MELISSOPALYNOLOGICAL ANALYSIS OF MULTIFLORAL HONEYS FROM THE SANDOMIERSKA UPLAND AREA OF POLAND

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1 Journal of Apicultural Science 65 MELISSOPALYNOLOGICAL ANALYSIS OF MULTIFLORAL HONEYS FROM THE SANDOMIERSKA UPLAND AREA OF POLAND E r n e s t S t a w i a r z, A n n a W r ó b l e w s k a Department of Botany, University of Life Sciences in Lublin, Akademicka 15, Lublin, Poland ernest.stawiarz@up.lublin.pl, anna.wroblewska@up.lublin.pl Received 05 May 2010; Accepted 20 May 2010 S u m m a r y The object of the study were 73 samples of multifloral honeys collected in the years in the Sandomierska Upland area of Poland. The material was found to contain pollen grains of 103 taxa (75 nectariferous and 28 non-nectariferous ones) belonging to 52 botanical families. Amongst the nectariferous plants, the main nectar flow was provided by the Brassicaceae (including Brassica napus), Prunus, Trifolium repens, Anthriscus, Aesculus, Salix, Taraxacum and Phacelia. Pollen grains of the taxa mentioned were characterized by a high pollen frequency ( %). Among the non-nectariferous plants, the highest pollen frequency (89.0%) was reported for the Poaceae (others). In particular, honey sample analyses found from 16 to 41 taxa of pollen grains, including 12 to 34 of the nectariferous plants and from 2 to 13 of the non-nectariferous plants. The colour of the honeys discussed in the study varied from creamy, through amber-like to dark brown. Keywords: multifloral honeys, nectariferous plants, pollen analysis, the Sandomierska Upland, Poland. INTRODUCTION Honey, apart from a variety of stable elements, contains grains of floral pollen that are collected by honeybees along with the nectar. The presence of the floral pollen grains affords the possibility of identifying the botanical and geographical origin of honeys. Those grains also help to determine honey variety. In Poland, microscopic analyses of honeys have been carried out since the 1950s. They were initiated by Demianowicz and Demianowicz (1955) and by Demianowicz (1961, 1964) and are currently conducted in a few research centres in Poland. Apart from a few types of specific honeys, the most frequently produced honeys in the Polish environmental surroundings also include multifloral honeys. The Sandomierska Upland is one of the largest regions of pomicultural and horticultural crops in Poland. This area is additionally abundant in numerous natural plant communities that have not been subject to chemical treatments. This is the reason that this area may constitute a valuable, ecologically-clean source of feed material for honeybees and other pollinating insects (Czarnecki, 1996; Myjak, 2000; Kęsik and Warzecha, 2000; Kęsik, 2003). The objective of this study was to evaluate the pollen composition of multifloral honeys and to identify the nectariferous flora which constitutes a source of nectar flow to honeybees in the investigated area. The knowledge of resources of the nectariferous flora is of great practical significance. Their size and appropriate utilization affects the proper development and functioning of a bee colony as well as the production yield of honey (Ostrowska, 1998). The quantity of nectar flow provided to insects is influenced by the plant species, its nectar yield and climatic conditions. Even plants

2 66 of the same taxon growing under different conditions may deliver variable quantities of feeding material (Zimmerman, 1988). MATERIAL AND METHODS Area and material of the study The experimental material were 73 samples of multifloral honeys collected in the years on the area of the Sandomierska Upland (southeastern Poland) (Fig. 1). The number of samples collected in the consecutive nectar flow seasons were: 30, 22 and 21. They originated from 14 districts located in the countryside of - Opatów (50 48' 18'' N, 21 25' 29'' E) and Sandomierz (50 41' N, 21 45' E). The number of samples from particular districts was varied and depended on the number of apiaries in a given area. The colors of the honeys collected were determined using a color key by Maerz and Paul (1950). Pollen analysis of honeys A microscopic pollen analysis of the honey samples was performed following the guidelines of the International Commission of Bee Botany (Louveaux et al., 1978) and the International Honey Commission (von der Ohe et al., 2004). Glycerol-gelatin preparations were made in duplicate for each honey. A pollen spectrum was evaluated by means of a Nikon Eclipse E 600 light microscope at 40x15 magnification. Fig. 1. Honey sample apiary locations

