STATISTICAL SEED CLASSIFIERS
Grillo, O., Mattana, E., Venora, G. and Bacchetta, G. (2010), Seed Sci. & Technol., 38, 455-476 Statistical seed classifiers of 10 plant families representative of the Mediterranean vascular flora
O. GRILLO1,2, E. MATTANA1, G. VENORA2 AND G. BACCHETTA1
1 Centro Conservazione Biodiversità (CCB). Dipartimento di Scienze Botaniche Università degli Studi di Cagliari. V.le Sant’Ignazio da Laconi, 13 - 09123 Cagliari, Italy (E-mail: [email protected]) 2 Stazione Sperimentale di Granicoltura per la Sicilia (SSG). Via Buganvillea, 20 - 95041 Caltagirone, Italy (E-mail: [email protected])
(Accepted April 2010)
Summary
The aim of this study was to implement a seed morphometric and colorimetric database in order to develop specific statistical classifiers for 10 families representative of the Mediterranean vascular flora. The 501 analysed accessions consisted of 274 taxa belonging to 161 genera and 52 families. Images were acquired by a flatbed scanner and then elaborated by a macro software program developed for the morpho-colorimetric measurements. The mean seed weight was also calculated for each accession. Statistical classifiers were elaborated for genera and specific and infraspecific taxa discrimination within the following families: Apiaceae, Asteraceae, Boraginaceae, Brassicaceae, Caryophyllaceae, Cistaceae, Fabaceae, Lamiaceae, Poaceae and Scrophulariaceae. Such classifiers, based on Linear Discriminant Analysis and checked by cross validation, showed a performance ranging from 90.4% to 98.5% and from 87.8% to 98.3% at genus and taxon levels respectively. A first application of this technique to gymnosperm seeds was also presented. The performance of correct classification (93.6%) obtained for the genus Juniperus suggests that this technology is also reliable for gymnosperms. Seed weight improved the accuracy of the classifier system, confirming that an extensive database of bio-morphological traits constituted by a large number of seed species may be useful for taxonomic identification.
Introduction
Although morphological seed traits such as shape, size and external ornamentation represent important diagnostic factors in plant taxonomy studies (e.g. Thellung, 1926; Valdés, 1970; Viano, 1978; Sutton, 1988), they are often not sufficiently considered in dichotomous keys of many families and genera. In fact they are observable in the field only for a short period, between the fruit and seed ripening and dispersal, when other important characters, such as flowers and leaves, often have already disappeared. However, the increasing availability of seeds collected from wild plants cultivated ex situ (e.g. botanic gardens) and, above all, stored in seed banks emphasizes the importance of seed macro and micro morphology studies in plant taxonomy. For example, Agulló et al. (1991) carried out a morphological seed analysis in 17 taxa belonging to 5 genera of the subfamily Papilionidae (Fabaceae) by evaluation of seed size, shape, external colour, weight and protein content to investigate their taxonomic relationships. González-Andrés
455 O. GRILLO, E. MATTANA, G. VENORA AND G. BACCHETTA et al. (1999) characterized three species belonging to the Medicago sect. Dendrotelis (Fabaceae), using 12 seed and 20 seedling morphometric characters, showing that they were more clearly differentiated on the basis of the seed characters. Kirkbride et al. (2004) created an interactive key accessible via internet for the seed identification of 683 commercially important legume genera. Fagundez and Izco (2003, 2004a, b, c, 2006, 2009) analysed seed micromorphology, size and colour to investigate the taxonomic position of European species of Calluna, Daboecia and Erica genera (Ericaceae). Anderberg et al. (2008) investigated seed shape, seed coat structure and surface patterns in 34 species of the genus Lysimachia and in 14 species and 2 subspecies of 6 additional genera (Anagallis, Ardisiandra, Asterolinon, Glaux, Pelletiera and Trientalis). Mapping the seed characters onto a recent phylogenetic tree, the authors identified potentially synapomorphic character states for various subclades of Lysimachia. Dadandi et al. (2009) elaborated an identification key based on macro and micromorphological properties of intact and mature seeds of 12 Turkish taxa belonging to the genus Nigella (Ranunculaceae) to assess the systematic position of these species. The application of image analysis techniques allows implementing quick, precise and repeatable measurements of colour, size and shapes of the objects inside a digital image (Serra, 1982). The cost reductions and the increased power of computer hardware and software packages for image processing have made image analysis systems more attractive in the automatic inspection of different aspects of quality evaluation and sorting of agricultural products (Chen and Sun, 1991). Seeds have a three-dimensional (3D) shape, while captured images displayed on a monitor or on printed pages are two-dimensional (2D) (Loomis et al., 1999). Nevertheless, several descriptors of seed size, shape and Red, Green and Blue (RGB) colour component densities can be easily estimated from 2D digital images (Dell’Aquila, 2007). In a review on new computer imaging techniques applied to seed quality testing and sorting, Dell’Aquila (2007) reported 2 main applications in seed science: (1) to monitor seed germination and radical growth (e.g. Jansen, 1995; McDonald et al., 2001; Dell’Aquila, 2006) and (2) define markers for seed identification and sorting (e.g. Liao et al., 1994; Venora et al., 2007, 2009a, b; Dana and Ivo, 2008; Zapotoczny et al., 2008). Bacchetta et al. (2008) characterized digital images of seed lots of wild plant species typical of the Mediterranean basin, stored at the Sardinian Germplasm Bank (BG-SAR) (Mattana et al., 2005), using an image analysis system at the Stazione Sperimentale di Granicoltura per la Sicilia (SSG). The analysed accessions referred to 148 taxa belonging to 102 genera and 47 families. Images of diaspores (fruits and seeds) were acquired by a flatbed scanner and elaborated by a specifically developed macro, to record 13 morphometric and colorimetric measurements of each seed in the acquired images. This method allowed the development of a database for the characterization of autochthonous germplasm upon entry into the seed bank and the realization of statistical classifiers for the discrimination of genera and species within the following families: Apiaceae, Boraginaceae, Caryophyllaceae, Cistaceae, Fabaceae and Scrophulariaceae. Such classifiers, based on the Linear Discriminant Analysis (LDA) technique and checked by cross-validation method, showed a performance ranging between 74.3% and 96.4%. This system was later improved for the genus Astragalus (Mattana et al., 2008) by adding
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20 new seed and fruit morphometric and colorimetric features. This new classifier had a better performance, discriminating with 100% accuracy between two closely related Astragalus species of Sardinia (A. maritimus and A. verrucosus) belonging to the subgenus Trimeniaeus. The aims of this study were: (1) to increase the size of the database developed in previous works and evaluate the classifier identification performance with a larger dataset and (2) to develop specific statistical classifiers at family level and test the system on the genus Juniperus as a first application on gymnosperm seeds.
