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Proc Natl Acad Sci U S A. 2001 June 5; 98(12): 6720–6724. Published online 2001 May 29. doi: 10.1073/pnas.111144598. | PMCID: PMC34419 |
Copyright © 2001, The National Academy of Sciences Evolution Rates of nucleotide substitution in sexual and anciently
asexual rotifers David B. Mark Welch and Matthew S. Meselson* Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138 Accepted March 26, 2001. The class Bdelloidea of the phylum Rotifera is the largest well
studied eukaryotic taxon in which males and meiosis are unknown, and
the only one for which these indications of ancient asexuality are
supported by cytological and molecular genetic evidence. We estimated
the rates of synonymous and nonsynonymous substitutions in the
hsp82 heat shock gene in bdelloids and in facultatively
sexual rotifers of the class Monogononta, employing distance based and
maximum likelihood methods. Relative-rate tests, using acanthocephalan
rotifers as an outgroup, showed slightly higher rates of nonsynonymous
substitution and slightly lower rates of synonymous substitution in
bdelloids as compared with monogononts. The opposite trend, however,
was seen in intraclass pairwise comparisons. If, as it seems, bdelloids
have evolved asexually, an equality of bdelloid and monogonont
substitution rates would suggest that the maintenance of sexual
reproduction in monogononts is not attributable to an effect of sexual
reproduction in limiting the load of deleterious nucleotide
substitutions. There are many hypotheses,
but no general agreement, as to why sexual reproduction is so
widespread in eukaryotes and why most populations that become entirely
asexual, even if initially successful, suffer early extinction ( 1– 10).
Although theoretical models have helped to define the conditions under
which various hypotheses might be valid, what has been lacking is an
experimental system with characteristics that would allow critical
testing of hypotheses and which might also suggest novel ones. A taxon
that has evolved asexually for many millions of years would therefore
be of great interest. The class Bdelloidea of the phylum Rotifera,
comprising some 360 described species, seems to be such a taxon. Bdelloid rotifers are abundant invertebrate animals of worldwide
distribution, mainly in fresh-water and moist-terrestrial habitats.
Individuals range from about 0.1 to 1 mm in length and have muscles;
ganglia; tactile and photosensitive sensory organs; structures for
feeding, swimming, and crawling; digestive and secretory organs; and
ovaries. Despite much study of field and laboratory populations since bdelloid
rotifers were described by Leeuwenhoek more than 300 years ago, the
class Bdelloidea seems to be entirely female, without males or
hermaphrodites ( 11– 13). Where oogenesis has been investigated, eggs
are produced from oocytes by two mitotic divisions, without chromosome
pairing and without reduction in chromosome number, with each oocyte
yielding one egg and two polar bodies ( 14, 15). Consistent with this
apparent lack of meiosis, chromosomes without morphological homologues
are present in bdelloid karyotypes ( 14– 16). The group most closely related to Bdelloidea is the rotifer class
Monogononta, comprising some 1,100 described species ( 17– 21). Like
bdelloids, monogonont rotifers are abundant free-living animals a few
tenths of a millimeter long with worldwide distribution, mainly in
fresh- and brackish-water habitats. Monogononts reproduce sexually and
asexually. Female monogononts are diploid and usually produce eggs from
oocytes by one mitotic division, giving a polar body and a diploid egg
that develops into an asexual female like its parent. In response to
environmental stimuli, such females produce diploid daughters that make
exclusively haploid eggs by meiosis. If unfertilized, these eggs
develop into highly reduced haploid males that, by mating with females
bearing haploid eggs, give rise to females that reinitiate the asexual
cycle ( 22, 23). Bdelloid remains have been reported in 30–40-million-year-old amber
( 24) and measurements of DNA sequence divergence suggest that bdelloids
and monogononts separated well before that ( 19, 20, 25). Bdelloids and
monogononts seem to be similar in genomic DNA content, with about
10 9 bp in the species that have been studied ( 26,
27). They also have comparable fecundity and generation times, with
16–32 oocytes per ovary and an average of ≈2–3 weeks between
generations under favorable conditions ( 26, 28– 32). Two other groups within the phylum are the Acanthocephala, with
≈1,000 species, and the Seisonidea, with only two known species ( 17,
33, 34). Acanthocephalans are large endoparasitic rotifers with a life
cycle that alternates between a vertebrate and an arthropod host.
