Two New Calciphytes from Western North America, Acarospora Brucei and Acarospora Erratica (Acarosporaceae)
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Opuscula Philolichenum, 17: 342-350. 2018. *pdf effectively published online 12October2018 via (http://sweetgum.nybg.org/philolichenum/) Two new calciphytes from Western North America, Acarospora brucei and Acarospora erratica (Acarosporaceae) KERRY KNUDSEN1* AND JANA KOCOURKOVÁ2 ABSTRACT. – Two new species that grow on calcareous rock, Acarospora brucei and A. erratica, are described from North America. Acarospora brucei was originally reported as A. complanata. It is proposed that the name A. complanata be removed from the North American lichen checklist. Sixty- four species of Acarospora and 99 species of Acarosporaceae are currently reported in North America. A protocol for reproducible iodine tests of hymenial and subhymenial substances in Acarosporaceae is provided. KEYWORDS. – Acarospora sparsa, Lugol’s, Mexico, nomenclature, taxonomy. INTRODUCTION The western United States is a center of diversity for Acarosporaceae and most of the 97 species of Acarosporaceae currently reported from North America north of Mexico occur there, particularly in the southwest (Esslinger 2018; Knudsen 2007; Leavitt et al. 2018; Magnusson 1929 & 1956). In this paper we describe two new species, both calciphytes, from western North America. MATERIALS AND METHODS Morphological and chemical studies. – Specimens were studied from OSC, UCR, UPS, and from the private herbaria of J. Hollinger (hb. Hollinger), K. Knudsen and J. Kocourková (hb. K & K), B. McCune (hb. McCune), T. Spribille (hb. Spribille) and T. Wheeler (hb. Wheeler), using standard microscopy and spot tests (Brodo et al. 2001). Hand sections were studied and measured in water. Hymenium measurements include the epihymenium. The amyloid reaction of the hymenial and subhymenium substances were tested with fresh undiluted IKI (Merck’s Lugol; see notes below for protocol). Ascus staining was studied in IKI, with and without pre-treatment in K (Hafellner 1993). Thin- layer chromatography (TLC) was used to verify the results of spot tests (Orange et al. 2001). Macrophotographs were taken with a digital camera Olympus DP72 mounted on Olympus SZX 7 stereomicroscope equipped with PRO-SZM1 - Focus Drive Motorization for stacking pictures and stacked using Olympus DeepFocus 3.4 module. Microphotographs were taken with a digital camera Olympus DP72 mounted on an Olympus BX51 Light Microscope fitted with Nomarski interference contrast and using Promicra QuickPhoto Camera 3.0 software. The figure plates were processed with QuickPhoto Camera 3.1 software fitted with Promicra Publisher Modul and eventually refined with Adobe Photoshop CS4 Extended ver. 11.0. 1KERRY KNUDSEN – Department of Ecology, Faculty of Environmental Sciences, Czech University of Life Sciences, Prague, Kamýcká 129, Praha 6 - Suchdol, CZ–165 00, Czech Republic. – e-mail: [email protected] *author for correspondance 2JANA KOCOURKOVÁ – Department of Ecology, Faculty of Environmental Sciences, Czech University of Life Sciences, Prague, Kamýcká 129, Praha 6 - Suchdol, CZ–165 00, Czech Republic. – e-mail: [email protected] 342 IKI reaction Euamyloid Hemiamyloid with Merck’s Lugol Type BB Type RR Type RB high iodine concentration blue red red low iodine concentration blue red blue shades Table 1. Three types of amyloidity can be distinguished when applying IKI to Acarosporaceae: RB is a type intermediate between BB and RR, but is subsumed under hemiamyloid because of the presence of the red IKI reaction when squashed (adapted from Barel 1987). Examination of hymenial reactions with Lugol’s (IKI). – For our studies of hymenial substances in Acarosporaceae we only use Merck’s Lugol (Iodine 3.4 grams per liter; Potassium iodine 6.8 grams per liter) stored in the dark in a dark glass bottle from the factory in an insulated foam box at tempatures between 15–25°C. From a stock solution, Lugol’s is poured into small dark glass lab bottles with droppers. It is stored in the dark at the same temperature as the stock solution when not in use at lab temperatures. It is changed once a month. It is shaken before use. Thin sections of the hymenium are placed in Lugol’s without water and squashed with a cover slip to insure saturation. Euamlyoid hymenial gel is always dark blue despite the age or concentration of Lugol’s used. The blue does not disappear if the slide is left for eight hours to dry, or when more Lugol’s is added. There are two possible reactions for hemiamyloid hymenial gel. In the first case, the hymenial gel does not contain the substance that reacts euamyloid to Lugol’s, and the reaction to Lugol’s is immediately and uniformly red. In the second case, the hymenial gel contains both substances and reacts euamyloid (blue) and hemiamyloid (red). If not squashed and saturated with Lugol’s the hymenial gel will usually have a blue or blue yellow to greenish blue reaction to Lugol’s. This reaction will usually remain