Pollen spectrum of Finnish honey

Pollen spectra were analysed on 160 honey samples representing the whole beekeeping area of Finland in the years 1977 and 1978. A semi-quantitative method of pollen analysis based on line counts was developed. It was found efficient for determining the relative numbers of pollen grains in the honey samples. On average, ca. 16 pollen types were identified per sample. The number of honeydew elements was low in all samples. Brassicaceae pollen was the dominant type. The other most frequent and abundant types were pollen grains of Trifolium repens+ T. hyhridum, Salix spp., "the Rosaceae group” (Prunus, Malus, Sorbus spp.), Apiaceae , and Filipendula uimaria. These pollen types constituted 90.8 % (1977) and 90.3 % (1978) of all the pollen examined. Between 1960—1963 and 1977—1978 the average proportion of Brassicaceae pollen has risen, while the proportion of T. repens + T. hyhridum pollen has decreased, evidently due to the changes in agricultural practices. The seasonal variation in the pollen spectrum was explained by the weather conditions. Regionally characteristic pollen spectra were not found, although Brassicaceae pollen was typical of the honeys of agricultural zones I and 11, due to cultivation of Brassica oilseed crops in thoseregions. The positive correlation of the proportion of Brassicaceae pollen with the total pollen count is discussed in connection with the problem of determining the botanical origin.


Introduction
In melissopalynology, the microscopic elements of honey are studied.These are mostly pollen grains of flowering plants, or algae and fungus spores and hyphae mainly present in the original nectar or honeydew.
The pollen spectrum is examined by counting the numbers of different kinds of pollen grains in the honey sample.In qualitative pollen analysis, the results are either presented as frequency classes as first described by ZANDER (1935), or as percentages if the statistical conditions are fulfilled.In quantita- tive pollen analysis, the absolute pollen counts in a given amount of honey are determined (LOUVEAUX et al. 1970(LOUVEAUX et al. , 1978)).
The objective of the study may be to find out the geographical origin of the honey, or the floral sources (botanical origin), to determine the season of production, or to identify honeydew honeys.The main problem in analysing the botanical origin of honey is that different nectar plants yield different amounts of pollen to honey in the same amounts of nectar.Thus, a direct correlation may not be assumed between the pollen and nectar contributions of a plant to the honey.MARTIMO (1945) published the results of pollen analysis of 21 Finnish honey samples and discussed their botanical origin.AARIO (1961) studied the pollen spectra of 85 Finnish honeys in order to describe the regional conditions for beekeeping.PUROKOSKI and FORSIUS (unpulished 1 ) analysed 120 samples in [1962][1963], their main interest being the chemical composi- tion.
The purpose of this study was to describe the average pollen spectrum of Finnish honey, and to analyse the most important factors responsible for its variation.The problem of determining the botanical origin is briefly discussed; this is still under study, and the results will be published later.

Material
The material originated from the years 1977 and 1978.The request for a honey sample, with instructions regarding the sampling procedure, was mailed to 83 beekeepers in 1977.They were selected from the registers of the beekeeping associations so as to provide regionally representative honey material (see Fig. 2).The provincial distribution of the honey samples did not differ from the distribution of active beekeepers reported by RIKALA (1976) (Goodness of fit test: X 2 = 13.1<X 2 os(v=8 )~15.5).
In 1977, 66 beekeepers replied (80 %).In 1978, in order to obtain samples comparable to those of the previous year, the request was repeated to these 66 active beekeepers, 45 of whom replied (68 %).In addition, five beekeepers participated in 1978 alone.Thus, 71 beekeepers with a total of 81 apiaries participated: 46 apiaries provided samples in both the years, 28 only in 1977, and 7 only in 1978.From each apiary, one to three honey samples and answers to a questionnaire about the apiary were received.

Honey samples
A total of 160 samples was collected: Each sample contained ca. 2 kg of honey.As a request was made for separate samples from each collection in the season, occasionally even three samples per apiary were received.The majority (75 %) of the beekeepers removed the honey only once, at the end of the season, and thus sent one sample only to represent the whole season.The samples were stored in a freezer at -15 °C.

