A melissopalynological map of the south and southwest of the Buenos Aires Province , Argentina

The aim of this work was to produce a melissopalynological map of the south and southwest of the Buenos Aires Province, Argentina, using pollen analysis data pertaining to 127 honey samples from the Pampa, Espinal (the Calden District), and Monte de Llanuras y Mesetas ecoregions, collected over the period 1992-2002. Using principal components and hierarchical cluster analysis, the different districts were grouped into five regions: I (Tres Arroyos, San Cayetano, Coronel Pringles and Coronel Dorrego), II (Guaminí, Saavedra, Coronel Suárez and Adolfo Alsina), III (Coronel Rosales, Monte Hermoso, Bahía Blanca and Villarino), IV (Patagones and Tornquist), and V (Puán). In Regions I, III and IV, 80% of honey samples were monofloral: Region I was characterized by the presence of 50% Helianthus annuus honeys and 10% clover honeys, Region III by 65% Eucalyptus sp. honeys, and Region IV by 30% Diplotaxis tenuifolia honeys. In Regions II and V, 50% of honeys were monofloral. Region II was distinguished by the presence of 50% H. annuus honeys, and Region V by 15% Larrea divaricata and 15% Vicia sp. honeys. The multifloral honeys of Region V included samples containing Condalia microphylla pollen. The families Fabaceae and Asteraceae provided the greatest diversity of pollen types. The association of Eucalyptus sp., Centaurea sp., and Diplotaxis tenuifolia characterised the honeys from all f ive regions. The natural variability of honey samples renders it very difficult to define the boundaries between the different regions. Additional key words: botanical origin, geographical origin, honey, melissopalynological, pollen analysis.


Introduction
Argentina is one of the world's main suppliers of honey, and in recent years local apicultural activity has grown appreciably.In 2005 the country exported 100,000 tons of this product (SAGPyA, 2006).Honey production in the Buenos Aires Province represents more than 50% of the national output (AACREA, 2003).Only about 8% of Argentinean honey is consumed domestically; most is exported in bulk.
The aim of this work was to construct a melissopalynological map of the south and southwest of the Buenos Aires Province, and to establish the geographical distribution of honeys according to their botanical origin.

Characteristics of the study area
The study area, the south and southwest of the Buenos Aires Province, includes the fifteen districts shown in Figure 1.The area is located in the south of the Pampa and Espinal ecoregions and includes a small area of the Monte de Llanuras y Mesetas ecoregion (Burkart et al., 1999).This area has a temperate cold, dry climate; snow occasionally falls in the hills.The mean annual temperature is 14ºC.The mean annual rainfall varies from 300 to 750 mm, with precipitations decreasing from the northeast towards the southwest (Cabrera, 1976).
The flat landscape of the Pampa ecoregion is only broken by the Ventania System which runs through the Puán, Tornquist, Saavedra, Coronel Pringles and Coronel Suárez districts.(Cabrera, 1976;Verettoni and Aramayo, 1976;Cabrera and Zardini, 1978;Lamberto et al., 1997).The south of the Pampa ecoregion also includes the San Cayetano, Monte Hermoso and Tres Arroyos districts, plus the north of the Puán, Tornquist, Bahía Blanca and Coronel Rosales districts.The Espinal ecoregion in Buenos Aires Province, includes the Patagones and Villarino districts, plus the south of the Puán, Tornquist, Bahía Blanca and Coronel Rosales districts.This region contains isolated specimens of Prosopis caldenia Burkart and Prosopis flexuosa AD. (left over from woodlands destroyed by anthropic action), and a herbaceous stratum rich in Poaceae.There are also dunes with psammophyllous vegetation and saline soils with halophyllous heathland and steppes.
The Monte de Llanuras y Mesetas ecoregion in the Buenos Aires Province includes a small part of the south of the Patagones district.The natural vegetation here is shrub steppe; the predominant species are Larrea sp., Disacaria americana and Chuquiraga sp.
The natural vegetation of the study area has been disturbed by agricultural activities.The most important crops are wheat (Triticum aestivum L.), sunflower (Helianthus annuus L.) and sorghum (Sorghum bicolor (L.) Moench.There are also horticultural crops along the lower valley of the Colorado River in the Villarino and Patagones districts.

