DNA metabarcoding assessment of Neotropical ichthyoplankton communities is marker-dependent.

The study of ichthyoplankton is paramount to understanding fish assemblages' reproductive dynamics. DNA metabarcoding has been applied as a rapid, cost-effective, and accurate taxonomy tool, allowing the identification of multiple individuals simultaneously. However, there remain significant challenges when using DNA metabarcoding, such as molecular marker choice according to the taxonomic resolution and length of the fragment to be sequenced, primer bias, incomplete reference databases, and qualitative inference incongruences. Here, 30 ichthyoplankton pools collected from a Neotropical river were identified at a molecular level using DNA metabarcoding to compare the resolution, sensibility, specificity, and relative read abundance (RRA) recovery of three molecular markers: the standard COI fragment (650 pb, with each end analyzed individually) and two short 12S rRNA genes markers (≅200 bp - NeoFish and MiFish markers). The combined use of the three markers increased the genera detection rates by 25%-87.5%, allowing an increased taxonomic coverage and robust taxonomic identification of complex Neotropical ichthyoplankton communities. RRA is marker-dependent, indicating caution is still needed while inferring species abundance based on DNA metabarcoding data when using PCR-dependent protocols.

Phylogenomics reconciles molecular data with the rich fossil record on the origin of living turtles.

Although a consensus exists that all living turtles fall within either Pleurodira or Cryptodira clades, estimating when these lineages split is still under debate. Most molecular studies date the split in the Triassic Period, whereas a Jurassic age is unanimous among morphological studies. Each hypothesis implies different paleobiogeographical scenarios to explain early turtle evolution. Here we explored the rich turtle fossil record with the Fossilized Birth-Death (FBD) and the traditional node dating (ND) methods using complete mitochondrial genomes (147 taxa) and a set of nuclear orthologs with over 10 million bp (25 taxa) to date the major splits in Testudines. Our results support an Early Jurassic split (191-182 Ma) for the crown Testudines with great consistency across different dating methods and datasets, with a narrow confidence interval. This result is independently supported by the oldest fossils of Testudines that postdate the Middle Jurassic (174 Ma), which were not used for calibration in this study. This age coincides with the Pangaea fragmentation and the formation of saltwater barriers such as the Atlantic Ocean and the Turgai Strait, supporting that diversification in Testudines was triggered by vicariance. Our ages of the splits in Pleurodira coincide with the geologic events of the Late Jurassic and Early Cretaceous. Conversely, the early Cryptodira radiation remained in Laurasia, and its diversification ensued as all its major lineages expanded their distribution into every continent during the Cenozoic. We provide the first detailed hypothesis of the evolution of Cryptodira in the Southern Hemisphere, in which our time estimates are correlated with each contact between landmasses derived from Gondwana and Laurasia. Although most South American Cryptodira arrived through the Great American Biotic Interchange, our results indicate that the Chelonoidis ancestor probably arrived from Africa through the chain islands of the South Atlantic during the Paleogene. Together, the presence of ancient turtle diversity and the vital role that turtles occupy in marine and terrestrial ecosystems underline South America as a chief area for conservation.

Ichthyoplankton DNA metabarcoding: Challenges and perspectives.

DNA metabarcoding has been widely used to access and monitor species. However, several challenges remain open for its mainstream application in ecological studies, particularly when dealing with a quantitative approach. In a From the Cover article in this issue of Molecular Ecology, Mariac et al. (2021) report species-level ichthyoplankton dynamics for 97 fish species from two Amazon river basins using a clever quantitative metabarcoding approach employing a probe capture method. They clearly show that most species spawned during the floods, although ~20% also spawned mainly during the receding period and some other year-round, but interestingly, species from the same genus reproduced in distinct periods (i.e., inverse phenology). Opportunistically, Mariac et al. (2021) reported that during an intense hydrological anomaly, several species had a sharp reduction in spawning activity, demonstrating a quick response to environmental cues. This is an interesting result since the speed at which fish species can react to environmental changes, during the spawning period, is largely unknown. Thus, this study brings remarkable insights into basic life history information that is imperative for proposing strategies that could lead to a realistic framework for sustainable fisheries management practices and conservation, fundamental for an understudied and threatened realm, such as the Amazon River basin.

Space-time dynamics in monitoring neotropical fish communities using eDNA metabarcoding.

