recovered nuLSU phylogeny for the higher Basidiomycota agrees well with previously published phylogenies and classifications (Hibbett et al. 2007; Kirk et al. 2008). The supported sister group relationship of Auriculariales with the remaining Agaricomycetes supports the basal position of this order (Weiss & Oberwinkler 2001; Hibbett et al. 2007). One order, Russulales, did not receive support but was nevertheless recovered as monophyletic. Albatrellaceae , previously placed in Polyporales , was confirmed in Russulales as found in other phylogenetic studies (Bruns et al. 1998; Hibbett and Binder 2002; Miller et al. 2006). The best-scoring ML topology suggests that Lepidodostromataceae is separate from the accepted orders in subclass Agaricomycetidae (Agaricales , Amylocorticiales , Atheliales, Boletales). The low backbone support in this area of the tree explains why inclusion of Lepidostromataceae in any of these orders cannot be rejected even at the 5 % level, but the same argument can be used to defend erecting a separate order for Lepidostromataceae, since also combining any of the existing orders does not result in significant rejection, even if each order is well-supported as a separate clade. The results of BLAST searches suggested either Atheliales or Gloeophyllales as closest relatives of Lepidostroma, for all species. However, while Atheliales forms part of the same subclass, Gloeophyllales falls outside Agaricomycetidae. It is also worth noting that a BLAST search performed on Sulzbacheromyces nuLSU did not return hits from any other members of Lepidostromataceae , and a BLAST search performed on Ertzia nuLSU did not return hits from Table 4 Results of the SH test implemented on the four classification constraints at ordinal level for Lepidostromataceae (nuLSU dataset) Constraint LH Unconstrained LH Constrained Difference LH 5% 2% 1% Agaricales +Lepidostromataceae Amylocorticiales +Lepidostromataceae Atheliales +Lepidostromataceae Boletales +Lepidostromataceae −17123.828480 −17123.828480 −17123.828480 −17123.828480 −17135.696587 −17127.756935 −17126.309605 −17124.796590 −11.868108±7.753102 −3.928455±9.575209 −2.481125±7.648238 −0.968110±10.359594 no no no no no no no no no no no no 172 Fungal Diversity (2014) 64:165–179 Fig. 3 Parsimony-informative sites for Lepidostromataceae extracted from the complete nuLSU alignment Sulzbacheromyces , although Sulzbacheromyces and Ertzia were the top two hits for the Lepidostroma query sequence. These BLAST results confirm previously expressed doubts on the algorithm’s utility in making taxonomic assignments for conserved rRNA sequences (e.g. Clémençon et al. 2004), as is increasingly done in next-generation sequencing studies (Margulies et al. 2005; Sogin et al. 2006; Rothberg and Leamon 2008; Buée et al. 2009; Amend et al. 2010; Lumini et al. 2010; Wallander et al. 2010; Nilsson et al. 2011; Dai et al. 2012; Hodkinson et al. 2012b). Since BLAST is based Table 5 Results from BLAST searches of Lepidostromataceae nuLSU sequences. Query sequences consisted of one representative of each genus in the family Lepidostromataceae. Hits for other members of Lepidostromataceae (excluding self-hits) and the ten best hits outside of the family are shown, considering only the single best hit for each genus Species Accession BLAST Hit ID Accession Order Max Idenity Ertzia akagerae FJ171733 Lepidostroma winklerianum FJ171738 Lepidostroma calocerum Piloderma fallax Boreostereum radiatum Tyromyces chioneus Chaetodermella luna Veluticeps abietina Columnocystis abietina Amylostereum areolatum Piloporia sajanensis Gloeophyllum sepiarium Gloeocystidiellum porosum Ertzia akagerae Sulzbacheromyces caatingae Piloderma fallax Cortinarius carbonellus FJ171737 GU187591 DQ679925 HQ659244 JN649328 HM536079 EU118619 AJ406498 HQ659239 JN649344 AF301101 FJ171733 KC170318 GU187591 GU233391 Lepidostromatales Atheliales Gloeophyllales Polyporales Gloeophyllales Gloeophyllales Gloeophyllales Russulales Polyporales Gloeophyllales Russulales Lepidostromatales Lepidostromatales Atheliales Agaricales 93 92 92 91 91 91 91 91 91 91 91 93 93 92 92 % % % % % % % % % % % % % % % Gloeocystidiellum porosum Pleurotus pulmonarius Rhodocybe spongiosa Termitomyces sp. Laccocephalum mylittae Jaapia argillacea Neolentinus kauffmanii AF310101 AY524787 GU384628 AB073537 