The genus Cladonia (Lichenized Ascomycota) from Restinga Vegetation of Espírito Santo state, Brazil: Supergroups Crustaceae and Perviae

In this paper we recognize a total of 11 species of Cladonia, five belonging to Supergroup Crustaceae and six to Supergroup Perviae, occurring in the Restinga vegetation of Espírito Santo state, Brazil. We confirm the occurrence of C. sprucei and C. rangiferina to Espírito Santo state Restinga, being their most austral record in Brazil so far. Four species are new records to the state: C. consimilis, C. dendroides, C. salzmannii, and C. sphacelata. Here we expand the number of known species of Cladonia of Supergroup Perviae from three to five, and of Supergroup Crustaceae from three to six in the studied environment. An identification key, comments and illustrations are provided.


Introduction
This work gives sequence to an investigation of the genus Cladonia P. Browne occurring in Restinga vegetation in Espírito Santo state, southeast Brazil (Fraga Jr. et al. 2017). Here, we discuss about the remaining Supergroups Crustaceae and Perviae, proposed by Stenroos et al. (2002).

Material and methods
The studied area, including map, its geographical and climatic data is described in Fraga Jr. et al. (2017). For the collection and preservation of the specimens the usual methodology in lichenology was followed. The specimens were deposited in the Herbarium of the Universidade Federal do Paraná (UPCB) and Universidade Federal do Espírito Santo (VIES). The morphological and anatomical analyses were performed under stereomicroscope (20-40×) and optical microscope (40-1000×). Spot tests (K, C, and KC), Thin Layer Cromatography (TLC) with solvent system C as well observation under UV Light (Orange et al. 2001) were performed to identify the lichen chemistry.

Results and Discussion
Five species belonging to the Supergroup Perviae were found: C. crispatula, C. polystomata Ahti & Sipman, C. turgidior, C. salzmannii Nyl., C. sphacelata Vain. The latter two being new records to Espírito Santo state, and C. polystomata is registered for the first time for the local Restinga vegetation, expanding its range of occurrence from the highlands (Ahti 2000) to the coastal region.
Six species of the Supergroup Crustaceae were found: C. confusa, C. consimilis Vain., C. dendroides (Abbayes) Ahti, C. rangiferina (L.) Weber ex F.H. Wigg., C. sprucei Ahti and C. substellata. We confirmed the occurrence of C. sprucei Ahti and C. rangiferina subsp. abbayesii (Ahti) Ahti & DePriest in Restinga vegetation from Espírito Santo state, which were mentioned by Marcelli (1998) but without a stored specimen, being their most austral record so far in Brazil. Cladonia dendroides and C. consimilis are also new records for the state.
Even though C. bahiana Ahti (Marcelli 1998), C. kalbii (Ahti) Ahti & DePriest and C. rugicaulis (Ahti 2000) were previously reported to this environment of Espírito Santo state, we were not able to find them in the fieldwork. Here we increase the number of known species of Cladonia of Supergroup Perviae from three to five, and of Supergroup Crustaceae from three to six in the Restinga vegetation of Espírito Santo. Cladonia confusa is a fair widely distributed species in the Neotropics, occurring from sea level up to the mountains (3000 m) (Ahti 2000). It's characterized by the totally ecorticate, yellowish, and much branched podetia, usually forming semiglobose heads in the top of the colonies, as well as by the production of usnic and perlatolic acids (Ahti 2000;Gumboski & Eliasaro 2012). In the specimens analysed we found a diverse secondary chemistry: homosekikaic and 4-O-demethylbarbatic acids, in addition, some unidentified compounds with Rfc approximately to 35,62 and 72 (Fraga Jr. 410 d). In one specimen (Fraga Jr. 406) we detected only production of perlatolic acid.
Cladonia evansii Abbayes and C. signata (Eschw.) Vain. are similar to C. confusa due the ecorticate and much branched podetia, without distinct main axis. However, in both species the ramification pattern is mostly dichotomic, and they can respectively produce atranorin and fumarprotocetraric acids, compounds absent in C. confusa (Vainio 1887;Ahti 2000).
This species occurs mainly in shaded places, bellow some shrubs, growing on sand or litter, often forming extensive colonies around four meters wide in Parque Natural Municipal de Jacarenema, growing mostly seldom or together with C. crispatula, C. dendroides or C. verticillaris (Raddi) Fr. Cladomia consimilis is characterized by the equally cylindrical and esorediate podetia, whitish, smooth, fairly dichotomously branched, ascyphose, with closed to open axils, red conidial slime, and by the production of thamnolic or barbatic acids (Vainio 1887; Ahti 2000).