Materials and methods
Seed material and selection of the plant families The 501 accessions analyzed in this study belonged to 274 taxa (species or lower ranks), 161 genera and 52 families, making a total of 47,493 fruits or seeds (hereafter referred to as seeds), mainly collected in Sardinia during the harvesting seasons 2004-2008 and stored at the BG-SAR (Appendix 1, see page 469). Some material collected elsewhere in the Mediterranean basin (e.g. Italian and Iberian peninsulas, Corsica, Balearic and Hyères, Crete and other Aegean islands) was also used, as well as seeds obtained by ex situ cultivation in the Botanic Gardens of Cagliari or provided by other scientific institutions. To create dedicated seed statistical classifiers, 10 plant families were selected based on their representativeness of the Mediterranean vascular flora and the amount of available seed lots stored at BG-SAR (table 1). These 10 selected families comprised more than 50% of the genera, taxa, accessions, and seeds in the total database. Taxonomic classifications were made according to the Angiosperm Phylogeny Group (APG II, 2003).
Table 1. Classifiers developed for 10 plant families representative of the Mediterranean vascular flora. The representativeness of the ten family classifiers respect to the General classifier is reported in brackets. Classifiers Families Genera Taxa Accessions Seeds General 52 161 274 501 47,493 Apiaceae 12 14 33 3,230 Boraginaceae 3 7 14 1,071 Caryophyllaceae 5 18 26 2,599 Cistaceae 4 13 25 2,510 Fabaceae 12 26 40 3,698 Scrophulariaceae 2 7 9 902 Asteraceae 24 33 49 4,682 Brassicaceae 7 9 18 1,811 Lamiaceae 8 15 22 2,169 Poaceae 11 14 22 2,064 Subtotal 88 (54.7%) 156 (56.9%) 258 (51.5%) 24,736 (52.1%)
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Data acquisition The images of seed samples were acquired according to Bacchetta et al. (2008) by a flatbed scanner (Epson GT-15000) with a resolution of 200 dpi and a scanning area not exceeding 1024×1024 pixel, before being dried at 15°C to 15% of R.H. to avoid any possible variation in dimension, shape and colour. For the analysis of every accession, a sub-lot of 100 seeds was randomly prepared; when an accession had fewer than 100 seeds, the analysis was done using the entire lot. As the system works with 2D images and is therefore unable to distinguish between globose and flattened seeds, the mean seed weight of each accession was also determined by weighing air dried seeds on a four decimal place balance, as an estimate of the thickness (3D) of the seed. A total of 34 seed characters were measured (table 2).
Table 2. List of thirty-four morphometric and colorimetric features measured on seeds. (*) indicates the new feature (seed weight) not considered in Mattana et al., 2008.
Feature Description
A Area Seed area (mm2) P Perimeter Seed perimeter (mm)
Pconv Convex Perimeter Convex perimeter of the seed (mm)
PCrof Crofton’s Perimeter Crofton perimeter of the seed (mm)
Pconv /PCrof Perimeter ratio Ratio between convex and Crofton’s perimeters
Dmax Max diameter Maximum diameter of the seed (mm)
Dmin Min diameter Minimum diameter of the seed (mm)
Dmin /Dmax Feret ratio Ratio between minimum and maximum diameters Sf Shape Factor Seed shape descriptor = (4 × π × area)/perimeter2 (normalized value) Seed roundness descriptor = (4 × area)/(π × max diameter2) Rf Roundness Factor (normalized value) Ecd Eq. circular diameter Diameter of a circle with equivalent area (mm)
EAmax Maximum ellipse axis Maximum axis of an ellipse with equivalent area (mm)
EAmin Minimum ellipse axis Minimum axis of an ellipse with equivalent area (mm)
Rmean Mean red channel Red channel mean value of seed pixels (grey levels)
Rsd Red std. deviation Red channel standard deviation of seed pixels
Gmean Mean green channel Green channel mean value of seed pixels (grey levels)
Gsd Green std. deviation Green channel standard deviation of seed pixels
Bmean Mean blue channel Blue channel mean value of seed pixels (grey levels)
Bsd Blue std. deviation Blue channel standard deviation of seed pixels
Hmean Mean hue channel Hue channel mean value of seed pixels (grey levels)
Hsd Hue std. deviation Hue channel standard deviation of seed pixels
Lmean Mean lightness ch. Lightness channel mean value of seed pixels (grey levels)
Lsd Lightness std. dev. Lightness channel standard deviation of seed pixels
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Table 2. continued
Smean Mean saturation ch. Saturation channel mean value of seed pixels (grey levels)
Ssd Saturation std. dev. Saturation channel standard deviation of seed pixels
Dmean Mean density Density channel mean value of seed pixels (grey levels)
Dsd Density std. deviation Density channel standard deviation of seed pixels S Skewness Asymmetry degree of intensity values distribution (grey levels) K Kurtosis Peakness degree of intensity values distribution (densit. units) H Energy Measure of the increasing intensity power (densitometric units) E Entropy Dispersion power (bit)
Dsum Density sum Sum of density values of the seed pixels (grey levels)
SqDsum Square density sum Sum of the squares of density values (grey levels) SW Seed weight (*) Mean seed weight (g)
Image processing The KS-400 V. 3.0 (Carl Zeiss, Vision, Oberkochen, Germany) image analysis system was used. To achieve the relative dimensions, shapes and colour of seeds (RGB – Red, Green, Blue and HLS – Hue, Lightness, Saturation channels), the macro developed for wild plant species characterization (Bacchetta et al., 2008) and later modified adding 20 new seed morpho-colorimetric features (Mattana et al., 2008) was used.
Classification statistics Statistical analyses were performed using the stepwise Linear Discriminant Analysis method (LDA) to determine the best classification variables. The procedure of cross validation was applied to verify the performance of the developed classifiers. This method, is useful for small populations of data without a broad group of new unknown cases. The LDA method tests individual cases and classifies them on the basis of all the others (SPSS release 15, SPSS Inc. 1989-2006). The statistical analysis was carried out in two distinct phases. The whole database was used to develop a general classifier to discriminate all the studied accessions, according to families, genera and infraspecific taxa. Specific classifiers were then developed for generic, specific and infraspecific taxa identifications, within each selected family.
