Comparative cytogenetics of Neotropical cichlid fishes (Nannacara, Ivanacara and Cleithracara) indicates evolutionary reduction of diploid chromosome numbers

Abstract A comparative cytogenetic analysis was carried out in five species of a monophyletic clade of neotropical Cichlasomatine cichlids, namely Cleithracara maronii Steindachner, 1881, Ivanacara adoketa (Kullander & Prada-Pedreros, 1993), Nannacara anomala Regan, 1905, N. aureocephalus Allgayer, 1983 and N. taenia Regan, 1912. Karyotypes and other chromosomal characteristics were revealed by CDD banding and mapped onto the phylogenetic hypothesis based on molecular analyses of four genes, namely cyt b, 16S rRNA, S7 and RAG1. The diploid numbers of chromosomes ranged from 44 to 50, karyotypes were composed predominantly of monoarmed chromosomes and one to three pairs of CMA3 signal were observed. The results showed evolutionary reduction in this monophyletic clade and the cytogenetic mechanisms (fissions/fusions) were hypothesized and discussed.


Introduction
Cichlids are a species-rich group of ray-finned fishes (Actinopterygii), distributed in tropical and subtropical freshwaters of Africa and South and Central America, Texas, Madagascar, the Middle East, India and Sri Lanka (Kullander 1998). As a third largest fish family (Eschmeyer and Fricke 2012) cichlids represent highly evolutionarily successful fish lineage and it is considered that no other family of vertebrates exceeds cichlids in a number of varieties, shapes, colors and especially in ecological and trophic specializations (Kocher 2004).
In general, genomes of ray-finned fishes are known for high evolutionary dynamics among vertebrates, which is reflected in huge genome-architecture variability (Mank and Avise 2006). The diploid chromosome number (2n) studied in 615 Actinopterygian species ranges from 22 to 250, but over a half of the species possess the conservative number of 2n = 48 -50 chromosomes (29.3% have 2n = 48 and 25.4% have 2n = 50; Mank and Avise 2006). The most frequent fish karyotype, i.e. 2n = 48 (n=24), is also recognized as an ancestral karyotype of the whole Teleostei (Ohno et al. 1969, Nakatani et al. 2007).
In total, over 190 cichlid species have been cytogenetically analyzed and the karyotype formula was determined for 157 of them (Arai 2011). Available cytogenetic data in cichlids show that the diploid chromosome numbers range from 2n=32 to 2n=60, but more than 60% of the examined species show the ancestral karyotype with 2n=48, which mostly dominates in the Neotropical cichlid lineage (Feldberg et al. 2003).
In the past only few species were analyzed and Neotropical cichlids were considered a karyotypically conservative group due to the frequent findings of 48 chromosomes (Thompson 1979, Kornfield 1984. Later, Marescalchi (2004) and Poletto et al. (2010) demonstrated much higher variability in the chromosome number and hypothesized that the ancestral karyotype of the Neotropical cichlids underwent significant changes in structure in several lineages, which led to extensive karyotype diversification. Further, many species possess the similar 2n=48, but differ in karyotype structures, which brings additional evidence of the karyotype differentiation due to the intra-chromosomal rearrangements like centromeric shifts (Feldberg et al. 2003). It is likely that at least some different lineages coincidentally converged to the same number of chromosomes from different ancestral stages but the mechanisms of why there is certain favorable number of chromosomes remains still unknown (Mank and Avise 2006).
Dwarf cichlids of the genus Nannacara Regan, 1905, and its relatives, genera Ivanacara Römer &Hahn, 2007 andCleithracara Kullander &Nijssen, 1989 represent a well-defined evolutionary lineage of acaras (NIC-clade of the tribe Cichlasomatini, Musilová et al. 2008) distributed mostly in rivers of the Guyana shield, as well as in the Rio Negro basin, and the Amazon and Orinoco deltas. This group includes seven known species, four in the genus Nannacara, then two species recently extracted from Nannacara to the genus Ivanacara (Römer and Hahn 2007), and the monotypic genus Cleithracara, which is basal to all the others. The cytogenetics of this clade remains poorly known since only two species of this group, Cleithracara maronii (Steindach-ner, 1881) with 2n=50 (Marescalchi 2004) and Nannacara anomala Regan, 1905 with 2n=44 (Thompson 1979) have been previously investigated.
In this study we present karyotypes and other chromosomal characteristics as revealed by CDD banding in five species of monophyletic clade of neotropical Cichlasomatine cichlids, namely Cleithracara maronii, Ivanacara adoketa (Kullander & Prada-Pedreros, 1993), Nannacara anomala, Nannacara aureocephalus Allgayer, 1983 andNannacara taenia Regan, 1912. We further mapped the results onto the phylogenetic hypothesis from molecular analyses based on four genes. We discuss possible scenario of the karyotype evolution of the clade of dwarf cichlids within the tribe Cichlasomatini.