Cladonia consimilis
Cladonia polytypa Vain. is morphologically very similar but exhibits a pronounced melanotic base of podetia and the conidial slime is hyaline (Ahti 2000).
Among the Cladonia species analysed in this study, C. consimilis can be easily confused with some slender morphs of C. crispatula and C. salzmannii. However, C. consimilis have thinner podetia, hardly reaching up to 1 mm thick, besides C. crispatula and C. salzmannii usually exhibits podetium over 1 mm thick, with a more scarcely branching pattern, predominantly dichotomic anisotomic, whereas in the previously mentioned species the branching pattern can be up to polytomic, with some isotomic tendency. Cladonia consimilis can exhibit some polytomic branching in the apices, which in some cases can be confused to C. salzmannii but this latter bears larger and conspicuous funnels, absent or very tiny in C. consimilis.
According to Ahti (2000), some specimens of C. crispatula can be confused with species of the Group Cladinae sensu Stenroos et al. (2002) by their secondary thallus size and branching pattern. However, C. crispatula exhibits traces of rudimentary cortex, occasionally some squamules, and produces thamnolic and decarboxythamnolic acid, which are absent in species of the Group Cladinae sensu Stenroos et al. (2002) (Ahti 2000).
Cladonia sipmanii Ahti is another similar species to C. crispatula, but exhibits slender podetia (0.5-1 mm thick), more brownish in color, with axils always closed, and its distribution is currently restricted to Venezuela and Guyana (Ahti 2000).
Some specimens displayed a notable morphological variation that even some that even approaches C. rugicaulis description aforementioned. However, none of the specimens displayed all those strict characteristics combined. We consider that the C. rugicaulis and C. crispatula of southeast and northeast Brazil are in need of a more detailed study to reveal a potential phenotypic plasticity of these species.
In this study, the majority of specimens collected correspond to C. crispatula s. str. in accordance to the descriptions of Ahti et al. (1993), Ahti (2000) and Gumboski & Eliasaro (2012).
Some variant specimens found (Fraga Jr. 553,702) are very similar to C. crispatula, exhibiting a more thickened podetia (2 mm), and short sided branches. Two specimens presented more isotomic branching pattern, also short side branches, with parallel running podetia, clearly smooth surface, wide openings mainly in apex, a constantly and distinct grayish layer up to half of the podetia, and forms flat topped colonies, rather than semiglobose. Some specimens yet exhibited a hyaline slime. We hypothesize that these morphologically variable specimens could represent distinct taxa closely related to C. crispatula, and thus, specific studies should be conducted with a larger number of specimens and employing molecular data to evaluate and clarify the taxonomic status of these distinct populations. The ecorticate podetia, densely isotomic dichotomic branched forming rounded heads on top of colonies, the blackened dead basal parts and the production of fumarprotocetraric acid and atranorin characterize this species (Ahti 2000).
According to Ahti (2000), is possible to confuse with C. kalbii, due the ecorticate and much branched thallus, but C. dendroides have a more isotomic branching pattern, forming denser heads. Besides, C. dendroides have a red conidial slime instead of purple like in C. kalbii (Ahti 2000).
Due the globose heads, this species can also be confused with C. confusa, but this latter has larger podetia, forming larger domes and produces usnic acid, giving a yellow tinge, whereas C. dendroides is ashy white, with small heads and have a hyaline slime instead red.
It's a conspicuous and very rare species, and only one specimen was found in the northern part of the state, at the borders of an open shrub formation, growing alone but near to C. clathrata and C. crispatula, on sand. It's a new record for Espírito Santo state. Ahti & Sipman, Fl. Neotrop., Monogr. 78: 294. 2000. Fig. 1e Descriptions: see Ahti (2000), Gumboski & Eliasaro (2012).
This species can be confused with C. sphacelata by the absence of soredia or granules, by the squamulose surface, and the production of thamnolic acid (Ahti 2000;Gumboski & Eliasaro 2012). However, the latter exhibits a developed, smooth and cartilaginous cortex, red conidial slime, closed axils and do not bear scyphoid funnels (Ahti 2000;Gumboski & Eliasaro 2012). Besides, C. polystomata is usually less squamulose in direct comparison than C. sphacelata.
It also can be confused with C. squamosa Hoffm., by the squamulose and ecorticate podetia, however, C. squamosa tends to be even more ecorticate and exhibits a granulose cover, which is absent in C. polystomata (Vainio 1887; Ahti 2000).

