Acta Botanica 1-2017 - za web.indd ACTA BOT. CROAT. 76 (1), 2017 1 Acta Bot. Croat. 76 (1), 1–8, 2017 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0039 ISSN 0365-0588 eISSN 1847-8476 Pollen morphology and the fl ower visitors of Chaerophyllum coloratum L. (Apiaceae) Marina Mačukanović-Jocić1, Danijela Stešević2, Dragana Rančić1*, Zora Dajić Stevanović1 1 Faculty of Agriculture University of Belgrade, Nemanjina 6, 11080 Zemun, Belgrade, Serbia 2 Faculty of Natural Sciences and Mathematics, University of Montenegro, Džordža Vašingtona put bb, 81000, Montenegro Abstract – The pollen features of Chaerophyllum coloratum L., endemic to the Dinaric Alps, have been ex- amined by both light microscopy and scanning electron microscopy in order to contribute to a better under- standing of the taxonomic status of the species. Flower visitors have also been observed and analyzed with the aim of clarifying certain pollination aspects of the species including fl ower attractiveness especially to honeybees, and also in order to ascertain its contribution to the bee pasture. The pollen grains of C. coloratum are isopolar, radially symmetrical and medium sized. Polar axis (P) is 26.83±1.77 μm length, and equatorial diameter (E) is 9.17±0.57 μm length. P/E ratio amounts 2.90±0.10 indicating a perprolate shape. In an equa- torial view, the grains are constricted in the equatorial region (bone-shaped), with obtuse polar caps. In polar view, they are triangular with obtuse angles and furrows in the sides of the triangle (interangular). The grains are tricolporate with three straight ectocolpi arranged regularly meridionally, of mean length 14.43±2.17 μm, each of which has one endopore. The characteristic internal thickenings around the protruding, clearly visible endopores (costae) in the constricted equatorial region are obvious in light microscopy. The ornamentation is psilate, irregularly rugulate (“cerebroid”), the exine surface is rather undulating. With regard to the observed fl ower visitors, the following pollination types occurred: melittophily, myophily, sapromyophily, cantharoph- ily, and phalaenophily, and the most frequent pollinator was the honeybee. Keywords: fl ower visitors, light microscopy, scanning electron microscopy, palynomorphology * Corresponding author, e-mail: rancicd@agrif.bg.ac.rs Introduction According to The Plant List (2010) the family Apiaceae belongs to the major group of Angiosperms, containing 347 genera and more than 3000 species and subspecies distri- buted worldwide (Tutin 1968). The genus Chaerophyllum L. (Apiaceae), the largest one in the subtribe Scandicinae belong ing to the tribe Scandiceae, comprises about 45 spe- cies, native to Eurasia, North Africa and North America (Pime nov and Leonov 2004, Yilmaz and Tekin 2013). As opposed to other Apiaceae genera such as Anethum, Ange- lica, Carum, Coriandrum, Daucus, Foeniculum, Levisti- cum, Peucedanum, Petroselinum, Pimpinella etc., species of which are widely used in the Dinaric or Balkan countries as spice, medicinal or culinary plants (Tucakov 1996, Pelagić 2001), the use of Chaerophyllum coloratum has not been reported. But there are several species of this genus growing elsewhere that are recognized as either medicinal or food plants: in Turkey C. bulbosum L. (Polat et al. 2013) and C. libanoticum Boiss. et Kotschy (Demirci et al. 2007), in Mongolia C. gracile Freyn. Sint. (WHO 2013), in India – western Himalaya C. villosum Wall ex DC. (Singh 2012), etc. The latest studies of Chaerophyllum species included their anatomy and morphology (Kowal et al. 1971, Kowal and Latowski 1973, Yilmaz and Tekin 2013, Reuther and Claßen-Bockhoff 2013), and the composition of essential oils and biological effects of physiologically active com- pounds (Gonnet 1985, Pedro et al. 1999, Baser et al. 2000, Dall’Acqua and Innocenti 2004, Nematollahi et al. 2005, Başer et al. 2006, Kürkçüoğlu et al. 2006, Kapetanos et al. 2008, Chizzola 2009, Lakušić et al. 2009, Razavi and Ne- jad-Ebrahimi 2010). A fair number of palynological studies of the family Apiaceae date back to the mid-1950s. The fi rst reports re- garding the pollen morphology of some species were pub- lished by Erdtman (1952). Many Apiaceae species, origi- nating from different regions or countries of almost all the continents, have already been palynomorphologically de- scribed. Thus, with a monograph about the pollen morpho- MAČUKANOVIĆ-JOCIĆ M., STEŠEVIĆ D., RANČIĆ D., DAJIĆ STEVANOVIĆ Z. 2 ACTA BOT. CROAT. 