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1 Contents / İçindekiler Erol Kodak, N.Münevver Pinar, Nezaket Adigüzel and Aydan Acar POLLEN MORPHOLOGY OF SOME TAXA OF GENUS TANACETUM L. (ASTERACEAE) IN TURKEY TÜRKİYE TANACETUM L. CİNSİNİN BAZI TAKSONLARININ POLEN MORFOLOJİSİ 2-10 Esra Demir POLLEN ANALYSIS OF HONEY SAMPLES COLLECTED FROM KOMATİ (ÇAMLIHEMŞİN) PLATEAU KOMATİ (ÇAMLIHEMŞİN) YAYLASI BALLARINDA POLEN ANALİZİ Fatih Dikmen and Ahmet Murat Aytekin NOTES ON ROPHITES ALGIRUS PÉREZ, 1895 AND ROPHITES QUINQUESPINOSUS SPINOLA, 1808 OF MEDITERRANEAN TURKEY WITH AN UPDATED LIST OF SUBFAMILY ROPHITINAE (HYMENOPTERA: HALICTIDAE) OF TURKEY TÜRKİYE DEKİ ROPHITINAE (HYMENOPTERA: HALICTIDAE) ALTFAMILYASININ GÜNCEL LİSTESİ İLE TÜRKİYE NİN AKDENIZ HAVZASINDAKİ ROPHITES ALGIRUS PÉREZ, 1895 AND ROPHITES QUINQUESPINOSUS SPINOLA, 1808 ÜZERINE NOTLAR Ömür Gençay Çelemli, Kadriye Sorkun, Bekir Salih CHEMICAL COMPOSITION OF PROPOLIS SAMPLES COLLECTED FROM TEKIRDAG-TURKEY TEKİRDAĞ-TÜRKİYE DEN TOPLANAN PROPOLİS ÖRNEKLERİNİN KİMYASAL İÇERİĞİ 28-32

2 MELLIFERA 12-24:2-10 (2012) HARUM RESEARCH ARTICLE 2 POLLEN MORPHOLOGY OF SOME TAXA OF GENUS TANACETUM L. (ASTERACEAE) IN TURKEY TÜRKİYE TANACETUM L. CİNSİNİN BAZI TAKSONLARININ POLEN MORFOLOJİSİ Erol Kodak*, N.Münevver Pinar*, Nezaket Adigüzel** and Aydan Acar* Summary:Morphological features of pollen of 8 Turkish taxa of the complex genus Tanacetum L. were examined using light (LM) and scanning electron (SEM) microscopy. The pollen is tricolporate, trisyncolporate or tricolpate. The shape is oblate- spheroidal. The exine is echinate. The ornamentations between spines are granulate, reticulate and rugulate-granulate. The results indicate that the value of pollen characters for taxonomic applications is limited for Tanacetum. Keywords: Asteraceae, Tanacetum, Pollen, LM, SEM, systematics, melissopalynology. Özet: Kompleks bir cins olan Tanacetum L. cinsinin Türkiye deki 8 taksonun polenlerinin morfolojik özellikleri ışık (LM) ve taramalı elektron (SEM) mikroskopları kullanılarak çalışılmıştır. Polenlerin trikolporat, trisinkolporat ve trikolpat oldukları bulunmuştur. Polen şekli oblat-speroidal dir. Ekzin ekinat dır. Spinler arasındaki ornamentasyonlar granulat, retikülat ve rugulat- granulat tır. Sonuçlar, taksonomik uygulamalar için polen karakterlerinin değerinin Tanacetum cinsi söz konusu olduğunda sınırlı olduğunu işaret etmektedir. Anahtar Kelimeler: Asteraceae, Tanacetum, Polen, LM, SEM, sistematik, melissopalinoloji. * Ankara University Faculty of Science, Department of Biology, Tandoğan Ankara, TURKEY **Gazi University Faculty of Science, Department of Biology, Ankara, TURKEY Corresponding Author [email protected]

3 3 Introduction Bees require large amounts of pollen for their own reproduction. The general view of pollen as an easyto-use protein source for flower visitors. That is Apis mellifera L. feed on pollen and nectar collected from blooming flowers. Tanacetum L. is also visited by Apis mellifera for pollen diets (Christophe et al. 2008; Hilty 2012). Knowledge about the pollen morphology of honey plants is important in the identification of plant species which contribute toward composition of honey (Howes 1953; Sodré et al. 2001). Silici and Gökçeoglu (2007) have presented that Tanacetum is an important bee plant in the Mediterranean region of Anatolia. T. vulgare L. has been reported as a minor element in honey by Sorkun (2008). The genus Tanacetum is one of the more than 100 genera in the tribe Anthemideae (Soreng and Cope 1991) which contains about 10% of the total genera and 15% of the species of Asteraceae (Heywood and Humphries 1977). About 150 species of Tanacetum are spreaded around the world. They are found throughout temperate, regions, particularly in the northern hemisphere even up to Northern Europe, Canada, Alaska, and Northern Russia (Hultén 1950; Hultén 1968; Heywood 1976; Heywood and Humphries 1977), although the center of diversity and probably also the origin for Tanacetum is South-West Asia and the Caucasus in the Old World (Heywood and Humphries 1977; Soreng and Cope 1991). In Turkey, Tanacetum is represented by 60 of which are 27 taxa (47%) endemic (Grierson 1975; Yildirimli 1989; Ekim et al. 2000). World (Heywood and Humphries 1977; Soreng and Cope 1991). In Turkey, Tanacetum is In this study, 8 taxa belonging to the genus Tanacetum were investigated; T. balsamita L. subsp. balsamita L., T. balsamita L. subsp. balsamitoides (Schultz Bip.) Grierson, T. argenteum (Lam.) Willd. subsp. flabellifolium (Boiss. End Heldr.) Grierson, T. argenteum (Lam.) Willd. subsp. argenteum (L.) All., T. argenteum (Lam.) Willd. subsp. canum (C. Koch) Grierson, T. depauperatum (Post) Grierson, T. haradjanii (Rech. Fil.) Grierson and T. tomentellum (Boiss.) Grierson. T. argenteum subsp. flabellifolium, T. argenteum subsp. argenteum, T. depauperatum, T. haradjanii are local endemic species. T. balsamita L. subsp. balsamita L., T. balsamita L. subsp. balsamitoides (Schultz Bip.) Grierson, T. argenteum (Lam.) Willd. subsp. canum (C. Koch) Grierson and T. tomentellum (Boiss.) Grierson are widely distributed, nonendemic taxa. The distribution map of the taxa is given in Figure 1. represented by 60 of which are 27 taxa (47%) endemic (Grierson 1975; Yildirimli 1989; Ekim et al. 2000). In this study, 8 taxa belonging to the genus Tanacetum were investigated; T. balsamita L. subsp. balsamita L., T. balsamita L. subsp. balsamitoides (Schultz Bip.) Grierson, T. argenteum (Lam.) Willd. subsp. flabellifolium (Boiss. End Heldr.) Grierson, T. argenteum (Lam.) Willd. subsp. argenteum (L.) All., T. argenteum (Lam.) Willd. subsp. canum (C. Koch) Grierson, T. depauperatum (Post) Grierson, T. haradjanii (Rech. Fil.) Grierson and T. tomentellum (Boiss.) Grierson. T. argenteum subsp. flabellifolium, T. argenteum subsp. argenteum, T. depauperatum, T. haradjanii are local endemic species. T. balsamita L. subsp. balsamita L., T. balsamita L. subsp. balsamitoides (Schultz Bip.) Grierson, T. argenteum Pollen morphology has provided an approach to the systematic relationships among the genera of Asteraceae (Wagenitz 1955; Stix 1960; Erdtman 1969; Pinar and Inceoglu 1996; Pinar and Adigüzel 1998; Pinar and Oybak Dönmez 2000; Punt and Hoen 2009). There are numerous publications on pollen morphology (Lam.) Willd. subsp. canum (C. Koch) Grierson and T. tomentellum (Boiss.) Grierson are widely distributed, nonendemic taxa. The distribution map of the taxa is given in Figure 1. Figure 1. Distribution of the eight investigated Tanacetum L. taxa in Turkey Figure 1. Distribution of the eight investigated Tanacetum L. taxa in Turkey Pollen morphology has provided an approach to the systematic relationships among the

