Melipona beecheii (Apidae) Propolis Floral Origin from La Gloria Cosautlán de Carvajal, Veracruz, Mexico

1. Introduction

Around the world there are various bee species belonging to the family Apideae [1], which are essential for the continued functioning of life on the planet [2], because of the pollination process, an activity that is part of the physiological development of vegetation [3], and it is estimated that 70% of plants require this process to survive [4,5,6].

In the tropical and subtropical regions of Africa, the Americas, and Australia, stingless bees are the most abundant and active pollinators [7] where a greater diversity is located in the Americas, with more than 400 described species. Actually, stingless bees nest products are culturally important in this continent, where pot honey, pollen and propolis are used in medicine, ancestral ceremonies [8], and social, economic, and religious activities [9,10,11]. There are 46 bee species described in Mexico, six are grouped into 16 genera, that are found in Oaxaca, Chiapas, Veracruz, Yucatan, and other states [12,13].

Melipona beecheii Bennett, Xunan Kab, also known as the honey lady, the earth bee is a species that is part of the cosmogony, mythology, and spiritual significance for the Mayan from the Yucatan peninsula and northern Guatemala [14]. This species is also valuable for their pot honey, which is associated with fertility and breast milk production increase [15,16,17], for heat-cold imbalances [18] and current studies indicated health benefits related to nutritional properties [19]. Melipona beecheii is found in a wide distribution in tropical areas of Mexico to Costa Rica [20,21], is also an important pollinator [2,22], and one of the higher stingless bee pot honey producers [11], and also for its wax, pollen and propolis [23].

Propolis is a resinous, balsamic material collected, that is processed by bees and is essential for the nest, its production involves collecting resins from tree bark and flower buds, which are mixed with enzymes added by the bees. Other materials found in propolis include balsams, waxes, essential oils, pollen, spores, algae, plant trichomes, silica, clay, and starch [24,25]. In the nest, propolis is used to seal cracks, protect it from moisture, and combat pathogens due to its antimicrobial and antioxidant properties [26,27,28].

In humans, propolis is being used since ancient times; the Egyptians used it to preserve certain foods and to embalm bodies [29], Trigona and Melipona propolis I is being used by native people from the southeastern Mexico, as a wound healer and for skin infections, ears, respiratory tract, and intestines, as well as gum inflammation. Its use as a wood preservative, pest repellent, and in human and veterinary medicine [3,27,30,31,32,33], Recent studies registered its antiviral, astringent, anti-inflammatory, analgesic, antioxidant, antitumor, immunoregulatory, and hepatoprotective properties; its biological activities are attributed to its diverse phytochemical compounds derived from the plant species from which it is obtained [34,35,36,37].

Chemical composition analysis revealed the relationship between its biological activity and the plant species involved [38]. e.g.; β-amirenone, a triterpene obtained from oleoresins of Bursera sp. and rosemary (Rosmarinus officinalis), has a positive effect on the treatment of skin conditions, hypertension, inflammatory, tumorous, and microbial reactions[39]. In the case of M. beecheii, a variety of pentacyclic triterpenes have been detected worldwide [40,41]. Considering that approximately 70% of propolis components are of plant origin, there is a need to identify the plant species of pollen grains involved in propolis, in order to determine the relationship between its phytochemical compounds and the flora [42,43,44,45,46]. Thus, the objective of this study was to determine and characterize the main botanical species that contribute to propolis from the native bee M. beecheii, in the community of La Gloria, municipality of Cosautlán de Carvajal, Veracruz, Mexico.

2. Materials and Methods

2.1. Study Area

The community of La Gloria belongs to the municipality of Cosautlán de Carvajal, Veracruz, Mexico, located in the central region, 32 km from Xalapa. It is situated at 19° 20' 56.0"N and -97° 00' 16.0"W, at an altitude of 1176 m above sea level. It has a temperate-humid climate (Cf) [47], with year-round rainfall, an average annual precipitation of 2001-3000 mm, and an average annual temperature of 18° to 22° C (Figure 1). The community is located in an area with tropical sub-deciduous forest and cloud forest vegetation, with seasonal agricultural activity [48]. These types of vegetation exist in Veracruz in discontinuous patches as a transition zone. They are generally dominated by deciduous trees and various woody climbing species, and members of the Bignoniaceae, Fabaceae, and Loranthaceae families can be observed [49]. Due to their proximity to these vegetation types, native bee nests can still be found in their natural habitat, and efforts are underway to conserve these species and the flora on which they depend [50].

