Molecular cytogenetic analyses of Epinephelus bruneus and Epinephelus moara (Perciformes, Epinephelidae)

Introduction

The family Epinephelidae comprises approximately 163 grouper species in 16 genera (Craig, Sadovy de Mitcheson & Heemstra, 2011). These species are of considerable economic value, especially in the coastal fisheries of tropical and subtropical areas (Heemstra & Randall, 1993). Taxonomic confusion in the Epinephelidae often occurs due to similarities of color patterns and ontogenetic changes in color (Heemstra & Randall, 1993; Craig, Sadovy de Mitcheson & Heemstra, 2011). Epinephelus bruneus (Bloch 1793) and E. moara (Temminck and Schiegel 1842) are two important aquaculture and commercial fish species. However, E. moara has long been considered a synonym of E. bruneus due to their similarities in coloration and overlapping in geographical distributions (Heemstra & Randall, 1993; Craig, Sadovy de Mitcheson & Heemstra, 2011). Designation of correctly identified new species is important not only to the study of phylogenetic relationships, but also to the identification of fry and parent in grouper aquaculture. Based on morphological characteristics and molecular comparisons, E. moara has been suggested as a valid species (Guo et al., 2008; Guo et al., 2009; Liu et al., 2013). The interspecific differences between them were identified mainly based on the skeleton system as well as the meristic and morphometric characteristics (Guo et al., 2008). Gene differentiation (Guo et al., 2009) and mitogenome analyses (Liu et al., 2013) partially provided molecular information confirming their taxonomic status. Chromosomes are the carriers of genetic information, and chromosomal recombination plays a vital role in genetic diversity. Therefore, more other evidences are needed to support the hypothesis that E. moara is a valid species, such as molecular cytogenetic analyses.

Chromosomes are hereditary elements of the complete nuclear genome. Molecular cytogenetic studies on chromosomes constitute important approaches for characterizing species and reconstructing phylogenetic relationships (Galetti, Aguilar & Molina, 2000; Ocalewicz, Woznicki & Jankun, 2008; Cioffi, Martins & Bertollo, 2010; Ruiz-Herrera, Farre & Robinson, 2012). Karyological features indicate the evolutionary distance between species of different taxonomic categories (Dobigny et al., 2004). The nucleolar organizer regions (NORs) were particularly significant in chromosomal evolutionary analyses (Miller et al., 1976; Fujiwara et al., 1998). Heterochromatin corresponding to C-bandings is normally associated with rearrangements, quantitative variation, and formation of new karyotypes (Miklos & Gill, 1982; Rocco et al., 2002). Many taxonomic studies were based on the variations and polymorphism of the chromosomes containing major 18S rDNA (both active and non-active) (Cioffi, Martins & Bertollo, 2010; Britton-Davidian, Cazaux & Catalan, 2012), and minor 5S rDNA (Fujiwara et al., 1998; Mazzei et al., 2004) by fluorescent in situ hybridization (FISH). Location of telomeric sequence (TTAGGG)_n provides direct evidence for cytotaxonomic studies and chromosomal evolution in fishes (Sola et al., 2003; Scacchetti et al., 2011). Therefore, molecular cytogenetic information has provided important contributions to the characterization of biodiversity and the evolution of ichthyofauna (Jesus et al., 2003; Vicari et al., 2008). The analyses of different methods above present a complete karyotypic picture for organisms.

The cytogenetic information provided by a variety of approaches will allow us to more fully explain the taxonomic and evolutionary statuses, and reveal the inherent differences of E. bruneus and E. moara. In this study, the karyotypic techniques, including Giemsa-staining, Ag-staining, C-banding and localization of 18S rDNA and telomere (TTAGGG)_n by FISH, were used to investigate the molecular cytogenetic characteristics of E. bruneus and E. moara. Cytogentic data provided a better definition of the specific epithet for these cryptic species. Molecular cytogenetic analyses were found to be applicable in differentiating between closely related species and reconstructing phylogenetic relationships in groupers.

