Subtribe Laeliinae (Orchidaceae) in a large and mature remnant of Brazilian Atlantic Forest

Orchidaceae is a megadiverse botanical family in Brazil, particularly in the Atlantic Forest where it is a priority group for conservation. Parque Nacional do Itatiaia (PARNA Itatiaia) was the first conservation unit established in Brazil, and represents a large and mature remnant of Atlantic Forest. Updated data on richness, distribution and conservation of the Neotropical and ornamental subtribe Laeliinae in the park recently disclosed the occurrence of presumed locally extinct species, but without a taxonomic approach. Thus, we present a comprehensive taxonomic treatment of Laeliinae in the PARNA Itatiaia. The subtribe is represented by six genera and 33 species, which corresponds to about 15% of the orchid flora of PARNA Itatiaia. Epidendrum is the richest genus, with 20 species, followed by Cattleya (6 spp.) and Prosthechea (4 spp.). Species of Laeliinae grow in shady and humid habitats, especially near rivers, at elevations ranging 750-1,200 m, with species richness decreasing with elevation. Several species form small populations (commonly < 10 individuals), which are in need of prompt conservation actions to avoid local extinction. Samples of Laeliinae from PARNA Itatiaia in collections of consulted herbaria were found to be scarce. Variable morphological characteristics, mainly of the lip, distinguishe species of Laeliinae.


Introduction
Orchidaceae is a megadiverse botanical family in Brazil with ca. 2,500 species and comprises some genera of horticultural importance for their ornamental features, such as Cattleya Lindl. and Encyclia Hook. (Nardy et al. 2016;Flora do Brasil 2020). More than 50% of Brazilian orchids occur in the Atlantic Forest (Stehmann et al. 2009).
Rodriguésia 72: e00082020. 2021 This phytogeographical domain may include up to 8% of all species of flora and fauna of the world, and is almost completely restricted to Brazil where it occurs in 17 states, most situated along the coast, with it being narrower to the north and wider to the south (Silva & Casteleti 2003;Ribeiro et al. 2009). Despite the wide geographical extension of the Atlantic Forest, this global biodiversity hotspot is one of the most threatened on the planet, having been reduced to ca. 15% of its original coverage (Mittermeier et al. 2004;Lima et al. 2015).
Orchids are a priority group for conservation in tropical forests (Solano-Gómez et al. 2016). World-wide, conservation threats to orchids are primarily of anthropogenic origin and are associated with habitat destruction, modification and fragmentation, as well as collecting (Batty et al. 2002). Species with reduced populations that are restricted to few areas are more susceptible to local extinction (Batty et al. 2002;Cardoso et al. 2016; Barberena et al. 2019), with maintenance of genetic diversity being a basic point of action plans (Borba et al. 2007). Nonetheless, an understanding of the basic biology of species and rigorous taxonomic scrutiny are first needed (Bortolus 2008;Broadhurst & Coates 2017).
Biodiversity of mountainous regions in Brazil must be studied, with emphasis on biological inventories and preservation (Martinelli 2007;Costa et al. 2015; Barberena et al. 2018). Maintenance of the remaining large mature forest fragments is one of the conservation priorities for the Atlantic Forest (Ribeiro et al. 2009). Established in 1937, Parque Nacional do Itatiaia (PARNA Itatiaia) was the first conservation unit in Brazil. It currently encompasses ca. 28,000 hectares and is fully inserted within the Atlantic Forest phytogeographic domain and the Serra do Mar biogeographic sub-region (Ribeiro et al. 2009;ICMBio 2014ICMBio , 2020. PARNA Itatiaia is located between two major metropolitan areas -the cities of Rio de Janeiro and São Paulo -and received about 140,000 visitors in 2017 (ICMBio 2019).
The first studies of Orchidaceae in the region probably date back to 1894 when Ernest Ule explored the higher areas of Serra do Itatiaia and collected specimens of 11 species and six genera (Ule 1895). Further study was performed by Dusén (1905) who documented 14 genera and 19 species, including a few species from the lower areas of the Serra. Porto (1915) was the first to present a detailed survey of Orchidaceae of PARNA Itatiaia, which was the result of intense field efforts to collect exclusively orchids. A total of 102 species and nine varieties belonging to 45 genera were documented. Subsequently, several naturalists and botanists continued studies on orchids in this region. Brade (1951) presented a taxonomic study for Habenaria Willd., and later he listed 84 genera of Orchidaceae for PARNA Itatiaia (Brade 1956). Ribeiro et al. (2007) reported four orchid species on rock outcrops on the Itatiaia Plateau, whereas Barberena & Fraga (2012) highlighted the occurrence of 20 species in high-altitude fields. Recently, Barberena (2010) provided a checklist of the orchid species occurring in PARNA Itatiaia, while Medeiros et al. (2013) presented photos of some orchids from Itatiaia and surroundings. The 135 years of collecting and studying orchids of the Itatiaia massif produced important results and expanded the knowledge its orchid flora with the description of at least 17 new species belonging to 10 genera (Anathallis Barb. Rodr., Cleistes Rich. ex Lindl., Campylocentrum Benth., Epidendrum L., Habenaria, Hapalorchis Schltr., Maxillaria Ruiz & Pav., Myoxanthus Poepp. & Endl., Octomeria R.Br., Pelexia Poit. ex Lindl., Pleurothallopsis Porto & Brade, and Stelis Sw.), including endemic species (e.g., Pessoa & Alves 2015;Barberena & Gonzaga 2016), whose type specimens are deposited in herbarium RB.
The very ornamental subtribe Laeliinae is Neotropical and characterized mainly by apical leaves, terminal (rarely lateral) inflorescences and compact, ceroid, laterally flattened pollinia (Pridgeon et al. 2005). Porto (1915) and Brade (1956) reported incipient data on the diversity of Laeliinae in PARNA Itatiaia (seven genera, 16 species and four varieties). Updated data on richness, distribution and conservation of Laeliinae in PARNA Itatiaia recently revealed the occurrence of 13 presumed locally extinct species and several possibly threatened species, but without a taxonomic approach (Barberena et al. 2018).
Accurate and reliable taxonomic studies are always desirable since detailed descriptions are essential for identifying unexpected remnant populations. They also provide information necessary for establishing conservation priority and appropriate actions for conservation and management of these species, particularly in conservation units. Similarly, taxonomic studies can encourage other investigations aimed at reintroducing presumably extinct and threatened species.
Floristic studies focused on a specific geographic area make valuable contributions to knowledge of the diversity, distribution and preferential habitats of taxa (Müller 2006;Heiden et al. 2009;Jordão et al. 2018). This is also true for Orchidaceae, a megadiverse and widely distributed family. Here, we present a comprehensive taxonomic treatment of the subtribe Laeliinae in PARNA Itatiaia, including an identification key, descriptions, illustrations and notes on the taxonomy of its species.

