Tree Genetics & Genomes (2019) 15: 81 https://doi.org/10.1007/s11295-019-1393-y

ORIGINAL ARTICLE

Genetic characterization of fragmented populations of officinalis L. (), a threatened of the northern Andean cloud forests

Augusta Cueva-Agila1 & Diego Vélez-Mora1 & Daniela Arias1 & Manuel Curto2 & Harald Meimberg2 & Chris Brinegar3

Received: 28 June 2019 /Revised: 1 October 2019 /Accepted: 4 October 2019 /Published online: 29 November 2019 # The Author(s) 2019

Abstract Cinchona officinalis L., a tree species endemic to the cloud forests of the northern Tropical Andes, has suffered from historical bark harvesting for extraction of antimalarial compounds and has also experienced recent demographic losses from high rates of deforestation. Most remnant populations are found in severely degraded habitat on the edges of pastures while a minority are protected in private reserves. The goals of our research were to assess the genetic diversities of fragmented populations of C. officinalis in the Loja province of southern Ecuador, characterize their phylogeographic distribution with respect to the region’s complex topography, and identify priority populations for conservation. Five nuclear microsatellite loci and the chlo- roplast rps16 intron were used to analyze six populations. Moderate levels of genetic diversity were found in all populations although the more remote southern population (Angashcola) had slightly higher heterozygosity and allelic richness. There were no indications of recent genetic bottlenecks although an rps16 intron haplotype was fixed in four populations. Genetic distance analysis based on microsatellite data placed the four easternmost populations in the same clade while the Angashcola population was the most divergent. Also, the most frequent rps16 intron haplotype in Angashcola was not found in any other population. Although each of the studied populations should be protected from further deforestation and agricultural expansion, the Angashcola population deserves highest conservation priority.

Keywords Cinchona officinalis . Tropical Andes . Microsatellites . rps16 intron . Genetic diversity . Ecuador

