The species E. smaragdus and E. pisonis have karyotypes with 2n = 46 acrocentric chromosomes (NF = 46), D. maculatus has 2n = 46 composed of 36sm + 4st + 6a (NF = 86), and G. guavina has 2n = 52 acrocentric chromosomes (NF = 52) (Fig. 1).
Figure 1. Karyotypes of E. smaragdus, E. pisonis, D. maculatus and G. guavina, after Giemsa staining, C-banding, and fluorescence in situ hybridization with 18S rDNA (red) and 5S rDNA (green) probes. The Ag-NORs and MM+/DAPI- regions (green) are showed in the boxes of the first column. The two rDNA arrays in the chromosomes of D. maculatus are highlighted in the larger box. Scale bar = 5 μm
The C-positive heterochromatin shows a diversified distribution and content among the species. In E. smaragdus, it occurs as conspicuous centromeric and terminal blocks in the chromosomes, in E. pisonis as small centromeric segments and in D. maculatus and G. guavina with an irregular distribution in centromeric, interstitial, and terminal blocks. In all species, some heterochromatic blocks occupy the interstitial regions or the entire arms of two-armed chromosomes (Fig. 1).
Ag-NORs sites are located on a single pair of chromosomes and are the only regions in the karyotypes exhibiting a MM+/DAPI- pattern (Fig. 1; in the boxes). These sites are localized in the terminal position on the long arms of pair 9 in E. smaragdus, in the interstitial position of the long arms of pair 21 in E. pisonis, in the terminal position of the short arms of pair 4 in D. maculatus, and in the interstitial region of the long arms of pair 19 in G. guavina (Fig. 1; in the boxes).
The 18S rDNA sites are congruent with the Ag-NORs signals in all species but located in non-homologous chromosomes. The 5S rDNA sites, in addition to numerical variation, also show large interspecific divergences in their chromosomal location (Fig. 1). In E. smaragdus, they have a proximal location on the chromosome pairs 7 and 14; in E. pisonis, they are interstitially co-located with the 18S rDNA site in pair 21; in G. guavina, they occupy an interstitial position on pair 4. In addition, D. maculatus exhibits a structural polymorphism. In this case, some individuals have 18S and 5S rDNA sites on the short arms of pairs 4 and 5, respectively, while others have only one homologue of pair 4 carrying an 18S rDNA site, the other homologue of this same pair carrying co-located 18S rDNA/5S rDNA sites, and a single homologue of pair 5 carrying a 5S rDNA site (Fig. 1).
The mapping of microsatellites (CA)15 and (CAA)10 was performed for E. pisonis, D. maculatus and G. guavina. The results show the distribution of these motifs in both heterochromatic and euchromatic regions (Fig. 2). The (CA)15 repeats occur in all chromosomes of the three species, mainly in the terminal region of their long arms. In E. pisonis, this motif additionally occurs in both arms of pairs 15 and 21. In D. maculatus, these sequences occupy the terminal regions of both arms of most chromosomes. On the other hand, in G. guavina, they have a very variable distribution, occurring exclusively in the terminal position of the short or long arms, in both arms of the chromosomes, or in the interstitial regions of a few chromosomal pairs (Fig. 2). In contrast, microsatellite sequences (CAA)10, do not occur in all chromosomes of any given species. In E. pisonis, they are mainly located in the terminal regions of the long arms in most of the chromosomes. However, in D. maculatus and in G. guavina, they occur in the terminal region of only one or both chromosome arms, but with a different distribution pattern along the chromosomes in each species (Fig. 2).
