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Volatile components variation in the Teucrium marum complex (Lamiaceae) from the Balearic Islands
Botanical Journal of the Linnean Society (2000), 132: 253–261. With 3 figures doi: 10.1006/bojl.1999.0298, available online at http://www.idealibrary.com on
Volatile components variation in the Teucrium marum complex (Lamiaceae) from the Balearic Islands ´ S SANZ JESU Instituto de Quı´mica Orga´nica, CSIC, c/Juan de la Cierva 3, E-28006, Madrid, Spain MAURICI MUS Bota´nica, Facultad de Ciencias, Universitat de les Illes Balears, E-07071 Palma de Mallorca, Spain ´∗ JOSEP A. ROSSELLO Bota´nica, Facultad de Biologı´a, Universidad de Valencia, E-46100 Burjassot, Valencia, Spain Received May 1999; accepted for publication July 1999
The volatile compounds of 18 samples from the western Balearic Islands belonging to three taxa of the Teucrium marum L. complex (T. marum subsp. marum, T. marum subsp. occidentale and T. marum subsp. drosocalyx) were fractionated by distillation-extraction and analysed by means of GC-MS. Seventeen components were characterized and the quantitative data were subjected to multivariate and clustering techniques. Two main groups appeared, one comprising T. marum subsp. occidentale samples and the other grouping T. marum subsp. marum and T. marum subsp. drosocalyx accessions. Chemical data and taxonomic groupings generally agreed, but quantitative and qualitative differences within and between taxa were found. Specimens from Minorca and Cabrera islands showing intermediate morphological features between T. marum subsp. marum and T. marum subsp. occidentale were included within the T. marum subsp. marum-T. marum subsp. drosocalyx group in the Principal Component Analysis. The origin of these intermediate specimens is discussed, and it is suggested that they are local variants of T. marum subsp. marum which had an independent origin throughout its area. 2000 The Linnean Society of London
ADDITIONAL KEY WORDS:—chemotaxonomy – endemic plants – infraspecific variation – western Mediterranean.
∗ Corresponding author: E-mail: [email protected]. 0024–4074/00/030253+09 $35.00/0
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CONTENTS
Introduction . . . . . . . Material and methods . . . Plant sampling . . . . Isolation and identification of Data analysis . . . . . Results . . . . . . . . Discussion . . . . . . . References . . . . . . .
. . . . . . . . . volatile . . . . . . . . . . . .
. . . . . . . . . . . . components . . . . . . . . . . . . . . . .
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INTRODUCTION
The Teucrium marum L. complex comprises small erect or pulviniform shrubs that grow from sea level up to 2000 m altitude in the western Mediterranean islands (Corsica, Sardinia, Hye`res and the Balearic archipelago). The last taxonomic revision based on morphological characters (Mus, 1992) has shown that, contrary to earlier claims, gross vegetative morphology is similar in all taxa included in the complex, at least under cultivation in a uniform environment. An accurate identification of the different entities is only possible when floral organs are available. The length of inflorescences and calyx, the shape of floral bracts, and the micromorphology of calyx hairs are the most reliable characters upon which specimen determination should be based (Castroviejo & Bayo´n, 1990; Mus, Rossello´ & Mayol, 1991; BiniMaleci & Servettaz, 1991; Servettaz, Bini-Maleci & Pinetti, 1992). Misidentification of several type specimens has added a further difficulty to the management of this complex. Mus (1992) pointed out that two out the three taxa formerly recognized as specifically distinct, i.e. T. marum L. and T. balearicum (Pau) Bayo´n & Castroviejo should be subsumed under a single entity. On the other hand, the name T. subspinosum Pourret ex Willd. should be applied to pulviniform plants of Minorca which were devoid of taxonomic recognition. Accordingly, it was proposed that T. marum should be split into three infraspecific taxa. Teucrium marum subsp. marum (=T. subspinosum) has a calyx covered by long eglandular hairs, and the leaves and the bracts have an identical shape and indument. Teucrium marum subsp. drosocalyx (Litard.) Mus, Rossello´ & Mayol features a calyx with long glandular hairs mixed with long eglandular hairs, and the bracts and leaves show a different shape and indument. Lastly, T. marum subsp. occidentale Mus, Rossello´ & Mayol (=T. balearicum) is characterized by its shorter calyx length, with adpressed short eglandular hairs; bracts and leaves are of similar shape and indument. This clear-cut morphological separation is often broken down since intermediates and puzzling specimens occur throughout their distribution range. We have undertaken a study of volatile compounds in order to discover if a different set of independent characters could provide new insights into the systematic framework of this complex. Previous phytochemical reports in the T. marum complex analysing the essential oils from plants gathered in Sardinia and the Balearic Islands (Servettaz et al., 1992) were not conclusive. The ambiguous nomenclature used as well as some misidentifications of the plants they studied clouded the results. Furthermore, since two different taxa grow together in the collecting sites they reported from Minorca (Balearic Islands), their analysis of the pooled populations could contain samples from both.
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T 1. Accessions of the Teucrium marum complex collected in the Balearic Islands. Island codes: Ma, Mallorca; Mi, Minorca; Ca, Cabrera Taxa T. marum subsp. occidentale
T. marum subsp. marum
T. marum subsp. drosocalyx T. marum subsp. occidentale – T. marum subsp. marum intermediate samples
Code
Place of collection
O1–O2 O3–O4 O5 O6
Ma: Ma: Ma: Ma:
Sierra de Alfabia, 900 m, open places on calcareous soils Castell d’Alaro´, 700 m, woody slopes with Pinus halepensis trees Penyal Fumat, 130 m, crevices on calcareous rocky places Esporles, towards Puigpunyent, 300 m, woody places
M1 M2–M3 M4 M5
Mi: Mi: Mi: Mi:
Favaritx, 10 m, open places on schists Barranc d’Algendar, 90 m, crevices on calcareous slopes Cala En Calde´s, 10 m, open places on schists E1 Toro, 325 m, crevices on calcareous slopes
D1 D2 D3
Mi: Favaritx, 10 m, open places on schists Mi: Canutells, 20 m, open areas on calcareous soil Mi: Biniancolla, 50 m, open areas on calcareous soil
I1 I2 I3–I4
Ca: Es Coll Roig, 80 m, open areas on calcareous soil Ca: Es Frare, 40 m, open areas on calcareous soil Mi: E1 Toro, 325 m, crevices on calcareous slopes
For these reasons we have tried to re-evaluate the usefulness of volatile compounds as chemotaxonomic markers in the T. marum complex. Since all taxa of the complex are present in the Balearic Islands and their morphological variability is well known there, we have restricted the study to this geographical area. The objectives of this paper include the evaluation of the variability of the essential oil composition among T. marum samples, the study of the relationships of the three taxa on the basis of its chemical composition, and the evaluation of chemical data in the study of the specimens showing intermediate morphological characters between T. marum subsp. marum and T. marum subsp. occidentale.
