A new view on the scenario of karyotypic stasis in Epinephelidae fish: Cytogenetic, historical, and biogeographic approaches

Abstract Epinephelidae (groupers) is an astonishingly diverse group of carnivorous fish widely distributed in reef environments around the world, with growing economic importance. The first chromosomal inferences suggested a conservative scenario for the family. However, to date, this has not been validated using biogeographic and phylogenetic approaches. Thus, to estimate karyotype diversification among groupers, eight species from the Atlantic and Indian oceans were investigated using conventional cytogenetic protocols and fluorescence in situ hybridization of repetitive sequences (rDNA, microsatellites, transposable elements). Despite the remarkable persistence of some symplesiomorphic karyotype patterns, such as all species sharing 2n=48 and most preserve a basal karyotype (2n=48 acrocentrics), the chromosomal diversification in the family revealed an unsuspected evolutionary dynamic, where about 40% of the species escape from the ancestral karyotype pattern. These karyotype changes showed a relation with the historical biogeography, likely as a byproduct of the progressive occupancy of new areas (huge diversity of adaptive and speciation conditions). In this context, oceanic regions harboring more recent clades such as those of the Indo-Pacific, exhibited a higher karyotype diversity. Therefore, the karyotype evolution of Epinephelidae fits well with the expansion and geographic contingencies of its clades, providing a more complex and diverse scenario than previously assumed.


Introduction
Reef regions are home to a huge diversity of fish (Bezerra and Silva, 2011), among which Epinephelidae (groupers) stand out for their exceptional diversity. The family and allies (Epinephelidae and Serranidae) include 593 species and 71 genera distributed around the world (Craig and Hastings, 2007;Vaini et al., 2019;Fricke et al., 2021), with the greatest species richness being concentrated in the Indo-Pacific region (Bawole et al., 2018).
Groupers present a broad reproductive strategy, including synchronous and asynchronous hermaphroditism (Pressley, 1981;Liu and Sadovy, 2004). Some species can reach up to more than 400 kg (Bright et al., 2016), making them an important target for commercial fishing and fish farming (Heemstra et al., 2002;Rimmer and Glamuzina, 2017). Commercial exploitation has placed groupers among the marine species most impacted by commercial fishing, with 12% of species under threat of extinction (Mitcheson et al., 2013). Some biological characteristics contribute to the low restoration of their populations such as slow growth, late maturation, high longevity (i.e., almost 40 years of life), and formation of large agglomerations during the reproductive period (Craig et al., 2011;Santos et al., 2019). However, some species such as the Atlantic goliath grouper (Epinephelus itajara) have responded to conservation measures (Giglio et al., 2014).
Molecular approaches have better clarified the phylogenetic relationships of the family (Minglan et al., 2014;Ma et al., 2016;Ma and Craig, 2018;Saad, 2019). In contrast, cytotaxonomic data are still extremely limited, comprising only 8% of the group representatives. In addition, most of the available information refers to Epinephelus species, and is restricted to conventional analyses of the karyotype (Arai, 2011;Pinthong et al., 2013;Paim et al., 2017).
Most Epinephelidae species have a karyotype composed of 2n = 48, with a predominance of acrocentric chromosomes (Arai, 2011;Tseng and Shih, 2018), suggesting the maintenance of a basal karyotype with a low evolutionary dynamic. However, chromosomal data of a larger number of representatives, considering their complex evolutionary biogeographical characteristic (Ma et al., 2016;Ma and Craig, 2018), have been entirely neglected, still missing pieces for inferences on the extent of the karyotype stability in the family (Motta-Neto et al., 2019). Amorim et al. 2 Thus, to understand the mechanism of karyotype evolution among Epinephelidae in depth, conventional cytogenetic analyses and chromosomal mapping of six repetitive DNA classes were performed in eight species from the Atlantic and Indian oceans. The data obtained were associated with a set of other available information, thereby providing a comprehensive view of the chromosomal evolution in a phylogenetic and geographic context.

Material and Methods
Samples, chromosomal preparations, and analyses Details of the size and location of the samples are presented in Table 1 and Figure 1. Individuals were subjected to a 24 h mitotic stimulation using intraperitoneal inoculation of a complex of fungal and bacterial antigens (Molina et al., 2010). Chromosome preparations were obtained from cell suspensions of the anterior region of the kidney using a shortterm culture as described by Gold et al. (1990). Chromosomes were stained using a standard 5% Giemsa solution (pH 6.8) and analyzed under an optical microscope at a magnification of 1000×. The nucleolus organizing regions (NORs) and C-positive heterochromatin were identified following Howell and Black (1980) and Sumner (1972), respectively.

