Next Article in Journal
The Fast Lane of Hypoxic Adaptation: Glucose Transport Is Modulated via A HIF-Hydroxylase-AMPK-Axis in Jejunum Epithelium
Next Article in Special Issue
Evolution of the Proto Sex-Chromosome in Solea senegalensis
Previous Article in Journal
Novel Potassium Channels in Kidney Mitochondria: The Hyperpolarization-Activated and Cyclic Nucleotide-Gated HCN Channels
Previous Article in Special Issue
Possible Phenotypic Consequences of Structural Differences in Idic(15) in a Small Cohort of Patients

Int. J. Mol. Sci. 2019, 20(20), 4994; https://doi.org/10.3390/ijms20204994

Article
Adaptive Radiation from a Chromosomal Perspective: Evidence of Chromosome Set Stability in Cichlid Fishes (Cichlidae: Teleostei) from the Barombi Mbo Lake, Cameroon
1
Laboratory of Fish Genetics, Institute of Animal Physiology and Genetics, Czech Academy of Sciences, 27721 Liběchov, Czech Republic
2
Zoological Institute, University of Basel, 4051 Basel, Switzerland
3
Department of Biological Sciences, University of Ngaoundéré, Ngaoundéré P.O. Box 454, Cameroon
4
Department of Management of Fisheries and Aquatic Ecosystems, University of Douala, Douala P.O. Box 2701, Cameroon
5
Department of Zoology, Faculty of Science, Charles University in Prague, 12844 Prague, Czech Republic
*
Authors to whom correspondence should be addressed.
Received: 30 August 2019 / Accepted: 7 October 2019 / Published: 9 October 2019

Abstract

:
Cichlid fishes are the subject of scientific interest because of their rapid adaptive radiation, resulting in extensive ecological and taxonomic diversity. In this study, we examined 11 morphologically distinct cichlid species endemic to Barombi Mbo, the largest crater lake in western Cameroon, namely Konia eisentrauti, Konia dikume, Myaka myaka, Pungu maclareni, Sarotherodon steinbachi, Sarotherodon lohbergeri, Sarotherodon linnellii, Sarotherodon caroli, Stomatepia mariae, Stomatepia pindu, and Stomatepia mongo. These species supposedly evolved via sympatric ecological speciation from a common ancestor, which colonized the lake no earlier than one million years ago. Here we present the first comparative cytogenetic analysis of cichlid species from Barombi Mbo Lake using both conventional (Giemsa staining, C-banding, and CMA3/DAPI staining) and molecular (fluorescence in situ hybridization with telomeric, 5S, and 28S rDNA probes) methods. We observed stability on both macro and micro-chromosomal levels. The diploid chromosome number was 2n = 44, and the karyotype was invariably composed of three pairs of meta/submetacentric and 19 pairs of subtelo/acrocentric chromosomes in all analysed species, with the same numbers of rDNA clusters and distribution of heterochromatin. The results suggest the evolutionary stability of chromosomal set; therefore, the large-scale chromosomal rearrangements seem to be unlikely associated with the sympatric speciation in Barombi Mbo.
Keywords:
karyotype; rDNA; chromosome banding; FISH; adaptive radiation; cytotaxonomy; chromosome stasis; African endemic fishes

1. Introduction

The intralacustrine speciation of fish species flocks has attracted the attention of biologists since the beginning of modern ichthyology, and lead to the discovery of rich and speciose lacustrine fish faunas, such as cichlids in African lakes [1,2], cottid sculpins in Lake Baikal [3,4], cyprinids of Lake Lanao in the Philippines [5], barbs of Tana Lake in Ethiopia [6], or coregonids in the Great Lakes in North America or northern Europe [7,8,9]. Besides these spectacular lacustrine fish faunas in ancient lakes [10,11,12], some smaller-scale fish species flocks have been discovered, such as the telmatherid sail-fin silversides in Malili lakes in Sulawesi [13], homalopterid genus Yunnanilus in the Yunnan province of China [14], killifishes of the genus Orestias in the Andean Altiplano [15,16], and the cichlids of Barombi Mbo crater lake in Cameroon [17,18], among many others. The differentiation and speciation of these lacustrine fish faunas have been assessed from various perspectives, but cytogenetic tools have been used only in a small number of cases. Using this approach, Symonová et al. [19] analyzed deep-water cisco species Coregonus fontanae from Lake Stechlin, Germany and its differentiation from ubiquitous, shallow-water European Vendace, Coregonus albula. Similarly, Dion-Côté et al. [20,21] studied incipient speciation process within various forms of Lake Whitefish Coregonus clupeaformis. In spite of the fact that cichlids are a textbook example and the focus of numerous studies on intralacustrine speciation, only a handful of studies aim to address karyotype evolution in African cichlids (e.g., [22,23]), and the comprehensive attempts to apply cytogenetic methods to understand details of the speciation process are still largely lacking.
The family Cichlidae includes more than 3000 species, comprising one of the most species-rich clades of freshwater euteleosts [24], distributed from Central and South America, across Africa to Madagascar and southern India [25,26]. The African (Pseudocrenilabrinae) and Neotropical (Cichlinae) cichlids are reciprocally monophyletic and sister taxa [27], with the divergence age estimated to 81–103 Mya (95% HPD 74–123 Mya [28,29]). The exceptional diversity and propensity to generate adaptive radiations have made cichlid fishes one of the most important vertebrate model systems for evolutionary biology research [2,25,30]. One such example of adaptive radiation is represented by a species flock from the Barombi Mbo Lake, the largest and the deepest volcanic crater lake in Cameroon. Despite the size of the lake not exceeding 7 km2 [31], it harbors an endemic monophyletic radiation of 11 cichlid species. This monophyletic species flock has most likely radiated in situ, via sympatric ecological speciation [32,33,34] from the common ancestor, probably Sarotherodon galilaeus (Oreochromini), which colonized the lake no earlier than one million years ago [17,18,35] when the last substantial geological activity was detected [36,37]. Cichlids from the Barombi Mbo Lake are classified in five genera, four of which are endemic to the lake (i.e., Konia, Myaka, Pungu, and Stomatepia), and one genus (Sarotherodon) is widely used also for other African mostly riverine cichlids [32,38]. The Barombi Mbo cichlid fishes are emerging as a model system for evolutionary biology, namely focusing on mechanisms of speciation, adaptive radiation, ecological diversification, or evolutionary history in general (e.g., [17,18,28,31,32,39,40]).
The intriguing question of sympatric speciation is how multiple species diversify in close geographic proximity without any physical barriers and with ongoing gene flow [41]. Karyotype differentiation and chromosomal rearrangements are one of the possible mechanisms able to cause postzygotic reproductive barriers, and therefore, may trigger or facilitate speciation (as postulated by [42,43] and reviewed in [44]). Such chromosomal speciation is broadly evidenced, e.g., from mammals (shrews [45], wallabies [46]), plants [47], or butterflies [48], however, cases in teleost fishes are generally more rare, such as in the swamp eels [49] or in chromosomal races in trahiras (Characiformes [50]). Smaller-scale chromosomal rearrangements not noticeable on the karyotype level (i.e., number and morphology of chromosomes), but detectable by the advanced techniques (such as rDNA staining) have been evidenced and hypothesized to be associated with speciation, for example, in Coregonus [19]. Similarly, in African cichlids, recent studies focused on the chromosome-level whole-genome assemblies of two species identified mostly intrachromosomal rearrangements [51], yet no clear case of speciation associated with the karyotype incompatibilities has been reported so far in cichlids. Here we present the first comparative cytogenetic research focused on the Barombi Mbo cichlid fishes in order to test if large-scale chromosomal rearrangements might have contributed to the speciation in the lake.
So far, karyotypes of almost 200 species of cichlids have been determined [22,23,52,53,54,55,56,57]. Diploid chromosome numbers range from 2n = 32 to 2n = 60, more than half of studied species possess karyotypes with 2n = 48 [52], which is the modal number for the Neotropical lineage, whereas African cichlids possess predominantly 2n = 44 (Figure 1, [23,52]). Even though cytogenetic data are known for a number of cichlid species, it still covers less than 10% of cichlid taxonomic diversity. In this study, we investigated karyotype differentiation in all 11 representatives of the Barombi Mbo Lake cichlid species flock to uncover trends in their chromosome and genome organization. Using molecular cytogenetic methods, we examined karyotypes and reconstructed the ideogram for the typical Barombi Mbo cichlid genome. We further identified the numbers and positions of minor and major rDNA genes on the chromosomes, and visualized the distribution of heterochromatic blocks and telomeric sequences. The main goal of this study was to address whether the sympatric speciation of this species flock may have been associated with karyotype or interchromosomal rearrangements, or finer-scale intrachromosomal variability, such as in the number of rDNA regions.

