**6. Application of molecular markers**

#### **6.1. Identification of Neotropical cryptic species**

mtDNA has been a marker of choice for reconstructing historical patterns of population demography, admixture, biogeography and speciation [88, 176] and can help identify cryptic individuals in many Neotropical fish species [177]. However, the main problem in genetic studies aimed at the maintenance of biodiversity is the difficulty of developing a method of species identification, since there are millions of unidentified and unknown species. The use of DNA barcodes, segments of approximately 600 bp of the mitochondrial gene cytochrome oxidase I (COI), has been considered an efficient technique to catalogue all biodiversity. The Neotropical freshwater ichthyofauna is considered the most diverse in the world, and very few fish species have been identified. It has been estimated that 30–40% of species have not been described, and genetic identification is a challenge, even with molecular techniques [176, 177].

Barcode research has already been performed in the São Francisco River Basin and provided evidence of the effectiveness of barcodes to catalogue the diversity of Neotropical basins by discovering new species and genera (*Hisonotus* sp.), expanding the range of known species (*Knodus moenkhausii*) and identifying overlooked species (*Bryconamericus stramineus*, *Piabina argentea* and *Poecilia* sp.) [178]. Analyses on 254 species of fish from the Upper Parana River Basin in Brazil correctly identified 252 species using their barcode sequences, including a large number of closely related species [179]. Moreover, comparative analyses using traditional morphological taxonomy and DNA barcoding of Neotropical ichthyoplankton from the Upper Paraná and São Francisco Rivers showed no conflicting results between the two techniques [180].

Advances in the use of barcodes have also been achieved in the Pampas plain region of Argentina and have shown that specimens of *S. brasiliensis*, *R. quelen*, *H. malabaricus*, *Synbranchus marmoratus*, *Australoheros facetus*, *O. jenynsii* and *Corydoras paleatus* differed by more than 3% from their conspecifics in other parts of South America. Overall, this study was able to highlight the likely occurrence of cryptic species, showing evidence of hidden diversity in the Neotropical region [181]. Although these results are important, more barcode research studies in Neotropical ichthyofauna are needed.

#### **6.2. Genetic variability**

Levels of genetic diversity between individuals in the same population and between populations are essential for species conservation in the face of environmental changes. In general, most of the wild populations tend to have high levels of genetic diversity [182]. This is largely due to formation of these groups by migratory fish, representing panmictic populations, since high gene flow and the size of the population reduce the effects of genetic drift [183].

Several factors that may interfere in the fragmentation of populations, or their migratory potential, may cause a population bottleneck and decrease the genetic variability. Bottlenecks reduce population size by making individuals subject to genetic drift and inbreeding, thereby reducing the species evolutionary potential [184].

Several studies carried out in the Paraná River Basin have already demonstrated a decline and genetic homogenisation among fish populations in this basin [185–188]. These studies indicate that the fragmentation of the basin due to the large hydroelectric dams installed in the Paraná River Basin, mainly in the Upper Paraná region, is one of the major factors affecting these populations.

Brazil is the third largest producer of hydroelectric power, accounting for up to 10% of total world production. The conversion of free-flowing tropical rivers into the regulated systems associated with hydroelectric dams is one of the major concerns for the conservation of freshwater Neotropical fish. In addition to the impact on water velocity and temperature, hydroelectric dams block the natural river flow that affects freshwater fish populations due to habitat fragmentation, with increased risks of population isolation and consequent destruction of gene flow. This has already been reported using microsatellite markers for *Prochilodus argenteus* in the São Francisco River [168] and *Brycon insignis* in the Brazilian southeast [189].

In order to mitigate the damage caused by hydroelectric dams, programmes to reintroduce affected species are a potential solution. However, lack of knowledge about the genetics of local species can have the opposite effect. Analyses of restocking programmes for *P. argenteus* indicate differences between stock populations and wild populations, and this differentiation represents a risk and interrupts the diversity of local genes [190].

In addition to hydroelectric dams and inappropriate programmes for genetic restocking, the inadequate management of cultivated populations may also interfere with the genetic variability of species. Fish escaping from aquaculture facilities may influence the level of genetic diversity in natural populations living in the vicinity of fish farms. The introduction of cultivated individuals to wild populations may result in a mixture of populations with different genetic characteristics that reduce the average genetic diversity (Wahlund effect), as has already been observed in many fish populations [191, 192].