4 MELLIFERA 4 of genus Tanacetum (İnceoğlu and Karamustafa 1977; Ramos & Mederos 2008; Punt and Hoen 2009). The aim of this study is to illustrate the range of variability in pollen characters of T. balsamita subsp. balsamita, T. balsamita subsp. balsamitoides, T. argenteum subsp. flabellifolium, T. argenteum subsp. argenteum, T. argenteum subsp. canum, T. depauperatum, T. haradjanii, T. tomentellum found in Turkey in order to establish their availability for future taxanomic and melissopalynological works. Material and Methods The material was collected from wild populations. The collectors and localities are provided in the Specimens examined for each taxon. The specimens are deposited in GAZİ (Gazi University Herbarium), AEF (Ankara University Farma Herbarium) and HUB (Hacettepe University Herbarium). Pollen slides were prepared using by the technique of Wodehouse (1935). LM studies were done with a Leitz-Wetzlar microscope. Measurements are based on at least 30 pollen grains for each taxon. For SEM studies, pollen grains were coated with gold for four minutes in a sputter-coater. Observations were made with a Jeol 100 CXII electron microscope. The pollen terminology follows Faegri-Iversen (1975) and Punt et al. (1994). The Simpson and Roe graphical test (Van der Pluym & Hideux 1977) was used for statistical calculations. Specimens examined The order of the species was adapted from Grierson (1975). All the specimens are deposied in GAZİ, AEF and HUB: T. balsamita L. subsp. balsamita L., Konya, Koyuncu AEF, Erzurum, Koyuncu AEF; T. balsamita L. subsp. balsamitoides (Schultz Bip.) Grierson, Hakkari Koyuncu AEF, Erzurum Koyuncu AEF, Sivas Çelik AEF, Gümüşhane, Kars Koyuncu AEF; T. argenteum (Lam.) Willd subsp. flabellifolium (Boiss. End Heldr.) Grierson, Konya Adigüzel GAZİ, T. argenteum (Lam.) Willd. subsp. argenteum (L.) All Kayseri Adigüzel GAZİ, Hakkari Koyuncu AEF; T. argenteum (Lam.) Willd. subsp. canum (C. Koch) Grierson, Tunceli Adigüzel GAZİ, Içel Çelik AEF, Erzincan Soner AEF; T. depauperatum (Post) Grierson, Hatay Adigüzel GAZİ; T. haradjanii (Rech.) Grierson Adana Çelik AEF; T. tomentellum (Boiss.) Grierson Bitlis, Peşmen HUB. Results Detailed pollen morphological features of the investigated taxa are summarized in Table 1 and Fig. 2 and representative pollen grains are illustrated in Fig Size, symmetry and shape The pollen grains are radially symmetrical and isopolar. The shape is generally oblate-spheroidal (the term according to Erdtman (1969) based on the P / E ratio, Table 1, with polar axes ranging from µm and equatorial axes from 25 to 34 µm, respectively. The largest pollen is found in T. balsamita subsp. balsamita (chromosome number 4n = 36), while the smallest pollen occurs in T. haradjanii. The outline is circular or subcircular in equatorial view and generally triangular-convex and sometimes circular or circular-convex in polar view. Amb intersemiangular. (Table 1, Fig. 2-4). Apertures Pollen grains of Tanacetum are operculate and usually tricolporate or rarely trisyncolporate and tricolpate. Some species have shown heteromorphic characteristics. For example; 2% tricolpate and 98% tricolporate in T. balsamita subsp. balsamita and T. argenteum subsp. argenteum and 2% trisyncolporate and 98% tricolporate in T. balsamita subsp. balsamitoides. Colpus, short or long ( µm) and broad (5-9 µm), and ora lalongated. The highest values were observed in T. balsamita subsp. balsamita and T. argenteum subsp. canum. Margins distinct, regular and ends acute in both of them. Colpus membrane more or less granulate. (Table 1 and Fig. 3,4). Exine The stratified exine has an overall thickness which ranges from 3 to 5.5 µm in. Ectexine is thicker than endexine without no costae and no cavea. Intratectal columellae very distinct under spines, but indistinct interspinal region. The spines are commonly conical with a broadened base and a tappered apical portion. The spine length varies between µm The width

5 5 of spines varies between µm. The base of the spines in almost all species studied has 3 or 4 irregular seriate perforations with larger holes which are often found distally. Also, large or small cavities are present. Number of cavities are Intine is thick ( µm) (Table 1 and Fig. 3,4). The pollen wall is provided with spines and its either granulate, reticulate and rugulate-granulate. In all of the species, the tectum surrounding the spine base is microperforate. Table 1 provides a summary of the tectal morphology. Discussion The grains of taxa of Tanacetum can be ascribed to the Anthemis type of Stix (1960), and Anthemis arvensis type of Punt and Hoen (2009). The results of our investigation show that the pollen dimensions (E and, P / E ratio), the thickness of the exine and intine, the shape of the polar and equatorial views and the aperture type of the Turkish taxa of Tanacetum were comparatively homogenous (Table 1 and Figure 2). Ramos and Mederos (2008) and Özmen et al. (2009) confirmed that the pollen morphologies of taxa of Tanacetum are homogenous. Most Tanacetum species are diploid, having 2n= 2x= 18 with the basic chromosome number x=9. The chromosome count (2n=36) of T. balsamita subsp. balsamita is also the report of the tetraploid level (Watanable 2011; Inceer and Hayırlıoğlu 2012). Chatuverdi et al. (1990) and Brochman (1992) report that pollen grain size strongly correlates with the level of polyploidy. In this study, the relationship between ploidy level or chromosome number and pollen size at the taxon level in T. balsamita subsp. balsamita is demonstrated statistically; higher ploidy levels correspond to an increase in pollen grain size (Table 1 and Fig. 2). Mesfin et al. (1995) said that the ornamentations between spines are important characters for Asteraceae. Ornamentations between spines are granulate in Tanacetum balsamita subsp. balsamita, T. balsamita subsp. balsamitoides and T. haradjanii, or reticulate in T. tomentellum, and rugulate-granulate in T. deparatum, T. argenteum subsp. argenteum, T. argenteum subsp. flabellifolium and T. argenteum subsp. canum. (Table 1 and Fig. 4). The general aperture form is tricolporate, but T. balsamita subsp. balsamita (2% tricolpate, 98% tricolporate), T. argenteum subsp. argenteum (2% tricolpate, 98% tricolporate) and T. balsamita subsp. balsamitoides (2% trisyncolporate and 98% tricolporate) show considerable aperture type variation (Table 1 and Fig. 3,4). Variations in pollen size and aperture type have been attributed to heteromorphy in pollen grains by Nair and Kaul (1965) and Inceoglu (1973). The taxa can be identificated by the sculpturing types in this study. But the other results of polar and equatorial axes, pollen shape, exine and intine thickness are generally similar to those taxa of the species.

6 MELLIFERA 6 Table 1. The palynological mesurements and observations of the eight inventigated Tanacetum L. taxa. Clg: Length of colpus, Clt: Latitude of colpus, Plg: Length of porus, Plt: Latitute of porus Polar axes (P) µm Equatorial axes (E) µm Colpus µm Pore (P)µm Spine µm Taxa Pollen Shape P/E Max Min Mean Max Min Mean Exine Intine Aperture type Clg Clt Plg Plt Length of spine µm Base of spine µm Ornamentation of between spines T. balsamita subsp. balsamita 0,97 24, , ,5 4,25 1 2% tricolpate 98% tricolporate 16, ,87 4,17 Granulate T. balsamita subsp. balsamitoide Grierson 0,95 23, , ,5 3,75 0,87 2% trisyncolporate 98% tricolporate 11, ,25 4,62 Granulate T. argenteum subsp. flabellifolium 0,82 20, , ,5 4 1,12 tricolporate 14, ,25 3,12 Rugulate Granulate T. argenteum subsp. argenteum 0,89 22, , ,5 1,25 2% tricolpate 98% tricolporate 14 7,5 7,5 7,5 3,75 4,25 Rugulate Granulate T. argenteum subsp. canum 0, , ,2 4 1,25 tricolporate ,25 2,25 Rugulate Granulate T. depauperatum 0, ,75 1 tricolporate ,25 2 Rugulate Granulate T. haradjanii 0,87 21, , ,7 1,07 tricolporate 14 6,5 6,5 6,5 3,25 3,75 Granulate T. tomentellum oblatespheraidal oblatespheraidal oblatespheraidal oblatespheraidal oblatespheraidal oblatespheraidal oblatespheraidal oblatespheraidal 0, , ,2 4,75 1,25 tricolporate ,25 3,25 Reticulate

7 7 Polar axes (P) µm T. balsamita T. balsamitoides T. flabellifolium T. argenteum T. canum T. depauperatum Taxa T. haradjanii T. tomentellum Max Min Mean A Equatorial axes (E) µm T. balsamita T. balsamitoides T. flabellifolium T. argenteum T. canum T. depauperatum Taxa T. haradjanii T. tomentellum Max Min Mean B Figure 2. A Polar axes (P), B Equatorial axes (E) Figure 2. A Polar axes (P), B Equatorial axes (E)

8 MELLIFERA 8 A B C D E F G H I K L M N O P R S T U V W X Y Z Figure 3. LM photos of Tanacetum species. A-C: T. balsamita L. subsp. balsamita L. A. Polar view B. Polar view and apertures C. Equatorial view. D-F: T. balsamita L. subsp. balsamitoides (Schultz Bip.) Grierson. D. Polar view. E. Polar view and apertures. F. Equatorial view. G-I: T. argenteum (Lam.) Willd. subsp. flabellifolium (Boiss. End Heldr.) Grierson G. Polar view. H. Polar view ornamentation. I. Equatorial view. K-M: T. argenteum (Lam.) Willd. subsp. argenteum (L.) All.. K. Polar view. L. Polar view of ornamentation. M. Equatorial view. N-P: T. argenteum (Lam.) Willd. subsp. canum (C. Koch) Grierson. N. Polar view O. Polar view and apertures P. Equatorial view. R-T: T. depauperatum (Post) Grierson. R. Polar view. S. Polar view and apertures. T. Equatorial view. U-W: T. haradjanii (Rech. Fil.) Grierson U. Polar view. V. Polar view ornamentation. W. Equatorial view. X-Z: T. tomentellum (Boiss.) Grierson. X Polar view. Y. Polar view and apertures. Z. Equatorial view

9 9 A B C D E F G H I K L M N O P R S T U V W X Y Z Figure 4. SEM photos of Tanacetum species. A-C: T. balsamita L. subsp. balsamita L. A. Equatorial view B. Polar view and apertures C. Ornamentation. D-F: T. balsamita L. subsp. balsamitoides (Schultz Bip.) Grierson. D. Equatorial view E. Polar view and apertures F. Ornamentation. G-I: T. argenteum (Lam.) Willd. subsp. flabellifolium (Boiss. End Heldr.) Grierson G. Equatorial view H. Polar view and apertures I. Ornamentation. K-M: T. argenteum (Lam.) Willd. subsp. argenteum (L.) All.. K. Equatorial view L. Polar view and apertures M. Ornamentation.. N-P: T. argenteum (Lam.) Willd. subsp. canum (C. Koch) Grierson. N. Equatorial view O. Polar view and apertures P. Ornamentation.. R-T: T. depauperatum (Post) Grierson. R. Equatorial view S. Polar view and apertures T. Ornamentation.. U-W: T. haradjanii (Rech. Fil.) Grierson U. Equatorial view V. Polar view and apertures W. Ornamentation.. X-Z: T. tomentellum (Boiss.) Grierson. X. Equatorial view Y. Polar view and apertures Z. Ornamentation.