2. Results

2.1. Karyotypes

Karyotypes of all 11 species invariably possessed the diploid number, 2n = 44 (Figure 2, Table 1) and fundamental number, NF = 50. The general Barombi Mbo cichlid karyotype, identical for all species, consisted of three pairs of submetacentric chromosomes (sm) and 19 pairs of subtelocentric-acrocentric (st/a) chromosomes. Karyotypes of all studied species also possessed a large subtelocentric chromosome pair, a characteristic marker for the karyotype of the model species Nile tilapia (Oreochromis niloticus [58]). Results of the karyological analysis together with results of the comparative cytogenetic analyses, respectively, are summarized in the ideogram representing the typical Barombi Mbo cichlid karyotype (Figure 3).

2.2. Telomere Mapping

We applied the FISH telomeric mapping method to test for signs of any putative chromosomal rearrangements. By using FISH with the conserved vertebrate telomeric repeat (TTAGGG)n, we detected signals only at the termini of all chromosomes (Figure 4 and Figure 5). We did not detect any interstitial telomeric sequences (ITSs) in chromosomes of any of the examined species.

2.3. C-Banding

We applied the C-banding technique to detect accumulations of heterochromatin throughout the chromosomes. This method revealed similar distribution patterns of constitutive heterochromatin blocks among all studied species. In all studied species, we observed C-positive bands only in the pericentromeric regions of all chromosomes. No other large accumulations of heterochromatin were observed in the studied species. (Figure 4 and Figure 5).

2.4. CMA3/DAPI Staining

Reversed fluorescence staining (CMA3/DAPI) revealed homogeneous patterns across chromosomes and extremely GC-rich signals were found in the pericentromeric regions of multiple chromosomes. Species differed slightly in numbers of these GC-rich signals (Figure 4 and Figure 5). The highest number of the signals (18) was observed in the chromosomes of Sarotherodon linnellii and Konia eisentrauti, whereas the lowest number (14) was observed in Pungu maclareni. All other studied species possessed the GC-rich signals in the pericentromeric region of 16 chromosomes.

2.5. Fluorescence In Situ Hybridization with rDNA Genes

The FISH (fluorescence in situ hybridization) experiments with rDNA probes showed the same numbers of clusters in the genomes of the nine studied species: three pairs of 28S rDNA hybridization signals and four pairs of 5S rDNA hybridization signals. The 28S rDNA probe hybridized at the pericentromeric region of two middle-sized acrocentric chromosomal pairs and at an interstitial position of one large acrocentric chromosomal pair. As for the 5S rDNA, two signals were located at the interstitial position of the largest pair, and six at the pericentromeric region of other three st/a chromosome pairs. One of the acrocentric chromosome pairs possessed both 28S and 5S rDNA loci (Figure 4 and Figure 5).