#### **6.3. Genetic structure**

that are considered one of the most captured species in the Neotropical region due to their high commercial value and potential in aquaculture [172, 173]. Other studies on SNPs (identified by the Pool-seq technique) refer to the evolutionary adaptation of species such as *Poecilia* 

studies regarding the identification of SNPs in the sex chromosomes of *Characidium gomesi* by

mtDNA has been a marker of choice for reconstructing historical patterns of population demography, admixture, biogeography and speciation [88, 176] and can help identify cryptic individuals in many Neotropical fish species [177]. However, the main problem in genetic studies aimed at the maintenance of biodiversity is the difficulty of developing a method of species identification, since there are millions of unidentified and unknown species. The use of DNA barcodes, segments of approximately 600 bp of the mitochondrial gene cytochrome oxidase I (COI), has been considered an efficient technique to catalogue all biodiversity. The Neotropical freshwater ichthyofauna is considered the most diverse in the world, and very few fish species have been identified. It has been estimated that 30–40% of species have not been described, and genetic identification is a challenge, even with molecular techniques [176, 177]. Barcode research has already been performed in the São Francisco River Basin and provided evidence of the effectiveness of barcodes to catalogue the diversity of Neotropical basins by discovering new species and genera (*Hisonotus* sp.), expanding the range of known species (*Knodus moenkhausii*) and identifying overlooked species (*Bryconamericus stramineus*, *Piabina argentea* and *Poecilia* sp.) [178]. Analyses on 254 species of fish from the Upper Parana River Basin in Brazil correctly identified 252 species using their barcode sequences, including a large number of closely related species [179]. Moreover, comparative analyses using traditional morphological taxonomy and DNA barcoding of Neotropical ichthyoplankton from the Upper Paraná and São Francisco Rivers showed no conflicting results between the two

Advances in the use of barcodes have also been achieved in the Pampas plain region of Argentina and have shown that specimens of *S. brasiliensis*, *R. quelen*, *H. malabaricus*, *Synbranchus marmoratus*, *Australoheros facetus*, *O. jenynsii* and *Corydoras paleatus* differed by more than 3% from their conspecifics in other parts of South America. Overall, this study was able to highlight the likely occurrence of cryptic species, showing evidence of hidden diversity in the Neotropical region [181]. Although these results are important, more barcode

Levels of genetic diversity between individuals in the same population and between populations are essential for species conservation in the face of environmental changes. In general,

S) in Mexico [174] and

*mexicana* in waters with high hydrogen sulphide concentrations (H<sup>2</sup>

**6. Application of molecular markers**

264 Biological Resources of Water

techniques [180].

**6.2. Genetic variability**

**6.1. Identification of Neotropical cryptic species**

research studies in Neotropical ichthyofauna are needed.

the RAD-seq technique with the aim of differentiating males and females [175].

As mentioned previously, Neotropical ichthyofauna is subject to many environmental factors that may affect their rate of retention in the environment of origin, including the destruction of their habitat and consequent fragmentation of populations. The effects on the spatial distribution of fish populations may result in genetic processes that affect gene frequency, including dispersive processes, gene oscillation and founder effects. These genetic processes intensify systematic migration, mutation and selection. Due to the high levels of polymorphisms and abundance throughout the genome, molecular markers are useful for genetic structure analyses in different populations [193]. Studies directed towards the verification of structures of Neotropical populations using microsatellite markers are concentrated on populations affected by the construction of dams.

stages or post-F1 generations, means they can only be differentiated using molecular marker techniques. Initially, non-PCR-dependent molecular methods (using allozymes and RFLPs) were used to identify hybrids of serrasalmid species [200]. These markers are not currently used due to the advantages of PCR techniques. Mendelian codominant PCR markers, such as microsatellites and SNPs, are suitable for hybrid identification and introgression events. However, more studies are required to define genetic markers, such as SNPs, that are essential for the identification of fish hybrids, together with production monitoring and management measures, particularly in detecting escaped fish hybrids in the natural environment [55].

Genetic Applications in the Conservation of Neotropical Freshwater Fish

http://dx.doi.org/10.5772/intechopen.73207

267

Alternative and less costly techniques, such as PCR-RFLP and multiplex-PCR, are easier to carry out and have proved to be efficient methodologies that can be quickly and inexpensively executed, allowing the identification by simple PCRs based on single nucleotide polymorphisms [55]. The PCR-RFLP method allows the analysis of DNA variation. Base substitutions in specific fragments formed at the enzyme recognition sites result in patterns of restriction fragments [202]. Multiplex-PCR uses species-specific primers for determining loci that differ between the analysed species by a few nucleotide substitutions, and two or more reactions can take place in the same tube [203]. PCR-RFLP and multiplex-PCR techniques are well established for the identification of hybrids between Neotropical species [55, 204]. However, genetic monitoring of hybridisation programmes should be applied in a routine way to verify whether the trade and management of hybrids are being performed correctly in fish breeding farms.

