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Heterochromatin distributon and chromosomal mapping of microsatellite repeats in the genome of Frieseomelita stingless bees (Hymenoptera: Apidae: Meliponini).

Hymenoptera includes bees, wasps, and ants, being regarded as the third largest order of insects (Michener 2007). Within this group, the family Apidae Latreille, stands out because of their remarkable species richness. This family is subdivided into 5 subfamilies, such as Apinae Latreille, which includes the tribe Meliponini (Moure 2015). This tribe is composed of social bees with reduced stngers and different colors, shape, and behavioral paterns, popularly known as stingless bees or meliponines (Nogueira-Neto 1997). These bees are efcient pollinators and thus essental to the conservaton of several plant species and their associated fauna (Kerr et al. 2001). The genus Frieseomelita (Hymenoptera: Apidae) comprises 16 valid species occurring in the Neotropical region, ranging from South America to Mexico (Camargo & Pedro 2003; Oliveira et al. 2011).

Cytogenetic reports are important to infer evolutonary processes and assess the genomic organizaton of species, thus being informatve to taxonomy and systematcs (Sumner 2003). Approximately 65 stngless bee species have been cytogenetically analyzed so far (Cristano et al. 2014), but only 5 of them include the genus Frieseomelita, in which only conventonal techniques (Giemsa staining, C-banding, and base-specifc fuorochrome staining), have been applied (Rocha et al. 2003; Carvalho & Costa 2011). Even though the number of cytogenetic studies in meliponines is underrepresented, the chromosomal data reported so far demonstrate a high amount of heterochromatn present in most species (Rocha et al. 2003).

The richness of heterochromatc regions might act as a hotspot for chromosomal rearrangements (Sumner 2003). A refned analysis of heterochromatn distributon and compositon is important to improve our knowledge of karyotype evoluton dynamics. However, detailed studies on the heterochromatn compositon in stingless bees are scarce, in part because there is no efectve fuorescence in situ hybridizaton technique procedure for this group. Indeed, the few available reports using fuorescence in situ hybridizaton are based on the mapping of ribosomal probes (18S and 5S) or chromosomal paintng with probes derived by microdissecton (Mampumbu and Pompolo 2000; Rocha et al. 2002; Brito et al. 2005; Fernandes et al. 2011; Martns et al. 2013; Lopes et al. 2014). Alternatvely, microsatellites probes already have been produced and successfully tested to evaluate heterochromatn compositon in several groups of animals and plants (Kubat et al. 2008; Martns et al. 2013; Parise-Maltempi et al. 2013; Lopes et al. 2014). Cytogenetic data in Frieseomelita are restricted to conventonal analyses (Giemsa-staining and C-banding) and staining with base-specifc fuorochromes (Rocha et al. 2003; Carvalho& Costa 2011). Once the accumulaton of repettve DNA sequences plays a key role in chromosomal evoluton (Charlesworth et al. 2005), serving as hotspots for chromosomal rearrangements, the chromosomal mapping of these sequences is useful to infer evolutonary pathways for distnct animal groups. Because the karyotype evoluton in Frieseomelita is inferred based on the Theory of Minimum Interacton, the visualizaton of chromosomal regions more susceptble to rearrangements is relevant to point out potental changes in chromosome structure.

Therefore, based on the lack of knowledge about karyotype evoluton and genome organizaton of Meliponini, we carried out cytogenetic analyses in 6 species of Frieseomelita using C-banding, base-specifc fuorochrome staining and fuorescence in situ hybridizaton with microsatellite probes to provide a refned analysis of the heterochromatn compositon and variaton among these species. Additonally, determining basic karyotypic aspects of Frieseomelita species will help us to beter understand their evolutonary relatonships.

Materials and Methods

The species Frieseomelita sp.n.; Frieseomelita varia Lepeleter; Frieseomelita dispar Moure; Frieseomelita francoi Moure; Frieseomelitta doederleini Friese; and Frieseomelita meadewaldoi Cockerell, were sampled in distnct localites of southeastern and northeastern Brazil (Table 1). Adult specimens were collected from each sampled colony for taxonomic identfcaton (by Profa. Dra. Favizia Freitas de Oliveira, Insttuto de Biologia, Departamento de Zoologia, Universidade Federal da Bahia). Thirty post-defecatng larvae were collected per colony for cytogenetic procedures.

The larvae were immersed in hypotonic 0.005% colchicine soluton and the chromosomes were obtained from cerebral ganglia, as described by Imai et al. (1988). Aferwards, the metaphase spreads were stained with Giemsa diluted in Sorensen bufer (1:30).

