Introduction
The genus Teucrium L. is represented by approximately 250 species whose centre of the diversity is in the Mediterranean region (Harley et al. 2004). The genus includes shrubs, subshrubs, and herbaceous perennials, rarely annual or biennial plants, mostly growing on open exposed rocky grounds, rock crevices and screes. Based on the characteristics of the calyx and life forms the genus Teucrium is separated into seven sections. The section Polium (Mill.) Schreb. comprises 19 species, where T. montanum L. and T. polium L. belong to the same phylogenetic lineage (Tutin and Wood 1972, Salmaki et al. 2016). As a result of the wide distribution of these two species, chromosome numbers vary greatly within this area. T. montanum is diploid or tetraploid with chromosome numbers 2n = 13, 22, 26, 26+0–2B, 26+0–7B, 30, 60. Chromosome numbers for T. polium are 2n = 26, 26+3–4B, 52, 78, and it can be found as diploid, tetraploid and hexaploid (Ranjbar et al. 2018).
These two taxa are semi-woody, evergreen small shrubs. T. polium is erect up to 45 cm high, whereas T. montanum is usually prostrate, growing up to 30 cm. The most prominent morphological differences of this species are observed in the indumentum and leaf shape. Teucrium polium is distinguished by a very dense indumentum composed of glandular and non-glandular multicellular, uniseriate, branched hairs, whereas T. montanum has a less dense indumentum composed of glandular and non-glandular multicellular, uniseriate and unbranched hairs (Jurišić Grubešić et al. 2007,Lakušić et al. 2010). Teucrium polium is characterised by a dense, hairy calyx, in contrast to the calyx of T. montanum whose calyx has fewer hairs. The calyx teeth of T. polium are moderately obtuse, while T. montanum has acute often setaceous teeth (Maurer 1967, Diklić 1974).
These two species are found in sympatry in certain parts of their distribution, with several hybridogenous taxa being described as: T. × castrense Verg., T. × bogoutdinovae Melinkov and T. × rohlenae K.Malý. The hybridogenous taxon between T. montanum and T. polium first described was collected in July 1907 and June 1908 near the town of Castres in south France by Verguin (Verguin 1908). Verguin named that plant T. × castrense, with T. polium ssp. polium Briquet and T. montanum L. as supposed parental species. Fifty years later, Maurer found individuals with morphological characteristics intermediate between the species T. montanum and T. polium on dunes near Lignano, North Italy (Maurer 1967). This was the first finding of T. × castrense in Italy (Maurer 1967). Another hybridogenous taxon has been described by Rohlena in the area near Kotor in Montenegro (Rohlena 1922). However, according to the Shenzhen Code and Art. H.2.1. (Turland et al. 2018) it was invalidly described as T. montanum × polium (and Malý validly renamed it as T. × rohlenae (Malý 1950). Subsequently, T. × bogoutdinovae from Moldova was described (Melnikov 2014). The species T. reuticum Bogoutdinova and T. polium were listed as parents for T. × bogoutdinove which was described on a rocky terrace of the River Reut (Bogoutdinova 1991). Given that T. reuticum is considered a heterotypic synonym of T. montanum (Eur+Med 2006), T. × bogoutdinove can also be treated as a hybrid between T. montanum and T. polium.
We conducted several field trips, during June 2018 in Bisko near Trilj in Croatia (Fig. 1), where T. montanum and T. polium grow sympatrically. There we found individuals with morphological characteristics intermediate between these two species, which a represent hybridogenous taxon.
The aim of our study is to compare morpho-anatomical and phytochemical characteristics of parents and their putative hybrid and to determine the most important characters of differentiation.
Materials and methods
Because of the small population sizes of T. montanum and the putative hybrid in their narrow hybridization zone (Bisko near Trilj, Croatia), and their presumed endangered status, destructive sampling was limited to a minimum, which resulted in a relatively limited number of analyzed individuals. A total of 29 specimens were selected and scored for analyses. Those include 10 specimens of T. polium, 13 specimens of T. montanum and six specimens of their putative hybrid. Specimens were deposited in the Herbarium of the Institute of Botany and Botanical Garden of the Faculty of Biology, University of Belgrade (BEOU – herbarium code followsThiers 2019).