The cytogenetic survey on Gobiiformes covered 139 species, and showed a diploid variation from 2n = 30 to 56 chromosomes, where 2n = 46 represents the most frequent condition, followed by 2n = 44 chromosomes, also present in high frequency and prevalent in some clades. Oxudercidae is the most representative family, with cytogenetic data available for 69 spp. Of these, 37% (25 spp.) have 2n = 44 chromosomes, 31% (21 spp.) have 2n = 46 chromosomes, and the remaining 32% (23 spp.) have diploid numbers varying from 2n = 38 to 2n = 56 chromosomes. The NF in this group ranges from 40 to 92. Gobiidae is the second group with the largest number of accessible cytogenetic data (50 spp.), among which, 42% (21 spp.) have 2n = 46 chromosomes, 24% (12 spp.) have 2n = 44 chromosomes, and the remaining 34% (17 spp.) have 2n = 30 to 2n = 50 chromosomes. The NF variation was shown to be extensive in this family, ranging from 38 to 98. Cytogenetic data for Eleotridae encompassed 11 species: 64% (7 spp.) with 2n = 46 chromosomes, and the others with 2n = 48 or 52 chromosomes. The NF ranges from 46 to 90, with NF = 46 being the most frequent. The Butiidae and Odontobutidae families are the least investigated: the former with five species analyzed exhibiting 2n = 46 or 2n = 48, NF from 48 to 58, and the only four species of the second having 2n = 44 acrocentric chromosomes.
The association between karyotype diversification and biological factors affecting the dispersive potential in Gobiiformes species revealed a greater divergence in 2n and NF in the benthic species (66% and 73%, respectively, of the karyotypes different from the basal pattern), while species with pelagic and bentho-pelagic habits share more conservative karyotype patterns. Similar trends occur in the Gobiidae and Oxudercidae families with the greatest samples (Table 1).
2n B (%) P (%) B/P (%) NF B (%) P (%) B/P (%) Gobiiformes < 46 43 (41.7) 2 (50.0) 11 (52.4) < 46 13 (13.0) - 4 (23.5) 46 35 (34.0) 2 (50.0) 10 (47.3) 46 22 (22.0) 1 (33.3) 8 (47.0) > 46 25 (24.3) - - > 46 65 (65.0) 2 (66.7) 5 (29.5) Total 103 4 21 100 3 17 Gobiidae < 46 22 (52.4) 1 (33.3) 4 (50.0) < 46 4 (10.8) 1 (25.0) 1 (7.1) 46 12 (28.6) 2 (66.7) 4 (50.0) 46 13 (35.2) 1 (25.0) 6 (42.9) > 46 8 (19.0) - - > 46 20 (54.0) 2 (50.0) 7 (50.0) Total 42 3 8 37 4 14 Oxudercidae < 46 22 (50.0) 1 (100.0) 6 (54.0) < 46 6 (14.0) - 2 (28.6) 46 16 (32.6) - 5 (46.0) 46 5 (11.6) - 1 (14.2) > 46 11 (22.4) - - > 46 32 (74.4) 1 (100) 4 (57.2) Total 49 1 11 43 1 7
Table 1. Frequency of diploid number (2n) and chromosome arms number (NF) in Gobiiformes species and families Gobiidae and Oxudercidae grouped according to benthic (B), pelagic (P) and bentho-pelagic (B/P) habitat categories
In another comparison, the 2n and NF data of the karyotypes of 124 Gobiiformes species were analyzed with respect to their preferential environments. Species with more than one habitat were divided into grouped categories. The results show that the chromosome variation is not precisely related to environmental categories (Table 2). However, the 2n of species from freshwater and freshwater/estuarine habitats are mainly equal to the considered basal karyotype (2n = 46) for the order. Similar results were obtained for the families Gobiidae and Oxudercidae.