The biodiverse Neotropical ecoregion remains insufficiently assessed, poorly managed, and threatened by unregulated human activities. Novel, rapid and cost-effective DNA-based approaches are valuable to improve understanding of the biological communities and for biomonitoring in remote areas. Here, we evaluate the potential of environmental DNA (eDNA) metabarcoding for assessing the structure and distribution of fish communities by analysing water and sediment from 11 locations along the Jequitinhonha River catchment (Brazil). Each site was sampled twice, before and after a major rain event in a five-week period and fish diversity was estimated using high-throughput sequencing of 12S rRNA amplicons. In total, 252 Molecular Operational Taxonomic Units (MOTUs) and 34 fish species were recovered, including endemic, introduced, and previously unrecorded species for this basin. Spatio-temporal variation of eDNA from fish assemblages was observed and species richness was nearly twice as high before the major rain event compared to afterwards. Yet, peaks of diversity were primarily associated with only four of the locations. No correlation between ß-diversity and longitudinal distance or presence of dams was detected, but low species richness observed at sites located near dams might that these anthropogenic barriers may have an impact on local fish diversity. Unexpectedly high α-diversity levels recorded at the river mouth suggest that these sections should be further evaluated as putative "eDNA reservoirs" for rapid monitoring. By uncovering spatio-temporal changes, unrecorded biodiversity components, and putative anthropogenic impacts on fish assemblages, we further strengthen the potential of eDNA metabarcoding as a biomonitoring tool, especially in regions often neglected or difficult to access.

Integrative approach detects natural hybridization of sympatric lambaris species and emergence of infertile hybrids.

Despite its relevance for ecology, evolution and conservation of species, natural hybridization and hybrids biology are still poorly studied in freshwater fish. Here, we tested the hypothesis that sympatric species Astyanax paranae and A. fasciatus are able to interbreed in the natural environment and presented evidence for the first record of hybridization between these species. We analyzed anatomical traits, gametogenesis, reproductive biology, and genetic variations of the COI and S7 genes of both species and putative hybrids. Intermediate morphometric and meristic features were observed in hybrids when compared to A. paranae and A. fasciatus. Overlap in reproductive season was showed for these species, with greater reproductive activity from August to January, but hybrids did not present any sign of gonadal maturation. Oogonia and perinucleolar follicles as well as spermatogonia and primary spermatocytes were found in hybrids, but previtellogenic and vitellogenic follicles, spermatids, and spermatozoa were absent. Moreover, several alterations in gametogenesis were detected, such as interrupted meiosis in both males and females, vacuolated and degenerated germ cells, increased interstitial tissue, and presence of immune cells. Molecular analyses supported the hypothesis of hybridization between A. paranae and A. fasciatus. Overall, our multidisciplinary approach also provides strong evidence that hybrids are infertile.

The complete mitochondrial genome of the threatened Neotropical catfish Lophiosilurus alexandri (Silurifomes: Pseudopimelodidae) and phylogenomic analysis indicate monophyly of Pimelodoidea.

Lophiosilurus alexandri is an endemic catfish from the São Francisco River Basin (Brazil) popularly known as pacamã, which has economic potential for aquaculture farming. The mitochondrial genome was sequenced for the threatened Neotropical catfish L. alexandri. Assembly into scaffolds using MIRA and MITObim software produced the whole, circularized mitochondrial genome, which comprises 16,445 bp and presents the typical gene arrangement of Teleostei mitochondria. A phylogenomic analysis was performed after the concatenation of all proteins obtained from whole mitogenomes of 20 Siluriformes and two outgroups. The results confirmed the monophyly of nine families of catfishes and also clustered L. alexandri as a sister group to the family Pimelodidae, thus confirming the monophyly of the superfamily Pimelodoidea. This is the first mitochondrial phylogenomics study for Pimelodoidea and the first mitogenome described for the Pseudopimelodidae family, representing an important resource for phylogeography, evolutionary biology, and conservation genetics studies in Neotropical fishes.

Integrative taxonomy supports new candidate fish species in a poorly studied neotropical region: the Jequitinhonha River Basin.

Molecular identification through DNA barcoding has been proposed as a way to standardize a global biodiversity identification system using a partial sequence of the mitochondrial COI gene. We applied an integrative approach using DNA barcoding and traditional morphology-based bioassessment to identify fish from a neotropical region possessing low taxonomic knowledge: the Jequitinhonha River Basin (Southeastern Brazil). The Jequitinhonha River Basin (JRB) has a high rate of endemism and is considered an area of high priority for fish conservation, with estimates indicating the presence of around 110 native and non-indigenous species. DNA barcodes were obtained from 260 individuals belonging to 52 species distributed among 35 genera, 21 families and 6 orders, including threatened and rare species such as Rhamdia jequitinhonha and Steindachneridion amblyurum. The mean Kimura two-parameter genetic distances within species, genera and families were: 0.44, 12.16 and 20.58 %, respectively. Mean intraspecific genetic variation ranged from 0 to 11.43 %, and high values (>2 %) were recovered for five species. Species with a deep intraspecific distance, possibly flagging overlooked taxa, were detected within the genus Pimelodella. Fifteen species, only identified to the genus level, had unique BINs, with a nearest neighbor distance over 2 % and therefore, potential new candidate species supported by DNA barcoding. The integrative taxonomy approach using DNA barcoding and traditional taxonomy may be a remedy to taxonomy impediment, accelerating species identification by flagging potential new candidate species and to adequately conserve the megadiverse neotropical ichthyofauna.

The complete mitochondrial genome of the southern purple-spotted gudgeon Mogurnda adspersa (Perciformes: Eleotridae) through pyrosequencing.

