Abstract

Groupers (Epinephelidae and Serranidae) have attracted special attention to fish farming, and their species offer good opportunities for successful hybridizations. Cytogenetic data allow a better understanding of the role of karyotypic diversification in the acquisition of post-zygotic reproductive isolation (RI). Thus, chromosomal analyses were performed on E. striatus (Caribbean Sea), E. coioides and E. tauvina (Indo-Pacific Region), using standard procedures and mapping of six repetitive DNA classes by the in situ hybridization. The three species have 2n=48 chromosomes. The karyotypes of E. coioides and E. striatus are composed only of acrocentric chromosomes (FN=48), while E. tauvina has 8 submetacentric chromosomes (FN=56). Heterochromatin has a preferential centromeric distribution, and the microsatellite repeats are dispersed throughout the chromosomes of all species. The 18S and 5S rDNA sites are unique but show a colocalization arrangement in E. tauvina and E. striatus. The chromosomal organization suggests that the three species still maintain a significant amount of syntenic regions. The range of the karyotype divergence and the RI levels showed low, but goes turn proportionally greater in relation to the divergence time between the parental species. The slow acquisition of postzygotic RI is consistent with the high karyotype homogeneity presented by Epinephelidae family.

Key words
Karyotype divergence; repetitive DNA; hybrids; post-zygotic barriers

INTRODUCTION

Interspecific hybridization promotes the arrangement of distinct genomes, which can result in hybrids with multiple adaptive traits (Abbott et al. 2013ABBOTT R ET AL. 2013. Hybridization and speciation. J Evol Biol 26: 229-246., Shivaramu et al. 2019SHIVARAMU S, VUONG DT, HAVELKA M, ŠACHLOVÁ H, LEBEDA I, KAŠPAR V & FLAJŠHANS M. 2019. Influence of interspecific hybridization on fitness-related traits in Siberian sturgeon and Russian sturgeon. Czech J Anim Sci 64: 78-88.). Natural hybridizations frequently occur among fish (Allendorf & Waples 1996ALLENDORF FW & WAPLES RS. 1996. Conservation and genetics of salmonid fishes, 1st ed., New York: Springer, 314 p., Rahman et al. 2013RAHMAN MA, ARSHAD A, MARIMUTHU K, ARA R & AMIN SMN. 2013. Inter-specific hybridization and its potential for aquaculture of fin fishes. Asian J Anim Vet Adv 8: 139-153.), mainly due to some conditions such as external fertilization, weak behavioral isolation, uneven abundance between parental species, loss of habitats, overlapping breeding areas, low frequency of sex chromosomes (Campton 1987CAMPTON DE. 1987. Natural hybridization and introgression in fishes: methods of detection and genetic interpretations. In: RYMAN N & UTTER F (Eds), Population Genetics and Fishery Management, Seattle: University of Washington Press, Seattle, USA, p. 161-192., Molina et al. 2014bMOLINA WF, MARTINEZ PA, BERTOLLO LAC & BIDAU CJ. 2014b. Evidence for meiotic drive as an explanation for karyotype changes in fishes. Mar Genomics 15: 29-34., Nagel et al. 2018NAGEL R, KIRSCHBAUM F, ENGELMANN J, HOFMANN V, PAWELZIK F & TIEDEMANN R. 2018. Male-mediated species recognition among african weakly electric fishes. R Soc Open Sci 5: e170443.), and the slow acquisition of post-zygotic reproductive isolation (RI) (Russell 2003RUSSELL ST. 2003. Evolution of intrinsic post-zygotic reproductive isolation in fish. Ann Zool Fennici 40: 321-329., Stelkens et al. 2010STELKENS RB, YOUNG KA & SEEHAUSEN O. 2010. The accumulation of reproductive incompatibilities in African cichlid fish. Evolution 64: 617-633.).

Combinations of different biological traits have, in many cases, increased the commercial value of hybrid fish, including the growth rate, environmental tolerance, resistance to cultivation, and production of monosexual stocks. (Rahman et al. 2013RAHMAN MA, ARSHAD A, MARIMUTHU K, ARA R & AMIN SMN. 2013. Inter-specific hybridization and its potential for aquaculture of fin fishes. Asian J Anim Vet Adv 8: 139-153., Rimmer & Glamuzina 2017RIMMER MA & GLAMUZINA B. 2017. A review of grouper (Family Serranidae: Subfamily Epinephelinae) aquaculture from a sustainability science perspective. Rev Aquac 11: 58-87., Shivaramu et al. 2019SHIVARAMU S, VUONG DT, HAVELKA M, ŠACHLOVÁ H, LEBEDA I, KAŠPAR V & FLAJŠHANS M. 2019. Influence of interspecific hybridization on fitness-related traits in Siberian sturgeon and Russian sturgeon. Czech J Anim Sci 64: 78-88.). Advantageous traits in artificial hybrids have been reported for several cultivated fish groups, such as catfish (Dunham & Smitherman 1983DUNHAM RA & SMITHERMAN RO. 1983. Crossbreeding channel catfish for improvement of body weight in earthen ponds. Growth 47: 97-103.), trout (Dorson et al. 1991DORSON M, CHEVASSUS B & TORHY C. 1991. Comparative susceptibility of three species of char and of rainbow trout × char triploid hybrids to several pathogenic salmonid viruses. Dis Aquat Organ 11: 217-224.), perch (Hooe et al. 1994HOOE ML, BUCK DH & WAHL DH. 1994. Growth, survival, and recruitment of hybrid crappies stocked in small impoundments. North Am J Fish Manag 14: 137-142.), carp (Kalsoom et al. 2009KALSOOM UME, SALIM M, SHAHZADI T & BARLAS A. 2009. Growth performance and feed conversion ratio (FCR) in hybrid fish (Catla catla x Labeo rohita) fed on wheat bran, rice broken and blood meal. Pak Vet J 29: 55-58.), sturgeons (Boscari et al. 2014BOSCARI E, BARMINTSEVA A, PUJOLAR JM, DOUKAKIS P, MUGUE N & CONGIU L. 2014. Species and hybrid identification of sturgeon caviar: A new molecular approach to detect illegal trade. Mol Ecol Resour 14: 489-498.), cichlids (Wohlfarth 1994WOHLFARTH GW. 1994. The unexploited potential of tilapia hybrids in aquaculture. Aquac Res 25: 781-788.) and groupers (Huang et al. 2016HUANG W, LIU Q, XIE J, WANG W, XIAO J, LI S, ZHANG H, ZHANG Y, LIU S & LIN H. 2016. Characterization of triploid hybrid groupers from interspecies hybridization (Epinephelus coioides ♀ × Epinephelus lanceolatus ♂). Aquac Res 47: 2195-2204.). Groupers of the Epinephelidae family are of particular economic interest (Mitcheson et al. 2013MITCHESON YS ET AL. 2013. Fishing groupers towards extinction: A global assessment of threats and extinction risks in a billion dollar fishery. Fish Fish 14: 119-136.), with around 50 species being exploited in fisheries or aquaculture (Rimmer & Glamuzina 2017RIMMER MA & GLAMUZINA B. 2017. A review of grouper (Family Serranidae: Subfamily Epinephelinae) aquaculture from a sustainability science perspective. Rev Aquac 11: 58-87., FAO 2019FAO GLOBAL CAPTURE PRODUCTION. 2019. http://www.fao.org/fishery/statistics/global-capture-production/query/en (August 5, 2019).
http://www.fao.org/fishery/statistics/gl... ). Some of their hybrids may even have a growth rate 50% higher than their parents (Sugama et al. 2014SUGAMA K, MUZAKI A, PERMANA IGN & HARYANTI H. 2014. Fluctuating asymmetry reflect the growth of hybrid grouper Epinephelus fuscoguttatus and Epinephelus polyphekadion. Indones Aquac J 9: 1-6.).

