Publications

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First author - 14 papers

Independent evolution of satellite DNA sequences in homologous sex chromosomes of Neotropical armored catfish (Harttia)

SASSI, F.M.C.; GARRIDO-RAMOS, M.A.; UTSUNOMIA, R.; DOS SANTOS, R.Z.; EZAZ, T.; DEON, G.A.; PORTO-FORESTI, F.; LIEHR, T.; CIOFFI, M.B.

Communications Biology, v. 8, 524, 2025

Abstract: The Neotropical armored catfish Harttia is a valuable model for studying sex chromosome evolution, featuring two independently evolved male-heterogametic systems. This study examined satellitomes—sets of satellite DNAs—from four Amazonian species: H. duriventris (X1X2Y), H. rondoni (XY), H. punctata (X1X2Y), and H. villasboas (X1X2Y). These species share homologous sex chromosomes, with their satellitomes showing a high number of homologous satellite DNAs (satDNAs), primarily located on centromeres or telomeres, and varying by species. Each species revealed a distinct satDNA profile, with independent amplification and homogenization events occurring, suggesting an important role of these repetitive sequences in sex chromosome differentiation in a short evolutionary time, especially in recently originated sex chromosomes. Whole chromosome painting and bioinformatics revealed that in Harttia species without heteromorphic sex chromosomes, a specific satDNA (HviSat08-4011) is amplified in the same linkage group associated with sex chromosomes, suggesting an ancestral system. Such sequence (HviSat08-4011) has partial homology with the ZP4 gene responsible for the formation of the egg envelope, in which its role is discussed. This study indicates that these homologous sex chromosomes have diverged rapidly, recently, and independently in their satDNA content, with transposable elements playing a minor role when compared their roles on autosomal chromosome evolution.

A state-of-art review of Loricariidae (Ostariophysi: Siluriformes) cytogenetics

SASSI, F.M.C.; CIOFFI, M.B.; MOREIRA-FILHO, O.

Neotropical Ichthyology, v. 22, p. 240050, 2024

Abstract: Loricariidae is a Neotropical fish family divided into six subfamilies and ranking in third among the most biodiverse fish groups. This study conducts an updated review of the cytogenetic investigations within the family, discussing the trends in chromosomal evolution and the main gaps and future directions for studies. Covering 125 publications that analyzed 234 species from all subfamilies except Lithogeninae, corresponding to about 21% of the valid species diversity, our study revealed samples from six different river basins in Argentina, Brazil, Ecuador, Paraguay, and Venezuela. There was a dearth of data for northeast Brazil, the Western Amazon, the Guianas Shield, and other Neotropical countries. In loricariids, there are seven different sex chromosome systems and a variety of diploid numbers (2n) ranging from 33 to 96 as a result of different chromosomal rearrangements such as fusions, fissions, translocations, and inversions. We recorded more simple nucleolar organizer regions (Ag-NOR) compared to multiple ones, and the fundamental number (FN) varied between 34 and 142. Populational studies have been conducted only in a few taxa, but a remarkable karyotype variation that includes B chromosomes is shown. Despite continuous efforts, cytogenetics still does not adequately capture the diversity of Loricariidae.

Homeology of sex chromosomes in Amazonian Harttia armored catfishes supports the X-fission hypothesis for the X1X2Y sex chromosome system origin

SASSI, F.M.C.; SEMBER, A.; DEON, G.A.; LIEHR, T.; PADUTSCH, N.; OYAKAWA, O.T.; VICARI, M.R.; BERTOLLO, L.A.C.; MOREIRA-FILHO, O.; CIOFFI, M.B.

Scientific Reports, v. 13, p. 15756, 2023

Abstract: The Neotropical monophyletic catfish genus Harttia represents an excellent model to study karyotype and sex chromosome evolution in teleosts. Its species split into three phylogenetic clades distributed along the Brazilian territory and they differ widely in karyotype traits, including the presence of standard or multiple sex chromosome systems in some members. Here, we investigate the chromosomal rearrangements and associated synteny blocks involved in the origin of a multiple X1X2Y sex chromosome system present in three out of six sampled Amazonian-clade species. Using 5S and 18S ribosomal DNA fluorescence in situ hybridization and whole chromosome painting with probes corresponding to X1 and X2 chromosomes of X1X2Y system from H. punctata, we confirm previous assumptions that X1X2Y sex chromosome systems of H. punctata, H. duriventris and H. villasboas represent the same linkage groups which also form the putative XY sex chromosomes of H. rondoni. The shared homeology between X1X2Y sex chromosomes suggests they might have originated once in the common ancestor of these closely related species. A joint arrangement of mapped H. punctata X1 and X2 sex chromosomes in early diverging species of different Harttia clades suggests that the X1X2Y sex chromosome system may have formed through an X chromosome fission rather than previously proposed Y-autosome fusion.

Turnover of multiple sex chromosomes in Harttia catfish (Siluriformes, Loricariidae): a glimpse from whole chromosome painting

SASSI, F.M.C.; DEON, G.A.; SEMBER, A.; LIEHR, T.; OYAKAWA, O.T.; MOREIRA-FILHO, O.; BERTOLLO, L.A.C.; VICARI, M.R.; CIOFFI, M.B. Frontiers in Genetics, v. 14, 1226222, 2023
Abstract: The remarkable fish biodiversity encompasses also great sex chromosome variability. Harttia catfish belong to Neotropical models for karyotype and sex chromosome research. Some species possess one of the three male-heterogametic sex chromosome systems, XY, X1X2Y or XY1Y2, while other members of the genus have yet uncharacterized modes of sex determination. Particularly the XY1Y2 multiple sex chromosome system shows a relatively low incidence among vertebrates, and it has not been yet thoroughly investigated. Previous research suggested two independent X-autosome fusions in Harttia which led to the emergence of XY1Y2 sex chromosome system in three of its species. In this study, we investigated evolutionary trajectories of synteny blocks involved in this XY1Y2 system by probing six Harttia species with whole chromosome painting (WCP) probes derived from the X (HCA-X) and the chromosome 9 (HCA-9) of H. carvalhoi. We found that both painting probes hybridize to two distinct chromosome pairs in Amazonian species, whereas the HCA-9 probe paints three chromosome pairs in H. guianensis, endemic to Guyanese drainages. These findings demonstrate distinct evolutionary fates of mapped synteny blocks and thereby elevated karyotype dynamics in Harttia among the three evolutionary clades.

Chromosomes of Asian cyprinid fishes: Novel insight into the chromosomal evolution of Labeoninae (Teleostei, Cyprinidae)

KHENSUWAN, S. and SASSI, F.M.C.; MORAES, R.L.R.; RAB P; LIEHR T; SUPIWONG W; SEETAPAN K; TANOMTONG A; TANTISUWICHWONG N; ARUNSANG S; BUASRIYOT P; TONGNUNUI S; CIOFFI MB. PLoS One, v. 19, e0292689, 2024
Abstract: The Labeoninae subfamily is a highly diversified but demonstrably monophyletic lineage of cyprinid fishes comprising five tribes and six incertae sedis genera. This widely distributed assemblage contains some 48 genera and around 480 recognized species distributed in freshwaters of Africa and Asia. In this study, the karyotypes and other chromosomal properties of five Labeoninae species found in Thailand Labeo chrysophekadion (Labeonini) and Epalzeorhynchos bicolor, Epalzeorhynchos munense, Henicorhynchus siamensis, Thynnichthys thynnoides (´Osteochilini´) were examined using conventional and molecular cytogenetic protocols. Our results confirmed a diploid chromosome number (2n) invariably 2n = 50, but the ratio of uni- and bi-armed chromosomes was highly variable among their karyotypes, indicating extensive structural chromosomal rearrangements. Karyotype of L. chrysophekadion contained 10m+6sm+20st+14a, 32m+10sm+8st for H. siamensis, 20m+12sm+10st+8a in E. bicolor, 20m+8sm+8st+14a in E. munense, and 18m+24sm+8st in T. thynnoides. Except for H. siamensis, which had four sites of 5S rDNA sites, other species under study had only one chromosome pair with those sites. In contrast, only one pair containing 18S rDNA sites were found in the karyotypes of three species, whereas two sites were found in that of E. bicolor. These cytogenetic patterns indicated that the cytogenomic divergence patterns of these labeonine species largely corresponded to the inferred phylogenetic tree. In spite of the 2n stability, diverse patterns of rDNA and microsatellite distribution as well as their various karyotype structures demonstrated significant evolutionary differentiation of Labeoninae genomes as exemplified in examined species. Labeoninae offers a traditional point of view on the evolutionary forces fostering biological diversity, and the recent findings add new pieces to comprehend the function of structural chromosomal rearrangements in adaption and speciation.

