Multivariate analysis of pollen frequency of the native species Escallonia pulverulenta (Saxifragaceae) in Chilean honeys

Análise multivariada da freqüência do pólen da espécie nativa Escallonia pulverulenta (Saxifragaceae) em méis do Chile

ARTICLES

Multivariate analysis of pollen frequency of the native species Escallonia pulverulenta (Saxifragaceae) in Chilean honeys

Análise multivariada da freqüência do pólen da espécie nativa Escallonia pulverulenta (Saxifragaceae) em méis do Chile

Gloria Montenegro¹; Raúl C. Peña; Rodrigo Pizarro

Pontificia Universidad Católica de Chile, Facultad de Agronomía e Ingeniería Forestal, Departamento de Ciencias Vegetales Casilla 366, Santiago-22, Chile

ABSTRACT

The aim of this work was the identification of geographic zones suitable for the production of honeys in which pollen grains of Escallonia pulverulenta (Ruiz & Pav.) Pers. (Saxifragaceae) can be detected. The analysis of botanical origin of 240 honey samples produced between La Serena and Puerto Mont (the IV and X Administrative Regions of Chile), allowed the detection of pollen grains of E. pulverulenta in 46 Chilean honeys. The geographic distribution of the honeys studied is presented together with their affinities, through factor analysis and frequency tables. The study was based on the presence of E. pulverulenta pollen. Escallonia pulverulenta pollen percentages oscillated between 0.24% and 78.5%. Seventeen of the studied samples were designated as unifloral - i.e. samples showing more than 45% pollen of a determined plant species. Two of these corresponded to E. pulverulenta (corontillo, madroño or barraco) honeys. The remaining unifloral honeys correspond to 8 samples of Lotus uliginosus Schkuhr (birdsfoot trefoil), 2 samples of Aristotelia chilensis (Molina) Stuntz (maqui) and 1 sample of Escallonia rubra (Ruiz & Pav.) Pers. (siete camisas), Eucryphia cordifolia Cav. (ulmo or muemo), Weinmannia trichosperma Cav. (tineo), Rubus ulmifolius Schott (blackberry) and Brassica rapa L. (turnip). Honeys with different percentages of E. pulverulenta pollen - statistically analyzed through correspondence analysis - could be associated and assigned to one of three geographic types, defined on the basis of this analysis. The geographical type areas defined were the Northern Mediterranean Zone (samples from the IV Region), Central Mediterranean Zone (samples from the V to the VIII regions including two samples of unifloral Escallonia pulverulenta honey), and Southern Mediterranean Zone (samples from the IX Region).

Key words: - Apis mellifera, Chile, Escallonia, Mediterranean type climates, unifloral honey

RESUMO

O objetivo do presente trabalho foi o de identificar as zonas geográficas preferidas para a produção dos méis que contêm grãos de pólen provenientes de Escallonia pulverulenta (Ruiz & Pav.) Pers. (Escalloniaceae), espécie essencialmente nectarífera. A análise da origem botânica de 240 méis, produzidos entre La Serena e Puerto Montt (IV e X regiões administrativas do Chile), detectou a presença de Escallonia pulverulenta em 46 destas amostras. A distribuição geográfica dos méis examinados é apresentada juntamente com suas afinidades morfopalinológicas, usando diagramas de freqüência e de análise de fatores (análise de correspondência). As porcentagens do pólen do E. pulverulenta variaram entre 0,24% e 78,5%. Dezessete das 46 amostras estudadas foram designadas como meis uniflorais, isto é, tem uma freqüência acima de 45% do pólen de uma determinada espécie de planta, mas somente duas corresponderam ao mel unifloral de E. pulverulenta. Dos méis uniflorais restantes, oito corresponderam a Lotus uliginosus Schkuhr (nome vulgar lotera, alfalfa chilota), dois a Aristotelia chilensis (Molina) Stuntz (maqui) e um a cada uma das espécies de Escallonia rubra ('siete camisas'), Eucryphia cordifolia Cav. (ulmo, muermo), Weinmannia trichosperma Cav. (tineo), Rubus ulmifolius Schott (mora) e de Brassica rapa L. (yuyo). Os méis com freqüência variável de pólen de E.pulverulenta, analisados estatisticamente usando a análise de correspondência, puderam ser associados e designados para cada uma das três zonas geográficas definidas com base nesta análise: a zona norte mediterrânea (amostras da IV região administrativa, a zona central mediterrânea (amostras entre a IV e VIII regiões administrativas, incluindo uma das amostras uniflorais de Escallonia pulverulenta e a zona sul mediterrânea (amostras da IX região administrativa).

