Skip to main content
×
Home

Information:

  • Access
  • Open access
  • Cited by 2
  • Cited by
    This article has been cited by the following publications. This list is generated based on data provided by CrossRef.

    Wijayawardene, Nalin N. Hyde, Kevin D. Rajeshkumar, Kunhiraman C. Hawksworth, David L. Madrid, Hugo Kirk, Paul M. Braun, Uwe Singh, Rajshree V. Crous, Pedro W. Kukwa, Martin Lücking, Robert Kurtzman, Cletus P. Yurkov, Andrey Haelewaters, Danny Aptroot, André Lumbsch, H. Thorsten Timdal, Einar Ertz, Damien Etayo, Javier Phillips, Alan J. L. Groenewald, Johannes Z. Papizadeh, Moslem Selbmann, Laura Dayarathne, Monika C. Weerakoon, Gothamie Jones, E. B. Gareth Suetrong, Satinee Tian, Qing Castañeda-Ruiz, Rafael F. Bahkali, Ali H. Pang, Ka-Lai Tanaka, Kazuaki Dai, Dong Qin Sakayaroj, Jariya Hujslová, Martina Lombard, Lorenzo Shenoy, Belle D. Suija, Ave Maharachchikumbura, Sajeewa S. N. Thambugala, Kasun M. Wanasinghe, Dhanushka N. Sharma, Bharati O. Gaikwad, Subhash Pandit, Gargee Zucconi, Laura Onofri, Silvano Egidi, Eleonora Raja, Huzefa A. Kodsueb, Rampai Cáceres, Marcela E. S. Pérez-Ortega, Sergio Fiuza, Patrícia O. Monteiro, Josiane Santana Vasilyeva, Larissa N. Shivas, Roger G. Prieto, Maria Wedin, Mats Olariaga, Ibai Lateef, Adebola Azeez Agrawal, Yamini Fazeli, Seyed Abolhassan Shahzadeh Amoozegar, Mohammad Ali Zhao, Guo Zhu Pfliegler, Walter P. Sharma, Gunjan Oset, Magdalena Abdel-Wahab, Mohamed A. Takamatsu, Susumu Bensch, Konstanze de Silva, Nimali Indeewari De Kesel, André Karunarathna, Anuruddha Boonmee, Saranyaphat Pfister, Donald H. Lu, Yong-Zhong Luo, Zong-Long Boonyuen, Nattawut Daranagama, Dinushani A. Senanayake, Indunil C. Jayasiri, Subashini C. Samarakoon, Milan C. Zeng, Xiang-Yu Doilom, Mingkwan Quijada, Luis Rampadarath, Sillma Heredia, Gabriela Dissanayake, Asha J. Jayawardana, Ruvishika S. Perera, Rekhani H. Tang, Li Zhou Phukhamsakda, Chayanard Hernández-Restrepo, Margarita Ma, Xiaoya Tibpromma, Saowaluck Gusmao, Luis F. P. Weerahewa, Darshani and Karunarathna, Samantha C. 2017. Notes for genera: Ascomycota . Fungal Diversity, Vol. 86, Issue. 1, p. 1.


    Lücking, Robert Hodkinson, Brendan P. and Leavitt, Steven D. 2016. The 2016 classification of lichenized fungi in the Ascomycota and Basidiomycota – Approaching one thousand genera. The Bryologist, Vol. 119, Issue. 4, p. 361.


    ×

Figures:

Actions:

      • Send article to Kindle

        To send this article to your Kindle, first ensure no-reply@cambridge.org is added to your Approved Personal Document E-mail List under your Personal Document Settings on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part of your Kindle email address below. Find out more about sending to your Kindle.

        Note you can select to send to either the @free.kindle.com or @kindle.com variations. ‘@free.kindle.com’ emails are free but can only be sent to your device when it is connected to wi-fi. ‘@kindle.com’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.

        Find out more about the Kindle Personal Document Service.

        Morphological, chemical and species delimitation analyses provide new taxonomic insights into two groups of Rinodina
        Available formats
        ×
        Send article to Dropbox

        To send this article to your Dropbox account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your Dropbox account. Find out more about sending content to Dropbox.

        Morphological, chemical and species delimitation analyses provide new taxonomic insights into two groups of Rinodina
        Available formats
        ×
        Send article to Google Drive

        To send this article to your Google Drive account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your Google Drive account. Find out more about sending content to Google Drive.

        Morphological, chemical and species delimitation analyses provide new taxonomic insights into two groups of Rinodina
        Available formats
        ×
Export citation

Abstract

Abstract

The genus Rinodina (Physciaceae), with approximately 300 species, has been subject to few phylogenetic studies. Consequently taxonomic hypotheses in Rinodina are largely reliant on phenotypic data, while hypotheses incorporating DNA dependent methods remain to be tested. Here we investigate Rinodina degeliana/R. subparieta and the Rinodina mniaraea group, which previously have not been subjected to comprehensive molecular and phenotypic studies. We conducted detailed morphological, anatomical, chemical, molecular phylogenetic and species delimitation studies including 24 newly sequenced specimens. We propose that Rinodina degeliana and R. subparieta are conspecific and that chemical morphs within the R. mniaraea group should be recognized as distinct species. We also propose the placement of the recently described genus Oxnerella in Physciaceae.

Introduction

Species classification and delimitation within the fungi received a strong impetus when molecular phylogenetic methods emerged (Blackwell 2011; Hibbett & Taylor 2013). Molecular methods have helped to solve several long-standing questions about higher-level taxonomic relationships among lichen-forming fungi (e.g. see James et al. 2006; Thell et al. 2012; Miadlikowska et al. 2014; Resl et al. 2015). However, applying molecular methods also creates new challenges and discussions are ongoing about how to crosslink phenotypic (including morphology, anatomy and secondary chemistry) information with DNA sequence data when drawing taxonomic conclusions such as the description of new lineages (Grube & Kroken 2000; Crespo & Lumbsch 2010; Hibbet & Taylor 2013).

The issue of reconciling molecular and phenotypic evidence to develop taxonomic hypotheses for individual species may be simplified to three different scenarios: 1) molecular and phenotypic evidence both support taxonomic conclusions; 2) taxonomic conclusions are supported by molecular but not phenotypic characters; and 3) phenotypic evidence supports taxonomic conclusions while molecular methods do not.

Not all characters may coincide with a taxonomic hypothesis. Nevertheless, a broad consensus exists that multiple independent lines of evidence in support of taxonomic conclusions should be the desired goal of every modern taxonomic study. Lichenologists, however, are often faced with the challenge of phylogenetic and phenotypic data being inconsistent (e.g. Baloch & Grube 2009; Altermann et al. 2014; Lücking et al. 2014; Singh et al. 2015). With more sequences becoming available, species boundaries traditionally circumscribed by phenotypic characters were challenged and it was proposed that extant taxa numbers were systematically underestimated when relying on phenotypic characters alone (Crespo & Lumbsch 2010; Lumbsch & Leavitt 2011). However, there are also cases where molecular methods have revealed that phenotypic differences might overestimate species diversity (Xanthoparmelia: Leavitt et al. 2011 a , b ; Bryoria section Implexae: Myllys et al. 2011; Velmala et al. 2014).

While phenotypic differences between species can be subtle (Spribille et al. 2014) they still represent a valuable source of information with which to formulate hypotheses on how to delimit species, especially in groups where phylogenetic data remain fragmentary. One such group, which has been subjected to rather few molecular phylogenetic studies, is the family Physciaceae (e.g. Lohtander et al. 2000, 2009; Wedin et al. 2000; Grube & Arup 2001; Helms et al. 2003; Crespo et al. 2004; Kaschik 2006; Nadyeina et al. 2010; Kondratyuk et al. 2014 a ). In this family, taxa are delimited mainly on the basis of secondary chemistry, asci and ascospores. The latter particularly have been emphasized as important and, consequently, ascospores are usually summarized into different ‘types’ (Poelt & Mayrhofer 1979; Mayrhofer & Moberg 2002; Sheard 2010). A thorough phylogenetic treatment of the Physciaceae including members of all known genera and sequences of multiple gene fragments to test these concepts is still missing. We focus here on two examples of closely related taxa with unclear taxonomy from the genus Rinodina with Physcia- and Physconia-type spores, and various secondary compounds. The combined analysis of phenotypic and molecular characters also allows us to provide integrated hypotheses of species taxonomy and nomenclatural status.

The first case was presented to us in the course of studying corticolous Rinodina specimens collected by GT and TT from Japan. Fertile, esorediate collections were found showing close morphological, anatomical and chemical similarities to the sorediate species Rinodina degeliana Coppins. Rinodina degeliana is scattered throughout the Northern Hemisphere. It occurs in Europe (Coppins 1983; Tønsberg 1992; Mayrhofer & Moberg 2002; Giavarini et al. 2009), North America (Sheard 2010; Lendemer et al. 2014) and Japan (GT, TT, J. W. Sheard, pers. obs.). The esorediate specimens were subsequently found to be phenotypically identical to the holotype of Rinodina subparieta (Nyl.) Zahlbr., a species considered to be endemic to Japan.

The treatment of otherwise identical sorediate or esorediate morphs has been discussed by Tønsberg (1992), and more recently by Brodo & Lendemer (2012), among others. For a full literature list and exhaustive discussions, please consult the references in these two papers. Suffice it to say that there is no consensus on how different morphs of lichen taxa should be recognized. Most instructive has been the molecular study of Tehler et al. (2009), of a single species aggregate within the genus Roccella where different taxa were found to be either variable or uniform with respect to their reproductive morphs. Therefore, it appears that all apparent pairs of fertile and vegetative morphs must be studied in detail in order to decide their taxonomic status. This leads us to question if the names Rinodina degeliana and R. subparieta should both continue to be recognized as distinct species distinguished by the presence or absence of soralia.