Chromosomes of Asian Cyprinid Fishes: Genomic Differences in Conserved Karyotypes of ´Poropuntiinae´ (Teleostei, Cyprinidae)

KHENSUWAN, S. and SASSI, F.M.C.; MORAES, R.L.R.; JANTARAT, S.; SEETAPAN, K.; PHINTONG, K.; THONGNETR, W.; KAEWSRI, S.; JUMRUSTHANASAN, S.; SUPIWONG, W.; RAB, PETR; TANOMTONG, A.; LIEHR, T.; CIOFFI, M.B.

Animals, v. 13, p. 1415, 2023

Abstract: The representatives of cyprinid lineage ‘Poropuntiinae’ with 16 recognized genera and around 100 species form a significant part of Southeast Asian ichthyofauna. Cytogenetics are valuable when studying fish evolution, especially the dynamics of repetitive DNAs, such as ribosomal DNAs (5S and 18S) and microsatellites, that can vary between species. Here, karyotypes of seven ‘poropuntiin’ species, namely Cosmochilus harmandi, Cyclocheilichthys apogon, Hypsibarbus malcomi, H. wetmorei, Mystacoleucus chilopterus, M. ectypus, and Puntioplties proctozysron occurring in Thailand were examined using conventional and molecular cytogenetic protocols. Variable numbers of uni- and bi-armed chromosomes indicated widespread chromosome rearrangements with a stable diploid chromosome number (2n) of 50. Examination with fluorescence in situ hybridization using major and minor ribosomal probes showed that Cosmochilus harmandi, Cyclocheilichthys apogon, and Puntioplites proctozystron all had one chromosomal pair with 5S rDNA sites. However, more than two sites were found in Hypsibarbus malcolmi, H. wetmorei, Mystacoleucus chilopterus, and M. ectypus. The number of chromosomes with 18S rDNA sites varied amongst their karyotypes from one to three; additionally, comparative genomic hybridization and microsatellite patterns varied among species. Our results reinforce the trend of chromosomal evolution in cyprinifom fishes, with major chromosomal rearrangements, while conserving their 2n.

Small body, large chromosomes: centric fusions shaped the karyotype of the Amazonian miniature fish Nannostomus anduzei (Characiformes, Lebiasinidae)

MORAES, R.L.R. and SASSI, F.M.C.; MARINHO, M.M.F.; RÁB, PETR ; PORTO, J.I.R. ; FELDBERG, E. ; CIOFFI, M.B. Genes, v. 14, p. 192, 2023
Abstract: Miniature refers to species with extraordinarily small adult body size when adult and can be found within all major metazoan groups. It is considered that miniature species have experienced severe alteration of numerous morphological traits during evolution. For a variety of reasons, including severe labor concerns during collecting, chromosomal acquisition, and taxonomic issues, miniature fishes are neglected and understudied. Since some available studies indicate possible relationship between diploid chromosome number (2n) and body size in fishes, we aimed to study one of the smallest Neotropical fish Nannostomus anduzei (Teleostei, Characiformes, Lebiasinidae), using both conventional (Giemsa staining, C-banding) and molecular cytogenetic methods (FISH mapping of rDNAs, microsatellites, and telomeric sequences). Our research revealed that N. anduzei possesses one of the lowest diploid chromosome numbers (2n = 22) among teleost fishes, and its karyotype is entirely composed of large metacentric chromosomes. All chromosomes, except for pair number 11, showed an 18S rDNA signal in the pericentromeric region. 5S rDNA signals were detected in the pericentromeric regions of chromosome pair number 1 and 6, displaying synteny to 18S rDNA signals. Interstitial telomeric sites (ITS) were identified in the centromeric region of pairs 6 and 8, indicating that centric fusions played a significant role in karyotype evolution of studied species. Our study provides further evidence supporting the trend of diploid chromosome number reduction along with miniaturization of adult body size in fishes.

Adding New Pieces to the Puzzle of Karyotype Evolution in Harttia (Siluriformes, Loricariidae): Investigation of Amazonian Species

SASSI, FMC; MOREIRA-FILHO, O; DEON GA; SEMBER, A; BERTOLLO, LAC; LIEHR, T; OLIVEIRA, VCS; VIANA, PF; FELDBERG, E; VICARI, MR; CIOFFI, MB. Biology, 10(9), 922 2021.
Abstract: A remarkable morphological diversity and karyotype variability can be observed in the Neotropical armored catfish genus Harttia. These fishes offer a useful model to explore both the evolution of karyotypes and sex chromosomes, since many species possess male-heterogametic sex chromosome systems and a high rate of karyotype repatterning. Based on the karyotype organization, the chromosomal distribution of several repetitive DNA classes, and the rough estimates of genomic divergences at the intraspecific and interspecific levels via Comparative Genomic Hybridization, we identified shared diploid chromosome numbers (2n = 54) but different karyotype compositions in H. dissidens (20m + 26sm + 8a) and Harttia sp. 3 (16m + 18sm + 14st + 6a), and different 2n in H. guianensis (2n = 58; 20m + 26sm + 2st + 10a). All species further displayed similar patterns of chromosomal distribution concerning constitutive heterochromatin, 18S ribosomal DNA (rDNA) sites, and most of the surveyed microsatellite motifs. Furthermore, differences in the distribution of 5S rDNA sites and a subset of microsatellite sequences were identified. Heteromorphic sex chromosomes were lacking in H. dissidens and H. guianensis at the scale of our analysis. However, one single chromosome pair in Harttia sp. 3 males presented a remarkable accumulation of male genome-derived probe after CGH, pointing to a tentative region of early sex chromosome differentiation. Thus, our data support already previously outlined evidence that Harttia is a vital model for the investigation of teleost karyotype and sex chromosome dynamics.

Can geometric morphometrics work for species clusterization on the armored catfish Hypostomus Lacépède, 1803 (Siluriformes, Loricariidae)?

SASSI, FMC; PEREZ, MF; SILVA, MA; KAVALCO, KF; PASA, R. Biodiversity Journal, v. 12, 1, 147-153 2021.
Abstract: Studies with geometric morphometrics are efficient to clusterize and delimitate fish species. The armored catfish family (Loricariidae) is the second with more species and the Hypostomus genus can be highlighted as the most representative. By that, we present a geometric morphometric analysis in seven Hypostomus species from Paranaíba river basin (Brazil), testing which view for this technique were able to clusterize more efficiently the species. We found that lateral, frontal and ventral views were capable to form species-clusters with few overlaps in the PCA analysis. With the dorsal view overlapping all species, except for Hypostomus nigromaculatus, we consider that this view cannot be used for the geometric morphometric analysis. Hypostomus presents a complex taxonomy, with several cryptic species, and geometric morphometrics can be used as a complementary tool on Hypostomus taxonomy. Therefore, further analyses were required to evaluate the contribution of each landmark in species clusterization.

High Genetic Diversity despite Conserved Karyotype Organization in the Giant Trahiras from Genus Hoplias (Characiformes, Erythrinidae)

SASSI, FMC; PEREZ, MF; OLIVEIRA, VCS; DEON, GA; SOUZA, FHS; FERREIRA, PHN; OLIVEIRA, EA; HATANAKA, T; LIEHR, T; BERTOLLO, LAC; CIOFFI, MB. Genes, v. 12, 252, 2021.
Abstract: In the fish genus Hoplias, two major general groups can be found, one of which is formed by the “common trahiras” (Hoplias malabaricus group) and the other by the “giant trahiras” (Hoplias lacerdae group, in addition to Hoplias aimara), which usually comprises specimens of larger body size. Previous investigations from the giant trahiras group recovered 2n = 50 meta/submetacentric chromosomes and no sex chromosome differentiation, indicating a probable conservative pattern for their karyotype organization. Here, we conducted comparative cytogenetic studies in six giant trahiras species, two of them for the first time. We employed standard and advanced molecular cytogenetics procedures, including comparative genomic hybridization (CGH), as well as genomic assessments of diversity levels and phylogenetic relationships among them. The results strongly suggest that the giant trahiras have a particular and differentiated evolutionary pathway inside the Hoplias genus. While these species share the same 2n and karyotypes, their congeneric species of the H. malabaricus group show a notable chromosomal diversity in number, morphology, and sex chromosome systems. However, at the same time, significant changes were characterized at their inner chromosomal level, as well as in their genetic diversity, highlighting their current relationships resulting from different evolutionary histories.

