Repetitive DNAs in scutellaris (: : Meliponidae): chromosomal distribution and test of multiple heterochromatin amplification in the Mariani Cristina Alves Piccoli, Vanessa Bellini Bardella, Diogo Cavalcanti Cabral-De-Mello

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Mariani Cristina Alves Piccoli, Vanessa Bellini Bardella, Diogo Cavalcanti Cabral-De-Mello. Repet- itive DNAs in (Hymenoptera: Apidae: Meliponidae): chromosomal distribution and test of multiple heterochromatin amplification in the genus. Apidologie, Springer Verlag, 2018, 49 (4), pp.497-504. ￿10.1007/s13592-018-0577-z￿. ￿hal-02120543￿

HAL Id: hal-02120543 https://hal.archives-ouvertes.fr/hal-02120543 Submitted on 6 May 2019

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 (2018) 49:497–504 Original article * INRA, DIB and Springer-Verlag France SAS, part of Springer Nature, 2018 DOI: 10.1007/s13592-018-0577-z

Repetitive DNAs in Melipona scutellaris (Hymenoptera: Apidae: Meliponidae): chromosomal distribution and test of multiple heterochromatin amplification in the genus

Mariani Cristina Alves PICCOLI, Vanessa Bellini BARDELLA, Diogo Cavalcanti CABRAL-DE-MELLO

Departamento de Biologia, Instituto de Biociências/IB, UNESP - Universidade Estadual Paulista, Rio Claro, São Paulo CEP 13506-900, Brazil

Received 13 November 2017 – Revised 31 March 2018 – Accepted 13 April 2018

Abstract – Melipona bees are remarkable due to the high contrast in heterochromatin amounts, making this group interesting for studying repetitive DNA amplification. Here, we performed the first efforts for the chromosomal localization of different repetitive DNAs in M. (Michmelia ) scutellaris and tested for unique or multiple hetero- chromatin amplification in Melipona subgenera. Our data revealed enrichment of repetitive DNAs in chromosomal heterochromatic arms demonstrated by C 0 t -DNA and DOP-PCR probe hybridization, although microsatellites and multigene families were located at terminal euchromatic regions. Analysis using C 0 t -DNA probe from M. scutellaris showed positive hybridization only in Michmelia , suggesting monophyletic amplification and sharing of heterochromatin sequences between species. However, the subgenus Melikerria , with a high amount of heterochromatin, probably underwent independent heterochromatin amplification or experienced sequence modification. bee / FISH / heterochromatin / multigene families / repetitive DNAs

1. INTRODUCTION chromosomal and genome organization and evolution in most diverse taxa, including some Eukaryote genomes are composed of the fol- groups. However, few studies have been lowing two types of repetitive DNAs: (i) in undertaken in bees (Brito et al. 2005; Rocha tandem arranged elements, including satellite et al. 2002; Lopes et al. 2014). DNAs, microsatellites, minisatellites, and sev- Stingless bees (Meliponini) comprise a diverse eral multigene families, and (ii) scattered ele- group of highly eusocial mostly occurring ments, which are representedbytransposons in the New World tropics (Michener 2007, and retrotransposons (Charlesworth et al. 2013;Camargo2013). They have great eco- 1994;Nowak1994, López-Flores and logical importance as pollinators of native and Garrido-Ramos 2012; Martins 2007). The agricultural plants (Heard 1999;Pedro2014). abundance, easy isolation, and facility in chro- The genus Melipona comprises at least 50 mosome mapping, allowed the frequent use of species distributed from Mexico to Argentina these sequences as markers to understand (Michener 2007). Melipona species have been the focus of ecological, behavioral, phyloge- netic, and genetic studies, but studies focused Corresponding author: D. Cabral-de-Mello, on their chromosomes are still scarce (Roubik [email protected] 2006; Rocha et al. 2007; Ramirez et al. 2010; Manuscript editor: Marina Meixner Tavares et al. 2017). 498 M. C. A. Piccoli et al.