3. Discussion

Cichlids are one of the most speciose fish families in freshwaters worldwide, with about 3000 recognized species [24], of which about 2000 evolved in the adaptive radiation flocks of East African great lakes, namely, Lake Malawi, Lake Victoria, and Lake Tanganyika [2]. These species flocks have provided a model system for the study of evolution for decades [2,25,26,59]. Much smaller examples of adaptive radiation in cichlids have evolved in the crater lakes of Cameroon, such as Barombi Mbo, which hosts an endemic species flock of 11 cichlid species [32]. This flock is evolutionary quite recent, sharing a most recent common ancestor with the riverine species Sarotherodon galilaeus between 1 and 2.5 million years ago [28,35]. Most of the species in the lake have evolved probably by adaptive ecological speciation [33,34] triggered by the ecological differentiation. For example, deep-water specialists have evolved twice independently within the lake (Konia dikume, Myaka myaka; [34,60,61]), a mechanism also recently described from the onset of speciation in Massoko crater lake cichlids [62]. Further, different trophic strategies have been established within the flock, which includes predators, planktivores, insectivores, pure herbivores, and spongivores [33,34,60,61]. Ecological speciation in sympatry is a well understood evolutionary mechanism for example in sticklebacks (e.g., [63]) and in Lake Malawi cichlids [64]. Additionally, differential seasonality in spawning, as observed in one species (Myaka myaka) [32], is another potential mechanism for achieving sympatric speciation, as observed in arctic chars [65] and coregonids [19]. Alternatively, chromosomal rearrangements and subsequent reproductive isolation are also known mechanisms, causing speciation for example in guppies [66], whitefishes [20], recently happening in swamp eels [49] and possibly also beginning in the trahiras [50]. In this study, we tested the hypothesis of chromosomal differentiation within the sympatric species flock, and we found that the karyotypes of all eleven species within the Barombi Mbo species flock are very stable and similar to each other. This is comparable to the findings in the lake whitefish (Coregonus clupeaformis) system [20], where species retained the same karyotypes, but hybrid incompatibilities led to a dramatic reduction in embryonic survival in first- and second-generation hybrids. In this study, we did not detect any dynamics of chromosomal evolution that could have contributed to or triggered speciation within the Barombi Mbo species flock.
Despite numerous genetic studies on lake cichlids, advanced karyotypic evolution has been studied only scarcely (e.g., [22,23]), and virtually nothing is known about karyotypic differentiation in the context of cichlid speciation. Karyotype data for cichlid fishes has been broadly published and almost 200 cichlid species have been cytogenetically analyzed [22,23,52,53,54,55,56,57]. Nevertheless, advanced molecular cytogenetic approaches have only been performed on a minority of the studied species. A lot of research attention has been focused on the Nile tilapia (Oreochromis niloticus), a model species closely related to the Barombi Mbo cichlids [23,67,68,69] or comparison with other African cichlids, recently including genomic data [51]. Both conventional and advanced molecular cytogenetic methods have been used to characterize genome evolution in cichlids (e.g., [70]). The ancestral diploid number of chromosomes (2n) for cichlids remain unclear (Figure 1), yet the modal 2n is different for different phylogenetic lineages. African cichlids have a modal 2n = 44 (but unclear ancestral 2n), whereas the Neotropical cichlids 2n = 48 [52] (and similarly, the ancestral 2n = 48; Figure 1). The chromosomal data published so far for the Pseudocrenilabrinae clade (= African cichlids) focuses mostly on the description of chromosome morphology and mapping of repetitive sequences [23,52]. Interestingly, B chromosomes have been identified in several species from lakes Victoria and Malawi in East Africa making the dynamics of chromosomal evolution even more complex [23,71,72]. No B chromosomes have been detected in this study or in Nile tilapia.
In this study, we provided cytogenetic analyses and description of chromosomal stability among the cichlid species of Barombi Mbo crater lake. All Barombi Mbo species are characterized by 2n = 44, the modal number of African cichlids [23], and the most prevalent number for the oreochromine lineage (Figure 1). Karyotypes of the Barombi Mbo cichlids are represented by three submetacentric chromosome pairs and 19 st/a chromosome pairs, including the large pair of subtelocentric chromosomes (Figure 2) characteristic also for karyotypes of many African cichlids, namely Nile tilapia (O. niloticus [58]). Three pairs of submetacentric chromosomes observed in the karyotypes of all Barombi Mbo species (Figure 2) is more than that in Nile tilapia with only one pair [23,68] (but see [67] who has also recognized three pairs); but lower than in, S. galilaeus (6 m/sm [73]) the closest related species to the Barombi Mbo cichlids [17]. This suggests that chromosomal rearrangements, such as centromeric shifts, possibly occurred in the ancestors of the flock to a certain extent. Previously, one representative from this species flock, St. pindu, was karyotyped [22], describing the same 2n = 44; however, the karyotype description itself differs slightly, possibly due to the different scoring of the (sub)metacentric and (sub)telocentric chromosomes. Similarly, even for the aforementioned model species, Nile tilapia, similar variation in scoring of sm vs st chromosomes is known (e.g., [23,68]—1 sm pair, [67]—3 sm pairs). We therefore did not aim to reach conclusions based on the comparison of different numbers of sm chromosomes, and rather focused on the chromosomal stability within the species flock.
Using advanced cytogenetic methods, we identified the similar number of clusters of 5S (four pairs of signals on four pairs of chromosomes) and 28S rDNA (three pairs of signals on three pairs of chromosomes) sequences among all nine studied species (Figure 3, Figure 4 and Figure 5). Additionally, one of the chromosomes possess both 5S and 28S rDNA signals (Figure 3). We observed the constitutive heterochromatin regions only in the pericentromeric regions of all chromosomes, the telomeric signals only in the telomeric regions, and no interstitial positions for the telomeric or heterochromatin signals were observed. All of the aforementioned analyses suggest that we can consider the karyotype of the Barombi Mbo species as very stable, with no signals suggesting recent chromosome rearrangement, or genomic modifications, such as massive accumulation of heterochromatin, or multiplication of the rDNA regions.
Interestingly, the number of the observed rDNA locations in the Barombi Mbo cichlids (14 signals per 2n; Figure 3), despite being invariable within the flock, is higher than the usual (and median) observed number for both 5S and 45S rDNA (i.e., including 28S rDNA) subunits. In ray-finned fishes, the rDNA is most commonly present as one single block (i.e., one pair of signals per 2n) for each type, i.e., 5S and 45S [74], although the observed numbers in this study (i.e., four pairs for 5S and three pairs for 28S/45S rDNA) are not outside the known range [74,75,76]. Even in African cichlids, variations in the number of rDNA gene clusters are frequently observed, and similarly, the presence of two clusters in homologous chromosomes is the most common pattern for both 5S and 28S rDNA traits [70,75]. The number of rDNA clusters per diploid genome ranges from 2 to 15 for 5S rDNA, and from 2 to 6 for 45S (28S) rDNA in cichlids [70]. The closely related Nile tilapia (O. niloticus) differs from the Barombi Mbo cichlids in the rDNA signals, possessing one 5S rDNA cluster less with no signal on the largest st/a chromosome pair (i.e., it has only three pairs, while Barombi Mbo cichlids have four pairs of 5S rDNA signals; [70,77], whereas the 28S rDNA signal is similar—three pairs in tilapia and Barombi cichlids). Variability in rDNA clusters from the Nile tilapia, and no observed variability within the Barombi Mbo cichlids therefore suggests that the genome of the Barombi Mbo cichlids possibly differentiated in the ancestor before the intralacustrine differentiation of the species flock, but not after.
Numbers of GC-rich regions with accumulated heterochromatin were the only analysis in which slight differences among species were observed. The highest numbers of signals (up to 18) were observed in metaphase chromosomes of Sarotherodon linnellii and Konia eisentrauti, while the lowest number (14) were observed in Pungu maclareni. However, we also observed substantial variability in the numbers of the GC-rich regions among individuals of the same species, and even among metaphase chromosomes within one individual. Such differences in the GC-rich scoring could be caused by different condensation of chromosomes and may, therefore, represent an artefactual observation. Like with rDNA, observation of multiple GC-rich regions is rare among genomes of teleost fishes, and has been observed in only a limited number of studies e.g., [78]. Since rDNA sites in eukaryotes are generally known as regions of substantial GC enrichment [79], multiple accumulation of GC-rich regions observed in this study may be simply associated with the multiple (14) repetitive rDNA signals observed in the cichlid chromosomes by applying FISH staining.
In conclusion, we integrated various comparative cytogenetic approaches, and we present the pilot cytogenetic study of the endemic Barombi Mbo cichlid species flock. Our results show karyotypic and chromosomal stability in these species, which have undergone rapid sympatric ecological speciation. Our results suggest that inter-chromosomal rearrangements followed by the karyotype incompatibility have likely not contributed to the speciation processes in the lake. Nevertheless, further advanced molecular cytogenetic techniques, such as chromosome painting or CGH (comparative genomic hybridization), will be required to verify detailed synteny of the chromosomal pairs across species, or more subtle differences undetectable by the approaches applied in this study. We finally consider our study as complementary evidence for any future molecular genomic studies focused on cichlids from the Barombi Mbo crater lake.

4. Materials and Methods

4.1. Specimens

We examined 29 individuals from 11 species as described in Table 1. Samples were collected and the research was conducted under research permits (numbers: 0000047,49/MINRESI/B00/C00/ C10/nye, 0000116,117/MINRESI/B00/C00/C10/C14, 000002-3/MINRESI/B00/C00/C10/C11, 0000032,48 -50/MINRESI/B00/C00/C10/C12) issued by the Ministry of Scientific Research and Innovation in Cameroon. Valid Animal Use Protocol issued by Ministry of Agriculture, Czech Republic (No. CZ 02386, approved 25th September 2014) was in force during this study in IAPG. Further information about numbers of examined cells are listed in Table 1. Due to the limited access to the studied material, we did not perform advanced stainings on chromosomes of S. caroli and K. dikume. In these species, only Giemsa-stained karyotypes are presented in this study.

4.2. Chromosome Preparation and Giemsa Staining, CMA3 Staining, and C-Banding

Metaphase chromosomes were prepared according to [80] with slight modifications. Briefly, fish were injected with 0.1% colchicine solution (1 mL/100 g of body weight) and euthanized after 45 min using an overdose of anaesthetic (phaenoxyethanol). The kidneys were dissected in 0.075 M KCl at room temperature. The cell suspension was hypotonized for 30 min in 0.075 M KCl, fixed in freshly prepared fixative (methanol: acetic acid 3:1, v/v), washed twice in fixative and spread onto slides. Alternatively, the chromosomes of the rare species, K. dikume, were obtained from the fin regenerate according to the protocol of [81], previously successfully applied to cichlids [55]. Chromosomal spreads were stained with Giemsa solution (5%, 10 min) to identify the number and morphology of chromosomes in all 11 species used in this study. To visualize the blocks of constitutive heterochromatin, the C-banding staining was performed according to [82], with slight modifications as described in [83]. After C-banding, the chromosomes were counterstained with Vectashield DAPI anti-fade medium (Vector Laboratories, Burlingame, CA, USA) to enhance the contrast, and the microphotographs were taken in the fluorescent regime and inverted. To reveal the GC genome composition, Chromomycin A3 (CMA3) staining was performed as described by [84] using Vectashield DAPI anti-fade medium as a mounting reagent (Vector Laboratories, Burlingame, CA, USA).