Results and discussion
General classifier The database of seed characteristics has grown since it was first published in 2008, with many additional accessions included (table 3). After each intermediate analysis the validity of the classification system and the representativeness of the database were evaluated (table 3). Values highlighted that the increase of samples and analysed taxa in the April 2009 database (501 accessions) led to a reduction of the general classifier performance, with a final percentage of correct identification of 54.2% at family level, 67.0% at genus
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Table 3. Trend over time of the database dimensions and performances of the general classifier. With (*) data from Bacchetta et al., 2008.
Elaborations Accessions Families Genera Taxa
May 2006 (*) 108 34 - 70.0% 67 - 80.3% 103 - 85.1% September 2006 (*) 179 45 - 63.0% 93 - 72.0% 146 - 75.3% January 2007 (*) 220 47 - 57.4% 102 - 66.5% 148 - 72.5% May 2008 319 51 - 65.1% 114 - 74.3% 182 - 76.4% April 2009 501 52 - 54.2% 161 - 67.0% 274 - 69.3% and 69.3% at taxa levels (table 3). The general classifier presented here achieved a lower performance than in previous studies because of the huge variability in the data. As considered in our previous report (Bacchetta et al., 2008), when the database becomes too large, it is better to divide it into separate databases and classifiers at the family level.
Statistical family classifiers To overcome the relatively low accuracy of the general classifier, 10 dedicated statistical classifiers were employed for individual families representative of the Mediterranean vascular flora (table 4). Performance of each developed classifier at genus and specific or infraspecific levels, including the previous results achieved by Bacchetta et al., (2008). Following APG (2003) classification, species belonging to the genera Digitalis, Erinus and Linaria that in the previous work (Bacchetta et al., 2008) were considered as belonging to the Scrophulariaceae family, were considered with the Plantaginaceae family in this study. For the Scrophulariaceae family, an additional statistical analysis was also carried out without considering the APG classification, to compare the achieved results with the previous data (table 4). Despite the number of accessions being more than doubled by increasing the number of genera, species and infraspecific taxa in the 6 comparable families (Apiaceae, Boraginaceae, Caryophyllaceae, Cistaceae, Fabaceae and Scropholariaceae), the classification performance was always higher than in the previous classifiers, except for the family Boraginaceae at genus level that had about a 1% of decrease in performance (table 4). The results achieved for this family may be explained by a reduction of its representativeness due to the addition of a new genus (Myosotis) represented by only one accession of one taxon. The Scrophulariaceae classifier (sensu APG) showed the largest improvement in classification performances both at genus and specific or infraspecific levels, notwithstanding the lower number of samples and analysed taxa (table 4). These results confirm the taxonomic distance between the genera belonging to Plantaginaceae (and previously considered as Scrophulariaceae) and the genera Scrophularia and Verbascum. In fact, the analysis carried out considering all these genera (table 4) achieved lower results (79.2% and 85.3% at genus and specific or infraspecific levels, respectively). Statistical classifiers were developed for four new families including Asteraceae, Brassicaceae, Lamiaceae and Poaceae, which reached performances of correct classification greater than 90.4% and 87.8% at genus and specific or infraspecific levels, respectively (table 4). In particular, high percentages of correct classification, both at generic (98.1%)
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Table 4. Performance percentages of family classifiers at genus and specific or infraspecific levels. With (*) data from Bacchetta et al., 2008. For the Scrophulariaceae family, the performance percentages, without considering the APG (Angiosperm Phylogeny Group) classification, are also reported in brackets.
Family classifier Genus Taxa
Apiaceae 95.6 (*) - 97.2 93.6 (*) - 94.0 Boraginaceae 96.4 (*) - 95.1 76.8 (*) - 88.3 Caryophyllaceae 89.9 (*) - 92.2 84.3 (*) - 97.0 Cistaceae 84.7 (*) - 93.3 88.8 (*) - 93.1 Fabaceae 82.6 (*) - 91.4 78.5 (*) - 91.3 Scrophulariaceae 74.3 (*)- (79.2) 91.9 74.4 (*) - (85.3) 96.9 Asteraceae 90.4 87.8 Brassicaceae 98.1 98.3 Lamiaceae 96.7 96.7 Poaceae 98.5 97.8 and specific or infraspecific (98.3%) levels, were achieved for Brassicaceae (tables 4, 5). Seed characters are considered some of the most useful diagnostic characters for the taxonomy in this family, as previously reported in the literature (e.g. Vaughan and Whitehouse, 1971; Bengoechea and Gomez Campo, 1975).
Key parameters Evaluating the contribution of all the variables used by the discrimination algorithm (LDA), it was possible to identify the features that, more than others, could be considered peculiar to each family (table 6). When there are many predictors (features), the stepwise method can be useful by automatically selecting the best variables to use in the model. This method starts with a model that doesn’t include any of the predictors. At each step, the predictor with the largest “F to enter” value that exceeds the entry criteria (F ≥ 3.84) is added to the model. The “Tolerance” value is the proportion of a variable’s variance not accounted for other independent variables in the equation. A variable with very low tolerance contributes little information to a model. The “F to remove” value is useful for describing what happens if a variable is removed from the current model. The features (predictors) included in the first five steps of the stepwise LDA method, are reported (table 6) together with the number of steps performed by the algorithm and the Tolerance and “F to remove” values in brackets. The feature Seed Weight, not included in the previous classifiers (Bacchetta et al., 2008; Mattana et al., 2008), showed here its discriminatory power by achieving the highest “F to remove” values. Indeed, this feature was always present in the first five parameters considered by the discrimination algorithm, in all families’ classifiers at least at one level (table 6). In particular, it was the first parameter at both levels for Lamiaceae and Poaceae, and at specific or infraspecific levels for Asteraceae, Boraginaceae, Caryophyllaceae and Cistaceae (table 6). Distinguishing between morphometric and colorimetric features