Traceability is the ability to identify species and their origin. It is considered important for the conservation of natural stocks and for certification of food quality. DNA-based methodology of traceability has greater reliability and accuracy and is an important tool for the conservation of threatened stocks of Neotropical fish. Furthermore, SNP arrays for species identification, or for identification of a specific population, can be used in processed fish samples that have been frozen, salted, cooked and canned with a high attribution power. This makes it possible to identify the origin of the fish consumed and avoid commercial fishing in places with threatened stocks. However, the fish traceability test alone is not sufficient to reduce the decline in fish numbers; rather, traceability techniques should be used in conjunction with sustainable fisheries, by-catch reduction and management-based policies [125, 154, 205]. Despite traceability research in fish populations worldwide to avoid predatory and indiscriminate overfishing, there is still a lack of important studies related to DNA traceability markers in freshwater Neotropical fish species.

The methodological advances and the development of sequencing technologies can enable an efficient applicability of molecular markers in the conservation of Neotropical fish. Despite the negative impact that human activities have had on fish from the Neotropical region (such as deforestation, construction of dams, overfishing and non-native species introduction to the basins), there are few genetic studies into population structure, genetic variability and hybrid

**6.5. Traceability of Neotropical fish**

**7. Conclusion**

identification.

Many freshwater fish species that inhabit Neotropical rivers have migratory behaviour and reproduce during the rainy season, when water levels increase and temperatures rise. Normally, fish migration occurs in the main river or its tributaries for the spawning of eggs that are subsequently carried downstream to the floodplains, where they find suitable conditions for development [19]. This ability to migrate long distances suggests that these fish species constitute a single panmictic population, as reported in several studies of *Prochilodus mariae* [194], *Brycon hilarii* [195] and *P. mesopotamicus* [196] using microsatellite analysis. Many studies have found genetic structuring on microsatellite loci in different migratory species. This is not necessarily a spatial structure, but a temporal structure between individuals that share the same habitat with overlapping reproductive periods, as shown for *Prochilodus costatus* [197] and *S. brasiliensis* [198] in different Neotropical hydrographic basins.

Some research has also been carried out using mtDNA for population structure analyses. D-loop regions were used to infer structural analyses of populations of pacu (*P. mesopotamicus*) from the Paraguay River and in four other main tributaries, showing high gene flow and the formation of a single panmictic population. This is due to the flood cycle that regulates the interconnectivity between different environments and allows gene flow between populations, forming metapopulations [42].

There are few studies that use SNPs for the conservation of Neotropical freshwater species, and there are insufficient data to evaluate the genetic structure of natural stocks. More genetic studies using SNP markers for species identification need to be conducted in order to better understand population structure and to develop management measures and conservation policies [172, 173].

#### **6.4. Identification of Neotropical hybrid fish**

In the Neotropical region, particularly in Brazil, serrasalmid and pimelodid hybrids represent important advances in aquaculture. Hybrid fish originating from serrasalmid species such as the pacu (*P. mesopotamicus*), tambaqui (*C. macropomum*) and pirapitinga (*P. brachypomus*), or from pimelodid species such as the pintado (*P. corruscans*) and cachara (*Pseudoplatystoma reticulatum*), are considered commercially valuable and have high growth rates and have other characteristics that are useful to the commercial sector such as resistance and better reproductive performance [199]. The interest in hybrid production may be due to the lack of knowledge about the pure species, mainly in genetic breeding approaches [200]. The tambacu (*C. macropomum* female crossed with *P. mesopotamicus* male) and patinga (*P. mesopotamicus* female crossed with *P. brachypomus* male) correspond to a large part of Brazilian aquaculture production [201]. However, the uncontrolled production of fertile hybrids, such as the patinga, can have negative consequences for the environment. Improper management may result in fish escaping into the rivers and threatening the genetic integrity of pure species with free occurrence of backcrossing.

Genetic technologies for hybrid fish identification include cytogenetic methods and PCR techniques. The morphological similarity of fish hybrids to their parental species, mainly in juvenile stages or post-F1 generations, means they can only be differentiated using molecular marker techniques. Initially, non-PCR-dependent molecular methods (using allozymes and RFLPs) were used to identify hybrids of serrasalmid species [200]. These markers are not currently used due to the advantages of PCR techniques. Mendelian codominant PCR markers, such as microsatellites and SNPs, are suitable for hybrid identification and introgression events. However, more studies are required to define genetic markers, such as SNPs, that are essential for the identification of fish hybrids, together with production monitoring and management measures, particularly in detecting escaped fish hybrids in the natural environment [55].