3 Journal of Apicultural Science 67 According to the recommendations of Moar (1985), at least 300 pollen grains were counted in each preparation. They were identified according to Zander s classification (1935, 1937) to the most possible exact taxon - species, genus, type of structure or family. Pollen grains within a given family whose genus or species were not determined, were referred to as others, e.g. amongst the Brassicaceae were distinguished pollen grains of Brassica napus, Sinapis alba, Matthiola and as well as amongst the Poaceae - Zea mays, Cerealia and Poaceae (others). The term type introduced after Zander (1935), e.g. Anthriscus type, Achillea type, Heracleum type, Taraxacum type, indicates all species or genera of plants represented by the same type of morphological structure of a pollen grain. In the microscopic pollen analysis, use was made of reference preparations and available keys (Zander, 1935, 1937; Hodges, 1952; Sawyer, 1981, 1988; Ricciardelli d Albore, 1998; Bucher et al., 2004). In the evaluation of the botanical origin of the honeys, pollen grains of nectariferous plants were separated in each sample. The following classification of pollen contribution was applied in the study: >45% dominant, 16-45% secondary, 3-15% important minor and <3% minor pollen. Records of the presence of pollen grains of non-nectariferous plants - entomophilous and anemophilous ones, were also kept. Based on the pollen spectrum of the honeys, groups of plants were distinguished which constituted a source of nectar flow to insects, i.e.: meadows and grazing lands, forests and scrubs, weeds, orchards and gardens as well as crops. RESULTS The honeys analyzed in the study were found to contain pollen grains of 103 taxa, including 75 from nectariferous plants and 28 from non-nectariferous plants. The list of more important taxa of the pollen of nectariferous plants is depicted in Fig. 2, whereas that of the non-nectariferous plants is given in Fig. 3. The identified pollen originated from plants that belonged to 52 botanical families, including 47 dicotyledoneous ones, 3 monocotyledoneous ones, and 2 gymnospermous ones. The most frequently represented families were Fabaceae and Rosaceae, with 11 taxa of pollen grains identified, as well as Asteraceae with 10 taxa. In the particular samples there occurred from 16 to 41 taxa total, including from 12 to 32 pollen grains of neactariferous taxa and from 2 to 13 of the non-nectariferous ones. The highest pollen frequency (100%) was noted for pollen grains of the and Prunus type. In turn, a pollen frequency of over 80% was observed in the case of Trifolium repens s.l., Rubus type, Anthriscus type and Aesculus. Pollen frequency of 50-80% - was noted in the case of Brassica napus, Salix, Taraxacum type, Tilia, Centaurea cyanus and Phacelia. A pollen frequency of over 20 to 30% was noted for 11 taxa. A pollen frequency of 30-50% was noted for 8 taxa, a frequency of 10-20% for 19 taxa, and a frequency of % for 23 taxa (Fig. 2). In contrast, the lowest pollen frequency (1.37%) was determined for pollen grains of: Boraginaceae (others), Echium, Euonymus, Fabaceae (others), Jasione, Ligustrum, Lysimachia, Potentilla, Rosaceae (others), and Spiraea. In the group of honeys discussed, the contribution of pollen from Brassicaceae (others) was found dominant in one sample (59.6%) and secondary in 5 samples ( %). Worth noticing are also other multifloral honeys with a high contribution of pollen of one taxon, including: 3 honeys from Robinia pseudacacia (27.7%, 27.8% and 28.9% of pollen in a sample), 2 honeys from Brassica napus (40.9% and 43.8%), 2 honeys from Galeopsis (44.5% and 40.4%), 2 honeys from Phacelia (42.1% and 42.4%) and one honey from both Salix (44.1%) and Taraxacum type (40.2%). In turn, two other samples of multifloral honeys were characterized by a high

4 68 Fig. 2. Pollen frequency of the most important nectariferous plants and its contribution in the microscopic view of multifloral honeys (%)

