A new remarkable Vanilla Mill. (Orchidaceae) species endemic to the Espinhaço Range, Brazil: its phylogenetic position and evolutionary relationships among Neotropical congeners

Emerson Ricardo Pansarin; Euler da Luz Fernandes Menezes

doi:10.3897/phytokeys.227.101963

Research Article

A new remarkable Vanilla Mill. (Orchidaceae) species endemic to the Espinhaço Range, Brazil: its phylogenetic position and evolutionary relationships among Neotropical congeners

Emerson Ricardo Pansarin^‡, Euler da Luz Fernandes Menezes^§

‡ University of São Paulo, Sao Paolo, Brazil

§ Federal University of Vales do Jequitinhonha e Mucuri, Diamantina, Brazil

Corresponding author: Emerson Ricardo Pansarin ( epansarin@ffclrp.usp.br )

Academic editor: Timothée Le Péchon

This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation: Pansarin ER, Menezes ELF (2023) A new remarkable Vanilla Mill. (Orchidaceae) species endemic to the Espinhaço Range, Brazil: its phylogenetic position and evolutionary relationships among Neotropical congeners. PhytoKeys 227: 151-165. https://doi.org/10.3897/phytokeys.227.101963

Abstract

During surveys conducted on Neotropical Vanilla, a new endemic species was found in the Brazilian campos rupestres of the Espinhaço Range. Here, this new remarkable Vanilla species, namely V. rupicola Pansarin & E.L.F. Menezes, is described and illustrated. A phylogeny for Vanilla is presented and the relationships between Neotropical species are discussed. The position of V. rupicola among Neotropical Vanilla is discussed within an evolutionary context. Vanilla rupicola is recognized by its rupicolous habit, its reptant stems, and its sessile and rounded leaves. This remarkable new taxon emerges in a clade that includes V. appendiculata Rolfe and V. hartii Rolfe. Vegetative and floral features support a close relationship between V. rupicola and sister taxa, mainly regarding the apical inflorescence (V. appendiculata), the type of appendages of the central crest of the labellum, and the labellar color pattern. Phylogenetic inference suggests that the circumscription of Neotropical Vanilla groups needs revision.

Key words

Atlantic forest, Brazilian campos rupestres, Cerrado vegetation, evolution, molecular phylogeny, Neotropics, orchids, Vanilleae, Vanilloideae

Introduction

An endemism area is defined as a geographic region that contains species that do not occur anywhere else (e.g. Platnick 1991). Among the Brazilian biodiversity hotspots, the Espinhaço Range of Minas Gerais (ERMG), situated between the Atlantic Forest and Cerrado Biomes, shows one of the richest floras in Brazil (Myers et al. 2000), with a high frequency of endemisms (Hensold 1988; Rapini et al. 2002; Versieux and Wendt 2007). Orchidaceae Juss. is one of the most diverse and important plant families in ERMG, with many endemic taxa having been described (Barros 1987; Barros and Pinheiro 2004; Azevedo and Van Den Berg 2007; Batista et al. 2016; Salazar et al. 2019), including members of the Vanilloideae Szlach. (Pansarin 2004).

Vanilloideae is divided into two tribes: Pogonieae Pfitzer ex Garay & Dunst. and Vanilleae Blume. With more than 100 species distributed throughout tropical regions of Asia, Africa and Americas, Vanilla Mill. is the most species-rich genus among Vanilleae (e.g. Cameron 2003). Vanilla is monophyletic (Dictyophyllaria Garay included; Pansarin 2010; Pansarin et al. 2012) with three well-supported clades, two of which occur throughout the Neotropics (Bouetard et al. 2010; Pansarin and Ferreira 2022). Among Vanilla, a Neotropical clade with thin and reticulate-veined leaves emerges as a sister to a clade that includes two subclades, an African/Asian/Caribbean group and a second, strictly Neotropical one. In the latter, the epiphyte and bird-pollinated V. palmarum (Salzm. ex Lindl.) Lindl. is positioned as a sister to a hemi-epiphytic clade whose species are almost completely pollinated by euglossine bees (Pansarin and Ferreira 2022).

Besides showing high diversity and ecological importance (Pansarin and Pansarin 2014; Pansarin et al. 2014), Vanilla is also the most economically important genus among the orchids since the fruits of the Neotropical V. planifolia Andrews and their relatives are a source of vanillin flavor. Efforts are currently underway to determine the diversity and the natural history of Neotropical Vanilla (Pansarin 2019, 2022; Pansarin and Ferreira 2022; Pansarin and Suetsugu 2022), and its potential use for gastronomy and industry (e.g. Oliveira et al. 2022). However, there is still much to know since new species have been found and described, notably in the Brazilian flora (e.g. Pansarin and Miranda 2016; Fraga et al. 2017).

In the course of the developing studies on Neotropical Vanilla, a new species has been found in Brazilian campos rupestres. A taxonomic description of this new remarkable Vanilla is presented here and a morphological comparison with other Neotropical taxa is provided. The phylogenetic position of V. rupicola within Vanilla and the relationships among Neotropical groups are discussed based on a molecular phylogeny for the genus.

Materials and methods

The fieldwork was performed in the municipality of Diamantina (approx. 18°14'17"S, 43°36'40"W; 1,288 m elevation) in the state of Minas Gerais, Southeastern Brazil. The Diamantina Plateau is situated in the southern region of the Espinhaço Range, at the boundaries of Serra do Cipó and Grão Mogol (Gonçalves et al. 2017). It is included in the Espinhaço Range Biosphere Reserve (UNESCO 2005), located between the Atlantic Forest and the Cerrado domains, two of the main global biodiversity hotspots in the world (Myers et al. 2000). The climate of the region is classified as “Cwb”, namely subtropical highland climate with dry winters (Köppen 1948).

