Comparative cytogenetics in three Melipona species (Hymenoptera: Apidae) with two divergent heterochromatic patterns

Abstract The genus Melipona is subdivided into four subgenera based on morphological characteristics, and two groups based on cytogenetic patterns. The cytogenetic information on this genus is still scarce, therefore, the goal of this study was to characterize Melipona paraensis, Melipona puncticollis, and Melipona seminigra pernigra using the following techniques: C-banding, DAPI/CMA3 fluorochromes, and FISH with an 18S rDNA probe. Melipona paraensis (2n=18) and M. seminigra pernigra (2n=22) were classified as high heterochromatin content species (Group II). Their euchromatin is restricted to the ends of the chromosomes and is CMA3 +; the 18S rDNA probe marked chromosome pair number 4. Melipona puncticollis (2n=18) is a low heterochromatin content species (Group I) with chromosome pair number 1 marked with CMA3 and 18S rDNA. Low heterochromatin content is a putative ancestral karyotype in this genus and high content is not a monophyletic trait (Melikerria presents species with both patterns). Differences concerning the karyotypic characteristics can be observed among Melipona species, revealing cytogenetic rearrangements that occurred during the evolution of this genus. Studies in other species will allow us to better understand the processes that shaped the chromatin evolution in Melipona.


Introduction
Species belonging to the Meliponini tribe are also known as stingless bees.These highly eusocial bees are of pantropical distribution and are important both economically and ecologically.They produce honey and propolis, pollinate a variety of cultivated and native crops, and play an important role as providers of ecosystem services (Kerr et al., 1996;Heard, 1999;Cortopassi-Laurino et al., 2006;Michener, 2007).In the Neotropics, Meliponini is composed of 33 genera with approximately 417 valid species (Camargo and Pedro, 2013).Among these genera, Melipona Illiger 1806 is the most species-rich genus in this tribe (Silveira et al., 2002), represented by 73 described species of which 43 occur in Brazil, and it is subdivided into four subgenera based on morphological characteristics: Eomelipona, Melipona stricto sensu, Michmelia, and Melikerria (Camargo and Pedro, 2013).It is important to highlight that a revision is needed, since Eomelipona is the only subgenus that was not recovered as a monophyletic clade in a molecular phylogenetic analysis (Ramírez et al., 2010;Rasmussen and Cameron, 2010).
Cytogenetic studies on 22 Melipona species indicate a preserved autosome diploid number of 2n = 18 chromosomes in most of the species studied so far, with Melipona seminigra merillae Cockerell, 1919 being the exception, showing 2n = 22 chromosomes (reviewed in Tavares et al., 2017).Despite the conservatism in the diploid number, the Melipona species have a divergent pattern regarding heterochromatin content, and defined through C-banding technique it can be subdivided into two groups: Group I is comprised of species with a low content of heterochro-matin, while Group II is comprised of species with a high heterochromatin content (Rocha and Pompolo, 1998;Rocha et al., 2002Rocha et al., , 2003;;Lopes et al., 2008Lopes et al., , 2011)).In this context, the subgenera Eomelipona and Melipona stricto sensu are comprised only of species with a low content of heterochromatin, Michmelia only of species with a high content, while Melikerria has species with both patterns.The cytogenetic data available on the genus Melipona re-garding chromosome number, C-banding, CMA 3 , and 18S rDNA patterns is revised in Table 1.
The goal of this study was to describe the cytogenetic characteristics (chromosomal number, heterochromatin content, DAPI/CMA 3 fluorochromes, and 18S rDNA patterns) of three Melipona species (Melipona paraensis Ducke, 1916, Melipona puncticollis Friese, 1902, and Melipona seminigra pernigra Friese, 1903), and to compile the cytogenetic data available for this taxon in order to identify the chromosomal variation that is characteristic for each Melipona Group (I and II), as well as to understand the role of these regions in the evolution of chromosomes in the genus.

Material and Methods
The three Melipona species (M.paraensis, M. puncticollis, and M. seminigra pernigra) were collected in Altamira, state of Pará, Brazil.The specimens were identified by Sílvia Regina de Menezes Pedro (Universidade de São Paulo, Ribeirão Preto, Brazil), and deposited in the scientific collection of the Apiário Central at Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil.Mitotic chromosomes were obtained from cerebral ganglia of larvae in the final defecation stage (Imai et al., 1988).The conventional staining was done using Giemsa diluted in Sorensen buffer in a 1:30 proportion, and at least 25 larvae of each species were analyzed.The chromosomes were classified following the arm ratios given by Levan et al. (1964).Heterochromatin was visualized through C-banding (Sumner, 1972) and digital images of the metaphases were taken in a BX53F Olympus microscope equipped with a DP73F Olympus camera, using CellSens imaging software.
Sequential staining with the fluorochromes 4'-6-diamindino-2-phenylindole (DAPI) and chromomycin A 3 (CMA 3 ) was performed following the method of Schweizer (1980).Fluorescent in situ Hybridization (FISH) followed the protocol described by Pinkel et al. (1986) using a ribosomal 18S rDNA probe isolated from M. mondury obtained through Polymerase Chain Reaction (PCR) using the following primers: 5'-TAATTCCAGCTCCAATAG-3' and 5'-CCACCCATAGAATCAAGA-3'.This probe was labeled by an indirect method using digoxigenin-11-dUTP (Roche Applied Science), and the signal was detected with anti-digoxigenin-rhodamine (Roche Applied Science).Digital images of the fluorescence images were captured in a BX53F Olympus microscope equipped with an MX10 Olympus camera using CellSens imaging software.An average of 10 metaphases was analyzed to determine the cytogenetic patterns revealed by the different techniques used in this study.

