Phytotaxa 340 (1): 086–092 http://www.mapress.com/j/pt/ Copyright © 2018 Magnolia Press ISSN 1179-3155 (print edition) Article PHYTOTAXA ISSN 1179-3163 (online edition) https://doi.org/10.11646/phytotaxa.340.1.7 A new species of Acarospora (Acarosporaceae, Acarosporales, lichenized Ascomycota) from the Falkland Islands (Islas Malvinas) DAG O. ØVSTEDAL1, LOUISE LINDBLOM1, KERRY KNUDSEN2 & ALAN M. FRYDAY3* 1 Department of Natural History, University Museum of Bergen, Box 7800, NO-5020 Bergen, Norway. Department of Ecology, Faculty of Environmental Sciences, Czech University of Life Sciences, Prague, Kamýcká 129, Praha 6 - Suchdol, CZ–165 21, Czech Republic 3 Herbarium, Department of Plant Biology, Michigan State University, East Lansing MI 48823, U.S.A. *corresponding author: fryday@msu.edu 2 Abstract Acarospora malouina Øvstedal & K. Knudsen is described from the Falkland Islands. It is morphologically very similar to the Antarctic species A. gwynnii but differs in chemistry, ecology and evidence from molecular data. Keywords: lichenized fungi, new species, South America, southern subpolar region. Introduction The crustose genus Acarospora is cosmopolitan and comprises several hundred saxicolous and terricolous species (Index Fungorum 2017); the exact number is impossible to estimate accurately due to the lack of a modern global monograph. Magnusson recognized 83 species with a yellow cortex resulting from the production of rhizocarpic acid, a pulvinic acid derivative, and/or epanorin (Magnusson 1929, 1956). In South America, ten yellow Acarospora species are currently recognized (Knudsen et al. 2008; Knudsen 2012). These species occur from the summits of the Andes to just above sea level in Ecuador (Galapagos Islands) and in Chile (Juan Fernandez Islands: Knudsen et al. 2008). Two species have been recognized from the Antarctic: A. flavocordia Castello & Nimis (1994: 284), which is characterized by the thallus having a yellow medulla and elongate marginal lobes (Castello & Nimis 1994, Øvstedal & Lewis Smith 2001), and A. gwynnii Dodge & Rudolph (1955: 144), which has a white medulla and lacks marginal lobes (Dodge & Rudolph 1955, Øvstedal & Lewis Smith 2001). In 2000, R. I. Lewis Smith collected a yellow Acarospora close to the sea on Saunders Island, one of the smaller islands in the Falkland Islands archipelago, that was morphologically similar to A. gwynnii and was, tentatively, assigned to that species. However, we were uncomfortable with this determination because only a small percentage (c. 7.5%, 27 species) of the lichen biota of the islands is also known to occur on the Antarctic continent (Fryday et al. in prep.) and, if this really was A. gwynnii, why was it found only on the north coast of Saunders Island, which is one of the most northerly of the Falklands Islands (Fig. 1) and among the places furthest away from Antarctica? In 2015 the fourth author (AMF) and another lichenologist (Alan Orange), visited the Falkland Islands for a total of five weeks as part of an extensive project to inventory and describe the bryophytes and lichens of the islands. Saunders Island was included in the itinerary of this inventory and further collections of the yellow Acarospora were made, thus permitting a molecular investigation of its relationship to A. gwynnii. This inventory also failed to find the yellow Acarospora at any other location on the islands. The aim of this paper is to determine whether the Falkland Islands’ population belongs to A. gwynnii (or any other known species in adjacent areas) or whether it is a new species. 86 Accepted by Christian Printzen: 20 Jan. 2018; published: 21 Feb. 2018 FIGURE 1. Map of Falkland Islands showing location of A. malouina () and Stanley (). Material and methods Four specimens of the Falkland Islands’ population, and nine specimens of A. gwynnii were examined. Microscopical details were obtained by examining hand-cut sections, mounted in 10 % KOH. Chemical content was obtained by thin layer chromatography (Orange et al. 2001). The reactions of the hymenial gel were tested with undiluted Lugol’s iodine on squashed thin sections. For definition of epicortex see Knudsen & Bungartz (2014). DNA extraction, amplification, and sequencing Molecular work was conducted in the Biodiversity Laboratories, DNA Lab, University of Bergen (UiB). Total DNA was extracted using the Dneasy Plant Mini Kit (Qiagen) following the manufacturer’s protocol. DNA extractions were diluted 10 x and used as template in the PCR. The complete nrITS fragment (nrITS1-5.8S-nrITS2, ca. 500 