461 O. GRILLO, E. MATTANA, G. VENORA AND G. BACCHETTA Total (100) (100) sd sd mean A A Sf S R H 98.1% (1810) (27; 0.008; 9.546) (29; 0.076; 80.414) (24; 0.107; 43.896) (29; 0.010; 45.905) (25; 0.010; 87.080 ) (29; 0.0083; 30.673) Spreng. Malcolmia 100.0% (100) (100)
sd sd sd mean mean . The number of steps, the tolerance S S L SW B H L. (24; 0.389; 49.319) (29; 0.095; 34.037) (29; 0.123; 44.555) (25; 0.086; 32.394) (27; 0.031; 37.430) (29; 0.003; 55.220) Thlaspi 100.0% (300) (300) DC. sd sd sd E S B H SW Ecd Carrichtera (25; 0.196; 7.596) (29; 0.123; 51.423) (27; 0.133; 94.565) (29; 0.019; 35.634) (24; 0.006; 204.346) (29; 0.599; 536.105) L. Iberis min 100.0% (200) (200) conv A E E mean P R EA (27; 0.019; 7.131) (25; 0.018; 12.308) (29; 0.002; 13.171) Mill. (24; 0.008; 275.175) (29; 0.474; 213.058) (29; 0.494; 163.297) Cakile 100.0% (100)
Crof Crof
sum 12345 /P /P E L. SW SW D conv conv P P (24; 0.120; 15.363) (27; 0.003; 33.439) (25; 0.546; 498.418) (29; 0.129; 105.528) (29; 0.282; 164.116) Erysimum 100.0% (100) (29; 0.584; 5,331.088) L. 1.0% (1) 99.0% (99) (100) Brassica 96.3% (876) 3.5% (32) 0.2% (2) (910) (genus level) (genus level) Classifier (genus level) (species or infrasp. level) (species or infrasp. level) (species or infrasp. level) DC. Spreng. L. L. L. Mill. L. to remove values are reported in brackets. F Boraginaceae Caryophyllaceae Table 6. The best five key parameters (see table 2 for the legend of features) correct classifications are reported and Apiaceae Brassica Erysimum Table 5. Cross validated performances for Brassicaceae. Data in brackets correspond to the number of seeds. Genus Cakile Carrichtera Thlaspi Iberis Malcolmia Overall
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max max sd sd sd Crof mean mean E A mean B R G SW SW SW P B D G EA EA (31; 0.126; 59.059 ) (12; 0.348; 89.877 ) (26; 0.010; 29.778 ) (31; 0.010; 83.517 ) (31; 0.034; 19.521 ) (30; 0.014; 16.888 ) (31; 0.008; 10.821 ) (29; 0.082; 79.749 ) (28; 0.017; 224.755) (21; 0.191; 811.971 ) (24; 0.008; 646.148 ) (28; 0.121; 505.188 ) (26; 0.025; 143.990 ) (29; 0.061; 216.520 )
min max sd sd sd sd sd mean mean mean mean mean mean mean S B R R B S L R R H G D EA EA (1; 0.015; 16.336) 31; 0.239; 77.794) (31; 0.015; 52.288) (29; 0.057; 74.343) (12; 0.239; 65.963) (21; 0.148; 26.030) (24; 0.002; 33.051) (28; 0.020; 35.265) (30; 0.006; 13.945) (26; 0.016; 336.206) (26; 0.019; 315.968) (29; 0.034; 102.497) (31; 0.021; 125.992) (28; 0.118; 168.874)
max sd sd sd sd sum mean E E mean mean Sf Rf S R B B SW D S R H EA (21; 0.003; 22.165) (26; 0.008; 59.257) (26; 0.015; 53.695) (29; 0.032; 51.356) (31; 0.003; 79.652) (24; 0.004; 53.704) (28; 0.030; 77.162) (31; 0.028; 15.099) (30; 0.103; 19.216) (31; 0.109; 41.242) (31; 0.056; 113.770) (12; 0.568; 249.120) (29; 0.235; 133.931) (28; 0.242; 112.283) Crof
max sd sum mean mean /P A E E E E mean mean H SW D S B H G EA conv P (28; 0.102; 8.674) (31; 0.008; 47.212) (28; 0.125; 17.731) (31; 0.268; 14.343) (31; 0.270; 21.797) (26; 0.161; 17.836) (26; 0.069; 26.227) (29; 0.047; 53.319) (12; 0.013; 30.652) (21; 0.017; 27.606) (31; 0.016; 61.423) (24; 0.193; 36.921) (30; 0.061; 43.812) (29; 0.180; 278.205)
max
sd E mean mean /D Sf Sf Sf S SW SW SW SW SW SW B R min D (31; 0.098; 65.570) (29; 0.147; 39.272) (29; 0.194; 42.348) (26; 0.041; 11.673) (21; 0.468; 15.970) (28; 0.207; 72.970) (24; 0.171; 88.543) (28; 0.480; 256.786) (31; 0.520; 416.915) (30; 0.345; 854.607) (12; 0.044; 220.113) (31; 0.673; 895.191) (26; 0.678; 487.142) (31; 0.738; 1,313.390) (genus level) (genus level) (species or infrasp. level) (genus level) (species or infrasp. level) (species or infrasp. level) (genus level) (species or infrasp. level) (species or infrasp. level) (genus level) (species or infrasp. level) (genus level) (genus level) (species or infrasp. level) continued Asteraceae Brassicaceae Lamiaceae Fabaceae Poaceae Table 6. Cistaceae Scrophulariaceae
463 O. GRILLO, E. MATTANA, G. VENORA AND G. BACCHETTA
(table 2), the latter were the more represented among the first five parameters, even if morphometric features were the first discriminant parameters at both levels for Apiaceae, Brassicaceae and Fabaceae (table 6). Cistaceae and Scrophulariacaeae were the only two families for which there were non-morphological parameters in the first step at specific or infraspecific levels. These data suggest that both types of measurements should be considered when trying to achieve high percentages of correct classification, confirming the findings of Bacchetta et al. (2008) and highlighting as well that the integration of mean seed weight values improved the performance of the system.
Gymnosperms seeds The percentage of correct identification of the species belonging to the genus Juniperus is reported (table 7). Using the images of 1,700 seeds of 8 different taxa, the cross-validated classification performance was 93.6%. Figure 1 shows the first three function scores used to distinguish the taxa belonging to the genus, explaining 96.4% of the variability. The high percentage of correct classification between the two subspecies of J. phoenicea (98.8%), confirmed the differentiation at subspecies level (Amaral Franco do, 1986; Conti et al., 2005; Jeanmonod and Gamisans, 2007) or species level (Rivas- Martínez et al., 1993; Lebreton and Pérez de Paz, 2001) for these two taxa (table 8). The 89.7% of correct classification of J. communis subsp. communis and J. communis subsp. alpina (table 9) confirmed the taxonomic distance between them. These two taxa have been considered by several authors as two different subspecies (Amaral Franco do, 1980, 1986; Jeanmonod and Gamisans, 2007) or species (Pignatti, 1982; Lebreton et al., 2000), but were recently considered as the same taxon by a few authors (Farjon, 2001; Adams, 2004). These findings for the genus Juniperus suggest that this seed classifier technology, based on quick and cheap acquisitions of morphometric and colorimetric feature measurements of seed accessions upon their entry to a seed bank, is also reliable for gymnosperms.