5 Journal of Apicultural Science 69 Table 1 Pollen contribution of major flow plants, and colour of some multifloral honeys from the Sandomierska Upland area of Poland ( ) Sample No Pollen percentage of plants exclusively nectariferous dominant >45% secondary 16-45% Taraxacum type Trifolium pratense Phacelia Robinia pseudacacia Phacelia Salix non-nectariferous in the total sample Honey colour** (Maerz & Paul Plate colour No.) 42.2* 12/E /E /J /F * 11/G Brassica napus Salix /E /E-3 24 Galeopsis /F * 11/E /C Prunus type Brassica napus Prunus type Brassica napus Aesculus Robinia pseudacacia Prunus type Salix Phacelia Verbascum Brassica napus Prunus type Robinia pseudacacia Brassica napus Galeopsis Phacelia Galeopsis /I /H /K /C /E /H /G /H /F-3 * probably with the addition of pollen bread **Color plates 9 and 10 cover light shades (from creamy to amber-like), whereas tables 6, 11 and 12 cover darker colors (from amber-like to dark brown)]

6 70 Fig. 3. Pollen frequency of non-nectariferous plants in multifloral honeys (%) percentage of pollen of the Prunus type (36.7% and 39.6%) (Tab. 1). Among the non-nectariferous plants, the highest pollen frequency (89.0%) was determined for Poaceae (others). A pollen frequency over the range of 50-70% was shown for pollen of Quercus, Rumex, Plantago and Filipendula. A pollen frequency over the range of 20-50% was found for 10 taxa, i.e.: Fragaria, Papaver, Betula, Anemone, Ranunculus, Artemisia, Zea mays, Chenopodiaceae, Cyperaceae and Pinus. The other 12 taxa reached pollen frequencies below 10% (Fig. 3). A fragment of the microscopic picture of the selected multifloral honeys is presented in Fig. 4. The results obtained indicate that in the area of the Sandomierska Upland the major source of flow were plants of forests and scrubs (30.7%) as well as those of meadows and grazing lands (28.4%). The additional feeding base of the discussed area were orchards and gardens (17.0%), weeds (16.4%) and crops (7.5%) (Fig. 5). The honeys analyzed in the study were characterized by high colour diversity - from creamy through amber-like to almost dark brown. This was due to various contributions of nectar flow originating from particular taxa of plants. The lightest honeys were those with a high contribution of flow from Brassica napus and other Brassicaceae, whereas the darkest ones were those with a contribution of Phacelia and Trifolium pratense (Tab. 1). DISCUSSION The results of the microscopic analysis of the multifloral honeys from the area of the Sandomierska Upland demonstrated the abundant and diversified pollen composition of the samples examined. Pollen grains of 103 taxa (including 75 nectariferous and 28 non-nectariferous) identified in the honey samples point to the high biodiversity of the area under scrutiny. The highest frequency ( %) and contribution (16-45%) was reported for the pollen of Brassicaceae (including Brassica napus), Prunus type, Trifolium repens s.l., Anthriscus type, Aesculus,

7 Journal of Apicultural Science 71 Fig. 4. A part of a microscopic view of some honeys Ach - Achillea type, Ae - Aesculus, An - Anthriscus type, B. nap - Brassica napus, Br - Brassicaceae (others), Car - Caryophyllaceae, Cent - Centaurea cyanus, Cy - Cyperaceae, Ga - Galeopsis, Maj - Majorana type, Sa - Salix, Ti - Tilia, Vi - Vicia Salix, Taraxacum type and Phacelia. Their presence in the honeys was noted in all experimental years, which confirmed the attractiveness of their nectar to honeybees. Attention should also be paid to taxa characterized by a lower frequency but a high contribution of pollen (over 40%), including: Heracleum type, Achillea type, Trifolium pratense, Solidago type, Robinia pseudacacia, Galeopsis, Po - Poaceae (others), Pr - Prunus type, Ra - Ranunculus, Ru - Rubus type, Fagopyrum, Verbascum, Myosotis and Sinapis alba. The presence of pollen of those taxa was in Polish honeys and those of other European countries, as demonstrated by various authors. Out of 25 multifloral honeys analyzed in the region of Lubelszczyzna (southeastern Poland) by Warakomska (1997), 100% frequency was noted for pollen grains of Brassica napus, Centaurea cyanus and Trifolium