4.3. Fluorescence in situ Hybridization with Telomeric Probe and rDNA Genes

FISH with Cy3-labelled telomeric PNA probe was performed according to the manufacturer’s instructions (Telomere PNA FISH Kit/Cy3, Dako, Denmark). Probes for rDNA FISH experiments were produced by PCR with the primer pairs as follows: (i) 28S rDNA: 5’-AAACTCTGGTGGAGGTCCGT-3’ and 5’-CTTACCAAAAGTGGCCCACTA-3’ [85]; (ii) 5S rDNA: 5’-TACGCCCGATCTCGTCCGATC-3’ and 5’-CAGGCTGGTATGGCCGTAAGC-3’ [86]. The PCR reactions were carried out as described in [87]. Probes were indirectly labelled with biotin-16-dUTP (Roche, Mannheim, Germany) and digoxigenin-11-dUTP (Roche) through PCR reamplification of PCR products. Reamplification was carried out under the same conditions as the previous PCR reaction. A hybridization mixture was made, consisting of labelled and precipitated PCR products of both genes, hybridization buffer [88], and salmon sperm blocking DNA (15 μg/slide; Sigma-Aldrich, St. Louis, MO, USA). The hybridization and detection procedures were carried out under conditions described by [88]. The biotin-dUTP-labelled probes were detected by Invitrogen Cy™3-Streptavidin (Invitrogen, San Diego, CA, USA; cat. no. 43-4315), the digoxigenin-dUTP-labeled probes were detected by anti-digoxigenin-rhodamine (cat. no. 11207750910). The slides were mounted with Vectashield DAPI anti-fade medium (Vector Laoratories, Burlingame, CA, USA).

4.4. Microscopy and Image Processing

Chromosomal preparations were examined by a ZEISS Axio Imager.Z2 epifluorescence microscope. Images of metaphase chromosomes were recorded with a CoolCube 1 camera (MetaSystems, Altlussheim, Germany). Analyses of images were performed in the IKAROS and ISIS imaging programs (MetaSystems, Altlussheim, Germany). The captured digital images from FISH experiments were pseudocolored and superimposed using Adobe Photoshop software, version CS5. For CMA3/DAPI staining, the CMA3 signal was inserted into the red and the DAPI signal into the green channel to enhance the contrast between these two types of signals. In karyotypes, chromosomes were ordered in decreasing size and the chromosomal categories were classified according to Levan et al. [89].

Author Contributions

Conceptualization, A.I., P.R. and Z.M. (Zuzana Musilova); data curation, A.I. and Z.M. (Zuzana Musilova); formal analysis, Z.M. (Zuzana Majtánová) and A.I.; funding acquisition, A.I. and Z.M. (Zuzana Musilova); investigation, Z.M. (Zuzana Majtánová), A.I., A.R.B.N. and Z.M. (Zuzana Musilova); methodology, Z.M. (Zuzana Majtánová); project administration, A.I. and Z.M. (Zuzana Musilova); resources, A.I. and A.R.B.N.; supervision, P.R. and Z.M. (Zuzana Musilova); validation, Z.M. (Zuzana Majtánová) and Z.M. (Zuzana Musilova); visualization, Z.M. (Zuzana Majtánová); writing—original draft, Z.M. (Zuzana Majtánová) and Z.M. (Zuzana Musilova); writing—review and editing, Z.M. (Zuzana Majtánová), P.R. and Z.M. (Zuzana Musilova).

Funding

The study was funded by the Czech Science Foundation, Project No. 16-09784Y (Z.Ma. and Z.Mu.), the Swiss National Science Foundation (PROMYS 166550; Z.Mu.), and Basler Stiftung für Biologische Forschung a Basler Stiftung für Experimentelle Zoologie (for Z.Mu. and A.I.). P.R. was supported by the project EXCELLENCE CZ.02.1.01/0.0/0.0/15_003/0000460 OP RDE.

Acknowledgments

The authors would like to express their gratitude to Walter Salzburger for his guidance and constant support. We would like to thank Šárka Pelikánová and Jana Kopecká for their help with the laboratory protocols. Thanks to Karl G. Moy for language correction.

Conflicts of Interest

The authors declare no conflict of interest. The founding sponsors had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript and in the decision to publish the results.

