First record of the genus Tropidia Lindl . ( Orchidaceae ) for Brazil

(First record of the genus Tropidia Lindl. (Orchidaceae) for Brazil). The genus Tropidia is composed of ca. 20-30 species distributed in southern Asia, South Pacific islands and northern Australia, and a single species occurring in America, from United States to Ecuador. The first record of the genus Tropidia for Brazil is presented here. It consists of Tropidia polystachya, a species found in the Cerrado biome, Midwestern Brazil. Description, illustration, photos, and distribution map of the species are provided. Additionally, the leaf anatomy of the species was studied and it is very similar to the leaves of other species of Tropidieae described so far. DNA sequences of T. polystachya are presented (plastid matK and rbcL and nuclear ITS) in order to assist future phylogenetic studies with the genus.


Introduction
The genus Tropidia Lindl (Orchidaceae) is composed of ca.20-30 species, distributed in southern Asia, South Pacific islands and northern Australia, and a single species occurring in America, from United States to Ecuador (Pridgeon et al. 2005, Govaerts et al. 2016).Early classifications (e.g.Dressler 1981) placed Tropidia as related to Spiranthoid orchids, especially due to its erect dorsal anther and soft sectile pollinia (Pridgeon et al. 2005).However, cladistic analysis based on anatomical and morphological characters positioned the genus within Epidendroideae (Stern et al. 1993, Freudenstein & Rasmussen 1999).On the other hand, molecular analyses, of the rbcL plastid gene, positioned Tropidia as part of an unresolved grade that is sister of most other Epidendroideae (Cameron et al. 1999).A recent phylogenetic study on the subfamily based on mitocondrial, nuclear and plastid regions combined (Freudenstein & Chase 2015) placed Tropidia as sister to Corymborkis Thouars, with strong support, among the other earlybranching epidendroids.
Tropidia comprises terricolous herbs, green or holomycotrophic; with erect stem, often branched; two or many plicate leaves; inflorescences axillary or terminal, racemose or paniculate, few-to manyflowered; flowers resupinate or non-resupinate, sometimes with a spur; and column with prominent rostellum, and a dorsal and erect anther with two elongated sectile pollinia (Pridgeon et al. 2005).
Tropidia polystachya (Swartz) Ames is commonly known in the United States as youngpalm orchid (Hammer 1997), and it is the only Neotropical species of the genus (Pridgeon et al. 2005).Detailed information about T. polystachya are scarce, however, is distributed from Southern Florida through the West Indies, Venezuela, Central America and Galapagos Islands (Pridgeon et al. 2005).
In this study we expand the known distribution of T. polystachya, registering the first record of the species for Brazil.Additionally, based on the analyses of the Brazilian specimen, we present a description, detailed illustration, photographs, distribution map, anatomical characterization of the leaves, and plastid genes (rbcL and matK) and nuclear (ITS) sequences of the species.