Until now, Melipona karyotypes had been de- slides were stained with 5% Giemsa for chromo- scribed for 24 species, displaying a very con- some counting and heterochromatin detection was served chromosome number of n =9 (2n = 18). performed according to Sumner (1972). This was observed in 23 species and only two Genomic DNAwas extracted from adults using M. seminigra subspecies showed n = 11, which the phenol/chloroform method (Sambrook and probably originated from fissions in two chromo- Russel 2001). Multigene families were amplified somes (revised by Tavares et al. 2017). Interest- by polymerase chain reaction (PCR) using specif- ingly, the heterochromatin amounts and chromo- ic primers for the 18S rDNA (Cabral-de-Mello somal distribution are highly divergent in et al. 2010) and U2 snDNA (Bueno et al. 2013). Melipona .AccordingtoRochaandPompolo Two strategies were used to obtain the repetitive (1998), they can be divided into two groups when DNA pools: (i) the reassociation kinetics tech- analyzing by C-banding. One group comprises nique (C 0 t ) and (ii) DOP-PCR. The reassociation species with low heterochromatic content (less kinetics technique (C 0 t ) was performed accord- than 50%) that is primarily distributed in ing to Zwich et al. (1997) with modifications. pericentromeric regions or in the short chromo- Approximately 8 μg of DNA was fragmented somal arms of their chromosomes (group I). The with DNAse (Sigma-Aldrich) for 1 min. The gen- other group includes species with high hetero- erated fragments were checked by 1% agarose gel chromatic content, which occupy almost the entire electrophoresis. The DNA samples were then de- chromosomes (group II). The heterochromatin in natured at 95 °C for 10 min and incubated on ice Melipona was studied until now by C-banding for 10 s. Then, the samples were subjected to a and fluorochrome staining providing only general reassociation temperature of 65 °C for 10 min. information about distribution and base pair en- After the samples were incubated at 37 °C for richment (reviewed by Rocha et al. 2007). 8 min with S1 nuclease, the reaction was stopped To better understand the chromosomal distri- by the addition of liquid nitrogen. The DNA was bution of repetitive sequences and their relation- purified with phenol/chloroform (1:1, v /v ) and ship with eu- or heterochromatin in the genus the results were confirmed by electrophoresis on Melipona , we characterized the chromosomes a 1% agarose gel. For amplification by DOP- from M. (Michmelia ) scutellaris , a species with PCR, a protocol proposed by Telenius et al. high heterochromatic content, using distinct (1992) using the degenerate primer 5′-CCGA probes for repetitive DNAs. Moreover, to test the CTCGACNNNNNNATCTGG was utilized. hypothesis of multiple or unique rounds of het- The repetitive DNA sequences were labeled erochromatin amplification and dispersion in with digoxigenin 11-dUTP or biotin 11-dATP by Melipona , we isolated the pool of repetitive PCR or nick-translation. The seven microsatellite DNAs (C 0 t -DNA fraction) from M. scutellaris probes were directly labeled with biotin-14 dATP and used as probe against genomic DNA at the 5′-end during their synthesis (Sigma, St from five other species belonging to the dis- Louis, MO, USA). Fluorescent in situ hybridiza- tinct subgenera and displaying the distinct tion (FISH) followed the protocol described by heterochromatin distribution. Pinkel et al. (1986), with modifications proposed by Cabral-de-Mello et al. (2010), and the probes 2. MATERIALS AND METHODS were detected using anti-digoxigenin-rhodamine (Roche) or Alexa-fluor-488-conjugated Melipona scutellaris larvae were obtained streptavidin (Life Technologies). All the prepara- from apiary colonies at the Bioscience Institute tions were stained with DAPI and mounted with at São Paulo State University, Rio Claro/SP. The Vectashield mounting medium (Vector, Burlin- mitotic chromosomes were obtained according to game, CA, USA). FISH signals were observed Imai et al. (1988) using the cerebral ganglia from using an Olympus BX61 microscope equipped post-defective larvae as the source. Slides were with a fluorescent lamp. prepared by maceration followed by drying on a To check whether the pool of repetitive DNAs heat plate (45 °C). For conventional analyses, the obtained by C 0 t -DNA from M. scutellaris is Repetitive DNAs in Melipona scutellaris 499

Figure 1. Fluorescence in situ hybridization using 18S rDNA (green) and U2 snDNA (red) as probes in Melipona scutellaris chromosomes. Note the hybridization signals are present in the terminal regions of the distinct chromosome pairs. The pair 1 is heteromorphic in size. shared with other species from the same genus, we Melipona , the diploid number conservation is performed dot-blot analysis according to the pro- recurrent in distinct genera, with slight variations tocol described by Anjos et al. (2016). The probe (revised by Tavares et al. 2017). was tested against genomic DNA from species The mapping of repetitive DNAs is scarce in with low amount of heterochromatin M. bees, and FISH mapping using multigene families (Eomelipona ) bicolor and M. (Melipona ) as a probe in Melipona is restricted to major quadrifasciata and with high amount of het- rDNAs in a few species (Rocha et al. 2002, erochromatin M. (Melikerria ) fasciculata , 2007). A common feature of the Melipona karyo- M. (Michmelia ) rufiventris ,andM. types is the heteromorphism in size for the chro- (Michmelia ) seminigra. The genomic DNA mosome 1, and in M. scutellaris ,thischromo- of M. (Michmelia ) scutellaris was used as some carries 18S rRNA at the terminal region positive control. (Figure 1). rDNA loci restricted to one chromo- somal pair were also observed in other species of 3. RESULTS AND DISCUSSION Melipona through FISH. Similar data was ob- served through silver nitrate staining (Rocha The 2n = 18 (female) observed in et al. 2002;Rocha2002). It could indicate a modal M. scutellaris (Figure 1) was previously reported pattern in the genus, but a higher number of spe- by Rocha et al. (2007). This condition was also cies should be studied. This pattern is also recur- reported in 22 other species from the same genus, ring in other Hymenoptera, such as parasitic and with variant diploid numbers only occurring in social wasps (Gokhman et al. 2014; Menezes et al. M. seminigra when considering the A comple- 2013) and (Imai et al. 2001). However, re- ment. Among Meliponini species including ports of multiple 18S rDNA signals were also

