Description and phylogenetic position of a new species of Nematanthus (Gesneriaceae) from Bahia, Brazil

Abstract Chautems, A. & M. Perret (2017). Description and phylogenetic position of a new species of Nematanthus (Gesneriaceae) from Bahia, Brazil. Candollea 72: 351–359. In English, English abstract. DOI: http://dx.doi.org/10.15553/c2017v722a13 Nematanthus exsertus Chautems, a new species of Gesneriaceae from the state of Bahia in Brazil, is described. It is easily distinguished from other Nematanthus Schrad. species by its pendent resupinate flowers with funnel-shaped and laterally compressed corolla combined with striking exserted stamens and style. According to the phylogenetic analyses based on nuclear and plastid DNA sequences, this species belongs to a clade including the morphologically distinct Nematanthus monanthos (Vell.) Chautems and four other species with a similar flower morphology but lacking exserted stamen and style. Field photographs accompany the description. The new species is known from one locality in the municipality of Wenceslau Guimarães in the southern part of Bahia state within the “região cacaueira” [cocoa producing area]. The new species is assigned a preliminary assessment of “Vulnerable” using the IUCN Red List Categories and Criteria. Received: May 31, 2017; Accepted: August 17, 2017; First published online: Septembre 27, 2017


Introduction
The genus Nematanthus Schrader includes 31 species, all endemic to the Brazilian Atlantic Forest (Chautems et al., 2005 ;Ferreira et al., 2016). Nematanthus species are epiphytic perennial herbs or subshrubs, less frequently growing on rocky substrates in humus pockets. Blossoms display a large range of morphologies among species, including short or long pedicelate, brightly colored flowers, with resupinate, or non-resupinate flowers. Flowers that are non-resupinate are pouched. Flowers that are resupinate are funnel-shaped and laterally compressed, but in some taxa, flowers are white and bell-shaped. Hummingbird pollination has been documented in the field for several species and is likely the dominant pollination mode in the genus, according to flower morphology (Serrano-Serrano et al., 2017). However, flowers matching the bee-pollination have also evolved in a clade of three Nematanthus species (Serrano-Serrano et al., 2015). Despite this large morphological variation, the monophyly of Nematanthus has been confirmed by phylogenetic data and traits such as flower resupination and bee syndrome have been identified as synapomorphies of specific clades (Ferreira et al., 2016 ;Serrano-Serrano et al., 2015).
The Brazilian Atlantic Forest is recognized as a global biodiversity hotspot and studies focused on forest remnants in the State of Bahia in eastern Brazil have revealed a higher species richness in trees, herbs, vines and epiphytes than in other areas from southeastern Brazil (Amorim et al., 2005(Amorim et al., , 2009Thomas, 2008). During fieldwork in the county of Wenceslau Guimarães in southern Bahia state, plants with resupinate red flowers and exserted anthers and style were fortuitously discovered by the first author while walking in a fragment of well-preserved rain forest. Here we recognize and describe this new species and used two nuclear loci and seven plastid DNA regions to investigate its phylogenetic placement within the genus. Morphology of the new species is discussed and compared with other closely related and sympatric species. Field photographs and distribution map are provided as well as a preliminary risk of extinction assessment using the IUCN Red List Categories and Criteria (IUCN, 2012).

Material and methods
Specimens of the newly identified species were collected during fieldwork carried out in May 2007 in the county of Wenceslau Guimarães, southern Bahia state, Brazil. All pressed material is deposited in the CEPEC and NY herbaria (acronyms according to Index Herbariorum, 2017). New collections were later registered from the same area in 2012 and material was deposited in the RB herbarium. The description and analysis of the new species were based on fresh and dried specimens. Data for comparisons with other species were taken from previous studies (Chautems, 1988, Chautems & Matsuoka, 2003 ; directly from herbarium specimens mostly at G, NY, RB ; from fresh specimens kept in cultivation at the Conservatoire et Jardin botaniques de la Ville de Genève ; or at Mauro Peixoto's private collection in sitio Primavera, Mogi das Cruzes, São Paulo, Brazil. The map was created using ArcGIS 10 (ESRI Inc.).
