Morphometric and Molecular Analysis of the Three Arbutus Species of Greece

Arbutus andrachne, A. unedo and A. × andrachnoides found in the Greek macchia are promising species for reforestations, ornamental use, as well as for medicinal use and the food industry. Μorphological traits and molecular markers (RAPD) were used to identify and distinguish these Arbutus species to facilitate their exploitation. Since there are no descriptors established for Arbutus spp., 23 qualitative morphological characteristics of crown, foliage, bark, flowering, fruiting, and four quantitative morphological characteristics of leaf and fruit were selected and used to define differences and similarities between sampled individuals of A. andrachne, A. unedo and individuals with intermediate characteristics sampled as A. × andrachnoides. Twenty eight individuals representative of three Arbutus taxa were sampled in two typical macchia forest areas of the prefecture of Attica, Greece. Cluster analysis based on morphological characteristics separated the individuals in three distinct groups, and this was confirmed by molecular analysis. Τhe intermediate form was indicated as A. × andrachnoides, a natural hybrid of A. andrachne and A. unedo. Fifteen 10-mer oligonucleotide arbitrary primers used to amplify genomic DNA generated 166 reproducible polymorphic fragments, which revealed that A. × andrachnoides has higher degree of genetic similarity with A. andrachne than A. unedo. The applied morphometric characteristics are suggested as a basis to develop a complete list of discriminating descriptors for Arbutus genus.


Introduction
Three Arbutus species (Arbutus andrachne L., Arbutus unedo L. and Arbutus × andrachnoides Link, f.Ericaceae), typical members of the Greek macchia, are promising for exploitation in forestry, landscape, horticulture and pharmaceutical industry.A. unedo (strawberry tree) and A. andrachne (Eastern or Greek strawberry tree) may be a vicariant pair, as it is isolated geographically from other Arbutus species (Torres et al., 2002;Blondel et al., 2010).A. unedo is used and A. andrachne has the potential for use as an ornamental shrub in the urban and suburban landscape (Bertsouklis and Papafotiou, 2013), while both are recommended species for reforestations in Mediterranean region, where fires are common, as they are characterized by vigorous re-sprouting after fire and overgrazing (Konstantinidis et al., 2006;Moreno-Jiménez, 2008;Kazanis et al., 2012).They both have high medicinal value (Diba et al., 2010;González et al., 2010;Afifi-Yazar et al., 2011;Mendes et al., 2011;Etxeberria et al., 2012;Abidi et al., 2015;Güler et al., 2015), while strawberry tree fruits are an excellent source of vitamin C, dietary fiber and total available carbohydrates, sugars, potassium and secondary metabolites, such as phenolic compounds, being poor in lipids and Na, with ascorbic acid and fat-soluble antioxidants (Ruiz-Rodríguez, 2011;Mosele et al., 2016) of higher values than other red small fruits (Guerreiro et al., 2013).They are also used for preparing liqueurs and aromatic traditional distillate (Soufleros et al., 2005;González et al., 2011;Botelho et al., 2015).Strawberry tree fruits can be stored at low temperature preserving their quality (Guerreiro et al., 2013;Fadda et al., 2015) and they could be introduced in the market as fruits and/ or as a source of bioactive compounds for dietary supplements or functional foods used as components in yoghurts, in cereal bars, in pastry making and confectionery, as flavors or in pieces, like other berries (Ayaz et al., 2000;Alarcão-e-Silva et al., 2001;Miguel et al., 2014).

Morphological traits
A total of 27 morphological traits of crown, leaf, flower and fruit were recorded for each individual; 23 qualitative descriptors (1-23) and five quantitative (24-27) were used (Table I) based on descriptors used for other woody plant species including those used by Lopes et al. (2012) for A. unedo.Hundred leaves from each individual (10 replications of 10 leaves each) were used to estimate length/width and leaf area (LI-3100 scanner, LI-COR Biosciences, USA).Mature, fully expanded leaves derived from nodes six to nine, of one year old shoots (15 cm length) were selected in early November from the SE-side of the shrubs.Hundred mature fruits derived from each individual were used to estimate fruit diameter.For 24 to 27 characteristics took place customization of characteristics (values 1, 2, 3).

DNA extraction
Young leaves were collected in early May from each individual, transported in dry ice and stored at -80 °C.Genomic DNA was extracted from 0.1 g of leaves using the Dneasy Plant Mini Kit (Qiagen).Amplification reactions were performed according to Stavrakakis and Biniari (1998), using the same PCR apparatus.Fifteen 10-mer oligonucleotide arbitrary primers were used (Table 2) for the amplification of RAPD sequences out of which 12 primers that could generate discernible and reproducible bands was selected for formal amplification.All reactions were repeated at least twice with independently isolated genomic DNA as templates.

Gel electrophoresis
Aliquots (20 μl) of the RAPD products were analyzed in 2% agarose gel electrophoresis in TAE buffer (40 mM Trisacetate and 1 mM EDTA, pH 8).After staining in ethidium bromide (1 μg ml -1 ) the gels were photographed on a Gel Doc 1000 (Biorad).A mix of λ DNA Hind III × Hae III was used as size marker.All of the reactions were repeated at least twice with independently isolated genomic DNA as templates.

