ID: 727 DNA barcoding for the identification of endangered with commercial use: spp. () from the Central Andes of Peru Susy J. Castillo1, 2 , Mónica Arakaki1 , Asunción Cano1 , José Roque1, Joaquina Albán1 & José Olivera3 1Universidad Nacional Mayor de San Marcos . Museo de Historia Natural. Lima – Perú. 2E-mail:[email protected] 3Instituto de Investigación de Bioquímica y Nutrición. Facultad de Medicina de la UNMSM. Lima – Perú. Abstract Gentianella (Gentianaceae), a genus with 74% of its species being endemic at the national level, includes at least four endangered species exploited by its medicinal properties, all together commercialized under Gentianella alborosea "hercampuri". As part of a larger phylogenetic study, barcode markers were used to resolve relationships, and define species boundaries, among Peruvian Gentianella. Even with low supports, Gentianella clades share affinities in their geographical distribution and growth form, with few exceptions.

Introduction G1 Gentianella, a genus with more than 300 species, is distributed worldwide, however most of the species diversity is found in the Andes of South America1, 2. Peru holds 100 species, being 74% of them G2 national endemisms, mostly distributed above 3800 m3,4,5,6,7,8,9. There are at least four species (G. alborosea, G. nitida, G. tristicha, G. G3 thyrsoidea) known as "hercampuri“ (Figs.1B, C, G, H), with significant demand for their medicinal properties in national and international G4 markets10, 11, 12(Figs.1.B, C, G, H). Even when exploitation requires state regulations, the mechanisms are not effective, mainly due the informal

extraction and incorrect taxonomic identification. G5 Our study is focused on resolving phylogenetic relationships among Gentianella from South America. At the same time, cover a necessity for enrichment of the DNA reference library for Gentianaceae. Intensive field work and morphological characterization has allowed the establishment of their real distribution, as well as to establish a hypothesis of relationships that is being tested, in the present work, with molecular markers traditionally used in barcoding (ITS, matK).

