Mycol Progress (2013) 12:231–269 DOI 10.1007/s11557-012-0830-1 ORIGINAL ARTICLE Phylogeny and taxonomy of the ‘manna lichens’ Mohammad Sohrabi & Soili Stenroos & Leena Myllys & Ulrik Søchting & Teuvo Ahti & Jaakko Hyvönen Received: 9 December 2011 / Revised: 17 May 2012 / Accepted: 23 May 2012 / Published online: 20 July 2012 # German Mycological Society and Springer 2012 Abstract We present ataxonomic revision of the ‘manna lichens’ based on morphological, chemical, ecological and molecular data. A large number of herbarium specimens and fresh collections were examined. Phylogenetic analyses were performed using nuclear ribosomal (nrITS, nrLSU) and mitochondrial small subunit (mtSSU) sequences. Some notable phenotypic characters were plotted on the phylogenetic tree, and the analysis reveals that some of these characters are useful for genus and species level distinction of certain ‘manna lichens.’ Phylogeny of the Megasporaceae was revised using a combined data set of nrLSU and mtSSU and performing parsimony and Bayesian analyses. Five genera (Aspicilia, Circinaria, Lobothallia, Megaspora and Sagedia) are recognized. Further, the relationships of five presumably closely related genera of ‘manna lichens’, namely Agrestia (vagrant), Aspicilia (crustose) Circinaria (crustose), Chlorangium M. Sohrabi : S. Stenroos : L. Myllys : T. Ahti Botanical Museum, University of Helsinki, P.O. Box 7, 00014 Helsinki, Finland M. Sohrabi : J. Hyvönen Department of Biosciences, University of Helsinki, P.O. Box 65, 00014 Helsinki, Finland M. Sohrabi (*) Department of Biotechnology, Iranian Research Organization for Science and Technology (IROST), P.O. Box 15815-3538, 15819 Tehran, Iran e-mail: mohammad.sohrabi@helsinki.fi M. Sohbari e-mail: msohrabi@myco-lich.com U. Søchting Department of Biology, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen Ø, Denmark (vagrant) and Sphaerothallia (vagrant) with different growth forms were analysed. The analyses revealed that ‘manna lichens’ do not form a monophyletic group but occur in different clades within the genus Circinaria. The genera Agrestia, Chlorangium and Sphaerothallia are assigned as new synonyms under the genus Circinaria and no vagrant or erratic species remain in the genus Aspicilia. The analyses also show that five new erratic, vagrant and crustose species can be recognized. In this study two ‘manna lichens’, viz. Circinaria rostamii sp. nov. (Azerbaijan, Iran and Turkey), and Circinaria gyrosa sp. nov. (Armenia, Azerbaijan, Iran, Turkey, Turkmenistan and Spain) are described as new to science. Three potentially new species with crustose and erratic forms need additional study. Aspicilia fruticolosofoliacea is reduced to synonymy under C. alpicola, and a lectotype is designated for C. aspera. Thirteen new combinations in Circinaria are presented. The phenomenon of vagrancy is briefly discussed, and the biogeography of the ‘manna lichens’ is outlined. Illustrations, distribution maps, and an identification key to the species are provided. Keywords Aspicilia . Circinaria . Eurasia . Iran . ‘Manna lichens’ . Sphaerothallia . Vagrant Introduction During the last two centuries, a group of vagrant species with subfruticose and subfoliose thalli in the genus Lecanora Ach., Sphaerothallia Nees and Aspicilia A. Massal. has been termed ‘manna lichens’. Examples of these species include, e.g., A. esculenta (Pall.) Flagey, A. fruticulosa (Eversm.) Flagey and A. vagans Oxner with their synonyms. The term vagrant (or ‘vagant’) has been applied by Büdel and Wessels (1986); Rosentreter (1993); Pérez 232 (1994, 1997a, b); Sohrabi and Ahti (2010), and Sohrabi et al. (2011a, b) to describe the peculiar morphotype or growth form of some lichens that have no proper substrate. Almost all ‘manna lichens’ are vagrant. The informal term ‘manna lichens’ is mostly associated with historical and ethnolichenological records rather than taxonomy. A brief history of ‘manna lichens’ and relevant references before 1901 were summarized by Elenkin (1901d), and several other references were also listed by Donkin (1980). In fact, this list is one of the most comprehensive lists of references related to ‘manna lichens’. In addition to the ‘manna lichens’, several species of plants, insects and even fungi have also been thought to be the source of the Biblical manna. The general circumscription of the ‘manna lichens’ was outlined by Sohrabi and Ahti (2010) and Sohrabi et al. (2011a, 2011b). In the nomenclatural evaluation it was shown that several generic names, viz. Agrestia J. W. Thomson (Thomson 1960), Chlorangium Link (Link 1849) and Sphaerothallia (Nees von Esenbeck 1831) have been used for ‘manna lichens’. Subsequently, all of these were united under the conserved generic name Aspicilia by Laundon and Hawksworth (1988). For ‘manna lichens’, the genus name Aspicilia has been widely used in many publications and checklists (e.g., Elenkin 1901a, b, c, d, 1907; Mereschkowsky 1911a, b; Oxner 1971; Andreeva 1987; Kulakov 2002, 2003; Litterski 2002; Seaward et al. 2008; Sohrabi and Ahti 2010; Sohrabi et al. 2010a, d; Urbanavichus 2010; Sohrabi et al. 2011a,b). The genus name Lecanora was used in the Catalogus Lichenum Universalis by Zahlbruckner (1921–1940) and by Poelt (1969). The genus Aspicilia was segregated from Lecanora based on a different reaction of the ascus with iodine (I), Hafellner (1991a, b) Recently, Nordin et al. (2010) revised the phylogeny of the Megasporaceae using a combined data set of nrLSU and mtSSU. They resolved the family as monophyletic within the Pertusariales, supporting the previous results of Schmitt et al. (2006), Lumbsch et al. (2007), and Lumbsch and Huhndorf (2007). In the same study by Nordin et al. (2010), the division of the Megasporaceae into the following genera was proposed: 1) Aspicilia A. Massal., 2) Circinaria Link, 3) Lobothallia (Clauzade & Cl. Roux) Hafellner, 4) Megaspora (Clauzade & Cl. Roux) Hafellner & V. Wirth, and 5) Sagedia Ach. Some important distinguishing characters of these genera were also tabulated. Their results were mainly based on fairly extensive sampling from the Megasporaceae, Pertusariaceae and Ochrolechiaceae. They were, however, in conflict with Miadlikowska et al. (2006), who proposed that Aspicilia belongs to the family Pertusariaceae. In Nordin et al. (2010) and Owe-Larsson et al. (2011) the genus Circinaria Link was resurrected after 200 years and several new combinations were proposed. For the first time the segregation of two ‘manna lichens’, viz C. emiliae (Tomin) A. Nordin, S. Savić & Tibell, and C. hispida (Mereschk.) A. Nordin, S. Savić & Tibell from the genus Mycol Progress (2013) 12:231–269 Aspicilia was presented. The newly revived genus Circinaria is the fifth but the oldest one so far introduced for ‘manna lichens’. In its resurrected form, the genus is quite heteromorphic and its generic type species C. contorta (Hoffm.) A. Nordin, S. Savić & Tibell (syn. Aspicilia contorta (Hoffm.) Kremp.) is saxicolous. The genus also includes several other saxicolous species, i.e., C. arida Owe-Larss., A. Nordin & Tibell., C. caesiocinerea (Nyl. ex Malbr.) A. Nordin, S. Savić & Tibell, C. calcarea (L.) A. Nordin, S. Savić & Tibell, C. contorta (Hoffm.) A. Nordin, S. Savić & Tibell, C. cupreogrisea (Th. Fr.) A. Nordin, S. Savić & Tibell, C. gibbosa (Ach.) A. Nordin, S. Savić & Tibell, C. elmorei (E.D. Rudolph) OweLarss., and C. leprosescens (Sandst.) A. Nordin, S. Savić & Tibell. All of these species, except C. elmorei, are morphologically very distinct from ‘manna lichens’. For example, the subfruticose species C. hispida and subfoliose species C. emiliae are distinguished from all of the above mentioned saxicolous species by having a well developed cortex (e.g., two layers in C. hispida) and thick medulla, and by having pseudocyphellae and lacking secondary metabolites. Subfruticose and subfoliose vagrant lichens (0 ‘manna lichens’) are morphologically very distinct from both crustose Aspicilia and Circinaria (sensu Nordin et al. 2010). For this reason Sphaerothallia, with its subfruticose vagrant type species S. esculenta (Pall.) Reichardt, might be assumed to be an appropriate generic name. This name has been used for ‘manna lichens’ by some authors (e.g., Szatala 1957; Follmann and Crespo 1974). It includes several subfruticose and subfoliose vagrant species such as S. affinis (Eversm.) Follmann & A. Crespo, (≡ Aspicilia vagans), S. aschabadensis (J. Steiner) Szatala, S. emiliae (Tomin) Follmann & A. Crespo, S. fruticulosa (Eversm.) Follmann & A. Crespo, S. hispida (Mereschk.) Follmann & A. Crespo, S. jussuffii (Link) Follmann & A. Crespo and S. lacunosa (Mereschk.) Follmann & A. Crespo. The genus also includes several crustose species such as S. aspera (Mereschk.) Follmann & A. Crespo, S. desertorum ‘(Kremp.)’ Follmann & A. Crespo, nom. illeg., S. foliacea (Elenkin) Follmann & A. Crespo and S. straussii (J. Steiner) Follmann & A. Crespo. All of these species are morphologically, ecologically and chemically different from the present circumscriptions of the crustose Aspicilia and Circinaria (sensu Nordin et al. 2010). Therefore, the term ‘sphaerothallioid’ is introduced here to unite this group of subfruticose and subfoliose vagrant or erratic species with few crustose species that are characterized by a well-developed cortex, rather massive medulla, large conidium size (c. 8–35 μm), distribution pattern of the algal cells, presence of pseudocyphellae, absence of aspicilin as a key lichen substance, as well as their ecological preference for arid climatic conditions. Büdel and Wessels (1986) stated that the facultatively unattached species should be termed ‘erratic’ while the term for the obligatory unattached lichens should be ‘vagrant’, Mycol Progress (2013) 12:231–269 and we follow them here. Therefore, the substrate preferences and growth forms of sphaerothallioid species are as follows: vagrant (0 obligatory unattached, seen only in vagrant growth form), erratic (0 facultatively attached, primarily crustose on pebbles or basally attached to soil, and in later stages partially vagrant, semivagrant or crustosevagrant), and crustose (0 obligately attached to soil or rocks). The term ‘manna lichen’ is accepted here in its strict sense, only for subfruticose and subfoliose sphaerothallioid species with vagrant morphotypes and excluding any crustose morphotypes. A saxicolous habit is not in accordance with the biblical definition of manna, which is supposed to be edible material and probably portable by wind (see also Sohrabi and Ahti 2010). In addition to the typical thallus morphologies of lichens, i.e., subfruticose, subfoliose and crustose some members of sphaerothallioid species develop a peculiar phenotype which cannot be easily classified into any of the commonly recognized types described in Grube and Hawksworth (2007). Therefore, the term ‘amorphous’ is used here as subdivision for subfruticose form to better describe the thallus shape of some species (e.g., C. rostamii sp. nov.). Sphaerothallioid species have never been comprehensively monographed. They are distributed throughout semiarid deserts and arid steppes in the Northern Hemisphere. They can be found in the mountain steppes of the large Irano-Turanian region (sensu Takhtajan 1986) as well as in the arid steppes of North Africa, the semiarid steppes of the Mediterranean region, the grasslands and shrublands of southern Europe, and the arid shrub-steppes of northwestern North America. Sphaerothallioid species are frequently reported from continental deserts and steppes with saline soils, low humidity, hot summers and cold winter with below-zero temperatures. It is assumed that their thallus morphology is able to transform in response to different environmental conditions, and thus that they can grow on the surfaces of small pebbles or else remain vagrant. However, within both life-forms they retain their physical mobility and are blown around by strong winds, washed into depressions by rain water, or attached to the hoofs of some animals (e.g., sheep and goats), in this way extending their distributions. Since the last century, the morphological and genetic variations shown by some crustose and vagrant growth forms have been a subject of debate. For instance, Mereschkowsky (1918), Klement (1950), Weber (1967, 1977) and Kunkel (1980) proposed that crustose and vagrant morphotypes might be genetically connected. The main goal of the present study is to clarify the generic and infrageneric status of the ‘manna lichens’, mainly those species commonly or periodically treated under Agrestia, Aspicilia, Chlorangium and Sphaerothallia, and lately also Circinaria. We also attempt to clarify their relationships to allied saxicolous species of Aspicilia and 233 Circinaria. Another aim is to assess the pattern of vagrancy among the sphaerothallioid species and also test the usefulness of molecular data for evaluating connections between selected crustose and vagrant morphotypes of the erratic species. Finally, we examine the consequences of the wider sampling of sphaerothallioid species for phylogeny of the Megasporaceae and the monophy of Sphaerothallia. Material and methods This study is based on approximately 500 herbarium and fresh specimens and includes evaluation of anatomical, morphological, chemical and sequence level characters. The specimens examined were mostly obtained from the following herbaria: ANES, ASU, B, C, CANB, CANL, E, F, FH, FR, G, CBFS, GFW, GZU, H, H-NYL, HAL, HMAS, IRAN, LE, M, MAF, MIN, MSK, O, PRA, S, SPR, TNS, TSB,TU, TUR, TUR-V, UPS, US, and W (all duplicates of Obermayer´s Lich. Graecenses No. 321 were examined before distribution; therefore, all herbaria where they were deposited are listed here). Herbarium acronyms follow Thiers (2011). Additional specimens were from the private herbaria of M. Sohrabi (hb. M. Sohrabi) and M.R.D. Seaward (hb. Seaward). The specimens were analysed by standard techniques using dissecting and compound microscopes. External morphology was studied under a dissecting microscope. Thallus anatomy (e.g., photobiont, conidia, ascospores and apothecia) was studied using a Leica Dialux 20 compact light microscope and photographed with a Leica DFC490 digital camera mounted on a Leica DM 2500 compact light microscope. For cortex observation pieces of thalli were cut using a razor blade. Microtome sections (8–12 and 16–20 μm thick) from thalli were prepared using a Leica CM 3050S freezing microtome. These sections were mounted in lactophenol cotton-blue or water. All microscopy measurements were made in water mounts. Measurements of external morphology, i.e. thallus mass size and branch thickness, were made with the digital vernier calliper. As a rule, ≥30 mature spores were measured from each specimen unless otherwise indicated. Measurements were taken as described in Sohrabi et al. (2010b). Exceptionally, in Circinaria emiliae, only a single apothecium was found and 10 ascospores were measured. The general description of the external morphology was based on dry material observed under a dissecting microscope. Chemical analyses of selected specimens were carried out using thin layer chromatography (TLC), following Orange et al. (2001) and using solvents A, B, and C. High performance liquid chromatography (HPLC) was used as described in Søchting (1997). Calcium oxalate was detected under the light microscope by adding a drop of 10 % H2SO4 to a piece of 234 Mycol Progress (2013) 12:231–269 thallus (0.3–1 mm) and placing a cover-slip over it after 1– 2 min, causing the crystals to dissolve and recrystallize to produce needle shaped crystals of calcium sulphate. The width of conidia was frequently equal to or less than 1 μm and thus smaller than the ocular scale of the microscope at ×1000 magnification. Occasionally they were found to be over 1.2– 1.5 μm. Therefore, only conidium length as well as the width and length of the ascospores were recorded according to the formula (min.–) arithmetic mean–SD–arithmetic mean + SD (–max.). The term paraphysoids follows Roux et al. (2011), Janex-Favre (1985) and Lumbsch (1997). Moniliform and submoniliform are used as defined by Magnusson (1939) and Owe-Larsson et al. (2007), and the presence of the epihymenium pigment ‘caesiocinerea-green’ follows Meyer and Printzen (2000). Apothecia in vagrant forms of sphaerothallioid species are rare. A species was considered fertile if any number of apothecia was observed on examined specimens, otherwise it was considered sterile. Distribution maps of species are based only on the vouchers examined. However, some literature records were evaluated, and omissions or corrections of doubtful records are proposed. The geographical names (mainly country and some provinces or cities) are in accordance with Room (2009), Brummitt (2001) or Merriam-Webster's Geographical Dictionary, 3rd rev. ed. 2001, when possible. Additional colour photographs and online distribution maps are available at the Myco-Lich website (www.myco-lich.com) edited by Sohrabi et al. (2010c). were prepared. The following reaction conditions were used: initial denaturation for 5 minutes at 95 °C, followed by five cycles of 30 seconds at 95 °C, 30 seconds at 58 °C, and 1 minute at 72 °C; in the remaining 30 or 35 cycles the annealing temperature was decreased to 56 °C. Following the last cycle a final extension step of 7 minutes at 72 °C was included. For the primer pairs ITS1F, ITS1LM and ITS4, ITS2KL, an annealing temperature of 56 °C in the first five cycles and 54 °C in the remaining cycles was also successfully used. For the primer pairs LR0R, LR7 and LR5 the annealing temperature was set to 55 °C during the first five cycles and 52 °C in the remaining cycles. With some minor variations, the annealing temperature for mtSSU1 and mtSSU3R was set to 52 °C during the first five cycles and 52 °C in the remaining cycles. DNA concentrations of the PCR products were measured using a Thermo Scientific NanoDropTM 1000 Spectrophotometer. The PCR products were sent to Macrogen Inc. (http://www.macrogen.com/) for sequencing. For the majority of the obtained PCR products purification was performed by Macrogen Inc. For some samples PCR products were cleaned using the GFX PCR DNA and Gel Band Purification Kit (GE Healthcare) following the protocol and eluting with 20–30 μl sterile water included with the kit. DNA concentrations of the uncleaned PCR products sent for sequencing were measured. The primers used for the PCR reactions were also used for sequencing. The obtained sequences were assembled with SeqMan II version 4.0 (DNASTAR). DNA extraction, PCR-amplification and sequencing Sequence alignments DNA was extracted from both fresh (up to 2 years old) and old (30–75 years) specimens. Generally the medulla or a small part of thallus was sampled. Total DNA was extracted using the DNeasy Blood and Tissue Kit or Plant Mini Kit (QIAGEN) according to the instructions given in the manufacturer’s protocol, except that the liquid nitrogen stage was omitted. Full details are provided in Sohrabi et al. (2010a, 2011b). For some specimens (e.g., Circinaria alpicola and C. cerebroides) the ITS1–5.8 S–ITS2 region was also obtained using direct PCR, following Arup (2006). The ITS1–5.8 S–ITS2 region was amplified using the primer pairs ITS1F (Gardes and Bruns 1993) and ITS4 (White et al. 1990), or ITS1LM (Myllys et al. 1999) and ITS2KL (Lohtander et al. 1998). For PCR amplification of the nrLSU region the primers LR0R, LR7 and LR5 (Vilgalys and Hester 1990), and for the mtSSU region the primers mtSSU1 and mtSSU3R (Zoller et al. 1999) were used. PCR reactions were performed using ‘Ready-To-Go’ PCR beads in 0.2 ml tubes (GE Healthcare). Twenty-five microlitre samples containing 19 μl of sterile water, 4 μl of DNA dilution, and 1 μl of each primer at 10 μM concentrations A number of nrITS, nrLSU and mtSSU sequences of specimens from the families Ochrolechiaceae, Pertusariaceae and Megasporaceae were obtained from GenBank (http:// www.ncbi.nlm.nih.gov). Altogether, 87 new sequences were generated for this study, including 54 sequences of nrITS, 16 nrLSU and 17 mtSSU. Voucher information is provided in Table 1. All new sequences have been deposited in GenBank. The combined analysis of the data set of nrLSU + mtSSU sequences included 67 (Fig. 1), and the nrITS data set 101 terminals (Fig. 2). The alignments were obtained using the MUSCLE 3.6 web server (Edgar 2004) with the default settings and then adjusted manually in PhyDE® (Phylogenetic Data Editor, Müller et al. 2005). Ambiguously aligned regions both in the nrLSU and mtSSU data sets were removed manually prior to the phylogenetic analyses. However, for the nrITS data sets ambiguous alignment positions were removed using the program Gblocks v. 0.91b, applying settings allowing for smaller final blocks, gap positions within the final blocks, and less strict flanking positions (Castresana 2000). Gblocks provides an objective and repeatable method for excluding poorly aligned regions within multiple sequence Mycol Progress (2013) 12:231–269 235 Table 1 Material used in this study. Vouchers, their geographical origin, and herbaria where vouchers are deposited are also listed. GenBank accession numbers of the newly obtained sequences are in boldface Species Locality and collector number (Herbarium) nrITS mtSSU nrLSU Aspicilia Aspicilia Aspicilia Aspicilia Aspicilia Aspicilia Aspicilia Aspicilia Aspicilia Aspicilia Aspicilia Aspicilia Aspicilia cernohorskyana cinerea cinerea cinerea cinerea cupreogrisea cyanescens dendroplaca dendroplaca dudinensis dudinensis epiglypta haeyrenii Iran, South Khorasan, ex dupl. TARI 2311 (B) Sweden, Östergötland, Nordin 5542 (UPS) Sweden, Uppland, Hafellner 37308 (GZU) Austria, Styria, Hafellner 40563 (GZU) Sweden, Dalarna, Hermansson 13275 (UPS) Sweden, Nordin 6046 (UPS) U.S.A., California, Owe-Larsson 9151 (UPS) Sweden, Torne Lappmark, Nordin 5952 (UPS) Finland, Enontekiön Lappi, Nordin 6366 (UPS) Sweden, Torne Lappmark, Nordin 6036 (UPS) Sweden, Torne Lappmark, Nordin 5971 (UPS) Sweden, Västergötland, Nordin 6303 (UPS) Sweden, Torne Lappmark, Nordin 5997 (UPS) – – AF332111 AF332110 EU057899 EU057903 – – – – – EU057907 – JQ797481 HM060696 – – HM060695 – HM060707 HM060706 HM060720 HM060710 HM060719 HM060718 HM060717 JQ797496 HM060734 – – HM060733 – HM060745 HM060744 HM060758 HM060748 HM060757 HM060756 HM060755 Aspicilia Aspicilia Aspicilia Aspicilia Aspicilia indissimilis laevata laevata mashiginensis nikrapensis Sweden, Torne Lappmark, Nordin 5943 (UPS) Finland, Åland, Nordin 5846 (UPS) Sweden, Uppland, Tibell 23659 (UPS) Sweden, Hälsingland, Nordin 5790 (UPS) Svalbard, Ebbestad SVL21 (UPS) EU057909 – EU057910 – – HM060708 HM060697 HM060692 HM060694 HM060746 HM060735 HM060730 HM060732 Aspicilia permutata Aspicilia rivulicola Aspicilia supertegens Aspicilia supertegens Aspicilia tibetica Aspicilia verruculosa Aspilidea myrinii Circinaria affinis Circinaria affinis Circinaria affinis Circinaria alpicola Circinaria alpicola Circinaria alpicola Sweden, Torne Lappmark, Nordin 6027 (UPS) Sweden, Torne Lappmark, Nordin 5957 (UPS) Norway, Troms, Owe-Larsson 9002 (UPS) Sweden, Torne Lappmark, Nordin 6023 (UPS) China, Tibet, Obermayer 04386 (H, isotype) Norway, Troms, Owe-Larsson 9007 (UPS) Sweden, Jämtland, Nordin 6205 (UPS) Russia, Astrakhan Region, Kulakov 1408 (M) China, Xinjiang, Abbas 20081364 (H) Russia, Astrakhan Region, Kulakov 1408B (M) Kyrgyzstan, Aksai-Tal, Ringel & Jaschhof 5183 (H) Kyrgyzstan, Tian-Shan, Ringel 5137 (H) Kyrgyzstan, SW Tian-Shan, Ringel 5241 (H) – – – – GU289915 – – HQ171237 HQ389194 HQ389196 JQ797524 JQ797552 JQ797554 HM060721 HM060709 HM060715 HM060704 HM060713 – HM060703 HM060716 – – JQ797492 – – – HM060759 HM060747 HM060753 HM060742 HM060751 – HM060741 HM060754 – – JQ797502 – – – Circinaria Circinaria Circinaria Circinaria Circinaria alpicola arida arida arida aschabadensis JQ797556 HQ406800 EU057905 HQ406801 – – – – – – – – Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria aschabadensis aspera caesiocinerea calcarea cerebroides cerebroides cerebroides contorta contorta contorta contorta contorta Kyrgyzstan, Maltabar Mt., Litterski 4848 (H) U.S.A., Arizona, Owe-Larsson 8759 (UPS) U.S.A., Arizona, Owe-Larsson 8770 (UPS) U.S.A., California, Knudsen 2046 (UPS) Turkmenistan, Kopet-Dagh Mt., Borisova s.n. (LE) Turkmenistan, Kopet-Dagh Mt., Borisova s.n.