In silico Characterization of Satellitomes and Cross-Amplification of Putative satDNAs in Two Species of the Hypostomus ancistroides Complex (Siluriformes, Loricariidae).

INTRODUCTION: The mapping of the satellite DNA on chromosomes is vital to understanding the distribution and evolution of repetitions in the genome since these chromosomal studies have shown the origin, evolutionary mode, and function of repetitive sequences. This study aimed to prospect the satellitome and determine its location in the genome of two cryptic species of Hypostomus, H. aff. ancistroides and H. ancistroides, with and without XX/XY sexual chromosome system. METHODS: Mitotic chromosomes and DNA extraction were obtained according to protocols. After the whole genome sequencing, the satDNAs were retrieved, amplified, and hybridized in chromosome preparations for male and female individuals. RESULTS: We found 30 satellite families (47 variants, two superfamilies) in H. ancistroides and 38 satellite families (45 variants, four superfamilies) in H. aff. ancistroides. The sequences varied from 14 bp to 2,662 bp in H. ancistroides and from 14 bp to 2,918 bp in H. aff. ancistroides. We did not observe any tandem repeats that were exclusive to each of the libraries; however, many sequences showed very different abundances and copy numbers between the libraries. Four satDNAs did not hybridize on the chromosomes of either species. Conversely, one satDNA hybridized in both species, HxySat1-80. However, the phenotypes found varied among species, populations, and in the same individual. There was no sign of HanSat3-464 and HanSat11-335 in any individuals of H. aff. ancistroides, but markings were in the chromosomes of H. ancistroides. HxySat12-1127 and HxySat8-52, on the other hand, were only hybridized in H. aff. ancistroides, while H. ancistroides had a negative sign. No hybridization of satDNAs was found in the X and Y sex chromosomes as they were mostly composed of euchromatin. CONCLUSION: We distinguish H. aff. ancistroides as genetically different from H. ancistroides, recognizing that such characteristics go far beyond morphological, karyotypic, and molecular data. Our data support the differential abundance and location of satellite DNAs and confirm that many organisms, including fish, have repetitive sequences that validate the library hypothesis. All found and validated satDNAs and the characterization of the satellitomes of the two species represent important contributions to cytogenomic studies of the genus Hypostomus.

Big jaw, small genome: first description of the mitochondrial genome of Odontomachus (Formicidae, Ponerinae): evolutionary implications for Ponerinae ants.

Mitochondrial DNA is a valuable tool for population genetics and evolutionary studies in a wide range of organisms. With advancements in sequencing techniques, it's now possible to gain deeper insights into this molecule. By understanding how many genes there are, how they're organized within the molecule, identifying the presence of spacers, and analyzing the composition of the D-Loop, we can better grasp the rearrangements that play a crucial role in the evolutionary dynamics of mitochondrial DNA. Additionally, phylogenetic analyses benefit significantly from having access to a larger pool of mtDNA genes. This wealth of genetic information allows for the establishment of evolutionary relationships with greater accuracy than ever before, providing a more robust framework than analyses based on a limited number of genes. Studies on mitogenomes belonging to the family Formicidae have proven promising, enabling the identification of gene rearrangements and enhancing our understanding of the internal relationships within the group. Despite this, the number of mitogenomes available for the subfamily Ponerinae is still limited, and here we present for the first time the complete mitogenome of Odontomachus. Our data reveal a gene duplication event in Formicidae, the first involving trnV, and new gene arrangements involving the trnM-trnI-trnQ and trnW-trnC-trnY clusters, suggesting a possible synapomorphy for the genus. Our phylogenetic analysis using the PCGs available for Formicidae supports the monophyly of the subfamily Ponerinae and sheds light on the relationship between Odontomachus and Pachycondyla.

Chromosomal Radiation: A model to explain karyotypic diversity in cryptic species.

