167
Continuing Evolution of Xenopus Genome
the other hand, males of the Xenopus interspecies hybrids
tested are sterile, and the number of sperm is remarkably
lower than non-hybrids (Kobel, 1996; Malone et al., 2007).
Nevertheless, they may produce some viable sperm with
the ability to fertilize (Kobel, 1996 ). The ploidy of these
sperm is unknown, but they are estimated to be aneuploid,
since endoreduplication has not been demonstrated in male
Xenopus hybrids (Kobel and Du Pasquier, 1986). In contrast,
it has been reported that the sperm of a hybrid between X.
laevis and X. muelleri appeared to be much larger than its
parental species (Malone et al., 2007), and they could be
diploid. If hybrids are able to produce diploid eggs as well
as diploid sperm, and if they fertilize, an allopolyploid (allotetraploid) individual may be produced simply in the next
generation.
Amphibians have a long history of being experimental animals, and numerous research techniques have been
developed over the years. Besides Xenopus, it was reported
that amphidiploids (allotetraploids) developed from heatshock treatment of Rana brevipoda eggs inseminated with
sperm of Rana nigromaculata (Kawamura and Nishioka,
1960). Another approach has also been reported in which
autotetraploid individuals of R. nigromaculata and amphidiploids (allotetraploids) of R. nigromaculata and R. brevipoda were generated (Kawamura and Nishioka, 1983).
These were made possible by utilizing “traditional” methods for amphibian embryo manipulation, namely applying heat shock to inhibit frst cleavage after insemination
to double the whole genome (2x to 4x, for example), or
early cold, heat, or high-hydrostatic pressure treatment
to suppress extrusion of the second polar body to double
the maternal genome (see references in Kawahara, 1978).
To produce allotetraploids, the frst step was to produce
autotetraploid R. nigromaculata by heat-shock treatment
of normally fertilized eggs. The sperm (2x) from this
male autotetraploid R. nigromaculata were then used for
fertilization of eggs from diploid female R. brevipoda,
followed by early cold treatment to double the maternal
genome. This enables the fertilized egg to possess two full
sets of paternal as well as maternal chromosomes. These
methods would also be applicable to generate allopolyploids of Xenopus species. Furthermore, application of
hydrostatic pressure or late cold shock inhibits f rst cleavage (Reinschmidt et al., 1979; Geach et al., 2012), and by
the former treatment, autooctoploid X. laevis individuals
(designated as tetraploid in the literature) have been produced (Reinschmidt et al., 1979). Normal fertilized hybrid
embryos subjected to these treatments would presumably
grow into allopolyploid individuals. Therefore, theoretically, application of any of these methods could generate
allooctoploids of X. laevis and X. borealis as well as allohexaploids using either of them and X. tropicalis, three
species whose whole genome sequence is/will be available. It would be interesting to use the hybrids or allopolyploids to detect the immediate effects of genomic shock
and subgenome dominance, making use of the genetic
information at hand.
All things considered, X. laevis and its related species
could be a model for evolutionary studies of the genome
and for polyploidy. With the increase in molecular information, experimental hybrid formation, or allopolyploidization
between two different Xenopus species, they are excellent
tools for analyzing genomic shock, such as asymmetric
changes of epigenetic marks and gene expression patterns.
These kinds of analyses will shed light on how asymmetric
evolution of subgenomes initially took place and may also
provide some clues to imagine what happened in the two
rounds of WGD in the common ancestor of vertebrates, as
well as in the third round of WGD in the common ancestor
of teleosts.
ACKNOWLEDGMENTS
The authors are grateful to Dr. Shuji Takahashi (Hiroshima
University) and Dr. Yoshikazu Haramoto (National Institute
of Advanced Industrial Science and Technology) for providing unpublished data. This work was supported in part by
Japan Society for the Promotion of Science KAKENHI grant
numbers: (15K14521 to M.K.) and (25251026, 18H02447,
and 21K06126 to M.T.).