10 MELLIFERA 10 References Brochman C Pollen and seed morphology of Nordic Draba (Brassicacea): phylogenetic and ecological implications. Nord J Bot, 12, Chatuverdi M, Yunus D & Nair PK Cytopalynological studies of Arachis L. (Leguminosae). Cultivated and wild species and their hybrids. Grana 29, Christophe J., Andreas M.,and Silvia D Specıalızed bees faıl to develop on non-host pollen: Do plants chemıcally protect theır pollen Ecology, Ekim T, Koyuncu M, Vural M, Duman H, Aytac Z, Adıguzel N Türkiye Bitkileri Kırmızı Kitabı, Ankara: Türkiye Tabiatını Koruma Derneği. Erdtman G Handbook of Palynology, Morphology, Taxonomy and Ecology. Munksgaard, Copenhagen. Faegri K & Iverson J Textbook of Pollen Analysis. 4th Edition. New York: Wiley. Grierson AJC Tanacetum L. (emend. Briq.) in P.H. Davis (Ed.) Flora of Turkey and the East Aegean Islands, Edinburgh: Edinburgh University Press, 5, , Heywood V.H Tanacetum. In: Tutin, T.G., Heywood, V.H., Burges, N.A., Moore, D.M. Heywood V.H. and Humphries C.J Anthemideae-systematic review. In: Heywood. Hilty J Insect Visitors of Illinois Wildflowers. World Wide Web electronic publication. illinoiswildflowers. info, version. Howes F.N Plantas melíferas. Barcelona, Espanha, Reverté, 35p. Hultén E Atlas of the distribution of vascular plants in NW Europe. Generalstabens Litografiska Anstalts Förlag, Stockholm, 512 p. Hultén E Flora of Alaska and Neighboring territories. A manual of the vascular plants.stanford University Press, Stanford, California p. İnceoğlu Ö. and Karamustafa F The pollen morphology of plant in Ankara region I. Compositae. Communications 21, İnceer H. Hayırlıoglu Ayaz S Karyological studies of some representatives of Tanacetum L. (Anthemideae-Asteraceae) from North-east Anatolia. Plant.Syst. Evol 298, İnceoğlu O Asyneuma canescens (W. K.) Griseb. & Schenk in polen morfolojisi ve heteremof polenler. Türk Biyoloji Derg 23, İrena E. La Serna Ramos and Miguel A.PadronMederos Polen morphology of endemic species of the Gonospermum Less., Lugoa DC. And Tanacetum L.complex (Asteraceae: Anthemideaae) in the Canary Islands (Spain), and its taxonomical implications. Grana 47, Mesfin T., Crawford d.j. and Smith E.B Pollen morphology of North Coreopsis (Compositae). Grana 34, Nair PK & Kaul KN Pollen grain in a gigantic of Rauwolfia serpentine. Current Sci 34, Özmen E., Kızılpınar İ., Özüdoğru B., Doğan C. and Erik S Pollen morphology of some taxa of aromatic genus Tanacetum L. (Asteraceae). Fabad J. Pharm. Sci. 34, Pınar N.M. and İnceoğlu Ö A Comparative study on the pollen morphology of Centaurea triumfettii All. groups A, B and C with light and electron microscopy. Turkish Journal of Botany, 20, Pınar N.M. and Adıgüzel N Pollen morphology of some Turkish Artemisia L. (Compositae) species. Ot Sistematik Botanik Dergisi, 5(2), Pınar N.M. and Oybak Dönmez E Pollen morphology of some Turkish endemic Helichrysum Gaertner species (Compositae). Pakistan Journal of Botany 32 (2), Punt W., Blackmore S., Nilson S. And Le Thomas A Glossary of polen an spore terminology LPP foundation, Utrecht. Punt W & Hoen PP The Northwest European Pollen Flora,70: Asteraceae-Asteroideae. Rev Palaeobot and Palyno 157, Ramos I.E. and Mederos M.A.P Pollen morphology of endemic species of the Gonospermum Less., Lugoa DC. and Tanacetum L. complex S. Silici & M. Keceoglu Pollen analysis of honeys from Mediterranean region of Anatolia Grana; 46, Sodré G.D., Marchini L.C., Moreti A.C. and Carvalho C Análises polínicas de méis de Apis mellifera L., 1758 (Hymenoptera: Apidae) do litoral norte do Estado da Bahia. Rev Agric, 76, Soreng R.J. and Cope E.A On the taxonomy of cultivated species of the Chrysanthemum Genus-Complex (Anthemideae; Compositae). Baileya, 23, Sorkun K Türkiye nin Nektarlı Bitkileri, Polenleri ve Balları. Palme Yayıncılık, Ankara. Stix E Pollenmorphologische Untersuchungen an Compositen. Grana Palynol, 2(2), Watanable W Index to chromosome numbers in Asteraceae. G asteraceae_e, updated. Wagenitz G Pollenmorphologie und Systematik in der Gattung Centaurea L. s.l. Flora, 142, Wodehouse PP Pollen Grains. New York: McGraw-Hill. Valentine D.H., Walters S.M. and Webb D.A. (eds). Flora Europaea, Plantaginaceae to Compositae (and Rubiaceae). Cambridge University Press, Cambridge, 4, Van der Pluym A & Hideux M Applications d une methodologie quantitative a la palynologie d Eryngium maritimum (Umbelliferae), Plant Syst Evol, 127, Yıldırımlı Ş Munzur dağlarının yeni, ilginc ve tukenen bitki turleri. Hacettepe Fen ve MühendislikBilimleri Dergisi, 10, 39-47, Aytuğ B İstanbul Çevresi Bitkilerinin Polen Atlası. İstanbul Üniversitesi Orman Fakültesi

11 MELLIFERA 12-24:11-16 (2012) HARUM RESEARCH 11 ARTICLE POLLEN ANALYSIS OF HONEY SAMPLES COLLECTED FROM KOMATİ (ÇAMLIHEMŞİN) PLATEAU KOMATİ (ÇAMLIHEMŞİN) YAYLASI BALLARINDA POLEN ANALİZİ Esra Demir* Summary: This study presents the pollen analyses of 10 floral honeys from Komati (Çamlıhemşin) Plateau in Rize. Samples were obtained from each beehive selected randomly from 10 different apiaries in the year All the honey samples were identificated under the light microscope. These samples examined Total Pollen Number in 10 g honey (TPN-10g). The pollen analyses revealed 1 unifloral honey and 9 multifloral honeys. The dominant group of pollen grains consisted of : Castanea sativa MILLER., Ericaceae, Fabaceae and Rosaceae. The amount of moisture in the samples of honey was identified between 15% and 19%. Keywords: Honey, Pollen Analysis, TPN-10g, Komati Plateau Özet: Bu çalışma Komati Yaylası ndan 10 bal örneğinin polen analizlerini içermektedir. Örnekler, 2011 yılında, rastgele belirlenmiş 10 farklı arılıktan seçilen birer kovandan toplanmıştır. Toplanan bütün bal örnekleri ışık mikroskobu altında teşhis edilmişlerdir. Bu örneklerde Toplam Polen Sayısı (TPS-10) incelenmiştir. Polen analizleri sonucunda 1 bal unifloral ve 9 bal multifloral olarak saptanmıştır. Dominant polen gruplarını Castanea sativa MILLER., Ericaceae, Fabaceae ve Rosaceae taksonları oluşturmaktadır. Bal örneklerindeki nem miktarı %15 ile %19 arasında tespit edilmiştir. Anahtar Kelimeler: Bal, Polen Analizi, TPS-10g, Komati Yaylası * Recep Tayyip Erdoğan University Faculty of Science, Department of Biology, Rize, TURKEY Corresponding Author [email protected]

12 MELLIFERA 12 Introduction Honey is great importance for commercial and importance source of nutriment for people. The taste, smell and color of honey is to change according to the nectar of the flowers (Kaya et al., 2005). Honey is the natural sweet substance produced by honeybees from the nectar of blossoms or from secretions of living parts of plants or excretions which honeybee (Apis mellifera L.) collect, transform and combine with specific substances of their own, store and leave in the honey comb to ripen and mature (Ünal and Küplülü, 2006). Beekeeping activity can be pursued only in such regions where bee flora is available. Therefore, identification of bee forage plants and their propagation help in improving the bee forage wealth and the concomitant efficacy of beekeeping industry and commercial honey production. The melissopalynological studies have significant application in the establishment of apiary industries. Analyses of pollen from honey and pollen loads provide relevant information for the pollen and nectar sources of an area. This knowledge is helpful for developing an apiary industry and commercial honey production (Bhusari et al., 2005). The first pollen analyses of honey were studied by Pfister (Kaya et al., 2005). In Turkey pollen analysis of honey was the first made by Qustiani (Stawiartz and Wreblewska, 2010). The first pollen analysis were carried out by Sorkun and İnceoğlu between 1979 and 1981 (Çam et al., 2010). In Turkey Başoğlu et al. (1996) were the first to determine honey quality by using the TPN-10 method. From 1996 and on, quality assessment trough the TPN-10 g as a melissopalynologic analysis has continued (Bölükbaşı, 2009). Pollen analysis were performed in 73 honey samples obtained from Sandomierska (Stawiardz and Wreblewska, 2010), 78 samples obtained from Muğla (Özkök, 2009), 25 honey samples obtained from Canary Islands (La-Serna Ramos et al. 1999), 14 samples obtained from Chubut (Forcone 2008), 13 samples obtained from Arıt (Mısır 2011), 20 samples obtained from Burdur (Taşkın 2009). Honey is hygroscopic; that is, it has excellent water absorbing properties. Thus honey will change in moisture content according to the surrounding atmosphere. This characteristic is important in storing honey because it will absorb water when exposed to high relative humidity (RH) and will give off water when exposed to low RH, until an equilibrium point is reached. High moisture honey will ferment (Sanford 1994). The amount of moisture that occurred in honey is a very important factor and the criteria that determine the quality of honey. The amount of honey is affected by plant source, temperature, rains, condition of bee glaze, the work during the marketing and degree of maturation of honey. Keeping the honey not soured is related to the amount of water in the structure of honey. According to Turkish food Codex, Notification of Honey, the amount of moisture of honey can not be more than the value 20% (MEB, 2012). The current study aimed at identifying nectar containing flower plants, which contribute to the formation of honey from Komati Plateau of Çamlıhemşin in Rize. Material and Method The honey samples were collected from different beehive the month of July- August in During the field studies, herbarium materials were collected. Reference pollen preparations was made from the herbarium materials. The preparation of the honey samples were done using the method defined by the International Bee Research Association (Von Der Ohe et al., 2004). Preparations were made from each honey samples for identification of pollen. After identification, 200 pollen samples were counted in each preparation. Source books (Aytuğ, 1971) were used during the pollen analyses. Olympus CX21 microscope was used for the analyses. Pollen types were classified into four categories: between 1% and 5% was considered as the rare group,

