Lecanora s.lat. (Ascomycota, Lecanoraceae) in Brazil: DNA Barcoding Coupled with Phenotype Characters Reveals Numerous Novel Species

We sequenced over 200 recent specimens of Lecanora s.lat. from Brazil, delimiting 28 species in our material. Many seem to represent undescribed species, some of which being morphologically and chemically similar to each other or to already described species. Here, we present a phylogenetic analysis based on ITS, including our specimens and GenBank data. We describe nine new species. The purpose of the paper is to illustrate the diversity of the genus in Brazil, not to focus on segregate genera. However, we found that all Vainionora species cluster together and these will be treated separately. Other Lecanora species with dark hypothecium clustered in several different clades. Species with the morphology of Lecanora caesiorubella, in which currently several subspecies with different chemistry and distribution are recognized, fall apart in different, distantly related clades, so they cannot be regarded as subspecies but should be recognized at species level. A key is given for the Lecanora species from Brazil.

The genus Lecanora initially comprised almost all crustose lichens with apothecia with a thalline (lecanorine) margin, regardless of the shape and colour of the ascospores. In the 20th century, the concept of the genus stabilized somewhat.
Most taxonomic work on Lecanora has been carried out in temperate or arctic environments. Only a few publications exist about Lecanora in the tropics [12][13][14][15][16][17], and only one of these [12] is exhaustive for a whole country.
Studies based on molecular data of problematic species and morpho-groups within Lecanora s.lat. have revealed several species complexes including within taxa that share

Phylogenetic Analyses
BLAST searches (NCBI) (https://www.ncbi.nlm.nih.gov, accessed on 10 October 2022) were performed for the newly generated sequences and after confirmation of their   Diagnosis: Similar to Lecanora caesiorubella Ach. but differing by the distinctly yellowish, very irregular margins, and much smaller apothecia.
Habitat and distribution: On exposed trees in Caatinga forest; known only from Brazil.
Additional sequenced specimens examined: BRAZIL, Sergipe, Monumento Natural Grota do Angico, Poço Redondo; 9 • 39 39.7 S, 37 • 41 09.7 W, on bark of unidentified tree, 24 November 2016, L.A. Santos s.n. (ISE-52389) Notes: This new species shares the chemistry of atranorin and protocetraric acid with Lecanora caesiorubella subsp. glaucomodes (Nyl.) Imshaug and Brodo but differs in the pale yellow and very irregular apothecial margins and the areolate thallus. We therefore consider the morphological differences in the apothecial margins and the thallus diagnostic.
Notes: Lecanora fluorosaxicola is most likely to be confused with L. vainioi Vänskä and L. kalbiana Lumbsch in the more or less brown to grey-brown apothecial disc and the substrate, as both are found on rock. The two species were described from Brazil: L. kalbiana was from Mato Grosso, currently only known from Brazil, and L. vainioi from Rio de Janeiro, restricted to the Neotropics. The new species differs in the thallus being mostly yellow, the apothecial discs with a yellowish green pruina, the hyaline hypothecium, and in the absence of zeorin. Lecanora ryanii T.H. Nash and Lumbsch is also superficially similar, but differs in the larger, sessile apothecia with epruinose disc; it is only known from Isla Margarita in México [42].
