(Hymenoptera, Apidae, Meliponini) by PCR+RFLP and Sequencing Rute Magalhães Brito, Maria Cristina Arias

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Mitochondrial DNA characterization of two Partamona species (Hymenoptera, Apidae, Meliponini) by PCR+RFLP and sequencing Rute Magalhães Brito, Maria Cristina Arias

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Rute Magalhães Brito, Maria Cristina Arias. Mitochondrial DNA characterization of two Partamona species (Hymenoptera, Apidae, Meliponini) by PCR+RFLP and sequencing. Apidologie, Springer Verlag, 2005, 36 (3), pp.431-437. hal-00892143

HAL Id: hal-00892143 https://hal.archives-ouvertes.fr/hal-00892143 Submitted on 1 Jan 2005

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Apidologie 36 (2005) 431–437 © INRA/DIB-AGIB/ EDP Sciences, 2005 431 DOI: 10.1051/apido:2005020 Original article

Mitochondrial DNA characterization of two Partamona species (Hymenoptera, Apidae, Meliponini) by PCR+RFLP and sequencing1

Rute Magalhães BRITO, Maria Cristina ARIAS*

Departamento de Biologia, Instituto de Biociências, USP, São Paulo, SP, 05508-090, Brazil

Received 5 March 2004 – Revised 3 December 2004 – Accepted 8 December 2004 Published online 9 August 2005

Abstract – We characterized the mitochondrial DNA of two stingless bee species of the genus Partamona. Partial restriction maps were obtained based on digestion of PCR amplified fragments with 8 restriction enzymes. Using Melipona bicolor mtDNA sequence as a model, we were able to amplify 12120 bp of P. mulata and 10300 bp of P. helleri, about 65.5% and 55.7% of their mitochondrial genome, respectively. The digestion assays showed 16 restriction sites for P. mulata and 20 for P. helleri, some of which were exclusive to the genus and others shared with other Meliponini species. The main mitochondrial genes could be mapped and through sequencing analysis we verified that the intergenic region that occurs between the genes CO I and CO II in Apis is absent in Partamona.

Partamona / Meliponini / mtDNA / pcr / rflp / restriction map / stingless bee

1. INTRODUCTION phological, ethological and cytogenetical analyses have been used to infer evolutionary rela- The tribe Meliponini is composed of sting- tionships within the group (Brito-Ribon et al., less bees which are distributed in the tropical 1999; Azevedo and Zucchi, 2000; Brito et al., and southern subtropical areas of the World. 2003; Pedro and Camargo, 2003). As a result The number of species is not well-defined four major phylogenetic groups have been although Michener (2000) estimated the occur- defined within the genus: bilineata/epiphy- rence of several hundred. Their ecological role tophila, musarum, nigrior and cupira (Pedro as natural pollinators is unquestionable, and and Camargo, 2003). The inclusion of nest some species have been domesticated for com- architecture information improved the resolu- mercial honey production (Nogueira-Neto, tion of the phylogenetic relationships within 1997). the cupira group, in which only P. helleri does This tribe presents a great diversity of nest not build nests associated with termites, thus architecture, intra colony population size, hab- being positioned as sister species (Camargo itat, morphology and behavior. The recently and Pedro, 2003). revised genus Partamona Schwarz comprises Mitochondrial DNA (mtDNA) analysis has 33 species and is broadly distributed, being been used widely in studies of population dynam- found from southern Mexico to southern Brazil ics, biogeography and genetic relationships (Pedro and Camargo, 2003). A number of mor- among species (Avise et al., 1987). However,

* Corresponding author: [email protected] 1 Manuscript editor: Walter S. Sheppard

