Introduction
The genus Lecidea Ach. has long been used as a catch-all to group all species with crustose thalli, lecideoid apothecia, chlorococcoid photobionts and simple, colourless ascospores following the treatments of Zahlbruckner (Reference Zahlbruckner1925, Reference Zahlbruckner1932). Studies on saxicolous lecideoid species proliferated in the second half of the 20th century, describing new genera for several groups with synapomorphies and making it clear that Lecidea Ach. s. str. is a predominantly saxicolous group (e.g. Hertel Reference Hertel1967, Reference Hertel1983, Reference Hertel1984, Reference Hertel1995, Reference Hertel2007; Hafellner Reference Hafellner1984, Reference Hafellner1993; Rambold Reference Rambold1989).
Epiphytic lecideoid taxa represent a polyphyletic group of species (Pérez-Ortega et al. Reference Pérez-Ortega, Spribille, Palice, Elix and Printzen2010; Schmull et al. Reference Schmull, Miadlikowska, Pelzer, Stocker-Wörgötter, Hofstetter, Fraker, Hodkinson, Reeb, Kukwa and Lumbsch2011; Miadlikowska et al. Reference Miadlikowska, Kauff, Högnabba, Oliver, Molnár, Fraker, Gaya, Hafellner, Hofstetter and Gueidan2014) whose taxonomy and systematics have been partially clarified in recent decades, giving rise to a large number of new genera, such as Japewia Tønsberg (Tønsberg Reference Tønsberg1990), Japewiella Printzen (Printzen Reference Printzen1999), Palicella Rodr. Flakus & Printzen (Rodríguez-Flakus & Printzen Reference Rodriguez-Flakus and Printzen2014), Ramboldia Kantvilas & Elix (Kantvilas & Elix Reference Kantvilas and Elix1994), Puttea S. Stenroos & Huhtinen (Stenroos et al. Reference Stenroos, Huhtinen, Lesonen, Palice and Printzen2009), Australidea Kantvilas et al. (Kantvilas et al. Reference Kantvilas, Wedin and Svensson2021) and Myochroidea Printzen et al. (Printzen et al. Reference Printzen, Spribille and Tønsberg2008).
The taxonomy of lecideoid epiphytic genera has been largely based on features of the excipulum and hamathecium, ascus type and the presence of certain secondary metabolites (Printzen et al. Reference Printzen, Spribille and Tønsberg2008; Rodriguez-Flakus & Printzen Reference Rodriguez-Flakus and Printzen2014; Rodriguez-Flakus Reference Rodriguez-Flakus2020).
Interestingly, it has been shown, with the aid of molecular characters, that many of these new genera belong to the family Lecanoraceae (Pérez-Ortega et al. Reference Pérez-Ortega, Spribille, Palice, Elix and Printzen2010; Schmull et al. Reference Schmull, Miadlikowska, Pelzer, Stocker-Wörgötter, Hofstetter, Fraker, Hodkinson, Reeb, Kukwa and Lumbsch2011; Miadlikowska et al. Reference Miadlikowska, Kauff, Högnabba, Oliver, Molnár, Fraker, Gaya, Hafellner, Hofstetter and Gueidan2014; Rodriguez-Flakus & Printzen Reference Rodriguez-Flakus and Printzen2014; Zhao et al. Reference Zhao, Leavitt, Zhao, Zhang, Arup, Grube, Pérez-Ortega, Printzen, Śliwa and Kraichak2016), with members characterized often by lecanorine apothecia and the presence of Lecanora-like asci. The systematics of the family Lecanoraceae is in continuous change, and although recent papers on systematics have been published dealing with the phylogenetic relationships among the major lineages of the clade, the deep relationships are far from being unravelled (e.g. Zhao et al. Reference Zhao, Leavitt, Zhao, Zhang, Arup, Grube, Pérez-Ortega, Printzen, Śliwa and Kraichak2016; Yakovchenko et al. Reference Yakovchenko, Davydov, Ohmura and Printzen2019; Ivanovich et al. Reference Ivanovich, Dolnik, Otte, Palice, Sohrabi and Printzen2021; Svensson et al. Reference Svensson, Haugan, Timdal, Westberg and Arup2022).
In spite of recent efforts made to determine the phylogenetic positions of lecideoid epiphytic species, many of them still remain unplaced. During studies by two of the authors on the lichenicolous members of Tremella Pers. in the Iberian Peninsula, a new species (Tremella diederichiana Pérez-Ort. et al.) was described growing on a puzzling lecideoid epiphyte which was tentatively identified as Lecidea aff. erythrophaea (Zamora et al. Reference Zamora, Millanes, Wedin, Rico and Pérez-Ortega2016). The species appears to be quite common, especially on branches of Cistus ladanifer in the central Iberian Peninsula, and had also been previously determined as Lecidea exigua Chaub. (Aragón et al. Reference Aragón, Sarrión and Martínez2004; Martínez & Aragón Reference Martínez and Aragón2004). Here, we introduce it as a new genus in the family Lecanoraceae, Nimisora Pérez-Ort., M. Svensson & J. C. Zamora, and a new species, Nimisora iberica Pérez-Ort., Turégano, M. Svenss. & J. C. Zamora, providing insights into its phylogenetic relationships based on ribosomal DNA data and morphology.
