Introduction
Acarospora is a large and cosmopolitan genus erected by Massalongo (1852) that contains over 200 species and is not monophyletic (Reeb et al. Reference Reeb, Lutzoni and Roux2004; Crewe et al. Reference Crewe, Purvis and Wedin2006). The type species of the genus is the yellow, terricolous Acarospora schleicheri (Ach.) A. Massal. The genus is characterized by polyspory (50 or more ascospores per ascus), simple hyaline ascospores, usually aspicilioid or pseudo-lecanorine apothecia, and bitunicate but non-fissitunicate asci with a nonamyloid tholus (Knudsen Reference Knudsen, Nash, Gries and Bungartz2007). Acarospora species are areolate to squamulose, brown or yellow, sometimes pruinose or effigurate, saxicolous or terricolous, incidentally and rarely lignicolous, with a small number of species lichenicolous with a parasitic juvenile phase (Magnusson Reference Magnusson1929; Clauzade & Roux Reference Clauzade and Roux1981; Knudsen Reference Knudsen, Nash, Gries and Bungartz2007).
Many species, including several yellow taxa, have a simple chemistry of gyrophoric acid usually with lecanoric acid (Knudsen Reference Knudsen, Nash, Gries and Bungartz2007). This is best studied under the microscope with the application of KOH followed by C, to mounted thin sections which will produce a pinkish-red reaction. Rarely is thin-layer chromatography necessary, but some species such as Acarospora nicolai B. de Lesd. often have very low concentrations of gyrophoric and lecanoric acid (Knudsen & Morse Reference Knudsen and Morse2009).
Acarospora bullata Anzi is a brown species with a usually effigurate margin and contains gyrophoric acid. In our continuing studies of the genus Acarospora it became apparent that there were possible problems of heterogeneity with Magnusson's concept of A. bullata which included specimens from Asia, Europe and California (Magnusson Reference Magnusson1929). It was also apparent that using an even broader concept of A. bullata that included South American specimens was unsatisfactory (Knudsen et al. Reference Knudsen, Nash, Gries and Bungartz2008). Molecular phylogenetic analysis to verify heterogeneity was therefore an appropriate tool in this case. We sequenced selected specimens identified as A. bullata from Asia, Europe, North and South America and used sequences of A. rugulosa Körb (Crewe et al. Reference Crewe, Purvis and Wedin2006) for phylogenetic reconstruction and established these species as heterogenic.
Acarospora rosulata (Th. Fr.) H. Magn. is an effigurate to non-effigurate brown species with gyrophoric acid known previously only from a small area of Norway (Magnusson Reference Magnusson1929). We discovered that specimens identified as A. bullata from western North America and Mongolia were conspecific with A. rosulata. Acarospora rosulata is revised in this paper.
Materials and Methods
Microscopy
Specimens were studied using standard microscopy and manually prepared sections. Measurements were made on material mounted in water. Specimens of A. bullata, A. hellbomii, A. mendozana, A. montana, A. rosulata, A. rugulosa and A. subcastanea were examined from B, DUKE, FH, GZU, KRAM, MSC, NY, S, UCR and UPS.
DNA preparation and sequencing
A total of 11 specimens identified as A. bullata from seven different countries (Austria, Bolivia, Germany, Iran, Italy, Mongolia, and USA) and one specimen of A. rosulata from Norway were sequenced over the internal transcribed spacer of the nuclear ribosomal DNA (ITS nrDNA), as well as five other specimens of Acarospora as outgroup (Table 1).
Specimens of Acarospora sequenced in this study.
Lichen tissues were frozen in liquid nitrogen and thawed three times, prior to total genomic DNA extraction using the DNeasy kit (Qiagen, Santa Clarita, CA, USA) following the manufacturer's protocol for plant tissue. Amplification of the c. 600-bp ITS nrDNA was performed using the primers ITS1F and ITS4 (White et al. Reference White, Bruns, Lee, Taylor, Innis, Gelfand, Sninsky and White1990; Gardes & Bruns Reference Gardes and Bruns1993) and the Herculase® II Fusion DNA polymerase from Stratagene (Cat. #600677). PCR conditions were as follows: initial denaturation at 94°C for 2 min, followed by 35 cycles at 94°C for 40 seconds, 52°C for 30 seconds, and 72°C for one and a half minutes, and a final extension period at 72°C for 5 minutes. PCR fragments were directly sequenced using the PCR primers and the BigDye™ terminator kit (PE-Applied Biosystems, Norwalk, CT, USA), then run on an ABI 3730 automated DNA sequencer at the Roy J. Carver Center for Comparative Genomics at the University of Iowa.
