NEOTROPICAL FRESHWATER FISHES: A dataset of occurrence and abundance of freshwater fishes in the Neotropics.

The Neotropical region hosts 4225 freshwater fish species, ranking first among the world's most diverse regions for freshwater fishes. Our NEOTROPICAL FRESHWATER FISHES data set is the first to produce a large-scale Neotropical freshwater fish inventory, covering the entire Neotropical region from Mexico and the Caribbean in the north to the southern limits in Argentina, Paraguay, Chile, and Uruguay. We compiled 185,787 distribution records, with unique georeferenced coordinates, for the 4225 species, represented by occurrence and abundance data. The number of species for the most numerous orders are as follows: Characiformes (1289), Siluriformes (1384), Cichliformes (354), Cyprinodontiformes (245), and Gymnotiformes (135). The most recorded species was the characid Astyanax fasciatus (4696 records). We registered 116,802 distribution records for native species, compared to 1802 distribution records for nonnative species. The main aim of the NEOTROPICAL FRESHWATER FISHES data set was to make these occurrence and abundance data accessible for international researchers to develop ecological and macroecological studies, from local to regional scales, with focal fish species, families, or orders. We anticipate that the NEOTROPICAL FRESHWATER FISHES data set will be valuable for studies on a wide range of ecological processes, such as trophic cascades, fishery pressure, the effects of habitat loss and fragmentation, and the impacts of species invasion and climate change. There are no copyright restrictions on the data, and please cite this data paper when using the data in publications.

Single-cell transcriptomes and runx2b-/- mutants reveal the genetic signatures of intermuscular bone formation in zebrafish.

Intermuscular bones (IBs) are mineralized spicules, present in the myosepta of many, but not all, teleost species. IBs are often small and sharp, and they consequently limit how the fish can be processed; the IBs may cause injury or trauma if lodged in consumers' throats or mouths, and therefore affect the appeal of the fish to many consumers. The development of IBs in teleosts is still not fully understood and the molecular basis of IB development remains to be established. Here, the characteristics of IB tissue are evaluated based on single-cell transcriptomics in wild-type zebrafish. The analysis defined 18 distinct cell types. Differentiation trajectories showed that IBs are derived from tendons and that a core tendon-osteoblast cell lineage is related to IB formation. In particular, the functions of 10 candidate genes were evaluated via CRISPR-Cas9 mutants. Among those, runx2b-/- mutants completely lost IBs, while swimming performance, growth and bone mineral density were not significantly different from runx2b+/+ zebrafish. Comparative single-cell RNA sequencing (scRNA-seq) analysis in runx2b-/- and runx2b+/+ zebrafish revealed the role of osteoblasts in IB formation. In addition, differentially expressed genes were enriched in the transforming growth factor ß/bone morphogenetic protein (TGF-ß/BMP) pathway after runx2b deletion. This study provides evidence for the crucial role of runx2b regulation in IB formation. Genetic breeding can target runx2b regulation and generate strains of commercial fish species without IBs, which can improve the safe consumption and economic value of many farmed fish species.

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.

Genome assembly and annotation of the tambaqui (Colossoma macropomum): an emblematic fish of the Amazon River Basin.

Colossoma macropomum, known as "tambaqui", is the largest Characiformes fish in the Amazon River Basin and a leading species in Brazilian aquaculture and fisheries. Good quality meat and excellent adaptability to culture systems are some of its remarkable farming features. To support studies into the genetics and genomics of the tambaqui, we have produced the first high-quality genome for the species. We combined Illumina and PacBio sequencing technologies to generate a reference genome, assembled with 39× coverage of long reads and polished to a consensus quality value (QV) of 36 with 130× coverage of short reads. The genome was assembled into 1269 scaffolds (a total of 1,221,847,006 bases), with a scaffold N50 size of 40 Mb, where 93% of all assembled bases were placed in the largest 54 scaffolds corresponding to the diploid karyotype of the tambaqui. Furthermore, the NCBI Annotation Pipeline annotated genes, pseudogenes, and non-coding transcripts using the RefSeq database as evidence, guaranteeing a high-quality annotation. A Genome Data Viewer for the tambaqui was produced, which will benefit groups interested in exploring the unique genomic features of the species. The availability of a highly accurate genome assembly for tambaqui provides the foundation for the discovery of novel ecological and evolutionary insights, and is a helpful resource for aquaculture.