Material and Methods
PARNA Itatiaia is located in southeastern Brazil (22°14'-22°27'S, 44°34'-44º46'W), within the limits of the states of Rio de Janeiro (municipalities of Resende and Itatiaia), and Minas Gerais (municipalities of Bocaina de Minas and Itamonte) (ICMBio 2014(ICMBio , 2020. A map of the park was produced using ARC-GIS software ver. 10.2. (Fig. 1). The elevation of PARNA Itatiaia ranges from 600 m in the south to 2,791 m at Agulhas Negras Peak of the Itatiaia Plateau (ICMBio 2020). The vegetation of the area is classified as dense ombrophilous forest and is subdivided into two phytophysiognomies based mainly on elevational gradient: montane at 500-1,500 m, and uppermontane above 1,500 m, including high-altitude fields mainly above 2,200 m ( Fig. 2) (Martinelli et al. 1989;Veloso et al. 1991). Data on local climate, relief and soil are available from IBDF (1982) and ICMBio (2014).
Intensive fieldwork was carried out from March 2008 to September 2009September , and occasionally in 2011September , 2014September , 2015September and 2016, by the walking survey method (Filgueiras et al. 1994). Specimens were georeferenced and processed according to Guedes-Bruni et al. (2002), and deposited in the herbaria RB and SP. Materials (including type specimens) from the herbaria ALCB, GUA, HB, R, RB, RBR, RFA, and SP (acronyms according to Thiers, continuously updated) were also examined, including specimens collected by P.C. Porto and A.C. Brade in the region of Itatiaia before the establishment of PARNA Itatiaia (Barberena et al. 2018). Exact collecting locations have been purposely omitted here since management actions have been discussed with the heads of PARNA Itatiaia and other researchers. The material was identified by comparison with specimens previously identified by experts and using the works of Pabst & Dungs (1975, 1977. To standardize descriptions, information from additional material and important references used for taxonomic identification was included as needed. The classification for Laeliinae followed van den Berg (2014). Morphological terminology for Orchidaceae follows Pridgeon et al. (1999). Data on geographical distribution and phytogeographic domains were based on Flora do Brasil (2020), whereas data on substrate were obtained during fieldwork and analysis of labels of specimens collected in PARNA Itatiaia, or sometimes from Flora do Brasil (2020). Data on conservation of Laeliinae in PARNA Itatiaia can be found in Barberena et al. (2018). In the key to the species presented below, presumed locally extinct species are marked with an asterisk, while species previously assessed (explicitly or implicitly) as possibly threatened in PARNA Itatiaia are marked with a hashtag, as already pointed out by Barberena et al. (2018).