MATERIAL AND METHODS
Plant sampling Samples from the three recognized subspecies of T. marum were collected in the Balearic Islands at the flowering stage (Table 1, Fig. 1). In addition, populations with individuals showing intermediate morphology between T. marum subsp. marum and T. marum subsp. occidentale from Minorca and Cabrera islands (hereafter called intermediate samples) were also included. Voucher specimens have been deposited in the herbarium of the University of the Balearic Islands. Isolation and identification of volatile components Individual samples were dried at room temperature. For each sample leaves and flowers were pooled and subjected to SDE (simultaneous distillation-extraction) using
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Figure 1. Distribution of the Teucrium marum complex in the Balearic Islands. Samples used in the chemical analysis are shown. (•) T. marum subsp. occidentale; (Ρ) lines: T. marum subsp. marum; (_) T. marum subsp. drosocalyx; (Ο) intermediate samples between T. marum subsp. marum and T. marum subsp. occidentale.
the equipment and conditions previously described (Godefroot, Sandra & Verzele, 1981) with pentane as solvent. SDS was used instead of chloroform extraction since it does not extract non-volatile compounds which can interfere with the gas chromatographic process. Extracts were concentrated by evaporation and directly injected into the GC column. Volatile fractions were analysed using a Hewlett-Packard 5890 gas chromatograph connected to a Hewlett-Packard 5971 mass detector. A fused silica capillary column (25 m×0.22 mm i.d.), coated with OV-1 methyl silicone was used. Column temperature was programmed for 70 to 230°C at 5°C min, and kept at 230°C for 20 min. Tentative identification was carried out by comparison with mass spectral data (Wiley library or published spectra, when available) and with published retention data, and confirmed by running standard compounds when available. Results were quantified directly from TIC peak areas, and expressed as percent concentration values in the volatile fractions.
Data analysis Principal components analysis (PCA) and clustering techniques were carried out using the BMDP statistical program package BMDP/Dynamic.
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T 2. Volatile compounds characterized in T. marum (% composition) Sample
O1 O2 O3 O4 O5 O6 M1 M2 M3 M4 M5 D1 D2 D3
Component A B C D E F G H I J K L M N O P Q
0.0 0.0 0.0 0.7 0.0 1.7 0.2 23.9 0.0 5.8 10.0 10.2 0.0 20.7 4.3 2.8 4.1
0.0 0.7 0.0 0.7 0.0 2.9 0.0 34.7 5.9 8.3 0.0 1.6 0.0 30.6 5.5 0.0 1.5
0.0 0.0 0.0 1.0 0.0 0.7 0.0 23.1 0.0 6.0 9.4 12.9 0.0 19.3 3.2 3.9 5.1
0.0 0.0 0.0 1.1 0.0 2.0 0.0 21.2 0.0 5.0 9.5 10.5 0.0 23.9 3.8 5.0 6.2
0.0 0.5 0.0 3.5 0.0 1.0 0.0 14.3 0.5 3.5 0.0 8.1 0.0 20.8 3.8 7.6 10.5
0.0 1.0 0.0 28.9 1.2 2.9 0.0 19.9 1.2 4.4 11.9 8.5 0.0 11.6 2.2 1.0 1.4
0.0 1.5 0.5 48.3 3.5 27.8 0.0 5.3 1.6 0.4 1.7 0.0 1.2 1.9 0.0 0.0 0.0
0.0 2.4 0.5 42.1 2.1 18.7 0.0 7.7 0.0 0.0 2.7 0.0 1.4 7.0 0.0 0.0 0.0
0.0 0.8 0.0 75.8 4.2 2.0 0.0 5.2 0.2 1.0 2.8 0.0 1.6 2.6 0.0 0.0 0.0
0.7 1.6 0.4 35.8 2.7 10.5 0.0 7.8 8.4 1.1 7.1 0.0 1.9 8.7 0.0 0.0 0.0
0.0 2.1 0.0 74.0 3.7 1.2 0.0 1.7 0.0 0.0 4.4 0.0 2.7 2.8 0.0 0.0 0.0
0.0 7.0 1.9 34.8 3.0 9.7 0.0 4.5 11.5 0.0 5.1 0.0 2.0 5.6 0.0 0.0 0.0
0.0 0.6 0.0 89.5 2.9 0.6 0.0 3.2 0.0 0.0 0.5 0.0 0.0 1.1 0.0 0.0 0.0
0.0 1.1 2.8 81.2 7.8 1.6 0.0 0.4 1.1 0.0 0.0 0.0 0.8 0.4 0.0 0.0 0.0
I1
I2
I3
I4
1.3 0.8 0.0 62.9 3.1 3.1 0.5 2.1 5.5 0.0 7.0 4.2 0.0 3.1 0.5 1.5 0.0
1.1 1.4 0.0 73.5 2.3 2.9 0.0 4.4 2.3 1.0 1.3 1.8 0.0 4.8 1.1 1.2 0.9
0.0 1.0 0.3 61.3 2.1 4.5 0.0 3.9 13.9 0.6 4.7 0.0 2.0 4.6 0.7 0.0 0.0
0.5 0.7 0.0 69.9 2.5 1.6 0.0 2.7 8.4 0.6 3.7 0.0 2.7 2.1 0.0 0.0 0.0
A: not identified (C8H16O); B: p-Allyl-anisole; C: not identified (C8H12O); D: dolichodial; E: epidolichodial; F: not identified (MW =168); G: not identified (MW=168); H: b-caryophyllene; I: a-bergamotene; J: a-humulene; K: b-bisabolene; L: d-cadinene; M: b-sesquiphellandrene; N: caryophyllene epoxide; O: humuladienone; P: c-cadinene; Q: a-bisabolol.
