Variability of tilapias (Oreochromis spp.) introduced in Mexico: morphometric, meristic and genetic characters

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1 J. Appl. Ichthyol. 20 (2004), 7 14 Ó 2004 Blackwell Verlag, Berlin ISSN Received: May 6, 2002 Accepted: August 20, 2002 Variability of tilapias (Oreochromis spp.) introduced in Mexico: morphometric, meristic and genetic characters By I. D. L. A. Barriga-Sosa 1, M. D. L. Jime nez-badillo 2,A.L.Iba n ez 1 and J. L. Arredondo-Figueroa 1 1 Planta Experimental de Produccio n Acuı cola, Departamento de Hidrobiologı a, Divisio n de Ciencias Biolo gicas y de la Salud, Universidad Auto noma Metropolitana-Iztapalapa, Mexico; 2 Centro de Ecologıá y Pesquerıás, Universidad Veracruzana, Carretera Xalapa-Las Trancas, Veracruz, Mexico Summary In Mexico, the tilapia Oreochromis provides one of the most important sources of animal protein and income in a wide variety of communities throughout the country, however, their culture and production face severe problems because of lack of management information. Evaluated in the present study is the degree of morphological, meristic and allozyme variation of two tilapia strains, two species and one hybrid from two important reservoirs in Mexico, two tilapia fry production farms (TFPF) and an experimental production system (EPS), to diagnose the actual status and degree of variation among them. Twenty-two presumptive loci were utilized to determine the genetic variation, structuring and distances among samples. Multivariate analyses of 11 meristic and 33 morphometric characters were also evaluated, including the distance from the end of the mouth opening to the most anterior dorsal fin (0.9894), distance from the anal fin base to the anterior part of the caudal fin (0.9845) and the base length of the dorsal fin (0.9839) which contributed to the separation of groups. The canonical discriminant functions for the morphometric and meristic variables show that the correct classification of the organisms in percentages and in the sites of origin was on average 72%. The tilapia from the reservoirs and the experimental production system had higher genetic variations than tilapia from the fry production farms (average H e 0.310, 0.062, for Metztitlan, Infiernillo and EPS, respectively, vs and for Los Amates and Zacatepec, respectively). The genetic data indicate that fishes of the TFPF should be monitored closely, as they are the main source of dispersion to the reservoirs. Present results show that these data could be a fast and reliable aid to the fisheries and management of tilapia in Mexico. Introduction The tilapia in Mexico is one of the three most important freshwater fisheries, representing a fifth of world production (FAO, 1997). First introduced to Mexico (Temascal, Oaxaca) in 1964 from Auburn University, Alabama, USA Tilapia rendalli, Oreochromis mossambicus and O. aureus, (Morales, 1974) became widely distributed throughout natural and artificial water reservoirs in tropical and temperate regions of the country (Arredondo, 1983). In 1978, the Nile tilapia (O. niloticus) from Panama was introduced to the same farm; in 1986, the first red strain of O. niloticus from Stirling University, Scotland, UK was introduced to the Centro de Investigacio n y Estudios Avanzados, Campus Merida in Yucatan from where the red strain was held at five government tilapia fry production farms in five states of the Mexican Republic. Since 1987, both national and local governments and the private sector introduced other tilapia strains including the Rocky Mountain (from Colorado, USA) albine strain and the red hybrid strain (from Puerto Rico) (Arredondo and Lozano, 1996). They were successfully cultivated by rural populations in the warm and humid areas of Oaxaca, Tabasco, Chiapas, Michoaca n, Veracruz and Sinaloa. However, there are few records on the origin of the introductions of these cichlids and of their strains, further compromising selective developments. The unmanaged distribution in the early years has resulted in some unfavourable traits, i.e. reduction of commercial size, early sexual maturation and deformities (Morales, 1991; Jimenez, 1999). Some consider that these traits may be the result of a too-small gene pool. The present account examines the genetic variation of two species, two important strains and one tilapia hybrid to investigate levels of genetic variation at the Infiernillo Reservoir, an important production site. Compared are also the variations among two other