Seisonid rotifers only are found attached to only a particular genus of
marine crustaceans. Acanthocephalans and seisonids are dioecious and
are obligately sexual. Sequencing studies of the hsp82 heat
shock gene in monogonont, acanthocephalan, and seisonid rotifers reveal
the presence in individual genomes of two closely similar copies of the
gene, as is characteristic of alleles in sexually reproducing diploids
(refs. 25 and 35 and unpublished data). Recently, additional evidence that bdelloid rotifers evolved asexually
has been obtained from sequence studies of hsp82 and other
genes in individual bdelloid and monogonont rotifers of diverse species
( 25). As would be expected if bdelloids evolved for many millions of
years without meiosis or homologous recombination, individual bdelloid
genomes seem to lack closely similar pairs of the gene and instead
contain highly divergent copies that may be assigned to one or the
other of two ancient lineages that separated before the radiation of
modern bdelloids and after the separation of bdelloids from
monogononts, during the interval when sex was presumably lost. Two such
ancient lineages, both represented in individual bdelloid genomes, also
are found for the gene for the TATA/binding protein. The conclusion that bdelloids are ancient asexuals is supported further
by the remarkable finding that, unlike other rotifers or any other
eukaryote tested, bdelloids lack retrotransposons, consistent with the
expectation that deleterious nuclear parasites will not be present in
ancient asexuals ( 36, 37). Here we report comparisons of nucleotide substitution rates in the
hsp82 gene of bdelloids and monogononts that bear on the
effect of asexual reproduction on the evolution of mutation rates and
on mutational hypotheses for the prevalence of sexual reproduction. Sequences Examined. Sequences of hsp82 from two acanthocephalan rotifers,
Oligacanthorhynchus tortuosa and Oncicola sp.
(order Oligacanthorynchida), were determined by direct PCR sequencing
of DNA provided by M. García-Varela (Universidad Nacional
Autónoma de México, Federal District, Mexico City). The
remaining sequences that were analyzed have been described ( 20, 25) and
are from the following species: Seisonida, Seison nebaliae;
Acanthocephala, Moniliformis moniliformis (order
Moniliformida); Monogononta, Brachionus plicatilis strain
AUS, Brachionus calyciflorus (order Ploima), Eosphora
ehrenbergi (order Ploima), and Sinantherina socialis
(order Flosculariacea); Bdelloidea, Philodina roseola
(family Philodinidae), Habrotrocha constricta (family
Habrotrochidae), and Adineta vaga (family Adinetidae). The
same 870- to 879-bp coding segment of hsp82, corresponding
to Drosophila melanogaster codons 13–302 ( 38), was examined
in all cases. For each bdelloid species, the sequences that were analyzed were those
present in the genome of a single individual, having been amplified and
cloned from the DNA of a small population recently grown from a single
egg. Individual bdelloid genomes contain multiple diverged copies of
hsp82 (four in P. roseola, three in H.
constricta, and three in A. vaga). Each copy may be
assigned to one or the other of two lineages, designated A and B ( 25),
that arose before the radiation of modern bdelloids. For bdelloids,
calculations of the ratio of synonymous to nonsynonymous substitution
rates (Table 1) were based on the 21 possible pairwise interspecies
comparisons of the eight hsp82 sequences of the A lineage:
Pr1, Pr2, Hc1, Hc2, Hc3, Av1, Av2, and Av3. There are no interspecies
pairs within the B lineage in these three rotifer species. All other
calculations used all 10 bdelloid hsp82 sequences.
| Table 1Ratios of synonymous to nonsynonymous substitution rates in
rotifers estimated from intraclass comparisons |
Most of the differences between rotifer hsp82 genes are
synonymous and all copies seem to be functional, being free of stop
codons and frame shifts. Approximately one-fifth of the sequenced amino
acid sites in bdelloids and in monogononts are polymorphic and are
largely the same sites in the two classes. The sequences display a
moderate degree of codon bias, with an effective number of codons ( 39)
ranging from 30 to 37, intermediate between the values for
hsp82 in D. melanogaster and humans ( 40). With
the exception of atypically high usage of codons with G or C at
degenerate sites in the monogonont B. plicatilis ( 20, 40),
there is little difference in codon usage between bdelloids and
monogononts. Excluding B. plicatilis, coding-strand
composition within the region of hsp82 analyzed is nearly
identical in bdelloids and monogononts, with average A, T, G, and C
composition of 40, 27, 19, and 14%, respectively. Computations. Sequences were aligned as described ( 20); regions with gaps were not
excluded. Numbers of nucleotide differences per site at 4-fold
degenerate sites between aligned sequences were counted by using the
procedure of Li ( 41) and Pamilo and Bianchi ( 42) as implemented in the
diverge program of the Wisconsin Package 10.0 (Genetics
Computer Group, Madison, WI) but without adjustment for multiple hits.
