Genetic and Morphological Characterization of the Endangered Austral Papaya Vasconcellea Chilensis (Planch

Genet Resour Crop Evol (2014) 61:1423–1432 DOI 10.1007/s10722-014-0143-0

NOTES ON NEGLECTED AND UNDERUTILIZED CROPS

Genetic and morphological characterization of the endangered Austral papaya Vasconcellea chilensis (Planch. ex A. DC.) Solms

B. Carrasco • R. Garcıá-Gonza´les • C. Dıáz • P. A´ vila • P. Caćeres • G. A. Lobos • H. Silva • P. D. S. Caligari

Received: 20 January 2014 / Accepted: 10 June 2014 / Published online: 9 July 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract The Austral papaya (Vasconcellea chilen- molecular markers, along with morphological trait sis) is an endangered species that has valuable analyses, suggest that these relict populations are the characteristics for introgression into other papaya result of a relatively recent fragmentation. This species. These characteristics include disease resis- implies that the fragmentation has not yet had its full tance, cold tolerance and latex with low proteolytic effect on the genetic variation and so emphasises the activity. It is a species that grows under extreme need for clear and urgent conservation measures to environmental conditions of drought, salinity and preserve the remaining genetic variation, particularly temperature; it is found growing naturally in Chile but for the most northern of the three populations which is today only as three remnant populations. The results presently unprotected. presented here, using inter simple sequence repeat

B. Carrasco C. Dıáz H. Silva Facultad de Agronomıá e Ingenierıá Forestal, Pontificia Laboratorio de Geno´mica Funcional y Bioinforma´tica, Universidad Cato´lica de Chile, Vicunã Mackenna 4860, Departamento de Produccioń Agrıćola, Facultad de Macul, Santiago, Chile Ciencias Agrono´micas, Universidad de Chile, Av. Santa e-mail: [email protected] Rosa 11315, 8820808 La Pintana, Santiago, Chile e-mail: [email protected] R. Garcıá-Gonza´les (&) P. Caćeres Facultad de Ciencias Agrarias y Forestales, Centro de P. D. S. Caligari Biotecnologıá de los Recursos Naturales (CENBio), Centre of Plant Breeding and Phenomics Center, Instituto Universidad Cato´lica del Maule, Av. San Miguel 3605, de Ciencias Biolo´gicas, Universidad de Talca, Talca, Chile 2 Norte 685, Talca, Chile e-mail: [email protected] e-mail: [email protected]

P. A´ vila Servicio Agrı´cola y Ganadero, 2 Poniente 1180, Talca, Chile e-mail: [email protected]

G. A. Lobos Facultad de Ciencias Agrarias, Plant Breeding and Phenomics Center, Universidad de Talca, 2 Norte 685, Talca, Chile e-mail: [email protected] 123 1424 Genet Resour Crop Evol (2014) 61:1423–1432