References

  1. Seehausen, O. Process and pattern in cichlid radiations—Inferences for understanding unusually high rates of evolutionary diversification. New Phytol. 2015, 207, 304–312. [Google Scholar] [CrossRef] [PubMed]
  2. Salzburger, W. Understanding explosive diversification through cichlid fish genomics. Nat. Rev. Genet. 2018, 19, 705–717. [Google Scholar] [CrossRef] [PubMed]
  3. Fryer, G. Comparative aspects of adaptive radiation and speciation in Lake Baikal and the great rift lakes of Africa. Hydrobiologia 1991, 211, 137–146. [Google Scholar] [CrossRef]
  4. Goto, A.; Yokoyama, R.; Sideleva, V.G. Evolutionary diversification in freshwater sculpins (Cottoidea): A review of two major adaptive radiations. Environ. Biol. Fishes 2014, 98, 307–335. [Google Scholar] [CrossRef]
  5. Kornfield, I.; Carpenter, K. The Cyprinids of Lake Lanao, Philippines: Taxonomic Validity, Evolutionary Rates and Speciation Scenarios. In Evolution of Fish Species Flocks; Echelle, A.A., Kornfield, I., Eds.; University of Maine Press: Orono, MN, USA, 1984; pp. 69–84. ISBN 089-1-0105-80. [Google Scholar]
  6. De Graaf, M.; Dejen, E.; Osse, J.W.M.; Sibbing, F.A. Adaptive radiation of Lake Tana’s (Ethiopia) Labeobarbus species flock (Pisces, Cyprinidae). Mar. Freshw. Res. 2008, 59, 391–407. [Google Scholar] [CrossRef]
  7. Præbel, K.; Knudsen, R.; Siwertsson, A.; Karhunen, M.; Kahilainen, K.K.; Ovaskainen, O.; Østbye, K.; Peruzzi, S.; Fevolden, S.-E.; Amundsen, P.-A. Ecological speciation in postglacial European whitefish: Rapid adaptive radiations into the littoral, pelagic, and profundal lake habitats. Ecol. Evol. 2013, 3, 4970–4986. [Google Scholar] [CrossRef]
  8. Hudson, A.G.; Lundsgaard-Hansen, B.; Lucek, K.; Vonlanthen, P.; Seehausen, O. Managing cryptic biodiversity: Fine-scale intralacustrine speciation along a benthic gradient in Alpine whitefish (Coregonus spp.). Evol. Appl. 2017, 10, 251–266. [Google Scholar] [CrossRef]
  9. Feulner, P.G.D.; Seehausen, O. Genomic insights into the vulnerability of sympatric whitefish species flocks. Mol. Ecol. 2019, 28, 615–629. [Google Scholar] [CrossRef]
  10. Brooks, J.L. Speciation in ancient lakes (concluded). Q. Rev. Biol. 1950, 25, 131–176. [Google Scholar] [CrossRef]
  11. Martens, K. Speciation in ancient lakes. Trends Ecol. Evol. Amst. 1997, 12, 177–182. [Google Scholar] [CrossRef]
  12. Cristescu, M.E.; Adamowicz, S.J.; Vaillant, J.J.; Haffner, D.G. Ancient lakes revisited: From the ecology to the genetics of speciation. Mol. Ecol. 2010, 19, 4837–4851. [Google Scholar] [CrossRef] [PubMed]
  13. Von Rintelen, T.; von Rintelen, K.; Glaubrecht, M.; Schubart, C.; Herder, F. Aquatic Biodiversity Hotspots in Wallacea: The Species Flocks in the Ancient Lakes of Sulawesi, Indonesia. In Biotic Evolution and Environmental Change in Southeast Asia; Gower, D., Johnson, K., Ridchardson, J., Rosen, B., Rüber, L., Williams, S., Eds.; Cambridge University Press: Cambridge, UK, 2012; pp. 290–315. ISBN 978-1-1070-0130-5. [Google Scholar]
  14. Kottelat, M.; Xin-Luo, C. Revision of Yunnanilus with descriptions of a miniature species flock and six new species from China (Cypriniformes: Homalopteridae). Environ. Biol. Fish. 1988, 23, 65–94. [Google Scholar] [CrossRef]
  15. Parenti, L.R. Biogeography of the Andean Killifish Genus Orestias with Comments on the Species Flock Concept. In Evolution of Fish Species Flocks; Echelle, A.A., Kornfield, I., Eds.; University of Maine Press: Orono, MN, USA, 1984; pp. 85–92. ISBN 089-1-0105-80. [Google Scholar]
  16. Vila, I.; Scott, S.; Lam, N.; Méndez, A.M. Karyological and Morphological Analysis of Divergence among Species of the Killifish Genus Orestias (Teleostei: Cyprinodontidae) from the Southern Altiplano. In Origin and Phylogenetic Interrelationships of Teleosts; Nelson, J.S., Schultze, H.-P., Wilson, M.V.H., Eds.; Verlag Dr. Friedrich Pfeil: New York, NY, USA, 2010; pp. 471–480. ISBN 3-89937-107-0. [Google Scholar]
  17. Schliewen, U.K.; Tautz, D.; Pääbo, S. Sympatric speciation suggested by monophyly of crater lake cichlids. Nature 1994, 368, 629. [Google Scholar] [CrossRef] [PubMed]
  18. Schliewen, U.K.; Klee, B. Reticulate sympatric speciation in Cameroonian crater lake cichlids. Front. Zool. 2004, 1, 5. [Google Scholar] [CrossRef] [PubMed]
  19. Symonová, R.; Majtánová, Z.; Sember, A.; Staaks, G.B.O.; Bohlen, J.; Freyhof, J.; Rábová, M.; Ráb, P. Genome differentiation in a species pair of coregonine fishes: An extremely rapid speciation driven by stress-activated retrotransposons mediating extensive ribosomal DNA multiplications. BMC Evol. Biol. 2013, 13, 42. [Google Scholar] [CrossRef] [PubMed]
  20. Dion-Côté, A.-M.; Symonová, R.; Ráb, P.; Bernatchez, L. Reproductive isolation in a nascent species pair is associated with aneuploidy in hybrid offspring. Proc. R. Soc. B-Biol. Sci. 2015, 282, 20142862. [Google Scholar] [CrossRef] [PubMed]
  21. Dion-Côté, A.-M.; Symonová, R.; Lamaze, F.C.; Pelikánová, Š.; Ráb, P.; Bernatchez, L. Standing chromosomal variation in Lake Whitefish species pairs: The role of historical contingency and relevance for speciation. Mol. Ecol. 2017, 26, 178–192. [Google Scholar] [CrossRef] [PubMed]
  22. Ozouf-Costaz, C.; Coutanceau, J.P.; Bonillo, C.; Mercot, H.; Fermon, Y.; Guidi-Rontani, C. New insights into the chromosomal differentiation patterns among cichlids from Africa and Madagascar. Cybium 2017, 41, 35–43. [Google Scholar]
  23. Poletto, A.B.; Ferreira, I.A.; Cabral-de-Mello, D.C.; Nakajima, R.T.; Mazzuchelli, J.; Ribeiro, H.B.; Venere, P.C.; Nirchio, M.; Kocher, T.D.; Martins, C. Chromosome differentiation patterns during cichlid fish evolution. BMC Genet. 2010, 11, 50. [Google Scholar] [CrossRef]
  24. Nelson, J.S.; Grande, T.C.; Wilson, M.V.H. Fishes of the World, 5th ed.; Wiley: Hoboken, NJ, USA, 2016; ISBN 978-1-118-34233-6. [Google Scholar]
  25. Kocher, T.D. Adaptive evolution and explosive speciation: The cichlid fish model. Nat. Rev. Genet. 2004, 5, 288. [Google Scholar] [CrossRef]
  26. Genner, M.J.; Seehausen, O.; Lunt, D.H.; Joyce, D.A.; Shaw, P.W.; Carvalho, G.R.; Turner, G.F. Age of cichlids: New dates for ancient lake fish radiations. Mol. Biol. Evol. 2007, 24, 1269–1282. [Google Scholar] [CrossRef] [PubMed]
  27. Smith, W.L.; Chakrabarty, P.; Sparks, J.S. Phylogeny, taxonomy, and evolution of Neotropical cichlids (Teleostei: Cichlidae: Cichlinae). Cladistics 2008, 24, 625–641. [Google Scholar] [CrossRef]
  28. Schedel, F.D.B.; Musilová, Z.; Schliewen, U.K. East African cichlid lineages (Teleostei: Cichlidae) might be older than their ancient host lakes: New divergence estimates for the east African cichlid radiation. BMC Evol. Biol. 2019, 19, 94. [Google Scholar] [CrossRef] [PubMed]
  29. Matschiner, M.; Musilová, Z.; Barth, J.M.I.; Starostová, Z.; Salzburger, W.; Steel, M.; Bouckaert, R. Bayesian phylogenetic estimation of clade ages supports trans-Atlantic dispersal of cichlid fishes. Syst. Biol. 2017, 66, 3–22. [Google Scholar] [CrossRef] [PubMed]
  30. Wagner, C.E.; Harmon, L.J.; Seehausen, O. Ecological opportunity and sexual selection together predict adaptive radiation. Nature 2012, 487, 366–369. [Google Scholar] [CrossRef] [PubMed]