(LE) Russia, Astarakhan Region, Owe-Larsson 9792 (H) Sweden, Uppland, Tibell 22612 (UPS) Sweden, Öland, Nordin 5888 (UPS) Kyrgyzstan, Terskej Alatau, Ringel & Jashhof 5180 (H) Kyrgyzstan, Innerer Tian-Shan, Ringel 5138 (H) Kyrgyzstan, Aksai-Tal, Ringel 5184 (H) Sweden, Öland, Nordin 5895 (UPS) Austria, Styria, Hafellner 43516 (GZU) Austria, Styria, Wilfling s.n. (GZU) Finland, Vantaa, Pykälä 30701 (H) Finland, Karjalohja, Pykälä 28872 (H) JQ797519 GU289916 JQ797531 – EU057898 JQ797529 JQ797534 JQ797553 EU057900 AF332109 AF332108 – – – – – HM060693 HM060705 – JQ797484 – – – – JQ797477 JQ797478 – – – HM060731 HM060743 – JQ797506 – – – – JQ797499 JQ797500 236 Mycol Progress (2013) 12:231–269 Table 1 (continued) Species Locality and collector number (Herbarium) nrITS mtSSU nrLSU – HQ171236 HQ171230 HQ389200 HQ406802 JQ797551 JQ797542 JQ797526 HQ389203 – JQ797512 JQ797513 JQ797479 – – – HM060689 – – – – HM060691 HM060690 – JQ797501 – – – HM060727 – – – – HM060729 HM060728 – JQ797511 JQ797510 JQ797535 HQ171226 HQ389195 JQ797485 – – – – JQ797493 – – – – JQ797486 – – – – HM060702 – – – – JQ797487 – – JQ797505 – – – – HM060740 – – JQ797504 – – – – Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria contorta digitata digitata elmorei elmorei s.lat. elmorei s.lat. elmorei s.lat. elmorei s.lat. elmorei s.lat. emiliae emiliae emiliae Finland, Lohja, Pykälä 22463 (H) Kyrgyzstan, Jangy-Jer Range, Ringel 5185-B (H) Kyrgyzstan, Jangy-Jer Range, Ringel 5185 (H) U.S.A., Nevada, Rosentreter 3689 (TU) Russia, Astarakhan Region, Owe-Larsson 9814 (UPS) Ukraine, Crimea, Vondrák 5671B (CBFS) Iran, East Azerbaijan, Sohrabi 10128 (IRAN) Iran, East Azerbaijan, Sohrabi 10405 C (IRAN) Iran, East Azerbaijan, Sohrabi 10205 (hb. M. Sohrabi) Kazakhstan, Atyrau, Kulakov 3798 (UPS) Kazakhstan, Atyrau, Kulakov 3702 (UPS) Kazakhstan, Atyrau, Kulakov 3702B (UPS) Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria esculenta esculenta fruticulosa fruticulosa fruticulosa fruticulosa Russia, Astrakhan Region, Owe-Larsson 9796 (UPS) Russia, Astrakhan Region, Owe-Larsson 9796 (UPS) Turkey, Anatolia prov., John 9538 (M) China, Xinjiang, Abbas 2008363-a (H) Iran, East Azerbaijan, Sohrabi 10405A (hb. M. Sohrabi) Kazakhstan, Tarbagatai, Lange 5186 (H) Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria fruticulosa fruticulosa fruticulosa fruticulosa gibbosa gyrosa gyrosa gyrosa gyrosa gyrosa gyrosa hispida s.lat. Russia, Astrakhan Region, Kulakov s.n. (M) Ukraine, Crimea, Vondrák 5670 (CBFS) China, Xinjiang, Abbas 940001(H) Ukraine, Crimea, Vondrák 5188 (CBFS) Sweden, Uppland, Nordin 5878 (UPS) Spain, Guadalajara, Printzen 8087 (FR) Iran, Golestan, Sohrabi 9496 (hb. M. Sohrabi) Iran, East Azerbaijan, Sohrabi 10085 (hb. M. Sohrabi) Spain, Guadalajara, MAF-Lich 15363 (H) Iran, East Azerbaijan, Sohrabi 10401A (hb. M. Sohrabi) Turkey, Kırıkkale, John 11984A (M) Iran, East Azerbaijan, Søchting 11187 (hb. M. Sohrabi) HQ171228 HQ171227 JQ797555 HQ171229 HQ389199 EU05790 JQ797514 JQ797539 JQ797540 JQ797557 JQ797528 JQ797532 JQ797558 Circinaria Circinaria Circinaria Circinaria Circinaria hispida hispida hispida hispida hispida s.lat. s.lat. s.lat. s.lat. U.S.A., Wyoming, Muscha & Rosentreter 121 (SRP) Russia, Kalmyk Region, Kulakov s.n. (M) Greece, Mayrhofer 15811B (GZU) Greece, Mayrhofer 15811A (GZU) HQ171234 HQ389201 JQ797523 JQ797522 – – – – – – – – Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria s.lat. s.str. s.str. s.str. s.str. s.str. hispida hispida hispida hispida hispida jussuffii jussuffii lacunosa lacunosa leprosescens leprosescens rogeri rogeri Iran, East Azerbaijan, Sohrabi 10405 (IRAN) Turkey, Malatya, Candan 11 (ANES) Russia, Kalmyk Region, Ochirova s.n. (LE) U.S.A., Idaho: Rosentreter 16233 & Cochrane (SRP) Iran, East Azerbaijan, Sohrabi 10212b (hb. M. Sohrabi) Iran, Golestan, Sohrabi 15099 (hb. M. Sohrabi) Morocco, Vězda: Lich. Sel. Exs. No. 2381 (H) Algeria, Esnault 2033 (GZU) China, Xinjiang, Abbas 940003 (H) Kazakhstan, South Peribalkhashya, Piregoudov s.n. (LE) Sweden, Uppland, Nordin 5906 (UPS) Sweden, Västergötland, Nordin 6059 (UPS) U.S.A., Wyoming, Rosentreter 16373 (SRP) U.S.A., Oregon, Rosentreter 16333 (SRP) JQ797509 HQ406806 HQ171235 HQ389198 HQ389197 HQ171233 JQ797521 JQ797518 JQ797517 JQ797520 EU05791 – HQ171231 HQ171232 – HM060722 – – – JQ797488 – JQ797489 JQ797490 – HM060711 HM060714 – – – HM060760 – – – JQ797503 – JQ797495 JQ797494 – HM060749 HM060752 – – Mycol Progress (2013) 12:231–269 237 Table 1 (continued) Species Locality and collector number (Herbarium) nrITS mtSSU nrLSU Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria Circinaria rostamii rostamii rostamii rostamii sp. 1 sp. 1 sp. 1 sp. 2 sp. 2 sp. 2 sp. 3 sp. 3 Iran, Semnan, Sohrabi 9364 (IRAN) Iran, East Azerbaijan, Sohrabi 10212 (IRAN) Iran, East Azerbaijan, Sohrabi 10095 (IRAN) Iran, East Azerbaijan Sohrabi 10212 (IRAN) Russia, Astrakhan Region, Owe-Larsson 9821 (UPS) Iran, East Azerbaijan, Sohrabi 4758 (H) Iran, East Azerbaijan, Sohrabi 10117B (hb. M. Sohrabi) Iran, Semnan, Sohrabi 9380b (IRAN) Iran, Semnan, Sohrabi 9380b (IRAN) Iran, East Azerbaijan, Sohrabi 10092A (IRAN) Iran, Semnan, Sohrabi 9347 (IRAN) Iran, Semnan, Sohrabi 9357 (IRAN) JQ797541 JQ797527 JQ797533 JQ797538 HQ389202 JQ797550 JQ797544 JQ797547 JQ797548 JQ797549 JQ797546 JQ797530 – – – JQ797491 – – – – – – – – – – – JQ797507 – – – – – – – – Circinaria Circinaria Circinaria Circinaria Circinaria sphaerothallina sphaerothallina sphaerothallina sphaerothallina sphaerothallina Iran, East Azerbaijan, Sohrabi 10117A (hb. M. Sohrabi) Iran, East Azerbaijan, Sohrabi 3679 (H) Armenia, Mayrhofer & Harutyunyan 13-491 (GZU) Iran, Semnan, Sohrabi 9369 (hb. M. Sohrabi) Iran, Tehran, Sohrabi 5083 (H) JQ797543 JQ797537 JQ797525 JQ797545 JQ797536 – – – JQ797476 – – – JQ797508 Coccotrema coccophorum Coccotrema maritimum Coccotrema pocillarium Lobothallia alphoplaca Lobothallia alphoplaca Lobothallia farinosa Lobothallia melanaspis Lobothallia radiosa Lobothallia recedens Lobothallia recedens Megaspora verrucosa Megaspora verrucosa Megaspora verrucosa Argentina, Prov. Neuquén, Messuti 2001(F) Canada, British Columbia, Brodo 30130 (CANL) U.S.A., Alaska, Printzen (ESS 20863) Iran, East Azerbaijan, Sohrabi 4362 (H) Iran, East Azerbaijan, Sohrabi 3677 (hb. M. Sohrabi) France, Rhône-Alpes, Roux 25286 (UPS) Sweden, Jämtland, Nordin 6622 (UPS) Switzerland, Lumbsch (F) Sweden, Nordin 6035 (UPS) Sweden, Dalarna, Nordin 6582 (UPS) Austria, Styria, Trinkaus (GZU) Austria, Styria, Hafellner 48544 & Ivanova (GZU) Iran, East Azerbaijan, Sipman 55434 (B) – – – JQ797515 JQ797516 – HQ259272 – HQ406807 – AF332121 AF332122 – – AF329168 AF329163 AF329166 – JQ797480 HM060723 HM060688 DQ780274 – HM060724 – – JQ797483 – AF274096 AF329164 AF274093 – – HM060761 HM060726 DQ780306 – HM060762 – – JQ797498 Megaspora verrucosa Megaspora verrucosa Ochrolechia oregonensis Ochrolechia parella Ochrolechia tartarea Turkey, Prov. Çorum, Kinalioglu 1679 (B) Sweden, Jämtland, Nordin 6495 (UPS) Canada, Schmitt (F) France, Brittany, Feige (ESS 20864) Scotland, Coppins (ESS 21493) – – – – JQ797482 HM060687 DQ780276 AF329173 JQ797497 HM060725 DQ780308 AF274097 Ochrolechia yasudae Ochrolechia yasudae Pertusaria albescens Pertusaria amara Pertusaria amara Pertusaria hemisphaerica Pertusaria hemisphaerica Pertusaria lactea Pertusaria ophthalmiza Pertusaria velata Sagedia mastrucata Sagedia mastrucata Sagedia simoënsis Culture 0217M (AKITA) AFTOL-ID 882 Czech Republic, Bohemia, Schmitt (ESS 20967) AFTOL-ID 379 Germany, Rheinland-Pfalz, Killmann (ESS 20865) AFTOL-ID 959 Germany, Nordrhein-Westfalen, Schmitt (ESS 21065) Germany, Rheinland-Pfalz, Schmitt (ESS 21070) Scotland, Coppins (ESS 21498) Australia, Archer (ESS 21500) Sweden, Lycksele Lappmark, Nordin 5481 (UPS) Norway, Troms, Nordin 5708 (UPS) Norway, Troms, Owe-Larsson 9000 (UPS) – – – – – – – – – – – EU057914 EU057913 – AY300899 DQ780282 – AF329175 AY584713 – DQ973000 – AF381564 AY567993 AY300906 M060699 HM060698 HM060701 AY300848 – DQ986776 AF329176 – AF274101 – AF381556 AF381557 AY568006 AY300855 HM060737 HM060736 HM060739 238 Mycol Progress (2013) 12:231–269 Table 1 (continued) Species Locality and collector number (Herbarium) nrITS mtSSU nrLSU Sagedia zonata Sagedia zonata Sagedia zonata Sweden, Nordin 6006 (UPS) Sweden, Nordin 5949 (UPS) Norway, Troms, Owe-Larsson 8942 (UPS) EU057952 EU057953 – – – HM060700 – – HM060738 alignments, a procedure that has been shown to improve phylogenetic accuracy in some cases (Talavera and Castresana 2007). The Gblocks-modified nrITS data sets were used for subsequent phylogenetic anlyses. Phylogenetic analyses Phylogeny of the Megasporaceae using a combined data set (mtSSU + nrLSU) In order to study generic relationships of the Megasporaceae we used a combined data set of nrLSU and mtSSU sequences. Phylogenetic analyses were performed using both parsimony and Bayesian inference. Selected taxa from the genera Coccotrema Müll. Arg., Ochrolechia A. Massal., Pertusaria DC. and Varicellaria Nyl. were chosen as outgroup terminals and the tree was rooted at Coccotrema maritimum Brodo. Bayesian analysis was performed using the software MrBayes 3.1.2 (Huelsenbeck et al. 2001; Ronquist and Huelsenbeck 2003). MrModeltest was used to determine the most appropriate model using AIC, with GTR + I + G found to be the best-fitting model of nucleotide evolution. Two independent runs, each with four incrementally heated simultaneous Markov chains and a temperature of 0.2 were executed and run for ten million generations. Trees were sampled every 1000 generations resulting in an overall sampling of 2002 trees. From these, the first 1001 trees were discarded (burnin 0 1000). A stationary phase was reached before the burn-in threshold, as revealed by the plot of the MrBayes cold chain likelihood values against the generation number. Trees were visualized using the program FigTree v1.3. (http://tree.bio.ed.ac.uk/software/figtree/). In order to obtain additional measures of branch support, we conducted parsimony analysis using TNT 1.0 (Goloboff et al. 2008a, b). Gaps were treated as missing data in the combined data matrix. Branch support was assessed with 1000 bootstrap replicates using tree bisection reconnection (TBR) branch swapping with 100 random addition replicates in each replicate and holding 20 trees in memory during each round of swapping. The topology of the parsimony and Bayesian trees were congruent and only the Bayesian tree is shown (Fig. 1). Phylogeny of ‘manna lichens’ and allied species using nrITS rDNA To examine the infrageneric relationships of ‘manna lichens’, nrITS rDNA sequences were obtained from several of the earlier sampled genera such as Aspicilia, Agrestia, Chlorangium and Sphaerothallia, and a data set with 101 terminals was assembled. Phylogenetic analysis was performed using Bayesian inference. Outgroup taxa were chosen from the genera Aspicilia, Lobothallia, Megaspora and Sagedia and the tree was rooted at Lobothallia melanaspis (Ach.) Hafellner. The genus Lobothallia was found to be the sister genus to Aspicilia, Circinaria, Megaspora and Sagedia in the analysis of Megasporaceae based on a combined data set of nrLSU and mtSSU sequences (see also Nordin et al. 2010). MrModeltest suggested SYM + I + G as the best-fit model of DNA substitution. For the Bayesian analysis, parameters similar to those used for the combined data set (mtSSU + nrLSU) were applied, and the resulting trees were visualized using the program FigTree. The majority-rule consensus tree is shown in Fig. 2. Branch support values (PP ≥0.50) are indicated above branches. Results The family Megasporaceae The final data set consisted of 67 terminals with 1357 nucleotide positions and included 923 constant characters, 102 variable parsimony uninformative characters, and 332 informative ones. The strict consensus tree from the parsimony analysis was congruent with the Bayesian tree. Twenty-seven internodes have bootstrap values ≥75 %, and 34 have posterior probabilities ≥0.95 (Fig. 1). Relationships of the major lineages within the Megasporaceae differ somewhat from those in Nordin et al. (2010). Sagedia was confirmed as the sister genus to Aspicilia. Furthermore, Aspicilia and Sagedia (sensu Nordin et al. 2010) were recovered as sister to Circinaria and Megaspora, and Lobothallia was placed as sister to all other genera within the Megasporaceae; this is in accordance with Nordin et al. (2010). Circinaria and Aspicilia are monophyletic and sister groups (Fig. 1). The ‘manna lichens’ Sphaerothallia esculenta (Pall.) Reichardt, Chlorangium jussuffii (Link) Link, Fig. 1 The majority-rule concensus trees inferred by Bayesian analy-„ sis of the combined data sets of the nrLSU and mtSSU gene regions and using the GTR + I + G parameter model. The numbers above the branches refer to bootstrap and posterior probability support values (BS/PPS). Five genera are defined and indicated by distinguishing morphological characters Mycol Progress (2013) 12:231–269 239 0.04 Circinaria ‘Sphaerothallioid lichens’ 1-4-6 rarely 8 spored ascus, subglobose, globose, some ellipsoid present Megasporaceae Megaspora absent aspicilin in some subfruticose, few crustose, medulla massive aspicilin absent thick, two layers in some crustose 6–12 µm 8–35 µm 100/1.00 Family Genera Sagedia Aspicilia 8 spored ascus, ellipsoid absent absent thin, one layer crustose, some radiating or lobate, medulla thin absent Lobothallia Spore per ascus, shape Thallus,medula mass Cortex thickness Conidia length 3–8 µm 8–12 µm 8–12 µm 99/1.00 11–40 µm 97/1.00 100/1.00 0.95 1.00 0.96 0.75 0.99 100/1.00 99/1.00 substictic acid present in some, aspicilin absent Pertusaria ophthalmiza Pertusaria albescens Pertusaria amara Varicellaria rhodocarpa 99/1.00 Pertusaria velata 0.82 Pertusaria hemisphaerica 100/1.00 Pertusaria lactea Ochrolechia parella 100/1.00 0.75 Ochrolechia tartarea 1.00 Ochrolechia oregonensis 0.75 Ochrolechia yasudae Aspicilia myrinii Lobothallia melanaspis 95/1.00 100/0.95 Lobothallia alphoplaca 82/1.00 95/1.00 Aspicilia cernohorskyana Lobothallia farinosa 0.55 Lobothallia radiosa 99/1.00 Lobothallia recedens 0.82 99/1.00 Sagedia mastrucata 99/1.00 Sagedia mastrucata Sagedia simoensis 0.99 Sagedia zonata 63/086 Aspicilia cinerea Aspicilia cinerea 99/1.00 100/1.00 Aspicilia dudinensis Aspicilia dudinensis 83/1.00 Aspicilia indissimilis Aspicilia laevata Aspicilia laevata Aspicilia haeyrenii Aspicilia permutata Aspicilia verruculosa Aspicilia supertegens 0.74 Aspicilia supertegens 86/1.00 Aspicilia nikrapensis 0.86 Aspicilia rivulicola 61/1.00 Aspicilia mashiginensis 77/1.00 Aspicilia dendroplaca 67/1.00 Aspicilia dendroplaca Megaspora verrucosa 100/1.00 Megaspora verrucosa 0.62 Megaspora verrucosa Circinaria gibbosa 99/1.00 79/1.00 Circinaria leprosescens Circinaria leprosescens Circinaria contorta Circinaria contorta 57/1.00 Circinaria contorta Circinaria caesiocinerea Circinaria calcarea 0.96 Circinaria elmorei s. lat. 1.00 Circinaria esculenta 0.60 Circinaria emiliae 85/1.00 Circinaria emiliae 71/1.00 Circinaria fruticulosa 94/1.00 Circinaria hispida s. str. Circinaria hispida s. str. 0.55 Circinaria gyrosa Circinaria cerebroides 0.75 Circinaria jussuffii 100/0.77 Circinaria sphaerothallina Circinaria rostamii 0.78 Circinaria affinis Circinaria lacunosa 68/0.94 100/1.00 Chemistry: substictic acid, aspicilin Pseudocyphellae, clustered algal cells Coccotrema maritimum Coccotrema pocillarium Coccotrema coccophorum 240 Mycol Progress (2013) 12:231–269 Lobothallia melanaspis HQ259272 Lobothallia alphopalca JQ797515 Lobothallia alphoplaca JQ797516 Aspicilia tibetica GU289915 Lobothallia recedens HQ406807 Megaspora verrucosa AF332121 Megaspora verrucosa AF332122 Sagedia zonata EU057952 Sagedia zonata EU057953 Sagedia mastrucata EU057913 0.52 Sagedia mastrucata EU057914 Aspicilia epiglypta EU057907 0.81 Aspicilia indissimilis EU057909 0.90 Aspicilia laevata EU057910 0.89 Aspicilia cinerea EU057899 Aspicilia cinerea AF332110 Aspicilia cinerea AF332111 Circinaria cupreogrisea EU057903 Circinaria gibbosa EU057908 Circinaria leprosescens EU057911 Circinaria arida HQ406801 Circinaria arida HQ406800 Circinaria arida EU057905 Circinaria calcarea EU057898 Circinaria calcarea HQ406804 0.80 Circinaria contorta EU057900 0.83 Circinaria contorta AF332108 0.60 Circinaria contorta AF332109 0.91 Circinaria sp HQ389202 C SP1 Circinaria sp JQ797544 V Circinaria sp JQ797550 C Circinaria alpicola JQ797556 V Circinaria alpicola JQ797554 V Circinaria alpicola JQ797524 C Circinaria alpicola JQ797552 V Circinaria sphaerothallina JQ797537 C 0.51 Circinaria sphaerothallina JQ797525 C Circinaria sphaerothallina JQ797536 C Circinaria sphaerothallina JQ797543 C Circinaria aschabadensis GU289916 V 0.98 Circinaria aschabadensis JQ797519 C Circinaria aspera JQ797531 C Circinaria jussuffii JQ797518 V 0.89 Circinaria jussuffii JQ797521 V Circinaria gyrosa JQ797532 Circinaria gyrosa JQ797528 Circinaria gyrosa JQ797540 Circinaria gyrosa JQ797539 Circinaria gyrosa JQ797557 0.86 Circinaria gyrosa JQ797514 Circinaria fruticulosa HQ171226 Circinaria fruticulosa HQ171229 Circinaria fruticulosa HQ171227 Circinaria fruticulosa HQ171228 Circinaria fruticulosa HQ389195 Circinaria fruticulosa JQ797535 Circinaria fruticulosa HQ389199 Circinaria fruticulosa JQ797555 Circinaria affinis HQ171237 V Circinaria affinis HQ389196 V Circinaria affinis HQ389194 V Circinaria sp JQ797549 C SP2 Circinaria sp JQ797547 V 63/0.84 Circinaria sp JQ797548 C Circinaria emiliae JQ797512 V Circinaria emiliae JQ797513 V Circinaria elmorei s.lat. HQ406802 C Circinaria esculenta HQ406803 V 0.85 Circinaria esculenta JQ797511 V Circinaria esculenta JQ797510 V Circinaria lacunosa JQ797517 Circinaria lacunosa JQ797520 Circinaria cerebroides JQ797553 Circinaria cerebroides JQ797534 0.59 Circinaria cerebroides JQ797529 0.80 Circinaria rostamii JQ797541 Circinaria rostamii JQ797533 0.98 Circinaria rostamii JQ797527 Circinaria rostami JQ797538 Circinaria elmorei s.lat. HQ389203 C 0.60 Circinaria digitata HQ171230 V Circinaria digitata HQ171236 V Circinaria elmorei s.lat. JQ797558 C 0.80 0.96 Circinaria elmorei s.lat. JQ797542 C Circinaria elmorei s.lat. JQ797526 C 0.89 Circinaria elmorei s.lat. HQ389200 C 0.70 Circinaria rogeri HQ171232 V Circinaria rogeri HQ171231 V Circinaria elmorei s.lat. JQ797551 C Circinaria sp JQ797530 C SP3 Circinaria sp JQ797546 C Circinaria hispida s.str. HQ406806 V 0.75 Circinaria hispida s.str. HQ171233 V Circinaria hispida s.lat. HQ389201C 0.56 Circinaria hispida s.lat. JQ797509 C Circinaria hispida s.str. HQ171234 V Circinaria hispida s.str. Q171235 V 0.96 Circinaria hispida s.str. HQ389197 V Circinaria hispida s.str. HQ389198 V Circinaria hispida s.lat. JQ797522 V Circinaria hispida s.lat. JQ797523 C 0.1 erratic or vagrant Circinaria vagrant erratic or vagrant 0.92 Sphaerothallioid species vagrant erratic or vagrant 0.57 0.77 0.93 0.55 0.70 0.94 crustose 0.58 Mycol Progress (2013) 12:231–269 The majority-rule concensus trees produced by the Bayesian analysis of the nrITS rDNA sequences and using the SYM + I + G parameter model. Numbers above branches indicate posterior probabilities obtained from analysis. Branches in bold indicate PP ≥95 %. Three species are designated as ‘Circinaria sp’ and will be described elsewhere ƒFig. 2 and Agrestia hispida (Mereschk.) Hale & W.L. Culb., were included in the analysis and these type species of the old genera were found to be nested within Circinaria. Therefore, these genera are synonymized under Circinaria. Vagrant and crustose sphaerothallioid species form a separate clade of their own (Fig. 1). Inside Circinaria clade some crustose species (C. caesiocinerea, C. calcarea, C. contorta, C. gibbosa and C. leprosescens) with certain morphological and chemical differences are distinguished from sphaerothallioid species and form a small group of their own. Aspicilia cernohorskyana (Clauzade & Vězda) Cl. Roux is nested in the Lobothallia clade. Its formal combination of Lobothallia cernohorskyana (Clauzade et Vězda) A. Nordin, Cl. Roux et Sohrabi was made in Roux (2012). 