Analysed samples are as follows:
montanum
CROATIA, Trilj, Bisko (43.579400° N, 16.695995° E), pseudomacchia, leg.: D. Lakušić, B. Lakušić, M. Zbiljić, 30.06.2018, (BEOU – 54028)
CROATIA, Trilj, Bisko (43.579400° N, 16.695995° E), pseudomacchia, leg.: D. Lakušić, B. Lakušić, M. Zbiljić, 30.06.2018, (BEOU – 54027)
CROATIA, Trilj, Bisko (43.579400° N, 16.695995° E), pseudomacchia, leg.: D. Lakušić, B. Lakušić, M. Zbiljić, 30.06.2018, (Teucrium × rohlenae K.Malý, BEOU – 54029)
CROATIA, Dalmatinska Zagora, Vrgorac, Mt. Matokit, Zavojane, Zekulić-Roč (43.244743° N, 17.264857° E), rocky ground pasture, leg.: M. Vukojević, 13.06.2015, (sub. T. montanum L., ZAGR – 40074).
For morphological analysis samples of stem with leaves (without terminal inflorescence), leaves, bracts, calyx and flowers from terminal inflorescences were stored in solution of glycerol and ethanol (1:1). Anatomical sections of leaves were preserved on permanent slides, prepared by the standard method for light microscopy. Cross-sections of leaves were cut on a Reichert sliding microtome (up to 15 µm thick). The sections were stained with safranin (1%, w/v, in 50% ethanol) and alcian blue (1%, w/v, aqueous). All slides were mounted in Canada balsam after dehydration (Lakušić et al. 2010). Morphometric analysis included 21 morphological and 23 anatomical characters.
Plant material (3-4 g) was extracted with n-hexane (1:10) for 24 hours at room temperature. The extraction was repeated, and the extracts were united and concentrated under the reduced pressure.
The qualitative and quantitative analysis of hexane extracts was done using the GC and the GC/MS method. The GC analysis was performed on an Agilent 6890N GC system with a 5975 MSD and FID detectors. The column was HP-5 MS (30 m × 0.25 mm, 0.25 μm film thickness). Two μL were injected and the injector temperature was 200 °C with a 10:1 split ratio. Helium was used as a carrier gas (1.0 mL min-1, constant flow mode). The column temperature was linearly programmed (60–280 °C, rate 3 °C min-1, 280 °C for 5 min). The transfer line was heated at 250 °C and the FID detector at 300 °C. EI mass spectra (70 eV) were obtained in the m/z range of 35–550. The retention indices (Kovat's retention index, RI) of essential oil components were experimentally determined relative to two series of n-alkanes (C8-C20 and C21-C40). Their spectra were obtained under the same chromatographic conditions. Identification of compounds was based on comparison of their retention indices (RI) and mass spectra with those from authentic samples and/or the NIST AMDIS software, Wiley, the Adams database and available literature (Adams 2007). Relative percentages of the identified compounds were computed on the basis of the peak areas obtained by FID detector.
The chemical analysis was performed on all compounds (28) of hexane extracts or only on terpene (17) compounds.
In order to describe the variability and significance of morphoanatomical and phytochemical differentiation of analysed groups Principal component analyses (PCA) and Canonical discriminant analysis (CDA) were used, as well as an UPGMA (unweighted pair group method with arithmetic mean) clustering analysis based on Mahalanobis distances for mopho-anatomical data and Pearson’s distances for chemical data in the UPGMA clustering method. Discriminant function analysis (DFA) was used to estimate the contribution of individual characters to the overall discrimination. Statistical analyses were performed using Statistica v.8.0 (StatSoft 2007).
Results
Multivariate analysis of the morpho-anatomical characters
Principal component analysis (PCA) and Canonical discriminant analysis (CDA) performed on morpho-anatomical characters revealed clear separation of T. polium and T. montanum, as well as clear intermediate position of the putative hybrid (Fig. 2A,B). Both analyses revealed a clear separation of three groups along the x-axis, whereas CDA revealed clear separation of putative hybrid along both discriminant axes.