Keywords Vasconcellea chilensis Austral papaya Vasconcellea sp. have a small genome, similar to Palo gordo Genetic diversity ISSR markers Carica papaya (between 442.5 and 625.9 Mb) orga- Genetic structure Introgression Genetic nized into 18 chromosomes, and a pair of primitive sex fragmentation chromosomes (Gschwend et al. 2013). In Chile only two species are known to exist, the first one is the introduced Vasconcellea pubescens (Lenne´ et C. Koch) Badillo which is cultivated in order Introduction to obtain canned fruit and juices. The second one is the endemic Vasconcellea chilensis (Planch. ex A. DC.) Caricaceae is a tropical and subtropical family Solms (Palo gordo, native papaya, Austral papaya), consisting of six genera (Carica, Vasconcellea, Ja- which is a small shrub 1–2 m tall; with a thick and caratia, Cylicomorpha, Jarilla and Horovitzia) with succulent stem (Fig. 1). Its shiny leaves display a very around 35 species. Most of them are found in the variable shape from an oval and deltoid to heart Americas and only the Cylicomorpha genus is native shaped. It has small purple flowers (5–6 mm) that to Africa (Stevens 2001; Gschwend et al. 2013). appear during winter. As most Caricaeae, V. chilensis According to Antunes-Carvalho and Renner (2012), is a polygamous species with male, female and the family belongs to subtropical and tropical regions hermaphrodite plants. The fruits are small with a of North and South America and Africa. The genus diameter around 1–3 cm; the pulp is minimal and the Cylicomorpha, is located in Central Africa (Antunes- latex has low levels of papain activity; the number of Carvalho and Renner 2012). The genus Carica is seeds per fruit ranges from 5 to 10 (Hechenleitner et al. represented by one species, probably the most culti- 2005). vated species of the family. The genus Jacaratia The natural populations of V. chilensis correspond includes seven tree species distributed in Mexico and to the most southerly distributed papaya so far South America (Antunes-Carvalho and Renner 2012). reported. Currently, the fragmented populations are There are two herbaceous genus, Horovitzia, repre- located between the Atacama (28°390S; 71°420W) and sented by Horovitzia cnidoscoloides (Lorence and Valparaiso (33°090S; 71°420W) Regions (Cuevas R.Torres) V. M. Badillo, and Jarilla which has three 1991; Jordan 2011). But in this range there are (to species (Antunes-Carvalho and Renner 2012). Vas- our knowledge) now only three remnant populations, concellea, or ‘‘highland papaya’’, has the largest with more than thirty plants, which is considered as a number of species (21), including a naturally occur- minimum number of individuals to detect alleles ring hybrid, Vasconcellea 9 heilbornii (V. M. Badillo) which are present at a frequency of 0.05 (Sjo¨rgren and V. M. Badillo (Antunes-Carvalho and Renner 2012). Wyo¨ni 1994; Llewellyn et al. 2003). Any other Vasconcellea species are mostly cultivated at high previous small populations are thought to have altitudes in South America, especially in Ecuador, disappeared due to habitat destruction, goat grazing Colombia, and Peru´ (Scheldeman et al. 2011). and harvest for firewood (Serra et al. 1986; Cuevas Because of the botanical similarity with Carica 1991). papaya L., and because they inhabit zones of high Usually the species is found growing under extreme altitude, they are known as highland papayas (National conditions throughout its natural range, such as living Research Council 1989; Badillo 2000; Van Dro- on soils with high salt concentration and subject to ogenbroeck et al. 2004; Scheldeman et al. 2007). drought. Also, it can support large fluctuations in day Botanically, Vasconcellea species have been and night temperatures. In its natural habitat, it shows described as herbaceous open pollinated species, in a very slow growth rate, scarce biomass production which it is possible to distinguish male, female and and its seeds are difficult to germinate. hermaphrodite plants (Storey 1976;Sańchez 1994). In the past the species was used for animal feed, Also they display a short generation time (1 year from which is still a current practice (Rıós 2004). In the seed to productive tree), a high diversity of growing main populations, goats used to feed from the natural habits, papain production, fruit shape, size and colour, populations during the summer, when other vegetation among other attributes (Antunes-Carvalho and Renner was sparse. However, the continuing use as animal 2012). In genetic terms, it is believed that feed is now very limited because the low number of 123 Genet Resour Crop Evol (2014) 61:1423–1432 1425

Fig. 1 Photographs of Austral papaya (Vasconcellea chilensis): A Isolated tree located on Valle del Encanto; B Fruits from V. pubescens (yellow fruited) and V. chilensis (green–brown fruited); C Vasconcellea chilensis leaf,and D fruit morphological variability individuals in natural population, the low rates of seed abiotic stresses in its natural habitats, means the production and lack of application of any agronomic species could be used as a genetic donor to improve its technology in the species. The fruit of the species is agronomic performance, such as cold tolerance and also considered of interest in terms of its fruit disease resistance (Ocampo et al. 2006; Jordan 2011). properties and papain content because its close At the same time, as with many other papaya relation with tropical papaya and highland papaya species, future industrial uses based on its fruits can be (Jordan 2011). Furthermore, its resistance to the expected if the species can be developed and planted 123 1426 Genet Resour Crop Evol (2014) 61:1423–1432

Fig. 2 Map of sampled 71° Vasconcellea chilensis 30° La Serena populations in the semi-arid Huachalalume regions of Coquimbo, Chile