Palavras-chave: - Apis mellifera, Chile, climas Mediterrâneos, Escallonia, mel unifloral

Introduction

Honey is produced by the honeybee Apis mellifera L. by the transformation of plant nectar and other sweet substances produced by the living parts of plants, or left on their surfaces by aphid insects (Codex Alimentarius Commission 1987, Bogdanov & Martin 2002). The botanical origin of a particular honey is one of the most important quality parameters which can add value to the product (Krell 1996, Montenegro et al. 2003). This can be determined using a melissopalynological method (Louveaux et al. 1978, Montenegro et al. 2003) which consists in the qualitative (identification) and quantitative (count and proportion calculation) analysis of the pollen grains content. Unifloral honeys - defined as those in which the dominant pollen grain is more than 45% of the total - have higher economical value than multifloral honeys since their sensorial properties (aroma, color and taste) and nutritive characters present greater consistency over time (Andrade et al. 1999, Montenegro et al. 2005a).

Applying the Chilean Regulation of Honey Denomination via Melisopalynological Analysis which was published recently in this country (Montenegro et al. 2005a), it is possible to classify a honey as unifloral, bifloral or multifloral.

Chile produces a limited number of unifloral honeys with native plant origin. These are mainly derived from Quillaja saponaria Molina (quillay) in the central zone, and Eucryphia cordifolia Cav. (ulmo) in the southern zone (Ramírez & Montenegro 2004, Montenegro et al. 2005b). Given their excellent qualities, these honeys are greatly appreciated in the international market and generate better returns than non differentiated honeys. However, unifloral honeys are also produced from other native Chilean plant species, such as Gevuina avellana Molina (avellano) and species of the genus Escallonia. For this reason the botanical origin of the honey produced by local beekeepers should be certified, identifying the species composition of the differentiated honey's pollen spectra. In Chile the genus Escallonia comprises thirteen species in the continental territories and one in the Juan Fernandez archipelago (Sleumer 1968). Various species of the genus (E. pulverulenta (Ruiz et Pav.) Pers., E. illinita K. Presl, E. revoluta (Ruiz et Pav.) Pers., E. rosea Griseb. and E. rubra (Ruiz et Pav.) Pers.) have been detected in the pollen spectrum of Chilean honeys. However the most important contribution is from E. pulverulenta (Montenegro et al. 2005a). The morphology of the pollen grain of E. pulverulenta - monad, isopolar, radiosymmetric, tricolporate, colpi length, recessed, pores short, circular, located in the area where colpi are mildly depressed, circular-subprolate, amb circular, exine ca. 1 μm in thickness, tectate, columellate, collumellae distinct, finely reticulate - is distinctive for the genus (Heusser 1971). The grain is commonly smaller than other Escallonia species, with 20-22 μm in equatorial axis and 19-21 μm in polar axis.