To assess the phylogenetic position of the new taxon, we performed a phylogenetic analysis based on DNA nucleotide sequence data. A DNA sample of the type collection was analyzed with sequences data obtained from other representative species of the genera Nematanthus Schrad. (1 of 2 species). Two outgroups, Drymonia serrulata ( Jacq.) Mart. and Chrysothemis pulchella (Donn ex Sims) Decne., were selected according to previous phylogenetic analyses (Perret et al., 2013). Phylogenetic relationsips among these taxa were reconstructed using seven plastid DNA regions (atpB-rbcL, matK, rps16, rpl16, trnT-trnL, trnL-trnF and trns-trnG) and two nuclear regions, the ribosomal internal transcribed spacer (iTs) and a portion of the plastid-expressed glutamine synthetase gene (ncpGs). New DNA sequences were generated for trnT-trnL, trns-trnG and for the new species, N. exsertus, following the procedure described in Perret et al. (2003) and Ferreira et al. (2016). All other sequences were obtained from previous works by Serrano-Serrano et al. (2015) and Ferreira et al. (2016). Newly acquired sequences have been deposited in GenBank. Voucher information and GenBank numbers for each sequence used in this study are provided in Appendix 1.
Newly acquired sequences were manually added to available alignments in Mesquite 3.03 (Maddison & Maddison, 2015). Phylogenetic analyses were performed on the CIPRES portal in San-Diego, USA [http ://www.phylo.org] using maximum likelihood (ML) and Bayesian Inference (BI). Maximum likelihood analyses were conducted using the software RAxML v.8.2.10 (Stamatakis, 2014) with a rapid bootstrap analysis followed by the search of the best-scoring ML tree in one single run. Bootstrap analysis stopped after 456 replicates, which were sufficient for getting stable support values according to the bootstrap convergence test using the extended majority-rule consensus tree criterion (autoMRE). The default model, GTRCAT, was used to perform the ML analyses. Bayesian inference analyses were performed using MrBayes 3.2.3 (Ronquist et al., 2012) following the procedure described in Serrano-Serrano et al. (2015). For each DNA region, the optimal substitution models were assessed using jModelTest2 (Darriba et al., 2012) according to the Akaike information criterion (AIC). The best-fit model was HKY + G for ncpGS, and GTR + G for all other genes. All the parameters values were unlinked across gene partitions and estimated during the Markov chain Monte Carlo (MCMC) runs. Two independent analyses were run from different random trees. The chains were run for 20,000,000 generations, with trees sampled every 1000th generation. We determined chain convergence and burn-in length (20% of the sampled generations) by examining trace plots of each parameter in Tracer v.1.6 (Rambaut et al. 2014). A consensus tree was calculated by removing the burn-in period and combining the two runs.

