The megadiverse Neotropical fish fauna lacks a comprehensive and reliable DNA reference database, which hampers precise species identification and DNA based biodiversity assessment in the region. Here, we developed a mitochondrial 12S ribosomal DNA reference database for 67 fish species, representing 54 genera, 25 families, and six major Neotropical orders. We aimed to develop mini-barcode markers (i.e. amplicons with less than 200 bp) suitable for DNA metabarcoding by evaluating the taxonomic resolution of full-length and mini-barcodes and to determine a threshold value for fish species delimitation using 12S. Evaluation of the target amplicons demonstrated that both full-length library (565 bp) and mini-barcodes (193 bp) contain enough taxonomic resolution to differentiate all 67 fish species. For species delimitation, interspecific genetic distance threshold values of 0.4% and 0.55% were defined using full-length and mini-barcodes, respectively. A custom reference database and specific mini-barcode markers are important assets for ecoregion scale DNA based biodiversity assessments (such as environmental DNA) that can help with the complex task of conserving the megadiverse Neotropical ichthyofauna.

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Hypomasticus copelandii is a Neotropical freshwater fish widely distributed across coastal drainages of southeastern Brazil, a highly impacted region of South America. The interspecific phylogenetic relationships within the genus and the taxonomic status of the species remain uncertain. Using two mitochondrial and one nuclear locus, we performed a phylogenetic, species delimitation, and time-calibrated analyses to test the hypothesis that H. copelandii is a species complex currently delimited by different Atlantic coastal systems. Results indicate that H. copelandii presents two well-delimited genetic lineages: one in the northern drainages of the Jucuruçu, Mucuri and Doce rivers, and the other in the southern region represented by the Paraíba do Sul River Basin. The time-calibrated phylogeny indicated a split between the two genetic lineages at around 2.8 million years ago (Ma), which might be related to headwater capture events during the Plio-Pleistocene. The discovery of a distinct genetic lineage for H. copelandii suggests distinct management plans for the northern and southern drainages. Such hidden diversity within the H. copelandii provides useful information for taxonomy and conservation across a severely impacted region of Brazil.

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 study of ichthyoplankton composition, abundance and distribution is paramount to understand the reproductive dynamics of local fish assemblages. The analysis of these parameters allows the identification of spawning sites, nursery areas and migration routes. However, due to the lack of characters in early life stages, the morphological identification of ichthyoplankton is often impractical and many studies identify only fish larvae. Additionally, its accuracy shows great variation between taxonomists and laboratories according to their experience and specialty. DNA barcoding emerged as an alternative to provide assertive identification of fish eggs and larvae, but it becomes too expensive and laborious when the study demands the processing of huge amounts of organisms. DNA metabarcoding can overcome these limitations as a rapid, cost-effective, broad and accurate taxonomy tool, allowing the identification of multiple individuals simultaneously. Here, we present the identification of a sample containing 68 fish eggs and another containing 293 fish larvae from a single site in the São Francisco River Basin, Eastern Brazil, through DNA metabarcoding. We used a low-cost saline DNA extraction followed by PCR amplification with three primer sets targeting the 12S rRNA gene: MiFish (~170bp), Teleo_1 (~60bp), and NeoFish (~190bp). The latter was recently developed by our research group specifically for the identification of Neotropical fishes. All the amplified samples were sequenced in a single multiplexed Illumina MiniSeq run. We performed the filtering steps and assigned Amplicon Sequence Variants (ASVs) using a DADA2/Phyloseq based pipeline and a custom 12S reference sequence database including 101 species and 70 genera from the Jequitinhonha and São Francisco basins. The species Cyphocharax gilbert, Leporinus taeniatus, Megaleporinus elongatus, Prochilodus argenteus, P. costatus and Psalidodon fasciatus were detected by all three primer sets in the larva pool, while Pterygoplichthys etentaculatus was detected solely by NeoFish (Fig. 1). Within the egg pool, all three markers detected the species Characidium zebra, Curimatella lepidura, M. elongatus, Pimelodus fur and P. costatus, but Brycon orthotaenia was detected only by NeoFish, P. maculatus only by Teleo, and P. pohli by MiFish and Teleo (Fig. 1). The consistency in species detection among all three markers underpins the credibility of this method to accurately describe the sample composition. Considering that most of species were exclusive to the larvae or egg pool, our experiment highlights the importance of including the identification of fish eggs in reproduction studies, as it can provide additional information about which species are spawning in an area. Furthermore, the application of DNA metabarcoding to the study of ichthyoplankton can help decision makers create more informed guidelines for conservation of economically and ecologically important fish species.

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 inferences 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 (650pb, with each strand analyzed individually) and two short 12S rRNA genes markers ( 200bp - NeoFish and MiFish markers). The combined use of the three markers increased the genera detection rates by 25% to 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 whilst inferring species abundance based on DNA metabarcoding data when using PCR-dependent protocols.

Species richness is a metric of biodiversity usually used in fish community assessment for monitoring programs. This metric is often obtained using traditional fisheries methods that rely on capture of target organisms, resulting in underestimation of fish species. DNA metabarcoding has been recognized as a powerful noninvasive alternative tool for fish biomonitoring and management. Despite the increasing popularity of this method for the assessment of aquatic megadiverse ecosystems, its implementation for studying the highly diverse Neotropical ichthyofauna still presents some challenges. One of them is to devise what primer set could reliably amplify the DNA of all fish species from a megadiverse river basin and have enough resolution to identify them. In order to identify and overcome these drawbacks, we have investigated the efficiency of the metabarcoding approach on Neotropical fishes using a mock sample containing genomic DNA of 18 fish species from the Jequitinhonha River basin, Eastern Brazil. We compared three primer sets targeting the 12S rRNA gene: two universal and widely used markers for fish metabarcoding [MiFish (~170bp) and Teleo_1 (~60bp)], and NeoFish (~190bp), recently developed by our research group specifically for the identification of Neotropical fishes (Milan et al., 2020). Two samples amplified using three primers were sequenced in a single multiplexed Illumina MiniSeq run, using normalized and non-normalized pools. Bioinformatic analyses were performed using a DADA2/Phyloseq based pipeline to perform filtering steps and to assign Amplicon Sequence Variants (ASVs). We used a custom 12S reference sequence database that included 190 specimens representing 101 species and 70 genera from the Jequitinhonha and São Francisco river basins. A total of 187 ASVs were recovered: 79, 66 and 42 for NeoFish, MiFish and Teleo_1, respectively. ASVs of unexpected species were identified for both pools (Fig. 1), though each of these ASVs had an abundance of less than 50 copies. In addition, species of the Hoplias and Prochilodus genera could not be identified at the species level, due to identical sequences within each genus, possibly because of the insufficient variation within the 12S region recovered by these primers’ amplicons. Unexpectedly, although a single individual of each species was placed in the pools, more than one ASV was identified for some species, likely caused by PCR biases. Overall, all primer sets displayed similar taxonomic resolution for the DNA pools and recovered all species, except for NeoFish, which could not detect Steindachneridion amblyurum due to an incompatibility in the 3’ of the NeoFish forward primer and Teleo_1, which could not identify Steindachnerina elegans. These results highlight the need of reliable databases in order to enable the full assignment of ASVs and OTUs to species level, and the importance of calibrating the DNA metabarcoding approach with mock samples to identify weaknesses and pivotal steps prior to the application on large scale DNA based biodiversity evaluation, that can help with the complex task of conserving the megadiverse Neotropical ichthyofauna.