Some fish hybrids present normal and fertile gonads, showing germ cells at different stages of maturation (Moron et al. 2018MORON S, PORTALETE JC, RAMOS AT, ONO EA & HELAYEL MA. 2018. Histology of the gonads of the hybrid Pseudoplatystoma punctifer x Leiarius marmoratus. Bol Inst Pesca 41: 279-286.). On the other way, although normal in size and structure, other hybrids produce abnormal gametes in morphology and/or chromosome sets, or even fertilizable but non-viable gametes (Hooe et al. 1994HOOE ML, BUCK DH & WAHL DH. 1994. Growth, survival, and recruitment of hybrid crappies stocked in small impoundments. North Am J Fish Manag 14: 137-142.). The highest degree of RI - the hybrid inviability - may result from the imperfect chromosome pairing during meiosis, a condition that can be overcome by the numerical and syntenic compatibility of chromosomes from homoploid parents (Yoshikawa et al. 2018YOSHIKAWA H, XU D, INO Y, YOSHINO T, HAYASHIDA T, WANG J, YAZAWA R, YOSHIZAKI G & TAKEUCHI Y. 2018. Hybrid sterility in fish caused by mitotic arrest of primordial germ cells. Genetics 209: 507-521.). Homodiploidy of parental groupers increases the chances of homologous pairing during meiosis in the hybrid genome and may minimize post-zygotic blocks derived from anomalous chromosome segregation. Under natural conditions, homodiploidy is reflected in evolution, making diploid reticulated speciation a fast track for the emergence of new species (Coyne & Orr 2004COYNE JA & ORR HA. 2004. Speciation, 1st ed., Sunderland, MA: Sinauer Associates, 545 p.). The reproductive strategies of groupers encompass one or more patterns of sequential hermaphroditism, mainly protogyny (female to male), but also protandry (male to female), and bidirectional sex changes (Mitcheson & Liu 2008MITCHESON YS & LIU M. 2008. Functional hermaphroditism in teleosts. Fish Fish 9: 1-43., Avise & Mank 2009AVISE JC & MANK JE. 2009. Evolutionary perspectives on hermaphroditism in fishes. Sex Dev 3: 152-163.). Although sequential hermaphroditism favors cultivation practices, the slow ontogenetic development in some species is a limiting factor and has stimulated the production of hybrids with faster growth rates (Tucker 1994TUCKER JW. 1994. Spawning by captive Serranid fishes: A review. J world Aquac Soc 25: 345-359., Mitcheson et al. 2013MITCHESON YS ET AL. 2013. Fishing groupers towards extinction: A global assessment of threats and extinction risks in a billion dollar fishery. Fish Fish 14: 119-136., Rimmer & Glamuzina 2017RIMMER MA & GLAMUZINA B. 2017. A review of grouper (Family Serranidae: Subfamily Epinephelinae) aquaculture from a sustainability science perspective. Rev Aquac 11: 58-87.). In addition to natural hybrids, a large number of artificial ones (about 20) have been reported in groupers, many of which are regularly used in fish farming (Table I).

The karyotype features and genome diversification of epinephelids have become better known in recent years (Wang et al. 2020WANG Y, WEN X, ZHANG X, FU S, LIU J, TAN W, LUO M, LIU L, HUANG H, YOU X, LUO J & CHEN F. 2020. Chromosome genome assembly of the leopard coral grouper (Plectropomus leopardus) with nanopore and Hi-C sequencing data. Front Genet 11: 876., Amorim et al. 2021AMORIM KDJ, COSTA GWWF, CIOFFI MB, TANOMTONG A, BERTOLLO LAC & MOLINA WF. 2021. A new view on the scenario of karyotypic stasis in Epinephelidae fish: Cytogenetic, historical, and biogeographic approaches. Genet Mol Biol 44: e20210122.). Epinephelidae species are characterized by an intermediate rate of karyotype changes regarding other Perciformes groups (Molina 2007MOLINA WF. 2007. Chromosome changes and stasis in marine fish groups. In: PISANO EC, OZOUF-COSTAZ F & FORESTI BG (Eds). Fish Cytogenetics, New York: CRC Press, New York, USA, p. 61-110., Molina et al. 2014bMOLINA WF, MARTINEZ PA, BERTOLLO LAC & BIDAU CJ. 2014b. Evidence for meiotic drive as an explanation for karyotype changes in fishes. Mar Genomics 15: 29-34., Motta-Neto et al. 2019MOTTA-NETO CC, CIOFFI MB, COSTA GWWF, AMORIM KDJ, BERTOLLO LAC, ARTONI RF & MOLINA WF. 2019. Overview on karyotype stasis in atlantic grunts (Eupercaria, Haemulidae) and the evolutionary extensions for other marine fish groups. Front Mar Sci 6: 1-12.). The diploid value (2n=48) is a symplesiomorphic trait, shared by all grouper species analyzed so far. Among them, about 60% have a basal karyotype composed of acrocentric chromosomes (FN=48). The remaining species have karyotypes diversified by structural rearrangements, with FN greater than 48 (Motta-Neto et al. 2019MOTTA-NETO CC, CIOFFI MB, COSTA GWWF, AMORIM KDJ, BERTOLLO LAC, ARTONI RF & MOLINA WF. 2019. Overview on karyotype stasis in atlantic grunts (Eupercaria, Haemulidae) and the evolutionary extensions for other marine fish groups. Front Mar Sci 6: 1-12., Amorim et al. 2021AMORIM KDJ, COSTA GWWF, CIOFFI MB, TANOMTONG A, BERTOLLO LAC & MOLINA WF. 2021. A new view on the scenario of karyotypic stasis in Epinephelidae fish: Cytogenetic, historical, and biogeographic approaches. Genet Mol Biol 44: e20210122.). To date, cytogenetic studies in groupers have focused on cytogenetic characterization aspects. Preliminars genetic divergences and cytogenetic characteristics of the epinephelids have been associated with the hybridization processes in this family (Rahman et al. 2013RAHMAN MA, ARSHAD A, MARIMUTHU K, ARA R & AMIN SMN. 2013. Inter-specific hybridization and its potential for aquaculture of fin fishes. Asian J Anim Vet Adv 8: 139-153., Tseng & Shih 2018TSENG MC & SHIH KW. 2018. Application of karyotype and genetic characterization analyses for hybrid breeding of Epinephelus groupers. Intechopen 3: 37-51.), however, the quantification of the karyotype divergences and its relation with the post-zygotic effects on the hybrids are unknown. Here, are presented the microestructural chromosome divergences among on three cultived species of groupers, E. coioides, E. striatus and E. tauvina, by chromosomal mapping of six repetitive DNA classes [18S and 5S rDNA, microsatellites (CA)₁₅, (GA)₁₅, (CAA)₁₀ and (CGG)₁₀], and the association between the karyotype divergences and the ontogenetic effects on epinephelid hybrids. These repetitive sequences have a fast evolutionary dynamics and offer a varied comparative set of chromosomal markers. The combined approach involving cytogenetical, phylogenetical and temporal divergence contributed to elucidate new aspects of the acquisition of post-zygotic barriers in these reef fishes.