Materials
The species included in the present study are listed in Table 1. Most of the individuals originated from aquarium trade from different breeders. Further, various collectors or ornamental-fish importers donated several samples for DNA analysis. Species were identified following Kullander and Nijssen (1989), Kullander and Prada-Pedreros (1993) and Staeck and Schindler (2004), and part of the analyzed fish was deposited in ICCU (Ichthyological Collection of Charles University, Prague). See Table 1 and Table 2.

Cytogenetic analyses
Chromosomes were obtained by non-destructive isolation procedure from caudal fin regenerates as developed by Völker et al. (2006) and modified by Kalous et al. (2010). This method is based on regeneration of the caudal fin tissue after cutting a small part (2-3mm) from its margin. After five to seven days the regenerated tissue was cut and incubated in the solution with colchicine for two hours at room temperature. A few drops of fixative (methanol, acetic acid 3:1) were added to the tissue after this incubation and it was placed for 30min at 4°C. The tissue was washed twice in fixative, always staying for 30min at 4°C after the wash. Next, the tissue was placed into a drop of 20% acetic acid and gently mashed through a fine sieve. The suspension was dropped on a slide on a hot plate (45°C). After 20 seconds the drop of suspension was sucked up from the slide and dropped to a different place in the slide. Metaphase chromosomes were stained in 4% Giemsa solution in phosphate buffer (pH=7). Generally 5-50 metaphases per individual were evaluated. Chromosomes were classified according to Levan et al. (1964), to be consistent with most of the recent studies on cichlid fishes (Marescalchi 2004, Fedlberg et al. 2003) and arranged to karyotypes by using ADOBE PHOTOSHOP, version CS7. The CDD fluorescent banding (Chromomycin A 3 /methyl green/DAPI) was performed following Mayr et al. (1985) and Sola et al. (1992).

Table 1.
Sample list for the present study. Details on individuals of cichlids investigated for the molecular genetics. Outgroup data were used from the original study (Musilová et al. 2008(Musilová et al. , 2009.

Molecular genetic analyses
DNA was extracted from the ethanol-preserved samples by the commercially available kits (QiaGen), and four target genes (cyt b, 16S rRNA, S7 first intron, RAG1) were amplified by PCR using primers according to Musilová et al. (2009). Sequences of the PCR products were obtained by commercial sequence-service company (Macrogen, South Korea, Netherlands). Sequences were aligned in BIO EDIT (Hall 1999) software and genes were concatenated for the bayesian analysis in MRBAYES 3.2. (Ronquist et al. 2012). Analysis parameters were: number of generations = 10,000,000, number of chains = 4, number of runs = 2, model set for every gene separately (and unlinked) based on the jModeltest (Posada 2008) results. Three additional species (Bujurquina vittata, Aequidens metae and Laetacara thayeri) from the same taxonomic tribus Cichlasomatini as Nannacara + Ivanacara were analyzed as well, and one species of the different tribus Geophagini (Geophagus brasiliensis) was determined as an outgroup for the phylogenetic analysis. Sequences were uploaded to GenBank (Table 1).

CDD fluorescence
In the karyotypes of four studied species, namely C. maronii, I. adoketa, N. anomala, and N. taenia, the CMA 3 -positive signals were found on one chromosome pair, although probably not homologous in different species. In C. maronii the CMA 3positive signals were located on terminal parts of the largest m-sm chromosome pair, whereas in I. adoketa and N. taenia the CMA 3 signals were located a chromosome pair from st-a group, terminal parts in N. taenia and around the centromere in I. adoketa.
In N. anomala the CMA 3 signals were found on the terminal parts of a chromosome pair from m-sm group, but not on the largest pair. Contrarily, in the karyotype of N. aureocephalus, the CMA 3 signals were located on three m-sm chromosome pairs including the largest chromosome pair in the centromeric region. See Table 3 for more detail about the karyotype formulas and CMA 3 phenotypes and Fig. 1 for representative metaphases and results of different staining steps.