Unadjusted numbers of amino acid differences per codon were counted
with the distances program. Substitutions per site at 4-fold degenerate sites, adjusted for
multiple hits and taking account of transition/transversion bias,
were determined by the methods of Li ( 41) and Pamilo and Bianchi ( 42)
implemented in the mega package ( 43). Differences at 4-fold
degenerate sites are slower to saturate and less sensitive to
transition-transversion bias than are differences at 2- and 3-fold
degenerate sites and their use requires no assumptions regarding the
relative weighting of codon degeneracy classes. The use of total
synonymous sites, however, provides more data for analysis and, for
maximum likelihood estimates, the problem of weighting degeneracy
classes does not arise. Substitutions per total synonymous (including 4-, 2-, and 3-fold) site
and per nonsynonymous site, adjusted for multiple hits and taking
account of transition/transversion bias, were determined by two
methods: ( i) the distance model of Yang and Nielsen ( 44)
implemented in PAML 3.0a ( 45), with equilibrium codon
frequencies calculated for each pairwise comparison from the average
nucleotide frequency at each of the three codon positions; and
( ii) the maximum likelihood model of Goldman and Yang ( 46)
implemented in paml, with equilibrium codon
frequencies calculated from the overall averages of the four nucleotide
frequencies, and with transition/transversion bias estimated from the
data. The ratio of synonymous to replacement substitutions was allowed
to vary among all branches of a phylogenetic tree that includes all 11
rotifer species listed above and is based on morphological and
molecular data (refs. 18– 21 and 25; Fig. ), and with
transition-transversion bias estimated from the data. Relevant
parameters in the program codeml were
runmode = 0, CodonFreq = 1, and model = 1. Relative-rate tests ( 47, 48) were used to compare the rates of
nucleotide and amino acid change during evolution from the common
bdelloid-monogonont ancestor to modern bdelloids and to modern
monogononts, using acanthocephalan rotifers as the outgroup. Referring
to the three-taxon tree of Fig. , the
rate for the path between the common ancestor and bdelloids is OB
= (BM + BA − MA)/2 and the average over all combinations of one
bdelloid, one monogonont, and one acanthocephalan sequence is
OB avg =
Σ[(B iM j +
B iA k −
M jA k)/2]/(n in jn k).
The monogonont B. plicatilis was not used in computing
synonymous rates because of its anomalously high GC content at
synonymous sites. Maximum-likelihood ratio tests of three-taxon trees
were performed on amino acid translations by using hyphy
0.71 [S. V. Muse (North Carolina State
University, Raleigh, NC) and S. L. Kosakovsky Pond (University of
Arizona, Tuscon, AZ)] with all amino acid changes weighted equally and
with changes weighted according to the Dayhoff matrix.
