Phytochemistry 98 (2014) 128–136 Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Micromorphological traits and essential oil contents of Micromeria kerneri Murb. and M. juliana (L.) Benth. (Lamiaceae) Dario Kremer a, Valerija Dunkić b,⇑, Mirko Ruščić b, Vlado Matevski c, Dalibor Ballian d, Faruk Bogunić d, Eleni Eleftheriadou e, Danijela Stešević f, Ivan Kosalec a, Nada Bezić b, Edith Stabentheiner g a Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovačića 1, HR-10000 Zagreb, Croatia Faculty of Sciences, University of Split, Teslina 12, HR-21000 Split, Croatia Faculty of Natural Sciences and Mathematics, Ss Cyril and Methodius University, 1000 Skopje, Republic of Macedonia d Faculty of Forestry, University of Sarajevo, Zagrebačka 20, BIH-71000, Bosnia and Herzegovina e Aristotle University of Thessaloniki, Faculty of Forestry and Natural Environment, GR-54124 Thessaloniki, Greece f Faculty of Natural Sciences and Mathematics, University of Montenegro, Džordža Vašingtona bb, 81 000 Podgorica, Montenegro g Institute of Plant Sciences, Karl-Franzens University, Schubertstrasse 51, A-8010 Graz, Austria b c a r t i c l e i n f o Article history: Received 14 June 2013 Received in revised form 4 December 2013 Available online 2 January 2014 Keywords: Essential oil Micromeria juliana Micromeria kerneri Pollen Plant trichomes a b s t r a c t The chemical composition of the essential oil (analysed by GC and GC–MS), the types and distribution of trichomes and pollen morphology (analysed by scanning electron microscopy) were investigated in two closely related species, Micromeria kerneri Murb. and Micromeria juliana (L.) Benth. (Lamiaceae) from Southeast Europe as a contribution to their taxonomy. The essential oil of M. kerneri was characterized by a high concentration of oxygenated sesquiterpenes, with caryophyllene-oxide as the major compound. Caryophyllene-oxide was also the major component of the essential oil of M. juliana from all localities, except from Mt Krivošije (Montenegro), where piperitone oxide was the major constituent. Non-glandular trichomes, peltate trichomes, and two types of capitate trichomes (type 1 composed of one basal epidermal cell, and one head cell with subcuticular space; type 2 composed of one basal epidermal cell, two stalk cells, and one head cell with subcuticular space) were observed on leaves, the calyx and on the stem. Pollen of both species had six apertures (hexacolpate) set in the equatorial pollen belt (zonocolpate) and showed medium reticulate ornamentation. Multivariate analysis (PCA and UPGMA) of essential oil components clearly separated the investigated M. kerneri and M. juliana populations, and confirmed the opinion that they are different taxa. On the other hand, micromorphological traits between these species were the same. Nevertheless, definitive conclusions about the taxonomic relationships among these species will require genetic analysis. Ó 2013 Elsevier Ltd. All rights reserved. Introduction The genus Micromeria Benth. (Lamiaceae) is a part of complex group of genera within the tribe Mentheae subtribe Menthinae (Bräuchler et al., 2005) that includes 54–70 (–90) herbs, subshrubs and shrubs with a distribution range extending from the Himalayan region to the Macronesian Archipelago and from the Mediterranean to South Africa and Madagascar (Bräuchler et al., 2005, 2008; Chater and Guinea, 1972; Erhardt et al., 2002). Twenty-two Micromeria species have been described for Europe, and about the half of these occur on the Balkan Peninsula (Chater and Guinea, 1972; Tan and Zielinski, 2001), many of which are endemic. To date, nine species have been recorded for Croatia (Šilić, 1979), five species for Bosnia and Herzegovina (Šilić, 1979), eight species for Montenegro (Petrović and Stešević, 2011; Šilić, ⇑ Corresponding author. Tel.: +385 21385133; fax: +385 21384086. E-mail address: dunkic@pmfst.hr (V. Dunkić). 0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2013.12.009 1979), six species for Republic of Macedonia (Micevski, 2002), and 14 taxa for Greece (three of them endemic to Crete) (Chater and Guinea, 1972; Euro+Med 2006–2013). M. kerneri Murb. is a dwarf shrub with few to many erect stems up to 30 cm high, patent-pubescent. Leaves are 3–9 mm long, 2– 5 mm wide, shortly pubescent at least beneath. Flowers are hermaphroditic, from 6 to 10 arranged in verticillasters. Bracts are half as long as the calyx. The calyx is about 3 mm long, sparsely hairy on veins and in throat, with lanceolate-subulate, unequal teeth. Corolla is purplish, 3–4 mm long. M. kerneri is an endemic Illyric-Balcanic species distributed in Bosnia and Herzegovina, Croatia, and Montenegro at altitudes of 5–250 m (Chater and Guinea, 1972; Šilić, 1979). M. juliana (L.) Benth. is a dwarf shrub with many erect stems up to 40 cm high, puberulent to pubescent throughout. Leaves are 3– 8 mm long, 1–2.5 mm wide, shortly pubescent at least beneath. Flowers are hermaphroditic, from 4 to 20 arranged in verticillasters. Bracts have the same length as the calyx. Calyx is 2.5– 129 D. Kremer et al. / Phytochemistry 98 (2014) 128–136 Table 1 Phytochemical composition (%), identification and major groups of chemical components of essential oil of Micromeria kerneri and M. juliana. For sample abbreviations see Table 3. No. Component RIa RIb Sample (yield in %) IM M. kerneri 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. Monoterpene hydrocarbons a-Thujene a-Pinene Verbenene Camphene Sabinene b-Pinene Myrcene d-3-Carene a-Terpinene p-Cymene b-Phellandrene Limonene (Z)-b-Ocimene c-Terpinene allo-Ocimene 30. 