Stingless bees (Apidae, Meliponini), a group of eusocial Hymenoptera, are exclusively tropical (Michener, 2013) and important pollinators of many flowering plants, both domesticated and wild (Heard, 1999). Some meliponine species have been semi-domesticated to provide honey and, more importantly, pollination services (Kwapong et al., 2010). A key aspect of this semi-domestication has been the development of colony splitting and propagation techniques (Aidoo, 2020). Meliponaries, where managed stingless bee colonies are established, can have relatively high relatedness and correspondingly decreased genetic diversity since meliponiculture leads to multiple daughter colonies arising from relatively few mother colonies.
Stingless bee colonies naturally reproduce when reproductive females (gyne) mate with males gathered in congregations that typically occur in close vicinity to the colony from which the virgin queen emerges (Wille, 1983). In
As with other meliponine species, there is an additional close relationship between mother and daughter colonies of the stingless bee
In prior studies with
To increase the number of colonies in a meliponary via artificial colony fission, a colony in good condition is split into two colonies at the correct time of year. One colony keeps the queen (the mother colony) while the other colony (the daughter colony) is transferred to a new hive box with brood, royal gyne cells, workers, pollen, and honey pots, resembling (in part) the natural process of colony reproduction. The virgin queen emerges from the daughter colony and then mates at a nearby male congregation (Guzmán Díaz et al., 2004). This illustrates the importance of wild stingless bee populations for maintaining the genetic variability and thereby the health of managed colonies (Moritz, 2002). In turn, managed stingless bee colonies, which are often transported from adjacent areas, provide males that help increase the genetic diversity of wild colonies by introducing alleles that might be absent or occur at low frequencies (Cortopassi-Laurino et al., 2006). Finally, the management of stingless bees has deep and ancient links with indigenous, cultural and economic practices (Cortopassi-Laurino et al., 2006; Quezada-Euán, 2018; Escareño et al., 2019). Thus, meliponaries have the potential to help conserve wild populations, enhance the pollination services that they provide, and preserve the rich cultural history of indigenous peoples (Hill et al., 2019).
Despite evidence for the high probability of mating between wild and managed individuals in stingless bees, no study has demonstrated experimentally that new alleles actually enter the genetic pool of managed colonies via the mating of managed queens with wild male stingless bees. We therefore sought to test this hypothesis and chose
Ten managed
Once colonies were ready for the procedure, ten identical wood boxes (22 × 22 × 40 cm) were prepared, and colony division was carried out as described by Guzmán Díaz et al. (2004). Essentially, the queen was retained in her old colony (mother colonies, M1–M10) and half of the workers, food pots, and combs were placed in another wood box to form new colonies (daughter colonies, D1–D10). It normally takes one to two months for virgin queens to emerge and breed (Guzmán Díaz et al., 2004). Each mother and daughter colony was labeled and maintained in the same meliponary. Queens from the mother colonies were painted on their thoraces with a POSCA PC5M marker to ensure that they were the original queens, not replacements, and then returned to their colonies. We allowed the virgin queen from the daughter colonies to mate freely. The queens of these daughter colonies were also distinctively paint-marked on their thoraces once they began laying eggs.
Exactly six months after splitting the colonies, we checked that all of them had painted queens. After confirming this, we collected from the colonies third instar larvae (which should be unambiguously genetically related to the queen) given that all larvae transferred from the mother colonies would have completed their development months beforehand. Five specimens per colony were obtained from the bottom combs in which older instar larvae are commonly found and stored in 95% reagent grade ethanol in labeled vials until analysis. We sampled five specimens because
We extracted DNA from the bees after first rinsing off the alcohol with 100 ml of double-distilled water for 30 min at room temperature. DNA was extracted with the HotSHOT method (Truett et al., 2000). Single locus PCR protocols with a final reaction volume of 5 μL (Solórzano-Gordillo et al., 2015) were used to genotype all specimens at six microsatellite loci: B124 developed for
First, we conducted descriptive genetic analyses of each colony. We then ran an AMOVA (Michalakis & Excoffier, 1996) using Genodive v2.0b27 (Meirmans & Van Tienderen, 2004) to determine any genetic differences between mother (M) and daughter (D) colonies that could arise due to mating with wild males. A power analysis was run using POWSIM v4.1 program as indicated by Ryman & Palm (2006). Finally, we used Matesoft v1.0 to determine the number of patrilines and the effective mating frequency in our colonies (Moilanen et al., 2004; Starr, 1984).
