Sex chromosome evolution from a heteromorphic to a homomorphic system by inter-population hybridization in a frog

Abstract

Sex chromosomes generally evolve from a homomorphic to heteromorphic state. Once a heteromorphic system is established, the sex chromosome system may remain stable for an extended period. Here, we show the opposite case of sex chromosome evolution from a heteromorphic to a homomorphic system in the Japanese frog Glandirana rugosa. One geographic group, Neo-ZW, has ZZ-ZW type heteromorphic sex chromosomes. We found that its western edge populations, which are geographically close to another West-Japan group with homomorphic sex chromosomes of XX-XY type, showed homozygous genotypes of sex-linked genes in both sexes. Karyologically, no heteromorphic sex chromosomes were identified. Sex-reversal experiments revealed that the males were heterogametic in sex determination. In addition, we identified another similar population around at the southwestern edge of the Neo-ZW group in the Kii Peninsula: the frogs had homomorphic sex chromosomes under male heterogamety, while shared mitochondrial haplotypes with the XY group, which is located in the east and bears heteromorphic sex chromosomes. In conclusion, our study revealed that the heteromorphic sex chromosome systems independently reversed back to or turned over to a homomorphic system around each of the western and southwestern edges of the Neo-ZW group through hybridization with the West-Japan group bearing homomorphic sex chromosomes.

This article is part of the theme issue ‘Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part II)’.

1. Introduction

Sex chromosome homologues are originally homomorphic in both sexes and have the potential to evolve toward a heteromorphic state in either sex. In almost all mammals and all birds, the sex chromosomes are heteromorphic in males and females, respectively [1,2]. By contrast, in poikillothermal vertebrates, the sex chromosomes take either form, homomorphic or heteromorphic, which depends on taxa [2]. All amphibians examined so far are genetic in sex determination and are unique in that the homomorphy of sex chromosomes is predominant, being observed in around 96% of species [3,4]. In the remaining 4% of species, the sex chromosomes evolved to heteromorphy after speciation or even differentiation of geographic populations within a single species [5–7]. This provides a rare opportunity to investigate the processes of sex chromosome evolution from homomorphy to heteromorphy and in genetic interactions associated with inter-species or inter-population hybridization [8,9].

The Japanese frog Glandirana rugosa is a good subject in which to study the mechanisms of sex chromosome evolution, because homomorphic and heteromorphic sex chromosome systems exist and they are separated into different geographic populations. This species comprises five major geographic groups: two with homomorphic sex chromosomes under male heterogametic sex determination (West-Japan and East-Japan groups; the sex chromosomes are not yet identified) and three with heteromorphic sex chromosomes under male (XY group in Eastern Central Japan) and female heterogametic sex determination (ZW and Neo-ZW groups in North-West Japan and Western Central Japan, respectively), of which sex chromosomes are all 7th largest in 13 haploid complements (figure 1; [10,11]).

Figure 1.

Heterogametic sex and haplotypes of mitochondrial cytochrome b in 16 populations in the Kinki district, central Japan. Male and female heterogameties are indicated by blue and red closed circles, respectively. Homozygosity for the sex-linked genes on chromosome 7 both in males and females under male heterogamety is indicated by white closed circles or squares. Numbers indicate the populations in table 1. Haplotypes of mitochondrial cytochrome b of West-japan, Neo-ZW and XY-groups, respectively, are shown by black, red and blue solid outlines of circles or squares. The Neo-ZW group comprises two sub-groups of Neo-ZW1 and Neo-ZW2. The latter was newly identified in our recent work [9] and is characterized by ZZ-ZW sex determination with mitochondrial haplotypes of XY-group and/or Neo-ZW1 subgroup. This subgroup is shaded in light purple and surrounded by a dotted purple line. The intermingling populations between the Neo-ZW2 subgroup and XY group are shaded in yellow. ki, ky and g indicate Kinomoto, Kyoto and Gifu, respectively, and the white dot in the blue XY group area of the Japan map at the bottom right is Hamamatsu (h) (electronic supplementary material, figure S1). (Online version in colour.)