bp) of the nuclear ribosomal DNA repeat was amplified by PCR using primers ITS1F and ITS4 (white et al. 1990, Gardes & Bruns 1993). PCR reactions contained 5 μl of DNA, 800 μM dNTPs (200 μM of each), 2.5 μl of 10x AmpliTaq buffer without MgCl2, 2.5 mM of MgCl2, 0.4 μM of each primer, 0.15 μl (5U/μl) of AmpliTaq (Applied Biosystems), and ddH2O to 25 μl reaction volume. PCR cycling was initiated by a denaturation step 4 min at 94°C, followed by six cycles: denaturation (1 min, 94°C), annealing (45 s, 62–56°C touchdown), elongation (1 min 45 s, 72°C), and 30 cycles: denaturation (30 s, 94°C), annealing (30 s, 56°C), and elongation (1 min 45 s +3 s each cycle, 72°C). Cycling finished with a 10 min elongation step at 72°C. PCR products were purified with a mix of Illustra Exonuclease I and Illustra Shrimp alkaline phosphatase (GE Healthcare). Sequencing was done at the Sequencing Facility (UiB). Voucher data and GenBank accession numbers for newly generated sequences are provided in Table 1. Alignment and calculations of genetic distances (observed p-distance) were carried out in Geneious v. 8.0.3 (Kearse et al. 2012). Observed p-distances were calculated as the proportion of nucleotide sites at which two sequences are different obtained by dividing the number of nucleotide differences by the total number of nucleotides compared (Nei & Kumar 2000). In addition to comparing the newly generated sequences with each other, we compared them with corresponding gene fragments of four yellow Acarospora species; two retrieved from GenBank: A. heufleriana (LN810774; Switzerland) and A. socialis (LN810802, U.S.A., California), and two made available to us by Lazzat Nurtai: A. fulva (MF134867, MF134868, MF134869; China), and A. stapfiana (MF134870, MF134871; China), to assess the general level of pairwise genetic distances between hypothetically closely related species. Comparison of these intra- and interspecific A NEw SPECIES OF ACAROSPORA Phytotaxa 340 (1) © 2018 Magnolia Press • 87 genetic distances was additionally used to assess the existence of a so-called barcode gap, which is indicated if there is no overlap of the two measures (for example Schoch et al. 2012, Meyer & Paulay 2005). TABLE 1. Newly generated DNA sequences (nrITS) used in this study, with their respective voucher information and GenBank accession numbers. Taxon Acarospora malouina Acarospora gwynnii Acarospora malouina Acarospora malouina Acarospora gwynnii Acarospora gwynnii Acarospora gwynnii Acarospora gwynnii Voucher information Falkland Islands, Fryday 11351 (MSC) Antarctica, N Victoria Land, Seppelt 25017(HO) Falkland Islands, Fryday 11346 (MSC) Falkland Islands, Fryday 11338 (MSC) Antarctica, S Victoria Land, Seppelt 25472 (HO) Antarctica, Mt Kyffin, Sancho (MAF-Lich 21272) Antarctica, Mt. Kyffin, Sancho (MAF-lich 21270) Antarctica, Mt. Kyffin, Sancho (MAF-Lich 21274) ITS MF138056 MF138057 MF138058 MF138059 MF138060 MF138061 MF138062 MF138063 Results Genetic distance New nrITS sequences were generated from five collections of A. gwynnii and three collections of the Falkland Islands’ entity collected in 2015. The total nrITS alignment was 486 nucleotides in length. The observed p-distance in total nrITS revealed 7.2–8.0 % difference between A. gwynnii and the Falkland Islands’ population. The variation, as calculated p-distance, within the three specimens of the Falkland Islands’ population was 0–0.4 % (that is, two sequences were 100 % identical, and the third differed in two nucleotide sites). The variation within the five specimens of A. gwynnii was 0–2.7 %. Comparisons with other yellow Acarospora species showed the calculated p-distance between the Falkland Islands’ population and A. fulva to be ca. 14 %, from A. heufleriana ca. 14 %, from A. socialis ca. 13 %, and from A. stapfiana ca. 11% (Table 2). Of these, more than one nrITS sequence was available only for A. fulva (3 sequences) and A. stapfiana (2 sequences), which had a within-species variation of 0.4 and 0 % respectively. TABLE 2: Observed p-distances between yellow species of Acarospora (n = number of sequences). A. gwynnii A. malouina A. fulva A. heufleriana A. socialis A. stapfiana A. gwynnii n=5 0–2.7 A. malouina n=3 7.2–8.0 0–0.4 A. fulva n=3 10.8–12.1 14.1 0.4 A. heufleriana n=1 8.7–9.9 13.7 12.8–13.3 n.a A. socialis n=1 9.7–10.7 13.2 12.4–12.8 7.3 n.a A. stapfiana n=2 7.4–9.1 11.2 13.7–14.1 9.2 9.1 0 The greatest intraspecific distance in our data set is 2.7 % (A. gwynnii), whereas the smallest interspecific distance is 7.2 % (A. gwynnii and the Falkland Islands’ population, Table 2). There is no overlap, and the difference strongly indicates barcode gaps between all compared species. Because the calculated p-distance between A. gwynnii and the Falkland Islands’ population was of the same order of magnitude as between each species and other yellow hypothetically closely related species of Acarospora, we conclude that the Falkland Islands’ population is most likely distinct from A. gwynnii, and describe it as a new species below. Acarospora malouina Øvstedal & K. Knudsen sp. nov. Mycobank no 824038 Diagnosis: Morphologically similar to Acarospora gwynnii, but differs in molecular characters, chemistry and ecology. Type:—FALKLAND ISLANDS, Saunders Island, north shore 200 m west of rockhopper penguin rookery,–51.308309° –60.122618°, 51 m., saxicolous on boulders, 5 November 2015. A.M. Fryday 11351 (holotype MSC!). (Figs 2, 3) thallus of contiguous squamules, 0.3–3 cm in diameter, 300–560 µm thick, indeterminate; squamules up to 2 mm wide, rugulose, fissured, continuously dividing. often forming large clusters of small squamules with 1 or 2 apothecia; 88 • Phytotaxa 340 (1) © 2018 Magnolia Press ØVSTEDAL Et AL. upper surface glossy yellow, lower surface pale brown with a central stipe; epicortex thin, less than 10 µm thick or almost absent, of gelatinized thin hyphae, cortex red-brown throughout, 20–60 µm thick, cells usually 2–4 µm wide, angular to round; algal layer uneven, interrupted by hyphal bundles 10–60 µm wide, forming algal palisades, 150–200 µm thick, algal cells 8–12 µm wide; medulla obscure, white to brownish, 200–300 µm thick of conglutinated anticlinal hyphae mostly 1–2 µm wide, continuous with stipe; apothecia 1–10 per squamule, disc reddish-brown, epruinose, 0.1– 0.4 mm wide, with or without a distinct parathecial ring, same color as the thallus or reddish brown, darker than disc; parathecium 10–100 µm wide at surface of disc, intergrading with cortex. Layer between parathecium and hymenium I+ blue, up to two thirds of distance to disc (referred to as an abscission layer in Castello & Nimis 1994); epihymenium ca. 20–25 µm, yellowish-brown, with thin hyaline gelatinous upper layer; hymenium 200–250 µm tall, hyaline I+ blue turning red, paraphyses thin, 1–1.5(–2.0) µm wide, apices not expanded, some branching; asci 100–150 × 15–30 µm, hundreds of ascospores, ellipsoid, 2.5–3.0 × 1.5–2.0 µm; subhymenium 20–30 µm tall, I+ blue; hypothecium 20–30 µm tall, I–; pycnidia not observed, but apothecial initials abundant. FIGURE 2: Habitat of Acarospora malouina: A: penguin rookery on north shore of Saunders Island. The rocks are dominated by crustose Teloschistaceae (e.g., Austroplaca hookeri (C.w. Dodge) Søchting, Frödén & Arup) but Acarospora malouina also occurs on the low rocks and boulders. B: rock with A. malouina (arrow). C: A. malouina (scale bar = 1 cm). Chemistry: rhizocarpic acid by tlc. Distribution and Habitat: The new species is known only from the upper surface of low siliceous rocks (quartz sandstone), close to penguin rookeries on the north shore of Saunders Island, north of west Falkland. Etymology: The species is named after the brave fishermen of St. Malo, who were the first to inhabit the Falkland Islands. A NEw SPECIES OF ACAROSPORA Phytotaxa 340 (1) © 2018 Magnolia Press • 89 FIGURE 3: Acarospora malouina (Fryday 11352—holotype); the surrounding black apothecia belong to Lecanora capistrata (Darb.) Zahlbr. (scale bar = 1.5 cm). Additional specimens of A. malouina studied. FALKLAND ISLANDS: Saunders Island, north shore between Rookery Cottage and rockhopper penguin rookery, –51.305938°, –60.112033°, 64 m, saxicolous on boulders, 5 November 2015, A. M. Fryday 11338 (MSC); ibid. –51.307733°, –60.118877°, 64 m, saxicolous on boulders, 5 November 2015, A. M. Fryday 11346 (MSC); ibid, east of The Neck, –51.30, –60.22, 50 m, rocks and stones on hillside above rockhopper penguin and black-browed albatross colony, 23 January 2000, R. I. Lewis Smith 11058 (AAS). Additional selected specimens of A.gwynnii studied (apart from those used in DNA studies–see table 1): ANTARCTICA, Dronning Maud Land, Vestfjella, Fossilryggen, 1979, Haugerud & Winsnes (BG); ibid., Vestfjella, 1985, K. M. Bratlien (BG); Ellsworth Land, Scotia Sector, Novocin Peak N, Hauberg Mountains, 1109 m, January 2000, P. Wickens 155 (AAS); MacRobinson Coast, Mawson, Feb. 1954, A. M. Gwynn s.n. (Holotype, FH). Victoria Land, Ross Sector, North end of Kay Island, wood Bay, 130 m, dry sandy terraces, R. I. Lewis Smith 10235 (AAS); Ross Dependency, Darwin Glacier, Diamond