Phylogenetic analysis and karyotype differentiation
Phylogenetic reconstruction based on the DNA sequences of up to four genes shows monophyly of the genus Nannacara (three species used in this study) and its sister relationship with the genus Ivanacara (one species present in our study). The monotypic genus Cleithracara (C. maronii) represents then basal lineage to the rest of Nannacara + Ivanacara (Fig. 2). The observed karyotype characteristics, i.e. the diploid chromosome number, the karyotype and the phenotype, were mapped on the phylogenetic tree and allowed reconstruction of the scenario of genome/karyotype evolution in the studied cichlids as well as to reconstruct as well as of the most likely hypothetical karyotype of an ancestor of the whole group. An ancestral karyotype of 2n = 48 was hypothesized as (16m-sm + 32 st-a) and was estimated as a basal stage for the clade by the most parsimonious reconstruction based on our material. The ancestor also had most likely only one pair of CMA 3 sites (Fig. 2).

Cytogenetic characteristics
Two of the five species presented within this study have been previously studied in Thompson (1979), Marescalchi (2004) and reviewed in Feldberg et al. (2003). The   Phylogenetic tree reconstructed based on the mitochondrial (cytochrome b, 16S rRNA) and nuclear (S7, RAG1) genes. Karyotype characteristics, such as diploid chromosomal number (2n), karyotype formula and CMA 3 phenotype were mapped on the tree and interpreted under the most parsimonious criterion. Ancestral karyotype of the group evolved from the ancestral cichlid karyotype 48st-a (Mank and Avise 2006) by increasing number of sub-metacentric chromosomes. One fission (in Cleithracara clade) and two fusion events (in the Nannacara clade) were detected, followed by at least one pericentric inversion in the latter case causing the decrease of the number of sub-metacentric chromosomes. Second pericentric inversion occurred in N. taenia, and another inversion leading to the multiplication of the CMA 3 regions occurred in N. aureocephalus.
karyotype of Nannacara anomala corresponds in both the chromosomal number (2n=44) and the karyotype (18m-sm+26st-a) to the results of Thompson (1979). The karyotype of C. maronii corresponds with various previous studies in chromosomal number (2n = 50; Marescalchi 2004, see Feldberg et al. 2003), but slightly differs in the karyotype description: while in our study we recognized seven pairs of sub-metacentric chromosomes (14m-sm+36st-a), Marescalchi (2004) found only six pairs of those. However, inspecting the study of Marescalchi (2004), we found one additional pair of sub-metacentric chromosomes in their original karyotype data as well, so it is fully comparable with our results. In the clade of Neotropical cichlids, three trends in karyotype differentiation can be distinguished (Feldberg et al. 2003). First trend -also called Karyotype "A" by Thompson (1979) -is characterized by maintaining the ancestral karyotype of 2n=48 with mostly subtelocentric-acrocentric elements (karyotype of 48st-a, although not exclusively) and evolved mostly by the pericentric inversions (during which the centromere is shifted from the central position of chromosome). Second evolutionary trend is similar to the previous one and additionally suppose the chromosomal breakage/fission events (Feldberg et al. 2003), leading to the increasing diploid chromosome number usually to the 2n=50 or 52, extremely up to 2n=60). This karyotype is dominated by uniarmed chromosomes. The third evolutionary trend -also called Karyotype "B" in Thompson (1979) -is represented by the opposite evolutionary scenario -mostly centric fusions played role in evolution from the ancestral karyotype, which lead to reduction of diploid chromosome number accompanied by increasing number of metacentric and submetacentric chromosomes (Thompson 1979. This trend of chromosome number reduction seems to be parallel to some other fish groups like it was uncovered in killifishes (Cyprinodontiformes, Nothobranchiidae) Völker et al. (2008).
All of the species within the studied evolutionary lineage have a higher proportion of sub-metacentric chromosomes in their karyotypes compared with the rest of cichlids . Especially considering the fact that the ancestral cichlid karyotype has been postulated as 2n=48 and 48st-a, i.e. no sub-metacentric chromosomes are present , the whole Nannacara -Ivanacara -Cleithracara clade seems to have evolutionary derived karyotype within cichlids. Based on Thompson's (1979) classification, the whole lineage possess the karyotype type "B" characterized by higher proportion of the sub-metacentric chromosomes, although not all the species have the lower number of chromosomes then the ancestral stage, which is usually characteristic for the karyotype "B" as well (Thompson 1979). Interestingly, the chromosome rearrangements and formation of karyotype "B" occurred several times independently in cichlid evolution, as from 41 examined Neotropical cichlids, the karyotype "B" has been found in three unrelated lineages: in the species Bujurquina vittata (Heckel, 1840) (tribe Cichlasomatini), in the genus Apistogramma Regan, 1913 (tribe Geophagini) and in the genus Symphysodon Heckel, 1840 (tribe Heroini; sister tribe of Cichlasomatini; Thompson 1979). Strikingly, the most similar karyotype formula possessed by all the species of the genera Apistogramma (22-24m-sm+16-22st-a) and Dicrossus Steindachner, 1875 (12m-sm+34st-a), which also represent another two unrelated lineage of the dwarf cichlids (Thompson 1979, Feldberg et al. 2003, and then a few other species like Cichlasoma paranaense Kullander, 1983 (14-20m-sm+28-34sta), Mesonauta festivus/insignis (Heckel, 1840) (12m-sm+36st-a), Crenicichla niederleinii (Holmberg, 1891) (14m-sm+34st-a) and Astronotus ocellatus (Agassiz, 1831) and Astronotus crassipinnis (Heckel, 1840) (12-18m-sm+30-36st-a, Feldberg et al. 2003). Note, that although the karyotype composed of mostly subtelocentric-acrocentric chromosomes is considered as ancestral for the cichlids, it is not generally ancestral trait for other fish groups. Therefore, the emergence of karyotype "B" (with more submetacentric chromosomes) probably represents secondary change back to the "common teleost karyotype" (Thompson 1979, Arai 2011.

