Orbilia beltraniae, a new succulenticolous species from the Canary Islands

Orbilia beltraniae is a new succulenticolous species from the Canary Islands associated with Euphorbia scrubs. Phylogenetic analyses based on rDNA sequences of ITS and partial LSU were conducted to determine the relationships of the new species to others in the genus. Macroand micromorphological, and ecology data are provided, as well as discussion in respect to closely related species. Orbilia beltraniae belongs to a strongly supported clade that includes non-nematophagous species of section Arthrobotrys, and its closest relatives are the European species O. rectispora and O. cotoneastri.


Introduction
Orbilia is by far the most specious genus of the Orbiliomycetes (Baral 2015, Baral in Jaklitsch et al. 2016). In the past its diversity was overlooked, mainly for the lack of exploration in drylands, as well as due to the morpho-taxonomical methods used (Baral 1992(Baral , 2015. Species occur in most ecosystems (from humid to arid, from subarctic to tropical) and most types of substrate (wood and bark, leaves, dung) and exposure (xeric, hygric, aquatic). In a monograph of Orbiliomycetes, more than 400 species are recognized in the genus (Baral 2015, Baral et al. in prep.).
Investigations on desiccation-tolerant fungi in their natural habitats are rare, although drylands occur on every continent and cover approximately 40% of the world's land area (UN 2011). Usually, such dry ecosystems have a low number of species, but a high amount of endemism (Lacoste and Salanon 1981, UN 2011, Davies et al. 2012. The studies done by E. Beltrán-Tejera and collaborators in drylands of Macaronesia have increased the knowledge of several groups, for example: (1) Basidiomycota with two new species and 23 new records, (2) Ascomycota with four new species and 10 new reports, and (3) Myxomycota with four new species and three new records (Beltrán-Tejera and Rodríguez-Armas 1999, Lado et al. 1999, Mosquera et al. 2000a, 2000b, 2003, Lado et al. 2007, Telleria et al. 2008, Beltrán-Tejera et al. 2010, Telleria et al. 2012, Quijada et al. 2012, Beltrán-Tejera et al. 2013, Quijada et al. 2014, 2015a, 2015b, 2015c. Euphorbia scrubs represents the native vegetation of drylands at lower elevations in the Canary Islands. These scrub lands are mainly composed of succulent plants (Aeonium Webb & Berthel, Ceropegia L., Euphorbia L., and Kleinia Mill) accompanied by other woody plants (Artemisia L., Periploca L., Rubia L.), with a high number of endemic species (>50%) (Del Arco et al. 2010). Since 2012, three new succulenticolous species of Orbilia have been published from this ecosystem and this type of substrate: Orbilia asomatica Baral, Quijada & Beltrán-Tej., O. pisciformis Baral, Quijada &Beltrán-Tej. andO. succulenticola Quijada, Baral &Beltrán-Tej. (Quijada et al. 2012, 2014). The aim of this investigation is to describe a new species of Orbilia that develops on the succulent remains of Euphorbia canariensis.

Methods
Specimens were collected in Tenerife (Canary Islands, Spain) during 2008-2014. Ten localities of Euphorbia scrubs were monitored in both the rainy season (September to May) and dry season (June to August), along an altitudinal transect (40-350 m) including both northern and southern slopes (Fig. 1). The sampling was restricted to the largest branches lying on the ground. Species of the following native succulent genera were sampled: Aeonium, Ceropegia, Euphorbia, and Kleinia.

Macro-and microscopic studies
Observations were made with a Motic stereomicroscope SMZ140, and with a Motic B1 light microscope. Microphotographs were taken with an USB Moticam 2500 camera and processed with the software Motic images Plus 2.0. Specimens were studied in both the living and dead state. Collection data and measurements followed methods of Quijada et al. (2012Quijada et al. ( , 2014. Cell walls were sometimes contrasted with Congo Red Figure 1. Monitored localities in Euphorbia scrubs: number of locality, place name for locality according to IDECanarias visor 3.0 (http://visor.grafcan.es/visorweb/), latitude, longitude, and altitude. In yellow the localities where Orbilia beltraniae was found.

DNA extraction, PCR amplification, and DNA sequencing
For complete details about DNA extraction, PCR amplification, PCR purification, and cycle sequencing see Baral et al. (2017b). Sequences were obtained of the complete Table 1. Specimens used in this study with voucher information and GenBank accession numbers. Orbilia beltraniae sequences are indicated in bold. Species with asterisk have at present unpublished names (Baral et al. 2017a).

Phylogenetic results
The alignment consisted of 943 bp characters, of which 322 were parsimony-informative, 415 were variable, and 528 were constant. Only the Bayesian consensus tree is shown (Fig. 2) because overall topologies of the ML and BI analyses were identical.

Discussion
Section Arthrobotrys will be proposed in the monograph of Orbiliomycetes (Baral et al. in prep.) as a subgroup of Orbilia to accomodate species with narrowly sickle-shaped, rod-shaped, or ellipsoid ascospores, desiccation-tolerant or -sensitive apothecia, and asexual states that either form various types of organs for trapping nematodes or other invertebrates or do not form trapping devices in culture when nematodes are added. Orbilia beltraniae is phylogenetically close to O. cotoneastri and O. rectispora, which belong to the group of section Arthrobotrys in which trapping organs are not formed, and that are currently referred to the asexual state genus Dactylella Grove. Orbilia cotoneastri has ascospores and spore bodies very similar to those of O. beltraniae, but differ in paraphysis morphology, showing uninflated or rarely capitateclavate apices; in O. beltraniae they are uninflated to medium lanceolate-lageniform with rounded tips. Orbilia rectispora has longer ascospores than O. beltraniae [up to 9.5(11) µm vs. up to 6.5(7) µm]. In addition, the three species have a very different ecology: O. beltraniae occurs on succulent substrates in semiarid places of the Canary Islands, O. cotoneastri occurs on wood, bark, and herbaceous stems in moist places of Europe, and O. rectispora occurs on leaves of monocots in wet places of Europe (Baral et al. in prep).
Orbilia cardui is phylogenetically more distantly related to the above species, but more similar to it in size of asci, ascospores, and spore bodies. Also here, the paraphyses shape distinguishes O. beltraniae from O. cardui which has slighty capitate paraphyses. Orbilia cardui has a wide ecological spectrum in Europe, growing in shady forests and open ruderal places or wetlands on wood, bark, herbaceous stems, and even on other fungi (Baral et al. in prep), whereas O. beltraniae is apparently restricted to wood of the Canarian endemic succulent Euphorbia canariensis.
Orbilia beltraniae is the fourth species so far found exclusively in the Euphorbia scrubs of the Canary Islands. Although it seems to be host specific on Euphorbia canariensis, the three previously described species (Orbilia asomatica, O. pisciformis, O. succulenticola) share this substrate but they also develop on other succulent species like E. balsamifera, E. lamarckii, and E. atropurpurea (Quijada et al. 2012E. lamarckii, and E. atropurpurea (Quijada et al. , 2014E. lamarckii, and E. atropurpurea (Quijada et al. , 2015c. Akaike H (1974)

Introduction
The lichen genus Graphina Müll. Arg. entailed an artificial concept of ascospore-based genera in Graphidaceae Dumort., including all graphidoid species with muriform, hyaline ascospores (Müller 1880). Based on phenotypic and molecular studies, a new classification of genera within the family was recently established (Staiger 2002;Rivas Plata et al. 2012). As a result, the genus Graphina was placed in synonymy with Thalloloma Trevis., based on the systematic affinities of its presumed type species, Graphina anguina Müll. Arg. [= Thalloloma anguinum (Mont.) Trevis.] (Staiger 2002). However, G. anguina had not actually been included in the protologue of Graphina, and therefore a new lectotype had to be selected, namely G. puiggarii Müll. Arg., which makes Graphina a synonym of Graphis Ach. (Lücking et al. 2007). Following the current generic concept of Graphidaceae (sensu Staiger 2002), many species of Graphina belong in Graphis s. str., whereas others have been moved into other genera based on phylogeny, apothecial morphology and/or anatomy. During our study of Chinese Graphidaceae, we attempted to resolve the status of all species reported with the genus name Graphina from China (Wei 1991;Aptroot and Seaward 1999;Aptroot andSipman 2001, Aptroot and. We found that 33 species were reported, which are here presented in the form of an updated 'checklist' and transferred to the corresponding genera, namely Carbacanthographis Staiger & Kalb, Diorygma Eschw., Fissurina Fée, Graphis Adans., Phaeographis Müll. Arg., Platygramme Fée, Platythecium Staiger and Thalloloma Trevis.

Materials and methods
Type material cited here was either obtained on loan from the herbaria in H, PC, and W or studied in the cited herbaria. Because many of the names discussed here have already been treated by Kalb et al. (2004), Staiger (2002), Nakanishi et al. (2003) and Lücking et al. (2009), we do not provide full synonymies and type specimen citations unless the name has not previously been treated or the type specimen is particularly relevant to the discussion. A dissecting microscope (Olympus SZX12) and a light microscope (Olympus BX51 and Nikon Eclipse-55i) were used for the morphological and anatomical studies. Measurements and illustrations were taken from the manual cross sections of fruit bodies in water. The amyloidy of the ascospores was tested using Lugol's solution. The lichen substances were detected and identified by thin-layer chromatography (Culberson and Kristinsson 1970;Culberson 1972;White and James 1985). Newly proposed taxonomic names and combinations were deposited in MycoBank.

