Download Letter The Evolution of Male–Female Sexual

The Evolution of Male–Female Sexual Dimorphism Predates the
Gender-Based Divergence of the Mating Locus Gene MAT3/RB
Rintaro Hiraide,1 Hiroko Kawai-Toyooka,1 Takashi Hamaji,2 Ryo Matsuzaki,1 Kaoru Kawafune,1 Jun Abe,3
Hiroyuki Sekimoto,4 James Umen,5 and Hisayoshi Nozaki*,1
1
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan
Department of Botany, Graduate School of Science, Kyoto University, Kyoto, Japan
3
Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Tokyo, Japan
4
Department of Chemical and Biological Sciences, Faculty of Science, Japan Women’s University, Tokyo, Japan
5
Donald Danforth Plant Science Center, St. Louis, Missouri
*Corresponding author: E-mail: [email protected].
Associate editor: Hideki Innan
New sequence data are deposited in the DDBJ database (accession nos. AB771928–AB771954).
2
Abstract
The molecular bases for the evolution of male–female sexual dimorphism are possible to study in volvocine algae because
they encompass the entire range of reproductive morphologies from isogamy to oogamy. In 1978, Charlesworth suggested the model of a gamete size gene becoming linked to the sex-determining or mating type locus (MT) as a mechanism for the evolution of anisogamy. Here, we carried out the first comprehensive study of a candidate MT-linked
oogamy gene, MAT3/RB, across the volvocine lineage. We found that evolution of anisogamy/oogamy predates the
extremely high male–female divergence of MAT3 that characterizes the Volvox carteri lineage. These data demonstrate
very little sex-linked sequence divergence of MAT3 between the two sexes in other volvocine groups, though linkage
between MAT3 and the mating locus appears to be conserved. These data implicate genetic determinants other than or in
addition to MAT3 in the evolution of anisogamy in volvocine algae.
Letter
Key words: gender-based divergence, gene conversion, male–female sexual dimorphism, MAT3/RB, mating type locus,
volvocine algae.
Sexual reproduction in the eukaryotes is classified according
to gamete size and motility, with three major types: isogamy
(equal sized gametes), anisogamy (large and small gametes),
and oogamy (large immotile eggs and small motile sperm)
(Bold and Wynne 1985). Anisogamous and oogamous organisms represent male–female sexual dimorphism that has
arisen repeatedly in evolution, presumably from simpler isogamous mating systems in ancestral unicellular species (Parker
et al. 1972; Kirk 2006). However, the molecular-evolutionary
bases for the transitions from isogamy to anisogamy to
oogamy are difficult or impossible to study in most extant
lineages due to the ancient origins of oogamy (Kirk 2006;
Nozaki et al. 2006). Volvocine algae or colonial Volvocales
are exceptional because they are a relatively young group,
yet they encompass the entire range of reproductive morphologies from isogamy to oogamy in an extant, phylogenetically coherent lineage (Nozaki 2003; Kirk 2006; Herron et al.
2009) (fig. 1).
In volvocine algae, sex is determined by a single mating
type locus (MT) with two haplotypes that specify sexual differentiation. Although MT segregates as a single Mendelian
trait, it is a complex genomic region that contains both shared
and sex-limited genes that are rearranged with respect to
each other and which do not undergo meiotic recombination
(Umen 2011). A recent comparative study of MT from an
oogamous volvocine species, Volvox carteri, with MT from an
isogamous species, Chlamydomonas reinhardtii, revealed
major differences in the size- and sex-based differentiation
of MT genes (Ferris et al. 2010). Volvox MT was found to be
about five times larger than Chlamydomonas MT, to contain
more genes than Chlamydomonas MT, and to show a much
higher degree of sex-based differentiation in its shared genes
(those with an allele in both mating haplotypes and sexes).
Shared genes that have become masculinized and feminized
in sequence and/or expression as occurred in V. carteri are
candidates for contributing to male–female sexual dimorphism. However, to date there has been no investigation of
MT genes in the context of the isogamy to anisogamy/
oogamy transition in volvocine algae besides the previously
described comparison of C. reinhardtii and V. carteri (Charlesworth D and Charlesworth B 2010; Ferris et al. 2010).
One candidate regulator of gamete size is the mating locus
gene MAT3. MAT3 encodes a homolog of the retinoblastoma
(RB) tumor suppressor protein. In Chlamydomonas, MAT3 is
tightly linked to MT and regulates cell size and cell cycle
progression (Umen and Goodenough 2001; Fang et al.
2006). Based on its repertoire of cell cycle regulatory proteins
that are orthologous to those in Chlamydomonas, a similar
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1038
Mol. Biol. Evol. 30(5):1038–1040 doi:10.1093/molbev/mst018 Advance Access publication January 30, 2013
Evolution of Mating Type Locus Gene MAT3/RB . doi:10.1093/molbev/mst018
MBE
FIG. 1. Simplified diagram for stepwise evolution of colonial volvocine algae and their transition from isogamy to anisogamy and oogamy (Nozaki 2003;
Herron et al. 2009).
function is predicted for the Volvox MAT3/RB pathway
(Prochnik et al. 2010). However, in contrast to Chlamydomonas where the minus and plus MAT3 alleles are nearly identical
and function interchangeably (Umen and Goodenough 2001;
Merchant et al. 2007), a high degree of male–female sequence
differentiation and sex-regulated alternative splicing was observed for V. carteri MAT3 (Ferris et al. 2010). This observation
led Ferris et al. (2010) to suggest that MAT3 homologs might
be related to control of gamete size in colonial volvocine algae
as predicted earlier by the gamete size regulator recruitment
model for the evolution of anisogamy/oogamy from isogamous mating types (Charlesworth 1978). If this model applies
to MAT3, then the degree of differentiation between MAT3
alleles from each mating type in isogamous species should be
much lower than in males–females from anisogamous and
oogamous species where MAT3 would have acquired sexspecific functions in gamete size control.
