Soil and Biota of Serpentine: A World View 2009 Northeastern Naturalist 16(Special Issue 5):39–55 Morphological Variation in Teucrium chamaedrys in Serpentine and Non-Serpentine Populations Dolja Pavlova* Abstract - Teucrium chamaedrys is the most variable species in the genus Teucrium, fitting Kruckeberg’s category of a bodenvag species. Six populations distributed on serpentine and 3 populations off serpentine were investigated, and 23 morphological features were studied by univariate and multivariate statistical analyses. The variation was higher for the vegetative features and not clearly expressed for the generative ones. In addition, metal concentration in the populations and soil were studied. The species tolerant to the serpentine conditions demonstrated specific morphological differences in stem length, stem length up to the first leave pair, and internode length between second and third leave pairs. Morphological differences, geographical isolation, and preliminary results on the karyology of T. chamaedrys in Bulgaria, suggest that the populations studied were different ecotypes. Introduction Teucrium chamaedrys L. (Wall germander) is a widely distributed species of Teucrium (Lamiaceae) found in Bulgaria and other countries in Central Europe. This perennial plant occurs in all floristic regions of Bulgaria up to 1500 m above sea level (Markova 1992, Peev 1989) and shows a high degree of morphological diversity. Reichinger (1941) considered it to be the most variable taxon in the Teucrium genus and described 15 subspecies, most of them locally distributed. These subspecies were not initially included in Flora Europaea (Tutin et al. 1972) but were later added by Greuter et al. (1986). Interest in T. chamaedrys is increased by the fact that it is one of 766 species of medicinal plants in the Bulgarian flora (Gussev 2005). The distribution and the degree of presence of medicinal plants are directly correlated to edaphic factors (Obratov-Petković et al. 2006). Also, the quantity of active substances in plant tissues depends on many ecological factors that affect the vegetative plant organs (Lombini et al. 1999). The species demonstrates a wide ecological tolerance as it is found in open grasslands, oak woodlands, pastures, calcareous and siliceous terrains, and on serpentine outcrops. Serpentine outcrops are probably the most extreme habitat conditions for this species. Serpentines and their soils are characterized by toxic quantities of heavy metals (particularly Ni and Cr), low Ca/Mg ratio, drought, and wide temperature fluctuations, and plant populations growing on serpentine sites are likely adapted at some level to these special edaphic conditions (Brooks 1987; Kruckeberg 1984, 1992). Many *University of Sofia, Faculty of Biology, Department of Botany, Boulevard Dragan Tzankov 8, 1164 Sofia, Bulgaria; pavlova@biofac.uni-sofia.bg. 40 Northeastern Naturalist Vol. 16, Special Issue 5 studies have documented variability of plant species growing on serpentine habitats (Bratteler et al. 2006; Mayer and Soltis 1994; Štepankova 1996, 1997; Westerbergh and Rune 1996; Westerbergh and Saura 1992; Wright et al. 2006). The aims of this study were to investigate natural serpentine and nonserpentine populations to: 1) document differences in morphological traits between-populations, and 2) compare the chemical composition in soils and plants from these populations. Material and Methods Sites and sampling Nine populations of T. chamaedrys originating from localities distributed in southern Bulgaria were investigated (Fig. 1, Table 1). Six sites were serpentine and three were non-serpentine. The non-serpentine localities were Figure 1. Map of Balkan Peninsula with distribution of the serpentines. Open circles indicate serpentine populations. Filled circles indicate non-serpentine populations. The numbers correspond to the localities described in Table 1. 