Development and Characterization of Fifteen Polymorphic Microsatellite Loci for Rare and Endangered Species within Luciobarbus Heckel, 1843 Genus in the Aral Basin and Their Conservation Application

: Biodiversity conservation entails not only the preservation of specific taxa but also genetic diversity. Despite the crucial role of molecular data in freshwater fish conservation management, there is a scarcity of information regarding the genetic diversity of Luciobarbus Heckel, 1843 (Actinopterygii, Cyprinidae) populations in the Aral system. Therefore, the primary aim of this study was to provide genetic information on two native species of the Luciobarbus genus found in the Aral system: L. conocephalus (Kessler, 1872) and L. brachycephalus (Kessler, 1872). These species, like many others in the Aral system, confront the imminent threat of extinction due to system alterations. However, genetic studies on these species at the nuclear level are challenging because Luciobarbus is an allote-traploid genus. Consequently, genetic investigations thus far have focused mainly on sequencing mitochondrial genes due to their haploid nature. This study has successfully developed fifteen new polymorphic microsatellite loci, which can prove to be valuable for population genetics, conservation, and other pertinent research on these species.


Introduction
The cyprinid family includes 152 genera and within freshwater fishes, this family stands out as the most diverse, comprising a remarkable 1695 species [1].Within the cyprinid fishes, the genus Luciobarbus Heckel, 1843 includes 51 medium to large-sized species distributed along rivers draining into the Persian Gulf, the Mediterranean, the Black, Caspian, and Aral Seas and into the Atlantic Ocean, from the Iberian Peninsula in Europe to Central Asia and North Africa [2][3][4].
The Aral Sea was once the world's fourth-largest inland body of water in terms of surface area [5].A lake basin, fed by two rivers, the Amu Darya and the Syrdarya, supports a diverse ecosystem and boasts a highly economically valuable fishery [6].Currently, the Aral Sea is facing severe environmental degradation primarily attributable to a rapid reduction in its water level.The main consequence of this dwindling water level is an increase in Fishes 2024, 9, 169 3 of 12 resources in one of the ecosystems, the Aral Sea, which has been severely impacted by human activities.

