Molecular Ecology (2001) 10, 2349–2354
Blackwell Science, Ltd
S H O RT C O M M U N I C AT I O N
Frequency and distribution of
t
-haplotypes in the Southeast
Asian house mouse (
Mus musculus castaneus
) in Taiwan
S . - W. H U A N G , * K . G . A R D L I E † and H . - T. Y U *
*Department of Zoology, National Taiwan University, Taipei 106, Taiwan, R.O.C. †Center for Genome Research, Whitehead Institute, Cambridge, MA 02139, USA
Abstract
t-haplotypes are a meiotic drive system found on the 17th chromosome of the house mouse (Mus musculus). They can be found in wild populations of all four genetically differentiated subspecies. The drive phenomenon is male-specific, such that heterozygous males (+/t) show non-Mendelian transmission and transmit the t-chromosome to > 90% of their off-spring. So far the most comprehensive studies on the frequencies of t-haplotypes in natural populations have been on just one of the subspecies (M. musculus domesticus). We applied molecular methods to accurately screen t-haplotypes in a large number of populations of a second subspecies (M. musculus castaneus) distributed in Taiwan. We found that the overall
t-haplotype frequency is low in M. m. castaneus (0.108), and the chromosomes are patchily distributed among its populations, closely resembling the situation found in M. m. domesticus. Further, we found the frequencies of t-haplotypes in our sample did not differ in relation to the sex or age of mice. This resemblance in the frequency and distribution among popula-tions of the two distinct subspecies suggests that similar general mechanisms might be responsible for the low frequencies in both subspecies.
Keywords: meiotic drive, Mus musculus, M. m. castaneus, t-haplotypes, transmission ratio distortion, wild mice
Received 15 December 2000; revision received 7 June 2001; accepted 7 June 2001
Introduction
t-haplotypes are natural mutant forms of chromosome 17 in the house mouse (Mus muculus) (Silver 1985, 1993; Hammer et al. 1989; Ardlie 1998). They show non-Mendelian transmission from heterozygous +/t males, such that 90% of their offspring inherit the t-chromosome. Despite this powerful selective advantage in favour of t-haplotypes, many surveys of t-haplotypes show frequencies, much lower than expected, of around 10–25% +/t mice (see Ardlie & Silver 1998), and the mechanisms maintaining t-haplotypes in wild mouse populations have been the focus of t-haplotype research for decades (Silver et al. 1987; Delarbre et al. 1988; Hammer et al. 1989; Morita et al. 1992; Hammer & Silver 1993).
Genetic data from allozymes, mitochondrial DNA (mtDNA), and single nucleotide polymorphism (SNP)
indicate that the house mouse contain four major genetic entities —M. m. musculus, M. m. domesticus, M. m. bactrianus, and M. m. castaneus (Boursot et al. 1993; Bonhomme et al. 1994; Yonekawa et al. 1994; Lindblad et al. 2000), which are largely concordant with their separate geographical ranges (Boursot et al. 1996; Din et al. 1996). Although house mice of two separate subspecies can interbreed in the laboratory, their genomes are not totally compatible with each other. The most striking illustration of this is the narrow hybrid zone (20–40 km) found in central Europe where the two subspecies, M. m. musculus and M. m. domesticus, come into contact (Sage et al. 1993).
In contrast with the general restriction of gene flow among subspecies, t-haplotypes appear to be capable of moving freely across subspecies boundaries. They are found in all subspecies, and t-chromosomes retrieved from the different subspecies show an extremely low level of nucleotide variation, even much lower than the variation seen among their wild-type counterparts (Silver et al. 1987; Morita et al. 1992; Hammer & Silver 1993). Based on the Correspondence: Dr Alex Hon-Tsen Yu. Fax: 886 2-23636837;
E-mail: ayu@ccms.ntu.edu.tw
2350 S . - W. H U A N G , K . G . A R D L I E and H . - T. Y U results, it has been suggested that all present-day t -haplo-types are descendants of a single ancestral chromosome that existed recently (subsequent to the divergence of the M. musculus subspecies), and this t-haplotype then spreads worldwide relatively rapidly, crossing the sub-species boundaries. The transmission bias in favour of t -haplotypes is presumed to have favoured their spread across these genetic boundaries, and yet, different genetic backgrounds are known to have a strong effect on the levels of transmission ratio distortion (TRD) in laboratory settings (Gummere et al. 1986). Thus, we are interested in determin-ing whether there is any variation in t-haplotype properties, such as the frequency distribution, in a subspecies other than M. m. domesticus. If similar selective forces are acting on t-haplotypes in different subspecific genetic back-grounds, one would expect to see similar patterns in their distribution and frequencies in populations of the different subspecies. So far, the most comprehensive knowledge of t-haplotypes in wild populations has been obtained from M. m. domesticus (Dunn et al. 1960; Anderson 1964; Petras 1967; Klein et al. 1984; Figueroa et al. 1988; Lenington et al. 1988; Ardlie & Silver 1998), with a few limited studies carried out on M. m. musculus/molossinus and M. m. bactrianus (Tutikawa 1955; Klein et al. 1984; Ruvinsky et al. 1991). No studies as yet have focused on the Southeast Asian sub-species, M. m. castaneus.
