United States National Science Foundation .
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This research is made possible by ongoing support from the
Eukaryotic Genetics Program of the United States National Science Foundation . |
The objective of our research is to understand why meiosis and
sexual reproduction occur in most eukaryotic species, and why the loss
of these processes usually leads to early extinction.
Although asexual lineages occasionally arise from sexual
populations, and initially may even thrive, they are almost invariably
short-lived on an evolutionary time scale. In consequence, very few
species of animals
and plants reproduce only asexually and those that do rarely comprise
an
entire genus, let alone a taxon of higher rank. Despite this and other
evidence
that most asexual species do not last long enough to evolve higher
taxa,
there is no agreed explanation of what causes their early extinction,
or
of why meiosis and sexual reproduction prevent the early extinction
suffered
by most asexual lineages. Indeed, why meiosis and sex exist is a
fundamental unsolved problem of biology.
Our approach to the problem is to ask if, contrary to the
fate of most asexual lineages, there is a high-ranking taxon that has
evolved without meiosis and sexual reproduction and, if so, to learn
what has allowed it to escape extinction. The Class Bdelloidea of the
Phylum Rotifera appears to be such an exception. Bdelloid rotifers are small fresh-water
invertebrates comprising four families, 19 genera, and some 360
described species. Despite much study of field and laboratory
populations since the first observation of bdelloids by Leeuwenhoek in
1677, the entire Class appears to be without males, hermaphrodites or
meiosis. Eggs are produced from oocytes by two mitotic divisions, with
no chromosome pairing and no reduction in chromosome number. Consistent
with a lack of meiosis,
bdelloid karyotypes contain chromosomes
without morphological homologs.
Although remarkable, these observations do not preclude rare
or unrecognized forms of sexual reproduction. We have therefore sought
more rigorous evidence that bdelloid rotifers have evolved asexually.
We have
obtained three types of molecular and cytological evidence that
strongly
support the indication from the observations summarized above that the
Bdelloidea
evolved without meiosis and sex.
Our first approach made use of the fact that the genome of a
sexually-reproducing diploid contains two sets of chromosomes that are
kept closely homologous by segregation of haplotypes and genetic drift.
In contrast, if reproduction is only mitotic and without segregation of
any kind, sequences that were allelic will remain together, each
gradually accumulating mutations, so that after many millions of years
individual genomes will contain highly divergent copies of former
alleles. In accord with this expectation, within individual genomes of
diverse bdelloid species we found
highly divergent copies of each of four genes examined (hsp82,
RNApolIII, tbp
, and tpi ) and no closely similar copies. Moreover, each copy
of
a given gene belongs to one or the other of
two ancient lineages , which differ by approximately 50% at
synonymous sites and which separated
before the bdelloid radiation, when meiosis and sexual reproduction
appear
to have been lost. In contrast, as expected for sexually-reproducing
diploids,
we found only pairs of
closely homologous copies in the genomes of individual rotifers of
two other classes within the
phylum, the facultatively sexual Class Monogononta and the obligately
sexual
Class Seisonida.
While the above results are in striking agreement with our
expectation that former alleles would have become highly divergent in
an anciently
asexual lineage, they do not rule out an alternative possibility:
Bdelloids
could be sexually reproducing tetraploids, descended from a common
tetraploid
ancestor, with extremely low divergence between alleles, which
therefore
could have gone undetected in our sequencing studies. This possibility
is
ruled out, however, by our finding by fluorescent in situ
hybridization
(FISH) that bdelloid nuclei do not contain closely homologous
chromosome
pairs. For each of several bdelloid probes we tested,
FISH to embryo mitotic nuclei labels only one chromosome, not pairs
of chromosomes.
A third and altogether different line of evidence for
asexual
evolution of the Bdelloidea is our finding that, unlike all
sexually-reproducing
species tested,
bdelloids lack both major families of transposable retroelements —
LINE-like and gypsy-like retrotransposons. The lack of these elements
is consistent with the expectation that active deleterious
vertically-transmitted elements will not persist in ancient asexual
lineages. Eventually, either the host lineage will be driven to
extinction by its increasing insertional load
or, if it is to persist, such elements must be eliminated, attenuated,
or
inactivated. In sexually reproducing populations, however, deleterious
mobile
elements can persist indefinitely if recombination is sufficiently
effective
in constituting less-loaded genomes from more-loaded ones, or if some
other
process dependent on meiosis limits the deleterious load.
Having established that the Bdelloidea have almost certainly
evolved without meiosis and sex, we have begun to use bdelloid rotifers
to test various hypotheses that seek to account for the maintenance of
sex. A principal class of hypotheses holds that sexual reproduction, by
either stochastic or deterministic processes, limits the accumulation
of deleterious nucleotide substitutions. This raises the possibility
that bdelloids may have an unusually low mutation rate. Using relative
rate tests and other methods of comparison, we found that the rates of
synonymous and nonsynonymous nucleotide substitutions
at hsp82 in bdelloids
are not significantly different from those in the facultatively
sexual rotifers of the class Monogononta.
We
have suggested that it may be the limitation of deleterious
insertions, rather than deleterious nucleotide substitutions that
accounts for the maintenance of meiosis and sex. This would be
consistent with the lack of retrotransposons in bdelloids and suggests
that bdelloids either lost such elements close to the time when sex was
lost, either by chance
or else by some active mechanism of suppression or removal. In order to
investigate these possibilities we are examining bdelloid genomes for
relict retrotransposons. Phylogenenetic analysis of such relict
sequences may allow us to determine the stage in bdelloid evolution
when they ceased to be active and may even provide information
regarding the mechanism of such inactivation.
Recently, we have found what may be such a relict and are isolating
and sequencing it from diverse bdelloid species. Moreover, these highly
unusual retroelements may have become domesticated and could play a
role in telomere maintenance, similar to Drosophila or Giardia. We are currently
analyzing bdelloid
telomeres and trying to find correlations between telomere structure
and the lack of meiosis.
We are developing methods to render bdelloid genomes
susceptible to genetic investigation. In addition to the introduction
of transgenes (under development), we have developed a protocol for
using RNAi to inactivate
specific bdelloid gene transcripts.
Using the
high-throughput sequencing capability of the Bay Paul Center , we have
begun a basic characterization of the genomes from the major bdelloid
orders. We will characterize and compare, inter alia, gene
density, intronic structure, intergenic regions, relict or pseudo genes
and insertions, discontinuities of sequence divergence, and Hox gene
structure
and organization. Moreover, in so unusual a genetic system as that of
bdelloid
rotifers, such genomic analysis may reveal important novel or
unanticipated
features.
Further opportunities for utilizing bdelloid rotifers in the
study of basic genetic problems include investigations of DNA repair,
genetic silencing, heterosis, population genetic diversity, and
response to selection. We welcome inquiries regarding joining or
collaborating with us.
References and further details can be found in the summaries
of our current grant support from the National Science Foundation:
