Despite the economical and conservational importance of fisheries resources, they have been severely compromised by human induced activities that affect the sustainable utilization of such resources (Mboya et al. 2004). These anthropogenic activities include habitat destruction, overfishing and unregulated fish transfers (Eknath and Hulata 2009). They have altered the natural genetic structure of different fish species especially Oreochromis species through admixture and hybridization (Tibihika et al. 2020). Therefore, it’s essential to understand the extent of genetic divergence of these fishes as this helps in the efficient management of wild fish populations and for aquaculture activities. This information can be achieved through differentiation of the tilapiines using high highly informative genetic markers especially microsatellite and mtDNA genotyping.
Genetic diversity and differentiation of Oreochromis niloticus populations
The populations of Oreochromis niloticus were highly genetically diverse both at mtDNA and microsatellite loci compared to other species. Heterozygosity compares the amount of genetic variation within different populations (Nagy et al. 2012; Gu et al. 2014; Kajungiro et al. 2019). In the current study, the overall observed heterozygosity was lower than the expected heterozygosity for OreochromiS niloticus populations. Different studies have shown lower observed heterozygosity and attributed this to factors like presence of null alleles, sample size, inbreeding levels of the different Oreochromis species as well as Wahlund (1928) (D’amato et al. 2007; Gu et al. 2014; Kajungiro et al. 2019). For example, studies by Kajungiro et al (2019) observed lower heterozygosity in Oreochromis niloticus populations and attributed this to the small sample size which was used to infer the findings. Similarly, Gu et al (2014) found that observed heterozygosity in six populations in the primary rivers of Guangdong province were lower than the expected heterozygosity and attributed it to inbreeding at many loci and in all of the Oreochromis niloticus populations. Therefore, the current lower heterozygosity among the Oreochromis niloticus could be could be partially attributed to the occurrence of non-random mating among the populations since sample size of the population in each beach was at least 30. This indicates that the Oreochromis niloticus population is not affected by smaller amounts of genetic drift as populations are generally bigger thus having higher effective population size and consequently higher genetic diversity (Martinez et al. 2018). Additionally, the low heterozygosity levels could be due to the Wahlund effect (Wahlund, 1928) which indicates that observed heterozygosity is reduced as populations diverge in an aquatic environment.
In comparison with other species, Oreochromis niloticus showed a higher heterozygosity which generally translates to higher genetic diversity than other species. The study utilized 136 samples of Oreochromis niloticus collected from different beaches which was far larger than other species: 60, 57 and 30 for Oreochromis leucostictus, Coptodon zillii and Oreochromis esculentus respectively. This indicates that the Oreochromis niloticus population is not affected by smaller amounts of genetic drift as populations were generally bigger thus having higher effective population size and consequently higher genetic diversity (Martinez et al. 2018).
The admixtures observed among the Oreochromis niloticus populations as evidenced by PCoA and Structure outputs is attributed to the chances/instances of uncontrolled movement/transfer of fish from one location to another as well as increased escapees from aquaculture (Ndiwa et al. 2014) since most of the sampled beaches are close to each other and some directly connected to the streams and rivers. To further explore the dynamics of admixture within these populations, the distribution of Oreochromis niloticus haplotypes in the study was considered. All the haplotypes of Oreochromis niloticus are close to each other with less than five mutational changes indicating the higher levels of admixtures. All these confirm that there is human mediated geneflow between Oreochromis niloticus populations in the sampled beaches.
It is also expected that aquaculture activities might be contributing to the observed gene flow between the populations in these beaches. The increased aquaculture activities in the Rift Valley region have enhanced fish transfer from one drainage system to another allowing mixing between populations and or species (Ndiwa et al. 2014). The Economic stimulus program (ESP) introduced in 2009 by the Kenyan government has tremendously led to the establishment of many fishponds of which some are constructed near the wetlands, streams, rivers, and lakes (Munguti et al. 2014; Opiyo et al. 2018). During the heavy rains, the ponds get flooded leading to fish escape into Lake Victoria. Most of these ponds are not isolated from streams and wetlands, thus farmed fish can easily escape and Oreochromis niloticus. hybridize with autochthonous (Angienda et al. 2011; Ndiwa et al. 2014).