In this study we screened a large number of M. m castaneus mice inhabiting rice granaries located all over Taiwan (see Chou et al. 1998) with recently developed molecular markers (Schimenti & Hammer 1990; Ardlie & Silver 1996). We investigate the geographical distribution and empirical frequencies of t-haplotypes in M. m. castaneus populations, and determine whether those populations containing t-chromosomes are related to one another by geographical location. The relationships of t-frequencies to two popula-tion parameters, sex and age, are examined as well.
Materials and methods
Sampling and age determination of mice
Since June 1995, we have collected a large number of house mice from rice granaries in 30 townships (Fig. 1; Table 1). We used body weight criterion to determine the age of all field-caught mice (see Chou et al. 1998). Mice weighing < 12 g are arbitrarily defined as ‘young’ mice, and ≥ 12 g as ‘old’ mice.
Molecular genotyping of
t
-haplotypes
Genomic DNA was prepared following standard protocols (Ausubel et al. 1995). We initially screened all mice for the presence of t-haplotypes by polymerase chain reaction (PCR). However, the Hba-ps4 PCR assay is known to incorrectly genotype a small fraction of mice due to gene
conversion. Thus, the genotypes of all individuals were further confirmed by restriction fragment length poly-morphism (RFLP) analysis (Hammer & Silver 1993; Ardlie & Silver 1996).
A region within the Hba-4 ps locus (alpha globin pseudo-gene) was amplified (Schimenti & Hammer 1990). PCR was performed in a 25 µL reaction containing 200 ng of
Fig. 1 Map indicating the location of the 39 mouse populations in our survey. Numbers correspond to the localities listed in Table 1. The relative positions of Jin-men (30) are shown in the inset panel. Asterisks indicate the populations with t-alleles. Light grey areas, 1000–2000 m; dark grey area, above 2000 m elevation.
t- H A P L O T Y P E F R E Q U E N C Y I N M O U S E P O P U L AT I O N S 2351
genomic DNA, 0.5 units of Taq polymerase (Promega), 1 µm of each primer, 250 µm dNTP, 12 mm MgCl2, and 2.5 µL of 10× reaction buffer. The reactions were amplified on a thermal cycler (MJ Research PTC-100) for an initial
cycle of 95 °C for 5 min, followed by 30 cycles at 95 °C for 30 s, 66 °C for 1 min, and 72 °C for 2 min. This assay amplifies 214 bp and 198 bp fragments on t-chromosomes and wild type chromosomes, respectively.
Geographic range Locality* Sample size no. of +/t
Northwestern 1. Ta-yuan 19 1 (0.05) 2. Gwan-yin 6 0 3. Hsin-wu† 15 1 (0.07) 4. Hsin-pu 10 0 5. Gwan-shi† 22 2 (0.09) Pooled 72 4 (0.06) Central 6. Tung-hsiao 4 0 7. Yuan-li 1 0 8. Ta-chia 5 0 9. Ta-an 1 1 (1.00) 10. Hou-li 2 0 11. Tsao-tun (#1) 9 0 (#4) 9 0 12. Shi-jou (?)‡ 16 0 (#7) 20 4 (0.20) (#8)† 30 7 (0.23) 13. Lin-nei (#3) 78 4 (0.05) (#9) 23 4 (0.17) Pooled 198 20 (0.10) Southwestern 14. Ma-dou 7 0 15. Jia-li 2 0 16. Shi-gang 18 4 (0.22) 17. Mei-nung 2 0 18. Ping-dung City 5 0 19. Wan-dan 2 0 Pooled 36 4 (0.11)
Southeastern 20. Tai-dung City 1 0
21. Guan-shan 13 0 22. Chr-shang 18 1 (0.06) 23. Shou-feng 35 12 (0.34) 24. Ji-an† 3 3 (1.00) Pooled 70 16 (0.23) Northeastern 25. Tung-shan (#3) 4 0 (#6) 12 2 (0.17) 26. San-hsing (#17) 5 0 (#18) 1 0 27. Wu-chiai (#2) 3 1 (0.33) (#6) 9 0 28. I-lan City (#10) 25 1 (0.04) (#13)† 16 1 (0.06) 29. Tou-cheng (#1) 20 0 (#2) 1 0 Pooled 96 5 (0.05)
Offshore isle 30. Jin-men† 19 4 (0.21)
Total 491 53 (0.108)
*Each number (1–30) represents one of the townships in our survey. Numbers in parentheses for some townships indicate different rice granaries and therefore different populations. †Include samples with unknown sex or body weight.
