NVEO 2017, Volume 4, Issue 4

CONTENTS

REVIEWS

1. Biological properties of essential oils and volatiles: Sources of variability / Pages: 1-13 A. Cristina Figueiredo

2. Systematic and Biogeographic overview of Lamiaceae in Turkey / Pages: 14-27 Ferhat Celep, Tuncay Dirmenci

3. Stamen construction, development and evolution in Salvia s.l. / Pages: 28-48 Regine Claßen-Bockhoff

ARTICLES

1. TLC-GC/MS Method for identifying and selecting valuable essential Oil Chemotypes from Wild Populations of Mentha longifolia L. / Pages: 49-61 Juan Antonio Llorens-Molina, Vicente Castell, Sandra Vacas, Mercedes Verdeguer

2. Chemical composition and antioxidant activity of essential oils of six Lamiaceae plants growing in Southern Turkey / Pages: 62-68 Hasan Maral, Murat Türk, Tuncay Çalışkan, Saliha Kırıcı

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REVIEW

Biological properties of essential oils and volatiles: Sources of variability

A. Cristina Figueiredo* Centro de Estudos do Ambiente e do Mar (CESAM Lisboa), Faculdade de Ciências da Universidade de Lisboa, Centro de Biotecnologia Vegetal (CBV), Edifício C2, Campo Grande, 1749-016 Lisboa, PORTUGAL

*Corresponding author. E-mail: [email protected]

Abstract Essential oils are a specific type of extracts that result either from hydro-, steam- or dry-distillation from any part of a duly identified botanical species, or by a mechanical process without heating, called expression, from the epicarp of Citrus fruit species. Any other type of extraction may provide a more, or less, volatiles-rich extract, but it is not an essential oil. Essential oils, per se, do not exist in the plant, since they result from a specific extraction procedure. In the plant there is a wide array of metabolites, only some of which will be extracted in the form of an essential oil, with variable composition even if just considering that they can either be obtained by distillation or expression. Man has used for long, both volatiles and essential oils, for a wide range of purposes in the pharmaceutical, food, beverage, cosmetic and perfumery industries, among others. The described biological activities of volatiles and essential oils are wide, but the results are not always congruent. Awareness of the factors that may influence the essential oil chemical variability and ultimately its biological activity, efficacy and safety, are thus very important. These comprise, a) the knowledge on the difference between plant volatiles and essential oils, b) the avoidance of botanical misidentification, c) getting information on plant parts chemical composition variability, the existence of chemotypes and essential oil components enantiomers, and also f) exploring different biological activity assay conditions, which was reviewed in this present work.

Keywords: Essential oils, volatiles, biological properties, chemical composition

Introduction Plants produce and/or accumulate a diverse assortment of metabolites that are generally defined as primary metabolites or secondary metabolites (Figueiredo & Barroso 2015). Volatiles are among the diverse constituents that plants produce and accumulate in specific secretory structures. All land plants, either bryophytes (liverworts, hornworts and mosses), or tracheophytes (vascular plants) produce volatiles. Plant volatiles correspond to (a) a complex mixture of compounds which have the property of naturally volatilizing under appropriate environment temperature and humidity conditions, allowing, for example, that we may smell the aroma of a rose, or of jasmine, or (b) include an equally complex mixture of compounds that are extracted from plants by different methodologies at home, laboratory or industrial level. These compounds can be composed only by carbon and hydrogen, or additionally have oxygen, nitrogen or sulphur in their molecules. In general, these compounds fall down in the chemical classes of terpenes, phenols, nitrogen-bearing compounds and other primary and/or secondary metabolites derived from diverse metabolic pathways (Figure 1). However, these substances are often present in the plant at reduced levels and/or different ratios which are species specific. Plants use volatiles as a part of their strategy to adapt to the environmental biotic and abiotic factors (Figueiredo and Barroso 2015), and man has learned to use both volatiles and essential oils for their diverse array of properties. In addition to the well-known therapeutic actions and positive effect on health and well- being, volatiles and essential oils find application in other fields such as food, beverages, cleaning, textile,

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perfume and cosmetic industries, to name a few. Likewise, many researchers devoted their studies to very broad subjects within this field of research.

Figure 1. Examples of volatile constituents isolated from plants. • Monoterpenes • Sesquiterpenes

HO OH

OH OH

Menthol Thymol Limonene Geranylgeraniol

Nerol Farnesene Farnesol HO D

Pulegone α-Pinene Germacrene-D Petasitene

• Diterpenes H H H

H H H H Abietadiene H Abietol HO Abietatriene H • Phenylpropanoids O O O O Cinnamaldehyde Safrole Anethole

• Polyacetylenes Falcarinol OH • Nitrogen-bearing • Sulphur-bearing H S S N N O N O Methyl 3- Ethyl 3- Indole Nicotine methylthiopropionate methylthiopropionate

O OH • Fatty acids Palmitic acid

• Others O O O Octanal trans-2-Decenal Methyl hexanoate In the great majority of the cases it is impossible to differentiate in an essential oil its diverse biological activities, and so its action, in a certain circumstance, is the result of the interaction of several of its components properties and, many times, of the way and amount used. For instance, essential oils may mitigate anticancer activity not because they act on the cancer itself but because of their antioxidant activity they lower the toxic effects of free radicals induced by several cancers, or even from anticancer agents. But being bioproducts, essential oils are also prone to variability, thus requiring assurance of constancy and quality, to guarantee efficacy and safety. On the other hand, the use of essential oils is not devoid of

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undesired side effects which are, in most cases, related with their misuse. Knowledge on the essential oil chemical composition is essential for understanding possible tolerance mechanisms and/or adverse effects. The knowledge on the biological properties of volatiles and essential oils is important, because they combine being a) natural and biodegradable, b) less toxic than chemical pesticides, and usually showing low toxicity to mammals, if accepted mean daily doses are respected, c) able to accomplish, simultaneously, the function of more than one of their synthetic equivalents and d) less expensive than some drugs. The reported biological activities of volatiles and essential oils are immense and to classify them is not an easy task. For simplicity, in Table 1 they are separated into therapeutic and non-therapeutic. Nevertheless, the properties intermingle, and many of the therapeutic uses support the non-therapeutic ones. For instance, essential oils and volatiles may have an external use as antiseptic and disinfectant, but they can be used in household products exactly for the same properties. On the other hand, whereas many studies focus on the Human or animal use, some studies also deal with a more ecological perspective, viewing to understand the importance of volatiles in plant-abiotic and plant-biotic relations.

Table 1. Some of the reported biological properties of volatiles, essential oils and their constituents. Therapeutic External: Antiphlogistic / anti-inflammatory, antiseptic and disinfectant, deodorant, hyperemic, among others. Internal: Analgesic / antinociceptive, antibacterial, anticancer, antioxidant, antiphlogistic / anti-inflammatory, anti-rheumatic, antispasmodic, antiviral, carminative, expectorant, hyperemic, penetration enhancer, sedative, among others. Non-therapeutic Antiseptic and disinfectant Absorption enhancer Preservative and spice Repellent Pesticide Toxic Defence and attraction in plant-abiotic and plant-biotic relations

A very large number of studies have been performed on this wide range of biological properties, and trying to summarize the gathered data is complex, because of the diversity of testing conditions used (some using commercial essential oils, others essential oils isolated from plants from the wild, cultivated plants, different plant parts, different extraction methods) and because, in some cases, results are different, or even contradictory. In view of this large amount of data, and results variability, this review will try to pinpoint some relevant aspects regarding the reasons for this variability, with some examples. Plant volatiles versus essential oils The isolation of plants active ingredients, at industrial or at laboratory level, may be carried out by different extraction methodologies, depending on the ultimate goal, using, among others: i) organic solvents, ii) supercritical fluids, iii) fats, iv) mechanical means, with or without temperature and solvent, v) specific selective procedures, and vi) distillation or expression (Figueiredo et al. 2014). Whereas these different methods can be used to extract volatile compounds from plants, only two are used to obtain an essential oil (Council of Europe 2010): a) hydro-, steam- or dry-distillation from properly identified botanical species or from its different parts, or b) a mechanical process without heating, termed expression, in the case of epicarp of Citrus fruit species, such as orange, lemon, mandarin, grapefruit, among others.

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It is noteworthy that an essential oil as such, with its characteristic chemical composition, does not exist in the plant, and it is a product of a suitable extraction process. In the plant there is a complex mixture of compounds, some of which are released naturally into the atmosphere, others may be extracted by distillation or expression, and there are others that are non-volatile and, as such, require other specific extraction methodologies. Thus, any phytochemicals extraction process will always give a partial perspective of the whole of the components that exist in the plant and the extraction methodology used should always take into account the characteristics of the type of compound(s) to be isolated and the goal of the study. Nevertheless, many times either in the literature or in commercial samples, the extracts are called, or the labelling mentions, an essential oil, when the extraction procedure used does not provide an essential oil, with obvious implications in the chemical composition and biological activity of the extract.

Table 2. Main components (≥5%) of commercial available labelled Helichrysum italicum essential oils (Hi 12 and Hi 13) and of published data (adapted from Ventura & Lima, 2017). Helichrysum italicum Hi 12* Hi 13* Essential Oils Components EINECS 289918-2 Corsica (France) Italy Portugal Bianchini et al. 2001 Satta et al. 1999 Costa et al. 2015 α-Pinene t 18.8 0.7-2.9 0.1-0.2 53.5 Limonene 1.2 1.9-7.5 1.2 0.5 Linalool t 1.0-2.8 9.1-14.9 0.1 4,6-Dimethyloctan-3,5-dione 1.0-11.3 Nerol 0.9 2.0-4.9 nd-10.7 Neryl acetate 11.6 11.4 15.8-42.5 nd-28.9 Italicene * 2.7 9.9 0.9-3.4 2.3-4.2 1.2 4,6,9-Trimethyldec-8-en-3,5-dione 0.8-5.6 Neryl propionate 1.6-6.7 ar-Curcumene 21.7 8.1 0.8-4.6 4.5-4.8 2.8 γ-Curcumene 8.1 0.8-12.9 11.4-18.2 27.4 δ-Cadinene 0.8-5.6 0.3 Eudesm-5-en-11-ol nd-5.1 Rosifoliol 3.9-20.2 * Commercial available labelled Helichrysum italicum essential oils from 2012 (Hi 12) and 2013 (Hi 13), EINECS 289918-2: Helichrysum leaf absolute CAS 8023-95-8 CoE225, nd: not detected, t: traces (<0.05%).

As an example, Ventura & Lima (2017) compared the chemical composition of commercial available labelled essential oils from Helichrysum italicum, with published data on the chemical composition of the essential oil from this species collected at different locations in the Mediterranean area, Table 2. Not only it is clear that the chemical composition varies with provenance, probably related to the subspecies used, the distillation length, plant developmental stage, plant part used, among other factors, but there is also a clear difference between the reported essential oils composition and that from the commercial samples. Indeed, although available as an essential oil, the label of the commercial samples also mentioned EINECS 289918-2. The presence of this code from the European Inventory of Existing Commercial Substances (EINECS) means that instead of being an essential oil, it was an absolute of Helichrysum, which partly explains the difference in chemical composition, which would have also an implication on the biological properties. So, when evaluating samples from commercial origin, it is important to certify that an essential oil is being used, or at least to state the correct type of extract used in the evaluation.

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Plant quality control Botanical misidentification is still a major problem, particularly when using wild collection of plant material and, sometimes, solely based on local common names. Accordingly, this will have obvious implications in the chemical composition as well as on the biological property of the isolated essential oil or of any other type of extract. Sullivan (2009) performed a comparative analysis of in-laboratory-isolated essential oils from commercially available herbal products of Eucalyptus globulus (eucalyptus), Foeniculum vulgare (fennel) and Mentha x piperita (peppermint), with the commercial essential oils counterparts, used in aromatherapy practice. Whereas only one out of the nine commercial E. globulus essential oils analysed showed some differences in the chemical composition, and some differences were found for essential oils isolated from F. vulgare probably because of using bitter instead of sweet fennel, the most striking discrepancy was found with the samples from M. piperita, Table 3.

Table 3. Minimum and maximum percentage range of main components (≥5%) from the essential oils isolated from M. piperita labelled herbal product, from commercial essential oils, and reference data from the Portuguese Pharmacopoeia (FP) and the International Organization for Standardization (ISO) (adapted from Sullivan, 2009). Mentha x piperita essential oil main components (≥5%) Commercial Specifications Components Herbal product Essential oils FP (2005) ISO (2006)* Min Max Min Max Min Max Min Max 1,8-Cineole 7.1 7.5 1.8 4.8 3.5 14.0 3.0 8.0 Limonene 6.0 7.1 1.8 4.8 1.0 5.0 1.0 3.0 Menthone 0.1 0.1 22.0 26.8 14.0 32.0 13.0 28.0 Isomenthone 0.3 0.3 3.1 5.7 1.5 10.0 2.0 8.0 Neomenthol t t 3.2 4.8 2.0 4.5 Menthol t t 36.4 53.7 30.0 55.0 32.0 49.0 Carvone 42.1 43.4 t t 1.0 0.5 3.0 Pulegone 21.1 21.7 0.4 2.4 4.0 FP: Farmacopeia Portuguesa (2005), ISO (2006): ISO 856:2006, * several types, t: traces (<0.05%).

The commercial essential oil samples of M. piperita analysed were all found to be within the assessment of quality specifications of the Portuguese Pharmacopoeia (Farmacopeia Portuguesa, 2005) and of the International Organization for Standardization ISO 856:2006, with menthol and menthone as dominant compounds. The chemical components cluster analysis from the five commercial essential oil samples analysed showed a very good level of correlation between them. They were, however, uncorrelated with those from the essential oils isolated from the commercial herbal product due to incorrect labelling and species identification. The analysis of the essential oil isolated from the commercial herbal product revealed carvone and pulegone as main components, suggesting that the herbal product was M. pulegium (pennyroyal) instead of M. piperita. The use of just common names, without a proper botanical certification, is also a major cause of plant misidentification and commercialization, both as herbal products and even in crop seeds. Common names often refer to different species, which, sometimes, do not even belong to the same plant family, Table 4.

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Table 4. Examples of some Portuguese plant common names and the plant species to which they may refer to. Portuguese common name May refer to, according the region of Portugal or abroad Family “Amieiro” Alnus glutinosa Betulaceae Frangula alnus Rhamnaceae Populus alba Salicaceae “Carqueja” Pterospartum tridentatum (Portugal mainland) Fabaceae / Leguminosae Ulex europaeus (Portugal, Madeira island) Fabaceae / Leguminosae Baccharis trimera (Brazil) Asteraceae “Cedro” Cedrus spp. Pinaceae Thuja spp. Cupressaceae “Erva-cidreira” Cymbopogon citratus Poaceae Melissa officinalis Lamiaceae / Labiateae “Loendro” Nerium oleander Apocynaceae Rhododendron ponticum Ericaceae “Macela” Chamaemelum spp., Matricaria spp., and even Helichrysum spp. Asteraceae / Compositae “Perpétua-das-areias” Helichrysum italicum and Helichrysum stoechas Asteraceae / Compositae “Rosmaninho” Lavandula spp. and Rosmarinus officinalis Lamiaceae / Labiateae

“Carqueja” is a highly appreciated plant in Portugal, both for culinary uses and because the infusion of the flowers is used for therapeutic properties (Grosso et al., 2007). Nevertheless, in the mainland Portugal “carqueja” refers to a Pterospartum species, whereas in the Madeira archipelago it refers to an Ulex species, and in Brazil, the same common name refers to a Baccharis species from a different family, Table 4. Recalling the previous example of H. italicum, both this species and H. stoechas, are known in Portugal by the same common name, “perpétua-das-areias” (everlasting). This may constitute an additional factor of chemical variability between plant extracts. These examples stress the importance of a correct plant identification, both at the academic, and at the essential oil producer level, as this will determine the chemical composition of the final product. Chemical composition, chemotypes and enantiomers

Phytochemcials in general, and volatiles or essential oils in particular, can be extracted from different plant parts, namely bark, flowers and flower parts, fruits, leaves, petioles, roots or rhizomes, seeds and/or stems. It is well known that the plant part can, in many cases, play a major role in the final composition of the essential oil (Figueiredo et al., 2008) as well as on its advised use (Ruppert-Aulabaugh, 2014). This different composition is many times related not only with the defensive or attractive role of the compounds in the plant, but also to where and how they are accumulated (Figueiredo & Barroso 2015) Plant metabolites in general are usually accumulated either in the vacuole, in plastids, in the cell wall, in the cuticle, and/or in specialized glandular structures (Figueiredo & Barroso 2015). Nevertheless, specialized glandular structures do not always have a regular distribution over the plant (Figueiredo et al., 2008), which, on itself, may determine different chemical profiles upon isolation. In addition, for instance metabolites accumulated in vacuole, are usually not volatile because they are accumulated in a soluble glycosylated form. To extract the volatile part of these molecules the glyosidic bound has to be broken prior to volatiles isolation (Belhattab et al. 2005). Ruppert-Aulabaugh (2014) reported two well known cases of essential oils isolated from the same plant, with different chemical profiles and different uses in aromatherapy. In one case the reason for this difference is the plant part used and in other the methodology of essential oil isolation used. Angelica archangelica

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provides a root essential oil and a seed essential oil. Whereas the seed essential oil is not considered a photosensitizer, the root essential oil may be, thus additional cautions should be taken on its use. Whereas the essential oil obtained, without heating, by the mechanical process of expression from the external part of the pericarp of Citrus × paradisi fruit is known to be photosensitizing, the distilled version is considered non-phototoxic. In addition to the variable chemical composition due to the use of different plant parts or different isolation procedures, the presence of chemotypes must also be evaluated for its influence in the biological activity of the essential oil. Chemotypes are chemically distinct groups within a species, that is, plants of the same species, which being phenotypically similar, differ in the type, or proportion, of their chemical constituents. The essential oils of several species such as Hypericum spp. (St. John's wort), Lavandula spp. (lavender), Mentha spp. (mint), Ocimum spp. (basil), Origanum spp. (oregano), Rosmarinus spp. (rosemary), Salvia spp. (salvia), or Thymus spp. (thyme), show several chemotypes. Althought not all essential oils show chemotypes, and only some chemotypes are available on the market, it is important to stress that different chemotypes may show diverse performance to a biological property, as shown with the example of Thymus caespititius essential oils chemotypes, Table 5. T. caespititius essential oils show four different chemotypes, carvacrol, sabinene, thymol and α-terpineol, although sabinene chemotype is much less frequent than the others (Figueiredo et al. 2008a). Dandlen et al. (2011), showed that from the three most common chemotypes, in general, the lowest acetylcholinesterase (AChE) inhibition activity was found with α-terpineol rich essential oils, Table 5. T. caespititius thymol and carvacrol rich essential oils were the most effective in AChE inhibition, although it is also clear from Table 5 that there are thymol and carvacrol rich essential oils which are less effective. This clearly shows that the effectiveness of an essential oil relies on the essential components as a whole and not just on its isolated constituents due to the existence of synergistic and antagonistic effects between its components. Data from Table 5 also shows that it is important to evaluate several samples, because the chemical composition will influence the final biological activity. Other studies (Faria et al. 2013), in which the essential oils were fractionated to evaluate the separate contribution of the fractions containing hydrocarbons or oxygen-containing molecules also highlighted distinct synergistic or antagonistic interactions between the essential oil components, influencing the overall activity.