Figure 2. Preliminary phylogenetic analyses based on Maximum Likelihood for ITS2+matK (A), and ITS2 (B, C), for species of Gentianella, plus outgroups, included in the present study. Bootstrap values (10000 replicates) are above the branches. Results and Discussion In our analyzes Gentianella species form a monophyletic clade that has Swertia (ITS) or Halenia (ITS2-matK) as the sister group (Fig.2). Gentianella campestris, type species of the genus, is found at the base. Preliminary results show low support for most of the branches, thus it is not possible at this point to establish a hypothesis of relationships among Peruvian Gentianella. However, in the ITS2-matK analysis (Fig.2,A), several of the groupings contain species with similar growth form and/or geographical distribution. Groups G1 and G2 are mostly perennial, robust, monocarpic or polycarpic, distributed in central- north Peru (Ancash and Cajamarca), being exceptions G. nitida, G. persquarrosa and Gentianella sp.1. Groups G3, G4 and G5 correspond Figure 1. A) Study area, B) Gentianella alborosea “hercampuri”, C) G. nitida “hercampuri”, D) Yanasinga mountain range (Ticlio, Lima), E) Sampling work, F) G. incurva, G) G. tristicha to species from central Peru (Junín, Lima, Pasco). G3 contains delicate “hercampuri”, H) G. thyrsoidea “japallanshacoc”, I) Imaging work in the laboratory. and robust small herbaceous, distributed very closely. G4 includes Species were identified with the help of specialized literature and examination of Type subshrubs, the exception being G. nitida. G. nitida, together with G. specimens from national (USM, MOL, HAO, HUT) and international (B, F, G, NY, P, US) herbaria. ruizii, have non-vascularized fimbriae in the corolla, a character only seen in these two species. G5 contains small caespitose herbs. The Methods highest percentages of support correspond to specimens of the same Study area & fieldwork. Samples from previous collections, plus newly species from different populations, for example G. thyrsoidea (99), and collected specimens were used. Additionally, we added samples and G. ruizii (99). results from previous Gentianella taxonomic treatments13 developed in the Central Andes (Fig. 1,A ). Most of the collections for the molecular Conclusions portion of the study were done during 2017. We obtained a total of 130 samples, belonging to 40 Gentianaceae species, between herbaria Preliminary results are concordant with what is known for the vouchers and samples recently preserved in silica gel. A few sequences Gentianeae tribe. Until this work, little work has been done with representing the type species of genera Gentianella, Gentiana and Gentianella of South America at the molecular level. Also, collections Halenia, from other regions, were obtained from GenBank. The project and treatments were scarce or incomplete. Our results show the counts with the corresponding permits from SERFOR, the national effectiveness of ITS2 in the differentiation of species and placement regulatory authority (Fig.1, E, F, I). within recognized clades, and concordant with patterns of DNA extraction, amplification and sequencing. DNA isolations were distribution and growth form. However, these results are not performed with a CTAB protocol14. Standard PCRs were carried out with sufficient to establish a phylogenetic hypothesis. A better sampling in primers ITS2F and ITS4 (for ITS), and KIM_3F and KIM_1R (for matK). specimens and molecular markers should improve our results. The PCR products were sequenced in both directions by Macrogen Inc. species with commercial use showed marked differences with the (Macrogen Inc., Seoul, Korea). Sequences were edited in Codon Code ones sold in markers, thus we see a good potential in the Aligner and aligned automatically. Maximum Likelihood analyses were development of identification protocols based on the data obtained. performed in IQ-tree15, with 10000 bootstrat replicates. References 1. Struwe L. & Albert V.A. (eds.), Gentianaceae: Systematics and Natural History. Cambridge University Press, Cambridge. 21–309. 13. Castillo S. J. 2013. Taxonomía y distribución de la familia Gentianaceae en el departamento de Ancash, Perú. Tesis para optar por el Título Profesional 2. Pringle, J.S. 2014a. Chapter 1: Morphological Characteristics of the Family Gentianaceae. In: Rybczyński, J.J., Michael, R.D. & Mikuła, A. de Bióloga. Universidad Nacional Mayor de San Marcos. (eds.). The Gentianaceae volume 1: characterization and ecology. Springer-Verlag Berlin Heidelberg. 1-12. 14. Doyle, J.J. & Doyle, J.L. 1987. Isolation of DNA from fresh tissue. Focus 12: 13–15. 15. Trifinopoulos J, Nguyen LT, von Haeseler A, Minh BQ. 2016. Nucl. Acids Res. 44 (W1): W232-W235 3. Fabris, H. 1958. Notas sobre Gentianella del Perú. Boletín de la sociedad argentina de botánica 7(2): 86-93. 4. Brako, L. & Zarucchi, J. 1993. Catálogo de las Angiospermas y Gimnospermas del Perú. Monographs in Systematic Botany from the Missouri Botanical Garden 45: 1-1286. 5. Pringle, J.S. 2011. Five new species of South American Gentianella (Gentianaceae). Novon 21(1): 78-89. Funding Sources And Acknowledgements 6. Pringle, J.S. 2014b. New species and nomenclatural transfers in South American Gentianella (Gentianaceae). Annales Botanici Fennici We thank Universidad Nacional Mayor de San Marcos (Lima-Peru) for financial support to 51(1–2): 117-124. 7. Pringle, J.S. 2016. New South American species of Gentiana and Gentianella (Gentianaceae). Novon 24: 389–398. 7. Pringle, J.S. & Grant, projects 021001101, 031001181, 041001201, B171001111; and to Programa Nacional de J.R. 2012. Two new species of Gentianella (Gentianaceae) from Peru. Brittonia 64(3): 282–288. Innovación para la Competitividad y Productividad (INNÓVATE PERÚ) for support to project 184- 8. Pfanzelt, S., Sylvester S.P., Ammann, L. & Sylvester, M.D.P.V. 2015. Gentianella viridiflora (Gentianaceae), a new species from the Peruvian Andes. Phytotaxa 222 (4): 283–289. PNICP-PIAP-2015. We greatly appreciate the collaboration of many taxonomists that facilitated 9. Castillo, S., Salinas, N., León, B. & Sánchez, I. 2007 (2006). Gentianaceae endémicas del Perú. Rev. peru. biol.13(2): 339s-354s collections, data and photographs, especially: H. Trinidad, H. Beltrán, P. Gonzales, G. Vadillo, M. 10. Castillo, S. J., J. Roque & M. Mendoza-Rojas. 2014. Especies de Gentianella (Gentianaceae) extraídas para el comercio en Ancash y Huánuco (Perú). XI Congreso Latinoamericano de Botánica 19-24 Octubre, Salvador, Bahía, Brasil Morales and S. Riva. 11. Soukup, J. 1980. Vocabulario de los nombres vulgares de la flora peruana y catálogo de los géneros. Ed. Salesiana. Lima Perú. 436 p. 12. SERFOR. 2015. Perú forestal en números año 2015. MINAGRI. Lima (Perú). 218 p.