In botanical terms, E. pulverulenta, as well as being a nectar source for honey production, is a main resource in the production of propolis. This plant is known, amongst other names, as barraco - which in Spanish means boar - due to its penetrating aroma. It is a shrub with resinous leaves and large inflorescences in spiciform racemes which are very attractive to pollinators such as A. mellifera (Bonod et al. 2003). It has been demonstrated that E. pulverulenta accumulates iridoids such as asperuloside, dafiloside and geniposide as well as other compounds which include the flavanone ether of 7-methylacacetine, kaempherol, rutine and chlorogenic acid (Plouvier 1956, Tomassini et al. 1993, Taskova et al. 2001). Given that E. pulverulenta is an endemic plant in Chile, the aim of this work was to analyze its pollen frequency in Chilean honeys, and undertake a multivariate analysis in order to determine the importance of this species in the geographical production of Chilean honeys.

Materials and methods

Hive locations - Of a total sample of 240 honeys, harvested from hives located between 30° and 39° S, and 70° and 72° W, whose botanical origin had been determined previously (Montenegro et al. 2003, Ramírez & Montenegro 2004, Montenegro et al. 2005b), 46 honeys which contained E. pulverulenta in their botanical composition were selected for analysis. The zone is characterized by a Mediterranean-type climate, with a dryness gradient from arid to perhumid trend (Di Castri & Hajek, 1976). This area has different anthropization degrees, with ample agrosilvicultural landscape, and the original flora reduced to patches or forest fragments (Grez et al., 2006). The apiaries were selected in basis to their location, in zones dominated by native flora.

The distribution of the forty-six honeys samples by administrative region was as follows (see tables 1-3 for numbers): six from the IV Region (samples 41 to 46), five from the V Region (samples 36 to 40), five from the Metropolitan Region (samples 31 to 35), eight from the VI Region (samples 23 to 30), five from the VII Region (samples 18 to 22), one from the VIII Region (sample 17), and sixteen from the IX Region (samples 1 to 16). The locations of the sampling apiaries are shown in figure 1.

Analysis of botanical origin of honeys - In order to analyze botanical origin, for each sample 20 g of honey were diluted with 20 mL of distilled water and the pollen concentrated by centrifuging at 2500 rpm for five min (Louveaux et al. 1978 modified). The pollen sediment was subsequently suspended with 0.1 mL of distilled water and of this suspension five aliquots of 20 μL were extracted in order to make five preparations for the optical microscope, using the method described in the literature (Louveaux et al. 1978), modified as in the Chilean Norm of Botanical Origin Denomination through melissopalynological method (Montenegro et al. 2004, Montenegro et al. 2005a, Montenegro & Pizarro 2006).

Frequency assessment was carried out qualitatively using comparison with palynotheque and pertinent references (Heusser 1971, Hodges 1984, Erdtman 1986) and quantitatively following Louveaux et al. (1978), Moar (1985), with our modifications (Montenegro et al. 1992, Montenegro et al. 2005a). A minimum of 400 pollen grains were counted for each honey sample.

Statistical Analysis - In order to see the relationships between each honey's pollen frequency and its geographical origin, a correspondence analysis was applied, using Statistica version 6^® (Stat Soft) software. For this analysis, all pollen types present in the honey's pollen spectra were considered. With the objective of verifying associations between pollen types present in the honeys' pollen spectra, a classification analysis was carried out using the cladistics program 'Hennig86' to recognize similarities between pollen types associations in the various analyzed honeys. The option 'multiple most parsimonious trees option' (mhennig*) was used. Pollen grain types percentages were coded as '0' (percentages less than 45%), '1' (ranging between 45 and 70%), and '2' (more than 70%). This analysis did not consider pollen types with percentages lower than 1%.

Results

The botanical origins of each of the 46 honey samples are shown in tables 1 to 3. Unifloral honey is considered if contains at least 45% from a single pollen types (Montenegro et al. 2005a), 17 of the 46 samples analyzed were identified as unifloral. Of these, eight were of Lotus uliginosus Schkuhr (lotera or alfalfa chilota), two of Aristotelia chilensis (Molina) Stuntz (maqui), two of E. pulverulenta (barraco), one of E. rubra (siete camisas), one of E. cordifolia (ulmo or muemo), one of Weinmannia trichosperma Cav. (tineo), one of Rubus ulmifolius Schott (mora) and one of Brassica rapa L. (yuyo).