8 72 Fig. 5. Contribution of pollen of various groups of plants in the honeys examined (%) repens s.l.. A high pollen frequency (88-96%) was additionally determined for Sinapis, Malus type, Melilotus, Salix and Trifolium repens s.l.. The Brassicaceae were also a valuable source of nectar flow in east central and northeastern Poland (Wróblewska, 2002; Wróblewska et al., 2006). In the multifloral honeys examined by the above-mentioned authors, a relatively high contribution (15-45%) was reported for pollen grains of Brassica napus, Salix, Centaurea cyanus, Trifolium repens s.l., Rubus type, Achillea type, Phacelia, Trifolium pratense, Anthriscus type, Galeopsis, and Malus type. The attractiveness of some taxa identified in the honeys from the Sandomierska Upland was also demonstrated by melissopalynological analyses conducted outside Poland. A high frequency and contribution of pollen of various Trifolium species was shown in the case of multifloral honeys originating from Italy (Floris et al., 2007) and from southern Croatia (Primorac et al., 2008). In the northeastern part of Italy (Gambon et al., 1995) as well as in Greece (Karabournioti et al., 2006) the dominant nectar flow was also provided by plants of the families: Asteraceae, Fabaceae and Rosaceae. The collection of honey flow from fruit trees and from Salix, Taraxacum, Rubus and Brassicaceae by honeybees was also demonstrated in studies of Austrian honeys (Fossel, 1968), German honeys (Zander, 1937; Hedtke, 1996) and Norwegian honeys (Maurizio, 1979). The Brassicaceae, as well as Trifolium repens, T. hybridum and Salix, have also been shown to constitute a valuable source of nectar for bees in Finland (Varis et al., 1982). In addition, the Finish honeys were reported to contain pollen of representatives of the family Rosaceae represented by Prunus type, Malus type and Sorbus. In contrast, in the southern regions of Finland, pollen of Brassicaceae was found to occur more frequently, which is linked with regionalization of rape crops. Plants of the family Rosaceae (Prunus and Rubus in particular) and additionally those of Trifolium repens and Aesculus also constitute a source of nectar flow in Ireland (Coffey and Breen, 1997). Amongst the major nectariferous plants of Canada, Louveaux (1966) and Adams et al. (1979) enumerate Salix, Aesculus, Trifolium repens and T. pratense, Tilia, Robinia pseudacacia, Cirsium as well as Arctium. The results obtained in the reported study confirmed the attractiveness of those taxa to honeybees. A pollen spectrum of the investigated multifloral honeys indicates that in the

9 Journal of Apicultural Science 73 area of the Sandomierska Upland, the main source of nectar flow were plants of forests and scrubs, whose pollen contribution in the samples examined accounted for 30.7%. Amongst nectariferous plants of that group, a high pollen frequency was reported for Salix, Tilia, Robinia pseudacacia, Rubus, Anthriscus and Galeopsis. In contrast, the non-nectariferous plants were mainly represented by pollen grains of Quercus, Betula and Anemone. Their presence in honeys is due to the high afforestation of the investigated area (Szafer et al., 1967; Czarnecki, 1996; Myjak, 2000). Amongst plants of meadows and grazing lands (28.4% share) Poaceae, Rumex and Filipendula predominated, followed by Trifolium repens and Taraxacum. Both meadows and pastures are highly typical communities of the areas of the Sandomierska Upland (Szafer et al., 1967; Czarnecki, 1996). In turn, orchards and gardens (17.0%) were mainly represented by pollen of Prunus, Malus and Aesculus. An additional feeding base of the Sandomierska Upland were weeds (16.4%) represented by Centaurea cyanus, Lamium, Artemisia and Chenopodiaceae. Most of them usually occur in farmfields of crops or on their outskirts. Still, they constitute a valuable source of nectar flow to pollinating insects. As shown in the study, the nectar flow was also provided by crops (7.5%), whose predominating representatives were Brassica napus, Fagopyrum and Sinapis alba. Important nectariferous plants also include Phacelia, cultivated as an aftercrop and sown especially for bees. A significant group of nectariferous plants in the area of the Sandomierska Upland are also non-nectariferous entomophilous taxa. In the analyzed honeys, they were represented the most frequently by pollen grains of Filipendula, Fragaria, Papaver, Ranunculus, Anemone and Hypericum. Flowers of these taxa possess a colorful perianthium and numerous androeacia. These features are found highly attractive to honeybees and other pollinating insects. Amongst the anemophilous plants identified in the material examined, a high frequency was reported for pollen grains of Poaceae, Quercus, Rumex and Plantago. It is difficult to state, however, whether pollen of those taxa occurred in the honeys analyzed as a result of being drifted into a hive or as a result of being collected by the bees. In various regions of Poland, the flora of forests and scrubs as well as that of meadows and grazing lands constitutes the major source of nectar flow. This has already been shown in studies by Warakomska (1996, 1997) conducted in the region of Wielkopolska (northwestern Poland) and that of Lubelszczyzna (southeastern Poland). Similar findings were reported by Wróblewska et al. (2006) in northeastern Poland and by the same author (Wróblewska, 2002) in the region of Podlasie (central-eastern Poland). CONCLUSIONS The Sandomierska Upland is characterized by diversified flora which constitutes a valuable source of nectar flow for honeybees and other pollinating insects from the early spring to the late fall. The major sources of nectar flow in the Sandomierska Upland area turned out to be: Brassicaceae (including Brassica napus), Prunus, Trifolium repens, Anthriscus, Aesculus, Salix, Taraxacum and Phacelia. REFERENCES Adams R. J., Smith M. V., Townsend G. F. (1979) - Identification of honey sources by pollen analysis of nectar from the hive. J. apic. Res., 18(4): Bucher E., Kofler V., Vorwohl G., Zieger E. (2004) - Das Pollenbild der Südtirolen Honige. Biologisches Labor der Landesagentur für Umwelt und Arbeitsschutz, Leifers (BZ). Czarnecki R. (1996) - The Sandomierska Upland Area. Eastern Part. Geographical landscape complexes. I, Warsaw, Author s own print.