Phylogenetic analyses
Phylogenetic analyses were carried out on a 27-taxon data set including sequences generated from this study and sequences obtained from the NCBI public database (Fig. 1). The outgroup was determined using a broad phylogeny of 93 taxa for the Acarosporaceae (unpublished data). ITS sequences were aligned under MacClade v4.07 (Maddison & Maddison Reference Maddison and Maddison2005). Ambiguously aligned regions that likely violate positional homology were assessed manually and excluded from further analysis. The model of sequence evolution for the 511 sites ITS dataset was determined using Modeltest 3.7 (Posada & Crandall Reference Posada and Crandall1998). Bayesian posterior probabilities (PP) were computed under the GTR + I + Γ model using the parallel version of MrBayes 3.1.1 (Ronquist & Huelsenbeck Reference Ronquist and Huelsenbeck2003). Two times four MCMCMC chains were run simultaneously for 2 million generations sampling trees every 100 generations. Stationarity in likelihood scores was determined by plotting the tree -lnL values against generations using Tracer v1.3 (Rambaut & Drummond Reference Rambaut and Drummond2005). All trees below the stationarity level were discarded (i.e., as ‘burnin’). A majority rule consensus tree was generated from the post-burnin trees. A maximum likelihood phylogeny was also inferred using RAxML v 7.0.4 (Stamatakis et al. Reference Stamatakis, Hoover and Rougemont2008) on the Cyber infrastructure for Phylogenetic Research (CIPRES) portal v 1.13 at http://www.phylo.org/. RAxML bootstrap analysis was done for 100 iterations using an estimated proportion of invariable sites. A majority rule consensus tree was calculated to determine the bootstrap proportion (BP) for each node.
Phylogeny of 27 Acarospora specimens inferred from maximum likelihood (RAxML) analysis of the internal transcribed spacer of the nuclear ribosomal DNA (ITS nrDNA). The phylogram shows the RAxML tree with the highest likelihood. Numbers above the branches are RAxML bootstrap proportions when ≥ 50%. Numbers below the branches indicate Bayesian posterior probabilities ≥ 95%. Numbers in parentheses correspond to NCBI GI numbers. Other terms in parentheses correspond to abreviations used for molecular work.
Results
Based on the molecular sequence data, the specimens identified as Acarospora bullata included in this study are distributed among three distinct clades (Fig. 1). Clade no. 1 (BP = 100% and PP = 100%) includes A. bullata from Europe (Austria, Germany, and Italy) and Iran, intermixed with A. rugulosa from Sweden (Crewe et al. Reference Crewe, Purvis and Wedin2006). Clade no. 2 is also well supported (BP = 96% and PP = 100%), including specimens identified originally as A. bullata from the US and Mongolia, as well as A. rosulata from Norway. Finally, in Clade no. 3, a specimen identified as A. bullata from Bolivia forms a sister relationship with A. austroshetlandica, but without support. Only the Clades nos 1 and 2 form monophyletic groups (without support), whereas the exact position of A. cf. bullata from Bolivia remains to be determined.
The Species
Acarospora rosulata (Th. Fr.) H. Magn
Monogr. Scand. Acar. 28: 121 (1924); type: Norway, Oppland, Vågå, Th. Fries, 1863 (UPS!—holotype)
Basionym: Acarospora fuscata discreta f. rosulata Th. Fr., Lich. Scand. 1: 218 (1871).
(Fig. 2)
Acarospora rosulata on granite, Eureka Peak, Joshua Tree National Park, California.
