Morc1 reestablishes H3K9me3 heterochromatin on piRNA-targeted transposons in gonocytes.

To maintain fertility, male mice re-repress transposable elements (TEs) that were de-silenced in the early gonocytes before their differentiation into spermatogonia. However, the mechanism of TE silencing re-establishment remains unknown. Here, we found that the DNA-binding protein Morc1, in cooperation with the methyltransferase SetDB1, deposits the repressive histone mark H3K9me3 on a large fraction of activated TEs, leading to heterochromatin. Morc1 also triggers DNA methylation, but TEs targeted by Morc1-driven DNA methylation only slightly overlapped with those repressed by Morc1/SetDB1-dependent heterochromatin formation, suggesting that Morc1 silences TEs in two different manners. In contrast, TEs regulated by Morc1 and Miwi2, the nuclear PIWI-family protein, almost overlapped. Miwi2 binds to PIWI-interacting RNAs (piRNAs) that base-pair with TE mRNAs via sequence complementarity, while Morc1 DNA binding is not sequence specific, suggesting that Miwi2 selects its targets, and then, Morc1 acts to repress them with cofactors. A high-ordered mechanism of TE repression in gonocytes has been identified.

Marsupial genome analysis suggests that satellite DNA formation from walb endogenous retrovirus is an event specific to the red-necked wallaby.

We recently identified walbRep, a satellite DNA residing in the genome of the red-necked wallaby Notamacropus rufogriseus. It originates from the walb endogenous retrovirus and is organized in a manner in which the provirus structure is retained. The walbRep repeat units feature an average pairwise nucleotide identity as high as 99.5%, raising the possibility of a recent origin. The tammar wallaby N. eugenii is a species estimated to have diverged from the red-necked wallaby 2-3 million years ago. In PCR analyses of these two and other related species, walbRep-specific fragment amplification was observed only in the red-necked wallaby. Sequence database searches for the tammar wallaby resulted in sequence alignment lists that were sufficiently powerful to exclude the possibility of walbRep existence. These results suggested that the walbRep formation occurred in the red-necked wallaby lineage after its divergence from the tammar wallaby lineage, thus in a time span of maximum 3 million years.

Na+/Cl- cotransporter 2 is not fish-specific and is widely found in amphibians, non-avian reptiles, and select mammals.

Solute carrier 12 (Slc12) is a family of electroneutral cation-coupled chloride (Cl-) cotransporters. Na+/K+/2Cl- (Nkcc) and Na+/Cl- cotransporters (Ncc) belong to the Nkcc/Ncc subfamily. Human and mouse possess one gene for the Na+/Cl- cotransporter (ncc gene: slc12a3), whereas teleost fishes possess multiple ncc genes, slc12a3 (ncc1) and slc12a10 (ncc2), in addition to their species-specific paralogs. Amphibians and squamates have two ncc genes: slc12a3 (ncc1) and ncc3. However, the evolutionary relationship between slc12a10 and ncc3 remains unresolved, and the presence of slc12a10 (ncc2) in mammals has not been clarified. Synteny and phylogenetic analyses of vertebrate genome databases showed that ncc3 is the ortholog of slc12a10, and slc12a10 is present in most ray-finned fishes, coelacanths, amphibians, reptiles, and a few mammals (e.g., platypus and horse) but pseudogenized or deleted in birds, most mammals, and some ray-finned fishes (pufferfishes). This shows that slc12a10 is widely present among bony vertebrates and pseudogenized or deleted independently in multiple lineages. Notably, as compared with some fish that show varied slc12a10 tissue expression profile, spotted gar, African clawed frog, red-eared slider turtle, and horse express slc12a10 in the ovaries or premature gonads. In horse tissues, an unexpectedly large number of splicing variants for Slc12a10 have been cloned, many of which encode truncated forms of Slc12a10, suggesting that the functional constraints of horse slc12a10 are weakened, which may be in the process of becoming a pseudogene. Our results elaborate on the evolution of Nkcc/Ncc subfamily of Slc12 in vertebrates.NEW & NOTEWORTHY slc12a10 is not a fish-specific gene and is present in a few mammals (e.g., platypus and horse), non-avian reptiles, amphibians, but was pseudogenized or deleted in most mammals (e.g., human, mouse, cat, cow, and rhinoceros), birds, and some ray-finned fishes (pufferfishes).