Forty six botanical families were identified in 46 honey samples with pollen grains of E. pulverulenta present in them. These include Fabaceae and Myrtaceae (represented in 14 samples); Rhamnaceae (in 8 samples); Rosaceae (in 4 samples); Anacardiaceae, Boraginaceae, Brassicaceae, Lamiaceae, Lauraceae and Solanaceae (in 3 samples each); and Asteraceae, Cunoniaceae, Euphorbiaceae, Gesneriaceae, Proteaceae and Vitaceae (in 2 samples each).

The correspondence analysis permitted the identification of three types and geographical associations of honeys (figure 2). The first group (geographic type 1) includes the six honey samples harvested in the extreme north of the study area (IV Region) in the Mediterranean climate zone with arid trends. The second group (geographic type 2) was composed of 24 honey samples produced between the V and VIII regions in the Mediterranean zone with semiarid, subhumid and humid trends. The third group (geographic type 3) corresponded to the sixteen honey samples obtained in the extreme south of the study area (IX Region), in the Mediterranean climate zone with perhumid trends.

By applying a correspondence analysis (figure 3) the dominant genera in each geographic type could be determined. The singular values, eigen values, inertia percentage, cumulative percentages and chi squared are shown in table 4. The cumulative percentages of the two first axis comprised 16.6 of the variability. The main inertias were exhibited by Heliotropium stenophyllum Hook. & Arn. for the samples of geographic type 1, Quillaja saponaria, Retanilla trinervia Hook. & Arn. and Raphanus sativus L. for geographic type 2, and Weimannia trichosperma, Aristotelia chilensis and Lotus uliginosus for samples of geographic type 3. The unifloral honeys of Escallonia pulverulenta appeared in the type which we define as geographic type 2, originated from the vegetation of the V region, and corresponding to the typical sclerophyllous matorral of Central Chile. The pollen fractions of these honeys contain 78.5% (sample 38) and 74.3% (sample 39) of E. pulverulenta pollen. An exploratory classification using the cladistics software "Hennig86" (figure 4) indicated that the species found in the honey samples form three clearly delimited groups. These groups are 1. Aristotelia-Amomyrtus. 2. Weinmannia-Eucryphia-Lotus, 3. Escallonia-Lithrea, Brassica-Raphanus-Schinus-Castanea-Cordia, Persea, Prunus and Retanilla.

Discussion

The analysis of 46 honey samples with presence of E. pulverulenta pollen collected in Chilean Mediterranean climate zones with different trends of aridity suggests that they can be subdivided into three geographic types, determined by their association with three different classes of botanical composition in their pollen spectra: 1. Aristotelia-Amomyrtus, 2. Escallonia-Lithrea, Brassica-Raphanus-Schinus, Castanea-Cordia, Persea, Prunus, Retanilla and 3. Weinmannia-Eucryphia-Lotus. Escallonia pulverulenta appears to be most strongly associated with plants of the second group (geographic type 2) (figure 3). This correlates well with its greater abundance, within its area of distribution, in the area of the Mediterranean climate zone with semiarid to humid trends (figure 1). In general the geographical types corresponded well with the different climate zones within the region with Mediterranean climate. The honey samples associated with geographic type 1, derived from the Mediterranean climate zone with arid trends; the honeys which determine the geographic type 2, derive from zones with semi arid, subhumid and humid trends; while geographic type 3 corresponded to honeys produced in the perhumid Mediterranean zone (di Castri & Hajek 1976).