Materials and Methods
Specimens of barbels were collected from the Aral Basin from May to September 2022.Along the Syrdarya River, specimens belonging to the genus Luciobarbus were found at five sampling points.The names and geographical coordinates of each locality are presented in Table 1.A map of the river basins (Figure 1) was constructed using QGIS 3.22.13(QGIS Development Team 2021).
Eighty-one individuals were collected from the sampling points using nets.Fin clips were preserved in 96% ethanol.All the samples were collected using the appropriate permits from the Ministry of Ecology and Natural Resources of the Republic of Kazakhstan.The specimens were released alive back into the river, with the exception of a small selection of 2 specimens from each population, and fin fragments are now deposited in the Collections and BioBank of the National Museum of Natural Sciences (MNCN-CSIC) in Madrid, Spain.For the first time, the developed microsatellite markers were tested in the MNCN-CSIC for the current populations analyzed.Total genomic DNA from the sampled tissues was obtained using Qiagen DNeasy 96 Blood and Tissue kits (QiagenTM, Venlo, Netherlands) following the manufacturer's recommendations.
PCRs were performed in a final volume of 12.5 µL containing 1 µL of DNA (10 ng/L), 6.25 µL of Type-it Microsatellite PCR Kit (Qiagen), 4 µL of PCR grade water, and 1.25 µL of the 10X primer mix.The optimal PCR protocol consisted of an initial denaturation step at 95 • C for 5 min: 30 cycles of 95  C for 90 s, 72 • C for 30 s; and a final extension step at 68 • C for 30 min.All the PCRs included a negative control to verify possible contamination.The tails of the oligonucleotides were attached to the 5 ′ ends of the primers for fluorescent labeling.The oligonucleotide tails used were the universal sequences M13 (GGA AAC AGC TAT GAC CAT), CAG (CAG TCG GGC GTC ATC) and T3 (AAT TAA CCC TCA CTA AAG GG).The oligonucleotide tails were labeled with PET, NED, and VIC dyes.The amplified PCR products were processed on an ABI Prism 3730 DNA Analyzer (250-500 LIZ size standards).Allele assignment was performed using GENEMAPPER 3.7 (Applied Biosystems, Foster City, CA, USA).The genetic diversity of each locus was estimated using the GenoDive v.3.0 program.[42], which allows the analysis of polyploid organisms.For this purpose, the following indices were estimated: the number of alleles per locus (N A ), the effective number of alleles per locus (A E ), the observed heterozygosity (H o ), the heterozygosity within populations (H s ), the total heterozygosity (H t ), the corrected total heterozygosity (H 't ), and the inbreeding coefficient (G is ).The genetic differentiation between populations was performed based on the G'st statistics [43].The significance value for this parameter was estimated using 999 permutations.Previously, MICRO-CHECKER v2.23 [44] was used to explore the existence of null alleles and evaluate their impact on the estimation of genetic differentiation.Eighty-one individuals were collected from the sampling points using nets.Fin clips were preserved in 96% ethanol.All the samples were collected using the appropriate permits from the Ministry of Ecology and Natural Resources of the Republic of Kazakhstan.The specimens were released alive back into the river, with the exception of a small selection of 2 specimens from each population, and fin fragments are now deposited in the Collections and BioBank of the National Museum of Natural Sciences (MNCN-CSIC) in Madrid, Spain.For the first time, the developed microsatellite markers were tested in the MNCN-CSIC for the current populations analyzed.Total genomic DNA from the sampled tissues was obtained using Qiagen DNeasy 96 Blood and Tissue kits (QiagenTM, Venlo, Netherlands) following the manufacturer's recommendations.
A total of 17 primer pairs were designed for microsatellite loci (Table 2).These primers were grouped into 4 sets of multiplex (1: M1287, M1417, M1182; 2: M2237, M2108, M2044, M2164, M2306; 3: M3230, M3264, M3318, M3444; 4: M4211, M4455, M4215, M4138, M4474) PCRs according to the primer properties, forward and reverse sequences, expected amplicon size, repeat motif, tail of the primer, and type of fluorescent marker used.Polymorphism information content (PIC) is a way to evaluate the ability of genetic markers to detect polymorphisms among individuals.This parameter was calculated using the following formula [45]: A model based on Bayesian clustering was used to study the population structure.The number of populations (K) with the highest posterior probability (mean lnProb [D]) was calculated using the program STRUCTURE 2.3.4 [46], assuming an admixed model and a uniform prior probability of the number of populations, K, and a ploidy of 4 (tetraploids).The MCMC simulations consisted of 1 × 10 6 burn-in iterations followed by 1 × 10 5 sampled iterations.Furthermore, the modal values of λ and ∆K [47] were also calculated to infer the best value of K in [48].
To determine the amount of genetic structuring among grouping levels, an analysis of molecular variance (AMOVA) [49] was carried out to estimate the percentage of variation in the allele frequency of the microsatellites among and within each population, grouping them by sampling location (population) for L. brachycephalus.The AMOVA was carried out in the GenoDive v.3.0 program [42].