13 13 between 6% and 20% was considered as the minor group, between 21% and 50% was considered secondary group and pollen exceeding 50% was called the dominant group (Doğan and Sorkun 2001). Based on the total number of plant elements, honeys are placed into one of the following classes (Sorkun 2008): Class I with less than 2000 pollen grains per gram of honey (includes unifloral honeys with underrepresented pollen); class II with pollen grains including most of multifloral and honeydew honeys and mixtures of flower and honeydew honeys; class III with pollen grains includes unifloral honeys with overrepresented pollen and honeydew honeys; class IV with between including unifloral honey with strongly overrepresented pollen and some pressed honeys; and class V with more than pollen grains (Dobre et al. 2012). The amount of moisture in honey samples which was stored at 20 C was measured with Portable Refractometer. Results The results of melissopalynologic analyses, 9 samples were identified multifloral honey, because they included pollen grains of multiple taxa. A sample which is unifloral, referred as chesnut honey. In the honey samples 1,3,4,5,7 and 10 no pollen was found in dominant amount. The sample number 6 Rosaceae pollen grains were found in dominant amount, whereas Ericaceae and Onobrychis pollen grains were found secondary amount. In the honey sample 8 Ericaceae pollen grains were found in dominant amount. In the honey sample 9 Castanea sativa MILLER. pollen grains were found in dominant amount while no pollen grains are found secondary and minor amount. Lauraceae and Poacaea pollen grains were found rare amount in the sample number 9 (Table I). In this study,tpn-10 g values ranged from to The sample number 9 contained the least number of TPN-10 g and sample number 1 contained the most number of TPN 10 (Table I). Moisture content of the honey samples were up to standart of honey notification of Turkish Food Codex Discussion The results of microscopic analyses revealed that taxa variability is greatest in the rare group, followed by minor, secondary and dominant groups (Table I). This seems to confirm the view that variability is always little amoung pollen taxa in dominant groups, while greater among rare, minor and secondary groups (Doğan and Sorkun 2001). It is determined that nectar contributing to formation of honey is obtained from plants with pollen grains included in dominant and secondary groups (Erdoğan et al. 2009). In the study that made on the honey samples from Komati Plateau pollen grains of Castanea sativa MILLER. Ericaceae and Rosaceae were determined in dominant group. In seven samples no pollen grains were found in dominant group. Castanea sativa MILLER. pollen grains were found to be dominant due to the prevalence of chestnust trees. In the multifloral honey samples were obtained pollen grains in seconder and minor group while in the unifloral honey sample no pollen grains were obtained in seconder and minor groups. Acknowledgements Techniqual support of this work by Kadriye Sorkun, Ömür Gençay Çelemli and Şeyda Turan is gratefully acknowledged.

14 MELLIFERA 14 Table I. Pollen Spectra, TPN-10 g Values (*Dominant pollen,**secondary Pollen,***Minor Pollen,****Rare Pollen) and the Amount of Moisture H. S. Number Pollen spectrum TPN-10 g The Amount of Moisture % 1 * - ** Castanea sativa. MILLER., Ericaceae, Rosaceae *** - **** Brassicaceae, Fabaceae, Pinaceae, Rumex * - ** Ericaceae, Rosaceae *** Castanea sativa MILLER., Fabaceae **** Dipsacaceae, Lamiaceae, Rumex sp., Salix sp., Tilia sp * - ** Castanea sativa MILLER., Ericaceae, Rosaceae *** Fabaceae **** Asteraceae, Boraginaceae, Brassicaceae, Poaceae * - ** Castanea sativa MILLER., Ericaceae *** Fabaceae, Rosaceae **** Brassicaceae, Lamiaceae, Poaceae, Ranunculus sp * - ** Castanea sativa MILLER. *** Rosaceae **** Ericaceae, Fabaceae, Salix sp * Rosaceae ** Ericaceea, Onobrichis sp. *** Castanea sativa MILLER. **** Salix sp * - ** Ericaceae, Rosaceae *** Fabaceae, Rumex sp. **** Brassicaceae, Lamiaceae, Pinaceae, Salix sp * Ericaceae ** Rosaceae *** Salix sp. **** Cistus sp., Lamiaceae, Onobrychis sp., Rumex sp * Castanea sativa MILLER. ** - *** - **** Lauraceae, Poaceae * - ** Castanea sativa MILLER., Ericaceae, Rosaceae *** Fabaceae **** Boraginaceae, Brassicaceae

15 15 Table 2. Number of Pollen in Honey Samples Sample No 1 Taxa Number of Pollen Ericaceae 69 Sample No Taxa Number of Pollen Rosaceae 103 Castanea sativa MILLER. 65 Ericaceae 47 Rosaceae 58 Onobrychis sp. 28 Fabaceae 6 6 Castanea sativa MILLER. 19 Brassicaceae 1 Salix sp. 3 Pinaceae 1 Ericaceae 85 Rumex sp. 1 Rosaceae 75 Ericaceae 95 Fabaceae 22 2 Rosaceae 57 7 Brassicaceae 9 Fabaceae 32 Salix sp. 5 Castanea sativa MILLER. 10 Lamiacaea 2 Lamiacaea 2 Pinaceae 1 Rumex sp. 2 Poaceaea 1 Tilia sp. 1 Ericaceae 106 Dipsacaceae 1 Rosaceae 66 Castanea sativa MILLER. 73 Salix sp. 22 Rosaceae 64 Cistus sp. 2 Ericaceae 42 8 Rumex sp. 2 Fabaceae 15 Onobrychis sp. 1 Brassicaceae 3 Lamiaceae Asteraceae 1 Castanea sativa MILLER. Poaceae 1 9 Poaceae 5 Boraginaceae 1 Lauraceaea 5 Castanea sativa MILLER. 90 Castanea sativa MILLER. Ericaceae 50 Rosaceae 60 Rosaceae 34 Ericaceae 41 Fabaceaea 12 Fabaceae 13 Brassicaceae Boraginaceae 4 Lamiaceae 1 Brassicaceae 1 Ranunculus Poaceae 1 Castanea sativa MILLER. 149 Rosaceae 40 5 Fabaceae 6 Ericaceae 3 Salix sp. 2

16 MELLIFERA 16 Figure I. Microphotographs of Pollen Grains a. Castanea sativa MILLER. b. Ericaceae c. Cistaceae d. Asteraceae e. Rumex sp. f. Lamiaceae g. Pinaceae h. Rosaceae 1µ : x100 2µ : x40 Figure I. Microphotographs of Pollen Grains References Aytuğ B İstanbul Çevresi Bitkilerinin Polen Atlası. İstanbul Üniversitesi Orman Fakültesi Başoğlu F. N., Sorkun K., Löker M., Doğan C. and Wetherilt H Saf ve Sahte Balların Ayırt Edilmesinde Fiziksel, Kimyasal ve Palinolojik Kriterlerin Saptanması. Gıda. 21(2), Bhusari, N. V., Mate, D. M., Makde, K. H Pollen of Apis honey from Maharashtra. Grana. 44: ISSN Bölükbası N. D., Melissopalynologıc Analysis of Pocked Honey. Mellifera. 9-18:2-8. Acknowledgements Çam B., Pehlivan S., Uraz G., Doğan C Pollen Analyses of Honey Collected from Various Regions of Ankara (Turkey) and Antibacterial Activity of These Honey Samples Against Some Bacteria. Mellifera :2-16. Dobre I., Alexe P., Escuredo O., Seıjo M. C Palynological evaluation of selected honeys from Romania. Grana. ifirst: 1 9. Doğan C., Sorkun K Pollen Analysıs of Honeys From Aegean, Marmara, Mediterranean and Black Sea Regions İn Turkey. Mellifera.1-1: Erdoğan N., Pehlivan S., Doğan C Pollen Analysis of Honeys from Sapanca-Karapürçek-Geyve and Taraklı Districts of Adapazarı Province (Turkey). Mellifrea. 9-17:9-18. Forcone A Pollen Analysis of Honey from Chubut (Argentinean Patagonia) Grana. 47: Kaya Z., Binzet R., Orcan N Pollen Analyses of Honeys From Some Regions in Turkey Apiacata. 40:10-15 La-Serna Ramos, I. E., MeÂndez PeÂrez, B. & Gomez Ferreras, C Pollen Characterization of Multiforal Honeys from La Palma (Canary Islands). Grana 38: 356±363. ISSN Mısır M., Arıt Bölgesi (Bartın) Ballarında Polen Analizi, Yüksek Lisans Tezi, Bartın. Özkök A The Microscopic, Organoleptic And Chemical Analysis Of Pine Honey And Propolis, Which Are Produced In Muğla Region. Doktora Tezi, Ankara. Sanford T. M Moisture in Honey.University of Florida IFAS Extension. Sorkun K Türkiye`nin Nektarlı Bitkileri, Polenleri ve Balları. Palme Yayıncılık. Stawiardz E., Wroblewska A Melissopalynolgıc Analysis Of Multifloral Honeys From The Sandomierska Upland Area Of Poland. Journal of Apicultural Science :54-1 Taşkın, D., Burdur Yöresi Ballarının Polen Analizi, Yüksek Lisans Tezi, Isparta Ünal C., Küplülü Ö Chemical Quality of Strained Honey Consumed in Ankara. Ankara Üniv Vet Fak Derg, 53, 1-4. Von Der Ohe W., Persano Oddo L., Piana M.L., Morlot M., Martin P Harmonized Methods of Melissopalynology. Apidologie. 35 S18 S25. Laboratuvar Hizmetleri Bal Analizleri-1 524LT MEB, Ankara.

