Genetic divergence in Tetragonisca angustula Latreille, 1811 (Hymenoptera, Meliponinae, Trigonini) based on rapd markers

ANIMAL GENETICS

RESEARCH ARTICLE

Genetic divergence in Tetragonisca angustula Latreille, 1811 (Hymenoptera, Meliponinae, Trigonini) based on rapd markers

Rosana de Cássia Oliveira^I; Francis de Morais Franco Nunes^I; Ana Paula Soraggi Campos^I; Soraya Matos de Vasconcelos^II; David Roubik^III; Luiz Ricardo Goulart^II; Warwick Estevam Kerr^II

^IUniversidade de São Paulo, Departamento de Genética, Ribeirão Preto, São Paulo, Brazil

^IIUniversidade Federal de Uberlândia, Instituto de Genética e Bioquímica, Uberlândia, Minas Gerais, Brazil

^IIISmithsonian Tropical Research Institute, Balboa, República do Panamá

^{Correspondence}

ABSTRACT

One of the commonest neotropical stingless bees is Tetragonisca angustula (Latreille, 1811), popularly known in Portuguese as jataí, which occurs in variable nesting sites from Mexico to Argentina. We used 18 primers to generate 218 RAPD markers which we used to determined the genetic distance between T. angustula populations from 25 localities in three different Latin America countries, using Tetragonisca weyrauchi from the Brazilian state of Acre and the common honey bee (Apis mellifera) as outgroups. Genetic distance, calculated as the Percentage of Dissimilarity (14%), based on all markers divided the T. angustula population into eastern (group 1) and western (group 2) groups. However, we were able to separate the two groups by using only two primers that have generated five specific molecular markers. The eastern group consists of T. angustula angustula which occurs from Panama to the Brazilian states of Maranhão and northern Minas Gerais and has spread through the Brazilian Atlantic Forest as far as the southern Brazilian state of Santa Catarina. Group 2 is made up of T. angustula fiebrigi which has a more southerly and western distribution, occurring only in the western Brazilian states of Mato Grosso and Mato Grosso do Sul as well as the west of some other Brazilian states (Goiás, Minas Gerais, São Paulo, Paraná and Santa Catarina) and northeastern Argentina.

Key words:Tetragonisca angustula, genetic divergence, molecular markers, UPGMA.

Introduction

One of the most common neotropical stingless bees is Tetragonisca angustula (Latreille, 1811) (Hymenoptera, Meliponinae), popularly known in Portuguese as jataí, which are generalist foragers and more efficient native pollinators of the American flora. Although T. angustula is widely distributed in the Americas from the south of Mexico to Argentina, it has not been observed in Andean cordillera, the scrubland (caatinga in Portuguese) of northeastern Brazilian and some regions of the Amazon.

There are two subspecies of T. angustula which can be separated morphologically, T. angustula angustula (Latreille) with a black mesepisternun and T. angustula fiebrigi (Schwarz) with a yellow mesepisternun (Castanheira, 1995). According to Nogueira-Neto (1970), T. a. angustula is distributed as described above (i.e. from Mexico to Argentina with some exceptions) while T. a. fiebrigi is restricted to sites in southern Brazil (the Paraná river valley in São Paulo state, part of the state of Paraná and the state of Santa Catarina) and Argentina and Paraguay.

The biological, behavioral, morphological and biochemical aspects of T. angustula have been studied since the beginning of the twentieth century. Castanheira (1995) investigated 228 T. angustula colonies from five Brazilian states (Mato Grosso, Bahia, Minas Gerais, São Paulo and Paraná) for genetic differences in six enzyme systems and found variation at the hexokinase, glycerol-3-phosphate dehydrogenase and malate dehydrogenase loci, the correlation between the degree of subspecies hybridization in the São Paulo populations and the frequency of the hexokinase Hk88 allele indicating that this allele is a marker for the T. a. fiebrigi subspecies. Diniz-Filho et al. (1998) studied geographical variation in morphology based on fifteen morphometric characters in eight local populations occurring in western-central and southeastern Brazil. These authors found significant differences in all the characters analyzed, with body-size showing a north-south clinal variation in which larger bees occurred mainly in the south, this being attributable to adaptation to a cooler climate (e.g. Bergmann's rule).

The study described in this paper used polymerase chain reaction - random amplified polymorphic DNA (PCR-RAPD) markers (Williams et al., 1990; Welsh and McClelland, 1990) to investigate the relationship between different T. angustula populations, the results providing interesting insights into future research on this species.