Placental Mammals Acquired Functional Sequences in NRK for Regulating the CK2-PTEN-AKT Pathway and Placental Cell Proliferation.

The molecular evolution processes underlying the acquisition of the placenta in eutherian ancestors are not fully understood. Mouse NCK-interacting kinase (NIK)-related kinase (NRK) is expressed highly in the placenta and plays a role in preventing placental hyperplasia. Here, we show the molecular evolution of NRK, which confers its function for inhibiting placental cell proliferation. Comparative genome analysis identified NRK orthologs across vertebrates, which share the kinase and citron homology (CNH) domains. Evolutionary analysis revealed that NRK underwent extensive amino acid substitutions in the ancestor of placental mammals and has been since conserved. Biochemical analysis of mouse NRK revealed that the CNH domain binds to phospholipids, and a region in NRK binds to and inhibits casein kinase-2 (CK2), which we named the CK2-inhibitory region (CIR). Cell culture experiments suggest the following: 1) Mouse NRK is localized at the plasma membrane via the CNH domain, where the CIR inhibits CK2. 2) This mitigates CK2-dependent phosphorylation and inhibition of PTEN and 3) leads to the inhibition of AKT signaling and cell proliferation. Nrk deficiency increased phosphorylation levels of PTEN and AKT in mouse placenta, supporting our hypothesis. Unlike mouse NRK, chicken NRK did not bind to phospholipids and CK2, decrease phosphorylation of AKT, or inhibit cell proliferation. Both the CNH domain and CIR have evolved under purifying selection in placental mammals. Taken together, our study suggests that placental mammals acquired the phospholipid-binding CNH domain and CIR in NRK for regulating the CK2-PTEN-AKT pathway and placental cell proliferation.

Hamster PIWI proteins bind to piRNAs with stage-specific size variations during oocyte maturation.

In animal gonads, transposable elements are actively repressed to preserve genome integrity through the PIWI-interacting RNA (piRNA) pathway. In mice, piRNAs are abundantly expressed in male germ cells, and form effector complexes with three distinct PIWIs. The depletion of individual Piwi genes causes male-specific sterility with no discernible phenotype in female mice. Unlike mice, most other mammals have four PIWI genes, some of which are expressed in the ovary. Here, purification of PIWI complexes from oocytes of the golden hamster revealed that the size of the PIWIL1-associated piRNAs changed during oocyte maturation. In contrast, PIWIL3, an ovary-specific PIWI in most mammals, associates with short piRNAs only in metaphase II oocytes, which coincides with intense phosphorylation of the protein. An improved high-quality genome assembly and annotation revealed that PIWIL1- and PIWIL3-associated piRNAs appear to share the 5'-ends of common piRNA precursors and are mostly derived from unannotated sequences with a diminished contribution from TE-derived sequences, most of which correspond to endogenous retroviruses. Our findings show the complex and dynamic nature of biogenesis of piRNAs in hamster oocytes, and together with the new genome sequence generated, serve as the foundation for developing useful models to study the piRNA pathway in mammalian oocytes.

Replacement of owl monkey centromere satellite by a newly evolved variant was a recent and rapid process.