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Table 5. Harvesting place, four main components and acetylcholinesterase (AChE) inhibition ability of Thymus caespititius essential oils, chemotypes carvacrol, α-terpineol and thymol (adapted from Dandlen et al., 2011). Thymus caespititius Essential Oil AChE inhibition

Harvesting place Four Main Components (%) (IC50 g/ml)* Caramulo (MP) α-Terpineol 40, p-cymene 14, γ-terpinene 5, τ-cadinol 5 5898.0 ±235.4 Covide (MP) α-Terpineol 35, p-cymene 17, γ-terpinene 9, τ-cadinol 6 4647.2 ±26.1 Lordelo (MP) α-Terpineol 24, p-cymene 16, γ-terpinene 12, τ-cadinol 7 1766.5 ±18.3 Óbidos (MP) α-Terpineol 52, p-cymene 15, γ-terpinene 7, τ-cadinol 6 12448.0 ±517.6 Outeiro (MP) α-Terpineol 41, p-cymene 14, γ-terpinene 9, β-caryophyllene 3 3000.6 ±125.3 Pico (A) Carvacrol 51, carvacryl acetate 19, p-cymene 6, γ-terpinene 4 567.4 ±12.8 Pico Verde (A) Carvacrol 32, thymol 23, carvacryl acetate 7, p-cymene 6 181.4 ±4.2 Planalto Central (A) Carvacrol 62, carvacryl acetate 12, α-terpineol 3, p-cymene 3 158.9 ±2.4 Ponta dos Rosais (A) Thymol 25, α-terpineol 19, p-cymene 12, γ-terpinene 10 3601.6 ±13.4 Serra do Cume (A) Thymol 35, carvacrol 13, p-cymene 8, thymyl acetate 8 139.1 ±2.5 Terras de Bouro (MP) α-Terpineol 24, γ-terpinene 14, p-cymene 12, γ-eudesmol 6 2579.1 ±2.3 Vilarinho das Furnas (MP) α-Terpineol 42, p-cymene 14, γ-terpinene 6, β-caryophyllene 4 4219.5 ±37.2 MP: Mainland Portugal. A: Azores archipelago, Portugal. * Concentration providing 50 % inhibition.

One other important point, often neglected, that contributes to chemical and biological activity variability is the chirality of essential oil components, when existing. Different enantiomers of chiral compounds often taste and smell differently and have different biological effects (Stahl-Biskup, 2002). Studies performed on the enantiomeric distribution of the chiral compounds present in high relative amount in T. caespititius essential oils, such as sabinene, terpinen-4-ol and α-terpineol (Pereira et al., 2000), or on the enantiomeric ratio of the monoterpene hydrocarbons -pinene, camphene, sabinene, β-pinene, limonene and of the oxygen-containing terpinen-4-ol, -terpineol and borneol in T. camphoratus essential oils (Miguel et al., 2009), showed clear enantiomeric polymorphism. Further enantioselective evaluation of essential oils may be of relevance in understanding the different biological performance of essential oils that show similar chemical composition. Biological activity assay conditions There is a large number of factors that additionally may affect the final biological activity of one essential oil, at and during the assay conditions, Table 6.

Table 6. Variability in biological activity assay conditions. Variability in assay conditions Different test types for one type of biological property evaluation For some biological properties, the diversity in organisms, strains or cell types to be evaluated Dose Way of application Moment of application Comparison with control substances Toxicological tests Direct contact assays do not necessarily reproduce the response in real environment

Not only can one essential oil be evaluated for the diverse reported biological properties, Table 1, but also each of these biological activities can be assessed in different ways. Considering any one of the reported biological properties of volatiles, essential oils and their constituents, there are always diverse ways of evaluating it, each of which will evaluate a particular characteristic performance, will deal differently with the essential oil volatility and poor water solubility.

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Table 7. Diversity of studies, and methodologies used, on the biological activity assessment of the essential oils, of different chemotypes, isolated from Thymus caespititius, grown in Portugal. Thymus caespititius Biological activity / Assay method Reference Antiacetilcolinesterase Carvacrol, thymol and AChE inhibition Dandlen et al. 2011a -terpineol chemotypes Thymol/carvacrol type AChE inhibition Aazza et al. 2016

Anti-hyperglycaemic Thymol/carvacrol type Inhibition of α-amylase and α-glucosidase Aazza et al. 2016

Anti-inflammatory Thymol/carvacrol type NO, 5-lipoxygenase assay Aazza et al. 2016

Antimicrobial Carvacrol, thymol and Growth inhibition of Helicobacter pylori (2 strains), Staphylococcus aureus, Dandlen et al. 2011 -terpineol chemotypes Salmonella enterica, Haemophilus influenzae, Streptococcus pneumoniae and Candida albicans (2 strains) by agar disc diffusion method and drop method for MIC -Terpineol type Growth inhibition of Aspergillus, Candida, Epidermophyton, Microsporum, Pinto et al. 2014 Tricophyton strains, by broth macrodilution method based on the Clinical and Laboratory Standards Institute (CLSI) reference documents M27-A3, S3, for MIC

Antioxidant -Terpineol type TBARS with and without ABTS Miguel et al. 2004 Carvacrol, thymol and DPPH•, hydroxyl and superoxide free radicals scavenging. TBARS assay Dandlen et al. 2010 -terpineol chemotypes Carvacrol, thymol and - ABTS and peroxyl free radicals scavenging Miguel et al. 2015 terpineol chemotypes Thymol/carvacrol type ABTS, ORAC, NO, Chelating, Liposomes, TBARS, 5-lipoxygenase assay Aazza et al. 2016

Antiproliferative / Cytotoxic Carvacrol type Cell line 23132/87 DSMZ nº ACC201, human gastric adenocarcinoma cell type, Dandlen et al. 2011 determined by Tetrazolium (MTT) method Carvacrol, thymol and THP-1 leukaemia cell line. Viability determined by Tetrazolium (MTT) colorimetric Miguel et al. 2015 -terpineol chemotypes assay

Nematicide Carvacrol type Direct contact assays against Bursaphelenchus xylophilus Barbosa et al. 2010 Carvacrol and -terpineol Direct contact assays against Bursaphelenchus xylophilus Faria et al. 2013 chemotypes Carvacrol, thymol and Meloidogyne chitwoodi hatching inhibition direct contact assays Faria et al. 2016 -terpineol chemotypes ABAP: 2,2’-azobis(2-amidinopropane). ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid). AChE: Acetylcholinesterase. DPPH: 2,2-diphenyl-1-picrylhydrazyl. MIC: minimal inhibitory concentration, MLC: minimal lethal concentration. NO: Nitric oxide. ORAC: Oxygen Radical Absorbance Capacity. TBARS: Thiobarbituric acid reactive species.

If, as an example, antinociceptive / analgesic evaluation tests are considered, studies may address a) transient pain, by applying thermal, mechanical, electrical and chemical stimulation, or b) continuous pain, by inducing polyarthritis. These may involve, the abdominal writhe test, capsaicin-injection into the mouse hind paw, carrageen induced paw oedema, formalin-induced nociception, hot-plate test, pain-induced functional impairment model in the rat (PIFIR model), tail-flick test, tail immersion test, among several others.

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Many times the results are not directly comparable, not all of them use controls, or test both the essential oil and essential oil constituents (Adorjan & Buchbauer, 2010; Lenardão et al., 2016). Combining the variability of biological properties possible, Table 1, the tests available and the chemical variability, it is easy to understand the diversity of data obtainable, even for less studied species, Table 7. The lower performance, or absence of activity when using one test does not mean that a positive results is not obtained with another type of test to evaluate a certain biological property, Table 8. The same essential oil can also exhibit different capabilities toward different strains of the same microorganism, Table 9.

Table 8. Antioxidant activity of Thymus caespititius essential oil thymol/carvacrol type assessed by different methodologies (adapted from Aazza et al., 2016). Antioxidant activity tests Thymus caespititius essential oil

thymol/carvacrol type [IC50 (µg/ml)] ABTS 10.0 ±0.0 ORAC* 2152.6 ±16.4* NO 300.0 ±0.1 Chelating - Liposomes 160.0 ±0.0 TBARS 130.0 ±0.0 ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid, ORAC: Oxygen Radical Absorbance Capacity, NO: Nitric oxide, TBARS: Thiobarbituric acid reactive species. * Micromoles Trolox equivalent (TE)/g

Table 9. Growth inhibition of two Helicobacter pylori strains with different samples of Thymus zygis essential oils (adapted from Dandlen et al., 2011). Thymus species essential oil Harvesting place EO main component (%) Helicobacter pylori strain [Inhibition zone (mm)] J99 26695 T. zygis sylvestris Alcanena Carvacrol 32 7.7 ±2.9 15.3 ±0.6 T. zygis sylvestris Condeixa p-Cymene 36 10.0 ±0.0 18.0 ±0.0 T. zygis sylvestris Covão do Coelho Carvacrol 35 10.3 ±0.3 17.3 ±1.5 T. zygis sylvestris Duas Igrejas p-Cymene 39 7.3 ±2.3 18.7 ±0.6 T. zygis zygis Rebordãos Carvacrol 44 16.0 ±1.7 20.8 ±1.4 Chloramphenicol / Amphotericin B 36.0 ±1.0 36.3 ±0.6

Other factors are also extremely important and determinant in the variability of results observed, Table 6, such as the dose of essential oil used, the way it is applied (pure, vapour phase or dispersion, in an oily carrier, in a wetting agent, in an organic solvent, in liposomes, among others), or the moment of application (for antimicrobial activity the growth phase and developmental stage of the microorganism is important). The use of positive control substances, whenever possible, is also relevant to asses not only a comparative performance, but also to evaluate if the test is working properly. Likewise, negative controls should be assessed, namely to discard secondary activities from any type of carrier, as well as toxicological tests to determine the toxic effect on non-target organisms. Most of the times the tests are performed in direct contact assays which do not necessarily reproduce the response in real environment, where other abiotic (pH, lixiviation, light, temperature, etc.) or biotic (biotransformation by cells or organisms) factors may drastically change the response to the essential oil. In conclusion, available data on the biological properties of essential oils is very broad, and, sometimes, results are not consistent. A number of factors which may be considered potential sources of this variability were addressed, namely type of extract, botanical certification, chemical polymorphism and assay conditions.

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Although the use of essential oils is Generally Regarded as Safe (GRAS), care should be taken on their use, since the way of action is still many times not fully understood, and/or unspecific, that is, both target and non-target cells or organisms may be affected. In view of this, after an initial screening of the effectiveness of essential oils, a further step should be taken to assess the toxicity to non-target cells. At this level, biotechnological models (Faria et al., 2015; Faria et al., 2016) may provide an intermediary step to evaluate the lowest effective concentrations that does not harm non-target cells, or the existence of mechanisms of detoxification and/or biotransformation, by either target and non-target cells or organisms.

Partially funded by Fundação para a Ciência e a Tecnologia under UID/AMB/50017/2013, FEDER PT2020-Compete 2020. Part of this work was presented in ISEO 2017, Pecs, Hungary.

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Stahl-Biskup, E. (2002). Essential oil chemistry of the genus Thymus – a global view. In Stahl-Biskup, E., Sáez, F. (Eds), Medicinal and Aromatic Plants – industrial profiles, vol. 17, pp. 75-124. Taylor & Francis, London. Sullivan, S. (2009). Caracterização química de óleos essenciais isolados de fitoterápicos e comerciais utilizados em aromaterapia: Eucalyptus globulus, Foeniculum vulgare e Mentha piperita. Relatório de Estágio Científico, Faculdade de Ciências da Universidade de Lisboa e Instituto de Medicina Tradicional. Ventura, C., & Lima, C. (2017). Óleos essenciais, produtos derivados e normalização. Estudo Orientado em Biologia Molecular e Genética, Faculdade de Ciências da Universidade de Lisboa.

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REVIEW Systematic and Biogeographic overview of Lamiaceae in Turkey

Ferhat Celep1,* and Tuncay Dirmenci2

1 Mehmet Akif Ersoy mah. 269. cad. Urankent Prestij Konutları, C16 Blok, No: 53, Demetevler, Ankara, TURKEY 2 Biology Education, Necatibey Education Faculty, Balıkesir University, Balıkesir, TURKEY

*Corresponding author. E-mail: [email protected]

Abstract Lamiaceae is the third largest family based on the taxon number and fourth largest family based on the species number in Turkey. The family has 48 genera and 782 taxa (603 species, 179 subspecies and varieties), 346 taxa (271 species, 75 subspecies and varieties) of which are endemic (ca. 44%) (data updated 1th February 2017) in the country. There are also 23 hybrid species, 19 of which are endemic (82%). The results proven that Turkey is one of the centers of diversity for Lamiaceae in the Old World. In addition, Turkey has about 10% of all Lamiaceae members in the World. The largest five genera in the country based on the taxon number are Stachys (118 taxa), Salvia (107 taxa), Sideritis (54 taxa), Phlomis (53 taxa) and Teucrium (49 taxa). According to taxon number, five genera with the highest endemism ratio are Dorystaechas (1 taxon, 100%), Lophantus (1 taxon, 100%), Sideritis (54 taxa, 74%), Drymosiphon (9 taxa, 67%), and Marrubium (27 taxa, 63%). There are two monotypic genera in Turkey as Dorystaechas and Pentapleura. Turkey sits on the junction of three phytogeographic regions with highly diverse climate and the other ecologic features. Phytogeographic distribution of Turkish Lamiaceae taxa are 293 taxa in the Mediterranean (37.4%), 267 taxa in the Irano-Turanian (36.7%), 90 taxa in the Euro-Siberian (Circumboreal) phytogeographic region, and 112 taxa in Unknown or Multiregional (14.3%) phytogeographical elements. In the Mediterranean phytogeographic region 61% of the taxa, in the Irano-Turanian phytogeographic region 50% of the taxa, and in the Euro-Siberian (Circumboreal) phytogeographic region 13% of the taxa are endemic. Some endemic taxa are widely distributed in the country, so their phytogeograhic elements are not clearly defined, the endemism ratio of these taxa are 13%. In time, species of Lamiaceae genera are migrated from one phytogeographic region to another one and specialised in specific habitats in the country. Salvia, Stachys, Sideritis, Phlomis, Teucrium, Thymus, Nepeta, Scutellaria, Origanum and Marrubium are species rich genera in Turkey. Particulary, most of Old Wold species of Salvia, Stachys, Origanum, Marrubium, Ballota, Lamium and Drymosiphon naturally grow in Turkey. These genera have also quite high endemism ratio with Phlomis, Scutellaria, Thymus, Nepeta and Satureja. Due to high taxon number and endemism ratio, different habitats, climates and soil types, high altitutinal range and diverse pollinators, it is clear that Turkey is a very good example for evolution and speciation of Lamiaceae family in the Old World.