Results
The diploid chromosome number of M. paraensis was defined as 2n = 18 (Figure 1a).C-banding revealed that 808 Cytogenetics in Melipona species the major part of each chromosome is comprised of heterochromatin.This hindered the visualization of centromeres, and hence it was not possible to define the karyotypic formula (Figure 1b).The DAPI/CMA 3 analysis indicated that the heterochromatin is DAPI + (Figure 2a and Figure S1a-c), while CMA 3 + marked all the extremities of the chromosomes corresponding to the euchromatin region (Figure 2b).FISH with 18S rDNA probe marked chromosome pair number 4 in its terminal position (Figure 3a).
The diploid chromosome number of M. puncticollis was defined as 2n = 18 and its karyotypic formula as 2n = 2m + 14sm + 2a (Figure 1c).C-banding indicated a low content of heterochromatin that is restricted to the pericentromeric region of chromosome pair numbers 1, 3, 6, 9, and the subtelomeric region of the long arms of chromosome pair numbers 2, 4, 5 and 7, while chromosome pair number 8 is completely euchromatic (Figure 1d).Sequential staining with DAPI/CMA 3 fluorochromes indicated strong DAPI + bands corresponding to the heterochromatin region (Figure 2c), while CMA 3 marked the interstitial region of chromosome pair number 1 (Figure 2d).The same result was found with the 18S rDNA FISH probe (Figure 3b).
The diploid chromosome number of M. seminigra pernigra was defined as 2n = 22 (Figure 1e).C-banding revealed that the majority of each chromosome is composed of heterochromatin, hindering the visualization of the centromeres, so it was not possible to define the karyotypic formula (Figure 1f).DAPI/CMA 3 analysis indicated that the heterochromatin is DAPI + (Figure 2e and Figure S1d-f).CMA 3 + marked all the extremities of the chromosomes corresponding to the euchromatin region, and we could identify one chromosome pair that strongly stained with CMA 3 fluorochrome (Figure 2f).FISH with the 18S rDNA probe marked chromosome pair number 4 in its terminal position (Figure 3c).
Regardless of the conservatism in the diploid number, differences concerning the karyotypic formula and heterochromatin content could be observed among species, re-vealing cytogenetic rearrangements that have occurred during the evolution of the genus.Changes in the karyotypic formula among species belonging to Group I indicate the occurrence of pericentric inversions that altered the number of metacentric and submetacentric chromosomes in this group: Melipona favosa Fabricius, 1798 (2n = 12m + 4sm + 2a) (Hoshiba, 1988), Melipona mandacaia Smith, 1863 (2n = 2m + 14sm + 2a) (Rocha et al., 2003), M. puncticollis (2n = 2m + 14sm + 2a) (present study), and Melipona quadrifasciata Lepeletier, 1836 (2n = 4m + 12sm + 2a) (Silva et al., 2012).In Group II species, the high heterochromatin content masks the position of the centromere, and therefore, makes it difficult to identify the morphology of the chromosomes to define the karyotypic formula of these species.This is a common trait among the Melipona species belonging to Group II, rather than an issue related to the quality of the metaphases (Rocha et al., 2002;Lopes et al., 2008Lopes et al., , 2011)).
In the species analyzed in this study, M. puncticollis is a low heterochromatin content species, while M. paraensis and M. seminigra pernigra are high heterochromatin content species (Figure 1).The first description of the C-banding pattern on M. seminigra merrilae indicated this subspecies as part of Group I, with low heterochromatin content (Francini et al., 2011), but analyzing the images from that publication, the pattern seems to be very similar to the high heterochromatin content species, as it was not possible to visualize the centromeres, and they had heterochromatin as the predominant constituent of the chromosomes.Ongoing cytogenetic studies on this subspecies confirm that M. seminigra merrillae, as well as M. seminigra pernigra are high heterochromatin content subspecies belonging to Group II (unpublished data).
In eusocial bees, the heterochromatin is usually AT-rich (DAPI + ) (Brito et al., 2003;Rocha et al., 2003;Lopes et al., 2011;Godoy et al., 2013), and this is a pattern shared by Melipona species with both low and high heterochromatin content (Figure 2).CMA 3 + positive bands are another characteristic used to distinguish Groups I and II in this genus (Table 1): Group I species have only one chromosome pair CMA 3 + marked in its interstitial position, and this chromosome pair is usually related to the nucleolar organizing region (NOR) (Rocha et al., 2002), indicating that the NOR is CG-rich in this group; Group II species have CMA 3 + terminal markings on all of the chromosomes corresponding to the euchromatin, indicating that these regions are CG-rich, and in some cases it is possible to identify one pair with the brightest mark as associated with ribosomal cistrons, as for instance in M. seminigra pernigra (Figure 2F) and other high heterochromatic content species (Lopes et al., 2008(Lopes et al., , 2011)).It is interesting to note that the solitary bee Melitoma segmentaria Fabricius, 1804 has the opposite pattern, as the euchromatic portion of the chromosomes are CMA 3 -and the heterochromatic ones are CMA 3 + (Cristiano et al., 2014).Other solitary bees, but they have unique CMA 3 accumulation patterns, showing that the heterochromatin is heterogeneous with respect to its composition, with some blocks rich in AT and others rich in CG (Fernandes et al., 2013), highlighting the diversity of the patterns observed among bees.