8 J. communis subsp. communis J. phoenicea subsp. phoenicea 6 J. phoenicea subsp. turbinata J. oxycedrus subsp. macrocarpa 4 J. communis subsp. alpina 2 J. sabina J. thurifera 0 -2 -4 -6
Discriminant Function 2 Function Discriminant -8 20 15 -10 10 0 50 5 Discriminant100 0 150 -5 Function200 1 250 -10 Discriminant Function 3 Figure 1. Graphic representation of the discriminating function scores for the genus Juniperus.
464 STATISTICAL SEED CLASSIFIERS Total (100) 93.6% (1700) 100.0% (100) (100) . Data in brackets correspond to the number of seeds. Juniperus 100.0% (400) (400) 97.0% (97) 3.0% (3) 12345678 0.5% (1)4.0% (4) 8.0% (16) 1.5% (3) 86.5% (173) 3.5% (7) (200) 2.0% (2) 94.0% (94) (100) 0.4% (2) 96.6% (483) 1.4% (7) 1.6% (8) (500) 84.0% (84) 1.0% (1) 15.0% (15)13.0% (26) 7.0% (14) 80.0% (160) (100) (200) ...... subsp subsp subsp . . . L L L. subsp L L. subsp subsp (Sibth. (Sibth. L. (8) (2) (6) (1) (Guss.) L. (7) elak (5) ˇ C J. communis communis J. sabina J. thurifera J. phoenicea phoenicea alpina J. oxycedrus oxycedrus J. communis turbinata Nyman (3) macrocarpa & Sm.) Neilr. (4) Table 7. Cross validated performances of the classifier developed for genus Taxa J. phoenicea J. oxycedrus Overall
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Table 8. Cross validated performances between the two subspecies of Juniperus phoenicea. Data in brackets correspond to the number of seeds. J. phoenicea L. subsp. J. phoenicea L. subsp. Taxa Total phoenicea turbinata (Guss.) Nyman Juniperus phoenicea L. 93.0% (93) 7.0% (7) (100) subsp. phoenicea Juniperus phoenicea L. 100.0% (500) (500) subsp. turbinata (Guss.) Nyman Overall 98.8% (600)
Table 9. Cross validated performances between the two subspecies of Juniperus communis. Data in brackets correspond to the number of seeds. Juniperus communis L. Juniperus communis L. Taxa Total subsp. communis subsp. alpina Cˇ elak Juniperus communis L. subsp. 84.0% (84) 16.0% (16) (100) communis Juniperus communis L. subsp. 8.0% (16) 92.0% (184) (200) alpina Cˇ elak Overall 89.7% (300)
Conclusions
This work validated the statistical classification system explored in previous works (Bacchetta et al., 2008, Mattana et al., 2008) by the implementation of a general database and the elaboration of a dedicated seed classifier for each of ten families representative of the Mediterranean vascular flora. These results confirm, as stated by Dell’Aquila (2007), that an extensive database of bio-morphological traits constituted by a large number of seed species may be applied also for sorting and taxonomy screening. A further step of this work will be to improve the classifiers adding other features related to the exact shape of the seeds with a morphological method based on the elliptic Fourier descriptors of approximation to closed contours in a two-dimensional plane. In addition, the availability of morphometric data should be helpful for ecological studies such as the prediction of seed persistence in the soil (e.g. Thompson et al., 1993; Cerabolini et al., 2003).
Acknowledgements
The authors wish to thank the BG-SAR staff for field work and help on image acquisition and all the institutions that kindly provided seeds or their images: CBNM de Porquerolles; DGMN de Murcia; CIEF Comunidad Valenciana; Jardí Botànic de la Universitat de València; Institut y Jardí Botànic de Barcelona; Museo di Storia Naturale di Livorno;
466 STATISTICAL SEED CLASSIFIERS
CNBF Peri; BGVA Cordoba; BG-MOL Università degli Studi del Molise; Dipartimento di Scienze Botaniche, Ecologiche e Geologiche Università degli Studi di Sassari and SCD Kew Gardens. The CCB is supported by the “Provincia di Cagliari - Assessorato Tutela Ambiente”. This research has also been supported by the P.R.I.N. 2007 project funds (2007JNJ7MX-002).
References
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468 STATISTICAL SEED CLASSIFIERS
Appendix: List of the analysed taxa grouped by family, following APG (2003). Taxa Family (APG II) Accessions Seeds (n) Achyranthes sicula L. (All.) Amaranthaceae 2 200 Chenopodium bonus-henricus L. Amaranthaceae 1 100 Halopeplis amplexicaulis (Vahl) Ces., Pass. & Gibelli Amaranthaceae 1 101 Salicornia dolichostachya Moss Amaranthaceae 1 101 Salicornia emerici Duval-Jouve Amaranthaceae 1 100 Salicornia patula Duval-Jouve Amaranthaceae 1 100 Sarcocornia fruticosa (L.) A.J. Scott Amaranthaceae 1 100 Suaeda vera J.F. Gmel. Amaranthaceae 1 100 Pancratium maritimum L. Amaryllidaceae 4 349 Bunium corydalinum DC. subsp. corydalinum Apiaceae 1 85 Bupleurum fruticosum L. Apiaceae 1 100 Crithmum maritimum L. Apiaceae 3 300 Daucus carota L. Apiaceae 1 100 Eryngium maritimum L. Apiaceae 1 100 Ferula arrigonii Bocchieri Apiaceae 9 900 Ferula communis L. s.l. Apiaceae 4 387 Ferula fontqueri