Correo 35 Sucursal 16, 5016 Córdoba, Argentina; 2Instituto Nacional de Enfermedades Virales Humanas,. Monteagudo 2510, 2700 Pergamino, Argentina;.
105
Genetica 101: 105–113, 1997. c 1997 Kluwer Academic Publishers. Printed in the Netherlands.
Oligoryzomys flavescens (Rodentia, Muridae): gene flow among populations from central-eastern Argentina Marina B. Chiappero1 , Gladys E. Calder´on2 & Cristina N. Gardenal1 1
C´atedra de Qu´ımica Biol´ogica, Facultad de Ciencias M´edicas, Universidad Nacional de C´ordoba, Casilla de Correo 35 Sucursal 16, 5016 C´ordoba, Argentina; 2 Instituto Nacional de Enfermedades Virales Humanas, Monteagudo 2510, 2700 Pergamino, Argentina; Author for correspondence Received 7 January 1997 Accepted 2 June 1997
Key words: Oligoryzomys flavescens, gene flow, isozymes, population genetics
Abstract In species acting as hosts of infectious agents, the extent of gene flow between populations is of particular interest because the expansion of different infectious diseases is usually related to the dispersal of the host. We have estimated levels of gene flow among populations of the sigmodontine rodent Oligoryzomys flavescens, in which high titers of antibodies have been detected for a Hantavirus in Argentina that produces a severe pulmonary syndrome. Enzyme polymorphism was studied by means of starch gel electrophoresis in 10 populations from the area where human cases of Hantavirus have occurred. Genetic differentiation between populations was calculated from FST values with the equation Nm = [(1=FST ) 1]=4. To assess the relative importance of current gene flow and historical associations between populations, the relationship of population pairwise log Nm and log geographic distance was examined. Low FST (mean = 0.038) and high Nm (15.27) values suggest high levels of gene flow among populations. The lack of an isolation by distance pattern would indicate that this species has recently colonized the area. The northernmost population, located on the margin of a great river, shows very high levels of gene flow with the downstream populations despite the large geographic distances. Passive transport of animals down the river by floating plants would promote unidirectional gene flow. This fact and the highest mean heterozygosity of that northernmost population suggest it is a center of dispersal within the species’ range.
Introduction The extent of genetic differentiation among local populations of a species largely depends on the magnitude of gene flow, as long as it counteracts the effects of mutation, genetic drift, and locally differing selection pressures occurring independently in each subpopulation. Thus, the knowledge of the amount of gene exchange among demes is important to understand the evolutionary forces responsible for the genetic structure of that species. Levels of gene flow among populations are usually assessed by indirect methods, based on spatial distribution of allele frequencies. One of the measures of genetic differentiation most frequently utilized is Wright’s FST , which is the standardized vari-
ance of allele frequencies among populations (Slatkin, 1987). Under an island model of population structure, at drift-migration equilibrium and assuming neutrality of alleles, Wright (1931) showed that the average number of migrants per population per generation (Nm) is inversely proportional to FST . This relationship is based on the assumption that gene flow is uniform among populations and that migrants comprise a random sample of genetic variation from all the other populations. Indirect methods give estimations averaged over long periods of time, which may not coincide with the actual levels of currently occurring gene flow (Slatkin, 1987). Slatkin (1993) proposed that estimation of Nm [(1/FST 1)]/4 between pairs of populations and its relationship with geographic distance
106 allow the analysis of different situations related to the equilibrium between genetic drift and migration. Knowledge of gene flow patterns and of the degree of isolation between subpopulations are of particular interest in species acting as hosts of infectious agents because the expansion of different viral infections has been related to the dispersal of the host (Sabattini & Contigiani, 1982; Weissenbacher et al., 1985). Recently, several human cases of Hantavirus Pulmonary Syndrome, which has a high mortality rate, have been detected in different regions of Argentina. Oligoryzomys flavescens is one of the rodent species presenting high titers of antibodies against the Hantavirus producing that disease. This species belongs to the subfamily Sigmodontinae and is widely distributed in the central-eastern plains of Argentina (‘humid Pampa’). It prefers habitats close to water streams and humid lowlands with dense vegetation, and stable environments of high weeds on road and field borders and along railroad tracks (Crespo, 1966; Polop, Mart´ınez & Torres, 1985; Mills et al., 1991). In order to estimate levels of gene flow and to determine dispersal patterns in this species, we analysed the allozymic polymorphism in populations of O. flavescens from the area where animals with antibodies against Hantavirus have been found.
