Parmotrema hydrium , a new species of Parmeliaceae in southeastern Brazil

Parmotrema hydrium is a new lichen species discovered during a survey of Parmeliaceae in the Cantareira mountain range of southeastern Brazil. In its medullary chemistry, the species contains several fatty acids and other substances, including small amounts of atranorin, typically restricted to the soralia and young lobes.

Here, we describe a new species containing undetermined several fatty acids (without most of the common acids found as medullary substances, except for what is probably atranorin in soralia and young lobes).This species was discovered during a survey of the Parmeliaceae species occurring in the Cantareira mountain range, located north of the city of São Paulo, within the state of São Paulo, in southeastern Brazil.The new species has rather rounded lobes, is marginally sorediate and has simple cilia.Only corticolous specimens were found.

Material and methods
Morphological characters were studied using standard stereoscopic and compound light microscopes.Anatomical sections were cut by hand with a razor blade.All five specimens examined lacked apothecia and pycnidia.The chemical constituents were initially checked by spot tests with potassium hydroxide (K), sodium hypochlorite (C) and para-phenylenediamine (P), as well as being examined under UV light (360 nm).Subsequently, chemical constituents were identified by thin-layer chromatography (TLC) using solvent C (Bungartz 2001), following the standard methods described in Elix & Ernst-Russell (1993) and Orange et al. (2001).Samples were also examined by toluene, ethyl acetate, acetic acid (6:4:1 v/v).The samples where submitted to TLC together with all substances commonly found in species with similar morphology (e.g., alectoronic acid, gyrophoric acid and psoromic acid).
For high-performance liquid chromatography (HPLC), pieces of the thallus (ca. 5 × 5 mm) were extracted with 200 μl of methanol at room temperature for 1 h.Extracts were filtered through 0.2 μm polytetrafluoroethylene filter plates (Acroprep Advance; Pall Corporation, East Hills, NY, USA) and diluted 10-fold with methanol.Samples were analyzed in a liquid chromatograph (1260; Agilent Technologies, Palo Alto, CA, USA) equipped with a quaternary pump, an incorporated degasser and diode array detection.Substances were separated on a Poroshell 120 EC-C18 column (2.7 μm, 3.0 × 50.0 mm; Agilent Technologies) regulated to 30°C, at a flow rate of 1.4 ml/min.Two solvent systems were employed.Solvent A was aqua bidest (LiChrosolv ® ; Merck, Darmstadt, Germany) containing 30% methanol and 0.0625% trifluoroacetic acid; and solvent B was 100% methanol.The methanol and trifluoroacetic acid were obtained from VWR International (HiPerSolv CHROMA-NORM ® ; BDH Prolabo, Lutterworth, UK).The HPLC system was equilibrated to solvent A for 2 min.After washing the needle automatically in 100% methanol, we injected 10 μl of extract.The run started with 100% solvent A and continued isocratically for 0.18 min at a flow rate of 1.4 ml/ min.After 0.18 min, solvent B was increased to 58% within 5 min, then up to 100% in 5 min, and then isocratically in 100% solvent B for an additional 0.82 min.At the end of the run, solvent A was increased to 100% within 0.5 min and the column was flushed with 100% solvent A for 2 min before a new run was started.
The compounds were detected at 210, 254, 280 and 310 nm and the spectra of each peak (λ = 190-650 nm, in 2-nm steps) were recorded automatically.Spectra and retention time were computer-matched against a library of spectra from authentic metabolites derived under identical conditions using OpenLAB CDS ChemStation software (Agilent Technologies).
Thallus: up to 7.0 cm wide, subcoriaceous, corticolous, greenish gray to dusky gray in the herbarium, lobate Lobes: irregularly branched, 1.5-6.Apothecia and pycnidia: not seen Spot reactions: upper cortex K+ yellow, UV−; medulla K− to variably K+ yellow, C−, KC−, P− to variably P+ sulfur yellow/yellow-orange at soralia or occasionally and randomly on younger lobes, UV−.The K+ and P + reactions might be due to concentrations of atranorin in different parts, particularly the young lobes and soralia.TLC: cortical atranorin; medullary unidentified substances, possible traces of terpenoids and sterols.HPLC: Each sample analyzed contained atranorin, chloroatranorin and lichesterinic acid.For some of the samples, additional substances were detected: SP264958 and SP264968 (protolichesterinic acid); SP416110 (unknown substance, possibly a depside); thuringione; protolichesterinic acid; unknown substance, SP265140 (thuringione and protolichesterinic acid) Additional specimens examined: Brazil, city and state of São Paulo, Cantareira mountain range, Sítio da Cachoeira, on tree exposed to direct sun, leg.M.P. Marcelli Comments: This species is morphologically somewhat similar to Parmotrema hababianum (Gyel.)Hale.The development of the soralia is somewhat similar, initially labriform and evolving to sublinear, unlike those of P. perlatum, which typically have a "pearl necklace" aspect.The medullary chemistry of the new species is also distinct.