Material and Methods

Bee samples and DNA extraction

We sampled worker bees from 26 T. angustula L. colonies from 25 sites (Table 1), the colony representing Pilar do Sul was in fact situated at Ribeirão Preto where it had been maintained for three years prior to sampling. For outgroups we used two Tetragonisca weyrauchi (Schwarz) colonies from the Brazilian state of Acre (09°58'S, 67°48'W) and an Apis mellifera colony from Uberlândia (18°54'S 48°16'W). In all cases, the samples consisted of a worker bee collected at the entrance of each colony and kept in absolute alcohol at -80 °C until DNA extraction (Post et al., 1993).

Each bee was washed three times in buffer (TrisCl 0.01 M, pH 8.0; NaCl 0.1 M and MgCl₂ 0.001 M) for 15 min (Dowton and Austin, 1994) and the DNA extracted as using a modified Shuster et al. (1992) protocol, i.e. incubation with proteinase K and Rnase, phenol-chloroform extraction and DNA precipitation using 70% ethanol.

RAPD-PCR amplification and separation and visualization of products

We used eleven decamer short primers (OPA 03, 09, 10, 13; OPF 13; OPL 04, 11, 13; OPO 06; OPV 07 and 10) from Operon Technologies, USA and seven 20 to 24 mer long primers (GOU 07, 10; MAU 101, B1, B2, 402 and 801) which we synthesized in our laboratory. Each amplification was performed in a total of 25 mL of reaction mixture consisting of 10 mM Tris-HCl, 50 mM KCl, 2 mM MgCl₂, 100 mM of each dNTP (dCTP, dATP, dGTP, dTTP), 10 pmoles of primer, 1U of Taq DNA polymerase and 10 ng of bee genomic DNA. Reactions were performed in a PTC-100 thermal cycler (MJ Research) for three cycles of 94 °C for 1 min, 35 °C for 1 min and 72 °C for 2 min, followed by 34 cycles of 94 °C for 10 s, 40 °C for 20 s and 72 °C for 2 min with a final cycle of 72 °C for 5 min. A negative control reaction was prepared for each primer used in all PCR amplifications to identify contamination of reactions with non-target template DNA. All amplification reactions were repeated twice in order to test reproducibility.

Amplified products were separated by electrophoresis in 1% (w/v) agarose gel for 1.5 h at 0.5 V cm^-2 using 0.5X TBE (tris- EDTA borate) buffer, stained with ethidium bromide and photographed using a VDS Image Master (Pharmacia Biosciences). A DNA molecular weight marker (Gibco-BR) was also included.

Amplicon analyses

Only those fragments that were consistently reproduced were used. A binary matrix was produced in accordance with the presence (1) or absence (0) of bands, the fraction of bands matching in two bees (M) being calculated using the formula M = N_AB/N_T where N_AB equals the total number of matches (i.e. both bands absent or both bands present) in individuals A and B and N_T is the total number of fragments scored. Genetic distance (GD) was calculated as GD = 1 - M and cluster analyses performed on the GD values using an unweighted pair-group method with averages (UPGMA) algorithm, the resultant dendrograms being generated using the version 4.5 of the Statistica program (Puterka et al., 1993).

Results and Discussion

Cluster analysis of T. angustula genetic distance values

The RAPD method has been reported to be an efficient tool to differentiate geographically and genetically isolated populations, and has been used to verify the existence of populations of species that have arisen either through genetic selection under different environmental conditions or as a result of genetic drift (Fuchs et al., 1998).

Samples of T. angustula analyzed using RAPD profiles generated by 18 primers resulted in 218 intense and reproducible polymorphic fragments. The cluster analysis dendrogram (Figure 1) divided the T. angustula population into two groups with 14% of genetic dissimilarity. One group clustered 12 populations (Panama, Mirador, Barra do Corda, Domingos Martins, Araxá, Bocaiúva, Canaã, São Francisco, Curitiba, Blumenau, Maricá and Pedreira), while the other group (Porangatu, Rio Verde, Grupiara, Campina Verde, Uberlândia 1 and 2, Campinápolis, Ladário, Ribeirão Preto, Pillar do Sul, Prudentópolis, Posadas, Aristóbulo del Valle and Cerro Azul) consists of 13 populations from the western side of South America. The genetic distance between groups 1 and 2 suggests that this species is undergoing genetic differentiation, a process which can be driven by ecological, evolutionary or historical factors. Genetic differentiation of T. angustula between the different groups of sampling sites is to be expected because the sites extend over a wide geographical area and are subject to different environmental factors, such factors including the temperature gradient from the equator to Argentina, vegetation patterns which vary from rain forest to cerrado (savanna-like vegetation) and the pampas of southern Brazil and Argentina, differences in rainfall and other, unknown, environmental factors and processes.