EU716114 AF518624 HM536073 Russulales Agaricales Agaricales Agaricales Polyporales Jaapiales 92 91 91 91 91 91 % % % % % % Tapinella panuoides Piloderma byssinum Boreostereum radiatum Veluticeps berkeleyi Gloeophyllum sepiarium Neolentinus kauffmanii Termitomyces sp. Heliocybe sulcata Columnocystis abietina Pseudomerulius curtisii Tapinella panuoides GU187605 DQ469282 HM536050 HM536081 JN649344 HM536073 AB073516 HM536069 EU118619 GU187589 GU187605 Gloeophyllales Boletales Atheliales Gloeophyllales Gloeophyllales Gloeophyllales Gloeophyllales Agaricales Polyporales Gloeophyllales Boletales Boletales 91 91 92 92 92 91 91 91 91 91 91 91 % % % % % % % % % % % % Sulzbacheromyces caatingae KC170318 Fungal Diversity (2014) 64:165–179 173 Fig. 4 Maximum-likelihood tree of rbc L sequences of selected Chlorophyta showing position of the photobionts of Lepidostroma and Sulzbacheromyces. Supported branches are indicated by thick lines and bootstrap support values are given 174 purely on sequence similarity, sequences with equivalent scores might end up in different clades of a phylogenetic tree, as here shown. This suggests that taxonomic identifications based on BLAST can be quite unreliable; therefore, higherlevel taxonomic assignments reported in next-generation sequencing studies based on certain sequence identity threshold levels should be evaluated with great care. The new order Lepidostromatales, introduced below, is in several ways a unique and remarkable lineage within Basidiomycota. It is the only entirely lichenized lineage at the family and order level, with no close, non-lichenized relative (Ertz et al. 2008; Hodkinson et al. 2012a; Sulzbacher et al. 2012). All other lichenized Basidiomycota have closely related nonlichenized relatives (Diederich and Lawrey 2007; Nelsen et al. 2007; Lawrey et al. 2009; Dal-Forno et al. 2013; Lücking et al. 2013). It is also the oldest lichenized lineage within Basidiomycota, with an estimated stem node age of 165 mya and a crown node age of 73 mya, corresponding to the middle Jurassic for its origin and the late Cretaceous for its initial diversification. The crown node age is only slightly surpassed by that of Lichenomphalia, which has a crown node age of 110 mya, but both stem node and crown node age of Lepidostromatales are substantially higher than those of Multiclavula, with 98 mya versus 7 mya (Lücking et al. 2013). This uniqueness is also underlined by the unique photobionts found in two of the three genera, one possibly representing a new genus and the other, Bracteacoccus, for the first time reported lichenized (Lewis 1997; Leliaert et al. 2012). In our phylogenetic reconstruction, which is based on the relatively conserved nuLSU region, the internal branches representing the three major lineages within Lepidostromatales and Lepidostromataceae are longer than most branches leading to genus-level clades in other lineages, such as Cantharellales, Atheliales, and Agaricales. This correlates well with morphological characters, since each of the three lineages of Lepidostromatales has a distinct thallus type, being either granulose-crustose, microsquamulose without medullary tissue, or distinctly squamulose with a welldeveloped medulla (see below for further details). Therefore, these lineages are here accepted as separate genera, with the name Lepidostroma retained only for the lineage including L. calocerum. In addition, the genetic differences between the two samples previously labeled L. calocerum correlate with thallus features, and a new species name is therefore introduced below for the material from Mexico. The five separate lineages of lichenized Basidiomycota exhibit different levels of evolution of thallus morphology and anatomy. Whereas Multiclavula has uniformly crustose, undifferentiated thalli, both Lepidostromataceae and Lichenomphalia show a similar evolutionary trend from crustose to squamulose thalli in which the squamules are differentiated into cortex, photobiont layer, and medulla (Petersen 1967; Oberwinkler 1970, 1984, 2001, 2012; Fungal Diversity (2014) 64:165–179 Redhead et al. 2002; Fischer et al. 2007; Ertz et al. 2008; Lawrey et al. 2009; Hodkinson et al. 2012a; Sulzbacher et al. 2012). This correlates with the aforementioned estimates for divergence dates for these lineages: while Multiclavula