Fresh and herbarium material of flowering and fruiting plants was used for the study. Photographs were based on specimens collected in the field. Measurements were made directly on floral structures using a Vernier Caliper. The vegetative structures, inflorescence and flowers were photographed with a Nikon D-SLR D800 camera and a Micro Nikkor 105 mm f2.8 lens. Floral details were analyzed with a Stereozoom Leica S8 APO stereomicroscope with an integrated photo output. Digitized images were used for diagramming a template over a black background, following the model presented by Hoehne (1945), using a Microsoft PowerPoint software. The final template was exported as a 600 dpi .TIFF file.

The terminology for describing shapes followed Radford et al. (1974). Features specific to Orchidaceae were based on Dressler (1993) and Pridgeon et al. (1999). The infrageneric classification of Neotropical Vanilla groups followed Soto-Arenas and Cribb (2010). The original description and digital images from holotypes of related species of Vanilla were consulted. Plant specimens were vouchered according to usual techniques (Fidalgo and Bononi 1989) and then deposited in the BHCB, DIAM, HDJF, LBMBP, SPFR, SPF and UEC herbaria, which were also examined in order to study the Brazilian diversity of Vanilla. Additional living specimens were collected in the field and are under cultivation at the Vanilla germplasm bank from the LBMBP Orchid House (Orchidarium of the Laboratory of Molecular Biology and Systematics of Plants), University of São Paulo (FFCLRP-USP), municipality of Ribeirão Preto (approx. 21°10′S, 47°48′W; 546 m a.s.l.), state of São Paulo, Brazil, available at https://www.lbmbplab.net/vanillacollection.

The conservation status of the new taxon was defined according to the IUCN red list categories and criteria and guidelines (IUCN 2012, 2016).

Taxon sampling for phylogenetic analysis

A total of 42 Vanilla accessions (37 species) were analyzed and are referred to here as the ingroup, which represents an increase of 10 Vanilla species (16 new accessions) over previous investigations (Pansarin and Ferreira 2022). Lecanorchis multiflora J.J. Sm. was selected as an outgroup according to previous phylogenetic studies on Vanilloideae (e.g. Pansarin and Ferreira 2022). A data matrix was built based on sequences available in the genbank database and obtained during the development of this study (Suppl. material 1). A list of ingroup and outgroup species, vouchers and GenBank accession numbers is given in Suppl. material 1.

DNA extraction, amplification and sequencing

DNA of Vanilla species was extracted from fresh material according to a modified CTAB method (Doyle and Doyle 1987). The amplifications were carried out using 50 µL PCR volumes. Relaxation of the DNA strands was achieved by the addition of a 5M betaine solution to the PCR preparations. Primers of the nuclear ribosomal transcribed spacer region (ITS), including the 5.8S gene (Sun et al. 1994) were used for amplification and sequencing. Taq DNA polymerase was added to the PCR mixture at 80 °C following a 10 min period of denaturation at 99 °C in the thermocycler. Thirty-five cycles were run according to the following program: denaturation, 1 min, 94 °C; annealing, 45 sec, 64 °C; extension, 1 min, 72 °C; final extension, 5 min, 72 °C. Amplified PCR products were purified using GFX PCR columns (GE Health Care). Sequencing reactions were prepared using Big Dye 3.1 (ABI), purified PCR products and the same aforementioned primers. Samples were dehydrated and re-suspended with loading dye. Sequences were obtained using an Applied Biosystems automated sequencer model 3100. Sequence Navigator and Autoassembler (Applied Biosystems) software was used for sequence editing and assembly of complementary and overlapping sequences. DNA sequences were aligned using the BioEdit version 5.0.9 software (Hall 1999). The sequence alignment is available upon request from the first author.

Phylogenetic analyses

A data matrix of ITS containing 43 taxa was used for phylogenetic analyses.

Maximum parsimony analysis (MP) was run with PAUP* version 4.0b5 (Swofford 2001) software. A heuristic search was conducted with 1000 replicates of random taxon entry additions, MULTREES option, and the tree bisection-reconnection (TBR) swapping algorithm, holding 10 trees per replicate and saving all the shortest trees. Support for clades was assessed using 1,000 bootstrap replicates (Felsenstein 1985). Bootstrap support (BS) values above 50% were calculated and mapped above the branches of the consensus tree. For bootstrap support levels, we considered bootstrap percentages of 50–70% as weak, 71–85% as moderate, and >85% as strong (Kress et al. 2002).

Maximum Likelihood (ML) analysis was run using the MEGA X tool (Kumar et al. 2018) with bootstrapping for 3,000 replicates. The analysis was based on the Kimura 2-parameter model (Kimura 1980). Initial trees for the heuristic search were obtained automatically by applying Neighbour-Joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with the superior log likelihood value.

Bayesian Inference (BI) was conducted using the MrBayes program, version 3.1 (Ronquist and Huelsenbeck 2003). The optimal model of sequence evolution for each partition was selected using MEGA X (Kumar et al. 2018) and Bayesian Information Criterion (BIC). The software selected the HKY+G as the best evolution model for the ITS region. Four Markov chains were run simultaneously for three million generations, with parameters sampled every 100 generations. The consensus tree was calculated after removal of the first 3,000 trees, which were considered to be “burn-in”. Posterior probability (PP) values above 0.5 were calculated and mapped below the branches of the consensus tree.

Results

Taxonomic treatment

Vanilla rupicola Pansarin & E.L.F.Menezes, sp. nov.

urn:lsid:ipni.org:names:77320774-1

Figs 1, 2

Type

Brazil. Minas Gerais: Mun. Diamantina, Distrito de Sopa, Afloramento rochoso ca. 2 km de Morrinhos, 18°11'43"S, 43°43'18"W, 817 m, 29 November 2022, E.R. Pansarin & E.L.F. Menezes 1561 (holotype: SPFR18105!).