Jury Apiaceae 1 58 Laserpitium siler L. subsp. garganicum (Ten.) Arcang. Apiaceae 1 100 Pastinaca divaricata Desf. Apiaceae 1 100 Pseudorlaya pumila (L.) Grande Apiaceae 1 100 Ptychotis sardoa Pignatti & Metlesics Apiaceae 3 300 Rouya polygama (Desf.) Coincy Apiaceae 1 100 Seseli praecox (Gamisans) Gamisans Apiaceae 5 500 Arum pictum L. f. subsp. pictum Araceae 1 100 Biarum dispar (Schott) Talavera Araceae 1 100 Helicodiceros muscivorus (L. f.) Engl. Araceae 3 301 Asphodelus fistulosus L. Asphodelaceae 1 100 Anthemis maritima L. Asteraceae 2 200 Artemisia campestris L. subsp. variabilis (Ten.) Greuter Asteraceae 1 99 Asteriscus maritimus (L.) Less. Asteraceae 1 100 Bellium crassifolium Moris Asteraceae 1 100 Buphthalmum inuloides Moris Asteraceae 1 100 Carduus fasciculiflorus Viv. Asteraceae 3 300 Carduus nutans L. s.l. Asteraceae 2 199 Carlina corymbosa L. Asteraceae 1 100 Centaurea corensis Vals. & Filigh. Asteraceae 1 100 Centaurea filiformis Viv. subsp. filiformis Asteraceae 1 94 Centaurea horrida Badarò Asteraceae 1 95
469 O. GRILLO, E. MATTANA, G. VENORA AND G. BACCHETTA
Appendix continued Taxa Family (APG II) Accessions Seeds (n) Centaurea magistrorum Arrigoni & Camarda Asteraceae 1 100 Crupina crupinastrum (Moris) Vis. Asteraceae 1 30 Dittrichia viscosa (L.) Greuter s.l. Asteraceae 1 100 Eupatorium cannabinum L. subsp. corsicum (Loisel.) P. Fourn. Asteraceae 1 100 Filago pygmaea L. Asteraceae 1 108 Helichrysum italicum (Roth) G. Don subsp. italicum Asteraceae 1 100 Helichrysum microphyllum Willd. subsp. tyrrhenicum Bacch., Asteraceae 1 101 Brullo & Giusso Helichrysum saxatile Moris subsp. morisianum Bacch., Asteraceae 1 100 Brullo & Mossa Lactuca longidentata Moris Asteraceae 1 100 Lamyropsis microcephala Moris Asteraceae 5 426 Limbarda crithmoides (L.) Dumort. s.l. Asteraceae 2 197 Otanthus maritimus (L.) Hoffmanns & Link subsp. maritimus Asteraceae 4 400 Ptilostemon casabonae (L.) Greuter Asteraceae 2 200 Ptilostemon chamaepeuce (L.) Less. Asteraceae 1 87 Pulicaria vulgaris Gaertn. var. sardoa Fiori Asteraceae 1 100 Rhaponticum coniferum (L.) Greuter Asteraceae 2 147 Robertia taraxacoides (Loisel.) DC. Asteraceae 1 99 Santolina corsica Jord. & Fourr. Asteraceae 1 100 Santolina insularis (Gennari ex Fiori) Arrigoni Asteraceae 3 300 Senecio cineraria DC. Asteraceae 1 100 Senecio rodriguezii Willk. ex Rodrig. Asteraceae 1 100 Tanacetum audibertii (Req.) DC. Asteraceae 1 100 Anchusa capellii Moris Boraginaceae 1 100 Anchusa crispa Viv. subsp. maritima (Vals.) Selvi & Bigazzi Boraginaceae 2 121 Anchusa formosa Selvi, Bigazzi & Bacch. Boraginaceae 3 220 Anchusa littorea Moris Boraginaceae 2 173 Anchusa montelinasana Angius, Pontecorvo & Selvi Boraginaceae 2 200 ex Bacch. et al. Borago pygmaea (DC.) Chater & Greuter Boraginaceae 3 177 Myosotis soleirolii Godr. Boraginaceae 1 80 Brassica insularis Moris Brassicaceae 8 801 Brassica tournefortii Gouan Brassicaceae 1 110 Cakile maritima Scop. subsp. maritima Brassicaceae 1 100 Carrichtera annua (L.) DC. Brassicaceae 1 100 Erysimum majellense Polatschek Brassicaceae 1 100 Iberis integerrima Moris Brassicaceae 2 200
470 STATISTICAL SEED CLASSIFIERS
Appendix continued Taxa Family (APG II) Accessions Seeds (n) Malcolmia ramosissima (Desf.) Gennari Brassicaceae 1 100 Thlaspi brevistylum (DC.) Mutel Brassicaceae 1 100 Thlaspi perfoliatum L. s.l. Brassicaceae 2 200 Campanula forsythii (Arcang.) Bég. Campanulaceae 2 201 Arenaria bertolonii Fiori Caryophyllaceae 1 100 Cerastium boisserianum Greuter & Burdet Caryophyllaceae 1 100 Cerastium soleirolii Duby Caryophyllaceae 1 100 Cerastium supramontanum Arrigoni Caryophyllaceae 1 100 Dianthus insularis Bacch., Brullo, Casti & Giusso Caryophyllaceae 2 200 Dianthus morisianus Vals. Caryophyllaceae 2 200 Dianthus sardous Bacch., Brullo & Giusso del Galdo Caryophyllaceae 1 100 Silene canescens Ten. Caryophyllaceae 1 100 Silene hifacensis Rouy Caryophyllaceae 1 100 Silene morisiana Bég. & Ravano Caryophyllaceae 1 100 Silene rosulata Soy.-Will. & Godr. subsp. sanctae-theresiae Caryophyllaceae 1 100 (Jeanm.) Jeanm. Silene salzmanii Badarò Caryophyllaceae 1 100 Silene succulenta Forssk. subsp. corsica (DC.) Nyman Caryophyllaceae 2 200 Silene valsecchiae Bocchieri Caryophyllaceae 2 200 Silene velutina Loisel. Caryophyllaceae 4 400 Silene vulgaris (Moench) Garcke subsp. vulgaris Caryophyllaceae 1 100 Silene vulgaris (Moench) subsp. prostrata (Gaudin) Caryophyllaceae 1 100 Schinz & Thell Spergularia macrorhiza (Loisel.) Heynh. Caryophyllaceae 2 199 Cistus albidus L. Cistaceae 2 200 Cistus creticus L. subsp. corsicus (Loisel.) Greuter & Burdet Cistaceae 1 100 Cistus creticus L. subsp. creticus Cistaceae 1 100 Cistus creticus L. subsp. eriocephalus (Viv.) Greuter & Burdet Cistaceae 4 400 Cistus crispus L. Cistaceae 1 100 Cistus ladanifer L. Cistaceae 1 100 Cistus monspeliensis L. Cistaceae 1 100 Cistus salviifolius L. Cistaceae 7 700 Halimium halimifolium (L.) Willk. subsp. halimifolium Cistaceae 2 210 Helianthemum aegyptiacum (L.) Mill. Cistaceae 2 200 Helianthemum caput-felis Moris Cistaceae 1 100 Helianthemum croceum (Desf.) Pers. Cistaceae 1 100 Tuberaria guttata (L.) Fourr. Cistaceae 1 100 Cornus sanguinea L. s.l. Cornaceae 1 100