Material and methods
Sampling -In 2012 and 2013, during field works in southwestern Mato Grosso State, in an area of the Cerrado biome, sterile individuals of T. polystachya were found.The population occurs in the Reserva Particular do Patrimônio Natural (RPPN) Vale do Sepotuba, located in the São Marcelo Farm (municipality of Tangará da Serra).The reserve occupies 1.200 ha of native semideciduous forest on the margins of the Sepotuba River, and presents a rhodic hapludox soil (Martins et al. 2010).This region is climatically classified as AW, according to the Köppen (1948) system, with an average annual temperature of 24.4 ºC, annual precipitation of 1.500 mm, and a relative humidity ranging from 50% to 80%.
Collected specimens were grown in the Catasetum Orquidarium at the campus Tangará da Serra of Universidade do Estado de Mato Grosso (UNEMAT).A specimen flowered in April 2013 and a voucher was pressed and deposited at Herbarium TANG (acronym according to Thiers 2015).
Species description -Flower measurements were taken from pickled flowers and all other measurements were made from the dried material.General morphological terms were based on Harris and Harris (1994), Radford et al. (1974) and Stearn (1983).The illustration was based on voucher material and pickled flowers.Map of distribution was made using the distribution presented in genera Orchidacearum (Pridgeon 2005) and from compiled data available in Govaerts et al. (2016).
DNA sequencing -DNA sequences from three regions (matK and rbcL plastid genes, and the nuclear ITS) were obtained in order to increase the knowledge of the phylogenetic placement of T. polystachya.Genomic DNA was extracted from fresh leaf material according to Doyle & Doyle (1987), using 2× CTAB extraction buffer, without the addition of RNase A and scaled to 2 ml tubes.Polymerase chain reaction (PCR) of all fragments was performed in 20 µl reactions (1× enzyme buffer, 2.5 mM MgCl 2 , 0.2 mM dNTPs, 0.5 µM of each primer, 10 ng BSA, 1 U of Taq DNA Polymerase (Invitrogen, Life Technologies Corporation), and 20-50 ng of genomic DNA.For the nuclear region 5 M betaine, 2% DMSO were added to the reaction.The nrITS region was amplified and sequenced with the primers described in Desfeux & Lejeune (1996); matK with the primers matK 3F_Kim f and matK 1R_Kim r (Ki-Joong Kim, pers.comm.); and rbcL with the primers rbcLa_f (Levin et al. 2003) and rbcLa_r (Kress & Eriksson 2007).Thermal cycling profile consisted of an initial step of 1 min at 94 °C and a final step of 5 min at 72 ºC, which was intercalated with 40 cycles of denaturation at 94 °C for 30 s, annealing at 51 °C for 40 s (nrITS), 53°C (matK and rbcL), and extension at 72 °C for 40 s.The PCR products were purified with polyethylene glycol (PEG) 10%, and the sequencing reactions were performed in the ABI3739XL genetic analyzer (Macrogen Inc., South Korea), using the same primers of the PCR amplification.The sequences were superimposed and edited with the Staden Package software (Staden et al. 2003).Statistics of the three genomic regions of T. polystachya were calculated in the MEGA 6 software (Tamura et al. 2013).The sequences obtained were compared with those from the GenBank database using the BLASTn tool from the National Center for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.nih.gov/) with default parameters.Sequences of plastid matK, rbcL and nrITS nucleotide obtained during this study were deposited in GenBank under accession numbers KR604798, KR604799 and KR604800.
Anatomical characterization -Two leaves of one individual were pickled in 70% ethyl alcohol or FAA 50 (Johansen 1940).Cross and paradermic sections from the medium third of the leaf blade were obtained.Histological sections were freehand sectioned with razor blade.Cross-sections were stained with astra blue and safranin 9:1 (Bukatsch 1972) and the paradermic sections were stained with toluidine blue as described by O' Brien et al. (1965).Semi-permanent slides were mounted with glycerin gelatin (Kaiser  1880).Observations and photomicrographs were conducted using an Olympus microscope with a SC30 camera and analySIS getIT image processing software.
Conservation status -Tropidia polystachya has a broad distribution from Florida to northern South America (B1 criteria) and a great number of known locations (B2 criteria), therefore the species is assessed as Least Concern (LC), according to the IUCN Red List Categories and Criteria (IUCN 2014).On the other hand, T. polystachya is known only in one locality in Brazil, and although found within a protected conservation unit, the species should be considered as Critically Endangered (CE; B1) for the country.
DNA sequencing -The genus Tropidia and Corymborkis are recognizable within the Tropidieae tribe, among the other early-branching Epidendroideae (Freudenstein & Chase 2015).According to the BLASTn tool, our nrITS sequence (KR604798), with 588 base pairs (bp), presented 99.3% identity with another sequence of T. polystachya (EU490674.1)deposited in the GenBank database of the NCBI; matK plastid sequence (KR604800), with 897 bp, presented 99% identity with T. graminea (EF079304.1);and rbcL plastid sequence (KR604799), with 561 bp, presented 100% identity with Tropidia sp.(Chase O-211).Unfortunately, due to the lack of more sequences from species of Tropidia deposited in the GenBank, it is not possible to draw any picture of the phylogenetic placement of T. polystachya within the genus at the moment.
Anatomical characterization -The leaf of T. polystachya presents, in front view, epidermal cells irregularly shaped, slightly curved anticlinal walls and tetracytic stomata (figure 4a).Additionally, stegmatas were observed close to the vascular bundles, as well as the presence of idioblasts with raphides in epidermal tissue (figure 4b), and uniseriate glandular trichomes scattered on the leaf surface (figure 4c).In cross section, the leaf blade shows a flat surface (figure 4d).The uniseriate epidermis cells are elliptical or isodiametric in shape, and the cells of the adaxial epidermis are higher than the cells of the abaxial epidermis (figure 4e).Presence of a thin cuticle covering the leaf blade was observed.Hypostomatic leaves show stomata at the same level of the other epidermal cells and present well developed substomatal cavity.Chlorenchyma is homogeneous, with isodiametric, rectangular or elliptical cells arranged from three to four layers horizontally disposed.Presence of collateral vascular bundles surrounded by a sclerenchymatic sheath was also observed (figure 4e).Midrib is constituted by a large collateral vascular bundle, whose adaxial region is surrounded by three to four layers of fibers, whereas the abaxial region of the bundle is surrounded by seven to eight layers of fibers arranged in an U shape (figure 4f).
The vegetative morphology and anatomy have proved to be great data sources to the understanding of relationships in groups within Orchidaceae, as opposed to reproductive characters, notably prone to selective pressures by pollinator groups and therefore considered more homoplastic (Cameron 2005, Salazar & Dressler 2011).Previously, leaf anatomy of Tropidia was only known from T. curculigoides Lindl., species distributed from the Himalaya to New Caledonia (Stern et al. 1993).Leaves of T. polystachya, from the individuals found in open areas of Cerrado are anatomically very similar to the leaves of other species of Tropidieae described so far (Stern et al. 2013(Stern et al. , 2014)), including the genus Corymborkis Thou.Thus, we believe that the leaf anatomical features may have low taxonomic significance for the tribe, since they remained unchanged even in very different habitats and species.The overall similarities with others basal Epidendroideae like Sobralia Ruiz & Pavón, Palmorchis Barb.Rodr., Xerorchis Schltr.and Eleanthus C. Presl indicated a conservative leaf structure and would probably be useful for the recognition of these basal groups of the subfamily in a broader context.

Figure 3 .
Figure 3. Distribution map of Tropidia polystachya including the new record for Brazil.

Figure 4 .
Figure 4. Anatomical characterization of the leaves of Tropidia polystachya.a. Abaxial epidermis with tetracytic stomata and irregularshaped epidermal cells (front view); b.Idioblasts containing crystals in the shape of druses (front view); c.Unisseriate tector trichome (front view); d.General view of the leaf (cross section); e. Epidermal cells, homogeneous mesophyll, stomata with substomatal chamber (arrow) and collateral vascular bundle (cross section); f.Middle vascular bundle partially surrounded by sclerenchyma fibres (cross section).