Vanilla rupicola differs from all Neotropical Vanilla species by its rupicolous habit, its reptant stem and its rounded leaves. The overall characteristics of V. rupicola resemble those of V. appendiculata Rolfe and V. hartii Rolfe. However, the remarkable new taxon (V. rupicola) is easily distinguishable from both related species by its leaves and flowers, which are smaller than those of V. appendiculata and larger than those of V. hartii (Table 1), its sessile leaves (vs. petiolate in V. appendiculata and V. hartii), and its papillose labellar protrusions (vs. finger-like in V. appendiculata and verrucose in V. hartii).

Table 1.

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Comparison of the morphological features of Vanilla rupicola and of closely related species. Morphological data from V. hartii were obtained from Ferreira et al. (2020), while characteristics of V. appendiculata were obtained from Engels and Ferneda Rocha (2016)^a and Barona-Colmenares (2018)^b.

Species characteristic	Vanilla rupicola	Vanilla appendiculata	Vanilla hartii
Habit	rupicolous	hemiepiphytic^{a, b}	hemiepiphytic
Stem	reptant	scandent^{a, b}	scandent
Leaves	3.2–9 × 2.8–5.2 cm	13–19 × 3.9–5.7 cm^a	6.5–8 × 2.5–3.5 cm
Leaves	3.2–9 × 2.8–5.2 cm	14–17.7 × 4.2–49 cm^b	6.5–8 × 2.5–3.5 cm
Leaves	sessile	petiolate^{a, b}	petiolate
Leaf blade	ovate to rounded	obovoid^a	elliptic
Leaf blade	ovate to rounded	spatulate^b	elliptic
Inflorescence	apical/lateral	apical^{a, b}	lateral
Sepals	5.8–6.3 × 1–1.3 cm	6.6 × 0.7 cm^a	4.9–5.3 × 0.8–1.1 cm
Sepals	5.8–6.3 × 1–1.3 cm	7.5–7.8 × 0.8–1.1 cm^b	4.9–5.3 × 0.8–1.1 cm
Petals	5.7–6.2 × 0.7–1.2 cm	6.5 × 0.6 cm^a	4.9–5.1 × 0.7–0.9 cm
Petals	5.7–6.2 × 0.7–1.2 cm	7.6–7.7 × 0.7–0.8 cm^b	4.9–5.1 × 0.7–0.9 cm
Labellum	5.6–6.3 × 3.2–3.5 cm	6.8 × 1.6 cm^a	4.4–4.7 × 2.3–2.4 cm
Labellum	5.6–6.3 × 3.2–3.5 cm	3.8 × 2.9 cm^b	4.4–4.7 × 2.3–2.4 cm
Labellar protrusions	papillous	finger-like^b	verrucose
Column	4.2–4.8 × 0.3–0.4 cm	5.4 × 0.2 cm^a	3.8–4 × 0.2–0.3 cm
Column	4.2–4.8 × 0.3–0.4 cm	6.1 × 0.3 cm^b	3.8–4 × 0.2–0.3 cm

Description

Rupicolous herbs up to 12 m in length. Roots axillary, 1.8–2.2 mm diam., yellowish-green, one per node. Stem reptant, flexuous, cylindrical, fleshy, glabrous, green to yellowish-green; internodes 40–130 × 3–13 mm. Leaves 3.2–9 × 2.8–5.2 cm, alternate, ovate to rounded, fleshy, glabrous, green to yellowish-green, apex acute to acuminate, sessile. Inflorescence 4.5–6 cm long, apical or axillary, racemose, with up to 10 flowers opening in succession; one flower opening each morning; bracts 5–9 × 3.5–7 mm progressively smaller toward the apex, alternate, triangular to ovate, coriaceous, adpressed to patent, concave, apex acute to acuminate. Flowers resupinate, whitish-green, pedicellate, abscission layer between perianth and ovary present; pedicel with ovary 2.8–3.2 × 0.40–0.52 cm, green, incurved, dilated to the apex, triangular in transverse section. Sepals 5.8–6.3 × 1–1.3 cm, free, oblanceolate, fleshy, slightly concave, pale green, margin entire, slightly incurved, apex acute to obtuse; dorsal sepal symmetric; lateral sepals asymmetric. Petals 5.7–6.2 × 0.7–1.2 cm, free, linear to slightly spatulate, asymmetric, membranous, pale green, apex acute to acuminate, adaxial surface with a longitudinal rib. Labellum 3-lobed, 5.6–6.3 × 3.2–3.5 cm, white, inner surface with longitudinal brown stripes, with a prominent central crest near the apex, and with a penicillate callus just below the anther; central crest ca. 4–5 mm wide, with yellow or white papillose protrusions arranged in five longitudinal lines near the apex; penicillate callus 5–6 × 5.2 mm, white; margins fused from the base to ca. ¾ of the column length forming a tubular nectar chamber; nectary chamber 2.2–2.6 cm in length; lateral lobes rounded, overlapping above the column, undulate margins; apical lobe rounded to emarginated, reflexed, undulate to slightly fimbriate. Column 4.2–4.8 × 0.3–0.4 cm, semi-cylindrical, slender, sinuous, white, attenuate base, dilated to the apex, with white-hyaline hairs close to the stigma, apex ending in a membranous ochrea; anther 4.9–5.1 × 3.9–4.2 mm, white, versatile; rostellum 3.8–4.1 × 3.9–4.0 mm, rectangular to trapezoidal, white. Fruits 9–13 × 1–1.8 cm, linear, tapering towards the apex, triangular in transverse section, green when immature. Seeds ovate, black.

Figure 1.