The Ratio of Synonymous to Nonsynonymous Rates Estimated from
Intraclass Comparisons. Intraclass comparisons of
hsp82 sequences reveal a large excess of synonymous over
nonsynonymous substitutions in bdelloids, monogononts, and
acanthocephalans (Table 1). Most nonsynonymous mutations therefore have
been eliminated by selection, consistent with the high degree of amino
acid sequence conservation in HSP82 in other eukaryotes ( 38, 49) and
with the preponderance of deleterious mutations over advantageous ones
in most proteins ( 35, 50). Each of the three methods of estimation give
a slightly greater value of the ratio of synonymous to nonsynonymous
rates for bdelloids as compared with monogononts, although in no case
is the difference greater than the average standard deviation of the
individual values (Table 1). The estimates for acanthocephalans,
however, are clearly higher than those for the other two classes of
rotifers. Synonymous and Nonsynonymous Rates Estimated from Interclass
Comparisons. The intraclass comparisons presented in Table 1 do not take account of
substitutions during the period before the separation of the most
distantly related sequences being compared and they allow comparison
only with respect to the ratio of synonymous to
nonsynonymous substitution rates. Therefore, relative-rate tests using
acanthocephalan rotifers as an outgroup were performed so as to obtain
estimates of the separate rates of synonymous and nonsynonymous
substitution in bdelloid and monogonont lineages during the interval
since they diverged from their common ancestor. To inspect the data without adjustment for multiple hits or codon
usage, relative-rate tests first were performed by using unadjusted
differences at 4-fold degenerate sites and differences at amino acid
sites. Fig. presents scatter plots of
the differences along lineages leading from the common ancestor to
bdelloids (OB) and to monogononts (OM). The symmetrical distribution of
data points indicates that the data set is free of conspicuously
anomalous 4-fold degenerate and amino acid differences along any
individual bdelloid or monogonont lineage. Although this simple
approach takes no account of multiple hits, the equal distribution of
data points on both sides of the line of bdelloid–monogonont equality
and the average values given in Table 2
reveal no consistent difference between bdelloid and monogonont
lineages in unadjusted 4-fold degenerate differences or in amino acid
differences.
| Table 2Synonymous and nonsynonymous substitution rates in rotifer
lineages estimated from relative-ratetests |
Neither is any clearly significant difference found between bdelloid
and monogonont lineages when multiple hits and codon usage are
considered in estimating synonymous and nonsynonymous substitution by
any individual method (Table 2). Along a given path (OB, OM, or OA),
all three methods of estimation give nearly the same values for
nonsynonymous substitution rates but more widely differing values of
synonymous rates, depending on how they deal with multiple hits and
codon usage. All three methods give slightly lower synonymous rates for bdelloids
(by about 20–30%), and two of the three estimates of nonsynonymous
rates are slightly lower for monogononts (by about 10–20%). This
trend may be seen in the differences between bdelloid and monogonont
branch lengths in the maximum likelihood phylogenetic trees of Fig.
. As mentioned above, however,
intraclass comparisons show the opposite trend (Table 1) and, being
based on less divergent sequences, are less dependent on methods of
adjustment for multiple hits.
Nonsynonymous rates along bdelloid and monogonont lineages were
compared further at the amino acid level by examining maximum
likelihood scores for each three-taxon
bdelloid–monogonont–acanthocephalan or bdelloid–monogonont–seison
tree with and without imposing the condition of equal rates. No tree
was found for which the constraint of equal bdelloid and monogonont
rates significantly reduced the likelihood scores. Using hsp82 coding sequences from three bdelloid
species (representing three of the four families of class Bdelloidea),
four monogonont species (representing two of the three orders of class
Monogononta), and three species of Acanthocephala (representing two
orders of Acanthocephala), we used intraclass sequence comparisons to
estimate the ratio of synonymous to nonsynonymous nucleotide
substitution rates in each rotifer class. We used relative-rate tests
to estimate synonymous and nonsynonymous substitution rates in
bdelloids and in monogononts during evolution from their common
ancestor, using three species of acanthocephalans as outgroups. In
addition, we obtained maximum likelihood estimates of the numbers of
synonymous and nonsynonymous substitutions in hsp82 along
each branch of a phylogenetic tree. Only small differences between bdelloid and monogonont substitution
rates were found by any method. The ratio of synonymous to
nonsynonymous substitutions was slightly less for bdelloids when
estimated from interclass comparisons, but the reverse was the case
when the ratio was estimated from intraclass comparisons. The ratio for
acanthocephalans is clearly greater than that for bdelloids and
monogononts and seems to result mainly from a higher synonymous rate.