‡The ‘?’ mark indicates rice granary number not specified.
Table 1 Frequencies of +/t heterozygotes in 39 wild populations of house mouse (Mus musculus castaneus) in Taiwan
2352 S . - W. H U A N G , K . G . A R D L I E and H . - T. Y U The PCR results were further conformed by RFLP analysis (Hammer & Silver 1993; Ardlie & Silver 1996). Approxim-ately 10 µg of genomic DNA was completely digested with TaqI restriction endonuclease (New England Biolabs). Digested DNAs were electrophoresed, blotted, and bound to nylon membranes (Amersham) by UV cross-linking. The membranes were then probed with 32P-labelled Bb-40
clones overnight at 65 °C. Bb-40 defines the locus D17Leh66b and reveals four t-specific TaqI fragments (Schimenti et al. 1987). Hybridization and washing of the blots were carried out following standard protocols (Ausubel et al. 1995). Only one PCR negative mouse turned out to be RFLP positive, and all other t-bearing mice were confirmed by both methods, suggesting satisfaction of the PCR screening protocol.
Results and Discussion
Frequency of
t
-haplotypes and population ecology
Four hundred and ninety-one mice from 39 different populations were screened for the presence of t-haplotypes (Table 1). Fifty-three mice were +/t heterozygotes and none were t/t homozygotes, yielding an overall +/t frequency of 0.108. The frequencies of +/t mice among the 39 populations ranged from 0.04 to 0.34 (Table 1), excluding those with frequencies of 0 and 1.The lower-than-expected frequency of t-haplotypes observed among wild mouse populations has long been a paradox (reviewed in Silver 1993; Ardlie 1998), and the population ecology of house mouse has been implied to play a major role in maintaining a low t-haplotype frequency in natural populations. However, it is hard to analyse much of the early data from the viewpoint of population ecology, for most of the surveys collected mice across a general region, which often included several disparate localities. In contrast, the mouse populations sampled here are well-defined as mice from the same granary and considered as one population. Our sample sizes reported here usually reflect the relative population sizes (Chou 1996; Chou et al. 1998). These granary populations were typically ephemeral and unstable due to regular turnover of grain within a period of 2–3 years (Chou et al. 1998). The t-haplotype frequencies in them were, therefore, highly variable and greatly affected by genetic drift.
Populations of Mus musculus castaneus containing t -haplotypes are rare and sparsely distributed in Taiwan, and a similar pattern was found by Ardlie & Silver (1998) for M. m. domesticus. In M. m. domesticus, the overall fre-quency of +/t mice was also low (0.062); approximately 55% of the populations did not contain any t-haplotypes; and t-haplotype-containing populations were scattered in the sampling realm. In addition, the reported t-haplotype frequencies for two other subspecies, M. m. bactrianus and
M. m. musculus, were low as well (0.172 and 0.273, respect-ively) (Ruvinsky et al. 1991). Hence, the low frequency of t-haplotypes in natural populations appears to be a consist-ent phenomenon across all subspecies of house mouse.
Geographical distribution of populations containing
t-haplotypes
To examine whether there was any general geographical or clinal variation in t-haplotype frequencies, we performed spatial autocorrelation analyses (Koenig & Knops 1998; Bjørnstad et al. 1999; Koenig 1999) for 10 western townships and eight eastern townships, respectively. In these analyses, frequencies were calculated for each township and small samples (n ≤ 5) were excluded. The analyses did not show synchrony declines with distance (P = 0.674 western town-ships; P = 0.265 eastern townships), suggesting no obvious geographical pattern of the t-haplotypes. However, when data were pooled according to geographical regions (Table 1), the +/t frequency of the southeastern populations was found to be the highest (0.23), significantly different from the other four regions combined (0.08) (P = 0.0002). Removal of the small samples (n ≤ 5) without t-alleles did not alter this conclusion (P = 0.0006). Finally, the offshore isle also showed a high frequency of +/t frequency (0.21).