Results and Discussion
The present study deals with 33 (Fig. 3). This total represents ca. 15% of the orchid flora of PARNA Itatiaia (Barberena 2010 Barb.Rodr., E.proligerum Barb.Rodr., and E.tridactylum Lindl.) have only been found as small populations (commonly < 10 individuals) and thus are in need of prompt conservation action to avoid local extinction (Barberena et al. 2018).
Consultation of herbaria revealed a significant scarcity of Laeliinae samples from PARNA Itatiaia, with only 162 specimens, even considering the new collections added to herbaria RB and SP. Only a few species have been systematically collected, such as Epidendrum secundum Jacq., the most collected species with 32 specimens, followed by Cattleya coccinea Lindl. with 23 specimens; the former is possibly the most common species of Laeliinae in the area, as well as one of the most common in Brazil. The present study resulted in the recollection of 15 species. Epidendrum ochrochlorum, E. ramosum Jacq. and E. tridactylum had not been collected for over 50 years, whereas E. filicaule had not been collected in the area for over 95 years. In addition, E. henschenii Barb.Rodr. was recorded for the first time in PARNA Itatiaia. Several other species were historically collected once or twice (e.g., Cattleya bicolor Lindl., C. crispa Lindl., E. saxatile Lindl., Isabelia virginalis Barb.Rodr.), including E. campos-portoi, a newly described endemic species of PARNA Itatiaia (Tab. 1). These data reinforce the importance of the present study and call for urgent conservation efforts. Although the conservation scenario is clearly alarming, it is possible that small subpopulations still exist in inaccessible or unknown restricted areas. Despite the data gathered so far, visitors and taxonomists are encourage to share photos and information about the location of orchid species through contacts available on official sites of conservation units (Barberena et al. 2019), including PARNA Itatiaia, in order to fill knowledge gaps or even spread floristic news.
Morphological characteristics of caulome, leaves, lip, column and rostellum are especially useful for distinguishing genera of Laeliinae of PARNA Itatiaia. The genera Isabelia and Scaphyglottis are represented in PARNA Itatiaia by one species each, and are easily distinguished from the others. Isabelia virginalis has the caulome covered by lax and fibrous sheaths, similar to thin cotton mesh, and filiform leaves, whereas Scaphyglottis modesta (Rchb.f.) Schltr. has the caulomes superposed and a column foot. Epidendrum has the column adnate to the lip up to the apex and the rostellum parallel to the axis of the column and split lengthwise. On the other hand, Cattleya, Encyclia and Prosthechea have an entire rostellum that is subperpendicular to the axis of the column. However, while Prosthechea is distinguished by having flowers that are not resupinate and the column adnate up to the middle portion, the other two genera have resupinate flowers and a free column or adnate to the lip only at the base. Encyclia is represented by only one species, which has remarkable pyriform pseudobulbs and inflorescences without spathaceous bracts, whereas species of Cattleya have subcylindrical, cylindrical, clavate, fusiform or ovoid caulomes, and inflorescences usually with spathaceous bracts (except for C. coccinea). The number of flowers and morphological characteristics of caulome, leaves, inflorescence, floral bracts, sepals, petals, and mainly of the lip are variable and allow the species of Laeliinae in PARNA Itatiaia to be distinguished.
Historically, C. coccinea has been considered morphologically very similar to C. mantiqueirae (Fowlie) Van den Berg. These species were differentiated by subtle features like the shape of pseudobulbs, the shape, size and color of the leaves, the color and size of the flowers, and different flowering periods. Nonetheless, currently both names supposedly represent the same species (revision in Rodrigues et al. 2015). Great plasticity in the shape of the pseudobulbs, leaves and floral traits was observed in PARNA Itatiaia. As we did not observe discrete sets of characters that could distinguish the two species, the oldest name C. coccinea is adopted here, according to the Shenzhen Code (Turland et al. 2018). Cattleya coccinea is distinguished from the other species of Cattleya at PARNA Itataia by its shorter pseudobulbs, leaves and lateral sepals.

