Morphological, genetic and phytochemical variation of the endemic Teucrium arduini L. (Lamiaceae)

Phytochemistry xxx (2015) xxx–xxx

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Morphological, genetic and phytochemical variation of the endemic Teucrium arduini L. (Lamiaceae) Dario Kremer a, Snjezˇana Bolaric´ b, Dalibor Ballian c, Faruk Bogunic´ c, Danijela Steševic´ d, Ksenija Karlovic´ b, Ivan Kosalec a, Aleš Vokurka b, Jadranka Vukovic´ Rodríguez a, Marko Randic´ e, Nada Bezic´ f, Valerija Dunkic´ f,⇑ a

Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovacˇic´a 1, HR-10000 Zagreb, Croatia Faculty of Agriculture, University of Zagreb, Svetošimunska 25, HR-10000 Zagreb, Croatia Faculty of Forestry, University of Sarajevo, Zagrebacˇka 20, BIH-71000, Bosnia and Herzegovina d Faculty of Natural Sciences and Mathematics, University of Montenegro, Dzˇordzˇa Vašingtona bb, 81 000 Podgorica, Montenegro e Public Institution Priroda, Grivica 4, HR-51000 Rijeka, Croatia f Faculty of Science, University of Split, Teslina 12, HR-21000 Split, Croatia b c

a r t i c l e

i n f o

Article history: Received 9 October 2014 Received in revised form 8 April 2015 Available online xxxx Keywords: AFLP Essential oil Morphology Phytochemistry Teucrium arduini

a b s t r a c t Analysis of the morphological traits of leaves, genetic variability (analyzed by AFLP) and chemical composition of essential oils (analyzed by GC–MS) was conducted on eleven populations of the endemic Illyric-Balcanic species Teucrium arduini L. in Croatia, Bosnia and Herzegovina, and Montenegro. Average blade length and width ranged from 20.00 to 31.47 mm and from 11.58 to 15.66 mm, respectively. Multivariate analysis (PCA, UPGMA) of morphological traits distinguished two continental Bosnian populations from the remaining populations. AFLP analysis separated the investigated populations into two groups based primarily on geographical distance. Essential oil analysis showed a total of 52 compounds, with two chemotypes distinguished based on the essential oil profile. The first was a sesquiterpene chemotype, with b-caryophyllene, germacrene D or caryophyllene oxide as the major compounds, while the second was an oxygenated monoterpene chemotype, with pulegone and piperitone oxide as the main components. The Mantel test showed a stronger correlation between the morphological traits and AFLP than between the essential oil profile and AFLP. The test also showed a stronger association between the essential oil profile and geographical position than between the morphological traits and geographical position. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction In Europe, 49 Teucrium L. species have been described, with the highest diversity observed in the southern European peninsulas (Tutin and Wood, 1972). In the northwestern Balkans, eleven species have been recorded in Croatia (Domac, 1994), five in Bosnia and Herzegovina (Bjelcˇic´ and Milanovic´, 1968; Šilic´ and Abadzˇic´, 1989; Šoljan et al., 2009), and eight in Montenegro (Rohlena, 1942; Pulevic´, 2005). Arduini’s germander (Teucrium arduini L.) represents an endemic Illyric-Balcanic floristic element distributed ⇑ Corresponding author. Tel.: +385 21385133; fax: +385 21384086. E-mail addresses: [email protected] (D. Kremer), [email protected] (S. Bolaric´), [email protected] (D. Ballian), [email protected] (F. Bogunic´), [email protected] (D. Steševic´), [email protected] (K. Karlovic´), [email protected] (I. Kosalec), [email protected] (A. Vokurka), [email protected] (J. Vukovic´ Rodríguez), [email protected] (M. Randic´), [email protected] (N. Bezic´), [email protected] (V. Dunkic´).

mainly in the mountains along the Adriatic coast of Croatia, Bosnia and Herzegovina, Montenegro, Kosovo, Serbia and northern Albania (Tutin and Wood, 1972; Lakušic´ et al., 2007). It is a semiwoody, branchy, erect or ascending dwarf shrub, 10–60 cm high, with whitish flowers that form simple, very dense inflorescences up to 16 cm in length. The species grows on calcareous rocks, rocky screes and crevices in an altitude range from 0 to 1600 m (Tutin and Wood, 1972; Lakušic´ et al., 2007). These habitats primarily belong to plant associations within the Dinaric endemic vegetation order Moltkietalia petraeae Lakušic´ 1968. Chemical investigations of T. arduini revealed the presence of volatile oils (Dunkic´ et al., 2011), tannins (Jurišic´ Grubešic´ et al., 2012; Kremer et al., 2012b), flavonoids (Harborne et al., 1986; Valant-Vetschera et al., 2003), phenolic acids (Šamec et al., 2010), phytosterols and bitter principles (Jurišic´ Grubešic´ et al., 2012), and macroelements and micronutrients (Kremer et al., 2012a).

http://dx.doi.org/10.1016/j.phytochem.2015.04.003 0031-9422/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Kremer, D., et al. Morphological, genetic and phytochemical variation of the endemic Teucrium arduini L. (Lamiaceae). Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.04.003

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D. Kremer et al. / Phytochemistry xxx (2015) xxx–xxx