2009 D. Pavlova 41 selected on both calcareous (Beledie Han) and siliceous (Rila) terrains. Random plants within each locality were collected during 2006–2007. Voucher specimens from all investigated populations were assigned a voucher number (Table 1) and placed in the Herbarium of University of Sofia (SO). Soil samples from five serpentine and two non-serpentine sites were collected to determine their chemical composition. Measurements The investigation of morphological differences between serpentine and non-serpentine plants was performed on 225 individuals (25 individuals per site) using 23 morphological characters (Var. 1 to Var. 23; Table 2). For each individual, a leaf from the second pair and a flower from the lower part of the inflorescence were measured. Vegetative characters were studied on dry plants, and flower characters were measured after boiling. One flower per individual was boiled in distilled water for 1–2 min to facilitate measuring. Table 1. List of the localities of the studied populations of Teucrium chamaedrys. # = number of individuals sampled, Abbr. = abbreviations used for the populations (see text). No Locality Voucher No # Abbr. Serpentine populations 1 Central Rhodope Mts. – Parvenetz village, southeastward on SO 103375 25 P the open stony slope, 277 m above sea level, N42°03.96', E24°39.44', UTM Grid -LG-06; D. Pavlova, August 2007 2 Eastern Rhodope Mts. – Fotinovo village westward from the SO 104608 25 F village, 421 m a.s.l., UTM Grid - LF-68; D. Pavlova, October 2007 3 Eastern Rhodope Mts. – Dobromirtzi village, southwestward SO 104607 25 D1 from the village, on the basis of the hill, left side of the road to Zlatodrad, 320 m a.s.l., N41°23.18', E25°12.24', UTM Grid - LF-58; D. Pavlova, October 2007 4 Eastern Rhodope Mts. – Dobromirtzi village, southwestward SO 104604 25 D2 from the village, on the top of the hill, left side of the road to Zlatodrad, 400 m a.s.l., UTM Grid - LF-58; D. Pavlova, October 2007 5 Vlahina Mts. – Murdzova chuka peak, northward of Stara SO 104902 25 Z Zeleznitza village, 720 m a.s.l., UTM Grid -FM-74; D. Pavlova & A. Nedelcheva, July 2006 6 Ograzden Mts. – easterward from the Gega village, SO 104513 25 G Black rocks locality, 600 m, a.s.l., UTM Grid - fl-69; D. Pavlova & A. Nedelcheva, July 2007 Non-serpentine populations 7 Rila Mts. – meadows after Rila Monastery, left site of the SO 104606 25 R road to Kirilova poljana, 1246 m a.s.l., N42°08.67', E23°21.7'; UTM Grid - FM-96; D. Pavlova & A. Nedelcheva, July 2007 8 Sofia region – Beledie han locality on calcareous terrain SO 104954 25 BH1 northern from Beledie Han village, 700 m a.s.l., UTM Grid - FN-74; N. Tzoneva & A. Nedelcheva, April 2007 9 Sofia region – Beledie han locality on calcareous terrain SO 104955 25 BH2 northern from Beledie Han village, 720 m a.s.l., UTM Grid - FN-75; N. Tzoneva & A. Nedelcheva, April 2007 42 Northeastern Naturalist Vol. 16, Special Issue 5 Statistical analysis Univariate and multivariate statistical procedures were applied to study morphological variation among the populations. Cluster analysis using Euclidean distances and unweighted pair group average (UPGMA) as computational criteria were used to examine the similarities between the populations (Fig. 2). Canonical discriminant analysis (CDA) was applied to discriminate the morphological variation and to differentiate serpentine and non-serpentine populations (P < 0.001). Principal component analysis (PCA) was used to show the loadings for each character and which character contributed most for differences between groups. Eigen values were extracted from the correlation matrix. Basic descriptive statistics, Tukey-test for independence of groups, one-way ANOVA, and cluster analyses were also used to determine the differences between the serpentine and non-serpentine populations. All statistics were performed using StatSoft - Statistica 7 program. Table 2. Characters investigated on the plant samples of Teucrium chamaedrys on the individuals from serpentine (S) and non-serpentine (NS) soils, and their number (Var.). Values shown are: mean and standard deviation (s.d.) for each soil type, P-level results of t-tests, and the standardized coefficients (Root 1 and Root 2) of the variables which contribute most to the group separation. Variable S NS P Character No mean s.d. mean s.d. levels Root 1 Root 2 Stem height [mm] Var. 1 76.93 37.24 119.69 47.72 0.00 -0.13 -0.39 Stem length from the bottom to Var. 2 23.45 16.45 37.59 21.54 0.00 0.15 -0.22 the first leaves pair [mm] Leaf length [mm] Var. 3 17.51 9.05 18.56 3.15 0.33 -1.26 0.09 Leaf width [mm] Var. 4 7.79 2.40 9.68 1.90 0.00 0.26 0.06 Ratio Var. 3:Var 4 Var. 5 2.33 1.24 1.96 0.35 