CMA 3 patterns
The CMA 3 signals represent usually the GC-rich DNA segments of heterochromatic regions, often correlated with the location of active or inactive NORs, usually represented by the rDNA regions in genome (Schmid and Guttenbach 1988, Ráb et al. 1999, but see Fontana et al. 2001, Gromicho et al. 2005or Saitoh and Laemmli 1994. The number of CMA 3 signals found within this study corresponds to what has been previously observed in cichlids -i.e. the most common number of NORs in Neotropical cichlids is one pair, but in some species were found up to three pairs (Feldberg et al. 2003). In the Nannacara -Ivanacara -Cleithracara clade, all species except for N. aureocephalus possess only one pair of CMA 3 signals in their karyotype. N. aureocephalus has three pairs of CMA 3 signals, which is usually interpreted as the result of inversion followed by the multiplication of the rDNA regions . Further, one of the observed CMA 3 regions in this species is located in the centromeric region.
After Feldberg et al. (2003), one pair of NORs on the larger pair of chromosomes represents the most common NOR phenotype for the whole family Cichlidae. Further, Hsu et al. (1975) suggested that species with the single pair of NORs should be considered as more primitive that the karyotype with several NOR pairs hinting that the ancestral karyotypes possess less NORs than the evolutionary derived. Multiplication of NORs is usually caused by the chromosomal rearrangements, such as translocation or inversion but recently an increasing number of studies has shown the cases of rDNA multiplication caused by the activity of transposable elements. (Cioffi et al. 2010, Symonová et al. 2013, Schneider et al. 2013. As summarized in Feldberg et al. (2003), five out of 15 analysed species of the subfamily Cichlasomatinae (tribes Heroini + Cichlasomatini) possess multiple NOR pairs, i.e. Caquetaia spectabilis (Steindachner, 1875) (Feldberg et al. 2003), Cichlasoma paranaense Kullander, 1983(Feldberg et al. 2003, Mesonauata insignis and M. festivus (Heckel, 1840) (Feldberg et al. 2003) and Symphysodon aequifasciatus Pellegrin, 1904(Feldberg et al. 2003.