Inquiry
A questionnaire was employed.In addition to the location of the apiary, the most important information required was: the date of removing the honey, the honey yield, the number of colonies, the race of the bees and the probable nectar sources.

Weather
The season, especially spring, was more favourable for bees in 1978 than in 1977 (Table 1).At the end of August, the growing season was one week more advanced in 1978 than in 1977, as measured by the effective tempera- ture sum (2 °C > + 5 °C).The effective temperature sum reached at the end of September in 1977 was 85 % of the normal value for the growing season, and in 1978 it was 95 %.

Pollen analysis
The honey sample was dissolved in water, and the solid particles were centrifuged for the microscopical analysis.The pollen grains and honeydew elements were identified and counted.The method was semiquantitative, modified from the quantitative method of Z. Demianowicz (LOUVEAUX et   al. 1978).
Preparation of slides.From the original honey sample a 10 g subsample was taken and dissolved in 20 ml of distilled water, and centrifuged for 10 min at 3 500 r/min, after which the supernatant liquid was drawn off.A further 10 ml of water was added to the sediment and centrifuged 5 min at 3 500 r/min, after which the liquid was drawn off.The sediment was transferred with a capillary pipette to a microscope slide, and dispersed evenly over an area of 20 mm X 20 mm.The slide was dried for 24 h at 30 °C.The mounting medium was glycerine jelly.
The reference slides of 135 species of flowering plants for identifying the pollen grains were prepared by the method described by LOUVEAUX et al. (1970).
Counting and identifying the microscopic elements.On each slide, all the pollen grains and honeydew elements were counted and identified on a 20mm line across the 20 mm X 20 mm square.The eyepiece micrometer (Leitz Wetzlar SM-Lux: eyepiece Periplan GF 12.3XM, objective Phaco NPI 40/ 0.65) was used as the line indicator.Particles that were only adjacent to the scale of the micrometer were also included.The line was always started at the same distance from the edge of the square (Fig. 1).The use of the same kind of line in each sample gave a basis for quantitative comparisons.This was considered to be an important advantage of the method.
From each honey sample two slides were prepared.The accuracy of the counts was evaluated by comparing the results of the parallel counts in a sample of 14 pairs of slides.The total numbers of pollen grains counted on the two parallel slides correlated statistically very significantly with each other (F(i,l3)=lo4.4: '' : '': '', r 2 =B9 %).Thus, two slides and counts per honey sample gave a reliable estimate of the pollen spectrum of the sample.
The pollen types were identified with the aid of the reference slides.The pollen of Myosotis spp. was not counted, because it is highly over-rep- resented (e.g.MAURIZIO 1949, DEMIANOWICZ 1961, LOUVEAUX et al. 1970).The occurrence of Myosotis pollen was noted, however.The other pollen types were grouped into the pollen of nectariferous and non-nectariferous plants, according to the information available in the literature (e.g.CRANE Fig. 1.The line method for counting the microscopic elements.HANSSON 1976, HOWES 1979).The honeydew elements were identified according to the instructions given by ZANDER and MAURIZIO (1975).
The results are given as means of the parallel counts.By the proportion of a pollen type is meant the percentage of its grains in the total pollen count.The mean of the proportions is called the average proportion of the type.As the proportions are of minor informative value and the average proportions may be misleading, the proportions were calculated only for comparisons with earlier studies in which they were used.By frequency of a pollen type the number (or percentage) of samples in which it was observed is indicated.