Escallonia belongs to a monogeneric family (Escalloniaceae) or to Saxifragaceae. Little information exists about the participation of species of the family Saxifragaceae in honey production in other regions with Mediterranean climate zones (Andrade et al. 1999), but most of the literature related to the botanical origin of honey from Mediterranean climate regions has not reported the presence of pollen types associated with Saxifragaceae in the honeys analysed (Serra-Bonvehi & Ventura-Coll 1995, Mateo & Bosch-Reig 1998, Mendes et al. 1998, Tsigouri & Passaloglou-Katrali 2000, Herrero et al. 2001, Serrano et al. 2004, Soria et al. 2004). Chile is a country where it is possible to produce unifloral honey of E. pulverulenta, and the most promising location for its production is the Mediterranean climate zone with semiarid trends (the fifth and the metropolitan administrative regions, located between 32° and 33° of South latitude, approximately). The promotion of certification of unifloral Escallonia honey, and thereby of its origin, is highly recommendable, as currently only unifloral honeys of Q. saponaria (quillay) and E. cordifolia (ulmo) are recognised in the international market. The next step towards obtaining unifloral honey of E. pulverulenta or of other native and endemic plants is to ascertain the optimal season for harvest. This could promote the conservation of determined plant species with apicultural utility, and under some degree of threat, by encouraging their economic exploitation in a sustainable way with minimal ecological impact through apicultural activity (Butz 1997). This is particularly valid for those species with narrow geographic distributions concentrated in areas with dense human population, the presence of which implies permanent habitat impacts and a degree of vulnerability; such is the case for the Mediterranean climate zone of Central Chile (Cincotta et al. 2000).

Through statistical analysis which included the whole spectrum of pollen types present in the samples, we obtained only one type of unifloral honey, compared with the three or four classes obtained by melissopalynological analysis, which only considers the percentage of the dominant pollen type. Generally, honey is characterized by the abundance of some pollen types and the infrequency of others. Although the honey samples were derived from a large area (North, Center and South of the country), representing Chile's Mediterranean climate zone, many pollen morphs from the total of 105 pollen types found in the pollen spectrum of all samples were shared among several of them. In turn, much of the inertia was also shared, requiring eight to twelve components in the correspondence analysis which made it difficult to define clusters. Different climatic trends are described within the study area: arid, semiarid, humid, and perhumid or very humid (di Castri & Hajek 1976), yet only the last of these was easily identified; in the factorial analysis by the grouping of components, and in the cladistic analysis by the allocation of associated species (Aristotelia and Amomyrtus, both typical of the southern zone) to the first two branches which separate from the main group of the cladogram (see figures 3 and 4). However, by associating them with the melissopalynological analyses these methods may be useful for identifying the biogeographical origin of a certain honey. Because the discriminative power of these methods is not based on dominant pollen type alone, which can be adjusted by the addition of pollen to the honey, but rather includes the total of all pollen types which appear in the pollen spectra, this could thereby help to prevent adulteration. Today the prediction of zones with apicultural potential is fundamental to the commercialisation of products with added value. Chile could be converted in a country unique in the production of endemic honeys such as E. pulverulenta honey, an attribute for which it could become recognised worldwide.

Lotus uliginosus "bird trefoil" a dominant pasture species in the South geographic zone requires further comments: first the sampling take into account Escallonia containing profiles, nevertheless in eight cases appear consistently Lotus-frequencies. Many of them may be typified as multifloral no-native in the Chilean Norm (NCh 2981. Of 2005) terms, or still as a multifloral mixture (Ramirez & Montenegro 2004, Montenegro et al. 2008). In this study pollen of this species appears consistently associated with Weinmannia and Eucryphia pollen grains as can see on cladistic analysis.

Aknowledgements - The authors are grateful to Prof. O. Monika Barth for the Portuguese translation. Funding by FONDECYT 1060535 and Corfo FDI-INNOVA 06CN12IAD-01 to G, Montenegro.

(received: July 04, 2007; accepted: October 14, 2010)

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Publication Dates

• Publication in this collection
  17 Oct 2011
• Date of issue
  Dec 2010

History

• Received
  04 July 2007
• Accepted
  14 Oct 2010