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Pollen diversity in honey collected from Lithuania's protected landscape areas/ Leedu looduskaitsealadelt kogutud mee oietolmu jaotus.

INTRODUCTION

In Lithuania honey is produced in different regions, including national parks, covering a wide range of terrains. Landscapes vary between western, southern, eastern, and northern Lithuania. A distinctive floral association can exist in different climatic terrains within the same country (Lincius, 2011). Particular floral association is a characteristic feature of different geographical areas (Ruoff and Bogdanov, 2004). Currently high importance is attached to the regions where honey is collected from the region-specific plants and its botanical origin is conspicuous. Lithuanian beekeepers living and producing honey in national parks, nature reserves, and other protected landscape areas have the privilege of marking their honey with the legal mark "Kokybe" (Lithuanian for quality), which is special only for their honey (Skirkevicius, 2010). In the honey from those terrains it is possible to identify area-specific pollen. Quality certificates of the tested honey are issued by the accredited laboratory in Lithuania; however, there is no accredited laboratory for the determination of honey origin in this country.

Monofloral rape honey is specific to Central Lithuania. Earlier the most abundant pollen of Brassica napus and Salix caprea were identified in the honey from Central Lithuania (Ceksteryte, 2002; Baltrusaityte et al., 2007). Monofloral willow honey is found in Italy, rape honey in Poland and Estonia (Persano Oddo and Piro, 2004; Wroblewska and Warakomska, 2009; Kirs et al., 2011). Geographical origin of a particular kind of honey is determined by a melissopalynological study of its pollen spectrum (Louveaux et al., 1978).

Melliferous plants produce various shapes, sizes, and surfaces of pollen grains. Their identification is complicated, therefore samples for comparison purposes (plant pollen collected by hand) to be used as reference material should be collected. Currently, depending on the research subject, laboratories use a host of microscopy techniques to study images of pollen grains.

Pollen grains examined under microscope can be seen in a polar or equatorial view. Polar position of pollen grains has one of various shapes: circular, angular, hexagonal, lobate, or semiangular, interangular, subangular. The semi-, inter-, and sub-forms are found also for lobate forms (Faegri and Iversen, 1964; Kremp, 1965; Hesse et al., 2009). In the equatorial view, the most typical shapes are rhomboidal, apiculate, rectangular, or oval. Pollen forms also can be characterized according to measures of grains, i.e. area, length of polar axis, and equatorial diameter (Erdtman, 1952; Ricciardelli D'Albore, 1998).

The present study was aimed to describe the most unique pollen found in the honey collected in protected landscapes of Lithuania and to show digital images of pollen grains found in honey.

MATERIALS AND METHODS

Sampling areas

Seventeen samples of honey were collected from Lithuania's protected landscape areas. Sampling was performed in protected areas in different parts of the country.

North-west Lithuania: Zemaitija National Park (involved in the programme NATURA 2000, the Europe-wide network of sites tasked with the preservation of natural heritage) in the Plunge District; the Varduva Scenic Landscape Reserve in the Mazeikiai District; and the Kretinga District in the Salantai Regional Park, which is close to Zemaitija National Park. The Salantai Regional Park was founded to preserve the endangered species of plants that grow in the hillside forests and natural flood plains of old valleys.

South Lithuania: Dzukija National Park in the Varena District.

South-west Lithuania: Lazdijai District. Commission Implementing Regulation (EU) No. 75/2012 of 30 January 2012 entering a name in the register of protected designations of origin and protected geographical indications 'Miod z Sejnenszczyzny/ Lozdziejszczyzny/Seiniu/Lazdiju krasto medus'. The regulation refers to Polish honey produced in Suvalki and Seiniai counties and in Lithuania in the Lazdijai District. The term 'Lazdiju krasto medus' is related to the honey produced in the Lazdijai District. The characteristic features of this district are a short plant growing season and clean environment.

Towards the north from the centre of Lithuania: Gomerta Landscape Reserve, Radviliskis District.

East Lithuania: Armona Geological Reserve, situated on the Aukstaiciai Upland and Plateau, Ukmerge District (Vilnius County).

Middle Lithuania: Krekenava Regional Park in the Kedainiai District.