Material and Methods
Morphological analyses
Specimens were examined using a Leica S9i dissecting microscope with an in-built digital camera. Hand-cut sections of apothecia and thalli were observed using either an Olympus BX51 with Nomarski differential interference contrast (DIC) or a Nikon Eclipse E200 microscope fitted with a set of polarized filters. Images were captured with a Leica DMC 4500 digital camera fitted to the Olympus microscope. Colour reactions were observed using 50% HNO3 (N), 10% KOH (K), 8% sodium hypochlorite (C), paraphenylendiamine (PD) and Lugol's iodine solution (256977.1609 PanReac AppliChem) (I), the latter both with (K/I) and without pretreatment with K. The measurements of ascospores were made on material mounted in water and are presented as minimum– (mean ± standard deviation) –maximum values followed by the number of measurements (n) and number of specimens (s). Thin-layer chromatography analyses were carried out following standard methods using solvent C (Orange et al. Reference Orange, James and White2001).
Molecular methods
Apothecial sections were excised from seemingly un-parasitized thalli and placed in microcentrifuge tubes. They were stored at −80 °C for 1 h after which they were pulverized using a Qiagen TissueLyser II and glass beads. DNA was extracted using E.Z.N.A ® Forensic DNA Kit (Omega Bio-Tek), following the instructions of the manufacturer. A fragment of the nuclear ribosomal DNA comprising the internal transcriber spacer (nrITS:ITS1, 5.8S, and ITS2) and domains D1–D3 of the nuclear large ribosomal subunit (nrLSU), as well as a fragment of the mitochondrial small subunit ribosomal DNA (mtSSU) of c. 800 bp, were amplified using the following primer pairs: ITS1-F (White et al. Reference White, Bruns, Lee, Taylor, Innis, Gelfand, Sninsky and White1990) and ITS4_KYO2 (Toju et al. Reference Toju, Tanabe, Yamamoto and Sato2012) for nrITS, LR0R and LR5 (Vilgalys & Hester Reference Vilgalys and Hester1990) for the nrLSU, and mrSSU1 and mrSSU3r (Zoller et al. Reference Zoller, Scheidegger and Sperisen1999) for the mtSSU. Amplification reactions were prepared for a 15 μl final volume containing 7.5 μl of MyTaq™ Red Mix (Bioline), 0.5 μl of each of the primers at 10 μM, 5.5 μl of H2O, and 1 μl of template DNA. PCR conditions for the amplification of the nrITS and nrLSU were as follows: 5 min at 95 °C; 10 cycles of 30 s at 95 °C, 30 s at 66 °C, 1 min 30 s at 72 °C; 34 cycles of 30 s at 95 °C, 30 s at 56 °C, 1 min 30 s at 72 °C; and 10 min at 72 °C. Conditions for the amplification of the mtSSU were: 10 min at 94 °C; 34 cycles of 45 s at 95 °C, 45 s at 50 °C, 1 min 30 s at 72 °C; 5 min at 72 °C. Additionally, DNA was extracted for one specimen of the new taxon and one of Japewiella tavaresiana (H. Magn.) Printzen and the nrITS and mtSSU amplified following the methods described in Svensson & Fryday (Reference Svensson and Fryday2022). PCR products were purified using Exo SAP-IT (USB Europe GmbH), following the manufacturer's instructions. The samples were sequenced with Sanger dideoxy-technology by Macrogen Inc. (Macrogen Europe, Madrid, Spain) using the same PCR primers. Contigs were assembled and edited using either AliView v. 1.1 (Larsson Reference Larsson2014) or Geneious® Prime v. 2020.0.3.
Sequence alignments
Preliminary BLAST searches (Altschul et al. Reference Altschul, Madden, Schäffer, Zhang, Zhang, Miller and Lipman1997) pointed to members of the Lecanoraceae as the closest hits for these sequences. Thus, to find the phylogenetic position within the family we aligned our data with sequences of the main genera and groups of Lecanoraceae (Rodriguez- Flakus & Printzen Reference Rodriguez-Flakus and Printzen2014; Zhao et al. Reference Zhao, Leavitt, Zhao, Zhang, Arup, Grube, Pérez-Ortega, Printzen, Śliwa and Kraichak2016). Multiple sequence alignments for each genomic region were performed using MAFFT v. 7.490 (Katoh et al. Reference Katoh, Misawa, Kuma and Miyata2002) as implemented on the CIPRES Science Gateway (Miller et al. Reference Miller, Pfeiffer and Schwartz2011) using default parameters. Ambiguous positions were removed using Gblocks v. 0.91b (Castresana Reference Castresana2000) at http://phylogeny.lirmm.fr/phylo_cgi/one_task.cgi?task_type=gblocks, using all the options available for the least stringent selection.
