Characterization and identification of old Camellia japonica L. cultivars growing at the Pazo de Gandarón, Pazo de Lourizán, Soutomaior Castle and at the urban gardens of Pontevedra and Santiago de Compostela (NW Spain)

Salinero M.C.*¹, Vela P. ¹, Couselo J.L. ¹, Sainz M. J. ², Neves A. ¹, González M.¹

¹Estación Fitopatolóxica do Areeiro, Deputación de Pontevedra, Subida a la Robleda s/n, 36153 Pontevedra, España
²Departamento de Producción Vegetal, Universidad de Santiago de Compostela,
Campus Universitario s/n, 27002 Lugo, España

Introduction

Ornamental camellias, mainly Camellia japonica and to a lesser extent C. reticulata and C. sasanqua, were introduced in Galicia (NW Spain) in the 18th century. In historic gardens, both public and private, and in the gardens of Pazos, there are ancient camellia trees of unknown cultivars. The ‘Pazos’, manor houses of the Galician nobility, which were built mostly between the 18th and 19th centuries, have in general a large walled estate, which includes, among other areas, a garden with many woody ornamental plants (Rodríguez and Izco, 1995), especially evergreen species, to maintain garden interest for the winter season (Izco, 2008). Some camellias growing in the gardens of the Pazos are included in the Galician Catalogue of Singular Trees (Xunta de Galicia, 2007) by Decree 67/1997 of 22 March, established to protect plant specimens of scientific, aesthetic and/or monumental interest, recognizing its value as cultural heritage. This is the case of Camellia japonica trees growing at the Pazo de Lens-Ames (Ames, A Coruña), Pazo de Santa Cruz (Vedra, A Coruña), Pazo de Torres Agrelo (Redondela, Pontevedra) and Pazo Quiñones de León (Vigo, Pontevedra), a tree of C. reticulata at the Pazo de Oca (A Estrada, Pontevedra) and a tree of C. sasanqua at the Pazo Torres Agrelo, some of them of unknown cultivars.

The Pazo de Gandarón (Pontevedra) was built in 1730-1795. It is believed that its garden was created in the last quarter of the 19th century, beginning then the collection of camellias that it has today, which includes 54 specimens (González et al., 2010). There are a few camellias of known cultivars, among which four specimens of C. japonica cultivars, 'Alba Plena', 'Fimbriata', ‘Pope Pius IX’ (also known as 'Prince Eugene Napoleon') and 'Oranda-kô' stand out; but for most of them only the Camellia species is known. Today it houses the research centre Misión Biológica de Galicia. The identification and characterization of this germplasm is important for knowing and preserving the diversity of cultivars of Camellia species in Europe and to protect them as natural heritage.

The Pazo de Lourizán (Pontevedra) has 54 hectares of garden, estate and woodland. The gardens were created at different stages during the 19th century. Around the house, exotic trees and shrubs were planted in a certain order and bordered by boxwood parterres that edged the walking areas and pathways. It is estimated that some magnolias and camellias (about 300 specimens) that surround the Pazo were planted between 1840 and 1850; these camellias came from the Escuela Práctica de Horticultura de la Caeira (Poio, Pontevedra) and the Estabelecemento Hortícola of Jose Marques Loureiro (Porto, Portugal). In addition, the camellia collection was expanded during the 20th century, with plants from Spanish nurseries and the Moreira da Silva nursery (Porto, Portugal). The Pazo has a large botanical garden, with exotic and native species, established in 1949 under the name of Arboretum, which includes species planted since the 19th century until today. Ancient camellias grow not only in Pazos and manor houses in Galicia, but also in the public gardens and streets of cities.

Soutomaior Castle is an impressive edifice that dates back to the 12th century. With an area of 35 hectares, it has a forest of indigenous species (chestnut and oak trees), a vineyard, pear and apple trees and a botanic park with an area of 6 hectares. This park featuers millennial chestnuts and trees from the five continents, as well as four hundred plants of camellias belonging to 23 species that are a reference for Galician nurserymen and camellia growers and enthusiasts. 