Hill, surroundings of the field camp, site 57, 495 m, on sandy soil over granite, 26.1.2009, R. türk 4463 (Herb. Türk). Discussion Acarospora malouina cannot be distinguished from A. gwynnii on morphological or anatomical characters. However, there are other differences that indicate that the two populations are genetically distinct and deserve to be recognized as separate species: A: chemistry A. gwynnii contains epanorin with rhizocarpic acid as a minor constituent (five specimens studied), whereas A. malouina has only rhizocarpic acid (four specimens studied). B: ecology On the nunataks of continental Antarctica, where A. gwynnii is found, lichens are exposed to temperatures as low as –60 to –70 °C in winter, and a summer diurnal range of from 15–20 °C to –40°C (Øvstedal & Lewis Smith 2001). Also, radiation is very high. 90 • Phytotaxa 340 (1) © 2018 Magnolia Press ØVSTEDAL Et AL. On the Falkland Islands, A. malouina grows on rocks close to penguin rookeries (Fig. 2). It is clearly a nitrophilous species because it has not been observed more than 500 m away from a penguin rookery and becomes more frequent and better developed closer to the rookery. The climate of the Falkland Islands is much milder than that where A. gwynnii occurs, with winter temperatures at Stanley (the only locality for which extensive climatic data are available) on the eastern extremity of East Falkland (Fig. 1) rarely falling below 0°C and with a record low of –11°C. The north shore of Saunders Island is likely to be even milder, which is supported by this locality being the only place on the islands where species with a more temperate distribution were recorded (e.g., Coenogonium sp.) C: molecular data The genetic distance supports the new species as distinct from A. gwynnii. The genetic distance between these two species is of the same magnitude as the distance between A. malouina and other species of Acarospora with a yellow thallus (Table 2). Ten yellow Acarospora species are reported from South America (Knudsen et al. 2008, Knudsen 2012, Santesson 1944), but in our opinion A. malouina cannot be identified as any of these on morphological, anatomical or chemical grounds. Also, they are mostly found in mountainous parts of tropical areas. Unfortunately, none of them have been submitted to molecular studies. Four of the South American species: A. boliviana Magnusson (1929: 92), A. ramosa Knudsen & Flakus (2009: 250), A. rouxii Knudsen (2007: 26), and A. theleomma Lamb (1948: 233), have orbicular, determinate and effigurate thalli rather than the indeterminate squamulose thallus of A. malouina (Knudsen 2012, Knudsen & Flakus 2009, Knudsen et al. 2008, Lamb 1948, Magnusson 1929). Hymenium height is an important character for distinguishing Acarospora species (Magnusson 1929), and four other South American species: A. catamarcae Magnusson (1947: 64), A. chrysops (Tuckerman 1858: 425) Magnusson (1929: 65), A. regnelliana Santesson (1944: 12) and A. xanthophana (Nylander 1861: 379) Jatta (1906: 10; misapplied name A. rhabarbarina Hue (1909: 117)) have a much lower hymenium height (65–120 µm) than that of A. malouina (200–250 µm) (Knudsen 2012, Knudsen et al. 2008, Knudsen & Flakus 2016, Magnusson 1929, Santesson 1944). One further species, A. brouardii Bouly de Lesdain (1922: 15), has a higher hymenium (130–170 µm tall) but this is still significantly lower than that of A. malouina (Knudsen et al. 2008). Acarospora brouardii also differs from A. malouina in having squamules with a black underside. The South American species most similar to A. malouina is A. congregata Knudsen & Flakus (2016: 149; misapplied name A. xanthophana auct.) (Knudsen et al. 2008, Knudsen & Flakus 2016) but that species differs in having a lower hymenium (130–200 µm), larger ascospores (3–5 × 1.5–2 µm) and also in having broadly attached areoles lacking a stipe whereas A. malouina has ascospores 2.5–3.0 × 1.5–2 µm and loosely attached squamules with stipes. Acarospora congregata is a common species in South America (Knudsen & Flakus 2016). Acknowledgments we thank the curators of AAS, FH, HO and MAF along with R. Türk (Salzburg) for the loan of the holotype and other collections of Acarospora gwynnii. M. whitelaw (Cambridge), J. Raggio Quilez (Madrid), G. Kantvilas (Hobart), and U. w. Ruprecht (Salzburg) are thanked for help with the provision of specimens of A. gwynnii. Lazzat Nurtai (Urumqi, China) is thanked for making her unpublished sequences of A. fulva and A. stapfiana available to us. 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