Article published by EDP Sciences and available at http://www.edpsciences.org/apido or http://dx.doi.org/10.1051/apido:2005020 432 R.M. Brito, M.C. Arias for stingless bees these studies are only begin- precipitated, and then digested with the second ning, with just a few species belonging to the enzyme. genera Plebeia Schwarz and Melipona Illiger The fragment amplified with the primer pair COI- having been studied so far (Francisco et al., IIF/mtD18 from both species (see Tab. I in Results) 2001; Francisco, 2002; Silvestre et al., 2002; was cloned and sequenced. The cloning was carried Weinlich et al., 2004). out using the M-13 vector (pGEM T-Easy – Promega) and the sequencing reactions were per- This present work reports the partial charac- formed using a Big Dye Terminator kit (Applied terization of the mitochondrial genome for two Biosystem), according to their respective protocols. Partamona species of the cupira group: P. hel- The single strand fragments originated were ana- leri (Friese) that has a large geographical dis- lyzed by an automated sequencer ABI-PRISM 310 tribution (from south to northeastern Brazil in (Perkin Elmer), and sequence alignments were per- the Atlantic Forest) and P. mulata (Moure) that formed with the programs Generunner V3.00 (Hast- is restricted to “cerrado” (savanna like) areas in ings Software) and MultiAlign 5.4.1 (Corpet, 1988). central western Brazil. Also, although they belong to the same species group (cupira), P. helleri is not associated with termite nests. 3. RESULTS AND DISCUSSION

3.1. Mitochondrial DNA fragments 2. MATERIALS AND METHODS of Partamona mulata and P. helleri Individuals from one natural nest of Partamona From the 116 primer pair combinations, mulata and one of P. helleri were collected, respec- 17 mtDNA regions amplified in P. mulata and tively, in Poconé, MT (16° 18’ LS, 56° 31’ LW) and 13 in P. helleri. Figure 1 presents the amplified Viçosa, MG (20° 45’ LS, 42° 52’ LW), Brazil, and used for the mtDNA analyses. fragments and their positions in comparison to the M. bicolor mtDNA genome (Silvestre, Total DNA was extracted using ten thoraces per tube following the methodology of Sheppard and 2002). Some amplified regions overlapped. McPheron (1991). For the mtDNA amplification, Table I summarizes those data and also the PCR primers designed for Apis mellifera (Hall and Smith, conditions determined for each primer pair. 1991; Arias et al., 1998), Melipona bicolor (Silvestre, The sum of the fragments amplified, dis- 2002) and others insects (Simon et al., 1994) counting overlapping stretches, is about 12120 were tested in a total of 116 combinations. The PCR µ bp for P. mulata and 10300 bp for P. helleri. reactions were performed in 50 L, containing Partamona 0.5 µL of DNA extraction, 5 µL of 10 X PCR buffer, Assuming that mtDNA has the 1.5 µL of each primer (20 µM), 5 µL of dNTPs same genome size (18500 bp) as M. bicolor µ and other Meliponini (Francisco et al., 2001; (2 mM each), 1.5 L of MgCl2 (50 mM) and 2.5 U of Taq DNA polymerase (Invitrogen). The PCR Silvestre, 2002; Fernandes-Salomão et al., amplifications followed the conditions described 2002; Weinlich et al., 2004), we were able to elsewhere (Francisco et al., 2001). However the amplify 65.5% and 55.7%, respectively (Fig. 1). annealing temperature and the cycle number were adjusted for each primer combination to improve the Some mtDNA regions did not amplify even reaction specificity (Tab. I). The PCR products, using several primer combinations and modi- 10 µL of each reaction, were submitted to electro- fications in the PCR conditions. One of the phoresis on 0.8% agarose gels. The gels were stained main regions absent in our map (Fig. 1) corre- with ethidium bromide, visualized and photo- sponds to the region encompassing the genes graphed under UV light with Polaroid film. ND5 and ND4, although a short incomplete Digestions of PCR fragments were carried out fragment was obtained for P. mulata. We using 13 different restriction enzymes: Bcl I; Bgl II; believe that sequencing the adjacent regions EcoR I; EcoR V; Hae III; Hind III; Pst I; Pvu II; Sca and designing specific primers for Partamona I; Xba I; Xho I (Boehringer Mannheim Biochemica); mtDNA would be the best strategy for further Cla I (Gibco BRL) and Nde I (New England sequencing attempts. A second area of absence Biolabs). The reactions were prepared with 3 to 5 µL of the PCR amplified DNA and 5 U of restriction in our map corresponds to the A+T rich region, enzyme following the manufactures instructions, or control region, the size of which is quite dif- and incubated overnight. For double digestions, the ficult to estimate since no data have been DNA was first digested overnight with one enzyme, obtained for any Meliponini species to date. MtDNA characterization of Partamona 433

Table I. PCR conditions and primer pairs that resulted in amplification of Partamona mtDNA. Gene content per region, fragment length (Fl) in base pair (bp) expected (according to Melipona bicolor) and observed, annealing temperature (At) in Celsius degrees and (number of PCR cycles) are shown. (-): Not amplified.