Preparation of honey slides

A honey sample of 10 g was weighed and dissolved in distilled water (20-40 [degrees]C) to the volume of 20 mL. This solution was centrifuged for 10 min at 1000g. After that the supernatant liquid was poured off. The sediment was dispersed with 20 mL of distilled water to completely dissolve the remaining sugar crystals and again centrifuged for 5 min at 1000g. The supernatant was decanted leaving only the sediment. The remaining excess liquid was taken up on absorbent paper. The sediment was collected with plastic Pasteur pipettes (volume 1 mL) and spread on a slide over an area of about 20 mm x 20 mm. The slide with the sediment of pollen was dried at 40 [degrees]C on a heating plate only for the time strictly necessary to dry.

The glycerine jelly (Kaiser's Glycerol Gelatine TM Merck) was liquefied by warming it to [less than or equal to]40 [degrees]C (either on a heating plate or in a water bath). The cover slips (22 mm x 22 mm) were warmed on the heating plate. One drop of glycerine jelly was deposited onto the cover slip and placed on the slide very slowly to avoid air bubbles. The pollen grain exine and shape were visualized under light microscope Nicon Eclipse E600. Pollen images were taken in two positions: polar and equatorial view, at 400x magnification, focusing on pollen wall and surface sculpture.

Expression of results

About 400-500 pollen grains were counted in each sample. The frequency of pollen of each melliferous plant is expressed as percentage of the total pollen sum. Honey considered as monofloral is mainly produced from one plant species or pollen content from one plant species is predominant (constituting more than 45.0%). The pollen content of other plant species is designated as follows: secondary pollen 16-45%; important minor pollen 3-15%; minor pollen < 3.0% (Louveaux et al., 1978).

Slide preparation of hand-collected pollen

Pollen collected by hand from 85 melliferous plants was prepared also for microscopy. Air-dried plant pollen was shaken from flowers, some part of it was applied on the slide and covered with a cover slip overlaid with liquefied glycerine jelly. These pollen samples are stored as reference. A catalogue of coloured digital images of Lithuanian melliferous plant pollen grains was created for comparison of images of pollen found in honey to those of known pollen collected manually (Ceksteryte, 2012).

RESULTS AND DISCUSSION

In the Varduva Scenic Landscape Reserve monofloral spring honey was collected. Pollen from Malus domestica accounted for 54.1% in sample I (Table 1). Pollen of Acer platanoides was found in the highest content (7.0%) compared to other kinds of honey. Pollen of Rubus fructicosus was identified only in honey sample I, where it accounted for 8.2%. Monofloral honey was produced also in the Salantai Regional Park with pollen of Carum carvi as dominant (50.19%) and Zemaitija National Park with Trifolium repens dominating (71.4%). Melissopalynological evaluation of honey pollen showed minor pollen of Trifolium repens (2.28%) in monofloral caraway honey (sample II) and Carum carvi (1.6%) in clover honey (sample IV).

In Dzukija National Park summer honey with the highest percentage of Tilia cordata (79.0%, sample V) and Fagopyrum esculentum (up to 100.0%, sample VII) pollen was collected. The composition of honey collected in the location of Gudakaimis (sample VI) was more diverse. This honey contained Salix caprea (44.6%) and Fagopyrum esculentum (26.5%). Pollen from Rubus idaeus, Frangula alnus, and Brassica napus varied in the range of 3.8-7.0% and Cerasus pollen was present as minor (2.1%). Pollen identified in those honey samples represents this region's melliferous flora from spring to the end of summer.

Monofloral spring honey from Salix caprea and Frangula alnus was collected in the Lazdijai District. Compared to other kinds of honey tested, the honey from Frangula alnus was conspicuous by its highest content (46.1%) of pollen of this plant. This honey was the only one having an abundant content of Frangula alnus pollen. A characteristic feature of honey from the Lazdijai District is monofloral honey from Onobrychis, which contains dominant pollen of this plant (52.1-54.4%), and polyfloral honey with Onobrychis pollen as secondary or an important minor (9.2-17.8%).

Three kinds of monofloral honey, lime (Tilia cordata), caraway (Carum carvi), and orchard, were identified in the samples from the Armona Geological Reserve. The dominant pollen was from Malus domestica (56.9%, sample XII), Tilia cordata (53.9%, XIII), and Carum carvi (82.8%, XIV) in those unifloral honey samples. Secondary pollen from Brassica napus (18.4%) was identified in monofloral Tilia cordata honey (sample XIII) and Salix caprea (29.0%) in the honey from Malus domestica (sample XII). In the latter honey, important minors were pollen from Frangula alnus and Acer platanoides, accounting for 4.9% and 4.6%, respectively.