Taxonomy
List of the Chinese species previously reported under the name Graphina Müll. Arg.
It agrees morphologically with Fissurina adscribens and its ascospore size falls in the range of the latter. Hence, we consider it a synonym to F. adscribens (see below). It is reported from Hunan (as Graphina olivascens, Zahlbruckner 1930Zahlbruckner , 1932.  Lücking et al. (2009), this taxon belongs in Graphis as G. oxyspora (Zahlbr.)

Chemistry. No substances present.
Notes. Because of the characteristics of thallus and lirellae, this species belongs to Graphis and is here recombined as G. lecanactiformis. It is similar to Graphis tenuirima (Shirley) A.W. Archer in anatomy, but differs in ascoma morphology (dussii morph according to Lücking et al. 2009) and the larger ascospores. In the world key to Graphis (Lücking et al. 2009), this species would key out at couplet 23 in Group 5.  Notes. Because the material of Graphina haloniata in W has the typical characteristics of Phaeographis, such as open discs and brownish ascospores, it is here transferred to Phaeographis. The reported differences between Graphina haloniata and G. plumbicolor were in ascospore size: 30-34 × 12-14 µm in G. haloniata and 29-30 × 10-11 µm in G. plumbicolor (Zahlbruckner 1933), but in the studied material these measurements largely overlap. The two names were only reported from their type locations in Taiwan (Zahlbruckner 1933(Zahlbruckner , 1940Lamb 1963;Wang and Lai 1973;Wei 1991).    Notes. The material of Graphina taiwanensis and f. obscurata in W shows the characteristics of Platygramme such as the distinctly labiate lirellae, closed discs, a laterally carbonized exciple and hyaline to grayish ascospores. Platygramme taiwanensis is most similar to P. platyloma, but the latter differs in having an inspersed hymenum, larger ascospores (more than 120 µm long) and lack of lichen substances. Platygramme pudica (Mont. & Bosch) M. Nakan. & Kashiw. differs in having an inspersed hymenum, larger ascospores (150-180 × 18-25 µm) and echinocarpic acid (Jia and Kalb 2013). The form obscurata only differs from the nominal taxonby the darker thallus, which is largely caused by the bark, and hence we include it in P. taiwanensis. The species was only reported from the type location in Taiwan (Zahlbruckner 1933;Lamb 1963;Lai 1973, Wei 1991 Morphologic and molecular data help adopting the insect-pathogenic nephridiophagids (Nephridiophagidae) among the early diverging fungal lineages, close to the Chytridiomycota Introduction Arthropods may be infected by a range of unicellular pathogens of disparate taxonomic affiliations (Lange and Lord 2012). The majority of entomopathogenic spore-forming protists belong to the supertaxa Opisthokonta (e.g. Microsporidia) and SAR (Alveolata with the Apicomplexa; Rhizaria with the Haplosporidia and Paramyxea; Adl et al. 2012). Nephridiophagids (Nephridiophagidae) are unicellular, spore-forming parasites previously of uncertain systematic position. They infect the Malpighian tubules of insects and are mainly found in the lumen of these tubules (e.g., Woolever 1966, Radek andHerth 1999). The life cycle of nephridiophagids includes a merogony phase with vegetative multinucleate plasmodia that divide into oligonucleate and uninucleate cells. Sporogonial plasmodia form internal, 5-10 µm long, oval, flattened spores, generally with one nucleus. Residual nuclei of the mother cell remain in the cytoplasm between the developing spores. The systematic position of the nephridiophagids has been discussed intensively. Morphologically, this lineage could not be assigned unambiguously to any of the known major taxa of spore-forming protists. Some authors place them with the haplosporidians (Ivanić 1937, Woolever 1966, Purrini and Weiser 1990 while others disagreed with this grouping (Toguebaye et al. 1986, Purrini and Rhode 1988, Lange 1993. With the aid of a light microscope, the nephridiophagid stages resemble microsporidians (Microsporidia), and by tradition some nephridiophagids have been given names in microsporidian genera (e.g., Nosema periplanetae and Pleistophora periplanetae; Lutz and Splendore 1903, Perrin 1906). A preliminary molecular analysis placed them within the Fungi, close to 'zygomycota' (Wylezich et al. 2004, White et al. 2006. Since then, the Microsporidia have been placed near the root of the fungal kingdom (Capella-Gutiérrez et al. 2012, Xiang et al. 2014) as have the Cryptomycota (Lazarus and James 2015). The genus Nephridiophaga was introduced by Ivanić (1937) for N. apis, which infects honey bees. Insects, which represent the metazoan group with the highest species richness, appear to be remunerative to screen for novel fungal taxa which were hidden in habitats insulated from the free environment (Hawksworth 2001).
The Fungi comprise upwards of 6 million extant species, of which some 135,400 have been described formally (Blackwell et al. 2011, Hibbett et al. 2011, Taylor et al. 2014; www.speciesfungorum.org as of May 2017). Although all true fungi are heterotrophs, they occupy a very wide range of niches and nutritional modes. About 1% of the described species -750-1,000 species from about 100 genera -are pathogens of insects. These entomopathogens are distributed over most fungal phyla, and their hosts are spread among 20 orders of insects (Araújo and Hughes 2016). All insect developmental stages from egg to adult may be subject to infection. Molecular data have increased our understanding of insect-fungal relationships considerably. A wide range of associations and infection types has been discovered, ranging from parasitic through commensal and even beneficial (Suh et al. 2005, Vega et al. 2012, Douglas 2015. High-throughput sequencing is rapidly gaining in popularity as a means of studying fungus-insect interactions, and published studies have uncovered surprising diversity even within single insect individuals (e.g., Dhami et al. 2013). This is in line with the results from other environmental fungal sequencing efforts, where tens to hundreds of previously unknown (or at least not sequenced) species are usually found in each new study undertaken (Nilsson et al. 2016). It is thus not speculative to assume that a significant number of insect pathogenic fungi await discovery and formal description.
Many early diverging fungi are associated with insects, however, this region of the fungal tree of life suffers from poor taxon sampling and phylogenetic resolution. The last few years have seen the description of numerous new species and lineages of early diverging fungi, even at the phylum level (e.g., James et al. 2006, Corsaro et al. 2014, Karpov et al. 2014a, b, Bauer et al. 2015. The nephridiophagids belong in this part of the fungal kingdom (Wylezich et al. 2004), but they have yet to be addressed using phylogenetic methods in the context of a rich taxon sampling of closely related taxa. The present study uses a molecular phylogenetic approach to examine the phylogenetic relationships of the nephridiophagids. We included three species of Nephridiophaga from cockroaches, viz. N. blattellae, N. blaberi, and a new species from the Madeira cockroach (Leucophaea maderae). Increasing the number of analyzed species we aim to clarify the relationships among the deep lineages of the Fungi. Our molecular, morphological, and ultrastructural results show that the nephridiophagids may represent a distinct clade at the root of the Fungi.

Animal material
Specimens of the Death´s Head Cockroach Blaberus craniifer, the German Cockroach Blattella germanica, and the Madeira Cockroach Leucophaea (Rhyparobia) maderae were retrieved from the Federal Environment Agency (UBA; https://www.umweltbundesamt.de/en) in Berlin, Germany. Cockroaches of different ages and sex were dissected, and their Malpighian tubules were removed and processed for further examination through light and electron microscopy as well as molecular analysis.

Light microscopy
For fresh preparations, parts of the tubules were ground with fine forceps in a drop of 0.6% NaCl solution. The infected tubules were then smeared on a microscopic slide, air dried, and fixed in methanol for 5 min prior to staining with Giemsa solution (Ac-custain, Sigma; 1:10 in tap water for 45 min). Dried smears were mounted in Entellan (Merck). Extracted bundles of Malpighian tubules were embedded in paraffin (Paraplast) for histological examination. Fixation was carried out in Bouin's fluid, modified after Dubosq-Brasil (Böck 1989). Sections of 7 µm were stained with hematoxylineosin (Böck 1989) and embedded in Malinol (Chroma). The chitinous spore walls of native spores were fluorescently labeled with 0.01% Calcofluor White M2R in a 50 mM phosphate buffer of pH 7.2 for 15 min. Photos were taken with a Zeiss Axiophot equipped with an Inteq digital camera and the software EasyMeasure 1.4.

Scanning electron microscopy
Cover glasses were coated with 0.01% poly-L-lysine to promote attachment of spores. Malpighian tubules were ground in a drop a fixative (1% OsO 4 , 2.5% glutardialdehyde, 0.1 M cacodylate buffer, pH 7.2) on the cover glasses and fixed for 1 h. After dehydration in a graded series of ethanol, the prepared cover glasses were critical point dried in a Baltec CPD 030 and sputtered with gold in a Baltec SCD 040. Images were taken with a Quanta 200 scanning electron microscope from FEI Company.

Transmission electron microscopy
Stages of N. blattellae were fixed (glutaraldehyde, reduced osmium) and embedded according to Radek and Herth (1999).