Habit of Vanilla rupicola Pansarin & E.L.F. Menezes on the rock outcrops of the Espinhaço Range, Minas Gerais, Brazil. Note the creeping stem on the rock.

Figure 2.

Vanilla rupicola Pansarin & E.L.F. Menezes A part of a flowering plant showing the stem and a lateral inflorescence with a typical-colored flower B detail of an apical inflorescence of an albino plant. Note the white flower C leaf and inflorescence D detail of a raceme. Note the adpressed floral bracts E dissected perianth. The detail shows the penicillate callus and the central labellar crest F detail of the adaxial surface of a petal showing the longitudinal keel G detail of a flower with a typical color showing the apex of the labellum, the penicillate callus and anther H detail of an albino flower showing the apex of the labellum, the penicillate callus and anther. Note that the labellum apex is more projected than in the typically-colored flower I detail of the apex of the labellum of a typically-colored flower showing the yellowish longitudinal lines and the yellow projections J detail of the apex of the labellum of an albino flower showing the whitish projections K labellum in lateral view L column in lateral view M apex of the column: in lateral view with an articulated anther (above), in lateral view with a disarticulated anther (mid), and in abaxial view (below) N immature fruit.

Distribution and ecology

The species has been reported for the campos rupestres vegetation of the Espinhaço Range of Minas Gerais (ERMG), municipality of Diamantina, Southeastern Brazil. In this locality, Vanilla rupicola shows a reptant habit on rock outcrops and rooting in rock clefts. The elevation is from 800 to 1300 m a.s.l. The flowers produce a sweet fragrance perceptible during the hottest hours of the day. Each flower lasts ca. 12 hours.

Phenology

Vanilla rupicola has been collected with flowers from late September to early November. The fruits ripen from May to June.

Etymology

The specific epithet (rupicola) refers to the rupicolous habit, uncommon among Neotropical Vanilla.

Conservation status

Vanilla rupicola is a rare species currently known to grow in a mountain-chain of Diamantina, in the ERMG. The populations found are composed of few specimens. According to the IUCN red list categories and criteria and guidelines, the species can tentatively be considered as Endangered (EN) due to its geographic range which is estimated to consist of 30 km², and fewer than 250 individuals were recorded in the occurrence area.

Additional specimens examined (paratypes)

Brazil. Minas Gerais: Mun. Diamantina, Parque Nacional das Sempre Vivas, Próximo a Serra do Landim, 17°53'27,6"S, 43°45'51.3"W, 1293 m elev., 22 October 2019, E.L.F. Menezes 133 (DIAM!). Município de Sopa. Estrada vicinal, 18°11'33.2"S, 43°47'07.5"W, 1210 m alt., 19 November 2022, E.L.F. Menezes 752 (HDJF!); Distrito de Sopa, Afloramento rochoso ca. 2 km de Morrinhos, 18°11'43"S, 43°43'18"W, 817 m, 29 November 2022, E.R. Pansarin & E.L.F. Menezes 1562 (BHCB!); Distrito de Sopa, Afloramento rochoso ca. 2 km de Morrinhos, 18°11'43"S, 43°43'18"W, 817 m, 29 November 2022, E.R. Pansarin & E.L.F. Menezes 1563 (UEC!); Distrito de Sopa, Afloramento rochoso ca. 2 km de Morrinhos, 18°11'43"S, 43°43'18"W, 817 m, 29 November 2022, E.R. Pansarin & E.L.F. Menezes 1564 (SP!); Distrito de Sopa, Afloramento rochoso ca. 2 km de Morrinhos, 18°11'43"S, 43°43'18"W, 817 m, 29 November 2022, E.R. Pansarin & E.L.F. Menezes 1565 (LBMBP!).

Morphological affinities

Vanilla rupicola is easily recognized by its uncommon rupicolous habit, by its reptant stems, and by its sessile and rounded leaves. These characteristics differ significantly from those of the remaining non-membranaceous Vanilla species, which are characterized by their hemiepiphytic or more rarely epiphytic habit and by their elliptic to lanceolate leaves. Floral features suggest a close relationship between V. rupicola and some species currently recognized in the V. planifolia group and V. trigonocarpa group mainly regarding the type of appendages of the central crest of the labellum, and labellar color pattern, with longitudinal brown lines over a white background that converge at the entrance of the nectary. In addition, species of the V. planifolia group show a white penicillate callus just below the gynostemium apex. Among the members currently recognized in the V. planifolia group, our data suggest taxonomic affinities between V. rupicola and the Amazonian V. appendiculata. In fact, both species share characteristics that are uncommon among Neotropical Vanilla, such as the production of apical inflorescences and ovate to obovoid leaf blades. The inflorescences of members of the V. planifolia group are lateral, and their leaves are elliptical to lanceolate. In addition, V. rupicola also shares some floral features with the Amazonian V. hartii (currently included in the V. trigonocarpa group), such as the lip lobes with undulate margins, labellar protrusions arranged on five longitudinal lines near the apex, and brown lines converging at the nectar chamber. The main differences between V. rupicola and related species (V. appendiculata and V. hartii) are summarized in Table 1.