diverged very recently in the late Miocene, both Lepidostromataceae and Lichenomphalia appear to have diverged much earlier in the late Cretaceous (Lücking et al. 2013). Taxonomic treatment Lepidostromatales Hodkinson & Lücking ordo nov. Mycobank 803215 Differing from Agaricales, Amylocorticiales, Atheliales, and Boletales in the clavarioid basidiomata combined with a lichenized basal thallus, and from Cantharellales in the lichenization with a chlorococcoid photobiont. Type: Lepidostromataceae Ertz, Eb. Fischer, Killmann, Sérus. & Lawrey [Lepidostroma Mägd. & S. Winkl.; Lepidostroma terricolens Mägd. & S. Winkl. = Lepidostroma calocerum (G. W. Martin) Oberw.]. Lichenized. Thallus crustose, microsquamulose or distinctly squamulose; photobiont chlorococcoid. Basidiomata clavarioid to club-shaped, unbranched or branched, pale yellow to orange or pink-salmon, with hymenial tissue covering the upper part of the basidiomes. Basidiomata internally composed of parallel, thin-walled, occasionally clamped hyphae forming a densely agglutinated central strand and a loosely organized ‘medullary’ tissue; cortex three-layered. Basidia with 2–4 sterigmata, thin-walled, hyaline. Sterile hymenophoral elements present, parallel, resembling sterile basidia, thinwalled, hyaline. Basidiospores ellipsoid-ovoid to lacryform or slightly reniform, thin-walled, hyaline, smooth. Lepidostromatales is here erected for the single family Lepidostromataceae. It is thus far the only entirely lichenized order in Basidiomycota. The order and family contain three genera, Ertzia, Lepidostroma, and Sulzbacheromyces, and six species (see below for new genus descriptions and key to genera and species). The order and family is known from tropical America and Africa, suggesting a Gondwanan distribution, but certainly species of this genus have been overlooked and might be present also in the eastern Paleotropics (southeastern Asia and Australia). The uniqueness of this clade and its independent lichenization is emphasized by the fact that the sequenced photobionts of two of the three genera are either phylogenetically unique (in Lepidostroma) or (in Sulzbacheromyces) belong to a genus of freshwater algae, Bracteacoccus (Lewis 1997; Leliaert et al. 2012), that has not yet been reported as lichenized. Ertzia Hodkinson & Lücking gen. nov. Mycobank 803216 Differing from Lepidostroma s.str. in the microsquamulose thallus forming contiguous glomerules with a cortex of Fungal Diversity (2014) 64:165–179 175 Fig. 5 Morphology and anatomy of thallus squamules of Lepidostroma winklerianum. a– b Fully grown and young squamules. c Section through squamule. d Photobiont cells. e Upper cortex in section view. f Upper cortex in surface view. Scale in A–B=1 mm, in C= 100 μm, in D–F=10 μm characteristic, jig-saw-puzzle-shaped cells and lacking a medulla. Type : Ertzia akagerae (Eb. Fisch, Ertz, Killmann & Sérus.) Hodkinson & Lücking comb. nov. [Mycobank 803221]; Multiclavula akagerae Eb. Fisch, Ertz, Killmann & Sérus. in Fischer et al., Bot. J. Linnean Soc. 155: 458 (2007); Lepidostroma akagerae (Eb. Fisch, Ertz, Killmann & Sérus.) Ertz, Eb. Fischer, Killmann, Sérus. & Lawrey in Ertz et al., Am. J. Bot. 95: 1553 (2008). Type specimen: Rwanda. Kibungo: Akagera National Park, foot of Mt. Mutumba, April 2005, Ertz et al. s.n. (holotype: BR; isotypes: KOBL, LG). Thallus microsquamulose, composed of distinct, contiguous glomerules with cortex formed by a layer of hyaline cells resembling the pieces of a jigsaw-puzzle. Photobiont not yet identified. Basidiomata clavarioid, unbranched to very rarely branched. Basidiospores ovoid. Other characters as in the family. It is a pleasure to name this new genus after Damien Ertz, for his important contributions to our knowledge of the evolution and classification of lichenized fungi. This new genus is here erected for the phylogenetically and morphologically distinct species, Ertzia akagerae . The distinctive feature of this species is the thallus being formed by contiguous glomerules with a cortex composed of cells resembling the pieces of a jigsaw-puzzle (Fischer et al. 2007; Ertz et al. 2008). This type of cortex is very similar to the hyphal sheath found around the cyanobacterial photobiont in the unrelated genera Cora , Corella , and Dictyonema in Agaricales (Parmasto 1978; Coppins and James 1979; Chaves et al. 2004; Lawrey et al. 2009; Yánez et al. 2012; Dal-Forno et al. 2013). However, as various phylogenetic studies including the one presented here show, these groups are only very distantly to Lepidostromatales. The thallus type found in the genus Ertzia is somewhat reminescent of the ‘Botrydina’ type found in Multiclavula and Semiomphalina and certain Lichenomphalia species (Oberwinkler 1970, 1984, 2001, 2012). However, the ‘Botrydina’ type has usually smaller granules and the hyphal sheath cells are isodiametricangular and not jigsaw-puzzle-shaped. 