Alpha satellite DNA is a major DNA component of primate centromeres. We previously reported that Azara's owl monkey has two types of alpha satellite DNA, OwlAlp1 and OwlAlp2. OwlAlp2 (344 bp) exhibits a sequence similarity throughout its entire length with alpha satellite DNA of closely related species. OwlAlp1 (185 bp) corresponds to the part of OwlAlp2. Based on the observation that the CENP-A protein binds to OwlAlp1, we proposed that OwlAlp1 is a relatively new repetitive DNA that replaced OwlAlp2 as the centromeric satellite DNA. However, a detailed picture of the evolutionary process of this centromere DNA replacement remains largely unknown. Here, we performed a phylogenetic analysis of OwlAlp1 and OwlAlp2 sequences, and also compared our results to alpha satellite DNA sequences of other primate species. We found that: (i) OwlAlp1 exhibits a higher similarity to OwlAlp2 than to alpha satellite DNA of other species, (ii) OwlAlp1 has a single origin, and (iii) sequence variation is lower in OwlAlp1 than in OwlAlp2. We conclude that OwlAlp1 underwent a recent and rapid expansion in the owl monkey lineage. This centromere DNA replacement could have been facilitated by the heterochromatin reorganization that is associated with the adaptation of owl monkeys to a nocturnal lifestyle.

Retrotransposons spread potential cis-regulatory elements during mammary gland evolution.

Acquisition of cis-elements is a major driving force for rewiring a gene regulatory network. Several kinds of transposable elements (TEs), mostly retrotransposons that propagate via a copy-and-paste mechanism, are known to possess transcription factor binding motifs and have provided source sequences for enhancers/promoters. However, it remains largely unknown whether retrotransposons have spread the binding sites of master regulators of morphogenesis and accelerated cis-regulatory expansion involved in common mammalian morphological features during evolution. Here, I demonstrate that thousands of binding sites for estrogen receptor α (ERα) and three related pioneer factors (FoxA1, GATA3 and AP2γ) that are essential regulators of mammary gland development arose from a spreading of the binding motifs by retrotransposons. The TE-derived functional elements serve primarily as distal enhancers and are enriched around genes associated with mammary gland morphogenesis. The source TEs occurred via a two-phased expansion consisting of mainly L2/MIR in a eutherian ancestor and endogenous retrovirus 1 (ERV1) in simian primates and murines. Thus the build-up of potential sources for cis-elements by retrotransposons followed by their frequent utilization by the host (co-option/exaptation) may have a general accelerating effect on both establishing and diversifying a gene regulatory network, leading to morphological innovation.

The genomic substrate for adaptive radiation in African cichlid fish.

Cichlid fishes are famous for large, diverse and replicated adaptive radiations in the Great Lakes of East Africa. To understand the molecular mechanisms underlying cichlid phenotypic diversity, we sequenced the genomes and transcriptomes of five lineages of African cichlids: the Nile tilapia (Oreochromis niloticus), an ancestral lineage with low diversity; and four members of the East African lineage: Neolamprologus brichardi/pulcher (older radiation, Lake Tanganyika), Metriaclima zebra (recent radiation, Lake Malawi), Pundamilia nyererei (very recent radiation, Lake Victoria), and Astatotilapia burtoni (riverine species around Lake Tanganyika). We found an excess of gene duplications in the East African lineage compared to tilapia and other teleosts, an abundance of non-coding element divergence, accelerated coding sequence evolution, expression divergence associated with transposable element insertions, and regulation by novel microRNAs. In addition, we analysed sequence data from sixty individuals representing six closely related species from Lake Victoria, and show genome-wide diversifying selection on coding and regulatory variants, some of which were recruited from ancient polymorphisms. We conclude that a number of molecular mechanisms shaped East African cichlid genomes, and that amassing of standing variation during periods of relaxed purifying selection may have been important in facilitating subsequent evolutionary diversification.

Coordinately Co-opted Multiple Transposable Elements Constitute an Enhancer for wnt5a Expression in the Mammalian Secondary Palate.