Phylogenetic relationships

Phylogenies obtained by analysis of the ITS1-5.8S-ITS2 region using distinct methods (BI, ML and MP) yielded trees with congruent topologies (ML (Fig. 3) is shown). In all analyses, the Neotropical Vanilla with reticulate-veined leaves (PP 1, BS 100%; Fig. 3) was recovered as sister to a large clade (PP .89, BS 100%) with two subclades: an Old-World/Caribbean clade (PP .97, BS 99%), and a well-supported clade including the remaining Neotropical Vanilla (PP .99, BS 100%; Fig. 3). Among the Neotropical reticulate-veined Vanilla, V. arcuata Pansarin & Miranda emerged as sister -without support- to a clade containing two sister groups, the Vanilla mexicana group, (V. inodora Schiede/V. paludosa Pansarin, J.M. Aguiar & A.C. Ferreira; PP 1, BS 100%) and the V. parvifolia group (V. diestschiana Edwall, V. angustipetala Schltr., V. edwallii Hoehne and V. parvifolia Barb. Rodr.; PP 1, BS 100%). Two well-supported clades were recovered within the latter group: V. diestschiana/V. angustipetala (PP 1, BS 94%), and V. edwallii/V. parvifolia (PP 1, BS 100%; Fig. 3). Within the Old-World/Caribbean Vanilla, the phylogenetic analyses (BI, ML and MP) recovered two poorly supported subclades: an Asian clade, and an African/Caribbean clade (Fig. 3). In the Asian Vanilla, the clade including V. siamensis Rolfe ex Downie and V. albida Blume (PP 1, BS 99%) emerged as sister to a clade comprising V. aphylla Blume/(V. borneensis Rolfe/V. griffithii Rchb. f.). In the African/Caribbean clade, V. africana Lindl. was nested as sister to two subclades: a West Indies clade that included V. claviculata Sw. and V. barbellata Rchb. f. (PP 1, BS 100%), and an African clade containing V. imperialis Kraenzl. /V. roscheri Rchb. f. (PP .84, BS 56%; Fig. 3). Within the thick-leaved Neotropical Vanilla (PP .99, BS 100%), species currently recognized among the V. palmarum group, i.e. the epiphytes V. palmarum and V. bicolor Lindl., emerged as sisters to a large clade whose species are mostly hemiepiphytes or nomadic vines (PP .84, BS 61%). Within the hemiepiphytic clade, the Amazonian V. trigonocarpa Hoehne emerged as sister to a clade including the remaining Neotropical taxa. The latter showed two subclades: one including the members of the V. pompona group (V. pompona Schiede, V. calyculata Schltr., and V. chamissonis Klotzsch, besides V. paulista Fraga & Pansarin (PP 1, BS 79%)), and the other a large and predominantly Amazonian clade that contained the remaining Neotropical taxa (Fig. 3). In the latter, a strongly supported clade (PP 1, BS 91%) including V. hartii (V. rupicola/V. appendiculata) emerged as sister to a large clade in which the members of the V. hostmanii group, V. dressleri Soto Arenas/V. cribbiana Soto Arenas (PP 1, BS 100%) were recovered as sister to a large clade that included two subclades: V. insignis Ames/(V. odorata C. Presl/V. helleri A.D. Hawkes) plus V. planifolia/(V. ribeiroi Hoehne/(V. phaeantha Rchb. f./V. bahiana Hoehne)) with moderate support (PP 1, BS 77%; Fig. 3).

Figure 3.

Maximum Likelihood analysis of Vanilla (Orchidaceae) based on ITS (nrDNA). Bootstrap values (%) >50 obtained by maximum parsimony analysis (MP) are given below the branches, while posterior probabilities values > 0.5 (BI) are given above branches. Vertical colored bars refer to the geographic distribution of Vanilla. The black arrow indicates the position of V. rupicola among the Neotropical thick-leafed Vanilla. The colored branches in the cladogram refer to Neotropical Vanilla groups according to the infrageneric classification presented in Soto-Arenas and Cribb (2010). AM = Amazonas, GO = Goiás, PA = Pará.

Discussion

Neotropical Vanilla species are represented by two distinct lineages, one including representatives with membranous leaves and the other including species with non-membranous, usually fleshy, leaves. Our results strongly agree with previous phylogenetic inferences based on the sequencing of the cpDNA (Bouetard et al. 2010) and nrDNA (Pansarin and Ferreira 2022) regions. The membranous reticulate-veined Vanilla (i.e. subgenus Vanilla; Soto-Arenas and Cribb 2010) includes members of two groups, i.e.: V. mexicana group and V. parvifolia (non V. parviflora) group. In fact, our data matrix including rare and endemic species from Brazil recovered both groups proposed for the subgenus Vanilla. However, although V. arcuata is a member of the membranaceous-leafed Vanilla (Pansarin and Miranda 2016), our data suggest further studies are needed in order to better determine the circumscription of infrageneric groups proposed by Soto-Arenas and Cribb (2010), as this Brazilian species emerges in a basal position within this Neotropical subgenus (Fig. 3).

Among the non-membranaceous Vanilla taxa, the basal taxa V. palmarum and V. bicolor, both occurring as epiphytes on palms (e.g. Soto-Arenas and Cribb 2010; Van Dam et al. 2010, E.R. Pansarin, pers. obs.), emerge as sisters to the hemiepiphytic and thick-leafed groups. Among the groups proposed by Soto-Arenas and Cribb (2010), the V. pompona group is recovered as monophyletic (V. paulista included). Within this clade, V. aff. chamissonis, which is found in semideciduous forests and Cerrado areas of Brazil, is more related to Vanilla calyculata (= V. columbiana Rolfe, Karremans et al. 2020) than to Vanilla chamissonis, which occurs along the Atlantic coast. Further studies involving members of this Vanilla group are needed in order to better understand their species boundaries. According to the classification of Soto-Arenas and Cribb (2010), V. appendiculata is a member of the V. planifolia group, while V. hartii has been placed in the V. trigonocarpa group. Our data suggest that realignment is necessary because V. trigonocarpa emerges in a basal position within the non-membranous hemiepiphyte Vanilla, while V. hartii emerges among the most derived clades in the genus. Indeed, the clade containing V. hartii, V. rupicola and V. appendiculata emerges as sister to the V. hostmanii group. Consequently, both the V. trigonocarpa group and V. planifolia group are polyphyletic (Soto-Arenas and Cribb 2010). According to the current Vanilla classification, V. rupicola emerges in an unrecognized group. As detailed in the results section (see Morphological Affinities and Table 1), V. rupicola shares a number of characteristics with V. hartii and V. appendiculata, mainly with regard to leaf shape, inflorescence position (i.e. apical, as in V. appendiculata), the type of appendages of the central crest of the labellum, and labellar color pattern (Ferreira et al. 2020; Engels and Ferneda Rocha 2016; Barona-Colmenares 2018).

