3.1. D-loop Haplotypes and Phylogenetic Relationships
A DNA sequence of 391 bp covering the hypervariable region (280 bp) and the first part of the central conserved region (111 bp) of the mitochondrial D-loop region was analyzed. There were 79 variable sites and 68 parsimony-informative sites. Twenty-seven haplotypes were identified among the 69 individuals sequenced (Table A1
). The first two haplotypes were exhibited among nearly 50% (N
= 32) of all the individuals from every site except Akosombo Tilapia Farm. Every geographic site, which exhibited haplotypes 1 and 2 included at least three individuals; except for Fujian Farm, in which a single individual exhibited haplotype 1. Of the remaining 25 haplotypes, 17 were private haplotypes observed in collections from wild sites only or farmed sites only. The private haplotypes from farmed sites were dominated by samples from two farms, Fujian Farm and Akosombo Tilapia Farm. One haplotype was shared by those two farms, Fujian Farm and Akosombo Tilapia Farm, and the Lower Volta River.
The results of the phylogenetic analysis of mitochondrial D-loop sequences showed two genetically distinct clusters with 100% posterior support (Figure 2
and Table A2
), which also were distinct from that of the outgroup species, C. zillii
. The first cluster, which we term the “native tilapia cluster”, contained 17 haplotypes, including haplotypes 1 and 2 (the haplotypes observed in the majority of both wild and farmed individuals), as well as seven “wild” haplotypes (of the eight observed) originating from the Black Volta River (haplotypes 17–23, Table A1
and Figure 2
). The “O. niloticus
” cluster also contained the sample from Cote d’Ivoire (Onilo_CD), the GenBank sequence Onilo_Kpa11 originally sampled from the Volta basin in Ghana by Falk et al. [48
] as part of a genetic study of black-chinned tilapia, Sarotherodon melanotheron
, and one sample from the U.S. grocery-store tilapia reference group (Onilo_WA1).
The second cluster, which can be described as the “non-native tilapia cluster”, further divided into two sub-clusters with 94% posterior support (Figure 2
). The first sub-cluster within the “non-native tilapia” cluster, which we term the “O. mossambicus
” cluster in Table A2
, contained all five GenBank sequences for O. mossambicus
, four private haplotypes from the Fujian Farm (haplotypes 10, 11, 14, and 16) and one private haplotype from the Akosombo Tilapia Farm (haplotype 27), one of the U.S. grocery-store tilapia samples (KB1), and the GenBank O. niloticus
_GIFT strain sequence.
The second sub-cluster, which we term the “O. niloticus
introgressed with O. mossambicus
” cluster in Table A2
, consisted of a GenBank sequence of an O. niloticus
× O. mossambicus
hybrid, five haplotypes, including two private haplotypes from three sites—Fujian Farm (haplotype 15), the Lower Volta River (haplotype 3), and Akosombo Tilapia Farm (haplotype 26); haplotype 4 shared by the Fujian Farm, Lower Volta River, and Akosombo Tilapia Farm; and haplotype 13 shared by Fujian Farm and Akosombo Tilapia Farm. The “O. niloticus
introgressed with O. mossambicus
” cluster also contained all five East African reference sequences; seven of the nine U.S. grocery-store tilapia reference samples, and the three GIFT-related strains (Egyptian strain, Filipino strain and the American strain).
Pairwise nucleotide Tamura-3 parameter distances provided further support for the clustering observed. The genetic distances were considerably larger between clusters than within clusters (Table A3
). For instance, the genetic distances (i.e., dissimilarity) between the “O. niloticus
” cluster and the “O. mossambicus
” cluster, and the “O. niloticus
introgressed with “O. mossambicus
” cluster ranged from about 14–16% and 13–15% respectively, compared to the largest within-group distance of 1.7% for the “O. niloticus
” cluster (Table A3
). Divergence based on fixed nucleotide differences at the variable sites further supported the inference that individuals in the “O. niloticus
” cluster were genetically distinct from individuals in the inferred “O. mossambicus
” and “O. niloticus
introgressed with O. mossambicus
” clusters (Table A2
3.3. Genetic Variability in Microsatellite Genotypes
All ten microsatellite loci screened were polymorphic. However, results from Microchecker analysis showed evidence of null alleles at locus UNH925 for several populations. Loci UNH130 and UNH925 consistently showed departure from HWE across all sites, and locus UNH130 showed evidence for segregation of null alleles in some populations. Thus, we excluded data from loci UNH925 and UNH130 from subsequent analysis. Significant departure from HWE was evident in the Akosombo strain (ARDEC) at loci UNH858 and UNH898; Lee’s Farm at UNH180 and UNH858; Volta Catch at UNH180 and UNH898; Fujian Farm at UNH858; Lower Volta River at UNH123, UNH180, and UNH858; and the Black Volta River at all loci except UNH991. We observed significant departures from linkage equilibrium in the Fujian Farm (one pair of loci), Black Volta River (four pairs of loci), and Lee’s Farm (seven pairs of loci) samples after Bonferroni correction.