0.01 1.19 0.10 Leaf teeth number Var. 6 5.04 0.96 4.71 0.84 0.01 0.00 0.16 Middle leaf teeth length [mm] Var. 7 2.19 0.82 2.51 0.72 0.01 0.16 -0.04 Upper leaf teeth length [mm] Var. 8 1.28 0.45 1.32 0.47 0.54 0.10 -0.29 Petiole length [mm] Var. 9 3.40 1.24 4.89 1.34 0.00 -0.21 0.02 Internode length between 2nd Var. 10 9.96 3.82 16.00 8.67 0.00 0.21 -0.51 and 3rd leaf pair [mm] Inflorescence length [mm] Var. 11 24.11 17.33 53.64 15.23 0.00 -0.09 -1.17 Inflorescence width [mm] Var. 12 13.31 9.00 14.48 2.33 0.52 -0.02 0.24 Ratio Var. 11:Var. 12 Var. 13 1.88 1.68 3.77 1.36 0.00 0.07 1.14 Number verticelasters in the Var. 14 4.11 1.71 7.60 1.26 0.00 0.37 0.14 inflorescence Peduncle length [mm] Var. 15 2.01 1.80 2.06 0.46 0.90 0.07 0.00 Calyx length [mm] Var. 16 5.03 1.21 6.06 0.46 0.00 0.32 -0.08 Calyx tooth length [mm] Var. 17 1.79 0.61 2.02 0.23 0.06 0.12 0.07 Corolla tube length [mm] Var. 18 4.84 1.54 5.26 0.66 0.18 -0.12 -0.12 Corolla lower lip length [mm] Var. 19 11.78 3.79 12.04 2.27 0.74 0.04 0.06 Corolla lower lip width [mm] Var. 20 4.87 1.74 4.94 0.78 0.85 0.25 0.34 Tooth of the lower lip [mm] Var. 21 2.78 1.71 2.86 0.44 0.82 0.24 0.14 Bract length [mm] Var. 22 9.99 3.08 14.04 2.32 0.00 0.27 -0.08 Bract width [mm] Var. 23 3.75 1.54 6.00 1.29 0.00 -0.12 -0.05 2009 D. Pavlova 43 Soil and plant analyses Seven composite soil samples were analyzed. Three soil cores per site at a depth of 0–15 cm were obtained near the plants collected for analysis and were composited for soil analyses. All plant samples were cleaned, dried, and ground to a fine powder. Vegetative and floral plant tissues used in pharmacy as Herba Teucrii chamaedri were analyzed for metal content. Element concentrations were determined following the standard No. 11466/1995 of extraction of trace elements soluble in aqua regia proposed by International Organization for Standards (ISO). The total concentrations of Cd, Co, Cr, Fe, Mn, and Ni in aqua regia extracts of soil were determined with flame and atomic absorption spectrophotometers. All concentrations were within the detection limits of the analyses used. Soil Ca/Mg ratio and soil pH (in H2O) were also measured. The analyses were conducted at the Institute of Soil Science “N. Poushkarov” in Sofia, Bulgaria. Results The statistical analyses found significant differences between plant populations growing on serpentine and those on non-serpentine soils. The range of variability of all morphological features in both groups of populations overlapped considerably. Most notable is the substantial variability in the vegetative set of characters compared to the generative parts of plants where Figure 2. Cluster diagram showing the groups of similarity among the populations investigated. 44 Northeastern Naturalist Vol. 16, Special Issue 5 variability was hardly detected. The groups of similarities, following the cluster analysis, are synchronous with groups distinguished by the discriminant and PCA analyses. The mean values and the standard deviation of the investigated morphological characters for the serpentine and non-serpentine population as well as P-values from t-tests of the differences between the means are presented in Table 2. The results from Tukey-tests indicated only 9 of the analyzed characters (leaf length, upper leaf teeth length, inflorescence width, peduncle length, calyx tooth length, corolla tube length, corolla lower lip length, corolla lower lip width, and length of tooth of the lower lip) were not significantly different (P < 0.05) between serpentine and nonserpentine individuals. Basic descriptive statistics and one-way ANOVA analyses demonstrated that the range of variability for most morphological features in populations overlapped considerably. Most of the characters unsuitable for discrimination of the populations were related to flower characteristics. Conversely, the difference between serpentine and non-serpentine populations was highly significant for the stem height, stem length up to the first leaf pair, leaf width, internode length between the second and third leaf pairs, length of flowering stem, number of flowers in verticelasters, and length and width of bracts. Cluster analysis A cluster diagram of similarity among the nine populations based on the 23 characters is presented in Figure 2. The