We present a concept that explains the pattern of occurrence of widely distributed organisms with large chromosomal diversity, large or small molecular divergence, and the insufficiency or absence of morphological identity. Our model is based on cytogenetic studies associated with molecular and biological data and can be applied to any lineage of sister species, chronospecies, or cryptic species. Through the evaluation of the karyotypic macrostructure, as the physical location of genes e satellites DNAs, in addition to phylogenetic reconstructions from mitochondrial and nuclear genes, per example, we have observed morphologically indistinguishable individuals presenting different locally fixed karyomorphs with phylogeographic discontinuity. The biological process behind this pattern is seen in many groups of cryptic species, in which variation lies mainly in the organization of their genomes but not necessarily in the ecosystems they inhabit or in their external morphology. It's similar to the processes behind other events observed in the distribution of lineages. In this work, we explore the hypothesis of a process analogous to ecological-evolutionary radiation, which we called Chromosomal Radiation. Chromosomal Radiation can be adaptive or non-adaptive and applied to different groups of organisms.

Phylogeography and karyotypic evolution of some Deuterodon species from southeastern Brazil (Characiformes, Characidae, Stethaprioninae).

Deuterodon is a genus of the subfamily Stethaprioninae, a group of Neotropical fish known as tetras. Deuterodon hastatus represents a species complex, which is supported by cytogenetic and molecular data. In this study, we show the results of comparative evolutionary analyses of the ATP synthase subunit 6 gene in four Deuterodon species, in addition to ribosomal markers (18S rDNA and 5S rDNA), of a new population of the D. hastatus species complex from the Angra dos Reis/RJ region. The study population comprised a new cytotype, which we refer to as cytotype D, in D. hastatus, with 2n = 50 = 6M+8SM+8ST+28A. We obtained three different clades of D. hastatus in our phylogeny, one of them composed only by specimens of cytotype D. By using molecular clock dating, we observed that the radiation of Deuterodon from southeastern Brazil seemed to be associated with neotectonic events that occurred during the Miocene-Pliocene and Pliocene-Pleistocene transitions, marked by the capture of headwater streams and marine transgressions. The results obtained reinforce the idea that D. hastatus is a species complex, and at least three evolutionary significant units were identified in this group.

Discovery of putative long non-coding RNAs expressed in the eyes of Astyanax mexicanus (Actinopterygii: Characidae).

Astyanax mexicanus is a well-known model species, that has two morphotypes, cavefish, from subterranean rivers and surface fish, from surface rivers. They are morphologically distinct due to many troglomorphic traits in the cavefish, such as the absence of eyes. Most studies on A. mexicanus are focused on eye development and protein-coding genes involved in the process. However, lncRNAs did not get the same attention and very little is known about them. This study aimed to fill this knowledge gap, identifying, describing, classifying, and annotating lncRNAs expressed in the embryo's eye tissue of cavefish and surface fish. To do so, we constructed a concise workflow to assemble and evaluate transcriptomes, annotate protein-coding genes, ncRNAs families, predict the coding potential, identify putative lncRNAs, map them and predict interactions. This approach resulted in the identification of 33,069 and 19,493 putative lncRNAs respectively mapped in cavefish and surface fish. Thousands of these lncRNAs were annotated and identified as conserved in human and several species of fish. Hundreds of them were validated in silico, through ESTs. We identified lncRNAs associated with genes related to eye development. This is the case of a few lncRNAs associated with sox2, which we suggest being isomorphs of the SOX2-OT, a lncRNA that can regulate the expression of sox2. This work is one of the first studies to focus on the description of lncRNAs in A. mexicanus, highlighting several lncRNA targets and opening an important precedent for future studies focusing on lncRNAs expressed in A. mexicanus.

Characterization of six new complete mitochondrial genomes of Chiasmodontidae (Scombriformes, Percomorpha) and considerations about the phylogenetic relationships of the family.

The fishes of the Chiasmodontidae family, known as swallower fishes, are species adapted to live in deep seas. Several studies have shown the proximity of this family to Tetragonuridae and Amarsipidae. However, the phylogenetic position of this clade related to other Pelagiaria groups remains uncertain even when phylogenomic studies are employed. Since the low number of published mitogenomes, our study aimed to assemble six new mitochondrial genomes of Chiasmodontidae from database libraries to expand the discussion regarding the phylogeny of this group within Scombriformes. As expected, the composition and organization of mitogenomes were stable among the analyzed species, although we detected repetitive sequences in the D-loop of species of the genus Kali not seen in Chiasmodon, Dysalotus, and Pseudoscopelus. Our phylogeny incorporating 51 mitogenomes from several families of Scombriformes, including nine chiasmodontids, recovered interfamilial relationships well established in previous studies, including a clade containing Chiasmodontidae, Amarsipidae, and Tetragonuridae. However, phylogenetic relationships between larger clades remain unclear, with disagreements between different phylogenomic studies. We argue that such inconsistencies are not only due to biases and limitations in the data but mainly to complex biological events in the adaptive irradiation of Scombriformes after the Cretaceous-Paleogene extinction event.

