118 urn:lsid:arphahub.com:pub:71cc5dc6-a767-5334-951f-ef6ae8936459 Plant Ecology and Evolution plecevo 2032-3913 2032-3921 Meise Botanic Garden and Royal Botanical Society of Belgium 10.5091/plecevo.154789 154789 Research Article Orchidaceae Molecular systematics Systematics Taxonomy South America Systematics of the Vanilla chamissonis clade (Orchidaceae): a study based on integrative taxonomy Pansarin Emerson R. epansarin@ffclrp.usp.br https://orcid.org/0000-0002-0355-8363 1 University of São Paulo, FFCLRP, Department of Biology, Ribeirão Preto, SP, Brazil University of São Paulo Sao Paulo Brazil

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

Academic editor: Brecht Verstraete

 2025 21 08 2025 158 2 260 278 004D6EAE-1C5B-5932-AE4A-422E1EE8B0D3 03 04 2025 13 06 2025 Emerson R. Pansarin 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.

Background and aims – The Brazilian biomes include the world’s greatest diversity of Vanilla. This richness has required studies on species boundaries, as many taxa have been described or erroneously synonymized. This is the case for V. argentina, a species described from the Dry Chaco synonymized under V. chamissonis. While studying the diversity of Neotropical Vanilla, differences between plants from the Cerrado/Dry Chaco, the Atlantic Forest, and the Caatinga became evident.

Material and methods – Descriptions of members of the Vanilla chamissonis clade, i.e. V. argentina, V. calyculata, and V. chamissonis are provided and a morphological comparison is given. An identification key and illustrations are provided. Geographic distributions and the reproductive strategies are presented. The phylogenetic relationships among the members of the V. chamissonis clade are discussed based on an ITS (nrDNA) phylogeny.

Key results – Plants occurring in the Cerrado and Dry Chaco correspond to V. argentina, while V. calyculata and V. chamissonis occur in dry environments and in the Atlantic Forest, respectively. Vanilla argentina is distinguished from the other species by several characteristics, such as the size of the leaves and the flower structures, and fruit features. Vanilla argentina is self-compatible, while V. calyculata and V. chamissonis have a reproductive system based on late-acting self-sterility. All three species are assessed as Endangered. The identities of V. carinata, V. gardneri, and V. vellozii are clarified. The name V. argentina is revalidated.

Conclusion – The evaluation of vegetative and reproductive characteristics of both living and dried specimens is crucial to understand the species delimitation among Vanilla. All evidence reveals that V. argentina is distinct from V. chamissonis. Vanilla chamissonis is endemic to Brazil, while V. argentina is distributed throughout Brazil, Argentina, and Paraguay. Vanilla calyculata occurs in dry regions of South and Central Americas. The Vanilla chamissonis clade comprises a lineage of three species within the Vanilla pompona group.

 Atlantic Forest Caatinga Cerrado Dry Chaco phylogeny taxonomy Vanilloideae Fundação de Amparo à Pesquisa do Estado de São Paulo 501100001807 https://ror.org/02ddkpn78 http://doi.org/10.13039/501100001807 Conselho Nacional de Desenvolvimento Científico e Tecnológico 501100003593 https://ror.org/03swz6y49 http://doi.org/10.13039/501100003593 Introduction

The genus Vanilla (Orchidaceae) encompasses more than 100 species distributed throughout tropical regions of Asia, Africa, and Americas (Cameron 2003; Pansarin 2024a; Pansarin and Menezes 2023). With more than 40 species, Brazil is the centre of diversity for Vanilla (Hoehne 1945; Pansarin 2010; Pansarin and Miranda 2016; Pansarin and Menezes 2023; Pansarin 2024a). Vanilla is monophyletic, with three main lineages, two of which occur throughout the Neotropics (Bouetard et al. 2010; Pansarin and Ferreira 2022; Pansarin and Menezes 2023; Pansarin 2024a). These phylogenetic approaches partially recover the infrageneric classification in groups by Soto Arenas and Cribb (2010). Among the infrageneric Vanilla groups, Soto Arenas and Cribb (2010) recognize six species in the Vanilla pompona group: V. calyculata Schltr., V. chamissonis Klotzsch, V. columbiana Rolfe, V. grandiflora Lindl., V. pompona Schiede, and V. vellozii Rolfe, as the name V. pseudopompona is not validly published. Recent studies involving Neotropical Vanilla have shown that the inclusion of specimens from the Cerrado (Brazilian savanna) areas in phylogenetic analyses make V. chamissonis paraphyletic. Specimens from the Cerrado are more related to V. calyculata, a species distributed throughout dry environments of South America, including the Caatinga (Brazilian dry forest), than to plants occurring in the Atlantic Forest (Pansarin and Menezes 2023; Pansarin 2024a). In addition, a phylogenetic inference using plants from the Dry Chaco (tropical dry forest) (Misiones, Argentina) recovered the paraphyly of V. chamissonis as well (Cascales et al. 2023). Besides molecular, geographical, and morphological evidences, preliminary results on the breeding systems reveal differences with regard to the reproductive strategies among species belonging to the V. chamissonis clade, as well as between plants from the Atlantic Forest (tropical rainforest) (i.e. V. chamissonis) and from the Cerrado. Vegetative and floral characteristics of specimens collected within the Cerrado strongly agree with those of V. argentina Hicken and V. chamissonis var. longifolia Hoehne, both taxa currently considered to be conspecific with V. chamissonis (BFG 2025). Vanilla chamissonis was described based on a specimen collected in the state of Santa Catarina, a region inserted in the Atlantic Forest. The name V. chamissonis var. longifolia was described based on a specimen collected in a Cerrado area in the municipality of Itú, São Paulo. However, the latter taxon was never validly published, as the diagnosis provided by Hoehne (1945) was written in Portuguese instead of Latin (International Code of Nomenclature for algae, fungi, and plants, Art. 39.1: Turland et al. 2018). Conversely, Vanilla argentina is a validly published taxon described based on specimens collected by J.A. Martinez in a Dry Chaco area in Formosa, northwestern Argentina (Hicken 1917).

Vanilla columbiana was described by Rolfe (1896) from a specimen collected in the Magdalena River valley, in Colombia (i.e. Goudot s.n.). Both the holotype in K (K000463745) and the isotype in P (P00367038) are degraded, unsatisfactory material (Flanagan et al. 2025). Based on the analysis of the holotype, Soto Arenas and Cribb (2010) stated that the dissected flower of the type is likely an immature bud. However, in a revision of several species, Karremans et al. (2020) proposed further synonyms. Amongst these species, V. calyculata has been synonymized under V. columbiana. Based on an accurate examination of herbaria specimens, including the types of Vanilla calyculata, V. columbiana, and V. phaeantha Rchb.f., the synonymization of V. columbiana under V. calyculata has been reassessed (Flanagan et al. 2025). This study strongly agrees with the previous assertion that the flower in the holotype of V. columbiana is an immature bud, as previously proposed by Soto Arenas and Cribb (2010). As a consequence, the name Vanilla calyculata has been revalidated, and V. columbiana synonymized under V. phaeantha (Flanagan et al. 2025).

Recent investigations have pointed out problems when forming conclusions about species delimitation based solely on herbaria samples in Vanilloideae (Batista et al. 2023; Pansarin 2024a, 2025). Species boundaries based on molecular, chemical, morpho-anatomical and ecological divergencies, besides geographical disjunctions (i.e. integrative taxonomy), have critically advanced the current appraisal and understanding of plant diversity (Barrett and Freudenstein 2011; Batista et al. 2023; Suetsugu et al. 2023; Pansarin 2024a, 2025). Here, I have used an integrative taxonomic approach to determine the species boundaries and the phylogenetic relationships within the Vanilla chamissonis clade. I investigated the species circumscription based on specimens from the Atlantic Forest (i.e. V. chamissonis) and dryer environments (i.e. V. argentina and V. calyculata) by using vegetative and reproductive data from both herbarium and fresh plant material, and also the analysis of breeding systems in order to elucidate their reproductive strategies. The data obtained here are discussed on light of a phylogenetic inference for Vanilla.

 Material and methods Plant features, habitat, and distribution

Fresh and herbarium material of flowering and fruiting plants were used for the investigations. The living material was collected from Brazilian populations and maintained in 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), Brazil). Flowering and fruiting specimens were vouchered according to usual techniques (Fidalgo and Bononi 1989) and then deposited in the LBMBP herbarium. In addition, I undertook a revision of Vanilla specimens present in the Brazilian herbaria: BHCB, CEN, CVRD, F, FLOR, FURB, HCF, HRCB, HUEFS, HUENF, HUNI, IBGE, ICN, JOI, LBMBP, MBM, MBML, RBR, RFA, SJRP, SP, UB, UEC, UPCB, and VIES. International herbaria were also consulted: AFP, AMES, BM, BIGU, BOG, COL, CUVC, FMB, HUA, ICESI, K, LAGU, MEDEL, MEXU, NY, UDBC, and US (acronyms according to Thiers 2024).

Plant structures were measured using a Vernier Caliper. The vegetative and reproductive structures were photographed with a Nikon D-SLR D800 camera and a Micro Nikkor 105 mm f2.8 lens. Floral details were analysed with a Stereozoom Leica S8 APO stereomicroscope with integrated photo output. Digitized images were used for diagramming a template over a black background, following the model presented by Hoehne (1945), using Microsoft PowerPoint. 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 the types of Vanilla chamissonis and related species were consulted.

The distribution map of all species belonging to the V. chamissonis clade was based on both herbaria material and living specimens from the Vanilla germplasm bank (LBMBP Orchid House). The map with the geographic distribution of Vanilla species was produced with QGIS v.3.32 (QGIS Development Team 2023). The conservation statuses of the Vanilla species were assessed according to the IUCN Red List categories and criteria, and guidelines (IUCN Standards and Petitions Committee 2024). The area of occupancy (AOO), estimated using a 2 × 2 km grid, and the extent of occurrence (EOO) were calculated using GeoCAT (Bachman et al. 2011).

 Taxon sampling for phylogenetic analysis

A total of 64 Vanilla accessions (41 species) were analysed and are referred to here as the ingroup. Lecanorchis multiflora J.J.Sm. was selected as an outgroup according to previous phylogenetic studies on Vanilloideae (e.g. Pansarin and Ferreira 2022; Pansarin and Menezes 2023). A data matrix was built based on sequences available in GenBank and obtained during the development of this study. 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 specimens were extracted from fresh material according to a modified CTAB method (Doyle and Doyle 1987). The amplifications were carried out using 50 µL PCR volumes. Denaturation of the DNA strands was facilitated by the addition of a 5 M betaine solution to the PCR reactions. Primers of the nuclear ribosomal transcribed spacer region (ITS), including the 5.8S gene, were used for amplification and sequencing (Sun et al. 1994). 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 s, 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 BigDye v.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) were used for sequence editing and the assembly of complementary and overlapping sequences. DNA sequences were aligned using BioEdit v.5.0.9 (Hall 1999).