The differences in chemistry and morphology are reflected in the molecular phylogeny (Figure 1), where L. fluorosaxicola represents a single taxon on a long branch in a subclade with absolute support, whereas L. vainioi, L. kalbiana and two sequences from Gen-Bank (Thailand) labeled L. vainioi, form another sub-clade. Both subclades fell in a clade with pruinose species (Figure 1).  15 (1966) Typus: Cuba, C. Wright Lich. Cub. II. 60 (H-NYL 27145, holotype) Description specimens examined: Thallus crustose, corticolous, up to 3.5 cm wide, c. 0.1 mm thick, areolate, rimose to verruculose, whitish grey to grey, dull, epruinose. Soredia absent. Prothallus absent or whitish grey. Apothecia adnate, 0.5-1.5 mm diam.; disc pale orange to brownish orange, carneous, whitish grey pruina, flat to convex; margin slightly paler than the thallus, prominent, becoming excluded or persisting, entire to flexuose, smooth. Amphithecium with small crystals which are dissolving in KOH (= allo-  ; disc dark grey to greyish black, with a thick, greenish yellow pruina, flat when young to strongly concave; margin concolourous with the thallus, becoming excluded, smooth. Amphithecium with small crystals which are dissolving in KOH (=melacarpella-type), parathecium with small crystals. Hymenium hyaline, 42.5-62.5 µm high; epihymenium green to yellowish-green, the pigment and with phana-type), parathecium with small crystals. Hymenium hyaline, 50-75 µm high; epihymenium brown to dark brown with pigment and small crystals dissolving in KOH (= chlarotera-type), c. 12 µm high. Hypothecium hyaline. Ascospores  Notes: This pruinose species is chemically characterized by the presence of protocetraric acid and atranorin. Given its phylogeny, it is here recognized at species level as Lecanora glaucomodes. If we consider all chemical variation recently found (see Brodo et al. [43]), this material agrees partially with L. caesiorubella subsp. glaucomodes. However, the chemical definition of L. glaucomodes is complex. Imshaug and Brodo [44] reported atranorin and protocetraric acid as a major compound, which was found in the isotype. Later, Lumbsch et al. [45] reported that the holotype of this species produces virensic acid as a major substance. In a recent review of the L. albella group, it was demonstrated that the definition of species or subspecies just observing the chemistry remains complex [43]. However, the new sequences generated in the present work form a clade distinct from Genbank sequences identified as L. caesiorubella s.str., from the USA and Australia ( Figure  1).  Synonym: Lecanora caesiorubella subsp. glaucomodes (Nyl.) Imshaug and Brodo, Nova Hedwigia 12(1+2): 15 (1966) Typus: Cuba, C. Wright Lich. Cub. II. 60 (H-NYL 27145, holotype) Description specimens examined: Thallus crustose, corticolous, up to 3.5 cm wide, c. 0.1 mm thick, areolate, rimose to verruculose, whitish grey to grey, dull, epruinose. Soredia absent. Prothallus absent or whitish grey. Apothecia adnate, 0.5-1.5 mm diam.; disc pale orange to brownish orange, carneous, whitish grey pruina, flat to convex; margin slightly paler than the thallus, prominent, becoming excluded or persisting, entire to flexuose, smooth. Amphithecium with small crystals which are dissolving in KOH (=allophana-type), parathecium with small crystals. Hymenium hyaline, 50-75 µm high; epihymenium brown to dark brown with pigment and small crystals dissolving in KOH (=chlarotera-type), c. 12 µm high. Hypothecium hyaline. Ascospores  Notes: This pruinose species is chemically characterized by the presence of protocetraric acid and atranorin. Given its phylogeny, it is here recognized at species level as Lecanora glaucomodes. If we consider all chemical variation recently found (see Brodo et al. [43]), this material agrees partially with L. caesiorubella subsp. glaucomodes. However, the chemical definition of L. glaucomodes is complex. Imshaug and Brodo [44] reported atranorin and protocetraric acid as a major compound, which was found in the isotype. Later, Lumbsch et al. [45] reported that the holotype of this species produces virensic acid as a major substance. In a recent review of the L. albella group, it was demonstrated that the definition of species or subspecies just observing the chemistry remains complex [43]. However, the new sequences generated in the present work form a clade distinct from Genbank sequences identified as L. caesiorubella s.str., from the USA and Australia ( Diagnosis: The new species can be distinguished from the somewhat similar species Lecanora coronulans Nyl. by the strongly irregularly and flexuose apothecium margin and larger ascospores. Description: Thallus crustose, corticolous, up to 3 cm wide, c. 0.1 mm thick, areolate, rimose to verruculose, white yellowish to whitish grey, dull, epruinose. Soredia absent. Prothallus absent or whitish grey. Apothecia adnate to sessile, 0.2-1.8 mm diam.; disc brown to dark brown, epruinose, flat to convex; margin slightly paler than the thallus, prominent, flexuose, smooth, entire, sometimes strongly flexuose, whitish to whitish grey. Amphithecium with small and large crystals (=melacarpella-type), parathecium with small crystals. Hymenium hyaline, 50-62.5 µm high; epihymenium dark red-brown to dark brown without crystals, pigmentation not altered by KOH (=glabrata-type), c. 12 µm high. Hypothecium reddish brown. Ascospores 8 per ascus, broad ellipsoid, (12-) 13-17(-18) × 7-10 µm. Pycnidia not observed.