Description of eight new mitochondrial genomes for the genus Neoarius and phylogenetic considerations for the family Ariidae (Siluriformes).

The genus Neoarius, known as marine catfish, is a group of the family Ariidae, composed of 10 species found in Oceania. None of the species in this genus have their mitochondrial genome described, which is highly valuable in phylogenetic and molecular evolution studies. For the present work, eight species from the Neoarius genus were selected: Neoarius utarus, Neoarius midgleyi, Neoarius graeffei, Neoarius leptaspis, Neoarius berenyi, Neoarius paucus, Neoarius pectoralis, and Neoarius aff. graeffei. DNA sequences of the eight species were obtained through the NCBI Sequence Read Archive (SRA) database, and the mitochondrial genomes were assembled using the NOVOplasty tool on the Galaxy platform, subsequently annotated with the MitoAnnotator tool. We then utilized the protein-coding genes from the mitogenomes to estimate the phylogenetic relationships within the group, including seven additional mitogenomes available in the NCBI. In all species, the mitochondrial genomes presented 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 1 D-loop.

Hidden Evolutionary Units and Its Implications on Conservation in a Vulnerable Species of a Freshwater Fish.

Brycon is a fish genus in the order Characiformes, distributed from southern Mexico to the La Plata River in Argentina. Several of its species, including Brycon nattereri, are threatened with extinction or considered vulnerable because they are highly sensitive to anthropogenic factors. The decline of these species may be related to the growth of agriculture and mining in the Brazilian Cerrado region, thus their recovery requires management plans. In this study, we use morphological, chromosomal, and genetic analysis to suggest that two distinct evolutionary units exist under the same denomination B. nattereri, indistinguishable by the descriptive morphological characters of the species until the present moment and show that the population of the São Francisco River is more diverse than that of the upper Paraná River basin. These results may help with future management and conservation programs of Brycon species in the Paraná and São Francisco river basins, two major Brazilian hydrographic basins.

Studies in two allopatric populations of Hypostomus affinis (Steindachner, 1877): the role of mapping the ribosomal genes to understand the chromosome evolution of the group.

Several cytogenetic markers show chromosomal diversity in the fish such as "armoured catfish". Although studies have characterized many species in the major genera representing these Siluridae, particularly in the genus Hypostomus Lacépède, 1803, trends in chromosome evolution of this group remain unclear. The Paraíba do Sul river basin contains the armoured catfish Hypostomus affinis Steindachner, 1877, which is unique because of its distribution of repetitive DNAs, the 5S and 18S rDNA. Identified samples and registered collections in Brazilian museums were identified as the same typological species, while we observed wide variations in the physical location of this gene in the karyotype based on fluorescent in situ hybridization results. In this study, we propose that these species can represent evolutionarily independent units, as these fish frequently undergo processes such as dispersion and vicariance and that the rDNA is associated with DNA that spreads in the genome, such as transposons. Additionally, the absence of gene flow due to the distance of the sample location could intensify evolutionary processes. The phenotypes found for the 18S rDNA showed minor changes in relation to the number of sites between the lower and upper drainage regions of Paraíba do Sul. The large difference in the number of sites found for the 5S rDNA entered the same region (upper drainage of the basin) and the literature data could represent a population dynamics where an expansion of the 5S rDNA sites provides an extinct or non-sampled cytotype in this work.

Chromosomal Diversification Higher Than Molecular Variation in Astyanax aff. fasciatus (Teleostei, Characidae).