76 (1), 2017 logy of Angiosperms, Cerceau-Larrival (1959) expanded the knowledge of the pollen features of Apiaceae of France and North Africa. From the same point of view, Ting (1961) described some American species, Cerceau-Larrival (1962a, b, 1963, 1965, 1981) Mediterranean species, Nilsson et al. (1977) North European species, Van Zeist et al. (1977) Asian species, Punt (1984) Northwest European species, Chester and Raine (2001) South European and North Afri- can species. In most cases, the researchers investigated spe- cies indigenous to a local area, region or country. For in- stance, Perveen and Qaiser (2006) studied species from Pakistan, Yousefzadi et al. (2006) and Amjad and Akkafi (2012) species from Iran, Pehlivan et al. (2009), Mungan et al. (2011), Dogan Guner et al. (2011) and Yilmas and Tekin (2013) those from Turkey and De Leonardis et al. (2008, 2009) species from Italy. However, only a few palynologi- cal studies concerning the genus Chaerophyllum, including the following 7 species have been carried out: C. astrantiae Boiss. & Balansa, C. aureum L., C. bulbosum L., C hirsu- tum L., C. refl exum Lindl., C. villosum Wall. ex DC. and C. temu lentum L. (Punt 1984, Chester and Raine 2001, Per- veen and Qaiser 2006, Yilmaz and Tekin 2013). According to the available literature data, there is only one published paper about C. coloratum, concerning the composition of essential oils of ripe fruits and umbels (Vajs et al. 1995). However, the pollen features of this species have previously been quite unknown. Pollination biology of the Apiaceae related to fl ower preferences of insects has also received little attention, and has been studied mostly in economic plants and only a few wild species: Heracleum sphondylium, Seseli farrenyi, Thaspium sp, Zizia sp. and Daucus carota (Bell and Lindsay 1978, Lindsey 1984, Lindsey and Bell 1985, Lamborn and Ollerton 2000, Lan- genberger and Davis 2002a, b, Rovira et al. 2004, Zych 2006). The detailed description of the pollen characteristics of this taxon in the present study contributes to taxonomic and melissopalynological research as well as to the pollen atlas of the region. By providing evidence about the fl ower visi- tors, the current study aims to examine certain pollination aspects of the species including fl ower attractiveness, espe- cially to honeybees, and also to establish its contribution as a melliferous plant to bee pastures. Material and methods Distribution of the study species and fl ower morphology related to pollinator attraction Chaerophyllum coloratum L. is an endemic plant, geo- graphically restricted to the Dinaric Alps (Croatia, Bosnia and Herzegovina, Montenegro, Kosovo, Albania), and grow- ing in sunny and dry habitats, amongst shrubs, on rocky grassland and similar Mediterranean-climate terrain (Šilić 1990). The plant is protected by the National Law of Mon- tenegro (Offi cial Gazette of RM, NO. 76/06), and is classi- fi ed as a species of international importance (Ste va nović et al. 1995). Based on the available literature data (Rohlena 1942, Šilić 1990, Bulić 1994, Karaman 1997, Tomović 2007, Lubarda 2013), relevant herbarium collections (Her- barium of the University of Montenegro – TGU, Herbarium of Tirana University – TIR, Herbarium of the Faculty of Forestry, University in Banja Luka), the Flora Croatica da- tabase (Nikolić 2014), and personal fi eld observations, a distribution map of the species has been prepared and is presented in Fig. 1. As a basic layer, the UTM grid map of the Balkans was used. C. coloratum L. is an annual to biennial aromatic herba- ceous plant, up to 1 m tall (Fig. 2). The species has a hol- low, furrowed, sparsely hairy, ribbed (branched in the upper part) stem, distinctive sheathing leaves pinnately divided 3 times into linear leafl ets, and intensively yellow fl owers ar- Fig. 1. Distribution map of Chaerophyllum coloratum, marked with dots. Fig. 2. Chaerophyllum coloratum, the general habitus of the study species. PALYNOMORPHOLOGY AND POLLINATORS OF C. COLORATUM ACTA BOT. CROAT. 