Materials and methods Specimens of O. flavescens were captured with Sherman live-traps in the following localities (Figure 1): M´aximo Paz (n = 30) and Oliveros (n = 18) in Santa Fe province; Pergamino (n = 20), San Pedro (n = 24), and Z´arate (n = 25) in Buenos Aires province; and Gualeguay (n = 30) and Lechiguanas island (n = 24) in Entre R´ıos province. This last site is one of the localities where human infection by Hantavirus and rodents with higher titers of antibodies have been detected. In M´aximo Paz, San Pedro, and Gualeguay, the seroprevalence was 0 and in the other localities it attained values up to 8.3% (Levis et al., 1995, 1996). Animals were killed by inhalation of methoxyfluorane. Liver and kidneys were removed and frozen in liquid nitrogen in the field and later stored at 70 C. Tissues were then submitted to gamma radiation to inactivate potential viral particles. We have observed that this treatment does not affect the activity and electrophoretic mobility of the enzymes analysed. A total of 16 enzymatic proteins affording information on 26 loci were studied. Preparation of
homogenates, electrophoresis, and staining to reveal enzyme activity were performed as described by Selander et al. (1971); Gardenal, Sabattini and Blanco (1980); and Gardenal and Blanco (1985) (Table 1). Genetic control of electrophoretic phenotypes was interpreted according to previous knowledge of similar variants in other species of sigmodontine rodents in which simple Mendelian inheritance has been demonstrated (Gardenal & Blanco, 1985; Gardenal, Sabattini & Blanco, 1980; Garc´ıa & Gardenal, 1989). Mean heterozygosity per locus, percentage of polymorphic loci (P95% and P99% criteria), and mean number of alleles per locus (A) were calculated. Agreement between observed and expected genotypic frequencies was analysed by a chi-square test. Genetic distance between populations was estimated by the D index (Nei, 1972). These values were used to construct a phenogram by the UPGMA method (Sokal & Michener, 1958); consistency of the clusters obtained was analysed by the bootstrap procedure. These data were calculated by using the BOOT program of PHYLIP package 3.55 version (Felsenstein, 1993). Genetic differentiation among populations was calculated by F statistics by using the corrected method of Weir and Cockerham (1984), which takes into account different sample sizes and the presence of multiple alleles in a locus. Significance of FST and FIS values was obtained by permutation procedures of alleles within the samples for FIS and of alleles and genotypes between samples for FST ; 200 permutations were performed in each case. This method obtains the distribution of the null hypothesis (FXX not > 0) and gives the probability of obtaining by chance a value as large or larger than the observed. The Fstat program of G`erˆome Goudet was used for these calculations. Gene flow between populations was estimated from FST values with the equation Nm [(1/FST ) 1]/4, where m is the migration rate and N the effective population size (Wright, 1931) and by Slatkin’s method based on the distribution of rare alleles, which has the advantage that is only weakly influenced by most kinds of selection affecting a locus (Slatkin, 1985; Slatkin & Barton, 1989). In this method, Nm is estimated by the formula:
Nm = e
ln[p(1)]+2:44 0:505
N
25
where p(1) is the average frequency of all alleles found in only one population and N is the average number of individuals sampled per population. Option 4 of
107
Figure 1. Location of collection sites. Shading indicates floodable area.
the GENEPOP program (Raymond & Rousset, 1995), which gives a corrected estimate of Nm (Barton & Slatkin, 1986), was used to perform the calculation. To estimate whether isolation by distance exists in this species, Nm between pairs of populations was calculated. The logs of these values were plotted against logs of the geographic distances between pairs of populations, and the correlation between the two matrices was calculated with the Mantel test.