To this date, there is only one report that has used FISH to confirm the position of the NORs with an 18S rDNA FISH probe; this was done in Melipona fasciculata Smith, 1854 (Rocha et al., 2002, revised in Tavares et al., 2017).Together with our study, it seems that only one pair of chromosomes labeled with this probe, which can be considered as a conserved characteristic in this genus (Figure 3).Studies applying ribosomal probes in bees are still scarce, but analyses combining Ag-NOR, CMA 3 + bands, and FISH 18S rDNA techniques have been used to identify NORs in different Meliponini species (Rocha et al., 2002;Brito et al., 2005;Duarte et al., 2009;Krinski et al., 2010;Lopes et al., 2011;Godoy et al., 2013;Miranda et al., 2013).Based on these three different techniques it can be inferred that having only one pair of NORs is a conserved characteristic in Melipona (Table 1).
Regardless of the conservatism in the number of markings, the position of the 18S rDNA cistrons can be used to differentiate Groups I and II in Melipona, as they are interstitial in low content species and terminal in high content ones (Table 1).Independent of the technique applied, the literature indicates pair number 1 as the NOR bearer in this genus.For: Melipona asilvai Moure, 1971, M. mandacaia, and Melipona marginata Moure, 1992 this was inferred by Ag-NOR (Maffei et al., 2001;Rocha et al., 2002Rocha et al., , 2003)).For Melipona bicolor Lepeletier, 1836, Melipona capixaba Moure and Camargo, 1994, Melipona mondury Smith, 1863, M. quadrifasciata, M. quinquefasciata, M. rufiventris, and Melipona subnitida Ducke, 1910 the identification was done by CMA 3 fluorochrome (Rocha et al., 2007;Lopes et al., 2008), and for M. fasciculata it was identified by FISH with a ribosomal probe (Rocha et al., 2002revised in Tavares et al., 2017).However, in our study only the low content species M. puncticollis had the first pair as the NOR bearer, while the high content species M. paraensis and M. seminigra pernigra had pair number 4 marked with the 18S rDNA probe (Figure 3), highlighting another distinct characteristic between Groups I and II.We argue that none of the cited studies above arranged the karyotype.Hence in metaphase cells, the terminal location of the probe in the high content species might have given the impression of a bigger chromosome (see Figure 3).
Despite being polyphyletic, basal Eomelipona species group together with Melipona stricto sensu (Ramírez et al., 2010), and this clade is composed of species with low heterochromatin content (Table 1), indicating that this is the plesiomorphic characteristic of the genus, while high heterochromatin content appeared more than once during the evolution and diversification of this taxon, emerging in both Melikerria and Michmelia subgenera.As we could observe both heterochromatin patterns in Melikerria (Table 1), the classification of the Melipona species into low and high heterochromatin content species did not form natural groups and did not represent monophyletic clades in the phylogenetic analysis.
The current study aimed to describe three Melipona species with divergent patterns of heterochromatin accumulation, arguing that a karyotype with low heterochromatin content is a putative ancestral karyotype in this genus and that high heterochromatin content is not a monophy-letic characteristic.We also contributed with new cytogenetic data on Groups I and II, highlighting the cytogenetic rearrangements that occurred during the chromosome evolution in this major stingless bee genus.Finally, we emphasize the importance of cytogenetic analyses to evidence the chromosomal rearrangements that occurred during the evolution of different species (Imai et al., 1994;Menezes et al., 2014).Studies in other species will allow us to better understand the processes that shaped chromatin evolution in Melipona.

Table 1 -
Cytogenetic data available on 22 Melipona species regarding their chromosome number (karyotypic formula), C-banding (high or low content), CMA 3 and 18S rDNA patterns.Species were assigned to subgenera based on the Moure's catalogue.
(Tavares et al., 2017)pressipes in the paper ofRocha et al. (2002)is indeed M. fasciculata(Tavares et al., 2017).‡ B chromosomes were reported in these two species.§ Reevaluated as high content.More details are given in the text.