water reservoirs, two tilapia fry production farms (TFPF) and one experimental production system (EPS). Materials and methods The reservoirs were chosen as follows: (1) a large reservoir (Infiernillo Reservoir, Michoacan-Guerrero) with records of tilapia introductions since 1969 (Rosas, 1976) and with the highest national yields; (2) and a small reservoir (Lake Metztitlan, Hidalgo) with recent introductions and also with high yields. The tilapia fry production farms (TFPF) are the main sources of tilapia in the country together with those from the experimental production system (EPS). Oreochromis niloticus Stirling strain (Ons) broodfish from TFPF Temascal, Oaxaca; O. niloticus red strain (Onr) from Martinez de la Torre, Veracruz, and the hybrid Rocky Mountain (O. niloticus O. aureus, Ona) from Zacatepec, Morelos (Arredondo et al., 1994), were selected and maintained at the EPS, in the Planta Experimental de Produccion Acuicola at UAM-Iztapalapa, Mexico City, from 1994 to 1999 and utilized as domesticated controlled strains. Samples from the TFPF in Los Amates, Veracruz, and Zacatepec, Morelos, and supplied by local farmers in 2000 were also analysed. Fish from Lake Metztitlan and Infiernillo Reservoir were considered to be ÔwildÕ. Samples from the lake were obtained from commercial catches in 1998 and 2000 using a 30 m long 1.5 cm mesh size gill net, and in 1999 and 2000 from U.S. Copyright Clearance Centre Code Statement: /2004/ $15.00/0

2 8 Barriga-Sosa et al. the reservoir with a 8.3-cm-mesh-size gill net. All samples were analysed for variation comparison. Samples used in this study are summarized in Table 1. Morphometric (M) and meristic (m) analyses Forty-four variables were used to assess morphological variation within samples: 33 morphometric (M), 25 according to Vreven et al. (1998) and eight to Jimenez (1999); and 11 meristic (m), seven according to Vreven et al. (1998) and four to Jimenez (1999). All M characters were measured to the nearest 0.1 mm using digital callipers (Fig. 1). The m variables registered were: number (no.) of spines and rays of dorsal fin (variables 1 and 2), anal fin (variables 3 and 4), pelvic fin (variables 5 and 6), no. of scales of the superior lateral series (variable 7), inferior lateral series (variable 8), transverse series (variable 9), no. of the pre-dorsal scales (variable 10) and no. of gill rakers (variable 11). Each category of data, M and m, was analysed separately. To eliminate differences related to size, morphometric data were transformed (Corti et al., 1988; Cuadras, 1991) according to three different criteria: (i) logarithmic transformation of each character; (ii) proportion, representing each character relative to length; and (iii) normalization of individuals of each group according to the method of Lombarte and Lleonart (1993). Also analysed were the original data (o) of each of the categories (M and m). The data were submitted to multivariate analyses with two descriptive and exploratory methods: principal component (PC) and reciprocal averaging (RA). Discriminate analyses (DA) were also utilized with both data sets to investigate discrimination among samples. Multivariate analysis was carried out with the computer program STAT- Plus (Manugistic Inc., Rockville, MD, USA) for windows version 5.0. GRAPHIC Allozyme analysis A mixture of liver, muscle and heart tissues, dissected from 360 organisms, each based on samples ( g) were minced and homogenized in chilled Tris ethylenediaminetetraacetic acid (EDTA) buffer or nicotinamide adenine dinucleotide phosphate (NADP) buffers ( ml), then centrifuged at g for 2 min at room temperature (RT). These extracts were used to perform horizontal cellulose acetate electrophoresis (Titan III gels; Helen Laboratories, Beaumont, TX, USA) for min at V and at RT. Selection of the allozyme markers and the allele designation was based on Barriga-Sosa et al. (2002). Thirteen enzymes were used: alcohol dehydrogenase (Adh, E.C ), carboxylesterase (Est, E.C ), glyceraldehyde-3-phosphate dehydrogenase (G3pdh, E.C ), glucose-6-phosphate isomerase (Gpi, E.C ), Glycerol-6-phosphate dehydrogenase (G6pdh, E.C. Table 1 Names and abbreviations of tilapias used in this study Numbers Oreochromis species, strains and hybrids (locality) (G) (M) (m) Year sampled 1 O. niloticus (Stirling strain, EPS ¼ OnsE) O. niloticus O. aureus (hybrid Rocky Mountain, EPS ¼ OnaE) O. niloticus (red strain, EPS ¼ OnrE) O. niloticus O. aureus (hybrid Rocky Mountain, Metztitlan ¼ OnaM) O. niloticus Stirling strain (Metztitlan, Hidalgo ¼ OnsM) O. aureus (Infiernillo Dam, Guerrero-Michoacán ¼ OaI)* O. aureus (Los Amates, Veracruz ¼ OaA) O. niloticus O. aureus (hybrid Rocky Mountain, Los Amates ¼ OnaA) O. niloticus O. aureus (hybrid Rocky Mountain, Zacatepec ¼ OnaZ) O. niloticus (Zacatepec, Morelos ¼ OnZ) N T *Includes samples Quebradora and Zicuirán referred to as OaQ and OaZir in the genetic analysis. EPS ¼ experimental production system, N ¼ sample size per analysis, G ¼ genetic, M ¼ morphometric and m ¼ meristic Fig. 1. The 33 morphometric measurements registered in each organism analysed. Modified from Vreven et al. 1998