DNA extraction
For molecular analysis of the nuclear small subunit (SSU, 18S) rRNA encoding rDNA sequences of microscopically identified Nephridiophaga species, dissected Malpighian tubules of Blattella germanica, Blaberus craniifer, and Leucophaea maderae were transferred into 1.5 ml PCR-clean reaction tubes (Eppendorf, Hamburg, Germany) with 50 µl of distilled water and stored at -20°C pending further analysis. Alternatively, the tubules were put into 50 µl of lysis buffer. (0.5% sodium dodecyl sulfate, 200 mM TRIS-HCl pH 8.0). For DNA extraction, specimens were centrifuged at 13,200 g for 5 min using the Eppendorf benchtop centrifuge 5415R with a F45-24-11 rotor. The supernatant was removed, and total DNA was extracted from the obtained pellets using the DNeasy Plant Mini Kit from Qiagen (Hilden, Germany). Briefly, each pellet was thoroughly resuspended in 400 µl of warm buffer AP1 and 4 µl RNase A (100 mg/ ml). Samples were incubated at 65°C for 10 min and 20 min at room temperature. Next, 130 µl of AP2 buffer was added and samples were incubated for 5 min on ice. Lysate was transferred into the QIA shredder column and the column was centrifuged for 2 min at 13,200 g. The flow-through was gently mixed with 1.5 volume AP3/E buffer, transferred to a DNeasy spin column, and centrifuged for 1 min at 6000 g. The column was placed into a new 2 ml collecting tube and washed with 500 µl AW buffer. The column was centrifuged for 1 min at 6,000 g, after which the flow-through was removed and the column was washed again with 500 µl AW buffer. Centrifugation was performed at 13,200 g for 2 min. Finally the column was placed into a 1.5 ml PCRclean reaction tube and DNA was eluted with 50 µl AE buffer. After 5 min incubation at room temperature, the column was centrifuged at 6,000 g for 2 min. The extracted DNA was stored at -20°C pending further analysis.
For amplification of the SSU sequences of N. blattellae, N. blaberi, and N. maderae, the eukaryotic universal primers published by Medlin et al. (1988) without polylinker were used that span the complete 18S (Table 1). Additionally, we designed a bridging Nephridiophaga-specific primer (Nephbla3 rv) based on the public SSU sequence of N. blattellae (NCBI GenBank accession no. AY603958) using PrimerBLAST (http:// www.ncbi.nlm.nih.gov/tools/primer-blast/). Since Nephridiophaga DNA was extracted from ground cockroaches, primer specificity was essential, so that the primers do not match the host. The tiny amount of fungal DNA compared to host DNA could lead to preferential amplification of cockroach DNA unless specific primers were used. The primers used targeted nucleotide position 1-21 (Euc Uni 18S fw), nucleotide position 872-891 (Nephbla3 rv and Nephbla3 fw) and nucleotide position 1787-1810 (Euc Uni 18S rv) of the complete 18S ribosomal RNA gene of N. blattellae (NCBI GenBank accession no. AY603958), resulting in sequences of 891 nt (Euc Uni 18S fw and Nephbla3 rv) and 939 nt (Nephbla3 fw and Euc Uni 18S rv) ( Table 1). All primer sets were synthesized by Eurofins MWG Operon (http://www.eurofinsgenomics.eu/).

PCR amplification of SSU rDNA
PCR amplification of the 18S rRNA gene was performed using HotStarTaq Plus DNA polymerase kit (Qiagen) and 10 mM dNTP mix (Peqlab, Erlangen, Germany) according to the manufacturers' protocols. PCR reactions were performed with an initial DNA denaturation step at 95°C for 5 minutes followed by 35 cycles of 94°C for 1 min, 59°C for 1 min for each primer set (Table 1), 72°C for 1 min, and a final elongation step at 72°C for 10 min. Amplification products were separated on a 1% agarose gel, stained with ethidium bromide, and visualized under UV light.

Sequencing of amplified the SSU rDNA
The PCR amplicons were purified using the QIAquick PCR Purification Kit from Qiagen. Briefly, 20 µl of each PCR-product was resuspended in 100 µl PB-buffer and transferred to a QIAquick DNA column, centrifuged at 16,100 g for 30 s and the flowthrough was aspirated. The column was washed with 750 µl PE-buffer and centrifuged at 16,100 g for 30 s. The flow-through was aspirated and the column was centrifuged at 16,100 g for 30 s to remove any residual ethanol. The column was placed into a 1.5 ml PCR-clean reaction tube (Eppendorf ), and 50 µl of warm EB-buffer was added onto the membrane. To elute the PCR amplicons, the columns were centrifuged at 8,000 g for 2 min. The purified PCR products were sent to Eurofins-Genomics (http://www. eurofinsgenomics.eu/) for sequencing. The short sequences (Table 1) were edited and processed using the VectorNTI software from Invitrogen TM Life Technologies (Darmstadt, Germany). Two new SSU rDNA sequences were generated, one each for N. blaberi (1,697 bases) and N. maderae (1,784 bases). These were deposited in GenBank (Benson et al. 2017) under accession numbers KU900289-KU900290. For further phylogenetic analysis we also used the N. blattellae SSU rDNA sequence AY603958 (1,807 bases) from GenBank.

Phylogenetic inference
The generated sequences were aligned against the SILVA SSU reference database (v119) using SINA (Pruesse et al. 2012), which accounts for secondary structures of the ribosomal RNA. We added the zygomycete sequences from White et al. (2006) to the reference database to increase the coverage of fungal lineages at the root of the fungal kingdom. For the general placement of Nephridiophaga into the eukaryotic tree of life we took the multiple sequence alignment of all 62k reference database entries and removed all overly short sequences as well as sequences with anomalies (Ashelford et al. 2005 (Nylander et al. 2004). Chain mixing and convergence were satisfactory (the latter approaching an average split frequency of 0.008). Sequence similarities were calculated based on Jukes and Cantor (1969) distances. The multiple sequence alignment and the phylogenetic trees were deposited in TreeBASE at https://treebase.org/ (study no. S19000).
In nine out of ten dissected Madeira cockroaches, the Malpighian tubules were infected by a spore-forming nephridiophagid. The degree of infection was generally low (6-10 sec of microscopy necessary before finding first stages). Two animals were infected more heavily (1-5 sec of microscopy). None of the infected cockroaches showed obvious symptoms of illness. In fresh smears, spore-containing plasmodia ( Fig. 1), vegetative multinucleate plasmodia (Fig. 2), and single spores were seen, which jointly form the typical stages of species from the genus Nephridiophaga. The number of spores in a sporogenic plasmodium varied between 6 and 26, with a mean number of 15 (n = 34). As long as the plasma membrane of the plasmodium is intact (Fig. 1, arrows), the spores are kept together in groups. Single spores have a flattened oval form, measuring 6.3-7.9 (7.2) x 3.1-4.7 (3.7) µm in fresh preparations (n = 50) and 4.8-7.5 (6.4) x 2. 4-4.5 (3.3) µm in Giemsa-stained smears (n = 50). Scanning electron micrographs reveal a centrally localized, plugged spore opening on the upper side (Fig. 4, left spore). The lower side has no opening but may be slightly folded (Fig. 4, right spore). The rim of the spore is thickened. In hematoxylin-eosin stained paraffin sections, the localization of the parasites in the lumen of the Malpighian tubules can be seen clearly (Fig. 5). Many cells attach to the microvilli border of the epithelial cells while others are free in the lumen. Only very rarely, intracellular vegetative plasmodia are found in the epithelial cells of the Malpighian tubules (Fig. 6). Giemsa staining of smears also reveals the different stages, viz. multinucleated vegetative plasmodia (Fig. 7 ), young spores whose interior can be stained (Fig. 8), and mature spores into which the stain cannot penetrate. Typical for nephridiophagids are the residual vegetative nuclei of the mother cell in the cytoplasm between the spores (Fig. 9). Characteristic ultrastructural features of the genus Nephridiophaga are demonstrated using the example of N. blattellae (Figs 10-13). Vegetative plasmodia have a variable cell form and contain one to several nuclei, numerous mitochondria, and an endoplasmic reticulum (Fig. 10). The mitochondria are of the tubular to sac-like type rather than of the cristae type. Sporogenic plasmodia internally form flattened-oval, thick-walled spores with one nucleus; residual vegetative nuclei remain in the cytoplasm of the mother cell (Fig. 11). The spores contain typical eukaryotic cell structures such as a nucleus, mitochondria, and an endoplasmic reticulum but no obvious extra elements (Fig. 12). A layer of small vesicles attaching to the lining of the developing The upper surface of the spore possesses a central spore opening (arrow, left spore) while the lower surface of the spore lacks an opening (right spore). 5, 6 Paraffin sections stained with hematoxylin-eosin. Generally, the plasmodia (pl) are found in the lumen of the Malpighian tubule but are often attached to the microvilli (mv) (5). Rarely, aggregates of vegetative plasmodia (arrow) occur in the epithelial cells of the Malpighian tubules (6). n = nuclei of epithelial cells. 7-9 Smears of macerated tubules stained with Giemsa depicting vegetative plasmodia (7), stained young spores (8), and unstained mature spores with residual nuclei (arrows) of the mother sporoplasm. Scale bars: 5 µm (1-4), 50 µm (5), 10 µm (6-9).
sporoblasts is probably involved in the formation of the spore wall (Fig. 12). The only structure apparently aiding in hatching of the sporoplasm is a central spore opening through which the sporoplasm can escape (Fig. 13). Calcofluor staining reveals the presence of chitin in the spore wall (Fig. 14).