Cattleya crispa
Lindl., Bot. Reg. 14: t. 1172. 1828.   Cattleya crispa is endemic to the Brazilian Atlantic Forest but there are no data yet on its location in PARNA Itatiaia, which corroborates the assumption of Barberena et al. (2018) that this species is locally extinct. Cattleya crispa can be confused with C. purpurata but is distinguished by having petals and central lobe of the lip with noticeably undulate margin (vs. petals with a slightly undulate to crenulate margin and central lobe of the lip with an entire margin), and oblong (vs. elliptical) and narrower petals (1-1.1 cm vs. 4.9-5.6 cm).

Cattleya purpurata (Lindl. & Paxton) Van den
Berg, Neodiversity 3: 10. 2008. Fig. 4g Epiphyte (Flora do Brasil 2020). Caulome 30-40 cm long, thickened in pseudobulb, fusiform, covered by congest sheaths not like a cotton mesh nor superposed, 1-foliated. Leaf 25-40 × 4-8 cm, apical, oblong. Solitary flower; spathaceous bract 10-17 cm long; floral bracts not seen. Flowers resupinate; pedicel+ovary 4-6 cm long; dorsal sepal ca. 8.5 × 2 cm, elliptical, apex acute, margin entire; lateral sepals 7.3-7.4 × ca. 1.9 cm, slightly elliptical, apex slightly acuminate, margin entire; petals ca. 8.1 × 4.9-5.6 cm, elliptical, apex acute, margin slightly undulate to crenulate; lip trilobed, lateral lobes ca. 5.1 × 1.7 cm, embracing the column, oblong, apex rounded, central lobe ca. 6.9 × 4.3 cm, oblong, apex slightly emarginated, margin entire; column ca. 3 cm long, equally thick from the base to the apex, free from the lip, column foot absent; rostellum subperpendicular to the axis of the column, entire; pollinia not seen. Capsules not seen. Cattleya purpurata is endemic to the Brazilian Atlantic Forest but there are no data yet on its location in PARNA Itatiaia, which supports the hypothesis of Barberena et al. (2018) that it is locally extinct. The description of the vegetative organs was taken from Cogniaux (1898Cogniaux ( -1902 and complemented with personal observations of individuals under ex situ cultivation in orchid greenhouses at Instituto de Pesquisas Jardim Botânico do Rio de Janeiro. It can be confused with C. crispa but differs mainly by having elliptical petals with a slightly undulate to crenulate margin (vs. oblong petals with noticeably undulate margin), which are also at least four times larger (4.9-5.6 cm vs. 1-1.1 cm), and the central lobe of the lip with an entire margin (vs. central lobe of the lip with noticeably undulate margin). Cattleya schofieldiana is endemic to the Brazilian Atlantic Forest. It has been recorded in montane vegetation in PARNA Itatiaia but is presumed locally extinct. Cattleya schofieldiana is morphologically similar to C. bicolor but can be distinguished by having a distinct trilobed lip (vs. obscurely trilobed), and prominent lateral lobes embracing the column (vs. reduced, not embracing the column).  Encyclia patens is endemic to Brazil (Atlantic Forest and Cerrado phytogeographic domains) and although it is restricted to montane forest in PARNA Itatiaia, it is one of the most common species of Laeliinae in the area. Pollination events tend to be rare in many species of the genus, resulting in low fruit formation, but self-pollination for most flowers was observed in some populations of E. patens (Pridgeon et al. 2005). In PARNA Itatiaia, each inflorescence produces up to 44 showy and odorless (rarely fragrant) flowers. High rates of fruits per inflorescence (up to 19 capsules) can be observed throughout the year. These observations suggest the occurrence of self-pollination or agamospermy, which may also explain the abundance of the species in the area. It is easily recognized by having a pyriform caulome.

Epidendrum pseudodifforme
Epidendrum pseudodifforme is endemic to the Brazilian Atlantic Forest. In PARNA Itatiaia it has been found in several localities in montane vegetation. This species could be confused with E. latilabrum (see comments under the latter species), but is distinguished by having following set of features: caulome uniformly thick from base to apex (vs. narrow at the base and thickening to apex), leaves of similar length along the caulome (vs. longer at the apex of the caulome than at the base), and lip measuring ca. 0.8 × 1.2 cm (vs. 1.4-1.7 × 2.6-3.7 cm) with two calli (vs. calli absent).

Epidendrum secundum
Epidendrum secundum is widely distributed throughout the Americas. It has been found in montane and upper montane formations in PARNA Itatiaia. It differs from the other species of Laeliinae in the park by having the inflorescence in subcorymb and non-resupinate flowers. Vegetative and reproductive buds can form on the inflorescence peduncle. Epidendrum strobiliferum occurs throughout the Americas. It has been recorded in montane forest in PARNA Itatiaia but is presumed locally extinct (Barberena et al. 2018). The species is recognized mainly by having leaves with emarginate-mucronate apexes.