RESULTS
Twenty-seven compounds, characterized from their chromatographic retention and mass spectra, have been detected in the volatile fractions of 18 samples of the Teucrium marum complex. Since ten of these compounds (1-octen-3-ol, a-muurolene, a-calacorene, b-ionone, b-farnesene, trans-farnesol, a-copaene, a dimethylbenzaldehyde isomer, a calacorenol isomer and an alkyl naphthalene isomer) are only present in a few samples in less than 0.5%, they have been excluded from the data analysis and will not be further considered. The tentative identity and relative concentration in the volatile fraction of the 17 remaining components are shown in Table 2. Components A and C could not be identified, but their elemental composition can be estimated from their mass spectra. Dolicholactones, previously found in T. marum (Pagnoni et al., 1976; Servettaz et al., 1992), were not detected. Components F and G elute closely to the dolichodial isomers, but their molecular weight is two units higher and their fragmentation pattern widely different, m/z 135 and 168 being the most important peaks. According to Servettaz et al. (1992) distillation destroys teucrein, a major component in T. marum extracts. Taking into account the molecular weight of F and G, and the fact that these compounds were not found by Servettaz et al. (1992) in T. marum, one could suggests that they arise from teucrein during the SDE distillation process. The values in Table 2 were first submitted to a cluster analysis of variables (not shown) in order to detect groups of compounds presenting a high correlation. The correlation coefficient was used as a measure of similarity between compounds, and the complete linkage method as the criterion for combining clusters. Three groups are formed when the correlation coefficient for all pairs of members in a group is higher than 0.75. A cluster includes b-caryophyllene, a-humulene, caryophyllene
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2 D1 M4 I1 I3
1 I4
O6
0 M1 M5
O3 O4
M2 I2
O5
D2
–1.5
O1
M3
–1
–2
O2
D3 –1.0
–0.5
0.0
0.5
1.0
1.5
2.0
Figure 2. PCA ordination of Teucrium marum samples obtained from a covariance data matrix calculated from the concentration values in Table 2 (x axis: first component, 84.9% of variance; y axis: second component, 11.8% of variance)
oxide and humuladienone; another group is formed by d-cadinene, d-cadinol and a-bisabolol, while the last cluster includes only the dolichodial isomers. A covariance data matrix calculated from the concentration values was then submitted to PCA (Fig. 2). The three first components accounted for the 98.3% of variance. The first component (84.9% of variance) is mainly related with dolichodial, the compound present in the highest concentrations. The second component (11.8% of variance) is associated with b-cariophyllene and b-cariophyllene oxide, and to a lesser extent with a-humulene, b-bisabolene and d-cadinene. Lastly, the third component (1.7% of variance) is mainly related with the unidentified compound F. The bivariate plot is dominated by the high relative weight associated with compounds present in high concentrations (dolichodial, cariophyllene and cariophyllene epoxide, b-bisabolene). For this reason, PCA was repeated using the correlation matrix in order to assign the same weight to each compound concentration. The first component (48.9% of variance) was positively related to most sesquiterpene compounds and negatively to dolichodial isomers. The second component (12.5% of variance) is positively related to the unidentified components A and G, and negatively to B, C and F. The third component (9.5% of variance) is positively related to abergamotene, b-bisabolene and components A, B and G. Figure 3 plots the sample scores using the first and third components, since the second component depends too strongly on the compounds A and G which are only present, although in very low concentrations, in samples I1 and I2. Both PCA analyses show two chemically distinct groups. One of them includes the T. marum subsp. occidentale samples, characterized by their usually lower content of dolichodial isomers and higher concentrations of b-caryophyllene, a-humulene, d-cadinene, caryophyllene oxide, humuladienone and a-bisabolol than the other Balearic populations of the T. marum complex so far examinated. The other group is composed of accessions belonging to T. marum subsp. marum, T. marum subsp. drosocalyx, and the intermediate samples. No clear gaps between these three entities were apparent in the multivariate space. Samples of T. marum subsp. drosocalyx are
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I1
3 2
I2
1 0 –1 –2 –1.5
D3 M1
D2 I4 M3 M5 M4 I3
O1 O3 O4
O6 O5
M2 O2
D1
–1.0
–0.5
0.0
0.5
1.0
1.5
2.0
Figure 3. PCA ordination of Teucrium marum samples from a correlation matrix calculated from the concentration values in Table 2 (x axis: first component, 48.9% of variance; y axis: third component, 9.5% of variance)
near some T. marum subsp. marum samples. Based on their volatile composition intermediate samples between T. marum subsp. marum and T. marum subsp. occidentale cannot be clearly distinguished from T. marum subsp. marum accessions. The most characteristic compounds of the intermediate samples are a-bergamotene (although samples D1 and M4 present also a high concentration of this compound) and component A, present only in very low concentrations. A classification of samples was carried out using cluster analysis (k-means method). The results (not shown) agree with those obtained by means of the PCA plots.