T. arduini is used in folk medicine in Bosnia and Herzegovina in the form of an infusion for stomach ailments (Redzˇic´, 2007). Antimicrobial investigations of the species have shown a broad spectrum of antimicrobial activity (Šamec et al., 2010; Vukovic´ et al., 2010; Kremer et al., 2012a). T. arduini is also a source of polyphenols and other antioxidants with radical-scavenging and chelating properties (Šamec et al., 2010; Kremer et al., 2013). There are no geographical studies of the genetic variation and variation in the chemical composition of the essential oil of T. arduini. The present study analysed populations from the northwest and central parts of the species distribution range. Focus was placed on morphological and genetic variation, and the variation of the essential oil composition, aimed at determining the specific geographical pattern of variation. Additionally, correlations between the investigated traits were analyzed using the Mantel test. Knowledge regarding essential oil variability and genetic relationships of populations is of great importance, both for the future research of medicinal properties and conservation activities of this endemic species. The present study represents a contribution to the knowledge of the poorly investigated Balkan Peninsula flora. Together with the Apennine and Iberian Peninsulas, the Balkan Peninsula was a major glacial refugium for temperate plant and animal species in Europe, providing a source for postglacial northward range expansion (Taberlet et al., 1998; Hewitt, 1999). The western Balkan Peninsula is a poorly investigated area with many endemic plant species. One such endemic plant with significant medicinal and horticultural potential is T. arduini. 2. Results and discussion 2.1. Morphological traits The morphological analysis of the leaf traits and one shoot trait in eleven T. arduini populations was performed. The results of the descriptive statistics are given in Table 1 (for abbreviations see Table 6). The Vo population had the shortest blade (length 20.00 mm), while the DG population had the narrowest (width 11.58 mm). The Or population had the longest blade (31.47 mm), while the Tr population had the widest (15.66 mm). According to Lakušic´ et al. (2007), the leaves of T. arduini from Montenegro and Kosovo were 19–45 mm long and 9–24 mm wide. It is evident that plants belonging to these analyzed populations have somewhat shorter leaves, which could be attributed to ecological conditions and/or genetic differences. The roundest leaves (length/width ratio 1.61) were seen in the Vo population, and the narrowest (length/width ratio 2.73) in the DG population. Teeth were shortest in the DG population (0.75 mm), and longest in the Tr population (1.63 mm).

The PCA of the analyzed morphological traits separated the T. arduini populations as shown in Fig. 1a. The first principal component (PC 1) explained 46.2% of the total variance, the second 28.3%, and the third component 10.6%. Thus the first three components accounted for 85.1% of the variance, highlighting the usefulness of the PCA. PC 1 was correlated with blade width, tooth length and tooth base width (Table 2). The second PC axis was correlated with blade length and petiole length, while PC 3 correlated with the number of leaves and internode length. The dendrogram based on leaf and shoot traits separated the T. arduini populations as shown in Fig. 1b. Cluster analysis gave similar results to the PCA, and three groups were distinguished. The four southern populations (Sn, Tr, Lo, Or) and the northernmost population (Uc) formed the first group. The second group was formed by the four populations (Su, SJ, VV, Vo) from the central area of the distribution, while the third group was formed by the two continental populations from Bosnia and Herzegovina (DG, Id). Kremer et al. (2012b) also separated the continental Bosnian populations of T. arduini from the Croatian coastal mountain populations based on calyx traits. Those results are similar to the results presented here, i.e. the two continental Bosnian populations of T. arduini (DG, Id) were separated from the Croatian coastal mountain populations (Uc, Su, SJ, VV, Sn, Vo).

2.2. DNA analysis To get insight into genetic variation among T. arduini populations the AFLP analysis was carried out. The dendrogram based on the AFLP analysis separated the T. arduini populations as shown in Fig. 2. Two groups of populations could be distinguished based primarily on geographic distance; the first was formed of the populations DG, Id, SJ, Vo, and Uc, and the second of the populations Or, Tr, Lo, Sn, Su, and VV. In the first group, the two continental and geographical closely related Bosnian populations (DG, Id) were clustered with the two Croatian populations from Mt Biokovo (SJ, Vo) and the northernmost population from Mt Ucˇka (Uc, Croatia). The second large group was composed of the four southern populations: three from Montenegro (Or, Tr, Lo) and the southernmost analyzed Croatian population (Sn). These southern populations were clustered with two populations from Mt Velebit, Croatia (Su, VV). The results obtained from the AFLP analysis are similar to the results of the morphological analysis. The greatest difference is in the position of the northernmost population (Uc, Croatia), which was clustered with the southernmost populations from Croatia (Sn) and Montenegro (Or, Lo, Tr) (Figs. 1 and 2). Both Bosnian populations (DG, Id) are situated in the Neretva River valley. The specific position of the Neretva Valley was also

Table 1 Descriptive statistics for the analysed morphological traits of Teucrium arduini. Minimum and maximum values are in bold. For abbreviations see Table 6. Locality

Uc Su VV Vo Sj Sn DG Id Or Lo Tr 1,2

Internodes length (mm)

Leaves number

Lamina

Teeth

Length (mm)

Width (mm)

Length/ width

On left side

28.97 ± 13.08 22.47 ± 9.24 22.15 ± 7.11 18.61 ± 6.31 25.19 ± 8.54 25.30 ± 12.00 24.32 ± 5.02 24.86 ± 7.34 27.36 ± 9.51 19.26 ± 6.70 24.92 ± 7.34

12.00 ± 1.63 16.40 ± 3.10 17.40 ± 1.35 15.60 ± 1.84 14.80 ± 2.35 16.00 ± 1.63 15.33 ± 1.63 14.40 ± 0.89 20.80 ± 2.35 14.20 ± 1.75 20.00 ± 2.24

25.45 ± 5.01 25.40 ± 6.18 22.66 ± 4.31 20.00 ± 4.03 25.60 ± 3.75 27.22 ± 6.70 23.12 ± 4.78 29.04 ± 5.19 31.47 ± 5.08 27.91 ± 5.26 28.48 ± 4.10

13.93 ± 3.17 14.11 ± 3.70 14.32 ± 3.63 11.58 ± 3.28 14.89 ± 2.97 16.06 ± 4.86 8.74 ± 2.61 12.82 ± 3.53 14.84 ± 2.45 14.87 ± 2.71 15.66 ± 2.80

1.88 ± 0.37 1.83 ± 0.29 1.61 ± 0.21 1.79 ± 0.30 1.76 ± 0.30 1.74 ± 0.21 2.73 ± 0.36 2.37 ± 0.46 2.14 ± 0.32 1.89 ± 0.26 1.86 ± 0.23

9.22 ± 1.69 12.32 ± 2.21 11.46 ± 1.87 11.28 ± 2.82 11.78 ± 2.77 11.23 ± 2.33 13.83 ± 2.45 15.35 ± 2.80 13.48 ± 2.96 10.14 ± 1.83 11.13 ± 2.21

1

On right side2

Length (mm)

Width (mm)

9.24 ± 1.58 12.13 ± 1.96 11.33 ± 1.47 11.02 ± 2.62 11.48 ± 2.54 11.13 ± 2.27 13.73 ± 2.35 15.22 ± 2.80 13.54 ± 2.96 11.27 ± 2.21 10.16 ± 1.91