  31. Musilová, Z.; Indermaur, A.; Nyom, A.R.B.; Tropek, R.; Martin, C.; Schliewen, U.K. Persistence of Stomatepia mongo, an endemic cichlid fish of the Barombi Mbo crater lake, southwestern Cameroon, with notes on its life history and behavior. Copeia 2014, 2014, 556–560. [Google Scholar] [CrossRef]
  32. Trewavas, E.; Green, J.; Corbet, S.A. Ecological studies on crater lakes in West Cameroon Fishes of Barombi Mbo. J. Fish Zool. 1972, 167, 41–95. [Google Scholar] [CrossRef]
  33. Baldo, L.; Pretus, J.L.; Riera, J.L.; Musilová, Z.; Bitja, A.N.; Salzburger, W. Convergence of gut microbiotas in the adaptive radiations of African cichlid fishes. ISME J. 2017, 11, 1975–1987. [Google Scholar] [CrossRef]
  34. Musilová, Z.; Indermaur, A.; Bitja-Nyom, A.R.; Omelchenko, D.; Kłodawska, M.; Albergati, L.; Remišová, K.; Salzburger, W. Evolution of visual sensory system in cichlid fishes from crater lake Barombi Mbo in Cameroon. Mol. Ecol. 2019. [Google Scholar] [CrossRef]
  35. Friedman, M.; Keck, B.P.; Dornburg, A.; Eytan, R.I.; Martin, C.H.; Hulsey, C.D.; Wainwright, P.C.; Near, T.J. Molecular and fossil evidence place the origin of cichlid fishes long after Gondwanan rifting. Proc. Biol. Sci. 2013, 280, 20131733. [Google Scholar] [CrossRef]
  36. Cornen, G.; Bande, Y.; Giresse, P.; Maley, J. The nature and chronostratigraphy of Quaternary pyroclastic accumulations from Lake Barombi Mbo (West-Cameroon). J. Volcanol. Geoth. Res. 1992, 51, 357–374. [Google Scholar] [CrossRef]
  37. Tchamabé, B.; Youmen, D.; Owona, S.; Issa; Ohba, T.; Németh, K.; Ngapna, M.; Asaah, A.; Aka, F.; Tanyileke, G.; et al. Eruptive history of the Barombi Mbo Maar, Cameroon Volcanic Line, Central Africa: Constraints from volcanic facies analysis. Open Geosci. 2014, 5, 480–496. [Google Scholar]
  38. Dunz, A.R.; Schliewen, U.K. Molecular phylogeny and revised classification of the haplotilapiine cichlid fishes formerly referred to as “Tilapia”. Mol. Phylogenet. Evol. 2013, 68, 64–80. [Google Scholar] [CrossRef] [PubMed]
  39. Dominey, W.J.; Snyder, A.M. Kleptoparasitism of freshwater crabs by cichlid fishes endemic to Lake Barombi Mbo, Cameroon, West Africa. Environ. Biol. Fish. 1988, 22, 155. [Google Scholar] [CrossRef]
  40. Richards, E.J.; Poelstra, J.W.; Martin, C.H. Don’t throw out the sympatric speciation with the crater lake water: Fine-scale investigation of introgression provides equivocal support for causal role of secondary gene flow in one of the clearest examples of sympatric speciation. Evol. Lett. 2018, 2, 524–540. [Google Scholar] [CrossRef] [PubMed]
  41. Coyne, J.A.; Orr, H.A. Speciation; Oxford University Press: Oxford, UK; New York, NY, USA, 2004; ISBN 978-0-87893-089-0. [Google Scholar]
  42. Dobzhansky, T. Genetics and the Origin of Species; Columbia University Press: New York, NY, USA, 1937; ISBN 978-0-231-05475-1. [Google Scholar]
  43. Rieseberg, L.H. Chromosomal rearrangements and speciation. Trends Ecol. Evol. 2001, 16, 351–358. [Google Scholar] [CrossRef]
  44. Navarro, A.; Barton, N.H. Chromosomal speciation and molecular divergence-accelerated evolution in rearranged chromosomes. Science 2003, 300, 321–324. [Google Scholar] [CrossRef]
  45. Searle, J.B.; Polly, P.D.; Zima, J. Shrews, Chromosomes and Speciation; Cambridge University Press: Cambridge, UK, 2019; Volume 6, ISBN 978-0-511-89553-1. [Google Scholar]
  46. Potter, S.; Bragg, J.G.; Blom, M.P.K.; Deakin, J.E.; Kirkpatrick, M.; Eldridge, M.D.B.; Moritz, C. Chromosomal speciation in the genomics era: Disentangling phylogenetic evolution of rock-wallabies. Front. Genet. 2017, 8, 10. [Google Scholar] [CrossRef]
  47. Livingstone, K.; Rieseberg, L. Chromosomal evolution and speciation: A recombination-based approach. New Phytol. 2004, 161, 107–112. [Google Scholar] [CrossRef]
  48. Kandul, N.P.; Lukhtanov, V.A.; Pierce, N.E. Karyotypic diversity and speciation in Agrodiaetus butterflies. Evolution 2007, 61, 546–559. [Google Scholar] [CrossRef]
  49. Supiwong, W.; Pinthong, K.; Seetapan, K.; Saenjundaeng, P.; Bertollo, L.A.C.; de Oliveira, E.A.; Yano, C.F.; Liehr, T.; Phimphan, S.; Tanomtong, A.; et al. Karyotype diversity and evolutionary trends in the Asian swamp eel Monopterus albus (Synbranchiformes, Synbranchidae): A case of chromosomal speciation? BMC Evol. Biol. 2019, 19, 73. [Google Scholar] [CrossRef] [PubMed]
  50. Bertollo, L.A.C. Chromosome Evolution in the Neotropical Erythrinidae Fish Family: An Overview. In Fish Cytogenetics; Pisano, E., Ozouf-Costaz, C., Foresti, F., Kapoor, B.G., Eds.; Science Publishers: Enfield, NH, USA, 2007; pp. 195–211. ISBN 978-1-5780-8330-5. [Google Scholar]
  51. Conte, M.A.; Joshi, R.; Moore, E.C.; Nandamuri, S.P.; Gammerdinger, W.J.; Roberts, R.B.; Carleton, K.L.; Lien, S.; Kocher, T.D. Chromosome-scale assemblies reveal the structural evolution of African cichlid genomes. Gigascience 2019, 8, 1–20. [Google Scholar] [CrossRef] [PubMed]
  52. Arai, R. Fish Karyotypes: A Check List, 2011 ed.; Springer: Tokyo, Japan; New York, NY, USA, 2011; ISBN 978-4-431-53876-9. [Google Scholar]
  53. Schneider, C.H.; Gross, M.C.; Terencio, M.L.; Artoni, R.F.; Vicari, M.R.; Martins, C.; Feldberg, E. Chromosomal evolution of neotropical cichlids: The role of repetitive DNA sequences in the organization and structure of karyotype. Rev. Fish Biol. Fisher. 2013, 23, 201–214. [Google Scholar] [CrossRef]
  54. Marescalchi, O. Karyotype and mitochondrial 16S gene characterizations in seven South American Cichlasomatini species (Perciformes, Cichlidae). J. Zool. Syst. Evol. Res. 2005, 43, 22–28. [Google Scholar] [CrossRef]
  55. Hodaňová, L.; Kalous, L.; Musilová, Z. Comparative cytogenetics of Neotropical cichlid fishes (Nannacara, Ivanacara and Cleithracara) indicates evolutionary reduction of diploid chromosome numbers. Comp. Cytogenet. 2014, 8, 169–183. [Google Scholar]
  56. Krajáková, L.; Musilová, Z.; Kalous, L. Karyotype Characterizations in Two South American Cichlasomatini Species (Perciformes, Cichlidae). In Proceedings of the Workshop on Animal Biodiversity, Jevany, Czech Republic, 7 July 2010; pp. 78–80. [Google Scholar]
  57. Valente, G.T.; de Andrade Vitorino, C.; Cabral-de-Mello, D.C.; Oliveira, C.; Souza, I.L.; Martins, C.; Venere, P.C. Comparative cytogenetics of ten species of cichlid fishes (Teleostei, Cichlidae) from the Araguaia River system, Brazil, by conventional cytogenetic methods. Comp. Cytogenet. 2012, 6, 163–181. [Google Scholar]
  58. Oliveira, C.; Wright, J.M. Molecular cytogenetic analysis of heterochromatin in the chromosomes of tilapia, Oreochromis niloticus (Teleostei: Cichlidae). Chromosome Res. 1998, 6, 205–211. [Google Scholar] [CrossRef]
  59. Kornfield, I.; Smith, P.F. African cichlid fishes: Model systems for evolutionary biology. Annu. Rev. Ecol. Syst. 2000, 31, 163–196. [Google Scholar] [CrossRef]
  60. Green, J.; Corbet, S.A.; Betney, E. Ecological studies on crater lakes in West Cameroon the blood of endemic cichlids in Barombi Mbo in relation to stratification and their feeding habits. J. Zool. 1973, 170, 299–308. [Google Scholar] [CrossRef]
  61. Thieme, M.L.; Abell, R.; Stiassny, M.L.J.; Skelton, P. Freshwater Ecoregions of Africa and Madagascar: A Conservation Assessment; Island Press: Washington, DC, USA, 2005; ISBN 978-1-55963-365-9. [Google Scholar]
