Chromosomal complements of some Atlantic Blennioidei and Gobioidei species (Perciformes)

Abstract A remarkable degree of chromosomal conservatism (2n=48, FN=48) has been identified in several families of Perciformes. However, some families exhibit greater karyotypic diversity, although there is still scant information on the Atlantic species. In addition to a review of karyotypic data available for representatives of the suborders Blennioidei and Gobioidei, we have performed chromosomal analyses on Atlantic species of the families Blenniidae, Ophioblennius trinitatis Miranda-Ribeiro, 1919 (2n=46; FN=64) and Scartella cristata (Linnaeus, 1758)(2n=48; FN=50), Labrisomidae, Labrisomus nuchipinnis (Quoy & Gaimard, 1824)(2n=48; FN=50) and Gobiidae, Bathygobius soporator (Valenciennes, 1837)(2n=48; FN=56). Besides variations in chromosome number and karyotype formulas, Ag-NOR sites, albeit unique, were located in different positions and/or chromosome pairs for the species analyzed. On the other hand, the heterochromatic pattern was more conservative, distributed predominantly in the centromeric/pericentromeric regions of the four species. Data already available for Gobiidae, Blenniidae and Labrisomidae show greater intra- and interspecific karyotypic diversification when compared to other groups of Perciformes, where higher uniformity is found for various chromosome characteristics. Evolutionary dynamism displayed by these two families is likely associated with population fractionation resulting from unique biological characteristics, such as lower mobility and/or specific environmental requirements.


introduction
Although karyotypic characteristics for some families of marine fish are already known, information on groups of Perciformes is still significantly disproportionate. Among these, suborders Blennioidei and Gobioidei stand out because of the large number of species they represent. Suborders Gobioidei, with 2,121 species, and Blennioidei with 732 species, are spread throughout the tropical zone, typically represented by small specimens with low mobility and the ability to withstand changes in temperature and salinity (Nelson 2006).
Species of Blennioidei and Gobioidei investigated (e.g. Cataudela et al. 1973;Garcia et al. 1987;Ene 2003) have shown sufficient chromosomal peculiarities for species discrimination and understanding of their evolutionary aspects. In some families, such as Blenniidae, Labrisomidae and Gobiidae, sharing cryptic morphological characteristics combined with poor knowledge of the biological characteristics for many species, contributes to the relative taxonomic inaccuracy of this group. As such, cytotaxonomic markers (Garcia et al. 1987;Caputo 1998;Caputo et al. 2001) and phylogenetic analyses based on molecular data (Wang et al. 2001;Thacker 2003;Gysels et al. 2004;Almada et al. 2005) have been increasingly used when assessing their kinship relations. Indeed, it has been suggested that phylogenetic analyses combine molecular and morphological data (Thacker 2003), as well as cytogenetic information. However, in light of the diversity in these groups, solid chromosome data are not yet sufficiently available, with only 7.5% of Bleniidae species and 4.5% of Gobioidei was karyotyped (Table  1). Despite the scarcity of data, a high degree of chromosomal polymorphism has been characterized among Gobiidae, primarily Robertsonian rearrangements (Caputo et al. 1999, Ene 2003, along with others such as tandem fusions and pericentric inversions ; Thode et al. 1985;Amores et al. 1990).

Cytogenetic analyses of Blenniidae species (Blennioidei)
Ophioblennius trinitatis showed 2n=46, with a chromosome formula equal to 6m+12st+28a (FN=64), irrespective of sex. Although chromosomes showed a gradual decline in size, the smallest acrocentric pairs corresponded to approximately one-third of the largest metacentric pairs. Nucleolar organizer regions are located in the terminal portions of the short arm on pair 9, the smallest subtelocentric pair. C-positive heterochromatin is discretely located in the centromeric/pericentromeric region of the chromosomes (Fig. 1a, b).
Scartella cristata showed 2n=48 chromosomes, with a chromosome formula equal to 4st+44a (FN=52). The karyotype also displays a gradual reduction in chromosome size. However, the largest chromosome pair exhibits only double the size in relation to the smallest karyotype pair. Ribosomal sites are located on the terminal portions of the short arms on chromosome pair 1. C-positive heterochromatin is also reduced and located in the centromeric regions of chromosomes (Fig. 1c, d).

