Page 2 - Thymus_BSE

L. Llorens et al. / Biochemical Systematics and Ecology 56 (2014) 246e254 247

fields including taxonomy, biogeography and medicine (for example, see S aez and Stahl-Biskup, 2002a; Giuliani and Maleci-
Bini, 2008; Jassbi et al., 2012; Lakusic et al., 2012) and citations.
One of the most important genera of the Lamiaceae in the Mediterranean region is the genus Thymus Benth., which in-
cludes approximately 110 widespread species (Morales, 1997). Thymus richardii Pers. (included in the section Serpyllum,
subsection Insulares, -Jalas, 1971-), is an example of a species having a relictual, fragmented and scattered distribution under.
It was first described in Majorca (Balearic Islands) by Persoon (1806), and later from Sicily (Gussone, 1843), Ibiza (Font Quer,
1935), Valencia (Bol os and Vigo, 1983), and Bosnia-Herzegovina (Jalas, 1971, 1972).
Based on morphological characteristics (occasionally determined using herbarium material), the Thymus taxon constitute
an aggregation of four infraspecific taxa (Jalas, 1971, 1972) distributed in few localities, where they generally form sparse and
isolated populations comprising few plants, including T. richardii subsp. richardii (Majorca, Spain; Bosnia-Herzegovina), T.
richardii subsp. ebusitanus (Font Quer) Jalas (Ibiza, Spain), T. richardii subsp. nitidus (Guss.) Jalas (Marettimo Island, Sicily, Italy)
and T. richardii subsp. vigoi Riera, Güemes & Rossell o (Valencia, Spain). However, in various studies some of these have been
treated as differentiated species, including Thymus nitidus Guss. (¼ T. richardii subsp. nitidus) and Thymus aureopunctatus Beck
(¼ T. richardii subsp. richardii, Bosnio-Herzegovinian populations).
From a phytosociological point of view the Bosnian populations form part of the Illyrian black pine woods (Orno-ericenion
“dolomiticum”), and the subspecies also occurs in xeric stands of oak (Konjic) and thermophilous beech woods (Borci). The
island of Marettimo (Sicily) is composed entirely of white and grey dolomite. Similar edaphic features occur in all the
populations of Ibiza. The populations at these two locations have a subrupiculous character, or grow on litosols.
Another feature is that in these two localities the plants are sometimes integrated into the Mediterranean scrubland
vegetation (Rosmarinetalia). In Valencia (La Safor) the plants only grow in this type of vegetation, which substitutes for Pinus
halepensis or Quercus ilex forest. However, in Majorca the populations (Puig Major) are exclusively found in rocky crevices on
the tops of mountains on the island, and have a clear rupiculous character.
The essential oils of Thymus species have been the subject of numerous studies (for example, see S aez and Stahl-Biskup,
2002b; Stahl-Biskup, 2002 and citation). Some studies have investigated the terpene composition and its variability in
relation to edaphic (Martonfi et al., 1994), environmental (Boira and Blanquer, 1998), altitudinal (Delazar et al., 2011), and
seasonal and cultural (Letchamo and Gosselin, 1995; Aziz et al., 2008; Sharafzadeh et al., 2011) conditions. However, few
studies have considered chemotaxonomic (Tzakou and Constantinidis, 2005) or biogeographic aspects (Blanco et al., 2012).
Wild Thymus species are highly chemically polymorphic, with variability in the essential oil composition being related to
various environmental and genetic factors (Thompson, 2002; Loziene and Venskutonis, 2005; Pluhar et al., 2007).
Most of the volatile terpenoids in Thymus oils belong to the monoterpene group, which usually comprise more than 90% of
the total. Sesquiterpenes are always present but, with only few exceptions, as a minor component (Stahl-Biskup, 2002). For
example, Thymus samius Ronniger (Tzakou and Constantinidis, 2005), determinate varieties of Thymus serpyllum (Miller)
Benth. (Paaver et al., 2008) and T. richardii Pers. subsp. nitidus (Guss.) Jalas (Bader et al., 2001) contain (E)-nerodiol, (E)-
caryophyllene, germacrene D and (very rarely) b-bisabolene as major sesquiterpenes.
Moreover, it seems that some species having phenol chemotypes (Thymus hyemalis Lange, Thymus mastichina L., Thymus
zygis L. and Thymus piperella L.) correspond to archaic forms with the original endemic distribution, while species having
geraniol are more evolved and have a more northern geographic distribution. This is consistent with previous studies
reporting that the abundance of phenolic compounds is associated with typical Mediterranean climatic and edaphic con-
ditions, and that the non-phenolic chemotypes among Thymus species must overcome environmental conditions that differ
from the above (e.g. cold winters) (Bruneton, 1995).
In defining chemotypes the biosynthetic precursors of phenolic compounds (g-terpinene and p-cymene) are commonly
taken into account. Correlations between the amounts of phenolic compounds and their biosynthetic precursors tend to
confirm the traditional pathway in which p-cymene is the immediate precursor of thymol and carvacrol (Thompson et al.,
2003). More recently it has been proposed that the formation of thymol and carvacrol is catalyzed by single P450s directly
from g-terpinene via a two-step oxidation, whereas p-cymene is a side product resulting from premature release of the
substrate from the enzyme active site (Crocoll et al., 2010). In relation to the phenolic nature of the thyme taxa, both pos-
sibilities lead to the consideration of thymol and carvacrol with their derivatives and precursors as a whole.
Knowledge of the terpene composition of the T. richardii group is restricted to two taxa: T. richardii subsp. nitidus (Bader
et al., 2001) and T. aureopunctatus (Cavara et al., 2009). The former is characterized by being rich in b-bisabolene (32.3%),
while the second is a thymol chemotype (34.5% thymol).
In the present study grex of taxa integrated into T. richardii were studied with the following objectives: 1) to determine the
terpene composition of the essential oils among the various populations studied; 2) to identify discriminating compounds to
differentiate chemical groups; and 3) to assess the relationship between chemical composition and biogeography, as well as
climatic and soil parameters.

2. Material and methods
2.1. Plant material

During summer 2011, samples of wild populations of the T. richardii group were collected in situ from their place of origin
(Fig. 1; Table 1). Sampled plants were in the full flowering stage, and were collected during in the sunniest time of the day. For