471 O. GRILLO, E. MATTANA, G. VENORA AND G. BACCHETTA
Appendix continued Taxa Family (APG II) Accessions Seeds (n) Bryonia marmorata Petit Cucurbitaceae 2 200 Juniperus communis L. subsp. alpina Cˇ elak. Cupressaceae 2 200 Juniperus communis L. subsp. communis Cupressaceae 1 100 Juniperus oxycedrus L. subsp. macrocarpa (Sibth. & Sm.) Neilr. Cupressaceae 4 400 Juniperus oxycedrus L. subsp. oxycedrus Cupressaceae 2 200 Juniperus phoenicea L. subsp. phoenicea Cupressaceae 1 100 Juniperus phoenicea L. subsp. turbinata (Guss.) Nyman Cupressaceae 5 500 Juniperus sabina L. Cupressaceae 1 100 Juniperus thurifera L. Cupressaceae 1 100 Cynomorium coccineum L. subsp. coccineum Cynomoriaceae 2 200 Carex pendula Huds. Cyperaceae 1 100 Cyperus capitatus Vand. Cyperaceae 4 400 Schoenus nigricans L. Cyperaceae 1 100 Scirpoides holoschoenus (L.) Sojak Cyperaceae 1 100 Cephalaria squamiflora (Sieber) Greuter subsp. Dipsacaceae 5 498 mediterranea (Viv.) Pignatti Scabiosa holosericea Bertol. Dipsacaceae 1 100 Sixalix atropurpurea (L.) Greuter & Burdet s.l. Dipsacaceae 1 100 Ephedra distachya L. subsp. distachya Ephedraceae 2 201 Ephedra nebrodensis Guss. subsp. nebrodensis Ephedraceae 1 100 Erica terminalis Salisb. Ericaceae 1 100 Euphorbia amygdaloides L. subsp. semiperfoliata Euphorbiaceae 1 100 (Viv.) Radcl.-Sm. Euphorbia gayi Salis Euphorbiaceae 1 13 Euphorbia paralias L. Euphorbiaceae 1 100 Euphorbia pithyusa L. subsp. pithyusa Euphorbiaceae 1 100 Euphorbia pithyusa L. subsp. cupanii (Guss. ex Bertol.) Euphorbiaceae 2 200 Radcl.-Sm. Euphorbia spinosa L. s.l. Euphorbiaceae 1 26 Anthyllis barba-jovis L. Fabaceae 3 300 Anthyllis hermanniae L. subsp. ichnusae Brullo & Giusso Fabaceae 1 100 Astragalus cavanillesii Podlech Fabaceae 1 24 Astragalus genargenteus Moris Fabaceae 2 200 Astragalus greuteri Bacch. & Brullo Fabaceae 1 100 Astragalus maritimus Moris Fabaceae 5 500 Astragalus oxyglottis Steven Fabaceae 2 200 Astragalus tegulensis Bacch. & Brullo Fabaceae 1 100 Astragalus terraccianoi Vals. Fabaceae 3 239
472 STATISTICAL SEED CLASSIFIERS
Appendix continued Taxa Family (APG II) Accessions Seeds (n) Astragalus thermensis Vals. Fabaceae 2 200 Astragalus tremolsianus Pau Fabaceae 1 100 Astragalus verrucosus Moris Fabaceae 1 100 Bituminaria morisiana (Pignatti & Metlesics) Greuter Fabaceae 2 200 Calicotome villosa (Poir.) Link Fabaceae 2 129 Colutea arborescens L. Fabaceae 1 100 Cytisus villosus Pourr. Fabaceae 1 100 Dorycnium hirsutum (L.) Ser. Fabaceae 1 100 Dorycnium pentaphyllum Scop. Fabaceae 1 100 Genista sardoa Vals. Fabaceae 1 44 Genista corsica (Loisel.) DC. Fabaceae 1 100 Genista linifolia L. Fabaceae 1 62 Genista valsecchiae Bocchieri Fabaceae 1 100 Lotus cytisoides L. subsp. conradiae Gamisans Fabaceae 1 100 Medicago arborea L. s.l. Fabaceae 2 200 Ononis variegata L. Fabaceae 1 100 Teline monspessulana (L.) K. Koch Fabaceae 1 100 Centaurium erythraea Rafn s.l. Gentianaceae 1 100 Gentiana lutea L. s.l. Gentianaceae 2 201 Erodium corsicum Léman Geraniaceae 1 100 Ribes multiflorum Kit. ex Roem. & Schult. subsp. multiflorum Grossulariaceae 1 100 Ribes multiflorum Kit. ex Roem. & Schult. subsp. Grossulariaceae 2 200 sandalioticum Arrigoni Ribes sardoum Martelli Grossulariaceae 4 42 Hypericum hircinum L. subsp. hircinum Hypericaceae 2 201 Hypericum scruglii Bacch., Brullo & Salmeri Hypericaceae 1 100 Crocus minimus DC. Iridaceae 1 100 Iris planifolia (Mill.) Fiori Iridaceae 1 100 Iris xiphium L. Iridaceae 2 200 Juncus maritimus Lam. Juncaceae 2 200 Juncus subulatus Forssk. Juncaceae 2 200 Clinopodium sandalioticum (Bacch. & Brullo) Lamiaceae 1 100 Bacch. & Brullo ex Peruzzi & F. Conti Micromeria filiformis (Aiton) Benth. subsp. cordata Lamiaceae 1 89 (Bertol.) Pignatti Micromeria graeca (L.) Benth. ex Rchb. s.l. Lamiaceae 1 100 Nepeta foliosa Moris Lamiaceae 1 100 Salvia aethiopis L. Lamiaceae 1 100
473 O. GRILLO, E. MATTANA, G. VENORA AND G. BACCHETTA