Data analysis
The matrix of average taxonomic distance for individuals was computed using the Euclidean distance coefficient.Cluster analysis was conducted on the taxonomic distance matrix with the Unweighted Pair Group Method based on Arithmetic Average (UPGMA) and the dendrograms were generated based on the genetic distance matrix.One-way ANOVA was used for determination of the differences between the mean values of the leaf traits.Genetic similarities were calculated using the Simple Match (SM) and Jaccard (JAC) coefficients described by Sokal and Sneath (1963).The RAPD data were used to generate two genetic distance matrices and dendrograms were generated using the same methods used with the morphologic data.landscape use it is recommended particularly for cut foliage production because its growth speed is higher than the other two species, has lower production of flowers and fruits and prolonged (90-d) vase life (Cervelli et al., 2012).Individuals with intermediate morphological characteristics are not necessarily hybrids.Morphological traits in combination with molecular markers (RAPDs) are used to assess genetic variability in plant species or to identify hybrids (Yüzbaşıoğlu et al., 2008;Hrṧak et al., 2011;Ferreyra et al., 2013;Song et al., 2015).In recent years RAPD have been used for studies between different A. unedo genotypes (Takhrouni and Boussaid, 2010;Lopes et al., 2012;Gomes et al., 2013).There are also a few references on morphological traits used for diversity studies in A. unedo in Italy (Mulas et al., 1998), Turkey (Celikel et al., 2008) and Portugal (Lopes et al., 2012).Koukos et al. (2015) studied the surface microscalpture and waxes of Greek A. unedo and A. αndrachne, this being the only information on genetic diversity of Greek Arbutus spp.
The objective of the present study was to test both morphological traits (quantitative and qualitative) and molecular markers in order to develop a set of proper descriptors to identify and distinguish Arbutus species found in Greece.The results could be used both for conservation strategies and cultivation of specific genotypes with particular characteristics.
In each area five A. unedo and five A. andrachne shrubs, as well as four shrubs with intermediate characteristics were selected being 2-3 m in height and 2-3 m in diameter (at 150 cm from the ground).The shrubs were re-sprouted after fires that occurred in 1990 at Kalamos and 1986 in Varympompi.The selected shrubs were at least 100 m apart from each other and were found along the existing paths (five -six paths) of each sampling area at 0-50 m distance left and right of the path.

Phenotypic classification to three groups
The identification of the sampled individuals was followed by the classification into three groups including individuals Principal coordinate analysis (PCA) was used in order to verify cluster analysis and to assist in visualizing the data.Mantel test (Mantel, 1967) was used to estimate the correlation between the distance matrices resulting from morphological, RAPD and combined analyses.Statistical analysis of morphological and RAPD markers was conducted by the software NTSYS-pc version 2.11f (Rolf, 1992).

Morphological characteristics
In the two study areas a few individuals with a mixture of intermediate morphological characteristics between A. unedo and A. andrachne were found, resembling more to A. andrachne.The distinction was mainly based on the morphology of the bark that was smooth and cinnamon-red in color, peeling in small strips, every year, with high rate, revealing a grey-green internal like the bark of A. andrachne, with the difference that A. andrachne bark was peeling in long strips (Table I).The bark of A. unedo was totally different being rough, ash-grey and occasionally peeling in small flakes revealing a chestnut-colored internal; A. unedo individuals were strong branching from base in contrary to other individuals.
The leaves of plants with intermediate morphological characteristics were leathery and elastic, shiny, with dark green colour on the upper side, like A. andrachne leaves; A. unedo leaves were membranaceous-rough, not shiny with green colour of upper side.Intermediate individuals had serrate leaves similar to A. unedo and their shape was either like A. unedo (elliptical to obovate) or A. andrachne (elliptical to oblong lanceolate).Petiole length and fruit diameter of intermediate individuals were also counted between A. andrachne and A. unedo.These characteristics of plants with intermediate characteristics match with descriptions of A. × andrachnoides the natural hybrid between A. andrachne and A. unedo (Arabatzis, 2001).
It was also observed that the flowering period of intermediate individuals was either like A. andrachne (January-April) or like A. unedo (October-January).Fruiting time was the same (October-January) in all species.Fruits of intermediate individuals were spherical, red-orange, with slightly granular surface like A. andrachne in contrast to granular surface of A. unedo.Fruit taste of intermediate individuals was sweet, like A. unedo, while A. andrachne was astringent.
Leaves of A. andrachne were larger than those of individuals with intermediate characteristics and A. unedo leaves were the smallest of all.Locality did not affect leaf length and leaf area (Table 3).Principal coordinate analysis confirmed the results and four components have been arranged in a decline series of their importance, explaining 98.92% of the total variability among the different individuals.All suggested descriptors (Table 1) that are involved in the same principal component are strong correlated.Descriptors 2 (vegetation density), 4 (branching), 5 (bark surface), 6 (external bark colour), 7 (bark peeling), 8 (peeling time), 9 (peeling rate), 10 (internal bark colour), 12 (leaf texture), 13 (boat-shaped leave), 16 (leaf colour of upper side), 21 (fruit surface) and 27 (fruit diameter) contributed better to variability compared to descriptors 17 (flowering) or 26 (fruit diameter); qualitative characteristics were strong correlated while quantitative characteristics were found to be more variable (Table 4, Fig. 1).
Cluster analysis separated the individuals according to their morphological characteristics in three branches; individuals H were found to be distinct from A or U but closer to A (Fig. 2) and the Mantel test comparing Euclidean values matrix and cophenetic matrix gave very high value (r=0.965).The analysis of the above data indicated that most probably the individuals with intermediate characteristics are the hybrid form A. × andrachnoides.