Keywords: Lamiaceae, Systematics, Biogeography, Turkey

Introduction

Turkey has a great variety of biotic and abiotic diversity, i.e. climatic, edaphic, geographic & geologic, and pollinator diversity. These factors leads to Turkey is one of the most important plant biodiversity centers in the world with over 10 000 plant species and ca. 35 % endemism ratio (Güner et al., 2012; Celep et al., unpublished data). In addition, three different phytogeographic regions are come together in the country, as Mediterranean, Irano-Turanian and Euro-Siberian (Circumboreal) phytogeographic regions (Zohary, 1973; Davis, 1975; Thomson, 2005). Areas surrounding the Mediterranean, Aegean, and Marmara Seas and a little further inside in mainland of Turkey show the characteristics of the Mediterranean phytogeographic region. The large part of Turkey including Central Anatolia, East Anatolia and Southeast Anatolia lies in the Irano-Turanian phytogeographic region. The Black Sea’s coastal areas (including norhern part of Black Sea geographic region and northeastern part of Marmara geographic region) lie in the Euro-Siberian (Circumboreal) phytogeographic region (Davis, 1971) (Figure 1). Endemic species are mostly found in the Mediterranean and Irano-Turanian

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phytogeographic regions. In regard to Lamiaceae, Turkey sits on one of the seven regions of high Lamiaceae diversity center as "Mediteranean and SW Central Asia" (Harley et al., 2004). Lamiaceae, the sixth largest Angiosperm family, contains more than 245 genera and 7886 species, and distributed worldwide (The Plant List, 2013, access 1th Feb 2017). It includes many economically and medicinally important species (Harley et al., 2004). In Lamiaceae, Harley et al. (2004) recognized seven subfamilies as Ajugoideae, Lamioideae, Nepetoideae, Prostantheroideae, Scutellarioideae, Symphorematoideae and Viticoideae. Recently, five new additional subfamilies have been described, namely Cymarioideae, Peronematoideae, Premnoideae, Callicarpoideae and Tectonoideae (Li et al., 2016; Li & Olmstead, 2017). In Turkey, there are five subfamilies as Ajugoideae, Lamioideae, Nepetoideae, Scutellarioideae and Viticoideae (Harley et al., 2004; Li et al., 2016; Li & Olmstead, 2017). In the family, the largest genera are Salvia L. (945 species), Scutellaria L. (360 species), Stachys L. (300 species), Plectranthus L'Hér. (300 species), Hyptis Jacq. (280 species), Teucrium L. (250 species), Vitex L. (250 species), Thymus L. (220 species) and Nepeta L. (200 species) (Harley et al., 2004; Will et al., 2015). After the publication of Flora of Turkey and the East Aegean Islands volume 7 (Davis, 1982), volume 10 (Davis et al., 1988) and volume 11 (Güner et al., 2000), which include Lamiaceae members, many new taxa have been published from Turkey (Güner et al., 2012; IPNI-The International Plant Name Index, access 1th February 2017). In addition, some taxonomic and nomenclatural changes have been done by classical and molecular taxonomists for last three decades (The Plant List, access 30 Sep. 2017). In this review, we have updated the latest taxonomic status of genera and species in the family for Turkish Lamiaceae members. In addition, we have provided some geographic and biogeographic/phytogeographic data based on species/taxon distribution pattern. Finally, we have suggested some hypothesis about origins of Turkish Lamiaceae members. Materials and Methods

This is a review study on Turkish Lamiaceae members. It was presented in International Symposium on Advances in Lamiaceae Science, April 26-29 2017, Antalya, Turkey, therefore the data used in this paper was prepared at 1th of February 2017. After presentation of data, two new endemic Lamiaceae species have been published from Turkey as Micromeria aybalae (Duman & Dirmenci, 2017) and Lamium bilgilii (Celep, 2017). When preparing the data set, we reviewed the current literature and related web sites (Davis, 1982; Güner et al., 2012; The Plant List, 2017; IPNI-The International Plant Name Index, access 1th February 2017). Based on the updated data set, we produced relevant informations and statistics for Turkish genera and species. In this data set, taxon or species which were only known from in the East Aegean Islands (Greece Islands) are exluded. Only taxa or species are known from Turkey mainland (Anatolia/Asian part of Turkey & Thrace region/European part of Turkey) are included. Phytogeographic regions of Lamiaceae taxa were mostly obtained from related literatures (Davis, 1982; Güner et al., 2012). If the phytogeographic element of an endemic taxon is not known, we determined it based on its biogeographic distribution pattern and habitat in this study. To preventing wrong placement of the phytogeographic region, we only determined new phytogeographic region for Turkish endemic taxa. Phytogeographic regions of Turkey is shown in Figure 1.

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Figure 1. Phytogeographic regions of Turkey (Davis, 1965; modified by Avcı, 1996).

Results and Discussion

According to our data set, there are 48 genera in Lamiaceae in Turkey with diverse habit, flower morphology, size and color (Figure 2 & 3). Recently, Drew et al. (2017) included some genera (Dorystaechas, Meriandra, Perovskia, Rosmarinus, and Zhumeria) in Salvia based on comprehensive molecular studies. On the other hand, Will and Classen-Bockhoff (2017) suggested to split Salvia s.l. into six genera with other closely related genera i.e. Dorystaechas and Rosmarinus. Although both studies (Drew et al., 2017, Will and Classen- Bockhoff, 2017) have similar data and results, Drew et al. (2017) and Will & Classen-Bockhoff (2017) reached different conclusions based on their point of view and interpretation. Due to ongoing molecular and taxonomic studies on generic status of Dorystaechas and Rosmarinus, we accepted them as still valid genera in this study as given in the Flora of Turkey. The genus Lophanthus Adans. has 23 species and mostly distributed in China, Mongolia, Afghanistan and Central Asia (Harley et al., 2004). Dirmenci et al. (2010) described Lophanthus turcicus Dirmenci, Yildiz & Hedge from eastern Anatolia. The genus represented only this endemic species in Turkey. Recently, Serpooshan et al. (2017) did a molecular phylogenetic study on Hymenocrater, Nepeta, Lophanthus and the other closely related genera. In this study, Serpooshan et al. (2017) made some taxonomic changes among studied genera, for example they transfered Lophantus turcicus into Nepeta as Nepeta turcica (Dirmenci, Yildiz & Hedge) Jamzad & Serpooshan. According to Serpooshan et al. (2017), a complete DNA sequencing of Lophanthus species with Nepeta and Hymenocrater may provide more resolved phylogenetic tree and more accurate interpretation on taxonomy of the genera. Until we will have more data about Lophanthus turcicus, we accepted it as a valid species in this study.

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Figure 2. Some Lamiaceae taxa from Turkey

A. Stachys ketenoglui B. Stachys thracica C. Salvia albimaculata D. Salvia argentea E. Ajuga vestita (Photos A, B, D, & E by T. Dirmenci, C by F. Celep).

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Figure 3. Some Lamiaceae taxa from Turkey

A. Origanum husnucan-baseri B. Lamium eriocephalum C. Lamium galeobdolon D. Sideritis trojana E. Teucruim alyssifolium F. Ziziphora taurica G. Nepeta cadmea (Photos A, D, E, F & G by T. Dirmenci, B by F. Celep).

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Lamiaceae in Turkey According to our data set which updated at 1th of February 2017, there are 782 Lamiaceae taxa in Turkey, 346 of which are endemic (44.2 % endemism ratio). According to species number, there are 603 Lamiaceae species in Turkey, 271 of which are endemic (44.9 % endemisim ratio). There are 23 hybrid species, 19 of which are endemic (82 % endemism ratio). The largest 15 genera based on taxon number in Turkey are given in Table 1.

Table 1. The largest 15 genera based on the taxon number in Turkish Lamiaceae Genera Taxon Species Endemic taxa number Endemic species number number number and endemism ratio (%) and endemism ratio 1 Stachys 118 90 53 (45%) 43 (48%) 2 Salvia 107 100 58 (54%) 53 (53%) 3 Sideritis 54 45 40 (74%) 36 (80 %) 4 Phlomis 53 33 30 (57%) 16 (48%) 5 Teucrium 49 36 17 (35%) 15 (42%) 6 Thymus 47 42 20 (43%) 20 (48%) 7 Nepeta 46 39 20 (43%) 17 (44%) 8 Scutellaria 39 17 17 (44%) 6 (35%) 9 Origanum 31 27 18 (58%) 18 (67%) 10 Marrubium 27 21 17 (63%) 11 (52%) 11 Lamium 26 15 5 (19%) 4 (27%) 12 Clinopodium 25 16 7 (24%) 3 (19%) 13 Ajuga 23 13 7 (30%) 6 (46%) 14 Ballota 18 12 11 (61%) 8 (67%) 15 Satureja 17 16 6 (35%) 5 (31%) Sideritis, Drymosiphon, Marrubium, Ballota, Origanum, Phlomis, Salvia have a large number endemic taxa and species. Their endemism ratio is over 54 % in Turkey. There are two monotypic genera in Turkey as Dorystaechas (endemic) and Pentapleura (non-endemic). Though Lophanthus has also only one species in Turkey, it is not a monotypic genus. Fifteen genera with the highest endemism ratio are given in Table 2.

Table 2. Fifteen genera with the highest endemism ratio in Lamiaceae in Turkey Genera Endemism ratio based on Endemism ratio based on taxa number (%) species number (%) 1 Dorystaechas 100 100 2 Lophantus 100 100 3 Sideritis 74 80 4 Drymosiphon 67 75 5 Marrubium 63 52 6 Ballota 61 67 7 Origanum 58 67 8 Phlomis 57 48 9 Salvia 54 53 10 Stachys 45 48 11 Scutellaria 44 35 12 Thymus 43 48 13 Nepeta 43 44 14 Teucrium 35 42 15 Satureja 35 31

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List of Turkish Lamiaceae Genera based on their Subfamilial Classification In Turkey, there are five subfamilies as Viticoideae, Ajugoideae, Scutellarioideae, Lamioideae, and Nepetoideae. We prepared subfamilial classification of Turkish Lamiaceae genera according to Harley et al., 2004; Li et al., 2016; Li & Olmstead, 2017. 1. Subfamily Viticoideae: Vitex. 2. Subfamily Ajugoideae: Ajuga, Teucrium, Clerodendrum. 3. Subfamily Scutellarioideae: Scutellaria. 4. Subfamily Lamioideae: Marrubium, Sideritis, Ballota, Eremostachys, Phlomis, Phlomoides, Stachys, Betonica, Prasium, Leonurus, Chaiturus, Galeopsis, Molucella, Lamium, Melittis. 5. Subfamily Nepetoideae: Tribe Elsholtzieae: Perilla, Elsholtzia Tribe Mentheae: Nepeta, Lycopus, Dorystaechas, Rosmarinus, Ziziphora, Salvia, Clinopodium, Drymosiphon, Hymenocrater, Lallemantia, Dracocephalum, Glechoma, Lophanthus, Prunella, Micromeria, Cyclotrichium, Melissa, Origanum, Mentha, Thymbra, Thymus, Hyssopus, Satureja, Pentapleura. Tribe Ocimeae: Lavandula, Ocimum. Phytogeographic distribution of Lamiaceae taxa in Turkey (total 782 taxa) According to our updated data, 287 taxa (36.7 %) are in the Irano-Turanian phytogeographic region, 293 taxa (37.4 %) are in the Mediterranean phytogeographic region, 90 taxa (11.5%) are in the Euro-Siberian (Circumboreal) phytogeographic region, and 112 taxa (14.3 %) are Unknown or multiregional element in Turkey. Though Irano-Turanian phytogeographic region covers larger area than the Mediterranean phytogeographic region in Turkey, both phytogeographic regions have about the same number taxa. The least number taxa are found in the Euro-Siberian phytogeographic region (Figure 4).

Figure 4. Phytogeographic distribution of Lamiaceae taxa in Turkey

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Phytogeographic distribution of Lamiaceae species in Turkey (total 603 species) Irano-Turanian element: 229 species, 37.9 % in total. Mediterranean element: 220 species, 36.4 % in total. Euro-Siberian (Circumboreal) element: 72 species, 11.9 % in total. Unknown or multiregional element: 82 species, 13.6 % in total. Phytogeographic distribution based on the hybrid species in Turkey (total 23 species) Irano-Turanian element: 8 species, 34.7 % in total. Mediterranean element: 10 species, 43.5 % in total. Euro-Siberian (Circumboreal) element: 2 species, 8.7 % in total. Unknown or multiregional element: 3 species, 13 % in total. Endemism in the Phytogeographic regions In the Irano-Turanian phytogeographic region: Total 287 taxa, 144 of which are endemic (50%). Total 229 species, 115 of which are endemic (50%). In the Mediterranean phytogeographic region: Total 293 taxa, 179 of which are endemic (61%). Total 217 species, 139 of which are endemic (64%). In the Euro-Siberian phytogeographic region: Total 90 taxa, 12 of which are endemic (13%). Total 72 species, 9 of which are endemic (12.5%). Unknown or multiregional element: Total 112 taxa, 12 of which are endemic (11%). Total 82 species, 9 of which are endemic (11%). According to the results, the highest number of taxa (293 taxa) and endemism ratio (64 %) are seen in the Mediterranean phytogeographic region. The second highest number of taxa (287 taxa) and endemism ratio (50 %) are seen in the Irano-Turanian phytogeographic region. The least number of taxa (90 taxa) and endemism (13 %) are seen in the Euro-Siberian (Circumboreal) phytogeographic region (Figure 5).

Figure 5. Total taxa in the each phytogeographic region (blue column), and endemic taxa number in each phytogeographic region (red column).

Phytogeographic properties of the largest 15 genera in Lamiaceae in Turkey are given in Table 3. Most taxa in Salvia, Nepeta, Thymus, Scutellaria and Marrubium are distributed in the Irano-Turanian phytogeographic region. Except for Nepeta (29 %), endemism ratio of these genera are over 52% in the Irano-Turanian

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phytogeographic region (Table 3). On the other hand, though Salvia, Nepeta and Marrubium has lower number taxa in the Mediterranean region, they have higher endemism ratio in the Mediterranean phytogeographic region than Irano-Turanian phytogeographic region (Table 3) in the country. Most taxa of Sideritis, Teucrium, Ballota and Origanum are distributed in the Mediterranean phytogeographic region with a high endemism ratio. Though Sideritis, Ballota and Origanum has lower number taxa in the Irano-Turanian phytogeographic region than Mediterranean phytogeographic region, they have over 75 % endemism ratio in the Irano-Turanian phytogeographic region (Table 3) in the country. Both Stachys and Phlomis have more or less equal number taxa in the Irano-Turanian and Mediterranean phytogeographic regions. While most Stachys endemic taxa (63%) are distributed in the Mediterranean phytogeographic region, most Phlomis endemic taxa (70%) are distributed in the Irano-Turanian phytogeographic region (Table 3).

Table 3. Phytogeographic distribution of the genera based on the taxon number Genera (Total Number of Number of Number of Euro- Number of Total endemic taxon number) Irano-Tur. Medit. Sib. Unknown or taxa number Element/ Element/ (Circumboreal) Multiregional/ and endemism Endemic taxa Endemic taxa Element/ Endemic taxa ratio (%) in the (%) in the (%) in the Endemic taxa (%) (%) in the genus region region in the region region 1 Stachys (118) 41/19 (46%) 46/29 (63%) 16/3 (%19) 15/3 (20%) 53 (45%) 2 Salvia (107) 65/35 (54%) 30/20 (67%) 7/1(%14) 5/2 (40%) 58 (54%) 3 Sideritis (54) 10/8 (80%) 38/29 (76%) 4/2 (50%) 2/1 (50) 40 (74%) 4 Phlomis (53) 23/16 (70%) 22/12 (55%) 1/1 (100%) 7/1 (14%) 30 (57%) 5 Teucrium (49) 13/4 (31%) 24/13 (54%) 5/0 (0%) 7/0 (0%) 17 (35%) 6 Thymus (47) 20/13 (65%) 12/5 (42%) 7/2 (29%) 8/0 (0%) 20 (43%) 7 Nepeta (46) 24/7 (29%) 17/13 (76%) 2/0 (0%) 3/0 (0%) 20 (43%) 8 Scutellaria (39) 21/11 (52%) 9/4 (44%) 4/0 (0%) 5/2 (40%) 17 (44%) 9 Origanum (31) 4/3 (75%) 20/15 (75%) 2/0 (0%) 5/0 (0%) 18 (58%) 10 Marrubium (27) 14/10 (71%) 8/7 (88%) - 5/1 (20%) 17 (63%) 11 Lamium (26) 6/1 (17%) 5/2 (40%) 8/2 (25%) 7/0 (0%) 5 (19%) 12 Clinopodium (25) 3/1 (33%) 5/2 (40%) 8/1 (13%) 6/0 (0%) 7 (24%) 13 Ajuga (23) 8/4 (50%) 7/3 (43%) 3/0 (0%) 5/0 (0%) 7 (30%) 14 Ballota (18) 5/4 (80%) 10/7 (70%) 2/0 (0%) 1/0 (0%) 11 (61%) 15 Satureja (17) 4/1 (25/) 6/3 (50%) 2/0 (0%) - 6 (35%)

Geographic distribution of Turkish Lamiaceae taxa Turkey has seven geographic regions and their subregions (Table 4, Figure 6). Results shown that most of Turkish Lamiaceae taxa are distributed in the Mediterranean geographic region, particularly in the eastern part of the Mediterranean geographic region with 283 taxa. The highest endemism ratio is found in the western part of the Mediterranean geographic region (43.5%). The other high taxon rich areas are western Aegean and western part of East Anatolia regions with 188 and 180 taxa, respectively. While Central Anatolian geographic region has lower taxa than the Black Sea geographic region, it has higher endemism ratio than the Black Sea geographic region (Table 4, Figure 6). Both the least number taxa and endemism ratio are seen in the Marmara geographic region, particularly in the European part of Turkey (Thrace region). Southeast Anatolia and southern part of East Anatolia have moderate number of taxa and endemism (Table 4, Figure 6).

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Table 4. Geographic distribution of Lamiaceae taxa in Turkey (see Figure 6 for Geographic regions and divisions). Geographic regions Geographic division Taxon number Endemic taxon number Endemism ratio (%) Marmara 1a 75 1 1.3 1b 101 4 4.0 1c 85 3 4.0 1d 120 13 10.8 Black Sea 2a 121 20 16.5 2b 131 20 15.2 2c 156 21 13.4 Aegean 3a 188 51 27.1 3b 118 31 26.2 Central Anatolia 4a 101 35 34.6 4b 117 45 38.4 4c 97 39 40.2 4d 97 30 30.9 East Anatolia 5a 180 65 36.1 5b 140 20 14.2 5c 145 27 18.6 5d 118 13 11 Mediterranean Region 6a 232 101 43.5 6b 283 109 38.5 Souteast Anatolia 7a 92 20 21.7 7b 105 15 14.2

Figure 6. Geographic regions and Taxon Distribution of Lamiaceae taxa in Turkey. (Arrows below show supposed taxon movement or migration routes, endemism ratio for each subgeographic region are given in the paranthesis).