MATERIALS AND METHODS

Samples and standard chromosomal analyses

E. coioides, one of the most economically important fish farmed in China and Southeast Asia, and E. tauvina and E. striatus groupers were analyzed in this study. E. coioides (n=5) and E. tauvina (n=5) were obtained, from the Andaman Sea (11°04’00”N and 95°44’34”E), and E. striatus (n= 10) were juvenile specimens of the coast of Florida – USA (25°09’40”N, 80°45’83”W) (Figure 1), and obtained from an experimental research laboratory.

Figure 1
Geographic distribution of (a) Epinephelus striatus (in green), (b) Epinephelus coioides and (c) Epinephelus tauvina, with a sympatric area indicated in red. The collection points are highlighted by black stars.

Individuals were previously submitted to mitotic stimulation by muscular and intraperitoneal injection of attenuated antigen complexes (Molina et al. 2010MOLINA WF, ALVES DE, ARAÚJO WC, MARTINEZ PA, SILVA MF & COSTA GWWF. 2010. Performance of human immunostimulating agents in the improvement of fish cytogenetic preparations. Genet Mol Res 9: 1807-1814.), for a period of 24 hours. Next, the animals were euthanized with an overdose of clove oil. Chromosome preparations were obtained by short-term culture (Gold et al. 1990GOLD JR, LI YC, SHIPLEY NS & POWERS PK. 1990. Improved methods for working with fish chromosomes with a review of metaphase chromosome banding. J Fish Biol 37: 563-575.) of the cell suspensions from anterior region of the kidney. The cell suspension were hypotonized with KCl 0.075M solution, preserved with methanol: acid acetic (3:1) fixative solution and dripped onto a slide covered with a film of distilled water heated to 60^oC. Chromosomes were stained with a 5% Giemsa solution diluted in phosphate buffer pH 6.8 for 8 min to determine the diploid chromosome number (2n) and the composition of the karyotype. The heterochromatic regions were analyzed using the C-banding method (Sumner, 1972SUMNER AT. 1972. A simple technique for demonstrating centromeric heterochromatin. Exp Cell Res 75: 304-306.), and the nucleolar organizer regions (NORs), by silver nitrate impregnation (Howell & Black 1980HOWELL WM & BLACK DA. 1980. Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: a 1-step method. Experientia 36: 1014-1015.).

Probes for chromosomal hybridization

The 5S (~200 bp) and 18S rDNA (~1400 bp) probes were obtained by PCR from the nuclear DNA of Rachycentron canadum (Rachycentridae), using the primers A 5’-TAC GCC CGA TCT CGT CCG ATC-3’ and B 5’-CAG GCT GGT ATG GCC GTA AGC-3’ (Pendás et al. 1994PENDÁS AM, MORAN P, FREIJE JP & GARCIA-VAZQUEZ E. 1994. Chromosomal mapping and nucleotide sequence of two tandem repeats of Atlantic salmon 5S rDNA. Cytogenet Genome Res 67: 31-36.) and NS1 5’-GTA GTC ATA TGC TTG TCT C-3’ and NS8 5’-TCC GGT GCA TCA CCT ACG GA -3’ (White et al. 1990WHITE TJ, BRUNS S, LEE S & TAYLOR J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, In: INNIS MA, GELFAND DH, SNINSKY JJ & WHITE TJ (Eds). PCR protocols a guide to methods and applications, London: Academic Press, London, UK, p. 315-322.), respectively. The 5S rDNA and 18S rDNA probes were labeled by nick translation, respectively, with biotin-14-dATP and digoxigenin-dUTP-11, according to the manufacturer’s specifications (Roche®, Mannheim, Germany). The oligonucleotides (CA)₁₅, (GA)₁₅, (CAA)₁₀ and (CGG)₁₀ were labeled with AlexaFluor 555 at the 5’ terminal position during synthesis (Invitrogen, Thermo Fisher Scientific, California, USA).

Chromosomal hybridization

FISH experiments were performed following Pinkel et al. (1986)PINKEL D, STRAUME T & GRAY JW. 1986. Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 83: 2934-2938.. Hybridization signals were detected using anti-digoxigenin rhodamine-conjugated, for the 18S rDNA probe, and streptavidin-FITC (Roche®, Mannheim, Germany), for the 5S rDNA probe. The chromosomes were counterstained with Vectashield/DAPI (1.5 μg/ml) (Vector Laboratories, Burlingame, CA, USA). The hybridization of the simple sequence repeats (SSRs) was performed according to Kubat et al. (2008)KUBAT Z, HOBZA R, VYSKOT B & KEJNOVSKY E. 2008. Microsatellite accumulation on the Y chromosome in Silene latifolia. Genome 51: 350-356..