Phylogenetic analysis
Single-locus trees were inferred using maximum likelihood (ML) in the IQ-TREE web server (Trifinopoulos et al. Reference Trifinopoulos, Nguyen, Von Haeseler and Minh2016). A visual inspection of the topologies revealed no incongruence between them, that is, there were no supported nodes that were incompatible with each other. Node support was calculated using 1000 ultrafast bootstrap replicates (BS), implying that only BS values > 95% should be considered indicative of compatibility (Hoang et al. Reference Hoang, Chernomor, von Haesler, Minh and Vinh2018). Thus, the single-locus alignments were concatenated into a single alignment using Geneious® Prime v. 2020.0.3 for subsequent analyses. This alignment was analyzed using ML and Bayesian phylogenetic inference (BI) methods. Maximum likelihood analysis was performed in RAxML-HPC2 v. 8.2.4 (Stamatakis Reference Stamatakis2014) as implemented on the CIPRES Science Gateway (Miller et al. Reference Miller, Pfeiffer and Schwartz2011), dividing the alignment into five partitions: nrITS1, 5.8S, nrITS2, nrLSU and mtSSU. The GTRGAMMA substitution model was used for all partitions. We searched the best-scoring ML tree and conducted a rapid bootstrap analysis with 1000 pseudoreplicates to evaluate nodal support in one single run. The BI analysis was performed in MrBayes v. 3.2.7a (Ronquist et al. Reference Ronquist, Teslenko, van der Mark, Ayres, Darling, Höhna, Large, Liu, Suchard and Huelsenbeck2012) as implemented on the CIPRES Science Gateway (Miller et al. Reference Miller, Pfeiffer and Schwartz2011). The same partitions used in the ML analysis were implemented in the BI analysis. We inferred the topology and sampled across the substitution model space with the reversible-jump Markov chain Monte Carlo (MCMC) method (Huelsenbeck et al. Reference Huelsenbeck, Larget and Alfaro2004). The analysis was run using default priors, two parallel runs of four MCMC chains over 20 million generations, starting with a random tree and sampling one tree every 5000 generations The first 25% of trees from each analysis was discarded as burn-in. Branch support (Bayesian posterior probability (PP)) was calculated using the consensus tree following the ‘50% majority rule’ of the remaining trees. Nodes with ML bootstrap value ≥ 70% and PP ≥ 0.95 were considered phylogenetically supported.
Results
We generated 10 new sequences: 4 nrITS, 2 nrLSU and 4 mtSSU (Table 1). The corresponding alignments were 632, 803 and 898 bp long, respectively; 368, 563, 545 of which remained after the Gblocks removal of ambiguously aligned regions and gaps. Maximum likelihood and Bayesian analyses retrieved single best and 50% majority-rule trees respectively with similar topologies, so only the tree recovered using maximum likelihood inference is shown in Fig. 1. Overall supported relationships among species were rather similar to those inferred in previous works with a limited number of loci (e.g. Pérez-Ortega et al. Reference Pérez-Ortega, Spribille, Palice, Elix and Printzen2010; Rodriguez-Flakus & Printzen Reference Rodriguez-Flakus and Printzen2014; Malíček et al. Reference Malíček, Palice, Vondrák and Tønsberg2020; Davydov et al. Reference Davydov, Yakovchenko, Hollinger, Bungartz, Parrinello and Printzen2021); that is, although most of the genera and morphological groups are well-supported, the relationships among them remain uncertain. All specimens from the new taxon form a well-supported (BP = 100, BPP = 1) monophyletic group sister to the species of the genus Ramboldia included in the analyses, although this relationship was not supported by either of the two inference analyses, so the exact phylogenetic position of Nimisora within the Lecanoraceae remains unclear.
Sequence data used for the phylogenetic analysis (Fig. 1), with GenBank Accession numbers and voucher information. Sequences generated in this study are in bold.
Most likely tree inferred by maximum likelihood (ML) analysis of the ITS, nrLSU and mtSSU regions of Lecanoraceae species. Sequences generated in this study and the new genus, Nimisora, are indicated in bold. Thick branches indicate nodes with phylogenetic support in both analyses (ML bootstrap values ≥ 70% / posterior probability ≥ 0.95). In colour online.
Taxonomy
Morphoanatomical, chemical and molecular data support the description of a new genus and species of lichenized fungus in Lecanoraceae.
Nimisora Pérez-Ort., M. Svenss. & J. C. Zamora gen. nov.
MycoBank No.: MB 848883
The genus is characterized by the following combination of characters: crustose thalli, lecideoid apothecia with a proper excipulum composed of thick-walled radiating hyphae, with widened terminal cells, hymenium of simple to branched paraphyses widened at the apex, brown to dark green K+ olivaceous pigment in the epihymenium and excipulum, an ascus similar to the Bacidia-type and simple, broadly ellipsoid, colourless ascospores.
Type species: Nimisora iberica Pérez-Ort., Turégano, M. Svenss. & J. C. Zamora
Etymology
The genus is named after Prof. Pier Luigi Nimis (University of Trieste) for his 70th birthday, and in gratitude of his great contribution to lichenology and especially to the knowledge of lichens in the Mediterranean region.
Nimisora iberica Pérez-Ort., Turégano, M. Svenss. & J. C. Zamora sp. nov.
MycoBank No.: MB 848884
An epiphytic species without a well-developed cortex, granulate to areolated crustose thalli, lecideoid apothecia with a proper excipulum composed of radiating thick-walled, sparingly branched hyphae and dark brown to dark green pigment, hymenium composed of simple to branched paraphyses with widened apices with brown caps, epihymenium with a brown to dark green K+ olive green pigment, ascus similar to the Bacidia-type, ascospores simple, broadly ellipsoid, colourless, usually straight, 10–15 × 4–7 μm, with no lichen substances detected by TLC.
Type: Spain, Madrid, Mataelpino, Cistus ladanifer thicket close to road M-617, 40°43ʹ57ʺN, 3°58ʹ16ʺW, 1135 m, on Cistus ladanifer, 16 September 2021, S. Pérez-Ortega 11512 (MA-Lichen 26675—holotype!) [TLC: nil, thallus with Tremella diederichiana].