241 paraplectenchymatous tissue that is indistinct in the inner part, mixed with prosoplectenchymatous tissue of the medulla. The results also show that C. elmorei and C. hispida s.str. are not monophyletic. These two species remain in need of more detailed study. Some taxa in this clade are crustose, growing on pebbles (e.g., C. elmorei s.lat., C. hispida s.lat.) while others are vagrant (e.g., C. digitata, C. hispida s.str., and C. rogeri). Circinaria sp. 3 is terricolous-crustose, and a vagrant form of this species has not been found. C. hispida s.str. in Fig. 2 refers only to the vagrant morphotype that is shown to be morphologically corresponding with the type specimen (TU). The C. hispida clade includes specimens that are morphologically distinct from each other, although these two forms do not form monophyletic groups. All undescribed crustose species in Circinaria are represented by multiple specimens. Since these species are primarily crustose they will be studied separately and in detail elsewhere, together with additional saxicolous species. Identifying sphaerothallioid species ‘Manna lichens’ and allied species This data set consisted of 101 terminals with 437 nucleotide positions and included 255 constant characters, 43 variable characters that were parsimony uninformative, and 169 parsimony informative ones. The majority rule consensus tree resulting from the Bayesian analysis with posterior probability support values is shown in Fig. 2. The analyses resulted in 37 internodes with posterior probabilities ≥0.95. The basal clades include the following crustose species, all of which lack pseudocyphellae and some of which contain aspicillin. These are Circinaria arida, C. calcarea, C. contorta, C. cupreogrisea, C. gibbosa and C. leprosescens. Sphaerothallioid species are divided into four clades. One of these consists of an undescribed species (Circinaria sp. 1), while another is formed by C. alpicola, C. aschabadensis, C. aspera, C. jussuffii and C. sphaerothallina. C. fruticulosa and C. gyrosa are combined in a clade of their own, with the rest of the species forming a single clade. The relationships of vagrant and crustose species remain unresolved. The largest clade includes several vagrant species (C. emiliae, C. cerebroides, C. esculenta, C. lacunosa and C. rostamii). This clade also includes an undescribed erratic species (Circinaria sp. 2), which is shown to be monophyletic, and also a crustose species which is placed in a clade together with C. elmorei and C. hispida. The results also show that species with a distinct two-layer cortex (outer part paraplectenchymatous, inner part prosoplectenchymatous) are not monophyletic. For example, C. digitata, C. fruticulosa, C. hispida and C. rogeri have two distinct layers in their cortices, although they group together with other crustose species that have single layered cortices. C. fruticulosa and C. gyrosa form a clade, while C. gyrosa has an uneven cortex layer with Important anatomical, chemical, ecological and morphological features for distinguishing the different sphaerothallioid species are: 1) thallus growth form, 2) presence of pseudocyphellae, 3) presence and shape of lobes/branches/subsquamulose, 4) cortex thickness: presence/absence of two different tissues (paraplectenchymatous and prosoplectenchymatous), 5) conidium size and 6) presence/absence of certain chemical compounds (aspicilin, stictic acid and hypostictic acid). Key to the world’s ‘manna lichens’ The first attempt to provide an identification key to the ‘manna lichens’ dates back to the middle of last century (Szatala 1957) and the resultant work has subsequently been improved by Poelt (1969), Oxner (1971) and Andreeva (1987). The present revision shows that 15 species of ‘manna lichens’ can be distinguished and a key is presented to cover them. Saxicolous forms are excluded from the key until further data becomes available. 1 Thallus vagrant, subfruticose or subfoliose, surface even, with shallow cracks, integrated, cortex thickness uneven.........................................................................................2 ─ Thallus vagrant, subfruticose, sometimes erratic (attached or imbedded in soil or invades pebbles), surface uneven and detached, cortex thickness even when thallus distinctly branched...............................................................5 2 (1) Thallus subfoliose, tube-like, wrinkled, c.10–40 (–50) mm wide, margin sometimes incised, often yellowish grey or muddy color, conidia (8–)9–11(–12) μm..............................................................C. emiliae (Fig. 4b) 242 ─ Thallus subfruticose, truffle-like, cerebriform, nodulose, strongly folded.............................................................3 3 (2) Thallus large, c.10–30(–40) mm wide; found in high mountains (alpine zone), hard, surface without large rounded pits, conidia (10–)12–13(–15) μm.......................C. cerebroides (Fig. 4c) ─ Thallus medium sized, c.10–20(–30) mm wide; below alpine zone...........................................................................4 4 (3) Thallus c.10–20(–30) mm wide, rather crumbly, surfaces with rounded pits, larg up to 0.5–1.5(–2) mm diam., conidia (8–)11–16(–21) μm, mostly found in lowlands..........................................C. lacunosa (Fig. 4a) ─ Thallus c. 10–25(–35) mm wide, rounded to irregular, folded in some parts, flexuose, often brain-like appearance, with rimose surfaces and areole-like lobes, conidia (8–)10– 12(–16) μm...................................C. rostamii (Fig. 3e, f, h) 5 (1) Thallus broadly ellipsoidal to globose, gyroid, comprises brain-like lobes or forming compact subsquamulose pieces, 5–40 mm wide.........................................................6 ─Thallus more or less subglobose distinctly branched, dwarfed form, 5–30 mm wide............................................13 6 (5) Thallus vagrant, subglobose to irregular, severely wrinkled, forming compact subsquamulose pieces, surfaces cracked, lobes pressing against one another........................7 ─ Thallus often vagrant, rarely found erratic gyroid, brain-like lobes....................................................................9 7 (6) Thallus forming compact pieces, small c. 3-15 mm wide.........................................................................................8 ─ Thallus forming undulating surface, large, c. 10–30(–40) mm wide..................................................................................9 8 (7) Thallus vagrant, small (c. 3–8 mm), yellowish to grey, apothecia abundant, pseudocyphellae uncommon, conidia (15–)17–22(–25) μm......................C. tominii (Fig. 4i) ─ Thallus vagrant, large (c. 3–15 mm), forming compact subsquamulose pieces, more or less elevated in some parts, often reddish brown (stictic acid and hypostictic acid present in some thalli), pseudocyphellae whitish spots, dotting the surface, conidia (8–)10–14(–16) μm........C. jussuffii (Fig. 4d) 9 (7) Thallus c. 10–40 mm wide, subsquamulose, forming undulating surface, in large pieces (3–8 mm wide), often angular, uneven, wavy or bent, pieces densely joined together by flexuose, thickened margins, apothecia and pycnidia rare, conidia (10–)18–30(–35) μm.....................C. esculenta (Fig. 4f) ─ Thallus c. 5–30 mm wide, gyroid, more or less irregular, surfaces forming short dumpy to conical lobes, undulating in some parts, verrucose..........................................10 10 (9) Thallus vagrant, forming brain-like lobes, pycnidia rare, without carbonized ostiole.........................................11 ─ Thallus often vagrant, sometimes erratic (invades pebbles), folded, forming short dumpy lobes, more or less conical lobes, pycnidia common often erumpent, with carbonized ostiole...................................................................12 Mycol Progress (2013) 12:231–269 11 (10) Thallus folded, composed of tiny (0.7–1.5 mm) dumpy lobes, somewhat depressed, conidia (15–)18–20 (–25) μm...................................................C. affinis (Fig. 5e) ─ Thallus more or less globose, dumpy lobes rather large (0.7–3 mm), densely jointed, conidia (8–)10–16(–18) μm.......................................................C. gyrosa (Fig. 3a, c) 12 (10) Thallus, medium sized, c. 5–12 mm wide, twisted in some parts, surface somewhat warty to verrucose, more or less areolate, apothecia and pycnidia abundant, conidia (8–)13–18 (–20) μm.............................................C. aschabadensis (Fig. 5d) ─ Thallus variable, c. 5–30 mm wide, appears more or less branching form with distinct conical lobes, thallus erratic, (sometimes on pebbles), composed of areoles, apothecia and pycnidia rare, conidia (7–)8.2–10.5 (–12) μm..............................................C. alpicola (Fig. 4g, h) 13 (5) Branching polytomic, isotomic or anisotomic, irregular, some branches occasionally dichotomic.............14 ─ Branching commonly dichotomic, regular, sometimes anisotomic with thin branches............................................15 14 (13) Thallus 10–20 mm wide, central branches often thick, pycnidia common with conspicuous black ostiole, conidia (7–)10–13(–16) μm..................C. rogeri (Sohrabi et al. 2011b. Fig 2) ─ Thallus 5–10 mm, branches more or less single and distinct, digitate, irregularly divided, pycnidia and conidia not seen......................C. digitata (Sohrabi et al. 2011a. Fig 2) 15 (12) Thallus c. 5–20(–30) mm, well-branched mostly dichotomic, often narrow to thick branches, radiating from central part, branchlets slender, usually with hollow or small depression on apices, pycnidia rare, usually located in depression at the uppermost part of branchlets, conidia (8–)9–12 (–13) μm............................................C. fruticulosa (Fig. 5f-h) ─ Thallus different, primarily terricolous (attached or imbedded in soil) later stages turning vagrant, appear both in vagrant and erratic forms...............................................16 16 (15) Thallus terricolous, with numerous branches, mostly anisotomic infrequently dichotomic, often narrow and cylindrical, with black tips on top of branches, conidia 8–12(–14) μm..............................C. hispida s.str. (Fig. 5a-c) ─ Thallus usually saxicolous (on rocks) or rarely terricolous (imbedded in soil) in later stages turning into vagrant or erratic forms...................................................................17 17 (16) Thallus distinctly areolate, thorn-like extensions elevated and elongated in some parts...........C. hispida s.lat. ─ Thallus more or less areolate, without thorn-like extensions (vagrant form not seen), conidia variable between 8 and 25 μm..................................................Circinaria spp. Note: Several additional saxicolous or erratic species are included in Circinaria spp., i.e. C. aspera, C. elmorei, Circinaria sp. 1, Circinaria sp. 2, Circinaria sp. 3, and Aspicilia (Circinaria) tortuosa (H. Magn.) N.S. Golubk., but will be studied in detail later. Mycol Progress (2013) 12:231–269 Fig. 3 a-d Habit and habitats of Circinaria gyrosa in Iran. a C. gyrosa (Sohrabi 9496, H, IRAN, hb. M. Sohrabi). b The habitat of the species in open Juniper forest in Shah Kuh and Jahan Nama district in Golestan Province. c C. gyrosa (holotype). d 32 km N of Marand towards Jolfa, habitat of C. gyrosa on low calcareous rock outcrops in Astragalus steppe. e-i Habit and habitats of C. rostamii in Iran. e-f Habit of C. 243 rostamii (holotype). g Habitat of C. rostamii in Jolfa district 1 km N of Asiab Kharabeh waterfall, Stachys steppe with gravelly, loamy soil and low calcareous outcrops. h-i Habit of C. rostamii (Sohrabi 9364, hb. M. Sohrabi) and Zygophyllum steppe with gravelly soil in Semnan, c. 80 km S of Shahrud 244 Mycol Progress (2013) 12:231–269 Fig. 4 a Circinaria lacunosa (Piregoudov, LE). b C. emiliae (Savicz: Lich. Ross. No. 115, H). c C. cerebroides (Ringel & Jaschhof 5184, H). d C. jussuffii (Rabenhorst, Lich. Europaei, No. 199, H). f C. esculenta (Savicz: Lich. Ross. No. 63 C, H). g C. alpicola (Litterski 4848, H). h C. alpicola (Ringel & Jaschhof 5183, H). i C. tominii (Baranov, H) The species 1907. ≡ Aspicilia alpinodesertorum f. esculenta-alpina Elenkin in Izv. Imp. S.-Peterburgsk. Bot. Sada 1: 36. 1901 (16 July), as ‘esculenta alpina’ or ‘esculenta (alpina)’. 0 Aspicilia alpinodesertorum f. fruticulosofoliacea Elenkin in Izv. Imp. S.-Peterburgsk. Bot. Sada 1: 27, 36, 39, tab. 2, rows IX & X, figs. 1-7. 1901, as ‘fruticuloso-foliacea’ ≡ Circinaria alpicola (Elenkin) Sohrabi comb. nov. (Fig. 4g-h). MB 563021 Basionym: Aspicilia alpicola Elenkin, Fl. Lishaynikov Sredney Rossii [Lichenes Florae Rossiae Mediae] 2: 222. Mycol Progress (2013) 12:231–269 245 Fig. 5 a-c Circinaria hispida s.str. (Sohrabi 15068, hb. M. Sohrabi, H). d C. aschabadensis (Borisova, LE). d C. affinis (Mereschkowsky, Lich. Ross. Exs. No. 19, H). f-h C. fruticulosa (Obermayer: Lich. Graecenses No. 321, GZU) Aspicilia fruticulosofoliacea (Elenkin) Sohrabi in Taxon 59: 627. 2010. Lectotype: designated by Sohrabi and Ahti (2010), Kyrgyzstan. ‘Ad terram argillosam in regione alpina montium Tian-Shan (Kaschgariae: Werchnij Syrt 12000 ft. ped)’, 1889, Roborowsky in Elenkin, Lich. Fl. Ross. No. 24d (H, isolectotypes LE 4 specimens). Thallus vagrant, subfruticose, consists of folding short dumpy lobes, 0.5–2 cm tall and 0.5–2(–3) cm broad, often irregularly shaped, rarely spherical, sometimes flat, rarely flattened, regularly and plainly lobate, lumps consists of short lobes, 0.2–0.6 cm, dense, compact, areole-like, verrucose, radiating from the central part. Axial part hard, variable in size, up to 1.5–5 mm in diam. Surface deeply cracked, and often covered by dust, otherwise dull brown, brownish grey to greyish green, sometime whitish grey, pale olive-brown to pale brown; surface in covered side usually darkish green, dark brown, greenish brown or almost dark green-brown, (sometime reddish brown when ferriferous sediments are present in soil). Pseudocyphellae common, ±white pit-like, conspicuous spots along the lobes. Cortex 246 thickness uneven, one layer, paraplectenchymatous, (40–) 60–90(–110) μm thick, cells (4–)6–7(–10) μm in diameter, inner part indistinct, more or less prosoplectenchymatous tissue, mixed with anticlinal hyphae of medulla. Epinecral layer 4–10(–15) μm thick. Photobiont chlorococcoid, cells 5–8(–22) μm in diam., clustered in small groups, each group up to 80–180×50–110 μm broad. Medulla prosoplectenchymatous with irregularly oriented hyphae (textura intricata), white, I-, containing crystals of calcium oxalate, visible in (H2SO2 10 % solution). Apothecia immersed, aspicilioid, rare, up to 0.5–1.5(–2) mm broad, among the branches in older parts. Disc black to brownish black, pruinose, concave to convex when young, becoming more flat when old. Thalline exciple flat to ± elevated and prominent in older apothecia, entire, concolorous with thallus or with a thin to thick white rim. True exciple (45–)65–85(–100) μm wide, ± I + medially blue, uppermost cell brown, ± globose, 4–5(–7) μm in diam. Epihymenium brown, K + fading from brown to light yellowish green, N + pale green (caesiocinerea-green). Hymenium hyaline, occasionally with few oil drops, (100–)110–120(–130) μm tall. Paraphysoids moniliform to submoniliform, with upper cells ± globose, 4–7 μm wide, in lower part 5–9 × 2–3 μm wide, branched. Hypothecium and subhymenium pale, (40–)50–65(–75) μm thick. I + blue. Asci broadly clavate, (75–)80–95(–100)× 20–30(–35) μm, with thick apical dome, up to (20–30 μm thick); 2–4(–5) spored. Ascospores hyaline, simple, globose to subglobose, (16–)19–[22.1]–24(–26)×(16–)19–[21.6]– 23(–25) μm (n030). Pycnidia rare, immersed, sometimes occur in the pseudocyphellae, single, stretch flask-shaped, ostiole frequently black to brownish but occasionally pale brown and slightly raised, pale, internal wall colourless. Conidia filiform, straight to very slightly curved (8–)9–11 (–13)×1–1.2 μm (n037). Chemistry: All spot-tests (K, C, KC, CK, P) negative both in the cortex and medulla. TLC and HPLC: No substances detected. UV: Negative. Ecology and distribution (Fig. 6c). Circinaria alpicola is widely distributed in Kyrgyzstan. It grows in vagrant form on the gravely soil, but some specimens have been found on pebbles as well. They are usually sparse forming scattered populations. All examined specimens originated from high altitudes in Tian-Shan Mountains that have a cold winter climate. Alpine plants such as Festuca and Kobresia spp. were reported as dominant in these habitats. According to the data derived from the labels of examined specimens, C. cerebroides and C. hispida are two other vagrant or erratic species growing in similar habitats in Kyrgyzstan. Remarks. The epithet ‘alpicola’ has been overlooked for many years and it was recently resurrected by Sohrabi and Ahti (2010). The lectotype of C. alpicola is poorly characterized (without fertile apothecium and conidia). It is aggregated into a compact, vagrant thallus and differs from the type of Aspicilia fruticulosofoliacea by lacking conical and Mycol Progress (2013) 12:231–269 short dumpy lobes. This type of dissimilarities of thallus form is particularly evident in the mixed collection of both species. Both taxa appear to be confined to alpine habitats in the Tian-Shan Mountains. Therefore, Sohrabi and Ahti (2010) recognized them as distinct taxa. In order to check the affinity of A. fruticulosofoliacea with C. alpicola we examined several collections, including fresh specimens, and found that the conidia, ascospores, and ecology of both species were identical. Nonetheless C. alpicola seems to be a very polymorphic taxon representing high rate of plasticity in some external features (e.g., lobe shapes). Molecular study was conducted on fresh specimens of both variants (see Figs. 3 and 4). These results supported the conclusion that the two taxa are conspecific, and the lobe morphology is not a reliable character to distinguish them. C. alpicola is morphologically similar to C. gyrosa, a new species which is described below. It seems that C. gyrosa has different habitat preference and distribution. So far C. gyrosa is known from steppes and shrub lands with outliers in the Mediterranean area in Spain and SW Asia (Armenia, Azerbaijan, Iran, Turkey and Turkmenistan) and it was recorded at lower altitudes at c. 1000–2000 m, while C. alpicola is an alpine species known from high altitudes of Tian-Shan Mountains, recorded at c. 2000–4000 m. Our nrITS analysis clearly showed that the two taxa are distinct (Fig. 2). Additional literature reports. In Spain, reports of Aspicilia fruticulosofoliacea Elenkin (nom. inval.) by Barreno et al. (1998) and Sancho et al. (2000) most probably refer to a species described as A. gyrosa. Some specimens of C. alpicola have been misidentified as A. fruticulosa (syn. C. fruticulosa) in Litterski (2002) and they are corrected here. Specimens examined. Kyrgyzstan. Depression of Sonköl Lake, 3023 m, 1970, Bredkina 445 (LE). Alai-Tal, S (Berhalb) Fluss Kysylcy, W Berg Maltabar, Matten, 3400 m, 1999, Litterski 4848 (H). Tian-Shan, Kashgaria, Roborowsky (LE). Central Tian-Shan, Moldo-Too, near River Mengoush, c. 3100 m, 1970, Bredkina 434 (LE). Vicinity of Bishkek, Ak-Say River, 2000 m, sine dato, Fetisov (H), Central Tian-Shan, reach At-Bashi, Crosing Kandi 3300 m, 1970, Bredkina (LE). Tian-Shan, NE AtBashi, c. 3 km from E Bosagu, 2700 m, 1970, Bredkina (LE). Tian-Shan, At-Bashi, Kyndy velley, 3300 m, 1970, Bredkina 558 (LE). Central Tian-Shan, 3820 m, 1949, Kozlova (H). Central Tian-Shan, Sothern slope, At-Bashi, valley of Ak-Say River, 4 km higher from Kyzyl-say River, 3300 m, 1970, Bredkina (LE). Tian-Shan, Samir Region, high mt., in place like ‘Nomadic camps’, Ak-Say, 1903, Lipsky (LE). Southwest Spor, of the Teskey Ala-Too Ridge (S of Sanköl Lake), Kala Fiorgo, Dolon, 3040 m, 1970, Bredkina (LE). Valley of Kalmak River, 3200 m, 1970, Bredkina (LE). Fergansky Mountain, Ken-köl, EspeeTawo, c. 3000 m, 1911, Tagantsev (LE). Tian-Shan, AtBashi-Gebirge, pass (Ottotisch) im SW des Gebiges Mycol Progress (2013) 12:231–269 247 Fig. 6 Known distribution of the ‘manna lichens’ in Eurasia and North Africa based on specimens of this study. The map outline was produced using PanMap software (http://www.pangaea.de/software/PanMap/) Hochgebirgssteppe, Grasland, 3300 m, 2000, Ringel 5137 (H). Alai-Gebirge, Paβ Talbyk, Matten, 3600 m, 1999, Litterski 4802 (H). SW Tian-Shan, NE Abhange des Fergana Gebirges, SW Chatyr-Kol, 3261 m, 2003, Ringel 5241 (H). Son-Köl-Plateau, Südufer der Son-Köl, 2007, Ringel & Jaschhof 5179 (H). Terskej Alataou, Arabel-Suu Hochebene, 3800 m, Ringel & Jaschhof 5181 (H). AksaiTal, Dshangy-Dsher, Karagerme Fluss, 3700 m, 2007, Ringel & Jaschhof 5183, 5186 (H). Issyk-Kulskaya Region, Sirtybakrovsky district, Karasay, 3200 m, 1957, Popova (LE). Tian-Shan, Verkhnii Syrt, 3600 m, 1889, Roborowsky (LE). Tian-Shan, Ak-Say, 3000 m, 1882, Fetisov (LE), ibidem, 3300 m, Fetisov (LE). Exsiccata. Kyrgyzstan. In valle Ak-Sai (10 000 ft. ped) jugi Tian-Schan, ad fines Kaschgariae, 1882, Fetisov, in Elenkin: Lich. Fl. Ross. No. 24i, as Aspicilia 248 alpinodesertorum f. fruticuloso-foliacea (H, LE, W). TianShan, the valley of Akh-Say River, 3300 m, 1862, Fetisov in Elenkin: Lich. Fl. Ross. No. 24a, as Aspicilia alpinodesertorum f. fruticuloso-foliacea (H, LE L1987) ‘Ad terram argillosam’ 1889, Roborowsky, in Elenkin: Lich. Fl. Ross. No. 24 h, as A. alpinodesertorum f. fruticuloso-foliacea (H, LE). Circinaria affinis (Eversm.) Sohrabi comb. nov. (Fig. 5e). MB 563034 ≡ Lecanora affinis Eversm. in Nova Acta Phys. Med. Acad. Caes. Leop. Carol. Nat. Cur. 15: 351. 