Eco-evolutionary factors that influence its demographic oscillations in Prochilodus costatus (Actinopterygii: Characiformes) populations evidenced through a genetic spatial-temporal evaluation.

The human activity impact on wild animal populations is indicated by eco-evolutionary and demographic processes, along with their survival and capacity to evolve; consequently, such data can contribute toward enhancing genetic-based conservation programs. In this context, knowledge on the life-history and the eco-evolutionary processes is required to understand extant patterns of population structure in Prochilodus costatus a Neotropical migratory fish that has been threatened due to loss and fragmentation of its natural habitat since 1960s promoted by the expansion of hydroelectric power plant construction programs. This study evaluated the eco-evolutionary parameters that cause oscillations in the demography and structure of P. costatus populations. An integrated approach was used, including temporal and spatial sampling, next-generation sequencing of eight microsatellite loci, multivariate genetic analysis, and demographic life-history reconstruction. The results provided evidence of the complex interplay of ecological-evolutionary and human-interference events on the life history of this species in the upper basin. In particular, spawning wave behavior might have ecological triggers resulting in an overlapping of distinct genetic generations, and arising distinct migratory and nonmigratory genetic patterns living in the same area. An abrupt decrease in the effective population size of the P. costatus populations in the recent past (1960-80) was likely driven by environment fragmentation promoted by the construction of the Três Marias hydropower dam. The low allelic diversity that resulted from this event is still detected today; thus, active stocking programs are not effective at expanding the genetic diversity of this species in the river basin. Finally, this study highlights the importance of using mixed methods to understand spatial and temporal variation in genetic structure for effective mitigation and conservation programs for threatened species that are directly affected by human actions.

Heterocyte production, gene expression, and phylogeography in Raphidiopsis (=Cylindrospermopsis) raciborskii.

Raphidiopsis (=Cylindrospermopsis) raciborskii was described as a subtropical-tropical cyanobacterium, later reported expanding into temperate regions. Heterocyte presence used to distinguish Cylindrospermopsis from the very similar Raphidiopsis, but recently the two genera were recognized as one and unified. This study aimed to investigate how heterocyte production is related to nitrogen (N) limitation in heterocytous and non-heterocytous strains of R.raciborskii. High N-concentrations did not inhibit heterocyte development in some strains, while prolonged N-starvation periods never stimulated production in others. RT-qPCR was used to examine the genetic background, through the expression patterns of nifH, ntcA and hetR. While gene expression increased under N-restriction, N-sufficiency did not suppress nifH transcripts as previously observed in other diazotrophyc cyanobacteria, suggesting that heterocyte production in R. raciborskii is not regulated by N-availability. Heterocytous and non-heterocytous strains were genotypically characterized to assess their phylogenetic relationships. In the phylogenetic tree, clusters were intermixed and confirmed Raphidiopsis and Cylindrospermopsis as the same genus. The tree supported previous findings of earlier splitting of American strains, while contesting the African origin hypothesis. The existence of two lines of Chinese strains, with distinct evolutionary patterns, is a significant addition that could lead to new hypotheses of the species biogeography.

Genetic evaluation of migratory fish: Implications for conservation and stocking programs.

Fish stocking programs have been implemented to mitigate the blockage of original riverbeds by the construction of hydropower dams, which affects the natural migration of fish populations. However, this method raises concerns regarding the genetic rescue of the original populations of migratory fish species. We investigated the spatial distribution of genetic properties, such as genetic diversity, population structure, and gene flow (migration), of the Neotropical migratory fish Prochilodus costatus in the Três Marias dam in the São Francisco River basin, Brazil, and examined the possible effects of fish stocking programs on P. costatus populations in this region. In total, 1,017 specimens were sampled from 12 natural sites and a fish stocking program, and genotyped for high-throughput sequencing at 8 microsatellite loci. The populations presented low genetic variability, with evidence of inbreeding and the presence of only four genetic pools; three pools were observed throughout the study region, and the fourth was exclusive to one area in the Paraopeba River. Additionally, we identified high unidirectional gene flow between regions, and a preferred migratory route between the Pará River and the upper portion of the São Francisco River. The fish stocking program succeeded in transposing the genetic pools from downstream to upstream of the Três Marias dam, but, regrettably, promoted genetic homogenization in the upper São Francisco River basin. Moreover, the data show the fragility of this species at the genetic level. This monitoring strategy could be a model for the development of conservation and management measures for migratory fish populations that are consumed by humans.

