Chromosome Science 24: 3-12, 2021 Uno 3

Review Article

Inference of evolution of vertebrate genomes and chromosomes from genomic and cytogenetic analyses using

Yoshinobu Uno

Received: December 28, 2020 / Accepted: January 15, 2021 © 2021 by the Society of Chromosome Research

Abstract this species can produce a large number of eggs all year round, and the sizes of eggs and embryos are large enough Amphibians, which first adapted to terrestrial life in to allow microsurgical manipulation and injection. This vertebrates, appeared around 350 million years ago was the experimental used by John Gurdon, (Mya) after the common ancestors of tetrapods (am- who was awarded the Nobel Prize for Medicine or Physiol- phibians, reptiles, birds, and mammals) diverged from ogy for his groundbreaking study on nuclear reprogram- ray-finned fishes around 410 Mya, and are therefore ming. Specifically, he showed that the nuclei from special- important model for understanding vertebrate ized X. laevis cells could be reprogrammed to give rise to evolution. To date, there are many cytogenetic reports a complete animal (Gurdon, 1962). However, X. laevis is of amphibians. Moreover, recent improvements in tech- difficult to apply with respect to genetic material because niques for cytogenetic and genomic analyses help accel- of an allotetraploid species (see section: Polyploid evolu- erate the accumulation of the cytogenetic and genomic tion in amphibians) and longer generation times than the data from amphibians. Inferred from recent genomic and other vertebrate models, including mice and zebrafish. cytogenetic analyses using amphibians, I review karyo- Since the 1990s, researchers have widely used the diploid type and chromosome evolution, including sex chromo- species related to X. laevis, the Western clawed frog (Xeno- somes, polyploidy, and origins of microchromosomes, in pus [formerly Silurana] tropicalis, Pipidae, Anura), whose not only amphibians but also entire vertebrates. generation time is shorter than X. laevis, as a novel animal model for biological research, including genetic analyses. Salamanders have contributed to biological research based Keywords: amphibians, chromosome, genome, evolu- on the discovery of the Spemann organizer (Spemann and tion, vertebrates Mangold, 1924). The Mexican axolotl (Ambystoma mexi- canum, Ambystomatidae, Caudata) is commonly used in Overview of Amphibians many regeneration laboratories to uncover the remarkable The class Amphibia occupies a phylogenetic position regenerative capability of this animal. between Actinopterygii (ray-finned fishes) and Amniota Since the 1980s, decreases in populations ow- (reptiles, birds, and mammals), and the ancestors of Tet- ing to invasive fungi and habitat destruction have been ob- rapoda split into the two main lineages of Amphibia and served around the world, which is one of the most critical Amniota roughly 350 million years ago (Mya) (Kumar et threats to global biodiversity (Alroy, 2015; O’Hanlon et al., al., 2017). The extant amphibians comprise three orders: 2018). Some researchers have estimated that almost one- Anura ( and toads), Caudata/Urodela (salamanders), third of amphibians are threatened with extinction (Stuart and (), which contain 54, 10, and et al., 2004); therefore, urgent action is needed to prevent 10 families, respectively, and the number of extant amphib- further declines and the extinction crisis in amphibians. ian species is approximately 8,200, of which nearly 88% Genome analyses using whole-genome sequences in con- are anurans (AmphibiaWeb, 2020) (as of November 2020) servation biology are known as powerful tools to highlight (Figure 1). Some amphibian species have been widely used the biological relationships of wildlife species at the ge- as experimental animals for life science research. The Afri- nome level and evaluate the potential response to chang- can clawed frog (Xenopus laevis, Pipidae, Anura) has been a ing environments (Steiner et al., 2013; Homola et al., 2019). particularly excellent animal model in developmental, cel- Revealing genome and chromosomal information on am- lular, and molecular biological research since the 1950s. X. phibians could contribute to not only a deep understanding laevis has unique advantages for experiments. For instance, of the evolution of vertebrate genomes and chromosomes but also the conservation of biological diversity in amphib- ians, many of which are threatened by extinction. Yoshinobu Uno (*) This review describes the characterizations of karyotypes Department of Life Sciences, Graduate School of Arts and Sciences, and genomes in amphibians and the amphibian sequenced The University of Tokyo, Tokyo, Japan. whole genomes published to date. Next, we present novel E-mail: [email protected] knowledge of genomic and chromosomal evolution in ver- Tel: +81-3-5454-6739 tebrates, including sex chromosomes, polyploid, and karyo- 4 Genomic and cytogenetic analyses using amphibians type evolution obtained by recent genomic and cytogenetic 2002; Matsui et al., 2006; Sessions, 2008; Venu et al., 2011) analyses using amphibians. (Figure 1). In particular, the karyotypes of anurans and sal- amanders have been the subject of numerous cytogenetic Karyotypes and genome sizes of amphibians studies, and karyotype data are available for most family Among Amphibia, karyotypes of diploid species have groups. Collectively, the karyotypes of diploid amphibian been reported for 1,193 (14.5%) of the ~8,200 known spe- species are commonly characterized by 20–26 bi-armed cies, namely 963 anurans, 209 salamanders, and 21 caeci- chromosomes. Some karyotype reports suggest that poly- lians (as of November 2020) (Morescalchi, 1973; Wake and ploidy may have been involved in some families (Schmid et Case, 1975; Kuramoto, 1990; Venkatachalaiah and Venu, al., 2015) (see section: Polyploid evolution in amphibians).