The second example is Rinodina mniaraea (Ach.) Körb., a terricolous taxon that has been studied in detail by Timpe (1990) and Ertl (2000). It is found in cold and moist alpine dwarf shrub and grassland habitats on bare soil, bryophytes and plant remnants (Mayrhofer & Moberg 2002; Sheard 2010). Within R. mniaraea several infraspecific taxa have been recognized, differing in chemistry (Mayrhofer & Moberg 2002). All these taxa regularly form apothecia with Lecanora-type asci, possess large Physcia-type ascospores and frequently contain variolaric acid. They have been accepted as varieties in a treatment of the genus Rinodina (Mayrhofer & Moberg 2002), although Sheard (2010) did not accept the use of varietal status. However, the names R. cinnamomea (Th. Fr.) Räsänen and R. mniaraeiza (Nyl.) Arnold already existed at the species rank for two of the varieties. How should these entities be referred to and which taxonomic rank is appropriate for them?

Here we present the results of a study using collections of R. degeliana and R. subparieta from multiple localities within their known Northern Hemisphere range and material of the three varieties of R. mniaraea from the European Alps and western North America. We performed detailed morphological, anatomical and chemical analyses of material from those species. Additionally, we reconstructed maximum likelihood and Bayesian phylogenies of numerous Rinodina taxa including R. degeliana, R. subparieta, R. mniaraea var. mniaraea, R. mniaraea var. mniaraeiza and R. mniaraea var. cinnamomea based on sequences from nuclear and mitochondrial gene fragments. We performed species delimitation methods using a Bayesian implementation of the General Mixed Yule Coalescence model (Reid & Carstens 2012) to find evidence for our two aims: 1) to examine species delimitation within these closely related sets of taxa in order to re-evaluate esorediate and sorediate morphs (in the case of R. degeliana/subparieta) and different chemotypes (R. mniaraea s. lat.), and 2) to decide on the status of the names R. degeliana, R. subparieta and the varieties of R. mniaraea s. lat.

Materials and Methods

SC, TS, GT, TT and James Lendemer provided fresh collections for molecular analysis. For Rinodina degeliana and R. subparieta, procedures for morphological and anatomical examination, and photography, follow those of Sheard et al. (2014). For R. mniaraea s. lat., we provide revised descriptions based on Timpe (1990). Spore measurements for R. subparieta/R. degeliana are quoted as the 25%–75% range around the median with the 5% and 95% outliers in brackets. For R. mniaraea s. lat the average spore size and the minimum and maximum range are given. Thin-layer chromatography was carried out according to the methods of Culberson & Kristinsson (1970) and Culberson (1972) with later modifications.

As a basis for our study we selected sequences from the dataset of Nadyeina et al. (2010) containing a broad range of Physciaceae species with different ascospore types and secondary metabolites. We were especially interested in the relationships of R. degeliana and R. subparieta with other Rinodina species possessing Physcia-type ascospores and the secondary compound atranorin. In addition to the ten samples from R. degeliana and two from R. subparieta, we also included eight specimens of R. mniaraea from its three varieties, all possessing Physcia-type ascospores and sometimes atranorin. Furthermore, we sequenced R. freyi H. Magn. and R. efflorescens Malme with Physcia-type spores, but both lacking atranorin. To complete our dataset we included samples of the previously unsequenced or rarely sequenced species R. trevisanii (Hepp) Körb. and Phaeorrhiza nimbosa (Fr.) H. Mayrhofer & Poelt, and also the hitherto unplaced Oxnerella safavidiorum S. Y. Kondr. et al. (Table 1).

Table 1 ID, origin, species and used loci of the present molecular studies. IDs indicate laboratory-tracking numbers and also correspond to numbers used in Fig. 1 and the main text. Newly published sequences generated in this study are indicated in bold

DNA isolation, PCR amplification and sequencing

Thallus squamules or apothecia were placed in 1·5 ml Eppendorf tubes, frozen at −80°C and ground to powder in a Retsch cell grinder. We directly applied lysis buffer to the sheared cells. DNA extraction followed the QIAmp DNA Investigator Kit protocol according to the manufacturer’s instructions. We eluted the nucleic acids in 50 µl elution buffer and used them undiluted for subsequent polymerase chain reactions (PCR). For each sample we sequenced the internal transcribed spacer regions 1 and 2 with the embedded 5.8S region of the ribosomal DNA (ITS) and the mitochondrial ribosomal small subunit (mtSSU). PCR was performed with PuReTaq Ready-To-Go PCR beads using primer pairs ITS1F/ITS4 (Gardes & Bruns 1993; White et al. 1990) and mtSSU1:mtSSU3R (Zoller et al. 1999) for ITS and mtSSU, respectively. PCR products were checked for the correct size on ethidium bromide-stained agarose gels and purified using the Omega E.Z.N.A Cycle Pure Kit according to the manufacturer’s instructions. Purified PCR products were sequenced by Microsynth (Vienna).

Alignment and phylogenetic reconstruction and tree selection

Raw sequences were subjected to manual quality control with BioEdit 7.2.5 (Hall 1999) and only high quality sequences were used for phylogenetic analyses. Sequences were aligned and concatenated using our in-house script pipeline (Resl et al. 2015; Resl 2015). We aligned sequences with MAFFT v.7 (Katoh & Standley 2013) employing the –genafpair algorithm. Alignments were checked manually with BioEdit 7.2.5 and only obvious errors (when MAFFT placed single nucleotides on either end of the alignments introducing long gaps) were corrected. These alignments were then used, without excluding any sites. Data matrices and trees are deposited at Treebase.org under study ID: 19205.

We created gene trees for the two gene fragments with RAxML v.8 (Stamatakis 2014). For each gene fragment we set RAxML to generate 500 fast bootstrap replicates. We partitioned the ITS dataset into the ITS1, 5.8S and ITS2 components and used PartitionFinder 1.1.1 (Lanfear et al. 2012) with a greedy search strategy under a BIC criterion to determine the optimal number of partitions and substitution models for RAxML. Accordingly, the dataset was divided into a 5.8S and ITS1/ITS2 partition with GTRGAMMA respectively. For the mtSSU dataset we used one partition and the GTRGAMMA substitution model. We identified topological conflicts among gene trees with compat.py (Kauff & Lutzoni 2002) based on a support threshold of 70. Conflict-free single locus datasets were combined in a concatenated RAxML analysis. Again we used PartitionFinder to identify the optimal number of partitions and substitution models. Accordingly we used two partitions (ITS1/ITS2/mtSSU and 5.8S) with a GTRGAMMA substitution model and generated 500 fast bootstrap replicates.

We also performed Bayesian phylogenetic reconstruction of the ITS dataset using BEAST 2.2.1 (Bouckaert et al. 2014). We determined the best site model and partitioning scheme for the ITS dataset (ITS1: GTR+G, 5.8S: TrNef+G, ITS2: GTR+G) with PartitionFinder 1.1.1 according to the BIC criterion and used a relaxed log-normal clock model for each partition. We linked the tree model and subjected it to a Yule tree prior. The MCMC chain was run for 107 generations and every 1000th tree was retained. Convergence of model parameters was assessed using Tracer 1.6 (Rambaut et al. 2014) and considered sufficient for ESS values larger than 200. After discarding the first 20% of trees from the posterior tree sample as burn-in, we created a maximum clade credibility tree with TreeAnnotator 2.2.1 included in the BEAST package and randomly selected 100 trees using bash scripts. The maximum clade credibility tree and the set of 100 random trees were used in all further analyses.

Species delimitation using General Mixed Yule Coalescence

We performed species delimitation with the statistical programming language R (R Development Core Team 2013) using the R package bGMYC (Reid & Carstens 2012). This package provides a Bayesian implementation of the general mixed Yule-coalescence model (Pons et al. 2006). Based on a given tree sample, bGMYC models the contribution of between-species divergence (Yule model component) and within-species coalescence events (Coalescent model component) (Pons et al. 2006), which give a hypothesis for species delimitation. To account for phylogenetic uncertainty in our dataset we performed all bGMYC analyses on a set of 100 randomly selected trees from the BEAST posterior tree sample (see above). We used the function bgmyc.multiphylo() to sample 50 000 steps with a burn-in of 40 000 and the thinning parameter set to 100 for each tree. We then created a co-assignment probability heat map as implemented in the bGMYC function spec.probmat(). To determine consensus partitions from the probability heat map, we employed a k-medoid clustering approach as described in Ortiz-Álvarez et al. (2015). We used the function pamk() in the R package fpc (Hennig 2013) to create groups from the bGMYC co-assignment matrix. Pamk() uses a partitioning around medoid (PAM) algorithm, which randomly selects data points from the co-assignment matrix as centres and clusters other data points around those centres according to their distance. The number of clusters is determined by optimum average silhouette width (Hennig 2013).

Results

Phylogenetic analyses

We obtained 40 sequences from 24 isolates. Together with the sequences downloaded from GenBank, our dataset consisted of 49 samples and a total of 84 sequences. All sequences used, including those newly published, are summarized in Table 1. Results from our maximum likelihood analyses are depicted in Fig. 1A–C. We identified one conflict between the ITS (Fig. 1B) and mtSSU (Fig. 1C) gene trees coming from specimen P280. As ITS is an important fungal barcode (Schoch et al. 2012) and our species delimitations were based on the ITS dataset, we excluded mtSSU sequences of these specimens from the concatenated phylogenetic analysis.

Fig. 1A A, maximum-likelihood phylogenetic hypothesis of the concatenated ITS and mtSSU dataset. Branch support is provided as bootstrap values. Branches with high support (bootstrap value ≥70) are indicated in bold.