Introduction Real montane forests ecoregion which contains up to 8000 species (Richter et al. 2009) and is dominated by ever- The Tropical Andes biodiversity hotspot is home to at least green broad-leaved forest communities, including cloud for- 20,000 endemic plant species (Myers et al. 2000). In Ecuador, ests at higher elevations. Southern Ecuador, particularly the this hotspot is composed primarily of the Eastern Cordillera provinces of Loja and Zamora-Chinchipe, has especially high species richness (Brehm et al. 2008). Communicated by F. P. Guerra Patterns of plant diversity are determined by multiple inter- related factors (Kreft and Jetz 2007). In southern Ecuador, the Electronic supplementary material The online version of this article high diversity can be explained by its special (https://doi.org/10.1007/s11295-019-1393-y) contains supplementary material, which is available to authorized users. and complicated topography with high mountains, valleys, and inter-Andean region (Richter 2003). The specific air cir- * Augusta Cueva-Agila culation patterns differ between the western and eastern [email protected] mountain ranges, conferring specific and heterogeneous pre- cipitation patterns to each side and between them, as well. The 1 Departamento de Ciencias Biológicas, Universidad Técnica existence of multiple dry and wet areas has resulted in many Particular de Loja, Loja, Ecuador different ecosystems suitable for multiple species (Richter and 2 Insititute for Nature Conservation Research, University of Natural Moreira-Muñoz 2005). In addition, southern Ecuador is locat- Resources and Life Sciences, Vienna, Austria ed within the Andean Depression, the lowest part of the 3 Division of Natural Sciences, University of Maine Farmington, Andean mountain range where the northern and central Farmington, ME, USA Andes merge (Richter et al. 2009). This low elevation might 81 Page 2 of 16 Tree Genetics & Genomes (2019) 15: 81 have allowed contact of coastal and Amazonian species, there- by Linnaeus in 1753 based on a specimen collected in by further increasing the region’s biodiversity (Beck and Cajanuma (near Loja, Ecuador) in 1737 by La Condamine Richter 2008). (Garmendia 2017). The distribution of C. officinalis is limited Furthermore, there are other evolutionary forces such as only to a small area in the Andean cloud forest. In Ecuador, ecological-geographical barriers that could explain the diver- C. officinalis L. is found in the southern provinces of Loja, El sity of this region (Hughes and Eastwood 2006; Prieto-Torres Oro, Cañar and Azuay at 1700–3100 m a.s.l. (Andersson et al. 2018). The convergence of the eastern and western cor- 1998). Brako and Zarucchi (1993) also reported the occur- dilleras of the Andes and the repeated Pleistocene glacial and rence of C. officinalis in the northern Andes of Peru. The interglacial periods in the Ecuadorian Andes would have cre- growth form of the species can be shrubby, but small ated several physical and climatic barriers to migration, in- up to 6 m tall are more common. Cinchona officinalis has cluding mountain ranges and low valleys in the Loja and distylous pinkish or purplish tubular flowers. (Andersson Zamora-Chinchipe provinces that likely contributed to the 1998) that promote outcrossing by hummingbirds (Tropicos current species richness of the region (Jørgensen et al. 1995; 2018). Its small wind-dispersed winged seeds are enclosed in Richter et al. 2009). Such impediments to gene flow raise the capsules (Andersson 1998). possibility that populations may have existed in isolation long During the conquest of the Americas in the 16th and enough for significant genetic divergence to have occurred. In 17th centuries, Spanish missionaries learned of addition, fluctuations in population size and repeated bottle- Cinchona bark’s medicinal properties from indigenous necks associated with ongoing Pleistocene climatic instability tribes and discovered its effectiveness in the treatment may be important factors that have affected the genetic diver- of malaria (Jaramillo-Arango 1947). Therefore, sity of populations inhabiting this region (Prieto-Torres et al. C. officinalis was probably an important source of qui- 2018). nine and other in the early years of Cinchona Habitat conditions can also affect the genetic flow harvesting but became less so as its small native popu- between populations (Coates et al. 2003). Deforestation lations near the early Spanish settlement of Loja were and habitat fragmentation have been the main pressures exhausted by the mid-18th century (Gray 1738). Early that are affecting the diversity of tropical forests bark harvesting methods were typically fatal to the trees (Hansen et al. 2013;Lambinetal.2003; Macedo (Arias 2017). Other, more abundant, Cinchona species et al. 2012; Tapia-Armijos et al. 2015). Also, some spe- were exploited over the next 200 years (Andersson cies have suffered from selective logging that intensifies 1998; Martin and Gandara 1945). Once Cinchona plan- the impacts on demographic structure of trees tations were established in Asia in the 19th century, the (Broadbent et al. 2008;Inzaetal.2012). Beginning in pressure on native Andean populations eased, but a sec- the late 20th century, the clearing of nearly half of the ond round of harvesting occurred during World War II native forests in southern Ecuador for pastureland and when the Allied source was cut off from the plantation silviculture (Tapia-Armijos et al. 2015)fur- Dutch East Indies and tens of millions of pounds of ther reduced and fragmented the remaining forests, Cinchona bark were exported to the United States many of which are on private, unprotected land. The (Hodge 1948; Cuvi 2011). The oldest map of combination of these disturbances in the landscape can Cinchona distribution in Loja is a hand-painted map reduce the forest into small remnants and can lead to deposited in the Archivo General de Indias, in which reduction in connectivity of populations with concomi- bark collectors and Cinchona trees are depicted tant decrease in gene flow, thus increasing genetic dif- (Crawford 2016).Accordingtothismap,Cinchona trees ferentiation and reducing genetic variability (Aguilar were widely distributed in the mountains of the Loja et al. 2008;Cascanteetal.2002;Loweetal.2005). province. However, the past overharvesting and the high Cinchona is a of the Rubiaceae family known rates of deforestation in southern Ecuador in recent de- for its medicinal importance as a source of quinine and cades (Tapia-Armijos et al. 2015) have threatened other alkaloids that are present in different quantities in C. officinalis. its 23 species. Cinchona is widespread throughout the Determining the genetic composition of remaining popula- central and northern Andes with a noticeable center of tions of C. officinalis can help plan effective conservation and diversity just north of the Huancabamba Deflection in reforestation measures for this important species. The main southernmost Ecuador. Most species of the Cinchona objectives of this research were (i) to characterize the genetic genus seem to have fairly restricted distributions, and diversity of the remnant C. officinalis populations in Loja prov- although they have a tendency to hybridize, their ranges ince of southern Ecuador by nuclear and chloroplast DNA do not typically overlap (Andersson 1998). analysis, (ii) to test our hypothesis that barriers to migration Cinchona officinalis was the first species of the genus to be and gene flow in southern Ecuador will have resulted in diver- described in detail (Andersson 1998). The species was named gent populations of C. officinalis in the more geographically Tree Genetics & Genomes (2019) 15: 81 Page 3 of 16 81 isolated parts of its distribution, and (iii) to identify priority by Curto et al. (2013). Fluorescently tagged M13-based populations for conservation. primers (VIC-5′-TAATACGACTCACTATAGGG-3′, PET- 5′-GATAACAATTTCACACAGG-3′,NED-5′-TTTC CCAGTCACGACGTTG-3′ or 6FAM-5′ -TGTA Materials and methods AAACGACGGCCAGT-3′) were included along with the locus-specific reverse primers (unmodified) and forward Population descriptions, sample collection, and DNA primers (modified with the appropriate M13 primer sequences isolation as 5′ tails as shown in Table 1). PCR was conducted on 10-μL reactions containing 5 Six populations of C. officinalis were sampled from forest μL of Multiplex PCR Master Mix (Qiagen, USA), 0.4 fragments in southern Ecuador (Fig. 1), all within Loja prov- μL of M13-tailed forward primer (1 pmol/μL), 0.4 μL ince. The majority of samples were collected from trees that of the reverse and fluorescent primers (10 pmol/μL), probably survived bark harvesting and deforestation due to and 3.8 μL of water. The reaction was promoted by their growth on steep slopes. Most of the individuals in the the following temperature profiles: 95 °C for 15 min; populations of El Madrigal, Vilcabamba, Yangana, and 11 cycles of denaturation at 95 °C for 30 s, a touch- Gonzanamá are scattered around the edges of pastures and down pattern for annealing from 60 to 55 °C, decreas- likely subsist because of continued local medicinal use (as ing 0.5 °C per cycle for 60 s, extension at 72 °C for 30 inferred from our occasional observations of limited bark re- s, followed by 19 cycles of denaturation at 95 °C for 30 moval). The most conserved populations were found in the s, annealing at 55 °C for 30 s and extension at 72 °C private nature reserves of Angashcola and El Cristal. The four for 30 s. Cycling was finished with a final extension populations located along the western slope of the Eastern step of 60 °C for 30 min. All successful PCR reactions Cordillera Real (i.e., the western border of Podocarpus were sent for fragment analysis to the Sequencing National Park) were at elevations of 2141–2271 m a.s.l. Service at the Ludwig Maximilian University Munich. whereas the Gonzanamá and Angashcola populations were Obtained chromatograms were analyzed in Geneious at 2643–2877 m a.s.l. using the Microsatellite plugin (Kearse et al. 2012). Samples were collected under a general permit (Contrato Marco de Acceso a Recursos Genéticos, Microsatellite PCR and genotyping número MAE-DNB-CM-2015-0016) issued by the Ecuadorian Ministry of the Environment. Permission to Two multiplex PCR reactions were performed for each sample collect was granted by landowners. Individual leaves using the three-primer method described above. Loci Coff8, from 25 to 31 trees in each population were desiccated Coff10, and Coff20 PCR products were amplified and labeled in plastic bags containing 10 g silica gel (Merck, with VIC, PET, and 6FAM, respectively, in a 10-μL multiplex Germany) and stored at room temperature. Total DNA reaction containing 1× ABI Master Mix (Applied Biosystems, wasisolatedfrom10to30mgofdryleaftissueusing USA), 5–20 ng genomic DNA and the following primers: the PureLink Genomic Plant DNA Purification Kit VIC-M13 (0.4 μM), PET-M13 (0.1 μM), 6FAM-M13 (0.1 (Invitrogen, USA) or by the CTAB method of Doyle μM), tailed Coff8-F (0.04 μM), tailed Coff10-F (0.01 μM), and Doyle (1987). DNA was quantified on a tailed Coff20-F (0.01 μM), Coff8-R (0.4 μM), Coff10-R (0.1 NanoDrop 2000 spectrophotometer (Thermo Fisher μM), and Coff20-R (0.1 μM). Loci Coff13 and Coff15 PCR Scientific, USA). products were amplified and labeled with NED and 6-FAM, respectively, in a 10-μL multiplex reaction containing 1× ABI Nuclear microsatellite marker development Master Mix, 5–20 ng genomic DNA and the following and screening primers: NED-M13 (0.1 μM), 6FAM-M13 (0.1 μM), tailed Coff13-F (0.01 μM), tailed Coff15-F (0.01 μM), Coff13-R Genomic DNA was isolated from a tree grown at the San (0.1 μM), and Coff15-R (0.1 μM). Both multiplex reactions Francisco Scientific Station (33 km east of Loja) using seed were run using a touchdown PCR procedure: initial denatur- obtained from a tree near San Pedro de Vilcabamba. DNAwas ation at 95 °C (15 min); 10 cycles of 95 °C (30 s), 62 °C (60 s), extracted from 20 mg of dry leaf material using the protocol of and 72 °C (30 s) with a 1 °C reduction in annealing tempera- Curto et al. (2013). This sample was then used for marker ture per cycle; 20 cycles of 95 °C (30 s), 52 °C (30 s), and 72 discovery using the Illumina MiSeq system (Illumina, USA) °C (30 s); final extension at 72 °C (30 min). For some samples and primer design following the approaches described in Deck with lower DNA concentrations, 22 cycles were performed at et al. (2016). This consisted of a three-primer assay using the the 52 °C annealing temperature instead of 20. multiplexing short tandem repeat polymorphisms with tailed PCR products (1 μL) were mixed with 0.15 μLof primers (MSTP) method of Oetting et al. (1995)asperformed GeneScan LIZ 600 size standards (Thermo Fisher Scientific) 81 Page 4 of 16 Tree Genetics & Genomes (2019) 15: 81