The C-banding was carried out according to Sumner (1972), with slight changes in the treatment with barium hydroxide [5% Ba(OH)2], as follows: 20 seconds for F. francoi and F. sp.n., 18 seconds for F. doederleini, and 25 seconds for F. varia, F. dispar, and F. meadewaldoi.

Base-specifc fuorochrome staining using Chromomycin A3 (CMA3) and 4'6-diamidino-2-phenylindole (DAPI) was used to detect GC- and AT-rich regions, respectively, while Distamycin A (DA) was added as counter stainer (Schweizer 1980).

Fluorescence in situ hybridizaton was performed as reported by Pinkel et al. (1986) with a stringency of 77%. The microsatellites repeats used as FISH probes in this study were: [(GA).sub.15], [(GC).sub.15], [(GAA).sub.10], [(CAA).sub.10], and [(GAG).sub.10]. These probes were directly labeled with [Cy.sup.3] during their synthesis as reported by Kubat et al. (2008). Two series of FISH experiments were performed; the frst involved hybridizaton of all probes in the 6 bee species. The second series included only the probes lacking signals in 1 or more species along with a positve control to confrm whether the failure of hybridizaton was related to technical artfact or to the lack of homologies between chromosomal and probe sequences. The chromosomes were counter-stained with DAPI (0.2 mg per mL) in Vectashield Mountng Medium (Vector Laboratories, Burlingame, California, USA).

At least 30 metaphase spreads per individual were analyzed to confrm the 2n, karyotype structure, and FISH results. The chromosomal spreads were analyzed and photographed using an Olympus BX-51 epifuorescence microscope equipped with image capture digital system (Image Pro Plus 6.1, Media Cybernetcs, Rockville, Maryland, USA). The chromosomal pairs were organized based on heterochromatn locaton (Imai 1991), into: Metacentric (M), Metacentric with pericentromeric and telomeric C-bands ([M.sup.t]), Acrocentric (A), and Pseudoacrocentric ([A.sup.M]).


All female specimens of Frieseomelita sampled here shared 2n = 30 chromosomes, as reported in other congeneric species (Rocha et al. 2003; Carvalho & Costa 2011). Similarly, the consttutve heterochromatn was distributed over centromeric, pericentromeric, and telomeric regions in the 6 analyzed species, with conspicuous C-bands in 1 arm of most chromosomes (Fig. 1).

In contrast, 5 karyotype formulae were defned, allowing differentatng F. varia and F. doederleini, F. sp.n. from F. meadwaldoi, F. doederleini, and F. dispar, F. francoi (Fig. 1, Table 1).

The karyotypic patern observed in F. varia and F. doederleini corroborates that reported by Rocha et al. (2003) because both are characterized by a high number of acrocentric and pseudoacrocentric chromosomes.

Frieseomelita sp.n. and F. meadewaldoi were cytogenetically analyzed for the frst time in this study, being the frst species characterized by the karyotype formula: 2K = 6M + 4A + 20[A.sup.M], and the second by the presence of [M.sup.t] chromosomes. As for the heterochromatn compositon, GC-rich sites ([CMA.sub.3+] / DAPI-) were exclusively observed at terminal heterochromatn on short arms of 5 chromosomal pairs in F. dispar (Fig. 2).

The karyotype formula of F. dispar (2K = 4M + 2M (t) + 6A + 18[A.sup.M]) herein described difers from that previously reported (Carvalho & Costa 2011).

The karyotype formula of F. francoi (2K = 4M + 2M (t) + 4A + 20[A.sup.M]) corroborates the report by Carvalho and Costa (2011).

As for the heterochromatn compositon, GC-rich sites ([CMA.sub.3+]/DAPI-) were exclusively observed at terminal heterochromatn on short arms of 5 chromosomal pairs in F. dispar (Fig. 2).

A GC-rich heteromorphic block was observed on short arms of a single chromosomal pair in F. meadewaldoi, since [CMA.sub.3+] signals were more conspicuous in one of the homologous chromosomes. The base-specifc fuorochrome staining in Frieseomelita sp.n., F. meadewaldoi, F. varia, and F. doederleini revealed no richness of either AT or GC segments.

The microsatellites presented a wide distributon in Frieseomelita species, besides interspecifc diferences in both amount and locaton of FISH signals (Fig. 3).

