Introduction
The Roccellaceae are a predominantly tropical group of lichens. The basal branch in the phylogeny contains the core group of Enterographa [including its type, E. crassa (DC.) Fée], as well as Erythrodecton (Ertz & Tehler Reference Ertz and Tehler2011). The genus Enterographa Fée is a predominantly tropical group of crustose Roccellaceae comprising c. 50 species (Sparrius Reference Sparrius2004; Ertz et al. Reference Ertz, Christnach, Wedin and Diederich2005; Sparrius et al. Reference Sparrius, Saipunkaew, Wolseley and Aptroot2006; Sparrius & Aptroot Reference Sparrius and Aptroot2007; Jagadeesh Ram et al. Reference Jagadeesh Ram, Sinha and Singh2008; Lücking Reference Lücking2008; Sparrius & Björk Reference Sparrius and Björk2008; Ertz Reference Ertz2009a , Reference Ertz b ; Seaward & Aptroot Reference Seaward and Aptroot2009; Yeshitela et al. Reference Yeshitela, Fischer, Killmann and Sérusiaux2009; Sipman Reference Sipman2011). Phylogenetic studies have shown the genus to be polyphyletic (Ertz et al. Reference Ertz, Miądlikowska, Lutzoni, Dessein, Raspe, Vigneron, Hofstetter and Diederich2009), and two species that were accepted in the genus by Sparrius (Reference Sparrius2004) were shown to fall into a different clade than the type species of Enterographa, E. crassa (DC.) Fée. These were transferred to the genera Opegrapha Ach. (Ertz et al. Reference Ertz, Miądlikowska, Lutzoni, Dessein, Raspe, Vigneron, Hofstetter and Diederich2009) and Fulvophyton Ertz & Tehler (Ertz & Tehler Reference Ertz and Tehler2011). It is likely that the many species remaining in the genus still do not form a monophyletic group.
The Atlantic rainforests and the Caatinga forests in NE Brazil are rich in lichen species but still incompletely explored (Cáceres Reference Cáceres2007; Menezes et al. Reference de Menezes, Xavier-Leite, Otsuka, de Jesus and Cáceres2011; Lima et al. Reference Lima, Mendoça, Maia, Aptroot and Cáceres2013). The Arthoniales, including the Roccellaceae, is one of the three dominant crustose lichen groups on bark in NE Brazil, and crustose Roccellaceae are common and sometimes abundant (Cáceres et al. Reference Cáceres, Lücking and Rambold2007, Reference Cáceres, Lücking and Rambold2008).
During ecological studies focusing on epiphytic crustose lichens in NE Brazil, an Enterographa-like lichen was found with bright orange stromata, due to the presence of an anthraquinone. It was found in a remnant of Atlantic transition forest in Sergipe. It was clear that it was an undescribed species, but as this would be the first Enterographa with anthraquinones, it was sequenced to ascertain that it really belongs to the genus Enterographa. However, it clustered with strong support in the sister group of the well-supported core group of Enterographa, together with species of Dichosporidium and Erythrodecton. Although it shares some characters with Erythrodecton in particular (the presence of an anthraquinone), it differs in several respects from both genera, markedly by the firm, not byssoid thallus, the absence of ascoma carbonization, and the ascospore type. Therefore, this new species is described here in the new genus Sergipea.
Also, a new species of the genus Enterographa was found as part of the ecological studies. Enterographa rotundata has solitary round apothecia, which is unusual in this genus with mainly elongated apothecia or punctiform apothecia arranged in lines. It was found in the Vale do Catimbau National Park, a Caatinga forest reserve, in Pernambuco State.
Material and Methods
Identification and descriptive work was carried out in Itabaiana, Universidade Federal de Sergipe, using a Leica EZ4 stereomicroscope and a Leica DM500 compound microscope, and also in Soest using an Olympus SZX7 stereomicroscope and an Olympus BX50 compound microscope with interference contrast, connected to a Nikon Coolpix digital camera. Sections were mounted in tap water, on which all measurements were also taken. The specimens from this study are preserved in ISE. The chemistry of the type specimens was investigated by thin-layer chromatography (TLC) using solvent A (Orange et al. Reference Orange, James and White2001).