Fig. 1 Locations of sampled C. officinalis populations. PNP, Podocarpus National Park; PNY, Yacuri National Park

and 10 μL of Hi-Di Formamide (Thermo Fisher Scientific). and scored in GeneMapper version 4.1 software (Applied Fragments were separated on a 3500 Genetic Analyzer Biosystems) using size bins for each locus. To avoid genotyp- (Applied Biosystems) according to manufacturer’s directions ing error when calling alleles, only peaks with amplitudes Tree Genetics & Genomes (2019) 15: 81 Page 5 of 16 81

Table 1 Cinchona officinalis microsatellite loci descriptions

Locus SSR motif GenBank Primer sequencesa Tm Allele size range Ab accession (bp)

Coff8(GA)3G2(GA)13 MH492482 F: 5′-TAATACGACTCACTATAGGGGGAAGGCTTCAAAC 60.6 157–181 11 AGAAATCA-3′ 57.5 R: 5′-CTTGTCTGTTTTGTGTCAGCA-3′ Coff10 (CT)16 MH492458 F: 5′-GATAACAATTTCACACAGGGAGCCGATCTTTTA 61.7 182–206 12 CCTGGGT-3′ 62.8 R: 5′-TCAATGTTGATGGGCTTTCTCA-3′ Coff13 (TAA)10 MH492461 F: 5′-TTTCCCAGTCACGACGTTGACTGTTCACCTCTT 58.0 215–271 19 TTTGCCT-3′ 58.2 R: 5′-GGTTGGATAGCTCCTTATCTCC-3′ Coff15 (ACC)8 MH492463 F: 5′-TGTAAAACGACGGCCAGTGAAGTGATGCCCAA 61.7 234–357 16 CAAAATTG-3′ 61.0 R: 5′-GGGATGGGTGAGGGATTAGA-3′ Coff20 (GGA)12AGA MH492467 F: 5′-TGTAAAACGACGGCCAGTGCTTGTACTTGTACTGGTCC 55.4 175–196 7 (GGA) A-3′ 63.7 R: 5′-TTCCCTCCACCCTCTGAACC-3′ a Underlined segments of the “tailed” forward primers are M13-based sequences (see text for details) b A = total number of observed alleles (6 populations, 162 individuals)

greater than 500 relative fluorescent units (RFU) were scored. FST matrix and the vegan package (Oksanen et al. 2019)to Samples with peaks below this threshold were reamplified compute the Mantel test. In both cases, 9999 random permu- until reliable consensus genotypes were obtained. tations were performed. BOTTLENECK v1.2.02 (Piry et al. 1999) was used to Statistical analysis detect signs of recent bottlenecks in the studied populations based on the heterozygosity excess method (Cornuet and Scored microsatellite genotypes were formatted in Luikart 1996; Luikart and Cornuet 1998). Since the CONVERT 1.31 (Glaubitz 2004) to facilitate conversion of Wilcoxon test gives most reliable results when less than 20 the data into other software formats. GENALEX 6.503 polymorphic loci are studied (Piry et al. 1999), the one-tailed (Peakall and Smouse 2006, 2012) was used to calculate allele Wilcoxon signed rank test was used with 10,000 iterations to frequencies, observed number of alleles (A), number of effec- test for significant excess of heterozygosity. Three mutation tive alleles (Ae), private alleles (PA), observed heterozygosity models were used: the infinite allele model (IAM), the step- (Ho), unbiased expected heterozygosity (He), overall FST wise mutation model (SMM), and the two-phase model (Wright 1951, 1965), F’ST (Meirmans 2006;Meirmansand (TPM). The IAM (Kimura and Crow 1964)andSMM(Ohta Hedrick 2011), GST (Nei 1973), G’ST (Hedrick 2005), Jost’s and Kimura 1973) are the extreme mutation models DEST (Jost 2008; Meirmans and Hedrick 2011), pairwise com- (Chakraborty and Jin 1992) while the TPM (Di Rienzo et al. parisons of population differentiation by FST and DEST,prob- 1994) is an intermediate model that more closely reflects mi- ability of identity [P(ID) and P(ID)sib] (Waits et al. 2001), and crosatellite mutation. Since bottlenecks also cause a reduction number of migrants per generation (Nm) based on overall FST. in low frequency (< 0.1) alleles, the qualitative graphical The number of migrants per generation (Nm) was also calcu- method of Luikart et al. (1998), available in the lated in GENEPOP on the Web based on the private allele BOTTLENECK software, was used to detect any mode-shift method of Barton and Slatkin (1986). Exact Hardy- in allele frequency distributions to more intermediate frequen- Weinberg global tests of heterozygote deficiency and inbreed- cies. Finally, the M-ratio approach developed by Garza and ing coefficients (FIS) were calculated in GENEPOP on the Williamson (2001), which is based on the ratio of the number Web (Raymond and Rousset 1995; Rousset 2008)and of microsatellite alleles to the range in allele size, was also FSTAT 2.9.3.2 (Goudet 2001), respectively. The program used to detect recent bottlenecks. FreeNA (Chapuis and Estoup 2007) was used to calculate null Bootstrapped UPGMA dendrograms were constructed in allele frequencies. Simple Mantel tests were performed using POPTREE2 (Takezaki et al. 2010) to visualize Nei’sDA dis- the Nei genetic pairwise distance matrix in GENALEX and tance (Nei et al. 1983; Takezaki and Nei 2008) with and with- the FST pairwise genetic differentiation matrix in R (R out null allele frequency corrections calculated by FreeNA Development Core Team 2004). The package hierfstat (Chapuis and Estoup 2007). Population structure was ana- (Goudet and Jombart 2015) was used to compute the pairwise lyzed using the Bayesian model-based clustering program 81 Page 6 of 16 Tree Genetics & Genomes (2019) 15: 81