The [(GA).sub.15] probe revealed specifc signals at terminal regions of some chromosomal pairs in F. varia (Fig. 3-1a). On the other hand, F. dispar was characterized by large blocks of this microsatellite, including 1 pair with FISH signals over the whole chromosome (Fig. 3-4a). This repeat also was abundant in the chromosomes of F. francoi (Fig. 3-5a), including some more conspicuous marks. In F. doederleini (Fig. 3-6a), it was accumulated at terminal region of all pairs. No fuorescence in situ hybridizaton signals were observed for the [(GA).sub.15] probe in F. sp.n. and F. meadewaldoi (Fig. 3-2a, 3a).

Interspecific variaton also was observed in relaton to the [(GC).sub.15] probe. In F. varia and F. doederleini, this microsatellite encompassed nearly all chromosomal pairs and entre chromosomes (Figs. 3-1b, -6b). In F. sp.n., this repettve DNA was dispersed, except for a single pair with signals through the whole chromosome (Fig. 3-2b). Only 2 pairs were positvely labeled with this probe in F. meadewaldoi (Fig. 3-3b) while no hybridizaton was observed in F. dispar (Fig. 3-4b). In F. francoi, positive signals were detected at terminal regions of 1 chromosomal arm in nearly all pairs (Fig. 3-5b).

In the case of the [(CAA).sub.10] probe, the FISH signals were reduced but widespread through the karyotype of F. varia, occupying the telomeres of all chromosomal and intersttal regions of some pairs (Fig. 3-1c). In F. sp.n., this microsatellite repeat was mapped at terminal region of 1 chromosomal arm of most chromosomes besides a chromosomal pair with signals through the whole chromosome extension (Fig. 3-2c). Two pairs were entrely marked in F. meadewaldoi with the same probe, while the other pairs were characterized by reduced signals at terminal regions (Fig. 3-3c). No hybridizaton signal was observed for this repettve DNA in F. dispar (Fig. 3-4c). In F. francoi, punctform signals were observed, except for 2 pairs with positve marks through the whole chromosomes (Fig. 3-5c). Instead, this microsatellite is widespread in the chromosomes of F. doederleini, once positve signals were detected in all pairs (Fig. 3-6c).

A few hybridizaton signals were observed for the [(GAA).sub.10] probe in Frieseomelita (Fig. 3). Reduced signals were observed at terminal and intersttal regions of F. varia (Fig. 3-1d). In F. sp.n., the FISH signals with this probe were located on a single arm of most chromosomes, through the entre extension of 1 pair and at terminal and intersttal regions of another pair (Fig. 3-2d). The signals in F. meadewaldoi were subtle and mainly restricted to terminal region of chromosomes (Fig. 3-3d). In F. dispar, the [(GAA).sub.10] signals were more conspicuous, being observed in most chromosomes (Fig. 3-4d), but they were absent in both F. francoi (Fig. 3-5d) and F. doederleini (Fig. 3-6d).

The hybridizaton with [(GAG).sub.10] probe revealed weak signals in studied species in relaton to other microsatellite repeats. In F. varia, positve signals were scatered through most chromosomes (Fig. 4-1e). In Frieseomelita sp.n., reduced terminal and intersttal signals were spread through all chromosomes (Fig. 4-2e). No signals were detected for F. meadewaldoi and F. dispar (Figs. 3-3e, 3-4e, respectively). In F. francoi, this microsatellite was detected in all chromosomes, except for a single pair (Fig. 3-5e). Positve signals were not detected in F. doederleini as well (Fig. 3-6e).


The diploid number found for all species in this study support the conservatve evoluton of chromosomal numbers in Frieseomelita, even though most trigonines have 2n = 34, except for Trigona fulviventris (Hymenoptera: Apidae) (2n = 32) (Rocha et al. 2003; Domingues et al. 2005; Miranda et al. 2013; Godoy et al. 2013).

The large number of acrocentric and pseudoacrocentric chromosomes found here corroborates the Theory of Minimum Interacton (Imai 1991; Imai et al. 1988, 1994) as the most parsimonious model of karyotype evoluton in Frieseomelita. This large number and the lack of large GC+/AT+ blocks in F. varia, F. sp.n., and F. doederleini suggest that these species might be closely related. Moreover, the karyotypic paterns also indicate that F. varia is highly differentated from F. francoi and F dispar, which is supported by morphological data that split these species into distnct clades (Camargo & Pedro 2003). Apparently, the [M.sup.t] chromosome characterized by the presence of heterochromatn blocks in pericentromeric telomeric region in 1 of the arms evolved from a rare case of pericentric inversion that consttutes a synapomorphy in F. dispar and F. francoi (Carvalho & Costa 2011). Therefore, cytogenetic data suggest that F. dispar and F. francoi, as well as F. meadwaldoi, are likely part of the same clade.