Spawning induction and embryonic development of Salminus hilarii (Characiformes: Characidae).

The present study aimed to evaluate two different methods of artificial reproduction induction, and characterize the embryonic development of Salminus hilarii. Different than observed for other tropical fish species, artificial reproduction induction followed by hand-stripping of gametes was considered unfeasible for S. hilarii, as no gamete fertilization was observed. However, females that were induced and spawned naturally presented a fertilization rate of 65.64 ± 0.54%. With a mean temperature of 26.20 ± 0.90ºC it was possible to clearly distinguish a large perivitelline space at 14 min after fertilization (AF) and at 49 min AF more than 50% of the embryos presented two blastomeres, and these cleavages occurred until 1 h 54 min AF. The gastrula phase was characterized at 3 h 56 min AF, and blastopore closure was observed at 8 h 31 min AF. At 9 h 1 min, organogenesis started, with a clear distinction of the yolk mass, embryonic axis, cephalic and caudal regions; at 11 h 51 min AF the embryos already had advanced segmentation and a free tail. Total hatching occurred at 21 h 17 min and after opening the mouth, which occurred at 33 h 9 min, the larvae of S. hilarii presented a strong and characteristic cannibalism. This information can be considered fundamental to improving S. hilarii production in captivity and for collaboration with a conservation programme in the upper Tietê river basin.

Quantitative analysis of black blotching in a crossbred red tilapia and its effects on performance traits via a path analysis methodology.

Red tilapia is a phenotype with potential value to the tilapia industry due to its attractive skin coloration. However, the presence of black blotches scattered on the body makes these fish less valuable than blemish-free red fish. The correlation between the degree of blotching and other economic traits is important for selective breeding programs including high-performance red fish without black blotches. Therefore, this study aimed to assess the magnitude and direct and indirect effects of blotching in crossbred red tilapia on the growth rate and body measure traits using path analysis methodology. Six genetic groups were evaluated in terms of body measurements, body weight rate, and proportion of black blotching over 250 days of growth: Chitralada and Red-Stirling; crossbred F1-♀ Red-Stirling × â™‚ Chitralada and ♀ Chitralada × â™‚ Red-Stirling, and the introgressive crossbreeding-♀ CR × â™‚ Chitralada and ♀ Chitralada × â™‚ CR (C × CR). All variables showed high and positive linear correlations. Individuals with black blotches had lower coefficients of correlation with other variables (body weight and measurements); the direct and indirect effects are near zero. The path analysis indicated a lack of genetic correlation between the degree of black blotches and the economic traits of red tilapia. This facilitates the development of high growth performance red fish with blotched-free reddish skin.

Development and characterization of 20 polymorphic microsatellite markers for Epinephelus marginatus (Lowe, 1834) (Perciformes: Epinephelidae) using 454 pyrosequencing.

The dusky grouper, Epinephelus marginatus, is a well-known and widespread marine fish assessed as endangered by the International Union for the Conservation of Nature. Analyzing the genetic diversity of this species is, therefore, of utmost importance and necessary for conservation purposes. Microsatellites are molecular tools with advantages that are ideal for population analyses. This study provides the first set of species-specific microsatellite loci for E. marginatus that can be applied when assessing both intra- and interpopulation genetic variation. Twenty microsatellite loci were isolated and characterized for the dusky grouper by genotyping 20 individuals obtained from the North Eastern Atlantic Ocean (n = 4) and from the South Western Atlantic Ocean (n = 16). The number of alleles per locus varied from 2 to 11, while the observed and expected heterozygosities ranged from 0.25 to 0.94 and 0.34 to 0.89, respectively. The polymorphic information content varied from moderately to highly informative. This suite of markers provides the first specific nuclear tools for E. marginatus and, thus, allows to assess with more specificity different populations' structures.