  62. Malinsky, M.; Challis, R.J.; Tyers, A.M.; Schiffels, S.; Terai, Y.; Ngatunga, B.P.; Miska, E.A.; Durbin, R.; Genner, M.J.; Turner, G.F. Genomic islands of speciation separate cichlid ecomorphs in an East African crater lake. Science 2015, 350, 1493–1498. [Google Scholar] [CrossRef]
  63. Schluter, D.; McPhail, J.D. Ecological character displacement and speciation in sticklebacks. Am. Nat. 1992, 140, 85–108. [Google Scholar] [CrossRef] [PubMed]
  64. Danley, P.D.; Kocher, T.D. Speciation in rapidly diverging systems: Lessons from Lake Malawi. Mol. Ecol. 2001, 10, 1075–1086. [Google Scholar] [CrossRef] [PubMed]
  65. Skúlason, S.; Snorrason, S.S.; Noakes, D.L.G.; Ferguson, M.M.; Malmquist, H.J. Segregation in spawning and early life history among polymorphic Arctic charr, Salvelinus alpinus, in Thingvallavatn, Iceland. J. Fish Biol. 1989, 35, 225–232. [Google Scholar] [CrossRef]
  66. Volff, J.N. Genome evolution and biodiversity in teleost fish. Heredity 2005, 94, 280–294. [Google Scholar] [CrossRef] [PubMed]
  67. Ferreira, I.A.; Poletto, A.B.; Kocher, T.D.; Mota-Velasco, J.C.; Penman, D.J.; Martins, C. Chromosome evolution in African cichlid fish: Contributions from the physical mapping of repeated DNAs. Cytogenet. Genome Res. 2010, 129, 314–322. [Google Scholar] [CrossRef] [PubMed]
  68. Valente, G.T.; Mazzuchelli, J.; Ferreira, I.A.; Poletto, A.B.; Fantinatti, B.E.A.; Martins, C. Cytogenetic mapping of the retroelements Rex1, Rex3 and Rex6 among cichlid fish: New insights on the chromosomal distribution of transposable elements. Cytogenet. Genome Res. 2011, 133, 34–42. [Google Scholar] [CrossRef] [PubMed]
  69. Mazzuchelli, J.; Kocher, T.D.; Yang, F.; Martins, C. Integrating cytogenetics and genomics in comparative evolutionary studies of cichlid fish. BMC Genom. 2012, 13, 463. [Google Scholar] [CrossRef]
  70. Nakajima, R.T.; Cabral-de-Mello, D.C.; Valente, G.T.; Venere, P.C.; Martins, C. Evolutionary dynamics of rRNA gene clusters in cichlid fish. BMC Evol. Biol. 2012, 12, 198. [Google Scholar] [CrossRef]
  71. Clark, F.E.; Conte, M.A.; Ferreira-Bravo, I.A.; Poletto, A.B.; Martins, C.; Kocher, T.D. Dynamic sequence evolution of a sex-associated B chromosome in Lake Malawi cichlid fish. J. Hered. 2017, 108, 53–62. [Google Scholar] [CrossRef]
  72. Clark, F.E.; Conte, M.A.; Kocher, T.D. Genomic Characterization of a B chromosome in Lake Malawi cichlid fishes. Genes 2018, 9, 610. [Google Scholar] [CrossRef]
  73. Feldberg, E.; Ivan, J.; Porto, R.; Antonio, L.; Bertollo, C. Chromosomal Changes and Adaptation of Cichlid Fishes during Evolution. In Fish Adaptation; Val, A.L., Kapoor, B.G., Eds.; Science Publishers: Enfield, NH, USA, 2003; pp. 285–308. ISBN 978-1-5780-8249-0. [Google Scholar]
  74. Sochorová, J.; Garcia, S.; Gálvez, F.; Symonová, R.; Kovařík, A. Evolutionary trends in animal ribosomal DNA loci: Introduction to a new online database. Chromosoma 2018, 127, 141–150. [Google Scholar] [CrossRef] [PubMed]
  75. Gornung, E. Twenty years of physical mapping of major ribosomal RNA genes across the Teleosts: A review of research. Cytogenet. Genome Res. 2013, 141, 90–102. [Google Scholar] [CrossRef] [PubMed]
  76. Cioffi, M.B.; Bertollo, L.A.C. Chromosomal distribution and evolution of repetitive DNAs in fish. Genome Dyn. 2012, 7, 197–221. [Google Scholar] [PubMed]
  77. Martins, C.; Wasko, A.P.; Oliveira, C.; Porto-Foresti, F.; Parise-Maltempi, P.P.; Wright, J.M.; Foresti, F. Dynamics of 5S rDNA in the tilapia (Oreochromis niloticus) genome: Repeat units, inverted sequences, pseudogenes and chromosome loci. Cytogenet. Genome Res. 2002, 98, 78–85. [Google Scholar] [CrossRef] [PubMed]
  78. Symonová, R.; Majtánová, Z.; Arias-Rodriguez, L.; Mořkovský, L.; Kořínková, T.; Cavin, L.; Pokorná, M.J.; Doležálková, M.; Flajšhans, M.; Normandeau, E.; et al. Genome compositional organization in gars shows more similarities to mammals than to oher ray-finned fish. J. Exp. Zool. B Mol. Dev. Evol. 2017, 328, 607–619. [Google Scholar] [CrossRef] [PubMed]
  79. Escobar, J.S.; Glémin, S.; Galtier, N. GC-biased gene conversion impacts ribosomal DNA evolution in vertebrates, angiosperms, and other eukaryotes. Mol. Biol. Evol. 2011, 28, 2561–2575. [Google Scholar] [CrossRef] [PubMed]
  80. Bertollo, L.A.C.; Cioffi, M.B.; Moreira-Filho, O. Direct Chromosome Preparation from Freshwater Teleost Fishes. In Fish Cytogenetic Techniques; Ozouf-Costaz, C., Pisano, E., Foresti, F., Foresti de Almeida-Toledo, L., Eds.; CRC Press, Inc.: Enfield, NH, USA, 2015; pp. 21–26. ISBN 978-0-3673-7755-7. [Google Scholar]
  81. Völker, M.; Ráb, P. Direct Chromosome Preparation from Regenerating Fin Tissue. In Fish Cytogenetic Techniques; Ozouf-Costaz, C., Pisano, E., Foresti, F., Foresti de Almeida-Toledo, L., Eds.; CRC Press, Inc.: Enfield, NH, USA, 2015; pp. 37–41. ISBN 978-0-3673-7755-7. [Google Scholar]
  82. Sumner, A.T. A simple technique for demonstrating centromeric heterochromatin. Exp. Cell Res. 1972, 75, 304–306. [Google Scholar] [CrossRef]
  83. Pokorná, M.; Rens, W.; Rovatsos, M.; Kratochvíl, L. A ZZ/ZW sex chromosome system in the thick-tailed gecko (Underwoodisaurus milii; Squamata: Gekkota: Carphodactylidae), a member of the ancient gecko lineage. Cytogenet. Genome Res. 2014, 142, 190–196. [Google Scholar] [CrossRef] [PubMed]
  84. Sola, L.; Rossi, A.R.; Iaselli, V.; Rasch, E.M.; Monaco, P.J. Cytogenetics of bisexual/unisexual species of Poecilia. II. Analysis of heterochromatin and nucleolar organizer regions in Poecilia mexicana mexicana by C-banding and DAPI, quinacrine, chromomycin A3, and silver staining. Cytogenet. Cell Genet. 1992, 60, 229–235. [Google Scholar] [CrossRef]
  85. Zhang, Q.; Cooper, R.K.; Tiersch, T.R. Chromosomal location of the 28S ribosomal RNA gene of channel catfish by in situ polymerase chain reaction. J. Fish Biol. 2000, 56, 388–397. [Google Scholar] [CrossRef]
  86. Komiya, H.; Takemura, S. Nucleotide sequence of 5S ribosomal RNA from rainbow trout (Salmo gairdnerii) liver. J. Biochem. 1979, 86, 1067–1080. [Google Scholar] [CrossRef] [PubMed]
  87. Majtánová, Z.; Moy, K.G.; Unmack, P.J.; Ráb, P.; Ezaz, T. Characterization of the karyotype and accumulation of repetitive sequences in Australian Darling hardyhead Craterocephalus amniculus (Atheriniformes, Teleostei). PeerJ 2019, 7, e7347. [Google Scholar] [CrossRef] [PubMed]
  88. Symonová, R.; Sember, A.; Majtánová, Z.; Ráb, P. Characterization of Fish Genomes by GISH and CGH. In Fish Cytogenetic Techniques; Ozouf-Costaz, C., Pisano, E., Foresti, F., de Almeida, L., Eds.; CRC Press: Boca Raton, FL, USA, 2015; pp. 118–131. ISBN 978-1-4822-1198-6. [Google Scholar]
  89. Levan, A.; Fredga, K.; Sandberg, A.A. Nomenclature for centromeric position on chromosomes. Hereditas 1964, 52, 201–220. [Google Scholar] [CrossRef]
Figure 1. Schematic evolution of cichlid karyotypes. Data reviewed from [22,23,52,53,54,55,56,57]. Modal chromosome numbers for African (2n = 44) and Neotropical (2n = 48) cichlids are highlighted in bold. Phylogenetic relationships after [38,39]. Note that Barombi Mbo cichlids belong in the Oreochromini tribe, and the phylogenetic position of this tribe among other African cichlids shows that they are only distantly related to the cichlids from the Tanganyika, Malawi and Victoria lakes. Photos show Konia dikume, Pungu maclareni, Stomatepia mongo, Sarotherodon linnellii and St. pindu.