1831. Lectotype: designated by Sohrabi and Ahti (2010), Kazakhstan. ‘Desertis Kirgisorum’ [1820], Eversmann in Elenkin: Lich. Fl. Ross. No. 24e (H). Thallus vagrant, subfruticose, almost subglobose, rarely irregular in shape, sometimes slightly bent or convoluted, or flattened, (0.5–)1–2.5(–3) cm in diam., wide, wavy, confluent, verrucose, with elevated areole-likes lobes, lobes at top depressed or flattened, varying in size, 0.5–1(–1.5) mm wide. Surface earthy, grey, grey-green, brick- to rustcoloured (when ferriferous sediments are presentin soil), rarely yellowish brown and matt; with shallow to deep cracks. Pseudocyphellae dot-like, rounded or extremely narrow, infrequently with whitish pruinose depressions. Cortex indistinctly delimited, thickness uneven, paraplectenchymatous, (40–)60–90(–100) μm thick, cells (4–)5–8(–11) μm in diam., ± brown, in some parts inner part indistinct, mixed with prosoplectenchymatous tissue of medulla and usually appearing as distinct layer (30–)40–80(–90) μm thick. Epinecral layer 4–15(–20) μm thick. Medulla white, I–, often muddy, depend on thallus size, 0.3–10 mm in central core, consists of calcium oxalate crystals. Photobiont chlorococcoid, cells 5–15(–22) μm in diam., often in groups up to 100–250×100–180 μm wide. Apothecia aspicilioid, rare, often single, up to 1–2 per lobe, deeply immersed. Disc black to brown, irregular in shape, rounded to fissure-like, white-pruinose, concave, surrounded by very thick margin, which is emergent, more or less flexuose, curved inside, surrounded by thin white rim. Thalline exciple 0.2–0.9 mm thick, entire or slightly cracked, more or less even or slightly flexuose, curved inside. True exciple black-brown, 45–60(– 70) μm wide. Epihymenium brown, 15–30 μm tall, K + colour fading from brown to yellowish green, contains insoluble crystals of calcium oxalat, N + pale green (caesiocinerea-green). Hymenium colourless, 100–125 μm tall, I + blue. Paraphysoids moniliform to submoniliform, tightly conglutinate in hymenial gelatine, with upper cells ± globose, 4–5 μm wide, in lower part with ± cylindrical cell 4– 6×1.5–2.5 μm wide; occasionally branched. Hypothecium and subhymenium colourless, 70–85 μm thick. Asci clavate, strongly thickened above, 95–120×25–35 μm, 2–3 (–4), with 1–4 spores. Ascospores almost globose, 18– 24 μm diam. Pycnidia common, immersed, sometimes occur in the pseudocyphellae, single, stretched flask- Mycol Progress (2013) 12:231–269 shaped, internal wall colourless, frequently with black to brownish ostiole. Conidia filiform, straight to very slightly curved (15–)16–20(–25) × 1–1.2 μm (n 049).. Chemistry: All spot-tests (K, C, KC, CK, P) negative both in cortex and medulla. TLC and HPLC: No substances detected. UV: Negative. Ecology and distribution (Fig. 6h). Occurs between small pebbles in stony steppes and deserts, sometimes on salty soil in plains and mountain slopes of Central Asia. It is often found in the same habitat with Circinaria esculenta and C. emiliae. Associated plants include: Anabasis salsa, A. turanica, Artemisia glauca, Atriplex cana, Camphorosma monspeliaca, Haloxylon aphyllum, H. persicum, Kochia prostrata, Nanophyton erinaceum and Salsola arbusculiformis. Remarks. In some publications the name Aspicilia vagans Oxner was used for this species (e.g., Oxner 1971, Andreeva 1987, Sohrabi and Ahti 2010, Urbanavichus 2010). This was because of homonymy between Aspicilia affinis Anzi and A. affinis (Eversm.) Mereschk. (see also Sohrabi and Ahti 2010). Morphologically C. affinis is somewhat similar to C. aschabadensis and C.gyrosa. All three species have short dumpy lobes, verrucose thallus surface, with uneven thickness in their cortex. Circinaria aschabadensis is fertile, often with several apothecia per thallus, pycnidia are abundant, erumpent, often with a carbonized ostiole, sometimes located in the pseudocyphellae, and its conidia are (8–)10–12(–16) μm. It is known from a very small area in Kopet-Dagh, the area close to the border of Turkmenistan and Iran. Circinaria affinis is often found sterile, and apothecia and pycnidia are very rare. Carbonized ostioles are lacking in the pycnidia and conida size is (15–)16–20(–25) μm. Its range is very wide, including many localities in Central Asia. Circinaria gyrosa produces irregular, large, dumpy lobes, is more or less brain-shaped, and has deep cracks and very large pseudocyphellae. Its conidia are rather small, (8–)10–16(–18) μm long. It is distributed in the Mediterranean area mostly in the shrub-steppes of the mountains of Spain, Turkey and Iran. Phylogenetic relationships of the three species are shown in Fig. 2. Additional literature reports. Circinaria affinis has been reported from Central Asia by (Eversmann (1831; Mereschkowsky 1911a, b)”. It is also reported from Mongolia by Klement (1966). Mongolia by Klement (1966). The record from Turkmenistan by Dzhuraeva (1978) needs to be confirmed. The record by (Crum 1993) from Iran is referred to as C. aschabadenis (see also Sohrabi and Ahti 2010). Circinaria affinis is also reported from Turkey (Berkeley 1856, as Lecanora affinis). The report by Berkeley (1856, 1857, as Lecanora affinis) most probably refers to C. gyrosa. Reports from Kyrgyzstan by Bredkina (1981) need to be confirmed. Some specimens of Aspicilia fruticulosofoliacea (Litterski 2002; No. Litterski 4848, 4876, and 5137; H) have been misidentified as A. vagans. They belong to C. alpicola. Mycol Progress (2013) 12:231–269 The record Aspicilia affinis (Eversm.) Mereschk. (syn. C. affinis) from Tunisian steppes by Seaward (1996) needs to be confirmed. Specimens examined. China. Xinjiang, Tacheng Toli, 1232 m, 2007, Abbas & Xahidin 20080364 (H). Kazakhstan. Akmola Region, valley of Shebdar, 1957, Andreeva (LE); ‘Asia Media, Deserto Kirkisorum’, 1822, Fischer (G, LE). ‘Deserto Kirgizorum’, 1820, Eversmann (G). Karagandinskaya Region, Aktogaysky district, KonurTobe, 1967, Leontieva (LE). Karkaralinsky district, mountain range Konur Tobe, 1967, Leontieva (LE). Central Khazakhstan, near Kanour Tobe, c. 25–30 km from village Kenistan on SE direction, 1967, Leontieva (LE). Mountain Berchogur, 1904, Dubiansky (LE). ‘Around the Caspian Sea’ (probably in N of the Caspian Sea in Russia), sine coll., in the same envelope with C. fruticulosa (UPS). Saarat Mountain, Cherichen, 1903, Keller (LE, W). Aqtöbe Region, East slope of Mughalzhar Mts., Birshogir, 1904, Dubiansky (LE). Mugoder, ‘Mugodzharskaya’ Mountain, N of Ust-Urt, near Koshkar-Ata, 1904, Dubiansky (LE). Pesky Bolshye Barsuky or Ust-Urt, 1904, Dubiansky (LE). Togayisk Region, 30 km N of Lake Chalkar, 1898, Krukov (LE). Mongolia. Altai, the road from Chench Somona to Bolghan Somon, c. 1500 m, 1981, Choy 5224 (LE). Govi-Altai aimag, ‘Gulin sum’ [no such sum any more], 3 km along Zavkhan River from ‘Gulin’, 1978, Biazrov 8389 (LE). Russia. Altai Republic, Tarkhatty River (tributary of Chuya), 1931, Rassadina (LE). Deserto Czuensi, Altai Austro-Orientalis, 1926, Baranov (S), ibidem, 1929, Baranov (S). Altai, Ovrutsky Region, near Tarkhaty River Chuy, 1931, Rasadina (LE). Astrakhan Region, Aktobinsky Region, vicinity of Lake Bashkunchak, 1926, Savicz (FH). Astrakhan, Berg Bogdo, 1927, Tomin (FH, S, US), ibidem, 1926, Tomin (FH, H, S, TUR, US). Berg Bogdo, 1910, Mereschkowsky (TU, US, W). Astrakhan Region, Bogdo Mt., semidesert, 1903, Keller (LE). Near the Lake Baskunchak, 1903, Keller (LE). Near Lake Baskunchak, W slope of mount big Bogdo, 1996, Kulakov 1408 (LE, M). Akhtuba District, E shore of Baskunchak Lake, Vakh-Tau Upland, 20 m, 1997, Kulakov (LE). Semipalatinsk Region, Zaysansky uyezd, river Ul-kut-Togas, 1914, Prasolov (LE); ‘Tartaria’, sine dato & coll. (determined by Nees von Esenbeck), in the same envelope with C. esculenta (UPS). Uzbekistan. Fergana, near airport, 1948, Schafeev (LE) Exsiccata. Kazakhstan. ‘Ad terram in deserto Kirgizorum’, 1820, Eversmann in Elenkin: Lich. Fl. Ross. 24e, as Aspicilia alpinodesertorum f. affinis (H, LE, W). Regio Actjubinskensis, montes Mugodzhary, prope ad terram cagatur, 1927, Krascheninnikov, in Savicz: Lich. Ross. 78, as A. affinis f. alutacea (LE). Russia. [Altay Republic], Altai Austro-Orientalis in Deserto Czuensi [Chuy Desert]’, 1926, Smirnow, in Gyelnik, Lich. No. 69, as A. esculenta f. altaica (H, TUR). Ad terram et inter lapides montis Bogdo prope lacu Baskuntschak, in gub. Astrachan, 120 m, 1910 249 Mereschkowsky in Mereschkowsky: Lich. Ross. Exs. No. 19, as A. affinis (B, H, LE, TU, W, hb. M. Sohrabi). ‘Regio Astrachanensis, in viciniis lacus Baskunczak in decliviis montis Bogdo’, 1926, Savicz in Savicz: Lich. Ross. No. 167 (H). Regio Astrachanensis, in viciniis lacus Baskunczak in decliviis montis Bogdo’, 1926, V.P. Savicz in Savicz: Lich. Ross. No. 77, as A. affinis f. affinis (H) Circinaria aschabadensis (J. Steiner) Sohrabi comb. nov. (Fig. 5d). MB 563023 Basionym: Lecanora esculenta subsp. aschabadensis J. Steiner in Ann. Mycol. 8: 227. 1910. Lectotype: designated by Sohrabi and Ahti (2010), Turkmenistan. ‘Regio transcaspica: Aschabad [Ashgabat] ad fines Persiae’, sine dato, Sintenis in Zahlbruckner, Lich. Rarior. Exs. No. 32 (error ‘39’) (W; isolectotypes B, LE, PC) Thallus vagrant, invades tiny pebbles, subfruticose, up to 1–2(–3) cm long, (0.5–)1–1.5(–2) cm wide, folding with tiny appressed warts forming areolete surface, lobes usually wider up to 1.5–5 mm in diam., convoluted, verrucose, with (0.3–)0.5–1(–1.5) mm in diam., dense, very compact, short and dumpy, radiating from the central axial. Surface mudcovered, whitish to yellowish-grey, dull brown, brownish grey to greyish green, sometimes pale olive-brown to pale brown, ±white spots along the branches and often also on top of the lobes. Pseudocyphellae common, white to grey, conspicuous, often located on tip of lobes. Cortex thickness uneven, one layer, paraplectenchymatous, (40–)60–90(– 110) μm thick, ± brown, cells (4–)5–7(–8) μm in diam., inner part prosoplectenchymatous, interrupted with anticlinal hyphae of the medulla. Photobiont chlorococcoid, cells 5–15(–18) μm in diam., clustered in small groups, each group up to 60–180×50–110 μm broad. Medulla prosoplectenchymatous, with irregularly oriented hyphae (textura intricata), white, I–, muddy in microtome sections, containing crystals of calcium oxalate. Apothecia aspicilioid (urceolate), sometimes appear crypto-lecanorine, deeply immersed and surrounded by whitish rim. Disc black to brown-black, concave to convex when young, becoming more flat when old, often pruinose or sometime with thin white pruina, 0.5–1 mm in diam. Thalline exciple flat to ± elevated and prominent in older apothecia, entire, concolorous with thallus or with a thin to thicken white rim. True exciple (25–)35–85(–90) μm thick, ± I + blue, uppermost cell brown ± globose, 4–5(–7) μm in diam. Epihymenium brown to dark brown, K + fading from brown to light yellowish green, N + pale green (caesiocinerea-green). Hymenium hyaline, occasionally with few oil drops, (100–)110–130(–150) μm thick. Paraphysoids moniliform to submoniliform, with upper cells ± globose, 4–7 μm wide, in lower part 5–9×2–3 μm wide, slightly branched. Hypothecium and subhymenium pale, (35–)45–70(–85) μm thick. I + blue. Asci broadly clavate, 2– 4 spored, more rarely 1–3–spored with thickened apex, I–, 70– 120 × 20–35(–45) μm Ascospores hyaline, globose to 250 subglobose, (15–)17.2–[21.0]–25.1(–29)×(14–)15.1–[18.6]– 22.1(–26) μm (n032). Pycnidia immersed, abundant, conspicuous (often occur in the pseudocyphellae), with black ostiole slightly raised, globose to somewhat pyriform, internal wall colourless, 0.1–0.2 mm in diam., occurring singly or a few together. Conidia filiform, straight to very faintly curved, (8–)10–12(–16)×1–1.3 μm (n030). Chemistry: All spot-tests (K, C, KC, CK, P) negative both in the cortex and medulla. TLC and HPLC: No substances detected. UV: Negative. Ecology and distribution (Fig. 6d). Circinaria aschabadensis grows as vagrant but may rarely invade tiny pebbles. It has been collected from two localities within a small area in the Kopet-Dagh Mountains between Iran and Turkmenistan, where there are arid and sub-arid mountains with steppe vegetation and shrub lands with peculiarly subdivided topography. This species most probably grows also in the Firyuza area, between Iran and Turkmenistan. According to Kamakhina (1994) the diversity of mountain habitats in the Firyuza drainage supports rich biological diversity still poorly known. This is an area in Turkmenistan where many rare species might be found. Remarks. This species was described by Steiner (1910) from Kopet-Dagh and after that there are no additional reports. This species is easily recognized by its peculiar characters such as whitish-grey colour, presence of black ostiole in its pycnidia, and abundance of apothecia. This species was first mistaken for other similar species, such as Circinaria affinis (e.g., Crum 1993). Circinaria aschabadensis differs from C. affinis by its shorter conidia (8–)13–18(–20) μm and lack of greenish to greenish brown colour and exposed pseudocyphellae. Further, C. affinis has a wider range in central Asia. Among Borisova’s collection (LE), a small number of individual thalli were found on tiny pebbles. It shows that some thalli uncharacteristically become crustose on pebbles. All important characters such as colour, conidia and spore size are exactly same as in vagrant thalli. The nrITS sequence of crustose specimen was identical with the sequence of vagrant specimen (see Fig. 2). Additional literature reports. Iranian report of Circinaria aschabadensis was provisionally excluded by Sohrabi et al. (2010d) as uncertain locality for the species. To confirm the report, further collections from the Iranian side of KopetDagh are needed. Specimens examined. Turkmenistan. Central part of Kopet-Dagh district, next to the border of Iran, stony part of mountain to the SW of Solukli, 1934, Borisova (LE). Circinaria cerebroides (Mereschk.) Sohrabi comb. nov. (Fig. 4c). MB 563025 Basionym: Aspicilia cerebroides Mereschk. in Ann. Mag. Nat. Hist., ser. 9, 8: 266. 1921. Type: Kyrgyzstan. ‘Turkestan, prope Kaschgariae (Rossia)’, ?Roborowsky (not seen, ?KAZ). Corresponding to original material: Icons in Elenkin, Izv. Imp. S.-Peterburgsk. Bot. Sada 1: p.38, tab. 1, row IV, figs. 6, 8 and Mycol Progress (2013) 12:231–269 row V, figs. 6, 7. 1901. Corresponding specimen: Kyrgyzstan. Tian-Shan, 12 000 ft. ped, 1889, Roborowsky (LE). Thallus vagrant, lumps subglobose to irregular, amorphous, resembles to tiny truffles, convoluted, variable in size, 1–3(–4) wide, (0.5–)1–2.5(–3) cm thick. Lobes folded forming large wide, entire, flexuose, rounded. Surface smooth, muddy brown, grey-brown, or reddish brown (when substrate soil contain ferriferous sediments), often with very shallow cracks at the surface, rarely partly depressed. Pseudocyphellae common, pale, greyish brown dot-like pits, rarely whitish or concolorous with thallus surface. Cortex thickness uneven, one layer, paraplectenchymatous, 35–60(–80) μm, consists of isodiametric cells, rounded to rounded-angular in section, thin-walled, with lumen about (2–)3–6 μm in diam., inner part indistinct, mixed with anticlinal hyphae of medulla and appears as prosoplectenchymatous tissue. Epinecral layer a yellowish brown zone, developed above cortex, 10–15(–30) μm thick. Photobiont chlorococcoid, cells 5–15(–17) μm in diam., clustered in small groups, each group up to 80–160×50– 110 μm broad. Medulla prosoplectenchymatous, irregularly oriented hyphae in the center and anticlinal hyphae between algal clusters, white, I–, (in microtome sections appear as white layer, very dence, up to 20 mm thick). Apothecia not seen. Pycnidia very rare, deeply immersed, 1–2 in a lobe, (65–)90–125(–140) μm in diam., punctiform, with pale ostiole (35–)50–70(–85) μm in diam. Conidia colourless, filiform, (10–)10–13(–15) × 1–1.2 μm μm (n 033). Chemistry: All spot-tests (K, C, KC, CK, P) on thallus and cortex negative both in the cortex and medulla. TLC and HPLC: No substances detected. UV: Negative. Ecology and distribution (Fig. 6a). Circinaria cerebroides grows on soil and is usually found among pebbles. Known from the alpine range of the high mountains in the central Tian-Shan. Grasses of the genera Festuca and Stipa have been given as main associated plants. Some other vagrant lichens such as C. alpicola and C. hispida occur together with C. cerebroides. Remarks. Circinaria cerebroides was first illustrated in a plate by Elenkin (1901a; LE). Additional information on this species is found in Mereschkowsky (1921). The species was examined closely and it is clearly distinguished from other species of the genus by its unique appearance (often resembles to small truffles). The species is at a first glance somewhat similar to C. lacunosa, but the latter has small rounded pits (1–2 mm wide) on the surface, and a flabby medullary layer. According to available material, C. lacunosa grows in the lowland regions (average elevation c. 200–1300 m) in Kazakhstan and China. Circinaria cerebroides grows at higher altitudes, frequently above 3000–4000 m. According to the analysis of nrITS data these two are distinct and group with other new species C. rostamii. Mycol Progress (2013) 12:231–269 Specimens examined. Kyrgyzstan. Tian-Shan, 12000 ft., 1889, Roborowsky (LE). Terskej Alatao, Arabel-SuuHochebene, 3800 m, Ringel & Jaschhof 5180 (H). AksaiTal, Dshangy-Dsher, Karagerme-Fluss, 3700 m, 2007, Ringel & Jaschhof 5184 (H). Innerer Tian-Shan, At-BashiGebirge, pass (Ottotisch), 3300 m, 2007, Ringel 5138 (H). Exsiccata. Kyrgyzstan. Ad terram argillosam regione alpina montium Tian-Shan (Kaschgariae: Werchnij Syrt 12000 ft ped.)", 1889, Roborowsky, in Elenkin: Lich. Fl. Ross. No. 24d, as Aspicilia alpinodesertorum f. esculenta alpina, in the same envelope with C. alpicola (W). Circinaria digitata (Sohrabi & Litterski) Sohrabi comb. nov. Figure in Sohrabi et al. (2011a). MB 563026 Basionym: Aspicilia digitata Sohrabi & Litterski, Lichenologist 43: 41. 2011. Holotype: Kyrgyzstan. JangyJer Range (Dshangy-Dsher), Jal-Jyr River (‘Dshal-dshirFluss’), mouth of Archaly River (‘Artschaly-Mündung’), on soil (‘epigäisch’), 41°18 15 N, 76°44 36 E, 2900 m, 6 July 2007, H. Ringel & C. Jaschhof 5185 (H). See full description in Sohrabi et al. (2011a). Circinaria emiliae (Tomin) A. Nordin, S. Savic & Tibell in Mycologia 102: 1346. 2010. (Figure. 4b). Basionym: Lecanora emiliae Tomin in Prir. Sel’sk. Khoz. Zasushl.-Pustyn. S.S.S.R. 2 (3): 58 [reprint p. 4.]. 1928. Lectotype: designated by Sohrabi and Ahti (2010), Kazakhstan. Ural’skaya gub, [(Ural) Oral Prov.], ‘Inderskoe ozero’, July 1927, Emilia Keller 60 (LE L91). Thallus vagrant, subfoliose, usually dorsiventrally flattened, diversely bent or folded marginally, occasionally rolled up almost to a tube 1–4(–6) cm long, (0.4–)0.50– 0.75(–1.5) mm thick, usually irregularly thickened, wavy, somewhat flattened in the middle, thin, margin thickened, knobby at places, sometimes incised, rolled down, and mostly tightly pressed to the lower side. Upper surface yellowish grey, brownish, muddy, yellow-orange, matt, smooth, slightly knobby here and there, with very low, flat protuberances, or somewhat wrinkled, covered (particularly in old specimens) with a net of shallow cracks (visible at 20–40× magnification), about 0.12–0.25(0.75) mm wide, irregular in shape, giving a somewhat scabrous appearance. Pseudocyphellae rare, usually inconspicuous, pale (±white), when getting wet appear as tiny dots. Cortex indistinctly delimited, in some parts in two layers, upper part paraplectenchymatous, 25–45(–55) μm thick, with globose or rounded cells, lumina 3.5–5(–6) μm wide, in inner part algal cells sinking deeper into the medulla, a prosoplectenchymatous layer develops over algal cells, usually 10–70 μm distanced from the upper part. Epinecral layer brownish, 10–25 μm thick. Photobiont chlorococcoid, cells 5–15(–20) μm in diam., arranged in an uneven to rather even layer and often clustered in small groups (70–)100–130(–150)×(90–)100–200(–250) μm broad. Medulla prosoplectenchymatous, with more or less irregular hyphae, white, I–. Apothecia very rare, all measurement 251 are based on a single apothecium in specimen at (hb. Seaward115150). Disc black to brownish black, pruinose, c. 0.5 mm broad. Thalline exciple flat to ± elevated, entire, concolorous with thallus. True exciple (25–)35–45(–60) μm wide, ± I + blue, uppermost cell pale brown, ± globose, 4–5(–6) μm in diam. Epihymenium brown, K + fading from brown to light yellowish green, N + pale green. Hymenium hyaline, (80–)100– 120(–130) μm. Paraphysoids submoniliform, upper cells ± globose, 4–7 μm wide, in lower part cells rectangular, 5–9× 2–3 μm wide, sometimes branched. Hypothecium and subhymenium pale, (30–)45–50(–65) μm thick. I + blue. Asci and ascospores not found as mature. Pycnidia rare, immersed, with whitish ostiole, flask-like to ± globose, inner part colourless. Conidia simple, bacilliform and ± straight, colourless, (7–)9–12 (–14)×1 μm (n039). Chemistry: All spot-tests (K, C, KC, CK, P) negative both in the cortex and medulla. TLC and HPLC: No substances detected. UV: Negative. Ecology and distribution (Fig. 6b). Circinaria emiliae is vagrant and mainly found between small pebbles and salty soils in semi-desert steppes of the central Asia. It has been collected from the lowlands near Volgograd Region in Russia, and from some localities near Almaty in Kazakhstan. Habitats of this species are rather poorly known, however several plant species, such as Artemisia glauca, Calligonum spp. and Haloxylon aphyllum are reported as dominant plants in the localities (see also Andreeva 1987). Remarks. The subfoliose thallus is conspicuous in the field. Thallus turns weakly brown or brownish-yellow to green when wet. It is somewhat similar to the deformed thalli of Dermatocarpon spp., but it can be easily distinguished by the absence of perithecia and other characters such thallus cortex layer and clustered algal cells. This is the only subfoliose member of ‘manna lichens’. The umbilicate Aspicilia oxneriana is somewhat similar in morphology and ecology with Circinaria emiliae, but it grows on calcareous rocks and is frequently fertile. Additional information on this species can be found in Savicz (1962). Additional literature reports. The record from Turkey by Candan and Türk (2008) needs further study. The reported specimen by Biazrov et al. (1989) from Mongolia is confirmed here. Specimens examined. Kazakhstan. Near Koshkarbay, 2009, Teshernyshev (hb. Seaward 115150). West Kazakhstan Region, Ura'sk ('Yaitskiy gorodok'), Kalmyk fort ('Krepost' Kalmykova'), 1769–1770, Pallas (H-NYL 3936, LE). Mongolia. Zavkhan [correctly Govi-Altai] aimag, Taishir sum, right bank of Zavkhan River, c. 3 km N of Taishir Town, 1978, Biazrov 8364 (LE). Exsiccata. Kazakhstan. Regio Gurjevensis, lacus jnder, 1927, G. S. Grigorijev, in Savicz: Lich. Ross. No. 115 (H, TU, hb. Seaward). Circinaria esculenta (Pall.) Sohrabi comb. nov. (Fig. 4f). MB 563027 252 Basionym: Lichen esculentus Pall., Reise Russ. Reich. 