3 Variability of Oreochromis spp. introductions in Mexico ), hexokinase (Hex, E.C ), isocitrate dehydrogenase (Idh, E.C ), L-lactate dehydrogenase (Ldh, E.C ), malate dehydrogenase (Mdh, E.C ), malic enzyme (ME, E. C ), phosphogluconate dehydrogenase (6Pgdh, E.C ), phosphoglucomutase (Pgm, E.C ) and xantin dehydrogenase (Xdh, E. C ). Four electrophoretic buffers were assayed: Citric acid morpholine ph ¼ 7.0 (CAAPM); Tris glycine ph 8.5 (TG); Tris EDTA, ph ¼ 8.0 (TE); and Tris EDTA NADP, ph ¼ 7.0 (TE- NADP). The staining procedures were those described by Hebert and Beaton (1989) with modifications by Barriga-Sosa et al. (2002). For each locus analysed, the allele frequencies, proportion of polymorphic loci (P 0.95 criteria), mean number of alleles per locus, and expected (H e ) heterozygosity were calculated for each species per strain. Deviations from the expected Hardy Weinberg proportions were tested using the Fisher s exact test in GENEPOP 3.1 (Montpellier, France) (Raymond and Rousset, 1995). Fisher s combined probability test (Sokal and Rohlf, 1995) was used to test the significance of allele frequency differences between species or strains (TFPGA 1.3, Miller, 1998). The probabilities that F indices (Weir and Cockerham, 1984) associated with each locus and sample were significantly different from zero were computed with GENEPOP 3.1. The estimator of Weir and Cockerham (1984) F-statistics was used to measure preliminary levels of genetic population structuring where data were available. The following parameters were estimated: f, the correlation of genes within individuals within populations and h S correlation of genes of different individuals in the same population (when data available). Estimates of the variance of these parameters were obtained by jack-knifing across loci (Weir and Cockerham, 1984). F-statistics were tested for differences from zero permuting (2000 replicates) alleles within samples (f) and alleles between samples (F and h) over all loci. The genetic relationships between pairs of species and calculated from the allele frequency data were estimated using Nei s genetic identity and distance estimates (I N /D N ) with the TFPGA (Flagstaff, AZ, USA) 1.3 program. Cluster analysis was performed with the UPGMA method of the same program using bootstrap sampling of loci and a consistency index (Felsenstein, 1985). Results Morphometric (M) and meristic (m) data analyses The cumulated percent of the variance resulting from PC and RA analyses is summarized in Table 2; it includes the cumulated percent for the first three components (I, II and III) with the transformed (i, ii and iii) and original (o) morphometric (M) and meristic (m) data. Meristic variables three, five and sixwere excluded from further analyses because they remained constant. The morphometric data for the first two components of the PC and RA analyses both for the original and transformed data (i ¼ logarithmic and ii ¼ proportion) show changes that relate to size. However, the PC and RA analyses with data iii (normalization) showed a correlation matrixwith different sign and magnitude, a pattern that expresses variation in shape (Corti et al., 1988; Cuadras, 1991). The PC analysis explained the highest percentage of the variance (Table 2). All organisms of the different localities were clearly differentiated through this analysis. Variables 1, 2, 3, 4, 6, 10, 11, 15, 18, 22 and 33 accounted for the highest percentage of the variance. Three variables contributed to the separation of groups: the distance from the end of the mouth opening to the most anterior dorsal fin (variable 2, ), the distance from the base of the anal fin to the anterior part of the caudal fin (variable 15, ), and the length of the base of the dorsal fin (variable 11, ). The first two discriminate functions resolved from those 11 morphometric variables showed a 69 and 29 relative percentage