76 (1), 2017 3 ranged in compound umbels. Tiny fl owers recognizable by typically infl exed petal apex, are produced in mid-spring, followed by linear, aromatic non-fl eshy fruits. Involucral leaves are absent, while leaves of involucelum are perma- nent and linear. Flowers in the outer and central part of the umbellet are perfect (hermaphrodite) and open fi rst, where- as the inner fl orets are functionally male and open next. The male stage is defi ned as the pollen-presenting phase and the female stage as the phase with receptive stigmas. In the post-fl oral stage, the ovaries of all hermaphrodite fl owers elongate to a certain degree irrespective of their fertilization status. Thus it is easy to distinguish even unfertilized her- maphrodite fl owers from functionally male fl owers, which may have rudimentary styles and ovaries, but never elon- gate after anthesis. The proportion of male fl owers varies between branch orders. In some cases fruit set in the termi- nal umbel is nearly 100% and it decreases towards the low- er branches (Reuther and Claßen-Bockhoff 2013). Periph- eral fl owers are usually slightly zygomorphic, while inner ones are actinomorphic. The stamens alternate to the petals, and arising from an epigynous nectary disk. Nectar is pro- duced in both fl ower phases. Sampling and analysis of pollen The plant material was collected in Montenegro (territory of Podgorica Municipality, 4222'46.08”N, 1913'20.8”E) in May 2013. The fl owers (anthers) were collected at full fl owering stage from 10 plants of wild populations. The voucher specimen (No 504520) was deposited in the her- barium collection of the Faculty of Natural Sciences and Mathematics, University of Montenegro TGU. The pollen morphology was examined by both light mi- croscopy (LM) and scanning electron microscopy (SEM). For light microscopy, the pollen grains, prepared according to the standard acetolysis method (Erdtman 1952), were mounted in glycerine jelly and observed with a Leica DMSL microscope equipped with a digital camera (Leica DC 300) and Leica IM1000 software. For SEM study, the pollen grains were covered with gold (in BAL-TEC SCD 005 Sputter Coater, 100 seconds in 30 mA) and observed using JEOL JSM- 6390 LV electron microscope at an ac- celeration voltage of 20 kV. Pollen grains were photo- graphed in polar and equatorial views, and observations and measurements were done on a sample of 50 or more grains for each morphological character. The following features describing pollen grains were examined: size, shape, ornamentation, apertures, polarity, symmetry, length of polar (P) and equatorial axis (E) (in SEM) and exine thickness (in LM). The terminology used to describe the pollen grains is based on Erdtman (1971) and Punt et al. (2007). Flower visitors With the intention of detecting the fl ower visitors of C. coloratum, plants were observed at fi ve sites at the territory of Podgorica Municipality (Zagorič 4227'35”N, 1915'40”E, Gorica 4227'00”N, 1916'49”E, Srpska gora 4222'46”N, 1913'19”E, Tuzi 4220'47”N, 1920'43”E, Bioče 4232'14”N, 1929'03”E), 3 times during anthesis, from 3rd to 20th May, during the daytime. Flower visitors were shot with digital camera Nikon Colorpix P500 and caught with an entomo- logical net and entomological exhauster for further identifi - cation. Results Pollen morphology The pollen grains of C. coloratum are isopolar, radially symmetrical and medium sized (Figs. 3, 4). The mean length of the polar axis (P) is 26.83±1.77 μm, and the mean of the equatorial diameter (E) is 9.17±0.57 μm. The ratio of the length of the polar axis to the equatorial diameter (P/E) averaged 2.90±0.10 making the pollen grain shape perpro- late. When observed in polar view, the grains are triangular with obtuse angles (Figs. 3B, 4B), and furrows in the sides of the triangle (interangular), in equatorial section fossaper- turate. In an equatorial view, the grains are bone-shaped, having equatorial constriction and obtuse polar caps (Figs. 3A and 4A). The grains are tricolporate, with three straight colpi, each of which has one endopore positioned in the indenta- tions between the mesocolpial lobes. Ectocolpi of mean length 14.43±2.17 μm are meridionally arranged, indis- tinctly tapering towards to the poles. They are narrow, slit- like, sunken and rather short ca 1/2 polar distance. In the side view, the inner and the outer contours of both mesocol- pial and colpial side are slightly concave, as can be seen in Fig. 3. Scanning electron micrographs of perprolate pollen grains of Chaerophyllum coloratum. In equatorial view, the pollen