Phylogenetic position of Nephridiophaga
Since we wanted to clarify the phylogenetic relationship of Nephridiophaga with respect to other spore-forming pathogens, we included members of the former Zygomycota as well as the Haplosporidia and Microsporidia (Suppl. material 1, Fig. S1). The genus Figures 10-14. Nephridiophaga blattellae, 10-13 transmission electron microscopy, 14 Calcofluor white staining. 10 Meront with several nuclei (n) and mitochondria (mi) in the lumen of Malpighian tubule. Inset: Mitochondrium with tubular to sac-like cristae. 11 Sporogenic plasmodium containing mature spores (sp), mitochondria (mi), and vegetative nuclei (n) in the cytoplasm. The plasmodium is anchored to the microvilli (mv) of epithelial cells (ep) of the tubule. 12 Young spore within the cytoplasm of a sporogenic plasmodium, surrounded by a layer of vesicles. The spore cytoplasm contains one nucleus (n), mitochondria (mi), and endoplasmic reticulum (er). 13 An infectious sporoplasm hatches through the central spore opening, leaving behind the spore wall of the emptying spore (sp). The nucleus (n) is squeezed through the tiny spore opening. 14 Calcofluor white stains the spore wall indicating the presence of chitin (bluish color). Scale bars: 1 µm (10-13), inset 0.1 µm (10), 5 µm (14).
Nephridiophaga is clearly positioned within the Fungi but does not cluster together with any of the long branches of Microsporidia (Cryptomycota), Haplosporidia (SAR group), or Dimargaris (Dimargaritales, Kickxellomycotina, 'zygomycota'). We further selected a representative set of entries from the Holomycota phyla and its sister clades in order to find the most probable position of Nephridiophaga in the backbone tree. Again we recovered strong support for the Nephridiophaga within the Fungi (Fig. 15). The clade could not be assigned to the Cryptomycota but instead formed a clade with the Chytridiomycota sensu lato. The fully supported branch leading to Nephridiophaga points to an independent lineage near the root of the fungal kingdom. In addition, the strong branch support obtained for data from each of the three samples of Nephridiophaga from different cockroach species supports the notion that the isolates indeed represent three distinct species. The new species Nephridiophaga maderae differed from described taxa by approximately 12-14% at the intrageneric level and by more than 20% at the inter-phylum level in its small subunit ribosomal DNA sequence (as referred to pairwise sequences similarity). The Nephridiophaga clade is not known from environmental sequences. Etymology and host. Named after its host, the Madeira cockroach, Leucophaea maderae.

Discussion
The phylogenetic position of Nephridiophaga has been a longstanding enigma in the systematics community. As a result of the re-appraisal of fungal phylogeny during the Deep Hypha project (Blackwell et al. 2006), Nephridiophaga blattellae was reported to cluster among the fungi and not among other eukaryotes as previously described using the SSU sequence generated by Wylezich et al. (2004). Nephridiophaga blattellae appeared to have some statistically supported phylogenetic relationship with the Kickxellales-Dimargaritales-Zoopagales clade among the zygomycetes (White et al. 2006). Here we report the generation of SSU sequence data from another species which was previously described, N. blaberi, and from the new species of Nephridiophaga in order to re-evaluate the phylogenetic position for the nephridiophagids. We were able to provide robust phylogenetic support (100%) for the position of the nephridiophagids near the root of the fungal kingdom.

The identification of Nephridiophaga maderae as novel species
All nephridiophagids found so far in cockroaches belong to the genus Nephridiophaga: N. archimandrita (Radek et al. 2011), N. blaberi (Fabel et al. 2000, N. blattellae (Crawley 1905, Woolever 1966, Radek and Herth 1999, N. lucihormetica (Radek et al. 2011), N. periplanetae (Lutz and Splendore 1903, Lange 1993, and N. tangae ). Characteristics of these species were compiled and tabulated by Radek et al. (2011) and the species studied so far seem to be host specific. Furthermore, they differ slightly in the size of spores and the number of spores within the sporogenic plasmodium. The localization of the life stages is mostly in the lumen of the Malpighian tubules, but in some species intracellular vegetative plasmodia have also been found. Due to these characters we believe that an as-yet unknown species of Nephridiophaga occurs in Leucophaea maderae. Woolever (1966) already mentioned the occurrence of a nephridiophagid in this host but did not provide any details. Our sequence data strongly support the existence of this un-named species and the general occurrence of different species of nephridiophagids in different hosts, indicating that speciation in Nephridiophaga is strongly linked to host speciation. The new species differs from N. blattellae by 12% and from N. blaberi by 14% in their nuclear small subunit ribosomal DNA which is a sufficient phylogenetic distance, according to Marshall and Berbee (2011), to justify a new species. Nephridiophaga maderae sp. nov. joins four other species of Nephridiophaga in having been recovered from cockroaches (Woolever 1966, Fabel et al. 2000, Radek et al. 2011, hinting at the unique and diverse life forms that can be found by investigating taxa inhabiting divergent host species.

Phylogenetic position of the genus Nephridiophaga
The results confirmed the finding of Wylezich et al. (2004) that Nephridiophaga belongs to the fungal kingdom rather than being related to non-fungal eukaryotes. It was, however, not possible to resolve the branching order of the non-Dikarya fungi in a robust way. We found a polytomy where the nephridiophagids were embedded within the flagellate fungi, the Chytridiomycota sensu lato (Voigt 2012). There is strong phylogenetic and ultrastructural support not to assign the nephridiophagids to any of the newly erected/redefined phyla Blastocladiomycota, Chytridiomycota sensu stricto, Monoblepharidomycota, and Neocallimastigomycota, all of which stem from the former phylum Chytridiomycota s.l. (see Voigt (2012) for an overview).
It is interesting that sequences from species of Nephridiophaga have never been recovered in studies based on environmental sequencing, although primer mismatches can be hypothesized to be the culprit (cf. Tedersoo et al. 2015). Alternatively, Nephridiophaga sequences may be rare enough to be below the detection limit in bulk environmental samples. So far, we have only recovered sequences from the cockroach clade (Blattodea), which is an early-diverging lineage. Thus, species of Nephridiophaga from other arthropods will be extremely helpful to retrace the evolutionary history of this cryptic and enigmatic group of fungi. During the past, the placement of one novel fungal group (Archaeorhizomycetes) discovered using DNA-based methods shifted when more characters from additional rDNA and protein-coding regions were added to the analysis (Rosling et al. 2011). Multiple markers such as the nuclear large subunit (LSU, 28S) ribosomal DNA (rDNA) in addition to the nuclear small subunit (SSU, 18S) ribosomal DNA sequences and protein coding genes will be very helpful for future phylogenetic efforts involving the Nephridiophaga clade. Indeed, the LSU has been proposed as a good genetic marker for non-Dikarya fungi (e.g., Letcher et al. 2006). Obtaining additional genes for Nephridiophaga is, however, very laborious and resource intensive, given the endobiotic nature of these minute fungi. Herein, we decided to opt for SSU rDNA sequences with the aim to expose the uniqueness of Nephridiophaga. Increased research interest in this genus and related lineages will hopefully bring about the developments needed in primer design to support the generation of additional genetic marker data, and even full genomes, to fully resolve the precise phylogenetic position of Nephridiophaga within the kingdom Fungi. We are in the process of generating additional ribosomal (ITS and LSU) and nuclear gene sequences (Elongation factor alpha) for Nephridiophaga.
While about 98% of the described fungi belong to the Dikarya, comprising the two phyla Ascomycota and Basidiomycota, the relationships among the remaining lineages of fungi are less well resolved (Carr and Baldauf 2011, Bauer et al. 2015). Our molecular analyses provide strong support for Nephridiophaga as a distinct lineage closely related to the Chytridiomycota s.l. Nephridiophaga probably originates from flagellate fungi but has secondarily lost its flagella. Morphological and ultrastructural characters that support the inclusion of Nephridiophaga in the Fungi include a heterotrophic lifestyle, propagation by spores, an intranuclear position of the spindle during nuclear division, the presence of chitin in the spore wall, and chitosome-like vesicles on the surface of the maturing spore (Radek et al. 2002).
Significant rDNA sequence divergence above 20%, distinctive morphology, and unique life cycle traits support the delimitation of Nephridiophaga from other fungus-like organisms -the ARM clade (Aphelida, Cryptomycota, and Microsporidia;cf. Karpov et al. 2014a, Corsaro et al. 2016) -found near the root of the kingdom Fungi. The ARM clade is presently not included in the Fungi. Nephridiophagids are different from Microsporidia by not possessing a polar tube, polaroplast, and posterior vacuole -structures that are involved in the hatching process of Microsporidia. Endoparasitic trophonts of the Aphelida and Cryptomycota (syn.: Rozellomycota) are able to phagocytose whereas nephridiophagids do not engulf particulate food (Powell 1984, Karpov et al. 2013. The morphology and the life cycle of Nephridiophaga deviate from the Chytridiomycota s.l., which possesses typical flagellate stages (zoospores) and centrioles. These structures are missing in nephridiophagids. The only microtubules detected in nephridiophagids so far are intranuclear spindle microtubules formed during nuclear division (Radek and Herth 1999). Thus, the morphology of the kinetosome-associated structures useful in the determination of families and genera of zoosporic fungi cannot be used for classification here (Powell and Letcher 2014). In general, the Blastocladiomycota, Chytridiomycota s.str., Neocallimastigomycota, and Monoblepharidomycota -the Chytridiomycota s.l. -develop posterior flagellate zoospores when free-living but lose the flagella when the life cycle is endoparasitic in all stages (James et al. 2006, Voigt 2012, Powell and Letcher 2014. The nephridiophagids appear to have lost the ability to produce flagella, much like the endoparasitic Microsporidia. This may be due to the completely endobiotic life style, which renders active motility less of a useful trait. In contrast to many other organisms at the root of the Fungi, the habitat of nephridiophagids is quite restricted -they represent one of the comparatively few groups of fungal endoparasites of arthropods known so far. Further morphological differences are the lack of mycelia (a thallus) and microbody-lipid complexes (MLCs). The MLCs, assemblages of lipid globules, endoplasmic reticulum, mitochondria, and microbodies in Chytridiomycota s.l. are suggested to be involved in the conversion of energy from lipids (Powell 1976, Powell andLetcher 2014). Lipid globules to date have not been identified in nephridiophagids, and the presence of microbodies is similarly unclear. In N. blattellae, opaque vesicles with homogenous content have been observed (Radek and Herth 1999), but catalase activity could not been shown (unpublished, RR). Compared to agile zoospores, the various life stages of nephridiophagids probably do not need much, if any, energy for motility. Nephridiophagids possess mitochondria with tubular to sac-like inner membrane structures, which stands in strong contrast to mitochondria with cristae in the other fungi. On the whole, there are no specific morphological traits in nephridiophagids that can be used to support a close relationship with chytridiomycete lineages. Molecular analyses presently seem to be the only clue to resolve their phylogenetic relationships -and the molecular results support the nephridiophagids as a distinct lineage among the early diverging fungi.