10 74 Coffey M. F., Breen J. (1997) - Seasonal variation in pollen and nectar sources of honey bees in Ireland. J. apic. Res., 36(2): Demianowicz Z. (1961) - Pollenkoeffizienten als Grundlage der quantitativen Pollenanalyse des Honigs. Pszczeln. Zesz. Nauk., 5(2): Demianowicz Z. (1964) - Charakteristik der Einartenhonigen. Annales de l Abeille, 7(4): Demianowicz Z., Demianowicz A. (1955) - A new method of pollen analysis of honeys. Pszczeln. Zesz. Nauk., 1: Floris I., Satta A., Ruiu L. (2007) - Honeys of Sardinia (Italy). J. apic. Res., 46(3): Fossel A. (1968) - Pollenersatzmittel im mikroskopischen Befund von Frühtrachthonigen. Zeischrift für Bienenforschung, 9(5): Gambon N., Renzo B., Frilli F. (1995) - La potenzialità apistica nell Alto Bacino del Tagliamento. L Ape Nostra Amica, 17(5): Hedtke C. (1996) - Pollen spectrum of honeys in Brandenburg. Pszczeln. Zesz. Nauk., 40(2): Hodges D. (1952) - The pollen loads of the honeybee. IBRA, London. Karabournioti S., Thrasyvoulou A., Eleftheriou E. P. (2006) - A model for predicting geographic orgin of honey from the same floral source. J. apic. Res., 45(3): Kęsik T. (2003) - Cultivation of orchards and vegetables in region of Sandomierz. In Proceedings of National Conference Horticulture of Sandomierska Land, Sandomierz, Poland, 17 November pp Kęsik T., Warzecha M. (2000) - The structure of arable cropland and horticultural cultures of Sandomierska Land. In Proceedings of Symposium Nature of the borderland between the Sandomierska Land and the Southeastern Poland, Sandomierz, Poland, 18 November pp Louveaux J. (1966) - Pollenanalyse einiger Kanadischer Honige. Zeischrift für Bienenforschung, 8(5): Louveaux J., Maurizio A., Vorwohl G. (1978) - Methods of Melissopalynology. Bee Wld., 59(4): Maerz A., Paul M. (1950) - A dictionary of color. McGraw-Hill Co., New York-Toronto- London. Maurizio A. (1979) - Beitrag zur Kenntins des Pollenspektrums norwegischer Honige. Apidologie, 10(4): Moar N. T. (1985) - Pollen analysis of New Zealand honey. N. Z. J. Agric. Res., 28: Myjak J. (2000) - Lipnik and the area (information on the Commune of Lipnik). PAIR, Sandomierz. Ohe von der W., Persano Oddo L., Piana M., Morlot M., Martin P. (2004) - Harmonized methods of melissopalynology. Apidologie, 35: Ostrowska W. (1998) - Apiarian management. Ed. V. PWRiL, Warszawa. Primorac L., Bubalo D., Kenjerič D., Flanjak I., Perl Pirički A., Mandič M. L. (2008) - Pollen spectrum and physicochemical characteristics of Croatian Mediterranean multifloral honeys. Deutsche Lebensmittel-Rundschau, 104(4): Ricciardelli D albore G. (1998) - Mediterranean melissopalynology. Ed. Univ. degli studi di Perugia, Fac. di Agraria, Perugia. Sawyer R. W. (1981) - Pollen identification for beekeepers. Ed. R.S. Pickard, Univ. College Cardiff Press; Cardiff, UK. Sawyer R. (1988) - Honey identification. Cardiff Acad. Press, Wales, UK. Szafer W., Kulczyński S., Pawłowski B. (1967) - Polish plants. PWN, Warszawa. Varis A. L., Helenius J., Koivulehto K. (1982) - Pollen spectrum of Finnish honey. Journal of the Scientific Agricultural Society of Finland, 54: Warakomska Z. (1996) - Pollen contents of some honeys orginating from Wielkopolska region. Pszczeln. Zesz. Nauk., 40(2):