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Supposed hypothesis on origins of Turkish Lamiaceae taxa After reviewing all available data on Turkish Lamiaceae taxa, we have proposed three hypotheses about origins of Turkish Lamiaceae members: 1. Manafzadeh et al. (2014) reported that the Irano-Turanian phytogeographic region represents one of the hotspots of evolutionary and biological diversity in the Old World, and serves as a source of xerophytic taxa. For this reason, we may assume that some Lamiaceae genera (i.e. Salvia, Nepeta, Thymus, Scutellaria and Marrubium) originated in the Irano-Turanian phytogeographic region and then they spread into mountainous Mediterrenean Phytogeographic region of Turkey, particularly in the northern face of Taurus Mountains. Anatolian Diagonal may played crucial role for migration of taxa for both directions from Irano-Turanian to the Mediterranean phytogeographic region. Although Salvia, Nepeta and Marrubium has lower number taxa in the Mediterranean phytogeographic region than the Irano-Turanian phytogeographic region in Turkey, their endemism ratio is higher in the Mediterranean phytogeographic region. Most of endemic or non-endemic taxa of these genera are locally distributed in the Mediterranean phytogeographic region at modetare (800-1500 m) or high altitute (1500- 3000 m). Taxa of these genera may penetrated into the Mediterranean phytogeographic region during Glacial times and then adapted to Mediterranean environment. When comparing species habitat of these genera in the Mediterranen phytogeographic region in Turkey, they grow relatively similar climate both in the Irano- Turanian and in the Mediterranean phytogeographic regions (Table 5).

Table 5. The genera which mostly distributed in the Irano-Turanian phytogeographic region. Genera (Total Ir.-Tur. Medit. Euro-Sib. Unknown or Total endemic taxon Element- Element- (Circumboreal) Multiregional- taxa number and number) Endemic taxa Endemic taxa Element-Endemic Endemic taxa endemism ratio (%) in the (%) in the taxa (%) in the (%) in the (%) region region region region

1 Salvia (107) 65/35 (54%) 30/20 (67%) 7/1(%14) 5/2 (40%) 58 (54%)

2 Nepeta (46) 24/7 (29%) 17/13 (76%) 2/0 (0%) 3/0 (0%) 20 (43%)

3 Thymus (47) 20/13 (65%) 12/5 (42%) 7/2 (29%) 8/0 (0%) 20 (43%)

4 Scutellaria (39) 21/11 (52%) 9/4 (44%) 4/0 (0%) 5/2 (40%) 17 (44%)

5 Marrubium (27) 14/10 (71%) 8/7 (88%) - 5/1 (20%) 17 (63%)

2. Some genera (Sideritis, Origanum, Teucrium) are mostly distributed and originated in the Mediterranean Phytogeographic region (Iestwaart, 1980; Greuter et al., 1986; Barber et al., 2007). It is also true for Turkey (Table 6). According to data, though Sideritis and Origanum have lower number taxa in the Irano-Turanian phytogeographic region than the Mediterranean phytogeographic region, they are mostly endemic and locally distributed in the Irano-Turanian phtogeographic region. Therefore, we may assume that these species could be migrated from the Mediterranean phytogeographic region to Irano-Turanian phytogeographic region and then specialized in there.

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Table 6. The genera which mostly distributed in the Mediterranean phytogeographic region. Genera (Total Ir.-Tur. Medit. Euro-Sib. Unknown or Total endemic taxon number) Element/En Element/En (Circumboreal) Multiregional/ taxa number and demic taxa demic taxa Element/Endemic Endemic taxa endemism ratio (%) in the (%) in the taxa (%) in the (%) in the (%) region region region region 1 Sideritis (54) 10/8 (80%) 38/29 (76%) 4/2 (50%) 2/1 (50) 40 (74%) 2 Teucrium (49) 13/4 (31%) 24/13 (54%) 5/0 (0%) 7/0 (0%) 17 (35%) 3 Origanum (31) 4/3 (75%) 20/15 (75%) 2/0 (0%) 5/0 (0%) 18 (58%)

3. Some cosmopolitan Lamiaceae genera are also distributed in Turkey. Some of their members are specialized in the specific habitats in Turkey in time. Stachys (Harley et al., 2004) and Phlomis (Mathiesen et al., 2011) have a fairly large number of taxa in Turkey. While Stachys and Phlomis taxa are distributed in all parts of the country, most of endemic and non-endemic taxa are localized in the Irano-Turanian and Mediterranean phytogeographic regions of Turkey (Table 7).

Table 7. Example for cosmopolitan Lamiaceae genera in Turkey. Genera Ir.-Tur. Meditterrenean Euro-Siberian Unknown or Total endemic (Total taxon Element- Element- (Circumboreal) Multiregional- taxa number number) Endemic Endemic taxa Element-Endemic Endemic taxa and endemism taxa (%) in (%) in the taxa (%) in the (%) in the ratio (%) the region region region region 1 Stachys (118) 41/19 (46%) 46/29 (63%) 16/3 (%19) 15/3 (20%) 53 (45%) 2 Phlomis (53) 23/16 (70%) 22/12 (55%) 1/1 (100%) 7/1 (14%) 30 (57%)

In the Euro-Siberian phytogeographic region (northern Turkey), cosmopolitan and non-endemic Lamiaceae taxa are mostly distributed. European part of Turkey (western Euro-Siberian phytogeographic region in Turkey) is similar to European Lamiaceae flora. On the other hand, northeastern Turkey (eastern Euro- Siberian phytogeographic region in Turkey) is similar to Caucasian Lamiaceae flora. To sum up, the results pointed out that Turkey is one of the species biodiversity centers in Lamiaceae in the Old World. Salvia, Stachys, Sideritis, Phlomis, Teucrium, Thymus, Nepeta, Scutellaria, Origanum and Marrubium are species rich genera in Turkey. Particulary, most of Old Wold species of Salvia, Stachys, Origanum, Marrubium, Ballota, Lamium and Drymosiphon naturally grow in Turkey. These genera have also quite high endemism ratio with Phlomis, Scutellaria, Thymus, Nepeta and Satureja. Taxonomic position of Dorystaechas hastata, Rosmarinus officinalis and Lophanthus turcicus are still under discussions. Their final taxonomic positions will be clarified with additional molecular and systematic studies. Most of the endemic and non-endemic Turkish Lamiaceae taxa are distributed in the Mediterranean (total 293 taxa, 179 taxa of which are endemic) and Irano-Turanian (total 287 taxa, 144 taxa of which are endemic) phytogeographic regions. Less and mostly non-endemic Lamiaceae taxa are distributed in the Euro-Siberian (Circumboreal) (total 90 taxa, 12 taxa of which are endemic) phytogeographic region. Mediterranean geographic region of Turkey has the most number endemic and non endemic taxa in Turkey (Figure 6). The other geographic regions where high endemism ratio are Central Anatolia, Aegean region and western part of Eastern Anatolia. The lowest number endemic and non-endemic taxa are found in the European part of Turkey (Figure 6).

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Due to its abiotic and biotic diversity (geology, climate, geography, phytogeography, edaphic, Anatolian Diagonal, pollinators), 782 Lamiaceae taxa (603 species), 346 taxa of which are endemic, are naturally grow in Turkey. We hypothesize that some Lamiaceae genera originated in the Mediterranean phytogeographic region (i.e. Sideritis, Origanum) and some others originated in the Irano-Turanian phytogeographic (i.e. Nepeta, Thymus) regions. In time, species of these genera migrated from one phytogeographic region to another one, and localised in specific habitats. Euro-Siberian Lamiaceae flora in Turkey is similar to European and Caucasus Lamiaceae flora. Interestingly, we have not seen a genus of Lamiaceae, which is mostly distributed with a high endemism ratio in the Euro-Siberian (Circumboreal) phytogeographic region of Turkey. In addition, ca. 112 taxa, which are unknown or present in multiregional phytogeographic regions in Turkey, are widely distributed in the country. In conclusion, Turkey is a very good example for evolution and speciation of the Lamiaceae family. Our hypotheses to explain origins of Lamiaceae in Turkey need to be further studies based on molecular phylogenetic, molecular dating and biogeographic studies with geological data.

ACKNOWLEDGMENT We thank Prof. Dr. Kemal Hüsnü Can Başer (Near East University, Cyprus) for inviting us to the International Symposium on Advances in Lamiaceae Science, April 26-29 2017, Antalya, Turkey, where part of this work was presented. We are grateful to Prof. Dr. Fatih Demirci (Anadolu University, Faculty of Pharmacy) for his works on the manuscript.

REFERENCES

Avcı, M. (1996). The floristic regions of Turkey and a geographical approach for Anatolian diagonal, Review of the Department of Geography, University of Istanbul, 3, 59-91. Barber, J. C., Finch, C. C., Francisco-Ortega J., Santos-Guerra A. & Jansen R. K. (2007). Hybridization in Macaronesian Sideritis (Lamiaceae): evidence from incongruence of multiple independent nuclear and chloroplast sequence datasets. Taxon, 56, 74-88. Celep, F. 2017. Lamium bilgilii (Lamiaceae), a new species from South-western Turkey (Burdur-Muğla). Phytotaxa, 312, 263-270. Davis, P. H. (Ed.) (1965). Flora of Turkey and the East Aegean Islands, Vol. 1, Edinburgh: University Press. Davis, P. H. (1971). Distribution Patterns in Anatolia with Particular Reference to Endemism, in Plant Life of South West Asia, P. H. Davis (Ed.), pp. 15–27, Botanical Society of Edinburgh. Davis, P.H. (1975). Turkey: Present State of Floristic Knowledge. Coll Int C N R S. 235, 93-113. Davis, P. H. (ed.) (1982). Flora of Turkey and the East Aegean Islands. Volume 7. Edinburgh: University Press. Davis, P.H. (ed.) (1988). Flora of Turkey and the East Aegean Islands, Volume 10. Edinburgh: University Press. Davis, P.H. (ed.) (2000). Flora of Turkey and the East Aegean Islands, Volume 11. Edinburgh: University Press. Dirmenci, T., Yıldız, B., Hedge, I. C., Fırat, M. (2010). Lophantus (Lamiaceae) in Turkey: a new generic record and a new species. Turkish Journal of Botany, 34, 123-129. Drew, B., Gonzáles-Gallegos, J. G., Xiang, C. L., Kriebel, R., Drummond, C. P., Walker, J. B. and Systsma K. J. 2017. Salvia united: The greatest good for the greatest number. Taxon, 66, 133-145. Duman, H. & Dirmenci, T. 2017. A new species of Micromeria (Lamiaceae) from Köyceğiz (Muğla, southwest of Turkey). Turkish Journal of Botany, 41, 383-391.

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Güner, A., Özhatay, N., Ekim, T., Başer, K. H. C. (2000). Flora of Turkey and the East Aegean Islands (Supplement 2), Vol. 11, University Press, Edinburgh. Güner, A., Aslan, S., Ekim, T., Vural, M., Babaç, M.T. (edlr.) (2012). Türkiye Bitkileri Listesi (Damarlı Bitkiler). Nezahat Gökyiğit Botanik Bahçesi ve Flora Araştırmaları Derneği Yayını. İstanbul. Harley, R.M., Atkins, S., Budantsev, A., Cantino, P.H., Conn, B., Grayer, R., Harley, M.M., Kok, R., Krestovskaja, T., Morales, A., Paton, A.J., Ryding, O. & Upson, T.( 2004). Labiatae. In: Kadereit, J.W. (ed.). The families and genera of vascular plants (Kubitzki, K.: ed.). Volume 7, pp. 167-275. Greuter, W., Burdet, H.M. & Long, G. (1986). Med-Checklist, volume 3. Geneva: Conservatoire et Jardin Botaniques., Ietswaart, J. H. (1980). A taxonomic revision of the genus Origanum (Labiatae). Leiden Botanical Series. No. 4. Netherlands: Leiden University Press. Li, B., Cantino, P. D., Olmstead, R. G., Bramley, G. L. C., Xiang, C. L., Ma, Z. H., Tan, Y. H. & Zhang, D. X. (2016). A large- scale chloroplast phylogeny of the Lamiaceae sheds new light on its subfamilial classification. Scientific Reports, 6, 34343. Li, B. & Olmstead, R. G. (2017). Two new subfamilies in Lamiaceae. Phytotaxa, 313, 222-226. Manafzadeh, S., Salvo, G. and Conti, E. (2014). A tale of migrations from east to west: the Irano-Turanian floristic region as a source of Mediterranean xerophytes. Journal of Biogeography, 41, 366–379. doi:10.1111/jbi.12185 Mathiesen, C., Scheen, A. C., Linqvist, C. (2011). Phylogeny and biogeography of the lamioid genus Phlomis (Lamiaceae). Kew Bulletin, 66, 93-99. Serpooshan, F., Jamzad, Z. Nejadsattari, T., Mehregan, I. (2017). Molecular phylogenetics of Hymenocrater and allies (Lamiaceae): New insights from nrITS, plastid trnL intron and trnL-F intergenic spacer DNA sequences. Nordic Journal of Botany, doi: [10.1111/njb.01600]. The International Plant Names Index (2012). Published on the Internet http://www.ipni.org [accessed 1 February 2017]. The Plant List (2013). Version 1.1. Published on the Internet; http://www.theplantlist.org/ (accessed 1st February 2017). Thompson, J.D. (2005). Plant evolution in the Mediterranean. Oxford, Oxford University Press. Will, M., Schmalz, N., Classen-Bockhoff, C.( 2015). Towards a new classification of Salvia s.l.: (re)establishing the genus Pleudia Raf. Turkish Journal of Botany, 39, 693-707. Will, M. & Classen-Bockhoff, R. (2017). Time to split Salvia s.l. (Lamiaceae) - New insights from Old World Salvia phylogeny. Molecular Phylogeny and Evolution, 109, 33-58. Zohary, M. (1973). Geobotanical foundations of the Middle East. volume 2, Stuttgart, Gustav Fischer.

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REVIEW

Stamen construction, development and evolution in Salvia s.l.

Regine Claßen-Bockhoff*

Institute of Organismic and Molecular Evolution (iomE), Johannes Gutenberg-University Mainz, 55099 Mainz, GERMANY

*Corresponding author. E-mail: [email protected]

Abstract Lever-like stamens are among the most important floral traits in Salvia s.l. Their morphology, function and diversity were repeatedly studied to understand the lever mechanism and to reconstruct stamen evolution. The present paper reviews current knowledge on stamen diversity in Salvia species and combines morphogenetic and phylogenetic data. Five stamen types are distinguished based on absence vs. presence of versatile anthers, bithecate vs. monothecate anthers, curved vs. straight connective growth and presence vs. absence of the lower lever arm. Type I (no versatile anthers) is restricted to the four species of the Californian sect. Echinosphace. Versatile anthers evolved at least five times: Type II (bithectae) is the most common one in all old world Salvia s.l. clades, but also appears twice in America. Type III (monothectae, curved connective) is derived from type II (ontogenetic abbreviation). It is not restricted to the Eurasian-African clade I (Salvia s. str.) and the E-Asian clade IV-A, but also underlies some bee-pollinated American species. The new function of the lower lever arm to serve as a sterile barrier goes along with novel growth centres (ontogenetic addition). Type IV (monothecate, straight connective) characterises the majority of the American species (subgen. Calosphace). Type V (no lower lever arm) is derived by ontogenetic abbreviation and most likely homoplastic. It evolved at least four times independently, i.e. twice in Salvia s. str., once in the Californian clade II-B (sect. Audibertia) and once or several times in the East Asian clade. The study shows that stamen diversity results from few developmental changes. The latter appear in different combinations and support subclades rather than clades.

Keywords: Inhibition, flower force, novel growth centers, ontogenetic abbreviation, ontogenetic addition, parallel evolution, phylogeny, staminal lever mechanism, stamen diversity

Introduction More than 200 years ago, Christian Konrad Sprengel (1793) published his epochal work on pollination biology including the first description and illustration of the staminal lever mechanism in Salvia. Since that time, the peculiar pollination mechanism represents one of the classic examples of dorsal pollination (Fig. 1). For decades, it supported the assumed monophyly of the genus. However, today we know that Salvia is not monophyletic (Walker et al., 2004; Will & Claßen-Bockhoff, 2017) and that parallel evolution has played a significant role. Not surprisingly, also the staminal lever mechanism evolved several times in parallel (Walker & Sytsma, 2007, Will & Claßen-Bockhoff 2014) again raising the question of its evolutionary significance. Interest in Salvia increased remarkably in the last 15 years. Due to the many species, the worldwide distribution and high ecological, structural and functional diversity (Figs. 2-5) the genus is an appropriate model system to study the function and evolution of floral traits (Claßen-Bockhoff & Speck, 2000; Claßen- Bockhoff et al., 2003; Speck et al., 2003). Among them, the lever-like stamens are of particular interest. Their reversible and precise movement contributes to pollen saving and pollen dispensing, their specific relative proportions to mechanical isolation and speciation (Claßen-Bockhoff et al., 2004b). Beyond, stamens served as morphological characters to group the diversity of Salvia species (Zalewska, 1928; Himmelbaur & Stibal, 1933; Hrubý, 1934) and are recently used to support phylogenetic clades (Walker & Sytsma, 2007; Walker et al., 2015; Will & Claßen-Bockhoff, 2014).