Fragment Fl ( bp) Fl (bp) Primer Primers Principal genes At (No. of cycles) (see Fig. 1) expected observed reference P. mulata P. helleri I mtD2 ND2, COI 2200 43.5 (45×)43 (45×) 2000 a mtD9 a II mtD7 COI, COII 1600 43.5 (45×)47 (35×) 1600 a COI-IIR b III COI-IIF COI, COII 900 43 (40×)43.5 (35×)900b mtD18 a IV mtD19 ATP8, ATP6 1100 42 (35×)42 (35×) 1200 a Seq11 c V Seq10 ATP8, ATP6 751 42 (35×)42 (35×)800c Seq11 c VI Seq24 COIII 600 42 (35×)- 600c mtD22 a VII 5612R COIII, ND3 1100 44 (35×)43 (40×) 1100 d tPheF c VIII Seq35 ND5, ND4 900 41.5 (35×)- 900c tHisF c IX Seq29R ND4 400 42 (35×)42 (35×)400c ND4F c X mtD24 ND4 667 42 (35×)42 (35×)700a ND4F c XI Seq38 ND6 1400 42 (35×) - 1200 c AMB16 d XII Seq38 ND6, cytB 1400 42 (35×)42 (35×) 1400 c mtD28 a XIII Seq38 ND6 1000 42 (35×) - 1100 c mtD27 a XIV mtD26 cytB, ND1 1700 42 (35×)42 (35×) 1700 a mtD30 a XV 16SF 16S 800 43.5 (35×)43 (35×)800b MEL 3 c XVI 16SF 16S 600 42 (35×)42 (35×)600b 16SR b XVII mtD36 16S/12S 1800 53 (35×)53 (35×) 1800 a 16SR b a - Simon et al. (1994); b - Hall and Smith (1991); c - Silvestre (2002); d - Arias et al. (1998). 434 R.M. Brito, M.C. Arias

Figure 1. Linearized restriction maps, PCR-amplified regions for Partamona mulata and P. helleri and the main gene positions, using the Melipona bicolor mitochondrial genome as a guide. Amplified fragments are represented by bars and roman numbers (see Tab. I). Regions not amplified are represented by dotted lines. The A+T rich region and tRNAs were not represented. B, Bcl I; G, Bgl II; C, Cla I; E, EcoR I; A, Hae III; D, Hind III; N, Nde I; P, Pst I. Italic letters represent restriction sites conserved between both species. ND2, NADH dehydrogenase subunit 2; COI, cytochrome c oxidase subunit 1; COII, cytochrome c oxidase subunit 2; 8, ATP F0 synthase subunit 8; 6, ATP F0 synthase subunit 6; COIII, cytochrome c oxidase subunit 3; ND3, NADH dehydrogenase subunit 3; ND5, NADH dehydrogenase subunit 5; ND4, NADH dehydrogenase subunit 4; ND6, NADH dehydrogenase subunit 6; cytB, cytochrome B; ND1, NADH dehydrogenase subunit 1; 16S, large subunit ribosomal RNA; 12S, small subunit ribosomal RNA.