REFERENCES
Aparicio, S., Chapman, J., Stupka, E., Putnam, N., Chia, J.M., Dehal,
P., Christoffels, A., Rash, S., Hoon, S., Smit, A., Sollewijn
Gelpke, M.D., Roach, J., Oh, T., Ho, I.Y., Wong, M., Detter,
C., Verhoef, F., Predki, P., Tay, A., Lucas, S., Richardson,
P., Smith, S.F., Clark, M.S., Edwards, Y.J.K., Doggett, N.,
Zharkikh, A., Tavtigian, S. V., Pruss, D., Barnstead, M., Evans,
C., Baden, H., Powell, J., Glusman, G., Rowen, L., Hood, L.,
Tan, Y.H., Elgar, G., Hawkins, T., Venkatesh, B., Rokhsar,
D., Brenner, S., 2002. Whole-genome shotgun assembly and
analysis of the genome of Fugu rubripes. Science 297, 1301–
1310. https://doi.org/10.1126/science.1072104
Berthelot, C., Brunet, F., Chalopin, D., Juanchich, A., Bernard, M.,
Noël, B., Bento, P., Da Silva, C., Labadie, K., Alberti, A., Aury,
J.M., Louis, A., Dehais, P., Bardou, P., Montfort, J., Klopp,
C., Cabau, C., Gaspin, C., Thorgaard, G.H., Boussaha, M.,
Quillet, E., Guyomard, R., Galiana, D., Bobe, J., Volff, J.N.,
Genêt, C., Wincker, P., Jaillon, O., Crollius, H.R., Guiguen,
Y., 2014. The rainbow trout genome provides novel insights
into evolution after whole-genome duplication in vertebrates.
Nat. Commun. 5, 3657. https://doi.org/10.1038/ncomms4657
Bewick, A.J., Anderson, D.W., Evans, B.J., 2010. Evolution of the
closely related, sex-related genes DM-W and DMRT1 in
African clawed frogs (Xenopus). Evolution (N. Y). 65, 698–
712. https://doi.org/10.1111/j.1558-5646.2010.01163.x
Bird, K.A., VanBuren, R., Puzey, J.R., Edger, P.P., 2018. The causes
and consequences of subgenome dominance in hybrids and
recent polyploids. New Phytol. 220, 87–93. https://doi.
org/10.1111/nph.15256
Brown, D.D., Dawid, I.B., 1968. Specifc gene amplif cation in
oocytes. Science 160, 272–280. https://doi.org/10.1126/science.
160.3825.272
Brown, D.D., Dawid, I.B., Reeder, R.H., 1977. Xenopus borealis
misidentifed as Xenopus mulleri. Dev. Biol. 59, 266–267.
https://doi.org/10.1016/0012-1606(77)90263-9
Continuing Evolution of Xenopus Genome
the other hand, males of the Xenopus interspecies hybrids
tested are sterile, and the number of sperm is remarkably
lower than non-hybrids (Kobel, 1996; Malone et al., 2007).
Nevertheless, they may produce some viable sperm with
the ability to fertilize (Kobel, 1996 ). The ploidy of these
sperm is unknown, but they are estimated to be aneuploid,
since endoreduplication has not been demonstrated in male
Xenopus hybrids (Kobel and Du Pasquier, 1986). In contrast,
it has been reported that the sperm of a hybrid between X.
laevis and X. muelleri appeared to be much larger than its
parental species (Malone et al., 2007), and they could be
diploid. If hybrids are able to produce diploid eggs as well
as diploid sperm, and if they fertilize, an allopolyploid (allotetraploid) individual may be produced simply in the next
generation.
Amphibians have a long history of being experimental animals, and numerous research techniques have been
developed over the years. Besides Xenopus, it was reported
that amphidiploids (allotetraploids) developed from heatshock treatment of Rana brevipoda eggs inseminated with
sperm of Rana nigromaculata (Kawamura and Nishioka,
1960). Another approach has also been reported in which
autotetraploid individuals of R. nigromaculata and amphidiploids (allotetraploids) of R. nigromaculata and R. brevipoda were generated (Kawamura and Nishioka, 1983).
These were made possible by utilizing “traditional” methods for amphibian embryo manipulation, namely applying heat shock to inhibit frst cleavage after insemination
to double the whole genome (2x to 4x, for example), or
early cold, heat, or high-hydrostatic pressure treatment
to suppress extrusion of the second polar body to double
the maternal genome (see references in Kawahara, 1978).