Comprising a large number of species, the genus Astyanax has been intensively studied by several approaches to elucidate its evolutionary relationships. Such studies have demonstrated that many nominal species are artificial clusters where distinct taxa are grouped under the same denomination. Astyanax aff. fasciatus stands out due to its high karyotypic diversity, since cytogenetic studies have reported three standard cytotypes (2n = 46, 48, and 50), as well as cases of sympatry between cytotypes, variant cytotypes, and B chromosomes. In this study, we attempted to evaluate the reliability of the chromosomal differences in relation to the analysis of the ATPase6/8 mitochondrial DNA (mtDNA) sequence, thereby providing subsidies to the evolutionary reconstruction of this group. Nine populations from four distinct hydrographic basins along Southeastern Brazil were analyzed. These are the first cytogenetic data collected for four of them. Fluorescent in situ hybridization with 5S rDNA probe evidenced the presence of a standard phenotype for the group and the existence of a new arrangement in the individuals from Ribeira de Iguape River. Besides the karyotypic variation, the genetic distance was low among the studied populations and some aspects of the evolutionary relationships among distinct cytotypes/populations could be ascertained by phylogeographic studies. The incipient molecular structuring of certain cytotypes in different hydrographic basins indicates the role of different evolutionary processes on the diversification of the group.

Cryptic diversity between two Imparfinis species (Siluriformes, Heptapteridae) by cytogenetic analysis and DNA barcoding.

Five Imparfinis mirini and one Imparfinis minutus populations were studied using basic cytogenetic and molecular techniques. Cytogenetic analysis showed that I. mirini individuals presented the same diploid number 2n=58 (FN=116). However, they presented two distinct karyomorphs: karyomorph A (36m+18sm+4st) for the Mogi-Guaçu and Paranapanema basin populations, and karyomorph B (42m+12sm+4st) for the Tietê basin populations. I. minutus populations from the Paraíba do Sul basin presented a karyotype identical to karyomorph A of I. mirini. C-banding also presented distinct patterns, with a greater amount of heterochromatin, most of which was pericentromeric and interstitial for karyomorph A I. mirini and I. minutus. There was a minor amount of heterochromatin in karyomorph B, most of which was terminal and interstitial. Simple and interstitial nucleoli organizer regions were located in the biggest metacentric pair of the complement in all populations with GC-rich nature, and this location was confirmed by the fluorescent in situ hybridization technique with 18S ribosomal DNA with 5S rDNA synteny. In molecular analysis by DNA barcoding, two other populations from the Tietê basin were added. The phylogram showed that the populations were more related to the intrabasin. Cytogenetic resemblance among specimens from distinct basins may be the result of either recent convergence or ancestral feature retention not followed by mutations in mitochondrial DNA.

Gene mapping of 18S and 5S rDNA genes in the karyotype of the three-spot gourami Trichogaster trichopterus (Perciformes, Osphronemidae).

Ornamental fish culture is important as an economic activity and for biodiversity conservation as well. The species of the genus Trichogaster (Perciformes, Osphronemidae), popularly known as three-spot gourami, are among the several commercial species raised around the world. In the present work, eight specimens of Thrichogaster trichopterus from aquarium trade facilities were analyzed. The karyotype was composed of 23 pairs of subtelo/acrocentric chromosomes. Fluorescent in situ hybridization allowed identifying the 18S ribosomal gene at telomeric region on long arms of the largest acrocentric pair. On the other hand, the 5S rRNA gene is located at a proximal region on a pair of medium-sized chromosomes. Such information is extremely useful in face of the risks of introduction and the development of ornamental fish trade, once many fish species can be identified only by genetic studies.

Astyanax hastatus Myers, 1928 (Teleostei, Characidae): A new species complex within the genus Astyanax?

Four populations of Astyanax hastatus Myers 1928 from the Guapimirim River basin (Rio de Janeiro State) were analyzed and three distinct cytotypes identified. These cytotypes presented 2n = 50 chromosomes, with 4M+8SM+10ST+28A (Cytotype A), 8M+10SM+14ST+18A (Cytotype B), 6M+8SM+4ST+32A (Cytotype C) and scanty heterochromatin, mainly located throughout pericentromeric regions of several chromosomal pairs. No homologies with the As-51 satellite DNA were observed in the three cytotypes, although all of them presented multiple 18S rDNA sites, as detected by both silver nitrate staining and FISH (fluorescent in situ hybridization). The application of the term "species complex" in Astyanax is discussed from a cytotaxonomic viewpoint.

Astyanax bockmanni Vari and Castro, 2007: an ambiguous karyotype in the Astyanax genus.