Figure 1. Chromosome number variation among 1,193 diploid species in Amphibia. The numbers of species with diploid chromosome numbers and with reported karyotypes were extracted from previous cytogenet- ic studies (Morescalchi, 1973; Wake and Case, 1975; Kuramoto, 1990; Venkatachalaiah and Venu, 2002; Matsui et al., 2006; Sessions, 2008; Venu et al., 2011). The numbers of extant amphibian species in individual suborders or families are shown in the parentheses based on an existing resource (AmphibiaWeb, 2020). The phylogenetic relationship is based on the existing literature (Wake and Koo, 2018; AmphibiaWeb, 2020). The divergence times between the three orders, Anura (frogs and toads), Caudata/Urodela (sala- manders), and Gymnophiona (caecilians), are cited from the existing literature (Kumar et al., 2017). Mya, million years ago. Uno 5

Amphibians are also characterized by extremely large ge- Sex chromosome evolution in amphibians nomes compared to other taxa of vertebrates and extensive Mammalian species exhibit a male-heterogametic ge- variation in genome size (C-value: picograms of DNA in netic sex-determination system with XX/XY sex chromo- haploid nuclei), which transcends variation in chromosome somes, and birds do female-heterogametic determination number and shape (Liedtke et al., 2018; Gregory, 2020). with ZZ/ZW sex chromosomes (Takagi and Sasaki, 1974; The genome sizes range from 0.95 to 11.50 pg, from 13.89 Vorontsov et al., 1980; Belterman and Boer, 1984). Both to 120.56 pg, and from 2.94 to 11.78 pg in Anura, Caudata, male heterogamety and female heterogamety have been and Gymnophiona, respectively. Crucially, the larger ge- found in reptiles (Olmo and Signorino, 2005). Most reptiles nome sizes compared to other vertebrates have prevented and all birds and mammals have differentiated sex chromo- whole-genome sequencing of amphibians. some pairs. On the other hand, teleost fishes and amphib- ians also exhibit both male heterogamety and female het- Whole-genome sequencing of amphibians erogamety, and most of these species have morphologically Genome sequencing projects are now ongoing for many undifferentiated sex chromosomes. Sex-determining genes vertebrate species, and the available information provides in several teleost fishes were identified using genetic and a new perspective on genome and chromosome evolution cytogenetic analyses, and these findings indicated that the in vertebrates. The draft genome assembly of X. tropicalis sex-determining genes and genetic linkages of sex chromo- was the first to be reported for amphibians in 2010 (Hell- somes were different not only within orders but also be- sten et al., 2010). A de novo genome of the Tibetan frog tween related species within genera (Kikuchi and Hamagu- (Nanorana parkeri, Dicroglossidae, Anura) was published chi, 2013; Bachtrog et al., 2014). This phenomenon results as the second draft genome assembly in amphibians (Sun from switches in the chromosome pairs used for sex deter- et al., 2015). The genomes of the two frogs were smaller mination, termed turnover of sex chromosomes. Sex chro- (1.7 Gb and 2.3 Gb, respectively) than those mammals (ap- mosomes are relatively young in organisms in which sex proximately 3 Gb) and sequenced using whole-genome chromosome turnover occurs; thus, sex chromosomes have shotgun methods based on Sanger sequencing or short- insufficient time to substantially differentiate from one an- read sequencing. Draft genome assemblies of organisms other. Accumulation of the cytogenetic and genomic data with larger genome sizes than these animals sequenced by of amphibian sex chromosomes, as shown subsequently, the only whole-genome shotgun methods have remained a has provided evidence that sex chromosome turnover be- major challenge, which yield highly fragmented assemblies tween related species occurs frequently in amphibians as that are insufficient for comparative analyses using chro- well as teleost fishes. mosome-scale genomes. Many researchers have conducted analyses of sex deter- Recent improvements in long-read sequencing technol- mination in amphibian species. However, the only identi- ogy and Hi-C, a genome-wide chromatin conformation fied sex-determining gene is amphibians to date is Dmw capture technology, have helped accelerate the release of (Yoshimoto et al., 2008). This gene, the ovary-determining high-quality assembly, termed chromosome-scale genome gene in X. laevis, is located on one of the homologous pairs assembly, of organisms with larger genomes (Burton et al., of chromosome 3S as the W chromosome. It might have 2013; Kaplan and Dekker, 2013; Dudchenko et al., 2017). evolved from gene duplication of Dmrt1, which can induce A genome assembly of the polyploid species, X. laevis, the masculinization of somatic cells in vertebrates (Yoshi- has been reported as the first chromosome-scale genome moto et al., 2008; Yoshimoto and Ito, 2011). Phylogenetic in amphibians using whole-genome shotgun methods in and genotypic analyses using Xenopus species revealed that combination with fluorescence in situ hybridization (FISH) Dmw was retained only in X. laevis and the more closely of 798 bacterial artificial chromosome clones (BACs) as related species (Bewick et al., 2011; Furman and Evans, well as in vivo and in vitro chromatin conformation capture 2016), suggesting that Dmw in X. laevis could be younger, analysis (Session et al., 2016) (see section: Polyploid evolu- approximately 13–34 Mya (Chain and Evans, 2006; Session tion in amphibians). A chromosome-scale assembly of the et al., 2016). To investigate the conservation of genetic link- X. tropicalis genome was reported by improving the previ- ages of sex chromosomes between X. laevis and