
==== Front
Genome Biol Evol
Genome Biol Evol
gbe
Genome Biology and Evolution
1759-6653
Oxford University Press UK

10.1093/gbe/evae179
evae179
Article
AcademicSubjects/SCI01130
AcademicSubjects/SCI01140
Correlation Between Subgenome-biased DNA Loss and DNA Transposon Activation Following Hybridization in the Allotetraploid Xenopus Frogs
https://orcid.org/0009-0005-4508-6498
Suda Kosuke Department of Bioscience, School of Science, Kitasato University, Kanagawa 252-0373, Japan

https://orcid.org/0009-0000-4772-344X
Suzuki Takahiro Department of Bioscience, School of Science, Kitasato University, Kanagawa 252-0373, Japan

https://orcid.org/0009-0000-5792-6064
Hayashi Shun Department of Bioscience, School of Science, Kitasato University, Kanagawa 252-0373, Japan
Amphibian Research Center, Hiroshima University, Higashi-Hiroshima 739-8526, Japan

https://orcid.org/0009-0002-4795-4293
Okuyama Honoka Department of Bioscience, School of Science, Kitasato University, Kanagawa 252-0373, Japan

https://orcid.org/0000-0001-8960-4975
Tsukamoto Daisuke Department of Bioscience, School of Science, Kitasato University, Kanagawa 252-0373, Japan

https://orcid.org/0000-0002-8030-2361
Matsuo Takuya Department of Bioscience, School of Science, Kitasato University, Kanagawa 252-0373, Japan

https://orcid.org/0000-0001-6195-5984
Tamura Kei Department of Bioscience, School of Science, Kitasato University, Kanagawa 252-0373, Japan

https://orcid.org/0000-0003-1881-6916
Ito Michihiko Department of Bioscience, School of Science, Kitasato University, Kanagawa 252-0373, Japan

Mueller Rachel L Associate Editor
Corresponding author: E-mails: michichikoito.ito@gmail.com; ito@sci.kitasato-u.ac.jp.
9 2024
21 9 2024
21 9 2024
16 9 evae17913 8 2024
21 9 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.
2024
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Abstract

In certain tetraploid species resulting from interspecific hybridization, one parent's subgenome is known to selectively undergo DNA loss. The molecular mechanisms behind this remain unclear. In our study, we compared the genomes of a standard diploid species with two allotetraploid species from the Xenopus genus, both possessing L (longer) and S (shorter) homoeologous subgenomes. We observed substantial gene losses and intergenic DNA deletions in both the S and L subgenomes of the tetraploid species. Gene losses were around 1,000 to 3,000 for L and 4,000 to 6,000 for S, with especially prominent losses in the S subgenome. Many of these losses likely occurred shortly after interspecific hybridization in both L/S subgenomes. We also deduced frequent large inversions in the S subgenome. Upon reassessing transposon dynamics using updated genome databases, we reaffirmed heightened DNA transposon activity during the hybridization, as previously reported. We next investigated whether S subgenome-biased DNA loss could be correlated with the activation of DNA transposons following hybridization. Notably, distinct patterns were observed in the dynamics of DNA transposons between the L and S subgenomes. Several DNA transposon subfamilies correlated positively with DNA deletions in the S subgenome and negatively in the L subgenome. Based on these results, we propose a model that, upon and after hybridization between two related diploid Xenopus species, the mixture of their genomes resulted in the derepression of DNA transposons, especially in the S subgenome, leading to selective DNA loss in the S subgenome.

interspecific hybridization
allotetraploidization
transposable element
inversion
subgenome
derepression
Ohsumi Frontier Science Foundation 10.13039/501100018732
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pmcSignificance

The genome of the allotetraploid frog, Xenopus laevis, experiences selective DNA loss in one of the two parental subgenomes, but the significance and molecular mechanisms of this phenomenon remain unclear. In this study, we demonstrated a correlation between biased DNA loss and the activation of DNA transposons through genomic analysis. This finding suggests that following interspecific hybridization, there may be an intergenomic conflict mediated by species-specific transposons.