Processing the data
The samples of 1977 and 1978 were mainly treated separately.The variation in the pollen spectra was analysed separately for the two seasons.The factors considered were: intercorrelations between pollen types, annual variation, regional variation, and variation connected with the bee race.
To study the annual variation, only those apiaries were included from which one or more honey samples were received in both 1977 and 1978.Thus, the annual comparisons were based on 62 samples in 1977 and 61 parallel samples in 1978.The regional division adopted in studying the regional variation is based on the length of the thermal growing season.It is used by the National Agricultural Extension Organisation in the recommen- dations for field crop varieties.The zones are numbered from south to north: Zone I, Zone 11, Zone 111 and Zones IV+V (Fig. 2).In the statistical analysis, three groups of bees were distinguished: Italian bees (Apis mellifera ligustica Spin.), dark bees (A. m. mellifera L.) and bees of mixed race.
The statistical analyses were done by the H-programmes of the Comput ing Centre, University of Helsinki.The inquiry: results and discussion Of the honey samples, 47 % were produced by Italian bees, 37 % by bees of mixed race, and the remaining 16 % mainly by dark bees (Table 2).According to the data of RIKALA (1976), the material was racially representa- tive of Finnish bees.
The average size of the participating apiaries was 6.6 colonies in 1977 (71 answers) and 6.0 colonies in 1978 (50 answers).In 1975 the average in Finland was 4.4 colonies per apiary (RIKALA 1976).
Sixty-five per cent of the honey samples were extracted in August (Fig. 3).The majority of the beekeepers removed the honey only once, at the end of the season: 77 % in 1977 and 70 % in 1978.The honey yield varied from 3 to 90 kg/colony.The average yield of the apiaries participating in both the years was 29 kg/colony in 1977 (50 answers) and 37 kg/colony in 1978 (41 answers).The difference between the years, 20.6 % units, was significant (t(89)=2.16:; ').As the weather was less favourable for bees in 1977 than in 1978, the honey yields were smaller.Weather is the key factor for the honey yield (RIKALA 1976).-The way in which the material was collected may be considered to have been selective for more active and interested beekeepers than average.This is probably reflected in the data on the number of colonies and the yield per colony.The honey yields showed significant regional variation, being lowest in Zone I and in Zone IV+V (Fig. 4).No generalizations should be made from these, as only two seasons were observed.However, it seems at least that southern Finland is not in a more advantageous position for beekeeping than the central parts of the country (see also RIKALA 1976).
According to the beekeepers, the most important honey-yielding plants in May were: Sali:c spp., Taraxacum spp.and Tussilago farfara L., and in June: Taraxacum spp., Rubus idaeus L. and Malus domestica Borkh., and in 1978 also the cultivated Ribes spp.In June the wild forest berries, Vaccinium spp., were often mentioned, but e.g.Prunus padus L. and Sorbus aucuparia L. were listed only seldom.
In July, the most important plants were said to be Epilobium angus- tifolium L. and Trifolium spp., followed by Rubus idaeus and in 1978 also by the cultivated oleiferous Brassica spp., rape and turnip rape.In August, the most important crop plants listed were Epilobium angustifolium (also in July), followed by heather Calluna vulgaris (L.) Hull and Trifolium spp.
Results of pollen analysis