nine populations formed four clusters. The first group of similar serpentine populations included Parvenetz (P), Dobromirtzi 2 (D2), and Zeleznitza (Z), and among them the highest similarity observed was for P and D2. The second group included the last three serpentine populations. The non-serpentine populations from Beledie Han (BH1 and BH2) demonstrated the highest similarity to each other and formed the third cluster. The cluster diagram shows that these two non-serpentine populations are more closely related to some of the serpentine populations than to other non-serpentine populations studied from Rila (R). The population from Rila formed a separate, fourth cluster in the diagram. The separation of this population was due to the highest stem length, internode length between second and third leaf pair, inflorescence length, and number of verticelasters in the inflorescence, which showed the highest values from all studied populations. The vegetative characters contributed the most to grouping the populations, while the generative traits were more conservative. Discriminant analysis Canonical discriminant analysis (CDA) was applied to evaluate the morphological variation between serpentine and non-serpentine populations belonging to T. chamaedrys, where population was used as a classification factor. The results are presented on a scatter diagram (Fig. 3). All characters used in this analysis were significant (P < 0.00005) with the exception of corolla tube length, for which P = 0.43. Three characters (inflorescence 2009 D. Pavlova 45 length, ratio of inflorescence length/width, and lower corolla lip length) were not included due to their P-levels exceeding the limit. The results from the analysis were obtained at Wilks’ Lambda = 0.0004, and P < 0.00005. The analysis was based on two discriminant functions that account for the most natural grouping of the population. The standardized coefficients of the variables that contributed most to the group separation are given in Table 2. The serpentine populations formed a relatively compact group that is clearly distinguished from the non-serpentine populations. Though more dispersed, the data from population Dobromirtzi (D1) was within the group of the serpentines sites. The three non-serpentine populations are distributed on the scatter diagram in two groups. The first group comprises the values of the classification factors from the region of Sofia (BH1 and BH2), which form a compact group, clearly distinguished and isolated from the group of serpentine populations. The second group, although more dispersed, represents the Rila (R) non-serpentine population. The position of this population was closer to the compact group of the serpentine populations, but the discriminant function 2 effectively separates it. The application of the serpentine and non-serpentine ecotype as a classification factor suggests that stem height, stem length up to the first leaf pair, leaf width, and internode length between the second and third leaf pairs are the features which contributed most effectively to the discriminant function. Figure 3. Scatter diagram of CDA of nine populations of Teucrium chamaedrys based on 23 measured characters. The abbreviations used for the populations are given in Table 1. S = serpentine populations, NS = non-serpentine populations. 46 Northeastern Naturalist Vol. 16, Special Issue 5 Principal component analysis Percentage eigenvalues and factor loadings on the axes are reported in Table 3. Ordination of the data set accounts for 51.9% with the first three components. Plotting the first against the second axis (Fig. 4) produces a two-dimensional scattergram where the loadings for each variable and their contribution to each of the PCs are shown. In this case, the numbers of leaf teeth and ratio between leaf length and leaf width with their positive coordinates and correlations with factor 1 contribute to population divergence. Most of the vegetative traits have positive coordinates and correlations with factor 2. They had a clear negative correlation with the ratio between leaf length and leaf width. The vegetative characters were well separated from the generative ones. It is obvious that vegetative traits were more variable, while some of the generative ones like corolla