Image processing

Approximately thirty mitotic metaphases of each individual were photographed using an OlympusTM BX51 epifluorescence microscope coupled to an Olympus DP73 digital capture system, using cellSens® software (Olympus Corporation, Ishikawa, Japan). Chromosomes were classified regarding the arms ratio (AR) in metacentric (m), with AR ranging from 1.00-1.70; submetacentric (sm), AR=1.71-3.00; subtelocentric (st), AR= 3.01-7.00; and acrocentric (a), AR>7.01 (Levan et al., 1964LEVAN A, FREDGA K & SANDBERG A. 1964. Nomenclature for centromeric position at chromosomes. Hereditas 52: 201-220.). The fundamental number (FN) (i.e. number of chromosome arms), was defined considering the m, sm and st chromosomes to have two arms, while the acrocentric chromosomes only one arm.

Mitochondrial 16S sequences

Partial sequences of the 16S mitochondrial gene from 24 Epinephelidae parental species of interspecific crosses were obtained from the GenBank (Supplementary Material - Table SI). The sequences were aligned using MUSCLE (Edgar 2004EDGAR RC. 2004. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 1792-1797.), and the average rates of genetic divergence (Kimura-2p model) were obtained using the MEGA 6 software (Tamura et al. 2013TAMURA K, STECHER G, PETERSON D, FILIPSKI A & KUMAR S. 2013. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30: 2725-2729.). The temporal divergence per million years was estimated from Domingues et al. (2005)DOMINGUES VS, BUCCIARELLI G, ALMADA VC & BERNARDI G. 2005. Historical colonization and demography of the Mediterranean damselfish, Chromis chromis. Mol Ecol 14: 4051-4063., considering 1.0% of genetic divergence by 2.4 My.

RESULTS

The three species analyzed have 2n=48 chromosomes, but with some variations in the karyotype formula. The karyotypes of E. striatus and E. coioides are exclusively composed of acrocentric chromosomes (FN=48), with a small differentiation in size among the sequential pairs, while the karyotype of E. tauvina is composed of 8sm+40a (FN=56) (Figure 2). The heterochromatin has a reduced amount, mainly located at the centromeric and pericentromeric regions of chromosomes. In E. striatus and E. coioides the Ag-NORs sites are situated on the short arms of pair 24. In E. tauvina they are also located on the short arms, but in a larger pair, the 20^th one (Figure 2).

Figure 2
Karyotypes of E. striatus, E. coioides and E. tauvina under Giemsa staining, C-banding and fluorescence in situ hybridization (FISH) with rDNA probes. Chromosome pairs bearing Ag-NORs/18S rDNA (red) and 5S rDNA (green) sites are highlighted in boxes. A syntenic 18S/5S rDNA array occurs on the 24 and 20 pairs of E. striatus and E. tauvina, respectively. Scale bar = 5μm.

A single locus of the 18S and 5S rDNA sequences was identified in all species. However, in E. coioides the 18S rDNA is located on the short arms of pair 24, while the 5S rDNA is located on the short arms of pair 23 (Figure 2). Differently, in E. striatus and E. tauvina the 18S and 5S rDNA sites are colocalized on the short arms of pairs 24 and 20, respectively (Figure 2). The microsatellites (CA)₁₅, (GA)₁₅, (CAA)₁₀ and (CGG)₁₀ have a dispersed chromosomal distribution in the three species (Supplementary Material - Figure S1).

DISCUSSION

The slow acquisition of RI among groupers agrees with the relatively low quantitative divergence of their karyotypes. In fact, several cytogenetic and biological conditions seem to favor a significant number of viable and fertile hybrids, thus highlighting a limited effect of post-zygotic barriers in Epinephelidae. Among these several cytogenetic features deserve to be highlighted, such as the sharing of homoploid karyotypes and the significant conservation of extensive syntenic and colinear stretches in the genome of the species (Wang et al. 2020WANG Y, WEN X, ZHANG X, FU S, LIU J, TAN W, LUO M, LIU L, HUANG H, YOU X, LUO J & CHEN F. 2020. Chromosome genome assembly of the leopard coral grouper (Plectropomus leopardus) with nanopore and Hi-C sequencing data. Front Genet 11: 876., Yang et al. 2021YANG Y, WU L, WENG Z, WU X, WANG X, XIA J, MENG Z & LIU X. 2021. Chromosome genome assembly of Cromileptes altivelis reveals loss of genome fragment in Cromileptes compared with Epinephelus species. Genes 12: 1873., Amorim et al. 2021AMORIM KDJ, COSTA GWWF, CIOFFI MB, TANOMTONG A, BERTOLLO LAC & MOLINA WF. 2021. A new view on the scenario of karyotypic stasis in Epinephelidae fish: Cytogenetic, historical, and biogeographic approaches. Genet Mol Biol 44: e20210122.). Chromosome homologies allow correct pairing, recombination, and uniform segregation. In addition, the asynchronous hermaphroditism minimizes the genomic divergences between sexes, including the differentiation of sex chromosomes, since the same genome transits between the two sexes during the ontogenetic history (Wright et al. 2016WRIGHT AE, DEAN R, ZIMMER F & MANK JE. 2016. How to make a sex chromosome. Nat Commun 7: 1-8.). In fact, the presence of differentiated sex chromosomes in one or both parents can alter the gene balance, promoting the sterility or infeasibility of heteromorphic sex in hybrids (Haldane 1922HALDANE JBS. 1922. Sex ratio and unisexual sterility in animal hybrids. J Genet 12: 101-109.).