31. 32. 33. 34. 35. 36. Oxygenated monoterpenes cis-Sabinene hydrate trans-Linalool oxide (furanoid) Linalool b-Thujone trans-Pinocarveol Camphor Pinocarvone Borneol Terpinen-4-ol a-Terpineol Myrtenol Verbenone trans-Carveol endo-Fenchyl acetate b-Cyclocitral Piperitone Linalyl acetate Bornyl acetate a-Terpenyl acetate Neryl acetate Piperitone oxide 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. Sesquiterpene hydrocarbons a-Copaene b-Bourbonene a-Gurjunene (E)-caryophyllene b-Copaene trans-a-Bergamotene (Z)-b-Farnesene a-Humulene allo-Aromadendrene b-Chamigrene Germacrene D Viridiflorene Bicyclogermacrene b-Bisabolene d-Cadinene 52. 53. 54. 55. 56. 57. Oxygenated sesquiterpenes Spathulenol Caryophyllene oxide c-Eudesmol a-Cadinol a-Bisabolol a-Bisabolol oxide 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. Phenolic compounds 924 938 960 962 971 982 992 1008 1016 1021 1025 1032 1052 1057 1128 1035 1032 – 1059 1128 1113 1174 1157 1251 1268 1213 1204 1218 1255 1351 M. juliana MkG (0.06) MkSP (0.05) MkKI (0.06) MkM (0.06) MjOD (0.08) MjS (0.08) MjL (0.09) 14.1 29.1 24.1 30.3 18.3 16.1 25.5 – 0.9 0.1 0.2 1.7 6.3 – – – – 1.1 1.7 – 2.1 – 0.3 4.2 0.7 0.9 4.9 9.1 0.9 – 0.2 tr 1.6 3.9 0.1 2.2 0.1 – 2.6 0.2 0.4 3.8 7.1 2.2 0.1 0.7 1.1 1.5 2.3 – 1.9 0.2 – 4.3 – 1.7 1.3 12.1 2.4 – 0.8 – 0.9 5.4 – 1.2 0.2 – 12.1 2.0 0.3 0.4 3.2 – – – – – 0.2 – – 0.1 – 2.9 1.9 0.5 0.2 7.0 0.3 – – – – 3.3 – – – – 11.0 2.3 2.1 1.6 8.3 0.1 – – – – 0.1 – – – 19.0 27.1 32.0 27.3 14.5 31.3 MjBR (0.07) MjNS (0.06) 13.2 18.7 – – – – – – – – – – – – – – 10.9 2.1 0.6 0.5 4.3 0.2 – – – – 0.1 – – tr 19.5 31.7 24.9 20.3 – – – – – – 1065 1561 2.2 1.7 1.5 2.5 1088 – 0.1 0.1 3.4 2.5 0.2 1.5 0.4 1099 1121 1147 1151 1160 1176 1184 1186 1197 1204 1215 1218 1548 1438 – 1499 0.3 2.1 0.1 1.2 1.1 – 5.7 3.1 0.2 2.2 4.2 1.1 0.2 0.9 0.3 0.4 3.2 – 6.2 2.7 0.4 2.5 6.3 1.9 0.6 0.8 0.2 1.5 4.1 – 4.7 2.3 0.2 1.5 3.2 1.1 – 5.4 – 0.3 – 1.2 0.2 0.5 0.1 0.2 0.3 0.1 – 7.8 – 4.1 0.6 4.1 3.3 0.3 0.2 1.1 1.1 0.5 – 5.3 – 0.6 – 1.3 4.2 – 0.1 0.2 0.3 0.4 – 1223 1250 1252 1285 1349 1358 1366 1629 1735 1553 1570 – 1692 – 0.9 0.2 0.2 1.1 – – – 3.2 0.3 0.4 0.2 – – – 1.1 – 0.3 0.2 0.1 – 0.1 2.2 0.1 0.3 – – – – 2.2 – – – – 3.8 – 2.4 0.6 1.5 – – 2.2 – 2.1 – 0.6 – 0.5 3.5 12.7 15.4 15.5 17.7 16.1 20.7 0.1 0.3 0.9 2.9 1.1 0.2 0.3 – 1.9 0.3 2.1 1.1 0.9 0.2 0.4 0.3 0.2 0.2 1.1 0.1 5.2 0.4 0.6 0.4 0.1 1.1 0.2 2.6 1.5 2.2 1.2 0.8 0.1 0.9 – 6.9 0.7 0.4 0.2 0.3 1.7 – 3.8 0.5 0.1 0.3 – 0.3 0.9 0.1 6.3 0.6 0.4 0.4 0.1 1.8 0.2 1.5 1.3 1.2 0.4 – 42.9 18.8 17.6 2.4 39.2 0.3 0.4 0.5 0.1 2.9 11.7 1.6 0.6 0.5 1.5 0.7 0.8 1377 1383 1407 1424 1429 1433 1454 1456 1465 1477 1481 1496 1500 1494 1517 1577 1581 1632 1655 1688 1748 1719 1611 1646 1782 – – – 1484 1508 1520 1585 – 1639 1654 1662 – 1692 1697 1718 1729 1745 2101 1955 2135 2208 2116 2511 – 0.2 4.3 – 0.9 3.3 0.1 2.1 2.2 0.9 – – MjK (0.08) 7.2 – 1.1 0.2 0.2 0.3 3.2 – – 0.6 0.2 1.1 0.3 6.5 0.4 1.1 1.1 4.1 0.7 0.2 6.2 – 4.5 – 0.3 – 0.2 2.2 1.1 – – 0.1 0.4 0.3 0.3 – – – – – – 1.1 RI, MS, S 5.3 RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, MS, S MS MS MS MS MS, S MS MS MS MS MS MS RI, RI, RI, RI, RI, RI, RI, MS MS MS MS MS MS MS RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, MS MS MS MS, S MS MS MS MS MS MS MS MS MS MS MS RI, RI, RI, RI, RI, RI, MS MS, S MS MS MS MS 0.2 – 1.9 0.3 0.1 0.2 1.1 2.4 0.2 0.3 0.3 – 0.1 – – 2.6 3.0 0.3 0.3 16.9 – – – – 14.5 – – – – 19.9 20.2 16.5 20.6 0.2 0.2 0.4 0.7 1.1 0.5 0.2 0.2 0.3 – 8.3 1.5 0.9 0.1 1.2 – – 2.3 – – 0.2 0.6 – 11.4 0.2 1.1 – 1.3 – – 4.9 – 0.1 0.3 1.2 – 10.3 0.7 0.1 – 2.7 1.2 – 3.1 – – 1.2 – – 11.1 0.5 tr – 1.9 – 0.3 2.7 0.8 – 0.7 0.4 20.1 23.0 20.6 13.7 3.9 12.8 0.2 – 0.7 – 4.2 13.5 0.8 0.7 0.3 0.6 – 18.5 0.8 0.3 1.1 2.3 – 18.3 1.1 0.8 0.2 0.2 6.9 0.5 6.8 4.2 – – – – 3.1 6.2 – 7.9 0.8 0.2 – 1.1 0.5 0.2 3.3 – – 8.3 1.1 – – 1.2 – – 4.9 2.1 – 0.5 1.3 0.9 1.6 17.7 25.9 21.8 – 10.1 0.9 0.2 0.4 2.1 – 14.7 0.6 – 2.1 0.3 – 23.3 0.6 0.6 0.9 0.5 0.1 17.3 2.1 0.1 0.3 1.9 3.4 3.4 – MS MS. S MS MS, S MS MS MS MS MS MS MS MS MS, S MS MS RI, MS – – RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, 2.3 (continued on next page) 130 D. Kremer et al. / Phytochemistry 98 (2014) 128–136 Table 1 (continued) No. Component RIa RIb Sample (yield in %) IM M. kerneri 58. 59. 60. Thymol Carvacrol Eugenol 1290 1298 1370 2198 2239 2175 61. 62. 63. 64. 