Results and discussion
Parmotrema hydrium was shown to have a distinctive medullary chemistry, containing several fatty acids as well as traces of medullary terpenoids and sterols, without the more common acids often found in Parmotrema species with similar morphological characteristics, e.g.alectoronic acid (P.rampoddense), psoromic acid (P.direagens), stictic acid (P.perlatum), gyrophoric acid (P.sancti-angelii) (Benatti & Marcelli 2009a, 2010;Brodo et al. 2001;Divakar & Upreti 2005;Elix 1994;Hale 1965;Nash & Elix 2002;Spielmann & Marcelli 2009;Swinscow & Krog 1988).Although it was not the main substance found in all specimens, some samples contained protolichesterinic acid, like the morphologically similar P. hababianum (norlobaridone and protolichesterinic acid) or P. grayanum (protolichesterinic acid only), which might indicate that the new species is associated with this group.The substances were tested together with the samples used in different solvents of TLC, and not even traces of other commonly found substances were detected, as confirmed by HPLC.
Positive K and P spot tests were irregular in Parmotrema hydrium, usually being positive only at the soralia and in random areas of younger lobes, and even variable on a single thallus.Even the yellowish reactions of these reagents differed from those normally observed in speci-mens of other Parmotrema species with atranorin.In P. hydrium, the yellow color of the K and P reactions were close to sulfur yellow, darkening to yellow-orange after some time.The reactions were often negative or weak in the older parts of the thallus, or even at some of the soralia.The possibility of atranorin concentrations at soralia and young lobes was discussed with colleagues, who also judged that this could be the only explanation for the positive K and P reactions, because the TLC and HPLC had revealed no other substances that could account for the reaction.Atranorin is an UV-negative substance that runs highest on the TLC plate and, because of this distance, can become quite diluted.
Because of the absence of clearly recognizable secondary metabolites in the TLC, the specimens were initially impossible to identify.They were in good condition, so the possibility that they were lacking substances due to deterioration could be ruled out.It was not possible to determine whether lichesterinic and protolichesterinic acid represented trace, minor or major substances, because of the limited quantity of material, which had already been sampled extensively in our attempts to elucidate the secondary metabolite composition.The following species are most similar: Parmotrema praesorediosum: The overall labriform aspect of the soralia is initially similar to those observed in P. praesorediosum, but soon becomes more linear.
Parmotrema hababianum: In P. hababianum, the marginal zone is mostly white at the lower cortex, whereas a white margin is seen in P. hydrium only at the lobes bearing soralia, as is common in most sorediate Parmotrema species.The medullary chemistry is distinct, P. hababianum containing medullary protolichesterinic acid, as well as norlobaridone (KC+, rose), and having no parts that react to K or P tests (Hale 1965).
Parmotrema grayanum: Although Parmotrema hydrium is somewhat similar to P. grayanum, the latter is a saxicolous species that also contains only protolichesterinic acid in the medullary chemistry and does not present K or P test positivity in any of its parts (Hale 1965;Divakar & Upreti 2005;Elix 1994;Swinscow & Krog 1988).The soralia of P. hydrium are closer to those of P. praesorediosum (more labriform than linear), whereas P. grayanum is ciliate, with coarse thick cilia.Overall, the new species is morphologically closer to Parmotrema hababianum, although somewhat chemically closer to P. grayanum.However, there are some issues regarding the specimens found: they all are corticolous, whereas all citations of P. grayanum mention only saxicolous specimens; K+ and P+ yellow-orange reactions were found for soralia and young lobes, such reactions being unknown for P. grayanum and atranorin being the only substance detected by both TLC and HPLC that could explain those reactions; and (according to the literature) P. grayanum contains only protolichesterinic acid as a medullary substance.
As described by Hale (1965), Parmotrema grayanum is a whitish gray, commonly pruinose, coriaceous lichen, with coarse, thick marginal cilia.The specimens of the new species found do not share these features.The type specimen of P. grayanum is purportedly in an herbarium in Paris, France (PC).Unfortunately, requests sent to the herbarium went unanswered.There are no known isotypes or paratypes.The type locality of P. grayanum is Neilgherries, India.
Among other similar species, Parmotrema ciliiferum differs from P. hydrium by its more irregular, linear marginal soralia, the medullary chemistry (P.ciliiferum containing constipatic acid and not atranorin, lichesterinic acid or protolichesterinic acid) and a total absence of positive spot test reactions in any of its parts.
Parmotrema mordenii is apparently the only other species that has medullary atranorin (the reactions are even more uniform throughout the medulla) but is an eciliate, saxicolous species (Marcelli & Benatti 2010b).
All Parmotrema hydrium specimens were corticolous.At present, this species is known only from the type region, a mountainous area of Atlantic Forest in somewhat open areas mostly at 1500-2200 m in elevation.The species is named after the Cantareira mountain range (Serra da Cantareira in Portuguese, literally translated as "Pitcher mountain range"), where the specimens analyzed were originally collected.