The current mtDNA results show a relatively higher haplotype diversity in Oreochromis niloticus compared to other tilapiines as more than five monomorphic haplotypes were observed. The current haplotype diversity for Oreochromis niloticus aligns with results (Hd: 0.800 in Oreochromis niloticus) obtained by Abdel-Hamid et al (2014). Since the introduction of Oreochromis niloticus dates back to 1950s in Lake Victoria, such time is enough for an introduced population to establish genetic divergence due to ecological tolerance and biological fecundity (Aloo 2003; Angienda et al. 2011; Firmat et al. 2013; Tibihika et al. 2020). Tibihika et al (2020) observed a higher diversity of Oreochromis niloticus and attributed this to the admixtures originating from several lineages as a result of multiple fish stockings. Therefore, the higher haplotype diversity of Oreochromis niloticus populations could be attributed to the widespread introductions and distributions of Oreochromis niloticus to different geographical areas within East African water bodies especially Lake Victoria. Additionally, the strong founder effects during colonization into new habitats may also contribute to the substantial genetic differentiation among the populations of Oreochromis niloticus.
The low genetic diversity of the other tilapiines in the study could be due to the hybridization levels with Oreochromis niloticus (Ndiwa. 2014). Haplotype 2 that was shared by Oreochromis niloticus from the field and Oreochromis niloticus sequences from the GenBank suggested that all these populations might be originating from similar maternal ancestors (Jiang et al. 2019). Therefore, the higher haplotype diversity indicates that the populations contain an abundant genetic resource for subsequent use in breeding or conservational measures.
Various approaches using both multivariate analysis; Principal Coordinates Analysis (PCoA) and Bayesian clustering algorithms (STRUCTURE) were used in this study to evaluate genetic structure of the Oreochromis species. The results of PCoA showed two main clusters with Oreochromis niloticus populations forming an independent cluster while other species (Oreochromis esculentus and Oreochromis leucostictus formed another cluster. Such independent clusters could be explained by the differences in mutation, selection associated with the evolutionary history of populations as well as drift and migration linked with the effects of fragmentation of populations and their demographic background (Martinez et al. 2018). For example, Oreochromis niloticus is primarily a phytoplankton feeder and dominates areas of dense algal stocks (Ndiwa et al. 2014; Laurent et al. 2020) while Oreochromis esculentus and Oreochromis leucostictus prefer habitats near papyrus fringes in littoral, shallow muddy bays, and lake inlets (Laurent et al. 2020). Probably such differences in habitat isolation contributes to the differences in PCoA and Structure outputs. Similarly, eight individuals of Oreochromis niloticus populations appeared in the cluster containing Oreochromis esculentus and Oreochromis leucostictus indicating that these individuals had some degree of admixtures. The presence of Oreochromis niloticus populations in the other cluster could be due to the potential misclassification of the species since admixed individuals resemble more Oreochromis niloticus and therefore they may have been misclassified (Angienda et al. 2011; Kariuku et al. 2021).
Oreochromis species from Lake Sare
Numerous studies on satellite lakes of Victoria basin have led to the discovery new fish species richness and genetic diversity which have not yet been sampled in the main Lake Victoria (Mboya et al. 2004; Abila et al. 2008; Angienda et al. 2011). Sare; a satellite lake provides refugia for different fish species. The lake is connected to main Lake Victoria by extensive Yala papyrus swamps that are anoxic to invasive predators like Nile perch and prevents entry of the Oreochromis niloticus species (Abila et al. 2008). In the current study, the obtained low heterozygosity of Oreochromis leucostictus and Oreochromis esculentus suggest low genetic variability for this species (López et al. 2007; Kajungiro et al. 2019) and could be attributed to bottlenecks caused by fast reduction of population size due to overfishing. Similarly, the haplotype and nucleotide diversity of the Oreochromis leucostictus and Oreochromis esculentus were lower (Hd=0.03; π=0.00 and Hd=0.00; π=0.00) respectively. Lake Victoria has been experiencing high fishing pressure leading to changes in the structure of the freshwater habitats, biodiversity, composition, and the productivity of the associated biota (Matsuishi et al. 2006). The loss of the fishing stocks has led to the loss of the genetic diversity of some fish stocks like Oreochromis esculentus and Oreochromis leucostictus (Mwanja et al. 2010; Angienda et al. 2011). This is because the increased fishing pressure results in phenotypic and genotypic changes in natural populations (Reznick and Ghalambor 2001). Contrary to our findings, studies by Angienda et al (2011) obtained a higher observed heterozygosity in Lake Kanyaboli compared to present findings where the observed heterozygosity was lower in Lake Sare. The differences could be attributed to the types of microsatellite markers used, may be the markers used by Angienda et al (2011) were more variable than the markers used in the current study. Secondly, Lake Sare being smaller than Kanyaboli limits the population size of the fish fauna thus low genetic diversity (Aloo 2003).