The sizes of the amplified fragments were was sequenced from both Partamona species similar to expected, according to the M. bicolor (GenBank accession numbers: AY497503 and mtDNA sequence (Silvestre, 2002) (Tab. I), AY497504), and a total of 924 bp was aligned. suggesting that the Partamona mitochondrial The similarity level between P. helleri and P. genome has the same main gene order. How- mulata was estimated at 95.15% and 17 trans- ever, the Partamona fragment amplified by the versions, 23 transitions and 5 indels were ver- primers mtD2/mtD9 was 200 bp less than in ified. Sequence comparison between P. mulata Melipona bicolor. This region comprises the and M. bicolor showed 90.7% similarity, 57 ND2, 5’CO I and several tRNA genes. Transfer transversions, 23 transitions, and 6 indels, and RNA genes are more likely to undergo translo- with P. helleri 92.1% similarity, 48 transver- cations in the mtDNA genome than protein sions, 22 transitions and 3 indels. The nucle- coding genes, a suggestion supported by the otide sequence corresponds to the 3’COI, finding that at least 8 tRNA translocations have tRNALeu and 5’COII genes, and no single been reported between A. mellifera and M. nucleotide was found between the tRNALeu and bicolor mtDNA (Silvestre, 2002; Silvestre 5’COII genes, which would characterize the et al., 2002). Thus this phenomenon may be intergenic region. In A. mellifera this region quite frequent in bees and explain the length was first described by Crozier et al. (1989), and differences observed, since this mtDNA region later Cornuet et al. (1991) verified that it has a comprises several tRNA genes. This needs to high level of length variation among A. mellif- be further investigated by sequencing. era subspecies (ranging from 200 to 650 bp). As described for Plebeia (Francisco et al., Moreover, Cornuet et al. (1991) proposed that 2001) and M. bicolor (Silvestre, 2002), the Par- this region may function as an extra mtDNA tamona species did not have an intergenic origin of replication, based on its secondary region between the COI and COII genes. The structure. The lack of an intergenic region in absence of the intergenic region in P. mulata Plebeia (Francisco et al., 2001), Melipona and P. helleri was inferred initially by a frag- (Silvestre, 2002; Weinlich et al., 2004) and ment length, that was 300 bp shorter than that Partamona (present work) suggests that it is a described in A. mellifera (but similar to those conserved evolutionary characteristic among observed in Plebeia and Melipona). This region the stingless bees. MtDNA characterization of Partamona 435