Figure 2. Mitotic metaphases of Melipona scutellaris . a Heterochromatin location revealed by C-banding. Note large amount of heterochromatin distributed along almost the entire chromosomes and euchromatin restrict to terminal of chromosomes. b Chromosomal mapping through fluorescence in situ hybridization (FISH) using as probe the C 0 t -DNA fraction of M. scutellatis . c FISH with the DOP-PCR probe obtained from the genomic DNA of M. scutellaris . Note the spread marks along the heterochromatic regions. 500 M. C. A. Piccoli et al. described in Hymenoptera, as in Partamona by high heterochromatin additions (Rocha and (Meliponini) (Brito et al. 2005)andsomeparasit- Pompolo 1998; Rocha et al. 2002; Rocha et al. oid wasps (Paladino et al. 2013; Gokhman et al. 2007). According to Rocha et al. (2002), hetero- 2014). The U2 snDNAwas located in pair 2 at the chromatin amplification could have a crucial role terminal region (Figure 1). This is the first effort to during chromosomal evolution in bees. The dif- locate this sequence in Hymenoptera. The pres- ferences in the heterochromatin amounts and ence of a unique chromosomal cluster of U2 distribution are remarkable aspects of Melipona , snDNA was also observed in true bugs (Bardella and based in these differences, Rocha and et al. 2016) and the grasshoppers Abracris Pompolo (1998) divided the genus into two flavolineata and Schistocerca gregaria (Bueno groups (see introduction). Species with high het- et al. 2013; Camacho et al. 2015), although mul- erochromatic contents also display larger genome tiple clusters were also noticed in other species, sizes, such as M. scutellaris , whose genome is like grasshoppers (Palacios-Gimenez et al. 2013; 1.08 pg (Tavares et al. 2010). The notion of the Castilho et al. 2017). The use of this probe in other amplification of repetitive DNAs, which is asso- Melipona and Hymenoptera will elucidate the ciated with large heterochromatic blocks, is rein- organization and evolution patterns of this se- forced in this work by the analysis of the DOP- quence, which could be a good tool for under- PCR and C 0 t -DNA fraction probe hybridization standing chromosome evolution. that labeled the same regions corresponding to The heterochromatin was distributed along the heterochromatin (Figure 2b, c). chromosomal arms, except in the terminal regions In contrast to the mapping with the C 0 t - (Figure 2a), which has been observed in speci- DNA and DOP-PCR probes, seven microsat- mens from other colonies and is putatively caused ellite repeats revealed enrichment in the

Figure 3. FISH with microsatellites probes in mitotic metaphases of Melipona scutellaris . Observe the stronger hybridization signals in the terminal euchromatic regions of chromosomes in comparison to heterochromatin. The microsatellite motifs are indicated directly in each image. Repetitive DNAs in Melipona scutellaris 501 euchromatic regions, although faint scattered M. scutellaris . It has been documented that the signals were also observed in the heterochro- genome size could correlate with microsatellite matin. No remarkable variability in density length and frequency. The presence of several was observed in the distinct chromosomes microsatellites contributes to increased genome (Figure 3). In other insects, euchromatic loca- sizes in many organisms, although it is not a tions for some microsatellite repeats have general rule (Hancock 1996, 2002; Butcher et al. been reported, such as in grasshoppers and 2000;Tóthetal.2000; Warner and Noor 2000; crickets (Milani and Cabral-de-Mello 2014; Comeron 2001). Considering the enrichment for Ruiz-Ruano et al. 2015;Palacios-Gimenez microsatellites in M. scutellaris euchromatin and and Cabral-de-Mello 2015). In the Hymenop- that the genome increase is related to heterochro- tera model species Nasonia vitripennis ,the matin amplification, we could speculate that the microsatellite composition, abundance, and microsatellite motifs mapped here play a minor density in distinct chromosomes were ana- role in genome size increase in this species. More- lyzed using genomic data. The data contrasted over, the microsatellite mapping indicates the with observations in this study based on the sharing of repetitive DNAs enriched in euchroma- density heterogeneity of microsatellites in the tin (C-negative regions), similar to what was ob- five chromosomes from this species. More- served for M. rufiventris heterochromatin (Lopes over, considering the heat map for the chro- et al. 2014). Putatively, the repetitive DNAs asso- mosomal distribution of microsatellites, there ciated with heterochromatin amplification in are no common regions with higher microsat- M. scutellaris are satDNAs and transposable ele- ellite density (Pannebakker et al. 2010)asob- ments, which are generally isolated in C 0 t -DNA served for the distal euchromatic regions in fraction. However, as C 0 t -DNA is anonymous,