 Phylogenetic analyses

Maximum parsimony analysis (MP) was run with PAUP* v.4.0b5 (Swofford 2001). For the ITS matrix, a heuristic search was conducted on 64 Vanilla accessions 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).

Additionally, an ITS data matrix containing 42 Vanilla accessions was analysed by Bayesian Inference (BI) using MrBayes v.3.1 (Ronquist and Huelsenbeck 2003). The optimal model of sequence evolution was selected using MEGA X (Kumar et al. 2018) based on the Bayesian Information Criterion (BIC). The software selected 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. Tracer v.1.7.2 (Rambaut et al. 2018) was used to assess chain convergence to check if the effective sample sizes (ESS) are above 200 for all parameters. The consensus tree was calculated after removal of the first 3,000 trees, which were considered to be the burn-in. Posterior probability (PP) values above 0.5 were calculated and mapped above the branches of the consensus tree.

 Breeding systems

Here, I carried out experimental pollinations in order to investigate the reproduction strategies of species belonging to the V. chamissonis clade. The specimens of V. chamissonis (N = 10) and V. argentina (N = 10) used for breeding system investigations were collected from natural populations of the southeastern Brazil (municipalities of Ubatuba and Itirapina, respectively) and cultivated at the LBMBP Orchid House. The LBMBP Orchid House is completely covered with 50% black shade cloth to prevent any possible insect visitors coming into contact with the experimental plants. The plants of V. chamissonis and V. argentina used in this study are part of the Vanilla germplasm bank of the LBMBP Orchid House. The experimental treatments for V. calyculata were performed from natural populations occurring in the municipality of Unaí, in the state of Minas Gerais, southeastern Brazil.

The following treatments were conducted to check the breeding system in members of the V. chamissonis clade: spontaneous self-pollination, manual self-pollination to test the occurrence of self-compatibility, cross-pollinations, and emasculations for apomixis. A total of 120 flowers (10 plants; 30 inflorescences; 30 flowers per treatment) were used. All treatments were randomly applied to each inflorescence using one-day flowers, as flowers of Vanilla commonly last one day (Pansarin 2022; Pansarin and Ferreira 2022). The number of manipulations per inflorescence varied depending on the availability of flowers. Treatments were performed in the morning hours (08:00–09:00 am), during the flowering periods of studied species; i.e. from October 2021 to December 2021, and from October 2022 to November 2022. The pollen content of an anther was used in each experimental pollination event (manual self- and cross-pollinations). Cross-pollinations were performed with previously emasculated flowers and using pollen from a distinct accession. Fruit set from experimental treatments was recorded when pods had ripened. Fruit set in successful treatment of manual self-pollination was compared with the treatment of cross-pollination using a t-test for independent samples with Statistica v.6.0 (StatSoft 2003).

 Results Taxonomic treatment Identification key to the species belonging to the <italic><tp:taxon-name><tp:taxon-name-part taxon-name-part-type="genus" reg="Vanilla">Vanilla</tp:taxon-name-part> <tp:taxon-name-part taxon-name-part-type="species" reg="chamissonis">chamissonis</tp:taxon-name-part></tp:taxon-name></italic> clade
1. Plants occurring in dry environments (dry forests and Caatinga vegetation), leaf blade linear to lanceolate, sepals 7.5–8 cm long, sepals revolute, petals linear-oblanceolate, labellum 8.8–9.5 cm long, nectar chamber 3.2–3.6 cm long, column 55–60 mm long, fruit dehiscent, mature fruit yellowish, pericarp hard V. calyculata
– Plants occurring in the Atlantic Forest, Dry Chaco, and Cerrado, leaf blade elliptic to oblong (rarely lanceolate), sepals < 7.5 cm long, sepals spreading, petals linear to oblong-elliptic, labellum < 8 cm long, nectar chamber < 3 cm long, column up to 40 mm long, fruit indehiscent, mature fruit brown, pericarp soft to half hard 2
2. Plants occurring in the Dry Chaco and Cerrado, leaf blade elliptic (rarely lanceolate), sepals 6.8–7.4 cm long, petals 6.6–7.1 cm long, linear, labellum 6.7–7.4 cm long, nectar chamber 1.8–2 cm long, column 38–40 mm long, pericarp half hard V. argentina
– Plants occurring in the Atlantic Forest, leaf blade elliptic to oblong, sepals 4.9–6.1 cm long, petals 4.9–6.1 cm long, oblong-elliptic, labellum 5.3–6.2 cm long, nectar chamber 1.4–1.6 cm long, column 36–38 mm long, pericarp soft V. chamissonis
Plantae Asparagales Orchidaceae FC850C2B-161D-5863-9A6E-B8FB6FDE65A6 Vanilla argentina Hicken (Hicken 1917: 235) Figs 1 , 4 , 6 , 5 , 7 , Table 1 Type

ARGENTINA – Formosa • Pilcomayo; 23 Sep. 1915; J.A. Domínguez 305; lectotype (designated here): SP! [SP45706B]; isolectotype: SI not found.

10.5091/plecevo.154789.figure1 55C9363F-3EEA-586C-A8D8-09330194B07A

Vanilla argentina Hicken. A. Part of a flowering plant showing the stem, leaves, and inflorescence. B. Flower in diagonal view. C. Flower in lateral view. D. Flower in front view. E. Dissected perianth. The details (dashed areas) show the central labellar crest (left) and the penicillate callus (right). F. Detail of the adaxial surface of a petal showing the longitudinal keel. G. Detail of the apex of the labellum showing the yellowish longitudinal crest with five longitudinal ribs and the lateral lobes overlapping the column apex. H. Pedicel/ovary, column, and labellum in lateral view. I. Pedicel/ovary and column in lateral view. J. Apex of the column: in lateral view with an articulated anther (above), in abaxial view (mid), and in lateral view with a disarticulated anther (below). K. Anther in dorsal view (left) and ventral view (right). L. Pollen mass in dorsal view (left) and ventral view (right). M. Mature fruit. N. Transversal section of a mature fruit. Note the hollow fruit cavity. Based on E.R. Pansarin 1570 (LBMBP).

https://binary.pensoft.net/fig/1393696 Description

Nomadic vines, long scandent. Roots axillary, one per node; terrestrial roots up to 8 mm diam., fleshy, whitish, with hyaline absorbing hairs; aerial roots 2.2–2.5 mm diam., whitish to brownish. Stem climbing, cylindrical, fleshy, straight to sinuous, glabrous, glaucous to dark green, strongly furrowed under arid conditions; internodes of ascendant stems 4–16 × 0.5–10 cm. Leaves 6–22 × 3–5.5 cm, alternate, distichous, elliptic to lanceolate, symmetric to asymmetric, fleshy, glabrous, green, pseudopetiolate, margin entire, base attenuate, apex acuminate; pseudopetiole 5.5–8 mm concave. Inflorescence axillary, racemose, with up to 14 flowers opening in succession; 1–2 flowers opening each morning; rachis 5–15.5 × 0.8–1.3 cm, terete, glaucous to dark green; bracts 5–9 × 4–7.5 mm, progressively smaller toward the apex, triangular to ovate, coriaceous, concave, green, erect to patent, apex acute, not incurved. Flowers resupinate, pedicellate, abscission layer between perianth and ovary present; pedicel with ovary 45–50 × 3–4 mm, trigonous in transverse section, white at the base, green to the apex, with a calyculus (6–6.5 × 2.5–3) at the apex. Sepals 6.8–7.4 × 1.2–1.4 cm, free, oblanceolate, fleshy, slightly concave, spreading, pale green to yellowish, margin entire to the apex and involute at the base, base attenuate, apex acute, somewhat thickened; dorsal sepal symmetric; lateral sepals asymmetric; base slightly incurved. Petals 6.6–7.1 × 1.2–1.5 cm, free, obliquely linear, asymmetric, lower margin more arcuate, membranous, pale green, base attenuate, apex acute to obtuse, adaxial surface with a central and longitudinally disposed keel. Labellum 1-lobed to slightly 3-lobed, 6.7–7.4 × 3.8–4.2 cm, tubular, deepening near the middle, yellowish to the base, white in distal portion, unguiculate, with a central crest from the unguiculus to the apex, and a penicillate callus just below the anther; unguiculus fused along the margins of the basal half (ca 30–35 mm) of column length forming a nectar chamber, nectar chamber 1.8–2 cm long, tubular; central crest yellowish from the nectar chamber to the penicillate callus, dark yellow to the apex; distal portion of the central crest swollen, low cushion, rugose-papillose at the apex, with a group of transversal yellow-orange scales near the penicillate callus, with five yellow longitudinal lines near the apex; penicillate callus 5.2–6 × 4.4–4.7 mm, made by yellow-hyaline lacerate-laciniate scales and clusters of trichomes; lateral lobes not much evident, rounded, overlapping the column apex, margin undulate; midlobe deeply emarginated; margin undulate. Column 38–40 × 4.5–5 cm, trigonous, arched to the base, forming an angle ca 90° with the ovary, strait to the apex, ventral surface flat with white to yellowish hyaline trichomes over the distal half, attenuate to the base, dilated to the apex, with two lateral wings; lateral wings rounded, bilobed, undulate. Stigma bilobed; rostellum 5–5.2 × 3.7–3.9 mm, trapezoidal, membranous, white. Anther 5.8–6 × 4.7–4.9 mm, rectangular to trapezoidal, white, versatile, apex truncate; pollen mass 3.2–3.3 × 3.5–3.8 mm, triangular, bipartite, yellowish. Fruits 11–16 × 2.3–2.8 cm, oblong, straight, transversally trigonous, fleshy, brown and indehiscent when mature, fragrant; pericarp half hard; fruit cavity hollow. Seeds ca 0.5 mm, ovoid, black.

Comparison of the morphological features of Vanilla argentina and the related species, V. chamissonis and V. calyculata.