tube length and corolla lip length and width were very closely correlated. An analysis using only floral characters shows that the calyx traits contributed much to each of the PCs and were more variable than the corolla traits. From the analysis of only the vegetative characters, the numbers of leaf teeth and the ratio between leaf length and leaf width were the most importance traits for separation of the populations and were negatively correlated with the inflorescence traits. Table 3. Percentage eigenvalues, variance explained by first three components, and factor loadings of the variables. Variable code 1 2 3 Eigenvalue (%) 28.84 14.52 8.54 Cumulative (%) 28.84 43.36 51.90 Variables Stem height StH -0.63 0.54 0.10 Stem length from the bottom to the 1st leaf pair StL -0.36 0.56 0.17 Leaf length Leafl-0.18 0.08 -0.91 Leaf width LeafS -0.46 0.49 -0.26 Ratio Var. 3:Var. 4 L/S 0.09 -0.23 -0.76 Leaf teeth number NTeeth 0.25 0.17 -0.11 Middle leaf teeth length MidLeafT -0.61 0.38 -0.23 Upper leaf teeth length UpLeafT -0.50 0.31 -0.30 Petiole length PetL -0.30 0.17 -0.23 Internode length between 2nd and 3rd leaf pair IntNode -0.71 0.43 0.04 Inflorescence length InflL -0.71 0.12 0.26 Inflorescence width InflS -0.19 -0.27 0.19 Ratio Var. 11:Var. 12 L1/S1 -0.58 0.12 0.24 Number verticelasters in the inflorescence fl-0.74 0.03 0.24 Peduncle length PedL -0.17 -0.28 0.03 Calyx length CaL -0.73 -0.32 0.01 Calyx tooth length CatL -0.38 -0.33 0.04 Corolla tube length Cot -0.62 -0.66 -0.03 Corolla lower lip length LipL -0.58 -0.67 -0.06 Corolla lower lip width LipS -0.54 -0.65 -0.07 Tooth of the lower lip TlipL -0.35 -0.48 -0.03 Bract length BrL -0.85 -0.03 -0.03 Bract width BrS -0.76 0.15 -0.05 2009 D. Pavlova 47 Soil and plant analyses The results of the analysis of metal concentrations in soil and plant samples of T. chamaedrys are given in Table 4. The total Ca concentration in serpentine soil samples varied from 1840 mg/kg in site Dobromirtzi 2 (D2) to 8758 mg/kg in site Gega (G). In the nonserpentine soil samples it ranged from 4569 mg/kg in site Rila (R) to 15,702 mg/kg in Beledie Han (BH1). Such high concentrations of Ca in Beledie Han (BH1) are in conformity with the calcareous bedrock of the area. The content of Mg was high in relation to Ca in all serpentine sites, and Ca/Mg ratios were <1. The serpentine sample from Gega (G) had the highest Mg level, with a value of 16,768 mg/kg. The soil samples from Rila and Beledie Han had concentrations of Ca and Mg which are typical for non-serpentine substrates and Ca/Mg ratio >1. While the amounts of Ca in the serpentine soils were low, the concentrations in T. chamaedrys leaves were much higher. The Ca/Mg ratio values in all plant samples were >1. Figure 4. Principal component analysis plot variables (vectors). Length of the vectors is proportional to the strength of the correlations between variables and one of the PCs (factors). Variable codes as in Table 3. 48 Northeastern Naturalist Vol. 16, Special Issue 5 The serpentine soils studied are relatively rich in the toxic metals Ni, Cr, and Co (Table 4). Nickel is known to be toxic in soils in quantities higher than 500 mg/kg (Allen et al. 1974). The concentrations of Ni in the soil samples were higher than 1000 mg/kg in all our sites except those in Gega, Rila, and Beledie Han. The concentrations of Ni in the plant samples from serpentines varied from 19.4 mg/kg to 28.4 mg/kg. The amounts of Cr in all the serpentine sites, except Gega, were more than 500 mg/kg and exceeded the upper limit for normal soils proposed by Allen et al. (1974) and Brooks (1987) and were similar to soil Cr levels reported by Karataglis et al. (1982) for northern Greece. Despite high total soil Cr, the amounts of Cr taken up by plants were small. The plant sample from Parvenetz (P) was the only sample where Cr (4.35 mg/kg) exceeded the limits proposed by Kabata-Pendias and Pendias (1984). The third element of this toxic group is Co. The amount of Co found in soil and plant samples was lower than the limits of normal concentrations proposed by Brooks (1987). Table 4. Chemical composition (mg/kg on dry weight) of soil and plant samples. Population abbreviations: F = Fotinovo, D1 = Dobromirtzi 1, D2 = Dobromirtzi 2, P = Parvenetz, G = Gega, R = Rila, BH 1 = Beledie Han. Populations