176 Sulzbacheromyces Hodkinson & Lücking gen. nov. Mycobank 803217 Differing from Lepidostroma s.str. in the entirely crustose, undifferentiated thallus lacking cortex and medullary structures. Type : Sulzbacheromyces caatingae (Sulzbacher & Lücking) Hodkinson & Lücking comb. nov. [Mycobank 803232]; Lepidostroma caatingae Sulzbacher & Lücking in Sulzbacher et al., Bryologist 115: 605 (2012). Type: Brazil. Piauí: Parque Nacional Serra das Confusões, 28 March 2011, Sulzbacher 235 (holotype: UFRN Fungos-1478; isotype: F). Thallus crustose, composed of minute, ecorticate granules. Photobiont a species of Bracteacoccus . Basidiomata clavarioid, unbranched. Basidiospores ellipsoid to slightly reniform. Other characters as in the family. We name this new genus after Marcelo Sulzbacher, a promising young Brazilian mycologist who discovered the type species (Sulzbacher et al. 2012). Sulzbacheromyces is characterized by its undifferentiated thallus lacking distinct cortical structures. In contrast to the ‘Botrydina’ type granules found in Multiclavula , Semiomphalina , and Lichenomphalia species, the hyphae associated with the minute granules do not form an enclosing layer. Thus, Sulzbacheromyces can be morphologically distinguished from Multiclavula by its chlorococcoid photobiont, the absence of an enclosing layer, and the more yellow-orange basidiomata (Sulzbacher et al. 2012). Lepidostroma winklerianum Hodkinson & Lücking spec. nov. Mycobank 803218 Differing from Lepidostroma vilgalysii in the smaller thallus squamules with single-layered upper cortex, and the 4-sterigmate basidia. Type : Mexico. Veracruz: Sierra Madre Oriental, just S of Xico above Coyopolán River; 19° 25′ N, 97° 01′ W, 1308 m; on roadside rocks and trees next to a coffee/banana plantation; 16 August 2007, Egan 18705 (holotype: OMA). Thallus distinctly squamulose, squamules 0.5–1.5(−2) mm diam., bright green when fresh, becoming dark green in the herbarium, their surface mottled with ligher areas resembling maculae, with raised, whitish margin (Fig. 5a–b). Photobiont a species of an unidentified, possibly undescribed genus near Diplosphaera in class Trebouxiophyceae. Rhizohyphae abundant, extending from the lower surface 1–3 mm under the squamules, composed of thin-walled, 3–5 μm thick, hyaline hyphae with clamps. Squamules in section 250– 350 μm thick thick, inversed, with upper and lower cortex, upper medullary layer, and basal photobiont layer (Fig. 5c–f); cortex single-layered, 7–10 μm thick, composed of rectangular cells (polygonal in surface view; Fig. 5e–f); medullary layer variably 100–200 μm thick, composed of loosely intricate, mostly anticlinal to irregular, 2–4 μm thick hyphae with scattered clamps embedded in a copious gelatinous matrix; photobiont layer variably 100–200 μm thick, with portions Fungal Diversity (2014) 64:165–179 forming pyramidal columns protruding up into medulla (actually forming ridges when seen in surface view); individual photobiont cells rounded to oval, 8–12×6–9 μm (Fig. 5d); lower cortex single-layered, 5–7 μm thick, composed of polygonal cells. Basidiomata club-shaped, unbranched, 8– 15 mm high, 0.4–0.7 mm diam., pale yellow to orange, with hymenium tissue covering the upper part. Basidiomata internally composed of parallel, thin-walled, clamped hyphae 3–4 μm diam., forming a densely agglutinated, orangecolored, central strand and a loosely organized ‘medullary’ tissue; cortex 20–30 μm thick, distinctly three-layered, composed of a thin, dense, brownish outer layer, a broader, hyaline middle layer leaving large interspaces, and a thin, dense, pale yellowish inner layer. Basidia 25–45×6–10 μm, subclavate to clavate, with 4 sterigmata, thin-walled, hyaline. Sterile hymenophoral elements