STRUCTURE 2.3.4 (Falush et al. 2003;Hubiszetal.2009; Results Pritchard et al. 2000) with the admixture ancestry model. Both allele frequency models (independent and correlated) were Microsatellite characteristics and discrimination run with length of burnin period and MCMC repetitions each power set at 10,000. Running five repetitions for each potential num- ber of clusters (K =3–7), the independent allele model gave The Illumina MiSeq run produced 273,042 paired reads from highly reproducible estimates of posterior probabilities of the which 271,118 passed the quality control and overlapped. data [LnP(D)]. However, in the correlated allele model, there Among those 20,957 contained microsatellites with flanking was significantly greater variability at some K values, requir- regions larger than 40 bp. A set of 26 primer pairs were de- ing an increase in burnin and MCMC repetition to 20,000. The signed from which 16 amplified in single reactions in the plateauing nature of the correlated allele K vs. LnP(D) graph sample used for marker development. However, after apply- made inference of the optimal K value difficult, but a ΔK ing them to the remaining samples, only five markers (two transformation of the data (Evanno et al. 2005)providedan dinucleotide and three trinucleotide repeats) provided reliable estimate that was consistent with the independent allele mod- and polymorphic PCR products, but these proved to be suffi- el. Results from STRUCTURE were graphically displayed cient for our study. Repeat motifs, primers and allele summa- using DISTRUCT (Rosenberg 2004) and were essentially ries are shown in Table 1. Coff13 and Coff15 were the most identical with either model. polymorphic (19 and 16 total observed alleles, respectively) Median-joining networks for chloroplast rps16 intron se- while Coff20 was the least polymorphic (7 total alleles). quence haplotypes were constructed in NETWORK 5.0 Although the original sequenced alleles from Coff13 and (Bandelt et al. 1999) using an epsilon value of 10 and weights Coff15 contained perfect trinucleotide repeats, the difference of 10 for substitutions and 5 for indels. Manual alignments of in sizes of observed alleles did not strictly follow a three base ahypervariableTnAn region were made in order to minimize pair pattern. the number of polymorphic sites. The five-locus microsatellite analysis was successful in identifying unique genotypes in 97.5% of individuals tested Chloroplast rps16 intron PCR and sequencing (Table 2). Two consecutively sampled trees in the Vilcabamba population had identical genotypes, as did two trees in The rps16 intron was initially amplified using the forward Yangana. For all populations, the mean standard probability

(rpS16F: 5′-AAACGATGTGGTARAAAGCAAC-3′) and re- of identity [P(ID)] was 0.00014 which is within the “reason- verse (rpS16R: 5′-AACATCWATTGCAASGATTCGATA- ably low” range of 0.01–0.0001 indicated by Waits et al. 3′) primers of Shaw et al. (2005). However, sequencing of a (2001). The mean probability of identity among siblings mononucleotide repeat (Gn)nearthe3′ end of the intron gave [P(ID)sib], i.e., the upper limit of probability that two individ- variable results depending on which primer was used for se- uals in a population will have the same multi-locus genotype, quencing. This problem was alleviated by designing a new averaged 0.023. Due to the high outcrossing rates of distylous reverse primer (rpS16R2: 5′-TCGGATCATAAAAA species, such as C. officinalis, it is likely that the standard P(ID) CCCACT-3′)inexon2ofrps16 located 56-bp downstream values are more accurate estimations and that the discrimina- of the original rpS16R priming site. The sequence of rpS16R2 tion power of the microsatellite panel was adequate for the was based on a Coffea arabica GenBank accession study. (EF044213.1). The 20-μL PCR reactions consisted of 1× GoTaq Buffer (Promega, USA), 1.5 mM magnesium chloride (included in Table 2 Probability of identity and number of unique genotypes for the the buffer), 0.2 mM each deoxyribonucleotide, 0.25 μg/mL six C. officinalis populations using five microsatellite loci μ bovine serum albumin, 0.5 M each of primers rpS16F and a b rpS16R2, 1 U GoTaq DNA Polymerase (Promega), and 20– Population P(ID) P(IDsib) Number Unique genotypes 50 ng template DNA. Amplifications were carried out at 94 °C El Madrigal 0.00010 0.021 26 26 (3 min) followed by 35 cycles of 94 °C (30 s), 50 °C (45 s), El Cristal 0.00012 0.026 27 27 and 72 °C (90 s). Final elongation was at 72 °C (10 min). PCR Vilcabamba 0.00016 0.024 25 23 products of approximately 940 bp were purified with the Yangana 0.00018 0.028 26 24 Wizard SV Gel and PCR Clean-up System (Promega) or the Gonzanamá 0.00009 0.023 31 31 Clean and Concentrator-5 Kit (Zymo, USA). Sequencing was Angashcola 0.00002 0.014 27 27 performed by Macrogen (South Korea) or the University of 162 158 Maine DNA Sequencing Facility (USA) using both PCR primers. Sequences were aligned using ClustalX2 (Larkin a P(ID) = standard probability of identity et al. 2007). b P(IDsib) = probability of identity among siblings Tree Genetics & Genomes (2019) 15: 81 Page 7 of 16 81

Genetic diversity number of private alleles was low (ranging from 1 to 3) where- as Gonzanamá and Angashcola, being more isolated, had 8 The average number of alleles per locus ranged from 5.2 to 7.6 and 6 total private alleles, respectively. Observed heterozygos- in the six populations (Table 3). In the four populations having ities (Ho) averaged over the five microsatellite loci ranged the least barriers to migration/pollination between them (El from 0.580 to 0.680 while overall expected heterozygosities

Madrigal, El Cristal, Vilcabamba and Yangana), the total (He) ranged from 0.616 to 0.717 (lowest in Yangana, highest

Table 3 Genetic diversity indicesa for six C. officinalis populations at five microsatellite loci

El Madrigal (n=26) El Cristal (n =27)