Acquisition of cis-regulatory elements is a major driving force of evolution, and there are several examples of developmental enhancers derived from transposable elements (TEs). However, it remains unclear whether one enhancer element could have been produced via cooperation among multiple, yet distinct, TEs during evolution. Here we show that an evolutionarily conserved genomic region named AS3_9 comprises three TEs (AmnSINE1, X6b_DNA and MER117), inserted side-by-side, and functions as a distal enhancer for wnt5a expression during morphogenesis of the mammalian secondary palate. Functional analysis of each TE revealed step-by-step retroposition/transposition and co-option together with acquisition of a binding site for Msx1 for its full enhancer function during mammalian evolution. The present study provides a new perspective suggesting that a huge variety of TEs, in combination, could have accelerated the diversity of cis-regulatory elements involved in morphological evolution.

Phylogenetic analysis of proteins involved in the stringent response in plant cells.

The nucleotide (p)ppGpp is a second messenger that controls the stringent response in bacteria. The stringent response modifies expression of a large number of genes and metabolic processes and allows bacteria to survive under fluctuating environmental conditions. Recent genome sequencing analyses have revealed that genes responsible for the stringent response are also found in plants. These include (p)ppGpp synthases and hydrolases, RelA/SpoT homologs (RSHs), and the pppGpp-specific phosphatase GppA/Ppx. However, phylogenetic relationship between enzymes involved in bacterial and plant stringent responses is as yet generally unclear. Here, we investigated the origin and evolution of genes involved in the stringent response in plants. Phylogenetic analysis and primary structures of RSH homologs from different plant phyla (including Embryophyta, Charophyta, Chlorophyta, Rhodophyta and Glaucophyta) indicate that RSH gene families were introduced into plant cells by at least two independent lateral gene transfers from the bacterial Deinococcus-Thermus phylum and an unidentified bacterial phylum; alternatively, they were introduced into a proto-plant cell by a lateral gene transfer from the endosymbiotic cyanobacterium followed by gene loss of an ancestral RSH gene in the cyanobacterial linage. Phylogenetic analysis of gppA/ppx families indicated that plant gppA/ppx homologs form an individual cluster in the phylogenetic tree, and show a sister relationship with some bacterial gppA/ppx homologs. Although RSHs contain a plastidial transit peptide at the N terminus, GppA/Ppx homologs do not, suggesting that plant GppA/Ppx homologs function in the cytosol. These results reveal that a proto-plant cell obtained genes for the stringent response by lateral gene transfer events from different bacterial phyla and have utilized them to control metabolism in plastids and the cytosol.

Coelacanth genomes reveal signatures for evolutionary transition from water to land.

Coelacanths are known as "living fossils," as they show remarkable morphological resemblance to the fossil record and belong to the most primitive lineage of living Sarcopterygii (lobe-finned fishes and tetrapods). Coelacanths may be key to elucidating the tempo and mode of evolution from fish to tetrapods. Here, we report the genome sequences of five coelacanths, including four Latimeria chalumnae individuals (three specimens from Tanzania and one from Comoros) and one L. menadoensis individual from Indonesia. These sequences cover two African breeding populations and two known extant coelacanth species. The genome is ∼2.74 Gbp and contains a high proportion (∼60%) of repetitive elements. The genetic diversity among the individuals was extremely low, suggesting a small population size and/or a slow rate of evolution. We found a substantial number of genes that encode olfactory and pheromone receptors with features characteristic of tetrapod receptors for the detection of airborne ligands. We also found that limb enhancers of bmp7 and gli3, both of which are essential for limb formation, are conserved between coelacanth and tetrapods, but not ray-finned fishes. We expect that some tetrapod-like genes may have existed early in the evolution of primitive Sarcopterygii and were later co-opted to adapt to terrestrial environments. These coelacanth genomes will provide a cornerstone for studies to elucidate how ancestral aquatic vertebrates evolved into terrestrial animals.

B chromosomes have a functional effect on female sex determination in Lake Victoria cichlid fishes.