grains are constricted in the equatorial region (bone-shaped), with short straight furrows (colpial side – A; mesocolpial side – B). In a polar view (B), the grains are triangular, with the furrows in the sides of the triangle (interangular). A detail of undulating exine surface showing psilate, irregularly rugulate (“cerebroid”) ornamentation (C). Fig. 4. Light microscopy micrographs of Chaerophyllum colora- tum pollen grains having protruding clearly visible pores in the constricted equatorial region: equatorial view of colpial side (A) and mesocolpial side (B). In polar view (B), the grains have trian- gular outline. MAČUKANOVIĆ-JOCIĆ M., STEŠEVIĆ D., RANČIĆ D., DAJIĆ STEVANOVIĆ Z. 4 ACTA BOT. CROAT. 76 (1), 2017 light microscopy (Fig. 4A). The characteristic internal thickenings surrounding the protruding clearly visible en- dopores (costae) in the constricted equatorial region are ob- vious in light microscopy (Fig. 4B). The sculpturing pattern, clearly visible in SEM, is psi- late, irregularly rugulate (“cerebroid”) (Fig. 3C). The exine surface is rather undulating and exine thickness averaged 0.95±0.15 μm. Flower visitors Flower visitors observed on this plant species could be classifi ed as primary and secondary pollinators or acciden- tal visitors (Figs. 5–7) belonging to all four insect orders: Hymenoptera (bees, wasps, and ants), Diptera (true fl ies), Lepidoptera (moths) and Coleoptera (beetles). Regarding the observed fl ower visitors, the following pollination types occurred: melittophily including pollination by honeybees (Fig. 5A), myophily (Figs. 5B, C, D) and sapromyophily including specialized and non-specialized fl y pollinators (Fig. 5E), phalaenophily (moth pollination) (Fig. 5F) and cantharophily (beetle pollination) (Figs. 6A, B, C, D). Snails (Fig. 7A), ants (Figs. 7B, C) and spiders (Fig. 7D) were also observed but they are not associated with the pollination process. The fl ower visitors of C. coloratum were hymenopter- ans (Apis mellifera, Fig. 5A; and ants: Camponotus vagus, C. aethiops, Fig. 7B, and Crematogaster sordidula, Fig. 7C), dipterans (mostly true fl ies: Calliphoridae – Lucilia sp., Fig. 5E; Syrphidae – Sphaerophoria sp. and Tabanidae – Tabanus sp., Dasyrhamphis sp., Fig. 5B) coleopterans (mostly Mordellidae – Mordella brachyura, Fig. 6D; Can- tharidae – Cantharis sp., Fig. 6B; and Cetoniidae – Cetonia aurata, Fig. 6C, and Oxytrea funesta Fig.6A), and the least frequent were butterfl ies – moths (Fig. 5F) and neuropter- ans. The majority of the visitors preferred the staminate phase of umbels, ants preferred the female phase, while co- leopterans and bees visited fl owers during both phases. Usually, infl orescences were routinely visited by certain in- sects individually, but in some cases, such is Mordella brachyura, many individuals were observed searching for nectar on a single infl orescence at the same time. Discussion Pollen grains of Apiaceae species are mostly stenopaly- nous, radially symmetrical, isopolar and prolate to perpro- late in shape (Yousefzadi et al. 2006, Erdtman 1952). With respect to aperturation, they are generally tricolporate pos- sessing three slit-like ectocolpi, each of which has one pore, and very distinctive and broad band-like costae (Punt 1984, Perveen and Qaiser 2006). As pointed out elsewhere, the tectum is commonly striate-rugulate or simple striate (Punt Fig. 5. The fl ower visitors of Chaerophyllum coloratum: A – Api- dae (Apis mellifera); B – Syrphidae (Sphaerophoria sp.); C, D – Tabanidae (C – Tabanus sp., D – Dasyrhamphis sp.); E – Calli- phoridae (Lucilia sp.); F – Lepidioptera (Tortricidae). Fig. 6. Coleopteran pollinators of Chaerophyllum coloratum: Ce- toniidae (A – Oxythyrea funesta, C – Cetonia aurata); B – Can- tharidae (Cantharis sp.), D – Mordellidae (Mordella brachyura). Fig. 7. Accidental visitors of Chaerophyllum coloratum: Gastrop- oda (A); Formicidae: Camponotus vagus (B) and Crematogaster sordidula (C); Aranea (D). PALYNOMORPHOLOGY AND POLLINATORS OF C. COLORATUM ACTA BOT. CROAT. 