Nimisora iberica (S. Pérez-Ortega 11512). A, habitus. B, thallus section. C, section of apothecium. D, detail of excipulum. E, detail of the hymenium. F, subhymenium. G, paraphyses. H, detail of ascus tip. I, mature ascus. J–M, ascospores. C–F, lactophenol cotton blue. H & I, KOH and Lugol's iodine solution. J–M, Lugol's iodine solution. G & I–M, differential interference contrast. Scales: A = 1 mm; B, C, E & F = 20 μm; D = 10 μm; G–M = 5 μm. In colour online.
Thallus crustose, episubstratal, granulose to minutely areolated, forming roundish to elongate patches up to 22 mm long and 10 mm wide, rarely coalescing. Granules roundish up to 350 μm diam., areoles roundish to elongate or angular in outline, average diam. of 0.03–0.42 mm, up to 0.55 mm in height reaching 0.7 mm when growing on Pinus branches, flattened to convex, greenish grey to light grey, or whitish, green when wet, matt, sometimes with a brownish tinge, without well-developed cortex, thallus hyphae I−. Soralia or isidia absent. Algal layer irregular, often expanded throughout the thallus and in contact with the substratum. Photobiont chlorococcoid, globose, algal cells up to 15 μm diam.
Apothecia common, more or less roundish in outline, sessile, immersed and with some thallus remains when very young, constricted at the base, single or in groups up to 7 or 8, usually 0.3–0.6 mm diam. (up to 0.9 mm). Disc very variable in colour, even within the same thallus, from light cream-brown to reddish brown or dark brown, flat to weakly convex, matt, epruinose, margin visible, usually darker than the disc, dark brown to black, thinner with age but almost always conspicuous, rarely excluded. Proper excipulum laterally 20–45 μm in section, composed of radially arranged septate hyphae, hyphae up to 6 μm diam., widening at the apex up to 10 μm, individual cells up to 10 μm long, lumina up to 2 μm wide; excipulum colourless within or with a dark green or dark brown pigment in the outer part of the hyphae, apical cells with a much darker pigmentation, K+ dark olive green or dark brown, N+ dark pink. Hypothecium, 15–35 μm high, of roundish to angular hyphae 4–6 μm wide, with very thin walls, colourless. Epihymenium olive green to light to dark brown, pigment present in the gelatinous matrix and the apical cell walls of paraphyses, 5–10 μm thick, K+ dark olive green, N+ dark pink, without granules or with scarce granules refracting polarized light. Hymenium hyaline, 35–50 μm thick, I+ dark blue. Paraphyses simple to branched, especially in the upper part, rarely anastomosing, 1.5–2.5 μm wide, lumina up to 1 μm wide, apical cells capitate, 3–4 μm wide, with dark brown pigmentation in upper internal walls, with a gelatinized sheath c. 1 μm thick around the caps, dissolving in K. Asci clavate, 32–45 × 8–10 μm when mature, tholus strongly amyloid, with poorly developed or no ocular chamber, with a pale conical axial mass usually with a dark layer around the axial mass (Bacidia-type). Ascospores simple, very rarely 1-septate, hyaline, ellipsoid to broadly ellipsoid, straight, rarely curved, with a single wall (c. 0.5 μm) and lacking gelatinous epispore, 10–(11.03 ± 1.07)–15 × 4–(5.71 ± 0.65)–7 μm (n = 35, s = 5).
Conidiomata not seen.
Chemistry
No substances detected by TLC.
Ecology
The species is common on twigs of Quercus rotundifolia in the centre of the Iberian Peninsula, and especially on twigs of Cistus ladanifer, being rarer in other areas of the supramediterranean belt of the Peninsula. It is accompanied by other species typical of twigs in the area such as: Blastenia xerothermica Vondrák et al., Evernia prunastri (L.) Ach., Lecanora carpinea (L.) Vain., L. chlarotera Nyl., L. varia (Hoffm.) Ach., Lecidella elaeochroma (Ach.) M. Choisy, L. euphorea (Flörke) Hertel, Melanohalea exasperata (De Not.) O. Blanco et al., Physcia adscendens H. Olivier, Rinodina pyrina (Ach.) Arnold, or R. sophodes (Ach.) A. Massal. The species is also the host of Tremella diederichiana, a lichenicolous basidiomycete which forms characteristic minute whitish galls on the thallus of N. iberica (Zamora et al. Reference Zamora, Millanes, Wedin, Rico and Pérez-Ortega2016).