17 MELLIFERA 12-24:17-26 (2012) HARUM RESEARCH 17 ARTICLE NOTES ON ROPHITES ALGIRUS PÉREZ, 1895 AND ROPHITES QUINQUESPINOSUS SPINOLA, 1808 OF MEDITERRANEAN TURKEY WITH AN UPDATED LIST OF SUBFAMILY ROPHITINAE (HYMENOPTERA: HALICTIDAE) OF TURKEY TÜRKIYE DEKI ROPHITINAE (HYMENOPTERA: HALICTIDAE) ALTFAMILYASININ GÜNCEL LISTESI ILE TÜRKIYE NIN AKDENIZ HAVZASINDAKI ROPHITES ALGIRUS PÉREZ, 1895 AND ROPHITES QUINQUESPINOSUS SPINOLA, 1808 ÜZERINE NOTLAR Fatih DİKMEN * and Ahmet Murat AYTEKİN * Summary: The study was conducted in the Mediterranean region of southern Turkey. Two Rophitinae (Halictidae: Hymenoptera) species, Rophites algirus Pérez, 1895 and Rophites quinquespinosus Spinola, 1808 were considered. Taxonomical identification keys, distribution maps and flower visits of these species were given. Detailed microscopic photos and drawings of some important morphological features were also revealed. Besides, the literature on the fauna of the subfamily Rophitinae was reviewed to establish an updated species list of Turkey. As a result of this overview eight species seem possibly endemic for Turkey. Key Words: Halictidae, Rophitinae, Fauna, Distribution, Turkey Özet: Çalışma Türkiye nin güneyindeki Akdeniz Bölgesi nde gerçekleştirildi. İki Rophitinae (Halictidae: Hymenoptera) türü, Rophites algirus Pérez, 1895 and Rophites quinquespinosus Spinola, 1808 ele alındı. Bu türlere ait taksonomik tanı anahtarları, yayılım haritaları ve bitki ziyaretleri belirtildi. Bazı önemli morfolojik karakterlere ait fotoğraflar ve çizimler de ayrıca gösterildi. Bunların yanında, Türkiye deki güncel tür listesinin elde edilebilesi için, Rophitinae altfamilyası faunası üzerine yapılmış olan literatür derlendi. Bu genel taslağın bir sonucu olarak, sekiz türün Türkiye için muhtemelen endemik olabilecekleri görüldü. Anahtar Kelimeler: Halictidae, Rophitinae, Fauna, Yayılım, Türkiye *Hacettepe University Faculty of Science, Department of Biology, Beytepe, Ankara, Turkey Corresponding Author [email protected] This study based on part of the PhD thesis of F. Dikmen submitted to Hacettepe University Institute of Science in

18 MELLIFERA 18 Introduction Halictidae (Apiformes: Apoidea: Hymenoptera) is one of the largest families of all bees (Pesenko et al. 2000; Michener 2007). It contains four subfamily (Rophitinae, Nomiinae, Nomioidinae, Halictinae) according to Michener (2007) and nearly 3500 species (Pesenko 2007) in the world. From these, Rophitinae is one the most interesting one because of their unique morphology and biological features. Rophitinae members can be easily separated from other groups by such brief characters; antennal sockets placed on lower half of face and clypeus shorter than labrum (Pesenko et al. 2000). Apart from these, they exhibit solitary life and they are mostly oligolectic unlike the other Halictidae members (Pesenko et al. 2000). Patiny and Michez (2006) reported that Systropha spp. are the most typical ones that show narrow plant choice and especially oligoleg on Convovulus L. spp. (Convolvulaceae). Moreover, Patiny et al. (2007) suggested that there should be an evolutionary tendency for Rophitinae species to specialize on the members of certain plant groups. This subfamily contains nearly 200 species in the world and half of them are distributed in Palaearctic region (Pesenko et al. 2000). Generally in West Palaearctic Region and also locally in Turkey it is represented by four genera: Dufourea Lepeletier, Morawitzia Friese, Rophites Spinola, and Systropha Illiger (Michener 2007). Among them, the most diverse genus is Dufourea and it has generally Holoarctic distribution. Whereas members of the genera Systropha, Morawitzia and Rophites show more likely restricted distributions (Pesenko et al. 2000; Michener 2007). On the other hand there are many tasks had to be done for exact evolutionary explanations. Especially there are still no sufficient and up to date knowledge on the distributions of Rophitinae groups for Turkey. The leading studies which also include scrappy data on the Rophitinae fauna of Turkey are Ebmer (1987; 1988; 1993) and Schwammberger (1976). Moreover the information about the members of the genus Rophites Spinola of Turkey is very scanty. For these reasons, this study aimed to make contributions on the Rophitinae fauna of Turkey. Rophites algirus Pérez, 1895 and Rophites quinquespinosus Spinola, 1808 were considered taxonomically. Secondly the scattered literature data were evaluated to figure out the updated faunistic list of the subfamily for Turkey. Materials and Methods The study was conducted at Mediterranean Region of Southern Turkey. Field studies were performed during the spring and summer seasons of 2008 and Random sampling protocol was used and vegetation boundaries were followed in collecting bees. Bee specimens were collected via hand nets and aspirators. Meanwhile, the plants that have been visited by bees were also recorded or collected for diagnosis. Captured bee samples and collected plants were properly prepared for collections. All of these specimens were deposited in the Department of Biology, Faculty of Science, Hacettepe University, Ankara (Turkey). GPS coordinates were taken by Garmin Etrex H. Materials were examined with stereoscopic microscopes for diagnosis. Identification of the bee specimens were made according to Warncke (1980), Ebmer and Schwammberger (1986) and Niu et al. (2005). Identification of plant specimens were made by Demet Töre (Department of Biology, Faculty of Science, Hacettepe University) and The International Plant Names Index (IPNI 2008) were followed for the author names of the plant taxa. Taxonomical identification key for two species were prepared. Some important morphological features including the dorsal view of genitalia of these species were also photographed. Ebmer and Schwammberger (1986) was followed for genitalia inspection. Ecoregion map (fig.1) was prepared with CFF 2.0 (Carto Fauna-Flora; Barbier and Rasmont 2000) and modified with Adobe Photoshop v7.0 for a better visualization. We followed the explanation of borders of West Palaearctic Region proposed by Patiny et al (2009). Distribution maps (fig. 4) for the Rophites algirus and R. quinquespinosus was also prepared with CFF 2.0 (Barbier and Rasmont 2000: Carto Fauna-Flora). Species lists for regions and subregions were prepared according to Schwammberger (1976), Ebmer (1987; 1988; 1993), Baker (1996); Pesenko (1998); Pesenko et al. (2000), Patiny (2003; 2004), Niu et al. (2005), Patiny and Michez (2006), Pesenko and Astafurova (2006), Ascher et al. (2009). The species are listed in tables are in the alphabetical order (tab.1 and tab.3).

19 19 Results Species identification key for female 1. Frons with thick spines medially, with 7 8 spines under median ocel and 8 10 spines laterally (fig. 2a) R. algirus Pérez 1. Frons with thick spines medially, with 4 5 spines under median ocel and 4 5 spines laterally (fig. 2b).. R. quinquespinosus Spinola Species identification key for male 1. S6 with a slight thin process longitudinally; distal lobe of S8 thin; gonostylus narrower and more cylindrical (fig. 2c, 2e, 3a) R. algirus Pérez 1. S6 with thick and obvious process longitudinally; distal lobe of S8 thick; gonostylus broader and more foliate like (fig. 2d, 2f, 3b). R. quinquespinosus Spinola Rophites algirus Pérez, 1895 Distribution: Turkey, Caucasus (Pesenko et al. 2000); Bulgaria, Morocco, France, Iran, Italy, Tunisia, Ukraine (Ascher et al. 2009). Inspected Material: 18-VII-2008, N, E, 1963 m, Söbüce Yayla, Antalya, 1 ; 22- VII-2008, N, E, 2225 m, Anamur - Ermenek road Gazipaşa cross, Anamur, Mersin, 1 ; 07-VI-2009, N, E, 1241 m, Gülnar - Ermenek road, 5 km to Akova, Mersin, 2 ; 23- VII-2009, N, E, 1752 m, Anamur - Ermenek road, Gazipaşa cross, Ermenek, Karaman, 1 (fig. 4a). Visited Flowers: Phlomis armeniaca Willd., P. monocephala P.H.Davis, P. sieheana, Stachys byzantina C. koch. (Lamiaceae). Rophites quinquespinosus Spinola, 1808 Distribution: Europe, China, Western Russia, Caucasus, Kazakhstan, Kirgizstan, Turkey (Ascher et al. 2009). Inspected Material: 23-VII-2009, N, E, 1752 m, Anamur - Ermenek road Gazipaşa cross, Ermenek, Karaman, 2 (fig. 4b). Visited Flowers: Ballota saxatilis Sieber ex C.Presl. (Lamiaceae). Biogeographical results The subfamily Rophitinae is represented by 95 species in Palaearctic region (Pesenko and Astafurova 2006). As a result of our literature research we can suppose that 29 of them (13 from Dufourea; 11 from Rophites; three from Morawitzia; and two from Systropha) are recorded from Turkey (tab.1). These numbers also constitute nearly 30% of Palaearctic fauna and also close with the richness of the fauna of Europe mainland (tab.2). Moreover, eight of them are possibly endemic for Turkey (tab.1). In addition to these data, twelve Rophitinae spp. were also listed according to their close distribution within adjacent boundaries (tab.3). Discussion During our field studies in Mediterranean Region of Turkey only two species belong to Rophites spp. were determined. However it was interesting that all specimens were captured at high elevation (between meters). Secondly it is important that Rophites algirus and R. quinquespinosus are captured just only on Lamiaceae members. This may be counted as a kind of clue about their oligolectic feeding behavior which was also mentioned in Pesenko et al. (2000). However because of the low sampling size it is difficult to make inference about the flower preferences and distributions of the Rophites species within Mediterranean Turkey. The biogeographical results showed that Turkey is one of the important reserves for the members of Morawitzia, Rophites and Dufourea. On the other hand Systropha members have Palaeatrctic and African distribution and they are hardly represented within Turkey (tab.2). The members of Morawitzia are generally endemic to Anatolian part of Turkey and Caucasia (Georgia and Armenia) and do not distributed in any other parts of the world. The endemism (nearly 20% of Palaearctic fauna) and the species richness (nearly 60% of the Palaearctic fauna) of the genus Rophites are also high in Turkey (Table.2). These findings generally supports that Turkey is one of the most important biogeographical zones for bees with considerably diversified fauna. The data presented here might exhibit a good ground base for bee conservation studies in Turkey and also would be helpful in such taxonomical studies focusing on the subfamily Rophitinae. Acknowledgements This study partly supported by Hacettepe University Research Foundation Project No:

20 MELLIFERA 20 Fig. 1: Subregions of West Palaearctic Region and Asia. BA: Balkans; CA: Central Asia; CAU: Caucasia; EA: Eastern Asia; ME: Middle East; MED: Mediterranean Basin; NA: Northern Asia; SA: Southern Asia; WP: West Palaearctic Region (including BA, CAU, MED, and ME regions). Fig. 1: Subregions of West Palaearctic Region and Asia. BA: Balkans; CA: Central Asia; CAU: Caucasia; EA: Eastern Asia; ME: Middle East; MED: Mediterranean Basin; NA: Northern Asia; SA: Southern Asia; WP: West Palaearctic Region (including BA, CAU, MED, and ME regions). 8

21 9 21 Fig. 2: a-b: Female head and the spines on frons; a: Rophites algirus and b: R. quinquespinosus; c-d: Sterna VI (S6) of male; c: Rophites algirus and d: R. quinquespinosus; Sterna VIII (S8) of male; e: Rophites algirus and f: R. quinquespinosus.

22 Fig. 2: a-b: Female head and the spines on frons; a: Rophites algirus and b: R. quinquespinosus; c-d: Sterna VI (S6) of male; c: Rophites algirus and d: R. quinquespinosus; 22 MELLIFERA Sterna VIII (S8) of male; e: Rophites algirus and f: R. quinquespinosus. Fig. 3: Male genitalia drawing and detailed microscopic photograph: a: Rophites algirus and b: R. quinquespinosus. gb: gonobase; gc: gonocoxite; gs; gonostylus; pv: penis valve; vs: volsella; S8: Sterna VIII. Fig. 3: Male genitalia drawing and detailed microscopic photograph: a: Rophites algirus and b: R. quinquespinosus. gb: gonobase; gc: gonocoxite; gs; gonostylus; pv: penis valve; vs: volsella; S8: Sterna VIII. 10

23 23 11 Fig. 4: Mediterranean distributions of a: Rophites algirus and b: R. quinquespinosus. Fig. 4: Mediterranean distributions of a: Rophites algirus and b: R. quinquespinosus. Table.1: The list of the Rophitinae Species of Turkey. 1: Ascher et al. (2009); 2: Ebmer (1987); 3: Pesenko (1998); 4: Ebmer (1988); 5: Ebmer (1993); 6: Schwammberger (1976); 7: Pesenko et al. (2000); 8: Baker (1996); Ref: References. Species Subgenus Ref. Distribution Dufourea armenia Ebmer, 1987 Cyprirophites 1; 2 Dufourea atrata (Warncke, 1979) Dufourea 1, 3 Endemic - Caucasia Dufourea caelestis Ebmer, 1987 Dufourea 1; 2 Endemic Dufourea cypria Mavromoustakis, 1952 Dufourea 1 East Mediterranean Dufourea graeca Ebmer, 1976 Halictoides 1; 2 Caucasia, Balkan Dufourea longicornis (Warncke, 1979) Cyprirophites 1 Caucasia East Mediterranean, Middle East, West Part of

24 MELLIFERA 24 Table 1: The list of the Rophitinae Species of Turkey. 1: Ascher et al. (2009); 2: Ebmer (1987); 3: Pesenko (1998); 4: Ebmer (1988); 5: Ebmer (1993); 6: Schwammberger (1976); 7: Pesenko et al. (2000); 8: Baker (1996); Ref: References. Species Subgenus Ref. Distribution Dufourea armenia Ebmer, 1987 Cyprirophites 1; 2 Caucasia Dufourea atrata (Warncke, 1979) Dufourea 1, 3 Endemic - Caucasia Dufourea caelestis Ebmer, 1987 Dufourea 1; 2 Endemic Dufourea cypria Mavromoustakis, 1952 Dufourea 1 East Mediterranean Dufourea graeca Ebmer, 1976 Halictoides 1; 2 Caucasia, Balkans Dufourea longicornis (Warncke, 1979) Cyprirophites 1 East Mediterranean, Middle East, West Part of East Asia Dufourea pontica (Warncke, 1979) Halictoides 1; 2 Caucasia Dufourea quadridentata (Warncke, 1979) Dufourea 1 Endemic Dufourea salviae Ebmer, 2008 Cyprirophites 1 Endemic Dufourea schmiedeknechtii (Kohl, 1905) Halictoides 1; 2; 3 North Asia; Caucasia; Europe Dufourea wolfi Ebmer, 1989 Dufourea 1 Balkans Morawitzia fuscescens Friese, 1902 Morawitzia 1 Endemic Morawitzia mandibularis Alfken, 1935 Morawitzia 1 Caucasia Morawitzia panurgoides Friese, 1902 Morawitzia 1 Caucasia Rophites algirus Pérez, 1895 Rophites 1; 4 West Palaearctic Rophites anatolicus (Schwammberger, 1975) Rophitoides 1 Endemic Rophites canus Eversmann, 1852 Rophitoides 1; 4 Trans-Palaearctic Rophites caucasicus Morawitz, 1875 Rophites 1; 5 Caucasia Rophites clypealis Schwammberger, 1976 Rophites 1; 6 Pontic Rophites foveolatus Friese, 1900 Rophites 1; 5 Caucasia, South Europe Rophites gusenleitneri Schwammberger, 1971 Rophites 1; 5 Endemic Rophites hartmanni Friese, 1902 Rophites 1; 4 East Europe, East Mediterranean Rophites heinrichi Schwammberger, 1976 Rophites 1, 6 Endemic Rophites leclercqi Schwammberger, 1971 Rophites 1, 7 Balkans Rophites nigripes Friese, 1902 Rophites 1; 5 East Mediterranean Rophites quinquespinosus Spinola, 1808 Rophites 1 WP, Middle East Rophites transitorius Ebmer, 1993 Rophites 1; 5 Endemic Systropha curvicornis (Scopoli, 1770) Systropha 1; 4 West Palaearctic and East Asia Systropha planidens Giraud, 1861 Systropha 1; 4; 8 Europe and Middle East

25 25 Table 2: Comparison of the Rophitinae fauna of Palaearctic Region, Europe and Turkey. Genus General Distribution Palaearctic (Number of Species) Turkey (Number of Species) Europe (Number of Species) Dufourea Holarctic (3 endemic) 17 Rophites Palaearctic (4 endemic) 10 Systropha Mainly Palaearctic and Africa 17 2 (no endemism) 2 Morawitzia Caucasia 3 3 (1 endemic) - Table 3: The list of the Rophitinae species of adjacent boundaries. 1: Ascher et al. (2009); 2: Ebmer (1987). Species Reference Distribution Dufourea alpina Morawitz, Mediterranean (South Europe) Dufourea bytinskii Ebmer, East Mediterranean Dufourea dentiventris (Nylander, 1848) 1 Europe, East Asia Dufourea goeleti Ebmer, East Mediterranean Dufourea halictula (Nylander, 1852) 1 Europe, Caucasia Dufourea inermis (Nylander, 1848) 1 Europe, Northeastern and East Asia Dufourea iris Ebmer, ; 2 Balkans Dufourea minuta Lepeletier, Europe, East Asia Dufourea paradoxa (Morawitz, 1867) 1 West Europe, Central Asia, East Asia Dufourea similis Friese, North Africa, East Mediterranean Dufourea trigonellae Ebmer, East Mediterranean Rophites schoenitzeri Dubitzky, Caucasia