Fray Jorge Ovalle

Valle del Encanto

31° on a commercial scale (Rodrı´guez et al. 2005), Materials and methods especially in areas where other crops would not grow readily. Sampling of plant material Currently, V. chilensis is considered as an endangered species being classified as ‘‘vulnerable’’ The three major remnant populations of Vasconcellea (Squeo et al. 2001); but it has not been given any chilensis were sampled over a 600 km2 range between economic importance. However, in reality, its latitudes 30° and 31° South, in the semi-arid regions of capacity for living in extreme environmental condi- Coquimbo Region, Chile (Fig. 2). The number of plants tions and low papain activity make it an important sampled was 20, 30 and 61 individuals respectively in wild resource. the three populations, dictated by the number of plants In this work, inter simple sequence repeats (ISSR) existing in each, as shown in Table 1. For genetic study markers were used to assess the genetic structure of the and morphological characterization, samples within V. chilensis populations. ISSR markers are a very each population were collected at random. useful tool for studies of genetic diversity (Wolfe et al. Young leaves collected from the 111 adult plants 1998; Gilbert et al. 1999). They are based on the use of were selected for DNA extraction. The collected microsatellite sequences as primers to generate multi- samples were frozen in liquid nitrogen and stored at ple band patterns. The necessity for previous knowl- -80 °C until DNA extraction. Additionally, mature edge of the genome is obviated in the design of fruits were collected from 37 female plants (12, 11 and primers, which makes this technique an attractive, 14 plants from Huachalaluma, Valle del Encanto and quick and economically attractive possibility Fray Jorge, respectively) for fruit trait analysis. (Zietkiewicz et al. 1994). Additionally, given the abundance of microsatellite sequences it is possible to DNA extraction and PCR amplification analyse a large number of loci, giving a high possibility of finding polymorphisms, even with DNA extraction and PCR amplification were carried closely related genotypes (Bornet and Branchard out according to Carrasco et al. (2009). A set of 36 2001; Arnau et al. 2003, Carrasco et al. 2009). To primers (ISSR set 100/8, Biotechnology Laboratory, our knowledge, this is the first investigation of the University of British Columbia, Vancouver) was organization of the genetic and phenotypic variability tested. The characteristics of the primers that gave of Austral papaya using ISSR’s and some important positive results are shown in Table 2. All PCR fruit characteristics. These results will help allow the products were checked by electrophoresis on 2 % establishment of a base line to assist future conserva- agarose gels, run in TAE 1X buffer and visualized by tion and breeding programmes of the Vasconcellea ultraviolet fluorescence after staining with ethidium and Carica genera. bromide (0.25 ll/ml staining solution). 123 Genet Resour Crop Evol (2014) 61:1423–1432 1427

Table 1 Vasconcellea chilensis populations City Population name Latitude Longitude Altitude Sample size

La Serena Huachalaluma 29°5900500 71°0900700 272–485 61 (10,000)a Ovalle Fray Jorge 30°3800600 71°3401600 170–600 30 (17,824)b Ovalle Valle del Encanto 30°4201200 71°2202200 215 20 (1,000)c The nearest city is given as a reference point along with: population name, actual geographical location (latitude, longitude and altitude) and sample size (population size) In bracket number estimated of individuals by population (a and c authors estimation; b Cuevas 1991)