Cytogenetic analyses of Labrisomidae and Gobiidae species (Gobioidei)
Labrisomus nuchipinnis (Labrisomidae) showed 2n=48 chromosomes with a chromosome formula of 2st+46a (FN=50), showing relatively more differentiated size between the largest and smallest chromosomes of the karyotype. Nucleolar organizer regions are in the terminal portions of the long arms on pair 2, corresponding to the largest pair of acrocentric chromosomes. C-positive heterochromatin was showed in the centromeric/pericentromeric region of all chromosome pairs, in relatively conspicuous blocks (Fig. 1e, f ).
Bathygobius soporator (Gobiidae) also displayed the karyotype composed of 2n=48 chromosomes, but with the chromosome formula distinct from that of L. nuchipinnis, specifically, 2m+6st+40a (FN=56). Size difference between the largest and smallest chromosomes of the karyotype was far less pronounced. Ribosomal sites were on the terminal portions of the short arms on chromosome pair 4. C-banding showed discrete heterochromatic regions in the centromeric regions of most chromosomes and telomeric regions of some acrocentric pairs (Fig. 1g, h).

Discussion
Though many perciform families display a conserved karyotype pattern, with 2n=48 acrocentric chromosomes, some groups demonstrate dynamic tendencies in relation to chromosome evolution (Molina 2007). Much of identifiable chromosome diversity is attributed to pericentric inversions, the most common mechanism of chromosome evolution in this order (Galetti et al. 2000(Galetti et al. , 2006. Representatives of the suborder Blennioidei (e.g., Carbone et al. 1987) and Gobioidei (e.g., Sawada 1974, 1975;Thode et al. 1988;Oliveira and Almeida-Toledo 2006) stand out for their greater karyotype variability and diversity. This includes species with conserved karyotyes and those that are highly diversified.
Within the Blennioidei, the Blenniidae, a monophyletic family, is divided into six tribes including Salariini and Parablenniini which, in turn, include the Atlantic species O. trinitatis and S. cristata respectively (Nelson 2006). Comparisons of mitochondrial DNA sequences in samples of Ophioblennius Gill, 1860 collected throughout the Atlantic suggest that the genus consists of six distinct lineages. One of these corresponds to species found in the Pacific, while the rest are recorded in the biogeographic provinces of the Atlantic: Brazilian, Caribbean, Mid-Atlantic, Sao Tome and Azores/Cape Verde (Muss et al. 2001). Chromosome characteristics reported here for O. trinitatis are the first for the genus, exhibiting 2n=46, 6m+12st+28a and FN=64. The relatively low diploid number and higher fundamental number in relation to the mean of other species of Blenniidae (Table 1), as well as the presence of large metacentric chromosomes, suggests pericentric inversion events and the occurrence of Robertsonian translocation involving two of its chromosome pairs. In turn, S. cristata, while also belonging to the family Blenniidae, has a distinct karyotype of 2n=48, 4st+44a and FN=52. Thus, S. cristata differs from O. trinitatis in that it contains an extra pair of chromosomes, lacks metacentric chromosomes and has different numbers of subtelocentric and acrocentric chromosomes in the karyotype. The karyotype of the S. cristata population studied here differs from the karyotypes previously described for the coastal population of Rio de Janeiro (SE Brazil), with 2sm+46st/a (Brum et al. 1994), and the Mediterranean population, with 2st+46a (Vitturi et al. 1986). Nevertheless, despite the growing number of discordant karyotype descriptions between populations on the NE and SE coasts of Brazil, one cannot rule out that these differences may arise from the difficulty in precisely defining types of cryptic chromosomes in the karyotype of this species.
In spite of displaying relative diversity in chromosome structure, only 18.5% of Blennioidei species exhibit differences in the basal diploid number, 2n=48 chromosomes. As shown in table 1, diploid numbers for representatives of this suborder vary between 2n=40, found in Dasson trossulus (Jordan & Snyder, 1902) (Arai and Shiotsuki 1974) and 2n=52 in Gobius niger Linnaeus, 1758 (Vitturi and Catalano 1989), but with a conspicuous modal value of 2n=48.
Among chromosome rearrangements involved in karyotypic differentiation of Gobiidae, Robertsonian fusions stand out, and are likely the most common event in this group (Amores et al. 1990;Galetti et al. 2000). However, other more complex changes in karyotypic structure (Thode et al. 1988;Vitturi and Catalano 1989;Caputo et al. 1997;Caputo et al. 1999), as well as the presence of different sex chromosomes (e.g., Pezold 1984;Baroiller et al. 1999), can also be observed, corroborating the high dynamic evolution that characterizes suborder Gobioidei. It has been suggested that the baseline/ancestral karyotype for Gobiidae would consist of 2n=46 acrocentric chromosomes (Vasil'ev and Grigoryan 1993), from which an increase in bi-brachial chromosomes would characterize more derived karyotypes. Based on this proposal, B. soporator (FN=56) would experience a greater number of structural rearrangements during its karyotypic evolution process in relation to L. nuchipinnis (FN=50).