and a canonical correlation of The strains from the EPS and the hybrid Rocky Mountain from Los Amates appear to form a group in the left section of the plot. The remainder of the strains from the other localities showed some degree of aggregation in the right section of the plot (Fig. 2). Most of the organisms from different localities showed differences using discriminate analysis. The Stirling strain from the EPS, O. aureus from Infiernillo (both samples) and the hybrid Rocky Mountain from Zacatepec were 100% correctly classified. The other strains from EPS (97 and 95%), the hybrid from Los Amates (81 and 89%) and that from Lake Metztitlan (58%, Table 3a) were not pure. The meristic data using RA explained the highest variance. Two groups were observed, one with fish from Lake Metztitlan, Los Amates and Zacatepec TFPF, and the second from the Infiernillo Reservoir, the hybrid Rocky Mountain and strains from the EPS. The same pattern appears using the discriminate analyses (DA) (Fig. 3) where the first function was significant with a relative percentage of 84% and a canonical correlation of 0.98, resulting in 100% of the Zacatepec tilapia being correctly classified. The tilapias from EPS and the Infiernillo Reservoir were 76% correctly classified; 24% were distributed between Stirling and red strains, and for the hybrid (O. niloticus O. aureus) 14% overlapped with O. aureus from the Infiernillo Dam. The two strains from Lake Metztitlan were completely separate from each other and 85% were correctly classified. The hybrid Rocky Mountain and the species from Los Amates were 93 and 67% correctly classified, respectively (Table 3b). Allozyme analyses. From the enzyme systems assayed, 22 presumptive loci were detected. The electrophoretic buffer Table 2 Cumulated percentage of the variance in three components for multivariate analyses (MA): reciprocal averaging (RA) and principal component (PC) Measure Data MA Component I Component II Component III Morphometric (M) Original (o) RA PC Log (i) RA PC Proportion (ii) RA PC Normalization (iii) RA PC Meristic (m) Original (o) RA PC

4 10 Barriga-Sosa et al. 13 OnsE Discriminant Function 2 (29.10) OnaE OnrE OnaM OnsM OaI OaA OnaA OnaZ Discriminant Function1(69.08%) OnZ Centroids Fig. 2. Plot of first and second discriminant functions of the discriminate analysis (DA) using 11 variables (M iii) and 10 samples Predicted group Actual OnsE OnaE OnrE OnaM OnsM OaI* OaA OnaA OnaZ OnZ (a) OnsE 100 OnaE OnrE OnaM OnsM OaI 100 OaA OnaA OnaZ 100 OnZ Table 3 Discriminate classification of tilapias based on (a) M iii and (b) m variables. Measurements are expressed in percentage (b) OnsE OnaE OnrE OnaM OnsM OaI OaA OnaA OnaZ 100 OnZ 100 *OaI ¼ O. aureus (includes both samples from the Infiernillo Reservoir). 9 OnsE Discriminant Function 2 (9.76%) OnaE OnrE OnaM OnsM OaI OaA OnaA OnaZ OnZ Discriminant Function 1 (84.53%) Centroids Fig. 3. Plot of first and second discriminant functions of the discriminate analysis (DA) using eight variables (m) and 10 samples CAAMP, resolved Idh and Est and TG, ph 8.5 resolved the remaining systems. Only one locus was detected for Adh, G3pdh, Hexand Pgm. Two loci were observed for Est, G6pdh, Idh, Ldh, Mdh, Me and Xdh, and three loci for Gpi. Allele frequencies and genetic variation The allelic frequencies of 10 polymorphic loci are shown in Table 4. Loci 1 and 2 for Adh, G6pdh, Idh, Mdh and Xdh were monomorphic in all samples, and so were loci 1 for

5 Variability of Oreochromis spp. introductions in Mexico 11 Table 4 Allelic frequencies of polymorphic loci for the tilapia samples analysed Sample Locus OnaE OnrE OnsE OnsM OnaM OaA OnaA OaQ* OaZic* OnZ OnIZ Fisher s P Est-1 (N) a b Est-2 a b Gpi-1 a b Gpi-2 a b c Gpi-3 a b c Ldh-1 a b Ldh-2 a b Mdh-1 a b c Mdh-2 (N) a b c d Pgm a b c P H e A locus is polymorphic if the frequency of the most common allele does not exceed 95%. N ¼ sample size; H e ¼ expected heterozygosity and P ¼ Fisher s P among samples. *Samples pooled as OaI for further analyses. G3pdh and Hex. Locus 1 for Me was polymorphic but was not included in the analysis because of inconsistencies in the resolving of band patterns. With the P 0.95 criteria, the higher total number of polymorphic loci were observed in ÔwildÕ samples from Lake Metztitlan, Hidalgo, followed by the domesticated tilapias maintained at EPS. The Infiernillo samples (OaQ and OaZic) had the lowest number of polymorphic loci under the same criteria. The organisms from Zacatepec and Los Amates had no polymorphic loci, except