Introduction

It is widely believed that interspecific hybridization between two closely related species plays a pivotal role in the evolution of life, encompassing aspects such as body evolution and species diversity. Examples of this phenomenon include genome duplication through allotetraploidization, which has the potential to enhance adaptability to various environments, as demonstrated by the phenomenon of heterosis. Ohno (1970) proposed that two rounds of whole-genome duplication (WGD) during vertebrate evolution led to diverse forms of body evolution, including the development of organs adapted to different environments (Holland et al. 1994; Spring 1997). Recent genomic studies suggest that these two rounds of WGD might have been the result of allotetraploidization followed by autotetraploidization (Simakov et al. 2020; Nakatani et al. 2021). However, interspecific hybridization often results in sterile progeny across species, from insects to vertebrates. One theory suggests that incompatibilities between transposons and piRNA from two distinct genomes might contribute to the observed infertility in Drosophila hybrids (Kotov et al. 2019).

Transposons, or transposable elements (TEs), are mobile DNA sequences capable of changing their position and proliferating within a genome. Their self-driven activities can potentially destabilize the genome in germline cells (Belancio et al. 2008). In animal germline cells, PIWI-interacting noncoding small RNAs (piRNAs) play a crucial role in suppressing TE activities, thereby safeguarding genome integrity (Iwasaki et al. 2015). The suppression of TEs often leads to the emergence of new TEs. Consequently, TEs and piRNAs evolve independently in reproductive populations, resulting in distinctive differences in composition and types among populations that have been reproductively isolated for extended periods.

Hybrids inherit two species-derived subgenomes, and in many cases, these subgenomes evolve asymmetrically. Subgenome dominance is commonly observed in plants (Cheng et al. 2018; Alger and Edger 2020; Wang et al. 2022). Reports indicate subgenome-biased gene losses between homoeologs/ohnologs in the allotetraploid frog Xenopus laevis (Hellsten et al. 2010; Session et al. 2016) and during the evolution of jawed vertebrates (Simakov et al. 2020; Nakatani et al. 2021). Furthermore, subgenome-biased DNA losses have been demonstrated across all chromosomes in X. laevis (Session et al. 2016). In the hybridogenesis of the European water frog, Pelophylax esculentus, formed by hybridizing P. ridibundus and P. lessonae, subgenome-specific exclusion has been observed in germline cells (Uzzell et al. 1980; Dedukh et al. 2019; Miura et al. 2021). However, the molecular mechanisms underlying such asymmetric subgenome evolution remain largely unexplored.

In this study, we compared the genomes of a standard diploid species with two allotetraploid species of Xenopus, each possessing L (longer) and S (shorter) homoeologous subgenomes. Our analysis revealed a bias toward gene loss and intergenic region deletions in the S subgenome, accompanied by extensive chromosomal inversions. Furthermore, we observed an increase in DNA transposon activity post-hybridization, particularly in the S subgenome, while retrotransposon dynamics remained consistent across both subgenomes. Additionally, we identified specific DNA transposon subfamilies that correlated with DNA deletions in each subgenome of X. laevis. Finally, we discuss the potential contribution of the derepression of specific DNA transposon subfamilies to the selective DNA loss in the S subgenome following hybridization.

Results

The Allotetraploid X. laevis Genome Has Retained Almost all Orthologs of X. tropicalis

Utilizing the new Xenopus genome databases (see Materials and Methods), we reanalyzed the genomes of one diploid X. tropicalis and two allotetraploid species, X. laevis and X. borealis. Both allotetraploid species possess L/S subgenomes that diverged from the ancestral allotetraploid frog. The overall results were consistent with the previous reports (Session et al. 2016; Furman et al. 2018). Specifically, when compared to the genome size of X. tropicalis, both allotetraploid species had an L subgenome of roughly the same size, while the S subgenome was more than 10% shorter (supplementary table S1, Supplementary Material online). Upon examining the genome composition, we found that the genomic occupancy rate of genes (exons + introns) was lower in the L/S subgenomes of X. laevis (30.2%/26.5%) compared to that of X. tropicalis itself (34.6%) (supplementary tables S2 and S3, and fig. S1, Supplementary Material online). Conversely, regarding DNA transposons and retrotransposons, their genomic occupancy rates were higher in the L/S subgenomes of X. laevis (L: 29.0% and 11.0%; S: 29.8% and 12.5%) compared to those of X. tropicalis (24.9% and 10.5%). For X. borealis, due to incomplete sequence contigs, approximately half of the L/S subgenomes consisted of DNA categorized as “other” (supplementary table S4 and fig. S1, Supplementary Material online).