The three species, E. striatus, E. coioides, and E. tauvina and all other karyotyped groupers (~ 50 species) share the same diploid value (2n=48). Among these species, 61% share structurally similar karyotypes formed entirely by acrocentric chromosomes (FN=48), as E. coioides and E. striatus, while the others, including E. tauvina, exhibit some additional structural changes in the chromosomes (FN=48-96) (Motta-Neto et al. 2019MOTTA-NETO CC, CIOFFI MB, COSTA GWWF, AMORIM KDJ, BERTOLLO LAC, ARTONI RF & MOLINA WF. 2019. Overview on karyotype stasis in atlantic grunts (Eupercaria, Haemulidae) and the evolutionary extensions for other marine fish groups. Front Mar Sci 6: 1-12., Amorim et al. 2021AMORIM KDJ, COSTA GWWF, CIOFFI MB, TANOMTONG A, BERTOLLO LAC & MOLINA WF. 2021. A new view on the scenario of karyotypic stasis in Epinephelidae fish: Cytogenetic, historical, and biogeographic approaches. Genet Mol Biol 44: e20210122.). In fact, similar to E. tauvina, more than 40% of Epinephelidae species show some karyotype diversification associated with pericentric inversions. The changes by inversions may be related with adaptive processes (Kirubakaran et al. 2016KIRUBAKARAN TG ET AL. 2016. Two adjacent inversions maintain genomic differentiation between migratory and stationary ecotypes of Atlantic cod. Mol Ecol 25: 2130-2143.), and act as post-zygotic barriers (Ortiz-Barrientos et al. 2016ORTIZ-BARRIENTOS D, ENGELSTÄDTER J & RIESEBERG LH. 2016. Recombination rate evolution and the origin of species. Trends Ecol Evol 3: 226-236.).

Recent data showed an increase in the karyotype diversification associated to the historical biogeographic expansion of groupers species. Indeed, in the Atlantic Ocean, 87% of the analyzed species have conserved 2n=48 basal karyotype, this pattern is reduced to 56% of the Pacific, 55% of the Indo-Pacific, and only to 33% of the Indian Ocean species (Amorim et al. 2021AMORIM KDJ, COSTA GWWF, CIOFFI MB, TANOMTONG A, BERTOLLO LAC & MOLINA WF. 2021. A new view on the scenario of karyotypic stasis in Epinephelidae fish: Cytogenetic, historical, and biogeographic approaches. Genet Mol Biol 44: e20210122.). Apparently, the progressive historical karyotype divergence observed in groupers (Amorim et al. 2021AMORIM KDJ, COSTA GWWF, CIOFFI MB, TANOMTONG A, BERTOLLO LAC & MOLINA WF. 2021. A new view on the scenario of karyotypic stasis in Epinephelidae fish: Cytogenetic, historical, and biogeographic approaches. Genet Mol Biol 44: e20210122.) was promoted by reach of new areas generating conditions for distinct evolutionary opportunities (Rohde & Muller 2005ROHDE RA & MULLER RA. 2005. Cycles in fossil diversity. Nature 434: 208-210., Carpenter et al. 2011CARPENTER KE ET AL. 2011. Comparative Phylogeography of the Coral Triangle and Implications for Marine Management. J Mar Biol 2011: 1-14.).

Despite this, the organization and distribution of repetitive sequences in the chromosomes still offer indications of chromosomal conservatism (Amorim et al. 2021AMORIM KDJ, COSTA GWWF, CIOFFI MB, TANOMTONG A, BERTOLLO LAC & MOLINA WF. 2021. A new view on the scenario of karyotypic stasis in Epinephelidae fish: Cytogenetic, historical, and biogeographic approaches. Genet Mol Biol 44: e20210122.).

The remarkable chromosomal conservatism in Epinephelidae species is particularly noteworthy when comparing P. leopardus (2n=48a) and E. akaara (2n=48a) karyotypes (Wang et al. 2020WANG Y, WEN X, ZHANG X, FU S, LIU J, TAN W, LUO M, LIU L, HUANG H, YOU X, LUO J & CHEN F. 2020. Chromosome genome assembly of the leopard coral grouper (Plectropomus leopardus) with nanopore and Hi-C sequencing data. Front Genet 11: 876.). These species have an estimated divergence time of more than 35 Mya (Ma et al. 2016MA KY, CRAIG MT, CHOAT JH & HERWERDEN VL. 2016. The historical biogeography of groupers: Clade diversification patterns and processes. Mol Phylogenet Evol 100: 21-30.), but still show a clear one-to-one relationship among their chromosomes, highlighting the synteny among of their 24 linkage groups (Wang et al. 2020WANG Y, WEN X, ZHANG X, FU S, LIU J, TAN W, LUO M, LIU L, HUANG H, YOU X, LUO J & CHEN F. 2020. Chromosome genome assembly of the leopard coral grouper (Plectropomus leopardus) with nanopore and Hi-C sequencing data. Front Genet 11: 876.). Indications of similar high genomic conservatism also occur between E. fuscoguttatus (2n=2sm+46a) and Plectropomus leopardus (2n=48a), whose divergence time is about 49.3 (32.5–65.9) million years ago (Yang et al. 2020YANG Y, WU LN, CHEN JF, WU X, XIA JH, MENG ZN, LIU XC & LIN HR. 2020. Whole-genome sequencing of leopard coral grouper (Plectropomus leopardus) and exploration of regulation mechanism of skin color and adaptive evolution. Zool Res 41: 328-340.).