Characteristic V. argentina V. chamissonis V. calyculata
Vegetation Cerrado, Dry Chaco Atlantic Forest Dry forests, Caatinga
Leaf blade elliptic to lanceolate elliptic to oblong linear to lanceolate
Rachis size (cm) 5–15.5 × 0.8–1.3 5–9 × 0.9–1.3 6–11 × 1.1–1.8
Sepal orientation spreading spreading revolute
Sepals size (cm) 6.8–7.4 × 1.2–1.4 4.9–6.1 × 0.9–1.3 7.5–8 × 1.1–1.4
Sepal shape oblanceolate oblanceolate oblanceolate to oblong-elliptic
Petals size (cm) 6.6–7.1 × 1.2–1.5 4.9–6.1 × 0.9–1.2 7.5–8 × 0.9–1.3
Petal shape linear oblong-elliptic linear-oblanceolate
Labellum size (cm) 6.7–7.4 × 3.8–4.2 5.3–6.2 × 3.3–4.2 8.8–9.5 × 3.7–4.5
Nectary length (cm) 1.8–2 1.4–1.6 3.2–3.6
Labellar crest five conspicuous lines three conspicuous lines inconspicuous lines
Column size (mm) 38–40 × 4.5–5 36–38 × 3.2–3.5 55–60 × 4–4.5
Fruit size (cm) 11–16 × 2.3–2.8 12–17 × 2.6–3.5 15–21 × 2–3
Fruit dehiscence indehiscent indehiscent dehiscent
Fruit transverse section trigonous subtrigonous terete to subtrigonous
Fruit pericarp half hard soft hard
Fruit colour brown brown yellowish
Fruit maturation 18 months 18 months 9 months
Breeding system self-compatible self-incompatible self-incompatible
Distribution and ecology

Vanilla argentina occurs in Cerrado areas in Argentina, Brazil, and Paraguay (Fig. 4). The species is commonly found in gallery forests, at elevation is from 657 to 850 m a.s.l. Vanilla argentina has a nomadic vine habit, commonly rooting on the forest litterfall, and climbing on tree trunks. The flowers are fragrant. Each flower lasts ca 1 day. The fruits ripen 18 months after pollination. The fruits are indehiscent and have an unpleasant aroma and a bitter flavour.

 Phenology

Vanilla argentina blooms from October to December. The fruits ripen from May to July.

 Preliminary IUCN conservation assessment

Endangered: EN B2ab(i,ii,iii). Vanilla argentina is an uncommon and rarely collected species currently known to grow in Cerrado areas of southeastern and central-western Brazil and Dry Chaco vegetation in Argentina and Paraguay. The populations are composed of few sparse specimens. The extent of occurrence (EOO) is estimated to be 1,344,031 km2, which falls within the limits for Least Concern (LC) under criterion B1, according to the IUCN Red List categories and criteria. The area of occupancy (AOO) is estimated to be 80 km2, which falls within the limits for Endangered (EN) under criterion B2. Considering that the Cerrado areas have been reduced to scattered fragments due to the expansion of soybean and sugar cane crops and the transformation of native areas into pastures lands, I project a continuing decline in (i) extent of occurrence, (ii) area of occupancy, and (iii) extent and/or quality of habitat for V. argentina. Based on these threats and the fact that the species is distributed in less than five locations, V. argentina is assessed as Endangered: EN B2ab(i,ii,iii).

 Additional material examined

ARGENTINA – Misiones • San Pedro, Catamarca; 26°37’45”S, 54°06’48”W; 548 m; Dec. 1916; T. Rojas 2040; SP.

BRAZIL – Distrito Federal • Brasília, Parque Nacional de Brasília, Cascalheira do Exército; 15°46’48”S, 47°55’45”W; 16 Sep. 2018; C.R. Martins 3054; CEN • FERCAL - APA da Cafuringa, a 38 km do CENARGEN; 15°46’48”S, 47°55’45”W; 910 m; 9 Aug. 1990; T.B. Cavalcanti 598; CEN • Taguatinga, Floresta Nacional de Brasília (Área 1); 15°50’00”S, 48°03’23”W; 1200 m; 23 Nov. 2007; J.H. Lima 32; UB. – Goiás • Campos Belos, Estrada velha de São Domingos para Campos Belos; 13°01’31”’S, 46°45’54”’W; 652 m; 22 Oct. 2001; M.L. Fonseca 3002; IBGE • Cristalina, Margem direita do Rio Preto; 16°46’4”S, 47°36’47”W; 1237 m; 12 Aug. 2002; A. Amaral-Santos 1367; CEN • Formosa, Ribeirão Bezerra; 15°32’13”S, 47°20’09”W; 911 m; 8 Oct. 2002; E. Tameirão Neto 3567; BHCB • Morrinhos, Rodovia Morrinhos-Caldas Novas; 17°43’16”S, 49°06’29”W; 768 m; 4 Sep. 1976; G. Hatschbach 38915; MBM. – Mato Grosso • Nova Xavantina, ca 70 km N of Xavantina; 14°22’S, 52°37’W; 300–400 m; 10 Oct. 1964; H.S. Irwin 6750; NY. – Mato Grosso do Sul • Campo Grande; 20°26’37”S, 54°38’52”W; 612 m; 15 Nov. 2023; E.R. Pansarin 1573; LBMBP. – Minas Gerais • Belo Horizonte, Campus da UFMG; 19°48’57”S, 43°57’15”W; 5 Nov. 1994; V.A. de Sousa s.n.; BHCB 24796 • Diamantina, Serra do Espinhaço; 18°14’17”S, 43°36’40”W; 975 m; 17 Apr. 1972; H.S. Irwin 28146; NY • Serra do Espinhaço, Mata Ciliar; 18°14’17”S, 43°36’40”W; 970 m; 25 Oct. 2023; E.R. Pansarin 1571; LBMBP • Mariana, Mina da Samitri; 20°22’41”S, 43°25’00”W; 718 m; 28 Nov. 2000; R.C. da Mota & L. Viana 570; BHCB • Paracatú, Cerrado, ca 5 km S.E. of Paracatú; 17°13’21”S, 46°52’31”W; 650m; 6 Feb. 1970; H.S. Irwin et al. 26210; UB • Pimhuí; 20°27’42”S, 45°56’45”W; 818 m; 1 Nov. 2023; E.R. Pansarin 1572; LBMBP • São Gonçalo do Rio Abaixo; 19°49’41”S, 43°22’55”W; 743 m; 21 Oct. 1993; E.L. Borba 79; BHCB. – São Paulo • Itirapina, Itaqueri da Serra, Mata de Galeria; 22°15’10”S, 47°49’22”W; 817 m; 22 Oct. 2023; E.R. Pansarin 1569; LBMBP • Itirapina, Itaqueri da Serra, Mata de Galeria; 22°15’10”S, 47°49’22”W; 817 m; 29 Oct. 2023; E.R. Pansarin 1570; LBMBP • Itú, Capão Sombrio; [23°15’57”S, 47°17’57”W]; 577 m; 20 Dec. 1897; A. Russel CGG3840; SP • São José do Rio Preto, Estação Ecológica do Noroeste Paulista; 20°43’36”S, 49°22’50”W; 410 m; 10 Oct. 1996; W. Forster 9; SJRP.

PARAGUAY • Cordillera de Altos; 25°16’55”S, 57°38’6”W; 7 Dec. 1902; K. Fiebrig 564; F.

 Notes

Vanilla argentina has been considered synonymous with V. chamissonis, the latter assumed to be a species widely distributed throughout South America. However, some authors have observed differences between specimens collected inland compared to those occurring on the coast. Such differences have resulted in the publication of two taxa, Vanilla argentina and V. chamissonis var. longifolia, the latter being invalidly published, as F.C. Hoehne providing a diagnosis in Portuguese instead of Latin (Turland et al. 2018). My data, based on specimens collected in Cerrado areas in the states of São Paulo, Minas Gerais, and Goiás, show that the plants of some populations have long and dark green leaves that are quite distinct from those from populations in the Gran Chaco. Specimens with long leaves strongly agree with those of V. chamissonis var. longifolia. However, the flowers and fruits are identical. In addition, ITS DNA sequences align perfectly with those of V. argentina. For this reason, the long- and short-leaved plants are treated here as single taxon, V. argentina.

 Morphological affinities

Vanilla argentina is distributed in the Dry Chaco and Cerrado (Fig. 4). The species is recognized by its commonly elliptic leaf blades (Fig. 5). Plants from some Cerrado areas in São Paulo, Goiás, and Minas Gerais (southwestern and central-western Brazil) commonly have long lanceolate leaves. However, this appears to be intraspecific variation, as populations with leaves morphologically similar to those plants found in the Dry Chaco are also found in the Brazilian Cerrado. Apart from vegetative variations, flowers from plants of both biomes are morphologically indistinguishable. Flowers of V. argentina are recognized from the remaining species of the V. chamissonis clade by its sepals 6.8–7.4 cm long, linear petals 6.6–7.1 cm long, labellum 6.7–7.4 cm long with a nectar chamber 1.8–2 cm long and a column 38–40 mm long, and fruits with a half hard pericarp (Figs 6–7). All morphological characteristics support V. argentina as a member of the V. chamissonis clade, which is corroborated by the phylogenetic analysis (see further).

 Plantae Asparagales Orchidaceae 864CA6A5-429F-5B9E-A70B-5FBCFB2092B3 Vanilla calyculata Schltr. (Schlechter 1920: 42) Figs 2 , 4 , 6 , 5 , 7 , Table 1 Vanilla esquipulensis Archila & Chiron (Archila and Chiron 2012: 6) – Type: GUATEMALA • El Barrial, Esquipulas, Chiquimula; 900 m; Apr. 2012; F. Archila 1300 ; holotype: BIGU!. Type

COLOMBIA – Cauca • 1000 m; M. Madero s.n.; holotype: B†. – Valle del Cauca • Tuluá, Corregimiento Mateguadua, Jardín Botánico Juan Maria Céspedes, laderas en vía de repoblación natural, 1100 m a.s.l.; 28 Sep. 1984; W. Devia 815; neotype (designated by Soto Arenas and Dressler 2010): TULV! [TULV003790]; isoneotype NY! [NY04170480].

10.5091/plecevo.154789.figure2 25FEA0D1-D583-5D1B-AFCF-B5AC06DABC94

Vanilla calyculata Schltr. A. Part of a flowering plant showing the stem, leaves, and inflorescence. B. Inflorescence in lateral view showing a mature bud. C. Detail of an inflorescence showing an open flower in diagonal view. D. Flower in upper view. E. Dissected perianth. F. Detail of the apex of the labellum showing the yellowish longitudinal crest. The detail (dashed area) shows the penicillate callus. G. Pedicel/ovary and column in lateral view. H. Apex of the column: in diagonal view with an articulated anther (above), in lateral view with an articulated anther (mid), and in ventral view with a disarticulated anther (below). I. Apex of the column in ventral view showing the bilobed rostellum. J. Anther in ventral view. K. Pollen mass in dorsal view. L. Mature fruit. M. Transversal section of a mature fruit. Note the hollow fruit cavity. Based on VAN 206 (Vanilla germplasm bank, LBMBP Orchid House).

