SARA VILLANUEVA-VIRAMONTES1, MARIANA HERNÁNDEZ-APOLINAR2, GERMÁN CAR- NEVALI FERNÁNDEZ-CONCHA1, 3, 4, ALFREDO DORANTES-EUÁN1, GABRIEL R. DZIB1, JAIME MARTÍNEZ-CASTILLO1, * Botanical Sciences 95 (2): 169-187, 2017 Abstract Background: Vanilla planifolia, a threatened species, is distributed naturally in semi-deciduous and evergreen rain for- DOI: 10.17129/botsci.668 ests of southeastern Mexico and parts of Central America. In the wild, it is difficult to diagnose from another sympatric Vanilla species, and individuals with reproductive structures are usually required. Copyright: © 2017 Villanueva-Vira- Questions: Can ISSR discriminate wild individuals of Vanilla planifolia from other sympatric species of the genus? Can montes et al. This is an open access article distributed under the terms of phylogenetic analyses of ITS recover the monophyly of V. planifolia and help identify Vanilla species? the Creative Commons Attribution Studied species: The vanilla (V. planifolia), the second-most important economically spice in the world. License, which permits unrestricted Study site and years of study: Plant material was collected during 2014 in the Mexican Yucatan Peninsula. use, distribution, and reproduction Methods: We analyzed 88 wild individuals of several Vanilla species. Individuals of five species ofVanilla reported for in any medium, provided the original the region were used as reference. 84 ISSR loci were analyzed using three clustering methods. A phylogenetic inference author and source are credited. using ITS was performed. Results: ISSR markers clearly discriminate wild Vanilla planifolia, finding definite genetic structure within the species. This research was conducted The three clustering methods identified genetic relationships with congruent patterns. Five groups were found and they corresponded with the species studied. Phylogenetic inference of ITS sequences supported the monophyly of the Vanilla at the Marcadores molecula- and the resulting cladograms were coherent with the clustering pattern observed in the ISSR studies. res laboratory of the Unidad Conclusions: Both ISSR and ITS analyses are able to identify V. planifolia. Molecular data suggest the presence of V. de Recursos Naturales, and pompona and a new species of Vanilla in the Yucatan Peninsula. Herbario CICY at the Centro Keywords: Genetic identification, molecular markers, phylogenetic analysis, vanilla, wild populations. de Investigación Científica de Yucatán, A. C. Vanilla planifolia silvestre y sus parientes en la Península de Yucatán, México: 1 Centro de Investigación análisis sistemáticos con ISSR e ITS Científica de Yucatán, A.C. Mérida, Yucatán, México. Resumen 2 Departamento de Ecología y Antecedentes: Vanilla planifolia es una especie amenazada que se distribuye naturalmente en las selvas tropicales perennifolias y subperennifolias del sureste de México y parte de Centroamérica. En estado silvestre, es difícil de dife- Recursos Naturales, Facultad renciar de otras especies simpátricas de Vanilla, y usualmente se requieren individuos con estructuras reproductivas. de Ciencias, UNAM. Cd. Preguntas: ¿Pueden los marcadores ISSR discriminar individuos silvestres de Vanilla planifolia de otras especies México, México. simpátricas de vainilla?, ¿Puede el análisis de ITS recobrar la monofilia de V. planifolia y contribuir a identificar las 3 Herbario CICY, Centro de especies de Vanilla? Investigación Científica de Especie en estudio: Económicamente, la vainilla (Vanilla planifolia) es la segunda especia más importante en el Yucatán, A.C. Mérida, Yuca- mundo. tán, México. Sitio de estudio y fecha: La colecta se realizó en 2014 en la Península de Yucatán. 4 Orchid Herbarium of Oakes Métodos: Se analizaron 88 de individuos silvestres de especies de Vanilla. Se utilizaron como testigos individuos de Ames, Harvard University cinco especies de vainilla reportadas para la región. 84 loci de ISSR fueron analizados usando tres métodos de agrupa- Herbaria, Cambridge, Mas- miento. Se realizó una inferencia filogenética con ITS. sachusetts, U.S.A. Resultados: Los ISSR discriminaron claramente a los individuos silvestres de Vanilla planifolia, detectando una es- * Corresponding author: tructura genética al interior de la especie. Los tres métodos de agrupamiento mostraron patrones congruentes, encon- [email protected] trándose cinco grupos que correspondieron con las especies estudiadas. El análisis filogenético de las secuencias de ITS mostró la monofilia del géneroVanilla , y los cladogramas obtenidos fueron coherentes con los resultados obtenidos con Corresponding author’s los ISSR. e-mail address: saravilvir@ Conclusiones: Tanto ISSR como ITS identificaron a Vanilla planifolia. Los análisis moleculares sugieren la presencia cicy.mx; mariana.hernandez. de