Epidendrum tridactylum occurs in South
America. In PARNA Itatiaia it has been found in montane vegetation, but is in need of conservation actions, as already pointed out by Barberena et al. (2018). This species is distinguished from other species of Epidendrum in the study area mainly by having the caulome thickened into a fusiform pseudobulb, glabrous inflorescence and conspicuously trilobed lip. Epidendrum vesicatum is endemic to Brazil (Atlantic Forest and Cerrado phytogeographic domains). In PARNA Itatiaia it has been recorded in montane vegetation but is presumed locally extinct. It is easily distinguished from other species of Laeliinae mainly by its pendant habit, and having imbricate and conspicuously concave leaves.  Isabelia virginalis is endemic to the Brazilian Atlantic Forest. In PARNA Itatiaia it has been recorded in montane vegetation but is presumed locally extinct (Barberena et al. 2018). It is easily distinguished by the presence of lax and fibrous sheaths that resemble a thin cotton mesh covering the rizome and pseudobulbs, and by the filiform leaves.  Prosthechea calamaria is endemic to Brazil (Atlantic Forest and Cerrado phytogeographic domains). There are no location data for the species in PARNA Itatiaia, which supports the hypothesis of it being locally extinct, as highlighted by Barberena et al. (2018). It differs from the other species of Prosthechea in the area mainly by having linear leaves and an oblanceolate dorsal sepal. Prosthechea fragrans occurs throughout the Americas. In PARNA Itatiaia it has been recorded in montane vegetation but is presumed locally extinct. It is morphologically similar to the other species of Prosthechea in the area but differs mainly by having oblong leaves, and lanceolate dorsal sepal.   According to Porto (1918) and Brade (1956), Cattleya × itatiayae is most likely a hybrid between C. loddigesii and C. guttata. Porto (1918) reported that the shape and color of the sepals and petals of a single specimen resemble C. loddigesii, whereas the consistency of floral parts, shape of the lip and macules of the sepals and petals are similar to C. guttata. Both authors also pointed out that both species were very common in the Itatiaia region, which corroborates the presumed origin.

Prosthechea pachysepala
The holotype (P. Campos Porto 201), collected in 1916 and comprising just one flower, was the only herbarium specimen found, and was identified by the collector himself as C. itatiayae (apparently not as a hybrid). Porto (1918) described the new taxon as having one to two maculate flowers. The number of flowers is congruent with the circumscription of C. loddigesii, whereas the presence of maculate sepals and petals is frequently observed in C. guttata.
The specimen belongs to Cattleya because the flower has a column free from the lip and the lateral lobes of the lip embrace the column. The possibility of Cattleya × itatiayae being a hybrid can not ruled out because the specimen has distinct floral characteristics that are present in both parental species. However, Porto (1918) did not cite any examined collection, and based the description on a cultivated specimen at Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Brazil, where it flowered.
Cattleya guttata occurs in the central region of the Brazilian coast (southern Bahia state to Rio de Janeiro state), from sea level to 300 m in elevation, and usually not far from the Atlantic Ocean (Zaslawski 2008). In this work, the author also stated that some orchid growers reported the occurrence of the species in the southern part of Brazil, in Santa Catarina state, and in the inland of the continent, in Minas Gerais state, where the plants occur at low altitudes and near water courses. This suggests the possibility that C. guttata occurs near PARNA Itatiaia, mainly in the area within Minas Gerais. Cattleya loddigesii occurs in the states of Rio de Janeiro and Minas Gerais (Flora do Brasil 2020) and has been observed in the buffer zone around the border of PARNA Itatiaia.
However, the occurrence of these two supposed parental species within PARNA Itatiaia remains unknown, and the supposed hybrid has not been collected in the area for almost 100 years. According to Porto (1918) and Brade (1956), Cattleya × itatiayae was collected at 600 m, at a locality that was greatly altered throughout over the 20 th century.
In PARNA Itatiaia, Cattleya × itatiayae differs from other species of Laeliinae by having maculate sepals and petals. However, it is considered a doubtful name based on the previous discussion, the absence of data in the literature and the lack of recent collections.

Author participation
FFVA provided all photos and collected, identified and described the specimens. JFAB and FB guided the work and provided significant suggestions during the study. All the authors contributed to preparation and critical revision of the manuscript and added intellectual content.