Figure 4. Dot-blot hybridization using M. scutellaris C 0 t -DNA fraction (repetitive DNA enriched fraction) as a probe in species from the Melipona genus groups, group I and group II. a Note the positive signals in M. rufiventris and M. seminigra , which belong to the subgenus Michmelia and have high heterochromatin contents. b Note the absence of signals in species belonging to the other subgenus, including a species with high heterochromatic content, M. (Melikerria ) fasciculata . Species with high heterochromatin amount are identified by (+HC) while species with low heterochromatin amount by (−HC). 502 M. C. A. Piccoli et al. specific sequences should be characterized to as- ACKNOWLEDGEMENTS certain it. Melipona species with high heterochromatin This study was partially supported by Fundação de amounts are placed into two distinct subgenera Amparo à Pesquisa do Estado de São Paulo (FAPESP) (Michmelia and Melikerria ). Based on phyloge- (process number 2014/11763-8), Coordenadoria de netic data, Lopes et al. (2011) proposed that het- Aperfeiçoamento de Pessoal de Nível Superior erochromatin amplification occurred more than (CAPES), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). DCCM was the re- once in Melipona. Our data using dot-blot hybrid- cipient of a research productivity fellowship from the ization shed light on this question. Membrane Conselho Nacional de Desenvolvimento Científico e hybridization using the C 0 t -DNA fraction of Tecnológico-CNPq (process number 304758/2014-0), M. scutellaris as a probe against the genomic VBB was a recipient of PNPD/CAPES scholarship DNA of some Melipona species (M. rufiventris , and MCAP was recipient of CAPES scholarship. M. seminigra , M. bicolor, M. fasciculate ,and M. quadrifasciata ) only showed positive hybrid- AUTHORS’ CONTRIBUTION ization to M. rufiventris and M. seminigra ,which are species belonging to the same subgenus DCCM conceived the study and designed the (Michmelia )(Figure4). These data support the experiments; MCAP and VBB performed the ex- presence of monophyletic heterochromatin ampli- periments; MCAP, VBB, and DCCM interpreted fication and conservation of sequence similarity the data and wrote the manuscript. All the authors among species of Michmelia. These findings also read and approved the final manuscript. suggest more than one round of heterochromatin amplification, i.e., independent amplification of distinct repetitive DNAs, in distinct subgenera of COMPLIANCE WITH ETHICAL Melipona or sequence diversification between STANDARDS subgenera. The occurrence of a polyphyletic sub- genus (Eomelipona ) and of species not assigned Conflict of interest The authors declare that they have no to a specific subgenus (Ramirez et al. 2010)isa conflict of interest. challenge for a more conclusive hypothesis. The analysis of other species and the better un- derstanding of and phylogenetic ADN répétitifs chez Melipona scutellaris (Hymenop- relationships between Melipona subgenera tera: Apidae: Meliponidae): distribution chromosomique et test d'amplification multiple de l'hétérochromatine could shed light on the hypothesis about dans le genre heterochromatin amplification. Even though the pool of repetitive DNAs is shared be- abeilles / FISH/ hétérochromatine / familles tween Michmelia species, the specific se- multigéniques / ADN répétitifs quences responsible for heterochromatin am- plification should be better investigated, Repetitive DNA in Melipona scutellaris (Hymenoptera: using for example, genome-sequencing data. Apidae: Meliponidae): Verteilung über die Chromosomen This is an interesting issue to be investigated und Test auf multiple Amplifikation von Heterochromatin in the future and will allow for a more innerhalb der Gattung conclusive hypothesis for the causes and the Biene / FISH/ Heterochromatin / Multigenfamilien / sequences involved in heterochromatin ampli- repetitive DNA fication in Melipona . These data together with the chromosomal mapping of repetitive DNAs studied here contribute to the im- REFERENCES provement of the hitherto poor knowledge of chromosomal organization of repetitive Anjos, A., Rocha, G.C., Paladini, A., Mariguela, T.C., DNAs among bees, mostly specifically in Cabral-de-Mello, D.C. (2016) Karyotypes and repeti- Melipona species. tive DNA evolution in six species of the genus Repetitive DNAs in Melipona scutellaris 503

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