Cladonia rangiferina subsp. abbayesii
In Brazil, the geographical distribution of this subspecies is quite enigmatic, with only Cladonia rangiferina subsp. abbayesii being registered for the country (Ahti et al. 1993;Ahti 2000). Ahti et al. (1993) mentioned its occurrence to northern states of Amazonas and Roraima, and the northeast states of Bahia and Sergipe. Marcelli (1998) mentioned its occurrence to the northern coast of Espírito Santo and other states above. Although, this same paper does not report any specimen, being field observations as written by Marcelli. Ahti (2000) records this subspecies only to northern Brazil (Roraima state), and refers similar lowland specimens to C. kalbii, and also affirms that C. rangiferina is securely absent from southeast Brazil, not mentioned directly Cladonia rangiferina subsp. abbayesii.
Besides this statement, the specimen found easily fits onto Ahti (1961Ahti ( , 2000 description of Cladonia rangiferina subsp. abbayesii, which is characterized by the mostly dichotomic tendency, with rather erect apices and more robust main axis, plus, the specimen found have clearly deflexed apices. In contrast, other species of C. rangiferina are hardly differentiated; being the branching pattern, color of podetia and geographical distribution the characteristics for its taxonomy. It is morphologically close to C. kalbii, in the branching pattern, with distinct main axis, and ecorticate arachnoid surface with smoothed apices (Ahti 2000). However, C. kalbii have a purple conidial slime, instead of hyaline, and the branching pattern in the apices is usually divaricate rather combed (Ahti 2000).
Moreover, even though C. kalbii was recorded to the local Restinga (Ahti 2000), we did not find any specimen with these strict characteristics. Since both are close related species, the presence of the unilaterally deflexed apices and the colour of slime were diagnostic characters about this consideration besides Ahti (2000) statement of its distribution.
Cladonia argentea (Ahti) Ahti & DePriest is also morphologically and chemically close related species related species, that resembles a robust morphotype of C. rangiferina, but do not exhibits any browned color and the conidial slime is also purple (Ahti 2000).
The specimen was found growing seldom in a well-lit area, on sand. It is locally a very rare species. Thus, we confirm the occurrence of this species for the Espírito Santo state. Cladonia salzmannii is characterized by its thin (0.5-2.0 mm) and fairly branched podetia, with distinct main axis, scyphoid openings at the apex in star like form, red conidial slime, and by the production of barbatic and/or thamnolic acids (Ahti 2000). We found specimens producing barbatic acid, thamnolic acid and one specimen producing both acids (Fraga Jr. 627).
In field it is easily confused with C. crispatula by the branched thallus and by the habitat. However, the star like apices with long proliferations and more dilated funnels are good features to separate them.
The analysed specimens exhibited a somewhat areolate and rugulose apex and a corticioid surface unlike the description of Ahti (2000), which describes with a smoothly corticated surface. We also observed a much more developed medulla (40-125 µm thick) than previously reported by the same author (10-15 µm thick).
This species grows mostly in decayed woods, sand, litter or organic soil, in fairly shaded areas. It forms distinct colonies in northern state, mainly in Área de Proteção Ambiental de Conceição da Barra. This is the first record of this species for Espírito Santo state. Cladonia sphacelata has a smoothly cartilaginous corticate podetia, rarely a little decorticate, also being moderately branched, squamulose, ascyphose without soredia or granules, and usually with closed axils, commonly producing thamnolic acid (chemotype I), other compounds such as squamatic (chemotype II), barbatic and didymic acids and related compounds (chemotype III) (Ahti 2000).