To investigate the pattern of gene loss in the L and S subgenomes of X. laevis and X. borealis, we compared each subgenome to the diploid X. tropicalis genome. Initially, we identified 15,022 protein-coding genes in the X. tropicalis genome, excluding tandem duplicated genes and those labeled as LOC. Synteny analysis revealed that the L and S subgenomes of X. laevis/X. borealis have 13,883/11,712 and 11,046/9,264 orthologs to X. tropicalis, respectively (Table 1). As expected, we observed a bias toward gene loss in the S subgenome of the two allotetraploid species. However, a significant number of gene losses also occurred in both L subgenomes (Fig. 1 and Table 1).

Fig. 1. Predicted number of lost protein-coding genes corresponding to X. tropicalis orthologs in X. laevis and X. borealis L/S subgenomes. The numbers of predicted lost protein-coding genes in the L and S subgenomes are shown in the Xenopus phylogenetic tree.

Table 1 The predicted number of retained and lost orthologs in X. laevis compared to X. tropicalis

	X. tropicalis	X. laevis	X. borealis	
L	S	L	S	
Gene	28,786	25,008	17,600	>21,318	>16,567	
Protein-coding gene	15,022a	13,883b	11,046b	>11,712b	>9,264b	
L/S total orthologs minus homeolog pair (retention %)	(100)	14,982
(99.7)	>13,449
(>89.5)	
Homeolog pair (retention %)	(100)	9,947
(66.2)	>7,527
(>50.1)	
Lost ortholog	…	1,139	3,976	<3,310	<5,758	
Species-specific L/S-common ortholog	…	40	>1,573	
Lost ortholog before divergence	…	<581	<2,546	<581	<2,546	
Lost ortholog after divergence	…	>518	>1,390	>1,156	>1,639	
aThe number of protein-coding genes excluding tandem duplicated genes and those registered as LOC.

bThe number of orthologs to the X. tropicalis 15,022 protein-coding genes.

Out of the 15,022 protein-coding genes in X. tropicalis, the corresponding orthologs exist in both the L and S subgenomes of X. laevis as 9,947 homeolog pairs, with a retention rate of 66.2% (Session et al. 2016). Importantly, when considering homeolog pairs as a single ortholog, the number of X. laevis orthologs corresponding to X. tropicalis is 14,982, which means that 99.7% of the 15,022 genes of X. tropicalis were retained as orthologs in the X. laevis genome (Table 1). This suggests that the two ancestral species with L and S genomes before hybridization likely had orthologs for approximately 15,000 genes of X. tropicalis. In this case, after hybridization, gene loss occurred in both the L and S subgenomes, with a greater loss biased toward the S subgenome.

Most Gene Losses Biased Toward the S Subgenome Likely Occurred Shortly After Xenopus Interspecific Hybridization

Our analysis of the two allotetraploid genomes, X. laevis and X. borealis, provided insights into the number of orthologous genes before and after the divergence of the two species. We discovered that 581 and 2,546 orthologs to X. tropicalis were likely lost in the L and S ancestral subgenomes, respectively, prior to speciation (Table 1 and Fig. 1). Gene losses in the L/S subgenomes continued independently in each species following speciation.

