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. 2019 Dec 4;23(1):100757. doi: 10.1016/j.isci.2019.100757

Parallel Evolution of Two dmrt1-Derived Genes, dmy and dm-W, for Vertebrate Sex Determination

Yusaku Ogita 1,7, Shuuji Mawaribuchi 2,7, Kei Nakasako 1, Kei Tamura 1, Masaru Matsuda 3, Takafumi Katsumura 4, Hiroki Oota 5, Go Watanabe 6, Shigetaka Yoneda 6, Nobuhiko Takamatsu 1, Michihiko Ito 1,8,∗
PMCID: PMC6941857  PMID: 31884166

Summary

Animal sex-determining genes, which bifurcate for female and male development, are diversified even among closely related species. Most of these genes emerged independently from various sex-related genes during species diversity as neofunctionalization-type genes. However, the common mechanisms of this divergent evolution remain poorly understood. Here, we compared the molecular evolution of two sex-determining genes, the medaka dmy and the clawed frog dm-W, which independently evolved from the duplication of the transcription factor-encoding masculinization gene dmrt1. Interestingly, we detected parallel amino acid substitutions, from serine (S) to threonine (T), on the DNA-binding domains of both ancestral DMY and DM-W, resulting from positive selection. Two types of DNA-protein binding experiments and a luciferase reporter assay demonstrated that these S-T substitutions could strengthen the DNA-binding abilities and enhance the transcriptional regulation function. These findings suggest that the parallel S-T substitutions may have contributed to the establishment of dmy and dm-W as sex-determining genes.

Subject Areas: Biological Sciences, Genetics, Evolutionary Biology

Graphical Abstract

graphic file with name fx1.jpg

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Highlights

  • •

    We detected parallel amino acid substitutions in two sex-determining genes, dmy and dm-W

  • •

    Both the substitutions from dmrt1 duplication are under positive selection

  • •

    These substitutions enhanced their DNA-binding activity as transcription factors

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    These substitutions might have contributed to the establishment of dmy and dm-W

Biological Sciences; Genetics; Evolutionary Biology

Introduction

Sexual reproduction in multi-cellular organisms results in the mixture of two types of genomes. Most bilaterian animals have two types of gonads, ovaries and testes. Gonadal sex determination can be defined as a decision whether bipotential gonads develop into ovaries or testes. In vertebrates, there are various sex-determining systems, which can be divided into two categories: environmental sex determination or genetic sex determination. In the latter system, sex chromosome(s) have sex-determining gene(s), which primarily induce a female or male gonad. Previously, we proposed a coevolution model for sex-determining genes and sex chromosomes, in which homomorphic (undifferentiated) sex chromosomes easily allowed the turnover of a sex-determining gene with another one, whereas heteromorphic (differentiated) sex chromosomes tended to be restricted to a specific sex-determining gene (Mawaribuchi et al., 2012). Most vertebrate species, except mammals and birds, have homomorphic sex chromosomes. Therefore, many sex-determining genes should be present in ectothermic vertebrates; however, only about 10 sex-determining genes have been identified so far (Ito and Mawaribuchi, 2013). The turnover of sex chromosomes and sex-determining genes has been evaluated in certain vertebrate species (Kitano and Peichel, 2012, Matsuda and Sakaizumi, 2016, Miura, 2017, Jeffries et al., 2018).

dmrt1 (doublesex- and mab-3-related transcription factor 1) belongs to the DM domain gene family, which encodes transcription factors characterized by a DNA-binding region called the DM domain. dmrt1 is a key gene in testis formation and/or gonadal somatic-cell masculinization in various vertebrate species, including the teleost fish Oryzias latipes, anuran amphibian Xenopus laevis, slider turtle Trachemys scripta elegans, and house mouse Mus musculus (Yoshimoto et al., 2010, Masuyama et al., 2012, Matson and Zarkower, 2012, Zhao et al., 2015, Ge et al., 2018). In the chicken Gallus, a Z-linked dmrt1 is required as a sex-determining gene for testicular formation (Smith et al., 2009). In addition, dmrt1 can also be involved in the germ-cell development from jawless vertebrates to mammals (Matson et al., 2010, Zarkower, 2013, Mawaribuchi et al., 2017a). In our recent study of the promoters and conserved noncoding sequences of dmrt1 orthologs during vertebrate evolution, we found that dmrt1 regulated germ-cell development in the vertebrate ancestor, acquiring another promoter to regulate somatic-cell masculinization during gnathostome evolution (Mawaribuchi et al., 2017a).