65. Carbonylic compounds 3-Octanol acetate Isobutyl hexanoate Butyl-n-hexanoate Isoamylhexanoate b-Ionone 1125 1155 1193 1256 1487 1376 – – 1457 1955 66. Aliphatic alcohol 1-Octen-3-ol 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. Hydrocarbons Eicosane Heneicosane Docosane Tricosane Tetracosane Pentacosane Hexacosane Heptacosane Octacosane Nonacosane Total identified (%) 974 1452 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 M. juliana MkG (0.06) MkSP (0.05) MkKI (0.06) 0.1 0.4 0.2 0.5 0.2 0.1 0.4 0.1 0.1 0.1 0.1 1.4 0.6 0.1 – 0.3 0.4 0.7 0.7 1.1 1.1 6.4 2.3 – 0.3 1.1 – 0.9 – – – – – 96.0 – – 0.9 5.4 – – – – – 0.1 – 96.9 MkM (0.06) MjOD (0.08) MjS (0.08) MjL (0.09) MjK (0.08) MjBR (0.07) 4.1 1.9 0.9 0.3 tr 0.2 4.1 2.7 – 0.9 3.3 – 2.3 1.1 – 2.5 0.9 – – – – 1.3 0.4 0.2 – 0.5 0.2 0.9 0.4 0.2 – – – – – – – – – – – – – – – – tr tr 1.4 tr – 0.4 0.2 0.1 0.3 0.2 – 0.1 0.1 98.8 – 0.2 0.1 0.3 0.5 0.3 0.1 – 2.2 0.1 – 0.4 0.1 0.3 – MjNS (0.06) – – – 0.2 0.2 – – – – 0.3 0.1 0.1 0.1 0.9 0.9 0.2 0.2 1.1 1.1 1.2 1.2 0.9 0.9 0.6 0.6 1.0 – – 0.3 0.1 – 0.2 0.1 0.3 – – 97.9 1.3 0.2 – – 0.2 – 0.4 0.5 – – – 80.9 2.6 – – – – 0.7 0.3 0.8 0.4 0.2 0.2 95.7 1.9 0.3 – – – – 0.3 0.9 0.3 0.1 – 85.3 2.2 0.4 – – – tr 0.1 – 0.2 0.7 0.8 84.1 1.6 0.2 1.4 0.2 – – – – – 0.3 0.5 0.6 – – – 83.4 0.4 0.4 – 0.4 – – – 85.9 RI, MS, S RI, MS, S RI, M, S RI, RI, RI, RI, RI, MS MS MS MS MS RI, MS RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, S S S S S S S S S S Retention indices determined relative to a series of n-alkanes (C8–C40) on the capillary column RIa, VF-5MS column, RIb, CP Wax 52 column; IM, Identification Method; RI, comparison of RIs with those listed in a homemade library, reported in the literature (Adams, 2007), and/or of authentic samples; MS, comparison of mass spectra with those listed in the mass spectral libraries NIST02 and Wiley 7; S, coinjection with reference compounds; tr, trace (less than 0.05%); –, component not identified. 3.5 mm long, glabrous in the throat, with subulate, rigid, slightly unequal teeth. Corolla is purplish, about 5 mm long. It is distributed in Portugal, southeast France, Corsica, Crete, Italy, Sicilia, Croatia, Bosnia and Herzegovina, Montenegro, Albania, Republic of Macedonia, Bulgaria, Greece and Turkey (Chater and Guinea, 1972; Šilić, 1979). M. kerneri and M. juliana are closely related species and according to Bräuchler et al. (2008), these two taxa might be conspecific. Phytochemicals with biological activity are found extensively at different levels in many plants. They are used in the food and cosmetics industries, in pharmacy, and in official and traditional medicine. Essential oil is among the most interesting plant bioactive constituents, possessing considerable antimicrobial (Ćavar et al., 2008), antifungal (Kremer et al., 2012a), antiviral (Vuko et al., 2012), and antioxidant (Amiri, 2011) activity. The family Lamiaceae comprises many species that accumulate commercially important essential oils. The essential oil composition of the genus Micromeria is variable (Duru et al., 2004; Formisano et al., 2007; Kremer et al., 2012b; Marinković et al., 2002; Sefidkon and Kalvandi, 2005; Slavkovska et al., 2005; Stojanović et al., 2006; Tzakou and Couladis, 2001; Vuko et al., 2012). Glandular trichomes are the site of essential oil biosynthesis, secretion and accumulation. The present taxonomy ascribes great value to the structure and distribution of both secretory (glandular) and non-secretory trichomes (Falciani et al., 1995). The structure of secretory trichomes in Lamiaceae has been studied by several authors (Corsi and Bottega, 1999; Fahn, 2000; Giuliani and Bini Maleci, 2008; Werker, 2000; Werker et al., 1985). On the other hand, palynology gives valuable data about the origin, evolution and taxonomy of the species, thus the pollen morphology of Lamiaceae was investigated by numerous authors (Abu-Asab and Cantino, 1992; Dunkić et al., 2012; Harley et al., 1992; Kremer et al., 2012b; Moon et al., 2008; Özler et al., 2011; Wagstaff, 1992). The objective of this study is to gain insight into the essential oil contents and micromorphological features of two closely related species, M. kerneri and M. juliana. To the best of our knowledge, there are no data on the essential oil composition and micromorphological traits of M. kerneri. Therefore, this study represents a contribution to the improved taxonomy of both investigated species, especially for the endemic M. kerneri. Results and discussion Gas chromatography and mass spectrometry (GC, GC–MS) The essential oils isolated by hydrodistillation from the aerial parts of M. kerneri and M. juliana were analyzed by GC and GC–MS with VF-5 ms and CP Wax52 column for the purpose of determining their possible similarities and the population-level variability in their chemical compositions. The general chemical profiles of the ten investigated essential oils, percentage contents, and retention indices of the components are given in Table 1. The identified components were