An insight into the chromosomal evolution of Lebiasinidae (Teleostei, Characiformes).

SASSI, FMC; HATANAKA, T; MORAES, RLR; TOMA, G A; OLIVEIRA, EA; LIEHR, T; RAB, P; BERTOLLO, LAC; VIANA, PF; FELDBERG, E; TURSELLINO, MN; MARINHO, MMF; SOUZA, JFSE; CIOFFI, MB. Genes, v. 11, p. 1-14, 2020.
Abstract: Lebiasinidae fishes have been historically neglected by cytogenetical studies. Here we present a genomic comparison in eleven Lebiasinidae species, in addition to a review of the ribosomal DNA sequences distribution in this family. With that, we develop ten sets of experiments in order to hybridize the genomic DNA of representative species from the genus Copeina, Copella, Nannostomus, and Pyrrhulina in metaphase plates of Lebiasina melanoguttata. Two major pathways on the chromosomal evolution of these species can be recognized: (i) conservation of 2n = 36 bi-armed chromosomes in Lebiasininae, as a basal condition, and (ii) high numeric and structural chromosomal rearrangements in Pyrrhulininae, with a notable tendency towards acrocentrization. The ribosomal DNA (rDNA) distribution also revealed a marked differentiation during the chromosomal evolution of Lebiasinidae, since both single and multiple sites, in addition to a wide range of chromosomal locations can be found. With some few exceptions, the terminal position of 18S rDNA appears as a common feature in Lebiasinidae-analyzed species. Altogether with Ctenoluciidae, this pattern can be considered a symplesiomorphism for both families. In addition to the specific repetitive DNA content that characterizes the genome of each particular species, Lebiasina also keeps inter-specific repetitive sequences, thus reinforcing its proposed basal condition in Lebiasinidae.

Multiple Sex Chromosomes and Evolutionary Relationships in Amazonian Catfishes: The Outstanding Model of the Genus Harttia (Siluriformes: Loricariidae)

SASSI, FMC; DEON, GA; MOREIRA-FILHO, O; VICARI, MR; BERTOLLO, LAC; LIEHR, T; OLIVEIRA, EA; CIOFFI, MB. Genes, v. 11, 1179, 2020.
Abstract: The armored Harttia catfishes present great species diversity and remarkable cytogenetic variation, including different sex chromosome systems. Here we analyzed three new species, H. duriventris, H. villasboas and H. rondoni, using both conventional and molecular cytogenetic techniques (Giemsa-staining and C-banding), including the mapping of repetitive DNAs using fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) experiments. Both H. duriventris and H. villasboas have 2n = ♀56/♂55 chromosomes, and an X1X1X2X2 /X1X2Y sex chromosome system, while a proto or neo-XY system is proposed for H. rondoni (2n = 54♀♂). Single motifs of 5S and 18S rDNA occur in all three species, with the latter being also mapped in the sex chromosomes. The results confirm the general evolutionary trend that has been noticed for the genus: an extensive variation on their chromosome number, single sites of rDNA sequences and the occurrence of multiple sex chromosomes. Comparative genomic analyses with another congeneric species, H. punctata, reveal that the X1X2Y sex chromosomes of these species share the genomic contents, indicating a probable common origin. The remarkable karyotypic variation, including sex chromosomes systems, makes Harttia a suitable model for evolutionary studies focusing on karyotype differentiation and sex chromosome evolution among lower vertebrates.

Comparative chromosomal mapping of microsatellite repeats reveals divergent patterns of accumulation in 12 Siluridae (Teleostei: Siluriformes) species

DITCHAROEN, S and SASSI, FMC; BERTOLLO, LAC; MOLINA, WF; LIEHR, T; SAENJUNDAENG, P; TANOMTONG, A; SUPIWONG, W; SUWANNAPOOM, C; CIOFFI, MB. Genetics and Molecular Biology, v. 43, e20200091, 2020.
Abstract: The freshwater family Siluridae occurs in Eurasia and is especially speciose in South and Southeast Asia, representing an important aquaculture and fishery targets. However, despite the restricted cytogenetic data, a high diploid number variation (from 2n=40 to 92) characterizes this fish group. Considering the large genomic divergence among its species, silurid genomes have experienced an enormous diversification throughout their evolutionary history. Here, we aim to investigate the chromosomal distribution of several microsatellite repeats in 12 Siluridae species and infer about their possible roles in the karyotype evolution that occurred in this group. Our results indicate divergent patterns of microsatellite distribution and accumulation among the analyzed species. Indeed, they are especially present in significant chromosome locations, such as the centromeric and telomeric regions, precisely the ones associated with several kinds of chromosomal rearrangements. Our data provide pieces of evidence that repetitive DNAs played a direct role in fostering the chromosomal differentiation and biodiversity in this fish family.

Chromosomal Evolution and Evolutionary Relationships of Lebiasina Species (Characiformes, Lebiasinidae)

SASSI, FMC; OLIVEIRA, EA; BERTOLLO, LAC; TURSELLINO, MN; HATANAKA, T; MARINHO, MMF; MOREIRA-FILHO, O; AROUTIOUNIAN, R; LIEHR, T; AL-RIKABI, ABH; CIOFFI, MB. International Journal of Molecular Sciences, v. 20, p. 2944, 2019.
Abstract: We present the first cytogenetic data for Lebiasina bimaculata and L. melanoguttata with the aim of (1) investigating evolutionary events within Lebiasina and their relationships with other Lebiasinidae genera and (2) checking the evolutionary relationships between Lebiasinidae and Ctenoluciidae. Both species have a diploid number 2n = 36 with similar karyotypes and microsatellite distribution patterns but present contrasting C-positive heterochromatin and CMA3+ banding patterns. The remarkable interstitial series of C-positive heterochromatin occurring in L. melanoguttata is absent in L. bimaculata. Accordingly, L. bimaculata shows the ribosomal DNA sites as the only GC-rich (CMA3+) regions, while L. melanoguttata shows evidence of a clear intercalated CMA3+ banding pattern. In addition, the multiple 5S and 18S rDNA sites in L. melanogutatta contrast with single sites present in L. bimaculata. Comparative genomic hybridization (CGH) experiments also revealed a high level of genomic differentiation between both species. A polymorphic state of a conspicuous C-positive, CMA3+, and (CGG)n band was found only to occur in L. bimaculata females, and its possible relationship with a nascent sex chromosome system is discussed. Whole chromosome painting (WCP) and CGH experiments indicate that the Lebiasina species examined and Boulengerella maculata share similar chromosomal sequences, thus supporting the relatedness between them and the evolutionary relationships between the Lebiasinidae and Ctenoluciidae families.

Coauthor - 26 papers

Repeatome Dynamics and Sex Chromosome Differentiation in the XY and XY1Y2 Systems of the Fish Hoplias malabaricus (Teleostei; Characiformes)

DE OLIVEIRA, M.P.B.; DEON, G.A.; SASSI, F.M.C.; SOUZA, F.H.S.; GOES, C.A.G.; UTSUNOMIA, R.; PORTO-FORESTI, F.; VIDAL, J.A.D.; SILVA, A.B.; EZAZ, T.; LIEHR, T.; CIOFFI, M.B. International Journal of Molecular Sciences, v. 26: 6039, 2025
Abstract: The wolf fish Hoplias malabaricus is a Neotropical species characterized by remarkable karyotypic diversity, including seven karyomorphs (KarA-G) with distinct sex chromosome systems. This study investigated the homologous XY (KarF) and XY1Y2 (KarG) sex chromosome systems present in this species by integrating cytogenetics and genomics to examine sex chromosomes’ composition through characterization of repeatome (satellite DNA and transposable elements) and sex-linked markers. Our analysis indicated that both karyomorphs are little differentiated in their sex chromosomes content revealed by satDNA mapping and putative sex-linked markers. Both repeatomes were mostly composed of transposable elements, but neither intra- (male versus female) nor interspecific (KarF x KarG) variations were found. In both systems, we demonstrated the occurrence of sex-specific sequences probably located on the non-recombining region of the Y chromosome supported by the accumulation of sex-specific haplotypes of HmfSat10-28/HmgSat31-28. This investigation offered valuable insights by highlighting the composition of homologous XY and XY1Y2 multiple sex chromosomes. Although homologous, the large Y chromosome in KarF corresponds to two separate linkage groups (Y1 and Y2) in KarG implying a specific meiotic arrangement involving the X chromosome in a meiotic trivalent chain. This scenario likely influenced recombination rates and, as a result, the genomic composition of these chromosomes.