Additional specimens examined
Spain: Madrid: Tres Cantos, Parque Central, Pinus radiata forest, 40°36ʹ7ʺN, 3°42ʹ16ʺW, 718 m, on Pinus radiata twig, 2021, S. Pérez-Ortega 12116 & E. Arróniz (MA-Lichen 26684) [TLC: nil]; Manzanares el Real, Cistus ladanifer in the surroundings of the reservoir, 40°44ʹ24ʺN, 3°50ʹ30ʺW, 901 m, on Cistus ladanifer, 2021, S. Pérez-Ortega 11207, M. Arróniz & E. Arróniz (MA-Lichen 26683) [TLC: nil]; San Agustín de Guadalix, Dehesa de Moncalvillo, siliceous outcrops (gneiss) and Cistus ladanifer thicket near La Sima creek, 40°42ʹ6ʺN, 3°38ʹ46ʺW, 843 m, on Cistus ladanifer, 2021, S. Pérez-Ortega 11260 (MA-Lichen 26682) [TLC: nil]; Olmeda de las Fuentes, Quercus rotundifolia forest, 40°20ʹ47ʺN, 3°12ʹ4ʺW, 821 m, on Q. rotundifolia twig, 2018, S. Pérez-Ortega 8790 (MA-Lichen 26881) [TLC: nil]; Madrid, Quercus rotundifolia forest near the road M-612 from Fuencarral to El Pardo, 40.511531°N, 3.750402°W, 679 m, on Q. rotundifolia twig, 2018, S. Pérez-Ortega 8753 (MA-Lichen 26680) [TLC: nil]; El Boalo, Cistus ladanifer thicket near San Isidro hermitage, 40°43ʹ46ʺN, 3°55ʹ12ʺW, 1000 m, on Cistus ladanifer, 2021, S. Pérez-Ortega 11404, M. Arróniz & E. Arróniz (MA-Lichen 26679) [TLC: nil, with Tremella diederichiana]; El Berrueco, Pradera del Amor, granitic boulders and Cistus ladanifer thicket close to M-127 road, 40°52ʹ55ʺN, 3°34ʹ38ʺW, 1013 m, on Cistus ladanifer, 2018, S. Pérez-Ortega 8183 & S. Prats i Font (MA-Lichen 26685) [TLC: nil]; ibid., S. Pérez-Ortega 8186 & S. Prats i Font (UPS) [TLC: nil, with Tremella diederichiana]; ibid., S. Pérez-Ortega 8185 & S. Prats i Font (G) [TLC: nil, with Tremella diederichiana]; Tres Cantos, Soto de Viñuelas, 40°37ʹ5ʺN, 3°40ʹ14ʺW, 689 m, on Q. rotundifolia twig, 2020, S. Pérez-Ortega 10797, M. Arróniz & E. Arróniz (MA-Lichen 26677) [TLC: nil]; Colmenar Viejo, gneiss outcrops in Cerro San Pedro, 40°43ʹ12ʺN, 3°43ʹ52ʺW, 1014 m, on Cistus ladanifer, 2021, S. Pérez-Ortega 11766, E. Arróniz & M. Arróniz (MA-Lichen 26678) [TLC: nil]. La Rioja: Sojuela, mixed forest of Q. pyrenaica and Q. rotundifolia, 42°21ʹ33ʺN, 2°33ʹ16ʺW, 814 m a.s.l., on Q. rotundifolia twig, 2019, S. Pérez-Ortega 8273 (MA-Lichen 26676) [TLC: nil]. Castilla- La Mancha: Ciudad Real, Retuerta del Bullaque, Cabañeros National Park, Viñuelas, Quercus pyrenaica forest, 30SUJ7159, 800 m, on dead Quercus pyrenaica, 1996, I. Fernández, F. J. Sarrión & J. A. Maroto 297 (MA-Lichen 14845); Ciudad Real, Villamanrique, 38.416507°N, −2.999149°W, on dead branches of Cistus ladanifer, 3 iii 2018, J. C. Zamora (UPS).
Other species examined
Lecanora symmicta (Ach.) Ach. Spain: Castile and León: Segovia, Riofrío de Riaza, Riaza valley, Majada Larga, 30TVL6564, 1720 m, on Juniperus nana, 1994, G. Aragón, I. Martínez & T. Rojas IMM 202/94 (MA-Lichen 4938).
Lecidella elaeochroma. Spain: Castilla y Len: Burgos, Torrecilla del Monte, Quercus rotundifolia forest with Juniperus oxycedrus and Quercus faginea, 42°4ʹ19ʺN, 3°42ʹ38ʺW, 948 m, on Quercus rotundifolia, 2018, S. Pérez-Ortega 9315 & A. Berlinches de Gea (MA-Lichen).
Lecidella euphorea. Spain: Madrid: Madrid, Quercus rotundifolia close to the road M-612, 40.511531°N, 3.750402°W, 679 m, on Quercus rotundifolia, 2017, S. Pérez-Ortega 10612 & M. Comte (MA-Lichen).
Pyrrhospora quernea (Dicks.) Körb. Spain: Islas Baleares: San Josep de sa Talaia, Pinus halepensis forests near the top of Sa Talaia, 38°54ʹ53ʺN, 1°16ʹ39ʺE, 445 m, on Pinus halepensis, 2017, S. Pérez-Ortega 5590 (MA-Lichen 26862).
Ramboldia elabens (Fr.) Kantvilas & Elix. Spain: Castilla-La Mancha: Toledo, Hontanar, Montes de Toledo, Estena River, Quercus rotundifolia forest, 30SUJ6380, 875 m, on dead trunk of Juniperus oxycedrus, 1995, G. Aragón 1006/95, J. L. Izquierdo & I. Martínez (MA-Lichen 7157).
Traponora varians (Ach.) J. Kalb & Kalb (sub Lecidea exigua Chaub.). Spain: Navarra: Oronoz Mugaire, Señorío de Bértiz, 30TXN1579, on Quercus robur branches, J. Etayo (MA-Lichen 3845). Galicia: Pontevedra, Niño do Corvo, 41°56ʹ58ʺN, 8°48ʹ37ʺW, 293 m, on Pyrus cordata, 15 viii 2021, D. Fernández-Costas & A. García-Morales (MA-Lichen 26863); Pontevedra, Niño do Corvo, Mirador de Tamuxe, 41°54ʹ59ʺN, 8°49ʹ34ʺW, 25 m, on Castanea sativa, 25 vii 2021, D. Fernández-Costas & A. García-Morales (MA-Lichen 26864).