provides the summary statistics calculated to show the variation across the eight loci included in the study. Mean observed and expected heterozygosities were moderate to high across sites, and ranged between 0.60 and 0.80, and 0.64 and 0.80, respectively (Table A4
). The Volta Catch, ARDEC, and Black Volta River collections exhibited the lowest, while the Fujian Farm, Akosombo Tilapia Farm, and Lee’s Farm collections showed the highest mean observed and expected heterozygosities. Similarly, numbers of alleles were moderate to high across sites and ranged between 6.63 and 9.75. Volta Catch recorded the lowest number of alleles, while the Lower Volta River, Black Volta River and Fujian Farm populations recorded the highest mean numbers of alleles (> 9.0).
Private alleles were observed in all, but the ARDEC population. The highest number of private alleles was observed in the Fujian Farm stock (N = 7), followed by Akosombo Tilapia Farm (N = 4). The Lee’s Farm and Volta Catch samples had three and two private alleles, respectively. The Fujian Farm and Akosombo Tilapia Farm stocks shared eight infrequent alleles. The two farmed populations also shared a number of infrequent alleles exclusively with either the ARDEC population or with Lee’s Farm population. The Lower Volta River population also shared infrequent alleles with the Fujian and Akosombo Tilapia farm stocks.
estimates revealed moderate to high genetic differentiation among the farmed and wild populations, which were significantly different from zero (Table A5
). In general, the Fujian Farm and Akosombo Tilapia Farm stocks were similar to one another, but differentiated from all the other farmed populations. The least differentiation was observed between Fujian Farm and Akosombo Tilapia Farm (FST
= 0.00), while the greatest was between Fujian Farm and Volta Catch (FST
= 0.21). The AMOVA also showed high differentiation among populations, with 11% of the variance explained by differences among populations (Table 3
). The Fisher’s exact G
test and the locus-by-locus FST
also showed highly significant differentiation (p
< 0.000) across all loci for all sites combined.
Structure analysis using the admixture model and the most probable number of K
selected using the Evanno et al. [40
] method revealed greatest support for K
= 2 clusters within and among the farmed and nearby wild populations analyzed, with high associated probabilities of assignment (Q
ranged between 0.95 and 0.99, Figure 4
). In general, individuals from Fujian Farm and Akosombo Tilapia Farm grouped into one cluster (shown in orange), while all other farmed populations and the reference to wild populations grouped into another cluster (shown in blue). On the other hand, the LnP
) values revealed the greatest support for K
= 6 clusters (Figure 4
, bottom plot). However, two distinct groups were evident within the six clusters. The first group comprised individuals in the ARDEC, Volta Catch, Lee’s Farm, Lower Volta River, and Black Volta River populations, while the second group comprised individuals from the Fujian and Akosombo Tilapia farms. These outcomes suggested hierarchical genetic structuring, with two high-level populations, a native Nile tilapia group and a non-native Nile tilapia group. Two individuals from the Lower Volta River (LV02 and LV03) (N
= 33) showed evidence of high levels of admixture and clustered with the non-native tilapia group from Fujian Farm and Akosombo Tilapia Farm (Q
= 0.86 and 0.88). In contrast, none of the Black Volta individuals (N
= 39) showed evidence of admixture (Q
> 0.98). The non-native tilapia group showed no admixture with the native populations. However, the Lee’s Farm stock contained several individuals apparently admixed with the non-native tilapia populations (Q
ranged between 0.11 and 0.74). Two individuals, one each in the ARDEC (Akosombo strain) and Volta Catch populations, appeared admixed with the non-native populations (Q