3 (2, Anhang): 760, tab. 1, fig. 4. 1776. Lectotype: designated by Sohrabi and Ahti (2010), [icon in] Pallas, Reise Russ. Reich. 3 (2, Anhang): 760, tab. 1, fig. 4 (dextra) (1776). Epitype: designated by Sohrabi and Ahti (2010) Russia. ‘Russia europaea, regio Stalingradensis [Volgograd Region], in viciniis lacus Elton, declivium collis Ulagan’ 1938, Leisle & Janischevsky, in Savicz: Lich. Ross. No. 63 C (H, isolectotypes LE, TU). Thallus vagrant, globose-angular, subglobose or irregular in shape, 1–3(–4) cm wide, formed undulating to compressed subsquamulose, squeezed in appearance, subsquamulose pieces 1–8 mm wide, 0.6–1.5 mm thick, edges flexuose, irregularly elevated, rounded, crenate, uneven, knobby or tuberculose, turned in or curved to wavy, closely adpressed by their margins in exterior parts and completely concrescent in interior parts. Surface olive-brown to grey-brown, or light grey, muddy, or more rarely rusty red (when ferriferous sediments are present in soil), rarely with some dove-coloured pruina on the swelling parts. Pseudocyphellae common, ±white, dot-like, normally visible on subsquamulose pieces. Cortex thickness uneven, one layer, paraplectenchymatous, (40–)60–80(–100) μm thick, ± brown, cells (4–)5–7(–10) μm in diam., inner part mixed with prosoplectenchymatous layer derived from medulla; sometimes anticlinal hyphae expand over algal cells making uneven layer up to 10–50 μm thicker. Epinecral layer 5–25(– 30) μm thick. Photobiont chlorococcoid, cells 5–20 μm in diam., clustered in small groups, each group up to 80–160× 90–150 μm broad. Medulla paraplectenchymatous with more or less irregular hyphae, white, pale, muddy color, containing crystals of calcium oxalate. Apothecia urceolate or aspicilioid, rare, up to 0.5–0.8(–1.2) mm wide, deeply immersed when young, very slightly elavated when mature, 1–3 (rarely 5) per subsquamulose, often located in older parts of the thallus. Disc black to brownish black, densely white-pruinose, weakly concave or flat, more or less rounded, rarely irregular, up to 0.5– 1 mm wide, surrounded by strongly emergent, whitish, crenulate rim. Thalline exciple thin, slightly flat, but ± elevated, entire, concolorous with thallus. True exciple (25–)35–45(– 85) μm wide, ± I + blue, uppermost cell light brown ± globose, 4–5(–7) μm in diam. Epihymenium brown, K + fading from brown to light yellowish green, contains insoluble crystals of calcium oxalat, N + pale green (caesiocinerea-green). Hymenium hyaline, (90–)110–135(–155) μm thick. Paraphysoids submoniliform or moniliform, upper cells ± globose, 3–7 μm wide, in lower part cells rectangular, 5–7× 2–2.5 μm wide, often branched. Hypothecium and subhymenium pale, (45–)65–95(–110) μm thick, I + blue. Asci broadly clavate, (80–)90–110(–120)×27–35 μm, with thick apical dome 20–30 μm tall, 1–3(–4) spored. Ascospores more or less globose, (20–)21.8–[25.3]–28.8(–33)×(19–)19.9–[23.0]–26.1 (–30) μm (n031). Pycnidia common, immersed, 1–2(–4) per subsquamulose, stretched flask-shaped, punctiform, internal Mycol Progress (2013) 12:231–269 surface colorless, often with black to brownish ostiole. Conidia filiform, straight to somewhat curved (10–)13–26(– 35)×1 μm (n0143). Chemistry: All spot-tests (K, C, KC, CK, P) negative both in the cortex and medulla. TLC and HPLC: No substances detected. UV: Negative. Ecology and distribution (Fig. 6f). This species is usually found on loamy soil, sometimes also on salty soil, in between small pebbles and stony places in the lowland steppes and deserts. It usually occurs at the average altitude of 200 m in certain areas NE of the Caspian Sea (e.g., Astrakhan area), where the soil is normally frozen for some months of the year. Other ‘manna lichens’ such as Circinaria emiliae, C. fruticulosa, C. hispida and C. affinis are reported from these same habitats. A list of additional associated lichen species was summarized in Kulakov (2002, 2003) and Shustov (2006) For example (Xanthoparmelia camtschadalis (Ach.) Hale, X. subdiffluens Hale, and X. desertorum (Elenkin) Hale are aften reported from same habitats. Some associated angiosperm species are Artemisia pauciflora, A. glauca, A. sublessingiana, Anabasis salsa, and Salsola arbusculiformis. Remarks. Circinaria esculenta was the first of the ‘manna lichens’ described by Pallas (1776). Although C. esculenta is a well-known ‘manna lichen’, it has a very complicated nomenclatural history. This is due to the earlier wide concept of ‘manna lichens’, in which many of what we presently now identify as vagrant species, including C.affinis, C. esculenta, C. fruticulosa, C. gyrosa and C. jussuffii, , as well as a variant of the saxicolous species Aspicilia ‘desertorum’, were included. We noticed that the material earlier referred to C. esculenta was not homogeneous. Many of the specimens were referred to the saxicolous A. ‘desertorum’ sensu Krempelhuber (1867), a crustose species with a massive medulla. A. ‘desertorum’ has often been reported as a common species in the semiarid areas of Iran (Seaward et al. 2008) and Central Asia (Mereschkowsky 1911a, b; Andreeva 1987; Dzhuraeva 1978). Recently C. elmorei, another saxicolous species with a massive medulla, was redescribed from arid regions of North America by OweLarsson et al. (2011). It is morphologically more or less identical to A. ‘desertorum’ sensu Krempelhuber (1867), and most probably the two are conspecific. The application of the name C. elmorei would reduce the nomenclatural confusion surrounding the crustose A. ‘desertorum’ sensu Krempelhuber (1867) and the vagrant A. esculenta. Our observations show that C. elmorei is heteromorphic and probably includes some morphologically poorly known or undescribed species. Therefore, we decided to retain this saxicolous group for the time being with a view to conducting more detailed studies using multiple approaches including analysis of morphological and molecular markers. Earlier reports of both A. ‘desertorum’ sensu Krempelhuber and A. esculenta need to be reviewed. Occasionally, the name C. esculenta has erroneously been used for the North African Mycol Progress (2013) 12:231–269 vagrant species C. jussuffii. This is due to the large variation present in both species. For example, when the subsquamulose lobes of C. jussuffii are wrinkled, they greatly resemble to C. esculenta. However, C. esculenta can be easily distinguished by its large thalli and subsquamulose lobes (c. 1– 5 mm), greenish grey colour and larger conidia (10–35 μm), as compared to the smaller reddish or brownish thalli and shorter conidia (up 8–16 μm) of C. jussuffii. In some thalli of C. jussuffii secondary metabolites were detected. Additional literature reports. The reports from Kyrgyzstan by Litterski (2002), Bredkina and Makarova (2005), and from Mongolia by Klement (1966), need to be confirmed. Some of the available historical collections from Iran and Turkey examined in this study, many collectively named Aspicilia esculenta, were found to be crustose, and are referred to Circinaria elmorei s.lat., a species currently under revision by Sohrabi et al. (in prep.). The vagrant manna specimens report from Iran by Göbel (1830 as Parmelia esculenta (Pall.) Spreng.) most probably referable to one of the other two newly described vagrant species C. gyrosa or C. rostamii). A disjunct population of C. esculenta was also found from the surroundings of the Don River in southwest Russia. A report by Léveillé (1842) and Kopachevskaya (1986) from the Ukrainian steppes need to be confirmed. Specimens examined. Kazakhstan. Prov. Ural [Oral] in deserto Kirghisorum, Ak-Kerege, 1904, Dubiansky (G). Mongolia. Zavkhan [correctly Govi-Altai] aimag, Taishir sum, right bank of Zavkhan River, c. 3 km N of Taishir Town, 1978, Biazrov 8370 (LE). Russia. Astrakhan, near the Lake Baskunchak, 1926, Tomin (FH, H, S). Berg Bogdo, 1926, Tomin (S, UPS, US, W), ibidem, 1928, (US). Berg Bogdo, 1910, Mereschkowsky (TU, UPS, US), ibidem,1920, Keller (UPS), ibidem, 1927, Keller (US, W). Near the Lake Baskunchak, W slope of mount big Bogdo, 1990, Sagalaev (M). Berg Bogdo, Tartary ‘Tartaria’ sine loc. & dato. (determined by Nees von Esenbeck), in the same envelope with C. affinis (UPS); Saratow Region, 1093, Keller (W); Volgograd Region, near Lake Elton, Ulagan Mt., 1938, Leisle & Yanishevskii (W). Uzbekistan. Bukhara, Zwockh (UPS). Exsiccata. Kazakhstan. ‘desertis Kirgisorum’ Eversmann 1820, in Elenkin: Lich. Fl. Ross. No. 24c, as Aspicilia alpinodesertorum f. esculenta-tesquina (FH, H, W). ‘regio Aktjubinskensis [Aktobe Prov.], montes cretacei Bish-tau in steppa deserta’, 1926, Iljin in Savicz: Lich. Ross. No. 64, as A. esculenta f. cretata (H, LE, TU, W). Regio Gurjevskensis, montis Inder prope Akssaj. 1926, Iljin and Grigorjeff, in Savicz: Lich. Ross. No. 65, as A. esculenta f. pallida (H, LE). Russia. ‘Regio Astrachanensis, mons Bogdo prope lacum Baskunczak, Declivia septentrionalia in steppa deserta paulum saxosa’ 1926, ‘Ad ripas lacus Baskunczak, prope p. Nizhnij Baskunczak, in steppa deserta ad terram salinam structuram habentem’. 1926, ‘Regio Satlingradensis (Volgograd Region) in viciniis lacus Elton, decliveium collis 253 Ulagan’, 1938, Leisle & Janischevsky in Savicz: Lich. Ross No. 63A-C, as A. esculenta (H, TU, W). Astrachan, in declivibus montis Bogdo prope lacum Baskunczak, in Krypt. exs. Vindobonensi. No. 3158, as A. esculenta (S, US). ‘Ad terram et inter lapides montis Bogdo prope lacu Baskuntschak, in gub Astrachan, 100 m, 1910, Mereschkowsky, in Mereschkowsky: Lich. Ross. Exs. No. 18, as A. esculenta (B, TU, hb. M. Sohrabi). ‘Ditio Astrachanensis. Distr. Wladimirskii, haud procul pag. Nizhnij Baskunczak’, Ad terram, in steppis, 1962, Kopaczewskaja & Zubec, in Vězda: Lich. Sele. Exc. No. 584, as A. esculenta (BM, H, S). ‘Regio Astrachanensis in viciniis lacus Baskunczak in decliviis montis Bogdo’. 1926, Savicz in Savicz: Lich. Ross. No. 66, as A. esculenta f. ferruginea (H, LE, TU). ‘Auf Kreidebergen des Don-Flusses ungefähr unter dem 48½ 0 nördl, Br. (Russland) Pitra in Charkow’, in Rabenhorst Lich. Eur. No. 825, as Chlorangium esculentum (B, FH, H, S, W). Circinaria fruticulosa (Eversm.) Sohrabi comb. nov. (Fig. 5f-h). MB 563028 Basionym: Lecanora fruticulosa Eversm. in Nova Acta Phys.-Med. Acad. Caes. Leop.-Carol. Nat.Cur. 15: 352, tab. 78A. 1831. Lectotype: designated by Sohrabi and Ahti (2010), Kazakhstan. ‘Desertis Kirgisorum’ [1820], Eversmann (H-NYL 25676). Thallus vagrant, subfruticose, convoluted, 10–20(–35) mm wide, subglobose, with numerous branches, variable in size (0.4)0.5–0.9(1.4) mm, often dichotomous, more or less rounded to cylindrical, radiating in different directions from the center, central axis sometimes up to 2–4 mm thick. In uppermost part of branchlets dot-like depressions. Surface blackish olive, greyish brown, earthy, sometimes rusty red (when soil contains ferriferous sediments). Pseudocyphellae common, white, conspicuous, often located at tip of branchlets. Cortex thickness even, distinctly two layered, exterior part paraplectenchymatous (10–)15–25(– 30) μm thick, ± brown, cells (4–)5–6(–8) μm in diam., inner part prosoplectenchymatous (25–)40–50(–65) μm thick. Epinecral layer 5–15(–19) μm. Photobiont chlorococcoid, cells 5–18(–22) μm, clusterd in groups of 100–250×90– 200 μm. Medulla I–, white, solid, sometimes wobbly, crystals of calcium oxalate not common. Apothecia cryptolecanorine, rare, in aggregates of 1–2(–3), at tips of short branchlets, immersed when young, constricted at base when mature, somewhat elevated, up to 2 mm wide. Disc brownblack, dark brown, with dense, thick dove-coloured pruina, rounded, more or less flat, c. 0.8–1.5(–2) mm in diam. Thalline exciple ± elevated and prominent in older apothecia, entire, concolorous with thallus or with a thin to thickened white rim. True exciple poorly developed, I + blue, 30–40 (–50) μm wide, evident only laterally, where it is colourless, of short-celled hyphae; sometimes exciple inconspicuous. Epihymenium brownish to dark brown, 15–30 μm tall, K ± fading from brown to light yellowish green, N + pale green 254 (caesiocinerea-green). Hymenium hyaline, sometimes few oil drop, 100–125 μm tall, colourless. Paraphysoids moniliform to submoniliform, very thin, lower parts joined with ellipsoid cells, 1.2–2 μm thick, uppermost cell broadly ellipsoid to globose, 4–5.5 μm wide, often branched. Hypothecium and subhymenium colourless, 30–50(–65) μm, I + blue, turbid of granules, muddy, with soluble crystal in KOH. Asci clavate, 75–100×25–33 μm, with thick apical dome 20–30 μm tall, 3–4 spored. Ascospores hyaline, simple, broadly ellipsoid to globose, (10–)17.2–[21.5]–25.9(–35)×(10–)16–[19.5]–23.9 (–30) μm (n035). Pycnidia rare, usually at tips of branchlets, sometimes on pseudocyphellae, 0.1–0.15 mm. Conidia filiform, needle-shaped, straight, (8–)8–12(–16)×1–1.3 μm μm (n039). Chemistry: All spot-tests (K, C, KC, CK, P) negative both in the cortex and medulla. TLC and HPLC: No substances detected. UV: Negative. Ecology and distribution (Fig. 6e). This true vagrant species is found on loamy or salty soil, sometimes in stony places in lowland deserts and mountain steppes. The overall distribution of Circinaria fruticulosa is outlined by Oxner (1971) including SE Ukraine, Russia (Bashkortostan, Volgograd and Astrakhan Region), Central Asia (Kazakhstan, Kyrgyzstan), Transcaspian Region (Turkmenistan), Caucasus regions and North Africa. The species is found from lowlands to highlands. In Russia, Ukraine and Kazakhstan it is most frequently found in the lowlands below the average altitude of 500 m. Its highest recorded elevation is c. 1500 m in the NW of Iran, Kyrgyzstan and China. The species has not been found in North America. The American species provisionally known as C. fruticulosa turned out to be a new taxon described as Aspicilia rogeri (≡ Circinaria rogeri) in Sohrabi et al. (2011b). Circinaria fruticulosa has been frequently collected in Central Asia together with other vagrant taxa, e.g., C. esculenta, C. hispida s.str. and C. affinis as well as saxicolous species of the C. elmorei s.lat. Several plant species have been reported as accompanying C. fruticulosa in Central Asia, e.g., Artemisia spp., Stipa capillata, Anabasis salsa, Salsola arbusculiformis, and Kochia prostrata (see also Andreeva 1987). Remarks. This member of the ‘manna lichens’ is characterized by thalli with dichotomous patterns of branching, and lacking the black tips or spots in the topmost part of the branchlets. Some of the branches are closely aggregated, rounded-cylindrical, slightly thickened at the apex. Due to this thallus surface looks finely verrucose to granulose. According to our observations, the average size of thalli in the lowland populations is up to 3 cm, which is generally larger than in highland populations, which are often only up 1–1.5 cm. The low amount of genetic variation found in the populations might suggest that the morphological differences observed be some kind of ecological adaptation for better surviving in higher altitudes. This is comparable to some alpine plants (dwarf forms). Mycol Progress (2013) 12:231–269 Circinaria fruticulosa can rarely be confused with other ‘manna lichens.’ However, during past decades the name C. fruticulosa (as Aspicilia fruticulosa) was used for some vagrant specimens in Spain (Sancho et al. 2000; Llimona and Hladun 2001). Our nrITS analysis clearly shows that Spanish and Eurasian C. fruticulosa are two genetically distinct taxa (Fig. 2). Spanish specimens together with other specimens from Iran and Turkey are described as new species (see C. gyrosa). Additional literature reports. Circinaria fruticulosa (as A. fruticulosa) from Turkey (Candan and Türk 2008) and Morocco (Egea 1996) need to be confirmed. It has been reported from Spain by numerous authors (see Llimona and Hladun 2001). In this study, some of the voucher specimens were examind (formerly reported in Follmann and Crespo 1974; Crespo and Barreno 1978; Follmann and Huneck 1968). Almost all of them were C. gyrosa. However, some of the specimens were not accessible and therefore their identification still needs to be confirmed. Specimens examined. Algeria. ‘(Constantine), Plateau du telegraphe, 1880’, Cosson & Rebaud (PC). China. Xinjiang, Tuo Li County, Laofeng Kou, 1994, Abbas 940001 (H). Xinjiang, Tacheng Toli, 1232 m, 2008, Abbas & Xahidin 20080363-a (H). Russia. Astrakhan Region, Bolshaya Bogdo, 1952, Savicz (LE). Omsk Oblast, Russkaya Polyana District, S of Stepanovka village, steppe, 1920, Knorring (LE), Berg Bogdo, 1910 Mereschkowsky (US, W), Berg Bogdo, 1926, Tomin (FH, H, US), ibidem, 1927, Tomin (S), ibidem, 1928 Tomin (S, US). Berg Bogdo, 1920, sine coll. (US). Near the Lake Baskunchak, 1926, Tomin (H, US). Bolshaya Bogdo, 1924, Kazekevish (LE). Bogdo Mt., 1926, Savicz (FH). Near the Lake Baskunchak, Vach-Tau, 1997, Kulakov (M). Bolshaya Bogdo, 1952, Nashli (LE). Berg Bogdo, 1910, Mereschkowsky (H, UPS, US); Volgograd, Lake El’ton, Ulagan Mt., 1938, Leisle & Yanishevskii (LE), Volgograd Region, Kalachovsky district, vicinity of Bolshegolubinsky garden. Northern slope of the River Bolshaya Golubaya, 1994, Kulakov (LE); Kalmyk Republic, Sarpinsky district, Kirowsky village, 2002, Ochirova (LE). Orenburg Region, Kovkenski district, near the village sosnovka, 2002, Merkulova (LE). Ad terram in viciniis Sarepta (Saratowsk Region), 1864, Becker (LE). Sarepta, sine coll. (H-NYL 25679). Kazakhstan, Akmolinskaya district, 12 km to the north Ladizhenka, 1954, Isachenko (LE). Akmolinskaya district, 25 km to the south of Akmola, 1954, Isachenko (LE). Akmola Region, Atbassar district, Togushkensky mt. along Ter Akken River, 1904, Gordiagin (LE). Akmola Region, Omsky district, 1920, Kroing (LE, US). Atbassar district, Arganat Mt. on the left bank of Bazay River, on the foot of Mukty-Yuy, 1904, Gordiagin (LE). Atbassar district, Karaganda Region, saline lands in semideserts by the military post Kokchetav [Kökshetau Town], on Ulutau-Atbasar road, 1914, Mycol Progress (2013) 12:231–269 Ganeschin (LE). Along the beach of Obala Lake, 1914, Ganeschin (LE). Karakaralinsky district, mountain range Konurtobe, 1967, Leontieva (LE). Aqtöbe Region, east slope of Mughalzhar Mts., Birshogir, 1904, Dubiansky (LE). Pavlodar Region [Prov.], Kaganovich [Assu] district, 120 km from Pavlodar, terraces of the Lake Altybai-Sor, 1938, Smirnova (MSK). Karaganda, Aktogaysky district, about 4– 5 km, SW of village Birlestyk, 1967, Leontieva (LE). ‘Deserto kirgizorum, Eversmann 1820, (B, FH, H-NYL 25676). Aktyubinsk, Aktyubinskaya Region, Chelkarskay district, Mountains Kizil-choku elong Sarlibay-Sago river, inflose into Chit-Irgiz, 1927, Krasheninnikov (LE); Tarbagatai, nordwest. Vorgebirge, c. 40 km E Stadt Tarbagatai, 1000 m, 2001, Lange 5186 (H). Iran. East Azerbaijan, Kaleybar district: 35 km S of Kaleybar along road to Ahar, 1750 m, 2007, Sohrabi et al. 10405A, ibidem, Sipman et al. 55488 (B, H, IRAN, UPS). Mongolia. Zavkhan [correctly Govi-Altai] aimag, Taishir sum, right bank of Zavkhan River, c. 3 km N of Taishir Town, 1978, Biazrov 8371 (LE). Turkey. Casikoporan, Surmalinsky district, Iravanskay Region, 1896, Koenig , filed in the same envelope with C. gyrosa (LE). Ukraine. Peninsula Taurica. ‘In decliviis Stepposis Tauriae borealis in viciniis pag. Monoj’, 1932, Kozlov (TUR); Crimean Peninsula, Alupka, Aj-Petrinskaja jajla c. 1 km SE of Bedene-kyr Mt., c. 1100 m, 2006, Vondrák 5188, 5258 (CBFS), ibidem, Vondrák 5670 & Šoun (CBFS), Crimea, Simpheropol steppes, 1910, Mereschkowsky (H); "Ad terram calcaream inter rupes calcareas e viciniis Theodossiae in peninsula Taurica, 1893, Lipsky (station G in envelope) (LE). Exsiccata Russia. Regio Astrachanensis, in viciniis lacus Baskunczak in decliviis