Although F. francoi shares the same karyotype formula and has been placed in the same clade of above-mentoned species according to C-banding (Carvalho & Costa 2011), the heterochromatn composition is different in this species. The presence of 4 pairs bearing GC+/AT-segments in the samples of F. francoi difers from that previously reported (Carvalho & Costa 2011).

Population karyotype variaton found in F. dispar might be related to isolaton by distance, since samples of this species were collected about 200 km from the locality sampled by Carvalho and Costa (2011). It is important to point out that both studies used the same tssue samples and similar procedures.

The presence of a reduced number of pseudoacrocentric ([A.sup.M]) chromosomes in F. dispar and F. meadewaldoi, when compared to the other congeneric species, could be related to the accumulaton of terminal heterochromatn, placing this species as karyotypically derived within Frieseomelita.

Polymorphism of GC+ bands found in F. meadewaldoi has been reported in bees of the genus Plebeia (Hymenoptera: Apidae) (Godoy et al. 2013), but it seems to represent an autapomorphy of F. meadewaldoi because these marks are absent in other congeneric species studied so far. Yet, the present data show that heterochromatn compositon in Frieseomelita is more variable than previously reported.

The microsatellite repeats used as probes for fuorescence in situ hybridizaton experiments have proved to be resolute markers in the analyzed species. So far, reports of molecular cytogenetics in stingless bees were restricted to chromosomal mapping of 18S and 5S rDNAs or chromosome paintng with probes derived by microdissecton (Mampumbu and Pompolo 2000; Rocha et al. 2002; Brito et al. 2005; Fernandes et al. 2011; Martns et al. 2013; Lopes et al. 2014).

Fluorescence in situ hybridizaton results indicate that that the genome of Frieseomelita species are mostly AT-rich because the [(GA).sub.15], [(CAA).sub.10], [(GAA).sub.10], and [(GAG).sub.10] probes revealed a high number of conspicuous signals on chromosomes of studied species. In fact, previous studies with fuorochrome staining (Rocha et al. 2003; Brito et al. 2003; Lopes et al. 2008) suggested that chromosomes of Meliponini are AT-rich, except for some terminal regions of pseudoacrocentric pairs (usually GC-rich), probably related to the presence of ribosomal genes. Similarly, Miranda et al. (2013) revealed richness of AT bases in Cephalotrigona Schwarz, 1940 (Hymenoptera: Apidae), which is in accordance with data presented here. In general, microsatellites are located within heterochromatn segments (Kubat et al. 2008). Albeit variable, the distributon patern of microsatellite repeats in Frieseomelita species was mostly coincident with heterochromatc regions. However, the distributon of microsatellites in some of the analyzed species encompassed the whole chromosome, including euchromatn. Most likely, this result is due to the presence of reduced segments of repettve DNA that could not be observed by C-banding, as already reported in beetles of genus Dichotomius (Scarabaeidae) (Cabral-de-Melo et al. 2011).

As revealed in the present study, most microsatellite repeats were widely dispersed over most chromosomal pairs, indicating variation in heterochromatin composition. This behavior also has been reported in other organisms, determining species-specific distribution patterns for each repetitive sequence (Kubat et al. 2008; Poltronieri et al. 2013). This scenario reveals unique evolutionary and recombination pathways in each taxon, thus generating cytotaxonomic markers, even among species with apparent similar karyotypes (Kubat et al. 2008; Poltronieri et al. 2013; Lopes et al. 2014).

In partcular, some studies have shown that accumulaton of microsatellites is strongly related to heterochromatnizaton, playing a key role in the differentaton of single sex chromosome systems in plants, fsh, and humans (Kubat et al. 2008; Poltronieri et al. 2013). Additonally, Bacolla and Wells (2004) stated that microsatellites (and heterochromatn) act as hotspots for chromosomal rearrangements such as deleton, duplicaton, transpositon, and inversions. Thus, the presence of these microsatellite repeats in Frieseomelita could drive structural chromosomal changes, accountng for their chromosomal differentaton and rate of karyotype evoluton, suggestng divergent evolutonary pathways.