Conclusion
The molecular, morphological, and ultrastructural evidence brought forward in this study point to the fact that the nephridiophagids form a distinct clade of fungi whose precise taxonomic affiliation cannot be settled at the present time. But we refrain from assigning a rank to this clade in the context of insufficient sampling and clade stability.
Fortunately, we have come far in the generation of molecular data from additional genes and genetic markers, such that we hope to be able to resolve the phylogenetic position of the nephridiophagids in the not too distant future. Studies closing in on the very root of the Fungi have the potential to cast light not only on those particular lineages, but also on the evolution of all extant fungal groups and their nutritional modes and biotic interactions. The increasingly ambitious sampling efforts undertaken by the mycological and molecular ecology communities leave no doubt that the next few years will witness substantial scientific progress in understanding and delimiting the root of the kingdom Fungi. Targeting the cockroach habitat will be worthwhile to consider for future environmental sequencing studies.

Introduction
The diversity of lichen-forming and lichenicolous fungi in Fennoscandia is often considered to be reasonably well-known, yet new species are discovered continuously. In 2004, the Fennoscandian checklist included 2414 lichen-forming species (Santesson et al. 2004), while the most recent one includes 2538 species (Nordin et al. 2017). Although new discoveries of macrolichens are indeed made (e.g., Arvidsson et al. 2012, Frödén and Thell 2010, Klepsland 2013, 2016, the main uncharted territory is found within the world of small, crustose lichens (e.g., Arup et al. 2014, Ekman 2015, Svensson and Palice 2009, Westberg et al. 2011, 2016. Crustose lichens comprise about two thirds of all lichens in Fennoscandia, but the taxonomic status, distribution and ecology of several hundred of these species are virtually unknown. The aim of this paper is to contribute to the understanding of Fennoscandian crustose lichen species by reporting a number of species as new to one or more Fennoscandian countries and by providing taxonomic and nomenclatural novelties, including new combinations, synonymizations and lectotypifications, on a series of crustose lichens and lichenicolous fungi.

Material and methods
Light microscopy measurements were made on material mounted in water using an oil-immersion lens, with a precision of 1 µm. Only well-developed ascospores lying outside the asci were measured. Measurements of asci and paraphyses of Micarea hylocomii were made on material cut to sections 12-18 µm thick using a freezing microtome and stained with lactophenol cotton blue. HPTLC was performed using the method described by Arup et al. (1993). Coordinates are in the WGS84 map datum unless otherwise stated.

Absconditella lignicola Vězda & Pišút
Nova New to Norway. Reported from much of the Northern hemisphere as well as Tasmania (Coppins 2009a, Urbanavichus 2010. In Norway, this species has usually been collected from the upper side of large, relatively recently decorticated logs of Picea abies, but once also on a stump (not cut). It has, however, also been collected on logs of Populus tremula and Pinus sylvestris. The species is mostly found in association with slimy biofilms, in Norway often accompa-
Variolaria torta Taylor  New synonyms. New to Norway. Previously known from the United Kingdom, Ireland, Sweden, the Netherlands, Luxembourg, Belgium, Germany, Austria, and the Czech Republic (Hulting 1872, Bouly de Lesdain 1910, van den Boom et al. 1999, Palice 1999, Berger and Türk 1993, Wirth et al. 2013, Diederich et al. 2014. Previously reported from Norway by Santesson et al. (2004), but the material on which this report was based has later been shown to be misidentified.
The types of Variolaria torta, Bacidia antricola, Lecidea carneoglauca, and L. byssoboliza are clearly conspecific. Variolaria torta, by far the oldest name, has after its introduction only been briefly mentioned by Adams (1909) and Zahlbruckner (1928) as a dubious name. Therefore, in the interest of nomenclatural stability, a proposal to reject Variolaria torta has been submitted (Ekman 2017). The type material of this name in BM consists of a small but well preserved and readily identifiable specimen with abundant pycnidia but no apothecia, sent to W. J. Hooker by Taylor. Among the remaining names, the combination Bacidia carneoglauca, introduced by Smith (1911) is currently the most widely used. However, Bacidia antricola was validly published in the dissertation of Johan Hulting no later than 27 May 1872 (Hulting 1872) and is consequently older than Lecidea carneoglauca. Bacidia antricola has been included in every subsequent lichen flora or checklist of Swedish lichens (e.g., Fries 1874, Forssell and Blomberg 1880, Magnusson 1936, Santesson 1984, Foucard 1990, Foucard 2002. Furthermore, there are reports under this name from Belgium (Bouly de Lesdain 1910), as well as a further Swedish record (Hulting 1925). Therefore, this name is adopted here. The lectotype of Bacidia antricola selected here is the largest and most well developed of the five available syntypes, with numerous pycnidia and several apothecia in various states of development.
Although geographically widely separated, both of the newly discovered Norwegian sites for B. antricola are situated close to the west-coast, with an oceanic climate. At both sites, the species grew on somewhat metal-enriched rocks in shady and humid situations, below overhanging cliffs and sheltered from rain. At the southernmost locality, the species was largely confined to steep or almost vertical rock walls at the entrance of an old (copper?) mine. The entrance to the cave is situated in a steep ESEfacing hillside, close to but well above the fjord, and is surrounded by a lush forest dominated by Corylus avellana, Fraxinus excelsior, and Ulmus glabra. At the northern locality, the species was mainly found on horizontal or slightly inclined rocks along a small stream, deep underneath an overhang at the bottom of a small but topographically uneven south-facing hill. The surrounding forest is dominated by Betula pubescens and Populus tremula, with scattered Corylus avellana. New synonym. Bacidia invertens was listed as an accepted species by Stenroos et al. (2016). The type material, however, consists of a well developed and typical specimen of Bacidia igniarii, and the former is consequently reduced into synonymy. There is some doubt whether the specimen in H was the only one available to Vainio at the time of description. Surprisingly, there does not seem to be any material of B. invertens deposited in TUR (Alava 1988). New to Norway. This species is distributed across Europe and eastern temperate North America (Ekman 1996). Reports from other areas of the world probably represent other species. Bacidia polychroa is red-listed as threatened or regionally extinct in a number of countries where it has been assessed, viz. Sweden (ArtDatabanken 2015), Finland (Jääskeläinen et al. 2010), Germany (Wirth et al. 2011), and the United Kingdom (Woods and Coppins 2012).
The Norwegian find of B. polychroa was made at the base of an old Acer platanoides situated in a narrow and rather deep ravine in a region of mixed temperate woodland composed of e.g. Corylus avellana, Fagus sylvatica, Fraxinus excelsior, Picea abies, Pinus sylvestris, Quercus robur, and Ulmus glabra. The site is sheltered and characterized by high humidity and minimal sun exposure. Several additional lichen species with oceanic preferences or a demand of high and stable air humidity grow in the vicinity,  Fig. 1A New to Sweden. Bacidia pycnidiata has been reported from Belgium, Poland, the Czech Republic, Slovakia, Lithuania, Estonia, Finland, Ukraine, and Russia (Republic of Mordovia and Republic of Adygea) in Europe, as well as the Republic of Buryatia south of Lake Baikal in Asian Russia (assuming that the watershed through Greater Caucasus is taken as the geographic border between Europe and Asia) (Czarnota and Coppins 2006, Suija et al. 2007, Ertz et al. 2008, Pykälä 2008, Motiejūnaitė et al. 2011, Dymytrova 2013, Malíček et al. 2014, Urbanavichus and Urbanavichene 2013, Urbanavichene and Palice 2016. The species is mostly found on trunks of deciduous trees and shrubs, either directly on the bark or over bryophytes, in more or less shady and humid habitats. The autecological amplitude seems to be wide, however, and there are scattered finds on coniferous trees, more or less moribund cyanolichens, soil, as well as stones (or bryophytes on stones) on the ground, including metal-rich waste (Vondrák et al. 2010, Czarnota and Hernik 2014 in addition to references above). It has been suggested that the species is favoured by anthropogenic impact (Czarnota and Hernik 2014), although its ecological repertoire also includes semi-natural old-growth forests (Suija et al. 2007).

