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.

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.

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.