Figure 1. Schematic evolution of cichlid karyotypes. Data reviewed from [22,23,52,53,54,55,56,57]. Modal chromosome numbers for African (2n = 44) and Neotropical (2n = 48) cichlids are highlighted in bold. Phylogenetic relationships after [38,39]. Note that Barombi Mbo cichlids belong in the Oreochromini tribe, and the phylogenetic position of this tribe among other African cichlids shows that they are only distantly related to the cichlids from the Tanganyika, Malawi and Victoria lakes. Photos show Konia dikume, Pungu maclareni, Stomatepia mongo, Sarotherodon linnellii and St. pindu.
Ijms 20 04994 g001
Figure 2. Karyotypes of all 11 cichlids from Barombi Mbo crater lake arranged from Giemsa-stained chromosomes. All 11 species from the lake possess an identical karyotype with 2n = 44 (NF = 50). (a) Sarotherodon steinbachi, (b) S. lohbergeri, (c) S. linnellii, (d) S. caroli, (e) Myaka myaka, (f) Stomatepia mariae, (g) St. pindu, (h) St. mongo, (i) Pungu maclareni, (j) Konia eisentrauti, and (k) K. dikume; sm, submetacentric and st/a, subtelocentric-acrocentric chromosomes. The enlarged subtelocentric chromosome pair, which is also characteristic of the Nile tilapia (Oreochromis niloticus) karyotype, is marked with an asterisk.
Figure 2. Karyotypes of all 11 cichlids from Barombi Mbo crater lake arranged from Giemsa-stained chromosomes. All 11 species from the lake possess an identical karyotype with 2n = 44 (NF = 50). (a) Sarotherodon steinbachi, (b) S. lohbergeri, (c) S. linnellii, (d) S. caroli, (e) Myaka myaka, (f) Stomatepia mariae, (g) St. pindu, (h) St. mongo, (i) Pungu maclareni, (j) Konia eisentrauti, and (k) K. dikume; sm, submetacentric and st/a, subtelocentric-acrocentric chromosomes. The enlarged subtelocentric chromosome pair, which is also characteristic of the Nile tilapia (Oreochromis niloticus) karyotype, is marked with an asterisk.
Ijms 20 04994 g002
Figure 3. Ideogram (schematic representation of the haploid chromosome set) of the Barombi Mbo cichlid fishes after cytogenetic protocols: C-banded chromosomal regions are marked by grey color; telomeric signals after PNA-telomeric FISH are marked by blue color, and rDNA signals are marked by red (28S rDNA) and green (5S rDNA). See Figure 4 and Figure 5 for the actual results of the methods visualized in the ideogram; abbreviations: sm, submetacentric and st/a, subtelocentric-acrocentric chromosomes. The enlarged subtelocentric chromosome pair, which is also characteristic of the Nile tilapia (Oreochromis niloticus) karyotype, is marked with an asterisk.
Figure 3. Ideogram (schematic representation of the haploid chromosome set) of the Barombi Mbo cichlid fishes after cytogenetic protocols: C-banded chromosomal regions are marked by grey color; telomeric signals after PNA-telomeric FISH are marked by blue color, and rDNA signals are marked by red (28S rDNA) and green (5S rDNA). See Figure 4 and Figure 5 for the actual results of the methods visualized in the ideogram; abbreviations: sm, submetacentric and st/a, subtelocentric-acrocentric chromosomes. The enlarged subtelocentric chromosome pair, which is also characteristic of the Nile tilapia (Oreochromis niloticus) karyotype, is marked with an asterisk.
Ijms 20 04994 g003
Figure 4. Comparative chromosome analyses of Sarotherodon steinbachi, S. lohbergeri, S. linnellii, and Myaka myaka. First column: DAPI-stained chromosomes (blue) with telomere repeat hybridization signals (red) located only in the telomeric region assuming no chromosome rearrangements. Second column: inverted DAPI-stained C-banding pattern highlights clusters with constitutive heterochromatin located in the pericentromeric regions. Third column: DAPI-stained metaphase chromosomes (green) with signals of GC-rich sites (red). Fourth column: DAPI stained metaphase chromosomes (blue), with six 28S rDNA (red, highlighted by arrows), and eight 5S rDNA (green, highlighted by arrowheads) hybridization signals in each species. Bar equals 10 µm.
Figure 4. Comparative chromosome analyses of Sarotherodon steinbachi, S. lohbergeri, S. linnellii, and Myaka myaka. First column: DAPI-stained chromosomes (blue) with telomere repeat hybridization signals (red) located only in the telomeric region assuming no chromosome rearrangements. Second column: inverted DAPI-stained C-banding pattern highlights clusters with constitutive heterochromatin located in the pericentromeric regions. Third column: DAPI-stained metaphase chromosomes (green) with signals of GC-rich sites (red). Fourth column: DAPI stained metaphase chromosomes (blue), with six 28S rDNA (red, highlighted by arrows), and eight 5S rDNA (green, highlighted by arrowheads) hybridization signals in each species. Bar equals 10 µm.
Ijms 20 04994 g004
Figure 5. Comparative chromosome analyses of Stomatepia mariae, St. pindu, St. mongo, Konia eisentrauti, and Pungu maclareni. First column: DAPI-stained chromosomes (blue) with telomere repeat hybridization signals (red) located only in the telomeric region assuming no chromosome rearrangements. Second column: inverted DAPI-stained C-banding pattern highlights clusters with constitutive heterochromatin located in the pericentromeric regions. Third column: DAPI-stained metaphase chromosomes (green), signals of GC-rich sites (red). Fourth column: DAPI stained metaphase chromosomes (blue), with six 28S rDNA (red, highlighted by arrows), and eight 5S rDNA (green, highlighted by arrowheads) hybridization signals in each species. Bar equals 10 µm.
Figure 5. Comparative chromosome analyses of Stomatepia mariae, St. pindu, St. mongo, Konia eisentrauti, and Pungu maclareni. First column: DAPI-stained chromosomes (blue) with telomere repeat hybridization signals (red) located only in the telomeric region assuming no chromosome rearrangements. Second column: inverted DAPI-stained C-banding pattern highlights clusters with constitutive heterochromatin located in the pericentromeric regions. Third column: DAPI-stained metaphase chromosomes (green), signals of GC-rich sites (red). Fourth column: DAPI stained metaphase chromosomes (blue), with six 28S rDNA (red, highlighted by arrows), and eight 5S rDNA (green, highlighted by arrowheads) hybridization signals in each species. Bar equals 10 µm.
Ijms 20 04994 g005
Table 1. Numbers of individuals and cells analyzed in this study.
Table 1. Numbers of individuals and cells analyzed in this study.
SpeciesNo. of IndividualsNo. of Cells Examined
GiemsaC-BandingCMA3TelomeresFISH
Konia eisentrauti1♂, 2♀3530303015
Konia dikume18n/an/an/an/a
Myaka myaka4♂3530303016
Pungu maclareni1♀3530303015
Sarotherodon steinbachi3 juveniles6030303012
Sarotherodon lohbergeri2 juveniles3530303015
Sarotherodon linnellii2♀, 1 juveniles4530303018
Sarotherodon caroli210n/an/an/an/a
Stomatepia mariae4 juveniles3530303010
Stomatepia pindu3 juveniles4030303013
Stomatepia mongo3 juveniles3030303012
♂: male individual; ♀: female individual.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Back to TopTop