The city of Pontevedra has an outstanding collection of camellias, distributed around the historic gardens and streets of the city as Vicenti Gardens, the Palace of the Diputación de Pontevedra, the Antonio Odriozola Walk, La Herrería Square, the School of Fine Arts, etc. Most were created in the last half of the 19th century, although some of their camellia specimens were planted during the first half of the 20th century. In these gardens there are more than 150 camellias.

In the centre of the city of Santiago de Compostela, between the University Campus and the historic old town, where the cathedral stands out, we find the Alameda Park, which is the most prominent public in the city park and its main lung. Its origin dates back to the Middle Ages, when in the twelfth century the ancient Church of Santa Susana was built, of which today there are only some remnants. The current composition of the park is the result of several enlargements and restorations that have shaped its magnificent architectural and botanical heritage. This garden has 66 camellia specimens planted in different areas.

The aim of this study was to identify and characterize, by morphobotanic descriptors and molecular markers, some historical camellias at the Pazo de Gandarón (5 plants) Pazo de Lourizán (5 plants), Soutomaior Castle (2 plants), the Alameda Park in Santiago de Compostela (13 plants) and some urban gardens in the city of Pontevedra (6 plants). These camellias were compared with 11 reference specimens corresponding to the cultivars 'Bella Romana', 'Clotilde', ‘Dom Pedro II’, ‘Federicci’, ‘Francesco Ferruccio’, ‘Incarnata’, ‘Magnólia Rosa’, ‘Malibran ‘, ‘Prince Eugene Napoleon’, ‘Sangre de Pichón’, and ‘Vilar d'Allen’ (Fig. 1).

 Material and Methods

Thirty-one camellia trees were selected at the historic gardens of Pazo de Gandarón, Pazo de Lourizán, and Soutomaior Castle, and the urban gardens of the cities of Santiago de Compostela and Pontevedra. The identification of species and cultivars was carried out using the information provided by morphobotanic descriptors and, when necessary, by photographic records, drawings and descriptive texts published in catalogues and facsimiles of old books. For the morphobotanic characterization, size and form of each tree, trunk diameter and tree crown diameter of the 31 camellia specimens were measured. For each specimen three layers of foliage (upper, medium, lower) were visually established, and 4 sectors for each layer, collecting samples of leaf and flower in each sector. The final sample per plant consisted of 10 mature leaves and 10 flowers fully developed. The specimens were characterized by 31 morphobotanic descriptors: one of the tree (form), nine of the leaf (length, width, leaf index, petiole length, blade shape, shape of apex, base shape, margin, and blade color) and 21 of the flower (shape and diameter of petals; quantity, margin, colour, colour distribution and venation, and form of external and internal petaloids: presence/absence, amount, disposition and variegation, in stamens: presence/absence, amount, layout, filament colour, anther colour, anther/filament union, anther dehiscence, and androecium/gynoecium relative height). The descriptors were selected from those proposed for C. sinensis by IPGRI (1997), for C. japonica by Corneo et al. (2000) and Salinero and Vela (2004), and from those studied by Luna and Ochotorena (2004) for Theaceae.

Fig. 1  Flowers of the reference specimens used for morphobotanical and genetic identification.

The specimens were grouped according to their morphobotanical features.  A reference specimen from the living collection of Diputación de Pontevedra growing at the Estación Fitopatolóxica do Areeiro was assigned to each group and their genetic profiles were compared using DNA microsatellite markers. For genotyping, the DNA was isolated from 50 mg of leaves with the EZNA HP Plant DNA Kit according to the manufacturer protocol (Omega Bio-Tek). A set of eight polymorphic microsatellites developed for C. japonica (Ueno et al. 1999; Abe et al. 2006) and C. sinensis (Freeman et al. 2004; Hung et al. 2008; Zhao et al. 2008), previously tested by Vela et al. (2009), were used. Polymerase chain reactions were carried out on Doppio (VWR) and TGradient (Biometra) thermal cyclers. The reaction was performed in 25 μL total volume containing 50 ng of genomic DNA. Cycling parameters were as follows: 5 min at 95ºC, 35 cycles for 1 min at 95ºC, 45 s of hybridization with corresponding Tm (Vela et al. 2009), 1 min at 72ºC, and a final step of 15 min at 72ºC. The fluorescent dye labels used were 6-FAM, NED, PET and VIC (Applied Biosystems). A volume of 0.1 μL of amplification products was added to 20 μL of formamide and 0.25 μL Genescan-500 LIZ size standard. The mixed solution was denatured at 95ºC for 3 min. The samples were run on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems).  Allele scoring was performed using the Genemapper 4.0 software (Applied Biosystems).