The endemic cichlid fishes in Lake Victoria are a model system for speciation through adaptive radiation. Although the evolution of the sex-determination system may also play a role in speciation, little is known about the sex-determination system of Lake Victoria cichlids. To understand the evolution of the sex-determination system in these fish, we performed cytogenetic analysis in 11 cichlid species from Lake Victoria. B chromosomes, which are present in addition to standard chromosomes, were found at a high prevalence rate (85%) in these cichlids. In one species, B chromosomes were female-specific. Cross-breeding using females with and without the B chromosomes demonstrated that the presence of the B chromosomes leads to a female-biased sex ratio in this species. Although B chromosomes were believed to be selfish genetic elements with little effect on phenotype and to lack protein-coding genes, the present study provides evidence that B chromosomes have a functional effect on female sex determination. FISH analysis using a BAC clone containing B chromosome DNA suggested that the B chromosomes are derived from sex chromosomes. Determination of the nucleotide sequences of this clone (104.5 kb) revealed the presence of several protein-coding genes in the B chromosome, suggesting that B chromosomes have the potential to contain functional genes. Because some sex chromosomes in amphibians and arthropods are thought to be derived from B chromosomes, the B chromosomes in Lake Victoria cichlids may represent an evolutionary transition toward the generation of sex chromosomes.

Functional Divergence in Solute Permeability between Ray-Finned Fish-Specific Paralogs of aqp10.

Aquaporin (Aqp) 10 is a member of the aquaglyceroporin subfamily of water channels, and human Aqp10 is permeable to solutes such as glycerol, urea, and boric acid. Tetrapods have a single aqp10 gene, whereas ray-finned fishes have paralogs of this gene through tandem duplication, whole-genome duplication, and subsequent deletion. A previous study on Aqps in the Japanese pufferfish Takifugu rubripes showed that one pufferfish paralog, Aqp10.2b, was permeable to water and glycerol, but not to urea and boric acid. To understand the functional differences of Aqp10s between humans and pufferfish from an evolutionary perspective, we analyzed Aqp10s from an amphibian (Xenopus laevis) and a lobe-finned fish (Protopterus annectens) and Aqp10.1 and Aqp10.2 from several ray-finned fishes (Polypterus senegalus, Lepisosteus oculatus, Danio rerio, and Clupea pallasii). The expression of tetrapod and lobe-finned fish Aqp10s and Aqp10.1-derived Aqps in ray-finned fishes in Xenopus oocytes increased the membrane permeabilities to water, glycerol, urea, and boric acid. In contrast, Aqp10.2-derived Aqps in ray-finned fishes increased water and glycerol permeabilities, whereas those of urea and boric acid were much weaker than those of Aqp10.1-derived Aqps. These results indicate that water, glycerol, urea, and boric acid permeabilities are plesiomorphic activities of Aqp10s and that the ray-finned fish-specific Aqp10.2 paralogs have secondarily reduced or lost urea and boric acid permeability.

A vertebrate-wide catalogue of T1R receptors reveals diversity in taste perception.

Taste is a vital chemical sense for feeding behaviour. In mammals, the umami and sweet taste receptors comprise three members of the taste receptor type 1 (T1R/TAS1R) family: T1R1, T1R2 and T1R3. Because their functional homologues exist in teleosts, only three TAS1R genes generated by gene duplication are believed to have been inherited from the common ancestor of bony vertebrates. Here, we report five previously uncharacterized TAS1R members in vertebrates, TAS1R4, TAS1R5, TAS1R6, TAS1R7 and TAS1R8, based on genome-wide survey of diverse taxa. We show that mammalian and teleost fish TAS1R2 and TAS1R3 genes are paralogues. Our phylogenetic analysis suggests that the bony vertebrate ancestor had nine TAS1Rs resulting from multiple gene duplications. Some TAS1Rs were lost independently in descendent lineages resulting in retention of only three TAS1Rs in mammals and teleosts. Combining functional assays and expression analysis of non-teleost fishes we show that the novel T1Rs form heterodimers in taste-receptor cells and recognize a broad range of ligands such as essential amino acids, including branched-chain amino acids, which have not been previously considered as T1R ligands. This study reveals diversity of taste sensations in both modern vertebrates and their ancestors, which might have enabled vertebrates to adapt to diverse habitats on Earth.