Comparative satellite DNA mapping in species of the genus Prochilodus (Teleostei, Characiformes) and its evolutionary implications

SANTOS, R.Z.; GOES, C.A.G.; STORNIOLI, J.H.F.; SASSI, F.M.C.; MORAES, R.L.R.; DERGAM, J.A.; PORTO-FORESTI, F.; CIOFFI, M.B.; UTSUNOMIA, R. Genome v. 68: e0085, 2024
Abstract: Satellite DNA (satDNA) sequences are dynamic components of the eukaryotic genome that can play significant roles in species diversification. The Prochilodontidae family, which includes 21 Neotropical fish species, is characterized by a conserved karyotype of 2n = 54 biarmed chromosomes, with variation in some species and populations regarding the presence or absence of B chromosomes. This study aimed to investigate whether the chromosomal distribution of specific satDNA sequences is conserved among three Prochilodus species (Prochilodus lineatus, Prochilodus costatus, and Prochilodus argenteus) regarding organization and number of loci, and to compare their genomes using comparative genomic hybridization (CGH). Our results demonstrated that most satDNA sequences share a similar distribution pattern across the three species, and CGH analysis corroborated that their karyotypes are very similar in terms of repetitive DNA distribution. We also identified a potential CENP-B box sequence within PliSat01, a satDNA located in the pericentromeric region of all analyzed species. In contrast, PliSat04 and PliSat14 displayed differential locations and variations in the number of loci per genome, underscoring the dynamic nature of repetitive sequences even in species with otherwise highly conserved genomes. These findings represent the first evidence of karyotype diversification in Prochilodus, highlighting the evolutionary dynamism of satDNA sequences.

Integrative morphological, cytogenetic and molecular characterization of the Andean climbing catfish Astroblepus mindoensis (Regan, 1916) (Siluriformes:Astroblepidae)

NIRCHIO, M. ; OLIVEIRA, C. ; CIOFFI, M. B. ; SASSI, F. M. C. ; RIZZI, F. P. ; BENAVIDES, S. W. N. ; BERRONES, A. J. C. ; ROMERO, J. F. R. ; DEON, G. A. ; KURANAKA, M. ; VALDIVIEZO-RIVERA, J. S. ; OLMEDO, J. C. C. ; ROSSI, A. R.Journal of Fish Biology: e15924, 2024
Abstract: Astroblepus species, commonly known as Andean climbing catfish, exhibit a unique challenge in species delimitation, leading to ongoing taxonomic debates. Here we report data on Astroblepus mindoensis, a vulnerable species endemic to Ecuador, obtained by an integrative approach that includes cytogenetic analysis, molecular identification of the specimens, and recording of morphological and morphometric characters useful for species diagnosis. Thus, this study aimed to associate the karyotype data of the specimens analyzed with morphological and molecular characters, improving and expanding the existing taxonomic information, thus contributing to the systematics of the species. Our morphology results, unlike Regan's original description, which is brief and ambiguous, provide a more detailed morphometric and meristic description. Molecular phylogenetic reconstruction and genetic distance based on a fragment of the cytochrome c oxidase subunit I (COI) showed that our samples constitute a well-supported and monophyletic clade within the A. grixalvii species complex. The cytogenetic analysis identified distinct chromosomal markers, including a single cluster of major ribosomal genes (on chromosome pair 3) and of minor ribosomal genes (on chromosome pair 12) with their localization differing from those reported in other Astroblepus species analyzed. Additionally, the presence of a heteromorphic chromosome pair in males suggests the presence of an XX/XY sex-determination system that has not been identified in other congeneric species. Further investigation is necessary to determine if these chromosomes are associated with the accumulation of repeated sequences, as typically occurs with sex chromosomes, and to assess their presence in other species of the genus.

Karyotypic stasis and its implications for extensive hybridization events in corallivores species of butterflyfishes (Chaetodontidae)

MOLINA, WAGNER FRANCO ; KHENSUWAN, S. ; MORAES, R. L. R. ; SASSI, F. M. C. ; COSTA, G. W. W. F. ; MIGUEL, D. Z. ; SUPIWONG, W. ; JANTARAT, S. ; PHINTONG, K. ; SEETAPAN, K. ; DITCHAROEN, SUKHONTHIP ; TANOMTONG, ALONGKLOD ; LIEHR, T. ; CIOFFI, M. B. Heliyon v. 10(6): e27435, 2024
Abstract: The butterflyfishes (Chaetodontidae), emblematic inhabitants of coral reef environments, encompass the majority of known coralivorous species and show one of the highest hybridization rates known among vertebrates, making them an important evolutionary model. The vast knowledge about their life history and phylogenetic relationships contrasts with scarce information on their karyotype evolution. Aiming to expand the cytogenetic data of butterflyfishes and evaluate their karyotype evolution in association with evolutionary aspects, we conducted an extensive cytogenetic analysis in 20 species (Heniochus pleurotaenia and 19 Chaetodon spp.) from the Atlantic and Indo-Pacific regions, comparing the karyotype macrostructure and the arrangement of the 18S and 5S rDNA repetitive DNA classes in their chromosomes. The results demonstrate that butterflyfishes underwent a period of karyotypic stasis, as evidenced by their homoploid and structurally identical basal karyotype, which has 2n = 48 acrocentric chromosomes and is shared by 90% of species. Only C. trifascialis (2n = 48; FN = 50) and C. andamanensis (2n = 48; FN = 52) stood out because they both had karyotypes that diverged due to pericentric inversions. The microstructural arrays of 18S rDNA and 5S rDNA sequences were primarily comprised by single and independent loci on homologous chromosomes, indicating that there was little reshuffling among sets of orthologue chromosomes of species. Geographical comparisons revealed similar karyotypes between individuals of C. striatus from the Greater Caribbean and those of the coast of Brazil, corroborating previous data of gene flow through Amazon/Orinoco plume. The conservative chromosomal patterns in the butterflyfishes, likely overcome the limitations related to segregation and pairing of heterospecific complements and reinforce their contribution to the high degree of hybrid viability and introgression in Chaetodon species.

Comparative Cytogenetics in Tyrannidae (Aves, Passeriformes): High Genetic Diversity despite Conserved Karyotype Organization

SARAIVA, D. M. ; SOUZA, M. S. ; TURA, V ; ROSSO, V. O. ; ZEFA, E. ; GARNERO, A. V. ; GUNSKI, R. J. ; SASSI, F. M. C. ; CIOFFI, M. B. ; KRETSCHMER, R.Cytogenetic and Genome Research 164 (1): 43–51, 2024
Abstract: Introduction: Passeriformes has the greatest species diversity among Neoaves, and the Tyrannidae is the richest in this order with about 600 valid species. The diploid number of this family remains constant, ranging from 2n = 76 to 84, but the chromosomal morphology varies, indicating the occurrence of different chromosomal rearrangements. Cytogenetic studies of the Tyrannidae remain limited, with approximately 20 species having been karyotyped thus far. This study aimed to describe the karyotypes of two species from this family, Myiopagis viridicata and Sirystes sibilator. Methods: Skin biopsies were taken from each individual to establish fibroblast cell cultures and to obtain chromosomal preparations using the standard methodology. The chromosomal distribution of constitutive heterochromatin was investigated by C-banding, while the location of simple repetitive sequences (SSRs), 18S rDNA, and telomeric sequences was found through fluorescence in situ hybridization. Results: The karyotypes of both species are composed of 2n = 80. The 18S rDNA probes hybridized into two pairs of microchromosomes in M. viridicata, but only a single pair in S. sibilator. Only the telomeric portions of each chromosome in both species were hybridized by the telomere sequence probes. Most of the SSRs were found accumulated in the centromeric and telomeric regions of several macro- and microchromosomes in both species, which likely correspond to the heterochromatin-rich regions. Conclusion: Although both species analyzed showed a conserved karyotype organization (2n = 80), our study revealed significant differences in their chromosomal architecture, rDNA distribution, and SSR accumulation. These findings were discussed in the context of the evolution of Tyrannidae karyotypes.