Discussion
The lecideoid epiphytic species described here has been treated equivocally in the literature as Lecidea exigua (=Traponora varians) (e.g. Martínez & Aragón Reference Martínez and Aragón2004) or L. aff. erythrophaea (e.g. Zamora et al. Reference Zamora, Millanes, Wedin, Rico and Pérez-Ortega2016). Records of these species from the Mediterranean region of the Iberian Peninsula should therefore be re-evaluated.
Our phylogenetic analyses showed that Nimisora iberica belongs to the family Lecanoraceae. A number of epiphytic lecideoid species have been shown to belong to the Lecanoraceae in recent years (e.g. Pérez-Ortega et al. Reference Pérez-Ortega, Spribille, Palice, Elix and Printzen2010; Rodriguez-Flakus & Printzen Reference Rodriguez-Flakus and Printzen2014; Schmull et al. Reference Schmull, Miadlikowska, Pelzer, Stocker-Wörgötter, Hofstetter, Fraker, Hodkinson, Reeb, Kukwa and Lumbsch2011). Unfortunately, our analyses have not been able to ascertain the closest relative of Nimisora, as its relationship to other groups within Lecanoraceae were not supported. Molecular data gathered in this study seem largely insufficient to build a solid phylogenetic hypothesis for the family, just as in previous studies on the systematics and taxonomy of the Lecanoraceae (e.g. Pérez-Ortega et al. Reference Pérez-Ortega, Spribille, Palice, Elix and Printzen2010; Rodriguez-Flakus & Printzen Reference Rodriguez-Flakus and Printzen2014; Davydov et al. Reference Davydov, Yakovchenko, Hollinger, Bungartz, Parrinello and Printzen2021).
Morphologically, the new genus shows clear similarities with many other genera of lecideoid epiphytic species but, in our opinion, the combination of the structure of the proper exciple, paraphyses, pigments and ascus type make Nimisora characteristic enough to be separated as a new independent genus.
Ascus types, defined by a combination of ascus characteristics, especially the staining patterns shown at the apex after KI application, have been widely used in the taxonomy and systematics of the Lecanoraceae and Lecideaceae for the last six decades (Hafellner Reference Hafellner1984; Rambold & Triebel Reference Rambold and Triebel1992). However, the interpretation of ascus type is sometimes not straightforward, as intermediate stages occur within the same groups and even at different developmental stages in the same species (Rambold Reference Rambold1995; Rodriguez-Flakus Reference Rodriguez-Flakus2020). This is the case for several of the taxa anatomically related to Nimisora, where the ascus types do not seem to conform exactly to one of the proposed ideal types (Hafellner Reference Hafellner1984). Likewise, the ascus of Nimisora is similar to the Bacidia-type; however, the strongly amyloid reaction of the tholus may make observation and interpretation of the ascus type difficult, and low concentrations of Lugol's often help with examination of the ascus tip structure.
Nimisora shares morphological and anatomical features with species from Lecidella Körb., Japewia, Japewiella, Palicella and Traponora Aptroot (Knoph Reference Knoph1990; Tønsberg Reference Tønsberg1990; Printzen Reference Printzen1999; Aptroot Reference Aptroot2009; Rodriguez-Flakus & Printzen Reference Rodriguez-Flakus and Printzen2014).
Lecidella is the most similar genus to Nimisora (Table 2). They share a number of characters, although the thalli of the two genera are characteristically different, with Lecidella species usually having well-developed verrucose to areolate thalli and much larger apothecia than Nimisora, with a distinctive black colour and showing well-developed thick margins when young. Anatomically, Nimisora and Lecidella have an excipulum composed of radiating hyphae which thicken at the apices. In addition, Nimisora and Lecidella have sparsely branched paraphyses, although in Nimisora they usually terminate in a widened cell surrounded by a dark brown pigment, which is not present in Lecidella. Lecidella species typically have the Cinereorufa-green pigment (Meyer & Printzen Reference Meyer and Printzen2000) in the epihymenium and the outer excipulum, reacting K+ vivid green, N+ purple, whereas Nimisora has an unknown dark brown or dark green pigment that reacts K+ dark olive green, N+ dark pink. The genera also apparently have different ascus types. It has been stated that Lecidella species have a Lecanora-type ascus (e.g. Cannon et al. Reference Cannon, Malíček, Ivanovich, Printzen, Aptroot, Coppins, Sanderson, Simkin and Yahr2022) or Lecidella-type ascus (Rodriguez-Flakus & Printzen Reference Rodriguez-Flakus and Printzen2014), a modification of the Lecanora-type in which the axial mass does not reach the upper part of the ascus tip and is usually broadened. However, according to our observations, species such as L. elaeochroma or L. euphorea show an ascus type similar to the Bacidia-type reported here for Nimisora. Following Ekman et al. (Reference Ekman, Andersen and Wedin2008), we do not recognize the Biatora-type since the presence of a darker layer surrounding the axial mass is a very variable character, even within species. Finally, Lecidella species often have atranorin as the major substance, usually accompanied by terpenoids and/or xanthones, whereas no TLC-detectable substance is known so far from Nimisora.