The rupiculous habit of V. rupicola is unique among the members of the non-membranaceous Neotropical Vanilla. The emergence of a rupicolous species within an entirely hemiepiphytic clade appears to be a result of evolutionary convergence (reversion), as some leafless Old-World species grow on rocks (e.g. Andriamihaja et al. 2020). Despite its rarity in Neotropical Vanilla, the rupicolous habit is widespread among Angiosperms distributed in the Espinhaço Range (e.g. Barros 1987; Azevedo and Van Den Berg 2007). Indeed, Neotropical Vanilla species are diverse and widespread throughout the Atlantic Forest and the Amazon Biome (Hoehne 1945). Based on the evidence that V. rupicola emerges in an essentially Amazonian clade, it seems plausible that the ancestor of this new taxon derived from an Amazonian ancestor adapted to the environmental conditions of the Espinhaço Range and evolved in this particular environment. Nowadays, there is consensus about the evolution of the Neotropical Biomes. During the Paleogene, the ancient Amazon Forest and Atlantic Forest were spatially interconnected and continuous (Morley 2000). The Espinhaço refuges evolved between the two Biomes as a consequence of extreme environmental conditions and climatic fluctuations during the Tertiary and Quaternary periods (Zappi 2008). Their unique geomorphological and climatic conditions resulted in a huge number of endemisms (Rapini et al. 2008; Zappi 2008), whose exclusive diversity is the consequence of a long process of evolution in this singular environment (Silva et al. 2008). Many orchids occur exclusively in the Espinhaço Range of Minas Gerais (e.g. Barros 1987; Azevedo and Van Den Berg 2007; Batista et al. 2016; Salazar et al. 2019). Here, an endemic Vanilla from the ERMG is reported for the first time. This is not surprising because Brazil, with more than 38 species, is the center of diversification for Neotropical Vanilla (Pansarin 2019). Furthermore, five species are endemic to the Brazilian Biomes (Hoehne 1945; Pansarin 2010; Pansarin and Miranda 2016; Fraga et al. 2017). Therefore, knowledge about the taxonomy and phylogeny of Brazilian Vanilla is fundamental in order to understand the evolution and natural history of this Pantropical orchid genus (Pansarin et al. 2012).

Acknowledgements

The first author thanks ICMBIO for permission to collect (Protocol SISBIO number 35178-1), and Elettra Greene for English correction. E.L.F.M. Thanks are due to Fabiane Nepomuceno da Costa (DIAM) and Evandro Luiz Mendonça Machado (HDJF) for logistical support and to FAPEMIG for granting a scholarship.

Additional information

Conflict of interest

No conflict of interest was declared.

Ethical statement

No ethical statement was reported.

Funding

Research supported by the São Paulo Research Foundation – FAPESP (Grant 2018/07357-5) and by CNPq (Productivity Research Grant 301773/2019-0).

Author contributions

Both authors have read and approved the manuscript: E.R.P. - concept of the study, field work, phylogenetic analysis, preparation of draft version of manuscript, and contribution to final version; E.L.F.M.- field work, and contribution to final version.

Author ORCIDs

Emerson Ricardo Pansarin https://orcid.org/0000-0002-0355-8363

Euler da Luz Fernandes Menezes https://orcid.org/0000-0002-1861-9552

Data availability

All of the data that support the findings of this study are available in the main text or Supplementary Information.

References

Andriamihaja CF, Ramarosandratana AV, Grisoni M, Jeannoda V, Besse P (2020) The leafless Vanilla species‐complex from the South‐West Indian Ocean region: A taxonomic puzzle and a model for orchid evolution and conservation. Research. Diversity (Basel) 12(12): 443. https://doi.org/10.3390/d12120443

Azevedo CO, Van Den Berg C (2007) Análise comparativa de áreas de campos rupestres da Cadeia do Espinhaço (Bahia e Minas Gerais, Brasil) baseada em espécies de Orchidaceae. Sitientibus. Série Ciências Biológicas 7: 199–210.

Barona-Colmenares AA (2018) Two new records in Orchidaceae (Vanillinae) from southernmost Colombian Amazonia: Vanilla javieri, a new species, and Vanilla appendiculata. Phytotaxa 375(4): 261–273. https://doi.org/10.11646/phytotaxa.375.4.2

Barros F (1987) Orchidaceae. In: Giulietti AM, Menezes NL, Pirani JR, Meguro M, Wanderley MGL, Flora da Serra do Cipó, Minas Gerais: caracterização e lista de espécies. Boletim de Botânica da Universidade de São Paulo 9(0): 1–151. https://doi.org/10.11606/issn.2316-9052.v9i0p1-151

Barros F, Pinheiro F (2004) Flora de Grão-Mogol, Minas Gerais: Orchidaceae. Boletim de Botânica da Universidade de São Paulo 22(2): 361–383. https://doi.org/10.11606/issn.2316-9052.v22i2p361-383

Batista JAN, Vale AA, Carvalho BM, Proite K, Ramalho AJ, Munhoz ACD, van den Berg C, Bianchetti LB (2016) Four new species in Habenaria (Orchidaceae) from the Espinhaço Range, Brazil. Systematic Botany 41(2): 275–292. https://doi.org/10.1600/036364416X691858

Bouetard A, Lefeuvre P, Gigant RL, Bory S, Pignal M, Besse P, Grisoni M (2010) Evidence of transoceanic dispersion of the genus Vanilla based on plastid DNA phylogenetic analysis. Molecular Phylogenetics and Evolution 55(2): 621–630. https://doi.org/10.1016/j.ympev.2010.01.021

Cameron KM (2003) Vanilloideae. In: Pridgeon A, Cribb P, Chase MW, Rasmussen F (Eds) Genera Orchidacearum. Oxford University Press, Oxford, 281–334.