Fig. 1B and C (continued). B, maximum-likelihood ITS gene tree. Branch support is provided as bootstrap values. Branches with high support (bootstrap value =≥70) are indicated in bold. C, maximum-likelihood mtSSU gene tree. Branch support is provided as bootstrap values. Branches with high support (bootstrap value =≥70) are indicated in bold. Scales show nucleotide substitutions per site.

The deepest split in our concatenated maximum likelihood tree (Fig. 1A) corresponded to the “Buellia” and “Physcia” groups already reported by Nadyeina et al. (2010). The “Buellia” group, here represented by Amandinea punctata (Hoffm.) Coppins & Scheid. and Buellia erubescens Arnold was well supported (100% bootstrap support (BS)). The “Physcia” group, which received high support (95% BS) was divided into two clades, hereafter referred to as “Physcia” clade 1 and 2 (Fig. 1A). “Physcia” clade 1 was only moderately supported (60% BS). It contained all samples of Rinodina mniaraea s. lat., which formed a well-supported monophyletic group (98% BS). All samples of the varieties R. mniaraea var. mniaraea and R. mniaraea var. cinnamomea, respectively, formed well-supported clades (>70% BS). The clade containing R. mniaraea var. mniaraeiza was supported with 65% BS. Another well-supported (98% BS) lower level clade of “Physcia” clade 1 consisted of Phaeophyscia ciliata (Hoffm.) Moberg, P. orbicularis (Neck.) Moberg, Rinodina bischoffii (Hepp) A. Massal. and Oxnerella safavidiorum. The latter was resolved as sister to Rinodina bischoffii with high support (82% BS). The third subclade of “Physcia” clade 1, containing Rinodina turfacea (Wahlenb.) Körb., Physconia muscigena (Ach.) Poelt, Phaeorrhiza nimbosa and Physconia distorta (With.) J. R. Laundon, received poor support (65% BS).

Physcia” clade 2 was well supported (87% BS) and contained all samples of R. degeliana and R. subparieta. They formed a monophyletic group (100% BS) with two subclades, both with high support (71% and 97% BS). Sister to the R. subparieta/degeliana clade we recovered R. capensis Hampe and R. confragosa (Ach.) Körb., which form a clade that was preceded by the divergence of Rinodina exigua (Ach.) Gray, all also containing atranorin.

Sister to the above group, the “Physcia” clade 2 also contained a moderately supported clade (69% BS) consisting of Rinodina milvina (Wahlenb.) Th. Fr., R. glauca Ropin, R. septentrionalis Malme, R. freyi, R. sophodes (Ach.) A. Massal., R. efflorescens Malme, R. olivaceobrunnea C. W. Dodge & G. E. Baker, R. orculata Poelt & M. Steiner, R. plana H. Magn, R. trevisanii (Hepp) Körb., Physcia aipolia (Ehrh. ex Humb.) Fürnr. and P. caesia (Hoffm.) Fürnr., all lacking atranorin except for the Physcia species.

The BEAST analyses of the ITS dataset led to stable parameter values (ESS>200) according to Tracer. We only present the BEAST maximum clade credibility tree here (Fig. 2), with posterior probability node support indicated by different colours.

Fig. 2 Phylogenetic hypothesis and species delimitation results of the ITS dataset according to the Bayesian General Mixed Yule-Coalesence model. The tree displayed is the maximum clade credibility tree from the BEAST analysis. Node support is provided as coloured circles. Probabilities of the bGMYC probability map are indicative for the chance that tips could be assigned to one species. The pamk clusters refer to grouping based on k-medoids. bGMYC and pamk clustering was based on a tree sample of 100 randomly selected trees from the BEAST posterior distribution of trees.

Species delimitation analysis

Based on the Bayesian mixed Yule-coalescent analysis for species delimitation (Reid & Carstens 2012), we detected significant levels of genetic structure in Rinodina degeliana and R. subparieta, as well as in R. mniaraea s. lat. The pamk clustering approach (see above) detected 21 clusters in the whole dataset. Rinodina degeliana and R. subparieta samples form a total of three clusters (cluster numbers 1–3; see Fig. 2). The two specimens of R. subparieta are assigned to cluster 1, together with three R. degeliana samples.

Rinodina mniaraea s. lat. also forms three clusters (cluster numbers 6–8; Fig. 2), corresponding to the chemotypes R. mniaraea var. mniaraea, R. mniaraea var. mniaraeiza and R. mniaraea var. cinnamomea.

Morphological investigation

We undertook an a posteriori examination of ascospore size and structure, and soredium size between the R. subparieta/R. degeliana clades (Fig. 2) distinguished by the molecular analysis. This was necessarily incomplete due to the absence of ascospores in non-fertile specimens.

The esorediate Japanese collections (P101, P235) had very well-developed thalli and, perhaps for this reason, ascospores were also fully developed (Physconia-type) and slightly longer ((20·5–)22·5–24·5(–25·5)×(10·5–)11·0–11·5(–12·5) µm, n=66, l/w ratio (1·8–)2·0–2·2(–2·3)) than those found in the type of R. subparieta ((19·5–)21·0–23·5(–24·5)×(10·0–)11·0–12·0(–13·0) µm, n=40, l/w ratio (1·7–)1·8–2·0). The Physconia-type internal ascospore structure, well-developed torus, and darkly pigmented and strongly ornamented ascospore walls are very similar in all these specimens. It should also be noted that Nylander (1900) measured even larger spores, up to 27 µm long, in the type. In addition, it is notable that the fully developed ascospores of the types of R. subparieta and R. degeliana are very similarly sized.

Fertile, sorediate Japanese specimens (representative in molecular study: P103) possessed ascospores that were on average smaller than those of esorediate specimens and apparently always belonged to the Physconia-type at maturity, measuring (17·0–)18·5–22·0(–23·5) × 10·0–11·0(–12·5) µm (n=55), l/w ratio (1·5–)1·7–2·0(–2·1). Immature ascospores have broad lumin canals (septal wall thickenings), some with additional apical wall thickening (Physcia-like, Fig. 3D). Young ascospores sometimes lose the apical thickening during further development and finally become constricted at the septum. Lumina are then uniformly rounded and thin walled (Milvina-like). Measurements for ascospores in specimens with incompletely developed, Physcia-type spores tended to be shorter and comparable with those given by Sheard (2010), (15·0–)18·5–19·5(–23·0)×(8·5–)10·0–11·0(–13·0) µm, for North America. The single fertile eastern North American specimen analyzed (Lendemer 43666; P229, Fig. 3A) had poorly formed apothecia with infrequent ascospores that were not measured since they were either too young or too old. Most of its apothecia had abnormally well-developed proper exciples, making them appear lecideine. However, the margins were thalline below, with typically deep cortices filled with atranorin crystals.

Fig. 3 Rinodina subparieta (A–G). Fertile sorediate morph (Lendemer 43666, GZU); B & C, fertile esorediate morph (Thor 28096); D, submature Physconia-type ascospores (arrow pointing to Physcia-like ascospore with narrow lumin canals and apical wall thickening) (Tønsberg 22502, BG); E, mature Physconia-type ascospores with broad lumin canals, no apical wall thickening and prominent tori at the septa in various optical sections (Tønsberg 22502, BG); F, labriform soralia arising from slightly upturned areole margins (Tønsberg 42540); G, sterile morph showing labriform soralia (Tønsberg 41921); H, R. mniaraea showing typical matt, rugose grey-brownish thallus. Scales: A–C, F & G=200 µm; D & E=10 µm; H=1 mm.

Alaskan specimens (P231, P232, P234, pamk cluster 3) were all sorediate and mostly sterile. The single fertile specimen (Tønsberg 42694; P231) analyzed from Alaska that yielded mature ascospores was anomalous in that part of the collection lacked atranorin (by TLC and lack of crystals in the cortex), the first such specimen recorded. Some ascospores were of the Physcia-type but many clearly trended towards the Physconia-type, although retaining some apical wall thickening at maturity. The small sample of ascospores measured from Alaskan material, (18·5–)22·0–24·0(–24·5) × (10·0–)10·5–12·5(–13·0) µm (n=19), l/w ratio (1·7–)1·8–2·1(–2·2), was comparable with the Japanese material cited above. All mature ascospores had a prominent torus but most had unusually light ornamentation. Another fertile, sorediate specimen from Alaska, which was not subject to molecular analysis, had normal chemistry with atranorin and zeorin present.

The Scandinavian and non-Alaskan western North American specimens (pamk cluster 2; Fig. 2) were sterile. However, ascospore sizes for Rinodina degeliana quoted by Mayrhofer & Moberg (2002) are comparable with those given above. The very complete type description (Coppins 1983) agrees with our observations of the holo- and isotype except that the ascospores, when fully developed, belong to the Physconia-type rather than the Dirinaria-type of the protologue, or the Physcia-type as reported by Mayrhofer & Moberg (2002) and Sheard (2010). Coppins (1983) reported ascospore measurements of 19–25×10–14 µm that were confirmed by our observations (20·5–25·0×10·5–13·0 µm, n=14) of the type specimens.

We found that specimens from all clades may possess soredia. Soredia were measured under the compound microscope to an accuracy of 5 µm but showed only a limited variation in size. Specimens from Norway and Washington (P102 and P233 respectively, pamk cluster 2) developed slightly smaller (median 25 µm diam.) soredia compared to 30 µm in other regions of the world ((20–)25–30(–40) µm, n=115). However, this small size difference is accommodated in the 25–75 of the above percentile range and is therefore not meaningful. Soralia develop marginally (somewhat labriform; Fig. 3F & G) on the areoles, and are of similar size in the different clades.

Concerning Rinodina mniaraea, Sheard (2010) noted that he searched in vain for morphological, anatomical and ascospore characters to separate the different chemotypes. Numerous collections by Imshaug (MSC) in the southern Rocky Mountains offered the opportunity to test ascospore sizes of different chemotypes from the same collections or from the same localities. Some significant differences were found but they were not consistent between the chemotypes. Therefore, Sheard (2010) was unable to recognize the chemical variants as being taxonomically distinct. Descriptions of the morphology of the taxa based on the chemical variants are found in the following section.