The Neo-ZW group, which has a different origin from the ZW group, was recently evolved from the XY group with heteromorphic sex chromosomes in eastern Central Japan [12]. The Neo-ZW group has been further classified into two sub-groups of Neo-ZW1 and Neo-ZW2, the latter of which has recently evolved by hybridization with the XY group that is geographically close to it. As seen in the history of sex chromosome evolution in this species, the boundary region between the geographic groups with different systems of sex determination and sex chromosomes is highly intriguing, because their hybridization showed us a unique evolution of a sex determination and sex chromosome system [9]. In this study, we genetically investigated the populations covering the boundary regions between the Neo-ZW group with heteromorphic ZZ-ZW sex chromosomes and the West-Japan group with homomorphic sex chromosomes under male heterogamety. We did not find any separation or sympatric distribution of the two groups at the boundaries; instead, they formed a new geographic group that has never been defined to date.

2. Material and methods

(a) . Frogs

In total, 129 frogs were collected from the western Kinki region and Kii Peninsula in Japan (table 1). Sex of the specimens was determined by inspection of gonads after euthanasia.

Table 1.

Sex-linked genotypes and mitochondrial haplotypes in 16 populations.

no.	population	prefecture	no. of frogs examined		genotype of sex-linked genesb		Cytochrome b haplotype	12S rRNA haplotypec
			male	female	male	female
			(for karyotype)
1	Shinonsen	Hyogo	7	0	homo	—	NE	West-J
2	Kamigoori	Hyogo	4	5	homo	homo	NE	West-J
3a	Toyooka	Hyogo	4	6	homo	homo	Neo-ZW	Neo-ZW
4a	Yasutomi	Hyogo	5(1)	3(1)	homo	homo	Neo-ZW	Neo-ZW
5a	Tatsuno	Hyogo	4(2)	5(2)	homo	homo	Neo-ZW	Neo-ZW
6	Fukuchiyama	Hyogo	10	1	homo	hetero	Neo-ZW	Neo-ZW
7	Sanda	Hyogo	5	5	homo	hetero	Neo-ZW	Neo-ZW
8a	Kawabe	Wakayama	3	8	homo	homo	XY	Neo-ZWc
9a	Tanabe	Wakayama	4(3)	9(4)	homo	homo	XY	Neo-ZWc
10	Ryujin	Wakayama	4	6	homo	hetero	Neo-ZW	Neo-ZW
11	Yanase	Wakayama	3	2	homo	hetero	Neo-ZW	Neo-ZW
12	Magari	Wakayama	3	1	homo	hetero	Neo-ZW	Neo-ZW
13	Nachi	Wakayama	4	4	homo	hetero	Neo-ZW	Neo-ZW
14	Isato	Wakayama	1	3	homo	hetero	Neo-ZW	Neo-ZW
15	Komatushima	Tokushima	2	2	homo	homo	NE	West-J
16	Anan	Tokushima	3	3	homo	homo	NE	West-J

NE, not examined.

^aSex-linked genes on chromosome 7 are homozygous in both sexes, while mitochodrial cytochrome b haplotpes are of Neo-ZW or XX-XY group.

^bSex-linked genes AAT, SF1 and Ar on chromosome 7 were examined using RFLP analysis.

^c12rRNA haplotypes do not discriminate the Neo-ZW from the XY group owing to no differences in sequence.

Homozygosity for sex-linked genes is shown in green and heterozygosity is in red.

Neo-ZW haplotype of mitochondrial cytochrome b is shown in red and XY haplotype is in blue.

Yellow shaded and italics indicate discordance of nuclear sex-linked genotype with cytochrome b haplotype.