Cladonia sphacelata
We found an uncommon chemistry, with high number of secondary metabolites, and even a combination of chemotype I and III, which is not yet reported. In addition, some unidentified compounds were observed: with Rfc approximately 0 (spot UV + blue), and gray spots with Rfc approximately 35, 39, 43 and a UV+ yellow with Rfc approximately 44.
This species is often confused with C. polystomata (Ahti 2000). Both have an esorediate and egranulose podetia covered with squamules, but C. sphacelata has a more continuous cortex and the axis is usually closed (Ahti 2000;Gumboski & Eliasaro 2012). They also differ in the color of slime, which is red in C. sphacelata and is hyaline in C. polystomata (Ahti 2000;Gumboski & Eliasaro 2012). In this study, besides the little morphological differences, C. sphacelata presented a higher number of secondary metabolites than C. polystomata (see discussion under C. polystomata).
Rodriguésia 71: e01612018. 2020 Besides this species being reported for the northeast to southern Brazil, mostly in the coastal region in Restinga vegetation (Ahti 2000;Gumboski & Eliasaro 2012), it was not yet recorded to Espírito Santo state. It was found colonizing bare sand or organic soil, in well-lit areas, forming conspicuous and browned colonies with about one meter wide. (Ahti) Ann. bot. fenn. 32: 01. 1961. Fig. 2a Description: see Ahti (2000. Chemistry: K+ yellow; C-; KC-; UV-; TLC: atranorin and fumarprotocetraric acid. Cladonia sprucei is characterized by forming dense and flat-topped colonies, with as anisotomic branching pattern, blackened apices on dead parts, red conidial slime and by the production of fumarprotocetraric acids and related compounds and atranorin (Ahti 2000).

Cladonia sprucei
According to Ahti (2000), this species is morphologically very similar to C. rangiferina subsp. abbayesii and C. kalbii, differing in the color of slime, which is hyaline and purple respectively, besides the more isotomic branching tendency and more compact and acuminate apices.
The presence of a red conidial slime and fairly compact apices were the diagnostic characteristics to separate C. sprucei from other species of the Group Cladinae sensu Stenroos et al. (2002) as C. rangiferina, and C. dendroides, in Restinga from Espírito Santo.
It is an uncommon species, growing on sand and litter in shaded to exposed areas. Marcelli (1998) mentioned its occurrence in Restinga vegetation from Espírito Santo, also without referring an herborized specimen. However, this species is only recorded to northern Brazil, in Amazonas state by Ahti (2000). So, we could confirm its occurrence in Restinga vegetation, being its first record for this environment and the most austral in the Neotropics so far.  Cladonia substellata is characterized by the slender and branched podetia, being pale yellow to grayish green, ascyphose, with closed to open axils, a corticioid surface, without propagules, with a fibrose stereome, and by the production of usnic and stictic acid often with related compounds (Vainio 1887;Ahti 2000;Charnei 2013). This species may occasionally lack stictic acid complex, producing fumarprotrocetraric instead (Ahti 2000;Stenroos et al. 2002). Besides the stictic acid, we also found one compound with Rfc approximately 14, probably related with stictic acid complex.
According to Ahti (2000), C. peltastica (Nyl.) Müll. Arg. is quite similar to C. substellata in gross podetia morphology. However, C. peltastica exhibits a corticated surface, does not produce stictic acid and is confined so far to the Amazon region (Ahti 2000).
In this investigation, we found some specimens in Parque Estadual Paulo Cesar Vinha that fits close to Ahti (2000) description of C. substellata. These specimens have a strongly adpressed secondary thallus, and the podetia are never squamulose, with smooth to clearly rugulose surface. However, they are anatomic and chemically similar, producing usnic and stictic acid, and related compounds. It's possible that some environmental conditions, such as sunlight exposure on white sand, salty spray winds and large dry periods led to that growing pattern. Due its morphological distinction, like in C. crispatula (see discussion under C. crispatula), specific studies should be conducted using a more comprehensive number of specimens and molecular data to evaluate the taxonomic status of these distinct populations.
It's a rare species, not really forming extensive colonies. It grows on bare sand, in open and well-lit areas around some bushes.  Ahti (1977Ahti ( , 2000. Chemistry: K+ yellow; C-; KC-; UV-; TLC: thamnolic and decarboxythamnolic acids. Cladonia turgidior is characterized by the turgid podetia, with much dilated openings, smooth and glossy surface in the upper half and rugulose to verruculose lower down, as by production of thamnolic acid (Ahti 1977(Ahti , 2000.
It is a distinct species in the field, forming fairly large clumps mainly in exposed areas. The thick podetia with large openings are characters that easily distinguish it from the other species. It grows mostly seldom, but few times was observed intermixed with thalli of C. crispatula.
Some underdeveloped specimens can resemble C. crispatula, but the floccose inner surface of podetia, and the glossy surface are diagnostic to separate them (Ahti 2000).