Ana Paula Vimieiro Martins

Description and comparison of two economically important fish species mitogenomes: Prochilodus argenteus and Prochilodus costatus (Characiformes, Prochilodontidae)

Abstract Prochilodus spp. are important Brazilian freshwater migratory fishes with substantial economic and ecological importance. Prochilodus argenteus and Prochilodus costatus are morphologically similar and a molecular species delimitation is impaired due to high degree of sequence identity among the available genetic markers. Here, the complete mitochondrial genome of P. argenteus and P. costatus and their comparison to the mitogenome of P. lineatus are described. The three species displayed a similar mtDNA annotation. A phylogenetic analysis was performed with other Characiformes species. The genus Prochilodus was recovered as a monophyletic group, as well as the family Prochilodontidae, both with high bootstrap probability.

Complete mitochondrial genome of Salminus brasiliensis (Characiformes, Characidae).

Abstract We report the complete mitochondrial genome of the fish Salminus brasiliensis, popularly known as dourado. It is a circular, 17,721 bp long DNA molecule, containing 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a non-coding Control Region of 2128 bp, a relatively large molecule when compared to other closely related fishes. All protein-coding genes are on the heavy strand, except for Nd6, and all have ATG as the start codon, except for Cox1 gene which has a GTG start codon. Seven protein-coding genes have incomplete stop codons (Nd2, Cox2, Atp6, Nd4, and Cytb have T– –, and Cox3 and Nd3 have TA–). TAG is the stop codon for Nd6 and AGG is the stop codon for Cox1.

Complete mitochondrial genome sequence of Prochilodus lineatus (Characiformes, Prochilodontidae)

Abstract Prochilodus lineatus (curimba) is an important Brazilian freshwater migratory fish with substantial economic importance in fishing. The complete mitochondrial genome of P. lineatus is 16,699 bp and contains 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes and one control region (D-loop). The mtDNA of P. lineatus is the first mitogenome of the Prochilodontidae family to be fully characterized. All of the PCGs in the mtDNA use the standard ATG start codon, with the exception of Cox1 that utilizes the GTG start codon. Six of the 13 PCGs contain TAA stop codons, two contain the incomplete stop codon TA– (Atp6 and Nd6), and five contain the incomplete stop codon T– – (Nd2, Cox2, Nd3, Nd4 and Cytb).

Microsatellite markers for Vellozia gigantea (Velloziaceae), a narrowly endemic species to the Brazilian campos rupestres

PREMISE OF THE STUDY Microsatellite primers were developed for the first time in Velloziaceae, in the endangered species Vellozia gigantea. METHODS AND RESULTS Using two different protocols, seven primer sets were characterized in three populations of V. gigantea. The primers amplified di- and trinucleotide repeats with six to 12 alleles per locus. These revealed high levels of genetic variation, presenting an average observed heterozygosity of 0.508 in V. gigantea. The seven primers were tested for cross-amplification in three Vellozia species. All primers successfully amplified in V. auriculata. Six primers amplified in V. compacta and three in V. hirsuta. CONCLUSIONS The new marker set described here will be useful for studies of population genetics of V. gigantea. The cross-amplification results indicate the utility of primers for studies in other Vellozia species.

High-Throughput Sequencing Strategy for Microsatellite Genotyping Using Neotropical Fish as a Model

Genetic diversity and population studies are essential for conservation and wildlife management programs. However, monitoring requires the analysis of multiple loci from many samples. These processes can be laborious and expensive. The choice of microsatellites and PCR calibration for genotyping are particularly daunting. Here we optimized a low-cost genotyping method using multiple microsatellite loci for simultaneous genotyping of up to 384 samples using next-generation sequencing (NGS). We designed primers with adapters to the combinatorial barcoding amplicon library and sequenced samples by MiSeq. Next, we adapted a bioinformatics pipeline for genotyping microsatellites based on read-length and sequence content. Using primer pairs for eight microsatellite loci from the fish Prochilodus costatus, we amplified, sequenced, and analyzed the DNA of 96, 288, or 384 individuals for allele detection. The most cost-effective methodology was a pseudo-multiplex reaction using a low-throughput kit of 1 M reads (Nano) for 384 DNA samples. We observed an average of 325 reads per individual per locus when genotyping eight loci. Assuming a minimum requirement of 10 reads per loci, two to four times more loci could be tested in each run, depending on the quality of the PCR reaction of each locus. In conclusion, we present a novel method for microsatellite genotyping using Illumina combinatorial barcoding that dispenses exhaustive PCR calibrations, since non-specific amplicons can be eliminated by bioinformatics analyses. This methodology rapidly provides genotyping data and is therefore a promising development for large-scale conservation-genetics studies.