The Ag-NOR sites are located in a single pair of chromosomes in the three analyzed species, in a medium-sized pair in E. tauvina and the smallest pair of the karyotype in E. striatus and E. coioides. The occurrence of ribosomal sites in the same position and on the smallest pair of chromosomes is also a significantly recurrent conservative condition among grouper species (Tseng & Shih 2018TSENG MC & SHIH KW. 2018. Application of karyotype and genetic characterization analyses for hybrid breeding of Epinephelus groupers. Intechopen 3: 37-51., Amorim et al. 2021AMORIM KDJ, COSTA GWWF, CIOFFI MB, TANOMTONG A, BERTOLLO LAC & MOLINA WF. 2021. A new view on the scenario of karyotypic stasis in Epinephelidae fish: Cytogenetic, historical, and biogeographic approaches. Genet Mol Biol 44: e20210122.). In general, the 18S and 5S rDNA sites are also not syntenic in groupers (Minglan et al. 2014MINGLAN G, WANG S, SU Y, ZHOU Y, LIU M & WANG J. 2014. Molecular cytogenetic analyses of Epinephelus bruneus and Epinephelus moara (Perciformes, Epinephelidae). PeerJ 2: e412., Paim et al. 2017PAIM FG, ALMEIDA LA DA H, AFFONSO PRAM, SOBRINHO-SCUDELER PE, OLIVEIRA C & DINIZ D. 2017. Chromosomal stasis in distinct families of marine Percomorpharia from South Atlantic. Comp Cytogenet 11: 299-307.). Therefore, the co-localization of 18S/5S rDNA in E. striatus and E. tauvina points to the potential evolutionary dynamism of these regions, which may eventually promote microstructural reorganizations in the chromosomes. However, different cytogenetic markers, including rDNA regions and other repetitive sequences (Amorim et al. 2021AMORIM KDJ, COSTA GWWF, CIOFFI MB, TANOMTONG A, BERTOLLO LAC & MOLINA WF. 2021. A new view on the scenario of karyotypic stasis in Epinephelidae fish: Cytogenetic, historical, and biogeographic approaches. Genet Mol Biol 44: e20210122., present study), support the substantial syntenic conservatism in grouper chromosomes. Comparative analyses of the repetitive sequences allow tracking its evolutionary dynamics in karyotypes, in view of their rapid evolutionary rates (Vicari et al. 2010VICARI MR, NOGAROTO V, NOLETO RB, CESTARI MM, CIOFFI MB, ALMEIDA MC, MOREIRA-FILHO O, BERTOLLO LAC & ARTONI RF. 2010. Satellite DNA and chromosomes in Neotropical fishes: Methods, applications and perspectives. J Fish Biol 76: 1094-1116., Cioffi & Bertollo 2012CIOFFI MB & BERTOLLO LAC. 2012. Chromosomal distribution and evolution of repetitive DNAs in fish. Genome Dyn 7: 197-221.), including fish groups with slow chromosomal divergence (Costa et al. 2013COSTA GWWF, CIOFFI MB, BERTOLLO LAC & MOLINA WF. 2013. Transposable elements in fish chromosomes: A study in the marine cobia species. Cytogenet Genome Res 141: 126-132., 2015COSTA GWWF, CIOFFI MDB, BERTOLLO LAC & MOLINA WF. 2015. Structurally complex organization of repetitive DNAs in the genome of cobia (Rachycentron canadum). Zebrafish 12: 215-220.). In the three Epinephelus species, the (CA)₁₅, (GA)₁₅, (CAA)₁₀ and (CGG)₁₀ repeats do not show clear differences in their genomic distribution, being equally dispersed in eu- and heterochromatic regions, without detectable accumulation points. This diffuse organization does not signal remaining rearrangements in the karyotypes. In fact, it may be a limiting factor for karyotypic alterations (Molina 2007MOLINA WF. 2007. Chromosome changes and stasis in marine fish groups. In: PISANO EC, OZOUF-COSTAZ F & FORESTI BG (Eds). Fish Cytogenetics, New York: CRC Press, New York, USA, p. 61-110.), mainly due to its small or non-close association with other repetitive elements (Piscor & Parise-Maltempi 2016PISCOR D & PARISE-MALTEMPI PP. 2016. Microsatellite organization in the B chromosome and A chromosome complement in Astyanax (Characiformes, Characidae) species. Cytogenet Genome Res 148: 44-51.).

The maintenance of chromosomal and genomic conservation over tens of millions of years plays a significant role in the slow acquisition of post-zygotic barriers among Epinephelidae fish. In fact, negative epistatic interactions and consequent RI increase are more likely to occur when there are divergences in the number and structure of chromosomes of the two hybridizing taxa (King 1993KING M. 1993. Species evolution: the role of chromosomal change, 1st edition, Cambridge: Cambridge University Press, 336 p., Cursino et al. 2014CURSINO MS, SALVIANO MB, ABRIL VV, SANTOS ZE & DUARTE JMB. 2014. The role of chromosome variation in the speciation of the red brocket deer complex: the study of reproductive isolation in females. BMC Evol Biol 14: 1-12., Moran et al. 2019MORAN RL, CATCHEN JM & FULLER RC. 2019. Genomic resources for darters (Percidae: Etheostominae) provide insight into postzygotic barriers implicated in speciation. Mol Biol Evol 37: 711-729.). The generalized homoploid condition of Epinephelidae fish overcomes blocks imposed by RI (Buggs et al. 2011BUGGS RJA, SOLTIS PS & SOLTIS D. 2011. Biosystematic relationships and the formation. Taxon 60: 324-332.), ensuring a greater hybrid viability (Rahman et al. 2013RAHMAN MA, ARSHAD A, MARIMUTHU K, ARA R & AMIN SMN. 2013. Inter-specific hybridization and its potential for aquaculture of fin fishes. Asian J Anim Vet Adv 8: 139-153.). Indeed, evidence of RI breaks is reported in Haemulidae (Marceniuk et al. 2019MARCENIUK AP, CAIRES RA, MACHADO L, CERQUEIRA NNCD, SERRA RRM & OLIVEIRA C. 2019. Redescription of Orthopristis ruber and Orthopristis scapularis (Haemulidae: Perciformes), with a hybridization zone off the Atlantic coast of South America. Zootaxa 4576: 109-126.), Lutjanidae (Batista et al. 2012BATISTA CHDO, CAMARGO GMB, OLIVEIRA PGV, VÉRAS DP, PINHEIRO PB, FERNANDES CAF, TRAVASSOS P & HAZIN FHV. 2012. Occurrence of the hybrid snapper between yellowtail snapper Ocyurus chrysurus (Bloch 1791) and lane snapper Lutjanus synagris (Linnaeus 1758) (Perciformes: Lutjanidae) in the Southwest Atlantic, Northeast Brazil. Panam J Aquat Sci 7: 45-49.), Pomacantidae (Pyle & Randall 1994PYLE RL & RANDALL JE. 1994. A review of hybridization in marine angelfishes (Perciformes: Pomacanthidae). Environ Biol Fishes 41: 127-145.) and Chaetodontidae (Montanari et al. 2012MONTANARI SR, HERWERDEN VL, PRATCHETT MS, HOBBS JPA & FUGEDI A. 2012. Reef fish hybridization: Lessons learnt from butterflyfishes (genus Chaetodon). Ecol Evol 2: 310-328.), all fish families showing a slower rate of karyotypic changes (Molina 2007MOLINA WF. 2007. Chromosome changes and stasis in marine fish groups. In: PISANO EC, OZOUF-COSTAZ F & FORESTI BG (Eds). Fish Cytogenetics, New York: CRC Press, New York, USA, p. 61-110., Molina et al. 2014aMOLINA WF, MARTINEZ PA, BERTOLLO LAC & BIDAU CJ. 2014a. Preferential accumulation of sex and bs chromosomes in biarmed karyotypes by meiotic drive and rates of chromosomal changes in fishes. An Acad Bras Cienc 86: 1801-1812.).