Serpentine Non-serpentine F D1 D2 P G R BH 1 Ca Soil 2120 1960 1840 2840 8758 4569 15,702 Plant 3600 3100 3500 3100 3368 8000 9420 Mg Soil 12,633 5589 5832 2916 16,768 2187 5182 Plant 2700 1100 840 600 2065 1900 2123 Ca/Mg Soil 0.17 0.35 0.32 0.97 0.52 2.08 3.03 Plant 1.33 2.81 4.16 5.16 1.63 4.21 4.43 Fe Soil 47,200 72,800 71,000 42,800 25,774 20,900 36,291 Plant 168 363 474 538 503 380 489 Ni Soil 1008 1428 1232 1312 101 24 39 Plant 19.4 23.9 28.4 21.7 24.8 0.8 1.1 Cr Soil 672 542 548 591 36.5 21.9 52.2 Plant 1.30 1.38 2.92 4.35 1.40 0.14 0.21 Co Soil 58 120 116 4 31 2 14 Plant 0.28 0.92 0.95 1.48 0.30 0.20 0.40 Cd Soil 0.46 0.41 0.41 3.91 0.01 0.25 1.90 Plant 0.05 0.10 0.07 0.12 0.01 0.05 0.09 Mn Soil 775 1895 1865 1505 1051 900 1994 Plant 18.0 26.3 30.0 30.0 18.8 52.0 64.0 pH 7.7 6.9 6.8 6.3 6.0 5.4 6.9 2009 D. Pavlova 49 The concentrations of Cd in the soils in all localities except Parvenetz (P) and Beledie Han (BH1) were below the limits given by Allen et al. (1974). The elevated Cd levels found in P and BH1 were probably caused by pollution from nearby road traffic rather than from a property of the parent soil. Regardless of soil Cd levels, Cd in T. chamaedrys plants were in the normal range. The concentrations of Mn in soils varied between 775 and 1994 mg/kg, and in plant tissues from 18 to 64 mg/kg. The high levels of Mn in the nonserpentine (BH1) soil and also in the D1 and D2 soils is atypical and could be caused by heavy road traffic in this area, the relief, the oak forest vegetation, and/or the large percentage of organic material in the sample. The Mn values in plants were within the range of the limits given by Kabata-Pendias and Pendias (1984). The concentration of Fe in all serpentine soil samples in this study were found to be high, which is common in serpentine soil from the Balkan Peninsula (A. Bani, Agricultural University of Tirana, Albania, pers. comm.; Konstantinou and Babalonas 1996; Obratov-Petkovic et al. 2008; Pavlova 2001; Pavlova and Alexandrov 2003; Ritter-Studnicka and Dursun-Grom 1973; Vergano Gambi 1992). The concentrations of Fe in two of the plant samples are slightly higher than the limit of 500 ppm proposed by Allen et al. (1974). According to Ritter-Studnicka and Dursun-Grom (1973), the content of Fe in plants should exceed 1000 ppm if they are considered to be as nonphysiological, something shown by comparative measurements in serpentine and non-serpentine soils. The pH values for serpentine soils are often high and range from 6.1 to 8.8 (Brooks 1987, Kruckeberg 1984). The pH values in this study ranged from 6.0 to 7.7 for the serpentine soils. Discussion The results of this study are similar to those provided by Štepankova (1996, 1997), Westerbergh and Rune (1996), and Westerbergh and Saura (1992) for other plant species. The variation in morphological traits in this study are an additional confirmation of Berg’s (1960) hypothesis that the size of the flowers in insect-pollinated plants should be selected to remain constant regardless of the size of vegetative structures. Data from the PCA clearly show that the correlations among floral traits and the correlations among vegetative traits were significantly greater than the correlations across these two groups of traits in T. chamaedrys populations. As Conner and Sterling (1996) pointed out, such positive correlations within the floral and vegetative trait groups do not indicate functional relationships among traits. These correlations are due at least in part to overall size relationship and probably reflect common developmental pathways. Teucrium chamaedrys is widely distributed without preferences to any type of rock and should be considered as a bodenvag species, which are those widely distributed in serpentine and non-serpentine habitats (Kruckeberg 1992). The significant ecological differences between population groups 50 Northeastern Naturalist Vol. 16, Special Issue 5 were related to the differences in edaphic conditions of the serpentine sites from the non-serpentine sites. In this study, T. chamaedrys populations were dense and well developed in mesophilous habitats on calcareous and siliceous terrains compared to serpentine populations. The growth habit of T. chamaedrys differed between serpentine and non-serpentine sites. The