several an- ously published draft genome assembly in 2019 (Mitros et urans, we performed comparative chromosome mapping al., 2019). Recently, a genome assembly for the Mexican ax- for four anuran frogs, X. laevis, X. tropicalis, the Japanese olotl was generated using long-read sequencing technology wrinkled frog ( [formerly Rana] rugosa, Rani- and optical mapping (Nowoshilow et al., 2018). Surpris- dae), and the endemic Japanese bell-ring frog (Buergeria ingly, the genome size of the salamander is 32 Gb, which is buergeri, Rhacophoridae) (Uno et al., 2008a, 2008b, 2015). more than 10 times larger than those in mammals and X. This result indicated that the sex chromosomes of X. lae- tropicalis, and axolotl had the largest genome ever fully se- vis, X. tropicalis, and G. rugosa were derived from differ- quenced at that point. To date, other than the two Xenopus ent chromosome pairs of their common ancestors (Figure frogs and axolotl, chromosome-scale genome sequences of 2). Recent genetic analyses by short-read sequencing for amphibians are available in NCBI databases for the three more than 20 anurans in Bufonidae, Hylidae, and Ranidae anurans (Bufo gargarizans, Leptobrachium leishanense, and also indicated diversity in the origins of anuran sex chro- Pyxicephalus adspersus) and three caecilians (Geotrypetes mosomes (Figure 2) and led to the inference of at least 10 seraphini, unicolor, and Rhinatrema bivit- and one turnovers of sex chromosomes among Ranidae tatum) (as of November 2020). Collectively, the currently frogs and between Bufo frogs, respectively (Brelsford et al., available improved sequence technology enables the con- 2013, 2016; Jeffries et al., 2018). The turnover of sex chro- struction of chromosome-scale genome assemblies of or- mosomes occurred in the lineage of R. blairi after diverging ganisms with genome sizes that are several times as large as from the related species less than 3.6 Mya, which is young- humans; therefore, chromosome-scale genome information er than interspecific turnovers reported in vertebrates to of numerous amphibians, including salamanders, will be our knowledge (Jeffries et al., 2018). acquired soon. Certain anuran species have mixed combinations of sex 6 Genomic and cytogenetic analyses using amphibians chromosome systems within species, which is rare in or- the sex chromosomes, Sox3, known to be expressed in oo- ganisms. G. rugosa has different sex chromosome systems cytes in the Xenopus frog (Koyano et al., 1997), was highly among populations separated geographically (Nishioka et expressed in undifferentiated ZW ovaries of this frog in the al., 1994; Miura, 2007). The seventh largest chromosome ZW group (Miura et al., 2009; Oshima et al., 2009). This in- pair, chromosome 7, which is homologous to X. tropicalis dicated that Sox3 is a strong candidate for sex-determining chromosome 8 (Uno et al., 2008b) (Figure 2), is the differ- genes as Ar in the ZW group of G. rugosa. However, there entiated ZW sex chromosomes in at least two populations, are no reports of gene expression patterns for Ar and Sox3 namely the ZW group and Neo-ZW group. In contrast, in different geographic populations of this frog. another population, the XY group, is characterized by dif- X. tropicalis is a diploid model system of amphibians em- ferentiated XY sex chromosomes derived from chromo- ployed not only for developmental biology but also genet- some 7 (Uno et al., 2008a). No morphological differences ics, and several laboratory strains of this frog have been es- were found between male and female karyotypes in the tablished. Natural populations of this frog exhibit a male- two other populations, the West-Japan group and East-Ja- heterogametic determination system, which includes the pan group. Consequently, this frog has experienced at least Y chromosome (Furman et al., 2020). However, recent three intraspecific turnovers of sex chromosomes result- genetic studies have demonstrated that the sex of X. tropi- ing from hybrids between populations within 1 Mya (Mi- calis laboratory strains was determined by more complex ura, 2007; Ogata et al., 2008). These events are, as far as we sex chromosome systems, including at least three differ- know, the most recent turnovers of sex chromosomes ob- ent sex chromosomes (Y, W, and Z) observed in YZ, YW, served in vertebrates. Interestingly, the Ad4bp/sf-1, Ar, and and ZZ males and in ZW and WW females because of ac- Sox3 genes, which play important roles in sexual differen- quisition of novel sex-determination mechanisms (Roco et tiation of vertebrates (Nagahama et al., 2020), are located al., 2015; Furman et al., 2020). These results also reflected on the ZW sex chromosomes in the ZW group and XY sex how the sex-specific regions on the Y, W, and Z sex chro- chromosomes in the XY group of G. rugosa (Uno et al., mosomes were located in the same region of chromosome 2008a). Among these three genes, gene expression of W- 7 of X. tropicalis; however, no sex-determining genes have linked Ar (W-Ar) was hardly expressed in ZW female em- been identified. Further genomic and cytogenetic analyses, bryos of this frog in the ZW group, and Z-linked Ar (Z-Ar) including whole-genome sequencing and identification of was upregulated in ZZ male gonads prior to the sex deter- sex-determining genes using several populations of G. ru- mination period (Yokoyama et al., 2009). Assessment with gosa and X. tropicalis, could help to understand the evolu- Ar-transgenic and knockdown frogs suggested that Z-Ar tionary mechanisms of sex chromosome turnovers and ac- might function as a male sex determinant in the ZW group quisition of sex-determining genes in vertebrates. of G. rugosa (Oike et al., 2017). Another gene located on