Locus Ho He HW FIS A Ae PA Null Ho He HW FIS A Ae PA Null

Coff8 0.615 0.632 ns 0.027 7 2.6 0 0.000 0.593 0.568 ns -0.044 4 2.3 0 0.000

Coff10 0.538 0.719 0.255 8 3.4 2 0.083 0.815 0.821 ns 0.008 6 5.2 0 0.000

Coff13 0.654 0.690 ns 0.054 7 3.1 0 0.016 0.815 0.705 ns -0.159 6 3.2 1 0.000

Coff15 0.731 0.758 0.037 6 3.9 0 0.055 0.704 0.772 ns 0.090 10 4.1 1 0.001

Coff20 0.731 0.546 ns -0.348 3 2.2 0 0.000 0.259 0.234 ns -0.110 3 1.3 0 0.000

Overall 0.654 0.669 0.023 6.2 3.0 2 0.031 0.637 0.620 ns -0.028 5.8 3.2 2 0.000

Vilcabamba (n=25) Yangana (n =26)

Locus Ho He HW FIS A Ae PA Null Ho He HW FIS A Ae PA Null

Coff8 0.480 0.662 ns 0.279 5 2.8 0 0.073 0.269 0.313 ns 0.142 5 1.4 1 0.053

Coff10 0.880 0.763 ns -0.157 6 4.0 0 0.000 0.846 0.831 ns -0.019 7 5.4 1 0.000

Coff13 0.800 0.710 ns -0.129 6 3.3 1 0.000 0.654 0.652 ns -0.002 6 2.8 0 0.005

Coff15 0.720 0.687 ns -0.049 6 3.1 0 0.000 0.615 0.735 0.165 7 3.6 1 0.076

Coff20 0.520 0.422 ns -0.238 3 1.7 0 0.000 0.692 0.548 ns -0.269 3 2.2 0 0.000

Overall 0.680 0.649 ns -0.049 5.2 3.0 1 0.015 0.615 0.616 ns 0.001 5.6 3.1 3 0.027

Gonzanama (n = 31) Angashcola (n= 27)

Locus Ho He HW FIS A Ae PA Null Ho He HW FIS A Ae PA Null

Coff8 0.645 0.665 ns 0.030 4 2.9 0 0.000 0.778 0.822 ns 0.055 9 5.2 1 0.000

Coff10 0.419 0.497 ns 0.158 4 2.0 0 0.037 0.741 0.685 ns -0.083 8 3.1 0 0.000

Coff13 0.677 0.774 ns 0.126 9 4.2 2 0.025 0.630 0.808 0.224 9 4.8 3 0.123

Coff15 0.645 0.810 0.206 12 4.9 5 0.090 0.444 0.747 0.410 6 3.7 0 0.162

Coff20 0.516 0.493 ns -0.047 5 1.9 1 0.000 0.593 0.524 ns -0.134 6 2.1 2 0.000

Overall 0.580 0.648 0.105 6.8 3.2 8 0.030 0.637 0.717 0.114 7.6 3.8 6 0.057 ns = not significant *P =0.05;**P = 0.01; ***P =0.001 a Observed heterozygosity (Ho), unbiased expected heterozygosity (He), exact test of heterozygosity deficiency (HW), inbreeding coefficient (FIS), number of observed alleles (A), number of effective alleles (Ae), number of private alleles (PA), estimate of null allele frequency (null). Overall values are averages for Ho, He, A, Ae, and null; total for PA; global for FIS 81 Page 8 of 16 Tree Genetics & Genomes (2019) 15: 81

in Angashcola). The number of effective alleles (Ae)was Population structure highest in Angashcola (3.8 vs. 3.0–3.2 for the other popula- tions), consistent with its greater expected heterozygosity and Wright’s FST (Wright 1951, 1965) is a traditional measure of average number of alleles. subpopulation genetic differentiation, but its current utility has Three populations (El Madrigal, Gonzanamá, and been questioned because its maximum value, i.e., complete Angashcola) showed significant deviation from Hardy- differentiation, can be severely reduced from 1 when using Weinberg equilibrium (heterozygote deficiency) over all loci, highly polymorphic markers such as microsatellites (Neigel but only Gonzanamá and Angashcola had relatively high pos- 2002;Jost2008; Meirmans and Hedrick 2011). The overall itive inbreeding coefficients (global FIS values > 0.10). Locus FST calculated from our microsatellite data is 0.180, and the Coff15 showed the highest estimated null allele frequency related GST (Nei 1973) value is similar (0.166). across all populations (average of 0.064), especially in the Standardization of these values to a scale of 0–1bydividing Gonzanamá and Angashcola populations (0.090 and 0.162, by their maximum values yields the more insightful measures respectively). Angashcola also had a relatively high estimated of F’ST (Meirmans 2006)andG’ST (Hedrick 2005). For the null allele frequency (0.123) at the Coff13 locus. The appear- C. officinalis populations, F’ST = 0.561 and G’ST = 0.542, ance of null alleles would be more likely in these two popu- values which imply a higher level of subpopulation differen- lations which are the most isolated from the source of genomic tiation. Jost’s D, which is claimed to be a much more reliable DNA (near Vilcabamba) that was used for microsatellite standardized measure of differentiation (Jost 2008), was screening (Chapuis and Estoup 2007). Therefore, Hardy- slightly lower (DEST = 0.451) although still an indication of Weinberg disequilibrium in Angashcola, and possibly relatively high subpopulation differentiation. The above Gonzanamá, could be attributed to the presence of null alleles values were derived from data without null allele correction, rather than to factors such as inbreeding or departure from but overall FST decreased by only 1.9% when the null allele random mating. correction was applied using FreeNA.