Preparation of samples and analysis
Melissopalynological data were obtained from 127 honey samples collected at apiaries in 15 districts of the south and southwest of the Buenos Aires Province over the period 1992-2002 (Valle et al., 1995(Valle et al., , 2000(Valle et al., , 2001(Valle et al., , 2004;;Andrada et al., 1998aAndrada et al., ,b, 2000a,b;,b;Andrada and Tellería, 2002).A melissopalynological map was constructed using the pollen identified after its separation from the honey by centrifugation.Figure 1 shows the districts of origin and the number of samples obtained from each.The material was prepared and analysed according to the methods recommended by the International Bee Research Association (Louveaux et al., 1978).
Qualitative analyses were undertaken by counting and attempting to identify up to 1,000 pollen grains per honey sample.Pollen grains were identified by comparing them with those of a pollen reference collection produced by the authors for the plants of the study area.Plant samples were deposited at the Regional Herbarium of the Agronomy Department of the Universidad Nacional del Sur, Bahía Blanca, and the pollen preparations deposited at the palynotheca of the same institution.Pollen from the collected plants and honey samples were acetolyzed (Erdtman, 1960), mounted in glycerine gelatine and sealed with paraffin.Pollen types were identified at the level of species, genus, tribe or family.Specialized literature was also used in this task (Erdtman, 1966;Kremp, 1968;Heusser, 1971;Markgraf and D'Anthoni, 1978;Tellería, 1995Tellería, , 2000;;Tellería and Forcone, 2002).Publications regarding the flora of the Buenos Aires Province were consulted regarding scientific nomenclature (Cabrera, 1968;Lamberto et al., 1997;Zuloaga and Morrone, 1999).
The frequencies of occurrence of the different types of pollen grain (recorded as a percentage) were deter- mined by counting the number of honey samples in which they appeared.The pollen grain frequency classes were designated as dominant (D), secondary (S), important minor (M), and trace (T < 3%) (Fig. 2) (Louveaux et al., 1978).Nectarless plant pollen was excluded when calculating percentages (Louveaux et al., 1978).
Ninety eight pollen types were identified, but only the 16 types with the highest frequency of occurrence (≥ 20%) were selected for further study.Pollen types that were non-representative of the studied region, with a frequency of occurrence < 20%, and in the T frequency class, were not taken into account in statistical analyses.

Statistical analysis
In order to group the districts in terms of species of similar importance, the mean value of the ordinal scale of the species for each district was first calculated.The resulting matrix (matrix H; 15 districts × 16 species) was subjected to multivariate analysis.The covariance matrix (using matrix H as the data source) was subjected to principal components analysis (PCA), and the districts represented graphically on the planes of the first two axes.Each axis was interpreted according to its correlation with each species.Hierarchical cluster analysis was used to group the districts as a measure of their Euclidean distances and complete linkage.The resulting groups (regions) were obtained by considering the division points to lie at the mid points of the maximum distances obtained.