DISCUSSION
Volatile compounds have been reported to be of great value to understand patterns of microgeographical differentiation at lower levels of systematic hierarchy (e.g. Vernet, Guillerm & Gouyon, 1977; Morgan, 1989). The analysis of volatile compounds of the T. marum complex has shown that all samples are chemically related. Nevertheless, chemical data clearly suggest the recognition of two groups that are related to taxonomic boundaries. Teucrium marum subsp. occidentale samples could be distinguished from the other Balearic accessions of both T. marum subsp. marum and T. marum subsp. drosocalyx. This is not in conflict with the available morphological evidence since the latter taxon can be mainly distinguished by the presence of glandular trichomes in the bracts and calyx. However, the results concerning T. marum subsp. drosocalyx should be taken with caution. First, since only three samples of this taxon were available for study, its volatile component variation may not been adequately sampled. In addition, the distribution of T. marum subsp. drosocalyx in Minorca is very restricted (Fig. 1) and large populations of T. marum subsp. marum are growing intermixed with it. Intermediate specimens linking both taxa are not rare, suggesting that the gene flow between both subspecies is not fully restricted. These statements, together with the chemical
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relatedness between their essential oil composition, pose the question of the extent to which the Balearic populations of T. marum subsp. drosocalyx are of an introgressive nature with T. marum subsp. marum. The results obtained from the few Balearic samples of the former taxon analysed do not constitute a sound basis on which this possibility could be ruled out. Chemical comparisons of plants of T. marum subsp. drosocalyx throughout its distribution range (Corsica, Sardinia, Hye`res islands) should be made to evaluate more accurately this possibility. Servettaz et al. (1992) studied the essential oils in plants from Sardinia apparently belonging to this taxon (called T. marum SE Sardinia in their paper), but their results are, unfortunately, by no means comparable. As emphasized in the experimental part of this work, neither the sampling methods nor the analytical procedure are concordant. Thus the disagreement between the two teams could be due to different methodological approaches. The populations of the T. marum complex from Cabrera and some from Minorca are difficult to ascribe to either of the infraspecific taxa. Individuals show a morphometry of the calyx closer to that of T. marum subsp. occidentale from Mallorca than to that of T. marum subsp. marum from Minorca (Mus et al., 1991). On the other hand, other key characters, such as the calyx indument and the number of flowers per inflorescence, approach these samples to T. marum subsp. marum. From their essential oil composition these intermediate specimens are nested with the subspecies marum and drosocalyx accessions in the multivariate ordination analyses (Figs 2, 3). With the data now available some possible explanations could be formulated to understand the origin of these puzzling specimens. One hypothesis suggests a hybrid origin for the deviating populations of Cabrera and Minorca, involving T. marum subsp. marum and T. marum subsp. occidentale as progenitors. As far as we know typical specimens of T. marum subsp. occidentale are not known on Cabrera and Minorca islands, whereas typical T. marum subsp. marum plants have not been reported from Cabrera. Claims supporting the presence in Minorca of T. marum subsp. occidentale, either as T. subspinosum var. balearicum Pau or as T. balearicum (Pau, 1914; Castroviejo & Bayo´n, 1990), are either based on deviating individuals of T. marum subsp. marum or on intermediate specimens between T. marum subsp. marum and T. marum subsp. occidentale (Mus, 1992). In order to accept this explanation it must be assumed that at one time T. marum subsp. occidentale was present in all the eastern Balearic Islands, a distribution concordant with those exhibited by several Balearic endemic taxa (Alomar, Mus & Rossello´, 1997) and that the past distribution in the archipelago of T. marum subsp. marum included, at least, Cabrera. However, the most important flaw in this hybridization hypothesis lies in explaining the selective extinction of the two (Cabrera) or one (Minorca) putative parents from these islands. A second hypothesis claims that the intermediate samples belong to a different sibling taxon not formerly recognized within the T. marum complex. The morphological data, however, do not support this. Neither of the deviating populations has a single distinctive character of its own; on the contrary, their features are only sorted characters from subspecies marum and occidentale. It is remarkable that although the specimens of the referred populations of Cabrera and Minorca combine attributes of the forementioned subspecies of T. marum, they are not morphologically half-way between them. In fact, the populations from Cabrera and Minorca are slightly different, each approaching in a particular fashion to the subspecies marum and occidentale respectively.
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Lastly, the discordant populations could be viewed as local variants of T. marum subsp. marum. Genetic polymorphism of some populations of the T. marum complex involving variations in chromosome numbers has been reported (Castroviejo & Bayo´n, 1990). However, the finding of three different chromosome numbers in the same root of a plant of T. marum collected in Minorca (Valde´s-Bermejo, 1981) cautions against the existence of true aneuploid individuals. Karyotype instability, if firmly demonstrated, could be a likely explanation for the presence and survival of the anomalous individuals. The assumption of an independent origin for the intermediate individuals could explain their morphological and chemical variability. This polymorphism could be related to an active process of microdifferentiation of T. marum subsp. marum throughout the area. In fact, plants from Sardinia reported as T. subspinosum (a binomial erroneously applied to the T. marum plant growing in Mallorca) are neither morphologically (Mus & Rossello´, unpublished data) nor chemically (Servettaz et al., 1992) identical to the Balearic populations. In our opinion, the last hypothesis best reconciles available data on the T. marum complex. Nevertheless, neither morphology nor the essential oil composition demands discarding the hybridization hypothesis. Unfortunately, the morphological and phytochemical approaches used are at the limits of resolution necessary to elucidate the taxonomic relationships of the complex. Both explanations are testable by means of molecular data (DNA-based methods) provided that substantial genetic differentiation between the taxa of the T. marum complex has been achieved during their evolutionary history.
REFERENCES
Alomar G, Mus M, Rossello´ JA. 1997. Flora ende`mica de les Balears. Palma: Consell Insular de Mallorca. Bini-Maleci L, Servettaz O. 1991. Morphology and distribution of trichomes in Italian species of Teucrium sect. Chamaedrys (Labiatae). A taxonomical evaluation. Plant Systematics and Evolution 174: 83–91. Castroviejo S, Bayo´n E. 1990. Notas sobre Teucrium marum L. y sus afines de las Islas Baleares. Anales del Jardı´n Bota´nico de Madrid 47: 507–509. Godefroot M, Sandra P, Verzele M. 1981. New method for quantitative essential oil analysis. Journal of Chromatography 203: 325–335. Morgan RK. 1989. Chemotypic characteristics of Thymus vulgaris L. in Central Otago, New Zealand. Journal of Biogeography 16: 483–491. Mus M. 1992. Estudio de la diversificacio´n en la flora ende´mica de Baleares. Aspectos taxono´micos y evolutivos. Ph.D. Thesis. University of the Balearic Islands. Mus M, Rossello´ JA, Mayol M. 1991. De flora balearica adnotationes (9). Candollea 46: 47–51. Pagnoni UM, Pinetti A, Trave R, Garanti L. 1976. Monoterpenes of Teucrium marum. Austrian Journal of Chemistry 29: 1375–1381. Pau C. 1914. Sobre algunas plantas menorquinas. Butlletı´ de la Institucio´ Catalana d’Histo`ria Natural 14: 135–142. Servettaz O, Bini-Maleci L, Pinetti A. 1992. Micromorphological and phytochemical characters of Teucrium marum and T. subspinosum (Labiatae) from Sardinia and Balearic Islands. Plant Systematics and Evolution 179: 129–139. Valde´s-Bermejo E. 1981. Nu´meros cromosoma´ticos de plantas occidentales 92–99. Anales del Jardı´n Bota´nico de Madrid 38: 259–263. Vernet P, Guillerm JL, Gouyon PH. 1977. Le polymorphisme chimique de Thymus vulgaris L. (Labie´e) I. Re´partition des formes chimiques en relation avec certains facteurs e´cologiques. Oecologia Plantarum 12: 159–179.