Habitat and distribution: on bark of tree in Itatiaia rain forest; known only from Brazil. Additional sequenced specimens examined: BRAZIL, Rio de Janeiro, Parque Nacional de Itatiaia, Itatiaia, 22 •  Notes: Lecanora irregularicrocea represents a hitherto undescribed member of the L. coronulans group. All species of this group have a pigmented hypothecium and occur exclusively in the tropics [46]. This new species is similar to an assemblage of taxa that occurs in Brazil: Lecanora concilianda Vain., L. concilians Nyl., L. coronulans Nyl., L. hypocrocina Nyl. All agree in the more or less brown to dark brown apothecial disc and the melacarpellatype amphithecium. Lecanora concilianda was described from Brazil; L. concilians originates from Colombia, whereas L. coronulans and L. hypocrocina were described from Cuba.
The differences between the five species are mainly morphological and in part chemical: Lecanora concilianda and L. concilians differ in spore size and thallus morphology; L. coronulans can be distinguished by its smaller apothecia and the presence of zeorin, and L. hypocrocina is different from all other species by the KOH+ red to purple reaction of the hypothecium (boryquinone). The new species somewhat resembles L. coronulans in the presence of zeorin, but differs in the strongly irregularly and flexuose apothecium margin and larger ascospores (13-17 × 7-10 µm vs. 9.5-14 × 5-8 µm).  Notes: This species can be recognized by the pale orange apothecial discs, the chlarotera-type epihymenium, and the greenish thallus. Lecanora helva Stizenb. is morphologically similar but can be distinguished by the smaller apothecia (0.4-1.0 mm diam. vs. 0.5-1.5 mm diam.) and smaller ascospores (5-7 × 10-13.5 µm vs. 12-15 × 5-7 µm). Lecanora stramineoalbida Vain. is another similar species, which differs in having a reddish brown hypothecium. Description specimens examined: Thallus crustose, corticolous, up to 7 cm wide, c. 0.1 mm thick, continuous, rimose to verruculose, whitish grey, dull, epruinose. Soredia absent. Prothallus absent or greyish black. Apothecia adnate, 0.5-2 mm diam.; disc pale yellow to brownish orange, carneous, whitish grey pruina, flat to convex; margin slightly paler than the thallus, prominent, becoming excluded or persisting, crenate. Amphithecium with small crystals which are dissolving in KOH (= allophana-type), parathecium The new species can be distinguished by the very green thallus, pale orange apothecial discs, larger apothecia and larger ascospores than in the Lecanora helva. The phylogenetic tree also indicate it forms an independent clade.