76 (1), 2017 5 1984) or, as stated by Perveen and Qaiser (2006), psilate to sparsely or densely granulate. There are different criteria for classifi cation of the pol- len grains of Apiaceae, such as P/E ratio, exine sculpturing pattern, etc. For instance, Cerceau-Larrival (1962) pro- posed 5 pollen types occurring within the family based on shape index: subrhomboidal (type 1, P/E: 1–1.5), subcircu- lar (type 2, P/E: 1–1.5), oval (type 3, P/E: 1.5–2), subrect- angular (type 4, P/E: 2), and equatorially constricted (type 5, P/E: over 2). According to this classifi cation, pollen grains of C. coloratum fi t into type 5. Perveen and Qaiser (2006) investigated the pollen of 50 species representing 27 genera of Pakistani Apiaceae and on the basis of tectum features distinguished three distinct pollen types: Bupleurum gilessii-type, Pleurospermum hookeri-type and Trachyspermum ammi-type. The authors analyzed two Chaerophyllum species – C. refl exum Lindl and C. villosum Wall. ex DC and classifi ed them into Bu- pleurum gilesii type characterized by a striate-rugulate tec- tum. Based on their results, pollen characters such as exine thickness, colpal membrane and the outline of a pollen grain seen in polar view (amb) are of little taxonomic value. The mentioned authors reported fossaperturate and perpro- late pollen grains in C. refl exum, but planaperturate and prolate grains in C. villosum. In view of the above, pollen grains of C. coloratum, described as psilate, irregularly ru- gulate (“cerebroid”) and fossaperturate, are more like those of C. refl exum. Punt (1984), who thoroughly studied the palynomor- phology of the North European Apiaceae, recognized 50 pollen types. The author pointed out that ornamentation features are of little value in pollen identifi cation among the family members. By analyzing exine structure, this author classifi ed Chaerophyllum pollen into 3 types: C. hirsutum type (including C. hirsutum and C. temulentum), C. bulbo- sum type (C. bulbosum) and C. aureum type (C. aureum), all three having a psilate, irregularly rugulate tectum. The fi rst type includes grains with distinct colpi 1/2 to 2/3 of polar distance, columellae shorter at the equator than at the poles, and the tectum smooth or very slightly undulating in the equatorial area, “cerebroid” in SEM. The second type, involving small grains of mean polar axis length of 28 μm, is characterized by indistinct and very small columellae in the mesocolpial region distincly increasing in size towards the poles, and a thin tectum distinctly undulating in the equatorial region, also “cerebroid” in SEM. In the third type, the endoaperture is a short colpus, columellae short but distinct at poles increasing in length and decreasing in width towards shoulders and equator, and tectum slightly undulating in equatorial area. The results of the present study concerning the characteristics of the exine surface of C. coloratum, correspond to those of Punt (1984) who found a similar sculpturing pattern in the C. bulbosum and C. hirsutum types, but to some extent a different pattern in C. aureum, whose tectum has also been described as psi- late, irregularly rugulate, but not designated as “cerebroid”. The characteristics of the pollen grains of C. coloratum with respect to type, number and position of apertures, as well as colpi length, correspond to those of Chaerophyllum species as described by Punt (1984). The main palynomor- phological features that distinguish Chaerophyllum species from each other are size and the shape index (P/E). Pollen grains of C. coloratum are more similar in size to those of C. temulentum (average 28.8 μm), C. aureum (average 26.5 μm) and C. bulbosum (average 28 μm), than to those of C. hirsutum, which are larger, with an average size of 35.5 μm. In a polar view, C. coloratum grains are triangular, as in the three above-mentioned pollen types, whereas in an equato- rial view, inner and outer contour of both, colpial and meso- colpial sides, is concave without any sexine extension above the endoapertures, as has been observed in C. hirsu- tum pollen type. Notwithstanding the generally recogniz- able shape at the family level, the P/E index may be an im- portant interspecifi c distinguishing parameter. Hence, in C. temulentum, C. bulbosum, C. aureum and C. hirsutum this ratio ranges from 2.16 to 2.46, while in C. coloratum it is higher (2.90), since the pollen is more constricted at the equator. This pronounced equatorial constriction contribut- ing to the higher P/E ratio, causes a distinctive bone-shaped outline that makes C. coloratum pollen recognizable. This shape is pronounced to a lesser extent in the majority of other species within the genus, as opposed