Group 1 (T. a. angustula) occurs in the east of the collection area, from Panama to southern Brazil, while Group 2 (T. a. fiebrigi) was distributed in the Brazilian states of Mato Grosso and Mato Grosso do Sul, the western parts of the Brazilian states of Goiás, Minas Gerais, São Paulo and Paraná, and northeastern Argentina (Figure 2). Our RAPD marker study shows that 56% of the 25 geographical sites were colonized by T. a. fiebrigi, showing that this subspecies is more widely distributed than is apparent when this subspecies is identified by mesepisternun color which identified T. a. fiebrigi in only 24% of the sites (Table 1). Previously (Nogueira-Neto, 1970), morphological data suggested that T. a. fiebrigi occurred only in southern Brazil in parts of São Paulo, Paraná and the River Paraná valley and the state of Santa Catarina, and in Paraguay and Argentina.

The wide geographic range of T. a. fiebrigi as suggested by RAPD markers may be the result of intraspecific hybridization and/or T. a. fiebrigi having no established allopatric limits.

The occurrence of T. a. fiebrigi could be the result of intraspecific hybridization. In our RAPD maker study there was only one possible intraspecific hybridization event, which can be seen in Figure 2 where the Pilar do Sul genotype (allocated to group 2 (T. a. fiebrigi) by our RAPD analysis) appears to be introgressing into group 1 (T. a. angustula). As explained in the materials and methods, the Pilar do Sul colony (morphologically T. a. angustula) was actually in Ribeirão Preto where it had been maintained for three years prior to sampling while the other colony sampled (also morphologically T. a. angustula) had always been in Ribeirão Preto, and it may have been that intraspecific hybridization occurred between these two colonies to produce T. a. fiebrigi. Intraspecific hybridization would be expected to result in co-dominant bands but this did not occur in this case, probably because RAPD markers usually yield dominant bands. Another way of demonstrating intraspecific hybridization would be by monitoring the introgression of RAPD markers in haplodiploid crossings, which also was not the case. However, Castanheira (1995) has demonstrated that rare alleles have been constantly introduced into the Ribeirão Preto T. a. angustula population due to importation of colonies of bees from other sites by beekeepers and researchers, isozyme markers of T. a. fiebrigi having been found in natural populations of Ribeirão Preto bees.

The zone of allopatry between these subspecies in the interior of South America has never been clearly established. The work related to the presence of subspecies of T. angustula dates from the beginning of the 20th century and has incorporated only one morphologic character for discrimination, the coloration of the mesepisternun. This seems unreliable because morphologic characters can be under quantitative, epistatic or pleiotropic genetic control, which can mask the geographic distribution of populations. The use of more discriminatory and complementary tools such as RAPD analysis and methods for the detection of enzymatic systems that reveal heterozygotes could be used to detect population polymorphisms and a systematic revision of the genus could clarify the results of more preliminary work. Our sampling procedure was too limited to establish range limits.

By removing specific markers belonging to the outgroups we produced two other dendrograms (not shown), one with all A. mellifera markers removed and the other with all A. mellifera and T. weyrauchi markers removed. However, neither of the dendrograms showed any alterations in the T. angustula genotype clusters for either of the groups, although the genetic distance between groups 1 and 2 slightly increased to 16.7% when the A. mellifera markers were removed and 24.5% when both the A. mellifera and T. weyrauchi markers were removed. It may be that the 24.5% genetic distance between T. angustula group 1 (T. a. angustula) and group 2 (T. a. fiebrigi) may be a more reliable distance estimate because the presence of data for other species (the outgroups) may influence the divergence analysis by minimizing distance within the same species.

Population markers

In our search for genetic markers we found two primers, MAU-B2 (GCCAGGCAGCA AGTTCTCAGTAAT) and OPL11 (ACGATGAGCC), each of which could differentiate between the two geographically well-characterized T. angustula clusters. The MAU-B2 primer (Figure 3-A) presents three informative polymorphic bands of approximately 470, 500 and 650 bp, with the 470 bp band identifying group 1 (T. a. angustula) and the 500 and 650 bp bands segregating together in group 2 (T. a. fiebrigi, found only in the southwest of the area studied). The OPL11 primer (Figure 3-B) was also able to distinguish the two T. angustula clusters based on two bands, a 1100 bp band occurring only in group 1 genotypes and a band of about 1700 bp present only in group 2 genotypes.