Karyotype divergence and ontogenetic effects in interspecific Epinephelidae hybrids

Inversions are the main detectable rearrangements in Epinephilidae karyotypes (Amorim et al. 2021AMORIM KDJ, COSTA GWWF, CIOFFI MB, TANOMTONG A, BERTOLLO LAC & MOLINA WF. 2021. A new view on the scenario of karyotypic stasis in Epinephelidae fish: Cytogenetic, historical, and biogeographic approaches. Genet Mol Biol 44: e20210122.). It is known that inversions can interfere with normal chromosomal pairing and recombination during meiosis (Rieseberg 2001RIESEBERG LH. 2001. Chromosomal rearrangements and speciation. Trends Ecol Evol 16: 351-358., Ortiz-Barrientos et al. 2016ORTIZ-BARRIENTOS D, ENGELSTÄDTER J & RIESEBERG LH. 2016. Recombination rate evolution and the origin of species. Trends Ecol Evol 3: 226-236.), and that even a single event can generate barriers driving to speciation (Ayala et al. 2013AYALA D, GUERRERO RF & KIRKPATRICK M. 2013. Reproductive isolation and local adaptation quantified for a chromosome inversion in a malaria mosquito. Evolution 67: 946-958.). However, inversions can be also related with adaptation processes (Wellenreuther & Bernatchez 2018WELLENREUTHER M & BERNATCHEZ L. 2018. Eco-evolutionary genomics of chromosomal inversions. Trends Ecol Evol 33: 427-440., Faria et al. 2019FARIA R, JOHANNESSON K, BUTLIN RK & WESTRAM AM. 2019. Evolving inversions. Trends Ecol Evol 34: 239-248.). In Gadus morhua (Gadidae), for example, inversions cover more than 6% of the genome, and are associated with eco-adaptations of widely migratory ecotypes (Kirubakaran et al. 2016KIRUBAKARAN TG ET AL. 2016. Two adjacent inversions maintain genomic differentiation between migratory and stationary ecotypes of Atlantic cod. Mol Ecol 25: 2130-2143., Wellenreuther & Bernatchez 2018WELLENREUTHER M & BERNATCHEZ L. 2018. Eco-evolutionary genomics of chromosomal inversions. Trends Ecol Evol 33: 427-440.).

Unfavorable effects of inversions do not seem to be significant among grouper species regarding the hybrid viability and fertility (Table I). The previous description of the karyotypes of E. coioides, which classified the Ag-NOR pair as submetacentric chromosomes (2sm+46a; Wang et al. 2010WANG S, SU Y, DING S, CAI Y & WANG J. 2010. Cytogenetic analysis of orange-spotted grouper, Epinephelus coioides, using chromosome banding and fluorescence in situ hybridization. Hydrobiologia 638: 1-10.), and E. lanceolatus (8sm+40a; Jiun & Mei 2009JIUN CS & MEI CT. 2009. Karyotype Identification of Epinephelus lanceolatus. In: 4th Academic Seminar, Department of Aquaculture, National Pingtung University of Science and Technology, Neipu, p. 33-37.) were considered in the estimates of karyotypic divergences of the species involved in interspecific crossings. Hybrids of E. coioides ♀ (48a/2sm+46a; FN=48/50) X E. lanceolatus ♂ (2n=6sm+42a/8sm+40a; FN=54/56), with at least four detectable pericentric inversions (ΔFN=4-8), reach maturity and normal gonadal development (Li et al. 2018LI S, LIU Q, XIAO L, TAO M, SHU H, ZHANG H, LIN H & ZHANG Y. 2018. Comparison of gonadal development in diploid and triploid hybrid groupers, Epinephelus coioides ♀ × Epinephelus lanceolatus ♂. J World Aquacult Soc 49: 328-337.). Hybrids from phylogenetically close lineages may even show a greater growth and adaptability than their parental species (Senoo 2006SENOO S. 2006. Hybrid production between tiger grouper Epinephelus fuscoguttatus x giant grouper Epinephelus lanceolatus (Fish Culture In Southeast Asia 64). Aquanet Magazine 12: 58-63., Liufu et al. 2007LIUFU YZ, ZHAO H & LIU X. 2007. Preliminary study on the hybrid red-spotted grouper (Epinephelus akaara) ♂ × orange-spotted grouper (Epinephelus coioides) ♀. Acta Sc Nat Brno 46: 72-75., Huang et al. 2016HUANG W, LIU Q, XIE J, WANG W, XIAO J, LI S, ZHANG H, ZHANG Y, LIU S & LIN H. 2016. Characterization of triploid hybrid groupers from interspecies hybridization (Epinephelus coioides ♀ × Epinephelus lanceolatus ♂). Aquac Res 47: 2195-2204.). Such a heterotic condition can even occur with some chromosomal diversification (Table I), probably due to sufficient levels of parental gene balance (Birchler & Veitia 2007BIRCHLER JA & VEITIA RA. 2007. The gene balance hypothesis: From classical genetics to modern genomics. Plant Cell 19: 395-402.), and the hybrid genome generating large adaptive effects (Dagilis et al. 2019DAGILIS AJ, KIRKPATRICK M & BOLNICK DI. 2019. The evolution of hybrid fitness during speciation. PLoS Genet 15: e1008125.). Some grouper hybrids, such as E. lanceolatus (2n=6sm+42a/8sm+40a) x E. fuscoguttatus (2n=2sm+46a), present a number of more favorable characters than their parental species, including incubation time, fertilization rates and hatching, growth, survival, adaptability and disease resistance (Ching et al. 2018CHING FF, OTHMAN N, ANUAR A, SHAPAWI R & SENOO S. 2018. Natural spawning, embryonic and larval development of F2 hybrid grouper, tiger grouper Epinephelus fuscoguttatus × giant grouper E. lanceolatus. Int Aquat Res 10: 391-402.). Therefore, favorable zootechnical characteristics (Senoo 2006SENOO S. 2006. Hybrid production between tiger grouper Epinephelus fuscoguttatus x giant grouper Epinephelus lanceolatus (Fish Culture In Southeast Asia 64). Aquanet Magazine 12: 58-63., Liufu et al. 2007LIUFU YZ, ZHAO H & LIU X. 2007. Preliminary study on the hybrid red-spotted grouper (Epinephelus akaara) ♂ × orange-spotted grouper (Epinephelus coioides) ♀. Acta Sc Nat Brno 46: 72-75., Huang et al. 2016HUANG W, LIU Q, XIE J, WANG W, XIAO J, LI S, ZHANG H, ZHANG Y, LIU S & LIN H. 2016. Characterization of triploid hybrid groupers from interspecies hybridization (Epinephelus coioides ♀ × Epinephelus lanceolatus ♂). Aquac Res 47: 2195-2204., Table I) demonstrate that hybridization is an important and effective strategy in grouper cultivation.