Results
The main characteristic of the honey samples from the study area was the association of Eucalyptus sp., Centaurea sp. and Diplotaxis tenuifolia pollen types in all samples (Fig. 2).Other pollens included those from Brassicaceae, Helianthus annuus, clovers (Melilotus sp., Lotus sp. and Trifolium sp.), Carduus sp., Cirsium sp., and Poaceae.
Table 1 shows the frequency of occurrence and frequency classes of the 98 pollen types identified in the 127 samples.The families Fabaceae and Asteraceae provided the greatest number of pollen types.
Figure 3 shows the PCA results.The distributions of the districts are shown on the plane of the first two PC axes.
Figure 4 shows the five regions obtained by cluster analysis.These regions are also shown in Figure 1.Region I includes the Tres Arroyos, San Cayetano, Coronel Pringles and Coronel Dorrego districts; Region II the Guaminí, Saavedra, Coronel Suárez and Adolfo Alsina districts; Region III the Coronel Rosales, Monte Hermoso, Bahía Blanca and Villarino districts; Region IV the Patagones and Tornquist districts; and Region V the Puán district (Fig. 4).
The pollen of Eucalyptus sp.showed a 60% frequency of occurrence in the honeys of the central region (Region III), a value not reached in other honeys from the Buenos Aires or La Pampa Provinces (Tellería, 1992(Tellería, , 1996a,b;,b;Naab et al., 2001) (Fig. 5).This can be explained by the widespread distribution of Eucalyptus sp. in Region III, where it is cultivated as a forest and ornamental species.
Although the eastern region (Region I) showed 50% sunflower honeys, this area is also characterized by 10% clover honeys; clover is characteristic of the humid prairies of the northeastern districts of the Buenos Aires Province (Tellería, 1992(Tellería, , 1996a,b),b).
The northwestern region (Region II) had 50% sunflower honeys and 50% multifloral honeys, with secondary pollen provided by Centaurea sp., Eucalyptus sp. and Condalia microphylla.The honey types of this region differ from those produced in the adjoining Utracán district of La Pampa Province (Naab et al., 2001) due to the absence of certain types of pollen typical of the Calden District flora, and to the presence of the sunflower honeys (attributed to agricultural activity).Clovers ( 6) Eucalyptus ( 8) (1) (1) (1) (2) Region IV showed 30% Diplotaxis tenuifolia honeys, while region V produced honeys from native species such as Larrea sp. and Vicia sp.These species were present in mixed honeys too, along with other representative species of the south of the Calden District such as Condalia microphylla and Prosopis sp.(Andrada, 2001).
The pollen spectrum of the honeys from the study area is characterised by the presence of pollen belonging to Diplotaxis tenuifolia, a perennial invading weed introduced into Argentina as a melliferous plant.It is now widespread in the southwest of the Buenos Aires and La Pampa Provinces (Rodríguez, 1974).This species is absent in honeys from the east and northeast of the Buenos Aires Province (Tellería, 1992(Tellería, , 1996a)).
The honeys from the study area resemble those from La Pampa Province because of the presence of Diplotaxis tenuifolia, but differ from them in the low frequency of native pollen types (Tellería, 1996b;Naab et al., 2001).
In general, the differences among the regions were not well defined, although the following conclusions can be drawn: i) the coastal districts, and their adjoining districts such as Tornquist and Coronel Pringles, are characterized by the presence of monofloral honeys; ii) in the northwestern districts, 50% of the honey samples are mixed; accompanying pollens came from Eucalyptus sp, Centaurea sp., and, in some samples, Condalia microphylla; with respect to the monofloral honeys, most were of Helianthus annuus although a few were of Larrea sp., and Vicia sp.(from the Espinal ecoregion); and iii) the natural variability of honey samples makes it very diff icult to def ine borders between the different honey regions.

)Figure 5 .
Figure 5. Percentage of monofloral and mixed honey samples collected in each region.

Table 1 .
The presence of taxa with S frequency class values, such as Eucalyptus sp., Helianthus annuus and clovers in Region I, Centaurea sp., Eucalyptus sp. and Condalia microphylla in Region II, Eucalyptus sp., Diplotaxis tenuifolia in Region III, Centaurea sp. and Eucalyptus sp. in Region IV, and Diplotaxis tenuifolia, Centaurea sp., Larrea sp., Condalia microphylla and Prosopis sp. in region V, is important in multifloral honeys (Fig. 2, Table 1).Frequency classes and frequency of occurrence of the 98 pollen types identified in the different samples

Table 1 (
cont.).Frequency classes and frequency of occurrence of the 98 pollen types identified in the different samples