Results The loci Mdh-1, Ldh-2, AcPk -2, Sod-1, Sod-2, Ndh, Adh, and Es-1 were monomorphic in all populations. Table 2 presents the allele frequencies of the polymorphic loci for each population. Observed phenotypic frequencies were not significantly different from those expected from the Hardy-Weinberg equilibrium. This
108 Table 1. Enzymes, loci, and tissues analysed Enzyme
Locus
Tissue
E.C. No.
Buffer system
Acid phosphatase (kidney)
Acpk -1 Acpk -2 Lap Ldh-1 Ldh-2 Acp1 Est-1 Est-2 Est-3 Est-4 Est-6 Sod-1 Sod-2 Pgm-2 Got-1 Got-2 Gpdh Adh Me Idh-1 Idh-2 Mdh-1 Mdh-2 6pgdh G6pdh Ndh
Kidney
3.1.3.2
I
Kidney Kidney
3.4.1.1 1.1.1.27
Liver Liver
3.1.3.2 3.1.1.1
Liver
1.15.1.1
Liver Liver
2.7.5.1 2.6.1.1
Liver Liver Liver Kidney
1.1.1.8 1.1.1.1 1.1.1.40 1.1.1.42
Kidney
1.1.1.37
Liver Liver Liver
1.1.1.44 1.1.1.49 –
Leucine aminopeptidase Lactate dehydrogenase Acid phosphatase (liver) Esterases
Superoxide dismutase Phosphoglucomutase Glutamate oxaloacetate transaminase
-Glycerophosphate dehydrogenase Alcohol dehydrogenase Malic enzyme Isocitrate dehydrogenase Malate dehydrogenase 6-phosphogluconate dehydrogenase Glucose-6-phosphate dehydrogenase NAD-linked nonspecific dehydrogenase
II
III
I: Tris-borate-EDTA, pH 8.6 (Markert & Faulhaber, 1965); II: Tris-citric, pH 6.3 (Gardenal y Blanco, 1985); III: Phosphate 0.1 M, pH 7 (Selander et al., 1971).
observation supports the validity of the genetic control proposed. Table 3 presents observed and expected mean heterozygosity values, proportion of polymorphic loci, and mean number of alleles per locus. There is a large difference between P95% and P99% in all populations due to the fact that the same alleles at several loci are present at low frequencies in all populations. Table 4 shows genetic distances (Nei, 1972) between pairs of populations. Figure 2 represents the topology resulting from 100 resamplings using the bootstrap technique. Numbers at the nodes indicate the number of times the populations to the right of the fork occurred in 100 bootstrap replications. San Pedro and Z´arate are the genetically closest populations and the node linking them was the only one appearing in a large number of the obtained trees. M´aximo Paz, Pergamino, and Gualeguay form another group with high similarity;
bootstrap percentages at nodes linking remaining populations were low. Table 5 presents FIS and FST values. Mean FST per 18 polymorphic loci was 0.035, ranging from 0.005 to 0.144. Theoretically, FST index is never negative, but it sometimes can take small negative values if corrections for sampling errors are applied (Trexler, 1988). In 7 of the 18 polymorphic loci significant FST values were obtained, suggesting that these loci or some linked to them could be subjected to selection. Mean Nm value was 15.27. Slatkin’s private alleles method yielded an estimate of 11.6. Table 6 shows mean FST values for polymorphic loci and Nm values between pairs of populations. The population from Oliveros showed high levels of gene flow with all other populations. Those of San Pedro, Z´arate, and Lechiguanas have a lower gene flow with the remaining populations (Nm between 3.06 and 6.86), whereas those from M´aximo Paz, Pergamino, and Gualeguay maintained high lev-