One hundred larvae from twenty colonies were fully genotyped at six loci, and, since at least one locus of each larva was heterozygous, none were considered male (Supplementary Material 1). The relative number of alleles between mother and daughter colonies is shown in Supplementary Material 2. Four new alleles were observed in daughter colonies, and thus clearly indicate that the queens of these colonies had mated with unrelated males (Supplementary Material 3). After 9999 permutations, the AMOVA (infinite mutation model) showed that there were no genetically significant differences between mother and daughter colonies, so they could be considered as coming from a single population (
Patrilines (Patril.) estimated in each colony, number of new alelles found in daughter colonies not found in any mother colony, and effective mating frequency (me), which was calculated as in Starr (1984):
Colony | Patril. | New alleles | |
---|---|---|---|
M1 | 1 | 0 | 1 |
D1 | 1 | 0 | 1 |
M2 | 3 | 0 | 1.69 |
D2 | 4 | 1 | 2.56 |
M3 | 3 | 0 | 1.92 |
D3 | 1 | 1 | 1 |
M4 | 1 | 0 | 1 |
D4 | 1 | 1 | 1 |
M5 | 2 | 0 | 2 |
D5 | 1 | 0 | 1 |
M6 | 1 | 0 | 1 |
D6 | 1 | 0 | 1 |
M7 | 3 | 0 | 1.92 |
D7 | 1 | 0 | 1 |
M8 | 1 | 0 | 1 |
D8 | 2 | 1 | 1.6 |
M9 | 1 | 0 | 1 |
D9 | 3 | 0 | 1.6 |
M10 | 5 | 0 | 1.47 |
D10 | 5 | 0 | 2.53 |
Mean (± SD) | 2.05±1.39 | 1.41±0.53 |
We tested the contribution of wild populations to the genetic pool of a managed meliponary by generating daughter colonies that could freely mate with wild and managed male stingless bees. Overall, queens mated with males of similar genotypes to the ones present at their meliponary, suggesting that the diversity of colonies established at a meliponary plays a strong role in its genetic diversity and, by association, fitness. However, the detection of four new alleles (12% of 33 alleles total) in daughter colonies that were not found in any mother colony, demonstrates the introduction of genetic material from wild male stingless bees. Moreover, multiple mated queens were detected, and thus polyandry in this species might be more frequent than previously stated. Our data therefore suggest that
In
Genetic analyses of male stingless bees in congregations near colonies from which gynes emerge demonstrate that these typically differ from male stingless bees raised in a
One of our key assumptions is that the wild population will have different alleles. Unfortunately, we do not know allele frequencies in the wild population because of the difficulty of finding all nearby wild colonies and sampling bees from them. Collecting males from nearby male congregations is more feasible, but again raises the issue of identifying males from wild versus managed colonies. Because this constrains our interpretations, we view this study as exploratory, with further research needed, perhaps using bee colonies transported from much greater distances and thereby with a higher likelihood of different alleles. However, this raises other problems, such as influencing the natural genetic diversity of nearby wild colonies and potentially introducing new pathogens or parasites. Detailed, landscape level studies of stingless bee genetic diversity and pathogen and parasite loads would be helpful for developing sustainable management practices that involve the transport of colonies across landscapes.
Seasonality and management effects could also account for differences in the relative proportion of wild to managed males. The production of brood and reproductives varies seasonally in stingless bees and connects with seasonal variation in food availability (Grüter, 2020). For example, in our study, queen mating occurred earlier in the year but the evaluation of male congregations by Mueller et al. (2012) occurred later in the year, during May and June. If wild colonies were just emerging from a food dearth during the early part of the year, one might expect them to produce fewer males as compared to managed colonies. Later in the year, when food is more abundant, wild colonies could produce more males, accounting for their increased frequency, as seen in Mueller et al. (2012). However, our colonies were only managed to the extent of being housed in wood boxes. Managed colonies did not receive supplemental food, and thus their production of reproductives should be similar to wild colonies nearby. In some cases, beekeepers may feed their managed colonies (Quezada-Euán et al., 2001) and thereby potentially encourage inbreeding by increasing the ratio of managed to wild males. This would be interesting to study in the future. Finally, we conducted this study in two separate years, 2017 and 2019. Although this may have contributed to variation in the production and ratio of managed to wild males between these years, we did not find any evidence for substantial variation between these years (based upon our AMOVA), and replication over more than one year in a field study is generally recommended (Lemoine et al., 2016).
To our surprise, nine out of twenty colonies had multiple-mated queens, which contradicts previous results by Palmer et al. (2002). Possibly our greater sample size (N=20 vs. N=5 in Palmer's work) allowed us to detect more patrilines. Another explanation is that there is variation in mating frequency among populations, as is known to occur in other highly social corbiculate bees. Within the highly polyandrous
Our results suggest that wild stingless bee populations can contribute to the genetic diversity of managed populations to a limited degree. These data show the importance of continuing to evaluate the contributions of wild and managed stingless bee populations and examining the factors that influence the genetic diversity of male stingless bee congregations. Our data also indicate that the genetic diversity of a meliponary plays a strong role in maintaining its diversity and its associated fitness, given that queens from managed colonies largely mated with males from managed colonies. Managed colonies may also provide alleles to the wild population, but we think that this contribution would be minor given that colonies of these species are typically not moved far from their original, natural location. Nonetheless, the artificial propagation of managed colonies may result in an increase in some alleles that are rare but that then become strong competitors of naturally abundant alleles. This hypothesis remains to be tested. Finally, increasing evidence for multiple mating in stingless bee species that were once considered singly mated might be due to increased testing and sampling of different populations. An intriguing question is whether polygyny is influenced by climate change or other anthropogenic factors. Both possibilities deserve future investigation.