Volatile components variation in the Teucrium marum complex (Lamiaceae) from the Balearic Islands ´ S SANZ JESU Instituto de Quı´mica Orga´nica, CSIC, c/Juan de la Cierva 3, E-28006, Madrid, Spain MAURICI MUS Bota´nica, Facultad de Ciencias, Universitat de les Illes Balears, E-07071 Palma de Mallorca, Spain ´∗ JOSEP A. ROSSELLO Bota´nica, Facultad de Biologı´a, Universidad de Valencia, E-46100 Burjassot, Valencia, Spain Received May 1999; accepted for publication July 1999
The volatile compounds of 18 samples from the western Balearic Islands belonging to three taxa of the Teucrium marum L. complex (T. marum subsp. marum, T. marum subsp. occidentale and T. marum subsp. drosocalyx) were fractionated by distillation-extraction and analysed by means of GC-MS. Seventeen components were characterized and the quantitative data were subjected to multivariate and clustering techniques. Two main groups appeared, one comprising T. marum subsp. occidentale samples and the other grouping T. marum subsp. marum and T. marum subsp. drosocalyx accessions. Chemical data and taxonomic groupings generally agreed, but quantitative and qualitative differences within and between taxa were found. Specimens from Minorca and Cabrera islands showing intermediate morphological features between T. marum subsp. marum and T. marum subsp. occidentale were included within the T. marum subsp. marum-T. marum subsp. drosocalyx group in the Principal Component Analysis. The origin of these intermediate specimens is discussed, and it is suggested that they are local variants of T. marum subsp. marum which had an independent origin throughout its area. 2000 The Linnean Society of London
ADDITIONAL KEY WORDS:—chemotaxonomy – endemic plants – infraspecific variation – western Mediterranean.
∗ Corresponding author: E-mail: [email protected]. 0024–4074/00/030253+09 $35.00/0
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CONTENTS
Introduction . . . . . . . Material and methods . . . Plant sampling . . . . Isolation and identification of Data analysis . . . . . Results . . . . . . . . Discussion . . . . . . . References . . . . . . .
. . . . . . . . . volatile . . . . . . . . . . . .
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INTRODUCTION
The Teucrium marum L. complex comprises small erect or pulviniform shrubs that grow from sea level up to 2000 m altitude in the western Mediterranean islands (Corsica, Sardinia, Hye`res and the Balearic archipelago). The last taxonomic revision based on morphological characters (Mus, 1992) has shown that, contrary to earlier claims, gross vegetative morphology is similar in all taxa included in the complex, at least under cultivation in a uniform environment. An accurate identification of the different entities is only possible when floral organs are available. The length of inflorescences and calyx, the shape of floral bracts, and the micromorphology of calyx hairs are the most reliable characters upon which specimen determination should be based (Castroviejo & Bayo´n, 1990; Mus, Rossello´ & Mayol, 1991; BiniMaleci & Servettaz, 1991; Servettaz, Bini-Maleci & Pinetti, 1992). Misidentification of several type specimens has added a further difficulty to the management of this complex. Mus (1992) pointed out that two out the three taxa formerly recognized as specifically distinct, i.e. T. marum L. and T. balearicum (Pau) Bayo´n & Castroviejo should be subsumed under a single entity. On the other hand, the name T. subspinosum Pourret ex Willd. should be applied to pulviniform plants of Minorca which were devoid of taxonomic recognition. Accordingly, it was proposed that T. marum should be split into three infraspecific taxa. Teucrium marum subsp. marum (=T. subspinosum) has a calyx covered by long eglandular hairs, and the leaves and the bracts have an identical shape and indument. Teucrium marum subsp. drosocalyx (Litard.) Mus, Rossello´ & Mayol features a calyx with long glandular hairs mixed with long eglandular hairs, and the bracts and leaves show a different shape and indument. Lastly, T. marum subsp. occidentale Mus, Rossello´ & Mayol (=T. balearicum) is characterized by its shorter calyx length, with adpressed short eglandular hairs; bracts and leaves are of similar shape and indument. This clear-cut morphological separation is often broken down since intermediates and puzzling specimens occur throughout their distribution range. We have undertaken a study of volatile compounds in order to discover if a different set of independent characters could provide new insights into the systematic framework of this complex. Previous phytochemical reports in the T. marum complex analysing the essential oils from plants gathered in Sardinia and the Balearic Islands (Servettaz et al., 1992) were not conclusive. The ambiguous nomenclature used as well as some misidentifications of the plants they studied clouded the results. Furthermore, since two different taxa grow together in the collecting sites they reported from Minorca (Balearic Islands), their analysis of the pooled populations could contain samples from both.
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T 1. Accessions of the Teucrium marum complex collected in the Balearic Islands. Island codes: Ma, Mallorca; Mi, Minorca; Ca, Cabrera Taxa T. marum subsp. occidentale
T. marum subsp. marum
T. marum subsp. drosocalyx T. marum subsp. occidentale – T. marum subsp. marum intermediate samples
Code
Place of collection
O1–O2 O3–O4 O5 O6
Ma: Ma: Ma: Ma:
Sierra de Alfabia, 900 m, open places on calcareous soils Castell d’Alaro´, 700 m, woody slopes with Pinus halepensis trees Penyal Fumat, 130 m, crevices on calcareous rocky places Esporles, towards Puigpunyent, 300 m, woody places
M1 M2–M3 M4 M5
Mi: Mi: Mi: Mi:
Favaritx, 10 m, open places on schists Barranc d’Algendar, 90 m, crevices on calcareous slopes Cala En Calde´s, 10 m, open places on schists E1 Toro, 325 m, crevices on calcareous slopes
D1 D2 D3
Mi: Favaritx, 10 m, open places on schists Mi: Canutells, 20 m, open areas on calcareous soil Mi: Biniancolla, 50 m, open areas on calcareous soil
I1 I2 I3–I4
Ca: Es Coll Roig, 80 m, open areas on calcareous soil Ca: Es Frare, 40 m, open areas on calcareous soil Mi: E1 Toro, 325 m, crevices on calcareous slopes
For these reasons we have tried to re-evaluate the usefulness of volatile compounds as chemotaxonomic markers in the T. marum complex. Since all taxa of the complex are present in the Balearic Islands and their morphological variability is well known there, we have restricted the study to this geographical area. The objectives of this paper include the evaluation of the variability of the essential oil composition among T. marum samples, the study of the relationships of the three taxa on the basis of its chemical composition, and the evaluation of chemical data in the study of the specimens showing intermediate morphological characters between T. marum subsp. marum and T. marum subsp. occidentale.