1.43 ± 0.39 1.19 ± 0.35 1.22 ± 0.36 1.29 ± 0.40 1.40 ± 0.38 1.59 ± 0.51 0.75 ± 0.23 0.95 ± 0.31 1.60 ± 0.34 1.27 ± 0.33 1.63 ± 0.54

2.42 ± 0.83 2.25 ± 0.64 2.20 ± 0.59 1.66 ± 0.45 2.24 ± 0.72 2.93 ± 1.03 1.51 ± 0.43 1.56 ± 0.62 2.17 ± 0.53 3.13 ± 0.79 2.88 ± 0.80

Petiole length (mm)

3.10 ± 1.55 3.06 ± 1.22 2.27 ± 0.69 1.87 ± 1.22 3.18 ± 1.68 3.45 ± 1.64 2.51 ± 1.15 3.26 ± 1.32 4.82 ± 1.24 4.78 ± 1.37 4.72 ± 1.31

Number of teeth on the left/right side of lamina.

Please cite this article in press as: Kremer, D., et al. Morphological, genetic and phytochemical variation of the endemic Teucrium arduini L. (Lamiaceae). Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.04.003

D. Kremer et al. / Phytochemistry xxx (2015) xxx–xxx

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split, and that gene flow has occurred across this deep but narrow valley (Surina et al., 2011). A similar relationship between genetically differentiated groups and their geographic origin is known from investigations of other species, including endemic Balkan plants. Based on AFLP analysis, Perny´ et al. (2004) obtained three genetically differentiated groups of Cardamine acris Griseb. (family Brassicaceae) populations that corresponded to their geographic origin on the Balkan Peninsula. According to Surina et al. (2011), E. serpyllifolius showed genetic differentiation into two to three geographically distinct groups, coinciding with isolated mountain ranges. A similar pattern has been reported for certain animal species in the western Balkan mountains (Kryštufek et al., 2007; Sotiropoulos et al., 2007). 2.3. Essential oils The essential oils from eleven populations of T. arduini were subjected to GC and GC/MS analyses in order to determine possible similarities and variability in the chemical composition depending on locality. The specifically identified compounds and their percentages are shown in Table 3. The total yield, based on the dry mass of samples, ranged from 0.3% to 0.4%. In total, 52 compounds were identified in all eleven investigated oils (83.1–97.5% of the total oil) and classified on the basis of their chemical structures into eight classes (Table 3). Sesquiterpene hydrocarbons (39.5–65.6%) were the main class of constituents of all T. arduini populations, except in the Idbar (Id) population (Bosnia and Herzegovina), where oxygenated monoterpenes (49.5%) were the major class, with piperitone oxide (39.1%) as the major compound. Such a high concentration of piperitone oxide has not been recorded in the previously studied species of the genus Teucrium. Also, oxygenated monoterpenes – piperitone oxide (10.3%) and pulegone (26.3%) – are important constituents of the investigated T. arduini essential oil from the Ucˇka (Uc) population (Croatia). Pulegone was also the main constituent of Teucrium scordium L. from Iran (Sharififar et al., 2010). The common presence of compounds piperitone oxide and

Fig. 1. PCA (a) and dendrogram (b) of investigated T. arduini populations for the analyzed morphological traits. For abbreviations see Table 6.

Table 2 Eigen vectors of the first three principal components obtained for the analysed morphological traits of the investigated T. arduini populations. Bold values indicate the highest contribution to each PC axis. Variable

PC 1

PC 2

PC 3

Number of leaves Internode length Blade length Blade width Blade length/width ratio Petiole length Number of teeth – left Number of teeth – right Teeth length Teeth base width

0.115889 0.073943 0.182842 0.423606 0.345008 0.276168 0.328536 0.330050 0.422400 0.421473

0.311393 0.294553 0.522293 0.113629 0.293971 0.407646 0.371680 0.367429 0.069389 0.003357

0.638215 0.656794 0.142005 0.072418 0.249718 0.014554 0.201974 0.165864 0.059431 0.038552

noted by Surina et al. (2011) during an investigation of Edraianthus serpyllifolius (Vis.) A. DC. and Edraianthus tenuifolius A. DC. (family Campanulaceae) populations. They concluded that the lower Neretva Valley does not coincide with any major phylogeographic

Fig. 2. Dendrogram of investigated T. arduini populations obtained from the AFLP analysis. For abbreviations see Table 6.

Please cite this article in press as: Kremer, D., et al. Morphological, genetic and phytochemical variation of the endemic Teucrium arduini L. (Lamiaceae). Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.04.003

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D. Kremer et al. / Phytochemistry xxx (2015) xxx–xxx

Table 3 Phytochemical composition (%), identification (%) and major groups of chemical components (%) of the essential oil of Teucrium arduini. Bold values indicate the main class content. For abbreviations see Table 6. Component

RIa

RIb

(Uc)

(Su)

(VV)

(Vo)

(SJ)

(Sn)

(DG)

(Id)

(Or)

(Lo)

(Tr)

IM

0.5 – – – – – 0.2 0.3

1.1 0.8 – 0.3 – – – –

3.2 – – – 0.1 0.9 2.2 –

3.1 – – – – 1.2 1.9 –

3.0 1.1 0.2 0.3 – 0.2 1.1 0.1

1.1 – – – – 0.9 0.2 –

3.9 – – – – 3.9 – –

2.9 – – – 0.7 1.2 0.8 0.2

2.0 – – – – 0.9 1.1 –

2.7 – – – – 1.4 1.3 –

RI, RI, RI, RI, RI, RI, RI,

MS, S MS MS, S MS MS, S MS MS

Monoterpene hydrocarbons a-Pinene Camphene b-Pinene Myrcene Limonene (Z)-b-Ocimene Terpinolene

938 962 982 992 1032 1052 1089

– – <1200 <1200 1204 1218 1286

1.1 – – – – 0.2 – 0.9

Oxygenated monoterpenes Linalool b-Thujone Camphor Borneol Terpinen-4-ol Myrtenol b-Cyclocitral Pulegone Piperitone Linalyl acetate Bornyl acetate a-Terpenyl acetate Piperitone oxide