https://binary.pensoft.net/fig/1393697 Description

Nomadic vines, long scandent. Roots axillary, one per node; terrestrial roots up to 10 mm diam., fleshy, whitish, with white-hyaline absorbing hairs; aerial roots 2.2–2.8 mm diam., creamy to brownish. Stem climbing, cylindrical, fleshy, sinuous, glabrous, green, strongly furrowed under arid conditions; internodes of ascending stems 7–16 × 0.6–1.2 cm. Leaves 8–21 × 2–4 cm, alternate, distichous, linear to lanceolate, symmetric, fleshy, glabrous, green, pseudopetiolate, margin often revolute, base rounded, apex acute; pseudopetiole 6–10 mm long, concave. Inflorescence axillary, racemose, with up to 20 flowers opening in succession; 1–3 flowers opening each morning; rachis 6–11 × 1.1–1.8 cm, terete, green; bracts 6.5–10 × 4.5–8 mm, progressively smaller to the apex, widely deltoid, coriaceous, concave, green, apex acute, involute. Flowers resupinate, pedicellate, abscission layer between perianth and ovary present; pedicel with ovary 42–56 × 4.5–5.5 mm, cylindrical, sulcate, whitish at the base, green to the apex, with a calyculus (7.5–8.5 × 3.5–4.5 mm) at the apex. Sepals 7.5–8 × 1–1.4 cm, free, narrowly oblanceolate to oblong-elliptic, slightly concave, revolute, white at the base, green to the apex, margin entire, base attenuate, apex subacute to obtuse, somewhat thickened; dorsal sepal symmetric; lateral sepals asymmetric. Petals 7.5–8 × 0.9–1.3 cm, free, obliquely linear-oblanceolate, asymmetric, lower margin more arcuate, membranous, white at the base, green to the apex, base attenuate, apex acute to rounded, adaxial surface with central and longitudinally disposed keel. Labellum 3-lobed, 8.8–9.5 × 3.7–4.5 cm, tubular, deepening near the middle, yellowish to the base, dark yellow at the distal half and with white lobes, unguiculate, with a central crest from the unguiculus to the apex, and a penicillate callus just below the anther; unguiculus fused along the margins of the basal half (ca 41–46 mm) of column length forming a nectar chamber; nectar chamber 3.2–3.6 mm long, tubular; central crest whitish from the nectar chamber to the penicillate callus, yellow to the apex; distal portion of central crest swollen, low cushion, rugose-papillose at the apex, with a group of transversal and yellow-orange hairs near the penicillate callus, longitudinal lines not much evident; penicillate callus ca 4.5 × 3.8 mm, made up by 8–10 flabellate, shortly lacerate-laciniate and retrorse scales; lateral lobes rounded, bilobed, overlapping the column apex, margins entire; midlobe deeply emarginate to bilobed, margins crenulate-undulate. Column 55–60 × 4–4.5 mm, subterete, subclavate, sigmoid, ventral surface flat with white to yellowish hyaline trichomes over the distal half, dilated to the apex, with two lateral wings; lateral wings with two triangular-flabellate protrusions, narrow, undulate. Stigma bilobed; rostellum 4.2–4.6 × 2.8–3.2 mm; trapezoid, flabellate. Anther 5.2–6 × 4–4.3 mm, rectangular, apex truncate to slightly emarginate; pollen mass 1.9–2.1 × 3.7–4.2 mm, triangular, bipartite, yellow. Fruits 15–21 × 2–3 cm, oblong, transversally terete to subtrigonous, fleshy, yellowish and dehiscent when mature, fragrant; pericarp hard; fruit cavity hollow. Seeds ca 0.5 mm, ovoid, black.

 Distribution and ecology

Vanilla calyculata is distributed throughout dry forests of Brazil, El Salvador, Guatemala, Honduras, and Colombia (Fig. 4). In Colombia, this species is widely distributed in the inter-Andean valleys in the western and central Andean cordilleras, at elevations between 500 and 1750 m a.s.l. When found at lower elevations, the vegetation is sub-xerophytic. In Brazil, this species occurs predominantly in the Caatinga.

 Phenology

Vanilla calyculata blooms from September to November in Brazil. The fruits ripen from June to July. Flowering in Colombia corresponds to two annual periods with higher precipitation: April–May and September–October. Flowers were recorded in April in El Salvador, Guatemala, and Honduras.

 Preliminary IUCN conservation assessment

Endangered: EN B2ab(i,ii,iii). Vanilla calyculata is a species distributed throughout dry forests of Brazil, Colombia, El Salvador, Guatemala, and Honduras. The species is rarely collected in Brazil and the populations are composed of few sparse specimens. The extent of occurrence (EOO) is estimated to be 4,145,682 km2, which falls within the limits for Least Concern (LC) under criterion B1, according to the IUCN Red List categories and criteria. The area of occupancy (AOO) is estimated to be 92 km2, which falls within the limits for Endangered (EN) under criterion B2. In Colombia, the tropical dry forest is distributed across the Caribbean and Eastern (Llanos) plains, and in the inter-Andean valleys in the Cauca and Magdalena watersheds. This ecosystem has been highly transformed for agriculture, cattle grazing, and urbanization, with only 8% of the original landcover remaining. The tropical dry forest ecosystem in Colombia is categorized as CR – critically endangered, with a representativity in the Colombia National Protected Area System (SINAP) of only 5%. In Brazil, the Caatinga vegetation has been reduced to the expansion of human activities in northeastern Brazil. Considering habitat fragmentation, besides the climatic changes that have reduced the raining period in the habitat of V. calyculata specially in Brazil, I project a continuing decline in (i) extent of occurrence, (ii) area of occupancy, and (iii) extent and/or quality of habitat for this species. Based on these threats and the fact that the species is distributed in less than five locations, V. calyculata is assessed as Endangered: EN B2ab(i,ii,iii).

 Additional material examined

BRAZIL – Bahia • Conceição do Coité, Salgadália. Próximo às casas populares; 28 Nov. 2012; D.N. Carvalho 173; HUEFS • s.loc.; 28 Oct. 2008; C.E. Ramos et al. 528; US. – Minas Gerais • Unaí, Ilha a montante do túnel de desvio, cerca de 500 m da ponte de madeira, Margem esquerda do Rio Preto; 12 Sep. 2002; A. Amaral-Santos 1508; CEN • Unaí, Fazenda Saco Grande, margem do córrego, próximo à casa do Sr. Teodorim; 16 Oct. 2019; A. Amaral-Santos 3758; CEN.

COLOMBIA – Cauca • El Socavón, Mercaderes; 1200 m; 3 Feb. 1990; M. Rocio Galindez 103; AFP • Piedrasentada, vereda Piedra Rica; 980 m; 4 May 2002; R.J.C. Muñoz 010; AFP • Vereda Potrerillo, Patía; 624 m; 25 Jan. 2015; G. Reina Rodriguez, I. Nichols, F. Lopez & J. Reyna 2122; FMB. – Cundinamarca • Finca “El Descanso”, vereda Chinauta, Fusagasugá; 1200 m; 3 Apr. 1988; F. Sarmiento 2140; BOG. – Huila • Between La Jagua & Altamira; 880 m; 23 Jul. 1961; L.A. Garay, C.E. McClennen & A. Kapuler 277; AMES. – Nariño • Vía Mojarras-Leyva, vereda Puerto Nuevo, Finca La Sortija, Leyva, Vegetación de Bosque seco; 576 m; 24 Jan. 2015; G. Reina-Rodriguez, I. Nichols, F. Lopez & J. Reyna 2101; FMB. – Santander • Reserva natural de la sociedad civil La Montaña Mágica – El Pole, Zapatoca; 1750 m; 17 May 2017; D. Díaz Rueda, R. Diaz Rueda & L. Rivera 1369; MEDEL. – Tolima • La Plata; 800–1500 m; 3 Dec. 1882; F.C. Lehmann 2263; BM. – Valle del Cauca • Hacienda El Carmen, carretera La Uribe-Sevilla; 1140 m; 19 Jul. 1994; P. Silverstone-Sopkin & N. Paz 6933; CUVC • Finca la Josefina, La Herradura, Bolivar; 1076 m; 5 Aug. 2010; G. Reina Rodriguez & M. Moreno 1344; FMB • Corregimiento Loboguerrero, Dagua; 730 m; 31 Jul.–4 Aug. 1998; W.G. Vargas 4643; COL, HUA • Atuncela, Corregimiento Loboguerrero, Dagua; 950 m; Jul. 1998; W.G. Vargas 6087; ICESI • Reserva Forestal Bosque Yotoco; 27 May 2007; F. Rojas 62; UDBC • Cultivated, Cali; E. Dryander 2379; BM • Roldanillo, Valle; 1000 m; F.C. Lehmann 8378; K.

EL SALVADOR – Depto. Morazán • Mpio. Arambala; 688 m; 7 Mar. 2018; Y. Ruiz s.n.; LAGU [LAGUJBL07763] • Mpio. Perquín, Crio. La Tejera; 1038 m; 19 Jun. 2018; Y. Ruiz 720; LAGU • La Palma, Finca El Refugio, Río Nanuapa; 1000 m; 3 Apr. 1969; O. Pank & F. Hamer 203; AMES.

GUATEMALA – El Barrial • Esquipulas, Chiquimula; 600 m; Mar. 1999; F. Archila s.n.; illustrated in Archila and Chiron (2012); BIGU • Río Jocotan, Chiquimula; 500 m; Apr. 2000; F. Archila s.n.; BIGU.

HONDURAS – Comayagua • Siguatepeque; 1050 m; 23 Jul. 1936; T.G. Yuncker, R.F. Dawson & H.R. Youse 6045; K, NY, AMES • Quebrada Santa Clara, ca 2 km al norte del Zamorano, Mpio de San Antonio de Oriente; 800 m; 19 Jun. 1996; J.L. Linares 3386; MEXU • El Paraiso, Quebrada El Cajocote, 8.7 km al N de Morocelí, por el camino a Mata de Plátano; 680 m; 29 Apr. 2004; J.L. Linares 7313; MEXU • Las Mesas region near Yuscarán; Aug. 1960; H.W. Pfeifer 1454; US.

 Notes

Vanilla calyculata has been considered synonymous with V. columbiana, a species described based on a specimen collected in the Magdalena River valley, Colombia. However, the holotype of V. columbiana is clearly a V. phaeantha with immature flowers. For this reason, the name V. calyculata has been re-established and V. columbiana has been synonymized under V. phaeantha (Flanagan et al. 2025). Vanilla calyculata is one of four species found in tropical dry forest ecosystems in Colombia. In the Cauca valley, it occurs in sympatry with V. odorata, V. phaeantha, and V. pompona. Herbarium specimens of V. calyculata have been historically referred to as V. phaeantha. However, V. calyculata can be distinguished from V. phaeantha by its long leaves larger than the internodes, its pendant flowers, by its pedicels, petals and sepals with white bases, and by its ovary ending in a calyculus.

 Morphological affinities

Vanilla calyculata occurs in dry forests. The species is recognized from the remaining species of the V. chamissonis clade by the following characters: long and linear to lanceolate leaf blades (Fig. 5), revolute sepals 7.5–8 cm long, labellum 8.8–9.5 cm long with a nectar chamber 3.2–3.6 cm long, column 55–60 mm long, and dehiscent fruit yellowish when mature with a hard pericarp (Figs 6–7). Several vegetative and reproductive characteristics support V. calyculata as a member of the V. chamissonis clade, such as the presence of an ovary ending in a calyculus, a labellum with emarginate midlobe, and a yellow-orange elevated central crest. The phylogenetic analysis supports V. calyculata as a member of the V. chamissonis clade (see further).