for sample OaA which has one polymorphic locus (Table 4). In the same manner, the H e indicated that the highest variation was found in samples from Lake Metztitlan (0.251 and 0.364, for OnsM and OnaM, respectively), followed by the tilapias kept at the EPS (0.107, and 0.206, for Onr, Ona, Ons, respectively). Lower H e values were found in samples from Infiernillo and Los Amates ( for OaQ and OaZic; for OaA and OnaA). Significant differences in allele frequencies within all samples were detected except for those from the Infiernillo Reservoir, thereof samples OaQ and OaZic were pooled for posterior analyses as OaI. Most samples showed significant departure from H W equilibrium at one or more loci, except for sample OnaA (Table 5). The significant results of most samples were due to a significant deficit of heterozygous individuals, which is characteristic of two or more assertively mating populations which hybridize to some extent (Campton, 1987; Gregg et al., 1998). The deficit of heterozygotes was stronger in samples from Lake Metztitlan (OnsM and OnaM), followed by samples from EPS (OnrE, OnsE and OnaE) and Infiernillo (OnaI). Genetic differentiation, distances and cluster analyses Preliminary Weir and Cockerham (1984) estimates of the F-statistics suggest genetic differentiation among samples. The average jack-knifed F, indicates an overall deficiency of heterozygotes in all samples (0.7177), and h S indicates significant genetic differentiation among the analysed samples (0.6322) (Table 6). Nei s unbiased estimates of genetic identities and distances (D N and I N ) indicated that the most genetically distant samples

6 12 Barriga-Sosa et al. Sample Locus OnaE OnrE OnsE OnsM OnaM OaA OaI Table 5 Fixation index (F IS ) for all samples analysed Est-1 )0.012* Est-2 )0.054* )0.032* )0.095* )0.054* )0.115* Gpi * Gpi-2 )0.043* 0.500* * Gpi-3 )0.062* 0.082* Ldh-1 )0.010* )0.689* )0.307* )0.508* )0.111* Ldh )0.075* Mdh-1 )0.034* ) )0.190* Mdh * Pgm )0.021* 0.357* 0.186* )0.111* Positive values indicate a deficiency of heterozygotes, F IS ¼ 0 for a randomly mating population. *Locus does not conform to Hardy Weinberg proportions, P ¼ Table 6 (a) F and h values for each polymorphic loci; (b) Weir and Cockerham (1984) overall estimates of genetic heterogeneity (h, f and F) in the analysed samples Parameter Enzyme h S h P F (a) Est Est Gpi Gpi Gpi Ldh Ldh Mdh Mdh Pgm Average (b) h S h P F f (0.0919) (0.1189) (0.1057) (0.1958) Standard deviation in parenthesis. P ¼ *99% confidence interval with bootstrapped replicates. were OnrE/OaA/OnaA followed by samples OaA/OaI/OnZ, whereas the sample from the Infiernillo Reservoir (OnaI) was the least genetically distant from the Zacatepec sample OnZ, followed by the distances among samples from the EPS (OnsE/ OnaE/OnrE) and OnsE/OnaM/OnsM. Although there were significant differences in the allelic frequencies between the samples from Zacatepec, no significant genetic distances were observed among them (data upon request). The UPGMA dendogram generated by using Nei s unbiased genetic distances (Fig. 4) clearly illustrates such relationships. Discussion In aquaculture, the worldwide cultivation of tilapia has been hindered because of diminishing catches, difficulties of identification because of hybridization processes, overfishing, disappearance of endemic species, habitat perturbation, changes in their trophic dynamics and the reduction of growth (Petr, 1987; Morales, 1991; Ochumba et al., 1992; Crul and Roest, 1995; Rognon et al., 1996; Lazard and Rognon, 1997; Marshall, 1997). Nevertheless, for O. aureus and O. niloticus, A.L. Iban ez (100%) (60%) OnaZ OnZ (100%) (50%) (40%) OaI OaA OnA OnsM OnaM OnaE OnsE OnrE Fig. 4. UPGMA dendogram derived from Nei s unbiased genetic distances and based on 10 polymorphic allozyme loci. Bootstrap values estimated from 1000 replications are reported when equal or higher than 50% (in prep.) reported higher growth rates in Mexico to those observed in African and Asian waters. National experiences in epicontinental waters had demonstrated growth rates up to 1 g/ day in eutrophic waters (A.L. Iban ez, op. cit.). In addition, the annual production of tilapia in Mexico and in the biggest reservoirs is on average tonnes, with more than 90% consumed locally (Arredondo and Lozano, 1996). This production has generated social and economical benefits to local rural areas of the country, estimated in a national income of 70 million US$. Studies describing the ÔoptimalÕ levels of genetic variability