1099 1121 1152 1176 1184 1197 1223 1234 1248 1252 1285 1349 1366

1548 1438 1499 1719 1611 1782 1629 1631 – 1553 1570 – –

37.4 0.2 – – – – – – 26.3 0.2 0.4 – – 10.3

13.2 5.6 0.2 – – – 0.3 0.2 3.5 0.9 0.1 – 0.1 2.3

13.7 5.6 1.5 0.1 – 0.4 – – 3.1 0.5 0.3 0.2 0.3 1.7

11.9 1.9 0.3 0.2 0.2 0.3 – – 4.1 2.7 – – – 2.2

11.0 3.4 0.2 – – – – – 2.5 1.9 0.1 0.1 tr 2.8

10.0 5.3 0.2 0.2 – 1.0 0.1 – 0.2 1.2 0.3 – – 1.5

9.1 1.7 0.2 0.1 0.2 0.1 – – 2.4 1.1 0.2 0.3 0.2 2.6

49.5 6.6 – – 0.3 0.1 – 0.1 2.7 0.3 0.3 – – 39.1

18 2.6 0.7 – – 1.2 – – 4.9 1.7 1.4 0.9 – 4.6

15.2 4.6 – – – – – – 1.2 0.3 – – 0.3 8.8

11.8 4.5 – – – – – – 2.8 1.2 – – – 3.3

RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI,

MS, MS, MS, MS, MS MS MS MS MS MS, MS, MS MS

b-Bourbonene b-Caryophyllene b-Copaene trans-a-Bergamotene (Z)-b-Farnesene a-Humulene allo-Aromadendrene Germacrene D b-Bisabolene d-Cadinene

1377 1383 1424 1429 1433 1454 1456 1465 1481 1494 1517

1484 1508 1585 – – 1639 1654 1662 1692 1729 1745

39.5 0.6 tr 22.1 0.2 – 0.8 0.9 1.1 11.9 1.5 0.4

49.4 0.3 – 29.1 0.5 0.1 0.2 – – 18.7 0.2 0.3

50.3 0.2 0.1 35.4 1.1 – 0.2 – 0.3 9.6 2.3 1.1

51.3 1.1 0.3 33.7 1.3 – – – 0.8 12.2 1.1 0.8

46.3 0.6 0.2 28.8 1.1 – – 2.7 – 12.5 0.4 0.2

48.6 – – 31.1 2.4 0.1 – 3.2 0.8 8.9 1.2 0.9

65.6 0.2 0.6 30.1 0.5 0.2 – 3.5 0.9 28.9 0.3 0.4

26.3 – – 8.2 – – – 2.2 0.3 15.2 0.4 –

54.9 – 0.3 29.2 1.2 0.2 0.6 4.2 0.6 17.3 1.3 –

44.8 – – 24.7 0.8 – – 1.5 0.1 16.6 1.1 –

51.5 – – 30.7 – – – 2.2 0.2 18.1 0.3 –

RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI,

MS MS MS, S MS MS MS MS MS MS MS MS

Oxygenated sesquiterpenes Spathulenol Caryophyllene oxide a-Cadinol a-Bisabolol Germacrone

1577 1581 1655 1685 1696

2101 1955 2208 – –

7.4 0.3 5.5 0.4 – 1.2

13.0 0.7 10.2 0.7 0.5 0.9

21.0 2.2 17.1 – 0.6 1.1

18.4 1.7 14.6 0.2 0.8 1.1

19.6 2.4 14.2 0.6 – 2.4

26.8 4.5 16.8 0.5 0.4 4.6

12.5 3.3 6.1 – 0.5 2.6

8.7 4.1 4.4 – – 0.2

15.1 2.3 8.9 – – 3.9

15.6 2.7 10.8 – – 2.1

12.1 1.4 7.2 0.3 – 3.2

RI, RI, RI, RI, RI,

MS, S MS, S MS MS MS

Phenolic compounds p-Vinyl-guaiacol Eugenol

1312 1370

– 2020

0.5 – 0.5

1.7 0.2 1.5

0.6 – 0.6

0.9 – 0.9

1.6 0.3 1.3

0.1 – 0.1

0.9 – 0.9

0.2 – 0.2

0.2 – 0.2

1.1 0.2 0.9

0.9 0.7 0.2

RI, MS RI, MS, S

Carbonylic compounds 3-Octanol acetate Hexyl isovalerate Isoamyl hexanoate

1125 1245 1256

1376 1409 1457

2 – 1.9 0.1

2.8 0.6 0.5 1.7

1.0 0.3 0.7 –

0.5 0.3 0.2 –

1.8 1.1 – 0.7

0.6 0.3 0.1 0.2

0.5 0.5 – –

3.1 3.1 – –

1.4 0.9 0.2 0.3

0.5 0.5 – –

1.6 1.4 0.2 tr

RI, MS RI, MS RI, MS

Hydrocarbons Eicosane Heneicosane Docosane Tricosane Pentacosane Heptacosane Octacosane Nonacosane

2000 2100 2200 2300 2500 2700 2800 2900

2000 2100 2200 2300 2500 2700 2800 2900

3.5 – 0.1 0.8 – 0.3 1.2 0.8 0.3

5.6 1.1 0.7 0.3 0.8 0.9 0.4 0.8 0.6

2.9 0.2 0.4 0.3 0.2 – 1.2 0.3 0.3

2.2 0.9 0.1 0.2 – – 0.3 0.5 0.2

3.5 0.8 0.6 0.3 0.5 – 1.3 – –

2.8 0.5 0.3 0.3 – – 1.7 – –

3.6 0.4 0.4 0.9 – 0.2 1.2 0.3 0.2

2.5 0.2 0.5 0.3 – – 0.9 0.2 0.4

2.9 0.3 0.2 1.2 – – 1.1 – 0.1

2.4 0.1 0.8 0.2 – – 0.4 – 0.9

1.9 0.2 0.2 0.3 – – 0.7 – 0.5

RI, RI, RI, RI, RI, RI, RI, RI,

1959 2054 2171

2476 – –

2.5 2.4 – 0.1 93.9 0.3

2.2 0.7 1.5 – 88.4 0.4

2.7 2.7 – – 93.3 0.4

1.3 1.3 – – 89.7 0.4

2.4 2.4 – – 89.5 0.4

1.6 1.6 – – 93.5 0.3

0.7 0.7 – – 94.0 0.4

0.5 0.5 – – 94.7 0.3

2.1 2.1 – – 97.5 0.3

1.5 1.2 0.3 – 83.1 0.3

0.9 0.3 0.6 – 83.4 0.3

Sesquiterpene hydrocarbons

a-Copaene

Fatty acids Hexadecanoic acid Heptadecanoic acid Octadecanoic acid Total identified (%) Yield (%)