Pollen of Phacelija tanacetifolia was identified only in the honey from the Gomerta Landscape Reserve. Pollen analysis showed that the honey was produced within different periods. The pollen of Phacelija tanacetifolia accounted for 19.5% in sample XV, while in sample XVI its content was 4.8%. The concentration of Brassica napus pollen reached 72.0% in the mono- floral rape honey from the same area (sample XVI). In sample XV the content of its pollen made up 36.1%.

The pollen of Robinia pseudoacacia is rarely found in Lithuanian honey; however, in honey sample XV it accounted for 8.5%. The presence of Salix caprea, Frangula alnus, Rubus idaeus, Trifolium repens, Centaurea cyanus, Arctium tomentosum, Carum carvi, and Fagopyrum esculentum pollen in those honey samples points to a wide diversity of melliferous plants within the area of the Gomerta Landscape Reserve.

Monofloral willow honey was collected from the Krekenava Regional Park in spring. An abundant content of pollen grains of Salix spp. (55.5%) was identified in this honey. Pollen from Brassica napus making up 31.3% was secondary and of Malus domestica and Frangula alnus, important minors, accounting for 5.7% and 3.3%, respectively.

Digital images of pollen grains of selected honeys from some parts of the microscopic view are presented in Figs 1-6. Pollen of Trifolium repens, Fagopyrum esculentum, and Salix spp. are shown in polar and equatorial views (Figs 1, 2, and 6, respectively); all Tilia cordata, Phacelija tanacetifolia, and Frangula alnus pollen in polar view (Figs 3, 5, and 6, respectively); and pollen of Carum carvi in equatorial view (Fig. 4). Polar and equatorial images of the same pollen are not similar. Small differences in pollen morphology cause difficulties in their identification (Sawyer, 1988; Lindbladh et al., 2002). The presence of specific blends of plant pollen in honey shows its geographical origin (Pupuleku et al., 2012). Therefore identification of pollen requires an expert person and a special methodology for the classification of pollen of plant species.

CONCLUSIONS

Lithuanian monofloral honey is produced in different locations. Buckwheat and cockshead honeys are characteristic of southern and south-western Lithuania. Buckwheat (Fagopyrum Mill.) is a traditional crop cultivated in southern Lithuania and cockshead (Onobrychis Mill.) in the south-western areas. Production of honey with a contribution from Phacelija tanacetifolia Benth. and Trifolium repens L. is also associated with traditional farming activities in northwestern Lithuania.

The data of melissopalynological analysis showed Tilia cordata Mill. pollen to be dominant (79.0%) in lime honey, where it was found at the highest counts in Dzukija National Park. This kind of honey was also harvested in the Armona Geological Reserve. Natural meadow is a distinct feature of the Armona Geological Reserve and of the Salantai Regional Park located close to Zemaitija National Park. Monofloral honey was produced from Carum carvi L. in those terrains. Pollen of Salix spp. was found in monofloral and polyfloral Lithuanian honeys harvested in different terrains.

doi: 10.3176/proc.2013.4.08

ACKNOWLEDGEMENT

This research was funded by a grant (No. SVE-01/2012) from the Research Council of Lithuania.

REFERENCES

Ceksteryte, V. 2002. Investigation of pollen composition and catalase activity in unifloral summary and polyfloral spring honey. Zemdirbyste, 80(4), 193-200 (in Lithuanian with English summary).

Ceksteryte, V. 2012. Plant Pollen Found in Lithuanian Honey. An Electronic Catalogue (in Lithuanian).

Baltrusaityte, V., Venskutonis, P. R., and Ceksteryte, V. 2007. Radical scavenging activity of different floral origin honey and beebread phenolic extracts. Food Chem., 101(2), 502-514.

Erdtman, G. 1952. Pollen Morphology and Plant Taxonomy. Angiosperms. Almquiste Wiksell, Stockholm.

Faegri, K. and Iversen, J. 1964. Textbook of Pollen Analysis. Munksgaard, Copenhagen.

Hesse, M., Halbritter, H., Weber, M., Buchner, R., Frosch-Radivo, A., and Ulrich, S. 2009. Pollen Terminology--An Illustrated Handbook. Springer, Wien.

Kirs, E., Pall, R., Martverk, K., and Laos, K. 2011. Physicochemical and melissopalynological characterization of Estonian summer honeys. Procedia--Food Sci., 1, 616-624.

Kremp, O. W. 1965. Morphologic Encyclopedia of Palynology. University of Arizona Press, Tucson.