Despite the widespread distribution of Astyanax bockmanni in streams from Upper Paraná River system in central, southeastern, and southern Brazil, just recently, it has been identified as a distinct Astyanax species. Cytogenetic studies were performed in two populations of this species, revealing conservative features. A. bockmanni shows 2n = 50 chromosomes, a karyotypic formula composed of 10 M + 12SM + 12ST + 16A and multiple Ag-NORs. Eight positive signals in subtelocentric/acrocentric chromosomes were identified by fluorescent in situ hybridization (FISH) with 18S rDNA probes. After FISH with 5S rDNA probes, four sites were detected, comprising the interstitial region of a metacentric pair and the terminal region on long arms of another metracentric pair. Little amounts of constitutive heterochromatin were observed, mainly distributed at distal region in two chromosomal pairs. Additionally, heterochromatin was also located close to the centromeres in some chromosomes. No positive signals were detected in the chromosomes of A. bockmanni by FISH with the As-51 satellite DNA probe. The studied species combines a set of characteristics previously identified in two different Astyanax groups. The chromosomal evolution in the genus Astyanax is discussed.

Astyanax hastatus Myers, 1928 (Teleostei, Characidae): a new species complex within the genus Astyanax?

Four populations of Astyanax hastatus Myers 1928 from the Guapimirim River basin (Rio de Janeiro State) were analyzed and three distinct cytotypes identified. These cytotypes presented 2n = 50 chromosomes, with 4M+8SM+10ST+28A (Cytotype A), 8M+10SM+14ST+18A (Cytotype B), 6M+8SM+4ST+32A (Cytotype C) and scanty heterochromatin, mainly located throughout pericentromeric regions of several chromosomal pairs. No homologies with the As-51 satellite DNA were observed in the three cytotypes, although all of them presented multiple 18S rDNA sites, as detected by both silver nitrate staining and FISH (fluorescent in situ hybridization). The application of the term "species complex" in Astyanax is discussed from a cytotaxonomic viewpoint.

Molecular cytogenetics of blind mexican tetra and comments on the karyotypic characteristics of genus Astyanax (Teleostei, Characidae).

Astyanax mexicanus is popularly known as the blind Mexican tetra or blind cave tetra and has been extensively studied regarding various aspects of its biology and genetics. Despite the identification of linkage maps of genes related to quantitative trait loci by many recent studies, only its diploid number was known from a cytogenetical point of view. With the purpose of providing a base for comparative studies and for the elucidation of physical maps for the species, cytogenetical studies were performed in a group of 10 blind specimens from Mexico. All the individuals presented 2n = 50 chromosomes and a karyotypic formula composed of 8M + 18SM + 12ST + 12A. A few specimens presented one or two B microchromosomes of the acrocentric type. Although simple argyrophilic nucleolar organizer regions (Ag-NORs) were evidenced, fluorescence in situ hybridization (FISH) with an 18S rDNA probe evidenced eight sites, and six sites were observed with a 5S rDNA probe. Little constitutive heterochromatin was observed, mainly related with the Ag-NORs and located close to the centromeres, including those from the B microchromosomes. A few pericentromeric heterochromatin regions were mainly constituted by GC, including the one from the Ag-NOR. Very subtle markings were observed by FISH with an As-51 satellite DNA probe. The B microchromosome did not present ribosomal genes or satellite DNA. Chromosomal aspects of the genus Astyanax are discussed.

Satellite DNA sites in four species of the genus Astyanax (Teleostei, Characiformes)

Cytogenetic data about satellite DNA distribution in four Astyanax species (Characidae) from the Paraitinga river, Paraíba do Sul river basin, Brazil, are presented. In order to characterize the constitutive heterochromatin, C-banding, chromomycin A3 and DAPI fluorescence staining, as well as fluorescence in situ hybridization (FISH) with the satellite DNA As-51 probe were performed. A. scabripinnis and A. parahybae presented 2n = 50 and 2n = 48 chromosomes, respectively. The heterochromatin was located in the pericentromeric and terminal regions of many chromosomes, corresponding to GC-positive regions and to the As-51 satellite DNA in terminal regions. A. intermedius and A. giton, both with 2n = 50 chromosomes, showed little heterochromatin, mostly restricted to the terminal and pericentromeric regions of a few chromosomes. No GC-positive regions, neither any correspondence between the scarce heterochromatin of these species and the As-51 satellite DNA was observed. AT-positive blocks were not detected in any of the species studied. Based on these and other available data, the hypothesis that Astyanax represents a polyphyletic group is discussed.