General description
Appendix 1 gives the annual means and standard deviations of the pollen counts, the frequencies of the pollen types in the material, and the zonal means of the counts for each pollen type and for the honeydew elements.
A total of 70 pollen types were identified.Of these, 26 were identified to the species level, 37 types to the generic level, and 7 to the family level.The number of new pollen types found decreased progressively with the number of samples examined (Fig. 5).The number of pollen types found in a sample ranged from 7 to 30, averaging 16 (1977) and 18 (1978).Most types represented nectariferous plants.One quarter represented non-nectariferous plants, the commonest of these being the wind-pollinated Alnus, Betula , 8 Fig. 4. Average honey yields in zones I-IV+V in 1977 and 1978 (shaded).In this and the following figures means followed by dif- ferent letters are significantly different at the 5 % level, Tukey-Kramer method.The vertical lines indicate standard deviations.
When the overall mean is taken as 100, the lowest total pollen count was 6 and the highest 414.Myosotis pollen was found in 5 % (1977) and 20 % (1978) of the samples.The numbers of honeydew elements found in a sample averaged 15 (1977) and 12 (1978); in all samples, the number of pollen grains exceeded the number of honeydew elements.
The nine clearly most frequent and numerous pollen types (Fig. 6) constituted 90.8 % (1977) and 90.3 % (1978) of all the pollen examined.Of this, the proportion of Salix pollen was 6 % in 1977 but 18 % in 1978.The Brassicaceae alone were represented by 47 % (1977) and 39 % (1978) of the grains counted.The count of Trifolium repens L. 4-T.hybridum L. was lower, but the frequency of this type was higher: it was found in every sample.Pollen grains of Prunus padus, Mains domestica, Sorhus ancuparia and Ruhus idaens (''the Rosaceae group") were numerous, but the species were difficult to distinguish from each other by the microscopic procedure.
The counts given for pollen types within the group should be considered estimates.The counts of Apiaceae pollen and pollen of Filipendnla uimaria (L.) Maxim, were almost the same in the two years.The deviations of the pollen counts were high due to the positive skewness of the frequency distributions.
The picture of the quantitative relationships of the pollen types obtained by averaging the pollen counts (Fig. 6) differed from that obtained by averaging the proportions (Fig. 7).E.g. the average proportion of Bras- sicaceae pollen was 36 % (1977) and 30 % (1978).
Total pollen count in relation to Brassicaceae pollen The total pollen count was positively correlated with the proportion of Brassicaceae pollen (Fig. 8).The coefficient of determination was 24 % (1977)  proportion of Brassicaceae pollen.The mean y is treated as 100. 95% confidence limits for the estimated mean of y are given.
and 16 % (1978).The regression line obtained in 1978 was similar to that obtained in 1977.
Relation of Salix pollen with other pollen types Especially in 1978 the counts of pollen of Alnus, Betula, Acer spp.and anemones were positively correlated with the count of Salix pollen (t-test, H o :q=0).The correlation coefficients and the levels of significance On average, the number of pollen types in the 1978 samples exceeded the number in the 1977 samples by three (t(ii4) = 3.98 :::; ' !; ').
Pollen of anemones was found only in 1978 samples, and the pollen of Caragana arhorescens Lam. and Liliaceae was also almost restricted to that year.The total count of pollen of non-nectariferous plants was higher in 1978 than in 1977 due to Rumex and Plantago pollen (App. 1).
The average count of Salix pollen was higher in 1978 than in 1977.Malus domestica pollen occurred in higher quantities, although less frequently, in 1977 than in 1978.Pollen of Trifolium repens + T. hybridum was best represented in the 1978 samples (Table 3).Trifolium repens + T. hyhndum 71.9 28.5 -43.4 2.26»

Regional variation
The total pollen counts of the honey samples (Fig. 9), and the counts and frequency of Brassicaceae pollen (Fig. 10) were highest in the southern samples and lowest in the northern samples.Especially in the southern samples, the annual variation in the occurrence of Salix pollen was evident (Fig. 11A).Regional differences in the average counts of Prunus padus pollen was Fig. 9. Zonal averages of the total pollen counts in 1977 and 1978 (shaded).5 % level, Tukey-Kramer method.almost the same in 1978 as in 1977, although the variation was not significant (Fig. 1 IB).Pollen of Rubus idaeus was most abundant in the samples from Zone 111 (Fig. 11C), and pollen of Sorbus aucuparia in the samples from the northernmost Zone IV+V in 1978 (Fig. HD).None of the pollen types was characteristic of only one region, and the number of pollen types found in the samples from the different regions tended to be the same.
Variation connected with the race of bees Compared with the honey produced by Italian bees, the honey produced by bees of mixed race had a total pollen count that was 7.0 % units higher in 1977 (t(74)=1.99*)and a Brassicaceae pollen count that was 57.5 % units higher in 1977 (t(?4)=2.76**)and 57.1 % units higher in 1978 (t(55)=2.22*).On the other hand, the Salix pollen count in the 1978 samples was 69.7 % units higher (t(55)=2.26*)in the honey of Italian bees, than in the samples produced by bees of mixed race.