Lecanora Notes: This taxon of the Lecanora cesiorubella morphotype with pruinose apothecia can be separated into two chemotypes. Chemotype I, characterized by the presence of norstictic acid and atranorin; and Chemotype II, with atranorin, protocetraric acid, and undefined fatty acids, but lacking norstictic acid. Additionally, phylogenetically ( Figure  1), the sequences generated for this material clustered into two distinct subclades, one with specimens that produce norstictic acid and atranorin, which agree with subsp. merrillii [43][44][45]. The other produces atranorin, protocetraric acid, undefined fatty acids (except the specimen ISE-52273) and lacks norstictic acid. Based on the chemistry, part of our material agrees with the L. albella (Chemotype I). However, Lecanora albella has smaller apothecia and ascospores [45], additionally, the new sequences generated in the present work form a clade distinct of Genbank sequences identified as L. albella (Figure 1). It is therefore recognized at species level. Therefore, we proposed the new name Lecanora neomerrilli, because there is already Lecanora merrillii Nyl, as, e.g., mentioned by Brodo et al. [43]. Description specimens examined: Thallus crustose, corticolous, up to 7 cm wide, c. 0.1 mm thick, continuous, rimose to verruculose, whitish grey, dull, epruinose. Soredia absent. Prothallus absent or greyish black. Apothecia adnate, 0.5-2 mm diam.; disc pale yellow to brownish orange, carneous, whitish grey pruina, flat to convex; margin slightly paler than the thallus, prominent, becoming excluded or persisting, crenate. Amphithecium with small crystals which are dissolving in KOH (=allophana-type), parathecium with small crystals. Hymenium hyaline, 50-62.5 µm high; brown to dark brown with pigment and small crystals dissolving in KOH (=chlarotera-type), c. 12 µm high. Hypothecium hyaline. Ascospores  Notes: This taxon of the Lecanora cesiorubella morphotype with pruinose apothecia can be separated into two chemotypes. Chemotype I, characterized by the presence of norstictic acid and atranorin; and Chemotype II, with atranorin, protocetraric acid, and undefined fatty acids, but lacking norstictic acid. Additionally, phylogenetically (Figure 1), the sequences generated for this material clustered into two distinct subclades, one with specimens that produce norstictic acid and atranorin, which agree with subsp. merrillii [43][44][45]. The other produces atranorin, protocetraric acid, undefined fatty acids (except the specimen ISE-52273) and lacks norstictic acid. Based on the chemistry, part of our material agrees with the L. albella (Chemotype I). However, Lecanora albella has smaller apothecia and ascospores [45], additionally, the new sequences generated in the present work form a clade distinct of Genbank sequences identified as L. albella (Figure 1). It is therefore recognized at species level. Therefore, we proposed the new name Lecanora neomerrilli, because there is already Lecanora merrillii Nyl, as, e.g., mentioned by Brodo et al. [43].  Notes: The new species is characterized by an areolate thallus and large, immersed apothecia with black disc and dark brown hypothecium. In the field, L. nigrilobulata can be confused with L. oreinoides (Körb.) Hertel and Rambold, but the latter differs by the shape of the areoles, which are becoming almost rounded, and the hyaline hypothecium. Lecanora nigrilobulata is also similar to L. subimmersa (Fée) Vain., but the latter has a non-areolate thallus surface, brown apothecial disc, and hyaline hypothecium. Notes: Lecanora notatictria can readily be identified by the pale yellow to brownish orange apothecial discs, the pulicaris-type amphithecium, a charotera-type epihymenium and the presence of atranorin, usnic acid, and notatic, isonotatic and subnotatic acids. Morphologically this species resembles Lecanora achroa Nyl., L. helva, L. leprosa Fée and L. parachroa L.A. Santos, J.G. Cavalcante and M. Cáceres, but is readily distinguished by its different chemistry. Notatic and subnotatic also occur in L. notatica Guderley, but this species has larger apothecia and in addition contains confluentic acid [47]. Notes: Lecanora notatictria can readily be identified by the pale yellow to brownish orange apothecial discs, the pulicaris-type amphithecium, a charotera-type epihymenium and the presence of atranorin, usnic acid, and notatic, isonotatic and subnotatic acids. Morphologically this species resembles Lecanora achroa Nyl., L. helva, L. leprosa Fée and L. parachroa L.A. Santos, J.G. Cavalcante and M. Cáceres, but is readily distinguished by its different chemistry. Notatic and subnotatic also occur in L. notatica Guderley, but this species has larger apothecia and in addition contains confluentic acid [47]. Diagnosis: The new species can be distinguished from the somewhat similar species Lecanora achroa by the paler orange disc.