 Plantae Asparagales Orchidaceae AEFC71E8-D18A-56EA-B723-3BE946E3AF78 Vanilla chamissonis Klotzsch (Klotzsch 1846: 564) Figs 3 , 4 , 6 , 5 , 7 , Table 1 Vanilla chamissonis var. brevifolia Cogn. (Cogniaux 1893: 149). Based on Epidendrum vanilla Vell. (Vellozo 1827: ic. 9, t. 1), nom. illeg. Vanilla gardneri Rolfe (Rolfe 1895: 177), syn. nov. – Type: BRAZIL • Rio de Janeiro, Morro do Flamengo, near Rio de Janeiro; G. Gardner 245; lectotype (designated by Soto Arenas and Dressler 2010): K! [K000463752, K000463753]. Vanilla vellozoi Rolfe (Rolfe 1896: 467), as “Vanilla vellozii”, syn. nov. – Type: BRAZIL • Rio de Janeiro; 1882; A. Glaziou 14302; lectotype designated by Soto Arenas and Dressler 2010): K! [K000940259]. Vanilla carinata Rolfe (Rolfe 1896: 468), syn. nov. – Type: BRAZIL • Rio de Janeiro, Organ Mountains; 1878; J. Miers s.n.; holotype: K! [K000463750]. Type

BRAZIL • Santa Catarina; A. von Chamisso s.n.; lectotype (designated by Christenson 1995: 33): LE! [LE00011144].

10.5091/plecevo.154789.figure3 238F19C0-68F9-5C3C-9285-58932D352CBA

Vanilla chamissonis Klotzsch. A. Part of a flowering plant showing the stem, leaves, and inflorescence. B. Flower in lateral view. C. Flower in diagonal view. D. Flower in front view. E. Dissected perianth. The details (dashed areas) show the central labellar crest (left) and the penicillate callus (right). F. Detail of the adaxial surface of a petal showing the longitudinal keel. G. Detail of the apex of the labellum showing the yellowish longitudinal crest with three longitudinal ribs and the lateral lobes of the labellum overlapping the column apex. H. Pedicel/ovary, column, and labellum in lateral view. I. Pedicel/ovary and column in lateral view. J. Apex of the column: in lateral view with an articulated anther (above), in abaxial view (mid), and in lateral view with a disarticulated anther (below). K. Anther in dorsal view (left) and ventral view (right). L. Pollen mass in dorsal view (left) and ventral view (right). M. Mature fruits. N. Transversal section of a mature fruit. Note the filled fruit cavity. Based on E.R. Pansarin 1579 (LBMBP).

https://binary.pensoft.net/fig/1393698 Description

Nomadic vines, long scandent. Roots axillary, one per node; terrestrial roots up to 8 mm diam., fleshy, whitish, with hyaline absorbing hairs; aerial roots 2.2–3.2 mm diam., whitish to brownish. Stem climbing, cylindrical, fleshy, straight to sinuous, glabrous, glaucous to dark green, strongly furrowed under arid conditions; internodes of ascendant stems 8–14 × 0.7–1.3 cm. Leaves 5.2–18 × 2.5–5.5 cm, alternate, distichous, elliptic to oblong, asymmetric, fleshy, glabrous, pale green to dark green, pseudopetiolate, margin entire, base attenuate, apex acuminate; pseudopetiole 5–10 mm concave. Inflorescence axillary, racemose, with up to 22 flowers opening in succession; 1–2 flowers opening each morning; rachis 5–9 × 0.9–1.3 cm, terete, pale green to dark green; bracts 4–7.5 × 4.5–8 mm, progressively smaller toward the apex, triangular/deltoid, coriaceous, concave, green, patent, apex acute, not incurved. Flowers resupinate, pedicellate, abscission layer between perianth and ovary present; pedicel with ovary 46–50 × 4.2–6 mm, triangular in transverse section, straight to incurved, whitish at the base, green to the apex, with a calyculus (6–6.5 × 3–3.5 mm) at the apex. Sepals 4.9–6.1 × 0.9–1.3 cm, free, oblanceolate, fleshy, slightly concave, spreading, pale green to yellowish, margin entire, involute at the base, base attenuate, apex acute to obtuse; dorsal sepal symmetric; lateral sepals asymmetric. Petals 4.9–6.1 × 0.9–1.2 cm, free, obliquely oblong-elliptic, asymmetric, lower margin more arcuate, yellowish at the base, pale green to the apex, base attenuate, apex obtuse to rounded, adaxial surface with a central and longitudinally disposed keel. Labellum 1-lobed to slightly 3-lobed, 5.3–6.2 × 3.3–4.2 cm, tubular, deepening near the middle, yellowish to the base, white in distal portion, unguiculate, with a central crest from the unguiculus to the apex, and a penicillate callus just below the anther; unguiculus fused along the margins of the basal half (ca 29–32 mm) of column length forming a nectar chamber, nectar chamber 1.4–1.6 cm long, tubular; central crest yellowish from the nectar chamber to the penicillate callus, dark yellow to the apex; distal portion of the central crest swollen, low cushion, rugose-papillose at the apex, with a group of transversal yellow-orange scales near the penicillate callus, with three yellow longitudinal lines near the apex; penicillate callus 5.1–5.5 × 3.8–4.2 mm, made by yellow-hyaline lacerate-laciniate scales and clusters of trichomes; lateral lobes not much evident, rounded, overlapping the column apex, margin undulate; midlobe deeply emarginated; margin undulate. Column 36–38 × 3.2–3.5 cm, trigonous, arched to the base, forming an angle ca 90° with the ovary, strait to the apex, ventral surface flat with white to yellowish hyaline trichomes over the distal half, attenuate to the base, dilated to the apex, with two lateral wings; lateral wings rounded, undulate. Stigma bilobed; rostellum 4–4.2 × 2.5–2.7 mm, trapezoidal, membranous, white. Anther 4.8–5.2 × 3.5–3.6 mm, rectangular to trapezoidal, white, versatile, apex truncate; pollen mass 2.9–3.2 × 2.9–3.1 mm, triangular, bipartite, whitish. Fruits 12–17 × 2.6–3.5 cm, oblong to clavate, arched, transversally subtrigonous, fleshy, brown indehiscent when mature, fragrant; pericarp soft; fruit cavity filled. Seeds ca 0.5 mm, ovoid, black.

 Distribution and ecology

Vanilla chamissonis is endemic to the Brazilian Atlantic Forest (Fig. 4). The species is a nomadic vine found both on tall trees in the restinga vegetation and on shrubs of the rocky outcrops close to the beach. The flowers are very fragrant and produce a small amount of nectar. The fruits have a bitter taste and an unpleasant odour. After 18 months from pollination, they turn brown and fall to the forest floor where they are consumed by agoutis.

10.5091/plecevo.154789.figure4 A21B5190-9312-5220-9588-66FAB56A572B

Occurrence map of Vanilla argentina, V. chamissonis, and V. calyculata.

https://binary.pensoft.net/fig/1393699 Phenology

Vanilla chamissonis blooms from November to January. The fruits ripen from April to June, 18 months from pollination.

10.5091/plecevo.154789.figure5 F6BB3CCC-44BE-5F84-A836-FB3BFD458C2D

Comparative morphology of leaves of related Vanilla species. A. V. argentina. B. V. calyculata. C. V. chamissonis. Scale bars = 2 cm. A from E.R. Pansarin 1570 (LBMBP), B from VAN 206 (Vanilla germplasm bank, LBMBP Orchid House), C from E.R. Pansarin 1579 (LBMBP).

https://binary.pensoft.net/fig/1393700 Preliminary IUCN conservation assessment

Endangered: EN B2ab(i,ii,iii). Vanilla chamissonis is distributed along the Brazilian coast. The species is particularly common in the Atlantic Rainforest of south and southeast Brazil. The populations are commonly composed of many individuals. The extent of occurrence (EOO) is estimated to be 229,334 km2, which falls within the limits for Least Concern (LC) under criterion B1, according to the IUCN Red List categories and criteria. The area of occupancy (AOO) is estimated to be 128 km2, which falls within the limits for Endangered (EN) under criterion B2. Considering that the Brazilian restingas have been reduced to scattered fragments due to the urban occupation, I project a continuing decline in (i) extent of occurrence, (ii) area of occupancy, and (iii) extent and/or quality of habitat of V. chamissonis. Based on these threats and the fact that the species is distributed in less than five locations, V. chamissonis is assessed as Endangered: EN B2ab(i,ii,iii).