Digital image processing
The best metaphases were photographed using an Olympus BX51 epifluorescence microscope coupled with an Olympus DP73 digital capture system using the cellSens ® software (Olympus). Chromosomes were defined as metacentric (m), submetacentric (sm), subtelocentric (st), and acrocentric (a), according to Levan et al. (1964). To count the chromosome arms (FN), the m, sm, and st chromosomes were considered with two arms and the acrocentric chromosomes with only one arm.
The 18S rDNA and the Ag-NOR sites were coincident and occupied a single locus in the karyotype of all species, always in the short arms of the chromosomes. In E. adscensionis, E. coeruleopunctatus, E. erythrurus, E. sexfasciatus, and C. fulva, they were localized in the acrocentric pair 24 (Figure 2), while were localized in the submetacentric pair 1 of E. itajara and C. formosa, and in the acrocentric pair 20 of R. saponaceus ( Figure 3). The 5S rDNA sequences also displayed a single site in the short arms of the chromosomes in all species. In E. adscensionis, E. coeruleopunctatus, E. erythrurus, E. sexfasciatus, E. itajara, C. formosa, and C. fulva they occurred in the acrocentric pair 23 and in the acrocentric pair 14 of R. saponaceus (Figures 2 and 3).
The microsatellites (CA) 15 and (GA) 15 had a scattered chromosomal distribution, with some more prominent clusters in the centromeric and terminal regions of some pairs (Figures 4  and 5). Tol2 transposons also showed a diffuse distribution, while Rex3 presented discrete accumulations in the centromeric and terminal chromosomal regions in all species, especially in E. itajara, in which more evident signals were detected (Figures 4 and 5).  (Table 2), including the ancient Plectropomus clade (~ 36 Mya) and recent lineages such as Alfestes (~ 5 Mya; Ma et al., 2016), supports 2n = 48a as the basal state for this family.

Discussion
The maintenance of this diploid number in all analyzed species represents a phylogenetic pattern in Epinephelidae. On the other hand, the karyotype macrostructure (2n = 48a; FN = 48), although still retained in most groupers, behaves as a more dynamic evolutionary trait. In fact, similar to E. itajara (2n = 48; FN = 54) and C. formosa (2n = 48; FN = 52), over 40% of the Epinephelidae species have some karyotype diversification associated with pericentric inversions, thereby increasing the number of chromosome arms (FN = 48-96) ( Table 2). This evolutionary trend, which has been better evidenced as chromosomal data increase, is considered as a moderate diversification and reveals an unexpected context for Epinephelidae.
A low rate of evolutionary changes is also evidenced in some repetitive DNA sequences, as highlighted by remarkable homeologies among the Ag-NOR/18S rDNA-bearing pairs in most Epinephelidae species. Indeed, in addition to five of the eight species analyzed (E. adscensionis, E. coeruleopunctatus, E. erythrurus, E. sexfasciatus and C. fulva), the localization of the major rDNA sites on the smallest pair of the karyotype (pair 24) is a symplesiomorphic array shared by a vast number of species (e.g. Martinez et al., 1989;Zou et al., 2005;Wang et al., 2012;Tseng and Shih, 2018), as indicated in Figure 6. In addition, non-syntenic arrays of the 18S and 5S loci, which are also frequent among teleost groups (Lucchini et al., 1993;Suzuki et al., 1996;Gornung, 2013), are present in all of the eight species analyzed, as well as in several other serranids (Sola et al., 2000;Wang et al., 2012;Paim et al., 2017) (Figure 6). However, in spite of this, some alternative arrangements such as multiple 18S rDNA sites (Minglan et al., 2014) or the co-localization of the 18S/5S sites in the same chromosome pair (Amorim et al., unpublished data) can occur, although not expressively. The distribution of heterochromatin also offers a little discriminatory condition, since it is commonly located in the centromeric/pericentromeric regions, as observed in all the species analyzed, as well as in many other Percomorpha groups (Sola et al., 2000;Motta-Neto et al., 2011;Minglan et al., 2014;Noikotr et al., 2014).
Karyotype conservatism is thought to be related to a high level of synteny, with chromosomal sharing similar gene organization and DNA classes arrays (Ellegren, 2010;Zhang et al., 2019). In this respect, the chromosomal prospecting of a diversified set of repetitive sequences allowed the estimation of evolutionary changes in different fish groups (Cioffi and Bertollo, 2012;Costa et al., 2015;Lima-Filho et al., 2015; Figure 3 -Karyotypes of Epinephelus itajara, Cephalopholis formosa, Cephalopholis fulva, and Rypticus saponaceus after Giemsa staining, C-banding, and fluorescence in situ hybridization with 18S (red) and 5S (green) rDNA probes. Chromosomes carrying Ag-NORs sites are highlighted in the boxes. Scale bar = 5 μm. Getlekha et al., 2016a). In the present study, (CA) 15 and (GA) 15 microsatellites showed a dispersed distribution among chromosomes, with sporadic clusters in the centromeric heterochromatin of some species. This pattern contrasts with that presented by several Percomorpha species (Costa et al., 2015), where conspicuous and diversified chromosomal clusters occur within the same species or among co-familiar species (Silva et al., 2020).
Transposable elements, which can act at different genetic levels, including epigenetic regulation, are important components of the genome of marine fish (Aparicio et al., 2002;Terencio et al., 2015;Xiao et al., 2020). In most of the analyzed species, Tol2 presented a dispersed distribution in the karyotype, except for some centromeric clusters in E. adscensionis. In turn, Rex3 showed a more discriminated distribution, with conspicuous accumulation in multiple centromeric and telomeric regions, mainly in E. itajara, a species displaying a more differentiated karyotype among the eight analyzed. This transposable element overlaps with heterochromatic regions, probably co-located with the microsatellites (CA) 15 and (GA) 15 , which suggests a shared evolution of both repetitive DNA classes, as also proposed for other fish species (Da Silva et al., 2002;Fischer et al., 2004;Costa et al., 2013).
Overall, the micro-and macrostructural profiles presented by grouper species indicate an intermediate evolutionary rate between clades with larger (Silva et al., 2020) and much lower (Getlekha et al., 2016b) degrees of chromosomal variation.