Taxonomy of Rinodina subparieta

Rinodina subparieta (Nyl.) Zahlbr.

MycoBank No.: MB816978

Cat. Lich. Univers. 7: 554 (1931).

Basionym: Lecanora subparieta Nyl., Acta Soc. Sci. Fenn. 26: 30 (1900); type: Japan, Itchigômé, 1879, Almquist (H-Nyl 28856––holotype!).

Rinodina degeliana Coppins, Lichenologist 15: 147 (1983); type: Sweden, Lule Lappmark, Kvikkjokk parish, south-east slope of Nammatj, 66°56'N, 17°42'E, alt. 520 m, on Salix bark in Picea woodlands, 1977, Coppins 6238 & Tibell (E––holotype!; UPS––isotype!).

(Fig. 3A–G)

Thallus epiphloeodal, thin, light grey, areoles to 0·50–0·80 mm wide, subangular or with minutely sublobate margins; areoles isolated around margin, rarely coalescing to become rimose-areolate; surface plane, sometimes becoming rugose, matt or somewhat shining; margin determinate; prothallus absent or very thin, sometimes black, visible between marginal areoles; soredia whitish (paler than thallus), 20–40 µm diam., arising from underside of slightly upturned areole margins to form labriform soralia (Fig. 3F & G), areoles sometimes dissolving into diffuse soralia, or soredia rarely absent.

Apothecia frequently absent, when present typically 1 per areole, becoming narrowly attached, numerous and often contiguous, to 0·50–0·90 mm diam. (Fig. 3A–C); discs dark brown or typically black, plane sometimes becoming convex, rarely half-globose; thalline margin 0·05–0·10 mm wide, entire (rarely sorediate in part), persistent or becoming excluded; excipular ring absent or visible when apothecia moistened, narrow and confluent. Thalline exciple 60–100 µm wide laterally; cortex 10–20 µm wide; sometimes with epinecral layer 5–10 µm wide; crystals in cortex (atranorin), sometimes in medulla (zeorin); cortical cells to 3·5–6·0 µm, not pigmented, mostly hidden by crystals; algal cells to 6·0–12·0 µm wide; thalline exciple expanding to 90–140 µm below, cortex 25–70 µm deep; proper exciple hyaline or very light brown, 5–10 µm wide laterally, expanding to 20–30 µm wide above; hypothecium hyaline to very light brown, 70–140 µm deep; hymenium (75–)90–120 µm high, not inspersed; paraphyses 2·0–2·5 µm wide, strongly conglutinate, apices 3·5–4·5 µm wide, brown capitate, immersed in dispersed pigment forming a red-brown epihymenium; asci 60–80×15–30 µm. Ascospores (rarely 4–)8 per ascus, type A development, Physcia- to Physconia-type (Fig. 3D & E), (18·0–)20·0–23·5(–25·5)× (10·0–)10·5–12·0(–13·0) µm (n=205), l/w ratio (1·6–)1·8–2·1(–2·2), thickened apical walls during early development and frequently arrested at this stage; torus present; walls mostly darkly pigmented, typically strongly ornamented (×1250).

Pycnidia rare, c. 0·05 mm diam., immersed in thallus, ostioles brown to black; pycnoconidia hyaline, bacilliform 3·5–4·5 ×1·0 µm.

Chemistry. Spot tests K+ yellow, C−, P+ bright lemon yellow (very rarely negative). Secondary metabolites: atranorin (very rarely absent) and zeorin. Cortical atranorin typically abundant as indicated by strength of P+ spot test.

Ecology. Corticolous, mainly on deciduous trees.

Notes. The revised description provided here is based on our studies of Rinodina degeliana from Europe (Coppins 1983; Tønsberg 1992; Mayrhofer & Moberg 2002), North America (Sheard 2010) (with the exception of the ascospore dimensions), the type of R. subparieta and fertile specimens from Japan collected by GT and TT. Rinodina subparieta is a variable species. In addition to having fertile sorediate, fertile esorediate (rare) and non-fertile sorediate morphs (the most frequent state), ascospore size and the development stage attained are also variable. Such ascospore variability is typical of many Rinodina species possessing vegetative diaspores (J. W. Sheard, pers. obs.).

The very detailed protologue of Rinodina degeliana (Coppins 1983) agrees with our observations of the types of that species. We found only one important difference: that fully developed ascospores belong to the Physconia-type, rather than the Dirinaria-type or Physcia-type as reported by Mayrhofer & Moberg (2002) and Sheard (2010), respectively. Sheard (2010) questioned the distinction between the Physcia- and Physconia-ascospore types, citing the many Rinodina species that have intermediate ascospore types. Rinodina subparieta is another such example, with specimens exhibiting the Physcia-spore type having arrested ascospore development, a very common feature of the species in Europe, North America and Scandinavia. The ascospore dimensions cited above are from the total of specimens examined in the present study.

The species is easily distinguished from other sorediate Rinodina species (such as R. efflorescens, R. griseosoralifera Coppins, R. sheardii Tønsberg and R. stictica Sheard & Tønsberg) by its typically light grey areoles with marginal, labriform soralia and whitish soredia, and chemically by the presence of abundant cortical atranorin (P+ strong lemon yellow) and zeorin in its medulla. Very rarely the thallus and the soredia may have a greenish tinge (e.g. specimen in Fig. 3A). We have noted two thalline morphotypes. Most common is the morph with a plane and somewhat shining (waxy) surface, the areoles more or less angular and 0·6–0·8 mm wide, to which the type specimens of R. degeliana belong. Very rarely such areoles coalesce to form a rimose-areolate thallus. The second morph, which may be more common in fertile specimens, has smaller areoles, rarely exceeding 0·5 mm in width, with a matt, slightly convex surface and minutely sublobate margins before breaking into soredia. It is notable that the esorediate form of R. subparieta has only been found at very high elevations on Honshu, Japan (1960–2550 m), whereas sorediate forms, widely distributed geographically in the Northern Hemisphere, are found at lower elevations, although at up to 1725 m on Honshu (J. W. Sheard, pers. obs.), and only at high elevations in the southern Appalachians (1502–1920 m, Lendemer 2528, 44770, 43873 NY).

Additional fertile, esorediate specimens examined. Japan: Honshu: Etchu Prov., Nakasinkawa-gun, 33 km ESE Toyama, Midagahara, along path to Tateyama crater 36°34'N, 137°34'E, alt. 1960–2000 m, subalpine open mixed deciduous Abies mariesii forest, on dead, standing, exposed Abies mariesii, 1994, Thor 12708 (UPS–duplicate in TNS, not seen); Kai Prov. (Yamanashi Pref.), Kofu City, along trail between Odarumi Pass and Mt. Kinpu (Kimpō), old-growth, subalpine forest dominated by Abies mariesii and Tsuga diversifolia, on Abies mariesii, alt. 2552 m, 35°52·163'N, 138°37·930'E (WGS84, ±10 m), 2012, Thor 27994 (UPS); Makioka-cho, Yamanashi City, Odarumi pass, near mountain hut, old-growth, subalpine forest dominated by Abies mariesii, on A. mariesii, alt. 2368 m, 35°52·171'N, 138°40·001'E (WGS84, ±100 m), 2012, Thor 28096 (UPS); on Sorbus sp., Thor 28120 (UPS).

Selected sorediate specimens examined. Japan: Hokkaido: Ishikari Prov., Kamikawa-gun, Kamikawa-cho, 0·5–1·5 km E of Obako Gorge tourist centre, along gravel road just N of Niseicharomappu-gawa stream, open and plane land with deciduous trees, on Abies sachalinensis, alt. 720 m, 43°42'N, 143°01'E, 1995, Thor 14624 (UPS); Kitami Prov., Esashi-gun, Esashi-cho, 2 km S of Honcho, just SW of road 238, plantation of Abies sachalinensis and Larix gmelinii var. japonica with scattered Quercus mongolica var. grosseserrata, on Larix, alt. 10–20 m, 44°55'N, 142°35'E, 1995, Thor 14251 (UPS); Roshiri Island, Oshidomari area, along the path from Rishiri-hokuroku campsite (2·5 km S of Sakae Town) to Mt. Pon (near the campsite), 45°13'N, 141°13'E, alt. 220 m, old-growth humid forest dominated by Abies sachalinensis, corticolous on Abies sachalinensis, 1995, Tønsberg 22289 (BG); Shari-gun, Shari-cho, Shiretoko National Park, 9 km NE of Utoro Village, along trail around Shiretoko-goko Lakes, 44°07'N, 145°05'E, alt. 260 m, old-growth, mixed deciduous/coniferous forest, on Betula, 1995, Tønsberg 22797 (BG); Kushiro Prov., Akkeshi-gun, Akkeshi-cho, 44 km E of Kushiro City, S Akkeshiko Lake, just S of road following coast, humid mixed Abies sachalinensis/deciduous forest, on Quercus mongolica var. grosseserrata, alt. 120 m, 42°60'N, 144°57'E, 1995, Thor 14486 (UPS); Hamanaka-cho, 55 km E of Kushiro City, 3 km E of Hichiripputo Lake, just S of road following coast, 43°02'N, 145°03'E, alt. 60 m, humid, mixed Abies sachalinensis/deciduous forest, corticolous on Sorbus, 1995, Tønsberg 22936b (BG); Nemuro Prov., Shiretoko Peninsula, Menashi-gun, Rausu-cho, just S of road 334 crossing Shiretoko Peninsula, 4 km WNW of Sakae City, Rausu hot spring, open deciduous forest, on Alnus sp., alt. 130 m, 44°02'N, 145°09'E, 1995, Thor 14406 (UPS); Teshio Prov., Rumoi-gun, Obira-cho, 21 km ENE of small town Obira at the coast, along the trail from parking area to Tengunotaki Waterfall, 44°04'N, 141°55'E, alt. 50–100 m, dense, old-growth, mixed deciduous forest with scattered Abies sachalinensis, corticolous on Betula, 1995, Tønsberg 21955 (BG); Tokachi Prov., Kato-gun, Kamishihoro-cho, 6 km S of Mikumi Tunnel through Mt. Mikumi-yama, just W of road 273, marsh with Abies sachalinensis/deciduous forest, on dead deciduous tree, alt. 680 m, 43°32'N, 143°09'E, 1995, Thor 14581 (UPS). Honshu: Bizen Prov. (Okayama Pref.), Maniwa-gun, Kawakami-mura, 1–2·3 km N of Hiruzen Research Institute, along road, on exposed tree at roadside, alt. 500–550 m, 35°18'N, 133°38'E, 1994, Thor 12212 (UPS); Etchu Prov. (Toyama Pref.), Nakashinkawa-gun (Nakaniikawa-gun), Tateyama-cho, 25 km ESE of Toyama, Bijodaira, path S of Bijodaira bus stop, mixed deciduous/Cryptomeria japonica forest, on old deciduous tree, alt. 960–980 m, 36°35'N, 137°28'E, 1994, Thor 12664 (UPS); Kai Prov. (Yamanashi Pref.), Makioka-cho, Yamanashi City, at road to Odarumi pass, open forest dominated by Larix, on Prunus sp., alt. 1726 m, 35°49·172'N, 138°38·963'E (WGS84, ±30 m), 2012, Thor 28169 (UPS); Shimotsuke Prov. (Tochigi Pref.), 20 km WNW of Nikko, 1 km S of Yumoto Village (36°48'N, 139°26'E), Yudaki Falls, on deciduous tree in open, mixed forest, alt. 1440–1480 m, 1994, Thor 12771 (UPS).