It was thus possible to distinguish between the two T. angustula groups using only two primers, a significant fact because RAPD markers are easy to obtain and make possible genetic evolutionary studies simpler to conduct. Suazo et al. (1998) have also detected specific RAPD markers which are able to distinguish between A. mellifera races from Africa and Europe, while Waldschmidt et al. (2000) used the same technique to identify a DNA marker present in Melipona quadrifasciata quadrifasciata but absent in M. q. anthidioides.

The findings reported in this paper have important implications for future research on T. angustula. The characterization and monitoring of natural species, using other molecular markers such as microsatellites, single-strand conformation polymorphism (SSCP-PCR) and sequencing of conserved genes will be of great value in establishing a clear picture of the distribution and dynamics of T. angustula in neotropical regions.

Acknowledgments

We thank Dr. João M. Franco de Camargo (USP-Ribeirão Preto, Brazil) for the identification of ours bees; Dr. Vera Lúcia Imperatriz-Fonseca, Dr. Robin F. A. Moritz and Dr. Maria Cristina Ramos Costa for valuable suggestions; and the beekeepers who supplied the samples used in this study. This work was supported by the Brazilian agencies CNPQ (RHAE), CAPES and FAPEMIG.

Editor: Horácio Schneider

Received: July 17, 2003;

Accepted: December 16, 2003.

• Castanheira EB (1995) Marcadores genéticos e sua utilizaçăo em estudos populacionais em Tetragonisca angustula e Plebeia droryana (Hymenoptera, Apidae, Meliponinae). Tese de Doutorado, Faculdade de Medicina de Ribeirăo Preto, Universidade de Săo Paulo, 86 pp.
• Diniz-Filho JAF, Balestra R, Rodrigues FM and Araújo ED de (1998) Geographic variation of Tetragonisca angustula angustula Latreille (Hymenoptera, Meliponinae) in Central and Southeastern Brazil. Naturalia 23:193-208.
• Dowton M and Austin AD (1994) Molecular phylogeny of the insect order Hymenoptera: Apocritan relationships. Proc Natl Acad Sci USA (91):9911-9915.
• Fuchs H, Gross R, Stein H and Rottmann O (1998) Application of molecular genetic markers for the differentiation of bream (Abramis brama L.) populations from the rivers Main and Danube. J Appl Ichthyol 14:49-55.
• Nogueira-Neto PN (1970) A criaçăo de abelhas indígenas sem ferrăo (Meliponinae). 2Şa. ed. Ed. Chácaras e Quintais, Săo Paulo, pp 74-76.
• Post RJ, Flook PK and Millest AL (1993) Methods for the preservation of insects for DNA studies. Bioch System and Ecology 21(1):85-92.
• Puterka GJ, Black IVWC, Steiner WM and Burton RL (1993) Genetic variation and phylogenetic relationships among worldwide collections of the Russian wheat aphid, Diuraphis noxia (Mordwilko), inferred from allozyme and RAPD-PCR markers. Heredity 70:604-618.
• Shuster DE, Kehrli ME, Ackermann MR and Gilbert RO (1992) Identification and prevalence of a genetic defect that causes leukocyte adhesion deficiency in Hostein cattle. Proc Natl Acad Sci USA 89:9225-9229.
• Suazo A, MacTiernan R and Hall HG (1998) Differences between African and European honey bees (Apis mellifera l.) in random amplified polymorphic DNA (RAPD). J Hered 89:32-36.
• Waldschmidt AM, Barros EG and Campos LAO (2000) A molecular marker distinguishes the subspecies Melipona quadrifasciata quadrifasciata and Melipona quadrifasciata anthidioides (Hymenoptera: Apidae, Meliponinae). Gen and Mol Biol 23(3):609-611.
• Welsh J and McClelland M (1990) Fingerprinting genomes using PCR with arbitrary primers. Nucl Acid Res 18:7213-7218.
• Williams JGK, Kubelik AR, Livak KJ, Rafalski JA and Tingey SV (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl Acid Res 18:6531-6535.

Publication Dates

• Publication in this collection
  20 July 2004
• Date of issue
  2004

History

• Received
  17 July 2003
• Accepted
  16 Dec 2003