Historical cytobiogeography and karyotype divergences
The Atlantic Ocean represents the probable origin center of the Epinephelidae family, from where lineages moved from its eastern region and colonized the Indian and Pacific Oceans by the Tethys Sea (Ma et al., 2016). During their extensive evolutionary history, estimated at 60 Mya (Ma et al., 2016), groupers experienced an extraordinary conservation of the diploid number (2n = 48; all currently analyzed species), followed by a less extensive conservatism of the chromosomal morphologies (~60% of species). Notably, the enlarged set of the karyotype patterns of the groupers, including the eight species investigated here, evidenced an increase in the karyotype diversification associated to the historical-geographic dispersion of their species. Indeed, while in the Atlantic Ocean, 87% of the analyzed species share the 2n = 48a basal karyotype (Table 2), this pattern is reduced to 56% of the Pacific, 55% of the Indo-Pacific, and only to 33% of the Indian Ocean species (Figure 6).
Until the Miocene, approximately 23 Mya, epinephelids had a low diversity in the Indian and Pacific oceans (Wilson and Rosen, 1998;Renema et al., 2008). When the invasion of the Indo-Pacific region occurred, historical tectonic processes promoted multiple reef habitats in that region, generating conditions for distinct evolutionary opportunities (Rohde and Muller, 2005;Carpenter et al., 2011). Indeed, sympatric and allopatric divergences in a short period of time, defined the contemporary diversity of the groupers (Craig et al., 2001;Ma et al., 2016;Ma and Craig, 2018), in agreement with the karyotype diversification of some groups.
Some features such as hermaphroditism, reproductive aggregations, high dispersive potential, and ecological plasticity are considered as gene flow maintainers and contributors to karyotype stability among groupers, as well as physical environment characteristics (Molina et al., 2014;Motta-Neto et al., 2019). In this case, the exploration and historical adaptation to new habitats may have had a disturbing effect on the modern grouper lineages, contributing to the disruption of the latent stability of the karyotype in the new colonization areas. Consequently, changes in the genome related to transposable elements (Schrader and Schmitz, 2019) and other repetitive sequences were established. In this context, adaptive pericentric inversions (Hoffmann and Rieseberg, 2008) could also be fixed as derived traits in some Epinephelidae species.
Notably, cytogenetic patterns of serranids have maintained a basal karyotype with 2n = 48 chromosomes for a long period since their origin. Chromosomal homeologies are also evidenced by similar physical and compositional patterns of repetitive sequences such as ribosomal DNA, microsatellites, and transposable elements. Despite this, evident divergences in the evolution of the karyotype also occur, especially among the more recent Epinephelidae lineages, suggesting a close correlation with the colonization of new habitats and evolutionary circumstances. In fact, the set of chromosomal data available showed a more extensive karyotype diversification associated with geographic expansion events (Ma et al., 2016) in the family. Therefore, the chromosomal evolution of the Epinephelidae proves to be more dynamic and diverse than supposed, with direct mediation of its historical and geographical contingencies.