The role of satellite DNAs in the chromosomal rearrangements and the evolution of the rare XY1Y2 sex system in Harttia (Siluriformes: Loricariidae)

DEON, G. A. ; SANTOS, R. Z. ; SASSI, F. M. C. ; MOREIRA-FILHO, O. ; VICARI, M. R. ; PORTO-FORESTI, F. ; UTSUNOMIA, R. ; CIOFFI, M. B. Journal of Heredity v. 115(5): 541–551, 2024
Abstract: The underlying processes behind the formation, evolution, and long-term maintenance of multiple sex chromosomes have been largely neglected. Among vertebrates, fishes represent the group with the highest diversity of multiple sex chromosome systems and, with six instances, the Neotropical fish genus Harttia stands out by presenting the most remarkable diversity. However, although the origin mechanism of their sex chromosome systems is well discussed, little is known about the importance of some repetitive DNA classes in the differentiation of multiple systems. In this work, by employing a combination of cytogenetic and genomic procedures, we evaluated the satellite DNA composition of H. carvalhoi with a focus on their role in the evolution, structure, and differentiation process of the rare XY1Y2 multiple-sex chromosome system. The genome of H. carvalhoi contains a total of 28 satellite DNA families, with the A + T content ranging between 38.1% and 68.1% and the predominant presence of long satellites. The in situ hybridization experiments detected 15 satellite DNAs with positive hybridization signals mainly on centromeric and pericentromeric regions of almost all chromosomes or clustered on a few pairs. Five of them presented clusters on X, Y1, and/or Y2 sex chromosomes which were therefore selected for comparative hybridization in the other three congeneric species. We found several conserved satellites accumulated on sex chromosomes and also in regions that were involved in chromosomal rearrangements. Our results provide a new contribution of satellitome studies in multiple sex chromosome systems in fishes and represent the first satellitome study for a Siluriformes species.

Chromosomal mapping of repetitive DNA and retroelement sequences and its implications for the chromosomal evolution process in Ctenoluciidae (Characiformes)

SOUZA, J. F. S. E. ; GUIMARAES, E. M. C. ; FIGLIUOLO, V. S. P. ; SOARES, S. C. ; CIOFFI, M. B. ; SASSI, F. M. C. ; FELDBERG, E. BMC Ecology and Evolution v. 24, 72, 2024
Abstract: Ctenoluciidae is a Neotropical freshwater fish family composed of two genera, Ctenolucius (C. beani and C. hujeta) and Boulengerella (B. cuvieri, B. lateristriga, B. lucius, B. maculata, and B. xyrekes), which present diploid number conservation of 36 chromosomes and a strong association of telomeric sequences with ribosomal DNAs. In the present study, we performed chromosomal mapping of microsatellites and transposable elements (TEs) in Boulengerella species and Ctenolucius hujeta. We aim to understand how those sequences are distributed in these organisms’ genomes and their influence on the chromosomal evolution of the group. Our results indicate that repetitive sequences may had an active role in the karyotypic diversification of this family, especially in the formation of chromosomal hotspots that are traceable in the diversification processes of Ctenoluciidae karyotypes. We demonstrate that (GATA)n sequences also accumulate in the secondary constriction formed by the 18 S rDNA site, which shows consistent size heteromorphism between males and females in all Boulengerella species, suggesting an initial process of sex chromosome differentiation.

Comparative karyotypic study of fifteen cyprinids (Cyprinidae, Cyprininae) species. An insight into the chromosomal evolution of the tribe Systomini

BUASRIYOT P; SASSI, F.M.C.; DONBUNDIT N; SUPIWONG W; KAEWSRI S; JUMRUSTHANASAN S; SEETAPAN K; PINTHONG K; TANOMTONG A; YANUKUN K; MUANGLEN N; LIEHR T; CIOFFI, M.B. PLoS One v. 19(2), 19(2): e0292689, 2024
Abstract: The family Cyprinidae is the largest freshwater fish group with 377 genera and over 3,000 described species. However, this group of fish has very limited cytogenetics and advanced molecular cytogenetics information. Therefore, in this study the karyotypes and other chromosomal characteristics of 15 species in the tribe Systomini (Cyprininae) were examined using Ag-NOR staining along with fluorescence in situ hybridization (5S and 18S rDNA). All species share a similar karyotype (2n = 50; NF = 88–100) in both sexes and no differentiated sex chromosome was observed. Chromosomes bearing NOR sites ranged from one to four pairs among the species, mostly mapped adjacent to telomeres in the short arms of distinct pairs in all analyzed species. This difference indicates an extensive rearrangement of chromosomes including genomic differences. The use of the 5S and 18S rDNA probe confirmed the Ag-NOR sites interstitially located in the telomeric regions of distinct chromosomes, characterizing an interspecies variation of these sites. In most of its analyzed species, the signals of 18S rDNA probe corresponded to the Ag-NOR regions, except in Barbonymus altus, B. gonionotus, B. schwanenfeldii and Puntius brevis having these signals on the same as Ag-NOR regions and other sites.

Chromosomal Rearrangements and Satellite DNAs: Extensive Chromosome Reshuffling and the Evolution of Neo-Sex Chromosomes in the Genus Pyrrhulina (Teleostei; Characiformes)

MORAES, R.L.R.; SASSI, F.M.C.; VIDAL, J.A.D.; GOES, C.A.G.; DOS SANTOS, R.Z.; STORNIOLI, J.H.F.; PORTO-FORESTI, F.; LIEHR, T.; UTSUNOMIA, R.; CIOFFI, M.B. International Journal of Molecular Sciences, v. 24, p. 13654, 2023
Abstract: Chromosomal rearrangements play a significant role in the evolution of fish genomes, being important forces in the rise of multiple sex chromosomes and in speciation events. Repetitive DNAs constitute a major component of the genome and are frequently found in heterochromatic regions, where satellite DNA sequences (satDNAs) usually represent their main components. In this work, we investigated the association of satDNAs with chromosome-shuffling events, as well as their potential relevance in both sex and karyotype evolution, using the well-known Pyrrhulina fish model. Pyrrhulina species have a conserved karyotype dominated by acrocentric chromosomes present in all examined species up to date. However, two species, namely P. marilynae and P. semifasciata, stand out for exhibiting unique traits that distinguish them from others in this group. The first shows a reduced diploid number (with 2n = 32), while the latter has a well-differentiated multiple X1X2Y sex chromosome system. In addition to isolating and characterizing the full collection of satDNAs (satellitomes) of both species, we also in situ mapped these sequences in the chromosomes of both species. Moreover, the satDNAs that displayed signals on the sex chromosomes of P. semifasciata were also mapped in some phylogenetically related species to estimate their potential accumulation on proto-sex chromosomes. Thus, a large collection of satDNAs for both species, with several classes being shared between them, was characterized for the first time. In addition, the possible involvement of these satellites in the karyotype evolution of P. marilynae and P. semifasciata, especially sex-chromosome formation and karyotype reduction in P. marilynae, could be shown.

Zebrafish (Danio rerio) Gynogenetic Production by Heat Shock: Comparison Between Mitotic and Meiotic Treatment

SETTI, P.G.; BORRA, R.C.; SASSI, F.M.C.; CIOFFI, M.B.; FUKUSHIMA, H.C.S. Zebrafish, ahead of print, 2023
Abstract: Inbred species are useful resources for a variety of biomedical research applications. To create isogenic zebrafish, it is feasible to stop meiosis II (repeatedly) or mitosis (two times) in a haploid embryo by applying pressure or by delivering a heat shock, respectively. In this study, to improve the repeatability, we suggest a less complicated approach based on sperm ultraviolet-C (UV-C) exposure for a shorter period followed by heat shock at various temperatures, eliminating the use of pressure in meiotic therapy since heat shock is more accessible to laboratories. In this study, the survivability rates of meiotic (Mei) and mitotic (Mit) gynogenesis offspring produced by various combinations of irradiation (28.5, 105, and 210 mJ/cm2) and temperature (Mei: 40.40°C, 40.60°C, or 40.90°C; Mt: 41.40°C, 41.90°C, or 42.45°C) were compared with diploid (C) and haploid (H) controls. Our findings demonstrated that 40.60°C and 41.90°C were the most suitable temperatures to produce meiotic and mitotic gynogenesis, respectively, whereas 28.5 mJ/cm2 was more successful in ensuring haploid embryos. As a result, we deduced that meiotic gynogenesis produces more viable offspring than the mitotic approach and requires a lower temperature to maintain the second polar body.