Bacidia pycnidiata Czarnota & Coppins
The Swedish find was made on bark of an old Acer platanoides in a semi-open stand of Quercus robur in a grazed field. The locality is situated at the outskirts of the town of Kalmar, and the surroundings consist partly of cultivated fields, partly of urbanized land (roads, housing, manufacturing, commerce, small airport etc.). Although frequently reported only in an anamorphic state, B. pycnidiata was found to produce abundant apothecia in the Swedish site.
Specimens examined: SWEDEN: Småland, Kalmar par., Hagbygärde, ekbacke S om Lantmännen, grov lönn i ekdominerad betad hagmark, 56.67492°N 16.30616°E, New to Fennoscandia. This species has been reported from the Netherlands, Belgium, United Kingdom, Ireland, Germany, Poland, Estonia, Czech Republic, Austria, Switzerland, France, Ukraine, Armenia, and Ecuador , Kubiak and Sparrius 2004, Aptroot et al. 2005, Aptroot and Honegger 2006, Vondrák 2006, Khodosovtseva 2009, Berger and Priemetzhofer 2010, Roux 2012, Gasparyan and Sipman 2016. In several instances, however, reports have been based on sterile material, a questionable practice given that the species does not produce any secondary substances. In an addendum to Smith et al. (2009) (available at http://www.britishlichensociety.org.uk/recording-mapping/downloads, accessed 21 November 2016), Bacidina adastra is considered rare and strongly overreported, being confused with crusts of free-living green algae. Morphologically, B. adastra is somewhat reminiscent of B. neosquamulosa (Aptroot & Herk) S. Ekman, which in its current delimitation may turn out to include more than one species. B. neosquamulosa in the strict sense forms imbricate, finely dissected microsquamules that may later disintegrate to form goniocysts. B. adastra, on the other hand, starts out as minute, sometimes somewhat flattened, granules that soon bud off new granules in a more or less coralloid manner, the end result being a thick, finely granular and pale green crust. In addition, the thallus surface tends to be more shiny in B. neosquamulosa than in B. adastra.
In Fennoscandia, Bacidina adastra is currently known from two sites in southern Skåne. The first find was made in a churchyard surrounded by houses in an otherwise open, agricultural landscape, where the species occurred in fair quantity and sparingly fertile on a young, planted Ulmus. The second find was made in the northern outskirts of the town of Lund, in public plantations with a variety of shrubs where the ground had been covered by a black fabric of non-woven polypropylene to prevent weeds from establishing. This fabric is colonized by a variety of lichens, mostly crustose lichens during the first years, whereas later successional stages are dominated by Peltigera didactyla (With.) J.R.Laundon and species of Cladonia. The crustose lichen flora is richest in species and individuals in slopes with moderate shade from shrubs. Slopes seem to be preferred because leaf litter does not easily accumulate on the fabric. The richest spots are downhill from fences cutting through the plantations, where the concentration of metal ions is probably high. Apart from large spots of abundantly fertile Bacidina adastra, other lichens encountered on the ground cover fabric were
New to Norway. Previously known from Sweden, Denmark, Scotland, France, Germany, Poland, the Czech Republic, Slovakia, Austria, Spain, Italy, Ukraine, Russia, and Rwanda (Vězda 1993, Palice 1999, Czarnota 2003, Killmann and Fischer 2005, Coppins et al. 2005, Sérusiaux et al. 2010, Knutsson 2014. The Norwegian finds were made on old and hard wood, in one case on the underside of a decorticated, leaning trunk of Sorbus aucuparia in a rain-sheltered site under an over-hanging rock, and in two other cases on wood of very old but living Taxus baccata, both on the underside of decorticated branches and on vertical surfaces inside a hollow trunk. All known localities consist of humid oldgrowth forests dominated by spruce and aspen or by birch and aspen. No apothecia were observed in the Norwegian sites, but numerous white and stalked pycnidia were present. Conidia in the Norwegian specimens measure c. 4 × 1.5 µm, and the photobiont is chlorococcoid, 6-15 µm diam. By comparison, conidia and photobiont cells in an isotype of Catillaria alba (UPS L-030528) measure 3.1-4.2 × 1.5-1.9 and 7-13 µm, respectively.
The Norwegian locality is situated close to the Barents Sea, just within the southern part of the arctic climate zone. The site is characterized by dwarf-shrub heath and sharp rocky ridges of layered, steeply inclined, metamorphic rocks of varying composition, both acid and base-rich. Catillaria scotinodes was found growing on a fairly exposed ridge of calciferous sandstone with layers of dolomite.
New to Norway. This species was recently described from two sites in the British Isles, one in Wales and one in Scotland (Fryday and Coppins 2012).
C. subtenerum was encountered at the Helgeland coast in central Norway, only 200 m from the coastline, where it occupied a shelf appearing on a roughly horizontal rock   Tehler et al. (2013).
New to Sweden. Dirina fallax is mainly distributed in the western part of the Mediterranean Region and along the Atlantic coast from northern Morocco to Scotland, with an outpost locality in the Canary Islands. Records from Baden-Württemberg in Germany and the South Bohemian and South Moravian Regions of the Czech Republic are geographically closest to the the Swedish locality (Tehler et al. 2013). However, Norwegian material determined as D. massiliensis Durieu & Mont. has not been examined by us and may partly represent D. fallax.
Dirina fallax was first collected in Sweden 1998 on Mt. Omberg in the province of Östergötland and was reported as D. massiliensis f. sorediata by Nordin and Hermansson (1999). They noted the siliceous substrate and the thin, dark thallus. The species was again observed at the same locality when visited by the Swedish Lichenological Society during an excursion in 2015 . The Swedish material is sorediate and lacks apothecia (see photograph in Westberg and Arup 2016, Fig. 3).
For a long time, Dirina fallax was treated as a synonym of D. massiliensis. Molecular data, however, show that they are distinct species, although closely related (Tehler et al. 2013). The shape and size of apothecia, ascospores and conidia as well as the secondary chemistry (erythrin, ± lecanoric acid and unidentified substances) are the same in both species. D. fallax, however, has a thinner and usually more brownish grey thallus compared to the thicker, whitish and chalk-like thallus of D. massiliensis (Tehler et al. 2013). The thallus and apothecial thalline margin of D. fallax vary considerably in colour, from dark brown over greyish to creamy white. D. fallax is confined to acidic rocks, D. massiliensis to calcareous rocks. Sorediate specimens of Dirina fallax are morphologically indistinguishable from sorediate specimens of D. canariensis Tehler & Ertz, which is considered endemic to the Canary Islands (Tehler et al. 2013).  New to Norway. Previously reported from central Sweden, central Germany (Ekman 2015), and Finland (Pykälä 2017).
The Norwegian finds are located c. 20 km apart in the area between the Oslofjord and lake Øyeren, in sheltered sites with old-growth bilberry-spruce forest. At both sites, the species was found exclusively on mineral-rich black biotite rock in deep shade, sheltered from rain and trickling water by overhanging rocks. The only associated lichen species recorded was Brianaria lutulata (  Remarks. The complicated nomenclature of this species was clarified by Jørgensen et al. (2002, see also Printzen 1995. In summary, the oldest name is Biatora furfuracea, validly and legitimately described in 1864, while B. amaurospoda is either an invalid or illegitimate name (depending on whether it is considered effectively published or not). Lecidea furfuracea (Anzi) Jatta is, however, not available in Lecidea because of the existence of an earlier homonym, L. furfuracea Pers., described in 1826. As pointed out by Jørgensen et al. (2002), the younger synonym L. pullata should therefore be used as long as the species is treated in Lecidea. However, when transferred to Frutidella, as was done by Schmull et al. (2011), the oldest epithet becomes available and F. furfuracea is consequently the correct name. Fig. 2B Folia Geobot. Phytotax. Bohemoslov. 1: 327 (1966). Gyalecta subscutellaris Vězda, Biológia, Bratislava 15: 173 (1960). -Type: Slovakia, Tatra Magna, in ascensu occid. alpis Ostrva, supra muscos destructos, 1750 m.a.s.l., 22 August 1958, A. Vězda (PRA-V-03129, holotype, not seen, PRA-V-05551, isotype, not seen; UPS L-093370, L-159273, isotypes, seen by AN and MW). New to Fennoscandia. When originally described, Gyalidea subscutellaris was placed in Gyalecta (Vězda 1960). It was found overgrowing mosses at a high-elevation locality in the Tatra Mountains of Slovakia. Later, it was reported from the Polish part of the Tatra Mountains (Flakus 2007) and in the United Kingdom ). The species is characterized by small apothecia (up to 0.5 mm diam., but usually smaller) with a dark brown to black rim and a brownish concave disc, developed on an inconspicuous thallus encrusting soil and bryophytes on basic, metal-rich (Britain) or slightly acidic ground (Tatra). According to Gilbert et al. (2009), the ascospores are muriform and measure (15-)17-20(-22) × 7-10 µm. The Swedish material agrees well with the isotypes at UPS, except that ascospores in Nordin 6631 are poorly developed and do not exceed 16 × 8 µm. In addition, the disc is black and concolourous with the rim in this specimen, a phenomenon potentially caused by environmental factors. In southern Sweden (Gotland and Uppland), G. subscutellaris was collected on calcareous ground, whereas the northern sites in Jämtland are situated on metal-rich soil at an old copper mine as well as on acidic ground.
The Norwegian specimen is typical of the species in having a coarsely papillate thallus surface. The papillae have a cortex and are larger than the blastidia in the otherwise similar L. erysibe (Ach.) Mudd. The material was collected in a steep, south-facing rock wall composed of calciferous meta-sandstone subjected to trickling water. The site is located close to the large river Lågen, near the bottom of the valley Gudbrandsdalen. This part of Gudbrandsdalen is one of the driest and most summer-warm places in Norway, with a weakly continental climate. Several saxicolous lichen species are, at least in modern times, largely confined to a limited inner section of this or a few neighbouring valleys, e.g. New to Norway. Apparently widespread in much of Europe, although with a concentration of finds in Central Europe and relatively few finds in eastern Europe (Mayrhofer 1988, Gavrylenko and Khodosovtsev 2009, Urbanavichus and Urbanavichene 2011.
Currently known from two sites in northern Norway, both in the county of Troms. At both sites, the species was found growing on calcareous rock under overhangs, on limestone and marble, respectively. Despite being sheltered from rain, both sites are fairly open and sun-exposed. The Balsfjord locality lies at the rim of a lake and is surrounded by birch forest, whereas the Lavangen locality is situated in the low-alpine zone.
Specimens New to Norway. Previously known only from two Swedish collections (Svensson and Thor 2011).
A small patch of this species was found growing on the upper side of a leaning (almost horizontal), moss-covered trunk of a living Sorbus aucuparia in an old-growth forest dominated by Betula pubescens and Populus tremula. The site lies close to the coast at the island Meløya in Nordland county, northern Norway. The site is further characterized by big boulders and a few vertical rock walls, which contribute to a sheltered and humid microclimate. M. capitata inhabited both Hylocomium splendens (Hedw.) Schimp and Hypnum cupressiforme Hedw. Another rare muscicolous lichen, Gyalideopsis muscicola P.James & Vězda, was found on the same trunk.