 Additional material examined

BRAZIL – Espírito Santo • Espírito Santo, Afonso Cláudio, Serra Pelada, comunidade de Palmital, propriedade da família Brandemburg, Pedra do Sol, Proximidades da Pedra da Lajinha. Inselberg; 20°00’36”S, 41°04’30”W; 760 m; 21 Oct. 2019; C.N. Fraga & D.R. Couto 3912; MBML • Guarapari, Village do Sol; 20°33’16”S, 40°24’35”W; 27 Oct. 1984; B. Weinberg 645; MBML • Guarapari, Parque Estadual Paulo César Vinha; 20°36’19”S, 40°25’19”W; 17 Nov. 1994; C. N. de Fraga et al. 59; MBML • Jaguaré; 18°54’20”S, 40°04’33”W; 78 m; 30 Nov. 2012; D.A. Folli 6934; CVRD • Santa Leopoldina, Rio das Farinhas, propriedade da Sra. Maria Knak Ule. Mata, dossel; 20°01’21”S, 40°36’29”W; 827 m; 15 Apr. 2008; A.P. Fontana, L. Kollmann & K.A. Brahim 4969; MBML • Vila Velha, Jacarenema; 20°19’46”S, 40°17’32”W; 25 May 1990; J.M.L. Gomes & O.J. Pereira 1129; VIES. – Paraná • Matinhos, ao pé do Morro do Tabaquara, perto de Matinhos; 25°49’02”S, 48°32’34”W; 3 Jan. 1967; J.C. Lindeman et al. 3855; MBM • Paranaguá, Ilha de Mel; 25°31’13”S, 48°30’33”W; 3–5 m; 28 Nov. 1970; G.G. Hatschbach 25679; NY • Paranaguá; 25°31’11”S, 48°30’33”W; 3–5 m; 23 Nov. 1994; J. Cordeiro 1201; HUEFS • Paranaguá, Ilha do Mel - Restinga da Praia Grande; 25°31’11”S, 48°30’33”W; 8 Nov. 1986; W.S. Souza 388; UPCB. – Rio de Janeiro • Angra dos Reis, Ilha Grande, Parcelas do Módulo Oeste do RAPELD Ilha Grande em Mata de restinga, Reserva Biológica Estadual da Praia do Sul; 23°10’36”S, 44°17’52”W; 13 Oct. 2016; A.C.R. Cruz & R.G. Diniz 2; RBR • Campos dos Goytacazes, Morro Itaoca - Morro do rato; 21°47’49”S, 41°26’52”W, 201 m; 23 Nov. 2008; A.S. Pessanha & M.T. Nascimento 48; HUENF • Maricá, Barra de Maricá, Área C1; 22°55’09”S, 42°49’06”W; 25 Oct. 1988; Occhioni et al. 9286; RFA • Macaé; 22°22’14”S, 41°47’12”W; 18 Sep. 2008; I.E. Santo & M.F. Castilhori 174; R • Rio de Janeiro, Restinga da Marambaia; 23°03’11”S, 43°35’03”W; 20 May 2009; B.S. Haiad, C. Novaes & I. Soares s.n.; HUNI [HUNI4402]. – Santa Catarina • Balneário Camboriú, Taquarinhas; 26°59’26”S, 48°38’04”W; 17 Nov. 2002; C. Hering-Rinnert 244; JOI • Bombinhas, Praia de Zimbros; 27°08’16”S, 48°31’01”W; 35 m; 11 Nov. 2006; M.G. Caxambú 1270; HCF • Garopaba, Ouvidor - Praia Vermelha; 28°06’42”S, 48°38’02”W; 23 m; 7 Jul. 2018; A. Kassner-Filho 3056; FURB • Florianópolis, Morro do Ribeirão, Ilha de Santa Catarina; 27°35’48”S, 48°32’57”W; 100 m; 23 Sep. 1970; R.M. Klein & A. Bresolin 8773; FLOR • Florianópolis, Lagoa Pequena, Rio Tavares; 27°39’20”S, 48°28’18”W; 11 m; 2 Nov. 2017; G.D.S. Seger & E. Bach 843; ICN • Imaruí, Forquilinha; 28°09’52”S, 48°52’10”W; 666 m; 28 Jan. 2010; J.L. Schmitt et al. 1111; FURB • Laguna, Praia do Sol, Dunas fixas; 28°28’57”S, 48°46’50”W; 3–5 m; 13 Dec. 2000; G. Hatschbach, A.C. Cervi & E. Barbosa 71869; BHCB • Paulo Lopes; 28°01’21”S, 48°42’20”W; 123 m; 18 Nov. 2022; M.E. Engels 10283; UPCB • Penha, J.B. World Entretenimentos S/A; 26°48’06”S, 48°36’58”W; 7 m; 26 Dec. 2018; A. Kassner-Filho & F.L.V. Bones 4490; FURB • São Francisco do Sul, Parque Estadual do Acaraí - Praia do Ervino; 26°20’58”S, 48°33’47”W; 5 m; 29 Nov. 2010; A. Korte & A.L. de Gasper 5252; FURB • São Francisco do Sul; 26°14’35”S, 48°38’17”W; 5 m; 5 Apr. 2008; L. Sevegnani s.n.; FURB [FURB7432]. – São Paulo • Cananéia, Parque Estadual da Ilha do Cardoso, Trilha morro das almas; 25°00’36”S, 47°55’11”W; 19 Mar. 2003; T.B. Breier 953; UEC • Cananéia, Ilha do Cardoso, Restinga do Pereirinha; 25°00’36”S, 47°55’11”W; 2 Dec. 1990; F. Barros & P. Martuscelli 1989; SP • Iguape, Estação Ecológica Juréia-Itatins, Estrada entre o rio Una do Prelado e o Rio Verde, 18 km do rio Una, restinga junto à desembocadura do rio Verde; 24°42’00”S, 47°32’59”W; 9 Dec. 1995; I. Cordeiro et al. 1595; SP • Peruíbe, Estação Ecológica Juréia-Itatins, Barra do Uma; 24°19’12”S, 46°59’24”W; 20 Nov. 1990; E.L.M. Catharino,et al. 1503; SP • Praia Grande; 24°00’00”S, 46°24’00”W; 14 Nov. 1929; A. Gehrt s.n.; SP24495 • Praia Grande; 24°00’00”S, 46°24’00”W; 14 Nov. 1929; A. Gehrt 24495; NY • Ubatuba, Picinguaba; 23°25’47”S, 45°04’12”W; 6 Nov. 1988; A. Furlan 598; HRCB • Ubatuba, Picinguaba; 23°25’47”S, 45°04’12”W; 28 Dec. 2024; E.R. Pansarin 1579; LBMBP • Ubatuba, Estação Experimental; 23°25’47”S, 45°04’12”W; 23 Nov. 1940; J.F. Cunha s.n.; SP [SP44817].

 Notes

Differences between specimens identified as Vanilla chamissonis from coastal populations and from the interior of Brazil have been recognized. The plants that occur inland are commonly referred to as “V. chamissonis mineira”, in reference to their occurrence in Minas Gerais, a southeastern Brazilian state inserted in the Cerrado. Morphological differences among inland and coastal plants were formally recognized twice: Hoehne (1945) published the invalid name V. chamissonis var. longifolia based on a plant collected in the municipality of Itú, southeastern Brazil. Itú has areas typical of an open Cerrado vegetation with many rocky outcrops, while Hicken (1917) described V. argentina based on a plant collected in the Formosa region, Argentina. The region of Formosa, besides Corrientes and Misiones, is characterized as Arid Chaco or Dry Chaco. The Dry Chaco vegetation consists of a mosaic formed by xerophytic forests, gallery forests, and savannas. Here, V. chamissonis, which occurs in the Atlantic Forest, is considered as a distinct species from V. argentina, which is distributed in the Brazilian Cerrado and Dry Chaco. Some species described based on plant material collected in the state of the Rio de Janeiro, at the Brazilian coast, clearly are synonyms of V. chamissonis. This is the case for Vanilla vellozoi (as V. vellozii). Although some authors consider V. vellozoi as an obscure taxon related to V. calyculata, both plant material collected in the Rio de Janeiro (hosted at K), i.e. A. Glaziou 11621 and A. Glaziou 14302 (the latter designated by Soto Arenas and Cribb (2010) as lectotype of V. vellozoi, as V. vellozii), strongly agree with V. chamissonis. Apart from the small size of the flower structures, the shape of the leaves (symmetrically elliptic) and the short and robust inflorescence with patent triangular/rhomboidal bracts leave no doubt that both plant material are V. chamissonis. Soto Arenas and Cribb (2010) consider V. argentina to be synonymous with V. vellozoi. However, as presented here, V. vellozoi is related to V. chamissonis, not to V. argentina. The second taxon is V. carinata. The holotype of V. carinata has been considered as inadequate and conspecific with V. planifolia (Hoehne 1945). However, the material J. Miers s.n. (K000463750) was collected on the Atlantic Coast, in the state of Rio de Janeiro, where V. planifolia does not occur. Although I agree that the holotype is not in good condition, since it lacks vegetative structures, analysis of the reproductive characteristics, such as the presence of a calyculus, the robust inflorescence rachis with patent triangular/rhomboidal bracts, and the presence of an evident labellar keel leaves no doubt that V. carinata is conspecific with V. chamissonis. A third obscure taxon is V. gardneri. Vanilla gardneri was described based on several materials belonging to at least three Vanilla species (Soto Arenas and Cribb 2010). However, the material G. Gardner 245 (K), collected at Morro do Flamengo, Rio de Janeiro, has been designated as the lectotype of V. gardneri (Soto Arenas and Cribb 2010). This exsiccate appears to contain a mixture of plant elements from two distinct species. While the vegetative portion and the flowers appear to be V. phaeantha, the fruit longitudinally sectioned strongly agree with those of V. chamissonis. This is corroborated by the fact that both species are sympatric in this region. Furthermore, the fruiting period of V. phaeantha, whose fruits ripen in nine months, does not overlap with its flowering period. On the other hand, the fruiting period of V. chamissonis, whose fruits take 18 months to mature, overlaps with the flowering period of both sympatric species, V. phaeantha and V. chamissonis. Therefore, based on the presence of a fruit of the latter species in the material G. Gardner 245 (K), V. gardneri was synonymized here under V. chamissonis.

 Morphological affinities

While V. calyculata and V. argentina occur in dryer environments, V. chamissonis is distributed throughout the Atlantic Forest sensu stricto (Fig. 4). Vanilla chamissonis is easily recognized by its elliptic to oblong and asymmetric leaf blades (Fig. 5), sepals 4.9–6.1 cm long, oblong-elliptic petals 4.9–6.1 cm long, labellum 5.3–6.2 cm long with a nectar chamber 1.4–1.6 cm long, column 36–38 mm long, and fruits whose pericarp is soft (Figs 6–7). Vegetative and floral features of V. chamissonis suggest a close relationship with V. calyculata and V. argentina. All three species show robust climbing stems with long fleshy ascendant leaves and lateral inflorescences with whitish resupinate flowers. Flowers of V. chamissonis and related taxa also share several characteristics, such as an unguiculate labellum with an emarginated apical lobe, and a yellow central crest with a penicillate callus just below the anther. In addition, the inner surface of the labellum base of V. chamissonis and related taxa is yellow, while the distal portion is white. In particular, fruit features suggest a close relationship between V. argentina and V. chamissonis. Both species produce large brown and indehiscent fruits that take 18 months to mature. Fruits have an unpleasant fragrance and a bitter flavour. The close relationships among V. argentina, V. chamissonis, and V. calyculata is corroborated by molecular data (see further).

10.5091/plecevo.154789.figure6 2BD66514-54BF-5448-A9AD-A4ADCE5B5F2B

Comparative morphology of perianth parts of related Vanilla species. A. V. argentina. B. V. chamissonis. C. V. calyculata. Lowercase letters on the right side of the floral parts are: (a) distended labellum, (b) labellum apex, (c) detail of the labellar crest, (d) petal. Scale bars = 2 cm. A from E.R. Pansarin 1570 (LBMBP), B from E.R. Pansarin 1579 (LBMBP), C from VAN 206 (Vanilla germplasm bank, LBMBP Orchid House).

https://binary.pensoft.net/fig/1393701 10.5091/plecevo.154789.figure7 986F48E1-DA48-5892-81EB-B13C3E8F5B7C

Comparative morphology of mature fruits of related Vanilla species. A. V. argentina. B. V. chamissonis. C. V. calyculata. Details in figures are transversal (A, B) or longitudinal (C) sections of mature fruits. Scale bar = 2 cm. A from E.R. Pansarin 1570 (LBMBP), B from E.R. Pansarin 1579 (LBMBP), C from VAN 206 (Vanilla germplasm bank, LBMBP Orchid House).

https://binary.pensoft.net/fig/1393702 Phylogenetic relationships

The phylogenetic analysis of the ITS region resulted in strongly congruent MP (Fig. 8) and BI (Suppl. material 2) trees. In both analyses, the Neotropical Vanilla clade with membranaceous leaves (clade A; PP 1, BS 100%; Fig. 8, Suppl. material 2) emerges as sister to a large clade (PP 0.87, BS 100%) with two subclades: a predominantly Old World/Caribbean clade (clade B; PP 0.97, BS 100%) and a clade including the remaining Neotropical Vanilla (clade C; PP 0.96, BS 100%; Fig. 8, Suppl. material 2). Within the non-membranaceous Neotropical Vanilla (clade C), the taxa currently recognized among the V. palmarum group, i.e. V. bicolor Lindl., V. palmarum (Salzm. ex Lindl.) Lindl., and V. sprucei Rolfe, emerge in a basal position to a large clade including the remaining Neotropical species (PP 0.94, BS 59%). In the latter, the Amazonian V. trigonocarpa Hoehne emerges as sister to a clade with two subclades: one clade including the members of the V. pompona group (PP 1, BS 88%), and the other large clade includes the remaining Neotropical Vanilla (Fig. 8, Suppl. material 2). Within the V. pompona clade, V. pompona emerges as sister to a strongly supported clade (PP 0.99, BS 100%) including V. chamissonis and V. calyculata/V. argentina (PP 0.99, BS 65%; Fig. 8, Suppl. material 2).