Appendix continued Taxa Family (APG II) Accessions Seeds (n) Salvia desoleana Atzei & Picci Lamiaceae 1 100 Satureja thymbra L. Lamiaceae 2 200 Stachys corsica Pers. Lamiaceae 1 80 Teucrium capitatum L. subsp. capitatum Lamiaceae 1 100 Teucrium flavum L. subsp. glaucum (Jord. & Fourr.) Ronninger Lamiaceae 4 400 Teucrium marum L. Lamiaceae 2 200 Teucrium massiliense L. Lamiaceae 1 100 Teucrium montanum L. Lamiaceae 1 100 Teucrium polium L. subsp. polium Lamiaceae 2 200 Vitex agnus-castus L. Lamiaceae 2 200 Linum muelleri Moris Linaceae 3 300 Lavatera arborea L. Malvaceae 5 437 Lavatera maritima Gouan s.l. Malvaceae 2 200 Lavatera plazzae Atzei Malvaceae 2 200 Lavatera triloba L. subsp. triloba Malvaceae 2 190 Anagallis monelli L. subsp. monelli Myrsinaceae 1 100 Paeonia corsica Sieber ex Tausch. Paeoniaceae 3 300 Glaucium corniculatum (L.) Rudolph subsp. corniculatum Papaveraceae 1 100 Glaucium flavum Crantz Papaveraceae 2 200 Pinus halepensis Mill. Pinaceae 4 368 Digitalis purpurea L. var. gyspergerae (Rouy) Fiori Plantaginaceae 3 300 Erinus alpinus L. s.l. Plantaginaceae 1 101 Linaria arcusangeli Atzei & Camarda Plantaginaceae 1 100 Linaria cossonii Barratte Plantaginaceae 2 199 Linaria flava (Poir.) Desf. subsp. sardoa (Sommier) A. Terracc. Plantaginaceae 2 200 Plantago subulata L. subsp. insularis (Gren. & Godr.) Nyman Plantaginaceae 1 100 Armeria pungens (Link) Hoffmanns. & Link Plumbaginaceae 1 100 Armeria sulcitana Arrigoni Plumbaginaceae 1 100 Limonium avei (De Not.) Brullo & Erben Plumbaginaceae 1 100 Limonium morisianum Arrigoni Plumbaginaceae 1 100 Limonium narbonense Mill. Plumbaginaceae 1 100 Limonium retirameum Greuter & Burdet Plumbaginaceae 1 100 Limonium virgatum (Willd.) Fourr. Plumbaginaceae 1 96 Ammophila arenaria (L.) Link. subsp. australis (Mabille) Lainz Poaceae 3 251 Ampelodesmos mauritanicus (Poir.) T. Durand & Schinz Poaceae 1 100 Arrhenatherum murcicum Sennen Poaceae 1 100 Dactylis glomerata L. subsp. hispanica (Roth) Nyman Poaceae 1 100 Elymus farctus (Viv.) Runemark ex Melderis subsp. farctus Poaceae 3 301
474 STATISTICAL SEED CLASSIFIERS
Appendix continued Taxa Family (APG II) Accessions Seeds (n) Elytrigia corsica (Hackel) J. Holub Poaceae 1 100 Phleum sardoum (Hack.) Hack. Poaceae 1 100 Piptatherum caerulescens (Desf.) Richter Poaceae 1 85 Piptatherum miliaceum (L.) Coss. s.l. Poaceae 4 400 Poa balbisii Parl. Poaceae 1 100 Sesleria insularis Sommier subsp. barbaricina Arrigoni Poaceae 2 200 Sesleria insularis Sommier subsp. insularis Poaceae 1 27 Sesleria insularis Sommier subsp. morisiana Arrigoni Poaceae 1 100 Trisetaria gracilis (Moris) Banfi & Arrigoni Poaceae 1 100 Polygala sardoa Chodat Polygalaceae 4 350 Polygala sinisica Arrigoni Polygalaceae 3 239 Rumex scutatus L. subsp. glaucescens (Guss.) Brullo, Polygonaceae 2 200 Scelsi & Spamp. Aquilegia barbaricina Arrigoni & E. Nardi Ranunculaceae 8 800 Aquilegia bernardii Gren. & Greuter Ranunculaceae 3 236 Aquilegia dumeticola Jord. Ranunculaceae 1 100 Aquilegia litardierei Briq. Ranunculaceae 3 218 Aquilegia nugorensis Arrigoni & E. Nardi Ranunculaceae 12 1188 Aquilegia vulgaris L. Ranunculaceae 3 245 Clematis flammula L. Ranunculaceae 1 100 Delphinium gracile DC. Ranunculaceae 1 100 Delphinium pictum Willd. s.l. Ranunculaceae 1 100 Helleborus lividus Aiton subsp. corsicus (Briq.) P. Fourn. Ranunculaceae 2 176 Nigella damascena L. Ranunculaceae 1 100 Thalictrum minus L. Ranunculaceae 1 100 Reseda luteola L. Resedaceae 1 100 Rhamnus alpina L. s.l. Rhamnaceae 1 100 Rhamnus lycioides L. subsp. oleoides (L.) Jahald. & Maire Rhamnaceae 1 100 Rhamnus persicifolia Moris Rhamnaceae 3 300 Pyracantha coccinea M. Roem. Rosaceae 1 100 Sarcopoterium spinosum (L.) Spach. Rosaceae 5 500 Sorbus aria (L.) Crantz s.l. Rosaceae 2 200 Asperula pumila Moris Rubiaceae 1 100 Crucianella maritima L. Rubiaceae 3 300 Galium corsicum Spreng. Rubiaceae 2 200 Galium minutulum Jord. Rubiaceae 1 99 Galium schmidii Arrigoni Rubiaceae 2 200 Rubia peregrina L. subsp. requienii (Duby) Cardona & Sierra Rubiaceae 1 100
475 O. GRILLO, E. MATTANA, G. VENORA AND G. BACCHETTA
Appendix continued Taxa Family (APG II) Accessions Seeds (n) Cneorum tricoccon L. Rutaceae 3 300 Ruta lamarmorae Bacch. & Brullo Rutaceae 1 100 Acer monspessulanum L. subsp. monspessulanum Sapindaceae 1 100 Scrophularia canina L. subsp. bicolor (Sm.) Greuter Scrophulariaceae 2 200 Scrophularia trifoliata L. Scrophulariaceae 2 200 Verbascum argenteum Ten. Scrophulariaceae 1 100 Verbascum boerhavii L. Scrophulariaceae 1 100 Verbascum conocarpum Moris Scrophulariaceae 1 102 Verbascum niveum Ten. subsp. garganicum (Ten.) Murb. Scrophulariaceae 1 100 Verbascum plantagineum Moris Scrophulariaceae 1 100 Lycium europaeum L. Solanaceae 1 100 Mandragora autumnalis Bertol. Solanaceae 2 200 Nicotiana glauca Graham Solanaceae 1 100 Solanum elaeagnifolium Cab. Solanaceae 2 200 Taxus baccata L. Taxaceae 3 300 Daphne oleoides Schreb s.l. Thymeleaceae 1 100 Centranthus amazonum Fridl. & A. Raynal Valerianaceae 8 360 Centranthus ruber (L.) DC. subsp. ruber Valerianaceae 6 522 Centranthus trinervis (Viv.) Bég. Valerianaceae 11 1098 Vitis vinifera L. subsp. sylvestris (C.C. Gmel) Hegi Vitaceae 1 100
Totals 501 47,493
476