Lincius, A. 2011. Lietuvos geologijos paveldo tyrimai ir issaugojimas [Lithuanian geological heritage investigation and protection]. BALTICA, 24, Special Issue, 65-68 (in Lithuanian).

Lindbladh, M., O'Connor, R., and Jacobson, G. L. Jr. 2002. Morphometric analysis of pollen grains for paleoecological studies: classification of Picea from eastern North America. Am. J. Bot., 89, 1459-1467.

Louveaux, J. Maurizio, A., and Vorwhol, G. 1978. Methods of melissopalynology. Bee World, 59(4), 139-157.

Persano Oddo, L. and Piro, R. 2004. Main European unifloral honeys: descriptive sheets. Apidol, 35, 38-81.

Pupuleku, B., Kapidani, G., Naqellari, P., Nikoleta Kallajxhiu, N., Turku, S., Gjeta, E., and Janc, A. 2012. Data on the palynological study of pollen grains of some Albania's honey plants of Leguminosae family. In BALWOIS, pp. 1-10. Ohrid, Republic of Macedonia.

Ricciardelli D'Albore, G. 1998. Mediterranean Melisso-palynology. Universita degli Studi di Perugia.

Ruoff, K. & Bogdanov, S. 2004. Authenticity of honey and other bee products. APIACTA, 38, 317-327.

Skirkevicius, A. 2010. Iskirtines kokybes biciu produktai ir misiniai [Exlusivity of the Quality of Bee Products and Mixtures]. Druka, Klaipeda (in Lithuanian).

Sawyer, R. 1988. Honey Identification. Academic Press, Cardiff.

Wroblewska, A. and Warakomska, Z. 2009. Pollen analysis of honey from Poland's Lubelszczyznza regions. J. Apic. Sci., 53(2), 57-67.

Violeta Ceksteryte (a) *, Bogumila Kurtinaitiene (b), and Jonas Balzekas (a)

(a) Institute of Agriculture, Lithuanian Research Centre for Agriculture and Forestry, Akademija, Kedainiai dist., Instituto aleja, LT-58344, Lithuania

(b) Institute of Biochemistry, Vilnius University, Mokslininku str. 12, LT-08662, Vilnius, Lithuania

* Corresponding author, violeta@lzi.lt

Received 10 April 2013, revised 21 August 2013, accepted 23 August 2013, available online 19 November 2013

Table 1. Pollen composition in honey from Lithuania's protected
landscapes

Sample    Village         Botanical composition of honey pollen, %
 No.
                             Varduva Scenic Landscape Reserve,
                                     Mazeikiai District

I        Dumciai      Malus domestica Borkh. 54.1; Salix spp. 16.7;
                        Brassica napus L. 8.9; Rubus fruticosus L.
                        8.2; Acer platanoides L. 7.0; Taraxacum
                        officinale L. 4.3; Rubus idaeus L. 0.8;
                        honeydew 0.8

                          Salantai Regional Park, Kretinga District

II       Balsiskiai   Carum carvi L. 50.19; Malus domestica Borkh.
                        15.21; Salix spp. 11.02; Brassica napus L.
                        8.74; Frangula alnus Mill. 4.18; Trifolium
                        repens L. 2.28; Taraxacum officinale L. 2.66;
                        Rubus idaeus L. 1.90; other 3.80

III      Balsiskiai   Salix spp. 27.5; Taraxacum officinale L. 13.6;
                        Malus domestica Borkh. 7.7; Brassica napus L.
                        4.2; Aesculus hippocastanum L. 3.1; Corylus
                        avellana L. 1.4; Carum carvi L. 1.4; Acer
                        platanoides L. 1.1

                          Zemaitija National Park, Plunge District

IV       Puckoriai    Trifolium repens L. 71.4; Rubus idaeus L. 8.9;
                        Brassica napus L. 8.0; Salix spp. 5.9; Carum
                        carvi L. 1.6; Malus domestica Borkh. 1.2;
                        Centaurea cyanus L. 0.9; Fagopyrum esculentum
                        M. 0.6; Taraxacum officinale L. 0.9; Frangula
                        alnus Mill. 0.6

                           Dzukija National Park, Varena District

V        Runge        Tilia cordata Mill. 79.03; Brassica napus L.
                        11.55; Carum carvi L. 5.47; Salix spp. 2.73;
                        Cerasus Mill. 1.21; honeydew 2.80

VI       Gudakaimis   Salix spp. 44.6; Fagopyrum esculentum M. 26.5;
                        Rubus idaeus L. 7.0; Frangula alnus Mill. 4.0;
                        Brassica napus L. 3.8; Centaurea cyanus L. 2.8;
                        Cerasus Mill. 2.1