The number of migrants per generation (Nm) across all loci Pairwise comparisons of population differentiation were was estimated to be 1.2 (FST method) or 1.4 (private allele calculated for both FST and Jost’s DEST (Table 4). Pairwise method), indicating that there is adequate gene flow between FST values ranged from 0.064 to 0.168 while Jost’s DEST populations to counter the negative effect of genetic drift on ranged from 0.220 to 0.763. The only relative difference was heterozygosity (Nason and Hamrick 1997). Bottleneck analy- that the El Madrigal population was slightly less differentiated sis of the data yielded no convincing evidence of recent bot- from Angashcola (0.130) than Gonzanamá (0.141) in the FST tlenecks (Supplementary Table S1). Of the three mutation comparison but slightly more differentiated from Angashcola models, the only significant result for heterozygosity excess (0.619) than Gonzanamá (0.587) in the Jost’s DEST compari- was for the Vilcabamba population using the IAM which is son. The latter comparison is more geographically consistent. the least conservative model and more prone to type I errors In both comparisons, the El Madrigal population is more ge- (Cornuet and Luikart 1996). All M ratios were above 1.7, well netically similar to the Vilcabamba population than to its above the critical value of 0.68 (Garza and Williamson 2001). neighboring El Cristal population. The Jost’s DEST compari- Similarly, there were no significant mode shifts in the allele son, in particular, shows a high degree of differentiation frequency distribution histograms that would be indicative of (0.619–0.763) between the four populations on the western low-frequency allele loss caused by bottlenecks. slope of the Eastern Cordillera Real and the Angashcola

Table 4 Pairwise population FST (below diagonal) and Jost’s DEST (above diagonal) comparisons. El Madrigal El Cristal Vilcabamba Yangana Gonzanamá Angashcola

El Madrigal 0.000 0.340 0.220 0.377 0.587 0.619

El Cristal 0.095 0.000 0.193 0.376 0.411 0.763

Vilcabamba 0.064 0.062 0.000 0.228 0.400 0.667

Yangana 0.105 0.113 0.072 0.000 0.442 0.701

Gonzanamá 0.141 0.115 0.107 0.123 0.000 0.548

Angashcola 0.130 0.168 0.144 0.159 0.122 0.000

Results were significant at P = 0.001 for all values (999 permutations) Tree Genetics & Genomes (2019) 15: 81 Page 9 of 16 81

Fig. 2 UPGMA dendrograms of C. officinalis microsatellite data. a Without null allele correction; b with null allele correction. Bootstrap percentages (1000 replications) are at nodes

population. As with overall FST, correction for null allele fre- admixture from the Angashcola cluster was found in any pop- quencies decreases the average pairwise FST only slightly ulation except for a few individuals in Gonzanamá. The least (2.0%). admixture was seen in the more isolated Angashcola popula- Because of the possibility of null alleles, distance trees tion. Although geographically located between the El were constructed from microsatellite data with and without Madrigal and Vilcabamba populations, El Cristal shows a dif- null allele frequency corrections (Fig. 2). In both cases, the ferent genetic background. The STRUCTURE results are con- group containing the populations located on the western slope sistent with the distance tree analysis (Fig. 2). of the Eastern Cordillera Real (El Madrigal, El Cristal, Using the Mantel test, correlation between the Nei genetic Vilcabamba, and Yangana) was well supported with high distance and geographic distances of population pairs was not bootstrap values although the positions of El Cristal and significant (r =0.045,p =0.181).WiththeFST genetic dis- Yangana were reversed and not well supported. The sister tance, the r value was slightly higher but only weakly signif- group of El Madrigal and Vilcabamba appeared in both den- icant (r =0.102,p =0.049). drograms. The positions of Gonzanamá and Angashcola were unaffected by the null allele corrections. Using data without Chloroplast haplotype variation and distribution null allele correction (Fig. 2a) gave a slightly more geograph- ically consistent dendrogram. Six haplotypes were detected at the chloroplast rps16 intron The program STRUCTURE was used to assess the micro- (Table 5). The four polymorphic sites included one base sub- satellite data by a Bayesian clustering approach (Fig. 3). stitution and three repetitive regions. Haplotype A, the pre- Although six nominal populations were sampled, the inferred sumptive ancestral sequence, was by far the most abundant cluster size (K) was determined to be five by either the inde- with an overall frequency of 0.877. Haplotypes D and E, with pendent or correlated alleles model with the admixture pat- frequencies of 0.006, were found in only one individual each. terns essentially the same between models. The majority of Haplotype distributions are shown in Fig. 4. El Madrigal individuals in the El Cristal, Yangana, Gonzanamá, and had the most haplotype diversity with five sequence variants Angashcola populations had ancestry within their unique clus- (A–E). However, the populations of El Cristal, Vilcabamba, ters, but the fifth cluster was composed mainly of individuals Yangana, and Gonzanamá were fixed at haplotype A. The from El Madrigal and Vilcabamba with significant admixture latter three populations occur in the most degraded habitat from the Yangana cluster and, to a lesser extent, El Cristal. and are the smallest (less than 50 individuals), suggesting that Very little admixture from the Gonzanamá cluster appeared in fixation may have occurred in this haploid genome through El Madrigal, El Cristal and Vilcabamba, and essentially no genetic drift. Angashcola was the only population containing

Fig. 3 Bayesian cluster analysis of C. officinalis microsatellite data from STRUCTURE (K =5) 81 Page 10 of 16 Tree Genetics & Genomes (2019) 15: 81

Table 5 Chloroplast haplotype sequence variation in the rps16 intron reflects that of the original forest, or if there has been an actual based on a 781 bp alignment reduction in heterozygosity over nearly 400 years of harvest- Polymorphic sitesa GenBank accession ing this species. However, compared to Angashcola, the larg- est and least impacted population in this study, the other five Haplotype 39 467– 579– 603– populations had expected heterozygosities that were 11% low- 480 588 610 er on average. Mean allelic richness and number of effective

AGT9A4 – T9 – A7 – MK071268 alleles for these five populations were 22% and 18% lower than Angashcola, respectively. Assuming that the Angashcola BGT10A4 T9 – A7 – MK071269 population analysis provides a minimum estimation of the CGT9A4 – T10 A7 – MK071270 original forest’s genetic diversity, these data support the con- DGT7A6 – T8 –– A7 – MK071271 clusions of Vranckx et al. (2012) that fragmentation may only ECT9A4 – T10 A7 – MK071272 have a slight negative impact on heterozygosity, but allelic FGT10A3 – T9 – A8 MK071273 richness is lost at a higher rate. Also, for microsatellites, a Dash = deletion changes in allelic diversity are considered to be more infor- mative than heterozygosity for assessing genetic erosion haplotype F and the only one where haplotype A was the least (Kang et al. 2008; Spencer et al. 2000). abundant. The latter observation is consistent with the previ- Loss of genetic diversity through genetic drift cannot be ous analyses which showed a greater genetic divergence of completely discounted in C. officinalis populations, but, ac- Angashcola relative to the other populations. cording to Young et al. (1996), our estimation of gene flow