Results and discussion

Table 1 lists some of the main morphobotanic features of the camellia flower that have been used to identify 31 ancient specimens from the gardens of Galicia. It also shows the features of the 11 reference specimens used for identification. All specimens showed morphobotanic characteristics similar to those of the corresponding reference specimens. However, due to the natural variability of the morphobotanic features within a single cultivar and the uncertain origin of the historical specimens, the genetic profiles of all specimens were analyzed using 8 DNA microsatellite loci (Table 2).

Genotyping confirmed the morphobotanic identification of 20 specimens (PP34, MB20, MB45, SC12, SC35, MB30, PP10, PP26, SC17, SC39, L5, L102, L85, BA9, V11, SC4, SC27, MB18, SC13 and SC34) but 11 samples (L78, L84, S04, S116, MB35, SC49, SC5, SC21, SC44, SC63, H9) showed different genetic profiles to the reference specimens and they could not be assigned to these cultivars.

The genotyping has allowed us to differentiate specimens of 14 cultivars of which 8 are known (‘Clotilde’, ‘Dom Pedro II’, ‘Francesco Ferruccio’, ‘Incarnata’, ‘Sangre de Pichón’, ‘Vilar d’Allen’, ‘Prince Eugene Napoleon’) while the other 6 are unknown. The unknown cultivars may correspond to cultivars already registered but not genetically characterized or could be new cultivars originated in the gardens of Galicia that have not been registered yet.

 Table 1. Main morphobotanic features of the 31 specimens of C. japonica from the historical gardens and the reference specimens (Bold). For each specimen, the label and the group that they were initially assigned by morphobotanic descriptors is indicated. The last column shows the cultivar that was assigned to each specimen after the genetic analysis.  Within each initial group, the same genotypes are shaded with the same color. Specimen labels, A: Areeiro, MB: Pazo de Gandarón, L: Pazo de Lourizán, SC: Alameda Park in Santiago de Compostela and PP,H,V, BA: urban gardens in the city of Pontevedra.

 All specimens initially assigned to the cultivars ‘Clotilde’ (PP34), ‘Dom Pedro II ‘(MB20, MB45, SC12, SC35), ‘Francesco Ferruccio’ (MB30, PP10, PP26), ‘Incarnata’ (SC17, SC39, L5, L102, L85), ‘Sangre de Pichón ‘(SC4, SC27), ‘Vilar d'Allen’ (MB18, SC13, SC34) were correctly identified. However, none of the specimens assigned to ‘Bella Romana’ (L78, L84, S04, S116), ‘Federicci’ (MB35, SC45), ‘Magnólia Rosa’ (SC5, SC21) and ‘Malibran’ (SC44, SC63) were properly determined by their morphobotanic features. The specimen H9 was also incorrectly assigned to cultivar ‘Prince Eugene Napoleon’ although BA9 and V11 showed the same genetic profile to the reference specimen of ‘Prince Eugene Napoleon’ (MB1).

 Table 2. Allelic profiles of the 31 specimens of C. japonica from the historical gardens and the reference specimens (Bold). For each specimen, the label and the group that they were initially assigned by morphobotanic descriptors is indicated. The last column shows the cultivar that was assigned to each specimen after the genetic analysis.  Within each initial group, the same genotypes are shaded with the same color. Specimen labels, A: Areeiro, MB: Pazo de Gandarón, L: Pazo de Lourizán, SC: Alameda Park in Santiago de Compostela and PP,H,V, BA: urban gardens in the city of Pontevedra.