Results
No evidence of shadow banding or genotyping errors was found, and the frequency of null alleles was low.Based on the allele frequencies observed in the analysis of 15 loci across 81 individuals, a total of 90 alleles were identified, accounting for a missing data percentage of 10.93%.The number of alleles per locus varied between 2 and 12, with a polymorphism information content (PIC > 0.5) of 0.774, indicating high polymorphism.Notably, one locus, M2108, exhibited a significant deviation from Hardy-Weinberg equilibrium (p = 0.0001).
The AMOVA revealed that the highest percentage of genetic variation for all the L. brachycephalus samples from four sampled populations was explained by the "within individuals" component (98.4%), while a low percentage of genetic variation explained the among individual and among population components at 0.3% and 1.3%, respectively.
For all measures of genetic diversity, including the average number of alleles per locus, the effective number of alleles, and heterozygosity, L. brachycephalus from the Basykara Dam consistently displayed higher values compared to all the other sampling locations.Within the examined populations of L. brachycephalus, the population closest to the Aral Sea estuary exhibited the lowest diversity.
The inbreeding coefficient values were statistically significant for all the studied populations, except for the population of L. brachycephalus from Rice Check, which is located in the headwaters of the Syrdarya River watershed (Table 3).Among the seventeen tested microsatellites, fifteen loci amplified successfully (Table 4) and were polymorphic; two of the loci (M3318 and M4455) were monomorphic and they were excluded from subsequent analyses.Among the fifteen amplified loci, M2044, M4215, and M1287 showed the greatest degree of polymorphism (Table 4).
Structural analyses did not reveal a clear genetic structure between the four populations of L. brachycephalus (Figure 2, clusters 1-4), which were separated from sampling site 5, represented by L. conocephalus (Figure 2).On detecting the population structure for the analyzed barbels from different sampling points in the Aral-Syrdarya Basin, bar plots showing the division of runs by mode for the optimal (K = 5) number of clusters were generated (Figure 2), although no genetic structure was found.Only two clusters of both species were separated: one with populations of the species L. conocephalus (sampling point 5) and the other with populations of L. brachycephalus (sampling points 1-4).However, some individuals belonging to L. conocephalus of the 2 and 4 at the sampling points collapsed into differentiated clusters (Figure 2).However, we assume that four specimens from sample points 2 and 4 (Kyzylorda region and Rice Check) are indeed L. conocephalus and not the result of introgression.Apparently, these four specimens were misidentified in the field.It is also worth noting that four specimens were caught in these places, although L. brachycephalus is traditionally found there.
Genetic introgression, also known as introgressive hybridization, is the transfer of genetic material from one species into the gene pool of another by repeated backcrossing of the interspecific hybrid to one of its parent species.Genetic introgression from native species is recognized as a detrimental impact resulting from biological invasions involving taxonomically similar invaders.The ecological consequences of genetic introgression from an invasive congener were tested using the endemic barbel populations from central Italy, where the invader was the European barbel Barbus barbus.The results indicate that the genetic introgression of an invasive congener from native species can result in substantial ecological consequences, including the potential for cascading effects [50].
However, our genetic analysis provided support for a low level of introgression of L. conocephalus into L. brachycephalus in the Bayesian cluster where L. brachycephalus was only present (one sampling point in Bairykum village), but not vice versa, indicating limited genetic exchange between the two species.
Bayesian structure analysis with microsatellites revealed differences between individuals of the two species; however, no evidence of intraspecific genetic structure was found within L. brachycephalus.
Basin, bar plots showing the division of runs by mode for the optimal (K = 5) number of clusters were generated (Figure 2), although no genetic structure was found.Only two clusters of both species were separated: one with populations of the species L. conocephalus (sampling point 5) and the other with populations of L. brachycephalus (sampling points 1-4).However, some individuals belonging to L. conocephalus of the 2 and 4 at the sampling points collapsed into differentiated clusters (Figure 2).However, we assume that four specimens from sample points 2 and 4 (Kyzylorda region and Rice Check) are indeed L. conocephalus and not the result of introgression.Apparently, these four specimens were misidentified in the field.It is also worth noting that four specimens were caught in these places, although L. brachycephalus is traditionally found there.
Genetic introgression, also known as introgressive hybridization, is the transfer of genetic material from one species into the gene pool of another by repeated backcrossing of the interspecific hybrid to one of its parent species.Genetic introgression from native species is recognized as a detrimental impact resulting from biological invasions involving taxonomically similar invaders.The ecological consequences of genetic introgression from an invasive congener were tested using the endemic barbel populations from central Italy, where the invader was the European barbel Barbus barbus.The results indicate that the genetic introgression of an invasive congener from native species can result in substantial ecological consequences, including the potential for cascading effects [50].
However, our genetic analysis provided support for a low level of introgression of L. conocephalus into L. brachycephalus in the Bayesian cluster where L. brachycephalus was only present (one sampling point in Bairykum village), but not vice versa, indicating limited genetic exchange between the two species.The studied species exhibit significant morphological similarities, making it rather challenging to differentiate between them.In cases where their geographical ranges overlap, their separation primarily relies on ecological factors related to habitat and lifestyle (including migratory and resident forms).Our nuclear study effectively distinguished between these two species without any ambiguities.It is worth noting that previous research [17] suggested the possibility of interbreeding between them.
These microsatellite loci can also be used for intrageneric genetic identification, according to the results obtained, since individuals of L. conocephalus show clear differentiation when divided into clusters.For this purpose, we also noted private alleles for L. conocephalus, such as M1447, M0244, and M4215 as well as M2237, which is only one locus that is not valid for individuals of this species.