To produce allotetraploids, the frst step was to produce
autotetraploid R. nigromaculata by heat-shock treatment
of normally fertilized eggs. The sperm (2x) from this
male autotetraploid R. nigromaculata were then used for
fertilization of eggs from diploid female R. brevipoda,
followed by early cold treatment to double the maternal
genome. This enables the fertilized egg to possess two full
sets of paternal as well as maternal chromosomes. These
methods would also be applicable to generate allopolyploids of Xenopus species. Furthermore, application of
hydrostatic pressure or late cold shock inhibits f rst cleavage (Reinschmidt et al., 1979; Geach et al., 2012), and by
the former treatment, autooctoploid X. laevis individuals
(designated as tetraploid in the literature) have been produced (Reinschmidt et al., 1979). Normal fertilized hybrid
embryos subjected to these treatments would presumably
grow into allopolyploid individuals. Therefore, theoretically, application of any of these methods could generate
allooctoploids of X. laevis and X. borealis as well as allohexaploids using either of them and X. tropicalis, three
species whose whole genome sequence is/will be available. It would be interesting to use the hybrids or allopolyploids to detect the immediate effects of genomic shock
and subgenome dominance, making use of the genetic
information at hand.
All things considered, X. laevis and its related species
could be a model for evolutionary studies of the genome
and for polyploidy. With the increase in molecular information, experimental hybrid formation, or allopolyploidization
between two different Xenopus species, they are excellent
tools for analyzing genomic shock, such as asymmetric
changes of epigenetic marks and gene expression patterns.
These kinds of analyses will shed light on how asymmetric
evolution of subgenomes initially took place and may also
provide some clues to imagine what happened in the two
rounds of WGD in the common ancestor of vertebrates, as
well as in the third round of WGD in the common ancestor
of teleosts.
ACKNOWLEDGMENTS
The authors are grateful to Dr. Shuji Takahashi (Hiroshima
University) and Dr. Yoshikazu Haramoto (National Institute
of Advanced Industrial Science and Technology) for providing unpublished data. This work was supported in part by
Japan Society for the Promotion of Science KAKENHI grant
numbers: (15K14521 to M.K.) and (25251026, 18H02447,
and 21K06126 to M.T.).
REFERENCES
Aparicio, S., Chapman, J., Stupka, E., Putnam, N., Chia, J.M., Dehal,
P., Christoffels, A., Rash, S., Hoon, S., Smit, A., Sollewijn
Gelpke, M.D., Roach, J., Oh, T., Ho, I.Y., Wong, M., Detter,
C., Verhoef, F., Predki, P., Tay, A., Lucas, S., Richardson,
P., Smith, S.F., Clark, M.S., Edwards, Y.J.K., Doggett, N.,
Zharkikh, A., Tavtigian, S. V., Pruss, D., Barnstead, M., Evans,
C., Baden, H., Powell, J., Glusman, G., Rowen, L., Hood, L.,
Tan, Y.H., Elgar, G., Hawkins, T., Venkatesh, B., Rokhsar,
D., Brenner, S., 2002. Whole-genome shotgun assembly and
analysis of the genome of Fugu rubripes. Science 297, 1301–
1310. https://doi.org/10.1126/science.1072104
Berthelot, C., Brunet, F., Chalopin, D., Juanchich, A., Bernard, M.,
Noël, B., Bento, P., Da Silva, C., Labadie, K., Alberti, A., Aury,
J.M., Louis, A., Dehais, P., Bardou, P., Montfort, J., Klopp,
C., Cabau, C., Gaspin, C., Thorgaard, G.H., Boussaha, M.,
Quillet, E., Guyomard, R., Galiana, D., Bobe, J., Volff, J.N.,
Genêt, C., Wincker, P., Jaillon, O., Crollius, H.R., Guiguen,
Y., 2014. The rainbow trout genome provides novel insights
into evolution after whole-genome duplication in vertebrates.
Nat. Commun. 5, 3657. https://doi.org/10.1038/ncomms4657
Bewick, A.J., Anderson, D.W., Evans, B.J., 2010. Evolution of the
closely related, sex-related genes DM-W and DMRT1 in
African clawed frogs (Xenopus). Evolution (N. Y). 65, 698–
712. https://doi.org/10.1111/j.1558-5646.2010.01163.x
Bird, K.A., VanBuren, R., Puzey, J.R., Edger, P.P., 2018. The causes
and consequences of subgenome dominance in hybrids and
recent polyploids. New Phytol. 220, 87–93. https://doi.
org/10.1111/nph.15256
Brown, D.D., Dawid, I.B., 1968. Specifc gene amplif cation in
oocytes. Science 160, 272–280. https://doi.org/10.1126/science.
160.3825.272
Brown, D.D., Dawid, I.B., Reeder, R.H., 1977. Xenopus borealis
misidentifed as Xenopus mulleri. Dev. Biol. 59, 266–267.
https://doi.org/10.1016/0012-1606(77)90263-9