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Figure 1. Pollen transfer in Salvia pratensis. A, longitudinal section showing the left lever-like stamen (from front view) and its movement (arrows). B, in the male flowering stage, a bee searching for nectar will be loaded with pollen (yellow) when releasing the lever mechanism. C, visiting a flower in the female flowering stage results in pollen transfer to the stigma, which is now in the position previously occupied by the anther.

The present paper reviews the recent knowledge on stamen diversity in Salvia. It uses developmental data to reconstruct the steps of diversification and illustrates the benefits of combining ontogenetic and phylogenetic data. Materials and Methods This present review is based on published and unpublished data collected during the last 15 years. Currently, there is a dispute about maintaining Salvia as a large genus including five genera into Salvia s.l. (Drew et al., 2017) or splitting the taxon into six genera (Will and Claßen-Bockhoff, 2017). The present paper is based on the second view, but (as a revision is still lacking) uses the traditional names to ease reading. The systematic position of the clades and the proposed generic names are given in Figure 10.

Results and Discussion Stamen construction The basal elements of the lever mechanism are the filaments, connectives and thecae of the two abaxial stamens. They vary in position, growth dynamics and size resulting in the well-known diversity of staminal forms.

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Figure 2. Bithecate anthers (types I, II, Table 1, Fig. 10). A-J, type II. A-B, S. officinalis. A, posterior thecae hidden below the upper lip; B, upper lip removed. C, D, S. scabra. C, lever-like conncetive exposed by a rather long filament; D, front view of the flower entrance showing theca arrangement for dorsal and lateral pollination. E, F, S. caespitosa. E, flower entrance only half closed by the anterior lever arms allowing small insects to steal nectar and pollen; F, imprecise pollen deposition (`smear effect´) onto the thorax of a bumble bee due to the small lever proportions. G. S. brachyodon. As in E, at the base a hole (arrow) bitten by bumblebees. H, S. whitehouseii. Extremely long corolla tube with small connective levers (longitudinal section, only one connective shown). I, J, S. roemeriana. Hummingbird-pollinated species. K: type I. S. carduacea. No lever mechanism. Clade affiliation (Fig. 10): A, B, E, F: I-D; C, D: I- A; H-J: I-B; K: II-B. Photos D, H-K: P. Wester, Mainz/Düsseldorf.

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Filaments The filaments of the two stamens are adnate to the lateral flanks of the corolla. They lack any appendages or outgrowths, but vary considerably in length, exit angle from the lower lip and stability. When the lever mechanism is released, the filaments transfer the exerted force to the corolla (Reith et al., 2007). This can result in a severe deformation of the flower influencing pollination (Ott et al., 2016). Furthermore, the filaments support the levers and place them in their final position. Being essential floral traits, their diversity and functional significance are not carefully investigated so far. Filament tip and joint formation Salvia species have versatile stamens (except type I, clade II-C, Fig. 10), i.e. the primary filament tip forms a very thin ligament allowing the connective to swing around the thus formed joint (Troll, 1929). In the majority of the species, tissue swellings in the shape of a secondary filament tip and a connective bulge cover the joint (Claßen-Bockhoff et al., 2004a; Fig. 6L-N). These structures closely interlock and restrict the free mobility of the anther like a hinge. Thereby, they increase and maintain precision during the reversible anther movement (Claßen-Bockhoff, 2004b; Thimm, 2008). The swellings, composed of large parenchymatic cells, may also play an essential role in the movement itself. Correns (1891) postulated torsion within the joint, but first histological studies (Thimm, 2008; unpubl. data) indicate that the joint remains stiff and gets passively twisted against the connective and filament protrusions. More histological and experimental investigations are needed to fully understand the biomechanics underlying the lever movement in Salvia s.l. Connectives and lever formation The remarkable extension of the connective separates the two thecae of each stamen and forms two lever arms. The upper (anterior) arm always ends in a fertile theca with two pollen-sacs, while the lower (posterior) arm is highly diverse producing pollen-sacs (bithecate anther; Fig. 2) or remaining sterile (monothecate anther; Figs. 3-4). Connectives are curved or straight and vary considerably in the proportions of their arms. The upper connective arm is rather uniform hiding the pollen-sacs below the upper lip (particularly in bee flowers; Fig. 3A) or exposing them out of the flower (many bird-pollinated species; Fig. 4B, G). The two neighbouring thecae are usually free, but may be fused increasing the precision of pollen deposition. The lower lever arms, in contrast, are highly diverse, often forming elaborate three-dimensional structures (e.g. Fig. 3C-F; Fig. 7E, F) or producing tooth-like outgrowths (e.g. Fig. 4J-M; Fig. 7A, B). They are free from each other (Fig. 2B, J), stick together at the rudimentary theca walls (Fig. 3C, F, M: arrows; Fig. 7F) or are connected with epidermal papillae or hairs along their whole length (Fig. 4B, D; Fig. 7B, D). They usually restrict access to nectar. Pollinators have to push back the barrier thereby releasing the lever mechanism (Fig. 1). As a consequence, the upper arm lowers down and transfers pollen onto the head or body of the visitor thus mediating dorsal (nototribic) pollen deposition (Müller, 1873; Correns, 1891; reviewed in Claßen-Bockhoff et al., 2003).

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Figure 3. Monothecate anthers with curved connective (type III, Table 1, Fig. 10). A-C, S. pratensis. A, longitudinal section: a, anther; al, anterior lever arm; n, nectary; pl, posterior lever arm; st, style. B, joint covered by secondary filament tip (sf) and connective bulge (cb). C, shovel-like lower lever formation. D-F, anterior lever formations. D, S. stenophylla. E, S. forsskoalei. F, S. argentea. G, S. austriaca, lateral pollination. H, S. africana-lutea, complete closure of flower entrance by paddle-like barrier. I, S. glutinosa, anterior lever arms with conspicuous rudimentary thecae. J, K, S. nutans. J, resupinate lever-like anther. K, hanging inflorescence. L, M, S. jurisicii. L, erect inflorescence with resupinated flowers. M, resupinate lever-like anther. Arrows in C, F, M: rudimentary theca. Clade affiliation (Fig. 10): A- C, F, G J-M: I-C; D, H: I-A; I: IV-A. Photos B-F, J, M: M. Crone, Mainz.

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Lever-movement Salvia s.l. is characterised by the staminal lever-mechanism. The group is extremely successful as to species number and world-wide distribution. Thus, one might assume that the pollen transfer mechanism contributes to speciation by selecting pollinators by physical force (as shown for Fabaceae in Córdoba & Cocucci, 2011). Only strong bees and birds would then get access to nectar, while weak insects were excluded. However, force measurements in more than 30 species representing the diversity in Salvia s.l. (Thimm, 2008; unpubl. data) show the very reverse. Forces are generally low ranging between 0.5 mN and 10(-40) mN. They are lower than the average force honeybees (14 mN) and bumblebee workers (25 mN) can afford (Reith et al., 2006) and comparable to forces needed to penetrate a hairy ring in the corolla tube (S. verticillata: 3.6 mN, Thimm, 2008; S. glutinosa: 2.5 mN, Reith et al., 2006). Obviously, forces do not exclude weak insects from nectar (Stöbbe et al., 2016). Forces needed to release the lever mechanism vary among species. Preliminary data indicate that the force depends more on the lever weight than on its size and that a higher force is demanded by bird- than by bee- pollinated species (Thimm, 2008). However, more data are needed to confirm the first results (in process). Forces also vary within and among flowers of the same individual. The first release usually demands the highest force particularly in species with upper lips enclosing the fertile thecae (e.g. S. pratensis). When repeatedly used (e.g.10-20 repetitions), the lever shows symptoms of wilting. The fertile thecae do not return completely to their previous position and the needed forces decrease (Thimm, 2008). In bee-pollinated species, the different force demands of young and old flowers may play an important role. Flowers are protandrous in this species and open in an acropetal sequence. As bees usually visit their food plants bottom- up (Harder et al., 2004), they first transfer foreign pollen to the stigma of an old flower and then take pollen from a younger flower. As these flowers demand more force, the pollinator may decide to leave the inflorescence, a behaviour increasing outcrossing in the population. Experiments with bumblebees and honeybees on artificial flowers show that both species are able to discriminate between a barrier-free model and a lever-model demanding 2 mN (Stöbbe et al., 2016). It is thus very likely that weak forces influence the selection of a food plant, the foraging behaviour of a bee and the degree of outcrossing. Lack and loss of lever function The majority of the Salvia species follows the general picture having a bilabiate flower, a reversible lever mechanism and a dorsal pollination. However, there are remarkable exceptions concerning joint formation, flower construction and pollen deposition site. No joint formation In the Californian section Echinosphace (4 sp.; Fig. 10: type I, clade II-C), the anther is not versatile. Instead, the connective is inflexibly fixed to the filament (Fig. 6H). Consequently, the species lack the lever mechanism completely (Himmelbaur & Stibal, 1933; unpubl. data) and does not belong to Salvia if defined by mobile anthers. However, as in Salvia species, the connective widens and separates the thecae presenting the anterior one out of the flower and the posterior one in the flower entrance (Fig. 2K). Both thecae contribute to pollen transfer, depositing pollen on the front and lower side of the insect pollinator (own obs.).

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Figure 4. Monothecate anthers with straight connective (type IV, Table 1, Fig. 10). A, S. involucrata, anterior lever arm blocks access to nectar. B, S. tubiflora, straight lever arms without any outgrowths. C, S. coccinea, joint covered by secondary filament tip (white), connective bulge small (not visible). D, S. splendens, joint largely uncovered. E, F, S. elegans, longitudinal sections. E, no space for lever movement, F, rare appearance of a rudimentary posterior theca. G, S. haenkei with Sappho sparganura. Pollen transfer without lever mechanism. H, S. cacaliifolia, no lever movement due to lever arm proportions. I, S. consobrina, view onto the staminal levers, lower lip removed. J-M, species with ventral outgrowths. J, S. stachydifolia, longitudinal section. K, L, S. rypara ssp. platystoma. K, frontal view showing the barrier in the flower entrance. L, longitudinal section. M, S. sophrona, longitudinal section. N-Q, species with dorsal outgrowths, longitudinal sections (N-P) and detail of the anterior lever arm (Q). N, S. corrugata. O, S. cusidata ssp. bangii. P, Q, S. uliginosa. Clade affiliation (Fig. 10): all II-A. Photos: A, D-H, J-L, N, O: P. Wester, Mainz/Düsseldorf.

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Reduction of lower lever arm A second group of species lacks the lever mechanism due to the extreme reduction of the lower connective arm. This reduction is found at least four times independently (Fig. 10, type V: orange colour) and goes along with a change in flower construction and pollination mechanism. In the S. verticillata group (4 spp., Will & Claßen-Bockhoff, 2017; Fig. 10: clade I), the lower connective arm forms a vermicular appendage (Fig. 5B). The fertile thecae hide the pollen-sacs below a movable upper lip which is pushed aside by the pollinator thus mediating frontal pollination (Fig. 5A; Correns, 1891; Claßen- Bockhoff et al., 2004b). In the majority of the species placed in sect. Audibertia (19 ssp.; Walker et al., 2015; Fig. 10: clade II-B), the reduction of the lower connective arm (Fig. 5C) is so extreme that almost no rudiment is left in the adult flower (Fig. 5D, E:*, Fig. 6J). Rather, the upper connective arm continues the filament in a straight-line way looking like a `normal´ stamen, however, with only a single theca. In S. apiana (Fig. 5E), pollen is exposed out of the flower but not accessible for pollen collecting bees. It is deposited to the bee’s body by corolla deformation passively lowering the fertile thecae (Ott et al., 2016).

Figure 5. Monothecate anthers with reduced anterior lever arm (type V, Table 1, Fig. 10). A, B, S. verticillata. A, longitudinal section showing the pollen-sacs hidden below the movable (arrow) upper lip, the reduced anterior lever arm (circle) and the style exposed out of the lower lip. B, detail with reduced anterior lever arm (al), part of the posterior lever arm (pl) and filament (fi). C, S. spathacea, with vermicular lower lever arm (circle), dorsal pollination. D, S. clevelandii, ventral pollination. E, S. apiana, lateral pollination. * in D and E: rudiment of anterior lever arm. Clade affiliation (Fig. 10): A, B: I S. verticillata-clade; C-E: II-C. Photos B: M. Crone, Mainz; C, E: P. Wester, Mainz/Düsseldorf.

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Loss of the lever mechanism The shift from bee to bird pollination is often associated with a dramatic change in flower construction. Wester & Claßen-Bockhoff (2007) listed 59 bird pollinated species (˃ 30% of all ornithophilous species) which transfer pollen without a lever mechanism. In these species, pollen and nectar are freely accessible as the thecae are exposed out of the flower and the lower lever arms do not block the flower entrance. Interestingly, some species still have a functional lever mechanism which is, however, not releasable due to the narrow flower tube (S. haenkei; Fig. 4G; Fig. 10: clade II-1). In other species, the non-use of the lever mechanism goes along with a reduction of the filament and connective protrusions around the joint (S. longistyla) or with a stiffening of the joint (S. elegans, Fig. 4E). In S. exserta, the lower lever arm blocks the flower entrance, but the joint is stiff. Here, the lever-mechanism is released because the force is transferred to the long and elastic filaments passively moving the lower lever arm (Wester & Claßen-Bockhoff, 2007). Loss of dorsal pollination While nototribic pollination is the common way of pollen deposition onto the head, thorax or bill of a pollinator, frontal pollination (S. verticillata; Fig. 5A), lateral pollination (S. austriaca; Fig. 3G) and abdominal pollination (S. glutinosa; Fig. 3I) appear dependent on the length and position of the upper lever arms (Claßen-Bockhoff et al., 2004b). In case of bithecate anthers, pollen from the anterior theca ensures dorsal and from the posterior theca lateral pollination (S. scabra, S. roemeriana; Fig. 2C, J). An exception is S. carduacea (Fig. 2K) with ventral and frontal pollination. In the Californian sect. Audibertia (Fig. 10: clade II- B), the monothecate anthers are in variable positions. The often-exposed fertile theca mediates dorsal pollination in S. spathacea (Fig. 5C), lateral pollination in S. apiana (Ott et al., 2016) and ventral pollination in S. clevelandii (Fig. 5D). It is likely that the different sites of pollen deposition on the pollinator contribute to mechanical isolation and speciation (Grant 1994). As tested in S. gravida (Wester et al., 2012), hanging inflorescences often respond to gravity and turn their flowers to ensure nototribic pollination. However, in S. nutans (Fig. 10: clade I-C), this resupination does not happen resulting in ventral (sternotribic) pollination (Fig. 3K). Sternotriby also characterizes S. jurisicii (Fig. 3L; Fig. 10: clade I-C). This species has erect inflorescences whose flowers turn down individually, thus acting like S. gravida but in the opposite direction. Both sternotribic species have well-developed staminal levers (Fig. 3J, M). Experiments with bumble bees (Bombus terrestris; unpubl. data) have shown that the insects can only handle the flower and release the lever mechanism when the upper lip serves as landing place thus illustrating that the whole flower construction has adapted to ventral pollination. Stamen development The monosymmetric flowers in Lamiaceae lack the median-adaxial stamen. Consequently, the pentamerous flowers only develop four stamens. Their primordia appear in a cycle (Fig. 6A, B), but in the adult flower, the thecae are usually placed below or in front of the upper lip (Fig. 8A, B). This dislocation of the abaxial pollen- sacs takes place during flower development. A common process to move the thecae in the right position for dorsal pollination is connective widening (Fig. 8C). It is not only found in Lamiaceae, but also in Gesneriaceae (Nemathathus), Orobanchaceae (Pseudosopubia), Scrophulariaceae (Calceolaria) and other families of the Lamiales, most likely representing a plesiomorphioc condition in the lineage. While all four anthers are presented in a row in the adult flower of Prunella vulgaris (Fig. 8B), Lamium species show a didynamic anther position with two pairs of anthers located one upon the other (Fig. 8D, E). With respect to the central position of the style, this pollen-sac arrangement is more effective. Interestingly, the same pattern is repeated in Salvia based on only two stamens with spatially separated thecae (Fig. 2D, J).

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Figure 6. Anther development and joint formation. A-C, flower development (S. viridis). Black stars: stamen primordial, white stars: staminode promordia. D, flower bud (S. leucantha): view onto the abaxial stamen. E, bithecate anther, type I (S. funerea). F, monothecate anther, type III (S. leucantha). G, monothecate anther, type V (S. apiana). H, anther not versatile, type I (S. greatae). I, anther versatile, type II (S. columbaria). J, joint stiff, type V (S. mellifera). K-N, S. glutinosa, type III. K, thin ligament between filament and connective (j: joint). L, coverage of the joint by filament and connective protrusions. M, secondary filament tip. N, connective bulge and outline of the joint (after removal). co, connective. f, filament. j, joint. lca, lower connective arm. lt, lower theca. uca, upper connective arm. ut, upper theca. Bars: A- C (same scale), 200µm. D, 40µm. E, G, K: 100µm. F: 50µm. H, I, J: 250µm. L-N: 200µm. Clade affiliation (Fig. 10): A, D, II-B. B, I-C. C, E, F, II-C. G-J, IV-A. Photos A-C: N. Daichent, Mainz. D, F, I, K-N. M. Crone, Mainz. E, G- J: S. Deobald, Mainz.