MS, MS, MS, MS, MS, MS, MS, MS,

S S S S

S S

S S S S S S S S

RI, MS RI, MS RI, MS

Retention indices determined relative to a series of n-alkanes (C8–C40) on the non-polar capillary column VF5-ms (RIa) and polar column. CP Wax 52 (RIb); IM, Identification Method: RI, comparison of RIs with those listed in a homemade library, reported in the literature (Adams, 2007), and/or authentic samples; MS, comparison of mass spectra with those in the mass spectral libraries NIST02 and Wiley 7; S, co-injection with reference compounds; –, component not identified; tr, trace, less than 0.05%.

pulegone is understandable due to their biosynthetic pathway, as described in Mentha  piperita L. (family Lamiaceae). This was developed as a model system for the study of monoterpene

metabolism, where the (+)-pulegone is a central intermediate in the biosynthesis of the Mentha  piperita essential oil (Lange et al., 2011).

Please cite this article in press as: Kremer, D., et al. Morphological, genetic and phytochemical variation of the endemic Teucrium arduini L. (Lamiaceae). Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.04.003

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et al., 2006), in T. scordium L. (Morteza-Semnani et al., 2007), in Teucrium royleanum Wall. ex Benth., and in Teucrium quadrifarium Buch.-Ham. ex D. Don (Mohan et al., 2010). On the other hand, germacrene D was identified as a major compound in the oil of T. chamaedrys from Turkey (Bagci et al., 2011), and in T. arduini from Montenegro (Vukovic´ et al., 2010). Therefore, it could be concluded that the eleven investigated T. arduini populations could be grouped into two chemotypes based on their essential oil composition. The first chemotype is a sesquiterpene chemotype, with b-caryophyllene, germacrene D or caryophyllene oxide as the major products. This chemotype encompasses the localities Su, VV, Vo, SJ, and Sn from Croatia, the locality DG from Bosnia and Herzegovina, and the localities Or, Lo, and Tr from Montenegro. The second chemotype is an oxygenated monoterpenes chemotype, with pulegone (locality Uc from Croatia) and piperitone oxide (locality Id from Bosnia and Herzegovina) as the main components. Variations in the essential oil profile among the populations are well known. Genetic factors, evolution, geographic variation, environmental conditions (i.e. harvest date, planting time), physiological variations (i.e. organ and leaf position), and developmental stage are known to affect the biosynthesis of the essential oil (Figueiredo et al., 2008; Rodrigues et al., 2013a,b). The PCA of the obtained essential oil components separated the T. arduini populations as shown in Fig. 3a. The first principal component (PC 1) explained 21.2% of the total variance, the second 17.2%, and the third component 16.4%, i.e. the first three components accounted for 54.8% of the variance. The axis PC 1 was most correlated with terpinen-4-ol, b-cyclocitral, b-copaene, nonacosane, and heptadecanoic acid (Table 4). The axis PC 2 correlated with limonene, caryophyllene oxide, b-caryophyllene, d-cadinene, a-cisabolol, and piperitone oxide, while PC 3 correlated with pulegone, hexyl isovalerate, terpinolene, and (Z)-b-farnesene. The dendrogram based on essential oil compounds separated the T. arduini populations as shown in Fig. 3b. The three southern populations from Montenegro (Tr, Lo, and Or) and one population from the central Croatian coast (Su) formed the first larger group. The second group was comprised of the four Croatian populations from the central coastal mountains (VV, Sn, Vo, and SJ). The DG population from continental Bosnia showed a higher degree of separation, while the most separated populations were Uc and Id, which belong to the second determined chemotype. According to the AFLP analysis (Fig. 2), the populations Uc and Id are grouped relatively closely, suggesting their genetic connections. Fig. 3. PCA (a) and dendrogram (b) of investigated T. arduini populations for the analyzed essential oil compounds. For abbreviations see Table 6.

Important essential oils constituents isolated from all populations were b-caryophyllene, germacrene D and caryophyllene oxide (Table 3). The analyses revealed that caryophyllene oxide showed concentrations lower than 7% in three populations (Uc, DG, Id), an outcome that could be to environmental factors (such as light, temperature and moisture status). Previous factor stated that these factors controlled the balance between the rate of formation and the rate of loss of volatile compounds such as monoterpenes, and their emission into the atmosphere (Lerdau et al., 1997). Previous analysis of the essential oils of Teucrium species (Teucrium polium L., Teucrium flavum L., Teucrium montanum L., and Teucrium chamaedrys L.), and T. arduini from other localities on Mt Biokovo (Croatia) indicated that the sesquiterpene hydrocarbons, i.e. bcaryophyllene and germacrene D, are the dominant group (Bezic´ et al., 2011; Kremer et al., 2012a, 2013). Additionally, b-caryophyllene was identified as a major compound in the oil of Teucrium orientale L. var. puberulens and T. chamaedrys ssp. lydium (Küçük

2.4. Correlations between morphological, phytochemical and molecular traits Correlations between the investigated traits of T. arduini were tested with the Mantel test (Table 5). The test showed a very weak correlation between the AFLP data and morphological traits (r = 0.19). A weak correlation was found between the morphological traits and geographical position of the populations. There was no correlation between the AFLP data and essential oil profile. On the other hand, a weak correlation was found between the AFLP and the five most represented oil compounds, AFLP and the second chemotype (oxygenated monoterpenes; represented by a high content of pulegone and piperitone oxide), and AFLP and the content of phenolic compounds. A somewhat higher association was found between the essential oil profile and geographic position. The strongest associations were between the content of carbonylic compounds and geographic position (r = 0.46), and between fatty acids content and geographic position (r = 0.52). On the other hand, a weak correlation was obtained between altitude and the investigated traits (Table 5).