11 Journal of Apicultural Science 75 Warakomska Z. (1997) - Pollen spectrum of multifloral honeys of Lublin region. In Proceedings of 1st National Scientific Conference Biology of flowering, nectar secretion and plant polination, Lublin, Poland, November pp Wróblewska A. (2002) - Flow flora of Podlasie in the light of pollen analysis of honeybee products. Publisher Agricultural University in Lublin, 264, 83pp. Wróblewska A., Warakomska Z., Koter M. (2006) - Pollen analysis of bee products from the north-eastern Poland. J. apic. Sci., 50(1): Zander E. (1935, 1937) - Beiträge zur Herkunftsbestimmung bei Honig. I Reichsfachgrupp Imker, Berlin; II Liedloff, Loth & Michaelis, Leipzig. Zimmerman M. (1988) - Nectar production, flowering phenology and strategies for pollination. In Lovett J. & Lovett L. (ed) Plant Reproductive Ecology. Patterns and Strategies. Oxford University Press, Oxford, UK, pp ANALIZA MELISSOPALINOLOGICZNA MIODÓW WIELOKWIATOWYCH Z TERENU WYŻYNY SANDOMIERSKIEJ S t a w i a r z E., W r ó b l e w s k a A. S t r e s z c z e n i e Obiekt badań stanowiły 73 próbki miodów wielokwiatowych uzyskanych w latach z terenu Wyżyny Sandomierskiej. Mikroskopową analizę pyłkową osadów przeprowadzono według wskazań Louveaux i in. (1978) oraz Moara (1985). Przy oznaczaniu ziaren pyłku zastosowano klasyfikację Zandera (1935, 1937). W analizowanym materiale wyróżniono ziarna pyłku 103 taksonów (75 nektarodajnych i 28 nienektarodajnych) z 52 rodzin botanicznych. Wśród roślin nektarodajnych głównego pożytku dostarczały Brassicaceae (w tym Brassica napus), Prunus, Trifolium repens, Anthriscus, Aesculus, Salix, Taraxacum i Phacelia. Ziarna pyłku wspomnianych taksonów charakteryzowały się wysoką frekwencją (52,0-100,0%). Wśród roślin nienektarodajnych najwyższą frekwencją (89,0%) odznaczały się Poaceae (inne). W poszczególnych próbkach miodów notowano ogółem od 16 do 41 taksonów ziaren pyłku, w tym nektarodajnych w liczbie od 12 do 34, a nienektarodajnych od 2 do 13. Barwa omawianych miodów wahała się od kremowej, poprzez bursztynową do ciemnobrązowej. Słowa kluczowe: miody wielokwiatowe, rośliny nektarodajne, analiza pyłkowa, Wyżyna Sandomierska, Polska.