Taxonomy of Rinodina mniaraea s. lat.

Rinodina cinnamomea (Th. Fr.) Räsänen

Ann. Acad. Scient. Fenn. 34(4): 137 (1931).

Basionym: Rinodina mniaraea var. cinnamomea Th. Fr., Nova Acta Regiae Soc. Sci. Upsal. 3(3): 228 (1860); type: Norway, Finnmark, Syd-Varanger, Pasvig [Pasvik], 1857, Th. Fries (UPS––lectotype! designated by Mayrhofer & Moberg 2002: 72).

Rinodina mniaraea var. chrysopasta (Lettau) Zahlbr., Cat. Lich. Univ. 7: 533 (1931).—Rinodina mniaraea f. chrysopasta Lettau, Hedwigia 60: 123 (1918); type: Switzerland, Engadin, am Morteratschgletscher bei Pontresina zwischen Gletscherzunge und dem Gasthaus, 1900 m, 1912, Lettau (B––holotype!).

Thallus thick, brown, dark brown to copper brown, continuous, rugose, matt.

Apothecia abundant, broadly attached, 0·3–1·5 mm diam.; disc dark brown to black-brown, rarely pruinose, plane to convex to strongly convex; thalline exciple 30–80 µm wide laterally; cortex indistinct; epinecral layer frequent, 10–30 µm wide; hypothecium hyaline or light brown at the base, 20–200 µm deep, inspersed with oil droplets; hymenium 80–130 µm high; epihymenium red-brown to brown. Ascospores Physcia-type, 19·0–27·5–35·0×10·0–12·5–15·0 µm, average l/w ratio 2·2.

Pycnidia not observed.

Chemistry. Orange to yellow pigment especially in the lower part of the medulla reacting K+ purple to violet; chemotype A: Skyrin, ±variolaric acid, ±cinnamomea unknown (5/3/5), ±atranorin; chemotype B: 1-O-methylemodin or 8-O-methylemodin, ±variolaric acid, ±cinnamomea unknown, ±atranorin.

Ecology and distribution. On soil, bryophytes and decaying plants often above slightly calcareous and acid bedrock in arctic-alpine habitats. Widespread across the Northern Hemisphere, scattered in more southerly mountains such as the Rocky Mountains, northern Scotland, Alps, Carpathian Mountains, Balkan Peninsula, Anatolian Mountains, Caucasus, Tibet, Himalayas (Mayrhofer & Moberg 2002; Strasser et al. 2015).

Notes. This species is characterized by a yellow to orange medulla. Chemotype A possesses the anthraquinone skyrin and chemotype B the anthraquinone 1-O-methylemodin or 8-O-methylemodin. Additional chemical compounds that may occur in different amounts are atranorin, variolaric acid and cinnamomea unknown. Based partly on the occurrence of atranorin, Mayrhofer & Moberg (2002) distinguished a total of four chemotypes. The type specimens of R. mniaraea var. cinnamomea and R. mniaraea var. chrysopasta belong to chemotype A.

Selected specimens examined. France: Dept. Hautes-Alpes: Dauphine, Umgebung des Col du Lautaret, Epaulement central de Combeynot, 2200–2600 m, 1957, Poelt 878 (GZU).—Germany: Bavaria: Allgäuer Alpen, Höfats-Gufel, 2000 m, 1918, Lettau (B).—Montenegro/Kosovo: Prokletije: Hajla, summit of Hajla mountain, 2403 m, 2010, Mayrhofer 464 & Stešević (GZU).—Norway: Finnmark: Mageröy, Skarvaag, 1864, Th. Fries (UPS).—Switzerland: Graubünden: Val Fenga, Ritzenjoch, 2500 m, 1967, Poelt & Vězda (BRA).—China: Tibet: Prov. Xizang, Ningjing Shan Mts., 9 km W Markam (=Gartog), c. 4000 m, 1994, Obermayer 3798 (GZU).—Georgia: Dzhava: Kaukasus montes Rachinski khrebet, in vicinitate pagi Tschordi, in monte Tagverula, 2300–2600 m, 1989, Vašak (GZU).—Nepal: Mahalangur Himal: Khumbu, Höhe westlich über Gorak Shep, 5300–5400 m, 1962, Poelt L905 (M).—Pakistan: W-Himalaya: Kaghan Valley, above Saiful-Muluk-Lake, 4070 m, 1990, Schickhoff 102/f4 (GZU).—Turkey: Provinz Bursa: Ulu Dag, 20 km SE von Bursa, 2150 m, 1976, Kalb 16068 & Plöbst (WIS).

Rinodina mniaraea (Ach.) Körb.

Syst. Lich. Germ.: 126 (1855).

Basionym: Lecanoramniaroea” Ach., Syn. Lich.: 339 (1814); type: [Switzerland], Helvetia, Schleicher (H-ACH 1136 “91a”––lectotype! designated by Mayrhofer & Moberg 2002).

Rinodina mniaroeoides (Nyl.) H. Olivier, Mém. Soc. Sci. Nat. Cherbourg 37: 162 (1909).—Lecidea mniaroeoides Nyl., Flora 53: 36 (1870); type: [Russia], Lapponia Kemensis, Muonio, Olostunturi, 1867, Norrlin (H-NYL 10657—lectotype! here designated).

Rinodina amniocola (Ach.) Körb., Parerg. Lich.: 73 (1859).—Lecanora amniocola Ach., Syn. Lich.: 156 (1814); type: [Switzerland], Helvetia, Schleicher (H-ACH 1187––holotype!).

Rinodina turfacea var. nuda Körb., Parerg. Lich.: 72 (1859); type: [Switzerland], Auf der Erde zwischen Moosen bei St. Moritz, Hepp [Hepp: Die Flechten Europas Nr. 83] (G––lectotype! here designated).

Note. Lecanora amniocola was described in the same publication as Lecanora mniaroea, the latter in the appendix. The index was given after the appendix, meaning that it was published at the same time in one issue. We therefore retain the name mniaraea, which was used earlier in Rinodina.

(Fig. 3H)

Thallus usually well developed, grey-brown to brown, continuous, surface usually rugose, rarely developing raised verrucae, matt.

Apothecia broadly attached and often contiguous, 0·7–1·1 mm diam.; disc dark brown to black-brown, sometimes pruinose especially when young, often becoming convex; thalline exciple 70–100 µm wide laterally; cortex 5–10 µm wide; epinecral layer frequent, 5–10 µm wide; hypothecium hyaline or light brown in specimens with dark thalli, 80–250 µm deep, inspersed with oil droplets; hymenium 80–130 µm high; epihymenium red-brown. Ascospores Physcia-type 21·0–27·5–35·0×10·0–12·5–15·0 µm, average l/w ratio 2·2.

Pycnidia immersed in thallus; pycnoconidia bacilliform, 4·0−5·0×1·0 µm.

Chemistry. Spot tests all negative, secondary metabolite±variolaric acid.

Ecology and distribution. On soil, bryophytes and decaying plants above calcareous and acid bedrock in arctic-alpine habitats, rarely descending into the subalpine forest zone. Widespread across the Northern Hemisphere, scattered in more southerly mountains such as the Rocky Mountains, Sierra Nevada, Pyrenees, Alps, Sudeten, Carpathians, Balkan Peninsula, Caucasus, central Asia, Karakorum and Himalayas (Mayrhofer & Moberg 2002; Mayrhofer et al. 2005; Sheard 2010). The record from the South Shetland Islands (Antarctica) by Olech (1989) needs confirmation.

Note. Rinodina mniaraea is characterized by the lack of other secondary metabolites apart from variolaric acid.