The time of divergence generally increases the rate of post-zygotic barriers among fish. Sterility in one or both sexes corresponds to the first level of RI, which progresses to the hybrid infeasibility when the average parental divergence reaches about ten million years (Russell 2003RUSSELL ST. 2003. Evolution of intrinsic post-zygotic reproductive isolation in fish. Ann Zool Fennici 40: 321-329.). Interspecific group hybrids have been obtained from parents bearing similar or structurally diversified karyotypes (Table I). The analysis of parental karyotypes and their divergence times allowed us to infer the ontogenetic development of the hybrids and RI. The divergence time between the parental species, estimated from the percentage differences in the 16S mtDNA sequences, ranged from 1.6% (E. costae x E. marginatus) to 7.0% (E. marginatus x E. aeneus) (4-17 Mya), or 1.4% in Cephalopholis (C. aurantia x C. spiloparaea - ~3.5 Mya). Data on hybrid biological traits (eg, fertilization and hatching rates) suggest that ontogenetic parameters are not directly affected by the parental time divergence for most of the interspecific crosses (Table I). Parental species with considerable hybrid production, such as E. fuscoguttatus x E. lanceolatus and E. fuscoguttatus x E. polyphekadion, show a genetic distance of 4.8%, indicating an evolutionary divergence of ~11 Mya (Table I). In all of these crosses, the hybrid products were viable. Although fertility aspects are not available for all crosses, some hybrids were also fertile. Hybrids of E. coioides x E. lanceolatus (5.4% genetic divergence) and E. lanceolatus x E. fuscoguttatus (4.8% genetic divergence) showed even greater growth, survival and adaptability to captivity than their parents (Table I). Regarding intergeneric crosses, the genetic distance ranged from 4.6% (C. fulva x P. furcifer - 11 Mya) to 18% (Epinephelus morio x Centropristis striata - 43 Mya). The later is the high value among the species pairs and resulted in hybrid inviability (Table I), with larval lethality three days after fertilization. Crosses among Epinephelus, Cromileptes and Cephalopholis species, with an evolutionary divergence between 14 to 24 Mya, indicated the occurrence of post-zygotic barriers regarding the performance of ontogenetic parameters. However, some crosses between Cephalopholis and Paranthias species, diverging around 11 Mya, can still produce viable larvae (Tucker 1994TUCKER JW. 1994. Spawning by captive Serranid fishes: A review. J world Aquac Soc 25: 345-359.). Likewise, hybrids between Cephalopholis and Epinephelus species (~11 Mya) may have shorter incubation time and higher growth than the parental species (Ching et al. 2018CHING FF, OTHMAN N, ANUAR A, SHAPAWI R & SENOO S. 2018. Natural spawning, embryonic and larval development of F2 hybrid grouper, tiger grouper Epinephelus fuscoguttatus × giant grouper E. lanceolatus. Int Aquat Res 10: 391-402.). On the other hand, crosses between E. morio × C. striata, with a very high evolutionary divergence time (~40 Mya), have resulted in few days of larval survival after hatching (Tucker 1994TUCKER JW. 1994. Spawning by captive Serranid fishes: A review. J world Aquac Soc 25: 345-359., Table I). But in this case, in addition to the divergence time, cannot be ruled out some influence of the significantly diversified karyotype of C. striata: 2n=24m+22sm+2a; FN=94 (Moran et al. 2019MORAN RL, CATCHEN JM & FULLER RC. 2019. Genomic resources for darters (Percidae: Etheostominae) provide insight into postzygotic barriers implicated in speciation. Mol Biol Evol 37: 711-729.).

The diversification of groupers was significantly influenced by major biogeographic barriers. The main barriers during the Pliocene and Pleistocene periods, which resulted from the sea level reduction at 5.3-0.01 Mya (Ma et al. 2016MA KY, CRAIG MT, CHOAT JH & HERWERDEN VL. 2016. The historical biogeography of groupers: Clade diversification patterns and processes. Mol Phylogenet Evol 100: 21-30.), were particularly preponderant. However, this period of divergence is much shorter than the estimated mean time for the acquisition of an effective RI (Russell 2003RUSSELL ST. 2003. Evolution of intrinsic post-zygotic reproductive isolation in fish. Ann Zool Fennici 40: 321-329.) (Figure 3), thus suggesting that allopatry or pre-zygotic reproductive barriers probably played a more important role in that process. The genetic cohesion of species is intrinsically related to their evolutionary histories and degrees of lineage relationships (Marques & Ferreira 2008MARQUES S & FERREIRA BP. 2008. Sexual development and reproductive pattern of the Mutton hamlet, Alphestes afer (Teleostei: Epinephelidae): a dyandric, hermaphroditic reef fish. Neotrop Ichthyol 9: 547-558., Papadaki et al. 2018PAPADAKI M, MAZZELLA D, SANTINELLI V, FAKRIADIS I, SIGELAKI I & MYLONAS CC. 2018. Hermaphroditism and reproductive function of hatchery-produced sharpsnout seabream (Diplodus puntazzo) under attenuated annual thermal cycles. Aquac 482: 231-240.). The ontogenetic developmental indices of grouper hybrids apparently support that the divergence time of the clades was not sufficient in establishing an effective RI yet.

Figure 3
Post-zygotic ontogenetic effects in interspecific Epinephelidae hybrids, under a phylogenetic and temporal perspective (adapted from Ma et al. 2016MA KY, CRAIG MT, CHOAT JH & HERWERDEN VL. 2016. The historical biogeography of groupers: Clade diversification patterns and processes. Mol Phylogenet Evol 100: 21-30.).

CONCLUSIONS

Groupers stand out as successful species for marine fish farming and hybrid production, but their level of introgression is still being better evaluated. In this study, in addition to new data on the karyotypic organization of some grouper species, ontogenetic effects of hybridization and the time of evolutionary divergence of the hybridizing species were also analyzed. Although the divergence time is a relevant factor for reproductive isolation, the general scenario that stands out in groupers is that post-zygotic reproductive isolation is not expressive yet. On the other hand, the high rate of karyotypic conservatism in this and other marine fish groups is consistent with their hybridization success. Therefore, the cytogenomic characterization of parental species stands out as a useful tool for analyzing hybridization and its traceability, as well as for the biological conservation and evolutionary approaches of groupers.

ACKNOWLEDGMENTS

The authors thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for the financial support (Process nº 442626/2019-3), and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for the scholarship granted to KDJA. They also thank IBAMA (Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis) for the license to collect the specimens (Process No. 19135-8) and UFRN (Universidade Federal do Rio Grande do Norte) for allowing this study to be carried out.

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