Session et al. (2016) reported that allotetraploidization and the speciation of X. laevis and X. borealis occurred 17 to 18 and 17 million years ago (Mya), respectively (Fig. 1a). As mentioned earlier, the current genome of X. laevis has orthologs corresponding to 99.7% of the 15,022 protein-coding genes of X. tropicalis (Table 1). Therefore, it is reasonable to assume that the two diploid ancestral species before hybridization also had approximately 15,000 corresponding genes. The number of protein-coding gene types in X. borealis was 13,499 (Table 1), suggesting that about 1,500 protein-coding genes (approximately 10% of 15,000) were not identified due to incomplete sequencing or other factors. Additionally, assuming that there were originally 15,000 protein-coding genes for both L and S, it can be estimated that X. borealis lost approximately 9,000 protein-coding genes, with about 900 (approximately 10% of 9,000) of these losses potentially attributable to incomplete sequencing or other factors. Specifically, we hypothesize that the 581/2,546 genes lost from the L/S subgenomes before species divergence were lost on a large scale in the relatively short period between hybridization and species divergence (Table 1). Interestingly, there is a stronger bias in gene loss in the S subgenome compared to the L subgenome, and this bias is believed to have persisted not only for a short period after hybridization but also after species divergence.

S Subgenome-biased DNA Loss in the Intergenic Regions of X. laevis

To investigate the relationship between intergenic regions and S subgenome-biased DNA losses, we first comprehensively selected adjacent orthologous gene pairs between the X. laevis L or S subgenome and the X. tropicalis genome. We then examined the interval sizes between these adjacent genes in each (sub)genome and compared them. The genome sequence of X. borealis was not included in this analysis due to insufficient contig information. We calculated the median interval size, excluding transposons, for each chromosome. Table 2 presents the ratio of the interval loss size to the total DNA loss for each chromosome in X. laevis, in comparison to its corresponding chromosome in X. tropicalis. The ratios in the L and S subgenomes ranged from −1.7% to 4.6% and from 5.6% to 23%, respectively. Notably, every chromosome derived from the S subgenome exhibited more DNA losses than its corresponding L chromosome, indicating that S subgenome-biased DNA loss also occurred in the intergenic regions following hybridization.

Table 2 Reduction rate of intergenic region lengths between orthologous genes on X. laevis L or S chromosomes compared to X. tropicalis

Chr	L (%)	S (%)	
1	0.9	5.6	
2	−1.7	7.8	
3	4.1	8.8	
4	1.2	11	
5	1.4	7.3	
6	−1.7	8.9	
7	4.4	14	
8	4.6	23	
9_10	2.8	7.2	

S-biased Chromosomal Inversions Prior to the Speciation of X. laevis and X. borealis

Session et al. (2016) reported that intrachromosomal rearrangements occurred more frequently in the S subgenome than in the L subgenome. For instance, they highlighted extensive inversions in X. laevis chromosomes 2S, 3S, 4S, 5S, and 8S. We further examined chromosomal inversions not only in X. laevis but also in X. borealis. Comparative analyses based on the chromosomes of X. tropicalis have revealed that the large inversions in chromosomes 2S, 3S, 4S, and 8S were shared by both X. laevis and X. borealis (Fig. 2; supplementary fig. S2, Supplementary Material online). An important point to note is that numerous common inversions were detected in the L subgenome, similar to those in the S subgenome. However, in comparison to the common inversions in the S subgenome, considerably smaller inversions were identified in the L subgenome (see Fig. 2). This indicates that these inversions, common to the S subgenome, occurred in the common ancestor of X. laevis and X. borealis. It implies that many S-biased chromosomal inversions occurred prior to the speciation of these two species.

Fig. 2. Relationships of orthologous genes among the X. tropicalis genome and the two subgenomes of X. laevis L/S, X. borealis L/S, X. laevis/X. borealis L, or X. laevis/X. borealis S. The locations of L/S-common, S-specific, and L-specific chromosomal inversions are depicted on the X. tropicalis genome.

S Subgenome-biased Activation of DNA Transposons From the Time of Hybridization to the Present

We previously reported the activation of DNA transposons through hybridization in Xenopus frogs approximately 17 to 18 Mya (Suda et al. 2022). Subsequently, we reconstructed the transposon landscapes from their variation frequencies using each new genome assembly (supplementary fig. S3, Supplementary Material online). As expected, we observed activation peaks of total DNA transposons around 17 Mya in both the L and S subgenomes of the two allotetraploid species, X. laevis and X. borealis. However, no such activation was seen in the diploid species X. tropicalis. The landscapes we obtained for both total DNA transposons and retrotransposons largely matched our previous results (Suda et al. 2022) but offered a more accurate depiction of the shapes and ratios of each (sub)genome.