In 2002, two groups independently reported the Y-linked gene, dmy/dmrt1bY, as a sex (male)-determining gene in O. latipes; this gene evolved for male determination from the whole duplication of dmrt1 and led to apparition of an XX/XY-type sex-determining system in the ancestor of this species (Matsuda et al., 2002, Nanda et al., 2002). In 2008, we identified the W-linked dm-W, as a sex (female)-determining gene in X. laevis; this gene evolved for female (anti-male) determination from the partial duplication of dmrt1, including the DM domain sequences, thus generating a ZZ/ZW-type sex-determining system in the ancestor of X. laevis (Yoshimoto et al., 2008, Yoshimoto et al., 2010, Yoshimoto and Ito, 2011). Notably, the two genes, dmy and dm-W, independently emerged for sex determination through neofunctionalization after the duplication of dmrt1, during species diversity in the Oryzias and Xenopus genera, respectively (Yoshimoto and Ito, 2011, Mawaribuchi et al., 2012). We also reported that dm-W established the ZZ/ZW-type system after allotetraploidization through hybridization between two closely related diploid Xenopus species around 17–18 million years ago (Session et al., 2016, Mawaribuchi et al., 2017b). Collectively, these findings indicate the convergent gene evolution from dmrt1 to dmy and dm-W for sex determination.

Almost all the sex-determining genes in vertebrates emerged independently during species diversification through neofunctionalization. These genes include dmy, dm-W, and a Y-linked gene, Sry, in therian mammals, which encode transcription factors (Mawaribuchi et al., 2012). Our previous evolutionary analyses showed that all the DNA-binding domain-coding regions in these three genes show higher substitution rates than their prototype genes, dmrt1 and Sox3 (Mawaribuchi et al., 2012). However, whether the emergence of such neofunctionalization-type sex-determining genes shared common evolutionary mechanisms remains to be elucidated.

In this study, we focused on the molecular evolution of dmy and dm-W, as both derived from dmrt1. In order to determine how dmy and dm-W evolved from dmrt1 for sex determination, we searched for shared common mechanisms in the convergent evolution of the dmrt1-derived sex-determining genes dmy and dm-W. In 2004, Zhang (2004) reported that the DM domain in DMY is under positive selection. In this study, we found a common amino acid substitution from serine (S) to threonine (T) at position 15 on the DM domains of both ancestral DMY and DM-W. Importantly, the parallel S15T substitutions in both DMY and DM-W are under positive selection. In addition, both the parallel substitutions were associated with increased DNA-binding and transregulation activities, suggesting a common mechanism for the molecular evolution of dmy and dm-W in sex determination.

Results

Amino Acid Substitutions Accumulate in the DM Domain of Sex-Determining Gene Products DMY and DM-W

Because dm-W and dmy exhibit higher substitution rates than their ancestral gene dmrt1 (Mawaribuchi et al., 2012), we phylogenetically analyzed the amino acid substitutions on the DM domain consisting of 56 or 41 amino acid residues for Oryzias DMY and DMRT1 or Xenopus DM-W and DMRT1, respectively. For Xenopus DM-W and DMRT1, only 41 amino acid sequences corresponding to the N-terminal region of the DM domains were obtained from several Xenopus species, except for X. laevis, in the database. Figure 1A shows the amino acid alignment of these sequences. Using the maximum likelihood method in MEGA6, we constructed two phylogenetic trees (Figures S1A and S1B, and Table S1) and inferred the ancestral amino acid sequence on each branchpoint (Figures 1B and 1C, and Table S2).

Figure 1.

Figure 1

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Phylogenetic Trees of the DM Domains of DMRT1 Subfamily Including DMY and DM-W in the Genus Oryzias and Xenopus and Estimated Amino Acid (aa) Substitutions on Their Ancestral Sequences

The DM domain sequences consisting of 56 aa residues from human, chicken, Xenopus laevis, Oryzias latipes DMRT1, O. latipes DMY, and X. laevis DM-W were aligned (A). Two maximum likelihood trees including DMY (B) and DM-W (C) were constructed from the alignments of the DM domains including 56 aa and 41 aa sequences, respectively, in DMRT1 subfamily. Each number and letter corresponds to the position from the 5′-terminal of the DM domain. One letter code denotes substituted amino acids. The only conserved substitution form S in DMRT1 to T in DMY and DM-W in the 15th position was displayed in red. Scale bar = 0.02 estimated amino acid substitutions per site.