Molecular assessment of Gymnotus spp. (Gymnotiformes: Gymnotidae) fishing used as live baitfish in the Tietê River, Brazil

The capture of live bait for sport fishing is an important activity for fishing communities. The main species used for this purpose are members of the genus Gymnotus, which comprises numerous species of cryptic nature that are difficult to identify based on external morphology. The aims of this work were to identify through partial sequences of the COI gene Gymnotus species fished in the Jacaré-Guaçu River, SP, and to develop a molecular diagnostic approach using PCR-RFLP to identify these species. Partial COI sequences were compared to those of other species deposited in GenBank. The sequences were assessed in the NEBCutter program to determine restriction sites in the sequence and the enzymes to be tested. Phenetic analysis performed by Neighbor-Joining method showed that the specimens sampled belong to two species preliminary identified here as G. cf. sylvius and G. cf. cuia, with G. cf. sylvius accounting for 95.2% of the individuals sampled. The enzymes NlaIII and SacI generated fragments that allowed distinguishing the Gymnotus species using PCR-RFLP. This analysis can be used to accurately identify these species, which is fundamental for monitoring Gymnotus fishing and assessing the conservation of this genetic resource.(AU)

A captura de iscas-vivas para a pesca esportiva constitui uma atividade importante em comunidades de pescadores. As principais espécies utilizadas para este propósito pertencem ao gênero Gymnotus, o qual compreende inúmeras espécies de natureza críptica que dificulta a identificação baseada na morfologia externa. Os objetivos deste trabalho foram identificar através de sequências parciais do gene COI, espécies de Gymnotus capturadas no Rio Jacaré-Guaçu, Ibitinga, SP, e desenvolver um diagnóstico molecular por meio de PCR-RFLP. Sequências parciais de COI foram comparadas com outras espécies depositadas no GenBank. As sequências foram analisadas no Programa NebCutter para determinar os sítios de restrição e definir as enzimas a serem testadas. A análise fenética pelo método de Neighbor-Joining mostrou que os espécimes pertencem a duas espécies identificadas preliminarmente aqui como G. cf. sylvius e G. cf. cuia, sendo que G. cf. sylvius representou 95,2% dos indivíduos amostrados. As enzimas NlaIII e SacI geraram fragmentos que permitiram discriminar as espécies por meio de PCR-RFLP. Esta análise pode ser usada na identificação precisa destas espécies, fundamental na proposição de monitoramento da pesca de Gymnotus na região e para medidas adequadas de conservação.(AU)

Large-scale SNP discovery and construction of a high-density genetic map of Colossoma macropomum through genotyping-by-sequencing.

Colossoma macropomum, or tambaqui, is the largest native Characiform species found in the Amazon and Orinoco river basins, yet few resources for genetic studies and the genetic improvement of tambaqui exist. In this study, we identified a large number of single-nucleotide polymorphisms (SNPs) for tambaqui and constructed a high-resolution genetic linkage map from a full-sib family of 124 individuals and their parents using the genotyping by sequencing method. In all, 68,584 SNPs were initially identified using minimum minor allele frequency (MAF) of 5%. Filtering parameters were used to select high-quality markers for linkage analysis. We selected 7,734 SNPs for linkage mapping, resulting in 27 linkage groups with a minimum logarithm of odds (LOD) of 8 and maximum recombination fraction of 0.35. The final genetic map contains 7,192 successfully mapped markers that span a total of 2,811 cM, with an average marker interval of 0.39 cM. Comparative genomic analysis between tambaqui and zebrafish revealed variable levels of genomic conservation across the 27 linkage groups which allowed for functional SNP annotations. The large-scale SNP discovery obtained here, allowed us to build a high-density linkage map in tambaqui, which will be useful to enhance genetic studies that can be applied in breeding programs.