The construction of a unique median-joining network of from microsatellite data (Nm =1.2–1.4) appears to be high haplotypes was not possible owing to the hypervariability of enough to counter genetic erosion in the nuclear genome the second polymorphic site (see Table 5) which allowed a caused by drift. Gene flow via long-distance pollination and number of possible alignments and networks (not shown). seed dispersal are mechanisms by which fragmented forest However, all networks placed haplotype A as the ancestral populations can maintain genetic diversity (Kramer et al. sequence. Depending on the alignment used, haplotype F 2008), and even isolated trees can act as “stepping stones” (unique to Angashcola) was either directly derived from hap- between fragmented populations (Lander et al. 2010;Lowe lotype A, indirectly derived from haplotype A via haplotype et al. 2005). Seed dispersal is an unlikely agent of gene flow B, or it shared a common (undetected) ancestral haplotype between fragmented C. officinalis populations because the with haplotype B. winged seeds of Cinchona species are probably incapable of dispersal over such long distances. According to Vittoz and Engler (2007), winged seeds in forest species can reach a Discussion dispersion distance of only 40–150 m. Due to their purple- pink coloration and tubular shape, the flowers of C. officinalis Habitat fragmentation can have potentially serious genetic are mainly pollinated by hummingbirds (Altshuler 2003; consequences, including reduction in genetic diversity and Sazima et al. 1996; Tropicos 2018) which are capable of for- gene flow, higher rates of inbreeding and greater inter- aging over long distances (Murcia 1996) although they typi- population divergence due to genetic drift (Lowe et al. cally forage within a narrow home territory (Renner 1998). 2005). Some tropical forest are particularly susceptible Some hummingbirds can persist in a matrix of forest frag- to the effects of habitat fragmentation and degradation owing ments and cattle pasture (Stouffer and Bierregaard Jr 1995), to their reliance on animal pollinators, complex self- but other studies have shown that open areas severely reduce incompatibility mechanisms, and high rates of outcrossing their visitations to fragments (Lindberg and Olesen 2001; (Hamrick and Murawski 1990). In a meta-analysis of more Magrach et al. 2012). Whether the large blocks of open pas- than 100 plant studies, Aguilar et al. (2008) generally found ture separating fragments of C. officinalis are impediments to significant negative effects of habitat fragmentation on expect- hummingbird pollination is likely species-dependent and re- ed heterozygosity, allele number, percent polymorphic loci, mains unstudied. and outcrossing rates whereas increased inbreeding was less Two populations in this study, Gonzanamá and common. In addition, the analysis showed that loss of hetero- Angashcola, had relatively high FIS values (0.105 and 0.114, zygosity was greater when more time had passed since respectively). We are hesitant to conclude that inbreeding is fragmentation. the cause. Instead, heterozygote deficiency is more likely ex- Since there are no known remaining extended forests of plained by the presence of null alleles, especially in locus C. officinalis, it is impossible to determine with certainty Coff15 which deviated significantly from Hardy-Weinberg whether the moderately high heterozygosity of the six equilibrium in four of the six populations. In fact, of all the fragmented populations (average He = 0.653) in our study populations, inbreeding would be least expected in Tree Genetics & Genomes (2019) 15: 81 Page 11 of 16 81

Fig. 4 Chloroplast rps16 intron haplotype frequency distributions of C. officinalis populations. PNP, Podocarpus National Park; PNY, Yacuri National Park