The complete mitochondrial genome of the Critically Endangered southern purple-spotted gudgeon, Mogurnda adspersa, was determined for the first time using 1/8 of a 454 pyrosequencing plate. The mitogenome was assembled using the bioinformatic software MIRA. The M. adspersa genome organization was very similar to most vertebrates, being 16,523 bp in length. It contained 13 protein-coding genes, 22 transfer RNA, 2 ribosomal RNA genes, and 1 non-coding control region. The current methodology was effective to assemble the whole mitogenome using 2008 mitochondrial reads representing 0.23% of total genomic reads produced and providing average mitogenome coverage of 17.4 reads per site. The whole mitogenome sequence provided here is deposited in NCBI with the accession number KJ130031 and may benefit systematics studies and conservation programs of M. adspersa.

Integrative taxonomy detects cryptic and overlooked fish species in a neotropical river basin.

The great freshwater fish diversity found in the neotropical region makes management and conservation actions challenging. Due to shortage of taxonomists and insufficient infrastructure to deal with such great biodiversity (i.e. taxonomic impediment), proposed remedies to accelerate species identification and descriptions include techniques that combine DNA-based identification and concise morphological description. The building of a DNA barcode reference database correlating meristic and genetic data was developed for 75 % of the Mucuri River basin's freshwater fish. We obtained a total of 141 DNA barcode sequences from 37 species belonging to 30 genera, 19 families, and 5 orders. Genetic distances within species, genera, and families were 0.74, 9.5, and 18.86 %, respectively. All species could be clearly identified by the DNA barcodes. Divergences between meristic morphological characteristics and DNA barcodes revealed two cryptic species among the Cyphocharax gilbert and Astyanax gr. bimaculatus specimens, and helped to identify two overlooked species within the Gymnotus and Astyanax taxa. Therefore, using a simplified model of neotropical biodiversity, we tested the efficiency of an integrative taxonomy approach for species discovery, identification of cryptic diversity, and accelerating biodiversity descriptions.

The complete mitochondrial genome of two recently derived species of the fish genus Nannoperca (Perciformes, Percichthyidae).

Here we report the complete sequence of mitochondrial genomes for two sister taxa of freshwater teleosts, the recently derived Yarra pigmy perch Nannoperca obscura and the southern pigmy perch Nannoperca australis. These represent the first complete mitochondrial genomes for Percichthyidae (Perciformes), a family mostly distributed in Australia. The de novo genome assembly of 316,430 pyrosequencing reads from 454 libraries has produced the entire mitochondria for N. obscura and a nearly complete version for N. australis. The mtDNA genome from the latter was completed through the design of one primer set and standard Sanger sequencing for genome finishing, followed by the hybrid assembly of reads with MIRA software using N. obscura sequence as reference genome. The complete mitogenomes of N. obscura and N. australis are 16,496 and 16,494 bp in size, respectively. Both genomes contain 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes and a control region. Several characteristics of mitochondria typically found in teleost fishes were detected, such as: (i) most genes found in the heavy strand, with the exception of ND6 and eight tRNA genes; (ii) avoidance of G as the third base of codons; (iii) presence of gene overlapping; (iv) percentage of bases usage. We found only eight indels and 197 nucleotide substitutions between these Nannoperca mitogenomes, consistent with a previous hypothesis of recent speciation. The data reported here provide a resource for comparative analysis of recent evolution of mitochondrial genomes.

Genetic characterization of native and introduced populations of the neotropical cichlid genus Cichla in Brazil.

A molecular phylogenetic analysis based on mitochondrial 16S ribosomal DNA and Control Region sequences from native and introduced populations was undertaken, in order to characterize the introduction of Cichla (peacock bass or tucunaré) species in Brazil. Mitochondrial DNA haplotypes found in introduced fish from Minas Gerais state (southeastern Brazil) clustered only with those from native species of the Tocantins River (Cichla piquiti and C. kelberi), thereby suggesting a single or, at most, few translocation acts in this area, even though with fish from the same source-population. Our study contributes to an understanding of the introduction of Cichla in regions of Brazil outside the Amazon basin, and adds phylogenetic data to the recently describe Cichla species, endemic from the Tocantins-Araguaia basin.

Genetic characterization of native and introduced populations of the neotropical cichlid genus Cichla in Brazil

A molecular phylogenetic analysis based on mitochondrial 16S ribosomal DNA and Control Region sequences from native and introduced populations was undertaken, in order to characterize the introduction of Cichla (peacock bass or tucunaré) species in Brazil. Mitochondrial DNA haplotypes found in introduced fish from Minas Gerais state (southeastern Brazil) clustered only with those from native species of the Tocantins River (Cichla piquiti and C. kelberi), thereby suggesting a single or, at most, few translocation acts in this area, even though with fish from the same source-population. Our study contributes to an understanding of the introduction of Cichla in regions of Brazil outside the Amazon basin, and adds phylogenetic data to the recently describe Cichla species, endemic from the Tocantins-Araguaia basin.