It seems unwarranted, however, to ascribe the difference to any
particular cause, because acanthocephalans differ greatly from
bdelloids and monogononts not only in reproductive mode, but also in
many other respects, being obligate parasites of enormous reproductive
capacity and, in the species we examined, having warm-blooded hosts. The close similarity of synonymous substitution rates in
bdelloids and monogononts taxa that are similar in many respects, is
noteworthy. Under deterministic conditions and assuming that mutations
are generally deleterious, selection against alleles that increase the
mutation rate will be stronger in asexuals than in sexuals, all else
being equal ( 51– 54). This increased selection is because, in asexuals,
such alleles remain linked to the genomes in which they cause mutation,
eventually driving clones containing them to extinction. In sexuals,
however, these alleles soon segregate away. The close similarity of
bdelloid and monogonont synonymous rates therefore suggests that,
despite presumably stronger selection against mutator alleles in
bdelloids, mutation rates in both rotifer classes may be close to some
lower limit, as would result from the physical inaccessibility or
steeply rising metabolic cost of further increasing the fidelity of DNA
replication ( 55– 58). Our results also have implications for hypotheses to explain the
preponderance of sexual taxa over asexual ones and for the early
extinction of most populations that become asexual. Most of these
hypotheses seek to explain the advantage of sex as an effect of genetic
exchange between individuals in accelerating the production of
advantageous new genotypes and facilitating adaptation to altered
selection or in limiting the accumulation of deleterious mutations ( 6,
7, 10). It is to the latter group of hypotheses that our estimates of
nucleotide substitution rates are relevant. If, as might be envisaged
under stochastic ( 59) or deterministic mutational models ( 6, 7, 60),
sexual reproduction limits the deleterious load of nucleotide
substitutions in monogononts and thereby prevents their extinction, how
can we explain the evolutionary success of the Bdelloidea? It might be supposed that bdelloids have reduced their nucleotide
mutation rate to a value substantially below that of monogononts. This
possibility, however, seems to be ruled out by our finding of little if
any significant difference between bdelloids and monogononts in
synonymous substitution rates. Alternatively, one might speculate that
deleterious nucleotide substitutions are better tolerated in bdelloids
than they are in monogononts, owing to some factor peculiar to
bdelloids. We see no indication of this possibility in estimates of
nonsynonymous substitution rates and know of no evident reason why it
should be so. If our results for hsp82 are applicable to
bdelloid and monogonont genomes in general, and if sexual reproduction
keeps monogononts from becoming extinct, it seems unlikely that it does
so by limiting the deleterious load of nucleotide substitution. We thank Ben Normark for discussions at an early stage of this
article; Irina Arkhipova, James Crow, Alex Kondrashov, Jessica Mark
Welch, and Richard Thomas for critically reading the manuscript; and
the National Science Foundation Eukaryotic Genetics Program for
support. D.M.W. was supported by a National Science Foundation Graduate