ISWI chromatin remodeling complexes recruit NSD2 and H3K36me2 in pericentromeric heterochromatin.

Histone H3 lysine36 dimethylation (H3K36me2) is generally distributed in the gene body and euchromatic intergenic regions. However, we found that H3K36me2 is enriched in pericentromeric heterochromatin in some mouse cell lines. We here revealed the mechanism of heterochromatin targeting of H3K36me2. Among several H3K36 methyltransferases, NSD2 was responsible for inducing heterochromatic H3K36me2. Depletion and overexpression analyses of NSD2-associating proteins revealed that NSD2 recruitment to heterochromatin was mediated through the imitation switch (ISWI) chromatin remodeling complexes, such as BAZ1B-SMARCA5 (WICH), which directly binds to AT-rich DNA via a BAZ1B domain-containing AT-hook-like motifs. The abundance and stoichiometry of NSD2, SMARCA5, and BAZ1B could determine the localization of H3K36me2 in different cell types. In mouse embryos, H3K36me2 heterochromatin localization was observed at the two- to four-cell stages, suggesting its physiological relevance.

The complete mitochondrial genomes of deep-sea squid (Bathyteuthis abyssicola), bob-tail squid (Semirossia patagonica) and four giant cuttlefish (Sepia apama, S. latimanus, S. lycidas and S. pharaonis), and their application to the phylogenetic analysis of Decapodiformes.

We determined the complete mitochondrial (mt) genomes of the deep-sea squid (Bathyteuthis abyssicola; supperfamily Bathyteuthoidea), the bob-tail squid (Semirossia patagonica; order Sepiolida) and four giant cuttlefish (Sepia apama, S. latimanus, S. lycidas and S. pharaonis; order Sepiida). The unique structures of the mt genomes of Bathyteuthis and Semirossia provide new information about the evolution of decapodiform mt genomes. We show that the mt genome of B. abyssicola, like those of other oegopsids studied so far, has two long duplicated regions that include seven genes (COX1-3, ATP6 and ATP8, tRNA(Asn), and either ND2 or ND3) and that one of the duplicated COX3 genes has lost its function. The mt genome of S. patagonica is unlike any other decapodiforms and, like Nautilus, its ATP6 and ATP8 genes are not adjacent to each other. The four giant cuttlefish have identical mt gene order to other cuttlefish determined to date. Molecular phylogenetic analyses using maximum likelihood and Bayesian methods suggest that traditional order Sepioidea (Sepiolida+Sepiida) is paraphyletic and Sepia (cuttlefish) has the sister-relationship with all other decapodiforms. Taking both the phylogenetic analyses and the mt gene order analyses into account, it is likely that the octopus-type mt genome is an ancestral state and that it had maintained from at least the Cephalopoda ancestor to the common ancestor of Oegopsida, Myopsida and Sepiolida.

Splashed E-box and AP-1 motifs cooperatively drive regeneration response and shape regeneration abilities.

Injury triggers a genetic program that induces gene expression for regeneration. Recent studies have identified regeneration-response enhancers (RREs); however, it remains unclear whether a common mechanism operates in these RREs. We identified three RREs from the zebrafish fn1b promoter by searching for conserved sequences within the surrounding genomic regions of regeneration-induced genes and performed a transgenic assay for regeneration response. Two regions contained in the transposons displayed RRE activity when combined with the -0.7 kb fn1b promoter. Another non-transposon element functioned as a stand-alone enhancer in combination with a minimum promoter. By searching for transcription factor-binding motifs and validation by transgenic assays, we revealed that the cooperation of E-box and activator protein 1 motifs is necessary and sufficient for regenerative response. Such RREs respond to variety of tissue injuries, including those in the zebrafish heart and Xenopus limb buds. Our findings suggest that the fidelity of regeneration response is ensured by the two signals evoked by tissue injuries. It is speculated that a large pool of potential enhancers in the genome has helped shape the regenerative capacities during evolution.