Discussion
In this article, we present the first results of a study with nuclear microsatellite markers for two Luciobarbus species inhabiting the Aral Basin.The nuclear markers analyzed in this study represent the first microsatellites isolated from Luciobarbus and increase the available molecular resources for these tetraploid species, which makes it difficult to study the nuclear genome.These microsatellites have been suggested to be informative for the analysis of the genetic diversity and structure of two Luciobarbus species inhabiting the Aral Basin and they will provide significant insights into species genetic diversity and population structure in other species of the allotetraploid genus Luciobarbus, assisting in conservation and other relevant studies for its more vulnerable species.
Most of the habitats of rare and endangered barbels in the Aral Basin in Kazakhstan were surveyed, and the sampling points covered were Bairykum village, the Kyzylorda region, the Basykara Dam, Rice Check, and the Badam River.The rarity of the species L. conocephalus compared with L. brachycephalus has attracted attention because this population has a very limited distribution area in the geographical context of the Aral-Syrdarya Basin within Kazakhstan.In addition to the drying of the Aral Sea and the erosion that this part of the basin has suffered, conservation measures should be implemented to prevent future actions that jeopardize the conservation of these two Luciobarbus species.
Of the 17 analyzed loci, 15 were polymorphic for all individuals; microsatellites were used to study the genetic structure and diversity of both L. brachycephalus and L. conocephalus.The number of alleles per locus is estimated to range from 2 to 12, namely M2044 and M4474-12, M1287-11, M4215-9, and M3444-8, with an average value of 6, in comparison with the number of alleles that fluctuate from 2 to 7 in other species [51][52][53][54].Additionally, at two points where L. brachycephalus is usually recorded, four individuals of L. conocephalus were also found, which was confirmed by our genetic results.
The contribution of this study, with fifteen newly developed microsatellites for Luciobarbus, is highly relevant given the precarious conservation status of the two species analyzed, especially L. conocephalus.The use of these microsatellites will allow us to gain a better understanding of the evolutionary history of the species within this genus and support the conservation of these rare and endangered species.
In this article, we embarked on a pioneering endeavor by isolating and employing microsatellite markers to unravel the enigmatic genetic landscape of the Luciobarbus genus within the confines of the Aral Basin.This landmark achievement significantly augments the molecular toolkit available for the study of Luciobarbus, a genus that has long confounded researchers due to its tetraploid nature.As such, the legacy of this research is poised to cast a long-lasting impact on the realms of ichthyology, genetics, and conservation biology.

Conclusions
In the conservation of biodiversity, the assessment of genetic diversity plays an important role.The conservation of a system of many genetically diverse local populations and their structure is necessary for the long-term survival of species and the functioning of ecosystems.
Therefore, the main goal of this study was to obtain genetic information about two native species of the genus Luciobarbus living in the Aral system: L. conocephalus (Kessler, 1872) and L. brachycephalus (Kessler, 1872).As a result of this research, nuclear markers for evaluating the genetic structure and diversity were successfully developed and applied.These microsatellite loci can also be used for intrageneric genetic identification.It should be noted that based on the level of genetic diversity, small, isolated populations are distinguished that show an average level of genetic diversity.
Despite the rare and endangered status of the barbels of the Aral Basin, in-depth studies to identify their population structure and assess their genetic diversity have not been carried out before this work.With the help of molecular genetic methods, it has been possible to largely solve this problem and make a significant contribution to the study of the genetic diversity of barbels, which has been unclear for more than 30 years.The results obtained from this study can be used to develop effective, science-based plans for the conservation of barbels and can be used by environmental, fishery, and other organizations whose activities are aimed at the protection, reproduction, and rational use of fish resources.
Reduced genetic diversity reduces the ability of a population to adapt to future environmental changes, such as biotic and abiotic changes, reducing the plasticity of population genomes.Further research is needed because these species, like many others in the Aral system, face an imminent threat of extinction due to changes in the drying water system.This emphasizes the need for continued investigation into these complexities and their implications.

Table 1 .
Sampling points and species of this study.

Table 3 .
Indices of genetic diversity for L. brachycephalus and L. conocephalus at each sampling point in the Aral-Syrdarya Basin.
a : number of alleles; N e : effective number of alleles; H o : observed heterozygosity; H e : expected heterozygosity; G is : inbreeding coefficient.

Table 4 .
Indices of genetic diversity per locus.
a : number of alleles; N e : effective number of alleles; H o : observed heterozygosity; H s : heterozygosity within populations; H t : total heterozygosity: H 't ; corrected total heterozygosity; G is : inbreeding coefficient.