The mtSSU of the holotype of Sergipea aurata and of two specimens of Dichosporidium nigrocinctum (Ehrenb.) G. Thor were sequenced, using primers mrSSU1 and mrSSU3R (Zoller et al. Reference Zoller, Scheidegger and Sperisen1999) and following the procedures cited by Ertz et al. (Reference Ertz, Miądlikowska, Lutzoni, Dessein, Raspe, Vigneron, Hofstetter and Diederich2009). For the phylogenetic analyses, 17 sequences were retrieved from GenBank in addition to our own sequences (Table 1). The outgroup species, Opegrapha vulgata Ach., was chosen based on Ertz & Tehler (Reference Ertz and Tehler2011).
Table 1. Specimens and DNA sequences used in this study, with their respective voucher information. GenBank accession numbers in bold refer to sequences (3) generated by this project. All other sequences (17 GenBank accession numbers) were obtained directly from GenBank.
The mtSSU sequences were aligned manually to the existing alignment of Ertz et al. (Reference Ertz, Miądlikowska, Lutzoni, Dessein, Raspe, Vigneron, Hofstetter and Diederich2009) using MacClade 4.05 (Maddison & Maddison Reference Maddison and Maddison2002). Ambiguously aligned regions and introns representing a total of 727 bp (mainly due to an intron in the sequence of Roccella fuciformis) were delimited manually and excluded from the analyses.
Maximum Parsimony (MP) analysis was performed in PAUP*4.0b10 (Swofford Reference Swofford2002) on the mtSSU matrix of 735 unambiguously aligned characters, including 251 variable characters of which 195 are parsimony-informative. Heuristic searches were used with 1000 random addition sequence replicates, MaxTrees set to autoincrease, tree-bisection-reconnection (TBR) branch swapping, MulTrees option in effect and gaps treated as missing data. The MP analysis yielded four most parsimonious trees (448 steps) with topological differences present only in the clade Lecanactis-Chiodecton. Bootstrap values (MP-bs) were obtained from 1000 replicates with three random addition sequences (all other parameters identical to the original MP search).
The best-fit model of DNA evolution GTR+G was chosen using the Akaike Information Criterion (AIC), as implemented in Modeltest v. 3.06 (Posada & Crandall Reference Posada and Crandall1998). Bayesian analyses were carried out using the Metropolis-coupled Markov chain Monte Carlo method (MCMCMC) in MrBayes v. 3.1.2 (Huelsenbeck & Ronquist Reference Huelsenbeck and Ronquist2001; Ronquist & Huelsenbeck Reference Ronquist and Huelsenbeck2003) on the CIPRES portal (Miller et al. Reference Miller, Pfeiffer and Schwartz2010). Analyses were run under the GTR model of nucleotide substitution, including a discrete gamma distribution with six rate categories. Two parallel MCMCMC runs were performed, each using four independent chains and 10000000 generations, sampling trees every 1000th generation. TRACER v.1.5 (Rambaut & Drummond Reference Rambaut and Drummond2007) was used to ensure that stationarity was reached by plotting the log-likelihood values of the sample points against generation time. Posterior probabilities (PP) were determined by calculating a majority-rule consensus tree generated from the last 15 002 of the 20 002 trees sampled.
The MP tree did not contradict the Bayesian tree topology for the strongly supported branches, and hence only the majority-rule consensus tree of the Bayesian analysis is shown here with the branch supports of the MP analysis. PP≥95% and MP-bs≥70% were considered to be significant.
The Species
Sergipea aurata M. Cáceres, Ertz & Aptroot gen. et sp. nov.
MycoBank No.: MB 803755 (genus) & 803756 (species)
Roccellaceae with non-carbonized ascomata immersed in stromata covered with orange anthraquinone, ascospores 7–9-septate, 35–40(–50)×5–6 µm, narrowly clavate, rather thick-walled with elongated lumina, thallus with lichexanthone, stromata with anthraquinone.
Type: Brazil, Sergipe, Areia Branca, Fonte da Bica, on tree bark, c. 400 m alt., 19 May 2012, M. E. S. Cáceres & K. A. Jesus 12539 (ISE—holotype).
(Fig. 1)
Fig. 1. Sergipea aurata, holotype. A, habitus; B, section through ascoma; C & D, ascospores. Scales: A=0·5 mm; B=50 µm; C & D=5 µm. In colour online
Thallus crustose, cracked, not corticate, dull, pale greenish grey, very thin and closely following the bark surface, patchily interspersed with slightly byssoid hypothallus (visible only at high magnification), surrounded by a brown prothallus line. Algae trentepohlioid.