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Table 4 Eigen vectors of the first three principal components obtained for the analyzed essential oil components of the investigated T. arduini populations. Bold values indicate the highest contribution to each PC axis. Variable

PC 1

PC 2

PC 3

a-Pinene

0.185991 0.153830 0.181207 0.127081 0.083449 0.049270 0.018743 0.082895 0.150947 0.180497 0.044789 0.238405 0.132324 0.217967 0.004846 0.090276 0.150668 0.145971 0.014467 0.105729 0.005268 0.101725 0.131575 0.217116 0.094186 0.056829 0.120473 0.173188 0.100752 0.193670 0.162403 0.113163 0.142236 0.085457 0.060511 0.180176 0.134353 0.155498 0.118476 0.001298 0.153188 0.071708 0.166642 0.111661 0.157662 0.178604 0.194870 0.112639 0.205726 0.172536 0.216305 0.000777

0.142808 0.089734 0.147068 0.113738 0.252314 0.073319 0.006374 0.039288 0.100781 0.150235 0.137295 0.011648 0.200696 0.075439 0.038895 0.085511 0.136296 0.119076 0.065233 0.217814 0.143404 0.046444 0.221074 0.159696 0.044176 0.050017 0.199344 0.050735 0.120786 0.066100 0.224118 0.102077 0.249100 0.202077 0.225420 0.025021 0.002298 0.187615 0.208757 0.047527 0.176942 0.219851 0.050238 0.157177 0.205733 0.146771 0.032533 0.129453 0.018407 0.084315 0.139350 0.033808

0.082884 0.100129 0.071926 0.046749 0.128617 0.134598 0.320269 0.226322 0.004657 0.086703 0.083759 0.038303 0.000746 0.007500 0.327165 0.084871 0.099539 0.035590 0.048733 0.026233 0.123291 0.013255 0.017444 0.103885 0.002801 0.307058 0.096255 0.172909 0.027819 0.118173 0.015791 0.241918 0.115847 0.048413 0.037597 0.089231 0.087488 0.009507 0.146716 0.320144 0.023550 0.081946 0.146783 0.174023 0.002544 0.131473 0.018918 0.239956 0.009167 0.145126 0.001502 0.311316

Camphene b-Pinene Myrcene Limonene (Z)-b-Ocimene Terpinolene Linalool b-Thujone Camphor Borneol Terpinen-4-ol Myrtenol b-Cyclocitral Pulegone Piperitone Linalyl acetate Bornyl acetate a-Terpenyl acetate Piperitone oxide a-Copaene b-Bourbonene b-Caryophyllene b-Copaene trans-a-Bergamotene (Z)-b-Farnesene a-Humulene allo-Aromadendrene Germacrene D b-Bisabolene d-Cadinene Spathulenol Caryophyllene oxide a-Cadinol a-Bisabolol Germacrone p-Vinyl-guaiacol Eugenol 3-Octanol acetate Hexyl isovalerate Isoamyl hexanoate Eicosane Heneicosane Docosane Tricosane Pentacosane Heptacosane Octacosane Nonacosane Hexadecanoic acid Heptadecanoic acid Octadecanoic acid

Several authors have investigated the association between morphological and molecular diversity. Liu et al. (2007) found a correlation between the morphological traits of Heptacodium miconioides Rehd. (family Caprifoliaceae) and the genetic variability revealed by RAPD analysis. On the other hand, according to Panahi et al. (2013) there was no association between agromorphological diversity and molecular diversity analysed by AFLP and RAPD in Carthamus tinctorius L. (family Asteraceae). Similar disparity between morphological traits and RAPDs was also observed in C. tinctorius (Johnson et al. (2007) and Camelina sativa (L.) Crantz (family Brassicaceae) (Vollmann et al., 2005). López et al. (2008) also did not find a correlation between phenotype and AFLP matrices in Coriandrum sativum L. (family Apiaceae) fruits. One reason for the lack of a strong correlation between AFLP and morphological traits could be that AFLP detects polymorphisms in both coding and non-coding regions of the genome, of

Table 5 Results of Mantel test for the analyzed Teucrium arduini traits. AFLP AFLP Morphology Essential oil 5 compounds Chemotype 1 Chemotype 2 Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Phenolic compounds Carbonylic compounds Hydrocarbons Fatty acids

– 0.19 0.08 0.23* 0.11 0.26** 0.04 0.11 0.08 0.04 0.28* 0.14 0.09 0.18

Morphology – – 0.05 0.38 0.39** 0.21 0.10 0.05 0.40* 0.07 0.03 0.11 0.36 0.18

GP

Altitude *

0.28 0.03 0.37* 0.26 0.17 0.29 0.08 0.17 0.00 0.18 0.03 0.46* 0.22 0.52**

0.16 0.13 0.16 0.09 0.19 0.12 0.11 0.30 0.24 0.06 0.16 0.17 0.08 0.04

Note. Essential oil: means of all identified compounds; 5 compounds: the five most represented compounds in oil; chemotype 1: b-caryophyllene, germacrene D and caryophyllene oxide; chemotype 2: pulegone and piperitone oxide; GP: geographical position (latitude, longitude). * P < 0.05. ** P < 0.01.

which only a small portion are the coding regions (Khan et al., 2008; Panahi et al., 2013). According to Persson and Gustavsson (2001), the relationship between molecular markers and phenotypic traits could be significant if the markers were linked to selected loci. Some authors have reported an association between volatile compounds and molecular diversity. In a study of Ophrys lupercalis Devillers-Tersch. et Devillers, Ophrys iricolor Desf. (family Orchidaceae) and their hybrids, no correlation was found between scent compounds and AFLP data using the Mantel test (Stökl et al., 2008). On the other hand, the Mantel test showed a correlation between scent compounds in Sorbus species (family Rosaceae) and the genetic variability revealed by AFLP (Feulner et al., 2014). A weak correlation was also found between AFLP and the essential oil profile of C. sativum L. fruits from different populations (López et al., 2008). 3. Conclusions In this study, the morphological traits of leaves, AFLP analysis and the essential oil profile were analyzed in eleven populations of the endemic Illyric-Balcanic species T. arduini growing in Croatia, Bosnia and Herzegovina, and Montenegro. The morphological investigations separated the two continental Bosnian populations from the populations from Croatia and Montenegro. Two chemotypes were distinguished based on the essential oil profile. The essential oil content of other continental Bosnian populations should be a subject of future study. Based on the data presented here and in the literature, it could be concluded that there may be a genetic basis for certain chemical compounds in the essential oil that could be observed with AFLP, although such a basis requires further clarification. This study represents a contribution to the knowledge of the poorly investigated flora of the western Balkan Peninsula, especially its endemic species T. arduini and the genus Teucrium in general. 4. Experimental 4.1. Herbal material Randomly selected samples of wild growing plants T. arduini were collected during the blooming period in June and July 2011 at eleven localities in Croatia, Bosnia and Herzegovina, and