if allowed to dry over eight hours. When squashed, the hymenial gel will remain blue if the Lugol’s has not been shaken, is diluted, or has lost its potency. If squashed, and the Lugol’s properly shaken, is not diluted, or has not lost its potency, the hymenial gel will quickly turn from blue to red. Thus, there are three types of reactions: blue (abbreviated BB; euamyloid), red-blue (abbreviated RB; containing euamyloid and hemiamyloid substances), and red (abbreviated RR; containing no euamyloid substances) (Baral 1987; summarized in Table 1 herein). For diagnostic purposes the BB or RR are the most important characters. The RB reaction is the most common reaction in Acarospora. The subhymenium in species is often euamyloid. Rarely, when the subhymenium is hemiamyloid (RB or RR), it can be useful as secondary character for identification (Knudsen & Kocourková 2017a). It must be recognized that Magnusson (1929, 1956) did not follow any standardized protocol when testing reactions of the hymenial gel with Lugol’s. As such his observations are unreliable, except in the few cases, where he reports strong color reactions, like a dark blue or a deep red reaction. Caution is advised in accepting some reports of euamyloid hymenial gel. For instance, in the description Acarospora austriaca H. Magn., Magnusson reports the hymenial gel as euamyloid but it was hemiamyloid (Magnusson 1935, Knudsen unpublished data). TAXNOMIC SECTION Acarospora brucei K. Knudsen & Kocourk., sp. nov. Mycobank #828143. FIGURE 1. TYPE: U.S.A. MONTANA. TETON CO.: on hill on W side of Pine Butte Swamp, in Pinus flexilis savanna, 47°50′N,112°36′W, 1480 m, viii.1985, on limestone outcrops, B. McCune 15165 (OSC!, holotype). Similar to Acarospora complanata but differing in producing gyrophoric acid and occurring on limestone instead of volcanic rock. DESCRIPTION. – Thallus rimose areolate, areoles 0.5–1.0 mm wide, 350–450 μm thick, outer areoles lobulate, lobes undivided, up to 1.5 mm long, widening to 1 mm, forming a single rounded lobe. Upper surface dark dull brown, with black hues, rugulose. Epicortex thin to indistinct. Cortex mostly 30–40 μm thick, upper layer reddish brown, thin, mostly a single cell layer thick, lower area hyaline, easily 343 Figure 1. Acarospora brucei (holotype, McCune 15165). A-B, morphology of thallus, areolate thallus with very slightly prolongated marginal lobes. C, detail of thallus with apothecia. All scales = 1.0 mm. 344 Figure 2. Acarospora complanata H. Magn. (UPS, isotype). The only surviving specimen of A. complanata identified by A.H. Magnusson. The rest of the specimens, probably including a large holotype with a well-developed determinate margin, are presumed to have been destroyed in the bombing of Dunkirk when Maurice Bouly de Lesdain’s herbarium was destroyed. observed in water, cells mostly round, mostly 4–5 μm wide, POL+ with crystals from secondary metabolite. Algal layer 60–100 μm thick, algal cells mostly 7–10 μm wide, continuous below apothecia, even, with a few slender hyphal bundles, less than 10 μm thick, but not distinctly interrupted. Medulla 200– 300 μm thick, medullary hyphae thin-walled, 2.0–2.5 μm wide, medulla obscured with substrate crystals, some POL+, the mycelial base thickening with age. Apothecia immersed, epruinose, rugulose, 1 to 3 per areole, 100–300 μm wide, disc reddish brown. Parathecium 10 μm wide, rarely wider. Epihymenium ca. 10 μm thick, reddish-brown and coherent. Hymenium 65–80(–100) μm high, paraphyses 1.5–2.5 μm wide, septate, hymenial gel IKI+ blue. Asci 40–50 × 10–17 μm wide, Acarospora type. Ascospores mostly 4–5 × 2.0–2.5 μm, broadly ellipsoid. Subhymenium 20–30 μm tall, IKI+ blue. Hypothecium thin, 10 μm wide, to indistinct. Pycnidia not seen. CHEMISTRY. – Gyrophoric acid in cortex (TLC, according to specimen annotation by Alphandary & McCune, 2015). Spot tests: cortex K-, C+ pink, KC+ pink, P-, UV-; medulla K-, C-, KC-, P-, UV-. ETYMOLOGY. – The species is named for the collector Bruce McCune in honor of his work as an ecologist and his study of lichen biodiversity in North America. He has made a major contribution to North American lichenology. DISTRIBUTION AND ECOLOGY. – Known only from its type locality in North America (Montana) on limestone, overgrowing the edge of another crustose lichen, at an elevation of 1480 m. DISCUSSION. – Only two described species with a determinate saxicolous thallus which produce gyrophoric acid have been reported in North America: Acarospora rosulata H. Magn. and A. tintickiana St. Clair, Newberry & S. Leavitt (Knudsen et al. 2010; Leavitt et al. 2018). Acarospora rosulata is common in western North America where the species was erroneously reported as A. bullata Anzi by Knudsen (2007) and later corrected by Knudsen et al. (2010). Acarospora brucei differs from A. rosulata in having a dull dark brown rugulose upper surface rather than a usually shiny brown smooth upper surface. Acarospora brucei usually has a hymenium that is not as tall as A. rosulata (65–100 μm vs. 80–150 μm).