Cluster analysis performed on all morpho-anatomical characters classified T. polium and the putative hybrid in the first cluster, while T. montanum belongs to the second cluster (Fig. 3A). On the other hand, the cluster analysis based on leaf anatomy revealed the hybrid individuals are closer to T. montanum (Fig. 3B).
Characters that predominantly contribute to the distinction among the groups are: coverage of adaxial indumentum, thickness of cuticle, number of teeth on the edge of a leaf, bract length, stem length, frequency of lateral tuft on stem nodes, distance between calyx base and tooth base, length of the narrow part of the calyx teeth and number of terminal inflorescences (Tab. 1).
Chemical analysis of volatile compounds
The chemical analysis of the aerial parts’ volatile compounds revealed that total hydrocarbons (saturated and unsaturated) were the main compounds in all investigated samples (82.9–90.7%). Sesquiterpene hydrocarbons and oxygenated sesquiterpenes were present in amounts that did not exceed 10.2% and monoterpene hydrocarbons only about 1% (Tab. 2).
The chemical compositions of the extracts of the studied groups were quite similar. Saturated and unsaturated hydrocarbons represented the main compounds (33.2-75.0% and 14.1-49.6%). Saturated hydrocarbons containing 25, 29 and 31 C atoms, pentacosane (33.6% and 46.5%), nonacosane (9.2-17.5), untriacontane (0.2-12.3%) as well as unsaturated untriacontene (11.4-48.4%) were dominant.
The cluster analysis of all compounds of hexane extracts (Fig. 4A) revealed a similarity in the composition of the volatiles of the aerial parts of T. polium and the putative hybrids. The extracts of T. polium and the putative hybrid were characterised by a pentacosane (46.5 and 33.6%), compound that was not present in the T. montanum volatiles. Unsaturated untriacontene (48.4%) was the main compound in the T. montanum extract.
On the other hand different results were obtained when only terpene compounds were used for cluster analysis (Fig. 4B). There was a certain similarity between the composition of monoterpenes and sesquiterpenes in extracts of T. montanum and the putative hybrid, mainly represented by higher germacrene D content (3.7% and 2.5%).
Discussion
Intermediate position of putative hybrid
According to the leaf shape and the edge of a leaf with one or more teeth, majority of the hybrid individuals resemble T. polium (Fig. 5). Indumentum of the leaf of the hybrid is less dense than T. polium leaf indumentum, but more dense than indumentum of leaf T. montanum. In addition, non-glandular uniseriate branched hairs that dominate in T. polium and are absent in T. montanum are present in the putative hybrid. At the same time, the hybrid individuals contain many of the non-glandular uniseriate unbranched trichomes present in T. montanum but absent in T. polium (Jurišić Grubešić et al. 2007,Lakušić et al. 2010, Fig. 5).
Also, some hybrid individuals have short dense tufts of lateral shoots on the stem nodes, features typical for T. polium, but absent in T. montanum (Fig. 5).
Important indicators of the transition in morphological characters of the putative hybrid are the colour and number of flowers and the consistency of the inflorescences. Teucrium polium has a compact inflorescence with a large number of small white flowers (between 13 and 51, on average 30); T. montanum has a lax inflorescence with a small number of large pale yellow flowers (between 6 and 16, on average 11), and the hybrid individuals has compact inflorescence with a small number of white flowers (between 7 and 20, on average 13) (Fig. 5). Calyx morphology clearly shows intermediate forms. In terms of calyx dimensions (calyx tube length, calyx tooth length and width) the putative hybrids are between the parents: smaller than T. montanum and larger than T. polium. Additionally, the putative hybrid possesses setae on the top of the acutiform setaceous calyx teeth that are shorter than the setae of T. montanum, while setae are missing in T. polium (Fig. 5).
Finally, and in its general habitus, the putative hybrid has a transitional character. Namely, flowering stems of hybrid individuals are erect, as in T. polium, while lateral vegetative shoots are decumbent, as in T. montanum.