Correlation Between Subgenome-biased DNA Loss and Activation of DNA Transposons in X. laevis

To investigate the types of transposons linked with the reduction of the L/S subgenomes in the short period following hybridization, we selected fragments from each transposon subfamily, focusing on those with a substitution rate between 0.04 and 0.05. Then we illustrated the correlation between the contraction of individual chromosomes and the activation of transposon subfamilies. This was achieved using approximate linear slope values (x-axis) and correlation coefficients (y-axis) on a 2D graph (Fig. 3). Notably, distinct patterns were observed in the dynamics of DNA transposons between the L and S subgenomes. Most subfamilies in the L subgenome displayed negative slope values, whereas those in the S subgenome predominantly showed positive values. In total, 8 and 15 subfamilies in the L and S subgenomes, respectively, exhibited correlation coefficients of 0.75 or higher. Seven of eight subfamilies in the L subgenomes had negative slope values, and five of them were classified as DNA transposons (supplementary table S5, Supplementary Material online). In contrast, all 16 subfamilies exhibited positive slope values, with 15 identified as DNA transposons (supplementary table S6, Supplementary Material online). When the same analysis was conducted with a substitution rate of less than 0.05, distinct patterns between the L and S subgenomes were similarly observed as with the 0.04 to 0.05 range (supplementary fig. S4, Supplementary Material online). However, regarding retrotransposons, there were more subfamilies with correlation coefficients of 0.75 or higher (supplementary tables S7 and S8, Supplementary Material online). This may be related to the activation of retrotransposons (supplementary fig. S3, Supplementary Material online) and gene loss due to decreased gene expression, which have occurred after a long time has passed since hybridization. Based on these results, we propose that the DNA loss biased toward the S subgenome in the short period after interspecies hybridization might be associated with the activation of DNA transposons.

Fig. 3. Scatter plot illustrating the relationship between DNA loss and the amount of transposons in X. laevis, characterized by the slope of the approximation straight line and the correlation coefficient. The values were determined by plotting the ratio of the decreased amount of DNA on each X. laevis chromosome relative to its corresponding X. tropicalis chromosome against the increased amount of each transposon subfamily on the chromosome. DNA fragments from each transposon subfamily were selected with a substitution rate between 0.04 and 0.05. Light and dark dots represent subfamilies from DNA transposons and retrotransposons, respectively.

Discussion

The subgenome-biased gene loss after interspecific hybridization is a classic yet unresolved issue and a critical problem from the perspective of species diversification and genome evolution. In considering this issue, there is a hypothesis suggesting that biased reduction in gene expression in subgenomes leads to gene loss (Woodhouse et al. 2014; Alger and Edger 2020; An et al. 2024). However, this study has shown a correlation between subgenome-biased DNA loss and DNA transposon activation following hybridization (Fig. 3). From this, it can be hypothesized that activated DNA transposons that induce double-strand breaks in the genome directly contributed to the biased DNA loss in the S subgenome of the allotetraploid Xenopus frogs. This hypothesis will be discussed in detail in the following paragraph.