Materials and Methods

Fish collection and identification

Twenty-five individuals of E. bruneus (standard length, L_S, 140–550 mm) and 24 individuals of E. moara (L_S, 188–650 mm) were collected alive from the coastal waters of Fujian, China, and reared in laboratory for one week before analyses. Specimen identification was based on external coloration (Fig. 1), skeleton and morphological characteristics established in previous studies (Guo et al., 2008).

Results

Karyotypes and banding patterns

A total of 172 metaphases of E. bruneus and 156 metaphases of E. moara were analyzed to determine the karyotype structure. All specimens of E. bruneus and E. moara invariably showed the same diploid number of chromosomes, 2n = 48. The karyotype formulas of E. bruneus and E. moara were 2m + 4sm + 42a, giving a fundamental number (NF) equaled to 54 (Figs. 2A and 2B), and 4sm + 44a, NF = 52 (Figs. 2C and 2D), respectively. Chromosomes pairs were numbered based on the relative length. The smallest chromosomes pairs No. 24 were submetacentric chromosomes (sm-3 for E. bruneus and sm-2 for E. moara, Figs. 2B and 2D). Chromosome pairs No. 9 in length were sm-2 for E. bruneus and sm-1 for E. moara. Chromosome pairs No. 2 were metacentric chromosomes m-1 for E. bruneus. Other chromosomes were acrocentric (a) chromosomes for both E. bruneus and E. moara.

Active NORs were identified on the terminal position of short arms or sub-centromere regions of those biarmed chromosomes. In E. bruneus, five actively transcribed NORs were located on the metacentric and submetacentric chromosomes (Fig. 3A). In E. moara, four Ag-NORs were found on the submetacentric chromosomes (Fig. 3B).

The constitutive heterochromatin was observed in the centromeric and/or pericentromeric region of most chromosomes for both E. bruneus and E. moara. And the biarmed chromosome pairs with positive Ag-NORs were coinciding with the positive heterochromatin C-bandings. While three pairs of acrocentric chromosomes were almost indiscernible in both fish species (Figs. 4A and 4B). However, the significant differences of heterochromatin were the heterochromatic blocks found in the interstitial region of the long arms of one pair of medium-sized acrocentric chromosome in E. bruneus (Fig. 4A).

FISH analyses

Multiple sites of 18S rDNA by FISH confirmed the data obtained by Ag-staining for NORs. In E. bruneus, six positive signals (both active and non-active) were identified, corresponding to metacentric (m) and submetacentric (sm) chromosomes (Fig. 5A). Four hybridization signals were observed on the short arms of submetacentric chromosomes in E. moara (Fig. 5B). 5S rDNA and 18S rDNA were found on different chromosomes. Two 5S rDNA sites were located on the arms of a medium-sized acrocentric chromosome pair in both E. bruneus and E. moara (Figs. 6A and 6B).

Telomeric repeats of (TTAGGG)_n showed the typically telomeric signals on both telomeres and/or centromeric region of all chromosomes in E. bruneus and E. moara. No positive signal was detected at interstitial sites (Figs. 7A and 7B). Ten chromosome pairs of E. bruneus were significantly stronger than the signals of the others (Fig. 7A). However, E. moara were characterized by uniform telomeric signals in strength and size (Fig. 7B).

Discussion

Our previous study has distinguished E.bruneus and E. moara as two species based on their morphometric and skeletal characteristics (Guo et al., 2008). The species-specific differences showed obviously on the bars of the body and stable skeleton characteristics of adult. And the pyloric caeca indicates their different feeding habits and digestive function, which means they could have different ecological niches. Mitogenome and molecular comparisons confirmed E. moara to be a valid species of the family Epinephelidae (Liu et al., 2013). Further, we developed a molecular method to differentiate both fish species (Guo et al., 2009). However, the cytogenetic backgrounds and evolutionary situation, which is very important to the cultivation and protection of fish resources, remains unclear for E. bruneus and E. moara. We here comprehensively analyzed the cytogenetic backgrounds, and reconstructed their phylogenetic relationships using molecular cytogenetic analyses.