to C. hirsutum whose grains are sometimes more elliptical. Within the large diversity of fl ower visitors, due to the open fl owers with exposed nectar-secreting gland and sexu- al parts, the family Apiaceae has usually been regarded as non-specialized in terms of pollination biology (Zych 2006). According to Niemirski and Zych (2011), Umbelliferae are mostly visited by fl ies, but may also be pollinated by bee- tles, bees or other hymenopterans. Flowers are visited by a wide range of insects, but not all of them are equally effec- tive at transferring and depositing pollen. Bell and Lindsay (1978) reported that only four species of bumblebees (Bom- bus spp.), and some other hymenopterans, were seen polli- nating Angelica species. Niemirski and Zych (2011) ob- served 72 insects from 7 taxonomic orders visiting fl owers of the same species, of which only 30% had special pollen- carrying structures. Grace and Nelson (1981) indicated that out of 80 insect taxa that visited Heracleum spondylium and H. montegazzianum fl owers, only 31 carried a signifi cant pollen load. Zych (2007) reported that the most effi cient pollinators of H. spondylium were dipterans (blow fl ies Lu- cilia sp.; hoverfl ies Eriozona syrphoides, Eristalis sp., and Meliscaeva cinctella, and true fl ies Phaonia angelicae and Thricops nigrifrons), bumblebee (Bombus terrestris), and beetles Stenurella and Dasytes. The current study suggests that C. coloratum fl oral characteristics, such as colour, scent, the availability of pol- len and nectar, fl ower arrangement in fl at-topped compound umbel, etc., serve as attractants to diverse insect pollinators belonging to the orders of Hymenoptera, Diptera, Lepidop- tera and Coleoptera. These taxonomic groups differ inpolli- nation effi ciency, the coleopterans being known as the least specialized pollinators (Mačukanović-Jocić 2010) despite the number and abundance of beetle species observed in the present study and some previous investigation carried out on Apiaceae (Zych 2006). Ants, snails and spiders were also observed on Chaerophyllum fl owers but, since they are not associated with the pollination process primarily due to MAČUKANOVIĆ-JOCIĆ M., STEŠEVIĆ D., RANČIĆ D., DAJIĆ STEVANOVIĆ Z. 6 ACTA BOT. CROAT. 76 (1), 2017 References Amjad, L., Akkafi , H., 2012: Pollen structure of Kelussia odorati- sima (Umbelliferae) from Iran. International Journal of Scien- tifi c and Engineering Research 3, 401–406. Baser, K. H. C., Tabanca, N., Özek, T., Demirci, B., Duran, A., Duman, H., 2000: Composition of the essential oil of Chaero- phyllum aksekiense A. Duran et Duman, a recently described endemic from Turkey. Flavour and Fragrance Journal 14, 287–289. Başer, K. H. C., Özek, G., Özek, T., Duran, A., 2006: Composition of the essential oil of Chaerophyllum macropodum Boiss. fruits obtained by microdistillation, Journal of Essential Oil Research 18, 515–517. Bell, C. R., Lindsay, A. H., 1978: The umbel as a reproductive unit in the Apiaceae. In: Actes du 2eme Symposium International sur les Ombelliferes, Contributions pulidisciplinaires ala Sys- tematique. Perpignan, France, 739–747. Bulić, Z., 1994: Flora and vegetation of Cijevna river canyon in Montenegro – ecological and phytogeographical study. Mas- ter’s thesis. Faculty of Biology, University of Belgrade (in Serbian). Cerceau-Larrival, M. T., 1959: Clé de determination d’Ombel li- fères de France et d’Afrique du Nord d’après leurs grains de pollen. Pollen et spores 1, 145–190. Cerceau-Larrival, M. T., 1962a: Le pollen dombelliferes mediter- raneennes. Pollen et spores 4, 955–104. Cerceau-Larrival, M. T., 1962b: Plantules et pollens d’Ombelli- fères. Mémoires du Muséum National d’Histoire Naturelle. Serie B. Botanique 14, 1–166. Cerceau-Larrival, M.T., 1963: Le pollen d’Ombellifères Méditer- ranéennes, II. Tordylinae Drude. Pollen et spores 5, 297–323. Cerceau-Larrival, M. T., 1965: Le pollen d’Ombellifères Méditer- ranéennes, III. Scandicinae Drude. IV. Dauceae Drude. Pollen et spores 7, 35–62. Cerceau-Larrival, M. T., 1981: World Pollen and Spore Flora 9. UmbeUiferae Juss. Hydrocotyloideae Drude/Hydrocotyleae Drude. Almqvist and Wiksell, Stockholm, 1–33. Chester, P. I., Raine J. I., 2001: Pollen and spore keys for quater- nary deposits in the nortern Pindos Mountains, Greece, Grana 40, 299–387. Chizzola, R., 2009: Composition of the essential oil of