In the genus Oryzias, two amino acid substitutions, F13L and S15T, at positions 13 and 15 on the DM domain (see Figure 1A), were detected on the branch of a common ancestral molecule between DMY and DMRT1 in the three species, O. latipes, O. sakaizumii, and O. curvinotus (Figure 1B). There were also 5, 6, or 9 amino acid substitutions from the ancestral DMY of the three species, O. sakaizumii, O. curvinotus, or O. latipes, respectively. In contrast, there may only be three or four amino acid substitutions from the common ancestral molecule between DMY and DMRT1 in O. sakaizumii or O. latipes, respectively (Figure 1B). In Xenopus, we detected four amino acid substitutions, S15T, M25I, K32N, and I37T, at positions 15, 25, 32, and 37 in the DM domain, on the branch of a common ancestor of DMRT1 and DM-W to a common ancestral DM-W among X. laevis, X. itombwensis, and X. clivi (Figure 1C). In addition, none, one, and two amino acid substitutions were found on the branches from ancestral DM-W to X. clivii, X. itombwensis, and X. laevis DM-W, respectively. In contrast, there may be none or only one amino acid substitution from the common ancestral molecule between DM-W and DMRT1 to X. laevis DMRT1.S or DMRT1.L, respectively (Figure 1C). These results suggest a higher nucleotide substitution rate for dmy and dm-W compared with dmrt1 (Mawaribuchi et al., 2012).

Remarkably, the common serine (S) to threonine (T) substitutions at position 15 on the DM domain were shared during the molecular evolution from the DMRT1 ancestors to the ancestor of DMY and DM-W (Figure 1). Importantly, S or T at position 15 represents an exclusive conservation in the dmrt1 gene of all jawed vertebrates or the dm-W gene in Xenopus and the dmy gene in Oryzias, respectively (Figure 1A). The S15T substitutions on DMY and DM-W must have occurred independently as dmy and dm-W emerged independently from the duplication of dmrt1.

Parallel Amino Acid Substitutions under Positive Selection in the Early Lineages of the Two Sex-Determining Genes, dmy and dm-W

Because more amino acid substitutions were found in the molecular evolution of dmy and dm-W (Figure 1), we examined whether dm-W or dmy evolved under positive selection during species diversification in Xenopus or Oryzias, respectively, through the dN/dS ratio test with codeml in PAML4.8 (see Transparent Methods). Then, we reconstructed two phylogenetic trees of the dmrt1 subfamily, containing dmy and dm-W, and denoted the branches of dmy and dm-W for each estimation as the foreground (“Figures S1C and S1D, and Table 1), which revealed positive selection in both lineages of dmy and dm-W (p < 0.05) (Table 1). Bayes empirical Bays (BEB) analysis demonstrated that positions 6 and 1 on the DM domain of the dmy and dm-W lineages, respectively, were under positive selection (see Figures S2 and S3). It is worth noting that these positive selection sites included the common S15T substitutions between DMY and DM-W. In other words, the parallel S15T substitutions under positive evolution may take place in the early lineages of dmy and dm-W during the convergent evolution of dmrt1-derived sex-determining genes.

Table 1.

Likelihood Ratio Test of Branch-Site Model for dmy and dm-W

Gene Fix Omega Parameter Estimated
 lnL 2ΔlnL (p valuea) BEB Analysis
Positively Selected Sites (DM Domain Positionb) Amino Acid Probablity ω > 1
Site Class 0 1 2a 2b
dmy Estimated Proportion 0.654 0.204 0.107 0.033 −2646 5.627 8 (–) R 0.991
Background ω 0.052 1 0.051 1 9 (–) P 0.952
Foreground ω 0.052 1 999 999 25 (14) K 0.979
26 (15) T 0.961
1 Proportion 0.546 0.173 0.212 0.067 −2649 (p < 0.01) 47 (35) Q 0.952
Background ω 0.05 1 0.05 1 49 (37) M 0.988
Foreground ω 0.05 1 1 1 50 (38) V 0.994
61 (49) D 1
dm-W Estimated Proportion 0.714 0.147 0.114 0.024 −2969 19.7 38 (15) T 0.951
Background ω 0.043 1 0.043 1 86 (–) Y 0.999
Foreground ω 0.043 1 9.418 9.419 107 (–) Q 1
1 Proportion 0.331 0.069 0.496 0.103 −2979 (p < 0.01)
Background ω 0.043 1 0.042 1
Foreground ω 0.043 1 1 1
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a

p-value was calculated by the 50:50 mixture of point mass 0 and χ12-test.