Ascomata not carbonized, c. 0·1 mm diam., globose, immersed and visible from above only by the ostiole, 5–25 clustered together in 1–2 mm wide, bright orange stromata. Excipulum hyaline, continuous below the hymenium. Epihymenium internally with pale brown pigmentation and externally with orange crystals. Hamathecium not inspersed, IKI+ blue (amyloid), paraphysoids 1·0–1·5 µm wide, anastomosing and often somewhat curled. Asci cylindrico-clavate, 73–89×21–27 µm. Ascospores 8 per ascus, hyaline, 7–9-septate, narrowly clavate, 35–40(–50)×5–6 µm, without gelatinous sheath, rather thick-walled with elongated lumina.
Pycnidia not observed.
Chemistry
Thallus partly UV+ yellow, partly UV−, C−, P−, K−, stromata K+ purple. TLC: thallus with lichexanthone, stromata with an unidentified anthraquinone that resembles parietin in Rf-value and KOH-reaction.
Ecology and distribution
On smooth bark of trees in Caatinga forest. Known only from Brazil. It grows together with the equally endemic Enterographa subquassiaecola M. Cáceres & Lücking (Cáceres Reference Cáceres2007), which is quite common in NE Brazil.
Discussion
As only one species is known in this genus, the description above refers to both the genus and the species (a so-called descriptio generico-specifica, ICBN Art. 38), as it would be impossible to discern generic and specific characters at this stage. The thallus and the internal structures, including the internal pigmentation, fit some species in the genus Enterographa well. It seems in habitus close to E. pertusarioides Sparrius (Sparrius Reference Sparrius2004), which however lacks anthraquinones, contains psoromic acid and has 3-septate ascospores of a much smaller size. Sergipea aurata is a truly remarkable and unexpected species.
Additional specimen seen. Brazil: same as the type, M. E. S. Cáceres & K. A. Jesus 12540 (ISE).
Enterographa rotundata E. L. Lima, M. Cáceres & Aptroot sp. nov.
MycoBank No.: MB 802592
Corticolous Enterographa species with round apothecia, ascospores (3–)7-septate, 50–60×3·5–5·0 µm, without substances.
Type: Brazil, Pernambuco, Buíque, Vale do Catimbau National Park, on bark of tree, c. 900 m alt., 3 February 2012, E. L. Lima 668 (ISE—holotype).
(Fig. 2)
Fig. 2. Enterographa rotundata, holotype. A, habitus; B, section through ascoma; C, hamathecium and young ascus; D–F, ascospores. Scales: A=0·5 mm, B & C=50 µm; D–F=5 µm. In colour online
Thallus crustose, continuous but often seemingly absent, not corticate, dull, pale pinkish to pale brownish, very thin and closely following the bark surface, without prothallus. Algae trentepohlioid.
Ascomata round or slightly ellipsoidal, dark brown to black, 0·1–0·2 mm wide, margin raised above the disc, ochraceous white, c. 0·05 mm wide. Excipulum hyaline to pale brownish. Epihymenium internally with pale brown pigmentation. Hamathecium not inspersed, IKI+ pale blue (weakly amyloid), paraphysoids 1·0–1·5 µm wide, anastomosing and often somewhat curled. Asci cylindrico-clavate, 65–79×15–23 µm, tholus amyloid. Ascospores 8 per ascus, hyaline, (3–)7-septate, narrowly clavate, 50–60×3·5–5·0 µm, surrounded by a 1·5–2·5 µm wide gelatinous sheath, lumina somewhat rounded.
Pycnidia not observed.
Chemistry
Thallus and apothecia UV−, C−, P−, K−. TLC: no substances detected.
Ecology and distribution
On smooth bark of trees in Caatinga forest. Known only from Brazil. It grows together with, for example, Dirinaria leopoldii (Stein) D. D. Awasthi, Lecanora leprosa Fée, L. leproplaca Zahlbr., Pertusaria ventosa Malme, Phaeographis ventosa Redinger, and Ramboldia haematites (Fée) Kalb et al.