Chaero- phyllum aromaticum (Apiaceae) growing wild in Austria, Nat- ural Product Communications 4, 1235–1238. their inability to fl y and ineptness at transferring pollen, they can be considered accidental visitors. In a similar study concerning the pollination of Heracleum sphondylium, Zych (2006) identifi ed Syrphidae and Calliphoridae as the most important pollinators. From the pollination point of view, C. coloratum, being an aromatic plant, is most ap- pealing to Apidae, followed by Syrphidae, and to a lesser extent to carrion fl ies. According to the present study, the most frequent visitor to Chaerophyllum fl owers was the honeybee, which was also registered as an important polli- nator of some other Apiaceae, such as Trachymene incise (Davila and Wardle 2002) and Carum carvi (Langenberger and Davis 2002b), but not in the case of Heracleum sphon- dylium (Zych 2006). Unlike Zych (2006) who noticed a few butterfl ies from the families Nymphalidae, Papilionidae and Pierideae visiting Heracleum fl owers, the present study mentions only moths with short mouthparts feeding on Chaerophyllum fl owers. The low visitation rate of butter- fl ies can be explained by their long proboscises, which are more suitable for nectar-sucking from deep fl oral tubes (Krenn et al. 2005). Due to the fact that the present study was not based on long-term monitoring of pollinators’ activity and pollen load analysis, questions addressing the diversity of fl ower visi- tors need to be answered. Another peculiarity of umbellifers is their dichogamy that leads to the presence of temporally unisexual fl owers. Such “temporal dioecism” may infl uence the behaviour of fl ower visitors (Zych 2007). In the case of Heracleum spondilum (Zych 2007), fl owers in the staminate phase were visited signifi cantly more often than those in the pistillate phase. This preference is especially noticeable in Dipterans. However, a study of fl y pollination of Angelica sylvestris indicated that these insects did not show any pref- erence regarding plant sexual phases (Niemirski and Zych 2011). Results of the current study on C. coloratum showed that a majority of the visitors preferred the staminate phase of umbels, ants preferred the female phase, while coleopter- ans and bees made visits during both phases. The current paper is a part of an extensive research pro- ject into apifl ora and its impact on the bee pasture of the select ed region. The provided palynomorphological infor- mation contributes towards a better understanding of the taxonomic status of C. coloratum. Additionally, the data obtained will be helpful in the forthcoming melissopalyno- logical analysis that will facilitate the identifi cation of the fl oral sources of different kinds of honey, and will enable the creation of the pollen atlas of the main taxa found in honey types produced in the Balkans. Qualitative analysis of pollen in honey originating from the research area would allow the determination of its botanical and geographical origin (assuming the entire pollen spectrum being consis- tent with the fl ora of a particular region). By determining the spectrum of pollen types in honey and calculating the relative frequency of C. coloratum as the respective per- centage with respect to the total number of pollen grains, it would be possible to confi rm or deny the attractiveness of this species to honeybees. Acknowledgements This research was fi nancially supported by the Ministry for Education, Science and Technological Development of the Republic of Serbia, Project No 46009 and TR31005 and EU Commission Project AREA, No 316004. The authors are grateful to Đorđije Milanović, for assistance in the prep- aration of distribution map of Chaerophyllum coloratum and providing data on the species distribution in Bosnia and Hercegovina, as well as Dr. Toni Nikolić, for providing data on the distribution of the species in Croatia, and also to Pro- fessors Alfred Mullaj and Alma Imeri, for providing distri- bution data in Albania. Thanks to Zoja Bećović for assis- tance in fi eld investigations. Sincere thanks should be extended to Prof. V. Pešić, Prof. S. Hrnčić, Dr. S. Malidžan, Dr. M. Karaman, Dr. A. Grill, MSc. B. Gligorović and Prof. P. Mazzei for their help in identifying insects. PALYNOMORPHOLOGY AND POLLINATORS OF C. COLORATUM ACTA BOT. 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