b

The selected sites are numbered from the N-terminal residues in the whole proteins and the DM domains. The number in parentheses shows that from the DM domain (see Figure S2).

Parallel S15T Substitutions in Both DM-W and DMY May Enhance Their DNA-Binding Activity

Next, we investigated the advantage of parallel S15T substitutions in the molecular evolution of both sex-determining genes dmy and dm-W. We investigated whether the S15T or T15S substitutions could affect the DNA-binding properties of DMRT1 or DMY and DM-W, respectively. As described in the Introduction section, X. laevis has allotetraploid genome consisting of L and S subgenomes (Session et al., 2016). It is considered that dm-W emerged from the S-subgenome-derived dmrt1.S after allotetraploidization (Bewick et al., 2011, Mawaribuchi et al., 2017b). Therefore, we used DMRT1.S in this experiment. We first constructed expression plasmids for four FLAG-tagged DMRT1 subfamily proteins (O. latipes DMRT1, O. latipes DMY, X. laevis DMRT1.S, and X. laevis DM-W) and their mutant proteins (O. latipes DMRT1(S15T), O. latipes DMY(T15S), X. laevis DMRT1.S(S15T), and X. laevis DM-W(T15S)), which contain one amino acid substitution. Each protein was produced and validated by in vitro transcription/translation, confirmed by immunoblotting with the anti-FLAG antibody (Figure S4), and then the DNA-binding affinity for the DMRT1-binding consensus DNA sequence (Murphy et al., 2007) was examined using an electrophoretic mobility shift assay (EMSA). Intriguingly, the shifted bands corresponding to the DNA-protein complexes from O. latipes or X. laevis mutant protein DMRT1(S15T), which had the DMY/DM-W-specific threonine at position 15 (15T), were significantly thicker than those corresponding to wild-type DMRT1 (Figure 2A). Conversely, the mutant proteins, DMY(T15S) or DM-W(T15S), which had a DMRT1-specific serine at position 15 (15S), exhibited weaker DNA-binding affinity than each corresponding wild-type protein. We detected similar amounts of proteins between the wild-types and the mutants by western blot analysis using the anti-FLAG antibody (Figure 2B) and confirmed that “15T” is involved in stronger DNA-binding activity than “15S” by quantification of the DNA-binding activity to protein content (Figure 2C). These reciprocal results indicate that the S15T substitutions in the DM domains of the two sex-determining gene's products, DMY and DM-W, may be responsible for the enhancement of both of their DNA-binding activities.

Figure 2.

Figure 2

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DNA-Binding of X. laevis (Xl) DMRT1.S, Xl. DM-W, O. latipes (Ol) DMRT1, Ol DMY, and Their S15T or T15S Mutant proteins to a Consensus DMRT1-Binding Sequence by EMSA

Each FLAG-tagged protein produced by in vitro transcription/translation was used for EMSA (A), and its amount was analyzed by western blotting (WB) using an anti-FLAG antibody (B). The densities of both the shifted bands (A) and the reacted bands with the antibody (B) were quantified using ImageJ, and the relative values were calculated (C). Vector, pcDNA3-flag empty vector; WT, wild-type.