2n Sets of aquatic environments M-M/E (%) F-F/E (%) E-F/M/E (%) NF M-M/E (%) F-F/E (%) E-F/M/E (%) Gobiiformes < 46 20 (54.1) 17 (37.0) 15 (36.7) < 46 6 (16.6) 9 (21.0) 3 (7.3) 46 7 (18.9) 21 (45.7) 19 (46.3) 46 7 (19.4) 15 (34.8) 9 (22.0) > 46 10 (27.0) 8 (17.3) 7 (17.0) > 46 23 (64.0) 19 (44.2) 29 (70.7) Total 37 46 41 36 43 41 Gobiidae < 46 10 (45.4) 4 (23.5) 6 (66.7) < 46 4 (17.3) 1 (6.2) 1 (9.0) 46 6 (27.3) 10 (58.8) 3 (33.3) 46 7 (30.4) 10 (62.5) 5 (45.5) > 46 6 (27.3) 3 (17.7) - > 46 12 (52.3) 5 (31.3) 5 (45.5) Total 22 17 9 22 16 11 Oxudercidae < 46 11 (78.6) 5 (34.0) 13 (40.6) < 46 2 (15.3) 4 (33.3) 2 (7.7) 46 1 (7.1) 7 (46.0) 13 (40.6) 46 1 (8.7) 2 (16.7) 2 (7.7) > 46 2 (14.3) 3 (20.0) 6 (18.8) > 46 10 (76.0) 6 (50.0) 22 (84.6) Total 14 15 32 13 12 26 M marine, F freshwater, E estuarine and presence in more than one aquatic environment
Table 2. Frequency of the diploid number (2n) and chromosome arms number (NF) in Gobiiformes species and in the families Gobiidae and Oxudercidae, grouped according to the type of aquatic environment
Karyotypes, C-, Ag- and DAPI/MM banding
The four Eleotridae species exhibited a conspicuous karyotype diversification regarding their fundamental number, chromosome morphology (Table 3), and organization of repetitive sequences on the chromosomes. These data are consistent with a wider evaluation of the karyotype evolution among Gobiiformes (Molina et al. 2014b).
Species 2n Karyotype NF Distribution References Dormitator latifrons 46 12 m+22sm+10st+2a 90 Eastern Pacific Uribe-Alcócer et al. (1983), Uribe-Alcocer and Ramirez-Escamilla (1989) D. maculatus 46 36sm+4st+6a 86 Western Atlantic Molina (2005), Present study D. maculatus 46 14 m+28sm+2st+2a ♀
13 m+28sm+3st+2a ♂
90 Western Atlantic Oliveira and Almeida-Toledo (2006) D. maculatus 46 34 m/sm+12st/a 80 Caribbean Maldonado-Monroy et al. (1985) Eleotris acanthopoma 46 46a 46 Indo-Pacific Arai and Sawada (1974) E. oxycephala 46 46a 46 Indo-Pacific Yu et al. (1987) E. picta 52 52a 52 Western Atlantic Uribe-Alcocer and Diaz-Jaimes (1996) E. pisonis 46 46a 46 Western Atlantic Molina (2005), Present study E. pisonis 44 2 m/sm+42st/a 46 Caribbean Uribe-Alcocer and Diaz-Jaimes (1996) Erotelis smaragdus 46 46a 46 Western Atlantic Present study Gobiomorus dormitor 48 2 m+4sm+42a 54 Western Atlantic Maldonado-Monroy et al. (1985) Guavina guavina 52 52a 52 Western Atlantic Present study Hypseleotris cyprinoides 48 48a 48 Indo-Pacific Suzuki (1996) Mogurnda mogurnda 46 6sm+40st/a 52 Western Pacific Arai (2011) m metacentric, sm submetacentric, st subtelocentric, a acrocentric chromosome, NF number of chromosome arms
Table 3. Cytogenetic data for Eleotridae species
The evolutionary history of some Eleotridae groups, such as Dormitator in the Atlantic, is recent (0.19-0.35 Ma) and linked with population fragmentation derived from some major geological and ecological events, such as the uplift of Central American Isthmus and regional isolation by climate and oceanographic changes (Galván-Quesada et al. 2016). These processes, on macro- or micro-scales, apparently had direct evolutionary effects on genomic diversification and on the fixation of chromosome rearrangements alongside their distribution limits (Molina 2005). D. maculatus has different regional karyotypes, such as in the Brazilian northeast (2n = 46; NF = 86) (Molina 2005, present data), and southeastern (2n = 46; NF = 90) (Oliveira and Almeida-Toledo 2006) coasts, in Western Atlantic and Caribbean (2n = 46; NF = 80) (Maldonado-Monroy et al. 1985). These karyotype divergences highlight a cryptic macroevolution pattern and support an under perceived scenario of profuse allopatric speciation in the Dormitator maculatus complex.