Chromosomal evolution of Suboscines: Karyotype diversity and evolutionary trends in Ovenbirds (Passeriformes, Furnariidae)

TURA, V; KRETSCHMER, R.; SASSI, F.M.C.; MORAES, R.L.R.; BARCELLOS, S.A.; ROSSO, V.O.; SOUZA, M.S.; CIOFFI, M.B.; GUNSKI, R.J.; GARNERO, A.V. Cytogenetic and Genome Research, v. 1, p. 1, 2023
Abstract: Furnariidae (Ovenbirds) is one of the most diversified families in the Passeriformes order and Suboscines suborder. Despite their great diversity of species, cytogenetic research is still in its early stages, restricting our knowledge of their karyotype evolution. We combined traditional and molecular cytogenetic analyses in three representative species, Synallaxis frontalis, Syndactyla rufosuperciliata, and Cranioleuca obsoleta, to examine the chromosomal structure and evolution of Ovenbirds. Our findings reveal that all the species studied had the same diploid number (2n= 82). Differences in chromosomal morphology of some macrochromosomes indicate the presence of intrachromosomal rearrangements. Although the three species only had the 18S rDNA on one microchromosome pair, chromosomal mapping of six simple short repeats revealed a varied pattern of chromosome distribution among them, suggesting that each species underwent different repetitive DNA accumulation upon their divergence. The interspecific comparative genomic hybridization (CGH) experiment revealed that the Furnariidae species investigated carry centromeric regions enriched in similar repetitive sequences, bolstering the Furnariidae family's karyotype conservation. Nonetheless, the outgroup species Turdus rufiventris (Turdidae) demonstrated an advanced stage of sequence divergence with hybridization signals that were almost entirely limited to a few microchromosomes. Overall, the findings imply that Furnariidae species have a high degree of chromosomal conservation, and also we could observe a differentiation of repetitive sequences in both Passeriformes suborders (Suboscines and Oscines).

Evolutionary tracks of chromosomal diversification in Trichopsis (Anabantiformes, Osphronemidae) fishes: New insights from a molecular cytogenetic perspective

MINGKWAN, B.; SASSI, F.M.C.; MUANGLENM, N.; PINMONGKHONKUL, S.; PINTHONG, K.; TONGNUNUI, S.; YEESIN, P.; TANOMTONG, A.; LIEHR, T.; CIOFFI, M.B.; SUPIWONG, W. Biodiversitas Journal of Biological Diversity, v. 24, p. 1551-1559, 2023
Abstract: This study investigated the chromosomes of three Trichopsis species (namely Trichopsis pumila, Trichopsis vittata, and Trichopsis schalleri) using conventional (Giemsa stain, Ag-NOR) and molecular cytogenetic techniques. Fluorescence in situ hybridization (FISH) with repetitive DNAs, including 5S and 18S rDNAs, and microsatellites as probes were also performed. Our results indicated a conserved diploid number of 46 (2n) for all analyzed species of both sexes, although varying in their karyotype structure. Furthermore, T. schalleri and T. vittata karyotypes were presented with only acrocentric chromosomes (46a), while T. pumila had a submetacentric pair in addition to acrocentric ones (2sm+44a). Positive Ag-NOR sites were differentially found in each species adjacent to the acrocentric chromosome's centromeric and/or interstitial sub-centromeric regions. Both 5S and 18S rDNAs vary between the analyzed species, but all were localized in multiple sites. However, the distribution pattern of microsatellites differed in each species, mostly scattering at pericentromeric and telomeric regions. These findings show that Trichopsis species, while having a conserved diploid number, differ significantly in the distribution of repetitive sequences in their karyotypes, particularly for rDNAs. These species-specific patterns can be useful for characterizing and identifying further different species and examining the evolutionary mechanisms that drove the evolution of these fishes' karyotypes.

Giant Sex Chromosomes in Omophoita Species (Coleoptera, Chrysomelidae): Structural and Evolutionary Relationships Revealed by Zoo-FISH and Comparative Genomic Hybridization (CGH)

VIDAL, J.A.D.; SASSI, F.M.C.; MORAES, R.L.R.; ARTONI, R.F.; LIEHR, T.; CIOFFI, M.B.; ALMEIDA, M.C.Insects, v. 14, p. 440, 2023
Abstract: The beetles of the subtribe Oedionychina (Chrysomelidae, Alticinae) are the only ones that have the atypical giant and achiasmatic sex chromosomes, which are substantially larger than the autosomes. Previous cytogenetic analyses suggest a large accumulation of repetitive DNA in the sex chromosomes. In this study, we examined the similarity of X and Y chromosomes in four Omophoita species and compared genomic differentiation to better understand the evolutionary process and the giant sex chromosomes origin. Intraspecific genomic comparation using male and female genomes of O. octoguttata and interespecific analyses using genomic DNA of O. octoguttata, O. sexnotata, O. magniguttis, and O. personata were performed. In addition, whole chromosome painting (WCP) experiments were performed with X and Y chromosome probes of O. octogutatta. CGH analysis revealed great genomic similarity between the sexes and a sex-specific region on the Y chromosome, and interspecific analysis revealed a genomic divergence between species. In contrast, WCP results revealed that the sex chromosomes of O. octoguttata have high intra- and interspecific similarity with the studied species. Our data support a common origin under the canonical evolution of the sex chromosomes in this group, as they have high genomic similarity between them.

Occurrence of sex chromosomes in fish of the genus Ancistrus with a new description of multiple sex chromosomes in the Ecuadorian endemic Ancistrus clementinae (Loricariidae)

NIRCHIO, M.; OLIVEIRA, C.; CIOFFI, M.B.; SASSI, F.M.C.; VALDIVIEZO, J.; PAIM, F.G.; BATISTA, L.S.; ROSSI, A.R. Genes, v. 14, p. 306, 2023
Abstract: Ancistrus Kner, 1854, is the most diverse genus among the Ancistrini (Loricariidae) with 70 valid species showing a wide geographic distribution and great taxonomic and systematic complexity. To date, about 40 Ancistrus taxa have been karyotyped, all from Brazil and Argentina, but the statistic is uncertain because 30 of these reports deal with samples that have not yet been identified at the species level. This study provides the first cytogenetic description of the bristlenose catfish, Ancistrus clementinae Rendahl, 1937, a species endemic to Ecuador, aiming to verify whether a sex chromosome system is identifiable in the species and, if so, which, and if its differentiation is associated with the presence of repetitive sequences reported for other species of the family. We associated the karyotype analysis with the COI molecular identification of the specimens. Karyotype analysis suggested the presence of a ♂ZZ/♀ZW1W2 sex chromosome system, never detected before in Ancistrus, with both W1W2 chromosomes enriched with heterochromatic blocks and 18S rDNA, in addition to GC-rich repeats (W2). No differences were observed between males and females in the distribution of 5S rDNA or telomeric repeats. Cytogenetic data here obtained confirm the huge karyotype diversity of Ancistrus, both in chromosome number and sex-determination systems.

Integrating Genomic and Chromosomal Data: A Cytogenetic Study of Transancistrus santarosensis (Loricariidae: Hypostominae) with Characterization of a ZZ/ZW Sex Chromosome System

TURSELLINO, M.N.; CIOFFI, M.B.; SASSI, F.M.C.; DEON, G.A.; OLIVEIRA, C.; KURANAKA, M.; VALDIVIEZO-RIVERA, J.; GONZALEZ, V.H.; ROSSI, A.R. Genes, v. 14, p. 1662, 2023
Abstract: The plecos (Loricariidae) fish represent a great model for cytogenetic investigations due to their variety of karyotypes, including diploid and polyploid genomes, and different types of sex chromosomes. In this study we investigate Transancistrus santarosensis a rare loricariid endemic to Ecuador, integrating cytogenetic methods with specimens’ molecular identification by mtDNA, to describe the the species karyotype. We aim to verify whether sex chromosomes are cytologically identifiable and if they are associated with the accumulation of repetitive sequences present in other species of the family. The analysis of the karyotype (2n = 54 chromosomes) excludes recent centric fusion and pericentromeric inversion and suggests the presence of a ZZ/ZW sex chromosome system at an early stage of differentiation: the W chromosome is degenerated but is not characterized by the presence of differential sex-specific repetitive DNAs. Data indicate that although T. santarosensis has retained the ancestral diploid number of Loricariidae, it accumulated heterochromatin and shows non-syntenic ribosomal genes localization, chromosomal traits considered apomorphic in the family.