A previous work showed that Teucrium species from the Balkan Peninsula usually contain small quantities of essential oil (Kovačević et al. 2001). Sesquiterpenes were the main compounds in the essential oils of the aerial parts of T. montanum and T. polium. In the investigated samples from Montenegro germacrene D (15.0%), α-pinene (12.4) and β-eudesmol (10.1%) were the most abundant in T. montanum, while β-pinene (19.8%) and germacrene D (11.9%) in the oil of T. polium (Kovačević et al. 2001). The essential oil from the aerial parts of T. montanum from Croatia contained germacrene D (17.2%), β-pinene (12.3%) and β-caryophyllene (7.1%), while β-caryophyllene (52.0%) was dominant in the oil of T. polium (Bezić et al. 2011). By contrast, germacrene D (31%) was dominant in the essential oil of T. polium from Serbia, while δ-cadinene (8.1%) and β-caryophyllene (5.1%) were the main compounds in T. montanum (Radulović et al. 2012). Furthermore, the most recent study of the variability of essential oil of different populations of T. montanum from the central and south Balkan Peninsula (14 populations from Serbia, Greece and Albania), revealed extremely large differences in the chemical composition of the essential oils of the aerial parts of T. montanum (Marčetić et al. 2018). This study showed that the composition of essential oils was quite variable but the main compounds in almost all oils were germacrene D (trace-45.5%), sabinene (trace-23.1%), α-pinene (trace-20.7%), limonene (trace-20.4%), (E)-caryophyllene (2.9-14.5%), γ-cadinene (trace-13.8%) and δ-cadinene (trace-12.0%).
These results show that the composition of the essential oils of the two species is not clearly different, given that they contain similar compounds and similar pattern of the variability. It indicates that it is not possible to distinguish them on the basis of the chemical composition of essential oils alone, which is in accordance with the results of several recent studies that showed that variation in the composition of essential oils within a species appears to be the rule rather than the exception, and that the geographical distribution of different types of essential oils of plants is strongly co-related with the environmental conditions including climate, geological, pedological and phytosociological characteristics of the habitats (Kuštrak et al. 1984,Franz 1993,Jug-Dujaković et al. 2012,Lakušić et al. 2012).
However, together with data of morphological and anatomical analysis, the data of the composition of the analysed essential oils also support the assumption of the hybridogenous origin of intermediate individuals.
Taxonomical treatment and distribution
Brief descriptions of hybridogenous taxa T. × castrense and T. × rohlenae largely overlap with features of collected hybrid individuals (Verguin 1908,Rohlena 1922,Malý 1950,Maurer 1967). Considering that the subspecies T. polium ssp. polium, one parent of T. × castrense, is distributed only in the western parts of the Mediterranean (Tutin and Wood 1972, Euro+Med 2006) we have excluded the possibility that hybridogenic individuals from Croatia belong to these taxa.
At the same time, given that T. polium ssp. capitatum (L.) Archangeli and T. polium ssp. vincentinum (Rouy) D.Wood are distributed in Croatia (Nikolić 2019), and that the plant from Trilj belongs to the subspecies T. polium ssp. capitatum also distributed in Montenegro (Euro+Med 2006) we suggest the hybrid be included in taxon T. × rohlenae, originally found and described in area near Kotor in Montenegro (Rohlena 1922,Malý 1950).
We have analysed ZAGR herbarium specimens of T. polium and T. montanum collected in Croatia, and we found specimens that perfectly match the appearance of the hybrid T. × rohlenae. These specimens were determined as T. montanum and were collected in 2015 on Mt Matokit near Vrgorac in Dalmatinska zagora.
Even though T. × rohlenae has been registered only in three locations (Montenegro: Kotor, Croatia: Trilj and Matokit,Fig. 1), it is likely that its distribution is much wider, so we can expect hybrid individuals in areas where T. montanum and T. polium grow sympatrically.
Acknowledgements
This work was supported by the Ministry of Education and Science of the Republic of Serbia (Grants No. 451-03-68/2020-14/200178 to D. Lakušić and 451-03-68/2020-14/200161).