montis Bogdo, 1926, Savicz in Savicz: Lich. Ross. No. 95, as Aspicilia fruticulosa (H, FH, TU, W, hb. Seaward). Regio Astrachanensis, in viciniis lacus Baskunczak in decliviis montis Bogdo, 1926 Savicz, in Savicz: Lich. Ross. No. 96, as A. fruticulosa f. ferruginea (FH, H, LE, TU, W). Ad terram et inter lapides montis Bogdo prope lacu Baskuntschak, in gub Astrachan, 1910, Mereschkowsky, in Mereschkowsky: Lich. Ross. Exs. No. 20 (H, TU, W). "Baschkirarss, districtus Zilairsky, collis (ssopka) invalle fluminis Tanalyk non procul p. Mambetova, ad terram inter lapides", 1929, Krascheninnikov, in Savicz. Lich. Ross. No. 54, as A. fruticulosa (FH, H, TU, US, W). "Ad terram in viciniis Sarepta (Gub. Saratowsk), 1864, Becker, in Elenkin: Lich. Fl. Ross. 24f, as A. alpinodesertorum f. fruticulosa (H, LE). "In den Wolgasteppen bei Sarepta", Wenck, Rabenhorst Lich. Eur. No. 874, as Chlorangium affine (H, FH, S, TUR-V 5751, W). Kazakhstan. Akmolinskaya Oblast (0Akmola Prov.), 20 km SE of the Tengiz Lake, banks of the river Kulanotpes, 4 km NNW of the town Kulanotpes, 340 m, Wagner (L-0070) in Obermayer: Lich. Graecenses No. 321 (H, all dupls. examined before distribution in ASU, B, C, CANB, CANL, E, G, GZU, HAL, HMAS, LE, M, MAF, MIN, O, PRA, TNS, UPS). Ukraine. Crimea (Krym), ‘ad terram regionum stepporum prope Simpheropolin, in Peninsula 255 Taurica’, 1910, Mereschkowsky in Mereschkowsky: Lich. Ross. Exs. No. 21 (B, H, LE, TU, hb. M. Sohrabi). "Ad terram calcaream et inter rupes calcareas e viciniis Theodossia in peninsula Taurica", 1893, Lipsky, in Elenkin: Lich. Fl. Ross. 24 g, as A. alpinodesertorum f. fruticulosa (H, LE, W). Turkey. Anatolia prov., Çorum, limestone hills near road from Merzion to Çorum, 30 km S of Merzifon, 950 m, 1997, John 9538, Yildiz & Zeybek, in Lumbsch and Feige: Lecanoroid Lich. Exs. No. 82 (BM, H, M). Circinaria gyrosa Sohrabi, Sipman, V. John & V.J. Rico, sp. nov. (Fig. 3a-d). MB 563029 Diagnosis. Thallus vagrant, more or less globose, brainlike, deeply divided into short and coralloid, surface dull brown, brownish grey, sometimes whitish grey, pale olivebrown to pale brown. Very similar to Circinaria affinis, but differentiated by its shorter conidia (10–15 μm), wider distribution in the highland (1000-2000 m) or steppe mountains of the Mediterranean region and monophyletic poistion of the nuclear ribosomal ITS phylogeny analysis. Type: Iran. East Azerbaijan, Marand district, 32 km N of Marand towards Jolfa, 38° 40.58' N, 45° 39.44' E., 1440 m, 2 Nov. 2007, M. Sohrabi 10085, H. Sipman, U. Søchting & M. R. Asef, (IRAN 14444, holotype; B, H, hb. M. Sohrabi, isotypes). Thallus vagrant, more or less globose, 0.5–2 cm tall and 0.5–2(–3) cm broad, brain-like, deeply divided into short and coralloid, flattened lobes or folds 1–3 mm broad, with rimose-verrucose surface, rather flattened on top. Surface dull brown, brownish grey, sometimes whitish grey, pale olive-brown to pale brown, on covered sides usually darkish green to dark brown, greenish brown or almost dark greenbrown (sometimes reddish brown when ferriferous components are present in soil). Pseudocyphellae very common, visible as whitish spots usually along or on top of the folded lobes. Cortex (40–)60–90(–110) μm thick, outer part paraplectenchymatous, ± brown, c. 2–3 cells thick, cells (4–)5–7 (–8) μm in diam., inner part indistinct, mixed with prosoplectenchymatous tissue of medulla, sometimes forming a distinct layer (30–)40–80(–90) μm tall (variable presence of algal cells in the medulla layer makes it uneven, poorly delimited and difficult to distinguish from the true cortex layer). Epinecral layer 1–5(–12) μm thick. Photobiont chlorococcoid, cells 5–22 μm in diam., clustered in small groups, each group up to 80–180 × 50–110 μm broad. Medulla white, I–, 0.3–4(–6) mm thick, containing crystals of calcium oxalate. Apothecia aspicilioid, rare, up to 0.5–1.5 (–2) mm wide, among the lobes in older parts. Disc black to brown-black, pruinose, concave to convex when young, becoming more flat when old. Thalline exciple flat to ± elevated and prominent in older apothecia, entire, concolorous with thallus or with a thin to thick white rim. True exciple (35–)45–75(–85) μm wide, I ± more or less blue, uppermost cells brown, ± globose, 4–5(–7) μm in diam. Epihymenium brown, K+, colour fading from brown to light 256 yellowish green, N + pale green (caesiocinerea-green). Hymenium hyaline, occasionally with few oil drops, (100–)110–140(–150) μm tall. Paraphysoids branched, apically moniliform to submoniliform, with upper cells ± globose, 4–7 μm wide, and lower cells cylindrical, 4–9×2– 3 μm wide, usually branched. Hypothecium and subhymenium pale, (35–)45–65(–85) μm thick, I + blue. Asci broadly clavate, (80–)90–100(–110)×25–35 μm, with thick apical dome 20–30 μm tall, 2–4(–5) spored. Ascospores hyaline, simple, globose to subglobose, (16–)19–[22.1]–24(–26)× (16–)19–[21.6]–23(–25) μm (n030). Pycnidia immersed (sometimes occurring in pseudocyphellae), single, stretched flask-shaped, internal wall colourless, with black to brownish ostiole. Conidia filiform, straight to very slightly curved, (8–) 10–14(–18)×1–1.2 μm (n0132). Chemistry: All spot-tests (K, C, KC, CK, P) negative both in the cortex and medulla. TLC and HPLC: No substances detected. UV: Negative. Etymology. The specific epithet ‘gyrosa’ refers to the brain-like lobes of the thallus. Ecology and distribution (Fig. 6c). The thalli of this species are found scattered on bare calcareous soil, mixed with gravel in several different habitats: in open grassland with isolated trees and shrubs, with a Mediterranean type climate, with mainly junipers and Astragalus spp. and in the sub-montane forest zone in Golestan province of Iran. In Spain it has been reported from same habitat types, however the main species composition and vegetation type is different and mixed with other Juniperus species. In NW Iran (Azerbaijan province) and in a similar habitats in SE Turkey the species has bee found in dry steppes with Artemisia spp. and Verbascum spp. In general, C. gyrosa appears to prefer very open habitats that are ephemerally moist in the winter or spring, and dry most of the year. The main associated plant communities include Artemisia spp., Poa spp. and Verbascum spp. Other associated lichen species on soil are Circinaria hispida, and C. fruticulosa, and on pebbles Caloplaca deceptoria (Flagey) J. Steiner, C. chalybaea (Fr.) Müll. Arg., C. ferrugineoides H. Magn., and Lecanora garovaglioi (Körb.) Zahlbr. Circinaria gyrosa is widespread and locally common, at elevations of c. 1000– 2000 m. Its range includes the Mediterranean and IranoTuranian regions. Remarks. Circinaria gyrosa is easily confused with the very similar vagrant species C. fruticulosa and C. affinis. In general Circinaria gyrosa is characterized by its large, 10– 30 mm wide, folded, vagrant thalli with brain-like morphology and its eye-catching light brown to dark grey-brown surface colour. Moreover, the conidia in C. gyrosa are up to 10– 15 μm long, and in C. affinis 15–25 μm. There are also ecological and distributional differences, C. affinis is so far known from low altitudes (from see level up to c. 1000 m) in Central Asia, while C. gyrosa grows in mountains, usually at 1000 to 2000 m. As to the differentiation from C. fruticulosa, Mycol Progress (2013) 12:231–269 C. gyrosa bears folded, rounded, flattened, dumpy lobes with an irregular pattern of radiation in the central part, whereas C. fruticulosa forms tiny, branched lumps with often dichotomous radiation. Microtome sections shave shown that Circinaria fruticulosa has a conspicuously two layered cortex, while the cortex in C. gyrosa consists of only one layer, is of very uneven thickness and is often interrupted by prosoplectenchymatous medulla tissue. Pseudocyphellae in C. gyrosa are very conspicuous and large (up to 0.6 mm), whereas in C. fruticulosa they are rather small (up to 0.3 mm) and usually located at the tips of the branchlets. Circinaria gyrosa is morphologically very similar to C. affinis. However, conidia in C. gyrosa are up to 10–15 μm long, and in C. affinis up to 15–25 μm. There are also some ecological and distributional differences between the two species, i.e., C. affinis is so far known from low altitudes (from see level up to c. 1000 m) in the Central Asia, while C. gyrosa grows in mountainous areas, usually at the altitudes of 1000 to 2000 m. Circinaria gyrosa is also rather similar to C. aschabadensis, with which it may grow together. Then its general appearance, e.g., reddish-brown colour, brain-shaped thalli, larger lobes, abundance of white spot-like pseudocyphellae, and lack of black ostioles in its pycnidia easily distinguish it from the latter species. In Sancho et al. (2008: Fig.3) a specimen presented as ‘Aspicilia fruticulosa’ is illustrated which greatly resembles to Circinaria gyrosa. The specimen had been sent to space for physiological research on lichens species (Sancho et al. 2008). The oldest specimen known from Iran was collected by Aucher-Éloy in 1825 from the surroundings of Hamadan and labeled as Lecanora esculenta. After 186 years it turned out to belong to our new species. A specimen of "L. esculenta" reported by Aucher-Éloy (1843) from Uromia (Reżā'iyeh) could not be located so that its true identity remains uncertain. Specimens examined and paratypes. Armenia. ‘Kurdistan’, [probably from South Armenia, NW Iran E Turkey and NW Iraq], Radde (LE). Near the Ararat mountain, 1872, Demidova (LE). Casikoporan, Surmalinsky district. Iravanskay Region, 1896, Koenig, in the same envelope with C. fruticulosa (LE). Azerbaijan. Baku, near railway station, Sumgait, 1901, Alexeenko 11214 (LE); Naxjivan (0 Naxjivan Autonomous Region), Ordubad district, 1864, Radde (LE). Iran. [Persia, ‘Elevant’], Hamadan, Alvand-Kuh, 1825–1830 Aucher 909 (PC). East Azerbaijan, Kaleybar district 35 km S of Kaleybar along road to Ahar, 1750 m, 2007, Sohrabi et al. 10405B (B, IRAN, hb. M. Sohrabi). Golestan, Gorgan district Jehan-Nema plain, c. 24 km S of Gorgan along minor road to Shahrad, 2250 m, 2007, Sohrabi et al. 9496 (H, IRAN, hb. M. Sohrabi). Spain. Prov. Soria, Hochfläche (Paramera) W Calatayud, Kurz W des Ortes Judes SE Arcos de Jalòn, c. 1200 m, 1983, Mayrhofer 3656 & Crespo (GZU), ibidem, Poelt (GZU). Guadalajara, Zaorejas, 1240 m, 2003, Printzen 8087 (FR). Mycol Progress (2013) 12:231–269 Olmedillas, junto al pueblo, 2007, ladera con caliza, en el suelo, I. González (MAF-Lich 15363, H). Turkey. Prov. Kırıkkale, 1100 m, 1990, Doumez (hb. Seaward); Prov. Kirşehir, Kizil Dağ NE of Mucur, 1300 m, 2001, John 11984B (M). Anatolia (0 Anatolian Prov.), sine coll. (H-NYL 25671); Harput [‘Charput, Karput’], (0 Elâzığ & Diyarbakır provinces). Harput, 1864, Haidinger (PC). Karput Mt., Diyarbakir, sine coll. (W). Diyarbakir, sine coll. (LE). ‘Kurdistan Region’ (0 East Turkey), Schehid duri 1863, Mayer (W). Niğde prov., Erdschias-Dagh, Argaeus, 1000 m, 1902, Zederbauer (W). Turkmenistan. Central part of Kopet-Dagh district, next to the border of Iran, Stony part of mountain, to the SW of Solukli, 1934, Borisova, packed under C. aschabadensis (LE). Exsiccata. Spain. Soria, plateau (Paramera) W of Calatayud, W of village Judes SE Arcos of Jalòn, c. 1200 m, 1983, Hafellner 17527, Crespo, et al. (GZU) in Obermayer: Lich. Graecenses: No. 211 (CANB, GZU, M, NY, UPS). ‘Prov. Soria, Prope vicum Judes Paramera’, c. 1250 m, 1983, Barreno et al. in Vězda: Lich. Sel. Exs. No 1904 (BM, US). Prov. Teruel, der Sierra de la Costera, Cañada Vellida, 1050 m, Follmann & Follmann-Schrag, 1973, in Follmann, Lich. Exs. Casselensi No. 99 (BM, H, TUR). Circinaria hispida (Mereschk.) A. Nordin, S. Savić & Tibell in Mycologia 102: 1346. 2010. (Fig. 5a-c). Basionym: Aspicilia hispida Mereschk. in Trudy Obshch. Estestvoisp. Imp. Kazansk. Univ. 43 (5): 10, 35. 1911. Lectotype: designated by Sohrabi and Ahti (2010), Russia. ‘Ad terram argilloso-calcaream montis Bogdo prope lacu Baskuntschak in gub Astrachan’, 50–120 m, 1910, Mereschkowsky in Mereschkowsky: Lich. Ross. Exs. No. 34 (TU; isolectotypes LE L1988, W). Note: for the moment the treatment below includes the whole C. hispida s.str., but excluding its crustose form. Thallus subfruticose, erratic, usually basally attached or imbedded in soil, in later stages become vagrant, (probably also rarly on pebbles, however this need further study), about 5–20 mm tall, 5–20(–30) mm broad, forming small tufts, branching irregular to dichotomous, main branches variable in width, (0.3–) 0.5–1.5(–2) mm in diam., but distinctly tapering and pointed at the tips. Surface grey, green–grey, olive–grey, yellow–grey, brown–grey to green, olive, olive–brown or almost brown, dull, at branch tips black. Pseudocyphellae whitish, round to elongated, 0.1– 0.8 mm in diam., common along the branches. Cortex two layered, outer part (30–)40–80(–90) μm thick, paraplectenchymatous, ± brown, c. 3–5 cells thick, cells (4–)5–7(–8) μm in diam., inner part prosoplectenchymatous c. 2–3 times as thick as the outer layer, cortex covered with a thin epinecral, amorphous layer 1–10(–15) μm thick. Photobiont chlorococcoid, cells 5–15(–20) μm in diam., clustered in small groups. Medulla white, I–, containing crystals of calcium oxalate. Apothecia not seen; for more information, see Sohrabi et al. (2011b). Pycnidia common, 257 immersed, sometimes occurring in the pseudocyphellae, often located along the branchlets, rarely at the apices, flask-shaped, with black ostiole. Conidia filiform, straight to slightly curved, (8–)10–12 (–14) × 0.8–1.2 μm. Chemistry: All spot-tests (K, C, KC, CK, P) negative both in the cortex and medulla. TLC and HPLC: No substances detected. UV: Negative. Ecology and distribution (Fig. 6g). The erratic species Cricinaria hispida is an example of a steppe element found in temperate and subtropical, semi–arid regions of the Northern Hemisphere. Circinaria hispida has been found in a wide range of ecological conditions in Eurasia. For instance, the type specimen was collected from lowland steppes in N Caspian Sea and other collections are reported from alpine areas in Italy (Hafellner et al. 2004) and TianShan Mountains in Kyrgyzstan. The species is often reported on ± calciferous soil in arid steppe or steppe–like habitats and is usually growing in open stony slopes. Vagrant forms accumulate in wind–deposited drifts. Some of the accompanying plants were listed by Andreava (1978), e.g., Anabasis salsa, Artemisia glauca, A. herba-alba, A. pauciflora, Atriplex cana, Calligonum spp., and Salsola arbusculiformis. Additional list of accompaning lichen species and plants can be found in Hafellner et al. (2004). The species is widespread, so far reported from southern Europe by Hafellner et al. (2004), from Russia by Kulakov (2002, 2003), from Ukraine by Mereschkowsky (1911a, b), Middle Asia by Andreeva (1987) and Iran by Seaward et al. (2008) and Sohrabi et al. (2010d). In North America it is known from Canada (Saskatchewan) and USA (eastern Oregon to eastern Montana and northern Great Plains, south to Utah, Colorado and Arizona; Owe–Larsson et al. 2007). Remarks. Circinaria hispida s.str., a vagrant morphotype sensu Mereschkowsky (1911b), is characterized by subfruticose, forming tiny, bushy, more or less Cladonia-like thallus with narrow cylindrical branches with black apices at tip of branchlets and scattered whitish pseudocyphellae along the branches. It initially grows on soil and later becomes vagrant. Therefore, it is classified as erratic. C. hispida s.str. has never been reported in fertile condition in Eurasia. So far, its fertile specimens have previously been reported from North America (see Thomson 1960; Brodo 1976; Sohrabi et al. 2011b). In the nrITS analysis, C. hispida is grouped with a few Eurasian saxicolous specimens that are fertile. Although this morphological difference between vagrant and crustose specimen of C. hispida is straightforward, the present result of nrITS analyses did not support the monophyly of vagrant and crustose morphotypes. Several specimens of vagrant morphotype, identical with the type specimen of C. hispida, were included in the study. However, they did not form a monophyletic group, either. Therefore, in this study vagrant morphotypes are accepted as C. hispida s.str. and saxicolous specimens with crustose 258 morphotype as C. hispida s.lat. Thus, the final conclusions concerning the status of C. hispida can not be made in the present study. According to Rosentreter (1998), Aspicilia californica Rosentr. and A. filiformis Rosentr., are two terricolous subfruticose species that are known only from northwestern North America. Up to this point, they have never been observed in vagrant form. Both species bear prostrate and patchy thalli and are clearly attached to soil. However, they somewhat differ from C. hispida s.str. mainly by the lack of pseudocyphellae and presence of secondary metabolites. Our attempts for DNA extractions from these two species were not successful. Additional literature reports. Circinaria hispida has been reported from several countries e.g., from Azerbaijan by Barkhalov (1952); from Iran by Seaward et al. (2008 as Aspicilia hispida); from Kyrgyzstan by Litterski (2002 as A. hispida); Turkey by Candan and Türk (2008 as as A. hispida) and from Turkmenistan by Dzhuraeva (1978 as as A. hispida). Specimens examined (C. hispida s.str.) Kazakhstan. Aklushenskaya district, Bayzhanshal limestone ridge, 1957, Andreeva (LE); Akmola Region, near Kökshetau Mts., S direction, 1957, Andreeva (LE). Kyrgyzstan. Northern side of Naryn-Too Mt., valley of River Naryn, 23 Km to the E. from Naryn Mt., 2250 m, 1970, Bredkina (LE). SW Tian-Shan, NE Abhange des Fergana-Gebirges, SW Chatyr-Kol, 3261 m, 2003, Ringel 5242 (H). SW TianShan, Susamyr, 2381 m, 2003, Ringel 5244 (H). Westl., Tian-Shan, Susamyr-Tal, c. 25 km W Susamyr, 2300 m, 1999, Litterski 5053 (H). Depression of Song-Köl Lake, 3023 m, 1970, Bredkina 445 (LE). Mountain Naryn-Too, near River Naryn, c. 23 km from Naryn, on the E direction from Naryn. 2250 m, 1970, Bredkina (LE). Iran. Hamadan, foothills above Gholi-Abad, c. 60 km N of Hamadan, 1800 m, 1974, Alava 14749-d (TUR). East Azerbaijan, Marand district, 32 km N of Marand towards Jolfa, 1440 m, 2007, Sohrabi et al. 10102 (hb. M. Sohrabi). Marand district, Zonuz, 20 km N of Marand towards Jolfa, 1800 m, 2007, Sohrabi et al. 10064 (hb. M. Sohrabi). Jolfa district 1 km S of Daran village, E of Hadishahr, 1700 m, 2007, Sohrabi et al. 10136 (hb. M. Sohrabi). Shabestar district W along road Tabriz-Marand, 7 km N of Sufiyan, 1450 m, 2007, Sohrabi et al. 10032, 10022 (hb. M. Sohrabi), Kaleybar district 35 km S of Kaleybar along road to Ahar, 1750 m, 2007, Sohrabi et al. 10407, 10405D (hb. M. Sohrabi). Golestan, Golestan National Park, Almeh valley, near the station, 1800 m, 2008, Sohrabi 15068, 15099 & Ghobad-Nejhad (hb. M. Sohrabi, H). Gorgan district Shah Kuh-e-Bala, c. 33 km S of Gorgan along minor road to Shahrud, 2600 m, 2007, Sohrabi et al. 9501 (hb. M. Sohrabi). Italy. Piemonte, Prov. Cuneo, Alpi Liguri, Cima di Pertega W above the village Úpega, just E below the summit, c. 2400 m, 2000, Hafellner & Hafellner 59353 Mycol Progress (2013) 12:231–269 (GZU, TSB). Piemonte, Prov. Cuneo, Alpi Cozie, crest SW above Colle dell’Agnello, c. 2830 m, 2000, Hafellner 59364 (GZU). Greece. Parnassus Mt., Fterolaka, near the cableway, 1850 m, 1989, Tretiach & Roux (TSB). Mongolia. Zavkhan [correctly Govi-Altai] aimag, Taishir sum, right bank of Zavkhan River, c. 2 km N of Taishir Town, 1978, Biazrov 8373 (LE). Russia. Astrakhan, near the Lake Baskunchak, 1926, Tomin (H), ibidem, 1926, Tomin 37 (FH, H). ibidem, 1927, Tomin 56 (FH, H). Mountain Bolshaya Bogdo, 1924, Kazakevich (LE). Near the Lake Baskunchak, W slope of mount Big Bogdo, 1998, Sagalaev (M). Bogdo Mt., northern slope, 1926, Savicz (FH, LE). ‘Russia’ sine loc., 1907, Djungalien (H). Berg Bogdo, 1910, Mereschkowsky (H, W). Saratow Region, Elshanke, 1926, Yanishewski (LE). Volgograd Region, Kalachovsky district, vicinity of Bolshegolubinsky house. Northern slop of river Bolshaya Golubaya, 1994, Kulakov (LE). Orenburg Region, to the northwest from Kutush village, beside the road, 1930, Sukhova (LE). Kalmyk Region, Elista town, ravin, close to the street Ulan Tug, 2003, Ochirova (LE). Kalmyk Region, Bergin village, E slope, 2002, Ochirova (LE). Spain. Prov. Soria, Hochfläche ("Paramera") W Calatayud, Kurz W des Ortes Judes SE Arcos de Jalòn, c. 1200 m, 1983, Crespo & Poelt (GZU). Calatañazor, 1979, Barreno & Crespo 2139 (hb. Seaward). Turkey. Malatya, Pınarbaşı mesire alanı ve çevresi, 931 m, 2003, Candan 11 (H). Ukraine. Crimea, Simpheropol steppes, 1910, Mereschkowsky (H). Exsiccata. Russia. Astrakhan, Ad terram argillosocalcaream montis Bogdo prope lacu, 50–120 m, 1910, Mereschkowsky, in Mereschkowsky: Lich. Ross. Exs. No. 34 (LE, TU). Regio Astrachanensis per declive (in parte superiore) montis Bogdo ad terram inter gramina, fruticulos lapidesque crescit, saepe libere vagature, 1926 Savicz, in Savicz: Lich. Ross. No. 97 (FH, GZU, H, W). Ukraine. Crimea (Krym), Supra Terram stepporum prope Simpheropolin, in Peninsula Taurica, 1910, Mereschkowsky, in Mereschkowsky: Lich. Ross. Exs. No. 35 (LE). Circinaria jussuffii (Link) Sohrabi comb. nov. (Fig.4d). MB 563030 Basionym: Placodium jussuffii Link in Bot. Zeitung (Berlin) 6: 666.1848. Lectotype: designated by Sohrabi and Ahti (2010), Algeria. ‘Du désert Algerien’, 1847, Jussuff (H-NYL 3312). Thallus vagrant, rarely invades small pebbles, folded, globose-angular in shape, convoluted subsquamulose, compact, forming areole-like surface, deeply cracked, hard, densely imbricate, interconnected by the central axis, up to 3–10(–25) mm diam. Surface smooth, concave to convex, often reddish brown, rusty