Thallus areolate, contiguous to dispersed, forming indeterminate to determinate patches to 7 cm, often confluent. Areoles round to angular, up to 3 mm diam. and 1·5 mm thick, broadly attached (over half of diameter), becoming subsquamulose, the mycelial base thickening, not forming a stipe, elevating the areoles (becoming gomphate), the areoles often lobulate, especially on the margin of thallus but also in the centre, the lobes generally round, 2 to 3 per areole, usually less than 1 mm long and less than 1 mm wide. Surface pale yellow brown to dark brown, usually shiny, smooth, epruinose. Upper and lateral cortices paraplectenchymatous to subparaplectenchymatous, 30–80 μm thick, cells 3–6 × 2·5–6 μm, globose to elongate, syncortex (sensu Knudsen Reference Knudsen, Nash, Gries and Bungartz2007) 4–30 μm thick rarely with visible periclinal hyphae; eucortex with reddish brown upper layer to 20 μm thick, and thicker hyaline lower layer. Lower surface usually ecorticate and white or rarely brown. Photobiont chlorococcoid green alga up to 10 μm diam., forming a continuous stratum 60–100 μm thick, the algal cells sometimes arranged in distinct vertical columns, often thinning beneath apothecia. Medulla prosoplectenchymatous, up to 600 μm thick, continuous with attaching hyphae, hyaline, mostly 3–4 μm diam., cells mostly 4–5 μm long.
Apothecia one to many per areole, immersed and punctiform, sometimes expanding to 1 mm diam., usually remaining immersed, occurring also on marginal areoles, rarely with a thin parathecial ring visible around the disc or elevated with thalline margin. Disc dark brown, epruinose, rough, round to irregular, rarely with umbo of sterile plectenchyma, sometimes reddish when wet. True exciple of radiating hyphae, 20–40 μm, expanding slightly around the surface of the disc, rarely forming parathecial crown. Hymenium 80–120 μm tall, epihymenium dark brown to reddish brown with diffused pigment, conglutinated, 10–20 μm thick; paraphyses lax in water, 1·5–2·5 μm diam., septate, sometimes with oil drops, apices often expanded, 2·5 – 4·0 μm wide, usually in brown pigmented caps. Subhymenium to 50 μm thick. Hypothecium 15–20 μm thick. Asci clavate, 60–100 × 15–28 μm, ascospores 100–200 per asci. Ascospores simple, hyaline, mostly 4–5 × 1·5–2 μm.
Chemistry. Spot tests: cortex, C+, KC+ pinkish red (best seen in mount); gyrophoric acid.
Ecology and substratum. On granite and siliceous rocks, schist, sandstone, rarely on limestone or calcareous rocks.
Distribution. Europe (Oppland, Norway), western North America and Asia (Mongolia).
Discussion. The thallus of Acarospora rosulata can be determinate, especially in hot arid locations in the desert or on smooth hard rock surfaces, but the outer and inner areoles can be lobulate and its areoles regularly become elevated and subsquamulose. The surface is shiny and always smooth.
Acarospora rosulata was recognized as a species by Magnusson from a small area of Norway in Oppland. Specimens of A. rosulata from California were included in A. bullata by Magnusson (Reference Magnusson1929) and Knudsen (Reference Knudsen, Nash, Gries and Bungartz2007). We do not recognize A. bullata as occurring in North America.
The discs of Mongolian specimens were very rough owing to an uneven build-up of conglutinated pigment and could, superficially, be mistaken for the rugulose discs of Acarospora bullata. Specimens from Norway often produce many apothecia on a single areole, which subsequently begins the process of vegetative division into separate areoles. Western North American specimens often produce the largest areoles usually with a single apothecium.