Number of pollen types
The number of honey samples studied was sufficient to reveal the commonest types of pollen in Finnish honey.Any further types found by increasing the number of samples would have been only occasional.As the number of types depends on the depth of taxonomic differentiation, it is not directly comparable with the results of previous studies.However, the average number of pollen types in Finnish honey does not differ essentially from that in other European honeys (see MAURIZIO 1978).
Change in the pollen spectrum from 1960 to 1978 Of the dominant pollen types in Finnish honey, Brassicaceae pollen and pollen of Trifolium repens + T. hybridum show changes in their proportions during the past two decades.Comparison with the results of AARIO (1961) and PUROKOSKI and FORSIUS (unpublished) fails to reveal any clear changes in the proportions of the other most common types (Fig. 12).Besides Brassicaceae pollen, the grains of ''the Rosaceae group", Apiaceae and Filipendula uimaria show slight rising tendencies in their frequencies.
The proportion of Brassicaceae pollen has grown together with the acreage of cultivated turnip rape, Brassica rapa L. ssp.rapa v. oleifera (Fig. 13).Winter turnip rape was earlier considered to be an important bee plant in Finland (AARIO 1961, PUROKOSKI and FORSIUS, unpublished).With the change to the later flowering spring varieties, the importance of turnip rape as a nectar yielder has evidently risen.Furthermore, the migratory beekeeping organised for pollinating the stands favours turnip rape and rape at the cost of other bee plants.Rape, B. napus L. v. oleifera, is also a good bee plant (e.g.HOWES 1979).However, the acreage of rape is ca. 10 % of the total acreage of oil plants in Finland, whereas that of turnip rape is ca. 90%.
The proportion of pollen of white and alsike clover, Trifolium repens and T. hybridum, has decreased in parallel with the increase in the proportion of Brassicaceae pollen.It is noteworthy that the frequency of clover pollen has not fallen.Current agricultural practices unfavourable for clover are: the use of rapidly growing, shading grass species in pastures, relatively heavy nitro- gen fertilization, and the general decrease in cattle farming and pastures, especially in southwestern Finland.ANON. 1962(ANON. , 1981)).Observation from the year 1937 by MARTIMO (1945), other years as in Fig. 12.
The total pollen count in honey has apparently increased together with the proportion of Brassicaceae pollen.Thus the decrease in the importance of white and alsike clover as bee plants is probably smaller than is suggested by the decrease in the proportion of their pollen.

Problem of botanical origin
The pollen grains are assumed to bear some linear relation to the nectar contributions of the plants.However, some plants may yield nectar without being represented by pollen in the honey (DEMIANOWICZ 1964).On the other hand, the relative abundance of pollen of non-nectariferous plants in the pollen spectrum of honey indicates that pollen of nectariferous plants may also be present in honey although the corresponding nectar did not occur in the raw material (for example Myosotis, see p. 406).
The positive correlation observed between the proportion of Brassicaceae pollen and the total pollen count also indicates that the growth in the proportion of Brassicaceae pollen was largely achieved by an increase in the number of Brassicaceae pollen grains, rather than by a decrease in the number of other kinds of pollen grains per unit honey weight.Consequently, the change in the proportion of Brassicaceae pollen does not indicate a directly corresponding change in the contribution of Brassicaceae nectar.Thus, the pollen spectrum alone is not a reliable index of the botanical origin of honey (see also DEMIANOWICZ 1961DEMIANOWICZ , 1968).Furthermore, the total pollen count varies from sample to sample and averaging the percentages obtained for the samples by qualitative analysis gives a misleading basis for deducing the botanical origin.This was clearly illustrated by the differences in the quan- titative relationships revealed by the average proportions and the average counts in the samples studied.