classified into eight classes on the basis of their chemical structures: oxygenated monoterpenes, monoterpene hydrocarbons, sesquiterpene hydrocarbons, oxygenated sesquiterpene, phenolic compounds, carbonylic compounds, aliphatic alcohol and hydrocarbons. The results presented here represent the first data on the essential oil composition for M. kerneri. The total yield of oils ranged from 0.05% to 0.06% (v/w) in M. kerneri and from 0.07% to 0.09% (v/w) in M. juliana based on dry mass of samples. In terms of the content of essential oils (<0.5), Slavkovska et al. (2005) classified M. juliana in the section Eumicromeria together with Micromeria croatica (Pers.) Schott., Micromeria parviflora (Vis.) Rchb., and Micromeria cristata (Hampe) Griseb. A total of 54 compounds were observed in the essential oil of M. kerneri from MkG, 60 from MkSP, and 64 from MkKI collected in Croatia. The fourth sample of M. kerneri, with 60 identified components, originated from the locality Mostar (MkM) in Bosnia and Herzegovina. Oxygenated sesquiterpene was the dominant class (42.9%) in M. kerneri from Gradina (Croatia) (MkG), with caryophyllene oxide as D. Kremer et al. / Phytochemistry 98 (2014) 128–136 the principal compound (39.2%). M. kerneri oils from MkSP, MkKI and MkM were also found to contain caryophyllene oxide as the major component, with a percentage of 11.7%, 12.8% and 13.5%, respectively (Table 1). Caryophyllene oxide was previously identified as the main or among the main components in the oil of M. croatica from Croatia and Serbia (Kremer et al., 2012b; Slavkovska et al., 2005), Micromeria graeca (L.) Benth. et Rchb. from Greece (Tzakou and Couladis, 2001), and M. cristata from Turkey (Tabanca et al., 2001) and Bulgaria (Kostadinova et al., 2007). The oils from the localities MkSP, MkKI and MkM contained higher concentrations of monoterpene hydrocarbons (29.1%, 24.1% and 30.3%), and oxygenated monoterpenes (27.1%, 32.0% and 27.3%) than the oil from the population MkG (14.1% of monoterpene hydrocarbons, and 19.0% of oxygenated monoterpenes). b-pinene (6.3%), docosane (5.4%), camphor (4.3%), and (E)-caryophyllene (2.9%) were the other main components of the essential oil of MkG. Camphor was previously noted as a major component in the essential oil composition of Micromeria fruticulosa (Bertol.) Šilić (Formisano et al., 2007) and M. cristata subsp. phrygia P. H. Davis (Tabanca et al., 2001). Among the samples investigated in this study, (E)-caryophyllene (6.9%, 6.3% and 5.2%), b-pinene (9.1%, 7.1% and 12.1%), borneol (5.7%, 6.2% and 4.7%), a-pinene (4.2%, 2.6% and 4.3%), and verbenone (4.2%, 6.3% and 3.2%) were abundant in the MkSP, MkKI and MkM populations, respectively. (E)-caryophyllene was the dominant compound in the essential oils of Micromeria myrtifolia Boiss. et Hohen. (Özek et al., 1992) and Micromeria cremnophila Boiss. et Heldr. (Baser et al., 1997) from Turkey, M. graeca from Greece (Tzakou and Couladis, 2001), M. croatica from Croatia (Kremer et al., 2012b), M. fruticulosa from Italy (Formisano et al., 2007), and Micromeria varia Benth. ssp. thymoides (Sol. ex Lowe) Pérez var. thymoides from the Madeira archipelago (Pedro et al., 1995). The oil of M. kerneri from MkKI contained a higher concentration of phenolic compounds (6.9%) than the oils from other populations, with thymol (4.1%) as the dominant component within this class. Also, Table 1 lists the chemical components of M. juliana essential oils. The total numbers of chemical constituents identified in essential oils were 41 from MjOD (Croatia), 45 from MjS (Croatia), 43 from MjL (Bosnia and Herzegovina), 49 from MjK (Montenegro), 37 from MjBR (Republic of Macedonia) and 42 from MjNS (Greece). Caryophyllene-oxide was the major component of the essential oil from all populations, except from MjK, where piperitone-oxide (16.9%) was the major compound. Piperitone-oxide (14.5%) was also an important compound in the oil from MjBR. Additionally, piperitone-oxide was identified as one of the major compounds in the oil of M. graeca (Wollenweber and Jay, 1988), Micromeria albanica (Griseb. ex K. Malý) Šilić, and Micromeria thymifolia (Scop.) Fritsch. (Slavkovska et al., 2005). The (E)-caryophyllene was the next most abundant constituent in all analysed M. juliana essential oils. The oil of M. juliana from MjL had virtually the same content of caryophyllene-oxide (10.1%) and (E)-caryophyllene (10.3%). Caryophyllene-oxide was also the dominant compound in the oil of M. juliana from Croatia (Mastelić et al., 2005), and in the oil of M. croatica from Serbia (Slavkovska et al., 2005). The proportion of the sesquiterpene hydrocarbon germacrene D in all investigated M. juliana populations varied between 2.7% and 4.9% (Table 1). Phenolic compounds thymol and carvacrol were found in the oils from all populations, except from the locality MjBR. Oxygenated sesquiterpenes (25.9%) were the main constituents of the MjBR population, followed by oxygenated monoterpenes (24.9%), sesquiterpene hydrocarbons (16.5%) and monoterpene hydrocarbons (13.2%). Carbonylic compounds, aliphatic alcohol and hydrocarbons were identified as a minor class in the essential oil composition of the investigated MjBR population, with a summary percentage of 2.9%. Furthermore, a-pinene was the main component of the essential oil from four localities, except from MjS and MjK. In the 131 Fig. 1. PCA (a) and UPGMA (b) of essential oil components in Micromeria kerneri and M. juliana. For abbreviations see Table 3. essential oils from these two populations, b-pinene prevailed over a-pinene. In Skaltsa et al. (1998), a-pinene and b-pinene were the dominant components in the oil of M. juliana from Greece. Also, the essential oil of M. juliana from Mt Sniježnica (MjS) had approximately the same content of sesquiterpene hydrocarbons (20.7%) and oxygenated sesquiterpene (20.6%). The class of oxygenated monoterpenes (31.3%) was dominant in this population, with linalool, trans-pinocarveol, pinocarvone, borneol and piperitone as the major components. The results obtained in this study are comparable to other published studies. In Mastelić et al. (2005), linalool was determined to be a major compound of M. juliana from Croatia. The essential oil of Micromeria pseudocroatica Šilić was characterized by a high concentration of oxygenated monoterpenes, of which borneol and camphor were the major compounds (Kremer et al., 2012c). The MjNS population (Greece) contained similar percentages of monoterpene (39.0%) and sesquiterpene (42.4%) groups. Groups of phenolic compound, carbonylic compounds, aliphatic alcohol and hydrocarbons represented 4.5% of the total oil from MjNS. Generally, the results showed a similar composition of the major compounds of the essential oils of two investigated Micromeria species. The quantity composition of the essential oils of M. juliana and M. kerneri populations were also similar. 132 D. Kremer et al. / Phytochemistry 98 (2014) 128–136 The PCA of identified essential oil components clearly separated M. kerneri from M. juliana populations (Fig. 1a). The first principal component (PC 1) explained 34.1% of the total variance, the second 14.7%, and the third component 13.1%. Thus the first three components accounted for 61.9% of the total variance, highlighting the usefulness of PCA. The most similar M. kerneri populations were MkM (locus classicus) and MkSP (Fig. 1a). The MkG population showed a slightly higher degree of separation. The most similar M. juliana populations were MjL and MjNS, while MjK and MjS showed a higher degree of separation. The content of spathulenol and (Z)-b-farnesene gave the highest contribution to the first PC axis. Furthermore, the content of butyl n-hexanoate and b-chamigrene contributed most to the second PC axis, while the maximum score for PC 3 was obtained from the heneicosane content. The UPGMA gave similar results to the PCA (Fig. 1b) and clearly separated populations into clusters of M. kerneri and M. juliana. UPGMA showed that the most similar populations of M. kerneri were MkM and MkKI, which formed one cluster at a Euclidean distance (DE) of 10.52. The MkG population was the most distant. The most similar populations of M. juliana were MjOD and MjBR (DE = 7.66), while MjK and MjS showed a higher degree of separation. In general, multivariate analysis of essential oil components separated the investigated populations of M. kerneri and M. juliana. The obtained results confirmed that M. kerneri and M. juliana are two different taxa, which corresponds to the findings of Chater and Guinea (1972), Šilić (1979), and Petrović and Stešević (2011), who also differentiated these two species. On the other hand, Bräuchler et al. (2008) suggested that these two taxa might be conspecific. Micromorphological traits Trichomes The type, occurrence and frequency of trichomes on all investigated plant parts (leaves, calyxes, and stems) of M. kerneri and M. juliana are shown in Table 2. In general, two types of trichomes (non-glandular and glandular) were observed in both species. Non-glandular (NG) trichomes were bi-cellular to multicellular, unbranched, uniseriate, folded at different levels, and with a warty appearance of the surface due to the occurrence of cuticular micropapillae (Fig. 2a and b). The length of NG trichomes varied distinctly from very short hairs on the adaxial leaf side (Fig. 2g) to moderately long hairs on the abaxial leaf side and on the calyx (Fig. 2n, o and p). According to Payne’s plant hair terminology (Payne, 1978), NG trichomes could be noted as attenuate hairs. Husain et al. (1990) described these hairs on the adaxial leaf Table 2 Occurrence and frequency of trichomes on aerial parts of Micromeria kerneri and M. juliana. Locality Year of collection M. kerneri Gradina (Croatia) 2011 Starigrad Paklenica (Croatia) 2011 Korčula Island (Croatia) 2011 Mostar (Bosnia and Herzegovina) 2011 M. juliana Mt Omiška Dinara (Croatia) 2011 Mt Sniježnica (Croatia) 2011 Lastva (Bosnia and Herzegovina) 2011 Mt Krivošije (Montenegro) 2011 Babuna River (Republic of Macedonia) 2012 Nomos Serron (Greece) 2012 Note: trichomes: – missing, ± rare, + present, ++ abundant. a Attenuate, non-glandular hairs. Trichome type Leaf Adaxial Abaxial Calyx Stem Attenuatea Peltate Capitate C1 Capitate C2 Attenuate Peltate Capitate C1 Capitate C2 Attenuate Peltate Capitate C1 Capitate C2 Attenuate Peltate Capitate C1 Capitate C2 +/++ + ± + ++ ± ± – +/++ ±/+ ± – +/++ ± ± – ++ +/++ + + +/++ + + ++ + + + ±/+ + + + – ++ + +/++ + +/++ + + +/++ ++ +/++ + + ++ + ±/+ +/++ + + ±/+ ±/+ + + + + +/++ + + + + + ± – Attenuatea Peltate Capitate C1 Capitate C2 Attenuatea Peltate Capitate C1 Capitate C2 Attenuatea Peltate Capitate C1 Capitate C2 Attenuatea Peltate Capitate C1 Capitate C2 Attenuatea Peltate Capitate C1 Capitate C2 Attenuatea Peltate Capitate C1 Capitate C2 ++ ± ± – ++ + + – ++ + – – + ± ± – + + ±/+ ± + + ± – ++ + +/++ ±/+ ++ + ++ ±/+ ++ + + +/++ + + + +/++ ++ + + + + + + + +/++ + + ±/+ ++ + + + + + + + ++ + + +/++ ++ + + ± ++ + + +/++ ++ + ± ± ++ + ±/+ + + + ± + + + ±/+ + + + + + + + + + D. Kremer et al. / Phytochemistry 98 (2014) 128–136 133 Fig. 2. SEM micrographs of the different trichome types of Micromeria kerneri (a, c, e, h, l, o, r) and M. juliana (b, d, f, g, k, m, n, p, s). Non-glandular trichomes (NG), peltate trichome (P), type 1 capitate trichomes (C1), type 2 capitate trichomes (C2) and their distribution on the adaxial (e, f, g) and abaxial leaf surface (d, l, m, n), on the outer side of calyx (a, b, c, o, p), and on the stem (h, k, r, s). surface in M. juliana as small to medium sized, unicellular hairs of conical shape with small granules distributed over the entire trichome surface. The same type of NG trichomes was also present on the abaxial leaf surface, though they were medium to large sized (Husain et al., 1990). These trichomes are also present in M. croatica (Kremer et al., 2012b), M. pseudocroatica (Kremer et al., 2012c), and M. fruticosa (L.) Druce (Werker et al., 1985). Two main types of glandular trichomes could be distinguished in M. kerneri and M. juliana, peltate and capitate. Peltate trichomes consisted of a basal cell, a short unicellular stalk, and a multicellular head with a large subcuticular space (Fig. 2a, b and g). In both species, they occurred on both the adaxial and abaxial leaf side, on the calyx and the stem. Peltate trichomes are commonly present in Micromeria species (Werker et al., 1985; Kremer et al., 2012b,c), and in other Lamiaceae (Corsi and Bottega 1999; Dunkić et al., 2012; Fahn, 2000; Giuliani and Bini Maleci, 2008; Kremer et al., 2012a; Werker, 2000). Husain et al. (1990) did not describe these hairs in M. juliana, although unclear SEM-micrographs showed peltate trichomes. Two types of capitate trichomes were observed on both species. Type one capitate trichome (C1) is composed of one basal epidermal cell and one elliptically formed head cell. These trichomes are not upright but could be described as clinging to the surface (Fig. 2a and c). They are found on both the adaxial and abaxial leaf side, the calyx and the stem (Table 2). According to Kremer et al. (2012c), these trichomes are common on the abaxial leaf surface, Fig. 3. SEM micrographs of Micromeria kerneri (a, c) and M. juliana (b, d) pollen after critical point drying. Equatorial and polar view (a, b); aperture and exine surface (c, d). calyxes and stem of M. croatica. Additionally, Kremer et al. (2012b) noticed these trichomes on both the adaxial and abaxial side of leaves and bracteoles, on the outer and inner side of calyx, and on the stem of M. pseudocroatica. Comparable trichomes are 134 D. Kremer et al. / Phytochemistry 98 (2014) 128–136 Table 3 Details on origin and collection data of investigated Micromeria kerneri and M. juliana samples. a Locality Voucher No. Latitude Longitude M. kerneri Gradina (Croatia) Starigrad Paklenica (Croatia) Korčula Island (Croatia) Mostar (Bosnia and Herzegovina)* HFK-HR-121-2011 HFK-HR-112-2011 HFK-HR-109-2011 HFK-HR-139-2011 43°450 55.90 0 44°170 35.10 0 42°570 04.50 0 43°200 36.60 0 N N N N 15°590 12.10 0 15°260 35.10 0 17°050 56.10 0 17°480 37.90 0 M. juliana Mt Omiška Dinara (Croatia) Mt Sniježnica (Croatia)a Lastva (Bosnia and Herzegovina) Mt Krivošije (Montenegro) Babuna River (Republic of Macedonia) Nomos Serron (Greece) HFK-HR-115-2011 HFK-HR-131-2011 HFK-HR-135-2011 HFK-HR-137-2011 HFK-HR-75-2012 HFK-HR 44-2012 42°260 50.90 0 42°320 54.40 0 42°410 46.00 0 42°290 48.10 0 41°410 02.10 0 41°160 10.80 0 N N N N N N 13°370 02.40 0 E 18°220 01.70 0 E 18°290 27.10 0 E 18°380 56.70 0 E 21°480 12.20 0 E 23°250 06.70 0 E E E E E Altitude a.s.l. (m) Year of collection Abbreviation 209 87 222 65 2011 2011 2011 2011 MkG MkSP MkKI MkM 65 506 389 190 175 187 2011 2011 2011 2011 2012 2012 MjOD MjS MjL MjK MjBR MjNS Sample for pollen analysis. also present in micrographs of Satureja thymbra L., Majorana syriaca (L.) Rafin. and Thymus