Characters distinguishing Nimisora from similar genera in the Lecanoraceae. Characters for genera mostly follow Aptroot (Reference Aptroot2009), Rodriguez-Flakus & Printzen (Reference Rodriguez-Flakus and Printzen2014), Rodriguez-Flakus (Reference Rodriguez-Flakus2020) and Cannon et al. (Reference Cannon, Malíček, Ivanovich, Printzen, Aptroot, Coppins, Sanderson, Simkin and Yahr2022).
Palicella also shows clear similarities with Nimisora (Table 1). It is noteworthy that all species of Palicella (except P. lueckingii Rodr. Flakus) and N. iberica have apothecia that show a great chromatic variation, often within the same specimen (Rodriguez-Flakus & Printzen Reference Rodriguez-Flakus and Printzen2014; Rodriguez-Flakus Reference Rodriguez-Flakus2018). Excipular hyphae in Palicella, although also radiating, do not have thick hyphal walls. Unlike Nimisora, dark-pigmented Palicella species have the Cinereorufa-green pigment in the epihymenium and outer excipulum. Furthermore, the paraphyses are not or only slightly apically thickened, except in P. lueckingii (Rodriguez-Flakus & Printzen Reference Rodriguez-Flakus and Printzen2014; Rodriguez-Flakus Reference Rodriguez-Flakus2018). In addition, the ascospores are narrowly ellipsoid (except in P. lueckingii), sometimes 1-septate, whereas in Nimisora they are broadly ellipsoid and extremely rarely septate. The species of Palicella have a Lecanora/Lecidella-type ascus, although Bacidia-like asci can also be found in young specimens (see Printzen & May Reference Printzen and May2002). Finally, Palicella species have atranorin as the major compound, whereas in P. lueckingii thiophanic acid is the major compound, often together with usnic acid or terpenoids (Rodriguez-Flakus & Printzen Reference Rodriguez-Flakus and Printzen2014; Rodriguez-Flakus Reference Rodriguez-Flakus2018). Holarctic species are known to contain aliphatic substances (paraensic acids; Pérez-Ortega et al. Reference Pérez-Ortega, Spribille, Palice, Elix and Printzen2010), confined apparently to apothecia (Palice et al. Reference Palice, Printzen, Spribille and Elix2011), and South American species have pannaric acid as crystals in the epihymenium (Rodriguez-Flakus & Printzen Reference Rodriguez-Flakus and Printzen2014; Rodriguez-Flakus Reference Rodriguez-Flakus2018).
The genus Japewiella is also anatomically similar to Nimisora, especially in terms of the excipulum and hamathecial structure. In both, the excipulum is formed by very thick-walled hyphae in a radial arrangement, although they are more branched and anastomosed in Japewiella than in Nimisora, in which the hyphae are unbranched or barely branched (Printzen Reference Printzen1999). Both genera have hamathecia composed of sparingly branched or anastomosed paraphyses with widened apical cells with brown walls. Regarding ascus type, it is stated in the literature that Japewiella has a Lecidella-type (Printzen Reference Printzen1999); however, plasticity there also exists and transitions to the Bacidia-type present in Nimisora are common. There are also differences in secondary chemistry: Japewiella species either contain atranorin and other depsides as major substances (Printzen Reference Printzen1999) or the β-orcinol depsidone norstictic acid (Allen & Lendemer Reference Allen and Lendemer2015), whereas Nimisora does not contain any lichen substance detectable by TLC. In addition, our phylogenetic analyses indicate a rather distant relationship between the two genera (Fig. 1).
Japewia may be superficially similar to Nimisora but it displays a large number of differences, such as an exciple not clearly different in structure from the hamathecium, which is composed of a network of highly branched and anastomosed paraphyses. Asci range from the Lecidella- and Bacidia-types (Tønsberg Reference Tønsberg1990), and ascospores have characteristic, thick multilayered walls (Tønsberg Reference Tønsberg1990; Printzen Reference Printzen1999; Malíček et al. Reference Malíček, Palice, Vondrák and Tønsberg2020).
Other groups in the Lecanoraceae which show some similarities with Nimisora are Ramboldia, Pyrrhospora quernea and the Lecanora symmicta group.
Ramboldia was described by Kantvilas & Elix (Reference Kantvilas and Elix1994) to accommodate a group of lignicolous species close to Lecidea elabens Fr., characterized by lecideoid apothecia, an unpigmented excipulum composed of radially branched and anastomosed hyphae, Lecanora-type asci and simple, hyaline, non-halonate ascospores. The group has later been revised to incorporate species formerly placed in Pyrrhospora, including saxicolous species and species with the anthraquinone russulone (Kantvilas & Elix Reference Kantvilas and Elix2007; Kalb et al. Reference Kalb, Staiger, Elix, Lange and Lumbsch2008). Nimisora differs from Ramboldia in the different ascus type and in the excipular hyphae which are much less branched and anastomosed than in Ramboldia, as well as in the very distinct chemistry of the latter genus.
Pyrrhospora quernea also has an excipulum with radiating hyphae, but they are usually not swollen at their ends and the tips are usually inspersed with red granules; it has the K+ purplish anthraquinone (7-chloroemodin) in the epithecium, a Lecanora-type ascus and ascospores typically turning yellow to brownish when mature.