(b) . Mitochondrial and sex-linked genes analysis

Total genomic DNA was extracted from blood cells using the DNA WB extraction Kit (Wako, Japan). Genotyping of sex-linked genes, ADP/ATP translocase (AAT) and steroid factor 1 (SF1) and haplotyping of mitochondrial 12S rRNA and cytochrome b genes were performed according to our previous studies [9,12]. PCR primers and conditions for the mitochondrial cytochrome b gene followed the method of Lee et al. [13]. The alignments of nucleotide sequence and construction of gene tree by the neighbour-joining method were performed using MEGA6 software [14]. Bayesian inference analyses were conducted with MrBayes v. 3.2.6 [15], using a HKY model as selected by Kakusan 4 [16]. Analyses were run using the following settings: ngen = 1000 000, samplefreq = 100 and burnin = 250 000.

(c) . Artificial crossing and sex reversal with testosterone

Ovulation was induced in females by injection of a pituitary gland solution of the frog Pelophylax nigromaculatus or G. rugosa, and eggs were artificially inseminated with sperm from the male according to the method of Ohtani et al. [17]. Sex-reversal experiments with testosterone were performed according to the method of Ogata et al. [18]. Tadpoles at stages III–VIII [19], which were anaesthetized with 0.0002% MS222 solution, were injected with 250 μg of testosterone propionate (25 mg of testosterone propionate [Enarmon] dissolved in 1 ml of sesame oil (Aska Pharmaceutical Co., Ltd.)) into abdominal cavities. As a control experiment, tadpoles were injected with equal volumes of sesame oil with no testosterone. One year after metamorphosis, the sex of the offspring was identified by inspection of gonads after euthanasia. Some of the males treated with testosterone were used for backcrossing with control females.

(d) . Principal coordinate analysis

Four nuclear genes (Tyrosinase, SLC8A3, POMC, and RAG1) were amplified from 29 individuals of six geographic groups (West-Japan, East-Japan, XY, ZW, Neo-ZW and Neo-West-Japan; electronic supplementary material, table S1). Primer sets are shown in the electronic supplementary material, table S2. Nucleotide sequences were determined with the ABI PRISM 370 genetic analyser (Applied Biosystems, USA) according to the manufacturer's instructions. DNA sequences were aligned using MEGA 6 [14]. For heterozygous sequences, we used DnaSP v. 5.1 [20] to determine haplotypes. To characterize the genetic relationships among 29 frogs, principal coordinate analysis (PCoA) by the covariance standardized approach of pairwise Nei's genetic distances was conducted in GenAlEx v. 6.5 [21,22]. STUCTURE (v. 2.3) [23] was run for 100 000 Markov chain Monte Carlo cycles following 100 000 burn in cycles, using an admixture model with independent allele frequencies. Ten replications were performed for each K in the range K = 1–10, and the optimal K was estimated using STRUCTURE HARVESTER software [24].

(e) . Chromosome preparation and banding techniques

Karyotypes were examined in frogs from the Yasutomi (P4), Tatsuno (P3) and Tanabe (P9) populations. The numbers of frogs investigated are shown in table 1. Mitotic metaphase chromosomes were prepared from blood cell culture [25,26]. Late-replication banding and C-banding techniques basically followed the methods of Takayama et al. [27] and Sumner [28], respectively.

3. Results

(a) . Discordance of mitochondrial haplotypes and nuclear sex-linked genotypes

To identify the precise distributions of the West-Japan and Neo-ZW groups of G. rugosa, we analysed haplotypes of mitochondrial 12S rRNA and cytochrome b in 16 populations covering their boundary. The 12S rRNA haplotypes of four populations (P1, 2, 15, 16) were of the West-Japan group, whereas those of the other 12 populations were of the Neo-ZW group (figure 1 and table 1). Analysis of cytochrome b with higher evolutionary rates further discriminated the two populations (P8 and 9) in the Kii Peninsula from the other 10 populations (P3–7 and 10–14) of the Neo-ZW group: the haplotypes of P8 and P9 were of the XY group with heteromorphic sex chromosomes (figure 1 and table 1; electronic supplementary material, figure S1). Analyses of the sex-linked genes, AAT and SF1, which are on the sex chromosomes, 7 out of 13 haploid complements in XY, ZW and Neo-ZW groups showed that genotypes of the eight populations (P2–5, 8, 9, 15 and 16) were homozygous in both sexes as in the West-Japan group, whereas those of the other seven populations (P6, 7, 10–14) were heterozygous in females and homozygous in males as in the Neo-ZW group (table 1; electronic supplementary material, figure S2). These results indicate that P3–5 share the cytochrome b and 12S rRNA haplotypes with the Neo-ZW group, whereas notably, the two genes on chromosome 7 were not sex-linked. Likewise, P8 and 9 in the Kii Peninsula shared cytochrome b haplotypes with the XY group, whereas the two genes on chromosome 7 were not sex-linked. In these five populations (P3–5 and P8, 9), the mitochondrial haplotypes belonged to the heteromorphic sex chromosomes group, Neo-ZW or XY, but the genotypes of sex-linked genes suggest that the chromosomes 7 are homomorphic in both sexes like those of the West-Japan group.