Similarly, karyotype divergences also occur among E. pisonis populations from the Brazilian (2n = 46; NF = 46) (Molina 2005, present data) and Caribbean coasts (2n = 44; NF = 46) (Uribe-Alcocer and Diaz-Jaimes 1996). As a whole, such karyotype variations also suggest the occurrence of cryptic species within the Eleotridae family (Molina 2005). However, despite exhibiting 2n variations (2n = 44-52), Eleotridae species most often have 2n = 46 chromosomes, a condition also found in E. pisonis, E. smaragdus and D. maculatus, suggesting that it may represent a basal trait for this family. Karyotypes with 2n > 46, as in G. guavina (2n = 52; the highest diploid value in the group), and NF > 46 (Table 3), indicate the importance of fission events, as well as pericentric inversions in the karyotype evolution of this fish group. Such rearrangements are also frequent in large marine groups as Percomorpha (Galetti et al. 2000).
Besides karyotype variations, marked intra- and interspecific heterogeneities in the amount and location of heterochromatin occur among the Eleotridae species. While a reduced and centromeric heterochromatic pattern occurs in G. guavina and E. pisonis, the C-positive heterochromatin is present in the interstitial and terminal regions of chromosomes of E. smaragdus and D. maculatus. This diversified heterochromatic organization is phylogenetically wide and has been recognized in several gobiiform groups (Caputo et al. 1997; Lima-Filho et al. 2012, 2014a), indicating an intense inner chromosomal reorganization of repetitive DNAs, probably associated with changes in the macrostructure of the Eleotridae chromosomes.
The mapping of rDNA sequences has shown a wide variation at both population and interspecific levels in Gobiiformes (Lima-Filho et al. 2012, 2014a, b; Ocalewicz and Sapota 2011). In Eleotridae, although only two Ag-NORs/18S rDNA sites occur, they show distinct size and location in conspicuously different chromosomal pairs among the species, thus suggesting the occurrence of disruptive events of the syntenic order in these chromosomes.
Evidence of significant internal reorganizations in the Eleotridae chromosomes is also provided by the differentiated distribution that the 5S rDNA sites have in this group. Location of the 18S and 5S rDNA sites in different chromosomes, like in G. guavina and E. smaragdus, is a common condition in several fish groups (Gornung 2013). However, syntenic arrays such as in E. pisonis, hitherto uncommon in Gobiiformes, constitute a derived condition. Indeed, collectively the rDNA sites create very exclusive species-specific patterns. The set of diversifications related to rDNA sequences and the bearing chromosome indicates that microstructural changes are frequent in Eleotridae and probably extend to other chromosomes of the species. Interestingly, D. maculatus exhibits a rDNA polymorphism related to the 18S and 5S sequences on pairs 4 and 5 of the karyotype comprising different arrangements which include a syntenic 18S/5S state in only one homologue of pair 4. This polymorphism reinforces the dynamic condition of the ribosomal DNAs among Eleotridae species and suggests a transient stage toward the colocalization of the 18S/5S sequences in the same chromosome pair.
Like the rDNA, microsatellite sequences are also evolutionarily dynamic, susceptible to high mutational rates in the genome (Oliveira et al. 2006), and can present independent evolutionary paths in chromosomes (Xu et al. 2017). In E. pisonis, D. maculatus and G. guavina, the (CA)15 and (CAA)10 microsatellites are clustered on different regions of the chromosomes, presenting an incomplete overlap with the C-banding regions. In these species, the heterogeneity of heterochromatin is identified by the heterochromatic and euchromatic regions harboring both, one or neither (CA)15 and (CAA)10 repeats. This level of heterogeneity suggests that these regions are evolutionarily less stable and potentially associated with the high karyotype changes in Eleotridae.
As a whole, the inter- and intraspecific diversification of the karyotypes, and the great potential for population fragmentation, make Eleotridae a target group for deeper taxonomic approaches in the search for the real meaning of its biodiversity.