Figure 2. Diversity of sex-determining loci in anurans. The schematic diagrams of X. tropicalis chromosomes are modified from our previous cytogenetic study using this frog (Uno et al., 2012). The sex-determining loci in 30 anurans species belonging to five families, which are taken from the existing literature data (Uno et al., 2008a, 2008b, 2015; Brelsford et al., 2013, 2016; Jeffries et al., 2018), are mapped to six different chromosome pairs of X. tropicalis. Homologous chro- mosomal locations of the sex-determining gene of X. laevis (Dmw), candidates of sex-determining genes of G. rugosa (Ar and Sox3), and six sexual differentiation genes (Ad4bp/sf-1, Amh, Cyp17a1, Cyp19a1, Dmrt1, and Sox9) on X. tropicalis chromosomes are shown to the right of each chromosome. Uno 7

Polyploid evolution in amphibians clature has been widely employed. Next, we sequenced the Polyploidization (whole-genome duplication: WGD), the whole genome of X. laevis using whole-genome shotgun presence of more than two chromosome sets, is an impor- methods, FISH mapping of 798 BACs, and in vivo and in tant event with respect to genome evolution (Ohno, 1970). vitro chromatin conformation capture analyses, and com- It has been thought that there is less polyploidization in pared it to that of the related diploid species X. tropicalis animals than in plants (Otto, 2007). In vertebrates, there (Session et al., 2016). As a result, we found two subgenomes are no reports of polyploid mammals or birds, though in extant X. laevis, which contain ‘fossil’ transposable ele- some lizards that reproduce by parthenogenesis and some ments, derived from two distinct diploid progenitors before fishes (salmonids, carps, and sturgeons) experienced WGD allopolyploidization (Figure 3C). These two homoeologous (Otto, 2007; Berthelot et al., 2014; Lien et al., 2016; Chen subgenomes, referred to as the L subgenome and S subge- et al., 2019; Du et al., 2020). In amphibians, polyploidy is nome, were distributed in nine pairs of L chromosomes and a widespread phenomenon in 15 and 4 families of the or- nine pairs of S chromosomes, respectively, in X. laevis, sug- ders Anura and Caudata, respectively (Schmid et al., 2015). gesting the absence of recombination between the two sub- Comparative genome analyses in vertebrates and genomes after polyploidization. Combined with genome- revealed that WGD occurred twice (2R-WGD) in the early wide transcriptome analyses, the findings revealed that the evolutionary history of vertebrates from 430 to 500 Mya, two subgenomes of X. laevis evolved asymmetrically (Ses- which is considered to have contributed to an increase in sion et al., 2016) (Figure 3C). The L subgenome has more genomic and phenotypic complexity in vertebrates (Putnam often preserved the ancestral state conserved in X. tropi- et al., 2008; Simakov et al., 2020). Construction of chro- calis; however, the S subgenome experienced more gene mosome-scale genome sequences for polyploid vertebrate loss, deletion, rearrangement, and reduced gene expres- species and comparative genomic and cytogenetic analyses sion. A recent comparison of chromosome-scale genome using polyploid species enabled the provision of important sequences between invertebrate amphioxus and advances in our understanding of the genome evolution of several vertebrate species implied that the second WGD of 2R-WGD in the basal lineage of vertebrates. However, little 2R-WGD, which early vertebrates experienced, was allote- is known about the process of genomic and chromosomal traploidization resulted from WGD after interspecific hy- rearrangements after WGD because many duplicate genes bridization, which was expected to exhibit an asymmetrical derived from WGD have been lost rapidly (Wolfe, 2001). distribution of gene losses as observed in X. laevis (Simakov For example, 88% of duplicated genes derived from WGD et al., 2020). More than 25 extant species classified into the were lost in yeast after WGD that occurred 80 Mya (Kel- genus Xenopus, including X. laevis and X. tropicalis, have lis et al., 2004) and 70% in Arabidopsis after WGD 86 Mya been reported, and it is suggested that these species, except (Bowers et al., 2003). Approximately 50% of duplicate genes for X. tropicalis, are polyploid (tetraploid, octoploid, and have been rapidly lost within roughly 75 million years af- dodecaploid) species with 36–108 chromosomes result- ter 3R-WGD, which occurred in the teleost fish lineage ap- ing from allopolyploidization events (Schmid et al., 2015). proximately 350 Mya after 2R-WGD (Jaillon et al., 2004; Whole-genome