26 MELLIFERA 26 References Ascher J.S., Rozen Jr. J.G. and Schuh T Discoverlife website. Apoidea species guide. Barbier Y. and Rasmont P Carto Fauna-Flora 2.0. Guide d utilisation. Université de UMH-Hainaut, UMH, Belgique, pp. 59. Baker D.B Notes on some palaearctic and oriental Systropha, with descriptions of new species and a key to the species (Hymenoptera: Apoidea: Halictidae). Journal of Natural History, 30: Ebmer A.W Die westpaläarktischen Arten der Gattung Dufourea Lepeletier 1841 mit illustrierten Bestimmungstabellen. Linzer Biologische Beiträge, 19: Ebmer A.W Kritische liste der nicht-parasitischen Halictidae Österreichs mit Berücksichtigung aller mitteleuropäischen Arten (Insecta: Hymenoptera: Apoidea: Halictidae). Linzer biol. Beitr, 20 (2): Ebmer A.W Die Bienengattung Rophites Spinola 1808 Erster Nachtrag. Linzer Biologische Beiträge, 25: Ebmer A.W. and Schwammberger K.H Die Bienengattung Rophites Spinola 1808 (Insecta: Hymenoptera: Apoidea: Halictidae: Dufoureinae). Illustrierte Bestimmungstabellen. Senckenbergiana biol., 66: IPNI The International Plant Names Index, org. Michener C.D The Bees of the World. 2nd edition. John Hopkins Univ. Press, Balitimor, USA. 953 p. Niu Z., Wu Y. and Huang D A taxonomic study of the four genera of the subfamily Rophitinae from China (Hymenoptera: halictidae). The Raffles Bulletin of Zoology. 53: Patiny S Revision of the subgenus Dufourea (Flavodufourea) Ebmer, 1984 (Hymenoptera, Halictidae, Rophitinae) and description of a new species D.(Flavodufourea) ulkenkalkana sp.nov. from Kazakhstan. Zootaxa, 255: 1-8. Patiny S Description of two new Systropha Illiger 1806 (Hymenoptera, Halictidae, Rophitinae). Linzer Biologische Beiträge. 36 (2): Patiny S. and Michez D Phylogenetic analysis of the Systropha Illiger, 1806 (Hymenoptera: Apoidea: Halictidae) and description of a new subgenus. Annales de la Société entomologique de France, 42(1): Patiny S., Michez D. and Danforth B.N Phylogenetic relationships and host-plant association within the basal clade of Halictidae (Hymenoptera, Apoidea). Cladistics, 23:1-15. Patiny S., Rasmont P. and Michez D A survey and review of the status of wild bees in the West Palaearctic region. Apidologie, 40 (2009): Pesenko Yu.A New and little known bees of the genus Dufourea Lepeletier (Hymenoptera, Halictidae) from the Palaearctic Region. - Ent. Obozrenie (St. Petersburg), 77 (3): [in Russian with English summary. English translation in Ent. Review, 78 (5): ]. Pesenko Yu.A The family Halictidae (Hymenoptera): General. In: A key to insects of the Russian Far East. Vol. IV, pt 5. Vladivostok (Dal nauka): [in Russian] Pesenko Yu.A., Banaszak J., Radchenko V.G. and Cierzniak T Bees of the Family Halictidae (Excluding Sphecodes) of Poland: Taxonomy, Ecology, Bionomics. Bydgoszcz, Poland. Bydgoszcz Press. p Pesenko Yu.A. and Astafurova Yu.V Contributions to the halictid fauna of the Eastern Palaearctic Region: subfamily Rophitinae (Hymenoptera: Halictidae). Entomofauna, 27(27): Schwammberger K.H Zwei neue Rophites-Arten aus der Türkei. Ent. Ztschr. 86: Warncke K Rophites quinquespinosus Spinola und R. trispinosus Pérez eine oder zwei Bienenarten? (Apidae, Halictinae). Entomofauna, 1/3:

27 MELLIFERA 12-24:27-32 (2012) HARUM RESEARCH 27 ARTICLE CHEMICAL COMPOSITION OF PROPOLIS SAMPLES COLLECTED FROM TEKIRDAG-TURKEY TEKİRDAĞ-TÜRKİYE DEN TOPLANAN PROPOLİS ÖRNEKLERİNİN KİMYASAL İÇERİĞİ Ömür Gençay Çelemli *, Kadriye Sorkun*, Bekir Salih** Summary: The aim of this study is to investigate the chemical compositions of propolis samples which were collected from Tekirdağ city of Turkey. A total of 92 different propolis samples collected from eight towns of Tekirdağ were examined by GC-MS (Gas Chromatography and Mass Spectrometry) to determine chemical composition and establish the chemical profile of Tekirdağ propolis. According to the GC-MS results, the compound; aldehydes, alcohols, aliphatic acids and their esters, flavonoids, hydrocarbons, carboxylic acid and their esters, cinnamic acids and their esters ketones, were determined in various amounts. Among these compounds the flavonoids were found in all samples and in higher amounts compare to the other compounds. Keywords: propolis, GC-MS, Tekirdağ, chemical profile, flavonoid Özet: Bu çalışmanın amacı Türkiye nin Tekirdağ ilinden toplanan propolis örneklerinin kimyasal içeriğini araştırmaktır. Tekirdağ ın sekiz ilçesinden toplanan toplamda 92 örneğin kimyasal içeriği GC-MS ile (Gaz Kromatografisi ve Kütle Spektrometresi) incelenmiş ve Tekirdağ propolisinin kimyasal profili oluşturulmuştur. GC-MS sonuçlarına göre aldehidler, alkoller, alifatik asit ve esterleri, flavonoidler, hidrokarbonlar, karboksilik asit ve esterleri, sinamik asit ve esterleri, keton bileşikleri değişik miktarlarda saptanmıştır. Bu bileşikler arasında flavonoidler tüm örneklerde belirlenmiş ve diğer bileşiklere göre daha yüksek oranlarda olduğu bulunmuştur. Anahtar kelimeler: propolis, GC-MS, Tekirdağ, kimyasal profil, flavonoid *Hacettepe University Faculty of Science, Department of Biology, Beytepe, Ankara, Turkey **Hacettepe University Faculty of Science, Department of Chemistry, Beytepe, Ankara, Turkey Corresponding Author [email protected] This study based on part of the PhD thesis of Ö. G. Çelemli.

28 MELLIFERA 28 Introduction Propolis or bee glue is a sticky dark-colored material that honey bees collect from plants and use it in the hive: they apply it to seal the walls, to strengthen the borders of combs, to line all cells inside, to embalm dead invaders (Bankova 2005). It is also well known that the propolis possesses antibacterial, antifungal and antiviral properties and many beneficial biological activities such as antiinflammatory, antiulcer, local anesthetic, hepatoprotective, antitumor, immunostimulating etc (Bankova et al. 2000). Bees use it, therefore, as a protective barrier against their enemies (Burdock 1998). However, propolis is being used in the traditional medicine since 3000 BC, in Egypt. For propolis production, bees use natural materials resulting from a variety of botanical processes in different parts of plants. These are substances actively secreted by plants as well as substances exuded from wounds in plants: lipophilic materials on leaves and leaf buds, gums, resins, lattices etc. The plant origin of propolis determines its chemical diversity. Bee glue s chemical composition depends on the specify of the local flora at the site of collection and thus on the geographic and climatic characteristics of this site (Bankova 2005). Propolis generally contains 50% resin, 30% wax, 5% pollen, 10% aromatic oils, and 5% other organic residues. Literature reported some important biological activities of propolis. The biological activities were verified due to the content of flavonoids, aromatic acids and esters present in the propolis (Lee et al. 2007). Material and Methods Propolis samples In the propolis samples were collected from the hives of Tekirdağ. The hives from eight towns (Çerkezköy, Çorlu, Hayrabolu, Malkara, Merkez, Muratlı, Saray, Şarköy) of Tekirdağ choosed according to the sampling method. By this method 92 bee farms were choosen to collect propolis. So the study carried on with 92 propolis samples. The number of beehives choosen by sampling method is given in Table 1. Propolis samples were collected from the edges of frames by scraping with a spatula. Extraction and sample preparation Each frozen propolis sample was grained and dissolved in ethanol (96%) with a ratio of 1/3. Then,the mixture kept in tightly closed bottle and in an incubator at 30 C for two weeks. After incubation period, the supernatant was filtered twice with Watman No 4 and No 1 filter papers. The final solution, (1:10, w/v), called Ethanol Extracts of propolis (EEP) was evaporated until completely dryness. About 5 mg of dry substance were mixed with 75 µl of dry pyridine and 50 µl bis (trimethylsilyl) trifluoroacetamide (BSTFA), heated at 80 C for 20 min and the final supernatant was analyzed by GC-MS (Gençay and Salih 2009). GC-MS analysis A GC 6890N from Agilent (Palo Alto, CA, USA) coupled with mass detector (MS5973, Agilent) was used for the analysis of EEP samples. Experimental conditions of GC-MS system was as follows: DB 5MS column (30 mx 0.25mm and 0.25 µm of film thickness) was used and flow rate of mobile phase (He) was set at 0.7 ml/min. In the gas chromatography part, temperature was kept for 1 min at 50 C and then increased to 150 C with 10 C/min heating ramp. After this period, temperature was kept at 150 C for 2 min. Finally, temperature was increased to 280 with 20 C/ min. heating ramp and kept at 280 C for 30 min. Organic compounds in propolis samples were identified using standard Willey and Nist Libraries available in the data acquisiton system of GC-MS, if the comparison scores were obtained higher than 95%. Otherwise, fragmentation peaks of the compounds were evaluated and the compounds were identified using our memorial background for the identification of the compounds appeared in GC-MS chromatograms. For the quantification of the compounds in the ethanol extract, no internal and external standards were used; only percentage reports of the compounds in the sample were used. This was the standard way to quantify most organic compounds in the propolis sample, thus reducing the relative error in <5%. Results and Discussion According to the GC-MS results aldehydes, alcohols, aliphatic acids and their esters, flavonoids, hydrocarbons, carboxylic acid and their esters, ketones, cin-