Here, we sequenced full-length coding regions of MAT3
from plus and minus mating types of isogamous Gonium
pectorale and Yamagishiella unicocca, and from males and
females of anisogamous Eudorina sp. and Pleodorina starrii,
and from males and females of oogamous V. africanus (supplementary fig. S1 and table S1, Supplementary Material
online). In contrast to V. carteri where the male and female
MAT3 alleles show large differences in structure and sequence
(Ferris et al. 2010), MAT3 homologs from the five colonial
species examined here had almost identical nucleotide sequences between the two sexes (fig. 2).
Our phylogenetic analysis of MAT3 sequences demonstrated that the extensive MAT3 divergence in the V. carteri
lineage might have occurred recently in the ancestor of the
three V. carteri forms after their divergence from the anisogamous lineage containing P. starrii and Eudorina sp. (fig. 3).
Therefore, the extreme, gender-based MAT3 divergence observed in V. carteri species may not be directly related to the
evolution of male and female dimorphism within the colonial
Volvocales as a whole because it is not observed in their
nearest anisogamous relatives (figs. 2 and 3). Moreover, in
the oogamous species V. africanus MAT3 showed only
modest levels of male–female divergence compared with
V. carteri male and female MAT3 (fig. 2; supplementary
table S2, Supplementary Material online).
FIG. 2. Gender-based divergence of MAT3 genes from six species of the
colonial Volvocales and Chlamydomonas reinhardtii (supplementary
table S2, Supplementary Material online). Bar graph depicting dN
(number of substitutions per nonsynonymous site) and dS (number
of substitutions per synonymous site) between MAT3 alleles from each
of the two mating types or sexes.
This study demonstrates that MAT3 genes from the two
sexes have been undergoing some form of genetic exchange
between mating types or sexes during most of their evolution
within the colonial Volvocales (fig. 3) that serves to preserve
homogeneity between alleles from each mating type. Gene
conversion as proposed recently (Teshima and Innan 2004;
Umen 2011) may contribute to sequence homogenization of
MT-linked genes such as MAT3 alleles that cannot undergo
crossover recombination. Gene conversion was also observed
in a recent study of the Cryptococcus neoformans mating
locus that is structured similarly to MT from volvocine
algae with two rearranged haplotypes (Sun et al. 2012), but
gene conversion has not been reported for volvocine algal MT
genes. It can be speculated that complete loss of recombination and gene conversion of MT genes in V. carteri was a
major contributor to male–female differentiation in this
sublineage.
Sex-specific expression and/or alternative splicing of MAT3
(Ferris et al. 2010) that might contribute to male–female
gamete size differences in anisogamous and oogamous volvocine algae were not detected in our reverse transcriptase
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MBE
Hiraide et al. . doi:10.1093/molbev/mst018
FIG. 3. Phylogeny of MAT3 proteins from the colonial Volvocales and Chlamydomonas. The analysis is based on 464 amino acid positions of slowly
evolving regions of the alignment (supplementary fig. S1 and alignment, Supplementary Material online). Branch labels indicate from left to right:
Posterior probabilities (0.90) from Bayesian inference/Bootstrap values (50%) obtained using 1,000 replicates with RAxML/Bootstrap values (50%)
using 1,000 replicates with PhyML/Bootstrap values (50%) using 1,000 replicates with maximum parsimony. For details, see supplementary information, Supplementary Material online.
polymerase chain reaction experiments for isogamous
Gonium pectorale, anisogamous Eudorina sp. and oogamous
V. africanus (supplementary fig. S2, Supplementary Material
online). Although our data do not completely rule out a role
for MAT3 in controlling gamete size in anisogamous and
oogamous volvocine algae, they raise the intriguing question
of what other MT genes are involved in specifying this trait.
Future molecular genetic studies aimed at characterizing MT
genes from anisogamous and oogamous volvocine species
will help to answer this question.
Materials and Methods
Volvocalean strains and MAT3 sequences used in this study
are listed in supplementary table S1, Supplementary Material
online. Amino acid alignment of MAT3 proteins is available in
supplementary alignment, Supplementary Material online.
Other details of Materials and Methods are described in supplementary information, Supplementary Material online.
Supplementary Material
Supplementary information, alignment, figures S1 and S2, and
tables S1 and S2 are available at Molecular Biology and
Evolution online (http://www.mbe.oxfordjournals.org/).
Acknowledgments
Dr H. Innan (Graduate University for Advanced Studies,
Hayama, Japan) kindly discussed the gene conversion.
Shotgun sequencing was carried out by Kazusa DNA
Research Institute (Kisarazu, Chiba, Japan). This research
was mainly done at the University of Tokyo. The alignment
of MAT3 is available (ID: S13735) via TreeBASE (http://www.
treebase.org/treebase-web/home.html). This work was supported by the Ministry of Education, Culture, Sports,
Science and Technology/Japan Society for the Promotion of
Science KAKENHI grants 24112707 and 24247042 to H.N. and
1040
22-40216 to H.K.-T. and the National Institutes of Health
grant GM078376 to J.U.
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