Phylogeny predicts the quantity of antimalarial alkaloids within the iconic yellow Cinchona bark (Rubiaceae: Cinchona calisaya) Maldonado Goyzueta, Carla Brenda; Barnes, Christopher James; Cornett, Claus; Holmfred, Else Skovgaard; Hansen, Steen Honoré; Persson, Claes; Antonelli, Alexandre; Rønsted, Nina Published in: Frontiers in Plant Science DOI: 10.3389/fpls.2017.00391 Publication date: 2017 Document version Publisher's PDF, also known as Version of record Document license: CC BY Citation for published version (APA): Maldonado Goyzueta, C. B., Barnes, C. J., Cornett, C., Holmfred, E. S., Hansen, S. H., Persson, C., ... Rønsted, N. (2017). Phylogeny predicts the quantity of antimalarial alkaloids within the iconic yellow Cinchona bark (Rubiaceae: Cinchona calisaya). Frontiers in Plant Science, 8, [391]. https://doi.org/10.3389/fpls.2017.00391 Download date: 08. Apr. 2020 ORIGINAL RESEARCH published: 22 March 2017 doi: 10.3389/fpls.2017.00391 Phylogeny Predicts the Quantity of Antimalarial Alkaloids within the Iconic Yellow Cinchona Bark (Rubiaceae: Cinchona calisaya) Carla Maldonado 1, 2 †, Christopher J. Barnes 1 †, Claus Cornett 3, Else Holmfred 3, Steen H. Hansen 3, Claes Persson 4, 5, Alexandre Antonelli 4, 6 and Nina Rønsted 1* 1 Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark, 2 Herbario Nacional de Bolivia, Universidad Mayor de San Andres, La Paz, Bolivia, 3 Analytical Biosciences, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark, 4 Department of Biological and Environmental Sciences, University of Gothenburg, Gothenburg, Sweden, 5 Gothenburg Global Biodiversity Centre, Gothenburg, Sweden, 6 Gothenburg Botanical Garden, Gothenburg, Sweden Edited by: Danièle Werck, Centre National de la Recherche Considerable inter- and intraspecific variation with respect to the quantity and Scientifique, France composition of plant natural products exists. The processes that drive this variation Reviewed by: remain largely unknown. Understanding which factors determine chemical diversity has Fernando Geu-Flores, University of Copenhagen, Denmark the potential to shed light on plant defenses against herbivores and diseases and Massuo Jorge Kato, accelerate drug discovery. For centuries, Cinchona alkaloids were the primary treatment University of São Paulo, Brazil of malaria. Using Cinchona calisaya as a model, we generated genetic profiles of leaf *Correspondence: Nina Rønsted samples from four plastid (trnL-F, matK, rps16, and ndhF) and one nuclear (ITS) DNA [email protected] regions from twenty-two C. calisaya stands sampled in the Yungas region of Bolivia. †These authors have contributed Climatic and soil parameters were characterized and bark samples were analyzed for equally to this work. content of the four major alkaloids using HPLC-UV to explore the utility of evolutionary Specialty section: history (phylogeny) in determining variation within species of these compounds under This article was submitted to natural conditions. A significant phylogenetic signal was found for the content of two Plant Metabolism and Chemodiversity, out of four major Cinchona alkaloids (quinine and cinchonidine) and their total content. a section of the journal Frontiers in Plant Science Climatic parameters, primarily driven by changing altitude, predicted 20.2% of the overall Received: 19 December 2016 alkaloid variation, and geographical separation accounted for a further 9.7%. A clade of Accepted: 07 March 2017 high alkaloid producing trees was identified that spanned a narrow range of altitudes, Published: 22 March 2017 from 1,100 to 1,350 m. However, climate expressed by altitude was not a significant Citation: driver when accounting for phylogeny, suggesting that the chemical diversity is primarily Maldonado C, Barnes CJ, Cornett C, Holmfred E, Hansen SH, Persson C, driven by phylogeny. Comparisons of the relative effects of both environmental and Antonelli A and Rønsted N (2017) genetic variability in determining plant chemical diversity have scarcely been performed Phylogeny Predicts the Quantity of Antimalarial Alkaloids within the Iconic at the genotypic level. In this study we demonstrate there is an essential need to do so Yellow Cinchona Bark (Rubiaceae: if the extensive genotypic variation in plant biochemistry is to be fully understood. Cinchona calisaya). Front. Plant Sci. 8:391. Keywords: alkaloids, Bolivia, Cinchona calisaya, climate, plant chemical defense, phylogeny, plant–climate doi: 10.3389/fpls.2017.00391 interactions, quinine Frontiers in Plant Science | www.frontiersin.org 1 March 2017 | Volume 8 | Article 391 Maldonado et al. Phylogeny Predict Plant Chemical Diversity INTRODUCTION mechanisms of plants response to environmental changes (Theis and Lerdau, 2003; Ramakrishna