present, parallel, resembling sterile basidia, basally 4–6 μm and apically 8–10 μm broad, thin-walled, hyaline. Basidiospores 7–12×3–5 μm, ellipsoid, thin-walled, hyaline, smooth, hilar appendix present, guttulate. This species is named after the late Sieghard Winkler, German ecologist, tropical biologist, and lichenologist, who first discovered the genus Lepidostroma together with Karl Mägdefrau (Mägdefrau and Winkler 1967). Lepidostroma winklerianum is very similar and closely related to L. vilgalysii (Hodkinson et al. 2012a). It differs in the smaller thallus squamules, the single-layered cortex, and the 4-sterigmate basidia. It is somehow intermediate between L. vilgalysii and L. rugaramae and its morphology and anatomy could be considered ancestral to that of the latter two species, whereas a multi-layered cortex (L. rugaramae) and 2-sterigmate basidia (L. vilgalysii) could be considered derived. The squamules of these species somewhat resemble the thallus surface of Marchantia liverworts. They could be compared to the ‘Coriscium’ thallus type found in certain species of Lichenomphalia (Oberwinkler 1970, 1984, 2001, 2012), but differ in the way the internal hyphae associated with the algal cells, lacking a distinct enclosing layer. When Mägdefrau and Winkler (1967) described L. terricolens (= L. calocerum) from Colombia, they mentioned a sample from Guatemala with larger squamules, which might represent either L. vilgalysii or L. winklerianum. We have not been able to locate that material. Key to genera and species 1a Thallus crustose, undifferentiated, lacking distinct cortical structures (Sulzbacheromyces); basidiomata clavarioid; Neotropics (northeastern Brazil).............................................. Sulzbacheromyces caatingae 1b Thallus microsquamulose to squamulose, with distinct cellular cortex; basidiomata clavarioid or clubshaped..............................................................................2 Fungal Diversity (2014) 64:165–179 2a Thallus microsquamulose, composed of contiguous glomerules with cortex formed by distinctly lobate, jigsaw-puzzle-shaped cells (Ertzia ), medulla absent; basidiomata clavarioid; tropical Africa (Rwanda)........................................Ertzia akagerae 2b Thallus distinctly squamulose, composed of scattered to dense, rounded to reniform squamules with cortex formed by polygonal or jigsaw-puzzle-shaped cells (Lepidostroma), medulla present; basidiomata clavarioid or club-shaped.................................................................3 3a Basidiomata clavarioid; squamules lacking raised margin and maculae; photobiont layer above medulla, more or less uniform, with scattered cells throughout medulla and in lower portion of squamules; photobiont cells lacking pyrenoids; Neotropics (Costa Rica, Colombia).......................Lepidostroma calocerum 3a Basidiomata club-shaped; squamules with conspicuous, slightly raised white margin, their surface maculate; photobiont layer below medulla, forming pyramidal columns protruding upwards; photobiont cells with pyrenoid(s).......................................................................4 4b Upper cortex with jigsaw-puzzle-shaped cells in surface view; tropical Africa (Rwanda)......................................... Lepidostroma rugaramae 4a Upper cortex with polygonal cells in surface view; Neotropics (Mexico)........................................................5 5a Squamules 1.5–3 mm diam.; upper cortex multi-layered; basidia 2-sterigmate...................Lepidostroma vilgalysii 5b Squamules 0.5–1.5(−2) mm diam.; upper cortex singlelayered; basidia 4-sterigmate............................................. Lepidostroma winklerianum Acknowledgments Funding was provided to BPH through an Explorers Club Diversa Award, a Duke University Graduate International Research Travel Award, an American Bryological & Lichenological Society Student Travel Award, and a Sigma Xi Grant-In-Aid of Research. Partial financial support for RL was provided by grant DEB 0841405 from the National Science Foundation to George Mason University: “Phylogenetic Diversity of Mycobionts and Photobionts in the Cyanolichen Genus Dictyonema, with Emphasis on the Neotropics and the Galapagos Islands” (PI: J. Lawrey; Co-PIs: R. 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