Selected specimens examined. Germany: Allgäuer Alpen: Obermädli Alpe, Rehm (W). Bayrische Alpen: Chiemgau, Hochfelln und Hochgern, Britzelmayr (PRM).—Switzerland: Bern: Oberaaralp–Bärenegg, 2400 m, 1920, Frey 9075 (G). Graubünden: Val Fenga, ad latera montis Heidelberger Spitze, 2500 m, 1967, Poelt & Vězda (BRA). Wallis: Zermatt, Gornergrat, Hochtälligrat, 3130–3150 m, 1961, Frey 27168 (G).—Georgia: Caucasus Magnus: region montis Elbrus, ad latera austro-occidentalia montis Čeget, supra vallem torrentis Baksan, 2800 m, 1980, Vězda 20659 (PRM).—Italy: South Tyrol: Hohe Tauern, Venediger Gruppe, Rieserferner-Ahrn Natur Park, SE of Kasern, Röttal, glacier forefield of the Rötkees, 2345 m, 2013, Bilovitz, Nascimbene, Tutzer, Wallner & Mayrhofer (GZU).—Mongolia: Archangai Aimak: Changai, Tarbagatai, 5 km vom Solon-Got-Paß, 2400 m, 1983, Huneck (GZU).—Nepal: Zentral-Himalaya: Langtang-Gebiet, Yala, oberes Langtang, 4830 m, 1986, Miehe & Miehe 4595 (GZU).—Pakistan: Karakorum: Shinghai Gali, 4580 m, 1990, Miehe & Miehe 1190 (GZU).—Turkey: Provinz Bursa: Ulu Dag, 20 km SE von Bursa, 2150 m, 1976, Kalb 15879, 16061, 16063 & Plöbst (WIS).

Rinodina mniaraeiza (Nyl.) Arnold

Flora 53: 469 (1870).

Basionym: Lecanoramniaroeiza” Nyl., Flora 53: 33 (1870); type: [Finland], Tavastia australis, Häme, Padasjoki, 1866, Norrlin (H-NYL 28734––lectotype! designated by Mayrhofer & Moberg 2002).

Rinodina mniaraea f. biatorina (Nyl.) Arnold, Verh. Zool.-Bot. Ges. Wien 37: 132 (1887).—Lecanora turfacea var. biatorina Nyl., Lich. Scand.: 151 (1861); type: Norway, Sør-Trøndelag, Dovre, Schimper (H-NYL 28717––lectotype! here designated).

Rinodina hookeri (Fr.) Dalla Torre & Sarnthein, Flecht. Tirol : 206 (1902).—Parmelia hookeri sensu Fr. non Lichen hookeri Borr. ex Sm., E. Fries, Lich. Eur.: 94 (1831); type: [Switzerland], Helvetia, Schleicher (UPS––lectotype!, here designated).

Thallus light grey, rarely pale brown to brown, continuous, rugose and matt.

Apothecia immersed to broadly attached, 0·6–0·9 mm diam.; thalline exciple 35–70 µm wide laterally; cortex indistinct; disc dark brown to black-brown, without pruina, plane to convex; hypothecium 70–250 µm deep, hyaline or light brown to dark brown at the base, inspersed with oil droplets; hymenium 85–150 µm high; epihymenium light brown. Ascospores Physcia-type, 20·0–25·0–31·5×9·0–11·5–15·0 µm, average l/w ratio 2·15.

Pycnidia immersed; pycnoconidia bacilliform, 3·8–4·5 × 1·0 µm.

Chemistry. Spot test K+ yellow, secondary metabolites atranorin, ±variolaric acid.

Ecology and distribution. On soil, bryophytes and decaying plants above calcareous and more rarely acid bedrock in arctic-alpine habitats. Widespread across the Northern Hemisphere, scattered in more southerly mountains such as the Rocky Mountains, Northern Scotland, Alps, Carpathians, Balkan Peninsula, Anatolian mountains and Karakorum (Mayrhofer & Moberg 2002; Strasser et al. 2015).

Note. Rinodina mniaraeiza is characterized by the presence of atranorin and can be distinguished from R. mniaraea and R. cinnamomea by the lighter thalli and by slightly smaller ascospores.

Selected specimens examined. Austria: Niederösterreich: Lunz am See, Scheiblingstein, 1500–1600m, 1995, Türk 20285 (GZU).—Germany: Bavaria: Wettersteingebirge, Hänge nördlich der Alpspitze, c. 2000 m, 1966, Poelt 13457 (GZU).—Switzerland: Bern: Grimsel, without date and collector, (BP); Fribourg, Simmental, Kaiseregg, östlicher Vorgipfel ob Waloalp, 2050 m, 1926, Frey 10010 (G). Graubünden: Unterengadin, Nationalpark, Buffalora Alp, 2140 m, Frey 10037 (G). Obwalden: Frutt, in den Schratten, 1920 m, 1921, Frey 28202 (G).—Pakistan: Karakorum: Baltistan, Haramosh Range, Alm Pakora SE Ganto La, 3700–3900 m, 1991, Poelt K91-171 (GZU). NW-Himalaya: Eastern Dersai Plateau, 3950 m, 1991, Poelt K91-267 (GZU).—Turkey: Provinz Bursa: Ulu Dag, 20 km SE von Bursa, 2150 m, 1976, Kalb 16048 & Plöbst (WIS).

Discussion

In the present study we have investigated the status of Rinodina degeliana, R. subparieta and R. mniaraea s. lat. By evaluating evidence derived from molecular phylogenetic, chemical and anatomical studies we provide hypotheses about the taxonomy and updated nomenclature for these crustose lichen lineages.

The status of Rinodina subparieta and R. degeliana

We recovered samples of Rinodina degeliana and R. subparieta in two well-supported clades in our concatenated maximum likelihood phylogeny (Fig. 1A). According to pamk-clustering based on the bGMYC results (Fig. 2), the two Rinodina subparieta samples were assigned to cluster 1 together with samples of R. degeliana. However, species delimitation also recovered Rinodina degeliana in two additional clusters (pamk cluster 2–3; Fig. 2), indicating multiple previously undiscovered lineages in R. degeliana.

The only reliable morphological character to separate Rinodina degeliana and R. subparieta was the lack of soralia in R. subparieta. We took an a posteriori approach to identify other possible synapomorphies informed by our phylogenetic hypothesis. Our morphological investigations, however, did not reveal any reliable differences between the R. degeliana clades or between R. degeliana and R. subparieta.

Fertile specimens were rarely observed in the material investigated and occasionally they lacked soralia. Ascospores from this material are variable in size and structure, even when immature and overmature ascospores were excluded from measurement. Such variable ascospores are typical for Rinodina species that tend to rely on vegetative reproduction (J. W. Sheard, pers. obs.). Young, not fully pigmented ascospores have Physcia-type lumina and mature into darkly pigmented Physcia-type ascospores or into Physconia-type ascospores also with darkly pigmented walls, a prominent torus and with rather heavily ornamented walls, although the latter character is sometimes less well developed. Asynchronous ascospore development was also frequent among specimens with Physconia-type spores; rarely only four ascospores in the ascus reach maturity.

The frequent development of Physcia-type ascospores into Physconia-type would seem to confirm the opinion of Sheard (2010) that these ascospore types should be regarded as developmental phases of a single ascospore type. Both are accompanied by a distinctive red-brown epihymenium colour in all clades, as are other species possessing these ascospore types (Ropin & Mayrhofer 1993). This coloration is derived from a dispersed red-brown pigment in the epihymenial gelatin together with the dark brown or black capitate pigmentation of the terminal cells of the paraphyses.

Conclusively, R. degeliana and R. subparieta (and also the different R. degeliana clades) were indistinguishable in respect to morphology, anatomy and chemistry. However, molecular results indicate multiple, although in part poorly supported, clades and clusters (Figs. 1A & 2). This could imply multiple species-level lineages in Rinodina degeliana. The specimens of R. subparieta available to us were nested in one of the R. degeliana clades and form a cluster with R. degeliana ITS sequences in bGMYC. Our molecular results and the lack of phenotypic characters to separate the two species thus lead us to conclude that R. subparieta and R. degeliana are conspecific.

We recognize that our interpretation of this species is broad and also includes specimens assigned to possible species-level lineages containing only R. degeliana specimens (pamk clusters 2 & 3; Fig. 2). This may represent an oversimplification of the taxonomic status of these lineages. At this point, the formal introduction of new names for any of the three clades would have to be based on molecular evidence only. It is generally possible to describe species based mainly on clade affinity in phylogenetic trees (Leavitt et al. 2013 a ), however we refrain from doing so in the case of Rinodina degeliana/R. subparieta.

Another solution to this problem, although probably impossible, would be to sequence the type specimens of R. subparieta and R. degeliana, ascertain which names apply to individual clades and adjust taxonomy accordingly. Since R. subparieta is nested in a clade (Fig. 1A) and cluster (Fig. 2) with R. degeliana samples and we lack morphological, anatomical and chemical characters to distinguish these lineages, we have been conservative in our interpretation. Further studies, particularly in view of a possible geographical separation, may help reveal the evolutionary history of the clades. Until additional data are available, we propose to use the name R. subparieta, which takes priority over R. degeliana.

The status of R. mniaraea s. lat.

We recovered Rinodina mniaraea in three clades (Fig. 1A) and detected three potential species-level lineages in the pamk clustering analysis (Fig. 2). The three clusters correspond to the three previously recognized varieties Rinodina mniaraea var. cinnamomea, R. mniaraea var. mniaraeiza and R. mniaraea var. mniaraea. Searches for morphological and anatomical differences between these lineages have been unsuccessful (Sheard 2010). All form Lecanora-type asci with eight Physcia-type ascospores, (20·5–)24·5–25·5(–30·0)×(9·5–)11·5–12·5(–14·5) µm, that have thick apical walls and septal wall thickenings separating the usually unequal-sized cells (Sheard 2010).

Differences between R. mniaraea var. cinnamomea, R. mniaraea var. mniaraeiza and R. mniaraea var. mniaraea are restricted to secondary chemistry (Mayrhofer & Moberg 2002): var. mniaraea possesses either no secondary compounds or variolaric acid while var. mniaraeiza possesses atranorin and sometimes variolaric acid; var. cinnamomea produces the anthraquinones skyrin and 1-O-methylemodin or 8-O-methylemodin, sometimes with atranorin and variolaric acid or both, and one additional unknown.

