All 15 of the proteins stained for provided adequate resolution or activity to be interpreted. Locus abbreviations and enzyme commission numbers along with the buffers and tissues yielding the best results are given in Table . The following protein products migrated cathodally: AAT-2, ADH, GAP, GPI-1, -2, MDH-2 and PGD-2.
Locus abbreviations, enzymes stained for, enzyme commission numbers (E. C. no.) as well as the tissues and buffers giving optimal results (buffer descriptions are given in Materials and Methods).
Nineteen (49%) of the 39 loci studied were polymorphic in one or more of the four populations analysed (Table ). These were: AAT-1
. This was somewhat higher than the previous 33% estimate in the preliminary study, and mainly due to the presence of the additional markers detected. Fixed allele differences between M. natalensis
and M. coucha
were detected at AAT-1
(blood; Fig. ), ADH
(liver; Fig. ), EST-1
(liver; Fig. ), PGD-1
(muscle; Fig. ), Hb-1
(blood; Fig. ). Locus differences were confirmed in the new material at GPI-2
. The differences at these last three loci were considered to be isozyme rather than allozyme differences due to the large separation distance between the bands [10
]. Thus these three loci remain useful in identifying individuals from either species, along with the new allozyme markers.
Allele frequencies, average heterozygosity per locus (H), mean number of alleles per locus (A) and percentage of loci polymorphic (P) for all populations studied with standard errors in parentheses.
Fixed allele differences between Mastomys coucha and M. natalensis at PGD-1.
Fixed allele differences between Mastomys coucha and M. natalensis at AAT-1.
Fixed allele differences between Mastomys coucha and M. natalensis at EST-1.
Fixed allele differences between Mastomys coucha and M. natalensis at ADH.
Fixed allele differences between Mastomys coucha and M. natalensis at Hb.
were not analysed in the La Lucia and Montgomery Park populations in the initial study. However, the results obtained via PAGE for the Hb
locus in the Vaal and Richards Bay populations, using the haemoglobin purification method described by Falk et al.
], are analogous to those by Gordon [8
]. Significant differences (FIT
= 0.936, P
< 0.05) were obtained for EST-1
in the earlier study, but the additional data from the new material and increased resolution at this locus allowed us to re-evaluate those results, and indicate this locus is useful as another marker (Fig. ). Results for the ADH
locus were also re-evaluated, as analysis of the new populations was performed on different buffer systems that provided better resolution than previously obtained (Fig. ). The most cost and labour effective analysis for the routine identification of these two species would appear to be to stain for the GPI-2
loci on an RW
buffer system running muscle and liver samples. While PAGE may provide better resolution, it is more expensive, requires more time to run and only one protein can be stained for at a time. The other markers at PGD-1
are on incompatible buffer systems, and the purified haemoglobin samples needed for the AAT-2
loci are rendered less economic by the additional time, materials and equipment needed for preparation.
The genotypes at the IDH-1 and LDH-2 loci in the Vaal (M. coucha) population and the PGM-1, -2 and -3 loci in the La Lucia (M. natalensis) population were close to the expected Hardy-Weinberg proportions for naturally occurring populations. Deficiencies of heterozygotes were observed at the following loci: AAT-2 (Richards Bay and Montgomery Park populations), ADH-1 (Richards Bay), EST-2 (Montgomery Park), GAP (Richards Bay), IDH-1 (Montgomery Park and La Lucia), IDH-2 (La Lucia and Richards Bay), LDH-2 (Montgomery Park) and PGM-4 (La Lucia). There were no deviations of allele classes from Hardy-Weinberg proportions in the Vaal population, which is surprising as this had the smallest sample size of all four sites.
) was 14.7 and 11.4% in the La Lucia and Richards Bay populations of M. natalensis
, whereas the values for Montgomery Park and Vaal populations of M. coucha
were 8.8 and 5.7% respectively, with average values of 13.1% for M. natalensis
and 7.3% for M. coucha
(Table ). These are comparable to values reported for other rodent species. Johnson and Selander [12
] obtained 7.9% in the Heteromyid Dipdomys
, 17.4% for Otomys irroratus
], 16.1% for Rhabdomys pumilio
], and 6.7% for Oryzomys palustris
]. The values for M. coucha
are lower than those for M. natalensis
, and although the lowest value of 5.7% for the Vaal population could be attributed to small sample size, one could expect that the much larger geographic range of M. natalensis
(from southern Africa right through to west Africa) and the greater diversity of biome type would give this species a somewhat larger degree of genetic variation.