(b) . Homomorphic sex chromosomes

To identify the sex chromosomes in the P4, P5 and P9 populations morphologically, we investigated the karyotypes of males and females, for which sex-linked genes on chromosome 7 are homozygous, although the cytochrome b haplotypes are of the Neo-ZW and XY groups, respectively (table 1). In these populations, no differences in karyotypes were identified between sexes in the late-replication and C-banding patterns (figures 2 and 3; electronic supplementary material, figures S3–S6). In particular, chromosome 7 was found to be subtelocentic in both sexes, a morphotype of the West-Japan group [6,29].

Figure 2.

Late replication banded karyotypes of male (a) and female (b) from Yasutomi population (P4). The chromosomes 7, which are the same as those of the West-Japan group in morphology and banding pattern, are sub-telocentric and boxed [6,18,29]. No morphological and banding differences are seen between the sexes. Bars, 10 µm.

Figure 3.

C-banded karyotypes of male (a) and female (b) from Yasutomi population (P4). The chromosomes 7 are sub-telocentric and boxed, which are the same as those of the West-Japan group in morphology and banding pattern. No morphological and banding differences are seen between the sexes. Bars, 10 µm.

(c) . Male heterogamety in sex determination

To identify the heterogametic sex in the P4 and P9 populations, we performed sex-reversal experiments with testosterone and backcrossing. If the heterogametic sex is female, the sex-reversed males are ZW and the offspring from mating with a normal ZW female should produce 25% males (ZZ) and 75% females (ZW and WW, if WW is alive). On the other hand, if the heterogametic sex is male, the sex-reversed males are XX and the offspring from mating with a normal XX female should produce 100% females (all XX). Almost all of the tadpoles treated with testosterone grew to males 1 year after metamorphosis (97.3% in P4 and 100% in P9; electronic supplementary material, table S3). Next, we crossed the four and eight testosterone-treated mature males of the P4 and P9 populations with control females of the respective populations. Offspring from four crossings using the two males of P4 (Nos. 1 and 2) and of P9 (Nos. 1 and 6), respectively, had a significant overrepresentation of females (87.5–100%; χ² = 6.815–58.17, p = 0.0001 or p < 0.0001), while the offspring from six crossings using the other two males and six males, respectively, gave males and females at ratios ranging from 42.9% to 70.6% (χ² = 0.008–1.774, p = 0.201–0.927; electronic supplementary material, table S4). One explanation could be that the two males from each of the P4 and P9 populations were sex-reversed XX males, and the unexpected males of their offspring were also sex-reversed XX males. Such XX sex-reversal from female to male in the offspring of XX males is reported in this species as well as in other Ranid frog species [6,25]. Thus, the males are heterogametic sex in these two populations, and this result will be further confirmed by another approach such as the identification of sex-linked SNPs.

(d) . Nuclear genome structures

To investigate the nuclear genome structures in the P3, P4 and P9 populations at the boundaries when compared to those of the other populations belonging to the five major groups, we performed PCoA analysis using SNPs identified in the sequences of four nuclear genes (electronic supplementary material, tables S1 and S2). The two ancestral groups of West- and East-Japan with homomorphic sex chromosomes (XX-XY), respectively, took separate positions from one another, while the derived (newly evolved) groups of XY and ZW with heteromorphic sex chromosomes occupied intermediate positions between the former two (figure 4). The Neo-ZW populations were located at a position between the West-Japan and XY groups, extending to the range of the former two. The P3, 4 and 9 populations at the boundaries were located in a wide range covering Neo-ZW and part of the XY group, but were outside the range of the West-Japan group (figure 4, dotted line; STRUCTURE histograms in electronic supplementary material, figures S7 and S8).