Discussion
One of very few Enterographa species with round apothecia and the third corticolous one with that character. Enterographa lecanoracea Sipman (Sipman Reference Sipman2011) differs by having larger ascomata (0·5–1·5 mm) that are sessile to nearly stalked, and a C+ red thallus. Enterographa mesomela Sparrius et al. is superficially similar to the new species but differs by a grey-green, slightly cracked to areolate thallus, slightly shorter ascospores (32–40 µm) and a different chemistry (confluentic acid in the thallus, and norstictic acid in the hypothecium; Sparrius et al. Reference Sparrius, Saipunkaew, Wolseley and Aptroot2006). The morphs of E. anguinella (Nyl.) Redinger [now Opegrapha anguinella (Nyl.) Ertz & Diederich] that were described as E. lecanoroides R. C. Harris also have nearly round apothecia (Sparrius Reference Sparrius2004), but they differ by nearly flush apothecia without a prominent margin and by a different chemistry (presence of psoromic acid). Three foliicolous species of Enterographa, viz. E. batistae Lücking & Sérus. (Lücking et al. Reference Lücking, Sérusiaux, Maia and Pereira1998), E. byssoidea Lücking (Lücking Reference Lücking1991) and E. perez-higaredae Herrera-Campos & Lücking (Herrera-Campos & Lücking Reference Herrera-Campos and Lücking2002), also have usually rounded apothecia (Lücking Reference Lücking2008). All differ by various characters from the new species, besides their foliicolous habitat, in each case also by very tiny ascomata which are smallest in E. batistae (c. 0·1 mm diam.); E. byssoidea differs also by the byssoid apothecium margin and E. perez-higaredae by the presence of psoromic acid. A superficially similar species is Lecanographa atropunctata Sparrius et al.; it differs from the new species by a thick dark brown to black excipulum and hypothecium, more septate ascospores (7–13-septate) and a different chemistry (presence of schizopeltic acid; Sparrius et al. Reference Sparrius, Saipunkaew, Wolseley and Aptroot2006).
Additional specimens examined. Brazil: same as the type, E. L. Lima 662, 665, 670 & 673 (ISE).
Discussion of the phylogenetic position of Sergipea and Dichosporidium
In our phylogenetic tree (Fig. 3), Sergipea aurata clusters close to the genera Dichosporidium and Erythrodecton in the basal branch of the Roccellaceae s.str. as defined by Ertz & Tehler (Reference Ertz and Tehler2011). It is included in a strongly supported clade sister to the genus Enterographa and thus cannot be described in this latter genus. The generic type of Dichosporidium, D. nigrocinctum, was here newly sequenced and our phylogenetic tree confirms the position of the genus close to Erythrodecton granulatum (Mont.) G. Thor, as first shown by Nelsen et al. (Reference Nelsen, Lücking, Grube, Mbatchou, Muggia, Rivas Plata and Lumbsch2009) for D. boschianum (Mont.) G. Thor. The relationships within the clade Erythrodecton-Sergipea are not supported. Therefore the generic position of the new taxon is uncertain within this clade.
Fig. 3. One locus (mtSSU) 50% majority-rule consensus tree produced by the Bayesian analysis and representing the proposed phylogenetic relationships among 20 specimens of Arthoniales. PP≥95% and MP-bs values≥70% are considered strongly supported and shown respectively above and below internal branches. The new genus and species Sergipea aurata is noted in bold.
Erythrodecton mainly differs from Sergipea by biclavate ascospores, a red pigment in the thallus medulla and internal carbonization of the ascomata. All species of Dichosporidium have internal carbonization of the ascomata, a different chemistry and a distinctly byssoid thallus, contrasting with Sergipea. Moreover, D. nigrocinctum differs, for example, by biclavate ascospores and D. boschianum by hooked ascospores. As a consequence, we prefer to describe a new genus to accommodate our new species.
It should be noted here that both species of Dichosporidium do not cluster together in our phylogenetic tree, suggesting that the genus might be paraphyletic. More data will have to be included in future phylogenetic studies to resolve the generic delimitation of these taxa.
The CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) is thanked for a research grant to MESC (Processo 501633/2009-0) and for financial support for the collecting trips (CNPq-Protax Process 562.330/2010-0 and INCT-Herbário Virtual Processo 573.883/2008-4). The Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco – FACEPE is also thanked for a Master's scholarship to ELL. AA is grateful to the Hugo de Vries-fonds for travel support. Leo Spier is thanked for performing thin-layer chromatography.
