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Vanilla sotoarenasii M.Pignal, Azofeifa-Bolaños & Grisoni sp. nov. urn:lsid:ipni.org:names:77160155-1 Figs 2, 3, 6 Diagnosis A Vanilla planifolia similis, sed folia caulesque breviores (folium: 7.6̅10.6 × 2.7̅3.3 cm versus 14.7̅14.9 × 4.8̅5 cm), laminae breviores, ellipticae-oblongaeque, Fores albiores, tubus Foris, sepala petalaque breviores, sepalum dorsale angustius, labellum angustiius cum papillis salientioribus, ovarium brevitius (3.8̅4.6 cm versus 5̅5.3 cm), fructus brevitior (11.4 cm versus 20.1̅20.3 cm) indehiscensque, sectio fructi cylindrica (versus trigona). Moleculae aromaticae fructi absimilis. Etymology This species is dedicated to Dr. Miguel Angel Soto Arenas (1963̅2009), authority in orchid Foristics and ecology, particularly in the Vanilla genus. Type material Holotype COSTA RICA: Cahuita (ex hort. parc E. Liais, from plants collected by B. Gosselin in 1993), 8 Oct. 1996, Pignal 396 b (holo-: P: P00075132). Paratypes COSTA RICA: Limón Province: Talamanca, Refugio Nacional Mixto de Vida Silvestre Gandoca- Manzanillo, 9.594° N, 82.602° W, altitude 2 m, 14 Nov. 2013 (originally collected), cultivated in BRC Vatel, Saint Pierre, La Réunion, France CRV2180, 28 Feb. 2016, M. Grisoni & J.B. Azofeifa-Bolaños V. sp. nov. V. Compounds / 100 % 100 g % Vanillin 0.26 a 10.9 2.28 b 68.0 Vanillic acid nd a 0.0 0.19 b 5.5 Vanillyl alcohol 0.73 a 31.0 0.32 b 9.6 Total vanillyl compounds 0.99 a 41.9 2.79 b 83.1 p -hydroxybenzaldehyde 0.20 a 8.5 0.18 a 5.4 p -hydroxybenzoic acid 0.37 a 15.8 0.37 a 11.2 p -hydroxybenzyl alcohol 0.27 a 11.6 0.01 b 0.3 Total p -hydroxybenzyl compounds 0.85 a 35.9 0.56 b 16.9 Anisyl alcohol 0.24 a 10.2 nd b 0.0 Anisaldehyde 0.03 b 1.4 nd b 0.0 Anisic acid 0.25 a 10.6 nd b 0.0 Total anisyl compounds 0.52 a 22.2 nd b 0.0 Total 2.36 a 100 3.35 b 100sotoarenasii planifolia g g dw g / dw JJMM01 (REU: REU13363); Canton of Talamanca, Refugio de Vida Silvestre Gandoca-Manzanillo (originally collected), cultivated in INISEFOR, Heredia, Costa Rica, 14 Nov. 2013, M. Grisoni, B. Azofeifa and J. García 2180 (CR 281507), cultivated at the same locality, B. Azofeifa and J. García 0 0 47 (CR 281508); Canton of Siquirres, Barra de Parismina (originally collected), cultivated in INISEFOR, Heredia, Costa Rica, 8 Feb. 2013, B. Azofeifa, J. García and A. Paniagua 0 0 0 2 (CR 281509). Description Hemiepiphytic vine up to 15 m high. Stems Fexuous, terete, smooth, green, 5̅6 mm thick; internodes, sometimes slightly curved apically, ca 10 cm long. Terrestrial roots pubescent, ramiFed, ca 2 mm thick; both attaching and free aerial roots terete, pubescent, ca 2 mm thick at base, ca 3.5 mm at middle. Leaves regularly alternate. Blade elliptic to obovate, slightly Feshy (ca 27 veins visible on dry specimens); base rounded to cuneate, shortly pseudopetiolate; apex shortly acuminate (acumen sharp, 12 mm long, 5 mm at base), slightly recurved; margin thinned; pseudopetiole canaliculate, 16 × 5 mm. InForescence: raceme, ca 10̅20-Fowered, 7̅13 cm, sometimes located on short axillary branches, sometimes lying on leafy stems. 3̅4 inForescence bracts, foliaceous, basal bract pseudopetiolate (40 × 20 mm), upper bract sessile and shorter (20 × 8 mm and 12 × 3 mm). Flowers at anthesis successively, ephemeral, 1̅2 simultaneously, white green, sepals forming an angle of approximately 45° with axis of column, petals more or less parallel to this axis. Ovary terete, smooth, slightly arcuate, white at base and green on upper ¾, 30 mm long and 4 mm in diameter. Parts of perianth with whitish inclusions (visible on dry specimens), longitudinally oriented, ca 0.1̅2 mm long, more numerous on petals, dorsal sepal narrow elliptic to oblanceolate, ca 11-veined, 40 × 10 mm, apex acute, rounded, base attenuate-clawed, slightly concave, canaliculate. Lateral sepals, elliptic asymmetric, slightly falciform, ca 11-veined, 38 × 10.5 mm, apex acute, slightly cupuliform, base attenuate, canaliculate, 4 mm wide. Petals elliptic asymmetric, slightly falciform, ca 12-veined, carinate dorsally (carena 1.2 mm wide), 38 × 9 mm, apex rounded, base attenuate, 3 mm wide (Fig. 6). Labellum attached to column along margins of 5/7 (ca 20 mm), funnel-shaped, spread apically (opening about 10 mm), trilobed, ca 30-veined ramiFed in distal third, margin crenulate. With 5̅6 Fmbriated scales, ca 1.5 × 1.5 mm, at about middle of