Honeydew
According to LOUVEAUX et al. (1978) the origin of honey may be determined from the pollen spectrum only if the ratio of honeydew elements to pollen grains is below one.According to this, the proportion of honeydew was not significant in the present honey samples.However, a more detailed study is required to find out the real importance of honeydew in Finland.
Seasonal changes in pollen spectra Bee plants flowering in spring.The annual variation in the count of Salix pollen was concentrated in the southern regions: towards the north the arrival of spring and the flowering of Salix become increasingly later and rapider, and the critical difference between the years in the progress of spring occurred as early as May.The importance of Salix spp. in providing pollen for the bees in spring is indicated by the correlation of the content of Salix pollen with the numbers of pollen grains of non-nectariferous plants flowering in spring, especially Alnus spp.The 1978 honey samples contained three times more Salix pollen than the 1977 samples.As the honey yields were also higher in 1978 than in 1977, this agrees with the assumption that a connection exists between the utilization of Salix pollen, the spring development of the bee colonies and their later productivity.
Pollen of Malus domestica was found more seldom, but in greater quantities in the less favourable season 1977 than in 1978.This indicates that, when needed and when its flowering is successful, Malus domestica (as well as e.g.Vaccinium spp.flowering at the same period) may substitute for Salix spp. as a pollen plant.
Mid season bee plants.The few species represented more often in the honey of 1977 than in the samples of 1978 were apparently compensatory sources for bees.Trifolium spp.seems to have a certain ability to even out the annual variation.It would be desirable to find out what bee plants are productive even in relatively poor weather conditions.
In 1978 the bees were able to visit the non-nectariferous pollen plants more intensively than in 1977.Plantago spp.are known as pollen plants (HANSSON 1976).The variation in the frequency and numbers of Rumex grains was similar to that of Plantago pollen.Also, bees have been reported to collect pollen intentionally from Rumex spp.(KÄPYLÄ and NIEMELÄ 1979), so it may have a more important function as a bee plant than is yet known.
Altogether, the supply of bee plants was richer in 1978 than in 1977.The importance of the main species was emphasized in 1977 at the cost of minor and occasional species.

Regional characteristics
Brassicaceae pollen was most abundant in the honey of the southern regions, as turnip rape, the main source of Brassicaceae pollen, is cultivated there (mainly in zones I and II).Turnip rape is evidently one of the main honey yielders wherever cultivated.On the other hand, Brassicaceae pollen is clearly over-represented in relation to the nectar that it contributes to the honey.Thus, the total pollen count of the honey samples was highest in the southern regions.In view of its over-representation and the fact that beekeeping in Finland is concentrated in the same regions as the cultivation of turnip rape, the predominance of Brassicaceae pollen in Finnish honey is understandable.
Characteristic pollen spectra clearly differentiating the honeys of the different regions did not exist.This result was expected, as the beekeeping area is a relatively small and phytogeographically uniform area.The regional differences found were merely quantitative and give only a rough indication of the geographical origin of honey within Finland.
Bee race and pollen spectrum As the spring development of Italian bees is more rapid in favourable conditions, in 1978 this race was able to utilize Salix spp.better than bees of mixed race.This may be partly due to the fact that the most skilful and active beekeepers mainly keep Italian bees.The average difference in honey yields, which was ca.20 % units in the favour of the Italian bees in both the years, was not significant, however.
If it is assumed that the amount of pollen entering honey with nectar does not vary with the race, then the Italian bees did not use turnip rape (indicated by Brassicaceae pollen) as much as the bees of mixed race.This was not due to differences in distribution, as the proportions of apiaries with the different breeds was the same in all the regions in both the years.A partial explanation may be that as the Italian bee has the longest tongue (glossa), it is able to utilize a greater variety of crop plants than the other races in Finland.
The number of apiaries of Caucasian bees or even dark bees participating in this study was so small that comparison of the result is not possible.   Berberidaceae, Prunus cerasus, Fragana, Rosa spp., Geum rivale, Melilotus, Geranium, Lonicera, Veronica, Ajuga, Galeopsis, Lamtum spp., Hyssopus officinalis, Campanula spp.

Fig. 2 .
Fig. 2. Distribution of the honey samples in the agricultural zones I-V.(The weather stations are numbered from 1 to 17, means given inTable 1.)

Fig. 3 .
Fig. 3. Distribution of the honey samples according to the time of removal of the yield.(Only samples with exact date are in- cluded.)

Fig. 5 .
Fig. 5. Number of the pollen types found in relation to the number of samples examined.

Fig. 8 .
Fig.8.Regression of the total pollen count on the proportion of Brassicaceae pollen.The mean y is treated as 100. 95% confidence limits for the estimated mean of y are

Table 1 .
Temperature (°C) and hours of sunshine in Finland inMay-August 1977 and 1978 (ANON.1977a,1978a), Average deviations form normal temperatures are weighted according to the distribution of the honey samples. (Means of 17 weather stations, see Fig.2.)

Table 2 .
Distribution of the apiaries and the honey samples by the race of the bees (%).