Complete mitochondrial genome of Sporophila maximiliani (Ave, Passeriformes)

Abstract Sporophila maximiliani, commonly known as Great-billed Seed-Finch or ‘bicudo’, is a trafficked bird in Brazil due to the species’ beauty and singing, which is appreciated by breeders and collectors. Generally, the Environmental Military Police and IBAMA maintain enforcement actions, rescue work, and seizure of illegally traded of ‘bicudo’ specimens. The genomic DNA of one specimen was sequenced on MiSeq (Illumina) sequencer. The reads obtained were analyzed, trimmed, and de novo assembled using CLC Workbench® v9.0 (CLC Bio-Qiagen). The mitochondrial genome of S. maximiliani consisted of 16,765 base pairs, 2 ribosomal RNA, 22 transporter RNA, 13 protein-coding genes, and 1 control region. The molecular phylogeny demonstrated that the mitochondrial genome of S. maximiliani diverged from others related Passeriformes.

Leporinus elongatus (Characiformes, Anostomidae): complete mtDNA sequence of an economically important fish from the Paraná and La Plata river basins

Abstract Leporinus elongatus is an important commercial fish found in the La Plata and Paraná River basins. Next-generation sequencing was used to sequence the mitochondrial DNA (mtDNA) of L. elongatus. The mtDNA was assembled using the CLC Workbench software v. 9.0 and subsequently aligned to other 10 complete fish mitochondrial sequences to enable phylogenetic analysis using MEGA 7.0. The complete mtDNA molecule had 16,784 bp and its GC content was 43%. The mtDNA structure was similar to that of other vertebrates: two ribosomal RNA, 22 transfer RNA, 13 protein-coding genes, and a D-loop region containing 1115 bp. Phylogenetic analysis yielded a tree with high bootstrap value that was coherent with the current phylogeny proposed for Characiformes.

Complete mitochondrial DNA of Hoplias intermedius (Günther, 1864) (Ostariophysi: Characiformes: Erythrinidae)

Abstract Mitochondrial complete genome (mtDNA) of Hoplias intermedius, commonly known as “traírão” is presented. DNA from a muscle tissue sample was sequenced by next-generation sequencing. To corroborate the results, phylogenetic analysis was performed with MEGA6 to compare the complete mitogenome of H. intermedius with genomes of other Characiformes species. The mtDNA molecule was circular and 16,629 bp in length. Gene content included 22 tRNA genes, 2 rRNA genes, 13 protein-coding genes and a D-loop noncoding region. All genes had ATG as the start codon. Six genes carried complete stop codons of AGG (COI), TGA (ND6) and TAA (ND1, ATPase 8, ND4L and ND5), while seven genes had incomplete stop codons. Phylogenetic relationships with other Ostariophysi species placed H. intermedius together with H. malabaricus as a monophyletic group belonging to the order Characiformes.

Complete mitochondrial genome sequence of Myrmecophaga tridactyla from Brazilian Savanna

Abstract Myrmecophaga tridactyla, popularly known as giant anteater, is a member of Xenarthra magnorder which is under the threat of extinction. Herein, we describe the complete mitochondrial genome of M. tridactyla. The circular DNA molecule is 16,546 bp long, contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and a non-coding Control Region of 1110 bp. All protein-coding genes are on the heavy strand, except for Nd6. Ten of the 13 PCGs contained an ATG start codon.

Complete mitochondrial genome sequence of the high-altitude Brazilian tree frog Bokermannohyla alvarengai (Anura, Hylidae)

Abstract The first complete mitochondrial genome (mtDNA) for the genus Bokermannohyla (Hylidae) is presented. The mtDNA contains 17,325 bp and is similar in size, gene content, and gene location to other hylid mitochondrial genomes described, with 2 rRNA, 22 tRNA, and 13 PCGs. The control region (D-loop) is shorter than in mtDNAs of hylids from Asia. A phylogenetic tree based on homologous genes did not corroborate the monophyly of Hylidae neither the recently proposed monophyly of Hyla and Dryophytes.