Based on the analysis and considerations in this study, we propose a model for the gene loss and asymmetric evolution mechanism of the subgenome from the birth of Xenopus tetraploidization via hybridization to the present (Fig. 4). Before 17 to 18 Mya during the hybridization between two distinct species with the ancestral L and S genomes, selfish DNA containing transposons evolved differently in each species, which could prevent pairing during meiosis between the L and S genomes. In other words, the production of the next diploid generation became impossible. However, allotetraploidization enabled the creation of a population. The mixture of different genomes in the hybrid germline cell caused the modulation of the piRNA-mediated transposon suppression system within each genome. In this hybrid genome mixture, the derepression of DNA transposons led to their activation in both the L and S subgenomes (Suda et al. 2022; supplementary fig. S3, Supplementary Material online). This scenario aligns well with the report by Elurbe et al. (2017) that the majority of newly emerged p300-bound enhancers specific to the L/S subgenomes are transposon-derived, and that derepression of DNA transposons occurs in experimental hybrids of X. tropicalis and X. laevis. The activation of cut-and-paste type DNA transposons increased the frequency of double-strand breaks on chromosomes and induced the loss of neighboring genes and intergenic regions. It may also have facilitated chromosomal inversions because the Galileo DNA transposon in Drosophila is involved in chromosomal inversions (Delprat et al. 2009). Therefore, some of the S-biased chromosomal inversions (Fig. 2 and supplementary fig. S2, Supplementary Material online) might have been influenced by the activation of the S-biased DNA transposons mediated by hybridization. Furthermore, the activation of DNA transposons also played a role in the emergence of the new sex-determining gene dm-W in the unstable situation of the sex determination system after hybridization (Mawaribuchi et al. 2017; Hayashi et al. 2022, 2023). In this context, shortly after hybridization, dynamic gene losses (Fig. 1) occurred in both the L and S subgenomes. Gene loss suggests that the population survived with DNA loss, where one ortholog remained, as evidenced by the 99.7% ortholog retention rate in X. laevis (Table 1). Figure 1 suggests that the rate of gene loss after speciation is higher in X. borealis compared to X. laevis. However, the genome assembly of X. borealis is significantly less complete than that of X. laevis, so there is no direct evidence that the rates of gene loss differ between the two. Nevertheless, the genome data of X. laevis is almost entirely complete, and it is observed that both genes and transposons in X. borealis have decreased in proportion within the genome, while the proportion of “Other” has increased (supplementary tables S3 and S4, Supplementary Material online). This suggests that DNA loss in X. borealis is relatively consistent throughout the genome. Based on these observations, it is considered that the genome of X. borealis has a higher rate of gene loss. This difference is speculated to be related to the differences in transposon activation after speciation.

Fig. 4. Proposal model for the asymmetric evolution of the L/S subgenome after Xenopus frog hybridization.

Furthermore, based on the results in Figs. 2 to 4, it is presumed that more chromosomal inversions and DNA loss occurred in the S subgenome mainly because there was more activation of DNA transposons derived from the S subgenome. Even after the divergence of the S ancestors of X. laevis and X. borealis, which occurred about 17 Mya, gene losses in the independent L/S subgenomes mainly due to DNA transposon activation (Figs. 3 and 4) continued slowly to the present (Fig. 1). In this case, the greater DNA loss in the S subgenome can be attributed to factors such as decreased intergenic complementarity and gene expression (Session et al. 2016) within the S subgenome. Verifying this model will be a challenge in the future.

In allopolyploids, subgenome-biased gene loss has been observed in several plant and animal species, similar to Xenopus in this study. However, some allopolyploids do not exhibit biased-gene loss between subgenomes (VanBuren et al. 2020; Xu et al. 2023). Considering the model for “subgenome-biased DNA loss by the derepression of DNA transposons in hybrids” proposed in this study, it is possible that in allopolyploids without differences in gene loss between subgenomes, there may not have been significant differences in the derepression of DNA transposons between subgenomes. Interestingly, in the allopolyploid fish mentioned above, there is maternal dominance in subgenome differentiation where no gene loss is observed (Xu et al. 2023). Although there may not be a direct relationship between the activation of DNA transposons involved in gene loss and maternal dominance without gene loss, it is suggested that both maternal dominance and transposon density contribute to subgenome differentiation in the allopolyploid fish. In the allotetraploid Xenopus frogs studied here, the diploid organisms derived from the L and S genomes are extinct, making it difficult to analyze maternal dominance. However, it would be extremely interesting to elucidate the relationship between mating patterns and transposons in various interspecies hybrid systems in the future.

Materials and Methods

Data Bases

Genomic sequence information from X. laevis and X. tropicalis was obtained from Xenbase (http://www.xenbase.org/entry/), which includes X. laevis v10.01 and X. tropicalis v10.0 genome assemblies. The genome assembly of X. borealis was retrieved from GenBank (https://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/024/363/595/GCA_024363595.1_UCB_Xborealis_1/). Gene annotation was performed using braker2 (https://github.com/Gaius-Augustus/BRAKER) with the –softmasking option, using the genome assembly of X. borealis, which had been soft-masked based on transposon annotation, and incorporated two types of RNA-seq data from tadpole mesonephros (SRR11844034) and adult liver (SRR11836343). The Xenopus transposon library was compiled using data from X. laevis and X. tropicalis from Repbase (https://www.girinst.org/repbase/).