Molecular evidence of two cryptic species of Stramonita (Mollusca, Muricidae) in the southeastern Atlantic coast of Brazil.

Snails of the genus Stramonita are commonly found in the rocky intertidal habitat of the western Atlantic Ocean coast. They belong to a monophyletic taxon that occurs along the tropical and warm-temperate Atlantic and eastern Pacific rocky shores. This genus comprises different valid species and members of the S. haemastoma complex. In the present study, samples of Stramonita were collected from three different regions of southeastern Brazil. Partial sequences of two mitochondrial genes, COI and 16S rRNA, were used to compare nucleotides sequences between Stramonita specimens. Levels of nucleotide divergence greater than 2% across the three sampled regions were used for differentiation at the species level. One of the identified species was S. brasiliensis, which has recently been described by molecular analysis; the other species may represent S. haemastoma, not yet described in the southeastern Brazilian coast.

Genetic structuring of Salminus hilarii Valenciennes, 1850 (Teleostei: Characiformes) in the rio Paraná basin as revealed by microsatellite and mitochondrial DNA markers

Genetic variation of Salminus hilarii was assessed by screening microsatellite loci and mitochondrial D-loop DNA across four sampling in the upper rio Paraná basin of Brazil. Genetic diversity - measured as mean expected heterozygosity (0.904) and mean number of alleles across populations (13.7) - was reasonably high. Differentiation of microsatellite allele frequencies among populations was shown to be low but significant by AMOVA Φ ST (0.0192), and high by D EST (0.185). D-loop variation was high, with haplotypic diversity of 0.950 and nucleotide diversity of 0.011. Mitochondrial DNA-based estimates for population differentiation were high, with an overall Φ ST of 0.173. The results of tests of nuclear and mitochondrial variation yielded no unequivocal inference of historical demographic bottleneck or expansion. Genetic differentiation observed among S. hilarii populations in the rio Grande may be caused by a combination of historical differentiation and recent gene-flow disruption caused by the dams followed by reproduction of isolated spawning assemblages in mid-sized tributaries of the respective reservoirs. We present spatially more intensive sampling of S. hilariipopulations across the rio Paraná basin in order to more effectively distinguish between historical and contemporary differentiation.

A variabilidade genética de Salminus hilarii foi avaliada por lócus microssatélites e sequências D-Loop do DNA mitocondrial em quatro populações da região da bacia do Alto Paraná. A diversidade genética - medida pela heterozigosidade média (0,904) e número de alelos médios das populações (13,7) - foi razoavelmente alta. A diferenciação das frequências alélicas entre as populações foi baixa, mas significativa pela AMOVA Φ ST (0,0192), e alta pelo D EST (0,185). A variação mitocondrial foi alta com uma diversidade haplotípica de 0,950 e uma diversidade nucleotídica de 0,011. Estimativas de diferenciação populacional baseadas no DNA mitocondrial foram altas, com um valor global de Φ ST de 0,173. Os resultados dos testes da variação nuclear e mitocondrial demonstram nenhuma inequívoca inferência histórica de contração e expansão demográfica. A diferenciação genética observada entre as populações de S. hilarii no rio Grande pode ter sido causada pela combinação de diferenciação histórica e interrupção recente do fluxo gênico causada pela construção de barragens seguida por um isolamento reprodutivo de populações em tributários de médio porte dos respectivos reservatórios. Nós apresentamos uma amostragem mais ampla e intensiva de populações de S. hilarii ao longo da bacia do alto rio Paraná para se efetivamente distinguir se a diferenciação genética das populações encontrada é histórica ou contemporânea.