Angashcola since it has the highest number of individuals. population used to construct the microsatellite library There would, however, be a higher probability of null alleles (Chapuis and Estoup 2007). Although the presence of null in Angashcola because of its greater divergence from the alleles can inflate FST values and genetic distances in 81 Page 12 of 16 Tree Genetics & Genomes (2019) 15: 81 significantly differentiated populations (Chapuis and Estoup with less historical cutting. Bayesian cluster analysis gave 2007), the difference between corrected and uncorrected over- similar results in that it separated El Cristal from the El all FST or average pairwise FST values in our study was mar- Madrigal-Vilcabamba cluster although there was some admix- ginal (≤ 2%). ture. El Cristal is located in the region of Cajanuma, an area in Many studies have shown that after forest fragmentation, which a royal decree in 1782 prohibited harvesting due to first generation progeny (seedlings and saplings) have reduced overexploitation of the tree (Arias 2017). However, there is genetic diversity and sometimes show signs of reduced fitness no subsequent documentation regarding unique management (Cascante et al. 2002; Fernández-M and Sork 2007; Lowe approaches in Cajanuma that might have influenced the spe- et al. 2005; Nason and Hamrick 1997). Therefore, Lowe cies’ genetic composition. Regardless of the reasons for this et al. (2015) have suggested that future studies be focused slight phylogeographic discrepancy, the Eastern Cordillera on the progeny of forest fragments. Such reductions in fitness Real populations appear to share a recent common ancestry could be due to environmental changes in the modified habi- and were probably part of a continuous population prior to tats as well as genetic factors (Cascante et al. 2002). Limited deforestation. studies have not shown evidence of reduced fitness of Since haploid genomes have smaller effective population C. officinalis seeds under controlled germination conditions: sizes than diploid genomes (Weising et al. 2005), chloroplast 70% germination with light exposure (Romero-Saritama and loci are more sensitive to genetic drift than nuclear markers Munt 2017), 87% with hydrogen peroxide and light exposure (Provan et al. 2001). Haplotype A of the C. officinalis chloro- (Armijos-González and Pérez-Ruiz 2016), and 71–83% in plast rps16 intron, the most common and widespread variant, sand and peat (Guáman et al. 2017). However, according to is presumably the ancestral form based on its abundance and Acosta (1947), germination of older seeds is greatly reduced. centrality in median-joining networks. Although there was no Further research is needed to determine whether post- evidence of genetic bottlenecks from the microsatellite data, fragmentation progeny of C. officinalis are indeed suffering fixation of haplotype A by genetic drift occurred in four out of from genetic erosion and reduced fitness. six of the C. officinalis populations. Despite the four rare The overall genetic differentiation of the C. officinalis pop- haplotypes (B–E) found at El Madrigal, haplotype A was still ulations is relatively high as measured by FST, GST, and Jost’s the most abundant in that population. However, haplotype F DEST, although the higher value of DEST is likely more de- was the most frequent in, and unique to, Angashcola. As with pendable since GST (and FST) typically underestimates differ- the microsatellite data, the chloroplast analysis underscores entiation when heterozygosity is high and Nm > 1 (Leng and the divergent nature of the Angashcola population relative to Zhang 2011). In a simulated study using microsatellite data the others. under the finite island model, Leng and Zhang (2011)sug- Both orogeny and glacial cycles undoubtedly played a role gested that one explanation for higher values (> 0.15) of GST in the phylogeographic history of C. officinalis.Accordingto and D is small effective population size (< 100). Pairwise Gentry (1982), much of the diversity of the Neotropics can be population comparisons, especially with Jost’s DEST,showed attributed to rapid species diversification caused by recent moderate genetic differentiation among the four populations Andean orogeny (< 10 MY). The complex mountain terrain located on the western slope of the Eastern Cordillera Real, of southern Ecuador combined with climatic fluctuations dur- but greater differentiation was seen between those populations ing glacial-interglacial periods created barriers to migration and the other two (Gonzanamá and Angashcola) where moun- that would have led to isolation and species divergence tainous terrain up to 3000 m or more would preclude easy (Beck and Richter 2008; Jørgensen et al. 1995). In fact, this gene flow. Angashcola, in particular, was highly differentiated general area of southern Ecuador is thought to be the center of from the Eastern Cordillera Real populations owing to its re- diversity for the genus Cinchona (Andersson 1998). mote location. This finding supports our original hypothesis The greater genetic differentiation of the Angashcola that the complex geological history of southern Ecuador population of C. officinalis implies that there was an an- would encourage divergence of some populations. cient split of an ancestral population, although the timing Distance-based tree analysis yielded similar results. The of its divergence (i.e., Pleistocene or late Miocene) re- only geographically inconsistent placement in the UPGMA mains unknown. The Gonzanamá population has genetic dendrogram was the El Cristal population which did not group characteristics that are intermediate between Angashcola between its neighboring populations (El Madrigal and and the Eastern Cordillera Real populations and may pro- Vilcabamba). Upon further investigation, we discovered that vide a clue as to the general location of the split. In ad- this population had not been cut over the past 100 years (G. dition to the mountain barrier (> 3000 m) separating the Samaniego, personal communication). Review of the micro- Eastern Cordillera Real from Angashcola, the large valley satellite data revealed that El Cristal had approximately twice east of Gonzanamá has also been an effective interglacial the percentage of rare alleles (frequency < 0.05) compared to barrier to migration of C. officinalis.Anorthwest- the average of the neighboring populations which is consistent southeast migration route along the mountain slopes Tree Genetics & Genomes (2019) 15: 81 Page 13 of 16 81 between Gonzanamá and Yangana is possible. The more performed in order to develop strategies (Menz et al. 2011) related populations on the western slope of the Eastern to reconnect C. officinalis fragments with native pollinators. Cordillera Real exist in a relatively narrow elevational Additionally, studies must be designed to detect and under- range of roughly 2100–2300 m. Much of the more acces- stand any reductions of natural regeneration or fitness. Finally, sible land below that elevation has been cleared for pas- efforts to educate the local population and landowners on the ture while growth above that range is limited by an abrupt importance of conserving the few remaining populations of climatic transition which favors páramo. Both the this threatened species should continue. Gonzanamá and Angashcola populations, however, have adapted to higher elevations (approximately 2600–2800 Data archiving statement The microsatellite dataset from the m) in inter-Andean mountain ranges further west. This current study is available from the corresponding author upon upward migration is likely due to warmer, less windy request. GenBank accession numbers for nuclear microsatel- conditions away from the Amazonian fronts that cross lite loci sequences and chloroplast haplotype sequences are the eastern cordillera. Mantel tests using Nei’s distance provided in Tables 1 and 4,respectively. and FST did not show strong evidence of isolation by distance as the r values were low and only one of the Acknowledgments The authors express their gratitude to Dr. Gustavo tests (F ) was weakly significant. In a review of the Samaniego, owner of Finca El Cristal, to Hugo Tapia, co-owner of ST Reserva El Madrigal, and to the Comuna Cochecorral, especially Sr. Mantel test, Guillot and Rousset (2013) referenced a num- Adán Gonzaga, administrators of Reserva Comunal Bosque de ber of studies that expressed concern about the test’s type Angashcola for access to their C. officinalis populations. We also thank Ierrorrate,sowehavelittleconfidenceinthemarginal María Fernanda Tapia for her assistance in creating the map shown in this significance (p = 0.049) of the test using F data. work, Carlos Manchego for collecting the leaf material used to develop ST the microsatellites, and Itziar Arnelas and Tim Breton for reading early Inadditiontothehistoric harvestingof C.officinalis forits versions of the manuscript. Chris Brinegar wishes to thank the Secretaría medicinal value, its habitat has also been severely degraded de Educación Superior, Ciencia, Technología e Innovación by deforestation and conversion to pasture or pine- (SENESCYT) of the government of Ecuador, the Fulbright eucalyptus plantations (Tapia-Armijos et al. 2015). Commissions of the United States and Ecuador, and Bonnie Brinegar for their financial support and also the University of Maine DNA However, despite the obvious decrease in population size Sequencing Facility. and continuing threats to the remaining habitat, the conser- vation status of this species has yet to be assessed by the Authors’ contributions Augusta Cueva-Agila conceived the study. IUCN (2018). Samples were collected by Diego Vélez-Mora., Augusta Cueva-Agila, In our study, all six fragmented populations of C. officinalis and Chris Brinegar. DNA isolation, PCR, and genotyping were conducted by Daniela Arias and Chris Brinegar. Microsatellite characterizations in Loja province were found to have moderate levels of ge- were performed by Manuel Curto and Harald Meimberg. Data were an- netic diversity. However, the more remote population of alyzed by Chris Brinegar and Augusta Cueva-Agila. The manuscript was Angashcola has the highest genetic diversity, is genetically written by Chris Brinegar, Augusta Cueva-Agila, Diego Vélez-Mora, and unique as measured by nuclear microsatellite and chloroplast Manuel Curto. All authors have reviewed the final version of the manuscript. analysis, and should therefore be given highest conservation priority. Fortunately, of all the populations studied, Compliance with ethical standards Angashcola has the largest number of individuals, the most intact habitat, and is managed as a private reserve. The El Conflict of interest The authors declare that they have no conflict of Madrigal and El Cristal C. officinalis populations are also interest. located in private reserves and therefore will have the oppor- tunity to recover. However, the populations of Gonzanamá, Open Access This article is distributed under the terms of the Creative Yangana, and Vilcabamba are very small and located on high- Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, ly degraded agricultural land. Without intervention, these pop- distribution, and reproduction in any medium, provided you give ulations are in danger of extirpation. appropriate credit to the original author(s) and the source, provide a link Remaining fragments of C. officinalis in southern Ecuador to the Creative Commons license, and indicate if changes were made. and northern Peru should be identified and studied in order to better understand the conservation needs of this species. Isolated individuals scattered at the peripheries of pastures or at higher elevations need to be evaluated for their value in References maintaining gene flow between populations. 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