serpentine populations had lower stems, smaller leaves, and shorter internodes compared to the non-serpentine populations. Obratov–Petković et al. (2006) also noted that T. chamaedrys growing on serpentines were less compact than those growing on limestone. Denser indumentum and longer simple or glandular hairs covering the stems, leaves, and bracts of the individuals growing on serpentines are also important characters. Most serpentine populations of T. chamaedrys studied in this work were densely covered by 3–5 celled glandular hairs. These hairs were not mentioned by Bini-Maleci and Servettaz (1991), Grubesic et al. (2007), or Navarro and El Qualidi (2000) for this species. Because hairs can play an important role in various aspects of plant physiology and ecology in Teucrium (Payne 1978), further investigations are needed. The chemical composition of the soil collected from serpentine substrates was similar to serpentine soils from other parts of the world (Brooks 1987), with concentrations of metals such as Ni and Cr uniformly high and Ca/Mg quotients relatively low. The serpentine soils had abnormally high contents of Ni, Cr, Cd, and Co. The ratio of Ca/Mg was <1 in the serpentine soils and >1 in non-serpentine soils. The levels of Ca and Mg found in these Bulgarian serpentine soils are typical for such soils from other areas (Brooks 1987; Kruckeberg 1984, 1992; Roberts and Proctor 1992). Similar to previous findings (Brooks 1987, Karataglis et al. 1982, Proctor 1971, Roberts and Proctor 1992, Walker 1954), while the amounts of Ca in the serpentine soils were small, the amounts of Ca taken up by the plant were higher. According to Proctor (1971), Ca is one of the elements contributing to the inhibition of the heavy-metal toxicity, and it is possible that the plant takes up Ca to compensate the toxic action of different toxic metals. Studying populations of Buxus sempervirens L. (Common Box) in Greece, Karataglis et al. (1982) suggested that the plant developed a mechanism to permit an excess soil Ca to be taken by plant. Normally, the high Mg of ultramafic soils is reflected in high Mg concentrations in plant tissues, but this result might also indicate an unusual Mg and low Ca requirement observed in some experiments (Proctor and Woodell 1975). The total Ni content in the soil was comparable to Italian (Vergnan Gambi et al. 1982), Greek (Babalonas et al. 1984), and Albanian (Shallari et al. 1998) serpentine areas. The accumulation of Ni, Cr, and Co in the above-ground plant parts for all serpentine populations was higher than in non-serpentine ones. These data are in accordance with results presented for T. montanum L. from Bulgaria (Pavlova and Alexandrov 2003), Serbia (Obratov-Petković et al. 2008), and Albania (Shellari et al. 1998). The concentrations of Ni found in plant samples from serpentines were high but not 2009 D. Pavlova 51 exceptional for T. chamaedrys, similar to findings of Shellari et al. (1998) and Obratov-Petković et al. (2008) for different Teucrium species. Ni amounts in T. chamaedrys were very low compared to the data for other plants growing on serpentines found by Karataglis et al. (1982), Vergnano Gambi et al. (1982), Konstantinou and Babalonas (1996), and Bani et al. (2007), but higher than levels normally considered to be toxic to most plants (Kabata-Pendias and Pendias 1984). Ni was taken up in higher quantities than Cr, and the Ni/Cr ratio in plant tissues was always much higher than in the soil. The low Cr concentrations in all studied plants corroborate the data of Bani et al. (2007) and Brooks (1987) that serpentine plants contain only trace quantities of Cr. At the same time, Cr concentrations were higher than Co in all plant samples suggesting that plants adapted to serpentine soil did not accumulate cobalt (Wallace et al. 1982). The ability of Buxus sempervirens to accumulate large quantity of calcium from the soil (Karataglis et al. 1982) may constitute one of the probable physiological mechanisms developed in order to compensate for the toxic action of heavy metals like Cr, Ni, Co, and Fe. Karataglis et al. (1982) consider toxic effects as almost imperceptible in regards to the low levels of Co found in the soil and the amounts taken up by the plants. Brooks and Yang (1984) found the concentration