2.2. Data Collection

At the invitation of the research project “Evaluation of the antimicrobial potential of propolis from stingless bees of Mexico” (PAPIIT) IN223719, of the Faculty of Higher Studies Cuautitlan, UNAM and, the relationship of the community of La Gloria, Cosautlán, with INANA A. C. “Initiatives for nature”, explorations begin in the region and, the stay from August 27 to 29, 2019, developing field activities in the community, through participant observation [51,52] and construction of dialogue of knowledge [53], with application of open questionnaires on regional meliponiculture and, carrying out a 500 m route for each cardinal point, around the meliponary of the community, base of collection [54] of 75 botanical species and taking samples of their palynomorphs of plants referred by meliponiculturists as foraged by M. beecheii; The connotations of abundant, moderate, scarce, and rare were assigned to each species based on observation [55,56]. Propolis was collected according to the Mexican Official Standard NOM-003-SAG/GAN-2017 [57], obtaining representative samples of the stored propolis throughout 2019; 85 g per nest “A” and “B”, preserved in amber glass jars. The floristic inventory was obtained by the botanical identification of the 75 collected species, based on specialized literature [49,58,59,60,61] validating the identification in “Plants of the World Online” (POWO) [62] and “The International Plant Name Index” (IPNI) [63], incorporating the botanical material into the Herbarium of the Faculty of Higher Studies Cuautitlan, Campus 4 of the UNAM.

The palynomorphs of the inventoried species were processed by using acetolysis, staining, and preparation of histological slides [64,65,66,67], representing each species and forming the initial part of a palynomorph collection at the Faculty of Higher Studies Cuautitlan, UNAM. Using a Carl Zeiss Axiostar Plus optical microscope with an integrated AxioCam ICc 3 camera and the ZEISS ZEN 3.5 software (Blue version). Each palynomorph of the inventoried species was described based on ornamentation type, shape, degree of association, apertures, and equatorial and polar position measurements [68,69,70,71,72], obtaining diagnostic micrographs.

The propolis underwent dilution, water bath, filtration, centrifugation, and decantation processes with xylene, 96% absolute ethyl alcohol, and 70% absolute ethyl alcohol [73]. The palynomorphs present were separated and subjected to acetolysis, staining, and preparation of histological slides [64,65,66,67,73]. This material revealed the species represented by their pollen.

From the histological slides of palynomorphs present in the propolis, 1500 pollen grains were counted, and the frequencies of the different palynomorphs were quantified. The botanical species constituting the propolis were determined based on pollen grain characteristics such as ornamentation, shape, size, degree of association, and apertures, in comparison with the same properties of palynomorphs from foraged flora, as well as with bibliographic support [74,75,76,77,78,79].

Pollen grain sizes, life forms, morphological characteristics, flowering periods, and bibliographic information of the species present in the propolis were analyzed to describe the tendency of M. beecheii in its formation [80,81].

3. Results

3.1. Foraging Flora Determination

The seventy-five (75) species collected and reported as foraged by native bees were identified and incorporated into the Herbarium of the Faculty of Higher Studies Cuautitlan (FESC). Histological slides of their pollen grains were also added to the Palinotheque of the FES Cuautitlan Botany Laboratory as a reference collection. The 75 species belong to 39 botanical families. Eight families are considered representative of the surrounding vegetation, as their species are abundant or frequent, with three or more species each: Asteraceae with seven species, Euphorbiaceae with three, Fabaceae with six, Fagaceae with three, Lamiaceae with six, Malvaceae with three, and Rubiaceae and Rutaceae with five each, totaling 38 species, which represents 50.6% of the sampled species. The Asteraceae family is primarily composed of weedy species such as Aldama dentata La Llave, Bidens pilosa L., and Melampodium perfoliatum (Cav.) Kunth, widely distributed in open areas. These plants flower for more than six months of the year and are excellent pollen producers, as their inflorescences contain numerous flowers. Fabaceae, with species like Inga vera Kunth, I. jinicuil Schltdl., and Mimosa albida Humb. & Bonpl. ex Willd., are abundant in the region. The first two provide shade for coffee plantations, while the third is a secondary vegetation species found along roadsides and hillsides. Lamiaceae is represented by species cultivated in family gardens, such as Agastache mexicana (Kunth) Lint & Eplin., Ocimum basilicum L., and Salvia leucantha Cav., which are used in traditional medicine for their essential oil production and their ability to attract bees. Rubiaceae, with Coffea arabica L., the main crop in the region, and others such as Crusea calocephala DC., Hamelia patens Jacq., and Palicourea padifolia (Roem. & Schult.) T.N. Taylor & Lorence, which form an important part of the understory. Rutaceae with species cultivated as fruit trees, such as Citrus aurantiifolia (Christm.) Swngle, C. × sinensis (L) Osbeck, and C. limetta Risso. The families Euphorbiaceae, Fagaceae, and Malvaceae, with three species each, form an important part of the tree and shrub layer of the vegetation.