V. pompona y de una nueva especie de Vanilla en la Península de Yucatán, México. [email protected]; car- Palabras clave: Análisis filogenético; identificación genética, marcadores moleculares, poblaciones silvestres, vainilla. [email protected]; badip@cicy. mx; [email protected] 169 SARA VILLANUEVA-VIRAMONTES ET AL. he genus Vanilla Mill. is composed of possibly more than 150 species. In 1954 Portères reported 110 distinct taxa but many more have been described since (e.g. Soto-Arenas & Dressler 2010), and several more still await formal taxonomic recognition. The genus features a Pantropical dis- tribution and it probably originated in America (Cameron 2000, Ramírez et al. 2007, Bouetard et al. 2010). Vanilla taxonomy has been hampered by several factors, including the following: • The difficulty of finding and collecting individuals with fertile structures in the field as a result of the ephemeral, gregarious flowering that usually happens high in the canopy of the forests (Schlüter 2002, Soto-Arenas & Dressler 2010). • Flowers, which are fundamental for identification of species, are poorly preserved in herbaria. Usually, herbarium species are sterile or include a single flower, often poorly preserved and irretrievably flattened (or altogether glued to the herbarium sheet). • The rarity or inaccessibility of many of the species. • The fact that many species are vegetatively indistinguishable. • The enormous vegetative variation and phenotypic plasticity associated with the hemiepi- phyte growth habit including leaves of different morphologies and sizes in the same indi- vidual as a response to age, vegetative development, and light exposure (Putz & Holbrook 1986, Ray 1990). Vanilla planifolia Andrews has great economic importance, being the source of approxi- mately 95 % of the world’s vanilla extract production (Lubinsky et al. 2008a). The vanilla is the second-most important spice in gastronomy and the cosmetic industry and is one of the most popular flavorings (Cid-Pérez & López-Malo 2011, Álvarez et al. 2013). Vanilla cultivation typically features vegetative propagation and hand-pollination, which ensures fruit production, albeit seeds are never harvested, and plants are never raised from seeds. Vegetative reproduc- Author Contributions Sara Villanueva-Viramontes: tion, plus the lack of new genotypes incorporated via seed, have in combination drastically collected most of the botani- reduced genetic variation in vanilla plantations (Smith et al. 1992, Soto Arenas 1999, Minoo cal material, conceived, de- et al. 2008). Because of this, FAO (1995) lists cultivated V. planifolia as species with a high signed, and performed the ex- degree of genetic erosion. Furthermore, wild populations of V. planifolia have been severely periments, analyzed the data, damaged by natural habitat destruction and illegal extraction for replenishment of commercial and drafted the first version of plantations, nearly driving the species to the brink of extinction, at least locally (Soto-Arenas the paper as well as reviewed 2006, Soto-Arenas & Solano-Gómez 2007, Menchaca 2010). Due to this, V. planifolia has a drafts of the manuscript. “special protection” status (SEMARNAT 2010) in Mexico. As a member of the Orchidaceae, it Mariana Hernández-Apolinar: is included in Appendix 2 of CITES (CITES 2016). provided botanical materials and reviewed drafts of the Vanilla planifolia is reported as native from southeastern Mexico, Guatemala, Belize, and manuscript. Costa Rica. However, a clear picture of its distribution is still uncertain (Soto-Arenas 1999) be- Germán Carnevali Fernández- cause relatively few wild individuals have apparently been collected (fewer than 30 individuals, Concha: provided botanical according to Soto-Arenas 2006 and Schlüter et al. 2007). There are also several vegetatively materials, conceived and similar species. To make matters worse, flowers in the herbaria are difficult to reconstruct; thus, designed parts of the experi- a significant portion of specimens are probably misidentified as V. planifolia in many herbaria ments and analyses. Translated (Soto-Arenas 2009, Soto-Arenas & Dressler 2010). These errors of identification have also been and reviewed drafts of the pa- reported in vanilla plantations in Mexico, as we have been able to corroborate in the Mexican per. Funded the ITS analyses. states of Quintana Roo (Villanueva-Viramontes, pers. obs.), and Oaxaca (M. Hernández-Apo- Alfredo Dorantes-Euán: Par- ticipated in the botanical col- linar pers. Obs.). The literature also reports this from other countries, such as Ecuador and lections, and reviewed drafts Guatemala (Soto-Arenas & Dressler 2010). Some of these plantations include, along with true of the first version. V. planifolia, individuals of