Integrating Cytogenetics and Population Genomics: Allopatry and Neo-Sex Chromosomes May Have Shaped the Genetic Divergence in the Erythrinus erythrinus Species Complex (Teleostei, Characiformes)

SOUZA, FHS; SASSI, FMC; FERREIRA, PHN; BERTOLLO, LAC; EZAZ, T; LIEHR, T; PEREZ, MF; CIOFFI, MB. Biology, 11 (2), 2022.
Abstract: Diversity found in Neotropical freshwater fish is remarkable. It can even hinder a proper delimitation of many species, with the wolf fish Erythrinus erythrinus (Teleostei, Characiformes) being a notable example. This nominal species shows remarkable intra-specific variation, with extensive karyotype diversity found among populations in terms of different diploid chromosome numbers (2n), karyotype compositions and sex chromosome systems. Here, we analyzed three distinct populations (one of them cytogenetically investigated for the first time) that differed in terms of their chromosomal features (termed karyomorphs) and by the presence or absence of heteromorphic sex chromosomes. We combined cytogenetics with genomic approaches to investigate how the evolution of multiple sex chromosomes together with allopatry is linked to genetic diversity and speciation. The results indicated the presence of high genetic differentiation among populations both from cytogenetic and genomic aspects, with long-distance allopatry potentially being the main agent of genetic divergence. One population showed a neo-X1X2Y sexual chromosome system and we hypothesize that this system is associated with enhanced inter-population genetic differentiation which could have potentially accelerated speciation compared to the effect of allopatry alone.

Tracking the evolutionary pathways among Brazilian Lebiasina species (Teleostei: Lebiasinidae): a chromosomal and genomic comparative investigation

LEITE, PPM; SASSI, FMC; MARINHO, MMF; NIRCHIO, M; MORAES, RLR; TOMA, GA; BERTOLLO, LAC; CIOFFI, MB. Neotropical Ichthyology, 20 (1), 2022.
Abstract: Despite several difficulties in chromosomal analyses of small-sized fishes, the cytogenetics of the Lebiasinidae was largely improved in the last years, showing differential patterns in the chromosomal evolution inside the family. In this context, it has been shown that genus Lebiasina preserves its karyotypic macrostructure, composed of 2n = 36 chromosomes, whereas the other genera generally present higher 2n. This study focused on the comparative cytogenetics of three Lebiasina species, one of them analyzed here for the first time, using conventional and molecular procedures. The results reinforced the differentiated evolutionary path of the genus Lebiasina while, at the same time, highlighted the genomic particularities that have accompanied the evolution of each species. In this sense, the repetitive components of the genome played a significant role in the differentiation of each species. It is also notable that L. minuta and L. melanoguttata, the two species that occur exclusively in the Brazilian territory, show greater chromosomal similarities to each other than to the trans-Andean sister species, L. bimaculata.

The Genetic Differentiation of Pyrrhulina (Teleostei, Characiformes) Species is Likely Influenced by Both Geographical Distribution and Chromosomal Rearrangements

FERREIRA, PHN; SOUZA, FHS; MORAES, RLR; PEREZ, MF; SASSI, FMC; VIANA, PF; FELDBERG, E; EZAZ, T; LIEHR, T; BERTOLLO, LAC; CIOFFI, MB. Frontiers in Genetics, 10.3389/fgene.2022.869073 2022.
Abstract: Allopatry is generally considered to be one of the main contributors to the remarkable Neotropical biodiversity. However, the role of chromosomal rearrangements including neo-sex chromosomes for genetic diversity is still poorly investigated and understood. Here, we assess the genetic divergence in five Pyrrhulina species using population genomics and combined the results with previously obtained cytogenetic data, highlighting that molecular genetic diversity is consistent with their chromosomal features. The results of a principal coordinate analysis (PCoA) indicated a clear difference among all species while showing a closer relationship of the ones located in the same geographical region. This was also observed in genetic structure analyses that only grouped P. australis and P. marilynae, which were also recovered as sister species in a species tree analysis. We observed a contradictory result for the relationships among the three species from the Amazon basin, as the phylogenetic tree suggested P. obermulleri and P. semifasciata as sister species, while the PCoA showed a high genetic difference between P. semifasciata and all other species. These results suggest a potential role of sex-related chromosomal rearrangements as reproductive barriers between these species.

Evolutionary breakpoint regions and chromosomal remodeling in Harttia (Siluriformes: Loricariidae) species diversification

DEON, GA; GLUGOSKI, L; HATANAKA, T; SASSI, FMC; NOGAROTO, V; BERTOLLO, LAC; LIEHR, T; AL-RIKABI, ABH; MOREIRA-FILHO, O; CIOFFI, MB; VICARI, MR. Genetics and Molecular Biology, 45 (2) 2022.
Abstract: The Neotropical armored catfish genus Harttia presents a wide variation of chromosomal rearrangements among its representatives. Studies indicate that translocation and Robertsonian rearrangements have triggered the karyotype evolution in the genus, including differentiation of sex chromosome systems. However, few studies used powerful tools, such as comparative whole chromosome painting, to clarify this highly diversified scenario. Here, we isolated probes from the X1 (a 5S rDNA carrier) and the X2 (a 45S rDNA carrier) chromosomes of Harttia punctata, which displays an X1X1X2X2/X1X2Y multiple sex chromosome system. Those probes were applied in other Harttia species to evidence homeologous chromosome blocks. The resulting data reinforce that translocation events played a role in the origin of the X1X2Y sex chromosome system in H. punctata. The repositioning of homologous chromosomal blocks carrying rDNA sites among ten Harttia species has also been demonstrated. Anchored to phylogenetic data it was possible to evidence some events of the karyotype diversification of the studied species and to prove an independent origin for the two types of multiple sex chromosomes, XX/XY1Y2 and X1X1X2X2/X1X2Y, that occur in Harttia species. The results point to evolutionary breakpoint regions in the genomes within or adjacent to rDNA sites that were widely reused in Harttia chromosome remodeling.

Chromosomal Rearrangements and Origin of the Multiple XX/XY1Y2 Sex Chromosome System in Harttia Species (Siluriformes: Loricariidae)

DEON, GA; GLUGOSKI, L; SASSI, FMC; HATANAKA, T; NOGAROTO, V; BERTOLLO, LAC; LIEHR, T; AL-RIKABI, ABH; MOREIRA-FILHO, O; CIOFFI, MB; VICARI, MR. Frontiers in Genetics, 10.3389/fgene.2022.877522. 2022.
Abstract: The Neotropical genus Harttia comprises species with extensive chromosomal remodeling and distinct sex chromosome systems (SCSs). So far, three different SCSs with male heterogamety have been characterized in the group. In some species, the presence of the XX/XY1Y2 SCS is associated with a decrease in diploid numbers and several chromosomal rearrangements, although a direct relation to sex chromosome differentiation has not been shown yet. Here, we aimed to investigate the differentiation processes that have led to the establishment of the rare XX/XY1Y2 SCS and track its evolutionary history among other Harttia species. For that, four whole chromosome painting probes derived from chromosome 1 of H. torrenticola (HTO-1), chromosomes 9 and X of H. carvalhoi (HCA-9 and HCA-X), and chromosome X from H. intermontana (HIN-X) were applied in nine Harttia species. Homeologous chromosome blocks were located in Harttia species and demonstrated that Robertsonian (Rb) fusions originated HTO-1, HCA-9, and HCA-X chromosomes, while Rb fissions explain Y1 and Y2 sex chromosomes. Specifically, in H. intermontana, HCA-X, HCA-9, and the NOR-bearing chromosome demonstrated that homeologous blocks were used in the HIN-X and metacentric pair 2 origins. Consequently, diploid numbers changed between the studied species. Overall, the data also reinforce the existence of unstable genomic sites promoting chromosomal differentiation and remodeling within the genus Harttia.

Tracking the evolutionary trends among small-size fishes of the genus Pyrrhulina (Characiformes, Lebiasinidae): new insights from a molecular cytogenetic perspective

MORAES, RLR.; SASSI, FMC; BERTOLLO, LAC; MARINHO, MMF; VIANA, PF; FELDBERG, E; OLIVEIRA, VCS; DEON, GA; AL-RIKABI, ABH; LIEHR, T; CIOFFI, MB. Frontiers in Genetics, 12:769984 2021.
Abstract: Miniature fishes have always been a challenge for cytogenetic studies due to the difficulty in obtaining chromosomal preparations, making them virtually unexplored. An example of this scenario relies on members of the family Lebiasinidae which include miniature to medium-sized, poorly known species, until very recently. The present study is part of undergoing major cytogenetic advances seeking to elucidate the evolutionary history of lebiasinids. Aiming to examine the karyotype diversification more deeply in Pyrrhulina, here we combined classical and molecular cytogenetic analyses, including Giemsa staining, C-banding, repetitive DNA mapping, comparative genomic hybridization (CGH), and whole chromosome painting (WCP) to perform the first analyses in five Pyrrhulina species (Pyrrhulina aff. marilynae, Pyrrhulina sp., P. obermulleri, P. marilynae and Pyrrhulina cf. laeta). The diploid number (2n) ranged from 40 to 42 chromosomes among all analyzed species, but P. marilynae is strikingly differentiated by having 2n = 32 chromosomes and a karyotype composed of large meta/submetacentric chromosomes, whose plesiomorphic status is discussed. The distribution of microsatellites does not markedly differ among species, but the number and position of the rDNA sites underwent significant changes among them. Interspecific comparative genome hybridization (CGH) found a moderate divergence in the repetitive DNA content among the species’ genomes. Noteworthy, the WCP reinforced our previous hypothesis on the origin of the X1X2Y multiple sex chromosome system in P. semifasciata. In summary, our data suggest that the karyotype differentiation in Pyrrhulina has been driven by major structural rearrangements, accompanied by high dynamics of repetitive DNAs.