the software, Structure (version 2.1) (Pritchard and Wen Table 2 Characteristics of the six ISSR primers used for the 2003). Ten independent runs of the algorithm were used, analysis of V. chilensis assuming values of K from 1 to 10. The run parameters Primer Sequence Total band Band size Annealing were performed with 1,000,000 Markov chain Monte sequence motif number range (bp) T° (°C) Carlo (MCMC) repetitions and a burning period of UBC811 (GA)8A 16 350–2,100 52 500,000, assuming an admixture population. The UBC814 (CT)8A 11 420–1,800 50 methodology implemented by Evanno et al. (2005) UBC836 (AG)8YA 15 270–1,400 52 was used to choose the optimal K. UBC841 (GA)8YC 17 280–1,500 54 The proportion of ancestry in a given cluster was UBC856 (AC)8YA 14 490–2,200 52 calculated as an assignment rate (q), where 0.8 is UBC873 (CT)8RC 13 520–2,000 48 accepted as a correct assignment to a single cluster. In the sequences of degenerated primers: B can be C, G or T; D can be A, G, or T; R can be either A or G; Y can be either C or Data analysis of fruit traits T The collected fresh fruit were evaluated for equatorial Data analysis from ISSR molecular markers and polar diameter (mm) (digital calliper, Starrett, 727 Series, USA), weight (g) (electronic scale, PG3001-S, From the agarose gels, each generated band was Mettler Toledo Inc., Switzerland), and number of seed considered as a locus and was designated as ‘‘present’’ per fruit. The total soluble solids of the fruits were (1) or ‘‘absent’’ (0) for each individual. The POP- estimated with a digital refractometer (°Brix) (Atago GENE version 1.31 software (Yeh et al. 1999) was Master M, Atago Co Ltd., Japan). used to calculate the percentage of polymorphic ISSR Data were analyzed using non-model-based multi- loci (P), Nei’s gene diversity (h), (Nei 1973) and variate approaches such as Principal Component and Shannon’s index (I) (Shannon 1948). Nei’s gene Linear Discriminant analyses. The null hypothesis that diversity is a simple measureP of genetic variability and there were no differences in fruit trait data among 2 is defined as: h = 1 - pi , where (in the case of population was tested using Wilks’ k test statistic. dominant molecular markers) pi is the population Additionally, ANOVA and Tukey’s test (HSD) frequency of each allele (1 and 0) at locus Pi (Nei 1978). (p B 0.05) were performed on each fruit trait in order Shannon’s index (I) is defined as: I =- pi ln (pi), to detect significant differences between populations. where pi is the proportion of the ith allele in the The analyses were carried out using R 3.0.0 population (Lewontin 1972). For each locus gene (R Development Core Team 2008). diversity produces values from 0 to 0.5 and Shannon’s index produces values ranging between 0 and 0.73 on a natural logarithm scale (Lowe et al. 2004). Results The hierarchical ISSR frequency distribution was analysed using AMOVA within the software GENAL- Genetic variability EX v. 6 (Peakall and Smouse 2006). Population structure within V. chilensis samples was inferred using Out of 36 ISSR primers studied, only six gave a Bayesian model-clustering algorithm implemented in reproducible band patterns. These primers gave 86 123 1428 Genet Resour Crop Evol (2014) 61:1423–1432

Table 3 Genetic diversity parameters for three populations of V. chilensis using 86 ISSR bands Population Shannon’s index (I) Gene diversity (He) Polymorphism (%)

Fray Jorge 0.34 (±0.032) 0.23 (±0.032) 62 Huachalaluma 0.33 (±0.031) 0.22 (±0.032) 67 Valle del Encanto 0.30 (±0.031) 0.20 (±0.032) 57 Standard deviation in brackets

Fig. 3 Bar plot obtained from Structure software. Cluster (HU) population. The individulas clustered in A and B, A shows 26 (87 %) and 16 (80 %) individuals from Fray Jorge displayed assignment values (q) C 0.8. Cluster C show admix- (FJ) and Valle del Encanto (VE) population, respectively. ure individuals with q values B0.8 Cluster B show 61(87 %) individuals belong to Huachalaluma recordable bands in the 111 samples of V. chilensis dell Encanto was smaller but still signiﬁcant studied (Table 2). Overall, 53 bands proved to be (/PT = 0.068; p B 0.05). polymorphic (p = 62 %) and the genetic diversity The best K value was obtained by applying a was intermediate. The gene diversity and Shannon’s Bayesian analysis of genetic structure (Structure Index (Table 3) were similar for Fray Jorge and software) using the criterion of Evanno et al. (2005). Huachalaluma, while Valle del Encanto was slightly The maximum DK value was revealed for K = 2 inferior for all the estimated parameters. (Fig. 3) under the admixture and correlated allele