MATERIAL AND METHODS
Plant sampling Samples from the three recognized subspecies of T. marum were collected in the Balearic Islands at the flowering stage (Table 1, Fig. 1). In addition, populations with individuals showing intermediate morphology between T. marum subsp. marum and T. marum subsp. occidentale from Minorca and Cabrera islands (hereafter called intermediate samples) were also included. Voucher specimens have been deposited in the herbarium of the University of the Balearic Islands. Isolation and identification of volatile components Individual samples were dried at room temperature. For each sample leaves and flowers were pooled and subjected to SDE (simultaneous distillation-extraction) using
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Figure 1. Distribution of the Teucrium marum complex in the Balearic Islands. Samples used in the chemical analysis are shown. (•) T. marum subsp. occidentale; (Ρ) lines: T. marum subsp. marum; (_) T. marum subsp. drosocalyx; (Ο) intermediate samples between T. marum subsp. marum and T. marum subsp. occidentale.
the equipment and conditions previously described (Godefroot, Sandra & Verzele, 1981) with pentane as solvent. SDS was used instead of chloroform extraction since it does not extract non-volatile compounds which can interfere with the gas chromatographic process. Extracts were concentrated by evaporation and directly injected into the GC column. Volatile fractions were analysed using a Hewlett-Packard 5890 gas chromatograph connected to a Hewlett-Packard 5971 mass detector. A fused silica capillary column (25 m×0.22 mm i.d.), coated with OV-1 methyl silicone was used. Column temperature was programmed for 70 to 230°C at 5°C min, and kept at 230°C for 20 min. Tentative identification was carried out by comparison with mass spectral data (Wiley library or published spectra, when available) and with published retention data, and confirmed by running standard compounds when available. Results were quantified directly from TIC peak areas, and expressed as percent concentration values in the volatile fractions.
Data analysis Principal components analysis (PCA) and clustering techniques were carried out using the BMDP statistical program package BMDP/Dynamic.
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T 2. Volatile compounds characterized in T. marum (% composition) Sample
O1 O2 O3 O4 O5 O6 M1 M2 M3 M4 M5 D1 D2 D3
Component A B C D E F G H I J K L M N O P Q
0.0 0.0 0.0 0.7 0.0 1.7 0.2 23.9 0.0 5.8 10.0 10.2 0.0 20.7 4.3 2.8 4.1
0.0 0.7 0.0 0.7 0.0 2.9 0.0 34.7 5.9 8.3 0.0 1.6 0.0 30.6 5.5 0.0 1.5
0.0 0.0 0.0 1.0 0.0 0.7 0.0 23.1 0.0 6.0 9.4 12.9 0.0 19.3 3.2 3.9 5.1
0.0 0.0 0.0 1.1 0.0 2.0 0.0 21.2 0.0 5.0 9.5 10.5 0.0 23.9 3.8 5.0 6.2
0.0 0.5 0.0 3.5 0.0 1.0 0.0 14.3 0.5 3.5 0.0 8.1 0.0 20.8 3.8 7.6 10.5
0.0 1.0 0.0 28.9 1.2 2.9 0.0 19.9 1.2 4.4 11.9 8.5 0.0 11.6 2.2 1.0 1.4
0.0 1.5 0.5 48.3 3.5 27.8 0.0 5.3 1.6 0.4 1.7 0.0 1.2 1.9 0.0 0.0 0.0
0.0 2.4 0.5 42.1 2.1 18.7 0.0 7.7 0.0 0.0 2.7 0.0 1.4 7.0 0.0 0.0 0.0
0.0 0.8 0.0 75.8 4.2 2.0 0.0 5.2 0.2 1.0 2.8 0.0 1.6 2.6 0.0 0.0 0.0
0.7 1.6 0.4 35.8 2.7 10.5 0.0 7.8 8.4 1.1 7.1 0.0 1.9 8.7 0.0 0.0 0.0
0.0 2.1 0.0 74.0 3.7 1.2 0.0 1.7 0.0 0.0 4.4 0.0 2.7 2.8 0.0 0.0 0.0
0.0 7.0 1.9 34.8 3.0 9.7 0.0 4.5 11.5 0.0 5.1 0.0 2.0 5.6 0.0 0.0 0.0
0.0 0.6 0.0 89.5 2.9 0.6 0.0 3.2 0.0 0.0 0.5 0.0 0.0 1.1 0.0 0.0 0.0
0.0 1.1 2.8 81.2 7.8 1.6 0.0 0.4 1.1 0.0 0.0 0.0 0.8 0.4 0.0 0.0 0.0
I1
I2
I3
I4
1.3 0.8 0.0 62.9 3.1 3.1 0.5 2.1 5.5 0.0 7.0 4.2 0.0 3.1 0.5 1.5 0.0
1.1 1.4 0.0 73.5 2.3 2.9 0.0 4.4 2.3 1.0 1.3 1.8 0.0 4.8 1.1 1.2 0.9
0.0 1.0 0.3 61.3 2.1 4.5 0.0 3.9 13.9 0.6 4.7 0.0 2.0 4.6 0.7 0.0 0.0
0.5 0.7 0.0 69.9 2.5 1.6 0.0 2.7 8.4 0.6 3.7 0.0 2.7 2.1 0.0 0.0 0.0
A: not identified (C8H16O); B: p-Allyl-anisole; C: not identified (C8H12O); D: dolichodial; E: epidolichodial; F: not identified (MW =168); G: not identified (MW=168); H: b-caryophyllene; I: a-bergamotene; J: a-humulene; K: b-bisabolene; L: d-cadinene; M: b-sesquiphellandrene; N: caryophyllene epoxide; O: humuladienone; P: c-cadinene; Q: a-bisabolol.