of Mg in plant tissue to be inversely proportional to the concentrations of other nutrients: Al, Fe, Co, Mn, P, and Na. These data clearly suggest that the uptake of Mg comes at a cost to the plant, as the uptake of other elemental nutrients is forfeited. According to Brooks and Yang (1984), the heightened level of Mg in serpentine soils and its antagonistic behavior toward other elements could be the most important factor in the serpentine syndrome. In contrast to other studies of serpentine plants (Bani et al., in press; Konstantinou and Babalonas 1996; Pavlova 2001; Pavlova and Alexandrov 2003; Ritter-Studnicka and Dursun-Grom 1973; Vergano Gambi 1992), Fe levels were not higher in plants. Babalonas et al. (1984) suggested that this characteristic should be given more attention because it may be applicable to all the serpentine flora of the Balkans. The soil from Gega (G), considered to be a serpentine site (Zidarov and Nenova 1995), had lower concentrations of Ni (101 mg/kg) and Cr (36 mg/kg) than the other serpentine soils in this study. Although total soil metal concentrations from G were intermediate between the serpentine and non-serpentine study sites, the concentrations of the plant tissues from G were similar to plant tissue concentrations at the other serpentine sites. This result could be related to the specificity of the G site from a geological point of view. The studied area is crossed by pegmatite-aplitic veins and as a result, metasomatic rocks similar to gabbros appear that follow the stage of syndeformation metamorphic recrystallisation and mark the beginning of the ultrabasic rock assimilation (Kozhoukharova 1999). Teucrium chamaedrys is a well-known herb with medicinal action and uses as a stimulant, tonic, diaphoretic, and diuretic. Teucrium chamaedrys acts as a 52 Northeastern Naturalist Vol. 16, Special Issue 5 slight aperient, as well as a tonic. For pharmaceutical purposes, above-ground plant parts (Herba Teucrii chamaedrys) are collected from natural and cultivated populations (Nikolov 2006). According to ECCE (1994), the proposed limits for average metal contents in plants are 0.4–4.0 mg/kg and 0.2–1.0 mg/kg for Ni and Cr, respectively. The data from the studied serpentine populations were considerably higher than these limits. Although T. chamaedrys is not a hyperaccumutator of heavy metals, its collection from serpentines for medicinal purposes should be avoided as proposed also by Obratov-Petković et al. (2008) and European Pharmacopoeia (2005). The species T. chamaedrys is tolerant of serpentine soils, and the established morphological variation of the vegetative parts of the plant can be a response to the unusual conditions provided by the serpentine substrate. Similar observations and ecotype differentiation for other species were presented by Karataglis et al. (1982), Proctor (1971), Rajakaruna and Bohm (1999), Štepankova (1996, 1997), etc., and in all cases, the plants reacted in different ways to the serpentine substrates. As Kruckeberg (1984) pointed out, the differences in morphology of serpentine races can be taken as an indication of genotypic response to physical stress. The serpentine population develops its own genetic pattern of adaptation to the serpentine environment. According to Kruckeberg and Rabinowitz (1985), various genes favorable for survival on the serpentine sites are accumulated through selection over time, while other genes of the originally non-serpentine population are eliminated by the selective action of the serpentine habitats. Morphological differences, the preliminary results on the karyology of T. chamaedrys in Bulgaria, a new chromosome number reported for the population of the species from serpentines in Dobromirtzi (D1) (Pavlova 2008), and the geographical isolation between studied populations give us reason to continue experimental work to obtain additional data to prove the existence of two ecotypes. Acknowledgments The author is thankful to Evgenia Stoimenova from the Institute of Mathematics, Bulgarian Academy of Sciences in Sofia for her help in the discriminant analysis. Bob Boyd from Auburn University, Auburn, AL, USA helped with useful comments and suggestions on an earlier version of the manuscript. 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