109 Table 2. Allele frequencies for polymorphic loci of O. flavescens Locus
Mdh-2 a b Idh-1 a b Idh-2 a b Ldh-1 a b c Acpk -1 a b c d Lap a b c Acp1 a b Got-1 a b c Got-2 a b Pgm-2 a b Me a b -Gpdh a b G6pdh a b 6Pgdh a b c
Populations Oliveros M´aximo Paz
Pergamino
San Pedro
Z´arate
Gualeguay
Lechiguanas
0.000 1.000
0.000 1.000
0.000 1.000
0.000 1.000
0.020 0.980
0.000 1.000
0.000 1.000
0.083 0.917
0.000 1.000
0.000 1.000
0.021 0.979
0.000 1.000
0.000 1.000
0.000 1.000
0.028 0.972
0.050 0.950
0.000 1.000
0.000 1.000
0.020 0.980
0.000 1.000
0.000 1.000
0.056 0.833 0.111
0.067 0.833 0.100
0.100 0.700 0.200
0.021 0.896 0.083
0.040 0.860 0.100
0.083 0.700 0.217
0.079 0.763 0.158
0.083 0.834 0.083 0.000
0.052 0.931 0.017 0.000
0.036 0.964 0.000 0.000
0.000 0.667 0.313 0.021
0.000 0.625 0.375 0.000
0.000 0.969 0.031 0.000
0.118 0.882 0.000 0.000
0.028 0.917 0.056
0.000 0.917 0.083
0.050 0.825 0.125
0.000 0.979 0.021
0.060 0.920 0.020
0.019 0.944 0.037
0.079 0.895 0.026
0.972 0.028
1.000 0.000
0.950 0.050
1.000 0.000
0.980 0.020
1.000 0.000
0.917 0.083
0.000 1.000 0.000
0.017 0.966 0.017
0.025 0.975 0.000
0.000 1.000 0.000
0.000 1.000 0.000
0.019 0.981 0.000
0.000 1.000 0.000
0.944 0.056
1.000 0.000
1.000 0.000
1.000 0.000
0.940 0.060
0.981 0.019
0.938 0.063
0.972 0.028
1.000 0.000
1.000 0.000
0.979 0.021
1.000 0.000
1.000 0.000
0.958 0.042
0.056 0.944
0.058 0.942
0.000 1.000
0.000 1.000
0.000 1.000
0.037 0.963
0.063 0.938
0.111 0.889
0.000 1.000
0.025 0.975
0.021 0.979
0.020 0.980
0.019 0.981
0.119 0.881
0.944 0.056
0.933 0.067
0.850 0.150
1.000 0.000
1.000 0.000
0.963 0.037
0.979 0.021
0.000 0.944 0.056
0.020 0.960 0.020
0.000 0.975 0.025
0.021 0.958 0.021
0.000 0.980 0.020
0.000 0.981 0.019
0.125 0.875 0.000
110 Table 2. Continued Locus
Est-2 a b Est-3 a b c Est-4 a b Est-6 a b c
Populations Oliveros M´aximo Paz
Pergamino
San Pedro
Z´arate
Gualeguay
Lechiguanas
0.083 0.917
0.017 0.983
0.125 0.875
0.000 1.000
0.020 0.980
0.033 0.967
0.000 1.000
0.028 0.972 0.000
0.125 0.875 0.000
0.100 0.900 0.000
0.042 0.958 0.000
0.040 0.940 0.020
0.017 0.983 0.000
0.042 0.958 0.000
0.972 0.028
0.967 0.033
0.950 0.050
1.000 0.000
1.000 0.000
1.000 0.000
1.000 0.000
0.361 0.583 0.056
0.467 0.533 0.000
0.475 0.525 0.000
0.354 0.604 0.042
0.400 0.540 0.060
0.500 0.483 0.017
0.261 0.739 0.000
Table 3. Levels of allozymic polymorphism for seven population samples of O. flavescens Population
Mean number of alleles per locus (A)
Percentage of polymorphic loci 95% 99% criterion criterion
Mean heterozygosis per locus Observed Expected
Oliveros M´aximo Paz Pergamino San Pedro Z´arate Gualeguay Lechiguanas
1.8 1.6 1.5 1.5 1.6 1.6 1.5
42.3 30.8 30.8 11.5 23.1 11.5 34.6
0.100 0.078 0.095 0.059 0.075 0.051 0.095
61.5 46.1 46.1 34.6 46.1 46.1 46.1
0.097 0.073 0.092 0.058 0.074 0.060 0.086
els of gene exchange among them (Nm between 52.69 and 80.03). Figure 3 is a plot of log Nm values against log of geographic distance between pairs of populations. There is no correlation between these values (r = 0.26; t = 0.94; P = 0.82).