10.5091/plecevo.154789.figure8 E3261D64-AF67-5DD3-86B4-7A62F1F9C4E3

Maximum parsimony analysis of Vanilla (Orchidaceae) based on ITS (nrDNA) showing the phylogenetic relationships among V. argentina from multiple populations (vertical bar) and Neotropical congeners. Bootstrap values > 50% are given on the branches. A = Neotropical Vanilla with membranaceous leaves, B = Old World/Caribbean clade, C = Neotropical Vanilla with non-membranaceous leaves. Acronyms are Brazilian states: AM = Amazonas, AP = Amapá, GO = Goiás, MG = Minas Gerais, MT = Mato Grosso, PA = Pará, PE = Pernambuco, SP = São Paulo, RJ = Rio de Janeiro. Names after the acronyms MG and SP are Brazilian municipalities of Minas Gerais and São Paulo, respectively.

https://binary.pensoft.net/fig/1393703 Breeding systems

Vanilla chamissonis and V. calyculata were obligatorily self-sterile, where V. argentina was completely self-compatible (Table 2). Flowers setting fruits were from 90% to 93.3% (n = 27–28) in cross-pollinations, and 93.3% (n = 28) in manual self-pollination (V. argentina). No fructification was recorded in either intact (bagged) or emasculated flowers, showing that a biotic pollen vector is necessary for pollen transference. Fruits were mature from nine months (V. calyculata) to 18 months after pollination (V. chamissonis and V. argentina). Among species yielding fruits through self-pollination (V. argentina), there was no significant difference (t-test = -1.734, d.f. = 0.1, p = 0.313) between the mean fruit set resulting from self- and cross-pollinations. Among self-sterile species (V. chamissonis and V. calyculata), the fruits aborted three months after manual self-pollination.

 Discussion

All evidence presented here, i.e. morphological, molecular, and ecological data, supports Vanilla argentina as a taxon distinct from its Neotropical congeners. Vanilla argentina emerges among the members from the “Vanilla pompona group” (Soto Arenas and Cribb 2010), in a clade including V. chamissonis and V. calyculata. Although Pansarin (2024a) and Pansarin and Menezes (2023) show V. paulista among the members of the “Vanilla pompona group”, the DNA sample on which their analyses were carried out was obtained from a juvenile specimen of V. chamissonis collected in the Atlantic Forest and cultivated in the LBMBP Orchid House. After flowering, the identification of the sample as V. chamissonis was confirmed. Actually, V. paulista emerges in a clade including V. dubia (Emerson R. Pansarin unpubl. data).

While widely distinguished by Brazilian orchidologists and vanilla producers, the species occurring in inland South America has been considered as a synonym of V. chamissonis (e.g. BFG 2025; Soto Arenas and Cribb 2010). However, this taxon was formally recognized as a distinct taxon from V. chamissonis by Hicken (1917) and Hoehne (1945). Vanilla chamissonis var. longifolia was never validly published, as Hoehne (1945) did not provide a diagnosis in Latin for this taxon. A Latin diagnosis became mandatory from 1935, i.e. Art. 39.1 (Turland et al. 2018). Conversely, V. argentina was validly published by Hicken (1917). Although V. argentina has been synonymized under V. chamissonis (e.g. BFG 2025), Soto Arenas and Cribb (2010) considered V. argentina as conspecific to V. vellozoi (as V. vellozii). Their assertion is based on the fact that the latter species differs from V. chamissonis by its shorter perianth parts and a broader subentire labellum. Vanilla vellozoi was described based on plants collected in restinga areas from Rio de Janeiro, the habitat of V. chamissonis. The material on which the description of V. vellozoi was based (Glaziou 11621, 14302), except for the length of flower segments, strongly agrees with those of V. chamissonis. Thus, V. vellozoi seems to be conspecific with V. chamissonis, not with V. argentina. Besides V. vellozoi, two other Vanilla names were clarified here. Although the holotype of V. carinata is not in good condition, the presence of a calyculus, the robust inflorescence rachis with patent triangular/rhomboidal bracts, and the presence of an evident labellar keel leave no doubt that V. carinata is conspecific with V. chamissonis. Another obscure taxon clarified here is V. gardneri. Although this species was described based on several materials belonging to at least three Vanilla species, Soto Arenas and Cribb (2010) selected the material G. Gardner 245 (K) as the lectotype of V. gardneri. The plant material was collected at Morro do Flamengo, Rio de Janeiro where two Vanilla species occur, V. phaeantha and V. chamissonis. While the vegetative portion and the flowers appear to be those of V. phaeantha, the fruit strongly agrees with those of V. chamissonis. It is very plausible, as the fruiting period of V. phaeantha, does not overlap with its flowering season (Pansarin 2022). Conversely, the fruits of V. chamissonis take 18 months to mature (Pansarin 2024b), overlapping with its flowering season.

The morphology of V. argentina suggests a close relationship with V. chamissonis. However, molecular data reveal that V. argentina is more closely related to V. calyculata than to V. chamissonis. The relationship between V. argentina and V. calyculata has been shown in previous studies involving Neotropical Vanilla (Pansarin and Menezes 2023; Cascales et al. 2023; Pansarin 2024a). Vanilla calyculata is clearly distinct from V. chamissonis (see Table 2). In addition, V. calyculata occurs in xeric environments (e.g. Soto Arenas and Dressler 2010; Flanagan et al. 2025), while V. chamissonis is distributed within the Atlantic Rainforest (Hoehne 1945). Vanilla calyculata has been assumed to be a synonym of V. columbiana, however, the latter is related to V. phaeantha, not to V. calyculata, as the description of V. columbiana was clearly based on a flower bud (Soto Arenas and Cribb 2010; Flanagan et al. 2025).

Results of the experimental pollinations in order to know the breeding systems of Vanilla argentina, V. chamissonis, and V. calyculata: percentage of fruit set per treatment. The fruit set/flowers is indicated between parentheses.

Treatment V. argentina V. chamissonis V. calyculata
Manual self-pollination 93.3% (28/30) - (0/30) - (0/30)
Spontaneous self-pollination - (0/30) - (0/30) - (0/30)
Emasculation - (0/30) - (0/30) - (0/30)
Cross-pollination 90% (27/30) 93.3% (28/30) 93.3% (28/30)

As far as known, the vast majority of Neotropical thick-leafed Vanilla are self-compatible but require a biotic vector for pollen transfer (e.g. Pansarin and Pansarin 2014; Pansarin 2023). However, most basal taxa among tick-leafed Vanilla, i.e. V. bicolor, V. lindmaniana Kraenzl., and V. palmarum (Pansarin 2024a, 2025; Pansarin and Ferreira 2022), set fruit by means of autonomous self-pollination (Gigant et al. 2011; Pansarin and Ferreira 2022; Pansarin 2025). Here, a breeding system based on total self-sterility is shown for the first time for Vanilla. Based on the fact that fruits of both V. chamissonis and V. calyculata abort 3–4 months from self-pollination, a late-action self-incompatibility can be involved. In a late-acting self-sterility system, the pollen from self-pollination successfully reaches the ovule, but embryos do not develop (Pansarin and Pansarin 2011).

 Conclusion

Here, it was possible to demonstrate that species that occur in the Cerrado and Dry Chaco (V. argentina) correspond to a taxon distinct from V. chamissonis, which is distributed along the Brazilian coast. Thus, as currently known, the Vanilla chamissonis clade includes three species: V. argentina, V. calyculata, and V. chamissonis. Beyond morphological comparisons of vegetative and reproductive characteristics that provide distinguishing features among the three Vanilla species, this study also includes phylogenetic inference with strong group representation, as well as ecological and biogeographic information. Each of these components contributed valuable data for making taxonomic decisions. A multisource approach (i.e. integrative taxonomy) plus extensive field work has been crucial to understand the species boundaries among Neotropical Vanilla. In addition, field investigations in the environments where the type specimens were collected has been critical to elucidate the circumscription of obscure Vanilla species.

 Acknowledgements

The author thanks ICMBIO for permission to collect (Protocol SISBIO number 35178-1), and Decio Chiracava for provide images from V. calyculata. Research supported by the São Paulo Research Foundation – FAPESP (Grant 2018/07357-5) and by CNPq (Productivity Research Grant 301773/2019-0).