The predominantly tropical genus Traponora also shows some similarities with Nimisora (Aptroot et al. Reference Aptroot, Diederich, Sérusiaux and Sipman1997; Aptroot Reference Aptroot2009). The main differences concern the anatomy of the apothecia, which in Traponora are irregular in shape, pruinose and surrounded by thallus remnants when young (Aptroot Reference Aptroot2009), in contrast to the more roundish and regular apothecia in Nimisora which only show scarce thallus remnants in some very young specimens and never show pruina. The excipulum in Traponora is composed of narrow radiating hyphae, not swollen at the tips, and it has a Lecanora-type ascus (Aptroot Reference Aptroot2009). Traponora is a genus with a mainly tropical distribution (Aptroot Reference Aptroot2009), but T. varians is a species with a temperate distribution in Europe and North America (see below).
The species of the Lecanora symmicta group also have excipular margins with radiating hyphae but they form a network through frequent anastomoses and are weakly swollen in the apex, and they also have granules in the outer part. In addition, the pale pigments in the epithecium are K−, and the thalli contain usnic acid as the major compound.
Nimisora iberica can be confused in the field with Lecidella elaeochroma and L. euphorea, two species with which it often co-occurs. Confusion is especially possible with the former since it may have thalli with coloration similar to N. iberica and apothecia, especially in shade forms, ranging from dark brown to light brown. However, both species are easily distinguished from Nimisora by the presence of whitish or creamy crustose continuous to areolate thalli, much larger apothecia and the characteristic Cinereorufa-green pigment in the epihymenium and external part of the excipulum, reacting a K+ vivid green.
There has previously been confusion in the literature between Nimisora iberica and two other species, namely Lecidea exigua Chaub. and L. erythrophaea Sommerf. The former taxon was recently put into synonymy with Traponora varians (Cannon et al. Reference Cannon, Malíček, Ivanovich, Printzen, Aptroot, Coppins, Sanderson, Simkin and Yahr2022), a species now having a disjunct distribution occurring on the Pacific Coast and Eastern Coast of North America (Hertel & Printzen Reference Hertel, Printzen, Nash, Ryan, Gries and Bungartz2004; León-González & Pérez-Pérez Reference León-González and Pérez-Pérez2020), as well as in several areas of Europe such as Great Britain (Cannon et al. Reference Cannon, Malíček, Ivanovich, Printzen, Aptroot, Coppins, Sanderson, Simkin and Yahr2022), Italy (Nimis Reference Nimis2023), France (Roux Reference Roux2012), and the Iberian Peninsula (e.g. Carballal & García-Morales Reference Carballal and García-Molares1991; Etayo & Gómez-Bolea Reference Etayo and Gómez-Bolea1992). Traponora varians, which according to our observations shares the same Bacidia-type ascus with N. iberica, can be distinguished from the new species by having a thallus usually surrounded by a black hypothallus, small lecideine apothecia which are orange to pale reddish brown, usually pruinose when young, and atranorin and unidentified xanthones as the main secondary metabolites (Cannon et al. Reference Cannon, Malíček, Ivanovich, Printzen, Aptroot, Coppins, Sanderson, Simkin and Yahr2022). In addition, in Europe, T. varians seems to prefer more humid localities than N. iberica. The relationships between T. varians and the tropical species of the genus, as well as the relationships between the North American and European populations, need to be investigated further.
Lecidea erythrophaea has larger apothecia than N. iberica (0.25–0.75(–1) mm), typically with reddish brown discs (Hertel & Printzen Reference Hertel, Printzen, Nash, Ryan, Gries and Bungartz2004). In addition, L. erythrophaea has a thin, white thallus, lacks a K+ green pigment in the epihymenium, contains reddish brown oily granules in the exciple and has a distinctly different ascospore shape, with comparatively more narrowly ellipsoid ascospores up to 18 μm long and 3–4.5(–5) μm wide (Aptroot et al. Reference Aptroot, Gilbert, Hawksworth, Coppins, Smith, Aptroot, Coppins, Fletcher, Gilbert, James and Wolseley2009; Wirth et al. Reference Wirth, Hauck and Schultz2013).
The mostly lignicolous Lecanora hypoptoides (Nyl.) Nyl. also shares some similarities with N. iberica, such as the very similar ascus type (Palice et al. Reference Palice, Malíček, Peksa and Vondrák2018). The two taxa can be differentiated by the poorly developed, often immersed, thallus in L. hypoptoides, as well as by the presence in this species of lecanorine apothecia when young, becoming biatorine with age (van den Boom & Brand Reference van den Boom and Brand2008; Cannon et al. Reference Cannon, Malíček, Ivanovich, Printzen, Aptroot, Coppins, Sanderson, Simkin and Yahr2022). In addition, the presence of paraensic C and D acids is diagnostic of L. hypoptoides (van den Boom & Brand Reference van den Boom and Brand2008; Palice et al. Reference Palice, Malíček, Peksa and Vondrák2018).
Acknowledgements
This study was partly funded by the grant PID2019-111527GB-I00 to SPO from the Spanish Ministry of Science and Innovation. André Aptroot is thanked for providing useful literature. We also thank two anonymous reviewers for their helpful comments on the manuscript.
Author ORCIDs
Sergio Pérez-Ortega, 0000-0002-5411-3698; Yolanda Turégano, 0000-0001-6948-2990; Måns Svensson, 0000-0003-1664-8226; Juan Carlos Zamora, 0000-0002-9243-2999.
Competing Interests
The authors declare none.
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