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Figure 7. Diversity of anterior levers. A, B, S. rypara ssp. platystoma. A, bud stage: ventral outgrowth close to the joint; *, position of reduced theca. B, adult stage: barrier formed by the outgrowths, straight lower lever arms connected by hairs. C, D, S. uliginosa. C, bud stage: dorsal outgrowth; *, position of reduced theca. D, adult stage: barrier formed by the curved posterior levers arms. E, S. argentea, adult stage: filament heel-like attached to the corolla, lower lever arm arched upwards with finger-like protrusions, connected with the neighboring lever at the position of the reduced theca (*). F, S. sclarea, adult stage: two spoon-shaped lower lever arms forming a hole by being connected at the reduced thecae walls. Bars: A-C: 500µm; D, E: 1mm: F, 1.5mm. Clade affiliation (Fig. 10): A-D: II-A; E, F: I-C. Photos E, F: M. Crone, Mainz.

In Salvia, only the two abaxial stamens develop while the adaxial ones remain inhibited and form minute staminodes (Fig. 6A, B: white stars). The thecae are placed below the upper lip by filament and connective elongation (Fig. 2B, C). Extended connectives combined with versatile anthers result in the formation of the staminal lever mechanism. Developmental studies in more than 30 Salvia species indicate that the basic steps in stamen development are similar even among highly diverse species (Troll, 1929; Trapp, 1956a, b; Claßen- Bockhoff et al., 2004b; unpubl. data).

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Figure 8. Anther position in Lamiaceae with four stamens. A-C, Prunella grandiflora (Lamiaceae). A, flower from the side, the four anthers located in a row below the upper lip. B, all anthers turned to enable dorsal pollination. C, anther with widened connective dislocating the theca for dorsal pollination. D, Lamium purpureum and, E, L. villosifolium, both species with didynamic anther position increasing precision of pollen transfer.

Thecae initiation and inhibition The initial stage of stamen development in Salvia is the formation of an asymmetric anther (Fig. 6D-G; Fig. 9A). In bithecate anthers, both thecae are initiated early, but differ in size, the abaxial (anterior) theca being larger than the adaxial (posterior) one. In monothecate anthers, the posterior theca is inhibited from the beginning (S. argentea; Fig. 6F) or clearly initiated and inhibited later (S. apiana; Fig. 6G). Interestingly, the time of theca inhibition is independent on the lower lever arm formation as S. argentea forms a highly elaborate sterile structure (Fig. 7E), while S. apiana lacks this lever arm completely (Fig. 5E). Furthermore, even if the posterior theca is early inhibited, its surface may differ from the connective epidermis in having the characteristic papillae cells of theca walls (Claßen-Bockhoff et al., 2004a). At these papillae, the two adjoined anterior lever arms are often postgenitally fused (Fig. 3C, F, M: arrows; Fig. 7E, F). Occasional theca formation and pollen production in monothecate anthers (Fig. 4F) illustrate that the clear difference between bithecate and monothecate anthers in the adult stage is developmentally only based on a gradual inhibition process. Connective widening Young anthers have a relatively broad connective, which starts widening before the filament originates. The upper lever arm always curves in a rectangular way locating the fertile theca in the longitudinal direction of the developing flower (Fig. 6I). In the adult stage, it only varies in its length ranging from rather short (Fig. 2B) to extremely long connective arms (Fig. 4H). In contrast, development of the lower lever arm is delayed.

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Figure 9. Stamen development in Salvia. A, asymmetric anther. B-E, developmental divergence resulting in bithecate (B) and monothecate anthers (C-E). B (type II), the lower connective arm curves due to increased growth at its distal side and presents the lower theca in the flower entrance. C (type III), as in B, but lower theca reduced and sterile lower connective arm forming a highly diverse barrier in the flower entrance. C1-C3’, cross sections and side views of sterile connective arms. D (type IV), straight extension of the lower connective arm; lower theca extremely reduced. D-D3’, cross sections and side view of sterile lever arm. E (type V), lower lever arm reduced, resulting in a one-armed connective. Stamen type I (no versatile anther) not included. Hatched areas: novel centres of growth. Dotted circle: position of the joint (from Claßen-Bockhoff et al., 2004b).

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Versatile anther formation In the next developmental stage, the filament elongates. In Salvia clade II-C (sect. Echinosphace; Fig. 10; type I), the anther is immobile as the transition from the filament to the connective remains stiff. In all other Salvia species (as far as known, see Himmelbaur & Stibal, 1933), a joint is formed by differential growth of the filament and filament tip. While the filament gets thicker with further development, the filament tip remains thin and forms a ligament (Fig. 6I, K: j). Very soon, swellings at both sides of the joint develop and cover the ligament (Fig. 6I, L-M; Claßen-Bockhoff et al., 2004a). Species that lost the lever function often show a stiff joint characterized by a broad contact between filament and connective (Fig. 6J). Such a stiff joint easily results from a less distinct difference among filament thickening and filament tip formation. Diversification of the lower lever arm In bithectae anthers, the connective arm always grows more intensively on its dorsal side resulting in a curved shape (Fig. 9B; Fig. 10: type II). This bending allows both thecae to be placed in the flower entrance and contribute to pollination. In monothecate anthers, the process of theca inhibition is either continued by the complete inhibition of the posterior connective arm (Fig. 9E; Fig.10: type V) or followed by an immense promotion of the sterile connective part. In old world Salvia s.l. species, the lower lever arm still grows more on its dorsal side blocking the flower entrance (Fig. 10: type III). The formation of three-dimensional barriers

(Fig. 3C-E; Fig. 7E, F) often goes along with novel growth centers at the connective (Fig. 9C-C3’). In many new world species, the lower lever arms elongate equally and gets a straight shape (Fig. 4A, D, E; Fig. 9D; Fig. 10: type IV). Tooth-like protrusions on the ventral side (Fig. 4J-M; Fig. 7A, B), and dorsal outgrowths (Fig. 4N-Q;

Fig. 7C, D) are based on novel cell division areas (Fig. 9 D-D3’). Evolution of stamen diversity: a functional phylogenetic view Stamen development in Salvia s.l. only includes few developmental processes the most important ones being inhibition (joint formation, theca and lower lever arm reduction), differential growth (curved vs. straight shape) and late developmental formations (proportion, fusion, appendages). The combination of connective widening and versatile anther attachment constitutes the origin of the staminal lever mechanism. A similar construction evolved in some Australian species of the tribe Westringieae (Lamiaceae subfam. Prostantheroideae; unpubl. data) and in the Scrophulariaceae, e.g. Pseudosopubia obtusifolia, Calceolaria pinnata and C. scabiosifolia (Trapp, 1956a, b). However, protrusions covering the primary filament tip and guiding the lever movement are only known from Salvia. The thereby gained precision of pollen deposition may be one of the main forces driving the evolution of the staminal level mechanism and, in consequence, speciation. Anther diversity was repeatedly used for systematic purposes (Correns, 1891; Zalewska, 1928: 206 sp.; Himmelbaur & Stibal, 1933: 430 sp.; Hrubý, 1934; Walker & Sytsma, 2007: 85 sp.; Table 1). While most authors used few `types´ indicating basic differences, Walker & Sytsma (2007) aimed to support phylogenetic clades with stamen morphological characters. They added relative proportions and secondary fusion of anthers and lower lever arms thereby increasing the number of `types´. The present situation is that the term `stamen type´ refers to two different levels, i.e. the levels of characters (referring to early developmental events like presence, shape and fertility of the lower lever arm, Claßen-Bockhoff et al., 2004a) and character states (including late developmental events like variable proportions and secondary fusion modifying the underlying type). Though it is helpful to use stamen morphology to support clades, the increase of stamen types due to character combinations is questionable.

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In the present paper, five stamen types are distinguished based on early developmental events (Claßen- Bockhoff et al., 2004a). Except the newly introduced type I, they largely correspond to the types introduced by Himmelbaur & Stibal (1933), Hrubý (1934) and Hedge (1974) (Table 1).

Table 1. Stamen types and distribution in Salvia. Himmelbaur & Stibal: types I-V, Aud, Audibertia. Walker & Sytsma: types A, B, E-I, M, N; free/fused refers to lower lever arms. Will & Claßen-Bockhoff 2017: clades I-IV (Fig. 10). NW, new world. OW, old world. S, ver., S. verticillata-clade.

Stamen types Clade affiliation

Present study Himmelbaur & Stibal, Walker & Sytsma, 2007 Will & Claßen-Bockhoff 2017 1933 anther OW NW OW NW OW NW anther not versatile bithecate Type I I* II-B connective angled anther versatile bithecate Type II I I A free G free I-A I-A connective curved M free I-D I-B N III II-C monothecate Type III II, V II B fused I-A connective curved N I-C anterior theca IV rudimentary Type IV III E fused II-A connective straight F ± free anterior theca lacking Type V IV Aud H I-A II-C connective straight I-S. ver. anterior theca and lever arm reduced * Walker & Sytsma (2007) did not consider the absence of the versatile anther.

Type I: Bithecate stamens lacking the lever mechanism This stamen type only appears in Salvia clade II-C including the four species of sect. Echinosphace (Fig. 10: symbol with grey connective). The filament is extremely short. The two connective arms are angled and of different length. The upper thecae are exposed out of the flower, while the lower thecae are placed at the flower entrance (Fig. 2K). Based on phylogenetic data (Walker et al., 2015; Fragoso-Martínez et al., 2017; Will & Claßen-Bockhoff, 2017), the American clades II-B/II-C (Ramona, i.e. subgen. Audibertia) and II-A (Lasemia (i.e. subgen. Calosphace) are sister to each other with the small SW-Asian genera Dorystaechas and Meriandra as closest relatives. It is thus reasonable to interpret the immobile stamen construction in clade II-C as a basal condition.

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Lacking versatile anthers, the species are more similar to their Asian relatives than to Salvia species only differing from the first by connective widening and different filament and connective proportions (late developmental steps).

Type II: Bithecate stamens with lever mechanism Versatile anthers with two fertile thecae represent the basal condition in the old world Salvia clades, i.e. clade I (Salvia s.str.; except clade I-B), III (Pleudia, Polakia) and IV Glutinaria and in the new world clades I-B and II-B (sect. Audibertia, Fig. 10: symbol with blue connective). Interestingly, also S. axillaris (Fig. 101), the basalmost species of clade II-A (subgen. Calosphace, Fragoso-Martínez et al., 2017), has bithecate anthers questioning the evolution of the straight stamens in this lineage. It is obvious that stamen construction II evolved several times in parallel. From the morphogenetic point of view this is not surprising as the two underlying components, i.e. connective widening and versatile anthers, are widely distributed among angiosperms. The peculiarity in Salvia is that both processes are combined resulting in a lever mechanism. From the evolutionary point of view, connective widening is under strong natural selection to enable dorsal pollination. Based on this precondition, the staminal lever mechanism might have evolved in bee-pollinated species to protect pollen against mass collection (Claßen-Bockhoff et al., 2004b; Westerkamp & Claßen- Bockhoff, 2007; Wester & Claßen-Bockhoff, 2007). As long as the thecae are hidden below the upper lip, pollen is safe. However, in large flowers, e.g. in many E-Asian and Mediterranean species (Fig. 2 E-G), insects may visit the flowers and drink nectar without touching the pollen sacs and getting loaded with pollen. In these cases, the lever mechanism is a useful construction to lower the thecae down onto the pollinators increasing the degree of successful pollen transfer and decreasing the number of nectar thieves. Except few bird-pollinated species in the American clade I-B (e.g. S. roemeriana, Fig. 2I, J) and the only known moth-pollinated species S. arborescens (Reith & Zona, 2016, clade II-A), Salvia species with bithecate anthers are bee-pollinated. The connective arms are usually short separating the thecae not too far from each other (Fig. 2). The functional constrain might be the same as in didynamic androecia (Fig. 8D, E), i.e., that a too large distance makes it impossible for the posterior pollen to get in contact with the stigma. This conflict limits the evolution of the staminal lever mechanism and may have triggered the evolution of monothecate anthers. Type III: Monothecate stamens with inhibited thecae and curved mobile connectives In old world Salvia species, monothecate anthers are derived from bithectae anthers (Fig 10: symbols with green connective). This is in accordance with the phylogenetic position of the species representing stamen type III (Walker & Sytsma, 2007; Will & Claßen-Bockhoff, 2014) and with stamen development interpreting monothecate anthers as ontogenetic abbreviations (Gould, 1977) of bithecate anthers. Once sterile, the posterior lever arm is free to adapt to its new function as a barrier in the flower entrance. In particular, species of Salvia s. str., e.g., clade I-C (Fig. 3A-C, F, G, J-M; Fig. 7E, F) and clade I-A (Fig. 3D, H), have evolved elaborate structures guiding the pollinators (Reith et al., 2007). But also representatives of the E-Asian clade IV-A show large and often conspicuous barriers (e.g. Fig. 3I; Fig. 10; Himmelbaur & Stibal, 1933). Morphogenetically, the new structures formed by the lower connective represent ontogenetic additions (Gould, 1977). Type IV: Monothecate stamens with reduced thecae and straight mobile connectives

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The new world clade II-A (Lasemia, i.e. subgen. Calosphace; Fig. 10), with approx. 500 species the largest lineage in Salvia s.l., is characterised by monothecate anthers. The only exception is S. axillaris (own observation) placed at the base of the lineage (Fragoso-Martínez et al. 2017). In contrast to the curved shape of the old world species, the lower lever arm is straight blocking access to nectar in a diagonal way (Fig. 4A). Many species form one or two tooth-like structures of unknown function close to the joint (Himmelbaur & Stibal, 1933). The sterile end of the lower lever can be broad but does not form any additional appendages as in type III. The adjacent lever arms may be free from each other or postgentially fused, the latter being a late ontogenetic process. While old world Salvia species are predominantly bee-pollinated, the American clade II-A includes more than 180 hummingbird pollinated species (Wester & Claßen-Bockhoff, 2011). The shift from bee to bird pollination has happened many times in parallel (Fragoso-Martínez et al., 2017) going along with multiple morphological changes in flower construction and loss of lever functions (Wester & Claßen-Bockhoff, 2007). Interestingly, the bee-pollinated species in this clade often (always?) form a ventral barrier compensating for the diagonal course of the posterior lever arm. Ontogenetic studies indicate that this barrier evolved differently (Baikova, 2002; Claßen-Bockhoff et al., 2004a). In S. rypara ssp. platystoma (Fig. 4K, L; Fig. 7A, B), the lower connective arm is straight and develops a ventral outgrowth blocking the flower entrance. In contrast, in S. uliginosa (Fig. 4P, Q; Fig. 7C, D) the lower lever arm is curved forming the barrier itself, while a dorsal outgrowth forms the straight part of the lever. This sword-like structure presses against the corolla when the lever mechanism moves. This increases the force, which is needed by the pollinator to get access to nectar, and the pressure by which pollen is loaded on the insect’s body. Functionally, the two stamen constructions fulfil the same service but, morphologically, they are different. The phenomenon of partial analogy (Claßen-Bockhoff et al., 2004a) has been largely disregarded though Himmelbaur & Stibal (1933) already noticed that in some stamens with `straight levers´ (e.g. S. uliginosa), the vascular bundle ends in a `ventral tooth´ (type III°, III°°° sensu Himmelbaur & Stibal, 1933). Beyond its assumed, but not tested adaptive value, it is urgently needed to study stamen development in more bee- pollinated species of clade II-A to identify the homologous vs. analogous formation of ventral barriers and straight levers. As to the evolution of stamen diversity in Salvia s.l., it has to be shown which development represents the ancestral condition and which the more common one and whether the two forms evolved only once or several times in parallel in clade II-A. Type V: Monothecate stamens with one-armed immobile connectives The early inhibition of the posterior theca is coupled with an almost complete inhibition of the lower connective arm in stamen type V resulting in monothecate anthers with minute appendages (S. verticillata, S. apiana; Fig. 5B, E; Fig. 9E). This stamen type is derived from stamen type II again through ontogenetic abbreviation. It has evolved at least four times in parallel in the clades I-A, I-S. verticllata-group, II-B and IV- A and/or IV-B. If stamen development is the same in all clades, stamen type V would be an excellent example for homoplastic similarity, meaning morphological and developmental identity (morphological homology) originating in different phylogenetic clades. Interestingly, monothecate anthers with an extremely reduced posterior theca also appear in Rosmarinus officinalis, which stands in a polytomy with Perovskia and Salvia s.s. (Fig. 10). However, despite its outer similarity, a joint is lacking in the anther of this species (Trapp, 1956a, b; Naghiloo et al., 2014; unpubl. data), thus representing just a monothecate anther as also found in some Australian Westringieae (unpubl. data) or Crossandra species and Chaetothylax umbrosus (both Acanthaceae; Trapp, 1956a, b).

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Figure 10. Stamen evolution in Salvia. Salvia s.l. includes five non-Salvia genera and six Salvia clades (shaded). Stamens are bithecate in Zhumeria, Meriandra, Dorystaechas and Perovskia and monothecate in Rosmarinus. They all lack versatile anthers as the stamens in Salvia sect. Echinosphace (clade II-C, type I) do (connectives grey). Versatile anthers evolved at least five times (red *) resulting in four mobile stamen types (Table 1). Stamen type III (green) and V (orange) derived several times independently from stamen type II (blue), stamen type IV (red) only appears in clade II-A with unclear relation to type III (grey query). Arrows: direction of pollen shedding. *, position of rudimentary/lacking theca. 1 S. axillaris. 2 S. columbariae. 3 S.namaensis and S. verticllata-group (4 ssp.). 4 S. subgen. Audibertia (14 ssp.). 5 S. scapiforme (possibly more ssp.). Light grey, old world clades: Salvia s.str. (I-A): Eurasia, Africa; Glutinaria (IV): E-Asia; Pleudia and Polakia (III): SW Asia to N-Africa. Dark grey, new world clades: Sect. Heterospace (I-B): N- /C-America; Lasemia (i.e. subgen. Calosphace, II-A): C-/S-America; Ramona (i.e. subgen. Audibertia, II-B, II-C): California. Phylogeny and clade names after Will and Claßen-Bockhoff, 2017.