labellum. Lateral lobes, obliquely triangular, ca 11-veined, 10 mm high and 7 mm wide, margins widely undulate. Midlobe quadrate, 20 mm wide and 6 mm high, about 12-veined, converging at apex. Papillae on four central veins, on apical half. Column trigonous-semicylindrical, 28 mm long, 2.5 mm wide, apically with 2 lateral auricles, crenulate, 2.5 mm high and 4 mm wide. Rostellum quadrangular, ca 3 × 3 mm. Stigma bilobed. Anther articulated, connective keel-shape, 2 × 2 mm, bicarinate on top. Operculum helmet-shaped, 2 × 3 mm. Pollinarium, 2. Fruit arcuate, banana-shaped, green, turning yellowish and then brown, 11̅16 × 1.5 cm, with cylindrical section. Phenology The Fowering period occurs from October to March in Costa Rica. IUCN status –Vulnerable The populations of V. sotoarenasii sp. nov. observed in the province of Limón showed strong vegetative development of vines and natural seed set was frequently observed. However, a small number of populations have so far been observed and only over a very limited area of Limón Province (10 populations over a coastal strip of about 50 km²), and its habitat is periodically submerged by the ocean. This makes us inclined to tentatively classify V. sotoarenasii sp. nov. as vulnerable D2 (IUCN 2012) until further data on distribution and population dynamics have been obtained. Discussion Using complementary approaches involving morphology of the reproductive and vegetative systems, molecular barcoding and the accumulation of secondary metabolites in fruits, we highlighted speciFc traits for the vanilla populations sampled on the Caribbean coast of Costa Rica which revealed a new taxon, V. sotoarenasii sp. nov. The morphology of the Fowers and leaves clearly assigned V. sotoarenasii sp. nov. within the V. planifolia group (Soto Arenas & Dressler 2010). Based on Fower morphology, V. sotoarenasii sp. nov. was more similar to V. planifolia and V. × tahitensis than to any other vanilla species of this group. However, V. sotoarenasii sp. nov. differs from the other two species by several characteristics (Table 6). Firstly, our data and previous data by Costantin & Bois (1915) and Portères (1953) showed that V. sotoarenasii sp. nov. has a signiFcantly smaller size for all organs measured (stem, leaf, Fower and fruit) compared to V. planifolia and V. × tahitensis, and had a distinct shape of the leaves: elliptic to obovate in the case of V. sotoarenasii sp. nov., elliptic to oblong for V. planifolia and narrowly oblong to lanceolate for V. × tahitensis (Table 6). In addition, the Fowers of V. sotoarenasii sp. nov. are more whitish, with a narrower label showing marked papillae, compared to those of V. planifolia, which are more greenish, with a wider label and smooth papillae. On the basis of HPLC quantiFcation of hydrolyzed volatile compounds in mature fruits, the aromatic precursors of V. sotoarenasii sp. nov. are very distinct from those of V. planifolia. In particular, fruits of V. sotoarenasii sp. nov. were characterized by less predominant vanillin content and the presence of anisyl compounds. They should develop, after over-maturation or curing, Favors extremely different from those of V. planifolia and more similar to those of V. pompona or V. × tahitensis (Pérez-Silva et al. 2006; Brunschwig et al. 2009; Maruenda et al. 2013). On the other hand, molecular analysis of nuclear DNA sequences (ITS) unambiguously separated the V. sotoarenasii sp. nov. group of plants from the V. planifolia group including 68 accessions originating from seven countries (Comoros, Costa Rica, Guatemala, Madagascar, Mayotte, Mexico, and La Réunion). This result is corroborated by ampliFed fragment length polymorphism (AFLP) analyses by Bory et al. (2008) that clearly separated V. sotoarenasii sp. nov. CR0068 from 303 V. planifolia genotypes of diverse origins with an average distance comparable to the distance between V. planifolia and V. × tahitensis. The facts that i) at the plastid DNA level (partial mat K sequence) V. sotoarenasii sp. nov. and V. planifolia share the same clade, and ii) in the ITS phylogeny V. sotoarenasii sp. nov. is in an internal position within the V. planifolia clade (like V. helleri within the V. odorata cluster), suggest the recent radiation of V. sotoarenasii sp. nov. from V. planifolia populations. So far, V. sotoarenasii sp. nov. has only been observed in the Limón Province of Costa Rica where it