Abrothallus tetrasporus (Etayo & Osorio) Etayo, Flakus & Rodr. Flakus, comb. et stat. nov.

Index Fungorum number: IF 900420; MycoBank number: MB 900420; Facesoffungi number: FoF 14175; Fig. 1.

Basionym – Abrothallus usneae var. tetrasporus Etayo & Osorio, Comunicaciones Botanicas del Museo Nacional de Historia Natural y Antropologia Montevideo 129(6): 4 (2004).

Holotype – H. S. Osorio 9638a

Lichenicolous. Parasite of various saxicolous and corticolous species of Usnea growing on main branches and laciniae (more rarely also apothecial disc) of Usnea (not changing color of the host thallus) and on galls of Biatoropsis usnearum s. lat. Sexual morph: Mycelium immersed, indistinct, I–, K/I–. Ascomata apothecioid, superficial, producing directly on thallus of Usnea or on galls of Biatoropsis usnearum s. lat., black, epruinose or rarely slightly yellowish pruinose, strongly convex, emarginate, single or aggregated, constricted at the base, 250–550 µm in diam. Epihymenium 5–10 µm, dark brownish violet, K+emerald greenish, N +violet (shortly). Exciple strongly reduced, hyaline to dark-brown or violet-brown to black in external part. Hymenium 40–90 μm high, hyaline to violet in upper part, K +emerald green,. Interascal filaments irregularly branched and sometimes anastomosed (especially in the upper part), 1–2 μm wide, apical cells slightly widened (up to 2–2.5 μm), brown pigmented. Hypothecium olive brown to dark brown in the upper part, 90–250 μm high. Asci bitunicate, I–, K/I–, narrowly clavate, apically widened with evident apical dome, 40–70×8–12 μm, consistently 4-spored. Ascospores 1-septated, medium to dark brown, distinctly verrucose, covered by gelatine coat when younger, broadly ellipsoid, 8.5–(x=12.6±1.4)–15×4–(x=5.2±0.5)–6.5 μm, l/b ratio 1.9–(x=2.4±0.3)–3 (n=77), not braking easily in semi-spores, composed of almost equal cells, strongly constricted near septum. Asexual morph: Conidiomata pycnidial, immersed in the host thallus (sometimes below ascomata), 80–160 μm in diam. Pycnidial wall pale to dark brown-violet (darker at the upper part), K + emerald greenish, N + violet (shortly), 10–20 μm wide. Conidiogenous cells holoblastic, 8–10 × 3–5 μm. Conidia simple, hyaline, obpyriform with truncated end, 4.5–(x=7.4±0.9)–9×4–(x=4.6±0.4)–5.5 μm, l/b ratio 1–(x=1.6±0.3)–2.3 (n=45) (Etayo and Osorio 2004).

Material examined – Uruguay, Rocha, Cerro Lechiguana, on Usnea densirostra on rocks, 34° 06′ S 53° 50′ W, 24 April 2003, H. S. Osorio 9638a & G. Geymonat (holotype); Bolivia, Department Cochabamba, Province Carrasco, Parque Nacional Carrasco, between Meruvia and Monte Punku, 17° 34′ 43″ S, 65° 15′ 25″ W, 3082 m, forest with Podocarpus sp., on corticolous Usnea sp., 26 November 2014, A. Flakus, (25621 KRAM, LPB); ibid. Wayra Mayu close to Monte Punku, 17° 33′ 30″ S, 65° 16′ 08″ W, 2750 m, lower montane Yungas cloud forest, on corticolous Usnea sp., 28 November 2014, A. Flakus (25854KRAM, LPB); Department La Paz, Province Nor Yungas, way from La Paz to Coroico towards Unduavi in the way to Chulumani 16º18′ 27″ S, 67º53′ 48″ W, 3210 m, on Usnea sp. on bushes in the cloud forest, 31 May 2011, J. Etayo (26948LPB, herb. Etayo); Department Santa Cruz, Province Caballero, near Siberia, 17° 49′ 38″ S, 64° 44′ 45″ W, 3950 m, On Usnea sp. on mossy branches, open Yungas cloud forest, 16 August 2012, J. Etayo 29613, 28616 (LPB, herb. Etayo); ibid, on dead Usnea gr. florida on branches, J. Etayo 29417, 29613 (LPB, herb. Etayo); Department Tarija, Province O’Connor, 90 km from Tarija on the way to Entre Ríos, 21° 25′ 32″ S, 64° 19′ 05″ W, 2250 m, Tucumano-Boliviano altimontano forest, with Alnus acuminata and Podocarpus sp., on Usnea with Biatoropsis on twigs, 9 August 2012, J. Etayo 29392 (LPB); 28 km from Entre Ríos, near Soledad, 21° 41′ 00″ S, 64° 07′ 29″ W, 1500 m, Tucumano-Boliviano montano forest, 11 Aug. 2012, J. Etayo 55–13 (LPB); Province Aniceto Arce, close to La Mamora between Tarija and Bermejo, 22° 09′ 51″ S, 64° 40′ 03″ W, 1320 m, disturbed TucumanoBoliviano forest with Tillandsia, Subandino (Basimontano), on disk of Usnea sp. on branches, 27 July 2015, J. Etayo 30407 (LPB, herb. Etayo).

GenBank numbers – LSU: OR133230, ITS: OR116441, OR116442, SSU: OR116445, OR116446, OR116447.

Notes – The species is known from several localities in Bolivia, Uruguay (Etayo and Osorio 2004) and Terceira Island in Azores (Etayo 2018). We included in our phylogenetic analyses (Fig. 2) three Bolivian sample of A. usneae v. tetraspora forming a strongly supported monophyletic clade, which is phylogenetically different from A. altoandinus, and only distantly related to A. usneae var. usneae (represented on the tree by Portugal sample determined as A. usneae s. str. by Pérez-Ortega et al. 2014). The significant phylogenetic distances between those three taxa growing on Usnea species, together with the evident anatomical differences, suggest that they represent three distinct species. Therefore, we proposed here to elevate A. usneae v. tetraspora (the morph producing 4-spored asci) to species level. A. tetrasporus can be easily distinguished form A. usneae s str. by its constantly 8-spored asci (Etayo and Osorio 2004).

Figure 1 – Abrothallus tetrasporus (Flakus 25621). a–c Habit of ascomata growing on Usnea sp. d Transversal section of ascoma (mounted in LPCB). e Strongly reduced exciple (mounted in LPCB). f Asci mounted in Lugol’s solution pretreated by 10% KOH. g Ascospores mounted in distilled water (upper row) and mounted in 10% KOH (lower row). h Habit of pycnidial conidiomata immersed in host thallus. i Transversal section of conidiomata (mounted in LPCB). j Conidia mounted in distilled water. Scale bars: a–c, h=250 µm, d=100 µm, e, i=25 µm, f, g, j=10 µm

Figure 2 – Phylogenetic reconstruction of the systematic position of Abrothallus altoandinus and A. tetrasporus inferred from BI analyses of combined dataset of LSU, ITS, SSU and tef-α dataset. Bold branches represent either bootstrap values≥than 70 and/or Bayesian posterior probabilities≥than 0.95