Doyle JJ, Doyle JS (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19: 11–15.

Dressler RL (1993) Phylogeny and classification of the orchid family. Dioscorides Press, Portland, 1–314.

Engels ME, Ferneda Rocha LC (2016) Vanilla appendiculata (Orchidaceae): Primeiro registro para o estado do Mato Grosso, Brasil. Rodriguésia 67(3): 855–858. https://doi.org/10.1590/2175-7860201667323

Felsenstein J (1985) Confidence limits on phylogenies: An approach using the bootstrap. Evolution; International Journal of Organic Evolution 39(4): 783–791. https://doi.org/10.2307/2408678

Ferreira AWC, Pansarin ER, Franken EP (2020) Confirmation of the presence of Vanilla hartii Rolfe (Orchidaceae, Vanilloideae) in Brazil. Check List 16(4): 951–956. https://doi.org/10.15560/16.4.951

Fidalgo O, Bononi VLR (1989) Técnicas de coleta, preservação e herborização de material botânico. Instituto de Botânica, São Paulo, 1–62.

Fraga CN, Couto DR, Pansarin ER (2017) Two new species of Vanilla (Orchidaceae) in the Brazilian Atlantic Forest. Phytotaxa 296(1): 63–72. https://doi.org/10.11646/phytotaxa.296.1.4

Gonçalves DJP, Shimizu GH, Yamamoto K, Semir J (2017) Vochysiaceae na região do Planalto de Diamantina, Minas Gerais, Brasil. Rodriguésia 68(1): 159–193. https://doi.org/10.1590/2175-7860201768124

Hall TA (1999) BioEdit: A user-friendly biological sequence alignment editor and analysis. Program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98.

Hensold N (1988) Morphology and Systematics of Paepalanthus Subgenus Xeractis (Eriocaulaceae). In: Anderson C (Ed.) Systemactic Botany Monographs, vol. 23. The American Society of Plant Taxonomists, Michigan, 1–150. https://doi.org/10.2307/25027709

Hoehne FC (1945) Orchidáceas. In: Hoehne FC (Ed.) Flora Brasílica, Fasc. 8 (Vol. XII, II; 13–43). Secretaria da Agricultura, Indústria e Comércio de São Paulo, São Paulo, 1–389. [+ 209 Tabs]

IUCN (2012) IUCN red list categories and criteria. Version 3.1. Second edition. Gland and Cambridge: International Union for Conservation of Nature and Natural Resources. http://www.iucnredlist.org/technical-documents/categories-and-criteria

IUCN (2016) Guidelines for using the IUCN red list categories and criteria. Version 12. IUCN, Gland, Switzerland and Cambridge, UK. http://www.iucnredlist.org/documents/RedListGuidelines.pdf

Karremans AP, Chinchilla IF, Rojas-Alvarado G, Cedeño-Fonseca M, Damián A, Léotard G (2020) A reappraisal of Neotropical Vanilla. With a note on taxonomic inflation and the importance of alpha taxonomy in biological studies. Lankesteriana 20: 395–497. https://doi.org/10.15517/lank.v20i3.45203

Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16(2): 111–120. https://doi.org/10.1007/BF01731581

Köppen W (1948) Climatologia: con un estudio de los climas de la tierra. Fondo de Cultura Económica, México, 478 pp.

Kress WJ, Prince LM, Williams KJ (2002) The phylogeny and a new classification of the gingers (Zingiberaceae): Evidence from molecular data. American Journal of Botany 89(10): 1682–1696. https://doi.org/10.3732/ajb.89.10.1682

Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution 35(6): 1547–1549. https://doi.org/10.1093/molbev/msy096

Morley RL (2000) Origin and evolution of tropical Rainforests. Wiley, New York, 384 pp.

Myers N, Mittermeier RA, Mittermeier CG, Fonseca GAB, Kent J (2000) Biodiversity hotspots for conservation priorities. Nature 403(6772): 853–858. https://doi.org/10.1038/35002501

Oliveira JPS, Garrett R, Koblitz MGB, Macedo AF (2022) Vanilla flavor: Species from the Atlantic forest as natural alternatives. Food Chemistry 375: 131891. https://doi.org/10.1016/j.foodchem.2021.131891

Pansarin ER (2004) Cleistes pusilla (Orchidaceae): A new species from Central Brazil. Kew Bulletin 59(4): 555–558. https://doi.org/10.2307/4110910

Pansarin ER (2010) Taxonomic notes on Vanilleae (Orchidaceae: Vanilloideae): Vanilla dietschiana, a rare south American taxon transferred from Dictyophyllaria. Selbyana 30: 198–202.

Pansarin ER (2019) The use of multiple data sources to elucidate the identity of Brazilian vanillas (Vanilloideae, Orchidaceae). Proceedings, 22^nd World Orchid Conference. Asociación Ecuatoriana de Orquideologia, Guayaquil, Ecuador, 162–167.

