**4. Genetic applications**

introduced to southern and southeastern regions of Brazil over 20 years (1970–1990) due to the difficulty of producing native species [71], a trend that has declined in recent years, though it still exists [21]. Hydroelectric companies have begun to produce native species for restocking (repopulation), but for this to be successful, evaluation of the efficiency and genetic quality of the parents is essential [21, 72]. In repopulation programmes, genetic monitoring is a fundamental step, since a reduction in genetic variability reduces the adaptability of the species to different environmental conditions and interferes significantly with the survival of young fish [66, 73]. The use of molecular markers has been shown to be effective for genetic management in order to maximise diversity and reduce inbreeding in the repopulation centres [67, 68, 74].

Aquatic organisms are fragile and sensitive to a wide range of stressors. Reproduction, growth and population survival are highly dependent on water quality. Environmental pollutants such as metals and pesticides present a serious risk to local ichthyofauna. The physiological effects of toxicants include disruption of hormonal, neurological and metabolic systems and elimination of behaviours that are essential to fitness and survival in natural ecosystems [75]. Studies into many Neotropical fish have corroborated this. *Prochilodus lineatus* subjected to 7 days of *in-situ* tests in a contaminated urban stream suffered a series of epithelial lesions, lamellar fusion and aneurysms in the gills [76]. Another study carried out with *Rhamdia quelen* exposed to herbicides (Roundup® Original, Primoleo® and Facet®) showed harmful effects in the gills, liver, kidneys and muscle [77]. *A. altiparanae* and *P. lineatus* exposed to biodegradable detergents in an urban lake showed various changes in gill function such as lamellar fusions, aneurysms, mucous and chlorine cell proliferation [78]. A study involving *A. altiparanae* showed that the level if DNA breaks was most sensitive biomarker to contamination with pesticides [79]. *P. lineatus* showed nickel accumulation in different tissues (kid-

Mining activities impact the aquatic ecosystem in the basins of Upper Paraguay and in the Colombian, Brazilian and Peruvian Amazon [6]. Mercury, the main compound released during gold mining, accumulates in the sediment and in the muscle and tissues of fish (bioaccumulation). This means that through the trophic chain, the predators that are high in the food chain tend to accumulate more metals (biomagnification). Fish in the rivers of Madre de Dios city (Perú), affected by illegal mining, revealed that the species *Calophysus macropterus*, *Pseudoplatystoma punctifer*, *P. fasciatum*, *H. malabaricus*, *Prochilodus nigricans*, *Hydrolycus pectoralis*, *Plagioscion squamosissimus* and *Zungaro zungaro* showed high levels of mercury accumulation in tissues that exceeded the maximum limit permitted by the World Health Organisation (500 μgHg/kg) [81]. The same was found in the Upper Pantanal (Brazil), in the Poconé and Nossa Senhora do Livramento regions (Upper Paraguay River), where piscivorous or detritivorous species such as *Pimelodus muculatus*, *P. fasciatum*, *P. lineatus*, *Salminus maxillosus*, *P. corruscans*, *Acestrorhynchus altus*, *Serrasalmus nattereri* and *H. malabaricus* also had high levels of mercury [82, 83]. Other metals that accumulate in fish organs are nickel and copper [80,

Mining can also lead to the collapse of dams, as occurred in Mariana city (Minas Gerais state, Brazil) in 2015 that was considered the biggest environmental disaster in Brazil that released

of mining waste directly into the watershed of the River Doce,

neys, liver, gills and muscle) with DNA damage [80].

256 Biological Resources of Water

84], causing significant damage to the fish species and its consumers.

approximately 55–62 million m3

Genetic tools are important resources for the conservation of Neotropical fish species. The biology and population dynamics of the species are still unknown due to insufficient research. In spite of the high diversity that characterises Neotropical fish, there are many species with a large geographical distribution and differing population structure. Along a hydrographic basin, one can find many populations, from panmictic populations of long-distance migratory species, characterised by large gene flow, to restricted populations of local organisms with well-defined population structures [88]. Research into the verification of variability and genetic structure of populations belonging to different river basins will aid the construction of policies and management measures for the maintenance of natural populations. In addition, genetic tools are increasingly being used to molecularly identify new species that was previously impossible due to morphological similarities. Furthermore, various anthropogenic activities in aquaculture and pollution have been increasingly studied at the molecular level, particularly with respect to research into hybrid fish and the effects of contaminants.

Biodiversity is conceptualised into distinct biological levels (genetic, species, community and ecosystem) that have each been impacted by human activities. The impact on genetic diversity is one of the biggest concerns, affecting species adaptation and taxa speciation [89, 90]. Knowledge of how the genetic diversity of Neotropical fish is maintained and how the populations are structured is important to determine how these species can be conserved. Many species of freshwater fish display genetic variation with adaptive traits that enhance survival and reproduction in particular environments and increase the capability of the organisms to adapt to environmental changes and anthropogenic activities [90].