Next, we examined the kinetics of association and dissociation between the DMRT1-binding consensus DNA sequence and the wild-type or mutant proteins by bio-layer interferometry (Table 2). Due to the large amounts of proteins needed to measure the kinetics, DMY, DM-W, and DMRT1 and their T15S or S15T mutant proteins were overexpressed in bacteria (Figure S5 and Table S3). Each purified protein was mixed with biotinylated dsDNA containing the DMRT1-binding consensus sequence to measure their association, followed by the immersion of the biosensor to measure their dissociation. The association and dissociation rate constants, Ka or Kd, respectively, were measured for the dsDNA and each protein. As expected from the EMSA results (Figure 2), X. laevis DMRT1.S or O. latipes DMRT1 had a lower Ka and a higher Kd than each S15T mutant protein (Table 2), whereas DMY and DM-W had a higher Ka and a lower Kd than each T15S mutant. These results indicate that 15T favors faster DNA association and slower DNA dissociation of DMRT1 subfamily proteins compared with 15S. Moreover, the values of the dissociation constant (KD), calculated as KD = Kd/Ka, were lower in the 15T proteins, O. latipes DMRT1(S15T), X. laevis DMRT1.S(S15T), DMY, and DM-W, than their corresponding 15S proteins, O. latipes DMRT1, X. laevis DMRT1.S, DMY(T15S), and DM-W(T15S), respectively.

Table 2.

Kinetics of Association and Dissociation between a DMRT1-Binding Consensus DNA Sequence and DMRT1 Subfamily Proteins or Their Mutant Proteins with One Amino Acid Substitution by Bio-layer Interferometry

Species Protein KD (M) Ka (Ms−1) Kd (s−1)
Trx-S-His 1.57 × 10−3 5.82 × 10−1 9.13 × 10−4
Xenopus laevis DMRT1.S 7.16 × 10−7 2.01 × 103 1.44 × 10−3
DMRT1.S (S15T) 3.11 × 10−7 4.13 × 103 1.29 × 10−3
DM-W 2.88 × 10−7 9.18 × 103 2.65 × 10−3
DM-W (T15S) 2.95 × 10−6 2.43 × 103 7.17 × 10−3
Oryzias latipes DMRT1 1.21 × 10−7 1.50 × 104 1.81 × 10−3
DMRT1 (S15T) 4.97 × 10−8 2.21 × 104 1.10 × 10−3
DMY 4.56 × 10−6 2.37 × 103 1.08 × 10−2
DMY (T15S) 1.45 × 10−5 1.64 × 103 2.38 × 10−2
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Ka, association rate constant; Kd, dissociation rate constant; KD, dissociation constant (Kd/Ka).

Parallel S15T Substitutions in Both DM-W and DMY May Contribute to Their Higher Transcriptional Regulation Abilities

The T15S substitutions decreased the DNA-binding abilities of DMY and DM-W (Figure 2 and Table 2). Consequently, we then verified whether the decrease in DNA-binding activity resulting from the substitutions in DMY and DM-W could influence their transcriptional regulation activity using a luciferase reporter assay. A DMRT1-driven luciferase reporter plasmid containing four tandem repeats of the consensus DMRT1-binding sequence (Yoshimoto et al., 2010) and expression plasmids for DMRT1, DMY, DM-W, and/or its mutant proteins were co-transfected in HEK293T cells. As expected, higher levels of luciferase activity were observed after the introduction of 10 ng of an expression plasmid for DMY than the same amount of an expression plasmid for its T15S substitution mutant, DMY(T15S) (Figure 3A). DM-W did not have a region corresponding to the transregulation domain of DMRT1; however, it did show transrepression functions against the transactivation induced by DMRT1 (Yoshimoto et al., 2008, Yoshimoto et al., 2010, Yoshimoto and Ito, 2011). We then examined the transrepression activity of DM-W against transactivation by DMRT1. The luciferase activity induced by the introduction of 10 ng of an expression plasmid for X. laevis DMRT1.S was downregulated by 44% or 61% compared with the same amount of an expression plasmid for DM-W or DM-W(T15S), respectively. There was a significant difference (p < 0.01) in the downregulation between the two proteins, indicating that DM-W(T15S) has a lower transrepression activity against DMRT1 transactivation than the wild-type DM-W (Figure 3B). Collectively, these findings demonstrate that parallel S15T substitutions in the early lineage of dmy and dm-W may play a role in their DNA-binding activity, resulting in an increase in their transregulation activity.

Figure 3.