Räsänen (1931) had already established R. cinnamomea (Th. Fr.) Räsänen and Arnold (1870) transferred Lecanoramniaroeiza” described by Nylander (1870) to Rinodina. It remains unclear if the lineages discovered by us are truly distinct species. We cannot rule out the alternative hypothesis that R. mniaraea varieties all belong to a single species. In the mtSSU gene tree (Fig. 1C) Rinodina mniaraeiza does not form a monophyletic group and we thus detected a topological conflict between gene trees. A topological conflict could indicate gene flow. However, at present the size of our molecular dataset of R. mniararea s. lat. prevents us from investigating this issue in detail. An alternative explanation could be recent speciation with still incomplete lineage sorting. Additional data are needed to resolve this issue. We failed to find anatomical or morphological characters to separate taxa in R. mniarea s. lat. in a situation similar to that of R. subparieta/R. degeliana. Secondary chemistry, however, is characteristic and corresponded to clade (Fig. 1A) and cluster (Fig. 2) affinity in all R. mniaraea s. lat. specimens in our dataset. This is similar to other studies where morphologically indistinguishable taxa were proposed to be chemically distinct species (Lendemer 2012; Leavitt et al. 2013 b ). Our consistent chemical, phylogenetic (but see mtSSU gene tree; Fig. 1C) and species delimitation results lead us to follow Räsänen and Arnold and propose that the varieties of R. mniaraea s. lat. should be recognized at the species level.

The placement of Oxnerella safavidiorum in Physciaceae

The monotypic genus Oxnerella was recently described from the Iranian province Esfahan (Kondratyuk et al. 2014 b ). Oxnerella safavidiorum possesses a crustose rusty brown thallus, lecanorine apothecia with a Biatora-type ascus and hyaline 1-septate ascospores (Kondratyuk et al. 2014 b ). Kondratyuk et al. (2014 b ) conducted a phylogenetic analysis based on ITS and mtSSU sequences but could not resolve the placement of Oxnerella. A possible explanation is that because Oxnerella possesses hyaline 1-septate ascospores, Kontratyuk et al. (2014b) assumed a position close to Lecania in the family Ramalinaceae.

Our phylogenetic analysis now resolves Oxnerella safavidiorum as sister to Rinodina bischoffii with high support (82% BS; Fig. 1A). This placement should be considered tentative as long as fresh material is unavailable to verify sequence identity and rule out contaminations of Oxnerella type material. The differences in ascus type and ascospores of Oxnerella and Rinodina are surprising. A verification of ascus and ascospore characters of Oxnerella also has to be postponed until additional material for study is available to us.

We thank Fernando Fernández-Mendoza for valuable discussions about the species delimitation analyses, Peter Kosnik for support with TLC, the curators of the herbaria B, BP, BRA, E, G, GZU, H, PRM, UPS and W, and Klaus Kalb for the loan of specimens. The herbarium of Klaus Kalb was recently deposited in WIS. We thank Peter Bilovitz for providing the image of R. mniaraea and James Lendemer for supplying a fresh collection for molecular analysis. Funding for this project comes from the Austrian Science Fund (FWF) in the projects P25078-B16 and P25237-B16. TT also thanks Bruce McCune for the invitation to do lichenological fieldwork in Alaska, the National Park Service (NPS), Southwest Alaska Network, Anchorage, for funding, and Amy Miller and James Walton (both NPS) for project coordination and for organizing and executing field logistics. We thank Jiří Malíček for bringing the Oxnerella problem to our attention. François Lutzoni and James Lendemer, and the anonymous reviewers, are thanked for valuable comments on the manuscript.