red (when ferriferous sediments are present in soil), yellowish brown, brown, more rarely red-brown. Pseudocyphellae very common, conspicuous, ±white spots along the subsquamuloes. Cortex one layer, (30–)50–75(–90) μm thick, paraplectenchymatous, ± Mycol Progress (2013) 12:231–269 brown, cells (4–)5–7(–10) μm in diam., inner part indistinct, more or less prosoplectenchymatous, mixed with anticlinal hyphae of medulla. Epinecral layer 5–15(–25)μm thick, usually brown to red. Photobiont chlorococcoid, cells 5–15 μm in diam., arranged and clustered in some small groups, each up to 80–170×80–150 μm wide, distance between groups 20– 100 μm. Medulla prosoplectenchymatous with irregularly oriented hyphae (textura intricata), white, I–, containing crystals of calcium oxalate, visible in (H2SO2 10 % solution). Apothecia aspicilioid, urceolate, immersed, not common, round, sometimes angular, 0.2–0.6 mm in diam., 1–2(–4) per subsquamulose. Thalline exciple flat to ± elevated, prominent in older apothecia, entire, concolorous with thallus or with a thin white rim. True exciple (30–)35–65(–85) μm, ± I + blue, uppermost cell brown, ± globose, 4–5(–7) μm in diam. Epihymenium brownish to dark brown, usually contain some crystals, K + colour fading from brown to light yellowish green, N + pale green (caesiocinerea-green). Hymenium hyaline, I + persistently blue, (80–)100–120(–135) μm tall. Paraphysoids moniliform to submoniliform, with upper cells ± globose, 4–5 μm wide, in lower part with ± cylindrical cell 6–7×2–3 μm wide; branched. Hypothecium and subhymenium pale, (35–)50–65(–90) μm thick, I + blue. Asci broadly clavate, with distinctly thickened apex dome (15–35 μm. thick), I–, 70–110×20–35 μm, 1–4(–6) spored. Ascospores hyaline, globose to subglobose, (15–)19.2–[22.5]–25.7(– 31)×(10–)15.8–[19.1]–22.3(–26) μm (n031). Pycnidia immersed, single, stretch flask-shaped, internal wall colourless, frequently with black to brownish ostiole. Conidia filiform, straight to very slightly curved, (8–)10–14(–16)×1–1.3 μm (n034). Chemistry: All spot-tests (K, C, KC, CK) negative both in the cortex and medulla, but in some thalli P±. TLC: Three common chemotypes containing (1) stictic acid and hypostictic acid, (2) only stictic acid, (3) no substances. Ecology and distribution (Fig. 6f). Circinaria jussuffii most commonly grows as vagrant on calcareous soil, or on gypsum crusts. It is very rarely found on small pebbles. The known distribution is restricted to North Africa, particularly in the Northern Saharan (Algeria, Libya and Moroco) steppes and semideserts areas. The total distribution of C. jussuffii is rather poorly known, but in North Africa the species seems to be very common, although overlooked, or misidentified as C. esculenta. The easternmost collection of the species was recently made from the Central Iran (Tabas district); see also Rabenhorst (1871). Additional literature reports. The first reports of Circinaria jussuffii from North Africa were made by Link (1848, 1849). Aspicilia esculenta was reported from Algeria by Flagey (1896). The name A. esculenta is still in use, rarely appears in North African lichen literature. However, after extensive evaluation of both herbarium material and recent publications (e.g., Donkin 1980, 1981; Crum 1993; Thor and Nascimbene 2010), it became clear that the reports 259 of C. esculenta from North Africa is dubious and might be referrabel to C. jussuffii. Remarks. Morphologically, Circinaria jussuffii is somewhat similar to C. esculenta. However, there are certain differences which can be highlighted. For example, C. esculenta is a lowland species (lower than c. 500 m), distributed in the northeast of the Caspian Sea and some parts of Central Asia. It can be easily distinguished from C. jussuffii by having wrinkled large subsquamulose thalli as well as longer conidia (10–)18–30(–35) μm. C. jussuffii is usually found in the open habitats from c. 100–1300 m and it has shorter conidia (8–)10–14(–16) μm, smaller subsquamulose with densely compact, more or less even thallus surface. Thallus colour may vary in both species. Occasionally C. esculenta grows on soil with ferriferous sediments and its surface colour changes to dark reddish-brown or rusty colour. Most of the herbarium specimens of C. jussuffii examined for this study were reddish-brown to rusty. Our nrITS analysis obviously revealed that the two taxa are distinct (Fig. 2). The records of A. esculenta in North Africa are probably based on C. jussuffii. Therefore, the exclusion of C. esculenta from the Libyan checklist (Thor and Nascimbene 2010) is proposed. Specimens examined. Algeria. Laghouat, 1851, E. Ripart (H-NYL 25672, UPS), ibidem, 1856, E. Ripart (UPS). "Sahara", 1857, Montagne, sine coll. (PC, UPS). Sahara, Laghouat, 1856, Jussuf & Rebaud (PC, W). ‘Der Sahara von der Umgegend von Laghouat’, Hohenacker, No. 722 (FH). Laghouat, 1857, Motelay 760 (PC). Auf steinigem boden in der Sahara bei Laghouat, 1856, Jussuf & Rebaud in Djelfa; No. 794 (FH, H-NYL 25670, PC, S, UPS, W). Environs de Laghouat, Janvier 1857, Rebaud (FH, US, W). Satara la Laghouat, 1856 Jussuf (W). Near Durandu, 1881, sine coll. ‘comm. Toepffer’ (W). ‘North Africa, in deserto Titteré’, sine coll. (UPS). Sahara. sine coll. (W). ‘Africa Borealis’ ("Algeria"), ibidem, Russel (S). Plaine du Zahrés, Région des Haute-plateux, a nord de Djelfa, 1857, Rebaud (H, UPS, W). E. Bourgeau, Pl. ďAlgerie, 1856, sine coll. (UPS). ‘Algeria’, sine coll. (UPS). El Aghwat im Norden der Sahara, Jussuf (UPS). ‘In deserto Titteré’, sine dato & coll. (PC, 6 specimens). ‘Sud de l' Algėriei", 1847, Jussuf (PC). Hauts-Plateaux, Pâturages pierreux du Serson, prês de Vfetor-Hugo, 9000–1000 m, Nom arabe, Oussekh-el ardh (crasse de la terr.), 1929, Maire (PC). ‘Sud de l' Algėriei", 1846, Guyon, (PC). Iraq. c. 130 km N of Ar Rutba, Fuggarai, 1966, Rami 34186 (BM). Iran. [‘Persia"], Yazd prov., near Tabas (see also Rabenhorst 1870), Stapff (W). Libya. Trigh el Abit, Slates, 1959, sine coll. (US). Exsiccata. Algeria. Environs de Laghouat, Janvier 1857, Rebaud. ‘Ex Herb. Durieu de Maisonneuve’, and Fragmenta Flora Algeriensis Exs. No. 500, as Parmelia esculenta (H, PC 2 specimens, UPS, W). ‘Sahara près de Laghouat’, Janvier 1856, Rebaud. [Flora Galliae et Germaniae exsiccati 260 de C. Billot.] No. 1997, as Placodium esculentum (LE, PC, S,). ‘Sahara près de Laghouat’, Janvier 1856, Rebaud (Lichen Helv. Exs. Schaer, No. 1160, and Hepp Flecht. Eur. No. 632 (FH, S, TUR-V 5824). ‘Sahara près de Laghouat’, Janvier 1856, Rebaud, Rabenhorst, Lich. Europaei, No. 199, as Chlorangium jussuffii (FH, H, S, US); N of Djolfa plaine du Zahries, 1856, Rebaud, in Bourgeau Fl. Algérie No. 247 (H, W). Morocco. ‘Regnum Maroccanum’ inter Taourirt et Taza, 75 km ad occidentem a Taza, 350 m, 1989, Hafellner & Follmann, in Vězda: Lich. Sel. Exs. No. 2381, as Aspicilia jussuffii (H, S, UPS, US, TSB). ‘Südmarokko, Distr. Ouârkziz, Hamadaformation bei Aouinet Torkoz’, 400 m, Follmann, in Follmann: Lich. Sel. Exs. No. 74 (TUR). Circinaria lacunosa (Mereschk.) Sohrabi comb. nov. (Fig. 4a). MB 563031 Basionym: Aspicilia lacunosa Mereschk. in Trudy Obshch. Estestvoisp. Imp. Kazansk. Univ. 43 (5): 11. 1911. Lectotype: designated by Sohrabi and Ahti (2010): Kazakhstan. Semipalatinsk Region [East Kazakhstan Prov.], Zaisan District, Keller (icon seen in H, W, voucher not found). Thallus vagrant, lumps subspherical to irregularly lobed, amorphous, truffle-like, rounded, variable in size, 0.5–2(–3) cm in diam. Surface flattened to uneven, folded, with narrow, thin cracks, broadly flexuose, obtuse, rounded at lobes, often grey, grey-olive, more rarely rust colour (when ferriferous sediments present in soil), ochre to bright red-orange, glabrous, often with rounded pores in different sizes, 0.5–1 (1.5) mm diam., occasionally surrounded by part of thallus. Pseudocyphellae conspicuous, numerous, small white dotlike spots. Cortex indistinctly delimited, (40–)50–100(–110) μm thick, paraplectenchymatous, consists of isodiametric, rounded to rounded-angular, thin-walled cells (3–)5–7(–8) μm in diam. Epinecral layer 3–10(–25) μm thick. Photobiont chlorococcoid, cells 5–15(–18) μm in diam., arranged in single layer and clustered in few small groups, each group up to 100–150×90–145 μm wide, often with 40–100 μm from each other. Medulla white, I–, irregularly developed, 0.8–2 mm thick, white, crumbly, when thallus broken hyphae appear giving tomentose appearance, arachnoid in the central part, occasionally with cavities, partially turbid due to rather numerous granules covering hyphae, containing crystals of calcium oxalate. Apothecia aspicilioid (urceolate), very rare, immersed, up to 0.5–0.8(–1) mm wide. Disc black to brown-black, pruinose, concave. Thalline exciple ± flat to slightly elevated, entire, concolorous with thallus. True exciple (25–)30–35(–40) μm wide, ± I + blue, uppermost cell brown ± globose, 4–5(–7) μm in diam. Epihymenium brown, K + colour fading from brown to light yellowish green, N + pale green (caesiocinerea green), Hymenium hyaline, with few oil drops, (110–)120– 130(–140) μm tall. Paraphysoids moniliform to submoniliform, with upper cells ± globose, 4–7 μm wide, in lower Mycol Progress (2013) 12:231–269 part 5–8 × 2–3 μm wide, more or less branched. Hypothecium and subhymenium pale, (35–)45–70(–90) μm thick. I + blue. Asci clavate, (70–)80–90(–95)×20–27 μm, with rather thick apical dome (10–15 μm), 2–4 spored. Ascospores hyaline, simple, globose to fairly subglobose, 14–18(–19)×(13–)15–17(–18) μm. Pycnidia immersed, single, stretched flask-shaped, internal wall colourless, frequently with black to brownish ostiole. Conidia filiform, straight to very slightly curved (8–)11–13(–16)×1–1.3 μm. Chemistry: All spot-tests (K, C, KC, CK, P) negative both in the cortex and medulla. TLC and HPLC: No substances detected. UV: Negative. Ecology and distribution (Fig. 6a). The ecology of Circinaria lacunosa is poorly known. It grows on gravely or sandy clayey soils in steppes and semideserts. Winddrifted thalli sometimes accumulate nearby the cushion plants. The following plant species have been reported as dominant in the habitats of C. lacunosa: Artemisia glauca, A. herba-alba, A. pauciflora, as well as Anabasis salsa, Atriplex cana, Calligonum spp., Kochia prostrata, and Salsola arbusculiformis. Remarks. Circinaria lacunosa is characterized by its subspherical to irregularly lobed, truffle-like, rounded, sometimes flattened, ± addpresed thallus, with rounded pits on the surface. Some of the thalli are narrowly flattened and they can be confused with subfoliose thalli of C. emiliae. However, C. lacunosa has a hole-like structure at some parts of the thallus and it has a corticated surface. Circinaria lacunosa may also greatly resemble to C. cerebroides but in the latter spec, hole-like structures are lacking and it grows in alpine-nival habitats (c. 2000–4500 m) of TianShan Mountains in the Central Asia. Circinaria lacunosa prefers lowlands and lower montane habitats (c. 100– 1100 m). For the first time, a fertile specimen was examined by Golubkova (1980) and the species description was somewhat improved. In the present study the same specimen was re-examined as the only fertile sample of C. lacunosa. Additional literature reports. Circinaria lacunosa was reported from several contries i.e., by Abbas and Wu (1998) from China; Schubert and Klement (1971) from Mongolia; Bredkina and Makarova (2005) from Kyrgyzstan, and Oxner (1971) from Uzbekistan. The report from Turkey by John and Türk (2006) referable to a new species Circinaria rostamii. However, additional specimens needs be confirmed. Specimens examined. China. Xinjiang, Tacheng Toli, 1050 m, 1994, Abbas 94003 (H). Kazakhstan. South Peribalkhashya, Kenes-Anarkhay, 1976, Piregoudov, ibidem, 1976, Piregoudov (LE). Betpak-dala, along the road to Kendyrlyk, 1957, Vasyagina (LE). SE Balkhash, 1951, Kazichas (LE). Syrdaria River, Mayon Kumski district, Koskaduski, Saksaoul village, 50-150 m from railway station Tchu, Saksaoul, near Orta khdok-Karman Kuduk, Mycol Progress (2013) 12:231–269 1929, Nikitin 3 (LE). Mongolia. Zavkhan [correctly Govi-Altai] aimag, Taishir sum, vicinity of Taishir, 1978, Biazrov 8375 (LE). Circinaria rostamii Sohrabi sp. nov. (Fig. 3e-i). MB 563032 Diagnosis. Thallus vagrant, brain-shaped, subspherical, surface even, more or less rimose, with shallow to deep cracks, dull brown, brownish grey to greyish green Very similar to Circinaria lacunosa and C. cerebroides, but differentiated by its more brain-like appearance, rimose surfaces, areole-like lobes, monophyletic poistion in the nuclear ribosomal ITS phylogeny analysis. Type: Iran. East Azerbaijan, Jolfa district, 1 km N of Asiab Kharabeh waterfall, S of road Hadi Shahr Siyahrud., 950 m, 4 November 2007, 38°51.89'N, 45° 51.56'E, M. Sohrabi 10212, H. Sipman, U. Søchting & M. R. Asef [IRAN 14442, holotype; F, GZU, H, hb. M. Sohrabi, isotypes] Thallus vagrant, lumps formed of folded lobes, brainshaped, subspherical, rounded, amorphous, occasionally flat, variable in size, 0.5–2.5 cm tall, 0.5–2(–3) cm wide. Surface even, more or less rimose, with shallow to deep cracks, dull brown, brownish grey to greyish green, sometimes whitish grey, pale olive-brown to pale brown, sometime reddish brown (when ferriferous sediments are present in soil). Pseudocyphellae very common, ±white, dot-like, usually visible on areole-like lobes. Cortex indistinctly delimited, one layer, (40–)60–90(–110) μm thick, paraplectenchymatous, ± brown, cells (4–)5–7(–8) μm in diam., inner part indistinct, mixed with prosoplectenchymatous tissue of medulla sometimes forming distinct layer of (30–)40–80(–90) μm thick, (floating of algal cells in the medulla make it uneven, without distinct margin to distinguish it from the real cortex). Epinecral layer 1–10(–18) μm thick. Photobiont chlorococcoid, cells 5–22 μm in diam., clustered in small groups, each group up to 80–180×50– 110 μm wide. Medulla white, I–, containing crystals of calcium oxalate. Apothecia aspicilioid, rare, up to 0.5–1.5 (–2.5) mm wide, often among the lobes in older parts of the thallus. Disc black to brown-black, pruinose, concave to convex when young, becoming more flat when old. Thalline exciple flat to ± elevated and prominent in older apothecia, entire, concolorous with thallus or with a thin to thick white rim. True exciple (40–)50–85(–90) μm wide, ± I + blue, uppermost cell brown, ± globose, 4–5(–7) μm in diam. Epihymenium brownish to dark brown, K + colour fading from brown to light yellowish green, N + pale green (caesiocinerea-green). Hymenium hyaline, occasionally with few oil drops, (95–)115–140(–155) μm tall. Paraphysoids moniliform to submoniliform, with upper cells ± globose, 4–7 μm wide, in lower part 4–8×2–3 μm wide, more or less branched. Hypothecium and subhymenium pale, (35–)50–65(–85) μm thick, I + blue. Asci broadly clavate, (80–)90–100(–110)×25– 261 35 μm, with thick apical dome (20–30 μm), 2–4(–5) spored. Ascospores hyaline, simple, globose to subglobose, (16–)19–[22.1]–24 (–26)×(16–)19–[21.6]–23(–25) μm (n0 30). Pycnidia usually on top of branches, immersed, single, stretched flask-shaped, internal wall colourless, frequently with black to brownish ostiole. Conidia filiform, straight to very slightly curved (8–)10–12(–16)×1–1.3 μm (n030). Chemistry: All spot-tests (K, C, KC, CK, P) negative both in the cortex and medulla. TLC and HPLC: No substances detected. UV: Negative. Etymology. The first author names this species after his father Rostam Sohrabi on the occasion of his 60th birthday, with thanks for the help provided during the initial stages of his interest in lichenology of Iran. Ecology and distribution (Fig. 6d). The thalli of this species can be found on bare calcareous soil mixed with gravel and big limestone outcrops. The species is found on dry steppes, mainly associated with xerophytic plant communities including Artemisia spp., Verbascum spp. and Zygophyllum spp. Other associated vagrant lichen species on soil is Circinaria hispida, and on pebbles Caloplaca chalybaea (Fr.) Müll. Arg., C. crenulatella (Nyl.) H. Olivier, C. polycarpoides (J. Steiner) M. Steiner & Poelt, Lecanora aff. dispersa, and L. garovaglioi (Körb.) Zahlbr. Some populations of C. rostamii can be mixed with C. gyrosa, the other vagrant species in the NW part of Iran. Circinaria rostamii has a wide range inside Iran, which extends from Kiamaky-Dagh Mts. in E. Azerbaijan province towards Semnan province in the Central Iran. The species is also known from Naxjivan, Ordubad district in Azerbaijan, where it is located in the border between Iran and Azerbaijan, very close (c. 10 km) to the type locality. It is also known from the dry steppes of Kurdistan Region in SE Turkey. Remarks. Circinaria rostamii more or less resembles to truffle-like thalli of C. cerebroides and C. lacunosa, and is characterized by shallow to deep cracks and areole-like lobes at the surface. C. rostamii differs from the alpine species of C. cerebroides (growing above alpine zone) by having smaller thallus lumps, having more or less areolelike surface and different ecology and habitat requirements. In C. lacunosa thalline lumps are often larger and it consists of flattened to wrinkled lobes, and possess hole-like structure in some parts of the thallus. The conidia size of all three species are more or less overlapped and minutely differentiated in size, up to c. 1–2 μm. However the monophyly of all three species was confirmed by using nrITS sequences (see Fig. 2). Examined specimens and paratypes. Azerbaijan. Naxjivan (0 Naxjivan Autonomous Region), Ordubad district, 1864, Radde (LE), Note: this specimens was illustrated by Elenkin (1901a). Iran. East Azerbaijan, Marand district, 32 km N of Marand towards Jolfa, 1440 m, 2007, Sohrabi et 262 al. 10095 ( H, IRAN, UPS, hb. M. Sohrabi). Semnan, Damghan district 80.5 km S of Shahrud along road to Torud, 1180 m, 2007, Sohrabi et al. 9364, (IRAN, hb. M. Sohrabi). Turkey. Kurdistan Region (0 SE Turkey), Schehid duri 1863, Mayer, in the same envelope with C. gyrosa (W). Circinaria tominii (Oxner) Sohrabi comb. nov. (Fig. 4i). MB 563033 Basionym: Aspicilia tominii Oxner in Novosti Sist. Nizsh. Rast. 9: 291. 1972. ≡ Lecanora esculenta f. altaica Tomin in Sist. Zametki Mater. Gerb. Tomsk. Univ. 5/6: 9. 1933. Lectotype:designated by Sohrabi and Ahti (2010): Russia. [Altai Republic], Deserto Czuensi [Chuy Desert], Altai Austro-orientalis, 24 September 1926, Baranov (H). Thallus vagrant, subfruticose, rounded, globose to subglobose, up to 3–10(–13) mm in diam., lumps forming tiny subsquamulose, 0.6–1.5(–2.5) mm long, 0.5–1(–1.5) mm wide, sometimes areole-like lobes, compact, tightly attached to each other. Surface even, often dusty, greyish to greyish green, matt, muddy, flexuose in some part, margins of subsquamulose more or less imbricate, somewhat elevated from the thallus surface. Pseudocyphellae conspicuous, tiny, round, withish to elongate pits, 1–2(–3) per subsquamulose, variable in size, up to 0.02–0.4(–0.7) mm wide. Cortex indistinctly delimited, uneven in thickness, paraplectenchymatous (35–)60–90(–100) μm thick, ± brown, with cells (4–)5–7(–8) μm in diam., inner parts often interrupted by prosoplectenchymatous tissue of medulla, in some parts becoming (30–)40–70(–95) μm thick. Epinecral layer dark brown, 5–18(–30) μm thick. Medulla white, I–, in sections often muddy, variable in different part of thallus, in central part 0.3–10 mm thick, containing crystals of calcium oxalate. Photobiont chlorococcoid, cells 5–18(–22) μm in diam., forming small clusters 120–180×80–150 μm wide. Apothecia crypto-lecanorine to aspicilioid, deeply immersed, common, 0.4–0.8(–1.5) mm wide, 1–2 per subsquamulose. Disc black, brown-black, whitish pruinose, at first rounded, later somewhat elongated or irregular in shape, sometimes surrounded by very thin white rim. Thalline exciple 0.2–0.6 mm thick, entire or slightly cracked, more or less even or slightly flexuose. True exciple (35–)40–60(– 65) μm wide in upper parts, at base 100–150 μm thick, ± I + blue, uppermost cell black-brown, ± globose, 4–5(–7) μm in diam. Epihymenium brown, 15–25 μm thick, K + colour fading from brown to yellowish green, contains insoluble crystals of calcium oxalat, N + pale green (caesiocinereagreen). Hymenium hyaline, (90–)100–110(–120) μm thick, I + blue. Paraphysoids moniliform to submoniliform, tightly conglutinate in hymenial gelatine, with upper cells ± globose, 4–5 μm wide, in lower part with ± cylindrical cell 5– 7×2–3 μm wide; branched. Hypothecium and subhymenium colourless (40–)45–60(–70) μm thick, I+blue, aften muddy in sections. Asci clavate, strongly thickened above, 90– 100×23–30 μm, with 2–3(4) spores. Ascospores hyaline, Mycol Progress (2013) 12:231–269 globose or broadly ellipsoid, (15–)18.8–[21.6]–24.4(–27)× (15–)18.4–[20.7]–23.0(–25) μm diam. (n032), spores often I + yellow to redish yellow. Pycnidia immersed, sometimes occur in the pseudocyphellae, 1–3 per subsquamulose, single, stretched flask-shaped, internal wall colourless, with more or less brownish ostiole. Conidia filiform, (15–)18– 23(–26)×1–1.2 μm (n046). Chemistry: All spot-tests (K, C, KC, CK, P) negative both in the cortex and medulla. TLC and HPLC: No substances detected. UV: Negative. Ecology and distribution (Fig. 6b). The ecology and habitat of Circinaria tominii remains poorly known. It has been reported from arid steppes of the Altai Region; an area between Russia, Kazakhstan and Mongolia. Andreeva (1987) gives some additional localities in Kazakhstan. Remarks. Circinaria tominii is somewhat similar to small thalli of C. esculenta, but differs by having consistent smaller thallus lumps (c. 3–10 mm), being rounded subsquamulose, abundant apothecia (high rate of thallus fertility), and