29 29 namic acids and their esters were determined in various amounts in the investigated 92 samples. Among these compounds flavonoids were the compounds observed at higher ratios. The flavonoids; 2-Propen-1-one,1-(2,6-dihydroxy- 4-metoxyphenyl)-3-phenyl (Pinostrobin, chalcone), 4, 5-Dihydroxy-7-methoxyflavanone, 4H-1-benzopyran-4-one, 5-hydroxy-2-(4-hydroxyphenyl)-7methoxy-(Tetrochrysin), 4H-1-Benzopyran-4-one,2,3-dihydroxy-5,7- dihydroxy-2-phenyl (Pinocembrin), 4H- 1-Benzopyran-4-one,5-hidroxy-7-methoxy-2-phenyl, 4H-1-Benzopyran-4-one,3,5,7-trihydroxy-2-phenyl (Galangin), 4H-1-Benzopyran-4-one 5,7 dihydroxy- 2-phenyl (Chrysin), 4H-1-Benzopyran-4-one,5,7- dihydroxy-2-(4-hidroxyphenyl) (Acacetin) were observed in Tekirdağ samples. The compound belong to the aldehydes group were observed in minor amounts. From aliphatic acids and their esters group mostly observed compounds were; Ethyl oleat, Hexadecanoic acid ethyl ester (palmitic acid ethyl ester), Octadecanoic acid ethyl ester (stearic acid ethyl ester), 9-Octadecanoic acid, Linoleic acid ethyl ester, 2-Butenoic acid, 2 methyl, Hexadecanoic acid, 9, 12-Octadecadienoic acid ethyl ester, Decanoic acid ethyl ester, Dodecanoic acid ethyl ester, Heptadecanoic acid, 15-methyl-,ethyl ester. From hydrocarbons group; 1,19-Eicosadiene, 10 heneicosene (c,t), 17-Pentatriacontene, 1-Docosane, 1-Heptadecane, 1-hexacosane, 1,13-Tetradecadiene, 1-Nonadecane 3-Eicosane, 7-Pentadecine, 9 Tricosane, (Z)-, 9,10 Antracenedione, 1-hidroxi 2 (hidroximethyl), 9,10-Anthracenedione, 1-hidroxy- 2-(hydroxymethyl)-, 9-Hexacosane, 9-Nonadecene Nonadecane, Octadecane, Pentacosane, Benzofuran, 2,3-dihydro-, Siclotetracosane, Docosane, Eicosane, Tricosene, Heneicosane, Heptacosene, Z-12-pentacosene, Z-5-Nonadecane, Siclotriacontane, 1,3,5,7-Cyclooctatetracane, Naphthalene, 1,2,3,5,6,7,8,8a octahydro 1, 8a dimethyl 7 ( 1 methylethenyl)-, [1R (1.alpha, 7. beta, 8a alpha.)]-, Naphthalene, 1,2,3,4-tetrahydro-1,6-dimethyl-4-(1- methylethyl)-, (1S-cis)-, Naphthalene,1,2,3,5,6,8ahexahydro-4,7-dimethyl-1-(1-methylethyl)-,(1S-cis), Naphthalene, 1, 2, 3,4,4a,5,6, 8a-octahydro-7-methyl-4-methylen-1-(1-methylethyl) (1.alpha., 4a.beta., 8a.alpha.) compounds were observed frequently but in minor amounts. As carboxylic acids and their esters ; Heptadecanoic acid,15-mehyl-ethyl ester, 4-Pentenoic acid, 5-phenyl, Benzoic acid, 4-pentenoic acid, 5-phenyl-cyclopropancarboxylic acid, 2-phenyl-, methyl ester, 2-butenoic acid, 2-methyl, pentadecanoic acid ethyl ester, 1,2-benzenedicarboxylic acid diis ooctyl ester were found to be main compounds. From cinnamic acid and its esters group; benzyl cinnamate, 2-Propenoic acid,3-phenyl- (cinnamic acid), Benzenepropanoic acid (Hydrocinnamic acid), Benzenpropanoic acid methyl ester, 2-propenoic acid, 3-phenyl-, methyl ester, 3,4-dimethoxycinnamic acid, 3-hidroxy-4-methoxycinnamic acid, Benzyl benzoate were observed in minor ratios. Discussion Among the determined compounds, the flavonoids were observed in quite higher amounts. In the most of the samples, chrysin a kind of flavonoid was determined. It has anti-tumor (Hladon et al., 1980), anti- Helicobacter pylori (Itoh et al., 1994), anti-inflammatory (Shin et al., 2009), anti-oxidant, antiviral, antidiyabetogenic, anti-axyoletic (Zheng et al., 2003) effects. So we can say that the 86 samples from Tekirdağ region, contain chrysin, may have valuable biological effects for further studies. Another kind of flavonoid; pinocembrin was determined in 86 Tekirdağ samples. This compound has bacterioteriostatic (Amoros et al., 1992), antimould (Miyakado, 1976), antimicrobial, anti-micotic (Metzner et al., 1979), anti-oxidant, anti-inflammatory (Gao et al.,2008) and local anesthesic effects. Also its anti microbial effect to Alternalis fungus was determined (Miyakado et al., 1976). Galangin is an other flavonoid found in 53 Tekirdağ samples. It has bacteriostatic (Amoros et al., 1992; Pepeljnjak, 1982), antimicrobial, anti-micotic (Metzner et al., 1979), anti-helicobacter pylori (Itoh et al., 1994) activities.

30 MELLIFERA 30 Comparing with previous studies in flavonoid contents base, similiar to our results galangin (in Bursa, Muğla, İzmir, Ankara samples) (Velikova et al., 2000), chrysin; (in Bursa, İzmir, Ankara, Elazığ and Erzincan, Erzurum samples) (Velikova et al., 2000; Kılıç et al. 2005; Silici and Kutluca 2005; Seven et al. 2005), pinocembrin (in Bursa, İzmir, Muğla, Ankara samples), 4,5-Dihidroksi-7-methoxyflavanon (in Ankara and Erzincan samples) (Kılıç et al., 2005), pinostrobin chalcone ( in Erzincan sample) (Gençay, 2004), acacetin (in Erzurum sample) (Silici ve Kutluca 2005) had been observed by other researchers. Benzaldehyde, 3-hydroxy-4-methoxy was found in Ankara and Erzincan samples by Kılıç et al. is similiar to our results (2005). According to the GC-MS results, octadecanoic acid from the frequently observed compounds belong to the aliphatic acids and their esters were found in Ankara and Bursa samples in previous studies. From identified aliphatic acids and esters in Tekirdağ samples, ethyl oleate was found in Ankara, Kemaliye-Erzincan samples by Kılıç et al. (2005), hexadecanoic acid in Ankara, Bursa, İzmir samples by Velikova et al (2000). Benzoic acid, the mostly observed compound from carboxylic acid and esters group in Tekirdağ samples, was found in the samples collected from Ankara, Bursa, İzmir, Muğla (Velikova et al., 2000), and Zonguldak samples (Girgin et al., 2009) by other researchers. This compound was identified in 77 Tekirdağ samples in our study and its bacteriostatic, antiseptic properties were showed by former researchs. Cinnamic acid, that is important for biological activities of propolis, was observed frequently (in 35 Tekirdağ samples) but in minor amounts in the investigated samples. This compound also has been observed in Ankara, İzmir, Muğla samples by Velikova et al.(2000). The determined amounts of flavonoid, hydrocarbon, carboxylic acids and their esters were compared by ANOVA-Duncan analysis in town base ( Table 2-4). According to the results of Anova-Duncan analysis, the amount of flavonoid in the propolis samples belong to the eight towns are very similiar and divided only two groups. As seen in table 2 values are very similiar to each other. Malkara samples contain the highest flavonoid ratios, Çerkezköy samples contain lower flavonoid content compare to the other towns. It is noticed from table 3 that, there is a big difference between Merkez and Şarköy samples according to the hydrocarbon contents. According to the results there is a big difference in the amount of carboxylic acids and esters between Şarköy and Saray samples (Table 4). As a result Tekirdağ propolis samples are valuable for further studies. All the samples are rich in flavonoid content and especially Malkara samples has the highest flavonoid content that can give antimicrobial, antioxidant, etc. activities to propolis samples. Also the samples may have anti viral, anti micotic, anti inflammatory, anti Staphylococcus aureus activities owing to the cinnamic acids and esters contents. Acknowledgement This research is supported by Hacettepe University Scientific Research and Development Office(Project Number: ).

31 31 Table 1. The number of collected propolis samples and collecting areas Number Towns The number of registered beehives (Nh) The number of samples that must be collect (nh) The number of collected samples 1 Çerkezköy Çorlu Ereğli Hayrabolu Malkara Merkez Muratlı Saray Şarköy TOTAL 9 TOWNS 497 BEEHIVES Table 2. The statistical comparing of eight towns of Tekirdağ city according to the Flavonoids Table 2. The statistical comparing of eight towns of Tekirdağ city according to the Flavonoids Table 2. amount The statistical comparing of eight towns of Tekirdağ city according to the Flavonoids amount amount *1; Çerkezköy samples, *1; *1; Çerkezköy 2; Çorlu samples, 2; 2; Çorlu 3; Hayrabolu, samples, 3; Hayrabolu, 4; Malkara, 4; Malkara, 5; 5; 5; Merkez, 6; 6; Muratlı, 6; Muratl, 7; Saray, 7; Saray, 8; Şarköy 8; 8; Şarköy Table Table The The statistical comparing of eight towns of of Tekirdağ city city according according to to the the Table 3. hydrocarbons hydrocarbons The statistical amount amount comparing of eight towns of Tekirdağ city according to the hydrocarbons amount *1; Çerkezköy samples, *1; Çerkezköy 2; Çorlu samples, samples, 2; Çorlu 3; Hayrabolu, samples, 3; Hayrabolu, 4; Malkara, 4; Malkara, 5; Merkez, 5; Merkez, 6; 6; Muratlı, Muratl, 7; 7; Saray, Saray, 8; Şarköy 8; Şarköy *1; Çerkezköy samples, 2; Çorlu samples, 3; Hayrabolu, 4; Malkara, 5; Merkez, 6; Muratl, 7; Saray, 8; Şarköy

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