Intraannual variations in the pollinic spectrum of honey from the lower valley of the River Chubut ( Patagonia , Argentina )

Thirty honey samples from the lower valley of the River Chubut, obtained by successive harvests in five apiaries over two consecutive apicultural periods (between 1997 and 2000) were analysed. Three hives per apiary were selected, from which three successive harvests were obtained at the end of the following periods: initial (September 1-December 20), middle (December 20-January 30) and final (January 30-March 15). Pollen types were identified by comparison with a reference collection. Both reference pollen and sample pollen were subjected to acetolysis. A total of fifty entomophilous pollen types were identified: thirty-six, thirty-five and thirty-seven in initial, middle and final periods, respectively. The most abundant pollen was similar over the whole season, due to the long flowering periods of the taxa producing dominant and secondary pollen. Tamarix gallica and Medicago sativa were main sources of nectar during the entire production period. Significant differences (P<0.001) in pollen content were detected when honey from the three production periods was compared. Most unifloral honeys were produced in Spring. In this season, the major contribution was recorded from the southern district of the Monte. Anemophilous pollen was present in all the samples. The number of pollen grains per gram of honey was less than 10,000 in the three periods considered.

Palabras clave: plantas melíferas, polen, melisopalinología periods of monofloral honey production to be identified, which have a greater commercial value.
Although few researches have focused on this issue, works at different latitudes have used melissopalynological analyses to study the nectariferous resources selected by A. mellifera during the apicultural period either by stepped harvests (Seijo et al., 1992a and b;Basilio, 1998) or by examining at regular intervals the nectar deposited in the honeycombs (Adams et al., 1979;Adams and Smith, 1981;Parent et al., 1990).
In Argentina, although melissopalynological studies have been carried out in some provinces, most have aimed at classifying honey according to its botanical and geographical origin.There is only one study of the intraannual variations of the pollinic spectrum of honey, which was carried out in the Paraná Delta, a floristically highly heterogeneous area within the pampean phytogeographic province (Basilio, 1998).
Apiculture is largely undeveloped in the Chubut province.The most important apiculture area corresponds to the lower valley of the River Chubut, with around 2,500 hives in production.The apiaries, most of which are small (20-50 hives), are mainly located in the areas of Trelew, Gaiman and Dolavon (Fig. 1).The apicultural period has a shorter duration than in the north of the country owing to climatic conditions, since hive production begins in November and finishes at the end of March, with an average yield approximate of 35 kg/year/hive.
From palynological analyses of the final harvests, 55 nectariferous plants were found to be used by A. mellifera in the lower valley of the River Chubut (Forcone and Tellería, 1998).The aim of this work was to detect the resources most used in three stages of honey production by studying the samples from successive harvests and the differences in the pollinic composition of honey that could justify harvesting in stages.

Characteristics of the area
The lower valley of the River Chubut (43-44°S and 65-66°W) is situated in the province of the same name between the Departments of Rawson and Gaiman.The region has a temperate cold semidesertic climate.The mean annual temperature is 12.7°C and the average annual rainfall ranges from 175 to 180 mm.The frost-free period covers 117 days from mid-November to mid-March (Walter et al., 1975;Arbuniez de Mc Karthy, 1994;Leon et al., 1998).From a phytogeographical viewpoint, the lower valley of the River Chubut is situated in the southern district of the Monte (Cabrera, 1971;Leon et al., 1998).Characteristic vegetation of this region is «jarillal» (Larrea divaricata Cav)., species accompanied among others by Larrea nitida Cav., Prosopidastrum globosum (Gillies ex Hook & Arn) Burkart, Prosopis alpataco Phil., Schinus johnstonii Barkley, Chuquiraga erinacea D. Don, Ch. avellanedae Lorentz, Lycium chilense Miers.ex Bertero, L. ameghinoi Speg., Junellia ligustrina (Lag.)Moldenke, Atriplex lampa (Moq.)D. Dietr., Cyclolepis genistoides D. Don and Suaeda divaricata Moquin.From this community only some species, mainly the halophiles, descend to the flood plain where saline soils with very low drainability are frequent (Soriano, 1950).Since over 100 years ago, seasonal irrigation from September-April has been carried out.The most important crops are foraging plants, mainly Medicago sativa L. To a lesser extent horticultural crops, fruit and cereals are grown.Among the forest crops, Populus spp.are predominant, especially P. nigra L., used to form wind breaks, and Salix spp.and Tamarix gallica L., the latter of which is naturalised and widespread in Patagonia (Rossow, 1988).