Phylogeny of Nannacara -Ivanacara -Cleithracara cichlids
The phylogenetic reconstruction of the Nannacara -Ivanacara -Cleithracara clade (also called NIC clade in Musilová et al. 2008Musilová et al. , 2009 corresponds to the results observed in the previous studies (Musilová et al. 2008(Musilová et al. , 2009). This suggests the basal position of the monotypic genus Cleithracara followed by the Ivanacara (one species) sister to the rest of fishes from the genus Nannacara (three species). Within Nannacara, the N. taenia has basal position and N. anomala + N. aureocephalus represent the sister species. In this study, we did not include two species of the studied clade, i.e. Nannacara quadrispinae and Ivanacara bimaculata, which we failed to obtain either as live individuals for cytogenetics, or as samples for DNA analysis. Especially I. bimaculata would be crucial for confirmation of monophyly of the genus Ivanacara, since I. bimaculata was previously found as closely related to the fishes of the genus Nannacara then to I. adoketa based on morphological data set (Musilová et al. 2009).
Within N. aureocephalus, more distinct forms are known; some of them were introduced into the aquarium trade under different names. So far no robust revision of Nannacara is available, and it is therefore difficult to make any taxonomic conclusion based on our data set. However, at least two different forms of N. aureocephalus are spread among the aquarium hobbyist within Central Europe (Germany, Poland, Czech Republic, Slovakia) -one of them called "blue" and the other one called "brown" both included in our analyses. These forms are not of artificial origin, as usually F1 progeny of the wild caught individuals has been studied. Intuitively, the blue morph shows more light-blue coloration with iridescent elements both on the face and body, while the "brown" form doesn't have the iridescent coloration and possess darker brown to dark-green coloration pattern. We have shown that those two morphs are genetically distinct; however, more detailed future work is necessary on this species/genus.

Karyotype differentiation
Cichlid karyotypes show some general common features -for example many species from African and Neotropical cichlids possess one pair of significantly larger chromosomes. Although the homology of the largest chromosome within the African lineage has been proved ) as well as high synteny conservation of African cichlid genomes (Mazzuchelli et al. 2012), it is, however, not yet clear to what extent is the homology present across the whole family Cichlidae (Valente et al. 2009).
Although all the studied species from the Nannacara -Ivanacara -Cleithracara clade are characterized by the karyotype "B" (Thompson 1979), they underwent different evolutionary paths in past. The phylogenetic reconstruction of the karyotype evolution suggests the following scenario: from the ancestral karyotype, first the karyotype of the Cleithracara maronii (2n = 50; 14mt-sm + 36 st-a) evolved by fission event of one sub-metacentric chromosome pair, falling apart into two additional pairs of subtelocentric-acrocentric chromosomes. While the karyotype of Ivanacara adoketa remained unchanged compared with the ancestral one, in the lineage of Nannacara, two fusions occurred decreasing chromosomal number to 2n = 44. These fusions were followed by pericentric inversions, which again decreased the number of sub-metacentric chromosomes. At least one pericentric inversion happened in the base of all Nannacara, and additional pericentric inversion happened in the N. taenia lineage. Finally, two inversion impacting CMA 3 regions happened in N. aureocephalus leading to the multiplicaiton of these signals.
The proposed mechanisms of chromosomal rearrangements are described in cichlids as well as in other fish species. Usually the sub-metacentric chromosome arises during the (centric) fusion, when two acrocentric-telocentric chromosomes fuse (Thompson 1979). However, the number of sub-metacentric chromosomes in karyotype is not evolutionarily stable. The sub-metacentric chromosome changes back to the acrocentric-subtelocentric chromosome by inversion, which involves the centromere, i.e. the pericentric inversion (Feldberg et al. 2003. Further, those pericentric inversions are considered as the main mechanism generally contributing to changes in chromosome arms size in various percomorph lineages (Galetti et al. 2000, Affonso 2005. In general, the taxon sampling within such comparative studies is however still too low to be able to make a strong conclusion about the general trends in cichlid karyotype evolution (Feldberg et al. 2003. To conclude, we aimed to provide a comparative study on a small scale of three genera combining molecular and cytogenetic approaches. Assuming that cytogenetic data provide additional information, which is undetectable by molecular genetics (Ráb et al. 2007), we expected a broad insight into the genome evolution of the studied group. In the dwarf cichlid genus Nannacara and its relatives (Ivanacara and Cleithracara), we reconstructed the phylogeny and we found substantial amount of karyotype characteristics, which we were able to interpret in the evolutionary context.