RESULTS
Twenty-seven compounds, characterized from their chromatographic retention and mass spectra, have been detected in the volatile fractions of 18 samples of the Teucrium marum complex. Since ten of these compounds (1-octen-3-ol, a-muurolene, a-calacorene, b-ionone, b-farnesene, trans-farnesol, a-copaene, a dimethylbenzaldehyde isomer, a calacorenol isomer and an alkyl naphthalene isomer) are only present in a few samples in less than 0.5%, they have been excluded from the data analysis and will not be further considered. The tentative identity and relative concentration in the volatile fraction of the 17 remaining components are shown in Table 2. Components A and C could not be identified, but their elemental composition can be estimated from their mass spectra. Dolicholactones, previously found in T. marum (Pagnoni et al., 1976; Servettaz et al., 1992), were not detected. Components F and G elute closely to the dolichodial isomers, but their molecular weight is two units higher and their fragmentation pattern widely different, m/z 135 and 168 being the most important peaks. According to Servettaz et al. (1992) distillation destroys teucrein, a major component in T. marum extracts. Taking into account the molecular weight of F and G, and the fact that these compounds were not found by Servettaz et al. (1992) in T. marum, one could suggests that they arise from teucrein during the SDE distillation process. The values in Table 2 were first submitted to a cluster analysis of variables (not shown) in order to detect groups of compounds presenting a high correlation. The correlation coefficient was used as a measure of similarity between compounds, and the complete linkage method as the criterion for combining clusters. Three groups are formed when the correlation coefficient for all pairs of members in a group is higher than 0.75. A cluster includes b-caryophyllene, a-humulene, caryophyllene
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2 D1 M4 I1 I3
1 I4
O6
0 M1 M5
O3 O4
M2 I2
O5
D2
–1.5
O1
M3
–1
–2
O2
D3 –1.0
–0.5
0.0
0.5
1.0
1.5
2.0
Figure 2. PCA ordination of Teucrium marum samples obtained from a covariance data matrix calculated from the concentration values in Table 2 (x axis: first component, 84.9% of variance; y axis: second component, 11.8% of variance)
oxide and humuladienone; another group is formed by d-cadinene, d-cadinol and a-bisabolol, while the last cluster includes only the dolichodial isomers. A covariance data matrix calculated from the concentration values was then submitted to PCA (Fig. 2). The three first components accounted for the 98.3% of variance. The first component (84.9% of variance) is mainly related with dolichodial, the compound present in the highest concentrations. The second component (11.8% of variance) is associated with b-cariophyllene and b-cariophyllene oxide, and to a lesser extent with a-humulene, b-bisabolene and d-cadinene. Lastly, the third component (1.7% of variance) is mainly related with the unidentified compound F. The bivariate plot is dominated by the high relative weight associated with compounds present in high concentrations (dolichodial, cariophyllene and cariophyllene epoxide, b-bisabolene). For this reason, PCA was repeated using the correlation matrix in order to assign the same weight to each compound concentration. The first component (48.9% of variance) was positively related to most sesquiterpene compounds and negatively to dolichodial isomers. The second component (12.5% of variance) is positively related to the unidentified components A and G, and negatively to B, C and F. The third component (9.5% of variance) is positively related to abergamotene, b-bisabolene and components A, B and G. Figure 3 plots the sample scores using the first and third components, since the second component depends too strongly on the compounds A and G which are only present, although in very low concentrations, in samples I1 and I2. Both PCA analyses show two chemically distinct groups. One of them includes the T. marum subsp. occidentale samples, characterized by their usually lower content of dolichodial isomers and higher concentrations of b-caryophyllene, a-humulene, d-cadinene, caryophyllene oxide, humuladienone and a-bisabolol than the other Balearic populations of the T. marum complex so far examinated. The other group is composed of accessions belonging to T. marum subsp. marum, T. marum subsp. drosocalyx, and the intermediate samples. No clear gaps between these three entities were apparent in the multivariate space. Samples of T. marum subsp. drosocalyx are
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I1
3 2
I2
1 0 –1 –2 –1.5
D3 M1
D2 I4 M3 M5 M4 I3
O1 O3 O4
O6 O5
M2 O2
D1
–1.0
–0.5
0.0
0.5
1.0
1.5
2.0
Figure 3. PCA ordination of Teucrium marum samples from a correlation matrix calculated from the concentration values in Table 2 (x axis: first component, 48.9% of variance; y axis: third component, 9.5% of variance)
near some T. marum subsp. marum samples. Based on their volatile composition intermediate samples between T. marum subsp. marum and T. marum subsp. occidentale cannot be clearly distinguished from T. marum subsp. marum accessions. The most characteristic compounds of the intermediate samples are a-bergamotene (although samples D1 and M4 present also a high concentration of this compound) and component A, present only in very low concentrations. A classification of samples was carried out using cluster analysis (k-means method). The results (not shown) agree with those obtained by means of the PCA plots.