sequencing along with genomic and cyto- Kasahara et al., 2007; Sato et al., 2009). Therefore, the anal- genetic analyses using other allopolyploid Xenopus frogs ysis of genomic structures for ‘young’ polyploid vertebrates will allow us to better understand the process and mecha- that retain most duplicated genes after WGD is desirable to nisms of genome evolution after 2R-WGD occurred in the trace the evolutionary process of genomes in ancestral ver- basal lineage of vertebrates. tebrates after 2R-WGD. X. laevis has experienced allotetraploidization (WGD ac- Evolution of ancestral karyotypes in vertebrates in- companying interspecific hybridization), which was esti- ferred from amphibian chromosomes mated to have occurred 17 Mya, after divergence from the Karyotypes of the majority of amphibians are character- lineage of diploid species of X. tropicalis 48 Mya (Bisbee ized by 20–26 bi-armed chromosomes. As an exception, et al., 1977; Session et al., 2016). Therefore, X. laevis and karyotypes, which comprise many chromosomes, includ- X. tropicalis are robust animal models for precisely under- ing many morphologically indistinguishable microchro- standing genomic and chromosomal rearrangements after mosomes, are observed in some species belonging to the WGD. To provide new insights into the process of chromo- families derived from each phylogenetically basal lineage somal rearrangements that occurred in polyploid species, of salamanders and anurans: Hynobiidae and Cryptobran- including the common ancestors of vertebrates, we per- chidae in suborder Cryptobranchoidea (2n = 56–78, more formed FISH mapping of 140 and 60 genes for X. tropicalis than 30 microchromosomes in most species) and Ascaphi- chromosomes (XTR) and X. laevis chromosomes (XLA), dae in suborder Leiopelmatoidea (2n = 46, 26 microchro- respectively, and constructed a comparative chromosome mosomes) (Morescalchi, 1973; Sessions, 2008) (Figure 1). map between these two Xenopus species (Uno et al., 2008b, In other vertebrates, microchromosomes are not observed 2012, 2013). This showed that the X. laevis karyotype com- in mammalian or teleost fish species as well as the majority prised nine sets of homoeologous chromosome pairs. The of amphibians. On the other hand, reptiles, birds, some car- eight sets correspond to XTR1–8, respectively, and the last tilaginous fishes, and the coelacanth (Latimeria chalumnae) set corresponds to a fusion of XTR9 and XTR10 (Figure retain many microchromosomes (Takagi and Sasaki, 1974; 3A, B). Based on these results, we proposed a new nomen- Bogart et al., 1994; Olmo and Signorino, 2005; Arai, 2011; clature for X. laevis homoeologous chromosomes based Uno et al., 2020). These observations led to the hypothesis on the relationship to XTR and the relative size of chro- that the common ancestors of tetrapods and those of ver- mosomes distinguished cytogenetically: XLA1L, XLA1S, tebrates might possess karyotypes with many microchro- XLA2L, XLA2S, and so on, in which the numbering of mosomes (Ohno et al., 1969; Bogart et al., 1994). Recent XLA corresponds to that in XTR and the postfixes, ‘L’ and ‘S’, comparative genomic and cytogenetic analyses using some stand for ‘long’ and ‘short’ chromosomes in the homoeolo- vertebrates, including two amphibians, X. tropicalis and the gous pairs (Matsuda et al., 2015) (Figure 3A). This nomen- Mexican axolotl, have provided increasing evidence to sup- 8 Genomic and cytogenetic analyses using amphibians port the hypothesis of the presence of many microchromo- The presumed karyotypes in the common ancestors of tet- somes in the common ancestors of bony fishes and those rapods and karyotype reports in amphibians also suggested of tetrapods (Nakatani et al., 2007; Voss et al., 2011; Uno et the possibility that fusions caused by the disappearance of al., 2012; Sacerdot et al., 2018; Simakov et al., 2020). If this microchromosomes might have occurred independently hypothesis is correct, the decrease in many microchromo- in the lineage of anurans after divergence from Leiopelma- somes by fusions between macro- and microchromosomes toidea around 210 Mya and in the lineage of salamanders and between microchromosomes occurred independently after divergence from Cryptobranchoidea roughly 200 Mya in the lineages of teleost fishes and mammals. On the oth- (Kumar et al., 2017). However, there is no available genome er hand, fusions with microchromosomes took place very information on these amphibian species with many micro- rarely or less frequently in the reptilian and avian lineages. chromosomes to prove this hypothesis.