is sympatric with V. planifolia, which has been reported as native to Costa Rica (Soto Arenas & Dressler 2010). However, recent introductions in the country of V. planifolia cultivars and hybrids have also been documented (Soto Arenas & Dressler 2010; Belanger & Havkin-Frenkel 2011; Varela Quirós 2011). Historical, genetic and phylogenetic data are insufFcient to decide whether V. sotoarenasii sp. nov. derived from natural or introduced populations of V. planifolia, nor how and when the radiation occurred. Orchid species are often interfertile, which allows them to create interspeciFc and even intergeneric fertile hybrids. Many interspeciFc hybrids between species of Vanilla have been produced in the last decades by botanists and agronomists (Knudson 1950; Theis & Jiménez 1957; Divakaran et al. 2006). In nature, however, interspeciFc hybrids are prevented by reproductive barriers that result primarily from the inability of pollinators to transfer pollen from one species to another.Within the limits of our sampling, we have never observed intermediary types, at the genetic or morphological level, in the Limón area, which suggests that a reproductive barrier may exist between V. planifolia and V. sotoarenasii sp. nov. Indeed, in the case of sympatric populations of the two very closely related species V. barbellata Rchb. f. and V. dilloniana Correll, having synchronous Fowering and the same pollinator in western Puerto Rico, intermediary types were observed, demonstrating natural interspeciFc hybridizations (Nielsen 2000). We hypothesize that the differences in Fower morphology observed between V. planifolia and V. sotoarenasii sp. nov. impede gene Fow from one species to another, which enhanced the radiation of the population in Limón. Vanilla pollinationis bee-dependent(Ackerman1986; Gigant et al. 2011)and Fower size difference is likely to constitute a reproductive barrier between species by selecting compatible pollinators. This has been observed for instance in Peru where, due to their small size, the Mellipona bees did not remove pollen when visiting V. grandiFora Lindl. Fowers, and therefore did not contribute to its pollination (Lubinsky et al. 2006). Given the signiFcant size reduction of the Fowers of V. sotoarenasii sp. nov. compared to those of V. planifolia, it is unlikely that a bee capable of pollinating one of the two species would be able to pollinate the other. In addition to size compatibility, the probability of pollinator visits is frequently increased by the aromatic metabolites emitted by orchid Fowers, which act as bee attractants (Ackerman 1986; Pansarin & Pansarin 2014). This is particularly important for vanilla Fowers that are open a single day, which reduces the chances of pollen-pollinator encounter. Analysis of the volatile compounds emitted by Fowers from various Vanilla species (unpublished data) has shown in particular that in V. planifolia (n = 4) the major compounds are a hydrocarbon monoterpene, (E)-β-ocimene (representing 17.10 ± 14.08% of all detected compounds), and an aromatic compound, 2,6-bis (1,1-dimethylethyl)- 4-(1-oxopropyl) phenol (11.59 ± 8, 04%), while in V. sotoarenasii sp. nov. (n = 1), only (E)-β-ocimene was predominantly found (63.26%). This difference in the nature of volatile emissions between the two species could also contribute to the speciFcity of the pollinators visiting the Fowers. Furthermore, the earlier Fowering of V. sotoarenasii sp. nov. compared to V. planifolia may also contribute to favor self or geitonogamous pollination as well as provide greater reproductive success to V. sotoarenasii sp. nov., as pointed out for non-rewarding orchids (Jersáková et al. 2006; Sun et al. 2009). All these factors hindering gene Fow between populations of V. sotoarenasii sp. nov. and V. planifolia may account for the radiation that led to the emergence of V. sotoarenasii sp. nov., a species that is genetically, morphologically, and biochemically distinct from V. planifolia. Finally, given its original aromatic content combining vanilyl and anisic notes, its high level of resistance (at least for CR0068) to the Fusarium root rot of vanilla (Koyyappurath et al. 2015), and its early and abundant Fowering in culture, V. sotoarenasii sp. nov. could be a promising genitor for breeding programs aiming to produce new vanilla varieties for the agroindustry.