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Conclusions

Walker & Sytsma (2007) for the first time mapped stamen types on a phylogenetic tree allowing an evolutionary interpretation of stamen diversity. They characterised each of their nine Salvia clades and subclades by a specific `type´. However, considering the description of their types, it is evident that they used character state combinations of the four types introduced by Himmelbaur & Stibal (1933) and, in part, by Hedge (1974). These types, supported by morphogenetic processes, are also used in the present paper and added by a fifth, non-versatile stamen type (Table 1). Walker & Sytsma’s (2007) stamen types A, G, M and N correspond to bithecate anthers (type II), B and N to monothecate anthers with curved connectives (type III) and E and F to those with straight connectives (IV). Stamen type H is characterised by a reduced lower lever arm (type V) and stamen type I (capital, no number) is immobile corresponding to the newly introduced type I (number, no capital; Table 1). The present study clearly illustrates that the diversification of stamens in Salvia s.l. is higher than expected by Walker & Sytsma (2007). Ramona (i.e., subgen. Audibertia; Fig. 10: clades II-B/II-C) has three instead of two stamen types. The same is true for Salvia s. str. (Fig. 10: clade I). Lasemia (i.e., subgen. Calosphace; Fig. 10: clade II-A) has two types based on different development. Little is known about their evolution and thus it is presently not possible to map them on individual subclades. Whether they correspond to the two types distinguished by Walker & Sytsma (2007) based on free (type F) or fused lower lever arms (type E) remains to be tested. Stamen type H (here type V) has evolved not only once but at least four times. The distribution of stamen types within Salvia s.l. clearly shows that the staminal lever mechanism has evolved several times in parallel. Drew & Sytsma (2012) expected at least three independent origins, but in fact, versatile anthers with widened connectives originated at least five times (Fig. 10: red *), while one group traditionally grouped into Salvia (sect. Echinosphace; clade II-C) has no lever mechanism at all. Interestingly, all lineages with a staminal lever mechanism are richer in species than their relatives. This indicates that precise pollen transfer by mobile stamens may have triggered speciation and adaptive radiation.

ACKNOWLEDGEMENTS I am very grateful to Prof. Dr. Hüsnü Can Başer for inviting me to the International Lamiaceae Symposium 2017 and initiating this special volume. I furthermore thank Dr. Petra Wester, Düsseldorf, Dr. Maria Will, Oldenburg, and Dr. Ferhat Celep, Ankara, for discussion and my former students and PhD students Nicole Daichent, Sarah Deobald, Michael Crone and Petra Wester for providing scanning electron microscopic pictures and photos.

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RESEARCH ARTICLE TLC-GC/MS Method for identifying and selecting valuable essential Oil Chemotypes from Wild Populations of Mentha longifolia L.

Juan Antonio Llorens-Molina1, *, Vicente Castell2, Sandra Vacas3 and Mercedes Verdeguer1 1 Mediterranean Agroforestry Institute, Universitat Politècnica de València, Valencia, SPAIN 2 Department of Plant Production, School of Agricultural Engineering and Environment, Universitat Politècnica de València, Valencia, SPAIN 3 Centre for Agricultural Chemical Ecology, (Mediterranean Agroforestry Institute), Universitat Politècnica de València, Valencia, SPAIN

*Corresponding author. Email: [email protected]

Abstract Wild populations of aromatic plants can be considered resources of natural biodiversity for selecting valuable genotypes in order to obtain well-characterized cultivars with defined essential oil (EO) profiles suitable for different purposes due to their biological activity. Often, their high intrapopulational variability requires preliminary screenings based on a significant number of individuals and, therefore, simple and fast methods to identify EO profiles are required. For this purpose, a TLC-GC/MS method has been proposed in order to identify chemotypes occurring in a selected wild population. Stolons of 50 individuals of Mentha longifolia L were planted and bred in greenhouse conditions before growing in experimental plots. Individual sampling and extraction of leaves were performed to determinate TLC individual profiles. They were validated by preparative TLC and GC/MS analysis of discriminant spots. Fresh material belonging to each defined TLC profile was collected in full flowering stage and subjected to SDE extraction and GC/MS analysis. Five chemotypes were characterized: A (piperitone and piperitenone oxides); B (piperitone oxide + pulegone); C (α- terpineol acetate + carvone acetate); D ((E) – dihydrocarvone) and E (pulegone + isomenthone + menthol). This TLC method was applied to individuals coming from another population. All of them could be clearly identified as belonging to chemotype E, which was afterwards confirmed by GC/MS analysis. Keywords: TLC, Mentha longifolia, essential oil, chemotype

Introduction Over the last decades, there has been a tendency to use essential oils (EO) and other natural products as healthier and ecological alternatives in food preservation, medicine, pest management, etc., based on their chemical composition (Newman & Cragg, 2007; Dias et al., 2012; Harvey et al., 2015). Both the occurrence of valuable compounds and the absence of those showing adverse effects demand to fulfil several stability requirements in order to be suitably standardized. This way, well-characterized cultivars with specific EO profiles are required. These cultivars can be obtained by means of conventional selection methods or new ones based on more recent advances in Biotechnology, such as micropropagation techniques or genetic engineering. With this aim, selecting and breeding valuable genotypes from wild populations as resources of natural biodiversity can be considered the first step. In relation to EOs, to study the chemical variability of wild germplasm can lead to identify promising chemotypes with regard to their biological activity or yield improvement. Many researches have been conducted in this way concerning different aromatic plant species: Thymus vulgaris L. (Delpit et al., 2009), Rosmarinus officinalis L. (Mulas et al., 2001), Agonis fragrans (Robinson, 2006), Achillea

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species (Rahimmalek et al., 2009), Lippia alba Mill. (Rufino et al., 2012), Lavandula multifida L. (Zuzarte et al., 2015), Origanum species (Goliaris et al., 2003; Turgut et al., 2016). Owing to the frequent high intrapopulational variability in EO chemical composition related to genetic factors, preliminary screenings of wild populations should be based on a significant number of individuals. Furthermore, studies on how each genotype is affected by environmental and ontogenetic factors are also required (Figueiredo et al., 2008). Consequently, simple and fast methods to process a great number of samples are demanded in order to obtain significant data allowing for well-defined chemotypes. Several methods have been reported for identifying EO chemical profiles such as vibrational spectroscopic methods (Schulz et al., 2003, Gudi et al., 2015) or the electronic nose (Vouillamor, 2009). Because of its simplicity, speed, reliability and economy, thin-layer chromatography (TLC) methods can be also useful (Pothier et al., 2001), mainly, in field research, away from well-equipped laboratories (Franz, 2010). In this work, a method combining TLC and GC/MS has been proposed to identify EO chemical profiles from individuals belonging to wild populations in order to be propagated for obtaining chemical homogeneous cultivars. It has been applied to Mentha longifolia L. As reported in the literature, EO from this species shows a well-known chemodiversity (Lawrence, 2006; Sharopov et al., 2012; Abedi et al., 2015). Two principal chemical profiles can be distinguished according to the molecular skeleton of their main components: Menthane skeleton oxygenated in C2. Samples containing carvone and dihydrocarvone (cis and trans isomers) as major compounds, as reported by Kokkini & Lanaras, (1995); Mastelic et al., (2002); Dzamic et al., (2010); Bertoli et al., (2011). Menthane skeleton oxygenated in C3. Two typical profiles related to alternative metabolic pathways involving the double bond C4-C8 reduction can be distinguished: (a) piperitenone, piperitone and their epoxides (Maffei, 1988; Saeidi et al., 2012; Segev et al., 2012; Jamzad et al., 2013; Moradalizadeh et al., 2014) and (b) pulegone, menthone or isomenthone, menthofuran and other menthol derivatives as well (Mkaddem et al., 2009; Hajlaoui et al., 2009; Segev et al., 2012). Furthermore, samples whose major components show other chemical structures have been also reported such as the menthane skeleton oxygenated in C8. For example, 1,8-cineol is usually found together with the above mentioned two molecular structures as relative major component (Koliopoulos et al., 2010; Moradalizadeh et al.,2014). It is even reported as main compound by Fleisher & Fleisher (1998). It is closely related to α-terpineol as it derives from it by means of an ether bond between C1 and C8. On the other hand, α-terpinyl acetate and terpinen-4-ol (menthane skeleton oxygenated in C4) are reported by Baser et al. (1999) as major components in samples from Turkey. Molecular structures based on the sabinene skeleton have been also reported. For example, (E)-sabinenhydrate has been found the principal compound in some samples from Crete (Kokkini & Papageorgiou, 1988) and Turkey, belonging to different subspecies (Baser et al., 1999). Samples rich in borneol (bornane skeleton) from Pakistan have been cited by Hussain (2010) and linalool (acyclic structure) as major compound has been also reported by Baser et al. (1999) in Turkish samples, as well. Taking as starting point the results of a previous research (Llorens-Molina et al., 2015), in which a noticeable intrapopulational variability was found in a population of Mentha longifolia L. located in Teruel (Spain). The aims of this work are the following; a) To develop and validate a TLC – GC/MS method for screening wild populations and to identify EO chemical profiles from individuals in order to be propagated for obtaining chemical homogeneous cultivars, b) To use this method as a quick way to identify individuals coming from any other accessions.

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Material and methods Material 50 small pieces of stolon from a wild population located in the confluence of Jiloca and Pancrudo rivers in Teruel (Spain) (40º 58’ 11.89’’ N, 1º 8’ 49.40’’ W) were cut during the winter period (Figure 1). They were planted in pots and bred in greenhouse conditions (Figure 2). Once these individuals were classified at the end of identification process, vouchers belonging to each one of chemotypes were kept at the herbarium of the Universitat Politècnica de València (VALA 9576-9580) and the individuals belonging to same EO profile were transplanted and grown together in field plots as seen in Figure 3.

Figure 1. Stolon pieces from wild population

Methods Sampling and extraction of mint leaves for Thin Layer Chromatography (TLC) analysis A non-destructive sampling was employed for isolating volatile and semivolatile components according to Wagner and Bladt, (2002). A small amount of material coming from each individual at flowering stage was subjected to solvent extraction: 2 mL of dichloromethane were added to 0.2 g of fresh material previously cut in small pieces in 5 mL vials. Then, they were placed on an orbital stirrer for 30 min. Afterwards, each extract was dried with a little amount of Na2SO4 and filtered with a syringe filter. Dried extracts were transferred to other vials and placed opened in fume hoods up to total solvent evaporation. Finally, 100 µL of toluene were added into each vial. They were sealed and kept in refrigerator at 4°C until TLC analysis. TLC analysis The extracts were applied on the TLC plates and developed according to Wagner and Bladt (2002) method for essential oils. Volumes of 10 µL were spotted with capillary tubes (Blaubrand intraMark, 10 µL) on the silica gel plates (DC-Fertigfolien Alugram Sil G/UV 254). In order to classify individuals with similar profiles, they were developed in duplicate using toluene:ethyl acetate (93:7) as mobile phase and further sprayed with sulfuric vanillin and sulphuric anisaldehyde (UV 265 nm), respectively, as visualization reagents, as described by Wagner and Bladt (2002). Five initial profiles were distinguished according the

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presence/absence of discriminant spots. With the aim of checking this first classification, a new TLC analysis was performed with three different individuals belonging to each one of the defined profiles. Retention factor

(Rf) values were calculated for these discriminant spots. TLC method validation TLC plates were spotted with 25 µL of sample extracts and developed in the same conditions but they were not sprayed with any visualization reagent. Silica gel layer matching to measured range for discriminant spots was scrapped and extracted with 0.5 mL of dichloromethane. The filtered extract was kept and sealed in 350 µL insert vials until GC/MS analysis. These extracts were analyzed by GC/MS in order to identify their composition. Chemotypes characterization Leaves coming from the plants in full flowering stage belonging to each profile were put together and homogenised. EO was extracted by Simultaneous Distillation Extraction (SDE) (Chaintreau, 2001) and analyzed by GC/MS. GC/MS analysis The analysis of samples was carried out by GC-MS using a Clarus 500 GC (Perkin-Elmer Inc., Wellesley, PA, USA) chromatograph equipped with a fused-silica capillary column ZB-5 (30 m × 0.25 mm i.d. × 0.25 μm film thickness; Phenomenex Inc, Torrance, CA, USA). The injection volume was 1 µL. Ionization source temperature was set at 200°C and 70 eV electron impact mode was employed. MS spectra were obtained by means of total ion scan mode (mass range m/z 45-500 uma). The GC oven temperature was programmed in the following way: 60°C for 5 min, raised at 3°C/min up to 180°C and then at 20°C/min up to 280°C, hold for 10 min. Helium was the carrier gas (1.2 mL min−1). For analysis of selected TLC spots, a shorter oven temperature program was used (60°C for 3 min, raised at 10°C/min up to 180°C and then at 20°C/min up to 280°C, hold for 3 min.) The total ion chromatograms (TIC) and mass spectra were processed with the Turbomass 5.4 software (Perkin-Elmer Inc.). Retention indices were determined by injection of C8–C25 n- alkanes standard (Supelco) under the same conditions. The EO components were identified by comparison of retention indices and mass spectra by computer library search (NIST MS 2.0) and available data from literature (Adams, 2007). Identification of the following compounds was confirmed by comparison of their experimental retention index (RI) with those of authentic reference standards (Sigma-Aldrich): α-pinene, β- pinene, camphene, myrcene, limonene, (Z)-β-ocimene, camphor, terpinolene, terpinen-4-ol, borneol and bornyl acetate. Application to individuals coming from other populations: Another way of using this method is to perform a first screening of nearby populations of the same species. An individual sample collected in Tuejar (Valencia) (39º 45’ 44’’ N; 1º 01’ 09’’ W) was processed with five samples (all of them were taken in vegetative stage) corresponding to identified profiles. All of them were processed according the method above described. Taking into account the presence/absence of discriminant spots, this new sample was classified according the five defined profiles. In order to check the applied method, this sample was also analyzed by GC/MS and its TIC chromatogram was compared with those taken as reference.

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Results and Discussion Preliminary TLC profiles According the first visual examination of the 50 individuals’ TLC profiles (one example is shown in Fig. 2) five patterns of composition were defined according the presence/absence of discriminant spots. Results of spraying with sulphuric vanillin and sulphuric anisaldehyde (UV 365nm) are displayed in Figure 3. The new TLC analysis based on three individuals of each defined profiles gave similar results (Figure 4a, 4b), so the Rf values were measured in order to apply them to find out by GC/MS the chemical identity of spots (Table 1).

Figure 2. Example of some individual profiles obtained by TLC, using sulphuric vanillin as spray reagent.

Figure 3. Five EO profiles were defined from the 50 individuals being tested.

Figure 4a. TLC analysis by triplicate for each one of defined profiles. Characterization of discriminant spots (sulphuric vanillin as spray reagent)

For each profile (A1-E1), the discriminant spots are specified as: capital letter, profile; small letter: spot in decreasing order of Rf. A2- E2 and A3-E3 are repetitions.

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Figure 4b. TLC analysis by triplicate for each one of defined profiles. Characterization of discriminant spots (sulphuric anisaldehyde –UV 365 nm- as spray reagent)

Table 1. Validation by GC/MS of TLC selection method. Rf, colour and composition for discriminant spots.

Discriminant spots features Main GC/MS peaks Other secondary GC/MS peaks

(Rf range) Reagent RI compound RI Compounds

Vanillin Anisaldehyde

Profile

(UV 365 nm) A Aaa: Reddish-brown (weak) Blue green 1365 Piperitenone oxide 1031 1,8-cineole 0.56-0.64 (strong) Ab: Reddish-brown (strong) Blue green 1263 Piperitone oxide 0.49-0.56 (strong) (Fig.6a) B Ba: Reddish-brown (weak) Blue green 1193 (Z)-dihydrocarvone 1031 1,8-cineole 0.39-0.56 1237 pulegone 1174 Menthol 1365 Piperitenone oxide C Ca: Purple Dirty-white 1348 α-terpineol acetate 0.68-0.76 (Fig. 6b) Cb: Orange Dark 1573 0.23-0.29 Carvone acetate D Da: Reddish (weak) Dirty-white 1193 (Z)-dihydrocarvone 1031 1,8-cineole 0.62-0.73 Db: Grey-violet (weak) Reddish 1201 Dihydrocarveol neo 1238 Dihydrocarveol neoiso 0.31-0.40 1337 Dihydrocarveol acetate Dc: Grey-violet (weak) Reddish 1205 Dihydrocarveol iso 0.15-0.25 (Fig. 6c) 1366 Dihydrocarveol acetate neoiso E Ea: Dark Blue green 1237 Pulegone (Fig. 6d) 1031 1,8-cineole 0.27-0.58 (strong) 1174 menthol a Capital letter: Profile; small letter: spot in decreasing order of Rf

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Validation of TLC analysis by GC/MS Examples of TIC chromatograms for some discriminant spots (marked in table 1) are displayed in Figure 5a- 5e. These results confirm the discriminant character of selected spots.

Figure 5a. TIC chromatogram of extract from the spot containing piperitone oxide.

Figure 5b. TIC chromatogram of extract from spot containing α-terpineol acetate.