Micarea capitata is perhaps most likely to be confused with M. hylocomii Poelt & Döbbeler (see note under that species below). Another species similar to M. capitata is M. olivacea Coppins, which was not discussed by Svensson and Thor (2011). Our observations indicate that M. olivacea differs from M. capitata by having apothecia without a clearly constricted base, a dark olivaceous K+ green pigment in the hymenium and hypothecium, and abundant pycnidia (unknown in M. capitata). M. olivacea has been found growing on lignum and on rock, not over bryophytes (Coppins 1983(Coppins , 2009b New to Fennoscandia. Initially described on material from Belgium and Great Britain (Coppins 1995), the distribution of M. deminuta has proven to be wide. Apart from additional European records (e.g., the Czech Republic, Palice 1999;Poland, Czarnota 2007), the species is now also known from Japan, North America, and Tasmania (Coppins 2009b, Czarnota 2004. The species was found colonizing an extensive area of soft wood on the upper side of a large, moderately to well decomposed log of Populus tremula. The site is an oldgrowth forest dominated by Picea abies and Populus tremula, between a lakelet in the east and a steep hill to the west, and consequently sheltered from direct sun. We also found an additional Norwegian specimen in UPS, where the species grew over plant debris, but any other ecological information is lacking.  Thallus forming small patches on leaves of Hylocomium splendens, thin, faint greygreenish grey, episubstratal. Photobiont cells regularly globose, 4-7 µm diam. (-10 µm according to Poelt and Döbbeler 1975), occurring in clusters inside the thallus. Apothecia numerous, scattered, immarginate, convex-hemispherical, ± adnate or sometimes with slightly constricted base, black or rarely grey (when young or when lacking green pigment), when wet often with a faint blue-green tinge, 0.06-0.12 mm diam. Epihymenium indistinct, light-dark blue-green, sometimes with dark brown tinges, c. 5 µm high, K-, C-, N+ red. Hymenium hyaline to light-dark blue-green in streaks, 19-35 µm tall, C-(blue-green pigment rapidly fading), N± red, I+ blue, K-, KI+ blue. Hypothecium hyaline to light brown without any red or purple tinge, K-, C-, N-(N± red if the blue-green pigment reaches the hypothecium). Paraphyses few and difficult to discern, simple or sparingly branched, colourless, 1-1.5(-2) µm wide, apices not or slightly thickened (-3 µm wide), hyaline. Exciple not seen, even in sections of young apothecia. Asci clavate, apically thickened, 8-spored, with wall KI+ blue throughout the length of the ascus, 18-32 × 8-13 µm. Ascospores narrowly ellipsoid, straight or slightly curved, 0-1-septate, (7-)8-10(-15) × (1.5-)2(-3) µm. Pycnidia not seen.
After noting some discrepancies between the Scandinavian material and the original description, we examined all available material of M. hylocomii, including the holotype. The main difference between our new description and the original one concerns the paraphyses, which Poelt and Döbbeler (1975) described as having spherical apices with dark brown or black pigment hoods. Generally, the extremely small size of the apothecia and the scarcity of paraphyses make these characters difficult to observe, but although the apices are slightly thickened in the Scandinavian material, no dark brown or black pigment hoods were seen. Subsequent examinations revealed that there are no such apical pigment hoods in the holotype either. However, the dark blue-green and brown pigments present in M. hylocomii are often concentrated to the upper part of the apothecium and seemingly adhere to the outer surface of the paraphyses, thus sometimes giving the impression of faint pigment hoods. Another discrepancy concerns the ascospores, which Poelt and Döbbeler (1975) described as 1-septate, but there are non-septate ascospores present in the holotype. There is generally some variation in the proportion of simple and 1-septate ascospores between the specimens, ranging from the exclusively simple ascospores in Svensson 725 to the mostly 1-septate ascospores in Svensson 1050.
The anatomy of the paraphyses as well as the uniformly KI+ blue ascus wall led Poelt and Döbbeler (1975) to suggest that M. hylocomii belongs in an undescribed genus, an opinion that was shared by Coppins (1983). As described here, however, the anatomy of the paraphyses is not clearly inconsistent with a placement in Micarea, which is true of most other characters as well (e.g. ascospores, size of the photobiont). Unfortunately we were, in spite of many attempts, unable to observe a well-developed apical apparatus. As noted by the original authors, however, the asci do seem somewhat unusual in displaying a strong, uniformly KI+ blue reaction throughout their length. Whether this is an indication of a different generic affiliation than Micarea should be further investigated using molecular methods.
In Norway and Sweden, Micarea hylocomii has always been collected on Hylocomium splendens, usually where the bryophyte is hanging down the vertical side of a boulder, though not in rain-protected situations. The species has been found on oneto three-year-old shoots of its host, indicating that its substrate is short-lived and that M. hylocomii is adapted to frequent dispersal. The ubiquitousness of its host suggests that M. hylocomii is likewise common. Jørgensen (1996) suggested that M. hylocomii could be a suboceanic species. Although the number of collections is too low to enable an evaluation of this suggestion, M. hylocomii may at least turn out to have quite specific requirements in terms of humidity, since most of the localities are quite humid, either because they are situated in swampy forests or close to a stream.
M. hylocomii is most likely to be confused with M. capitata, which also inhabits Hylocomium splendens. M. capitata, however, differs from M. hylocomii by having larger apothecia (0.10-0.35 mm diam.) with a more clearly constricted base, broader ascospores (-4 µm), and by possessing a blue-green pigment that does not fade rapidly in C (Svensson and Thor 2011). Furthermore, M. capitata has numerous, branched and anastamosing paraphyses, while paraphyses are scarce and difficult to discern in M. hylocomii. Other Micarea species with a thin or immersed thallus and minute (-0.2 mm diam.), black apothecia, such as M. contexta Hedl., M. deminuta Coppins, M. eximia Hedl., and M. olivacea, may also be confused with M. hylocomii, although none of them is known to grow on H. splendens (Coppins 1983, Czarnota 2007. M. deminuta is readily distinguished by its dark brown pigment in the hypothecium and broader (3-6 µm wide) ascospores (Coppins 1995). More care is needed to separate the other three species, since they too have a green, N+ red pigment in their apothecia. M. contexta differs in having constantly 1-septate ascospores with one cell larger than the other. Also, it has a dark green and/or a dark purple pigment in the hypothecium, reacting K+ green (Coppins 1983). M. eximia has a light reddish brown, K+ green hypothecium (Coppins 1983). M. olivacea has numerous paraphyses, mostly 1-septate ascospores, and a dark olivaceous or olive brown hypothecium that reacts K+ green (Coppins 1983 New to Sweden. M. lynceola was described from Norway in 1874, but has so far not been correctly reported from Sweden. The species has also been recorded from Ireland, United Kingdom, the Netherlands, Belgium, Germany, Austria, the Czech Republic, Poland, Finland, and the Murmansk Region of Russia (Palice 1999, Aptroot and van Herk 1999, Ertz et al. 2008, Urbanavichus et al. 2008, Coppins 2009b, Czarnota 2011. M. lynceola is a pioneer species of siliceous rocks and the Swedish collection was made on a loose rock on a road-bank. It is easily confused with M. polycarpella (Erichsen) Coppins & Palice, which has similar ecology and to which earlier Swedish records of M. lynceola belong (Palice 1999). M. lynceola, however, has a well-developed, 30-40 µm wide exciple which is readily distinguished as a non-amyloid zone after treatment with KI, while M. polycarpella has 7-10 µm wide excipular rim of pigmented hyphae that does not contrast with the hymenium in KI (Palice 1999 New to Fennoscandia. This recently described species was originally reported from Poland and the Czech Republic (Guzow-Krzemińska et al. 2016). It belongs to the Micarea prasina group and is characterized by having distinct soralia and containing micareic acid. In Sweden it has been found in the nature reserve Fiby urskog near Uppsala, where it occurs on decaying logs in an old-growth forest dominated by conifers, and in one locality in the outskirts of Uppsala, where it grew on wood of Salix.
Specimens examined. SWEDEN: Uppland, Husby-Ärlinghundra par., Östra Steninge, along jogging trail c. 500 m NW of the Syrian Orthodox Church, on dead mossy boughs of Salix on the ground, 59. in Alstrup et al., Fróðskaparrit 40: 96 (1994). Lecidea subconfusa Nyl., Flora 52: 84 (1869). -Type: Faeroe, Strömsö, "Torshavn", August 1867, E. Rostrup (C-L-76663, lectotype, selected by Alstrup in Alstrup et al. 1994 [ICN Art. 9.9 New to Fennoscandia. Micarea subconfusa is a rarely recorded species, currently known from Ireland, Scotland, and the Faeroe Islands (Alstrup et al. 1994, Coppins 2009b, Coppins and James 1992. M. subconfusa belongs to the M. assimilata group and inhabits acid rocks in the lowlands. It is similar to the alpine M. paratropa (Nyl.) Alstrup, but lacks K+ violet pigmentation in the hymenium and has a K -hypothecium. The Swedish specimen grew on wood of an old pilework close to the seashore, which likely represents a case of a primarily saxicolous species occasionally growing on dust-enriched wood. Due to superficial similarities with other, not closely related saxicolous lecideoid lichens, M. subconfusa is possibly an overlooked species. Alstrup in Alstrup et al. (1994) referred to the collections C-L-76662 and C-L-76663 as the "holotype" of Lecidea subconfusa, thus effectively designating both as lectotype. We here further specify this by designating the specimen C-L-76663 as lectotype. This specimen has the words "specimen primarium" written with red ink on the sheet to which it is glued, as well as an indication that the specimen has been sent to Nylander ("a Rostrup Nylandro missum"). According to Alstrup et al. (1994), the handwriting is that of Rostrup. Syntypes of Lecidea submoestula are available in BM and H-NYL. The specimen H-NYL 19033 only gives the locality as "route de Westport" and the year as 1876. Two collections in BM are possible duplicates of the Nylander specimen, but give the date as February 1876 and March 1876 respectively, which means that it cannot be ascertained which constitutes a duplicate of the specimen in H-NYL. Consequently, the specimen H-NYL 19033 is chosen here as lectotype of L. submoestula.
New synonym. Bacidia atrolivida was listed as an accepted species by Stenroos et al. (2016). The type material, however, consists of typical Mycobilimbia tetramera, and the former is consequently reduced into synonymy. According to Vainio (1922), Bacidia atrolivida is supposed to differ from 'Bilimbia obscurata' (i.e., Mycobilimbia tetramera) in having a sparsely sorediate thallus, an observation we were unable to confirm. The type material in TUR-V is cited here as the holotype, because it appears to have been the only specimen available to Vainio at the time of description (Alava 1988). Lecidea sanguinaria var. lecanoroidea Nyl., Lichenes Japoniae: 77 (1890). -Type: Japan, Itchigômé, 1879, E. Almquist (H-NYL 10912, syntype, seen by TS).