3.2. Partial restriction maps probe with high specificity, considerably more of Partamona mulata and P. helleri sample DNA to conduct the restriction digestions and more time and labor. Taq polymerase Of the 12 restriction enzymes used on all errors are easily resolved by repeating doubtful fragments, three (EcoR V; Sca I, and Xho I) did results, digesting PCR products from different not cut in either species. The enzyme Pvu II was reactions or even digesting other fragments tested only on the mtD36/16SR fragment and that overlap with the suspect one. The potential no cleavage was observed. Sixteen restriction of PCR+RFLP to assess genetic variability sites were detected in P. mulata and 20 in has been demonstrated in various studies on P. helleri, corresponding to 94 and 118 base birds, lagomorpha, rodents, primates and fishes pairs, respectively. Partial restriction maps are (Lovette et al., 1999; Mamuris et al., 2001; shown in Figure 1. Some restriction sites were Perwitasari-Farajallah, 2001; Ittig et al., 2002; found only in Partamona and not in Melipona Papasotiropoulos et al., 2002). We plan to fur- and Plebeia. These included: Bgl II; Cla I; ther use this technique to analyze mitochon- Hind III; Nde I, in fragment II (COI-COII) drial haplotypes in population genetics studies (Fig. 1); Bcl I; Bgl II; Pst I in IV (COII/ATPase 6 of the Meliponini. Nde XIV and 8) and I in (cytB and ND1). More- The data obtained here constitutes the first over, some of those above mentioned sites step toward haplotype recognition that will be were exclusive for P. helleri; such as Bgl II and useful in population genetic studies of Parta- Hind III in II; and Bgl II in IV (Fig. 1). How- mona helleri and P. mulata. The variability ever, restriction site differences that appear found in the mtDNA of these species also sug- fixed must be considered premature until sur- gests that this methodology can be used to veys of population samples are completed. investigate phylogenetic relationships within Comparing our results to mtDNA restriction and among Partamona groups. maps previously described for A. mellifera (Crozier and Crozier, 1993), Plebeia (Francisco et al., 2001) and Melipona (Weinlich et al., ACKNOWLEDGEMENTS 2004), we verified that some restriction sites seem to be very conserved within corbic- We are grateful to Núcleo de Estudo da Fauna ulate bees, such as: Bcl I sites in fragment II; (NIEFA) – Universidade Federal de Mato Grosso EcoR I and Cla I in XIV and Pst I in XV for the great cooperation; to Dr. Lucio A.O. Cam- (Fig. 1). pos of Universidade Federal de Viçosa for provi- ding Partamona helleri specimens; to Mr. João Some restriction sites were shared exclu- Losano (Piuval Farm, Poconé – MT – Brazil) for sively among bees from the tribe Meliponini allowing us to collect P. mulata specimens at his such as: Cla I site in fragment III (but not in private property; to Susy Coelho for technical sup- Melipona rufiventris and P. mulata); Hind III port; and to Dr. Walter Steven Sheppard for his comments and English revision. This work was in fragment VII except for Melipona melano- financially supported by FAPESP. venter and M. rufiventris; and Hae III in fragment XVII. We also found Partamona sites Résumé – Caractérisation de l’ADN mitochon- that were shared with one other species or drial de deux espèces de Partamona (Hymenop- genus: Bcl I and Nde I in fragment III with A. tera, Apidae, Meliponini) par PCR + RFLP et mellifera; Nde I in fragment IV with Plebeia séquençage. Le genre Partamona comprend except Plebeia saiqui; and one Cla I site on 33 espèces et sa large répartition s’étend du sud du fragment II shared only with Plebeia sp. Brésil et du Paraguay jusqu’au Pérou et au sud du Mexique (Pedro et Camargo, 2000). On a essayé de The PCR+RFLP technique employed here tirer des conclusions sur l’évolution de ce groupe provides the first molecular characterization of d’abeilles, bien que le faible niveau de variabilité the mitochondrial genome of Partamona spe- trouvé n’autorise que la détermination des groupes cies. We were able to show that, in spite of Taq phylogénétiques principaux au sein du genre. Les polymerase errors, it is possible to use this tech- analyses d’ADN mitochondrial (ADNmt) ont pour- nique to map restriction sites as an alternative tant été utilisées dans des études de parenté généti- que parmi les espèces appartenant à la tribu des to Southern blot procedures. This PCR based Meliponini et ont montré une variabilité aux niveaux method has some clear advantages over South- intergénérique, interspécifique et intraspécifique. ern blot mapping, as the latter can require a Le but du présent travail était de caractériser 436 R.M. Brito, M.C. Arias l’ADNmt de deux espèces d’abeilles du genre Par- bei P. mulata und 20 bei P. helleri (Abb. 1). Einige tamona. En utilisant comme modèle la séquence de Schnittstellen waren auf die Gattung beschränkt, l’ADNmt de Melipona bicolor, nous avons pu einige waren auch bei andere Arten der Meliponini amplifier 12120 pb de d’ADNmt de P. mulata et vorhanden. Wir glauben, dass die molekulare Ana- 10300 pb de celui de P. helleri, ce qui correspond lyse der mtDNA zusätzliche Daten aufzeigen wird, respectivement à 65,5 % et 55,7 % de leur génome um die genetische Verwandtschaft innerhalb und mitochondrial total (Tab. I et Fig. 1). L’absence de zwischen den Partamona Arten besser darzustellen. région intergénique entre les gènes cytochrome oxydase I (COI) et cytochrome oxydase II (COII) a Partamona / Meliponini / mtDNA / pcr / rflp / été confirmée par le séquençage. Cette absence chez Restriktionskarten / Stachellose Biene Partamona et chez d’autres Meliponini suggère qu’il s’agit d’un caractère phylogénétique conservé parmi les abeilles sans aiguillon. Des cartes partiel- REFERENCES les ont été construites d’après la digestion par 8 enzymes de restriction des fragments amplifiés Arias M.C., Francisco F.O., Silvestre D., Weinlich R., par PCR. Les tests de digestion ont montré l’exis- Sheppard W.S. 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