Although some families have only one or two species, they are often visited by numerous bees, as is the case with Psittacanthus calyculatus (DC) G. Don and Struthanthus sp., Loranthaceae; Trema micrantha* (L.) Blume, Cannabaceae; and Cecropia obtusifolia Bertol., Urticaceae, just for mentioning a few, which are considered as foraged plants. Many of the above species are propagated in the “El Chinini” nursery, which belongs to the Demanos family in the community, who propagate around 110 species [82] as a resource of Melipona bees and wildlife, an activity that complements the rescue of native bee nests, from trees felled by logging and deforestation, or others that are at risk in the region surrounding the community, becoming part of the INANA meliponary network, the nursery is considered as a refuge space for six species of stingless bees such as Plebeia spp., Scaptotrigona spp., Trigona spp., among others.

3.2. Propolis Floral Origin

From propolis of M. beecheii in the community of La Gloria Cosautlán we identified 25 species of palynomorphs with different percentage of participation (Table 1).

3.3. Palynomorphs Role

In order to evaluate the potential of the plant resource, only palynomorphs that contribute 2.0% or more to the propolis composition were considered. Seven species meet this percentage, two of which are considered significant: Miconia xalapensis (Bonpl.) D. Don ex DC. (Melastomataceae) and Mimosa albida Humb. & Bonpl. ex Willd (Fabaceae), which contribute 75.8% of the pollen in the propolis composition. According to [83], the propolis in this study is a monofloral resource of M. albida (Figure 2), as the dominant species, ruling out the possibility of classifying this resource as multifloral; M. xalapensis is the secondary pollen; minor pollen includes Sambucus nigra L., Coffea arabica L., Struthantus aff. quercicola, Trichilia havanensis Jacq., and Pimenta dioica (L.) Merr.; the remaining 18 species as pollen present, due to their low percentages of participation [84,85,86,87].

3.4. Palynomorphs Size

Melipona beecheii preferentially visits plant species whose pollen grains range from 9 to 18 µm, M. albida, with the most abundant pollen grain, is the smallest, followed by M. xalapensis; other less abundant species include S. nigra, C. arabica, S. aff. quercicola, T. havanensis, and P. dioica, whose size varies between 15 and 35 µm (Figure 3). Therefore, for propolis production, M. beecheii opts for small or slightly medium-sized pollen grains [65].

3.5. Palynomorphs from the Main Propolis Species

Palynomorph species with more than 2.0% representation in the propolis of M. beecheii, in La Gloria, Cosautlán, Veracruz (Figure 4 and 4A).

Mimosa albida ((Figure 4A and 4B). “Spherical tetrads 9–11 µm in diameter. Asymmetric, apolar, inaberturate grains. Exine: < 0.5 µm” [88].

Miconia xalapensis (Figure 4C and 4D). D. Oval, heterocolpate pollen, with three colpores and three subsidiary colpi. Transverse colp reduced to a circular to slightly elongated endopore 1.0–1.5 µm, with ribs. Exine: tectate, psilate, 1.0 µm thick with a slight thickening towards the equator and apertures. Monad isopolar, radiosymmetric. Prolate; CP: interhexagonal, CE: elliptic. P: 18.8 (18.2-20.0) µm, E: 13.1 (12.8-15.0) µm [89].

Sambucus nigra (Figure 4E and 4F). “Spherical, tricolporate pollen with radial symmetry, isopolar, reticulate exine, grain size 14 to 22 µm” [90]. DP (polar diameter 18-21 µm), DE (equatorial diameter 22-24 µm).

Coffea arabica (Figure 4G and 4H). Polymorphic, radiosymmetric grains, isopolar or heteropolar, tricolporate, stephanocolporate (four colpi) or pericolporate, sometimes syncolpate, oblate-spheroidal to prolate-spheroidal, circular or quadrangular in shape; DE (equatorial diameter) 32-40 µm, DP (polar diameter): 30-38 µm. Exine: 2 µm, granular, perforated, foveolate, fossulate, areolate or reticulate; pores elongated, irregular or circular, sometimes annulate (2-8 × 3-11 µm). Colpi with irregular margins and of variable length, 3-7 µm wide; sometimes latitudinal colpi occur in the polar or equatorial area [88].