The involvement of gonadotropins and gonadal steroids in the ovulatory dysfunction of the potamodromous Salminus hilarii (Teleostei: Characidae) in captivity.

Potamodromous teleosts that require migration to reproduce show dysfunctions that block ovulation and spawning while in captivity. To understand the physiological basis of these reproductive dysfunctions, follicle-stimulating hormone b subunit (fshb) and luteinizing hormone b subunit (lhb) gene expression analyses by real-time quantitative PCR, together with measurements of estradiol (E 2), 17α-hydroxyprogesterone (17α-OHP) and 17α,20ß-dihydroxy-4-pregnen-3-one (17α,20ß-DHP) levels, were carried out throughout the reproductive cycle of the potamodromous Salminus hilarii. The following reproductive stages were evaluated in captive and wild females: previtellogenic (PV), advanced maturation/mature (AM) and regression/spent (REG/SPENT). In the wild females, fshb expression decreased from the PV to the AM stage, and the opposite pattern was detected for E 2, which increased from the PV to the AM stage. fshb was expressed at lower levels in captive than in wild females, and this difference did not change during the reproductive cycle. lhb expression also increased from the PV to the AM stage in both groups, but the wild females at the AM and REG/SPENT stages showed higher lhb expression levels than the captive females. The concentrations of 17α-OHP did not change during the reproductive cycle, and the levels were higher in the captive than in the wild females at all reproductive stages. 17α,20ß-DHP levels did not change between wild and captive females. However, in captive females, the transition from PV to AM stage was followed by an increase in 17α,20ß-DHP levels. These data indicate that dysfunctions in the gonadotropins and steroids synthesis pathways cause the ovulation failure in captive S. hilarii.

Genetic relationship among Camponotus rufipes Fabricius (Hymenoptera: Formicidae) nests by RAPD molecular markers / Relação genética entre ninhos de Camponotus rufipes Fabricius (Hymenoptera: Formicidae) usando marcadores moleculares RAPD

Random amplified polymorphic DNA (RAPD) markers were used to investigate the genetic relationship among nests of the carpenter ant, Camponotus rufipes, located in the same area. Five random oligodecamers were used to amplify DNA from 108 ant workers collected from six nests. A total of 47 RAPD markers were identified, which revealed low levels of genetic differentiation among nests (Φst = 0.00218) and a low average Shannon index (0.3727) among workers within nests. These results together suggest that the C. rufipes nest may be formed by a single, once-mated queen and that nests produced by queens that are genetically related tend to keep their nests in close proximity to one other.

Marcadores moleculares RAPD (Random Amplified Polymorphic DNA) foram usados para investigar a relação genética entre os ninhos da formiga carpinteira, Camponotus rufipes, localizados em uma mesma área. Cinco oligodecâmeros aleatórios foram utilizados para amplificar o DNA de 108 operárias coletadas em seis ninhos. Um total de 47 marcadores RAPD foi identificado, indicando baixa diferenciação genética entre os ninhos (Φst = 0,00218) e um baixo índice de Shannon (0,3737) entre operárias de um mesmo ninho. Os resultados sugerem que a colônia de C. rufipes pode ser formada por uma única rainha e, ninhos produzidos por rainhas geneticamente relacionadas possuem tendência de serem fundados em locais próximos.

Microsatellite markers developed for Utricularia reniformis (Lentibulariaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed to study the genetic diversity and population structure of the carnivorous bladderwort Utricularia reniformis, which is endemic to the Atlantic Forest of southern and southeastern Brazil. Cross-species amplification was tested in U. gibba, U. neottioides, U. subulata, and Pinguicula benedicta. METHODS AND RESULTS: The U. reniformis genome was sequenced in a 454 GS FLX sequencer, and eight primer sets were developed based on the microsatellites identified from the reads. All loci are polymorphic, showing 1.6 to 4.8 alleles per population. Preliminary results show that primer sets are suitable for population-level studies. Cross-species amplification was successful in three other Utricularia species and one Pinguicula species. CONCLUSIONS: Markers developed in this study provide tools for analyses of intra- and interpopulation genetic diversity in Utricularia and Pinguicula.