Discussion
Figure 2. Topology of the tree obtained using the bootstrap technique under the UPGMA method and based on Nei’s (1973) genetic distance values. The lengths of branches are not correlated with genetic distance.
O. flavescens presents important degrees of genic variability. P and H values (26.37% and 0.077 respectively) are higher than those mentioned by Nevo, Beiles, and Ben-Shlomo (1984) as an average for 814 mammalian species (P = 19.1%; H = 0.041). The important difference between P95% and P99% values indicates that there is a large number of alleles that are maintained in low frequencies in all populations. This suggests that
111 Table 4. Genetic distance coefficient (Nei, 1972) between populations of O. flavescens
1 2 3 4 5 6 7
Oliveros M´aximo Paz Pergamino San Pedro Z´arate Gualeguay Lechiguanas
1
2
3
4
5
6
7
**** 0.0019 0.0034 0.0033 0.0042 0.0030 0.0028
**** 0.0021 0.0048 0.0059 0.0016 0.0044
**** 0.0083 0.0088 0.0019 0.0058
**** 0.0007 0.0058 0.0061
**** 0.0064 0.0078
**** 0.0049
****
Table 5. FIS , FST , and Nm values in O. flavescens Locus
FIS
FST
Nm
Mdh-2 Idh-1 Idh-2 Ldh-1 Acpk -1 Lap Acp1 Got-1 Got-2 Pgm-2 Me -Gpdh G6pdh 6Pgdh Est-2 Est-3 Est-4 Est-6
0.000 0.052 0.020 0.056 0.037 0.073 0.043 0.003 0.038 0.010 0.036 0.071 0.080 0.053 0.068 0.066 0.028 0.045
0.000 0.047 0.010 0.011 0.144 0.009 0.024* 0.005 0.015 0.002 0.009 0.037 0.038* 0.024* 0.037 0.009 0.014 0.013
– 5.06 24.75 22.48 1.49 27.53 10.17 – 16.42 – 27.53 6.51 6.33 10.17 6.51 27.53 17.61 18.98
Mean
0.027
0.035
15.27
P < 0.05; P < 0.01.
population sizes in this species are large, reducing the influence of genetic drift in the determination of allelic frequencies. The small degree of differentiation among populations is shown by the low values of genetic distances (DN ). It is also evident from the poor consistency of UPGMA clusterings. To infer the genetic structure of these populations, F statistics (Wright, 1931) were applied to observed spatial distribution of genotypes. The FIS index measures the reduction of heterozygosity due to nonrandom mating. Almost all FIS calculated for O. flavescens are negative, but none is significantly different from zero. This would indicate a small but systematic heterozygote excess com-
pared with expectations when there is random mating. Although O. flavescens usually nests on the surface, it has been frequently found in burrows during winter. In these habitats, it forms groups composed of one or more adult females and youngs of both sexes. Oligoryzomys flavescens tends to form social groups in winter, integrated by mothers and the litters born by the end of autumn. At the beginning of the breeding season (spring), these groups disperse (Crespo, 1966). This is in agreement with the finding of negative FIS values, which would indicate that matings between closely related individuals would be infrequent in populations of O. flavescens. In other rodent species of the same subfamily, avoidance of consanguineous mating has been observed (Theiler, 1997). Mean FST for 18 polymorphic loci was 0.035 and mean Nm value was high, 15.27 individuals per generation. Use of FST to estimate Nm does not tell us whether the lack of differences in allele frequencies among populations is due to a current high gene flow or to historical events (Slatkin, 1987, 1993). Slatkin (1993) proposed that even with high levels of gene flow, it is possible to detect a pattern of isolation by distance, provided that the long-distance dispersal is smaller than that at short distance, and that colonization is old enough to allow isolation to become evident. Lack of correlation between log Nm and log of geographic distance would indicate that colonization of the studied area by O. flavescens is relatively recent and the time elapsed not sufficient to attain an equilibrium between gene flow and genetic drift. However, our data and those from other investigators on ecological characteristics of this species indicate that important current gene flow cannot be discarded, especially among some of the analysed populations. High values of P and H support the assumption of a large effective population size. By using Slatkin’s private alleles approach, we also obtained high values of Nm (11.6). If FST and Nm values between popula-