Sampling procedure
The pollinic contents of 30 honey samples obtained sequentially in five apiaries during two consecutive apicultural periods were studied.The apiaries were selected for having an area and honey production representative of the study area, with a mean size of 50 hives.In all cases hives were of the Dadant type.The locations of the different apiaries are indicated in Figure 1.In the G (Gaiman), D (Dolavon) and A (ABEPA) hives, harvests were carried out in the seasons 1997-1998and 1998-1999, and in apiaries B (Siguero) , and in apiaries B (Siguero) and C (Neira) harvests were obtained in the seasons 1998-1999 and 1999-2000.Three hives per apiary were randomly selected and successive harvests were carried out in each one, corresponding to three apicultural periods: initial, from the start of the season (1 September) until the first operculation (20 December); middle, 20 December-30 January; and final, 30 January-15 March.Three empty labelled frames were placed in the hives at the start of each period, which were removed and replaced at the end of each of these steps as described in Seijo Coello et al. (1992a).The honey from the frames removed from all three hives was extracted together by centrifugation obtaining one sample per apiary for each period.

Qualitative analysis
In order to determine the percentage representation of each taxon in the pollen contents of the honey, the methodology proposed by Louveaux (1978) was followed.Pollen types were identified by comparing them with a reference collection obtained using plants from the area; this collection was deposited in the palynotheque of the Facultad de Ciencias Naturales of the Universidad Nacional de la Patagonia (Sede Trelew).Pollen from the samples and from the reference collection was acetolysed.
To determine frequency distribution classes, 500 pollen grains were counted per sample and anemophilous pollen was deducted.Pollen types, according to their percentages, were classified into: >45%, dominant pollen (D); 15-45%, secondary pollen (S); 3-15%, pollen of minor importance (M); <3%, pollen traces (T).The samples in which one pollen type represented >45% were classified as monofloral, and those in which no pollen type reached this percentage were classified as mixed (Louveaux, 1978).This criterion was not used in the classification of monofloral honeys of Medicago sativa and Eucalyptus spp., types under and overrepresented, respectively, in the pollen contents of honey.In these plants, honeys were considered to be monofloral of M. sativa when 20% of the pollen was from this species and as monofloral honey of Eucalyptus spp., when 75% of the pollen was from this latter species according to Maurizio and Louveaux (1961) and Serra Bonvehi and Cañas Lloria (1988).

Quantitative analysis
To determine the absolute number of pollen grains per gram of honey the methodology proposed by Stockmarr was followed (1971).In all samples, the number of honeydew elements were counted and the HDE/P index (ratio of the number of honeydew elements/number of pollen grains) was calculated.
Palynological analysis was complemented with observations of A. mellifera activity on vegetation and records of the flowering phenology were carried out according to Anderson and Hubritch (1940).

Statistical analysis
The data of the qualitative palynological analysis of the harvests of each period were analysed by applying Chi-squared test with P<0.001.The 16 pollen types with the highest frequencies were considered.The samples from each harvesting period were compared and then the mean values for each period (Steel and Torrie, 1981).

Final period
The dominant pollen in the three production periods corresponded to Tamarix gallica and Medicago sativa, types that presented a frequency of occurrence of 100%.In the initial period Rosaceae and Prosopidastrum globosum were also found in this category.
The most abundant accompanying pollen types (with a percentage>10%) found over the whole production period were: Astereae, Brassicaceae, Eucalyptus spp.and Malvella leprosa; the latter two with a frequency of occurrence of 90% in the three stages studied.In the initial period Prosopis, Larrea, Lycium, Trifolium and Carduus spp.were also found; in the middle period, Centaurea, Melilotus, Taraxacum, Lycium and Carduus spp.; and in the final period, Centaurea, Melilotus, Cichorium, Larrea and Cressa spp.(Fig. 2).
The pollen composition of honeys was heterogeneous in all (initial, middle and f inal) periods (P<0.001).On the other hand, when comparing pollen composition of honeys from the three periods, we found highly significant differences (χ 2 = 2426.3;P< 0.001) (Table 2).
Anemophilous pollen was detected over the whole honey production period.In Spring, this mainly came from Plantago spp., and in Summer from Plantago spp.and Chenopodiaceae-Amaranthaceae.Other anemophilous types detected were Typha spp., Poaceae, Cyperaceae, Artemisia absinthium L., Juglans regia L., Zea mays L. and Ambrosia tenuifolia Spreng.
The number of pollen grains per gram of honey was low in all the samples (Fig. 3) and most were, therefore, placed in Group I described by Louveaux et al. (1978).The HDE/P index was lower than 1 in all cases.
Flowering periods of the taxa identified in the dominant and secondary pollen are shown in Figure 4.
Spring was the season with the greatest production of monofloral honey and most came from Tamarix gallica, coinciding with the optimum flowering of this species; the others were from fruit trees (Rosaceae), from the native shrubland (Prosopidastrum globosum) and from Medicago sativa.The latter started the phenophase of full flowering at the end of November, when there was a strong nectar flow to the hives.In this season too, the greatest contribution was made by plants from the Monte: Larrea spp., Lycium spp., Prosopidastrum globosum and Prosopis spp.