Selected specimens examined. Mongolia: Khovad, Altai Mountain, west of Ulaantology, 46°54′32.0″ N 92°15′12.9″ E, 2098 m, 2004, Reeb [VR 17-VII-04/1] & Zavarzin (DUKE); Töv, Gokhi-Terelj Nature Reserve beside a river, 47°50′08.3″ N 107°28′42.6″ E, 1420 m, on cliff, 2004 Reeb [VR 30-VII-04/4] & Zavarzin (DUKE).—Norway: Oppland: Lom Parish, På muren vid kyrkan, 1952, Per-Olaf Lindahl (UPS); [Krypt Exs. No. 2867] Magnusson (S); ibid., 1948 Ahlner (S); ibid., 2008, Westberg 08-156 (S); Vågå Parish, 5·5 km WSW of Vågå, 61° 52′ N, 7°99′ E, 580 m, 1985, Tibell 15834 (UPS); ibid., 2008, Westberg 08-192, 08-193, 08-238 (S).—USA: California: Inyo County, Little Lake Station, 1911, 2 specimens, Hasse (FH); near Bishop along US 395, 5400 ft, 1966, Wetmore 14871 (FH); Orange County, Santa Ana Mountains, Fremont Canyon, 33° 48′ 05″N, 117° 41′ 59″W, 387 m, on sandstone, 2007, Knudsen 9358 (UCR); Riverside County, east side of San Jacinto Mountains, Pinon Flats area, 33° 34′ 35″N, 116° 29′ 14″W, 1276 m, in full sun on granite, 2004, Knudsen 929 (NY, UCR); off Burnt Canyon Road, 33° 34′ 00″N, 116° 36′ 07″W, 1415 m, on granite boulder, 2008, Knudsen 9509 w/ Lendemer (S, UCR); San Bernardino County, east side of San Bernardino Mountains, Cactus Flats, 34° 18′ 22″N, 116° 47′ 43″W, 1858 m, on granite and limestone, 2005, Knudsen 3351 (UCR). Colorado: summit between Engineer & Gravel Mts., 1954, Imshaug 17082 (MSC). Utah: Sevier Co, 5 miles east of Salina, 1957, Shushan 14848 (FH); San Juna Co., La Sal Mts., summit of Mt. Mellenthin, 12890 ft., 1954 Imshaug 16899 (MSC).
Conclusion
Our study, while revising A. rosulata, does not revise A. bullata and A. rugulosa in Europe and Asia. The types of A. rugulosa could not be found at Leiden (G. Thijsse, pers comm.). The German specimens of A. bullata have rugulose thalli, the types of A. bullata are smooth and are similar to determinate and indeterminate specimens identified as A. rugulosa in Fennoscandia. Specimens in the bullata-rugulosa Clade no. 1 identified as A. rugulosa (Crewe et al. Reference Crewe, Purvis and Wedin2006) have indeterminate thalli with no lobes or rudimentary lobes and are apparently conspecific with Magnusson's concepts of A. montana H. Magn. and A. hellbomii H. Magn. (Magnusson Reference Magnusson1929). The only characters uniting all specimens we studied from Europe were a rugulose disc, usually with a parathecial crown and a distinct thalline margin and thin paraphyses, mostly 1 μm wide, not wider than 2 μm, usually branching, lax in water, apices not expanded or slightly expanded to 2 μm. Multi-gene analysis is needed to verify the results that A. bullata and A. rugulosa are conspecific and a neotype for A. rugulosa needs to be selected.
The first author synonomized A. subcastanea (Nyl.) Hue and A. mendozana H. Magn. from South America with A. bullata (Knudsen Reference Knudsen, Nash, Gries and Bungartz2007) but we do not recognize this synonymy as valid. We think A. mendozana is probably synonymous with the older name A. subcastanea based on type material and specimens from S and H. Nonetheless we had only one specimen from South America collected by Adam Flakus in Bolivia which we could sequence and which was most similar to specimens identified as A. mendozana. This specimen was not related to A. bullata or A. rosulata. At this time, the Antarctic endemic A. austroshetlandica (C.W. Dodge) Øvstedal (Øvstedal & Lewis Smith Reference Øvstedal and Lewis Smith2001) appears distinct from the Flakus collection from Bolivia but probably related to the South American taxon. The revision of effigurate or subeffigurate species of brown Acarospora with gyrophoric acid in South America needs additional study and more specimens included in molecular phylogenetic analyses.
We would like to thank the curators of B, DUKE, FH, GZU, KRAM, L, MSC, S and UPS for sending specimens. We also thank G. Thijsse (L) for help in trying to locate the types of A. rugulosa and R. Muertter for the photograph in Fig. 2. We are grateful for the constructive comments of Christian Printzen and an anonymous reviewer. This research was partly supported by a grant to DB from the National Science Foundation Assembling the Tree of Life program (EF 04-31117).