Figure 3

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Effects of One Amino Acid Substitution T15S in DMY or DM-W on DMRT1-Driven Transactivation by Luciferase Reporter Assay in HEK293T Cells

Here, 100 ng of DMRT1-driven firefly luciferase reporter plasmid; 5 ng of the internal control Renilla luciferase plasmid pRL-TK; and 10 ng of each expression plasmid for O. latipes DMY, DMY(T15S) (A), or X. laevis DMRT1.S (B) were co-transfected into HEK293T cells as indicated. In the case of (B), 10 ng of an additional expression plasmid for X. laevis DM-W or DM-W(T15S) was also co-transfected, as indicated. As a negative control, 20 ng of pcDNA3-FLAG empty vector was used (left lanes in A and B). Twenty-four h post-transfection, luciferase activities were measured. The data are shown relative to the luciferase activity in the case of O. latipes DMY (the second left lane in A) or X. laevis DMRT1.S (the second left lane in B). The results are expressed as the mean ± SEM for three independent experiments, performed in technical triplicate. One-way ANOVA and Tukey's HSD test were used for statistical analysis. Different letters indicate significant differences (p < 0.01). WT, wild-type.

Discussion

Transcription-factor-encoding sex-determining genes in vertebrates include Y-linked Sry in therian mammals, Z-linked dmrt1 in chickens, W-linked dm-W in X. laevis, Y-linked dmy in O. latipes, and Y-linked sox3y in O. dancena (Ito and Mawaribuchi, 2013, Takehana et al., 2014). Except for dmrt1 in chickens, these genes are considered to have emerged as a result of gene duplication or allelic mutations and neofunctionalization for sex determination. The molecular evolution of promoters and enhancers must have been essential for the establishment of the four genes, Sry, sox3y, dm-W, and dmy, as sex-determining genes from their prototype genes, sox3 and dmrt1 (Mawaribuchi et al., 2012, Takehana et al., 2014). Moreover, the coding regions of Sry, dm-W, and dmy have a higher substitution rate than those of their prototype genes, resulting in several amino acid substitutions (Mawaribuchi et al., 2012). Here, we detected two and four amino acid substitutions in the DM domains of the early lineage of DM-W and DMY, respectively (Figure 1). Among them, common S15T substitutions were found and were estimated to result from positive selection (Table 1). Most importantly, the parallel S15T substitutions in the early lineage of DMY and DM-W may enhance their DNA-binding activity (Figure 2 and Table 2), which may contribute to the establishment of dmy and dm-W as sex-determining genes. Compared with S15T, we found that the other substitutions 13L or 25I, 32N, and 37T in the DM domains of the early lineage of DM-W or DMY, respectively (Figure 1), are not under positive selection (Table 1). However, we could not deny the possibility that these substitutions contributed to the establishment of dmy or dm-W as sex-determining gene. In contrast, we detected seven and two amino acid sites in DMY and DM-W, respectively, different than S15T, which are under positive selection (Table 1). It is possible that the substitutions, which were likely to occur after the early lineage of DMY and DM-W, contributed lineage-specific neofunctionalization of DMY or DM-W.

Serine at position 15 of the DM domain of DMRT1s may play an important role in determining DNA-binding properties, as serine is completely conserved in all vertebrate species except for Agnatha fish (Mawaribuchi et al., 2017a) (Figure 1A). Surprisingly, except for DMY and DM-W, there are no DM domains with T at position 15 in the DMRT family, including invertebrate-sex-determination-related proteins DSX and MAB-3. Murphy et al. (2015) found that S15 on the human DMRT1 does not come into contact with the specific-binding DNA sequence or other DM domains in its homodimer or trimer form in crystallography and nuclear magnetic resonance. These findings suggest that T15 has unique and important roles in both DMY and DM-W for sex determination, despite S and T sharing the most characteristics among the 20 amino acid residues. Based on the three-dimensional structural information of human DMRT1 (Murphy et al., 2015), we compared the stability of complexes between the specific DNA and human DMRT1 or its S15T substitution mutant protein by root-mean-square deviation and found that the substitution DMRT1(S15T) protein had a slightly higher stability with DNA than its wild-type version (data not shown). In addition, the in vitro binding analysis and transactivation assay in cultured cells, as shown in Figures 2 and 3 and Table 2, suggest that the S15T substitution of DMRT1 could modulate the DM domain structure, leading to alternation in its DNA-binding activity.