Discussion
The families most represented in pollen contents of the honey in the three periods considered were Fabaceae and Asteraceae.These families have been shown to be important sources of nectar at different latitudes (Crane, 1991) and in different honey-producing regions of Argentina (Tellería, 1988(Tellería, , 1992(Tellería, , 1996;;Tellería and Forcone, 2000;Basilio and Romero, 1996; An-  Tellería, 2002).Although both families predominated over the whole production period, the majority of species belonged to Fabaceae in Spring and to Asteraceae in Summer.
Although statistical differences were found when comparing pollen contents of the three production periods, the main components of pollen were the same over the whole season in accordance with the long flowering periods of the main sources of nectar.Tamarix gallica and Medicago sativa were the species that made the greatest contribution of nectar over the whole apicultural period and were the main components in monofloral honey.
The fact that the greatest number and diversity of monofloral honeys were obtained at the end of the Spring demonstrates the benefit of harvesting the honey produced at this time.Current practise in the study area is to harvest only once at the end of the season storing the honey on the hives and extracting a mixture of honeys from different flowerings.
The low pollen contents of the honey is related to the supply of nectar from Medicago sativa, present in all the samples.Similarly, some species frequently visited for nectar had very little or almost no presence in the honey pollen as occurred with Salix spp.and Glycyrrhiza astragalina Gillies ex Hook.& Arn.The low representation of these taxa is attributed to the pre-dominance of the female foot in the most widespread Salix spp.(S. fragilis L., S. alba L. and S. babilonica L.) and to the small pollen production of G. astragalina (personal observations).
The low presence of the willows and osiers, the abundance of tamarisk and the presence of pollen types characteristic of the south of the Monte (Lycium spp., Prosopidastrum globosum, Larrea spp.) distinguishes the Spring honey in the study area from that produced in the Paraná Delta, where Salix spp., Rosaceae, Cytrus spp., Myrtaceae and Amorpha fruticosa L. are the most abundant types at this time of year (Basilio, 1998).On the other hand, although summer honey from both areas is distinguished by its dominant types and by the presence of pollen associations specific to the different regions, a common characteristic is abundant pollen from Astereae, mainly represented by Baccharis spp.and Solidago spp. in the Paraná Delta and by Grindelia tehuelches and Baccharis spp. in the lower valley of the River Chubut.
The abundance of anemophilous pollen is a constant feature of extra-Andean Patagonian honeys studied to date (Forcone andTellería, 1998, 2000;Tellería and Forcone, 2000).The presence of this pollen has been attributed to different factors, but mainly to contamination with corbicular loads (Louveaux, 1958;Fernández and Ortiz, 1994).However, this does not  seem to be a very likely origin in honeys from the lower valley of the River Chubut since these usually present low values of pollen per gram.Probably, the anemophilous pollen results from contamination of the nectar with pollen particles in the air, especially owing to the incidence of wind in the region.

Figure 1 .
Figure 1.Location of the lower valley of the River Chubut.A, B, C, D, G: apiaries sampled.Grey area: phytogeographical province of the Monte.

Figure 4 .
Figure 4. Flowering periods of the taxa found as dominant and secondary pollen.

Table 1 .
Frequency distribution classes and frequency of occurrence of entomophilous pollen found in three periods of the apicultural season in the lower valley of the River Chubut Frequency classes: D (dominant), S (secondary), M (minor), T (traces).The values indicate the percentage of samples in which the pollen types appear in each class.Frequency of occurrence (FO): percentage of samples in which the pollen types appears.* Native plants.

Table 2 .
Comparison of pollen composition of honeys of the three harvesting periods using the χ 2 test

Lyc Mal Mel Med Pros Pro Ros Tam Tar Tri Others
Classification of the samples according to the absolute number of pollen grains per gram of honey.