Highly Rearranged Karyotypes and Multiple Sex Chromosome Systems in Armored Catfishes from the Genus Harttia (Teleostei, Siluriformes)

DEON, GA; GLUGOSKI, L; VICARI, MR; NOGAROTO, V; SASSI, FMC; CIOFFI, MB; LIEHR, T; BERTOLLO, LAC; MOREIRA-FILHO, O. Genes, v.11, 1366, 2020.
Abstract: Harttia comprises an armored catfish genus endemic to the Neotropical region, including 27 valid species with low dispersion rates that are restricted to small distribution areas. Cytogenetics data point to a wide chromosomal diversity in this genus due to changes that occurred in isolated populations, with chromosomal fusions and fissions explaining the 2n number variation. In addition, different multiple sex chromosome systems and rDNA loci location are also found in some species. However, several Harttia species and populations remain to be investigated. In this study, Harttia intermontana and two still undescribed species, morphologically identified as Harttia sp. 1 and Harttia sp. 2, were cytogenetically analyzed. Harttia intermontana has 2n = 52 and 2n = 53 chromosomes, while Harttia sp. 1 has 2n = 56 and 2n = 57 chromosomes in females and males, respectively, thus highlighting the occurrence of an XX/XY1Y2 multiple sex chromosome system in both species. Harttia sp. 2 presents 2n = 62 chromosomes for both females and males, with fission events explaining its karyotype diversification. Chromosomal locations of the rDNA sites were also quite different among species, reinforcing that extensive rearrangements had occurred in their karyotype evolution. Comparative genomic hybridization (CGH) experiments among some Harttia species evidenced a shared content of the XY1Y2 sex chromosomes in three of them, thus pointing towards their common origin. Therefore, the comparative analysis among all Harttia species cytogenetically studied thus far allowed us to provide an evolutionary scenario related to the speciation process of this fish group.

Chromosomes of Asian cyprinid fishes: Variable karyotype patterns and evolutionary trends in the genus Osteochilus (Cyprinidae, Labeoninae, “Osteochilini”)

PASAKORN, S; SUPIWONG, W; SASSI, FMC; BERTOLLO, LAC; RAB, P; KRETSCHMER, R; TANOMTONG, A; SUWANNAPOOM, C; REUNGSING, M; CIOFFI, MB. Genetics and Molecular Biology, v. 43, e20200195, 2020.
Abstract: The Cyprinidae family is a highly diversified but demonstrably monophyletic lineage of cypriniform fishes. Among them, the genus Osteochilus contains 35 recognized valid species distributed from India, throughout Myanmar, Laos, Thailand, Malaysia, Indonesian archipelago to southern China. In this study, karyotypes and other chromosomal characteristics of five Osteochilus species occurring in Thailand, namely O. lini, O. melanopleura, O. microcephalus, O. vittatus and O. waandersii were examined using conventional and molecular cytogenetic protocols. Our results showed they possessed diploid chromosome number (2n) invariably 2n = 50, but the ratio of uni- and bi-armed chromosomes was highly variable among their karyotypes, indicating extensive chromosomal rearrangements. Only one chromosome pair bearing 5S rDNA sites occurred in most species, except O. melanopleura, where two sites were detected. In contrast, only one chromosomal pair bearing 18S rDNA sites were observed among their karyotypes, but in different positions. These cytogenetic patterns indicated that the cytogenomic divergence patterns of these Osteochilus species were largely corresponding to the inferred phylogenetic tree. Similarly, different patterns of the distributions of rDNAs and microsatellites across genomes of examined species as well as their different karyotype structures indicated significant evolutionary differentiation of Osteochilus genomes

Cytogenetics of the small-sized fish, Copeina guttata (Characiformes, Lebiasinidae): Novel insights into the karyotype differentiation of the family

TOMA, GA; MORAES, RLR; SASSI, FMC; BERTOLLO, LAC; OLIVEIRA, EA; RAB, P; SEMBER, A; LIEHR, T; HATANAKA, T; VIANA, PF; MARINHO, MMF; FELDBERG, E; CIOFFI, MB. PLoS One, v. 14, p. e0226746, 2019.
Abstract: Lebiasinidae is a small fish family composed by miniature to small-sized fishes with few cytogenetic data (most of them limited to descriptions of diploid chromosome numbers), thus preventing any evolutionary comparative studies at the chromosomal level. In the present study, we are providing, the first cytogenetic data for the red spotted tetra, Copeina guttata, including the standard karyotype, C-banding, repetitive DNA mapping by fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH), providing chromosomal patterns and novel insights into the karyotype differentiation of the family. Males and females share diploid chromosome number 2n = 42 and karyotype composed of 2 metacentric (m), 4 submetacentric (sm) and 36 subtelocentric to acrocentric (st-a) chromosomes. Blocks of constitutive heterochromatin were observed in the centromeric and interstitial regions of several chromosomes, in addition to a remarkably large distal block, heteromorphic in size, which fully corresponded with the 18S rDNA sites in the fourth chromosomal pair. This overlap was confirmed by 5S/18S rDNA dual-color FISH. On the other hand, 5S rDNA clusters were situated in the long and short arms of the 2nd and 15th pairs, respectively. No sex-linked karyotype differences were revealed by male/female CGH experiments. The genomic probes from other two lebiasinid species, Lebiasina melanoguttata and Pyrrhulina brevis, showed positive hybridization signals only in the NOR region in the genome of C. guttata. We demonstrated that karyotype diversification in lebiasinids was accompanied by a series of structural and numeric chromosome rearrangements of different types, including particularly fusions and fissions.

Uso do Role-Playing Game (RPG) como complemento didático no ensino de imunologia

LAUREANO, MOL; CAMPOS, MLCB; SASSI, FMC; ALMEIDA, MEF. Journal of Biochemistry Education, v. 15, p. 64, 2017.
Abstract: RPG is defined as a Role-Playing Game, in which participants can play against each other, or work together towards a objective. Thereby, an RPG game was developed to work on the content of Immunology with children and adolescents (5th to 8th grade) where medieval battles, involving fantastic creatures, are associated with elements that make up the Immune System, such as a shaman representing an T lymphocyte. To teach Immunology through the RPG is primarily needed a master, will guide the participants to teach the content in a playful way, throughout a "war" within a person. The students who will control the characters responsible for attacking and defending the body (infectious agents and white blood cells, respectively) having a dice for the scrolling of their actions in the game. As a result, it is expected with this work that Immunology content will be more easily fixed in a fun and enjoyable way.

Book chapters - 3 chapters

Chapter 24: FISH—in Fish Chromosomes. In Thomas Liehr (editor) Cytogenetics and Molecular Cytogenetics, p. 281-297. CRC Press.

SASSI, FMC; TOMA, GA; CIOFFI, MB. 2022.

Chapter V.1: Ensino e ciência na era do digital: construção de podcast cenográfico para a divulgação científica no ensino superior. In Tomás S. Martínez, Marina G. Carmona, Arturo F. Cabrera, Antonio-Manuel Rodríguez-García, Jesús L. Belmonte (Eds.) Innovación Educativa en la Sociedad Digital, p. 815 - 826

SILVA, MM; BARONAS, RL; SASSI, FMC; PEREIRA, L. 2019.

Chapter 4: Natural hybridization between chromosomal discrepant species and the role of hybrid speciation in the genus Astyanax. In Naomi Norris and Carmen Miller (Eds.) An Essential Guide to Cytogenetics, p. 123 - 137. Nova Science Publisher.

SASSI, FMC; RESENDE, SV; PASA, R; KAVALCO, KF. 2018.

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