We describe a new vanilla species growing in sympatry with Vanilla planifolia Jacks. ex Andrews (Orchidaceae) in the province of Limón, Caribbean coast of Costa Rica. The morphology of the reproductive and vegetative organs observed on vines cultivated under shade-house, the nuclear (Internal Transcribed Spacer) and plastid (matK) nucleotide sequences, as well as the contents of aromatic compounds measured in ripe fruits, show that this species is close to but distinct from V. planifolia. The name V. sotoarenasii M.Pignal, Azofeifa-Bolaños & Grisoni sp. nov. is proposed for this new Vanilla species endemic in Costa Rica. It is especially distinguished from V. planifolia by a reduction of about 30% of the size of the fruits and flowers, by a divergence of ITS sequences for at least two species-conserved nucleotides compared to seven other species of the V. planifolia group, and by the presence of anisic compounds and low content of phenolic compounds (including vanillin) in the fruits. These results confirmed the extension of the area of distribution of V. planifolia southward to Costa Rica, where a recent speciation process occurred. Because of its particular agronomic and aromatic properties, V. sotoarenasii sp. nov. could represent a valuable biological resource for the vanilla industry. Describimos una nueva especie de vainilla que crece en simpatía con Vanilla planifolia Jacks. ex Andrews (Orchidaceae) en la provincia de Limón, costa caribeña de Costa Rica. La morfología de los órganos reproductores y vegetativos observados en vides cultivadas bajo sombra, las secuencias nucleotídicas nucleares (Internal Transcrito Spacer) y plastidios (matK), así como el contenido de compuestos aromáticos medidos en frutos maduros, muestran que esta especie es cercano pero distinto de V. planifolia. El nombre V. sotoarenasii M. Pignal, Azofeifa-Bolaños & Grisoni sp. nov. Se propone para esta nueva especie de vainilla endémica en Costa Rica. Se distingue especialmente de V. planifolia por una reducción de aproximadamente el 30% del tamaño de los frutos y flores, por una divergencia de secuencias de ITS para al menos dos especies de nucleótidos conservados en comparación con otras siete especies del grupo de V. planifolia. , y por la presencia de compuestos anísicos y bajo contenido de compuestos fenólicos (incluida la vainillina) en los frutos. Estos resultados confirmaron la extensión del área de distribución de V. planifolia hacia el sur hasta Costa Rica, donde ocurrió un reciente proceso de especiación. Por sus particulares propiedades agronómicas y aromáticas, V. sotoarenasii sp. nov. podría representar un valioso recurso biológico para la industria de la vainilla. Descrevemos uma nova espécie de baunilha que cresce em simpatia com Vanilla planifolia Jacks. ex Andrews (Orchidaceae) na província de Limón, costa caribenha da Costa Rica. A morfologia dos órgãos reprodutivos e vegetativos observados em vinhas cultivadas à sombra, as sequências de nucleotídeos nucleares (espaçador de transcrição interno) e plastídeos (matK), bem como o conteúdo de compostos aromáticos medidos em frutos maduros, mostram que esta espécie está próxima, mas diferente de V. planifolia. O nome V. sotoarenasii M. Pignal, Azofeifa-Bolaños & Grisoni sp. Nov. É proposto para esta nova espécie endêmica de baunilha na Costa Rica. É especialmente distinta de V. planifolia por uma redução de aproximadamente 30% no tamanho de frutos e flores, por uma divergência de sequências ITS para pelo menos duas espécies de nucleotídeos conservados em comparação com outras sete espécies do grupo V. planifolia. , e pela presença de compostos anísicos e baixo teor de compostos fenólicos (incluindo vanilina) nos frutos. Esses resultados confirmaram a extensão da área de distribuição de V. planifolia ao sul até a Costa Rica, onde ocorreu um recente processo de especiação. Devido às suas propriedades agronômicas e aromáticas particulares, V. sotoarenasii sp. Novembro pode representar um valioso recurso biológico para a indústria de baunilha. Universidad Nacional, Costa Rica Université de La Réunion, Francia Instituto Tecnológico de Tuxtepec, México Instituto Tecnológico de Veracruz, México Universidad Autónoma de México, México Benemérita Universidad Autónoma de Puebla, México Universidad de San Carlos de Guatemala, Guatemala. Instituto de Investigación y Servicios Forestales Escuela de Ciencias Agrarias Escuela de Ciencias Biológicas