Use of complete mitochondrial genome sequences to identify barcoding markers for groups with low genetic distance.

Complete mitochondrial sequences can be rapidly obtained and are widely available, providing a great source of species information and allowing for the discovery of new specific molecular markers. However, for some taxonomic groups, traditional approaches for species delimitation are impaired by the low genetic distance values. In these cases, other species-level markers are used. For Prochilodus, which includes important neotropical fish species, species-level delimitation usually results in poor phylogenetic resolution when using mitochondrial COI/cytB genes as barcoding markers because of low genetic variability and low species-level resolution. Thus, in this study, we developed an approach to design and validate new barcoding markers with high species-level resolution obtained from the D-loop region, using Prochilodus spp. as a model. For the new barcoding marker validation, the amplicon region was used to infer the phylogenetic relationships of Prochilodus spp. through three distinct methods: Bayesian inference (BI), Neighbor-Joining method (NJ), and Maximum Likelihood method (ML). The phylogenetic relationships of Prochilodus spp. revealed high resolution at species-level, nonoverlapping clades, and high branch support. The genetic distance results allied to two different clustering methods (Bayesian Poisson tree processes and automatic barcode gap discovery) revealed the existence of a barcoding gap, thus, validating the use of the barcoding markers designed in this study. The approach proposed here may, therefore, be expanded to other taxa to access and validate new barcoding markers with higher resolution at the species level.

Complete mitochondrial genome sequence of Piaractus mesopotamicus (Holmberg, 1887).

The migratory species Piaractus Mesopotamicus, popularly known as the pacu, was determined to have a complete mitochondrial genome of 16,722 bp with 45% GC content. The genome contained 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and a 1048 bp Control Region (D-loop). Almost all the PCGs used the standard ATG start codon, except for Cox1 that used a GTG start codon. Five of the 13 PCGs had a TAA stop codon, two had the incomplete stop codon TA- (Atp6 and Cox3), and five had the incomplete stop codon T-- (Nd2, Cox2, Nd3, Nd4, and Cytb). AGG was the stop codon of Cox1.

Complete mitochondrial genome sequence of Brycon orbignyanus (Characiformes, Bryconidae).

We report the whole mitochondrial genome of the Brycon orbignyanus, commonly known as the piracanjuba. The mitogenome was determined to be a circular, 16,800 bp DNA molecule, containing 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes and one 1160 bp noncoding control region. Twelve of the PCGs were located on the heavy strand, and one PCG (Nd6) was located on the light strand. The most common start codon was ATG; however, the Cox1 gene displayed the GTG start codon. Seven PCGs had incomplete stop codons: specifically, Cox2, Cox3, Nd3, Nd4, and Cytb contained the T- - codon, and Nd2 and Atp6 contained the TA- codon. The Cox1 gene contained the AGG stop codon.

Pimelodus maculatus (Siluriformes, Pimelodidae): complete mtDNA sequence of an economically important fish from the São Francisco river basin.

Pimelodus maculatus is an important commercial fish found in the São Francisco and Paraná river basins. NGS was used to sequence the mtDNA of P. maculatus. The mtDNA was annotated and aligned with that of 25 other fish species to enable phylogenetic analysis. The complete mtDNA molecule had 16,561 bp and its GC content was 43.7%; the structure was similar to that of other vertebrates: 2 rRNA, 22 tRNA, 13 protein-coding genes, and a D-loop region containing 914 bp. Phylogenetic analysis yielded a tree with a high bootstrap coefficient that was coherent with the actual phylogeny of the species.