Pansarin ER (2022) Vanilla flowers: Much more than food-deception. Botanical Journal of the Linnean Society 198(1): 57–73. https://doi.org/10.1093/botlinnean/boab046

Pansarin ER, Ferreira AWC (2022) Evolutionary disruption in the pollination system of Vanilla (Orchidaceae). Plant Biology 24(1): 157–167. https://doi.org/10.1111/plb.13356

Pansarin ER, Miranda MR (2016) A new species of Vanilla (Orchidaceae: Vanilloideae) from Brazil. Phytotaxa 267(1): 84–88. https://doi.org/10.11646/phytotaxa.267.1.9

Pansarin ER, Pansarin LM (2014) Floral biology of two Vanilloideae (Orchidaceae) primarily adapted to pollination by euglossine bees. Plant Biology 16: 1104–1113. https://doi.org/10.1111/plb.12160

Pansarin ER, Suetsugu K (2022) Mammal-mediated seed dispersal in Vanilla: Its rewards and clues to the evolution of fleshy fruits in orchids. ESA Ecology e3701. https://doi.org/10.1002/ecy.3701

Pansarin ER, Salatino A, Pansarin LM, Sazima M (2012) Pollination systems in Pogonieae (Orchidaceae: Vanilloideae): A hypothesis of evolution among reward and rewardless flowers. Flora (Jena) 207(12): 849–861. https://doi.org/10.1016/j.flora.2012.09.011

Pansarin ER, Aguiar JMRBV, Pansarin LM (2014) Floral biology and histochemical analysis of Vanilla edwallii Hoehne (Orchidaceae: Vanilloideae): an orchid pollinated by Epicharis (Apidae: Centridini). Plant Species Biology 29(3): 242–252. https://doi.org/10.1111/1442-1984.12014

Platnick NI (1991) On areas of endemism. Australian Systematic Botany 4: 11–12.

Pridgeon AM, Chase MW, Cribb PJ, Rasmussen FN (1999) Genera Orchidacearum, Vol. 1. General Introduction, Apostasioideae, Cypripedioideae. Oxford University, Oxford, 1–197.

Radford AE, Dickison WC, Massey JR, Bell CR (1974) Vascular Plant Systematics. Harper & Row Publishers, New York, 891 pp.

Rapini A, Mello-Silva RD, Kawasaki ML (2002) Richness and endemism in Asclepiadoideae (Apocynaceae) from the Espinhac¸ o Range of Minas Gerais Brazil – a conservationist view. Biodiversity and Conservation 11(10): 1733–1746. https://doi.org/10.1023/A:1020346616185

Rapini A, Ribeiro PL, Lambert S, Pirani Jr (2008) A flora dos campos rupestres da Cadeia do Espinhaço. Megadiversidade 4: 16–24.

Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics (Oxford, England) 19(12): 1572–1574. https://doi.org/10.1093/bioinformatics/btg180

Salazar GA, Batista JAN, Meneguzzo TEC, Cabrera LI, Figueroa C, Calvillo-Canadell L, do Vale AA, Jiménez-Machorro R (2019) Polyphyly of Mesadenus (Orchidaceae, Spiranthinae) and a New Genus from the Espinhaço Range, Southeastern Brazil. Systematic Botany 44(2): 282–296. https://doi.org/10.1600/036364419X15562054132974

Silva JA, Machado RB, Azevedo AA, Drumond GM, Fonseca RL, Goulard MF, Moraes Júnior EA, Martins CS, Ramos Neto MB (2008) Identificação de áreas insubstituíveis para conservação da Cadeia do Espinhaço, estados de Minas Gerais e Bahia, Brasil. Megadiversidade 4: 273–309.

Soto Arenas MA, Cribb P (2010) A new infrageneric classification and synopsis of the genus Vanilla Plum. ex Mill. (Orchidaceae Vanillinae). Lankesteriana 9: 355–398.

Sun Y, Skinner DZ, Liang GH, Hulbert SH (1994) Phylogenetic analysis of Sorghum and related taxa using internal transcribed spacers of nuclear ribosomal DNA. Theoretical and Applied Genetics 89(1): 26–32. https://doi.org/10.1007/BF00226978

Swofford DL (2001) PAUP: Phylogenetic analysis using parsimony (and Other Methods), version 4.b.8. Sinauer Associates, Sunderland.

UNESCO [United Nations Educational, Scientific and Cultural Organization] (2005) Espinhaço Range Biosphere Reserve, Brazil. https://en.unesco.org/biosphere/lac/espinhaco [Accessed 30 Oct. 2022]

Van Dam AR, Householder JE, Lubinsky P (2010) Vanilla bicolor Lindl. (Orchidaceae) from the Peruvian Amazon: Auto-fertilization in Vanilla and notes on floral phenology. Genetic Resources and Crop Evolution 57(4): 473–480. https://doi.org/10.1007/s10722-010-9540-1

Versieux LM, Wendt T (2007) Bromeliaceae diversity and conservation in Minas Gerais state, Brazil. Biodiversity and Conservation 16(11): 2989–3009. https://doi.org/10.1007/s10531-007-9157-7

Zappi DC (2008) Fitofisionomia da Caatinga associada à Cadeia do Espinhaço. Megadiversidade 4: 34–38.

Supplementary material

Supplementary material 1

Species of Vanilla included in the molecular study, their locations, vouchers and GenBank accession numbers

Emerson Ricardo Pansarin, Euler da Luz Fernandes Menezes

Data type: docx. file

Explanation note: VAN = Vanilla germplasm bank, LBMBP Orchid House, Department of Biology, FFCLRP-USP, University of São Paulo, Brazil available at https://www.lbmbplab.net/vanillacollection.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

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﻿Abstract

Key words

﻿Introduction

﻿Materials and methods

﻿Taxon sampling for phylogenetic analysis

﻿DNA extraction, amplification and sequencing

﻿Phylogenetic analyses

﻿Results

﻿Taxonomic treatment

﻿ Vanilla rupicola Pansarin & E.L.F.Menezes, sp. nov.