Molecular analysis
For the molecular analysis and the identification of the individuals studied, 12 primers were used which proved to be highly polymorphic (63.5% polymorphism) producing a 426 total of 166 amplified markers.Higher percentage of polymorphism was presented by primers ΟPM-18 and ΟPM-14 (76.19% and 71.43% respectively, Table 2).Primers could classified in three groups (Table 2) with:   The primers 1225 and 1227 proved much more useful in differentiating individuals as they generated more polymorphic DNA fragments.As expected there was genetic variation among the individuals studied and they were grouped in three different branches (Fig. 3, 4) consistently with our observations based on morphological characteristics.Individuals with intermediate morphological characteristics from Kalamos (I=0.98-1.00)grouped in one branch, something that allows the hypothesis that belong to another species, at least for the primers that were used, possibly A. × andrachnoides (Fig 1).The genetic similarity between A. andrachne individuals from two different sampling areas was high (I=0.94).The higher genetic similarity found between individuals with intermediate characteristics and A. andrachne individuals (I=0.69-0.73)was consistent with the bigger   (2012).
Association between matrices of morphological and genetic distances were investigated (Mantel test) and the results of the tests showed significant correlation (r=0.958 and r=0.951 for SM and JAC, respectively).
Thus, individuals sampled as A. × andrachnoides showed both a mixture of A. andrachne and A. unedo morphological characteristics and intermediate characteristics that complicated their identification as hybrids.However the suggested descriptors in combination with molecular markers proved a useful tool to assess the hybrid identity of these individuals.Combination of morphological traits with RAPD has been successfully used for differentiation of other natural hybrids and clones, too (Yüzbaşıoğlu et al., 2008;Stavrakaki and Biniari, 2016).The results of this research are supported by the fact that plants derived by micropropagation of individuals with intermediate characteristics defined as A. × andrachnoides (Bertsouklis and Papafotiou, 2011) were morphologically identical after three years ex vitro growth to the mother plant found in Kalamos (unpublished data) revealing that the hybrid was defined correctly.
A total of morphological characteristics that proved for first time suitable for identification of the hybrid form of the two Greek Arbutus species, RAPD profiles, the resulting similarity and the dendrogram, led to the conclusion that most probably individuals with intermediate morphological characteristics from Kalamos is another species, possibly the one reported in the bibliography as a natural hybrid between A. unedo and A. andrachne, named A. × andrachnoides.All three Arbutus species found in Greece have or could be introduced for various uses in forestry, landscape, medicinal and horticultural Furthermore the in vitro propagation of these species has been successfully achieved both by axillary bud proliferation of adult plant (Mereti et al., 2002;Bertsouklis and Papafotiou, 2009;Gomes et al., 2010;Mohamed El-Sayed et al., 2010;Bertsouklis and Papafotiou, 2011) and from seedlings (Papafotiou et al., 2013), so the most suitable clones could be propagated.All these techniques would be very useful tools for breeding programs involving Arbutus species or for developing effective conservation strategies.Santiso et al. (2015) reported that A. unedo maintains the ability to evolve despite low genetic differentiation and stabilizing selection.The present study, proposing appropriate morphological characteristics in combination with selected molecular markers for distinguish individuals of three Arbutus species, is a step in achieving these goals for Arbutus spp. in Greece and other counties, particularly those in which Arbutus species are endangered or threatened, as A. andrachne in Ukraine, Albania, Jordan and Georgian SSR (Al-Tellawi, 1989;Garzuglia, 2006;Melia et al., 2012), Α. unedo in Slovenia (Garzuglia, 2006) and Α. × andrachnoides in Croatia (Šatović, 2004).

Conclusions
The present work provides both morphological traits and molecular markers (RAPD) to differentiate the three Arbutus species found in Greece, particularly useful for identification of the hybrid A. × andrachnoides from the parental species A. andrachne and A. unedo, and in this way to facilitate their exploitation in horticultural and pharmaceutical industry.The applied morphometric characteristics are suggested as a basis to develop a complete list of discriminating descriptors for Arbutus genus.

Fig. 1 .
Fig. 1.Evaluation of the descriptors and their contribution to the variability of the individuals studied

Table 1 .
Morphological traits of Arbutus individuals

Table 2 .
Primer sequences, number of bands per primer, number of polymorphic bands per primer and the approximate band size range

Table 3 .
Leaf traits of 28 individuals selected from two different areas (mean values)

Table 4 .
Evaluation of the descriptors and their contribution to the variability of the individuals studied