Microsatellite variation and population genetic structure of a neotropical endangered Bryconinae species Brycon insignis Steindachner, 1877: implications for its conservation and sustainable management

Piabanha (Brycon insignis) is a freshwater fish species from the drainages in Southeastern Brazil. During the 1950s, it was an important economic and food resource for local populations, but dramatic and continuous environmental degradation seriously jeopardized the B. insignis populations in the region. Microsatellite markers were used to assess the genetic structure of wild populations of B. insignis and compare the genetic variability and integrity of the wild populations with a captive population. Samples of DNA from 208 specimens from geographically isolated populations were analyzed. Population genetic structure was investigated using F ST, R ST estimates as well as AMOVA. All five loci used in this study were polymorphic with observed heterozygosity ranging from 0.77 (± 0.15) to 0.88 (± 0.07) in the wild population and 0.90 (± 0.09) in the captive population and the allelic richness average were 7.56 (± 0.27) and 5.80 (± 1.02), respectively. Overall genetic differences were significantly partitioned among populations (F ST = 0.072, p = 0.034). Evidence of a genetic bottleneck was found in some of the wild populations, but especially in the captive population. The results showed that genetic variability still can be found in B. insignis populations which are currently structured possibly due to anthropic actions. The implications of these findings for the management and conservation of B. insignis populations are discussed.(AU)

Piabanha (Brycon insignis) é uma espécie de peixe de água doce oriunda de drenagens da região sudeste do Brasil. Durante os anos de 1950, esta espécie foi um importante recurso econômico para populações locais. Contudo, a intensa e contínua degradação ambiental afetou seriamente as populações de B. insignis na região. Marcadores microssatélites foram usados para avaliar a estrutura genética de populações selvagens de B. insignis e comparar a variabilidade genética e integridade das populações selvagens com uma população de cativeiro. Amostras de DNA de 208 espécimes de populações geograficamente isoladas foram analisadas. A estrutura populacional foi investigada usando-se estimadores de F ST e R ST bem como AMOVA. Todos os loci usados neste estudo foram polimórficos com heterozigosidades observadas variando de 0.77 (± 0.15) a 0.88 (± 0.77) em populações selvagens e 0.90 (± 0.09) na população de cativeiro e a riqueza alélica média foi de 7.56 (± 0.27) e 5.80 (± 1.02), respectivamente. A maioria das diferenças genéticas foi significativa entre populações (F ST = 0.072, p = 0.034). Evidências de efeito gargalo foram observadas em algumas populações selvagens e especialmente também na população de cativeiro. Os resultados do presente estudo mostraram que as populações de B. insignis ainda apresentam variabilidade genética e que estas populações estão atualmente estruturadas geneticamente provavelmente devido a ações antrópicas. As implicações destes achados para o manejo e conservação das populações de B. insiginis são discutidas.(AU)

Molecular cloning and sequence analysis of growth hormone cDNA of Neotropical freshwater fish Pacu (Piaractus mesopotamicus)

RT-PCR was used for amplifying Piaractus mesopotamicus growth hormone (GH) cDNA obtained from mRNA extracted from pituitary cells. The amplified fragment was cloned and the complete cDNA sequence was determined. The cloned cDNA encompassed a sequence of 543 nucleotides that encoded a polypeptide of 178 amino acids corresponding to mature P. mesopotamicus GH. Comparison with other GH sequences showed a gap of 10 amino acids localized in the N terminus of the putative polypeptide of P. mesopotamicus. This same gap was also observed in other members of the family. Neighbor-joining tree analysis with GH sequences from fishes belonging to different taxonomic groups placed the P. mesopotamicus GH within the Otophysi group. To our knowledge, this is the first GH sequence of a Neotropical characiform fish deposited in GenBank.