One possible explanation of the significantly higher t-frequency of southeastern populations than other parts of Taiwan is that they harboured a higher diversity of t-haplotypes (complementary groups). In natural popula-tions where a greater variety of lethal and semi-lethal t-haplotypes coexist, higher frequencies of t-haplotypes can be found (Ardlie & Silver 1998), because t/t homo-zygotes can survive and such females can also breed, trans-mitting t-haplotypes to their offspring. It has been shown that there is considerable population structure at the level of six geographical regions (Table 1) (Peng 1998). In particular, the southeastern populations stand out as being most dif-ferentiated from those of the other regions. Furthermore, mitochondrial D-loop sequence data have shown that the southeastern region harbours a high frequency of an older mtDNA lineage, which is distinct and separated by a deep branch from the other mtDNA haplotypes (Yang 1998). As a more ancient and the other newly intrusive mouse lin-eages coexist, it is plausible that southeastern Taiwan may have a higher diversity of t-haplotypes than that of other parts of Taiwan. Currently the types and diversity of t-haplotypes in Taiwanese M. m. castaneus are undetermined and will be pursued in future studies.
Frequencies of +/t heterozygotes in relation to sex and age
Since reduced fitness in +/t heterozygotes is often implicated to contribute to the low t-haplotype frequency in nature (Young 1967; Johnston & Brown 1969; Durand et al. 1997),t - H A P L O T Y P E F R E Q U E N C Y I N M O U S E P O P U L AT I O N S 2353 we measured two major population parameters, sex and
age, to determine their effect on t-haplotype frequency. Our data do not show any evidence for differences in t-haplotype frequencies between two sexes in 16 of 17 populations (one population, Ji-an, sex unrecorded) con-taining t-haplotypes. In eight of these 16 populations, the +/t frequencies were higher in males, while in the other eight populations the +/t frequencies were higher in females. The mean frequencies of t-haplotypes in two sexes (for the 16 populations with t-alleles) were the same, which were 0.14 for both males (n = 177) and females (n = 177). The goodness-of-fit test showed no significant difference in +/t frequency between sexes (P = 0.9700), implying no differential fitness existing between +/t males and +/t females as suggested by Lenington et al. (1988).
We then examined whether there was any relationship of +/t frequency with age. All mice were placed into one of two age categories (young or old) based on their body weight. The overall frequency of +/t heterozygotes among old mice was 0.18 (25/142), which was slightly higher than the frequency of 0.11 (22/193) among young mice; however, the difference was not significant (P = 0.2678). Therefore, it suggested that if any age-related fitness components act against t-haplotypes, they must be manifested prenatally, such as embryonic lethality or fertility differences (e.g. Johnston & Brown 1969; Lenington et al. 1994; Ardlie & Silver 1996) and not as postnatal survival rates.
Origin and rapid expansion of t-chromosomes
in house mouse
Here we have shown a considerable similarity in the low t-haplotype frequency, in two genetically differentiated subspecies of the house mouse: M. m. domesticus and M. m. castaneus. The similarity, which involved populations of two subspecies inhabiting disparate geographical ranges, suggests that not only are these chromosomes of the mouse subspecies closely related, but also the selection forces on them are similar. There may exist general mechanisms to prevent the spread of t-haplotypes in natural populations despite of their high transmission advantages.
Acknowledgements
We thank Schmenti for offerring DNA probes, Hwei-yu Chang, Chuck Langley, Wen-Hsiung Li, Manyuan Long, Chung-I Wu, three anonymous reviewers and the Subject Editor, Robert Wayne, for helpful comments on the manuscript. This research was supported by the National Science Council of the Republic of China (86– 2311-B-002–030-B17, 87–2311-B-002–016-B17, 88–2311-B-002–051).
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Shiao-Wei Huang is a PhD candidate in the Department of Zoology, National Taiwan University. Her doctoral thesis is focused on the population genetics of the t-haplotypes and major histocompatibility complex in the house mouse in Taiwan. Hon-Tsen Yu is an associate professor in National Taiwan University. His laboratory is working on the evolution of the house mouse in Taiwan and neighbouring areas, using molecular markers to address several evolutionary aspects of the species. Kristin G. Ardlie has studied the evolution of the t-haplotypes in Mus musculus domesticus for many years. Currently she is working at Genomics Collaborative.