and Ravishankar, 2011), and Bark from Cinchona trees (Cinchona L., Rubiaceae) of the Andean as with other plant chemical defenses, have likely developed mountain forests produce quinine alkaloids, which were the over different evolutionary timescales (Becerra et al., 2009). only effective treatment of malaria for more than four centuries Evolutionary approaches have been successfully implemented (Honigsbaum, 2001; Kaufman and Ruveda, 2005). The medicinal in predicting plant phytochemical composition, accelerating the value of Cinchona bark was first discovered in Loxa (now Loja, discovery, and exploration of plant-based medicines (Bohlin Ecuador) in the seventeenth century by Jesuit monks, and soon et al., 2010; Zhu et al., 2011; Rønsted et al., 2012). However, exports of different varieties of Cinchona pubescens Vahl (red other studies have found inconsistency of specialized metabolite bark) from South America to Europe were reaching half a profiles at various taxonomic levels (e.g., Wink, 2003; Wink and million kilograms bark per year (Roersch van der Hoogte and Mohamed, 2003). While it is established that different plants Pieters, 2015). Import could not meet demand, and a quest began produce different specialized metabolites, the underlying causes for the most productive source of Cinchona trees to establish determining these differences remain unknown. Two major plantations by the British, Dutch, and French empires. The hypotheses have been proposed, as outlined below. Bolivian Cinchona calisaya Wedd. (yellow bark, Figure 1) proved The “escape-and-radiate” hypothesis (ERH) predicts to be the most productive species known to date (Greenwood, sequential cycles between plant and herbivore/pathogens, 1992; Nair, 2010). with plants increasing in chemical complexity over evolutionary C. calisaya is one of 23 species of trees in the genus time, and the evolution of novel traits that promote speciation Cinchona described to date, which produce varying amounts are incremental and directional throughout the diversification of alkaloids. The four major Cinchona alkaloids (quinine, process (Ehrlich and Raven, 1964; Berenbaum, 1983; Agrawal, quinidine, cinchonine, and cinchonidine) (Figure 2a) all possess 2007). antimalarial activity but have different pharmacological profiles The “resource availability hypothesis” (RAH) predicts that (Taggart et al., 1948; Hill, 1963; Bruce-Chwatt, 1990). Since plants will invest more in defense when the cost of tissue the first isolation of quinine in 1820 over 30 minor and less replacement is high (Janzen, 1974; Coley et al., 1985; Fine et al., studied Cinchona alkaloids have been described from the genus 2006). Therefore, in harsher, nutrient poor environments, there (Kacprzak, 2013). Bark and roots are the main source of Cinchona will be a greater investment in physical and chemical defense alkaloids, whereas cinchophyllines are reported from leaves mechanisms over evolutionary time. For example, sparsely (Aerts et al., 1991). The site of production of the alkaloids vegetated harsh soil is associated with increase in environmental has not been established. In addition, Cinchona type alkaloids stresses from greater exposure to droughts and herbivores, and have also been found in the related genera Ladenbergia Klotzsch is also expected to increase investment in chemical defenses or and Remijia DC (Okunade et al., 2001; Ruiz-Mesia et al., 2005; other morphological or physiological adaptations such as crypsis, Cosenza et al., 2013). escape, mimicry, etc. (Strauss and Cacho, 2013; Cacho et al., Cinchona bark and its alkaloids remained the most efficient 2015). treatment of malaria until the 1940s when chloroquine and other Contrary to the ERH, the RAH was developed using different synthetic antimalarial compounds were developed (Newman species rather than a single species. Furthermore, there is little et al., 2000; Kaufman and Ruveda, 2005). With the development evidence of this process within species under natural conditions. of resistant malaria strains (Bruce-Chwatt, 1990) the quest for Early experiments within plantations of C. calisaya in Java, new antimalarial compounds is continuing, and the discovery of outside the natural habitat (Winters et al., 1947; Loustalot artemisinin from a Chinese herbal medicine based on Artemisia and Winters, 1948), found alkaloid content to increase with annua L. (Tu, 2011), was rewarded with the Nobel prize in increasing soil nitrogen, while low levels of soil moisture caused medicine in 2015. a decrease in alkaloids, a clear contrast to the RAH (Arechavaleta Quinine content does not only vary among species (Nair, et al., 1992; Malinowski et al., 1998). 2010), but also among populations from different sites, Variation