Identification of TEs in the Genome

The genomic locations of transposons were identified using RepeatMasker (http://www.repeatmasker.org/). Transposons were aligned to consensus sequences from Repbase using MAFFT (https://mafft.cbrc.jp/alignment/software/) with the options –retree 2 –reorder. CG sequences were removed using a customized Python script. Subsequently, transposon diversity was recalculated using FastTree (http://www.microbesonline.org/fasttree/) with the -nt option. Another customized Python script was employed to overlap the locations of transposons, giving priority to those with low diversity scores. TE landscapes were generated using RepeatLandscape (github.com/caballero/RepeatLandscape) to calculate the Kimura distance as a measure of age.

Analysis of Orthologous Protein-coding Genes in Xenopus Frogs

For the ortholog analysis of protein-coding genes, we initially extracted annotated genes from the X. tropicalis genome database (X. tropicalis v10.0). We then excluded tandem-duplicated genes, genes with uncertain functions (LOC), and noncoding RNA genes from consideration. Subsequently, in order to identify orthologous genes in both X. laevis and X. borealis, we conducted a blastn search within each of the L/S subgenomes and identified homologous genes. Using the information from homologous genes, we selected candidate orthologous exons based on their bitscore, and total bitscores were calculated for one or several exons. The L or S gene with the highest bitscore (≥300) between X. tropicalis and either X. laevis or X. borealis was then chosen as the ortholog. To further confirm the allotetraploid orthologous L or S genes to X. tropicalis, we conducted a synteny analysis. This analysis involved assessing the synteny between X. tropicalis and either X. laevis or X. borealis using the L/S subgenomes in regions spanning 1 Mbp around the candidate genes. Synteny was defined by the conservation of three or more neighboring genes between X. tropicalis and either X. laevis or X. borealis.

Chromosomal Inversion

In our analysis, when we observed that the gene strand, preserving synteny, was inverted relative to X. tropicalis, we classified those genes as inversions. To categorize these inversions, we grouped together genes that were inverted and located within a 20 Mbp span. This grouping allowed us to identify inversions in each subgenome and evaluate overlaps between different species. Our ultimate goal was to characterize inversions that did not overlap with inversions found in other subgenomes as species-specific inversions.

Correlation Between DNA Loss and Variation in Transposon Quantity on Chromosomes

To determine DNA loss, we employed a customized Python script that measured the interval sizes between adjacent orthologous gene pairs, which were comprehensively selected by synteny, between corresponding chromosomes in X. tropicalis and X. laevis subgenomes. We calculated the proportion of the reduced length after excluding transposons, establishing the median ratio for each chromosome as the chromosome loss ratio. To assess the increase in transposons on each chromosome, we selected fragments from each transposon subfamily, focusing on those with a substitution rate below 0.05 or between 0.04 and 0.05. We then measured the coverage depth where these fragments overlapped with the consensus sequence of their respective transposon subfamilies. Subsequently, we normalized the peak coverage by the chromosome length, excluding transposons, and referred to this normalized peak coverage as the increment for each transposon. We derived both the correlation coefficient and the slope of the approximate linear trend by analyzing the loss ratios and increments of transposons across chromosomes. These metrics were determined for transposons extending 30,000 bp or longer, falling within the substitution rate below 0.05 or between 0.04 and 0.05.

Supplementary Material

evae179_Supplementary_Data

Supplementary Material

Supplementary material is available at Genome Biology and Evolution online.

Author Contributions

K.S. and M.I. designed the study. K.S., T.S., S.H., H.O., D.T., T.M., K.T., and M.I. analyzed the data. K.S. and M.I. wrote the paper.

Funding

This work was partially supported by Ohsumi Frontier Science Foundation to M.I.

Conflict of Interest

The authors declare no competing interests.

Ethics Statement

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Kitasato University. Written informed consent was obtained from the owners for the participation of their animals in this study.

Data Availability

This study includes no data deposited in external depositories.
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