Struthanthus aff. quercicola (Figure 4I and 4J). Pollen 15–30 µm, oblate, isopolar diplosyndecolporate, with well-developed endoapertures [91]. DP (30–33 µm), DE (39–40 µm).

Trichilia havanensis Jacq. (Figure 4K and 4L). Aperture: tetracolporate or pentacolporate. Long meridional colpus 27 × 2 µm. Colpus membrane smooth, transverse colpus elliptic-circular 3–4 × 5–7 µm. Median polar area (0.27). Exine: tectate, psilate with a microreticulate pattern, exine 2 µm thick. Monad isopolar; radiosymmetric. Shape: prolate spheroidal; CP circular; DE elliptic-circular. Size: 34.7 µm (30-40 µm) E (32.5 7 µm) [92].

Pimenta dioica (L.) Merr. (Figure 4M and 4N). Pollen: Oblate, 11(14.5)17 µm × 20(21.6)23 µm. P/E 0.67. Triangular view, 19(20.5)22 µm in diameter. Exine: 1.5 µm thick, sexine equal to nexine. Tectate, psilate, with a microreticulate pattern. Apertures: Tricolporate, syncolpate, heteropolar: Colpi 5(9.2)14 µm long × 1(1.6)3 µm wide, with psilate membranes. The endoaperture is elongated, measuring 3(4)7 µm long × 1(1.4)2.5 µm wide. On the face opposite the syncolpia, the distance between the colpi is 2(3.2)5 µm. The pollen availability index (PAI) is 0.15, small [74].

3.6. Resource Availability

The pollen resources used by M. beecheii to produce its propolis are collected according to their availability throughout the year. Two species are available in bloom for the longest period: M. albida and S. aff. quercicola, being the first one the most abundant pollen resource. M. xalapensis, the second most important component, is available for only two months of the year; however, the bee has access to other species that alternate their flowering periods, covering the entire year (Table 2).

3.7. Characteristics of Mostly Foraged Species

The most intensively foraged resources correspond to the shrub layer (75.8%). The species that support M. beecheii have numerous inflorescences with inconspicuous or small flowers, generally less than 15 mm long, with small to medium-sized pollen. M. albida is distributed on slopes and roadsides, M. xalapensis and T. havanensis in open spaces and along roadsides, S. aff. quercicola is a hemiparasite present on different species; all belong to secondary vegetation and are common in the area. S. nigra, an introduced species, is scarce; one tree was found in the surveyed area. P. dioica, a species of primary vegetation, is common; its distribution is favored by its fruit, which is harvested and sold C. arabica, widely distributed, is the main crop of economic importance in the region.

The genus Mimosa plays an important role in ethnomedicine due to its nutritional, pharmaceutical, and toxic properties [93]. M. albida, enriches and protects the soil where it grows, and contains phytochemical constituents such as triterpenes, steroids, flavonoids, lactones, alkaloids, anthocyanins, naphthoquinones, tannins, phenols, catechins, saponins, reducing sugars, fats, and amino acids [94], making it a widely used species in traditional medicine for cardiovascular, gastrointestinal, renal, and inflammatory problems, as well as in cases of diabetes, cancer, and anxiety. It possesses antioxidant and antibiotic properties and has been reported to have non-fatal toxicity [93,95].

Miconia xalapensis offers significant advantages in the area where it grows, contributing to regeneration, protecting the soil, promoting the germination rate of other species, and fostering increased diversity. This is due to the production of abundant fruits, which are consumed by birds that disperse the seeds with high germination rates. The establishment of the species reduces habitat fragmentation and contributes to the arrival of new species of both primary and secondary vegetation, which is crucial for passive restoration [96,97]. Its analgesic, anti-inflammatory, antimicrobial, antiparasitic, antitumor, antimutagenic, and antioxidant activity has been evaluated, due to the presence of triterpenes, coumarins, benzoquinones, hydrolyzable tannins, phenolic acids, polyphenols, anthocyanin-type flavonoids and flavone glycosides, as well as cyanogenic glycosides in smaller proportions [98,99,100,101].

Sambucus nigra, a species introduced to Mexico, is cultivated in Europe, Canada, Chile, Germany, and New Zealand for its flowers and fruits, which are rich in nutrients, vitamins, and essential oils. These are used in the nutraceutical food industry[102] and in pharmaceuticals for their bioactive properties due to the presence of polyphenols, flavonols, and phenolic acids such as quercetin, kaempferol, and isorhamnetin, along with proanthocyanidins, anthocyanins, terpenes, lecithins, and potentially toxic cyanogenic glycosides. These compounds are present in the young fruits and degrade into cyanidin upon ripening. The species is used in folk medicine for a variety of ailments due to its antioxidant, antimicrobial, antitumor, hepatoprotective, neuroprotective, cardioprotective, and immunostimulatory properties, among others. In Europe, it is an important medicinal species [98,103,104,105].