 References ArchilaFChironGR (2012) Addition à la flore du Guatemala: Vanilla esquipulensis (Orchidaceae), espèce des forêts xérophytes.Richardiana13: 3–12. BachmanSMoatJHillAWde la TorreJScottB (2011) Supporting Red List threat assessments with GeoCAT: geospatial conservation assessment tool.ZooKeys150: 117–126. https://doi.org/10.3897/zookeys.150.2109 BarrettCFFreudensteinJV (2011) An integrative approach to delimiting species in a rare but widespread mycoheterotrophic orchid.Molecular Ecology20(13): 2771–2786. https://doi.org/10.1111/j.1365-294X.2011.05124.x BFG (2025) Flora e Funga do Brasil. Jardim Botânico do Rio de Janeiro. https://floradobrasil.jbrj.gov.br [accessed 11.06.2025] BatistaJANCastroCSambinACruz-LustreGPansarinER (2023) Clarifying the identity of the Cleistes rosea complex (Orchidaceae) based on integrative taxonomy.Systematics and Biodiversity21(1): 2207575. https://doi.org/10.1080/14772000.2023.2207575 BouetardALefeuvrePGigantRLBorySPignalMBessePGrisoniM (2010) Evidence of transoceanic dispersion of the genus Vanilla based on plastid DNA phylogenetic analysis.Molecular Phylogenetics and Evolution55(2): 621–630. https://doi.org/10.1016/j.ympev.2010.01.021 CascalesJParisCIGottliebAM (2023) The fragrant orchid Vanilla chamissonis: an appraisal of the genetic structure of wild populations from Osununú Natural Reserve (Misiones, Argentina).Lankesteriana23(3): 433–456. https://doi.org/10.15517/lank.v23i3.56432 CameronKM (2003) Vanilloideae. In: PridgeonACribbPChaseMWRasmussenF (Eds) Genera Orchidacearum.Oxford University Press, Oxford, 281–334. CogniauxCA (1893) Vanilla chamissonis var. brevifolia. In: Martius C (Ed.) Flora Brasiliensis, vol. 3, part 4. R. Oldenbourg, Monachii et Lipsiae [Munich & Leipzig], 149. https://www.biodiversitylibrary.org/page/136899 [accessed 24.06.2025] ChristensonEA (1995) Type specimens of Orchidaceae conserved at the Komarov Institute, St. Petersburg, Russia (LE).Brittonia47: 31–43. https://doi.org/10.2307/2807246 DoyleJJDoyleJS (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue.Phytochemical Bulletin19: 11–15. DresslerRL (1993) Phylogeny and Classification of the Orchid Family. Dioscorides Press, Portland, 1–314. FelsensteinJ (1985) Confidence limits on phylogenies: an approach using the bootstrap.Evolution39(4): 783–791. https://doi.org/10.2307/2408678 FidalgoOBononiVLR (1989) Técnicas de Coleta, Preservação e Herborização de Material Botânico. Instituto de Botânica, São Paulo, 1–62. FlanaganNSNavia-SamboniAVargasWGDíaz-RuedaDMCruzJYSRPansarinERCastañoA (2025) Some taxonomic clarifications in Vanilla subgenus Xanata (Orchidaceae), and the resurrection of Vanilla calyculata.Kew Bulletin80: 83–96. https://doi.org/10.1007/s12225-024-10210-7 GigantRLBorySGrisoniMBesseP (2011) Biodiversity and evolution in the Vanilla genus. In: OscarGGianfrancoV (Eds) The Dynamical Processes of Biodiversity: Case Studies of Evolution and Spatial Distribution.Intechopen, Rijeka, 1–26. HallTA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis. Program for Windows 95/98/NT.Nucleic Acids Symposium Series41: 95–98. HickenCM (1917) Vanilla argentina Hicken. In: Estudios Botánicos: Presentados en la Primera Reunión Nacional de la Sociedad Argentina de Ciencias Naturales, Tucumán, 1916. Coni, Buenos Aires, 233–237. https://www.biodiversitylibrary.org/item/37165#page/35/mode/1up [accessed 24.06.2025] HoehneFC (1945) Orchidáceas. In: HoehneFC (Ed.) Flora Brasílica, Fasc.8, vol. 12(2). Secretaria da Agricultura, Indústria e Comércio de São Paulo, São Paulo, 13–43. IUCN Standards and Petitions Committee (2024) Guidelines for Using the IUCN Red List Categories and Criteria. Version 16. Prepared by the Standards and Petitions Committee. https://www.iucnredlist.org/documents/RedListGuidelines.pdf [accessed 14.04.2025] KarremansAPChinchillaIFRojas-AlvaradoGCedeño-FonsecaMDamiánALéotardG (2020) A reappraisal of neotropical Vanilla with a note on taxonomic inflation and the importance of alpha taxonomy in biological studies.Lankesteriana20(3): 395–497. https://doi.org/10.15517/lank.v20i3.45203 KlotzschJF (1846) Ueber die Arten der Gattung Vanilla.Botanische Zeitung4: 561–567. https://www.biodiversitylibrary.org/page/32615469 [accessed 24.06.2025] KressWJPrinceLMWilliamsKJ (2002) The phylogeny and a new classification of the gingers (Zingiberaceae): evidence from molecular data.American Journal of Botany89(10): 1682–1696. https://doi.org/10.3732/ajb.89.10.1682 KumarSStecherGLiMKnyazCTamuraK (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms.Molecular Biology and Evolution35(6): 1547–1549. https://doi.org/10.1093/molbev/msy096 PansarinER (2010) Taxonomic notes on Vanilleae (Orchidaceae: Vanilloideae): Vanilla dietschiana, a rare south American taxon transferred from Dictyophyllaria.Selbyana30(2): 203–207. https://www.jstor.org/stable/41760364 [accessed 24.06.2025] PansarinER (2022) Vanilla flowers: much more than food-deception.Botanical Journal of the Linnean Society198(1): 57–73. https://doi.org/10.1093/botlinnean/boab046 PansarinER (2024a) Rediscovery and revalidation of the Brazilian endemic Vanilla schwackeana Hoehne (Orchidaceae): its distribution and phylogenetic position.Plant Ecology and Evolution157(1): 32–41. https://doi.org/10.5091/plecevo.110331 PansarinER (2024b) Specialized seed dispersal in Neotropical Vanilla reveals fruit unpalatability to omnivores.Plant Biology26(7): 1185–1192. https://doi.org/10.1111/plb.13726 PansarinER (2025) Vanilla lindmaniana and V. palmarum (Orchidaceae) are distinct allopatric species.Plant Ecology and Evolution158(1): 53–62. https://doi.org/10.5091/plecevo.134103 PansarinERFerreiraAWC (2022) Evolutionary disruption in the pollination system of Vanilla (Orchidaceae).Plant Biology24(1): 157–167. https://doi.org/10.1111/plb.13356 PansarinERMenezesELF (2023) A new remarkable Vanilla Mill. (Orchidaceae) species endemic to the Espinhaço Range, Brazil: its phylogenetic position and evolutionary relationships among Neotropical congeners.Phytokeys227: 151–165. https://doi.org/10.3897/phytokeys.227.101963 PansarinERMirandaMR (2016) A new species of Vanilla (Orchidaceae: Vanilloideae) from Brazil.Phytotaxa267(1): 84–88. https://doi.org/10.11646/phytotaxa.267.1.9 PansarinERPansarinLM (2011) Reproductive biology of Echinodorus grandiflorus (Alismataceae): evidence of self-sterility in populations of the state of São Paulo.Rodriguésia62(1): 213–221. https://doi.org/10.1590/2175-7860201162114 PansarinERPansarinLM (2014) Floral biology of two Vanilloideae (Orchidaceae) primarily adapted to pollination by euglossine bees.Plant Biology16: 1104–1113. https://doi.org/10.1111/plb.12160 PridgeonAMChaseMWCribbPJRasmussenFN (1999) Genera Orchidacearum, vol. 1. General Introduction, Apostasioideae, Cypripedioideae. Oxford University, Oxford, 1–197. QGIS Development Team (2023) QGIS Geographic Information System. Version 3.32. Open Source Geospatial Foundation Project. https://www.qgis.org [accessed 08.07.2025] RambautADrummondAJXieDBaeleGSuchardMA (2018) Posterior summarization in Bayesian phylogenetics using Tracer 1.7.Systematic Biology67(5): 901–904. https://doi.org/10.1093/sysbio/syy032 RadfordAEDickisonWCMasseyJRBellCR (1974) Vascular Plant Systematics. Harper & Row Publishers, New York, 1–891. RolfeRA (1895) Vanillas of commerce.Bulletin of Miscellaneous Information, Royal Gardens, Kew1895(104): 169–178. https://www.biodiversitylibrary.org/page/40984721 [accessed 24.06.2025] RolfeRA (1896) A revision of the genus Vanilla.The Journal of the Linnean Society, Botany32: 439–478. https://www.biodiversitylibrary.org/page/265061 [accessed 20.07.2023] RonquistFHuelsenbeckJP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models.Bioinformatics19(12): 1572–1574. https://doi.org/10.1093/bioinformatics/btg180 Schlechter (1920) Die Orchideenfloren der südamerikanischen Kordillerenstaaten. II. Colombia. Repertorium specierum novarum regni vegetabilis.Beihefte7: 1–301. https://www.biodiversitylibrary.org/page/36342778 [accessed 24.06.2024] Soto ArenasMACribbP (2010) A new infrageneric classification and synopsis of the genus Vanilla Plum. ex Mill. (OrchidaceaeVanillinae).Lankesteriana9(3): 355–398. https://doi.org/10.15517/lank.v0i0.12071 Soto ArenasMADresslerRL (2010) A revision of the Mexican and Central American species of Vanilla Plumier ex Miller with a characterization of their ITS region of the nuclear ribosomal DNA.Lankesteriana9(3): 285–354. https://doi.org/10.15517/lank.v0i0.12065 StatSoft (2003) STATISTICA (Data Analysis Software System), Version 6. StatSoft, Tulsa, Oklahoma, USA. SuetsuguKHirotaSKHayakawaHFujimoiSIshibashiMHsuT-CSuyamaY (2023) Spiranthes hachijoensis (Orchidaceae), a new species within the S. sinensis species complex in Japan, based on morphological, phylogenetic, and ecological evidence.Journal of Plant Research136: 333–348. https://doi.org/10.1007/s10265-023-01448-6 SunYSkinnerDZLiangGHHulbertSH (1994) Phylogenetic analysis of Sorghum and related taxa using internal transcribed spacers of nuclear ribosomal DNA.Theoretical and Applied Genetics89: 26–32. https://doi.org/10.1007/BF00226978 SwoffordDL (2001) PAUP*: phylogenetic analysis using parsimony (and other methods), version 4.b.8. Sinauer Associates, Sunderland. ThiersB (2024) Index Herbariorum: a global directory of public herbaria and associated staff. New York Botanical Garden’s Virtual Herbarium. https://sweetgum.nybg.org/science/ih/ [accessed 20.12.2024] TurlandNJWiersemaJHBarrieFRGreuterWHawksworthDLHerendeenPSKnappSKusberW-HLiD-ZMarholdKMayTWMcNeillJMonroAMPradoJPriceMJSmithGF (2018) International Code of Nomenclature for algae, fungi, and plants (Shenzhen Code) adopted by the Nineteenth International Botanical Congress Shenzhen, China, July 2017. RegnumVegetabile 159. Koeltz Botanical Books, Glashütten, 1–245. https://doi.org/10.12705/Code.2018 VellozoJMC (1827) [1831] Petro nomine ac imperio primo Brasiliensis imperii perpetuo defensore... Florae Fluminensis Icones nuc Primo Eduntur, Vol. 9. Senefelder, Paris, tabs 1–128. https://www.biodiversitylibrary.org/item/15450#page/2/mode/1up [accessed 08.07.2025] Supplementary materials 10.5091/plecevo.154789.suppl1 55956E46-675C-5BA3-A05A-95ECF9FCEA90

Taxa of Vanilla included in the molecular study, their locations (State and country), vouchers, and GenBank accession numbers. VAN = Vanilla germplasm bank, LBMBP Orchid House, Department of Biology, FFCLRP-USP, University of São Paulo, Brazil.

 https://binary.pensoft.net/file/1393704 10.5091/plecevo.154789.suppl2 31770566-5ADB-5A20-9E7E-4D8118456FB4

Bayesian inference analysis of Vanilla (Orchidaceae) based on ITS (nrDNA) showing the phylogenetic relationships among members of the V. chamissonis clade and Neotropical congeners. Note the position of V. argentina (arrow). Posterior probability values > 0.5 are given on the branches. A = Neotropical Vanilla with membranaceous leaves, B = Old World/Caribbean clade, C = Neotropical Vanilla with non-membranaceous leaves. The acronym MG is the Brazilian state Minas Gerais.

 https://binary.pensoft.net/file/1393705