Figure 5c. TIC chromatogram of extract from spot containing dihydrocarveol.

Figure 5d. TIC chromatogram of extract from spot containing pulegone.

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Chemotypes characterization

GC/MS TIC chromatograms of defined profiles are shown in Figure 6a-6e (major compounds are marked)

Figure 6a. Essential oil profile of chemotype A

Figure 6b. Essential oil profile of chemotype B

Figure 6c. Essential oil profile of chemotype C

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Figure 6d. Essential oil profile of chemotype D

Figure 6e. Essential oil profile of chemotype E

Piperitone oxide and piperitenone oxide are the major compounds in profile A. This composition is similar to that reported by Baser et al. (1999) in Turkey and Singh et al (2008) in India. Profile B is characterized by (E) dihydrocarvone, pulegone and piperitone oxide as main compounds. Chemotypes rich in (E)-dihydrocarvone have been reported in samples from Serbia (Mimica-Dukic et al, 1991; Matovic & Lavadinovic, 1999; Dzamic et al, 2010). Chemotypes similar to profile C (α-terpineol acetate + carvone acetate) have not been reported so far except for samples coming from the same and nearby populations (Llorens-Molina et al.,2015). Profile D has been found mainly rich in dihydrocarvone isomers and minor amounts of carveol isomers and derivatives. It may be also considered a new specific chemotype given that no similar composition has been reported. Lastly, profile E has been found rich in menthone, menthol and pulegone. A very similar composition has been reported by Hajlaoui et al., (2009) in Tunisia. Other chemotypes rich in menthone and pulegone have been also reported from South Africa (Asekun et al., 2007; Oyedeji & Afaloayan, 2006). Concerning the usefulness of the proposed TLC-GC/MS method, the results related with the identification of samples coming from Tuejar are promising. TLC profiles of this new sample matches with E chemotype. GC/MS TIC chromatogram confirms its identity (Figure 7).

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Figure 7. New sample coming from Tuéjar compared with the five TLC profiles defined. GC/MS analysis confirm the identification of this new sample as belonging to chemotype E.

Conclusions The proposed methodology contributes with an easy and reliable procedure allowing the screening of wild plant populations in several concerns: a) a first overview of intrapopulational variability, b) identification of chemical profiles potentially useful for their possible biological activities, c) the possibility of propagating these specific individuals in order to obtain chemical homogeneous cultivars. To the extent that this method can be applied to more populations, a greater number of profiles could be defined, thus increasing its usefulness. On the other hand, the improvement of the photographic techniques used when the plates are developed with UV light, would allow to dispense with the validation by GC/MS, whose necessity is justified especially when the discriminant spots have very close Rf or very similar fluorescence colors.

ACKNOWLEDGEMENTS Part of this work was presented in ISEO 2017, Pecs, Hungary. REFERENCES

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RESEARCH ARTICLE Chemical composition and antioxidant activity of essential oils of six Lamiaceae plants growing in Southern Turkey

Hasan Maral1, *, Murat Türk2, Tuncay Çalışkan3, Ebru Kafkas4, Saliha Kırıcı3

1 Ermenek Vocational School, Karamanoğlu Mehmetbey University, Karaman, TURKEY. 2 Ceyhan Vocational School of Higher Education, Çukurova University, Adana, TURKEY, 3 Department of Field Crops, Agricultural Faculty, Çukurova University, Adana, TURKEY. 4 Department of Horticulture, Agricultural Faculty, Çukurova University, Adana, TURKEY.

*Corresponding author. E-mail: [email protected]

Abstract Plants of the Lamiaceae family are rich in polyphenolic compounds, and a large number are well known for their antioxidant properties. In the present study, the essential oils of six species were evaluated for their chemical composition and antioxidant activity. The species used in the present study were namely Origanum dubium, Origanum vulgare subsp. hirtum, Thymbra spicata, Thymus sipyleus, Satureja cilicica and Ziziphora clinopodioides, respectively. The essential oil contents for Origanum dubium was 4.6%, for Origanum vulgare subsp. hirtum 3.4%, Thymbra spicata 1.28%, Thymus sipyleus 1.78%, Satureja cilicica 1.64% and for Ziziphora clinopodioides was 0.35%. The essential oils compositions were determined by GC-MS. Major identified components were carvacrol for O. dubium, O. vulgare subsp. hirtum, and T. spicata; α-pinene for T. sipyleus; and borneol for Z. clinopodioides. Total phenolic contents of essential oils have a very high equivalent amount compared to gallic acid, however, low radical scavenging properties.

Keywords: Antioxidant, Lamiaceae, Essential oils, Chemical composition

Introduction The Lamiaceae family is spread all over the world, mostly in North-Western Asia and the Mediterranean region, and comprises mostly herbs and shrubs consisting of annual or perennial aromatic plants. Members of the Lamiaceae can grow in almost all habitat types and all altitudes (Watson & Dallwitz, 1978). This family with about 220 genera and 3500, is represented by 45 genera, 565 species and 735 taxa in our country (Başer, 1994; Davis, 1988; Morgariset al., 1982; Watson and Dallwitz, 1978). Lamiaceae is the family with the highest number of endemic species in Turkey and the endemism rate is 45% (Güner at al, 2000). At the intersection of different climate zones, Turkey is a very rich country in terms of plant species and diversity. Herbs and spices of the Lamiaceae constitute an important proportion of the export items to the world markets generating an important trade income. Plants of this family are also used for various purposes, mostly as tea or spices (Baytop, 1999). In addition, Turkey is an important gene center for Lamiaceae members, which have been used in traditional medicine for very long times (Kocabaş & Karaman, 2001; Özkan, 2007). Biological and pharmacological activities of species belonging to this family have been known for years. The bioactivity of these plants are also associated to their essential oil components (Bozinet al., 2006). Members of this family have also been the focus of research in terms of plants with antioxidant activity in recent years. Rosmarinus officinalis and Salvia spp. are currently known and used in the world (Göger, 2006). Their main use is in tea form. Lamiaceae plants are among the most known and studied plants for essential oils all over the world (Başer, 1994; Göger, 2006).

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Antioxidant supplements or antioxidant-containing foods may be used to help the human body to reduce singlet oxidative damage (Halliwell & Gutteride, 2007). There are two basic categories of antioxidants namely synthetic and natural ones. However, the use of these synthetic antioxidants has been restricted in some countries, mainly because they are suspected to be carcinogenic (Bartosz, 1997). Many researchers have focused on natural antioxidants, and in the plant kingdom numerous crude extracts and pure natural compounds have previously been reported to have antioxidant properties. Therefore, the development and utilization of more effective antioxidants of natural origin are desired. The aim of this study was to determine chemical composition and antioxidant potential of the essential oils of Origanum dubium, Origanum vulgare subsp. hirtum, Thymbra spicata, Thymus sipyleus, Satureja cilicica, and Ziziphora clinopodioides growing in southern Turkey. Materials and Methods Plant Material The aerial parts of Origanum dubium, Origanum vulgare subsp hirtum, Satureja cilicica, Thymbra spicata, Thymus sipyleus and Ziziphora clinopodioides were collected at bloom stage in July, 2012 from Ermenek (Southern part of Turkey). The plant specimens were identified at the Department of Agronomy of Agricultural Faculty of Çukurova University. Vouchers of the specimens are deposited. Essential Oil Isolation Air- dried plant material (25 g) was hydrodistilled for 3 h using a Clevenger type apparatus. The resulting oils were kept in sealed vials at 4 °C until analysis. GC–MS Analysis Essential oils were analyzed by GC-MS. A Perkin Elmer apparatus equipped with CPSil5CB (25 m × 0.25 mm i.d., 0.4 μm film thickness) fused-silica capillary column. The flow rate of helium as carrier gas was 1 mL/min. The injector temperature was 250ºC, set for splitless injection. The column temperature was 60ºC/5ºC/min/260ºC for 20 min. Mass spectra were taken at 70 ev. Mass range was between m/z 30–425. A library search was carried out using the Wiley GC-MS Library and the Flavor Library of Essential Oil Constituents. The mass spectra were also compared with those of reference compounds and confirmed with the aid of retention indices from published sources. Relative percentage amounts of the separated compounds were calculated from total ion chromatograms by the computerized integrator. DPPH Radical Scavenging Assay The hydrogen atom or electron donation ability of the corresponding extract and some pure compounds were measured from the bleaching of a pure –colored methanolic solution of DPPH. This spectrophotometric assay uses the stable radical 2,2’–diphenylpicrylhydrazyl (DPPH•) as a reagent (Hwang et al., 2001). In brief, 0.1ml of extract or essential oil solution was mixed with 3.9 ml of DPPH• solution.The mixture was incubated for 30 min at 25 oC. After a 30 min incubation period at room temperature the absorbance was read against a blank at 517 nm. Inhibition of free radical DPPH (%I) was calculated as;

I%= Ablank – Asample / Ablank x 100 ; where Ablank is the absorbance of the control reaction (containing all reagents except the test compound) and Asample is the absorbance of the test compound. The extract concentration providing 50% inhibition (IC50) was calculated from graph of inhibition percentage against extract concentration. Tests were carried out in triplicate.

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Assay for Total Phenolics Total phenolic content was estimated by the Folin-Ciocalteu method (Singleton and Rossi, 1965). 100 microliters of 1:10 diluted sample were added to 1 ml of 1:10 diluted Folin-Ciocalteu reagent. After 4 min, 800 µL of sodium carbonate (75 g/L) were added. After 2 h of incubation at room temperature, the absorbances at 765 nm were measured. Gallic acid (0-500 mg/L) was used for calibration of a standard curve. The results were expressed as gallic acid equivalents (GAE) / g dry weight of plant material. Triplicate measurements were taken and mean calculated. Results and Discussion

The essential oil content of O. dubium hydrodistilled by Clevenger apparatus was determined as 4.6 %. Eighteen compounds, representing 96.96 % of the total oil, were characterized with carvacrol (63.1%) and gamma-terpinene (11.1%) being the main compounds whereas other significant compounds were pcymene (9.3%), alpha-phellandrene (2.7%) and myrcene (2.6%) as listed in Table 1. It was reported that the chemical composition of Origanum essential oils were very variable (Scheffer et al. 1986; Barataet al. 1998; Baser et al. 2003). Our finding is in agreement with Baser et al., 2011, who reported that Origanum dubium oil was rich in carvacrol (78.27%). The essential oil content of O. vulgare subsp. hirtum was determined as 3.4%. GC-MS analysis of O. vulgare subsp. hirtum resulted in the identification of seventeen compounds, representing 96.82% of the oil. The results showed that the essential oil was rich in carvacrol (60.8%), linalool (9.2%), p-cymene (7.5%), - terpinene (6.1%), myrcene (3.4 %) and isoeugenol (2.8%) as main components. According to Russo et al. (1998), there are two main chemotypes of oregano (either the thymol or the carvacrol chemotype), but there are also two intermediary chemotypes (thymol/carvacrol and carvacrol/thymol). The environmental conditions are able to influence which biosynthetic pathway (thymol or carvacrol) may be dominant. The results reported in the present work suggest that the oregano that we analyzed belonged to a carvacrol/thymol chemotype. The essential oil content of T. spicata was determined as 1.28%. sixteen components were identified representing 97.44% of total oil. The major constituents were carvacrol (27.4%), α-terpinene (27.0%), p- cymene (14.5%), thymol (13.1%) and alpha- terpinene (4.1%). Our results verify that, in previous studies the major essential oil components of T. spicata were carvacrol, γ-terpinene and p-cymene (Kizil, 2013; Barakat et al., 2013; Ravid and Putievsky, 1985; Baydar et al., 2004; Unlu et al., 2009; Markovic et al., 2011; Kizil et al., 2015). Barakat et al. (2013) found that the main components of T. spicata oil of Lebanese origin were carvacrol (16.1–62.9%), α-thujene (1.7–4.8%), myrcene (1.1–5.1%), γ-terpinene (11.4–24.1%) and p-cymene (8.1–46.8%), respectively. Seventeen compounds, representing 88.87 % of the total oil, were identified in T. sipyleus essential oil, where α-pinene (20.9%), (Z, E)-farnesol (16.1%) and 1,8-cineole (9.5%) were the main compounds (Table 1). The other important compounds were determined as limonene (7.6%), linalool (6.6%), α-terpineol (5.2) and isoborneol (isomer 1) (4.6%). The compositional data shows that α-pinene was the main compound of essential oil of T. sipyleus. The major constituents of the Z. clinopodioides essential oil were borneol (24.4%), followed by α-pinene, (17.7%), camphene (8.9%), camphor (8.6%) and cis-verbenol (4.7%), isoborneol (21.2%). Meral et, al. reported that main component of the essential oils of Z. clinopodioides is borneol. Pulegone is the major component of several Ziziphora species (Sezik et al., 1991; Meral et al., 2002; Başer 2002; Shahla, 2012).

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Table 1. GC/MS analysis results (relative %) of essential oils RT Compound 1* 2* 3* 4* 5* 3.28 Tetradecane - - - - 0.45 7.02 α-phellandrene 2.74 2.09 3.10 - - 7.24 α-Pinene 1.38 1.10 1.10 20.88 17.65 7.76 Camphene 0.25 0.16 0.20 4.29 8.86 8.84 β-Pinene 0.24 - 0.24 1.99 0.97 8.84 sabinene - 0.20 - 1.08 0.48 9.53 Myrcene 2.59 3.40 2.39 1.86 - 10.00 α -phellandrene 0.36 0.26 0.40 0.71 - 10.52 α-terpinene 2.36 1.30 4.06 - - 10.87 p-Cymene 9.25 7.53 14.47 0.41 1.71 11.03 Limonene 0.61 0.49 0.70 7.59 2.98 11.13 1,8-cineole (Eucalyptol) 0.25 - - 9.49 3.32 12.42 α - terpinene - - 26.95 - - 12.39 γ-Terpinene 11.08 6.08 - - 0.43 12.70 Linalylbutyrate 1.00 - - - - 12.70 -3-carene - 0.91 0.30 - - 14.27 Linalool 0.23 9.20 0.32 6.61 0.43 15.30 α-Methylbenzylalcohol - - - 0.28 - 16.08 Farnesol - - - 16.10 - 16.21 ocimene - - - 4.26 0.48 16.58 trans- Sabinene - - - - 2.04 17.09 Isoborneol 0.302 0.182 0.22 4.60 24.42 17.66 terpinene 0.425 0.235 - 0.27 - 18.29 α-Terpineol 0.189 - - 5.24 - 18.35 Camphor - - - - 8.57 20.73 Isoeugenol - 2.834 - - - 21.79 Verbenol - - - - 3.33 22.64 cis-Verbenol - - - - 4.67 22.98 Thymol - - 13.14 - - 23.46 Carvacrol 63.09 60.57 27.42 - - 28.06 β-Caryophyllene 0.631 0.283 2.39 3.21 - Total (%) 18 17 16 17 16 Number of components 96.96 96.82 97.44 88.87 80.34

1*Origanum dubium, 2* Origanum vulgare subsp. hirtum, 3* Thymbra spicata, 4* Thymus sipyleus, 5* Ziziphora clinopodioides

Antioxidant Activity DPPH radical-scavenging activity: The DPPH radical is a stable free radical, commonly used as a substrate, to evaluate in vitro antioxidant activity of extracts of fruits, vegetables and medicinal plants. Antioxidants can scavenge the radical by hydrogen donation, which causes a decrease of DPPH absorbance at 517 nm. Table 2 shows the DPPH radical-scavenging activity of the different the essential oils. The concentration of sample at which the inhibition percentage reaches 50% is defined as the IC50 value. Thus, IC50 value is negatively related to the antioxidant activity, the lower the IC50 value, higher is the antioxidant activity of tested sample. The radical scavenging effect with the DPPH method was found to be too low for each essential oil. Their 50%

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inhibition concentration is very high. Compared with essential oil extracts, the highest radical cleaning feature showed essential oil extract of Origanum vulgare subsp. hirtum. Origanum dubium and Thymbra spicata are slightly less than Origanum vulgare subsp. hirtum. Ziziphora clinopodioides and Thymus sipyleus have the lowest radical scavenging effect.

Table 3. Radical scavenging activity of Lamiaceae essential oils in terms of IC50 values (g/mL)*1000 Essential Oil Radical Scavenging Activity Thymus sipyleus > maximum concentration Origanum dubium 1340 Origanum vulgare subsp. hirtum 8886 Thymbra spicata 1319 Ziziphora clinopodioides > maximum concentration Satureja cilicica 196800 *Values are mean of six different samples of each Lamiaceae species, analyzed individually in triplicate.

In the present study, the Folin-Ciocalteu reagent was used to obtain a crude estimate of the amount of phenolic compounds present in the extracts. As results, essential oil extracts showed very high total phenolic content. Equivalent amounts of gallic acid are given on Table 3. The order of the extracts is given on the table. Origanum dubium, O. vulgare subsp. hirtum, and Thymbra spicata have shown high total phenolic effect because carvacrol and other oxygenated compounds are abundant in these species.

Table 4. Phenolic contents of Lamiaceae essential oils Essential Oil mg-GA/mg-extract Thymus sipyleus 75,91 Origanum dubium 344,24 Origanum vulgare subsp. hirtum 307,92 Thymbra spicata 302,18 Ziziphora clinopodioides 41,18 Satureja cilicica 232,84

Figure 1. Antioxidant activity

Standard curve of gallic acid

3 y = 0,0051x 2,5 R² = 0,9943 2 1,5 1 0,5 0 0 100 200 300 400 500 600

ACKNOWLEDGEMENTS The authors would like to thank Çukurova University Scientific Research Projects Number: 2F2011020 for supporting this work.

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