Figure 4.

PCoA analysis on SNPs of four nuclear genes of the frogs from five geographic groups including P3, 4 and 9 populations at the boundaries. The two ancestral groups, West-Japan and East-Japan, take distinct positions (black and brown, respectively), and the derived groups (blue and red, respectively) of XY and ZW take the intermediate positions between the former two. Neo-ZW (pink) and the three populations P3, 4 and 9 are located between the XY/ZW and West-Japan groups, extending to the range of the XY.

4. Discussion

The Japanese frog G. rugosa comprises five major geographic groups based on the sex chromosome and sex-determining systems. Here, we identified a new geographic group: the populations are located at boundaries between the Neo-ZW group and the West-Japan group (figure 1). The mitochondrial genes are derived from the Neo-ZW or XY group with heteromorphic sex chromosome 7 of ZZ-ZW or XX-XY, respectively. However, the genes on chromosome 7 are not sex-linked, and in fact, the chromosomes 7 are subtelcentric, a type of the West-Japan group, and are homomorphic in both sexes. Also, no heteromorphic sex chromosomes were identified in either sex and the males are heterogametic in sex determination. The nuclear genes covered the range between Neo-ZW/XY and the West-Japan groups.

The homomorphic sex chromosomes under male heterogamety and the subtelocentic chromosome 7 are common to the populations of West-Japan group. Thus, our conclusion is that the new group may have evolved by hybridization between the West-Japan group and either the Neo-ZW or the XY group. The new group (P3–5) in the Chugoku district is geographically located between the Neo-ZW group in the east and the West-Japan group in the west. This region around the boundary is known to separate genetically wild insects and animals species into two distinct geographic groups within a species [30–32]. Thus, it is likely that in G. rugosa, after the geographic barrier to separate the West-Japan from Neo-ZW was dissolved, the two groups secondarily contacted and hybridized with one another to produce the new group.

On the other hand, the other new group (P8 and 9) at the southwestern edge of the Kii Peninsula is located close to Neo-ZW group (Neo-ZW1), which separates the new group from the XY group, while the sea (Wakayama bay) separates the new group from the West-Japan group (figure 1). Based on our previous study [12], it is assumed that the XY group had been originally distributed over the Kii Peninsula before the evolution of the Neo-ZW group. In addition, the southern edge region of the Kii Peninsula is known to be where ancestral lineages of mammals have been isolated from others and survived [33,34]. Thus, taking the geographic distribution of the West-Japan group over the sea in Shikoku into consideration, we hypothesize that the remnant of the XY group on the southwestern edge of the Kii Peninsula secondarily contacted with the West-Japan group emigrating from Shikoku island (P15,16) through a land bridge occurring during the previous glacial era [30,35] and produced the present new group. Consequently, the populations at the two distant locations (P3–5 and P8–9) evolved independently by inter-population hybridization and converged on the homomorphic sex chromosomes under male heterogamety. Here, we define the new group as the ‘Neo-West-Japan group’. It is necessary to identify the locus of male determination in the Neo-West-Japan as well as the original West-Japan group to know whether the homomorphic sex chromosomes are shared with one another or turnover occurred between the two groups.