Figure 3. The evolution of X. laevis genome. (A) A circos plot (Krzywinski et al., 2009) using 5,044 1-to-2 orthologs between X. tropicalis genome and X. laevis subgenomes showing con- served synteny of diploid species X. tropicalis chromosomes (colored, left) and L and S chromosomes in allotetraploid species X. laevis (white, right). Numbers of X. laevis chromosomes correspond to the new nomenclature (Matsuda et al., 2015). Each genetic linkage of X. tropicalis chromosomes, except for chromosomes 9 and 10, has been conserved in two pairs of X. laevis homoeologous chromosomes with no inter- chromosomal translocations after allopolyploidization. (B) Chromosomal localization of BAC clones to metaphase chromosome spreads of a female X. laevis. The BAC clones of the Nfatc2.L (green) and Ccnyl1.L (red) genes, which were located on X. tropicalis chromosome 10 and chro- mosome 9, respectively, were localized to X. laevis chromosome 9L (9_10L). Arrows and arrow heads indicate hybridization signals. A scale bar represents 10 µm. (C) A schematic scenario of the evolution of the X. laevis genome. The decrease in chromosome number by a fusion event between two chromosomes homologous to X. tropicalis chromosomes 9 and 10 occurred in the ancestor of X. laevis, which diverged from the common ancestor of Xenopus frog 48 Mya. The ancestral extinct diploid species, termed progenitor L and progenitor S, had derived 34 Mya, and allotetraploidization occurred that resulted from interspecific crossing between the two species and WGD 17 Mya. After poly- ploidization, the S subgenome distributed in S chromosomes derived from the diploid progenitor S experienced the loss of more genes and more frequent chromosome rearrangements compared to the L subgenome in L chromosomes from the progenitor L. The divergence times are cited from the existing literature (Session et al., 2016). Mya, million years ago. Uno 9

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