Location and frequency of Ag-NOR sites are efficient cytotaxonomic markers in many groups of fish (Caputo 1998). Among species of Gobiidae, at least six different arrangement patterns for nucleolar organizer regions have been identified (Fig. 2), which supports the occurrence of intense karyotypic diversification mechanisms in this group. Thus, Ag-NOR sites can be found (a) in the telomeric region on the short arm of a single pair of acrocentric chromosomes, as in Gobius fallax Sarato, 1889 (Thode et al. 1983) and Gobius paganellus Linnaeus, 1758 (Caputo 1998); in the telomeric region on the long arm of a single pair of acrocentrics, such as in Zosterisessor ophiocephalus (Pallas, 1814) (Caputo 1998); (c) in the interstitial/pericentromeric region on the long arm of a single pair of acrocentric chromosomes, as seen in Proterorhinus marmoratus (Pallas, 1814) (Ráb 1985) and Gobius cobitis Pallas, 1814 (Caputo 1998);(d) in the telomeric region on the short arm of a single subtelocentric pair, described in B. soporator; (e) in the interstitial/pericentromeric region on the long arm of a single metacentric pair, observed in N. eurycephalus (Ene 2003); and (f ) in the telomeric regions on the short arms of two acrocentric chromosome pairs, recorded in Gobiusculus flavescens (Fabricius, 1779) (Klinkhardt 1992). Ag-NOR phenotypes a-f described in species of Gobiidae. Ag-NORs sites described in the karyotypes of Gobiidae species were found a in the telomeric region on the short arm of a single pair of acrocentric chromosomes b in the telomeric region on the long arm of a single pair of acrocentrics c in the interstitial/pericentromeric region on the long arm of a single pair of acrocentric chromosomes d in the telomeric region on the short arm of a single subtelocentric pair e in the interstitial/pericentromeric region on the long arm of a single metacentric pair and f in the telomeric regions on the short arms of two acrocentric chromosome pairs. Few data are available on ribosomal sites for Labrisomidae. Ag-NORs in L. nuchipinnis exhibit the phenotype (b) described above, in addition to both species of Blenniidae, O. trinitatis and S. cristata, which may suggest an ancestral condition for this location.
In contrast, other chromosome characteristics, such as C-positive heterochromatin distribution, may be more conserved. This occurs in several species of Percifomes where discrete blocks are preferentially located in the centromeric/pericentromeric regions of chromosomes (Molina 2007). This pattern is repeated in S. cristata, O. trinitatis and L. nuchipinnis, as well as in some Gobiidae, such as G. cobitis, Z. ophiocephalus and N. eurycephalus (e.g. Caputo et al. 1997;Ene 2003). In B. soporator, in addition to centromeric/pericentromeric regions, heterochromatic sites are also observed in terminal regions of some chromosomes. This arrangement has already been described for other Gobiidae, including G. paganellus and G. niger, where pericentromeric and telomeric heterochromatic regions are distributed among almost all chromosomes (Amores et al. 1990;Caputo et al. 1997).
Moreover, karyotypic diversity present in Gobioidei is increased by the occurrence of chromosome polymorphisms frequently observed in this group. This is particularly evident in several examples of intraspecific karyotypic variability, as well as polymorphisms involving different types of chromosome rearrangements, such as in G. niger (Vitturi and Catalano 1989;Caputo et al. 1997) and G. fallax (Thode et al. 1988). Data obtained for the paedomorphic Gobiidae Aphia minuta (Risso, 1810) also show variations in the diploid number and chromosome formula, resulting in five different cytotypes (2n=41-44 and FN=42-44) (Caputo et al. 1999). Similar karyotypic variability was reported in N. eurycephalus, where three specific cytotypes (2n=30, 31 and 32) were associated to the occurrence of centric fusions (Ene 2003).
All these examples demonstrate clear chromosomal dynamism, with possible transitions to new karyotype patterns.
In fact, karyotypic diversity among Blennioidei and Gobioidei seems to accompany phyletic diversification of these groups. This is a result of vicariant factors ) and could be favored by their low dispersive potential (Fanta 1997), as well as ecological specificities that favor population fractionation in this family (Huyse et al. 2004). The present study also highlight the importance of ribosomal sites as effective chromosomal markers in the further cytogenetic studies in gobiids species.