In 1967, Ohno proposed an evolutionary theory of sex chromosomes using outstanding examples of snakes in which the sex chromosomes differentiate from a primary homomorphy to an evolved heteromorphy [36]. Since then, his theory has been well accepted by cytogeneticists studying plants and animals [37–39]. By contrast, our study identified the opposite case of sex chromosome evolution from a heteromorphic to a homomorphic system. In the Neo-West-Japan group, which may have originated from hybridization between the West-Japan and the Neo-ZW or XY group, the heteromorphic sex chromosomes deriving from one parental group were lost and instead have been replaced with homomorphic ones (the homomorphic sex chromosomes could be homologous to sex chromosomes 7 or be turned over to another chromosomes). A comparable case was reported in the Australian dragon lizard [40]. The species has differentiated heteromorphic sex chromosomes of ZZ-ZW type, but sex determination is also sensitive to temperature: high temperature during egg incubation changes the phenotypic sex of ZZ individuals to female. In fact, ZZ sex-reversed females were discovered in the wild deserts around the centre of Australia. This suggests that the system of ZZ-ZW heteromorphic sex chromosomes could be changed to a temperature sex determination system without W chromosomes, an evolution from heteromorphy to homomorphy. However, the real population, of which reproduction works between only ZZ males and ZZ females, has yet to be discovered and confirmed, and thus this case remains a theory.

By contrast, the Neo-West-Japan group of the frog G. rugosa found in this study is a documented case in nature. In the primary hybridized populations, the W or Y chromosomes may have been lost through WW or YY embryos produced by backcrossing between the W or Y chromosomes-carrying males and females, because WW or YY embryos have been experimentally shown not to survive over early developmental stages and die owing to lethal genes accumulated on the W or Y chromosome [9,11]. Although the reason why sex determination converged on male heterogamety remains unsolved, it is plausible that homomorphy of sex chromosomes is dominant against heteromorphy if the two systems meet and hybridize. Here, it is interesting to see the constitutions of sex chromosome systems in amphibians and fish. In both animals, homomorphy of sex chromosomes is predominant over heteromorphy [2]; it is particularly noteworthy in amphibians, of which 96% species have homomorphic sex chromosomes [3,4]. One theory says that the abundance of homomorphy is owing to spontaneously, rarely born sex-reversed XY females, in which recombination between the X and Y chromosomes occurs and resets their genetic differences, except for the male determining gene [41]. By contrast, Pennell et al. [42] noted that statistically, the reverse transition from heteromorphy to homomorphy is much more likely in fish and amphibians, although the rate is not significant. Our present study suggests a mechanism to return a heteromorphic back to or be turned over to a homomorphic system and thereby contribute to the predominance of homomorphy of sex chromosomes. If an original system of homomorphic sex chromosomes remains somewhere in geographic populations, the heteromorphy system that evolved in another could be reversed back to homomorphy by hybridization with the homomorphy system. Losing degenerated and deleterious mutations accumulated on the differentiated W and Y chromosomes would strongly benefit the population. This benefit is also obtained by recycling the X chromosome to a new W chromosome discovered in this species [9]: the original W chromosomes were lost from the hybridized populations between the Neo-ZW and XY groups both with heteromorphic sex chromosomes, and then the X chromosomes were recycled to new W chromosomes, which re-started the history of W chromosome evolution from the beginning with no degenerated genes. Consequently, the reverse sex chromosome evolution from heteromorphy to homomorphy may be an effective strategy for eukaryotes to shed genetic loads that have been accumulated by the ‘Muller's ratchet’ [43] during heteromorphic sex chromosome evolution.

Ethics

Animal care and experimental procedures were conducted under approval of the Committee for Ethics in Animal Experimentation at Hiroshima University (permit no.: G18-2).

Data accessibility

Sequence data of mitochondrial cytochrome b gene and four nuclear genes (Tyrosinase, SLC8A3, POMC and RAG1) from this article have been deposited with the DDBJ data libraries under accession nos LC030018–LC030021/LC545395–LC545398 and nos LC554836–LC554889 (electronic supplementary material, table S2), respectively.

Authors' contributions

M.O. and I.M. conceived the study. M.O., K.S. and Y.Y. collected the animals and M.O. and I.M. analysed the data. M.O. and I.M. wrote the manuscript with input and revision from all coauthors.

Competing interests

The authors have no conflicts of interest to declare.

Funding

This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan awarded to I.M. (grant no. 19K06788). The funder had no other role in the preparation of the data or manuscript.