The significant diversification of chromosomal numbers and karyotypic formulas (Arai 2011), distinguishes Gobiiformes from other large groups of marine fish with a clear 2n = 48 conservatism (Motta-Neto et al. 2019). Phylogenetic relationships (Betancur-R et al. 2013; Thacker 2009) indicate a higher frequency of karyotypes with 2n = 46 acrocentric chromosomes distributed from basal clades to recent lineages of this order. While in families Eleotridae and Butiidae 2n = 46 acrocentric chromosomes (NF = 46) is a prevalent condition, Oxudercidae shows a greater frequency of 2n = 46 chromosomes, but with NF > 46. Apart from the Odontobutidae, which possess 2n = 44 chromosomes, other families of Gobiiformes, with ancient or recent divergence, have some species with 2n = 46 chromosomes. The presence of a high incidence of karyotypes with 2n = 46 chromosomes in Apogonidae (Araújo et al. 2010), a family closely related to Gobiiformes (Betancur-R et al. 2017), suggests that 2n = 44 chromosomes is a homoplasic and recurrent trait in some groups of Gobiiformes. In addition, Gobiiformes also include variations in intraspecific diploid number (Caputo et al. 1999; Prazdnikov et al. 2013), in 5S rDNA sites (Lima-Filho et al. 2012; present data), in karyotypes of congeneric species (Caputo et al. 1997; Grigoryan and Vasiliev 1993; Thode et al. 1988), and in the emergence of sex chromosomes (Lima-Filho et al. 2014b; Pezold 1984).
This diversified scenario is also supported by the high evolutionary variation of the ribosomal sequences, indicating a massive internal reorganization in the chromosomes. Although generally present on a single pair of chromosomes, the present study shows that 18S rDNAs can be found in different positions and on different chromosomes among gobiiform species, which is consistent with the findings of Lima-Filho et al. (2012) and Ocalewicz and Sapota (2011). Similar reorganizations are also found for 5S sites in parallel to large numerical variations. In addition, syntenic arrangements such as those in E. smaragdus, or complex polymorphic arrangements showed in D. maculatus, along with their location on the sex chromosomes (Lima-Filho et al. 2014b), complement the evolutionary dynamism of these sequences.
Some biological characteristics of Gobiiformes, such as particular habitats and reproductive strategies, seem to act on the dispersive potential of the species, thus supporting population stratifications and the fixation of chromosomal rearrangements. Some divergent cytogenetic patterns are found in marine species, contrasting with the more obvious biogeographic stratification of freshwater species. This is in accordance with to the patterns of genetic variability in Gobiiformes, whose pelagic species have a more homogeneous genetic structure than the benthic ones (Giovannotti et al. 2009).
The extensive variation in NF values among the Eleotridae species (NF = 46-90; Table 4), and in Gobiiformes generally (NF = 40-96; Arai 2011) indicates a significant participation of pericentric inversions in the karyotype evolution of these groups. Genomic-based studies revealed that large inversions are common in fishes and keep favorable allelic combination involved in local environmental adaptations (Kess et al. 2020; Kirubakaran et al. 2016; Pearse et al. 2014). Inversions are central to the evolution of many species (Faria et al. 2019), which the eco-evolutionary effects are extensive, encompassing morphological, physiological, behavioral adaptations and phyletic diversification (Ayala et al. 2017; Berg et al. 2016, 2017; Wellenreuther and Bernatchez 2018). In the order Gobiiformes, the reorganization of genomic architecture promoted by inversions possibly favored fine‐scale adaptation to the several environments and salinity gradients occupied, and it is likely that such mechanisms have played an equally important role in the evolution of the lineages within this group. Despite offering an apparent chance for greater gene flow among populations, marine environments are large and subdivided by extensive ecosystems that become progressively occupied during species colonization. The available data illustrate the unusual chromosomal diversity found in Eleotridae and other Gobiiformes fishes, offering a new example of congruence of phyletic and karyotype diversification within the marine ichthyofauna.
Species Sampling Site N Dormitator maculatus Pium River (5° 56′ 51.2″ S, 35° 14′ 09.2″ W) 10 (8♂, 2♀) Eleotris pisonis Pium River (5° 56′ 51.2″ S, 35° 14′ 09.2″ W) 25 (15♂, 10♀) Erotelis smaragdus Curimataú River (6° 19′ 15.50″ S, 35° 2′ 29.31″ W) 15 (10♂, 5♀) Guavina guavina Potengi River (5° 41′ 07.2″ S, 35°14′ 28.1″ W) 4 (1♂, 3♀)
Table 4. Collection sites and the sample sizes (N) of the Eleotridae genera