Habitat and distribution: On exposed trees in Caatinga forest; known only from Brazil.
Additional sequenced specimens examined: Sergipe, Monumento Natural Grota do Angico, Poço Redondo, 9°39'39. Notes: Lecanora parahelva is chemically characterized by the presence of alectoronic acid in addition to atranorin, 2-chloro-6-O-methyl-norlichexanthone and 4,5 dichloronorlichexanthone. Anatomically it can be recognized by the presence of yellow thallus, large apothecia with margins slightly paler than the thallus.  Notes: Lecanora saepiphila is characterized by the small apothecia with orange brownish apothecial discs, which are UV+ green. Morphologically L. achroa and L. parachroa. However, both species have slightly larger asco ver, those two species can be readily distinguished from L. saepiphila by chemistry; in addition to atranorin, L. achroa produces usnic acid and L. pa dentified fatty acid [22].   Notes: Lecanora saepiphila is characterized by the small apothecia with pale orange to orange brownish apothecial discs, which are UV+ green. Morphologically it is similar to L. achroa and L. parachroa. However, both species have slightly larger ascospores. Moreover, those two species can be readily distinguished from L. saepiphila by their different chemistry; in addition to atranorin, L. achroa produces usnic acid and L. parachroa an unidentified fatty acid [22].

Discussion
The circumscription of species within of Lecanora s.lat. remains complex and man studies focusing on delimitation of groups within this genus have been inconclusiv [1,8,18,21,48]. The aim of this study was to illustrate the diversity of the genus in its broa sense in Brazil, not to discuss segregation of smaller genera and the delimitation o Lecanora s.str. We therefore treat this group as Lecanora s.lat.
The few tropical Lecanora specimens sequenced so far are mainly from the Paleotrop ics [7,49] and a recent study with molecular data focusing on Bolivia [50]. With additiona sequences from Brazil, we found that some of the species' identifications of publishe sequences are incongruent; for example, the GenBank (GB) sequences identified as L. vain ioi from Thailand were clustered in a distinct branch separate from the Brazilian materia the type of L. vainioi being is from Brazil [51]. Consequently, the Brazilian specimens ar likely to represent L. vainioi s.str. and the two Thai GB sequences are something else. Ou phylogenetic analyses shown that some morphologically similar specimens were recov ered in separate strongly supported lineage and/or clades (Figure 1). Diagnosis: The new species can be distinguished from the somewhat similar species Lecanora coronulans Nyl. by the verrucose thallus and apothecial margins and smaller ascospores.
Habitat and distribution: On exposed trees in Caatinga forest; known only from Brazil. Additional sequenced specimens examined: BRAZIL, Sergipe, Monumento Natural Grota do Angico, Poço Redondo; 9 • 39 39.7 S, 37 •  Notes: Lecanora xanthoverrucosa represents another undescribed member of the L. coronulans group. It resembles L. coronulans, L. hypocrocina and L. egranulosa Nyl., but can be readily distinguished by the pigmentation of the epihymenium, the verruculose thallus, and the presence of a xanthone.

Discussion
The circumscription of species within of Lecanora s.lat. remains complex and many studies focusing on delimitation of groups within this genus have been inconclusive [1,8,18,21,48]. The aim of this study was to illustrate the diversity of the genus in its broad sense in Brazil, not to discuss segregation of smaller genera and the delimitation of Lecanora s.str. We therefore treat this group as Lecanora s.lat.