The AMOVA indicated (/PT = 0.10, p \ 0.001), frequencies models. that the major portion of the genetic diversity existed These two genetic groups showed a high assign- within groups (90 %) while a smaller but signiﬁcant ment rate (q C 0.8). Cluster A grouped 87 and 80 % of (10 %) of the genetic diversity was distributed analysed individuals from Fray Jorge and Valle del between the three populations. Huachalaluma popu- Encanto, respectively. While cluster B grouped 87 % lation was signiﬁcantly different from Fray Jorge and of the individuals belonging to the Huachalaluma

Valle del Encanto (/PT = 0.10–0.11; p B 0.05), population. Out of 111 individuals analyzed only 17 while the difference between Fray Jorge and Valle showed assignment values inferior to 0.8, therefore

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Table 4 Means and standard deviations for fruit characteristics in the samples from the three populations Population N ED LD SS NS FW

Huachalaluma 12 1.3 ± 0.25ns 2.3 ± 0.37ns 6.1 ± 1.58ns 6.7 ± 1.16a 0.4 ± 0.46ns Valle del Encanto 11 1.4 ± 0.29ns 2.6 ± 0.32ns 7.4 ± 1.29ns 5.2 ± 0.98b 0.7 ± 0.49ns Fray Jorge 14 1.3 ± 0.23ns 2.3 ± 0.42ns 6.7 ± 1.16ns 4.9 ± 1.45b 0.4 ± 0.48ns Prob. Level 0.51 0.23 0.09 0.002* 0.38 N sample size, ED equatorial diameter (mm), LD mm longitudinal diameter (mm), SS soluble solids (°Brix), ns number of seeds and FW fruit weight (g) Means followed by the same letter within a column are not significantly different (Tukey’s test, p B 0.05). ns = not significant; * = significant at p \ 0.05; ±=standard deviation they were considered as admixture. Mantel test was or as animal feed in extreme environments as well as, not able to verify a geographic pattern for the three more immediately, because of its physiological char- populations (r = 0.022, p = 0.14). acteristics and its relationship to the economically important tropical species such as Carica papaya L. Fruit variability (tropical papaya), Vasconcellea 9 heilbornii cv. Babaco (babaco) among others. In Chile, the Austral Principal component analysis (PCA) was performed papaya has developed specific eco-physiological on the fruit data to provide an easy visualization of the characteristics that have allowed it to adapt to grow 37 female plants in a reduced dimension. However, it under the most stressed environmental conditions was not possible to distinguish any pattern in the PCA known to the Caricacea species (Rodrı´guez et al. plot, even when discriminate analysis was carried out 2005). Therefore, its conservation and utilization (data not shown). requires that genetic and phenotypic variability be Nevertheless, it is important to point out the PCA well characterized. showed that 91.4 per cent of the observed variance In general terms, the genetic structure of natural could be explained by the first three components. PC1, populations is affected mainly by the mating systems, PC2 and PC3 accounted for 53.6, 22.8 and 14.9 % of ecological factors of their habitats and historical total variability, respectively. PC1 was represented events. As long-lived, dioecious angiosperms, the mainly by fruit weight and equatorial diameter, which levels of genetic variation should be relatively high showed negative values for the correlations. Soluble within populations and relatively low between popu- solids explained PC2 with a positive value for the lations (Nybom 2004). Nevertheless, fragmentation, correlations. Finally, PC3 was explained by the isolation between populations and inbreeding for a number of seeds which displayed negative values for long time can produce decreased genetic variability the correlation. and increased differentiation among populations. The analysis of variance for equatorial diameter, In this study a medium level of ISSR variation longitudinal diameter, soluble solids and fruit weight within populations was observed (I = 0.3–0.34; did not show any differences among the populations h = 0.2–0.23; p = 57–67 %). These results are sim- (Table 4). Only the number of seeds was significantly ilar to the average diversity reported for plants using different between the three populations studied dominant markers (h = 0.19–0.23) (Nybom 2004). (F = 7.53; p B 0.05). However, the genetic diversity was superior to those reported for Vasconcellea pubescens, the cultivated highland papaya (I = 0.16; h = 0.01; p = 55.3 %) Discussion (Carrasco et al. 2009). Most of the genetic diversity in the Austral papaya The endangered Austral papaya is an interesting was found within populations (90 %) but a significant, phytogenetic resource because there is a percieved low to moderate genetic differentiation between future potential for its growth either for industrial use populations, was detected (/PT = 0.10; p B 0.05).