Genetic variability in populations can be measured by the allele number and heterozygosity [91]. Intrapopulation variability is influenced by factors such as mutation, genetic drift and natural selection. Genetic variation originates from mutations and decreases in genetic drift that increases the interpopulation differentiation due to a finite population size, with gene flow occurring between populations [92]. Conversely, natural selection can reduce genetic variation by allele fixation [93]. Anthropogenic activities, such as habitat fragmentation, increase the risks of genetic drift and gene flow reduction, diminishing the genetic variability of populations and interrupting flow of the adaptive genes leading to extinction of some species [90]. Molecular genetic markers have emerged as a powerful tool to identify genetic variability in populations [94] and have had a substantial impact on the fields of ecology, evolution and conservation [95].

The identification of cryptic species is an important genetic application for the ecology and conservation of Neotropical freshwater fish. This taxonomic challenge has been overcome due to the advent and availability of rapid DNA sequencing for detecting and differentiating morphologically similar species [22]. The destruction and disturbance of river basins, especially those caused by human interference, have led to the threat of complete extinction of several fish species [96]. However, many species exposed to these threats are still undescribed, and efforts to catalogue and identify these fish are increasingly important. Most species have been described by morphological and typological characteristics [97]. However, speciation is not always accompanied by differences in morphology, and due to the difficulty of identification, the actual number of existing fish species is greater than previously described [22].

markers, high genome coverage markers [single nucleotide polymorphisms (SNPs)] and

Genetic Applications in the Conservation of Neotropical Freshwater Fish

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Allozymes were considered the first molecular marker, discovered in the 1960s in enzymes. When DNA sequences of two or more alleles in the same locus are divergent, and the corresponding RNA encodes different amino acids, multiple variants of the same protein are created. However, not every mutation in a DNA sequence results in changes to the amino acid sequences, and this is one of the disadvantages of using an allozyme as a molecular marker [107]. Other disadvantages include heterozygote deficiencies due to null alleles and the amount and quality of tissue samples required [108]. The limitations and disadvantages of

In the 1980s, the first DNA-based molecular markers were developed. They can be classified into dominant and codominant markers. It is not possible to identify heterozygotes in dominant markers, whereas in codominant markers, this differentiation can be determined, and it is possible to estimate allele frequencies. Molecular markers can also be classified into those with known function (type I markers) or with anonymous regions (type II markers) [108].

RFLP markers were the first markers discovered that were based on DNA sequences [109]. They are considered codominant markers and are type I or type II. They are based on bacterial enzymes that recognise specific DNA sequences. The DNA is then cut into fragments where these sequences are found. The digestion of DNA by restriction enzymes results in fragments that vary between individuals, populations and species. The fragments can be analysed using the polymerase chain reaction (PCR), and the PCR products are digested by restriction enzymes. RFLP markers have low potential in determining genetic variation when compared to new, recently discovered molecular markers, mainly due to the low level of polymorphism. In addition, sequence information of the specimen is required, making it difficult to determine markers in species without molecular information. However, one advantage of these

RAPD techniques use PCR amplification of random anonymous segments of genomic DNA with identical pairs of primers at 8–10 bp in length. Unlike RFLP markers, RAPD does not require any knowledge of DNA sequences of the organism. Therefore, nearly all RAPD markers are dominant, and it is not possible to distinguish whether a DNA segment is amplified from a heterozygous or homozygous locus [110]. The primers used are short and anneal at low temperatures, amplifying multiple products from different loci. Due to the fact that most of the nuclear genome is non-coding, most amplified loci are neutral. Genetic variation is assessed by considering each band as a bi-allelic locus, with the presence or absence of the amplified product generated by PCR. One disadvantage of this technique is the intensity

these markers led to the development of DNA-based genetic markers.

**5.2. Restriction fragment length polymorphisms (RFLP)**

markers is that they are codominant [108].

**5.3. Randomly amplified polymorphic DNA (RAPD)**

maternal inheritance markers (mtDNA).

**5.1. Allozymes**

DNA sequencing has introduced a new method of species discovery known as DNA barcodes [98]. DNA barcodes are short and standardised sequences from a part of the mitochondrial genome that can be used to distinguish different species. This differentiation can easily be determined when genetic variation between species exceeds that within species [99]. The barcode sequence from each unknown specimen is then compared with a library of reference barcode sequences derived from individuals of known identity. Research has been carried out to evaluate the effectiveness of this technique in identifying cryptic species in insects [100], birds [101] and plants [102]. The diversity Neotropical freshwater ichthyofauna is the richest in the world and make up around 25% of the total freshwater fish fauna on Earth [5]. However, the lack of knowledge of their diversity makes taxonomic identification a great challenge.

Genetic methods facilitate the identification of cryptic species and species with few identifiable phenotypic characteristics. The presumed neutrality of some molecular markers, in conjunction with phylogenetic methods, provides a new perspective on species identification, especially in hierarchical relatedness and relative rates of evolution. The increased frequency with which cryptic species can be discovered with DNA sequence data, and often subsequently confirmed with morphological and/or ecological data, suggests that molecular data should be routinely incorporated into taxonomic research.

Another major problem for the natural populations of Neotropical fish (that can be reduced or controlled using genetic resources) is accidental or deliberate release of non-native fish species [103]. Hybridisation is the mating of genetically differentiated individuals and may involve individuals within a species or between species [104]. Conventional approaches to detect interspecific hybridisation include morphometric and molecular analyses. In recent years, DNA polymorphisms have been used for investigating fish hybridisation [105]. Nuclear genetic markers, in particular, allow hybrid species identification because contributions to the hybrid genome of both the father and mother can be identified [106].