Numbers of alleles per locus (A
) values were similar, ranging from 1.06 for the Vaal population to 1.21 for the La Lucia population, whereas the Richards Bay and Montgomery Park populations were calculated to be 1.11 and 1.12. Individual heterozygosity (h
) values for the Montgomery Park population ranged from 0.064 (LDH-2
) to 0.278 (IDH-1
), for the Vaal population, 0.105 (LDH-2
was the only heterozygous locus within this population), La Lucia from 0.069 (PGM-4
) to 0.594 (IDH-1
) and from 0.124 (IDH-2
) to 0.500 (AAT-2
). The mean H
value for M. coucha
was 0.018, and 0.032 for M. natalensis
. These values put these two species in the lower range of rodent variation (Figure ). A much higher mean heterozygosity (0.073) was obtained by Mahida et al.
] in their genetic assay of the grassland specialist, Rhabdomys pumilio
. This is interesting, as Rhabdomys
is a species that prefers a more pristine habitat than the opportunistic and pioneering multimammate mice. Indeed, M. coucha
and M. natalensis
tend to be replaced over time by Rhabdomys
species in areas that are recovering from habitat damage of some sort [7
values determined by Taylor et al.
] for O. irroratus
were also high (mean = 0.071). One would expect that generalist species like M. coucha
and M. natalensis
might possess more variation in the genome to deal with more challenging types of habitats, especially human-disturbed, but it would appear that this is not the case. H
for M. natalensis
was calculated to be nearly double that of M. coucha
, but the low value for the Vaal population was probably due to the small sample size, which resulted in that species' average being brought down. On the whole, M. natalensis
does appear to possess more genetic variation than M. coucha
, perhaps because of the much greater range of biome types spanned by the former species, as mentioned above.
Comparative heterozygosities (H) for various species of Rodentia, along with the mammalian mean.
values of Wright [16
] are useful to estimate the amount of genetic differentiation between species (Table ). High values of FST
are considered to reflect substantial differences at any given locus, and are expected when studying separate species or populations that have diverged to a certain degree. Wright [16
] used the following groupings for the evaluation of FST
values: the range 0 to 0.05 is considered to reflect little genetic differentiation; 0.05 to 0.15 is indicative of moderate differentiation; 0.15 to 0.25 indicates great genetic differentiation and values greater than 0.25 reflect very great genetic differentiation. All of the measures have increased slightly with the addition of the new material, with pair-wise FST
values between the species of 0.882 (Vaal and Richards Bay populations) and 0.743 (La Lucia and Montgomery Park populations) indicating large amounts of differentiation between the two species by the abovementioned criteria (Table ). As expected, this is higher than the FST
value of 0.375 for populations of O. irroratus
] and 0.459 for populations of the striped field mouse R. pumilio
]. These are comparable to the FST
values of 0.558 between the two M. coucha
populations, and 0.228 between the two M. natalensis
populations. Pair wise Χ2
probability totals between both sets of populations (Vaal-Richards Bay and Montgomery Park-La Lucia) were zero (Table ), indicating significant (p = 0) differentiation as expected for the two species.
Summary of F-statistics between the Richards Bay and Vaal populations, the Montgomery Park and La Lucia populations (bold) at all loci analysed, as well as contingency Χ 2 analysis for the pooled data of the two species.
Genetic distance (D
) is a measure of the amount of genetic divergence that has occurred between two species since a hypothetical separation point sometime in their evolutionary past. The D
value of Nei [17
] is specially adapted for small sample sizes and the mean value, between M. coucha
and M. natalensis
was 0.26, with a D
of 0.31 between the Richards Bay and Vaal populations and 0.21 between the La Lucia and Montgomery Park populations. The discrepancy between these values is the result of the additional allozyme markers detected in the new material. This value is obviously slightly larger than those obtained from intraspecific calculations of D
between populations of R. pumilio
= 0.189, [14
]) and O. irroratus
= 0.117, [13
Murid rodents are biologically interesting subjects for genetic analysis, due to their rapid evolutionary radiation, and cytogenetic differences are common between genera and species [1
]. It would seem that the sibling species of M. coucha
and M. natalensis
are more examples of this phenomenon, with a chromosomal mutation within M. natalensis
giving rise to the possibly younger M. coucha
, which has been subsequently isolated reproductively. It is unknown as to why these two species, which have so much in common biologically, do not act as reservoirs for the same pathogens, although it is possible that differing physiological biochemistry may be the reason. Still, multimammate mice remain the most important pest species of African rodents [18
] both medically and agriculturally, and the isozyme markers reported here, as well as the new allozyme markers are reliable methods of distinguishing between them. This is certainly an aid to identify various disease threats from either species in areas like the Kruger National Park, where the two species occur in sympatry, as well as assisting in determining the eventual distribution of both species.