Please cite this article in press as: Kremer, D., et al. Morphological, genetic and phytochemical variation of the endemic Teucrium arduini L. (Lamiaceae). Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.04.003

D. Kremer et al. / Phytochemistry xxx (2015) xxx–xxx

Montenegro (Table 6). Voucher specimens of herbal material from Croatia, and Bosnia and Herzegovina were deposited in the Fran Kušan Herbarium at the Department of Pharmaceutical Botany with Fran Kušan Pharmaceutical Botanical Garden at the Faculty of Pharmacy and Biochemistry, University of Zagreb, Croatia (HFK-HR). Herbal material from Montenegro was deposited in the Herbarium of the Faculty of Natural Sciences and Mathematics, University of Montenegro, Podgorica, Montenegro (TGU). In the morphological and DNA investigations, each population was represented by ten randomly selected plants. From each plant, one healthy, fertile shoot with completely developed leaves and inflorescence was selected for the measurement of all leaves and internode lengths. Eight leaf traits (number of leaves on shoots, blade length and width, number of teeth on the left and right blade side, longest tooth length, longest tooth base width, petiole length) and one shoot trait (internode lengths) were measured. Additionally, these data were used to calculate the blade length/ width ratio (a higher ratio indicated a narrower blade, while a lower ratio indicated a rounder blade). During the collection of plant material, several leaves were removed from each plant, placed in silica-gel to dry, and stored at 80 °C until analysis. For investigation of essential oils, the above ground parts of several dozen randomly selected plants were harvested from mature plants on a dry day, mixed to obtain a randomly selected sample, and air-dried in a single layer and protected from direct sunlight for 15 days in a well-ventilated room at 60% humidity and room temperature (22 °C). The dried aerial parts (100 g) were subjected to hydro distillation for 3 h in a Clevenger type apparatus. The obtained essential oil was dried over anhydrous sodium sulfate. 4.2. Chemicals Silica-gel (Merck, Germany), 5%-phenyl-95%-dimethylpolysiloxane (Varian Inc., Lake Forest, CA, USA), EcoRI and MseI restriction enzymes (New England BioLabsÒ, Ipswich, MA, USA), 6-FAM fluorescent dyes (Applied Biosystems, Foster City, CA, USA).

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the binary matrix, AFLP fragments with sizes between 50 and 500 bp were recorded as either present (1) or absent (0). The AFLP binary matrix was used as the data set for calculating of Nei’s pairwise genetic distance matrix between geographical populations (Lynch and Milligan, 1994). 4.4. Gas chromatography and mass spectrometry (GC, GC/MS) Gas chromatography (GC) analyses were performed according to Dunkic´ et al. (2011) on a gas chromatograph (model 3900; Varian Inc., Lake Forest, CA, USA) equipped with flame ionization detector (FID), mass spectrometer (model 2100T; Varian Inc.), non-polar capillary column VF-5MS (30 m  0.25 mm i.d., coating thickness 0.25 lm), and polar column CP Wax 52 (30 m  0.25 mm i.d., coating thickness 0.25 lm). In short, the VF-5MS column temperature was programmed at 60 °C isothermal for 3 min, and then increased to 246 °C at a rate of 3 °C min1 and held isothermal for 25 min. The CP Wax 52 column temperature was programmed at 70 °C isothermal for 5 min, then increased to 240 °C at a rate of 3 °C min1 and held isothermal for 25 min. Other chromatographic conditions were: carrier gas helium; flow rate 1 mL min1; injector temperature 250 °C; volume injected 1 lL; split ratio 1:20; FID detector temperature 300 °C. MS conditions were: ionization voltage 70 eV; ion source temperature 200 °C; mass scan range: 40– 350 mass units (Dunkic´ et al., 2011). Individual peaks were identified by comparison of their retention indices (relative to C8–C40 n-alkanes for VF-5MS and CP Wax 52) to those from an inhouse library, literature (Adams, 2007) and/or authentic samples, as well as by comparing their mass spectra with literature (Adams, 2007), Wiley 7 MS (Wiley, New York, NY, USA) and NIST02 (Gaithersburg, MD, USA) mass spectral databases. An inhouse library was created from authentic compounds obtained commercially and from the main components of many essential oils obtained during previous studies. The component percentages were calculated as mean values from the GC and GC–MS peak areas using the normalization method (without correction factors). 4.5. Statistical analysis

4.3. DNA analysis Genomic DNA from 10 genotypes per population was separately isolated from dried leaves in silica gel using DNA isolation methods for medicinal and aromatic plants, protocol No. 2 as described by Pirttilä et al. (2001). The AFLP analysis was carried out according to the method described by Vos et al. (1995) using EcoRI and MseI restriction enzymes. For preselective amplification EcoRI and MseI primers that consisted of a core sequence and an enzymespecific sequence according to Vos et al. (1995) with one additionally selective nucleotide (EcoRI + A, MseI + C) were used. To identify primer combinations giving the optimal number of polymorphic bands in selective amplifications, three very geographically distant populations were selected and analyzed using a total of 23 randomly selected primer combinations (primer screening) to detect the number of polymorphic bands. On the basis of the numbers of scorable polymorphic bands (data not shown), three highly polymorphic primer combinations were selected for selective amplifications and used to generate AFLP fragments for all investigated populations (Table 7). Selective amplification was carried out using EcoRI and MseI primers with three additional selective nucleotides. Each of the forward primers (EcoRI primers) was labeled with 6-FAM fluorescent dyes. Separation of the AFLP fragments was carried out in a fourcapillary 3130 Genetic Analyzer (Applied Biosystems) using the POP-7 polymer and 36-cm capillaries, and analyzed using GeneMapper V 4.0 software (Applied Biosystems). To construct