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These results are lower than those indicated by Nybom analysed tended to be quite homogeneous, supporting

(2004) for plants in general (/PT = 0.34–0.35). the idea that fragmentation of the Austral papaya However, low genetic differentiation among natural populations is a recent event. populations also has been reported for Carica papaya However, only the number of seeds per fruit was in Costa Rica based on microsatellite results (Fst = significantly different between populations. The num- -0.008 to 0.107) (Brown et al. 2012). ber of seeds from Fray Jorge and Valle del Encanto Bayesian clustering indicated that the three popu- were similar to each other but significantly inferior to lations were grouped into two genetic clusters, one those collected from Huachalaluma. These results are group included Fray Jorge and Valle del Encanto, are in agreement with two genetic groups identified by the geographically separated from each other by about Bayesian clustering. 30 km. A second group included only individuals The main goal in conservation is try to retain as from Huachalaluma which is 110 km away from the much as possible of the genetic and phenotypic former populations. diversity. The description of the genetic variation However, the Mantel test supports the conclusions between and within populations of rare and endan- that there is no significant correlation between genetic gered species, like Austral papaya, is crucial for the distance and geographical distance. Admixed individ- development of adequate conservation strategies uals detected by ‘‘Structure’’ could explain this low (Frankel and Soule 1981). Today, out of three major correlation. remnants of Austral papaya, only two of them are According to Schaal et al. (1998), the level of protected, the population located in the National Park genetic diversity and genetic differentiation of plant Fray Jorge (17, 824 individuals; Cuevas 1991) and the populations can reflect interactions among demo- small population located in the National Archeolog- graphic shifts (modification in natural distribution, ical Monument Valle del Encanto (around 1,000 habitat fragmentation and population isolation) and individual; author´s estimation). Unfortunately, the biological and genetical events (mating systems, Huchalaluma population (the most northerly located mutation, selection, genetic drift, and gene flow). population) is not protected and is suffering from The medium level of genetic diversity and its mod- constant pressure of goat grazing and its harvest for erate differentiation among populations could there- firewood. The important level of genetic and pheno- fore suggest that the current strong habitat typic diversity found in Huachalaluma (around 10,000 fragmentation and reduction of population size are individuals) and its differentiation from the other two recent events and that they have not yet impacted populations, make it an urgent candidate for the genetically on the Austral papaya populations. Previ- implementation of conservation strategies. Also, it is ous reports (Cuevas 1991) mentioned that the Austral imperative to study any isolated individuals or small papaya grows discontinuously over 600 km2, and it is populations that can be found to the North of probable these are fragments of an earlier single Huachalaluma, if are any still exist. This would population. Unfortunately there are no paleontological potentially permit the broadening of the genetic or historical studies which can support this hypothesis; diversity, not only for genetic improvement purposes however, there could be a possible association with but also to reinforce the in situ conservation of the human settlements in Northern and Central Chile. natural populations. The analysis of fruit traits was carried out only for a small sample of trees because the Austral papaya is Acknowledgments This work was partly supported by the considered a scarce, uncommon and seriously endan- project ‘‘Insercioń de Investigadores Postdoctorales’’ granted from CONICYT (Project N° 10). The authors also thanks to gered species. The multivariate analysis (principal Gobierno Regional del Maule, Chile, for partially supporting component analysis and discriminant analysis) this study through the project VITROTECH I (BIP 3303689-0). showed, however, that the fruit traits studied were homogenous among populations (Fig. 1) with a ten- dency to small fruits, low soluble solids and few seed, References as detected by the principal component analysis. It was not possible to detect any specific pattern or grouping Antunes-Carvalho F, Renner SS (2012) A dated phylogeny of by using multivariate techniques; the female plants the papaya family (Caricaceae) reveals the crop’s closest 123 Genet Resour Crop Evol (2014) 61:1423–1432 1431

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