112 Table 6. FST (below diagonal) and Nm (above diagonal) between populations of O. flavescens
1 2 3 4 5 6 7
Oliveros M´aximo Paz Pergamino San Pedro Z´arate Gualeguay Lechiguanas
1
2
3
4
5
6
7
**** 0.0046 0.0085 0.0188 0.0232 0.0124 0.0063
54.00 **** 0.0047 0.0469 0.0537 0.0044 0.0352
29.28 52.69 **** 0.0756 0.0695 0.0031 0.0392
13.01 5.09 3.06 **** 0.0000 0.0629 0.0581
10.54 4.41 3.35 – **** 0.0624 0.0674
19.87 56.08 80.03 3.73 3.76 **** 0.0463
39.23 6.86 6.13 4.05 3.46 5.15 ****
Figure 3. Plot of log Nm against log geographic distance between O. flavescens population pairs.
tion pairs are analysed, it can be observed that levels of gene flow are not homogeneous. Despite the short geographic distance separating San Pedro and Z´arate from Lechiguanas, Nm values are rather low, whereas those populations show little differentiation with Oliveros, which is the farthest located site (Table 6). This could be explained by passive unidirectional transport of animals along the river. The formation of large aggregates of aquatic plants close to the margins is common in the Paran´a River. These ‘floating packs’ of plants frequently detach from the river banks and are carried downstream. They frequently are the vehicle for the dispersal of plant and animal species (Tur, 1972). Because O. flavescens prefer humid habitats, like river borders, individuals of these species may be transported on these ‘vegetal rafts,’ promoting gene flow. A similar pattern has been described in Oligoryzomys microtis from the Juru´a river (a major tributary of the Amazon River) (Patton, Da Silva & Malcolm, 1996). It is possible that, in this case, the estimated gene flow reflects current levels. Although O. flavescens capacity for dispersal is unknown, Busch and Kravetz (1992) pointed out that this species is restricted to habitats with a well
structured plant cover and low levels of human disturbance, for example high weeds along railroads, road sides, and field borders. Those environments represent a continuous link between distant areas in the same region (Crespo, 1966). The competitive inferiority of O. flavescens with respect to other rodent species of the same habitats would promote its dispersion (Busch & Kravetz, 1992). However, determination of migration patterns with mark and recapture techniques are needed to accurately assess current levels of migration among O. flavescens populations. Oliveros is the only population with very high Nm values with respect to all others. This population, located in the center of the species’ geographic range (Redford & Eisenberg, 1984), also showed the highest values of A, H, and P. Mayr (1970) proposed that populations in the central area of the species’ distribution range possess greater intrapopulation variability than those located at the periphery. These populations, originated from a few colonizers coming from the central area, may lose low frequency alleles (founder effect) and, if the effective population size is maintained low for several generations, they may undergo reduction
113 of variability by genetic drift. This could be the case for populations like San Pedro and Gualeguay, while Oliveros could be a focus of dispersal toward the other sites occupied by the species in the studied area.
Acknowledgements We thank Drs. A. Blanco and M.S. Sabattini for their critical revision of the manuscript, and H. L´opez and D. Olivera, who collected the samples. This work has been supported, in part, by grants from the Consejo Nacional de Investigaciones Cient´ıficas y T´ecnicas of Argentina (CONICET), Consejo de Investigaciones Cient´ıficas y Tecnol´ogicas (CONICOR) the Subsecretar´ıa de Ciencia y Tecnolog´ıa of C´ordoba Province and from the Antorchas Foundation. M.B.C. is a Fellow of CONICOR and C.N.G. is a Career Investigator of CONICET.
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