In the present study, the PCoA results showed intermediate clustering of the two Oreochromis species. The intermediate clustering could be associated with the hybridization which makes the species close to each other as they occupy similar ecosystem (Angienda et al. 2011; Laurent et al. 2020; Kariuku et al. 2021). Studies show that Oreochromis leucostictus and Oreochromis esculentus prefer habitats near papyrus fringes in littoral, shallow muddy bays, and lake inlets (Laurent et al. 2020). Therefore, this enhances their ability to randomly mate thus admixtures among the species. Mwanja et al (2010) observed that the populations of Oreochromis esculentus and Oreochromis leucostictus were close to each other than to Oreochromis niloticus and Oreochromis variabilis. This pattern of phylogenetically intermediates ‘mixed’ populations observed in the study provides strong evidence for the occurrence of hybridization and indicating the direction of introgression.
Oreochromis leucostictus
In the present study, the distinct populations of Oreochromis leucostictus from Sare and Victoria could be attributed to the physical isolation created by the main road and Yala swamp. The two lakes are separated by a natural wetland, the eco-physiological properties of these two lakes are different, which could also limit gene flow between the two populations by local adaptation or physiological barriers (Crispo and Chapman 2008). Although Oreochromis leucostictus from Victoria and Sare is generally differentiated based on the PCoA output, some samples occupied an intermediate position. Since these lakes are so close to each other and only separated by the main road and Yala swamp (Aloo 2003), it is clearly possible that multiple stockings/ fish transfers might be happening or happened which contributes to the observed gene-pool indicated by the intermediate populations. Oreochromis leucostictus populations of Lake Sare formed a subcluster and this is an indication that a pure stock still exists in the lake.
Hybridization between different species as a result of introductions
While hybridization events often occur among tilapiines following non-native species introductions into the natural environment, cases of hybridization between sympatric indigenous species are limited (Shechonge et al. 2018). In the present study, hybridization levels can be evidenced by the presence of the individuals of Oreochromis niloticus in a cluster containing Oreochromis leucostictus and the occurrence of haplotypes shared by Oreochromis niloticus and Oreochromis lecucostictus as indicated in other studies (Mwanja et al. 2010; Angienda et al. 2011; Deines et al.2014; Shechonge et al. 2018).
The current mtDNA results reveals that haplotype; 3, 15, 16 and 17 are shared among Oreochromis niloticus and Oreochromis leucostictus suggesting mtDNA introgression (Ndiwa et al. 2014) with a possibility of genetic admixture at the population phase. Other studies have indicated that Oreochromis niloticus has hybridized with other Oreochromis species (Deines et al. 2014; Blackwell et al. 2020; Diedericks et al. 2021) Elsewhere, studies by Ndiwa et al (2014) observed low hybridization levels between Oreochromis niloticus and Oreochromis leucostictus in Lake Naivasha Kenya. He also indicated that the haplotype of Oreochromis leucostictus from Lake Naivasha had been previously described to occur in Oreochromis niloticus from Lake Baringo population. Recent studies in Lake Edward-George system indicate that hybridization between Oreochromis niloticus and Oreochromis leucostictus is happening though rarely (Diedericks et al. 2021). Study by Deines et al (2014) found out that the native cichlids Oreochromis macrochir and Oreochromis andersonii hybridize in presence of the Oreochromis niloticus. The two native species do not naturally hybridise when in sympatry, suggesting that the presence of the non-native species facilitates hybridization events (Deines et al. 2014). In Tanzania, studies by Blackwell et al (2020) observed introgression levels between Oreochromis niloticus and Oreochromis korogwe Nambawala as well as hybrids between Oreochrmois urolepis and Oreochromis korogwe MlinganOreochromisThey attributed this to the introduced Oreochromis niloticus populations in Lake Nambawala that hybridized with the native species (Shechonge et al. 2018; Blackwell et al. 2020). Similarly, the genetic differentiation of the tilapiines based on the FST values indicates that Oreochromis niloticus is closer to Oreochromis leucostictus. This perhaps implies that gene flow is prominent among populations of Oreochromis niloticus with Oreochromis leucostictus and Oreochromis esculentus without physical barriers because of unregulated fish translocations and aquaculture activities (Shechonge et al. 2018; Laurent et al. 2020). Therefore, the present results contribute further evidence to the hypothesis that there is a low level of hybridization from Oreochromis niloticus into other Oreochromis species which likely threatens the conservation of these species.