Kangaroo endogenous retrovirus (KERV) forms megasatellite DNA with a simple repetition pattern in which the provirus structure is retained.

The kangaroo endogenous retrovirus (KERV) was previously reported to have undergone a rapid copy number increase in the red-necked wallaby; however, the mode of amplification was left to be clarified. The present study revealed that the long terminal repeat (LTR) (0.6 kb) and internal region (2.0 kb) of a provirus are repeated alternately, forming megasatellite DNA which we named kervRep. This repetition pattern was the same as that observed for walbRep, megasatellite DNA originating from another endogenous retrovirus. Their formation process can be explained using a simple model: pairing slippage followed by homologous recombination. This model features that the initial step is triggered by the presence of two identical sequences within a short distance; the possession of LTRs by endogenous retroviruses fulfills this condition. The discovery of two cases suggests that formation of this type of satellite DNA is one of non-negligible effects of endogenous retroviruses on their host genomes.

Reverse evolution in RH1 for adaptation of cichlids to water depth in Lake Tanganyika.

Reverse evolution is a widespread phenomenon in biology, but the genetic mechanism for the reversal of a genetic change for adaptation to the ancestral state is not known. Here, we report the first case of complete reverse evolution of two amino acids, serine and alanine, at a single position in RH1 opsin pigment for adaptation to water depth. We determined RH1 sequences of cichlid fishes from four tribes of Lake Tanganyika with different habitat depths. Most of the species were divided into two types: RH1 with 292A for species in shallow water or 292S for species in deep water. Both types were adapted to their ambient light environments as indicated by the absorption spectra of the RH1 pigments. Based on the RH1 locus tree and ecological data, we inferred the ancestral amino acids at position 292 and the distribution of the depth ranges (shallow or deep) of ancestral species of each tribe. According to these estimates, we identified two distinct parallel adaptive evolutions: The replacement A292S occurred at least four times for adaptation from shallow to deep water, and the opposite replacement S292A occurred three times for adaptation from deep to shallow water. The latter parallelism represents the complete reverse evolution from the derived to the ancestral state, following back adaptive mutation with reversal of the RH1 pigment function accompanied by reversal of the species habitat shift.

Retroposon analysis and recent geological data suggest near-simultaneous divergence of the three superorders of mammals.

As a consequence of recent developments in molecular phylogenomics, all extant orders of placental mammals have been grouped into 3 lineages: Afrotheria, Xenarthra, and Boreotheria, which originated in Africa, South America, and Laurasia, respectively. Despite this advancement, the order of divergence of these 3 lineages remains unresolved. Here, we performed extensive retroposon analysis with mammalian genomic data. Surprisingly, we identified a similar number of informative retroposon loci that support each of 3 possible phylogenetic hypotheses: the basal position for Afrotheria (22 loci), Xenarthra (25 loci), and Boreotheria (21 loci). This result indicates that the divergence of the placental common ancestor into the 3 lineages occurred nearly simultaneously. Thus, we examined whether these molecular data could be integrated into the geological context by incorporating recent geological data. We obtained firm evidence that complete separation of Gondwana into Africa and South America occurred 120 +/- 10 Ma. Accordingly, the previous reported time frame (division of Pangea into Gondwana and Laurasia at 148-138 Ma and division of Gondwana at 105 Ma) cannot be used to validate mammalian divergence order. Instead, we use our retroposon results and the recent geological data to propose that near-simultaneous divisions of continents leading to isolated Africa, South America, and Laurasia caused nearly concomitant divergence of the ancient placental ancestor into 3 lineages, Afrotheria, Xenarthra, and Boreotheria, approximately 120 Ma.