Hybridization between native and nonnative fish species is a major conservation issue, especially in ecosystems with high levels of endemism, such as Iberian streams. To date, hybridization with the invasive bleak Alburnus alburnus has been reported for the Iberian chub Squalius alburnoides and S. pyrenaicus and in scattered locations only. However, the bleak is spreading in the region, potentially increasing the risks of hybridization with other Squalius species. To gather a more comprehensive picture on the current geography of hybridization, we compiled records on hybrids between bleak and chub in Portugal and conducted genetical assessments of hybrids between bleak and S. carolitertii. We found that hybridization with bleak is widespread throughout Portuguese river basins and involves at least S. alburnoides, S. pyrenaicus and S. carolitertii. Hybridization with bleak may not only cause waste of reproductive effort and damage the genetic integrity of these endemic species but also promote shifts in the reproductive dynamics of the S. alburnoides hybrid complex, which includes individuals with various ploidy levels and combinations of parental genomes, reproducing sexually and asexually. We recommend that future studies characterize the fitness of bleak hybrids and their ecological and genetic interactions with native fish, in order to design effective conservation measures.

Freshwater fish diversity is highly imperiled due to environmental change, habitat destruction, over-fishing and invasive species. Consequently, it is crucial to develop efficient and easy-to-standardize tools to describe these communities. Traditional fishing methods might not be able to capture all-size and well-hidden organisms, which can lead to an underestimation of biodiversity. In theory, it is possible to detect all species present in a certain locality by sequencing DNA traces left in the environment (eDNA). This approach is especially promising in freshwater systems, which may function as eDNA reservoirs of their respective drainage area. In this context, eDNA metabarcoding is the established method where amplicons for a specific locus are sequenced. However, primers affinity towards certain taxa can lead to false negatives and biased species abundances estimates. In this study, we tested eDNA ability and efficiency in detecting fish species in a threatened and highly endemic ecosystem: the Portuguese area occupied by the Tagus Basin. DNA present in the collected water samples from the Tagus River was amplified with 12S rRNA gene primers and sequenced with Illumina MiSeq technology. Primer choice was previously carried out by in silico PCR test which evaluates primers taxonomic coverage and species identification reliability. The obtained sequences were converted to 312 Amplicon Sequence Variance (ASV), filtered to decrease contamination errors, and compared to different reference databases. Obtained results were compared with conventional methods, such as electric fishing, considering that differences might shed light on which factors constrain species detection using eDNA metabarcoding. Preliminary results show congruences in fish species distributions between electrofishing and eDNA metabarcoding. However, eDNA metabarcoding detected exotic species that had not been previously detected in the Tagus basin, but which are known to exist in other Iberian basins: the South American cichlid—ustraloheros facetus—and a European leuciscid—Squalius cephalus. Nevertheless, traditional methods were more efficient in describing fish communities at some sites along the basin, showing the importance of using both approaches in a complementary way.

Biological invasions are considered one of the main extinction drivers of native species worldwide. Invasive species have detrimental effects on local ecosystems by means of competition, predation, habitat modification and nutrient cycling, as well as disease spreading. Along with ecological impacts, there are socio-economic consequences to human populations dependent on the services provided by these ecosystems. One of the most fundamental steps towards understanding the influence of invasive species is to determine their role on the local food web. The Mozambique tilapia (Oreochromis mossambicus) is an extremely aggressive opportunistic feeder that has a high biological and ecological plasticity, including flexible reproductive strategies (including mouthbrooding) and tolerance to a wide range of temperature, salinity and dissolved oxygen conditions. Its biological traits coupled with being used in aquaculture have made it a successful invader, widely distributed outside its native range. Yet, its populations have rarely been studied in the wild; hence, its potential impacts remain largely unknown. In this study, we investigate the diet of the invasive Mozambique tilapia in two mangroves of the oceanic island of São Tomé. We applied metabarcoding to simultaneously identify multiple taxa in tilapia gut content samples, using high-throughput sequencing. To achieve a greater taxonomic coverage, we combined the use of two barcodes, the 18S ribosomal RNA and the cytochrome C oxidase subunit I genes, to target phytoplankton and animal species, respectively. We found a total of 251 amplicon sequence variants belonging to 96 taxa. The results revealed diet differences between specimens from the two mangrove locations, not only regarding the level of biodiversity, but also in the frequencies of the occurrence of certain functional groups. Some taxa, such as diatoms, green algae and rotifers were found in the gut contents at both mangroves, whereas others, such as arthropods and mollusks, were almost exclusive to one of them. These findings provide useful insights into the ecological implications of the biological invasion of vulnerable island ecosystems, offering some specific guidance on how to minimize the impact of tilapia on the mangroves of the São Tomé Island.

Freshwater fish communities are highly threatened by human activities, making it necessary to establish methodologies able to efficiently monitor possible change. Recently, environmental DNA (eDNA) has shown to be one of the most promising sources of biodiversity information, especially when combined with high throughput sequencing approaches, such as DNA metabarcoding, allowing a simplified and efficient description of fish communities. However, metabarcoding relies on PCR amplification of eDNA, which has some limitations, such as priming biases, exclusion of shorter target eDNA fragments, limited taxonomic range, and limited taxonomic information in a single barcode. Alternatively, metagenomics sequences all eDNA fragments present in a sample without enriching for a specific taxonomic group, locus, or fragment length, overcoming these limitations. So far, eDNA metagenomics has mostly been implemented to describe microorganism communities and its applicability to large metazoans, such as fishes, is still understudied. In the current study, we test the power of this method to describe freshwater fish communities using water samples from the Ave and the Tagus rivers. Metagenomics is able to describe the whole freshwater biome by detecting taxa across the tree of life. When compared with eDNA metabarcoding, eDNA metagenomics provides more information in two aspects: First, a higher detection ability for fish species represented by low abundant and highly degraded DNA. Second, it differentiates between local and transported eDNA, which is not possible with eDNA metabarcoding. Our results also show a higher detectability for taxa represented by a whole genome sequence in the reference database. Therefore, some of the diversity is still being missed, since not all organisms have a genome available. Efforts are being made to produce genomic resources for all eukaryotes, meaning that in the future metagenomic data produced now will still be useful for the description of new diversity and characterization of community change.