In conclusion, C-banding, base-specifc fuorochrome staining, and mapping of microsatellites repeats by FISH in Frieseomelita all together facilitated evaluaton of the compositon and distributon of repettve DNA in the genome of 6 species of Frieseomelita and determinaton of interspecifc variaton. These analyses should be extended to other meliponines in order to infer the evolutonary mechanisms underlying the microstructural differentaton in karyotype of stingless bees.


We thank Dra. Favizia Freitas de Oliveira (Universidade Federal da Bahia) for species identfcaton, Jamille Araujo Bitencourt, and the students for cytogenetics laboratory work. This study was funded by Fundacao de Amparo a Pesquisa e Inovacao do Estado da Bahia (FAPESB, Brazil) and Conselho Nacional de Desenvolvimento Cientfco e Tecnologico (CNPq, Brazil).

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Jadilla Mendes dos Santos (1), Debora Diniz (1), Tecavita Ananda Santos Rodrigues (2), Marcelo de Bello Cioff (2), Ana Maria Waldschmidt (1,*)

(1)Universidade Estadual do Sudoeste da Bahia, Departamento de Ciencias Biologicas, Jequie, Bahia, Brazil; E-mail: (J. M. S.); E-mail: (D. D.); E-mail: (A. M. W.)

(2)Universidade Federal de Sao Carlos, Departamento de Genetca e Evolucao, Sao Carlos, Sao Paulo, Brazil; E-mail: (T. A. S. R.); E-mail: (M. B. C.)

(*) Corresponding author; E-mail:

Caption: Fig. 1. C-banded karyotypes of females of Frieseomelitta varia (a), Frieseomelitta doederleini (b), Frieseomelitta sp. nov. (c), Frieseomelitta meadewaldoi (d), Frieseomelitta dispar (e), and Frieseomelitta francoi (f). (M = metacentric, A = acrocentric, MT = metacentric with centromeric, and telomeric C-bands, AM = pseudoacrocentric).

Fig. 2. Metaphase spreads of females of Frieseomelitta varia (a, g), Frieseomelitta

Caption: sp. n. (b, h), Frieseomelitta meadewaldoi (c, i), Frieseomelitta dispar (d, j), Frieseomelitta francoi (e, k), and Frieseomelitta doederleini (f, l) after basespecific fluorochrome staining. The arrows indicate the GC-rich regions.

Caption: Fig. 3. Metaphase spreads of females of Frieseomelitta species after fluorescence in situ hybridization with microsatellite probes.
Table 1. Studied species of Frieseomelita, collecton sites in Brazil,
diploid number (2n) and karyotypes.

Species                    Collecton site

Frieseomelita varia        Lontra - MG
Frieseomelita sp. n.       Jequie - BA
Frieseomelita meadewaldoi  Nova Ibia - BA
Frieseomelita dispar       Jequie - BA
Frieseomelita francoi      Santa Ines - BA
Frieseomelita doederleini  Nova Ibia - BA

Species                    Geographic coordinates

Frieseomelita varia        15.9033[degrees]S, 44.3050[degrees]W
Frieseomelita sp. n.       13.8575[degrees]S, 40.0836[degrees]W
Frieseomelita meadewaldoi  13.8100[degrees]S, 39.6255[degrees]W
Frieseomelita dispar       13.8575[degrees]S, 40.0836[degrees]W
Frieseomelita francoi      03.6666[degrees]S, 45.3800[degrees]W
Frieseomelita doederleini  13.8100[degrees]S, 39.6255[degrees]W

Species                    2n       Karyotype

Frieseomelita varia        2n = 30  2K = 4M + 4A + 22[A.sup.M]
Frieseomelita sp. n.       2n = 30  2K = 4M + 4A + 22[A.sup.M]
Frieseomelita meadewaldoi  2n = 30  2K = 6M + 2M (t) + 4A + 18[A.sup.M]
Frieseomelita dispar       2n = 30  2K = 4M + 2M (t) + 4A + 20[A.sup.M]
Frieseomelita francoi      2n = 30  2K = 4M + 2M (t) + 4A + 20[A.sup.M]
Frieseomelita doederleini  2n = 30  2K = 2M + 2M (t) + 4A + 12[A.sup.M]

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Author:dos Santos, Jadilla Mendes; Diniz, Debora; Rodrigues, Tecavita Ananda Santos; Cioff, Marcelo de Bell
Publication:Florida Entomologist
Article Type:Report
Geographic Code:3BRAZ
Date:Mar 1, 2018
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