Research grant. This article is dedicated to DeLill Nasser. 1. Darlington C D. The Evolution of Genetic Systems. Cambridge, U.K.: Cambridge Univ. Press; 1939. 2. Mayr E. Animal Species and Evolution. Cambridge, MA: Harvard Univ. Press; 1963. 3. White M J D. Modes of Speciation. San Francisco: Freeman; 1978. 4. Bell G. The Masterpiece of Nature: The Evolution and Genetics of Sexuality. Berkeley, CA: Univ. of California Press; 1982. 5. Berbee M L, Taylor J W. In: The Fungal Holomorph: Mitotic, Meiotic, and Pleomorphic Speciation in Fungal Systematics. Reynolds D R, Taylor J W, editors. Wallingford, U.K.: CAB International; 1993. pp. 67–78. 6. Kondrashov A S. J Hered. 1993;84:372–387. [PubMed] 7. Crow J F. Dev Genet. 1994;15:205–213. [PubMed] 8. Raikov I B. Eur J Protistol. 1995;31:1–7. 9. Judson O P, Normark B B. Trends Ecol Evol. 1996;11:41–46. 10. Hurst L D, Peck J R. Trends Ecol Evol. 1996;11:46–52. 11. Leeuwenhoek A. Philos Trans R Soc London. 1677;12:821–831. 12. Hudson C T, Gosse P H. The Rotifera or Wheel-Animalcules. Green, London: Longmans; 1886. 13. Ricci C. Hydrobiologia. 1987;147:117–127. 14. Hsu W S. Biol Bull. 1956;111:364–374. 15. Hsu W S. Cellule. 1956;57:283–296. 16. Mark Welch J L, Meselson M. Hydrobiologia. 1998;387/388:403–407. 17. Wallace R L, Snell T W. In: Ecology and Classification of North American Freshwater Invertebrates. Thorp J H, Covich A P, editors. San Diego: Academic; 1991. pp. 187–248. 18. Melone G, Ricci C, Segers H, Wallace R L. Hydrobiologia. 1998;387/388:101–107. 19. García-Varela M, de Leon P P, de la Torre P, Cummings M P, Sarma S S S, Laclette J P. J Mol Evol. 2000;50:532–540. [PubMed] 20. Mark Welch D B. Invert Biol. 2000;119:17–26. 21. Sørensen M V, Funch P, Willerslev E, Hansen A J, Olesen J. Zool Anz. 2000;239:297–318. 22. Gilbert J J. In: Reproductive Biology of the Invertebrates. Adiyodi K G, Adiyodi R G, editors. Vol. 1. New York: Wiley; 1983. pp. 181–209. 23. Wallace R L. In: Encyclopedia of Reproduction. Knobil E, Neill J D, editors. Vol. 4. San Diego: Academic; 1998. pp. 290–301. 24. Poinar G O, Jr, Ricci C. Experientia. 1992;48:408–410. 25. Mark Welch D B, Meselson M. Science. 2000;288:1211–1215. [PubMed] 26. Pagani M, Ricci C, Redi C A. Hydrobiologia. 1993;255/256:225–230. 27. Mark Welch D B, Meselson M. Hydrobiologia. 1998;387/388:395–402. 28. Ricci C. Atti Soc Sci Nat Museo Civ Stor Nat Milano. 1976;117:144–148. 29. Ricci C. Mem Ist Ital Idrobiol. 1978;36:109–116. 30. Amsellem J, Ricci C. Zoomorphology. 1982;100:89–105. 31. Ricci C. Hydrobiologia. 1983;104:175–180. 32. Ricci C. Hydrobiologia. 1991;211:147–155. 33. Crompton D W T, Nickol B B, editors. Biology of the Acanthocephala. Cambridge, U.K.: Cambridge Univ. Press; 1985. 34. Ricci C, Melone G, Sotgia C. Hydrobiologia. 1993;255/256:495–511. 35. Li W-H. Molecular Evolution. Sunderland, MA: Sinauer Associates; 1997. 36. Hickey D A. Genetics. 1982;101:519–531. [PubMed] 37. Arkhipova I, Meselson M. Proc Natl Acad Sci USA. 2000;97:14473–14477. [PubMed] 38. Blackman R, Meselson M. J Mol Biol. 1986;188:499–515. [PubMed] 39. Wright F. Gene. 1990;87:23–29. [PubMed] 40. Mark Welch D B. Dissertation. Cambridge, MA: Harvard; 1999. 41. Li W-H. J Mol Evol. 1993;36:96–99. [PubMed] 42. Pamilo P, Bianchi N O. Mol Biol Evol. 1993;10:271–281. [PubMed] 43. Nei M, Kumar S. Molecular Evolution and Phylogenetics. Oxford: Oxford Univ. Press; 2000. 44. Yang Z, Nielsen R. Mol Biol Evol. 2000;17:32–43. [PubMed] 45. Yang Z. Comput Appl Biosci. 1997;13:555–556. [PubMed] 46. Goldman N, Yang Z. Mol Biol Evol. 1994;11:725–736. [PubMed] 47. Sarich V M, Wilson A C. Science. 1973;179:1144–1147. [PubMed] 48. Wu C-I, Li W-H. Proc Natl Acad Sci USA. 1985;82:1741–1745. [PubMed] 49. Gupta R S. Mol Biol Evol. 1995;12:1063–1073. [PubMed] 50. Endo T, Ikeo K, Gojobori T. Mol Biol Evol. 1996;13:685–690. [PubMed] 51. Leigh E G., Jr Genetics. 1973;Suppl. 73:1–18. [PubMed] 52. Kimura M, Muruyama T. Genetics. 1966;54:1337–1351. 53. Kondrashov A S. Genet Res. 1995;66:53–70. 54. Johnson T. Genetics. 1999;151:1621–1631. [PubMed] 55. Sturtevant A. Q Rev Biol. 1937;12:464–467. 56. Blomberg C. J Theor Biol. 1985;115:241–268. [PubMed] 57. Blomberg C. J Theor Biol. 1987;128:87–107. [PubMed] 58. Kirkwood T B L, Rosenberger R F, Galas D J, editors. Accuracy in Molecular Processes: Its Control and Relevance to Living Systems. London: Chapman & Hall; 1986. 59. Muller H J. Mutat Res. 1964;1:2–9. [PubMed] 60. Kondrashov A S. Nature (London). 1988;336:435–440. [PubMed]
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