Coffea arabica, a species widely cultivated worldwide for its beverage used in food, is recognized for its important use in traditional medicine due to its psychoactive, neurological, antibacterial, antioxidant, anticancer, antiglycemic, anti-arteriosclerosis, anti-inflammatory, and genotoxicity-reducing activity, as well as its ability to control numerous ailments due to its bioactive components derived from phenols and terpenes in its essential oil, with the presence of palmitic, oleic, linoleic, and stearic acids; throughout the plant it presents polyphenols such as chlorogenic acid, diterpenes such as cafestol and kahweol, alkaloids such as caffeine and theobromine, tannins, flavonoids such as quercetin, anthocyanins, isoquercitin, kaempferol, and rutin, terpenoids, cafesterol, amino acids, and histidine [106,107,108,109].

Struthanthus quercicola Schltdl. & Cham.) G. Don, a hemiparasitic species with medicinal uses for various ailments, is recognized for its immunoregulatory, antibacterial, antiviral, antioxidant, antineoplastic, and hypoglycemic activity. It does not cause hepatotoxicity and inhibits cell proliferation. It contains numerous bioactive components such as terpenoids, alkaloids, flavonoids, saponins, and tannins; the proportion of these components varies depending on the species it parasitizes [110,111].

Trichilia havenensis, is a food source for birds, which promotes its distribution. It contributes to soil and water conservation and is used as a living barrier on slopes, roads, riparian corridors, and for the restoration of degraded areas. Its wood is used in carpentry, packing crates, and sculpture carving [112]. The bark has potential as an organic pesticide, acting as a molluscicide and insecticide against larvae and insects, due to the presence of limonides, monoterpenes, sesquiterpenes, triterpenes, diterpenes, steroids, coumarins, flavonoids, glycosylates, and lignans, contributing to antibacterial and anticarcinogenic activity [2,113].

Pimenta dioica, a species of economic importance for the production of allspice, is a spice used in food. Its essential oil, the source of its aroma and flavor, exhibits antioxidant and antimicrobial activity against fungi and bacteria, attributed to its high phenol content [29,114,115].

4. Discussion

According to the results, M. beecheii, when producing its propolis, does not forage for species present solely based on their frequency or flowering periods, since species from the Asteraceae, Fabaceae, or Lamiaceae families, common in the region with abundant flowering over a prolonged period, do not have significant or representative percentages of their pollen present in the propolis.

Melipona beecheii in La Gloria, Cosautlán, Veracruz, is selective for trees and shrubs of the regional flora with inconspicuous or small flowers, numerous inflorescences, and small pollen, such as M. albida, whose dominant percentage of pollen in its propolis classifies it as monofloral [83], a result similar to [116] for a honey study in Teocelo, a municipality near La Gloria. The phytochemical components present in species with a 2% or higher concentration in propolis are associated with biological activities such as antibacterial, antiviral, antifungal, antioxidant, anti-inflammatory, and immunoregulatory properties, among others [117,118]. The chemical composition and properties of M. beecheii propolis in the La Gloria community are influenced by the interdependence and coevolution through mutualism between species [119]. Melipona beecheii is an effective pollinator; the presence of at least 25 palynomorph species in its propolis indicates its involvement in the pollination of both wild and cultivated plants. This contributes to the natural regeneration of vegetation, benefiting biodiversity conservation and serving as an indicator of the ecosystem's conservation status [2,113].

The knowledge and management of their populations by the meliponiculturists in La Gloria Cosautlán, Veracruz, incorporated into their culture as an ecosystem service, contributes to the conservation of the human population in their place of origin [17,120] and provides valuable information to science for the management of resources for the benefit of humanity [29,114,115].

5. Conclusions

Melipona beecheii selects Mimosa albida from the diverse flora, the dominant pollen species in its propolis, resulting in a monofloral propolis. It is also a good pollinator, foraging on twenty-four other species and thus contributing to the regeneration and conservation of the region's biodiversity. The most frequently visited species exhibit biological activity due to their phytochemical components, which will be significantly reflected in the activity of its propolis. The knowledge and management of its populations, incorporated into the meliponiculture practices as an ecosystem service, contributes to the conservation of the human population in its place of origin. The collected botanical specimens increased the Herbarium's collection; the histological slides and the Palinotheque of the Botany Laboratory, both collections of the Faculty of Higher Studies Cuautitlan, UNAM.