The few tropical Lecanora specimens sequenced so far are mainly from the Paleotropics [7,49] and a recent study with molecular data focusing on Bolivia [50]. With additional sequences from Brazil, we found that some of the species' identifications of published sequences are incongruent; for example, the GenBank (GB) sequences identified as L. vainioi from Thailand were clustered in a distinct branch separate from the Brazilian material, the type of L. vainioi being is from Brazil [51]. Consequently, the Brazilian specimens are likely to represent L. vainioi s.str. and the two Thai GB sequences are something else. Our phylogenetic analyses shown that some morphologically similar specimens were recovered in separate strongly supported lineage and/or clades ( Figure 1).
Species having a dark hypothecium did not cluster in a monophyletic group, as also demonstrated previously [7]. Nine sequenced species in our material have a dark hypothecium, and three of these are new species.
Lecanora caesiorubella is not monophyletic, as also demonstrated previously [7,8]. Our phylogeny shows a separation of the studied specimens into two subclades, both with two species (Figure 1), although all fit some traditionally recognized subspecies of L. caesiorubella [44,45]. Two of them resemble each other both chemically and morphologically, viz,. subsp. merrillii and subsp. glaucomodes. Curiously, all our sequences of pruinose specimens are positioned in a distinct branch of GB sequences from L. caesiorubella, viz. with L. albella, L. farinacea, and L. subcarnea; similar to Medeiros et al. [50]. Reports of L. caesiorubella Ach. From Brazil are probably all referable to L. neomerrillii.
Our data thus confirm that ITS provides good resolution for species delimitation in the genus Lecanora.
The genus Lecanora has a wide geographic distribution in Brazil (https://www.gbif. org/occurrence/map?country=BR&taxon_key=2569863, accessed on 10 December 2022), except for the North region. During a lecanoroid lichen investigation in Brazil, all regions and ecosystem of the country were visited. Of these areas, twenty-eight taxa of Lecanora are treated here, of which 10 are new to science. Despite being a highly fragmented ecosystem [52], most of the Lecanora specimens were collected in areas of the Caatinga, also including new species. Interestingly, the majority of the new species are restricted to the ecosystem areas where they were initially found. This study shows morphological and molecular data that demonstrate the species richness and provide data for a better delimitation between the species of Lecanora from Brazil.

Key to Species of Lecanora in Brazil
This key is not exhaustive; further species have been reported or described from Brazil, but their identity is not known to us, and most are probably wrong identifications. Species referable to Vainionora are excluded from this key; they all share a dark hypothecium and the presence of an UV+ orange to red xanthone in thallus and/or apothecium. Our data thus confirm that ITS provides good resolution for species delimitation in the genus Lecanora.
The genus Lecanora has a wide geographic distribution in Brazil (https://www.gbif.org/occurrence/map?country=BR&taxon_key=2569863, accessed on 10 December 2022), except for the North region. During a lecanoroid lichen investigation in Brazil, all regions and ecosystem of the country were visited. Of these areas, twenty-eight taxa of Lecanora are treated here, of which 10 are new to science. Despite being a highly fragmented ecosystem [52], most of the Lecanora specimens were collected in areas of the Caatinga, also including new species. Interestingly, the majority of the new species are restricted to the ecosystem areas where they were initially found. This study shows morphological and molecular data that demonstrate the species richness and provide data for a better delimitation between the species of Lecanora from Brazil.

Key to Species of Lecanora in Brazil
This key is not exhaustive; further species have been reported or described from Brazil, but their identity is not known to us, and most are probably wrong identifications. Species referable to Vainionora are excluded from this key; they all share a dark hypothecium and the presence of an UV+ orange to red xanthone in thallus and/or apothecium. structure and equipment to carry out DNA and secondary metabolite analyzes, respectively. Thanks also to the Administration of Santuário do Caraça RPPN by the permits and its employees for help during the field. The reviewers are also acknowledged for their valuable contributions.

Conflicts of Interest:
The authors declare no conflict of interest.