for adequate production farming have increased in the past two decades, however, all are related to species or strains in Asian and African countries (Cruz et al., 1982; McAndrew and Majumdar, 1983; Feresu-Shonhiwa and Howard, 1988; Van Der Bank et al., 1989; Eknath et al., 1991; Macaranas et al., 1995; Sugunan, 1995; Rognon et al., 1996; Lazard and Rognon, 1997; Morales et al., 1998; Vreven et al., 1998). Since the first introductions during the 1960s, genetic studies in Mexico are reduced to the description on banding patterns of some of the introduced species (Uribe-Alcocer et al., 1989; Tenorio, 1995). This study is the first attempt to describe the morphological and genetic variability on tilapia species and strains that had been used in Mexico for aquaculture practices. Morphometric and meristic variation The high variation and clear separation among different water reservoirs (i.e. Infiernillo and Lake Metztitlan) as shown by

7 Variability of Oreochromis spp. introductions in Mexico 13 morphometric results suggest that environment has a direct influence in shape differentiation, as reported by Vreven et al. (1998). There is no clear shape differentiation between strains; for instance, the Lake Metztitlan samples (OnaM and OnsM) overlap in the discriminant classification. The meristic results support this separation according to water reservoirs as does the morphometric analysis; furthermore, m data allow a clearer separation among samples (strains and species), except for the hybrid OnaE which shows an overlap with OaI, and for the samples OnaA and OaA from Los Amates. This is not surprising as the hybrid Ona is a product of the cross of O. aureus O. niloticus, and shows a trend towards the dominance of the former species over O. niloticus, as reported in natural environments by Payne and Collinson (1983) who found that the most reliable difference between O. niloticus and O. aureus was the dorsal spine number (17 and 16, respectively). Genetic variation The genetic results and the morphometric and meristic data all show that the Metztitlan samples had the greatest variation, and that the data sets for EPS, Infiernillo, and the TFPF were less. Similar patterns of genetic variation are known for cultured and ÔwildÕ populations in Africa (Rognon et al., 1996; Lazard and Rognon, 1997). The high genetic variation (H e )in fish from Lake Metztitlan could be explained by their recent introduction and constant re-stocking from two TFPF (Los Amates and Zacatepec) in the past 4 years. Hybridization or introgression in Lake Metztitlan may have taken place and deficiency of heterozygotes in one or more loci indicates this may have happened (Campton, 1987; Gregg et al., 1998). Introgression may also be taking place within the hybrid Rocky Mountain (O. niloticus O. aureus) and the O. niloticus Stirling strain used in this study, and should be tested by analysing pure populations with other genetic markers. The intermediate levels of H e observed in the domesticated tilapias OnsE, OnaE and OnrE are similar to results observed in controlled stocks (Macaranas et al., 1995) where levels of variation are considered intermediate; it is also known that years of inbreeding had to have reduced variability. Thus, the contrasting low levels of genetic variation of fish from Los Amates and Infiernillo Reservoir in relation to cultured tilapias in other areas of the world might represent a real and drastic case of a bottleneck, an event that could be explained by several factors. The species O. aureus from the Infiernillo Reservoir was introduced in 1969 (Jimenez, 1999) and actual stocks were derived from those original broodfish; therefore, no gene flow from new genetic pools is documented and local inbreeding could be expected as considerably high. Similarly low levels of heterozygosity of African O. niloticus have been reported by other authors (McAndrew and Majumdar, 1983; Macaranas et al., 1995). In addition, drastic reduction and total lack of heterozygosity in the cultured tilapias from the tilapia fry production farms at Veracruz and Morelos indicate that managing strategies are inadequate and that these farms require better and more accurate strategies for their culture. Acknowledgements We thank the people of the TFPF included in the study, and V. Espinosa Lemus for supplying fish. I. Mora-Souza, E. Torres- Frı as, B. Torres-Jua rez and L. Ferna ndez-bringas assisted in the fish identification and laboratory work. 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