Alternative and less costly techniques, such as PCR-RFLP and multiplex-PCR, are easier to carry out and have proved to be efficient methodologies that can be quickly and inexpensively executed, allowing the identification by simple PCRs based on single nucleotide polymorphisms [55]. The PCR-RFLP method allows the analysis of DNA variation. Base substitutions in specific fragments formed at the enzyme recognition sites result in patterns of restriction fragments [202]. Multiplex-PCR uses species-specific primers for determining loci that differ between the analysed species by a few nucleotide substitutions, and two or more reactions can take place in the same tube [203]. PCR-RFLP and multiplex-PCR techniques are well established for the identification of hybrids between Neotropical species [55, 204]. However, genetic monitoring of hybridisation programmes should be applied in a routine way to verify whether the trade and management of hybrids are being performed correctly in fish breeding farms.

#### **6.5. Traceability of Neotropical fish**

structure analyses in different populations [193]. Studies directed towards the verification of structures of Neotropical populations using microsatellite markers are concentrated on popu-

Many freshwater fish species that inhabit Neotropical rivers have migratory behaviour and reproduce during the rainy season, when water levels increase and temperatures rise. Normally, fish migration occurs in the main river or its tributaries for the spawning of eggs that are subsequently carried downstream to the floodplains, where they find suitable conditions for development [19]. This ability to migrate long distances suggests that these fish species constitute a single panmictic population, as reported in several studies of *Prochilodus mariae* [194], *Brycon hilarii* [195] and *P. mesopotamicus* [196] using microsatellite analysis. Many studies have found genetic structuring on microsatellite loci in different migratory species. This is not necessarily a spatial structure, but a temporal structure between individuals that share the same habitat with overlapping reproductive periods, as shown for *Prochilodus costa-*

Some research has also been carried out using mtDNA for population structure analyses. D-loop regions were used to infer structural analyses of populations of pacu (*P. mesopotamicus*) from the Paraguay River and in four other main tributaries, showing high gene flow and the formation of a single panmictic population. This is due to the flood cycle that regulates the interconnectivity between different environments and allows gene flow between populations,

There are few studies that use SNPs for the conservation of Neotropical freshwater species, and there are insufficient data to evaluate the genetic structure of natural stocks. More genetic studies using SNP markers for species identification need to be conducted in order to better understand population structure and to develop management measures and conservation

In the Neotropical region, particularly in Brazil, serrasalmid and pimelodid hybrids represent important advances in aquaculture. Hybrid fish originating from serrasalmid species such as the pacu (*P. mesopotamicus*), tambaqui (*C. macropomum*) and pirapitinga (*P. brachypomus*), or from pimelodid species such as the pintado (*P. corruscans*) and cachara (*Pseudoplatystoma reticulatum*), are considered commercially valuable and have high growth rates and have other characteristics that are useful to the commercial sector such as resistance and better reproductive performance [199]. The interest in hybrid production may be due to the lack of knowledge about the pure species, mainly in genetic breeding approaches [200]. The tambacu (*C. macropomum* female crossed with *P. mesopotamicus* male) and patinga (*P. mesopotamicus* female crossed with *P. brachypomus* male) correspond to a large part of Brazilian aquaculture production [201]. However, the uncontrolled production of fertile hybrids, such as the patinga, can have negative consequences for the environment. Improper management may result in fish escaping into the rivers and threatening the genetic integrity of pure species with free occurrence of backcrossing. Genetic technologies for hybrid fish identification include cytogenetic methods and PCR techniques. The morphological similarity of fish hybrids to their parental species, mainly in juvenile

*tus* [197] and *S. brasiliensis* [198] in different Neotropical hydrographic basins.

lations affected by the construction of dams.

266 Biological Resources of Water

forming metapopulations [42].

**6.4. Identification of Neotropical hybrid fish**

policies [172, 173].

Traceability is the ability to identify species and their origin. It is considered important for the conservation of natural stocks and for certification of food quality. DNA-based methodology of traceability has greater reliability and accuracy and is an important tool for the conservation of threatened stocks of Neotropical fish. Furthermore, SNP arrays for species identification, or for identification of a specific population, can be used in processed fish samples that have been frozen, salted, cooked and canned with a high attribution power. This makes it possible to identify the origin of the fish consumed and avoid commercial fishing in places with threatened stocks. However, the fish traceability test alone is not sufficient to reduce the decline in fish numbers; rather, traceability techniques should be used in conjunction with sustainable fisheries, by-catch reduction and management-based policies [125, 154, 205]. Despite traceability research in fish populations worldwide to avoid predatory and indiscriminate overfishing, there is still a lack of important studies related to DNA traceability markers in freshwater Neotropical fish species.