Although the definition of a cultivar refers to morphobotanic features, these results highlight the difficulty of distinguishing many cultivars of C. japonica using morphobotanical descriptors alone. There are many cultivars with distinctive morphobotanic features but in other cases differences in morphobotanic features are unclear so proper identification could be complicated. In addition, some features are significantly unstable within the same cultivar that complicates the identification (Fig. 2). Genotyping is a useful tool for the identification of the cultivars of C. japonica with similar and/or unstable morphobotanical features as the DNA is independent to the phenotype. Currently, the genotyping approaches are used to characterize plants of forestry, agricultural and ornamental interest (Becher et al. 2000), as they complement the morphobotanical characterization of the variety registration and avoid the synonymy. Our results show that they are also a useful tool in the management of historic gardens, especially when the original documentation of the garden project has been lost.

Fig. 2  Variability of the flowers of the reference specimen MB1, cultivar Prince Eugene Napoleon

Acknowledgements

This work was funded by Xunta de Galicia (grant PGIDIT03RAG60301PR).

 References

Abe H., Matsuki R., Ueno S., Nashimoto M., Hasegawa M. 2006. Dispersal of Camellia japonica seeds by Apodemus speciosus revealed by maternity analysis of plants and behavioral observation of animal vectors. Ecological Research 21: 732-740.
Becher S.A., Steinmetz K., Weising K., Boury S., Peltier D., Renou J.-P, Kahl G., Wolff K. 2000. Microsatellites for cultivar identification in Pelargonium. Theor Appl Genet 101:643–651.
Corneo A., Remotti D. Acatti E. 2000. Camelia dell’Ottocento nel Verbano. Regione Piemonte. Turín, Italia.
Escandón A., Zelener N., Pérez de la Torre M., Soto S. 2007. Molecular identification of new varieties of Nierembergia linariaefolia (Graham), a native Argentinean ornamental plant.  J Appl Genet 48(2): 115–123.
Freeman S., West J., James C., Lea V., Mayess S. 2004. Isolation and characterization of highly polymorphic microsatellites in tea (Camellia sinensis). Molecular Ecolgy Notes 4: 324-326.
Gong L., Deng Z. 2011. Development and characterization of microsatellite markers for caladiums (Caladium Vent.). Plant Breeding 130: n^o doi: 10.1111/j.1439-0523.2011.01863.x
González M., Malvar R.A., Ordás A., Salinero C., Vela P. 2010. El pazo de Gandarón (Pontevedra). Camelia 17: 16-22.
Hung C.Y., Wang K.H., Huang C.C., Gong X., Ge X.J., Chiang T.Y. 2008. Isolation and characterization of 11 microsatellite loci from Camellia sinensis in Taiwan using PCR-based isolation of microsatellite arrays (PIMA). Conservation Genetics 9: 779-781.
IPGRI. 1997. Descriptores para el té (Camellia sinensis). Instituto Internacional de Recursos Fitogenéticos, Roma, Italia.
Izco J. 2008. Claves y tendencias en la jardinería de la Ibería Atlántica. Actas de Horticultura 52: 21-29.
Luna I., Ochotorena H. 2004. Phylogenetic relationships of the genera of Theaceae based on morphology. Cladistics 20:223-270.
Rodriguez C., Izco J. 1995. Diversidad florística de los jardines pacegos de Galicia. Rev. Real Acad. Galega de Ciencias 14: 81-116.
Salinero C., Vela, P. 2004. La camelia en la Diputación de Pontevedra. Ed. Diputación Provincial de Pontevedra, Pontevedra.
Ueno S., Yoshimaru H., Tomaru N., Yamamoto S. 1999. Development and characterization of microsatellite markers in Camellia japonica L. Molecular Ecology 8: 335-346.
Xunta de Galicia 2007. Decreto 67/2007, 22 de marzo. Catálogo Galego de Árbores Senlleiras. Consellería de Medio Ambiente e Desenvolvemento Sostible.
Zhao L.P., Liu Z., Chen E.L., Yao E.M.Z., Wang E.X.C. 2008. Generation and characterization of 24 novel EST derived microsatellites from tea plant (Camellia sinensis) and cross-species amplification in its closely related species and varieties. Conservation Genetic 9: 1327–1331.