Results and discussion
The topology of the phylogenetic trees resulting from the ML and BI analyses are identical and are summarized in Fig. 1. The result is also congruent with the maximum clade credibility tree described in Serrano-Serrano et al. (2015) showing that Nematanthus species are clustered in five main clades exhibiting contrasting flower morphologies. Species with resupinate flowers and hummingbird syndrome belong to clades I and III, whereas the three species with white non-resupinate flowers and bee pollination syndrome belong to clade II (Fig. 1). The Nematanthus species with non-resupinate flowers  and hypocyrtoid (or pouched) corolla are found in clades IV and V (Fig. 1). Our analysis support that the new species, N. exsertus, nests in a well-supported clade (bootstrap support, BS = 90 %, Bayesian posterior probability, PP = 1) with four other resupinate species (N. brasiliensis, N. crassifolia, N. fluminensis  and N. monanthos, Fig. 1). Nematanthus exsertus is sister to N. monanthos, but interspecific relationships within this clade are not supported (BS < 50%). Although the new species can be readily distinguished from N. monanthos, it presents morphological affinities with N. brasiliensis, N. corticola, N. crassifolia and N. fluminensis. All five species in this group are characterized by the presence of pendent flowers with elongate pedicels with funnel-shaped corolla tubes that appear laterally compressed in the throat. However, all of the other species have inserted stamens and pistil. In contrast, N. exsertus (Fig. 2) is unique by the presence of exserted stamens and pistil. Scarlet-red corollas are found in N. exsertus, N. corticola and N. crassifolius, whereas N. brasiliensis displays a yellow corolla with red stripes and N. fluminensis a plain yellow corolla. However, none of these species have stamens and pistil as exserted as in N. exsertus (Fig. 2). Scarlet-red corolla is found in N. exsertus, N. corticola and N. crassifolius, whereas N. brasiliensis displays a yellow corolla with red stripes and N. fluminensis a plain yellow corolla. The only closely related species that is sympatric with N. exsertus is N. corticola (Fig. 3). These two species are easily differentiated when fertile or sterile. When sterile, they can be differentiated by the presence of villous indumentum on the stem in N. exsertus in contrast to the glabrous stems in N. corticola. In flower, N. exsertus is differentiated by the presence of exserted stamens and style and shorter corolla tube with narrower gibbosity (Table 1).
Distribution, ecology and phenology. -Nematanthus exsertus is currently known by two collections made in the "Estação Ecológica Estadual Nova Esperança" in the municipality of Wenceslau Guimarães in the state of Bahia (Fig. 3). The individuals were observed growing epiphytically on small trees at 1-2 m above the ground in the understory of a fragment of wet and dense forest on flat grounds. Specimens were collected with flowers in January and May. According to flower morphology, the species is likely pollinated by hummingbirds. Pollination by hermit hummingbirds has been documented for the related species N. crassifolia and N. brasiliensis presenting similar pendent and resupinate flowers with laterally compressed corolla (SanMartin-Gajardo & Vianna, 2010 ;Wolowski et al., 2013).
Conservation status. -Nematanthus exsertus is known only from a single location of a dozen individuals in an area of preservation created in 1997 and extended in 2,000, covering 2,418 ha. of Atlantic Rain Forest ("Estação Ecológica Estadual Nova Esperança"). This area is nevertheless subject to local preservation conflicts, like deforestation, illegal hunting and illegal occupation [http://www.inema.ba.gov.br/gestao-2/ unidades-de-conservacao/estacao-ecologica/estacao-ecologicade-wenceslau-guimaraes]. Therefore, with a restricted AOO, plausible threats that could rapidly push the species to "Critically Endangered", N. exsertus is assigned a preliminary assessment of "Vulnerable" [VU D2] using the IUCN Red List Categories and Criteria (IUCN, 2012).
Notes. -Our first thought, at the time of discovering this species, was that we had found an individual of Nematanthus corticola with ill-formed stamens and style, because this taxon occurs frequently in the southern Bahian rain forest (Fig. 3). After having observed few individuals, each bearing several flowers, it was readily apparent that we had discovered a new species.
The striking exserted position of stamens and style are a unique feature in the genus Nematanthus. Nematanthus exsertus resembles N. corticola with similar pedicel, calyx lobes, corollas, but differs by a villous indumentum on stem and petioles, as well as shorter and more ventricose corollas with strikingly exserted stamens and style (see Table 1).
The total number of known species for the genus Nematanthus is now 32. The new species should be added to the recent checklist that established a high endemism rate of epiphytic plants within the eastern Brazilian Atlantic rain forest (Freitas et al., 2016). It is also an addition to the number of Gesneriaceae species that have been registered so far for the state of Bahia, the total number of registered species reaches now 29 (Chautems, 1991 ;Araujo et al., 2017).   Chautems (star) showing also the occurrences of morphologically related species N. corticola Schrad. (circles) and N. crassifolius (Schott) Wiehler (triangles).