Convergent evolution is the independent evolution of similar features in two or more lineages. Convergence also occurs at the protein level, which can be separated into two types: convergent and parallel amino acid substitutions. The latter have a higher probability to occur by chance than the former (Zhang and Kumar, 1997). Zhang (22) reported that the molecular evolution of dmy from dmrt1 duplication is under positive selection. The author predicted that a single amino acid substitution from DMRT1 to DMY may be responsible for the establishment of dmy as a sex-determination gene in medaka fish (Zhang, 2004). This study could support the validity of the prediction; we found the positive selection of S15T in medaka DMY, which might be involved in the sex-determining activity (Figures 2 and 3, Tables 1 and 2). In addition, we detected positive selection at the same position, S15T of the DM domains, in the early lineage of Xenopus dm-W after dmrt1 duplication (Figure 1 and Table 1). Such parallel amino acid substitutions have been reported in several species, including three voltage-gated sodium channel genes in garter snakes for tetrodotoxin resistance (McGlothlin et al., 2014) and two testis-specific duplicates of the broadly expressed aldehyde dehydrogenase gene in different Drosophila lineages (Chakraborty and Fry, 2015). To our knowledge, this is a first example of parallel amino acid substitutions for sex determination.

Were the parallel S15T amino acid substitutions involved in homologous recombination suppression? It is believed that dmy and dm-W have evolved under recombination suppression. We previously reported that recombination suppression might lead to accumulation of DNA replication errors during population and species diversification (Mawaribuchi et al., 2016). Therefore, it is possible that amino acid substitutions might occur with increased frequency under the suppression of recombination.

Based on these findings, we propose a model for the molecular evolution of dmy and dm-W (Figure 4). In some ancestor of Oryzias or Xenopus, the whole or partial duplicate of dmrt1 was inserted into another chromosome via transposable element, resulting in the emergence of the prototype dmy and dm-W genes (Matsuda et al., 2002, Nanda et al., 2002, Yoshimoto et al., 2008, Herpin et al., 2010, Bewick et al., 2011, Yoshimoto and Ito, 2011, Matsuda and Sakaizumi, 2016, Mawaribuchi et al., 2017b). Each prototype gene happened to obtain a (additional) promoter and enhancer(s) for expression in gonadal somatic cells before gonadal differentiation, leading to the appearance of the ancestor dmy or dm-W genes for sex determination (Herpin et al., 2010, Mawaribuchi et al., 2012). After obtaining this new promoter/enhancer system, the dmy or dm-W genes mutated, causing the T15S substitution, which may enhance DNA-binding ability (Figures 1 and 2, and Table 2). As a result, these reinforced dmy or dm-W genes were established as sex-determining genes in Oryzias or Xenopus.

Figure 4.

Figure 4

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A Proposed Model for the Establishment of dmrt1-Derived Sex-Determining Genes, dm-W and dmy

Limitations of the Study

This work indicated that the parallel amino acid substitutions from S to T on ancestral DMY and DM-W could enhance their DNA-binding activity and transcriptional regulation function, which might have independently supported the establishment of the dmrt1-type sex-determining genes, dmy and dm-W. However, it remains unknown whether each S15T substitution was involved in the enhancement of the sex-determining function in the ancestor of the fish O. latipes or frog X. laevis. It would be helpful for the question to investigate an effect of the S15T substitution in DMRT1 or T15S substitution in DMY/DM-W on sex determination in knock-in transgenic O. latipes and X. laevis individuals.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.

Acknowledgments

This work was supported by Grant-in-Aid for Scientific Research, Japan Society for the Promotion of Science (22132003 and 18K06389) to MI and Takahashi Industrial and Economic Research Foundation, Japan to MI.

Author Contributions

Y. O., S. M., and M. I. designed the study; Y. O., S. M., and K. N. performed the experiments; Y. O., K. T., M. M., T. K., H. O., G. W., S. Y., and N. T. analyzed the data; and Y. O. and M. I. wrote the paper.

Declaration of Interests

The authors declare no competing interests.

Published: January 24, 2020

Footnotes

Supplemental Information can be found online at https://doi.org/10.1016/j.isci.2019.100757.

Supplemental Information

Document S1. Transparent Methods, Figures S1–S5, and Tables S1–S3
mmc1.pdf (5.4MB, pdf)

References

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Associated Data

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Supplementary Materials

Document S1. Transparent Methods, Figures S1–S5, and Tables S1–S3
mmc1.pdf (5.4MB, pdf)

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