shorter conidia (15–25 μm). In C. esculenta thalline lumps are often larger (10–40 mm), it consist of wrinkled subsquamulose, deep cracks occur in between the subsquamules, and it has larger conidia (10–35 μm). Unfortunately, DNA extraction from the 80 years old herbarium collection (see also Sohrabi et al. 2010a) was not successful. However, C. tominii might be yet another close relative of C. esculenta. It is also found in the same habitats with C. affinis, but these species have different type of thallus mophology and are easily distinguished even in the field. Circinaria affinis has a gyroid thallus and bears large, conspicuose pseudocyphellae and it is often sterile. Additional literature reports from Kazakhstan by Andreeva (1987). Specimens examined. Russia. Altai Austro-Orientalis, in deserto Czuensi, 1926, Baranov (H, S); Deserto Czuensi, Altai Austro-Orientalis, (Kosh-Agach, Altay), 1929, Baranov (FH, LE, S), ibidem, 1929, Baranov (TUR). Discussion The results of the two analyses are only partially comparable because of the differences in included species. Due to difficulties in getting fresh material of the key species for complete amplification of selected loci (nrITS, nrLSU and mtSSU) and missing data, especially from saxicolous species, two independent analyses were performed. One was aimed at the delimitation of genera within the family Megasporaceae, the other at species delimitation of ‘manna lichens’. Generic delimitation The position of the sphaerothallioid species in the genus Circinaria, and their relationship to other allied genera of the Megasporaceae is still controversial. In order to study this group of species a combined data Mycol Progress (2013) 12:231–269 set of mtSSU and nrLSU sequences was assembled. Our analysis, compared to earlier analyses of the family Nordin et al. (2010), included many additional specimens from vagrant sphaerothallioid species, viz., C. affinis, C. cerebroides, C. esculenta, C. fruticulosa, C. hispida, C. gyrosa, C. lacunosa, C. jussuffii and C. rostamiias well as two crustose sphaerothallioid species viz., C. elmorei s.lat. and C. sphaerothallina (see Fig. 1). The results indicated a monophyletic Circinaria and supported Nordin et al. (2010). In this study the type species of Chlorangium (C. jussuffii) and Sphaerothallia (C. esculenta) were included in addition to the type species of Agrestia (C. hispida) that was included in Nordin et al. (2010). The results show that all of these genera are within Circinaria. According to our results the genus can be divided into two main groups and is quite heteromorphic. The sphaerothallioid species form their own clade separate from several saxicolous species (e.g., C. caesiocinerea, C. calcarea, C. contorta, C. gibbosa and C. leprosescens; see Fig. 1) All of these saxicolous species differ from sphaerothallioid species by having primarily crustose growth form, thin medulla and cortex layers, and shorter conidium length (6–12 μm). They are without pseudocyphellae and some of them contain aspicilin. Hafellner (1991) stated that pseudocyphellae, conidia, ascocarp structure and secondary metabolites are important taxonomic characteristics of the Hymeneliaceae (including current Megasporaceae) and noted that as the number of ascospores per ascus decreases there is often an increase in the size of individual spores. This is also true for sphaerothallioid species. Species delimitation The sphaerothallioid group consists of 15 subfruticose and subfoliose vagrant species worldwide. According to our results they all belong to the genus Circinaria. Owing to the remarkable morphological plasticity in some populations of the vagrant sphaerothallioid species, problems in defining monophyletic species were expected. Likewise, the lack of informative morphological characters in some species complexes, as well as the occurrence of intermediate forms of thalli between several species, has made accurate delimitation of these taxa difficult. For these reasons several specimens of each species were included, whenever possible, in the analysis of the nrITS data set (Fig. 2). Species with a vagrant morphotype were prioritized in sampling efforts. Some of the vagrant and crustose specimens formed distinct clades but the morphology of these specimens is not uniform. They show both vagrant and crustose morphotypes and are referred to as erratic species. They comprise several taxa such as C. alpicola, C. aschabadensis, C. elmorei, C. hispida, Circinaria sp. 1 and Circinaria sp. 2 that occasionally seem to shift their substrate preference. Only two predominantly saxicolous species, C. aspera and C. sphaerothallina were included in the sphaerothallioid clade. It must be mentioned that still our 263 current knowledge of their morphological variation is deficient and more field observations are required. The only terricolous species in the material is Circinaria sp. 3. which grows on soil in the arid steppes of central Iran and for which no vagrant morphotype has yet been observed. As shown in Fig. 2, vagrant and crustose species of Circinaria are not monophyletic. Vagrant species include C.affinis, C. cerebroides, C. emiliae, C. esculenta, C. fruticulosa, C. gyrosa, C. lacunosa and C. rostamii , and they are distributed among crustose species. The monophyly of C. esculenta and C. jussuffii is of particular interest, because the application of these names has been confused. Therefore, two samples were included from North Africa and three from Eurasia and it was confirmed that the two species are distinct (Fig. 2). Genetic similarity of crustose and vagrant morphotypes The specimens of some erratic species included in the analysis are from the same collection site and they turned out to be microscopically identical. In the nrITS analyses crustose and vagrant morphotypes of Circinaria alpicola and C. aschabadensis confirm the conspecificity of the morphotypes (see Fig. 2). In C. aschabadensis the nrITS sequences of morphotypes differ from each other only in one base pair, and in C. alpicola by three positions. This suggests that identification of erratic species based on external morphology might be misleading. The other example of an erratic species is the heteromorphic C. hispida that grows on soil (see Fig. 5c) or develops as a vagrant. This species has never been reported in saxicolous form. The nrITS sequences were obtained from seven vagrant specimens (matching the type species of C. hispida in LE) and three crustose ones. Although they form a monophyletic entity vagrant and crustose specimens are not monophyletic but instead mixed with each other. This result is somewhat congruent with the findings of Owe-Larsson et al. (2011), which show that C. hispida is in a group that includes several taxa earlier recognized as A. ‘desertorum’ sensu Krempelhuber (1867). This implies that C. hispida s.lat. is a rather complicated species that includes vagrant, terricolous (attached on soil) and saxicolous specimens and it might comprise several distinct taxa. Therefore, more extensive sampling and additional molecular markers from several geographically unrelated populations are required to resolve this problem. Chemistry Only a few studies have been conducted on the chemistry of sphaerothallioid species. Follmann and Huneck (1968) traced stictic acid in some North African specimens of Circinaria jussuffii. Esnault (1985) was able to detect one additional substance, hypostictic acid. In the present study no substances were detected in any of the examined species except C. jussuffii. Here we confirm the earlier reports on the chemical variation of North African specimens. Stictic and hypostictic acids typically do not occur in all collections 264 of C. jussuffii. We analyzed several herbarium specimens and detected that they were not chemically uniform even in the same envelope. It is still not clear whether occurances of stictic and hypostictic acids are reflecting at infraspecific level or not. This issue needs more detailed study. Note: Recently a new terricolous species, Aspicilia mansourii Sohrabi, was described in Lumbsch et al. (2011). It contains aspicilin, and therefore its systematic position needs to be tested and compared with other species in Circinaria, in particular C. arida, C. caesiocinerea, C. calcarea, C. contorta, C. gibbosa and C. leprosescens, all of which contain aspicilin. Circinaria sp. 3 is also a terricolous species but differs from A. mansourii by having large lobes and pseudocyphellae and lacking aspicilin. Biogeography and distribution patterns Sphaerothallioid taxa as a whole show a Holarctic distribution pattern. Most species are widespread in the Irano-Turanian region in Asia, the Mediterranean region in North Africa and southern Europe, and the Madrean region in North America (sensu Takhtajan 1986). In Eurasia, the largest proportion of vagrant species are Irano-Turanian (e.g., Circinaria affinis, C. alpicola, C. aschabadensis, C. cerebroides, C. digitata, C. emiliae, C. esculenta, C. fruticulosa, C. lacunosa, C. rostamii and C. tominii). Only C. jussuffii and C. gyrosa can be considered as Mediterranean species, both with fairly wide ranges. The same pattern of distribution is also known for many plants and isoften referred to as a Mesogean distribution (including large areas of the Mediterranean, IranoTuranian and Saharo-Arabian regions, sensu Quézel 1978). Circinaria rogeri is known exclusively from North America. Circinaria hispida is widespread both in Eurasia and North America, but has not yet been reported from North Africa. Circinaria alpicola, C. aschabadensis, C. cerebroides, C. digitata, and C. tominii have restricted distributions in localities with a particular vegetation type. Circinaria affinis, C. esculenta, C. emiliae, C. fruticulosa and C. lacunosa are all relatively widespread in the arid steppes of Central Asia. Based on altitude, vagrant species occuring in the IranoTuranian region can be divided into three groups: 1) species found in the lowlands (0–700 m) such as Circinaria affinis, C. esculenta, C. emiliae, C. fruticulosa, and C. lacunosa; 2) species largely distributed at mid-elevations (below the alpine zone) on mountains (1000–2000 m) such as C. aschabadensis and C. rostamii; and 3) species with high alpine to nival zone (2500–4500 m) range, such as C. alpicola, C. cerebroides, C. digitata and even C. hispida s.str. The Caspian lowland steppes are at the meeting point of the Middle East, Europe and Asia. It is a semidesert area that becomes increasingly arid from north to south and from west to east (Laity 2008). This is the area where six species namely C.affinis, C. esculenta, C. emiliae, C. fruticulosa, C. Mycol Progress (2013) 12:231–269 hispida and C. lacunosa, can be found. Several other subfruticose or subfoliose vagrant lichens have been reported from such habitats by Kulakov (2002, 2003). In the early Miocene (c. 22 Mya) due to the dehydration of the eastern part of the Tethys Sea, desert habitats expanded in Central Asia (Reichert 1953, Guo et al. 2002). Favourable conditions for xerophytic species were thus formed across large areas. The other aridification event in the Central Asia took place during the Middle Pleistocene (c. 1- 0.5 Mya; Dennell 2004). We can assume that some ‘manna lichens’ might have expanded their range during these aridification periods and their diversification was possibly accelarated in such favourable conditions. The possible endemism of the ‘manna lichens’ is still poorly known. Based on the data available it seems that some species are restricted to certain geographical regions. Circinaria alpicola, C. cerebroides and C. digitata are restricted to high altitudes in the Tian-Shan Mountains (Sohrabi 2011a) and are exclusively found at around 3000 m. Circinaria aschabadensis appears to be another ‘endemic’, its distribution restricted to Kopet-Dagh Mountain on the border of Iran and Turkmenistan, while C. tominii is known only from the Altai Region, although our knowledge of its distribution is still very deficient. In North America C. rogeri might be another example of ‘endemic’ species (see Sohrabi et al. 2011b). In this study many vagrant species were found sterile and have not been observed to produce apothecia. Apothecia were found in the following vagrant species: C. affinis, C. alpicola, C. aschabadensis, C. emiliae, C. esculenta, C. fruticulosa, C. gyrosa, C. jussuffii, C. lacunosa, C. rogeri, C. rostamii and C. tominii. Sufficient information is lacking on the fertility of C. cerebroides, C. digitata and Eurasian C. hispida s.str. In contrast to vagrant species, the crustose morphotypes of the erratic species often produce fertile thalli. The scarcity of sexual reproduction in vagrant species raises questions about their dispersal. Many species probably disperse by fragmentation. It has been stated in several studies (e.g., Bailey 1976, Rosentreter 1993, Leavitt et al. 2011) that fragmentation might be the only method of reproduction in vagrant species. Reproduction by fragmentation apparently restricts dispersal ability and genetic exchange among populations (see also Leavitt et al. 2011). During the summer, thalli of vagrant species can dry out very rapidly, becoming light in weight, and can then easily be distributed by wind. They are very brittle and their thalli frequently have fractured or scraped apices, as observed for example in C. hispida. As far as we know, none of the vagrant Circinaria species produce isidia, soredia or other specialized propagules. The sole widespread erratic species is C. hispida s.lat., which can be found both in North America and Eurasia (Sohrabi 2011a, b). Such a wide distribution might be due to an older continuous range in Laurasia. There are several examples of lichens with such distributions, Mycol Progress (2013) 12:231–269 notably in the genera Toninia and Caloplaca (Weber 2003). Finally, according to the present information and extensive evaluation of literature from other parts of the world, it is clear that none of the sphaerothallioid species (particularly ‘manna lichens’) have been reported from deserts or dry lands in Australasia (Eldridge and Rosentreter 1997), the Far East (Oxner 1971, Urbanavichus 2010), South America (Redon 1982) and Namib in Africa (Lalley and Viles 2005). Vagrancy among the sphaerothallioid species The vagrant growth form of lichenized fungi has evolved in several distantly related fungal lineages, including Lecanoraceae (Rhizoplaca Zopf), Megasporaceae (Circinaria), Parmeliaceae (Cetraria Ach., Masonhalea Kärnefelt and Xanthoparmelia (Vain.) Hale) and Verrucariaceae (Dermatocarpon Eschw.). Elenkin (1901a, b, c, d, e), Mattick (1951), Weber (1967, 1977), Bailey (1976), Rogers (1977), Hale (1990), Rosentreter (1993), Arup and Grube (2000) and Honegger (2001), Sohrabi and Ahti (2010), Sohrabi et al. (2011a, b) have all briefly discussed vagrancy in traditional Aspicilia (including current genus Circinaria). In the study by Elenkin (1901c: 24, 31) the formation of the vagrant growth form was explained. Elenkin´s assumptions about this formation process remained tentative and need to be tested in the light of our current understanding of thallus formation among the lichenized fungi. In the most recent studies by Honegger (2001), it is stated that ‘a large number of morphologically less advanced vagrant lichens do not roll and unroll during drought stress and rehydration events, but instead have a more or less globose shape’. Sphaerothallioid species show different types of substrate preference, and they can be divided basically into three groups. Some species are vagrant (obligatorily unattached), growing and reproducing without proper attachment to a substrate, i.e., C.affinis, cerebroides, C. esculenta, C. fruticulosa, C. gyrosa, C. jussuffii, C. lacunosa andC. rostamii. The other type of substrate preference is found in the erratic species (facultatively unattached) C. alpicola, C. aschabadensis, C. hispida s.lat., Circinaria sp. 1. and sp. 2. These species show two different morphotypes, probably associated with initially being attached to rocks and then later developing a vagrant growth form. The third type is represented by crustose (obligatorily attached) species. Our nrITS analysis shows that several saxicolous species belong to sphaerothallioid species. Some of them grow on large stones, some on tiny pebbles. Our results show that vagrancy has evolved several times in distantly related lineages of the sphaerothallioid species and several species are shown to be existed both in vagrant and crustose morphotypes (Fig. 2). This occurance of such erratic species indicates morphological convergence and might be due to ecological adaptation. The reason for the high plasticity in external morphology of vagrant and crustose species remains unknown. 265 Allied saxicolous sphaerothallioid species Several saxicolous or erratic species such as Circinaria alpicola, C. aschabadensis, C. aspera, C. elmorei C. hispida s.lat., C. sphaerothallina and Circinaria sp. 1-3, are shown here to belong to the sphaerothallioid clade. They differ from subfruticose vagrant species only by their external morphology. Based on literature re ports (e.g., Steiner 1919; Mereschkowsky 1911a, b; Magnusson 1940; Werner 1956, 1958; Oxner 1971; Kondratyuk and Zelenko 2002; Seaward et al. 2008) several other saxicolous species are documented that are morphologically and ecologically associated with sphaerothallioid species (traditional ‘manna lichens’, or the C. esculenta group). For example, Aspicilia albosparsa (Werner) S.Y. Kondr., A. auricularis (Werner) S.Y. Kondr., A. ferruginea (J. Steiner) Szatala, A. oxneriana O.B. Blum, A. ochraceoalba (H. Magn.) Golubkova, A. rhizophora (Müll. Arg.) Hue, A. straussii (J. Steiner) Sohrabi, A. syriaca (J. Steiner) Szatala, A. tortuosa (H. Magn.) N.S. Golubk., A. transbaicalica Oxner and the ‘Aspicilia desertorum’ complex have been presented as being related to ‘manna lichens’. Their relationship to Circinaria has not yet been studied using sequence level data. Obtaining fresh or recently collected material of these species would be essential for further study of this group. New names and combinations As a consequence of a revised generic concept of Aspicilia and Circinaria several new combinations are necessary, and these are listed below. Circinaria rogeri (Sohrabi) Sohrabi comb. nov. MB 563035 ≡ Aspicilia rogeri Sohrabi, The Bryologist 114: 182, 2011. Type: U.S.A. Oregon: Wallowa Co., The Nature Conservancy’s Zumwalt Prairie Nature Preserve, NE of Summer Camp, barren rocky ephemeral seepage area surrounded by Oregon Palouse Prairie, 1380 m (4528 ft.), 45.578 N, 116.983 W, 7 August 2007, Rosentreter 16333 (holotype: SRP, fertile specimen; isotypes: H, fertile specimen & IRAN). Circinaria aspera (Mereschk.) Sohrabi & Şenkard. comb. nov. MB 563036 ≡ Aspicilia desertorum var aspera Mereschk. in Trudy Obshch. Estest-voisp. Imp. Kazansk. Univ. 43 (5): 13. 1911. Lecanora aspera (Mereschk.) Tomin, Prir. Sel'sk. Khoz. Zasushl.-Pustyn. Obl. S.S.S.R. 1/2: 4. 1927., nom. illeg., non. L. aspera Stizenb. (1890). Lectotype: here designated! Azerbaijan ‘In rupibus calcareis e gub. Baku in Caucaso’, 1893 Lipsky in Elenkin: Lich. Fl. Ross. No. 24b, [(LE, L2017 lectotype & L2016, L 2018, H isolectotypes). Note: The specimen in position (A) is Aspicilia alpinodesertorum f. foliacea Elenkin and belongs to a subumbilicate species in Aspicilia s.lat. Its status will be discussed together with that 266 of Aspicilia oxneriana in a forthcoming paper by Sohrabi (in prep). Further study is required of the variants Aspicilia aspera var. evoluta Oxner and A. aspera var. hispidoides (Mereschk.) Tomin. Circinaria sphaerothallina (J. Steiner) Sohrabi comb. nov. MB 563037 ≡ Aspicilia calcarea var. sphaerothallina J. Steiner, Ann. Naturhist. Hofmus. 20. 379. 1907. Additional study is required before a lectotype specimen for this species can be designated. In the analyses of the nrITS sequences (Fig. 2) A. tibetica is included, is sampled in the nrITS analyses (Fig. 2). It was confirmed that it is not belong to sphaerothallioid species and is nested within the Lobothallia group. Its systematic positions and nomenclature will be studied in the revision of this genus (Sohrabi et al. in prep.). Acknowledgments The senior author is grateful to O. Vitikainen, A. Sennikov and N. Bell (all from Helsinki), H. Sipman (Berlin), V. Rico (Madrid), V. Jhon (Bad Dürkheim), U. Arup (Lund), A. Nordin and B. Owe-Larsson (both from Uppsala), F. Lutzoni (Durham, NC), A. Şenkardeşler (Izmir) and W. Obermayer (Graz) for valuable information and discussions. Special thanks are due to B. Litterski (Greifswald), A. Abbas and H. Xahidin (both from Urumqi), V. Wagner (Halle), M. Seaward (Bradford), M. Candan (Eskişehir), R. Rosentreter (Boise, Idaho), M. Andreev (St-Petersburg) and L. Biazrov (Moscow) for providing fresh material for examination. Masoomeh Ghobad-Nejhad (Helsinki) is acknowledged for her kind help during the lab work. We also thank Pirkko Harju and Eija Rinne (both from Helsinki), who helped us to make microtome sections for some samples, and also the laboratory technicians at the Department of Biology (Copenhagen) who helped us with HPLC. This study was supported by awards from the Iranian Ministry of Science and Technology to the senior author enabling him to study at the University of Helsinki. MS wishes to thank the Societas pro Fauna et Flora Fennica for financial support of this study. We also thank the curators of the indicated herbaria for their help in searching and lending specimens and are indebted to the anonymous reviewers for their critical advice and helpful suggestions. References Abbas A, Wu JN (1998) Lichens of Xinjiang. 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