DISCUSSION
Volatile compounds have been reported to be of great value to understand patterns of microgeographical differentiation at lower levels of systematic hierarchy (e.g. Vernet, Guillerm & Gouyon, 1977; Morgan, 1989). The analysis of volatile compounds of the T. marum complex has shown that all samples are chemically related. Nevertheless, chemical data clearly suggest the recognition of two groups that are related to taxonomic boundaries. Teucrium marum subsp. occidentale samples could be distinguished from the other Balearic accessions of both T. marum subsp. marum and T. marum subsp. drosocalyx. This is not in conflict with the available morphological evidence since the latter taxon can be mainly distinguished by the presence of glandular trichomes in the bracts and calyx. However, the results concerning T. marum subsp. drosocalyx should be taken with caution. First, since only three samples of this taxon were available for study, its volatile component variation may not been adequately sampled. In addition, the distribution of T. marum subsp. drosocalyx in Minorca is very restricted (Fig. 1) and large populations of T. marum subsp. marum are growing intermixed with it. Intermediate specimens linking both taxa are not rare, suggesting that the gene flow between both subspecies is not fully restricted. These statements, together with the chemical
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relatedness between their essential oil composition, pose the question of the extent to which the Balearic populations of T. marum subsp. drosocalyx are of an introgressive nature with T. marum subsp. marum. The results obtained from the few Balearic samples of the former taxon analysed do not constitute a sound basis on which this possibility could be ruled out. Chemical comparisons of plants of T. marum subsp. drosocalyx throughout its distribution range (Corsica, Sardinia, Hye`res islands) should be made to evaluate more accurately this possibility. Servettaz et al. (1992) studied the essential oils in plants from Sardinia apparently belonging to this taxon (called T. marum SE Sardinia in their paper), but their results are, unfortunately, by no means comparable. As emphasized in the experimental part of this work, neither the sampling methods nor the analytical procedure are concordant. Thus the disagreement between the two teams could be due to different methodological approaches. The populations of the T. marum complex from Cabrera and some from Minorca are difficult to ascribe to either of the infraspecific taxa. Individuals show a morphometry of the calyx closer to that of T. marum subsp. occidentale from Mallorca than to that of T. marum subsp. marum from Minorca (Mus et al., 1991). On the other hand, other key characters, such as the calyx indument and the number of flowers per inflorescence, approach these samples to T. marum subsp. marum. From their essential oil composition these intermediate specimens are nested with the subspecies marum and drosocalyx accessions in the multivariate ordination analyses (Figs 2, 3). With the data now available some possible explanations could be formulated to understand the origin of these puzzling specimens. One hypothesis suggests a hybrid origin for the deviating populations of Cabrera and Minorca, involving T. marum subsp. marum and T. marum subsp. occidentale as progenitors. As far as we know typical specimens of T. marum subsp. occidentale are not known on Cabrera and Minorca islands, whereas typical T. marum subsp. marum plants have not been reported from Cabrera. Claims supporting the presence in Minorca of T. marum subsp. occidentale, either as T. subspinosum var. balearicum Pau or as T. balearicum (Pau, 1914; Castroviejo & Bayo´n, 1990), are either based on deviating individuals of T. marum subsp. marum or on intermediate specimens between T. marum subsp. marum and T. marum subsp. occidentale (Mus, 1992). In order to accept this explanation it must be assumed that at one time T. marum subsp. occidentale was present in all the eastern Balearic Islands, a distribution concordant with those exhibited by several Balearic endemic taxa (Alomar, Mus & Rossello´, 1997) and that the past distribution in the archipelago of T. marum subsp. marum included, at least, Cabrera. However, the most important flaw in this hybridization hypothesis lies in explaining the selective extinction of the two (Cabrera) or one (Minorca) putative parents from these islands. A second hypothesis claims that the intermediate samples belong to a different sibling taxon not formerly recognized within the T. marum complex. The morphological data, however, do not support this. Neither of the deviating populations has a single distinctive character of its own; on the contrary, their features are only sorted characters from subspecies marum and occidentale. It is remarkable that although the specimens of the referred populations of Cabrera and Minorca combine attributes of the forementioned subspecies of T. marum, they are not morphologically half-way between them. In fact, the populations from Cabrera and Minorca are slightly different, each approaching in a particular fashion to the subspecies marum and occidentale respectively.
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Lastly, the discordant populations could be viewed as local variants of T. marum subsp. marum. Genetic polymorphism of some populations of the T. marum complex involving variations in chromosome numbers has been reported (Castroviejo & Bayo´n, 1990). However, the finding of three different chromosome numbers in the same root of a plant of T. marum collected in Minorca (Valde´s-Bermejo, 1981) cautions against the existence of true aneuploid individuals. Karyotype instability, if firmly demonstrated, could be a likely explanation for the presence and survival of the anomalous individuals. The assumption of an independent origin for the intermediate individuals could explain their morphological and chemical variability. This polymorphism could be related to an active process of microdifferentiation of T. marum subsp. marum throughout the area. In fact, plants from Sardinia reported as T. subspinosum (a binomial erroneously applied to the T. marum plant growing in Mallorca) are neither morphologically (Mus & Rossello´, unpublished data) nor chemically (Servettaz et al., 1992) identical to the Balearic populations. In our opinion, the last hypothesis best reconciles available data on the T. marum complex. Nevertheless, neither morphology nor the essential oil composition demands discarding the hybridization hypothesis. Unfortunately, the morphological and phytochemical approaches used are at the limits of resolution necessary to elucidate the taxonomic relationships of the complex. Both explanations are testable by means of molecular data (DNA-based methods) provided that substantial genetic differentiation between the taxa of the T. marum complex has been achieved during their evolutionary history.
REFERENCES
Alomar G, Mus M, Rossello´ JA. 1997. Flora ende`mica de les Balears. Palma: Consell Insular de Mallorca. Bini-Maleci L, Servettaz O. 1991. Morphology and distribution of trichomes in Italian species of Teucrium sect. Chamaedrys (Labiatae). A taxonomical evaluation. Plant Systematics and Evolution 174: 83–91. Castroviejo S, Bayo´n E. 1990. Notas sobre Teucrium marum L. y sus afines de las Islas Baleares. Anales del Jardı´n Bota´nico de Madrid 47: 507–509. Godefroot M, Sandra P, Verzele M. 1981. New method for quantitative essential oil analysis. Journal of Chromatography 203: 325–335. Morgan RK. 1989. Chemotypic characteristics of Thymus vulgaris L. in Central Otago, New Zealand. Journal of Biogeography 16: 483–491. Mus M. 1992. Estudio de la diversificacio´n en la flora ende´mica de Baleares. Aspectos taxono´micos y evolutivos. Ph.D. Thesis. University of the Balearic Islands. Mus M, Rossello´ JA, Mayol M. 1991. De flora balearica adnotationes (9). Candollea 46: 47–51. Pagnoni UM, Pinetti A, Trave R, Garanti L. 1976. Monoterpenes of Teucrium marum. Austrian Journal of Chemistry 29: 1375–1381. Pau C. 1914. Sobre algunas plantas menorquinas. Butlletı´ de la Institucio´ Catalana d’Histo`ria Natural 14: 135–142. Servettaz O, Bini-Maleci L, Pinetti A. 1992. Micromorphological and phytochemical characters of Teucrium marum and T. subspinosum (Labiatae) from Sardinia and Balearic Islands. Plant Systematics and Evolution 179: 129–139. Valde´s-Bermejo E. 1981. Nu´meros cromosoma´ticos de plantas occidentales 92–99. Anales del Jardı´n Bota´nico de Madrid 38: 259–263. Vernet P, Guillerm JL, Gouyon PH. 1977. Le polymorphisme chimique de Thymus vulgaris L. (Labie´e) I. Re´partition des formes chimiques en relation avec certains facteurs e´cologiques. Oecologia Plantarum 12: 159–179.