For E. bruneus and E. moara, species-specific characteristics presented in karyotype, NORs, C-banding and telomere distribution patterns. Karyotype variation appears to parallel speciation events in many groups of vertebrates (Morescalchi et al., 2007; Ruiz-Herrera, Farre & Robinson, 2012). Variations of NOR constituted a strong cytotaxonomic character in fishes (Fujiwara et al., 1998; Galetti, Aguilar & Molina, 2000). Many species in genus Epinephelus showed the same karyotypic characteristics, such as karyotype formula and NORs (Wang et al., 2012). However, karyotype formula and NORs were different between E. bruneus and E. moara (Table 1). Further, interstitial C-bandings were observed in E. bruneus, but not in E. moara. Similar interstitial heterochromatin was also found in E. coioides (Wang et al., 2010) and Diplectrum radiale (de Aguilar & Galetti, 1997). The distribution patterns of (TTAGGG)_n were different obviously between E. bruneus and E. moara. Cytogenetic differences were inter-specific, because E. bruneus and E. moara showed a similar geographical distribution (Heemstra & Randall, 1993; Guo et al., 2009), and coupled with heterogeneously morphological characteristics (Guo et al., 2008) and chromosomal structure.

The cytogenetic analyses suggested that the lineages of E. bruneus and E. moara recently derived within the genus Epinephelus. Both fish species share a uniform number of chromosomes to other species in the genus Epinephelus (Wang et al., 2012). However, they contained more biarmed chromosomes such as metacentric and/or submetacentric chromosomes (Table 1). In fishes, 48 uni-armed chromosome types like acrocentric chromosomes represented the ancestral complement of diploid origin (Ohno, 1974; Vitturi et al., 1991; Sola et al., 2000). In addition, most species in genus Epinephelus showed a conserved, NOR-bearing chromosome pair No. 24 (Table 1), while E. bruneus and E. moara showed additional NORs on chromosome pairs. For most vertebrates, the presence of a single NOR pair seems to be an ancestral character state (Hsu & Pardue, 1975; Schmid, 1978; Galetti, Molina & Affonso, 2006). Both E. bruneus and E. moara show even more constitutive heterochromatin (related to chromosomal rearrangements or variation) than other species in Epinephelus (Sola et al., 2000; Molina, Maia-Lima & Affonso, 2002; Phillips & Rab, 2001; Wang et al., 2012).

Moreover, E. moara exhibited more plesiomorphic features than E. bruneus. The fundamental number (FN) of E. bruneus is larger than that of E. moara. Species with a larger FN are more derived in evolutionary terms (Martinez et al., 1989; Ghigliotti et al., 2007). Chromosomal rearrangements and genomic modifications were more obviously in E. bruneus compared to those in E. moara. Interstitial C-bandings appeared in E. bruneus imply the karyotypic rearrangement (Galetti, Aguilar & Molina, 2000), robertsonian rearrangements and/or reciprocal translocations (Eler et al., 2007). Despite the conservation of (TTAGGG)n sequence and location, slight changes in the telomeric sequences have occurred during vertebrate evolution (Meyne et al., 1990). Uniform telomeric distribution in E. moara is similar to other species in Epinephelus (Table 1) (Sola et al., 2000; Wang et al., 2012). However, remarkably high repetitions of telomere sequences seem to exist on ten chromosome pairs with stronger signals in E. bruneus, which appear to involve complex homologous or/and non-homologous recombination.

18S rDNA could be simultaneously applicable in the taxonomic and evolutionary analyses of groupers. The 5S rDNA seems to be unsuitable in the phylogenetic resolution, because the order percomorpha in the NJ trees were not recovered as monophylum. Distribution patterns of 5S rDNA of E. bruneus and E. moara are similar to other species, while that of 18S rDNA were different among species in genus Epinephelus (Sola et al., 2000; Wang et al., 2010; Wang et al., 2012). In addition, the different distributions of highly conserved18S rDNA and telomere suggest the distinct genomes and evolutionary situation of the closely related species E. bruneus and E. moara.

In summary, many useful cytogenetic charateristics are available to distinguish E. bruneus from E. moara, such as karyotypes, NORs, C-banding, 18S rDNA and telomere (TTAGGG)_n distribution patterns. Moreover, the lineage of E. bruneus and E. moara seems to be derived recently, and E. moara exhibits more plesiomorphic features than E. bruneus. Molecular cytogenetic analyses could be applicable in identification of closely related species and reconstruct their phylogenetic relationships in groupers.