capitatus (L.) Hoffmanns. (syn. Coridotymus capitatus (L.) Rchb. f.) presented by Werker et al. (1985). Type two capitate trichome (C2) is upright and composed of one basal epidermal cell, a distinct stem of two cells and a smaller, unicellular, rounded head with a subcuticular space (Fig. 2a and d). Trichomes comparable to this type are more abundant in Lamiaceae. According to Kremer et al. (2012c), these trichomes are common on the abaxial leaf surface, on calyx and stem of M. croatica. These trichomes were also noticed on both the adaxial and abaxial side of leaves and bracteoles, on the outer and inner side of calyx, and on the stem of M. pseudocroatica (Kremer et al., 2012b). This hair type was also reported by Werker et al. (1985) for Micromeria fruticosa. Trichomes comparable to C2 trichomes were also observed in certain Salvia L. (Werker et al., 1985) and Satureja L. (Dunkić et al., 2012) species. Pollen Pollen grains of M. kerneri and M. juliana are single (monad pollen) and isopolar with more or less elliptical equatorial outline (Fig. 3a and b). According to Kremp’s classification (1965), M. kerneri has small pollen (20–25 lm) with a polar axis of 23.64 ± 1.29 lm, and equatorial diameter of 27.81 ± 1.11 lm. Minimum and maximum for the polar axis lengths are 21.9 and 25.7 lm, respectively, while minimum and maximum for the equatorial diameter are 26.9 and 29.9 lm, respectively. According to the P/E ratio (0.85), it has oblate-spheroidal shape (Ricciardelli D’Albore, 1997). M. juliana also has small pollen, with a polar axis of 23.55 ± 1.29 lm, and equatorial diameter of 26.55 ± 1.23 lm. The minimum and maximum lengths of the polar axis are 21.8 and 26.3 lm, respectively, while the minimum and maximum for the equatorial diameter are 25.1 and 28.6 lm, respectively. According to the P/E ratio (0.89), it has oblate-spheroidal shape. According to Moon et al. (2008), the pollen of Micromeria marginata (Sm.) Chater has oblate, suboblate or oblate-spheroidal shape and is middle-large pollen with a polar axis of 26.6 lm and an equatorial diameter of 35.2 lm. The pollen of M. croatica has an oblatespheroidal shape and is also middle-large pollen with a polar axis of 25.8 lm and an equatorial diameter of 28.5 lm (Kremer et al., 2012c). In general, the pollen of both species was very similar without visible differences (Fig. 3). The polar view in both species shows a circular shape with visible ends of the apertures, while the equatorial outline is more or less elliptical. The pollen has six apertures (hexacolpate or stephanocolpate), which are set in the equatorial pollen belt (zonocolpate). Apertures are long and rather narrow, widest in the middle and gradually narrower towards the poles, with sharp edges, acute ends (Fig. 3a and b), and ornamented membranes (Fig. 3c and d). The apocolpium is quite small, while the mesocolpium is rather large (Fig. 3a and b). The exine has medium reticulate ornamentation (Fig. 3c and d) with unequal reticulum meshes (hetrobrochate). The surface of the muri is smooth. The lumina are varied in size. They are narrower or sometimes approximately the same width as the muri, and have an irregular shape with obtuse angles. In general, the appearance of the apertures and ornamentation of both M. kerneri and M. juliana are similar to the apertures and ornamentation of M. marginata and M. croatica described by Moon et al. (2008) and Kremer et al. (2012c), respectively. Conclusions In this study, the chemical composition of the essential oil and micromorphological traits of trichomes and pollen of both M. kerneri and M. juliana were investigated. The main constituent in both species was caryophyllene oxide with the exception of one M. juliana sample, where piperitone oxide was the major compound. Multivariate analysis of essential oil components clearly separated populations of M. kerneri and M. juliana, confirming that they are different taxa. Micromorphological investigations showed the presence of attenuate non-glandular trichomes, and two types (peltate and capitate) of glandular trichomes on the leaves, calyx and stem. The pollen of both species was very similar. In general, there were no differences between M. kerneri and M. juliana samples based on the investigated micromorphological traits. Nevertheless, definitive conclusions about the taxonomic relationship between M. kerneri and M. juliana will require genetic analysis. This study represents a contribution to the taxonomy of both investigated species, especially of the endemic M. kerneri and the genus Micromeria in general. Experimental Herbal material Randomly selected samples of M. kerneri Murb. and M. juliana (L.) Benth. were collected from the wild during the blooming period in July 2011 and 2012 (Table 3). Voucher specimens of herbal material were deposited in the Fran Kušan Herbarium (HFK-HR), Department of Pharmaceutical Botany with Fran Kušan Pharmaceutical Botanical Garden, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia (Table 3). The aboveground parts of several dozen randomly selected plants were harvested from plants on a dry day and mixed to obtain a randomly selected sample. Samples were air-dried for ten days in a well-ventilated room at 60% relative air humidity and room temperature (22 °C), single-layered and protected from D. Kremer et al. / Phytochemistry 98 (2014) 128–136 135 direct sunlight. Dried aerial parts (100 g) were subjected to hydrodistillation for 3 h in a Clevenger type apparatus. The obtained essential oil was dried over anhydrous sodium sulphate. For micromorphological investigation of trichomes, samples (leaves, flowers, and stems) of seven plants per locality were fixed in FAA (formalin/96% ethanol/acetic acid/water – 5/70/5/20; V/V/ V/V). After three days, samples were transferred to 70% (V/V) ethanol. PC axes. The populations were used as operational taxonomic units (OTUs) in PCA (Sneath and Sokal, 1973). Additionally, cluster analysis using the Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) based on Euclidean distances (DE) was performed to confirm the results of PCA. Each variable was standardized prior to the cluster analysis (Mook et al., 1992; Vandeginste et al., 1998). Statistical analyses were performed using the Statistica 7 software package (StatSoft Inc., Tulsa, OK, USA). Gas chromatography and mass spectrometry (GC, GC–MS) Acknowledgment Gas chromatography (GC) analyses were performed on a gas chromatograph (model 3900; Varian Inc., Lake Forest, CA, USA) equipped with flame ionization detector (FID), mass spectrometer (MS) (model 2100T; Varian Inc.), non-polar capillary column VF5 ms (30 m  0.25 mm i.d., coating thickness 0.25 lm) and polar CP Wax 52 (30 m  0.25 mm i.d., coating thickness 0.25 lm). The VF-5 ms column temperature was programmed at 60 °C isothermal for 3 min, and then increased to 246 °C at a rate of 3 °C min 1 and held isothermal for 25 min. The CP Wax 52 column temperature was programmed at 70 °C isothermal for 5 min, and then increased to 240 °C at a rate of 3 °C min 1 and held isothermal for 25 min. The other chromatographic conditions were as follows: carrier gas was helium; flow rate was 1 mL min 1; injector temperature was 250 °C; injected volume was 1 lL; split ratio was 1:20; FID detector temperature was 300 °C. The MS conditions were: ionization voltage 70 eV; ion source temperature 200 °C; mass scan range: 40–350 mass units. The individual peaks were identified by comparison of their retention indices (relative to C8–C25 n-alkanes for VF-5 ms and CP Wax 52) to those of authentic samples and literature (Adams, 2007), as well as by comparing their mass spectra with the Wiley 7 MS library (Wiley, New York, NY, USA) and NIST02 (Gaithersburg, MD, USA) mass spectral database. The percentage composition of the samples was computed from the GC peak areas using the normalization method (without correction factors). The component percentages were calculated as mean values from duplicate GC and GC/MS analyses. This work was supported by the Ministry of Science, Education and Sports of the Republic of Croatia (projects no. 006-00000003178 and 177-1191192-0830). Micromorphological traits For SEM-investigation, samples of leaves, flowers and stems were transferred from 70% (V/V) ethanol to 70% (V/V) acetone, then dehydrated (70%, 90% and 100% acetone) and subjected to critical point drying using CO2 as the drying medium (CPD030; Baltec). Samples were then sputter coated with gold (Sputter Coater, AGAR) and examined under the scanning electron microscope XL30 ESEM (FEI) with 20 kV acceleration voltages in high vacuum mode. The occurrence and frequency of the different trichome types was qualitatively assessed ( missing, ± rare, + present, ++ abundant). Pollen from several flowers per plants (seven plants per species) was removed from anthers after critical point drying and mixed to obtain random samples. The length of 30 pollen grains was measured. Common terminology was used in the description of micromorphology (Payne, 1978; Punt et al., 2007). Statistical data processing To obtain insight into the general pattern of variation between the two species, the results of GC and GC–MS analysis were subjected to the principal component analysis (PCA). PCA calculation was based on the correlation matrix between the values of the characteristics (chemical components) meaning that the contribution of each variable was independent of the range of its values (Massart et al., 1998; Vandeginste et al., 1998). The results of PCA were displayed on a three-dimensional plot for the first three Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2013. 12.009. References Abu-Asab, M.S., Cantino, P.D., 1992. Pollen morphology in subfamily Lamioideae (Labiateae) and its phylogenetic implication. In: Harley, R.M., Reynolds, T. (Eds.), Advances in Labiatee Science. Royal Botanic Gardens, Kew, pp. 97–112. Adams, R.P., 2007. 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