VII      Zervynai     Fagopyrum esculentum M. 100

                                      Lazdijai District

VIII     Leonardavo   Salix spp. 63.7; Onobrychis Mill. 17.8; Brassica
                        napus L. 8.9; Taraxacum officinale L. 5.9;
                        Malus domestica Borkh. 2.2; Melilotus (L.)
                        Mill. 1.5; honeydew 9.4

IX       Runge        Onobrychis Mill. 54.4; Salix spp. 12.2; Sinapis
                        arvensis L. 10.7; Trifolium repens L. 7.4; Acer
                        platanoides L. 4.7; Brassica napus L. 4.0;
                        Pyrus L. 2.7; Trifolium pratense L. 1.3; Rubus
                        idaeus L. 1.3; Melilotus (L.) Mill. 1.3;
                        honeydew 5.7

X        Avizieniai   Onobrychis Mill. 52.1; Malus domestica Borkh.
                        14.7; Salix spp. 17.2; Aesculus hippocastanum
                        L. 4.9; Carum carvi L. 4.3; Sinapis arvensis L.
                        2.5; Rubus idaeus L. 1.8; Acer platanoides L.
                        1.3; Frangulaalnus Mill. 1.2

XI       Seirijai     Frangula alnus Mill. 46.1; Salix spp. 21.1;
                        Brassica napus L. 13.2; Onobrychis Mill. 9.2;
                        Rubus idaeus L. 5.2; Pyrus L. 5.2;
                        honeydew 33.9

                         Armona Geological Reserve, Ukmerge District

XII      Baraucizna   Malus domestica Borkh. 56.9; Salix spp. 29.0;
                        Frangula alnus Mill. 4.9; Acer platanoides L.
                        4.6; Brassica napus L. 2.0; Taraxacum
                        officinale L. 2.0; Rubus idaeus L. 0.6

XIII     Baraucizna   Tilia cordata M. 53.9; Brassica napus L. 18.4;
                        Rubus idaeus L. 9.8; Carum carvi L. 5.3;
                        Trifolium repens L. 5.3; Salix spp. 5.0; Ribes
                        L. 1.6; Sinapis arvensis L. 0.7

XIV      Baraucizna   Carum carvi L. 82.8; Brassica napus L. 4.6; Malus
                        domestica Borkh. 4.2; Salix spp. 3.1; Rubus
                        idaeus L. 2.2; Centaurea cyanus L. 1.8;
                        Frangula alnus Mill. 1.2; honeydew 1.5

                       Gomerta Landscape Reserve, Radviliskis District

XV       Debeikiai    Brassica napus L. 36.1; Phacelija tanacetifolia
                        Benth. 19.5; Rubus idaeus L. 16.7; Robinia
                        pseudoacacia L. 8.5; Carum carvi L. 5.7; Salix
                        spp. 4.3; Trifolium repens L. 3.2; Prunus
                        domestica L. 2.5; Fagopyrum esculentum M. 0.7;
                        Frangula alnus Mill. 1.4; Carum carvi L. 0.7;
                        Centaurea cyanus L. 0.7

XVI      Debeikiai    Brassica napus L. 72.0; Salix spp. 7.3; Phacelija
                        tanacetifolia Benth. 4.8; Arctium tomentosum
                        Mill. 4.2; Trifolium repens L. 3.9; Rubus
                        idaeus L. 3.4; Fagopyrum esculentum M. 3.1;
                        Carum carvi L. 0.5; Malus domestica Borkh. 0.8

                         Krekenava Regional Park, Kedainiai District

XVII     Uzupe        Salix spp. 55.5; Brassica napus L. 31.3; Malus
                        domestica Borkh. 5.7; Frangula alnus M. 3.3;
                        Sinapis arvensis L. 1.0; Taraxacum officinale
                        L. 0.3; Centaurea cyanus L. 0.3; Tussilago
                        farfara L. 0.3; Aesculus hippocastanum L. 0.4;
                        Acer platanoides L. 0.5; Trifolium repens
                        L. 0.4; Rubus idaeus L. 0.9
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Title Annotation:PALYNOLOGY
Author:Ceksteryte, Violeta; Kurtinaitiene, Bogumila; Balzekas, Jonas
Publication:Proceedings of the Estonian Academy of Sciences
Article Type:Report
Geographic Code:4EXLT
Date:Dec 1, 2013
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