Mycoblastus sanguinarioides Kantvilas
New to Finland and Sweden. This species was described from Tasmania, Australia (Kantvilas 2009), but has later been shown to be widespread in the Northern Hemisphere (Canada, Japan, Russia, USA; Spribille et al. 2011). There is one collection each from Finland and Sweden in herbarium UPS. Both localities are apparently very humid (near a waterfall and a rapid, respectively). The Swedish locality harbours several rare lichens, such as Pannaria conoplea (Ach.) Bory, Pilophorus robustus Th.Fr., Placopsis gelida (L.) Lindsay, and Ramalina thrausta (Ach.) Nyl. (herbarium material in UPS). The Fennoscandian localities are in keeping with the occurrence of the species in humid regions in eastern Eurasia and coastal western and eastern North America.
Mycoblastus sanguinarioides is similar to M. sanguinarius (L.) Norman but can be distinguished by often having flat apothecia surrounded by a thin ring of whitish thalline tissue. In contrast, small apothecia of M. sanguinarius are usually distinctly convex with a constricted base. Furthermore, the hymenium of M. sanguinarioides contains birefringent hymenial crystals, visible in polarized light (see Spribille et al. 2011, Fig. 2). The chemistry of the two Fennoscandian specimens (bourgeanic acid and atranorin) agrees with the chemistry of M. sanguinarioides elsewhere in the Northern Hemisphere. Both compounds occur in M. sanguinarius as well, but always together with one or several additional compounds. M. sanguinarius has four chemotypes (Spribille, unpublished data), three of which are found in northern Europe: (1) rangiformic acid and atranorin (common, northern), (2) bourgeanic acid, caperatic acid and atranorin (mainly in the south), (3) bourgeanic acid, rangiformic acid and atranorin (northern) monotypic, but Stenroos et al. listed several other candidates for inclusion, of which two were later combined into the genus: P. exsequens (Nyl.) Printzen & Davydov (Davydov and Printzen 2012) and P. caesia (Fr.) M. Svensson & T.Sprib. (Dillman et al. 2012). P. duplex is distinct from the other three species by having 16-24 ascospores per ascus, but otherwise fits well in Puttea on account of having minute, pale apothecia, asci with a KI+ blue tholus penetrated by a canal that slightly widens towards the apex, and crystals that dissolve in K in the epihymenium and hymenium.
According to Coppins and Aptroot (2008), the exciple of P. duplex is paraplectenchymatous, which would be consistent with a placement in Fellhanera (Lücking 2008), while Puttea margaritella (the type species of that genus) has a strongly gelatinized exciple composed of branched, parallel hyphae (Stenroos et al. 2009). Although the exciple of P. duplex is often poorly developed and difficult to observe, we found that it is in fact quite similar to that of P. margaritella, being strongly gelatinized and consisting of dichotomously branched hyphae with narrowly cylindrical cell lumina.
The Swedish specimen was found on bark of Betula in a mature coniferous production forest. The specimen differs from the original description in having longer ascospores (-9 µm versus -5 µm) and by growing directly on bark and not over bryophytes. However, as the original description of F. duplex was based on only three specimens, the range of variation in ascospore size is possibly larger than indicated there and the ecology of the species may likewise be broader. Since the Swedish specimen agrees well with the holotype in other respects, we prefer to include it in P. duplex pending further studies.
New to Finland. Previously known from the Alps, Sarcogyne algoviae was recently reported from Sweden and Norway .
The newly discovered specimen was collected on calcareous rock in northernmost Finland. The species is characterized by apothecia with a strongly carbonized margin, a colourless hypothecium, and narrowly ellipsoid ascospores . pochlorella and T. subnitida, mentioning that they differ only in the hymenium being entirely green in the former, whereas the latter has a bluish epihymenium. There are, however, additional differences. In L. hypochlorella, the hypothecium contains a mixture of green and dull brown pigments, which contrast to the strongly darker proper exciple. In T. subnitida, on the other hand, the hypothecium and proper exciple are very similar in hue (dark red-brown) and do not contrast. Furthermore, ascospores in L. hypochlorella are 1(-2)-celled, whereas they are consistently 2-celled in T. subnitida. The material from Torne lappmark in Sweden (UPS L-785614) represents L. hypochlorella. The Norwegian specimens of Toninia subnitida had been misidentified as Bacidia coprodes (Körb.) Lettau and were discovered while revising material filed under that species (Ekman 2014). Kilias (1981) reported T. subnitida (as Catillaria tristis) also from Sweden, Finland, Russia, Germany, Czech Republic, Switzerland, Austria, and Italy. It has later been recorded also from Spain and Montenegro (Hladun andGómez-Bolea 1982, Knežević andMayrhofer 2009). Reports from North America are doubtful, as the name was introduced in the checklist of Egan (1987) with reference to Kilias (1981). The latter author, however, does not mention any North American finds.