References

S. Altermann , S. D. Leavitt , T. Goward , M. P. Nelsen & H. T. Lumbsch (2014) How do you solve a problem like Letharia? A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPs from multilocus sequence data. PLoS ONE 9: e9755614.
F. Arnold (1870) Lichenologische Fragmente. X. Flora (Regensburg) 53: 465488.
E. Baloch & M. Grube (2009) Pronounced genetic diversity in tropical epiphyllous lichen fungi. Molecular Ecology 18: 21852197.
M. Blackwell (2011) The fungi: 1, 2, 3 ... 5.1 million species? American Journal of Botany 98: 426438.
R. Bouckaert , J. Heled , D. Kühnert , T. Vaughan , C.-H. Wu , D. Xie , M. A. Suchard , A. Rambaut & A. J. Drummond (2014) BEAST 2: a software platform for Bayesian evolutionary analysis. PLoS Computational Biology 10 (4): e10035376.
I. M. Brodo & J. C. Lendemer (2012) On the perplexing variability of reproductive modes in the genus Ochrolechia: notes on O. africana and O. arborea in eastern North America. Opuscula Philolichenum 11: 120134.
B. J. Coppins (1983) A new corticolous sorediate Rinodina from Swedish Lappland. Lichenologist 15: 146150.
A. Crespo & H. T. Lumbsch (2010) Cryptic species in lichen-forming fungi. IMA Fungus 1: 167170.
A. Crespo , O. Blanco , X. Llimona & Z. L. Ferencová (2004) Coscinocladium, an overlooked endemic and monotypic Mediterranean lichen genus of Physciaceae, reinstated by molecular phylogenetic analysis. Taxon 53: 405414.
C. F. Culberson (1972) Improved conditions and new data for the identification of lichen products by a standardized thin layer chromatographic method. Journal of Chromatography 72: 113125.
C. F. Culberson & H. Kristinsson (1970) A standardized method for the identification of lichen products. Journal of Chromatography 46: 8593.
R. Ertl (2000) Die Flechtengattung Rinodina in Zentralasien unter besonderer Berücksichtigung der muscicolen Arten. Diploma thesis, University of Graz.
M. Gardes & T. D. Bruns (1993) ITS primers with enhanced specificity for basidiomycetes – application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113118.
V. Giavarini , P. W. James & O. W. Purvis (2009) Rinodina (Ach.) Gray (1821). In The Lichens of Great Britain and Ireland (C. W. Smith, A. Aptroot, B. J. Coppins, A. Fletcher, O. L. Gilbert, P. W. James & P. A. Wolseley, eds): 812825. London: British Lichen Society.
M. Grube & U. Arup (2001) Molecular and morphological evolution in the Physciaceae (Lecanorales, lichenized Ascomycotina), with special emphasis on the genus Rinodina . Lichenologist 33: 6372.
M. Grube & S. Kroken (2000) Molecular approaches and the concept of species and species complexes in lichenized fungi. Mycological Research 104: 12841294.
T. A. Hall (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 9598.
G. Helms , T. Friedl & G. Rambold (2003) Phylogenetic relationships of the Physciaceae inferred from rDNA sequence data and selected phenotypic characters. Mycologia 95: 10781099.
C. Hennig (2013) fpc: flexible procedures for clustering. R package version 2.1-5. Available at: https://cran.r-project.org/web/packages/fpc/index.html
D. S. Hibbett & J. W. Taylor (2013) Fungal systematics: is a new age of enlightenment at hand? Nature Reviews Microbiology 11: 129133.
T. Y. James , F. Kauff , C. L. Schoch , P. B. Matheny , V. Hofstetter , C. J. Cox , G. Celio , C. Gueidan , E. Fraker , J. Miadlikowska et al. (2006) Reconstructing the early evolution of fungi using a six-gene phylogeny. Nature 443: 818822.
M. Kaschik (2006) Taxonomic studies on saxicolous species of the genus Rinodina (lichenized Ascomycetes, Physciaceae) in the Southern Hemisphere with emphasis in Australia and New Zealand. Bibliotheca Lichenologica 93: 1162.
K. Katoh & D. M. Standley (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution 30: 772780.
F. Kauff & F. Lutzoni (2002) Phylogeny of the Gyalectales and Ostropales (Ascomycota, Fungi): among and within order relationships based on nuclear ribosomal RNA small and large subunits. Molecular Phylogenetics and Evolution 25: 138156.
S. Y. Kondratyuk , L. Lőkös , J. A. Kim , M.-H. Joeng , A. Kondratiuk , S.-O. Ou & J.-S. Hur (2014 a) Kashiwadia gen. nov. (Physciaceae, lichen-forming Ascomycota), proved by phylogenetic analysis of eastern Asian Physciaceae . Acta Botanica Hungarica 56 (3–4): 369378.
S. Y. Kondratyuk , L. Lőkös , J. Kim , A. Kondratiuk , M. H. Jeong , B. Zarei-Darki & J. S. Hur (2014 b) Oxnerella safavidiorum gen. et spec. nov. (Lecanoromycetidae, Ascomycota) from Iran (Asia) proved by phylogenetic analysis. Acta Botanica Hungarica 56 (3–4): 379398.
R. Lanfear , B. Calcott , S. Y. W. Ho & S. Guindon (2012) PartitionFinder: combined selection of partitioning schemes and substitution models for phylogenetic analyses. Molecular Biology and Evolution 29: 16951701.
S. D. Leavitt , L. Johnson & L. L. St. Clair (2011 a) Species delimitation and evolution in morphologically and chemically diverse communities of the lichen-forming genus Xanthoparmelia (Parmeliaceae, Ascomycota) in western North America. American Journal of Botany 98: 175188.
S. D. Leavitt , L. A. Johnson , T. Goward & L. L. St. Clair (2011 b) Species delimitation in taxonomically difficult lichen-forming fungi: an example from morphologically and chemically diverse Xanthoparmelia (Parmeliaceae) in North America. Molecular Phylogenetics and Evolution 60: 317332.
S. D. Leavitt , F. Fernández-Mendoza , S. Pérez-Ortega , M. Sohrabi , P. Divakar , H. T. Lumbsch & L. L. St. Clair (2013 a) DNA barcode identification of lichen-forming fungal species in the Rhizoplaca melanophthalma species-complex (Lecanorales, Lecanoraceae), including five new species. MycoKeys 7: 122.
S. D. Leavitt , T. L. Esslinger , M. P. Nelsen & H. T. Lumbsch (2013 b) Further species diversity in Neotropical Oropogon (Lecanoromycetes: Parmeliaceae) in Central America. Lichenologist 45: 553564.
J. C. Lendemer (2012) A tale of two species: molecular data reveal the chemotypes of Lepraria normandinoides (Stereocaulaceae) to be two sympatric species. Journal of the Torrey Botanical Society 139: 118130.
J. C. Lendemer , E. A. Tripp & J. Sheard (2014) A review of Rinodina (Physciaceae) in Great Smoky Mountains National Park highlights the growing significance of this “island of biodiversity” in eastern North America. Bryologist 117: 259281.
K. Lohtander , M. Källersjö , R. Moberg & A. Tehler (2000) The family Physciaceae in Fennoscandia: phylogeny inferred from ITS sequences. Mycologia 92: 728735.
K. Lohtander , L. Myllys , M. Källersjö , R. Moberg , S. Stenroos & A. Tehler (2009) New entities in Physcia aipoliaP. caesia group (Physciaceae, Ascomycetes): an analysis based on mtSSU, ITS, group I intron and betatubulin sequences. Annales Botanici Fennici 46: 4353.
R. Lücking , M. Dal-Forno , M. Sikaroodi , P. M. Gillevet , F. Bungartz , B. Moncada , A. Yánez-Ayabaca , J. L. Chaves , L. F. Coca & J. D. Lawrey (2014) A single macrolichen constitutes hundreds of unrecognized species. Proceedings of the National Academy of Sciences of the United States of America 111: 1109111096.
H. T. Lumbsch & S. D. Leavitt (2011) Goodbye morphology? A paradigm shift in the delimitation of species in lichenized fungi. Fungal Diversity 50: 5972.
H. Mayrhofer & R. Moberg (2002) Rinodina . In Nordic Lichen Flora Volume 2. Physciaceae (T. Ahti, P. M. Jørgensen, H. Kristinsson, R. Moberg, U. Søchting & G. Thor, eds): 4169. Uddevalla: Nordic Lichen Society.
H. Mayrhofer , C. M. Denchew , D. Y. Stoykov & S. O. Nikolova (2005) Catalogue of the lichenized and lichenicolous fungi in Bulgaria. Mycologia Balcanica 2: 361.
J. Miadlikowska , F. Kauff , F. Högnabba , J. C. Oliver , K. Molnar , E. Fraker , E. Gaya , J. Hafellner , V. Hofstetter , C. Gueidan et al. (2014) A multigene phylogenetic synthesis for the class Lecanoromycetes (Ascomycota): 1307 fungi representing 1139 infrageneric taxa, 317 genera and 66 families. Molecular Phylogenetics and Evolution 79: 132168.
L. Myllys , S. Velmala , H. Holien , P. Halonen , L.-S. Wang & T. Goward (2011) Phylogeny of the genus Bryoria . Lichenologist 43: 617638.
O. Nadyeina , M. Grube & H. Mayrhofer (2010) A contribution to the taxonomy of the genus Rinodina (Physciaceae, lichenized Ascomycotina) using combined ITS and mtSSU rDNA data. Lichenologist 42: 521531.
W. Nylander (1870) Addenda nova ad lichenographiam Europaeam. Continuatio tertia decima. Flora (Regensburg) 53: 3338.
W. Nylander (1900) Lichenes Ceylonensis et additamentum ad lichens Japoniae. Opus posthumum. Acta Societatis Scientiarum Fennicae 26: 133.
M. Olech (1989) Lichens from the Admiralty Bay region, King George Island (South Shetland Islands, Antarctica). Acta Societatis Botanicorum Poloniae 58: 493512.
R. Ortiz-Álvarez , A. de los Ríos , F. Fernández-Mendoza , A. Torralba-Burrial & S. Pérez-Ortega (2015) Ecological specialization of two photobiont-specific maritime cyanolichen species of the genus Lichina . PLoS ONE 10: e0132718.
J. Poelt & H. Mayrhofer (1979) Studien über Ascosporen-Typen der Flechtengattung Rinodina . Beiheft zur Sydowia 8: 312331.
J. Pons , T. Barraclough , J. Gomez-Zurita , A. Cardoso , D. Duran , S. Hazell , S. Kamoun , W. D. Sumlin & A. P. Vogler (2006) Sequence-based species delimitation for the DNA taxonomy of undescribed insects. Systematic Biology 55: 595609.
R Development Core Team (2013) R: A Language and Environment for Statistical Computing, reference index version 3.0.2. R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org
A. Rambaut , M. A. Suchard , D. Xie & A. J. Drummond (2014) Tracer v1.6. Available at: http://beast.bio.ed.ac.uk/Tracer. Accessed 24 August 2015.
V. Räsänen (1931) Die Flechten Estlands. Mit einer Bestimmungstabelle der wichtigsten Nord- und Mitteleuropäischen Flechtenarten und Varietäten. I. Annales Academiae Scientiarum Fennicae 34 (4): 1163.
N. M. Reid & B. C. Carstens (2012) Phylogenetic estimation error can decrease the accuracy of species delimitation: a Bayesian implementation of the general mixed Yule-coalescent model. BMC Evolutionary Biology 12: 196.
P. Resl (2015) phylo-scripts: python scripts for phylogenetics, v0.1. Available at: http://github.com/reslp doi:10.5281/zenodo.15983
P. Resl , K. Schneider , M. Westberg , C. Printzen , Z. Palice , G. Thor , A. Fryday , H. Mayrhofer & T. Spribille (2015) Diagnostics for a troubled backbone: testing topological hypotheses of trapelioid lichenized fungi in a large-scale phylogeny of Ostropomycetidae (Lecanoromycetes). Fungal Diversity 73: 239258.
K. Ropin & H. Mayrhofer (1993) Zur Kenntis corticoler Arten der Gattung Rinodina (lichenisierte Ascomyceten) in den Ostalpen und angrenzenden Gebieten. Herzogia 9: 779835.
C. L. Schoch , K. A. Seifert , S. Huhndorf , V. Robert , J. L. Spouge , C. A. Levesque & W. Chen , Fungal Barcoding Consortium (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences of the United States of America 109: 62416246.
J. W. Sheard (2010) The Lichen Genus Rinodina (Ach.) Gray (Lecanoromycetidae, Physciaceae ) in North America, North of Mexico. Ottawa: NRC Research Press.
J. W. Sheard , B. McCune & T. Tønsberg (2014) A new corticolous species of Rinodina (Physciaceae) and two interesting range extensions for species collected in Katmai National Park, Alaska. Bryologist 117: 253258.
G. Singh , F. Dal Grande , P. K. Divakar , J. Otte , S. D. Leavitt , K. Szczepanska , A. Crespo , V. J. Rico , A. Aptroot , M. E. S. Cáceres et al. (2015) Coalescent-based species delimitation approach uncovers high cryptic diversity in the cosmopolitan lichen-forming fungal genus Protoparmelia (Lecanorales, Ascomycota). PLoS ONE 10: e0124625.
T. Spribille , P. Resl , T. Ahti , S. Pérez-Ortega , T. Tønsberg , H. Mayrhofer & H. T. Lumbsch (2014) Molecular systematics of the wood-inhabiting, lichen-forming genus Xylographa (Baeomycetales, Ostropomycetidae) with eight new species. Symbolae Botanicae Upsalienses 37 (1): 193.
A. Stamatakis (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30: 13121313.
E. A. Strasser , J. Hafellner , D. Stešević , F. Geci & H. Mayrhofer (2015) Lichenized and lichenicolous fungi from the Albanian Alps (Kosovo, Montenegro). Herzogia 28: 520544.
A. Tehler , M. Irestedt , F. Bungartz & M. Wedin (2009) Evolution and reproduction modes in the Roccella galapagoensis aggregate (Roccellaceae, Arthoniales). Taxon 58: 438456.
A. Thell , A. Crespo , P. K. Divakar , I. Kärnefelt , S. D. Leavitt , H. T. Lumbsch & M. R. D. Seaward (2012) A review of the lichen family Parmeliaceae – history, phylogeny and current taxonomy. Nordic Journal of Botany 30: 641664.
E. B. Timpe (1990) Untersuchungen an muscicolen, arktisch-alpin verbreiteten Arten der Gattung Rinodina (Physciaceae, Lichenes). Diploma thesis, University of Graz.
T. Tønsberg (1992) The sorediate and isidiate, corticolous, crustose lichens in Norway. Sommerfeltia 14: 1331.
S. Velmala , L. Myllys , T. Goward , H. Holien & P. Halonen (2014) Taxonomy of Bryoria section Implexae (Parmeliaceae, Lecanoromycetes) in North America and Europe, based on chemical, morphological and molecular data. Annales Botanici Fennici 51 (6): 345371.
M. Wedin , H. Döring , A. Nordin & L. Tibell (2000) Small subunit rDNA phylogeny shows the lichen families Caliciaceae and Physciaceae (Lecanorales, Ascomycotina) to form a monophyletic group. Canadian Journal of Botany 78: 246254.
T. J. White , T. Bruns , S. Lee & J. Taylor (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications (M. A. Innis, D. H. Gelfand, J. J. Sninsky & T. J. White, eds): 315322. New York: Academic Press.
S. Zoller , C. Scheidegger & C. Sperisen (1999) PCR primers for the amplification of mitochondrial small subunit ribosomal DNA of lichen-forming ascomycetes. Lichenologist 31: 511516.