Descriptive statistics were analyzed for all measured morphological traits and the one calculated trait. Results were evaluated using multivariate analyses: Principal Component Analysis (PCA) and Cluster Analysis (CA). Initially, PCA was performed to show the overall pattern of variation and relationships among the populations. PCA was carried out based on the correlation matrix between the values of the traits; as a result, the contribution of each variable was independent of the range of its values (Miller and Miller, 2000). Dendrograms of Euclidean distances using the Ward’s and Unweighted Pair-Group Method with Arithmetic Means (UPGMA) method for populations were constructed to check their affinities obtained in previous PCA analysis. Prior to running analyses, all data obtained by measuring morphological traits were standardized due to different scales of character scoring (Quinn and Keough, 2009). Descriptive statistics, PCA and CA were performed using the software Statistica 7 (StatSoft Inc., Tulsa, OK, USA). The extent of genetic diversity among the genotypes within each population was assessed using the average Nei’s gene diversity and the estimated proportions of polymorphic loci were obtained with AFLP-SURV software (Vekemans et al., 2002). The computed pairwise genetic distance matrix between populations was estimated using AFLP-SURV with bootstrapping (1000 replicates) over AFLP loci and then the matrix was used to compute bootstrap confidence values on the tree branches using the PHYLIP software package, ver. 3.69 (Felsenstein, 1993).

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Table 6 Details on the origin and collection data of the investigated Teucrium arduini populations. Locality Ucˇka (Croatia) Šušanj (Mt Velebit, Croatia) Veliki Vaganac (Mt Velebit, Croatia) Vošac (Mt Biokovo, Croatia) Sv Jure (Mt Biokovo, Croatia) Mt Snijezˇnica (Croatia) Diva Grabovica (Bosnia and Herzegovina) Idbar (Mt Prenj, Bosnia and Herzegovina) Mt Orjen (Montenegro) Mt Lovc´en (Montenegro) Trebjesa (Montenegro)

Voucher No. HFK-HR-36-2011 HFK-HR-12-2011 HFK-HR-14-2011 HFK-HR-21-2011 HFK-HR-22-2011 HFK-HR-33-2011 HFK-HR-25-2011 HFK-HR-27-2011 TGU-475036 TGU-475038 TGU-475030

Latitude 0

Longitude 00

45°17 32.9 N 44°310 33.800 N 44°190 46.400 N 43°180 46.000 N 43°190 08.500 N 42°340 08.000 N 43°350 59.000 N 43°370 06.000 N 42°330 35.100 N 42°230 19.300 N 42°460 02.700 N

Table 7 Nucleotide sequences of primer combinations for selective amplifications. Primer combination (50 ? 30 ) E0 -AAG/M00 -CTG E-AAG/M-CAT E-AGA/M-CTA E0 – primer core of EcoRI adaptor 50 -GAC TGC GTA CCA ATT C-30 . M00 – primer core of MseI adaptor 50 -GAT GAG TCC TGA GTA A-30 .

The Mantel (1967) test was used for the estimation of the correlation between genetic (AFLP) distances and geographic, morphological and essential oil compounds distance matrices. Mantel’s test was performed using the program NTSYS-pc 2.21L (Rohlf, 2008). Acknowledgement This work was supported by the Ministry of Science, Education and Sports of the Republic of Croatia (Projects Nos. 006–0000000– 3178 and 177–1191192–0830). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2015. 04.003. References Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy, fourth ed. Allured Publishing Corp., Carol Stream, IL, USA. Bagci, E., Yazgin, A., Hayta, S., Cakilcioglu, U., 2011. Composition of the essential oil of Teucrium chamaedrys L. (Lamiaceae) from Turkey. J. Med. Plants Res. 4, 2588– 2590. Bezic´, N., Vuko, E., Dunkic´, V., Rušcˇic´, M., Blazˇevic´, I., Burcˇul, F., 2011. Antiphytoviral activity of sesquiterpene-rich essential oils from four Croatian Teucrium species. Molecules 16, 8119–8129. ˇ Bjelcic´, Zˇ., Milanovic´, S., 1968. Flora of Unca River valley. Glasnik Zem muzeja (PN) 7, 193–208. Domac, R. 1994. Teucrium L. – dubacˇac. In: Croatian Flora. Zagreb, Školska knjiga. pp. 289–290. Dunkic´, V., Bezic´, N., Vuko, E., 2011. Antiphytoviral activity of essential oil from endemic species Teucrium arduini L. Nat. Prod. Commun. 6, 1385–1388. Felsenstein, J., 1993. PHYLIP – Phylogeny Inference Package (Version 3.2). Cladistics 5, 164–166. Feulner, M., Pointner, S., Heuss, L., Aas, G., Paule, J., Dötterl, S., 2014. Floral scent and its correlation with AFLP data in Sorbus. Org. Divers. Evol. 14, 339–348. Figueiredo, A.C., Barroso, J.G., Pedro, L.G., Scheffer, J.J.C., 2008. Factors affecting secondary metabolite production in plants: volatile components and essential oils. Flavour Fragrance J. 23, 213–226. Harborne, J.B., Tomás-Barberán, F.A., Williams, C.A., Gil, M.I., 1986. A chemotaxonomic study of flavonoids from European Teucrium species. Phytochemistry 25, 2811–2816. Hewitt, G.M., 1999. Post-glacial re-colonization of European biota. Biol. J. Linn. Soc. 68, 87–112. Johnson, R.C., Kisha, T.J., Evans, M.A., 2007. Characterizing safflower germplasm with AFLP molecular markers. Crop Sci. 47, 1728–1736.

0

00

14°12 28.5 E 15°060 45.100 E 15°260 45.000 E 17°030 07.700 E 17°030 15.200 E 18°210 27.600 E 17°410 04.300 E 17°510 01.700 E 18°420 17.900 E 18°540 38.600 E 18°570 24.000 E

Altitude a.s.l. (m)

Abbreviations

1189 604 670 1295 1377 1152 251 669 672 743 608

Uc Su VV Vo SJ Sn DG Id Or Lo Tr

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Please cite this article in press as: Kremer, D., et al. Morphological, genetic and phytochemical variation of the endemic Teucrium arduini L. (Lamiaceae). Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.04.003