Hidenori Nishihara

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.

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, ≈120 Ma.

The evolution of two partner LINE/SINE families and a full-length chromodomain-containing Ty3/Gypsy LTR element in the first reptilian genome of Anolis carolinensis.

Transposable elements have been characterized in a number of vertebrates, including whole genomes of mammals, birds, and fishes. The Anolis carolinensis draft assembly provides the first opportunity to study retroposons in a reptilian genome. Here, we identified and reconstructed a number of retroposons based on database searches: Five Sauria short interspersed element (SINE) subfamilies, 5S-Sauria SINE chimeras, Anolis Bov-B long interspersed element (LINE), Anolis SINE 2, Anolis LINE 2, Anolis LINE 1, Anolis CR 1, and a chromodomain-containing Ty3/Gypsy LTR element. We focused on two SINE families (Anolis Sauria SINE and Anolis SINE 2) and their partner LINE families (Anolis Bov-B LINE and Anolis LINE 2). We demonstrate that each SINE/LINE pair is distributed similarly and predict that the retrotransposition of evolutionarily younger Sauria SINE members is via younger Bov-B LINE members while a correlation also exists between their respective evolutionarily older SINE/LINE members. The evolutionarily youngest Sauria SINE sequences evolved as part of novel rolling-circle transposons. The evolutionary time frame when Bov-B LINEs and Sauria SINEs were less active in their retrotransposition is characterized by a high retrotransposition burst of Anolis SINE 2 and Anolis LINE 2 elements. We also characterized the first full-length chromoviral LTR element in amniotes (Amn-ichi). This newly identified chromovirus is widespread in the Anolis genome and has been very well preserved, indicating that it is still active. Transposable elements in the Anolis genome account for approximately 20% of the total DNA sequence, whereas the proportion is more than double that in many mammalian genomes in which such elements have important biological functions. Nevertheless, 20% transposable element coverage is sufficient to predict that Anolis retroposons and other mobile elements also may have biologically and evolutionarily relevant functions. The new SINEs and LINEs and other ubiquitous genomic elements characterized in the Anolis genome will prove very useful for studies in comparative genomics, phylogenetics, and functional genetics.

Identification of four Engrailed genes in the Japanese lamprey, Lethenteron japonicum

We have isolated four homologs of Engrailed genes from the Japanese lamprey, Lethenteron japonicum, an agnathan that occupies a critical phylogenic position between cephalochordates and gnathostomes. We named these four genes LjEngrailedA, LjEngrailedB, LjEngrailedC, and LjEngrailedD. LjEngrailedA, LjEngrailedB, and LjEngrailedD share a major expression domain in the presumptive midbrain–hindbrain boundary region of the central nervous system, although their levels and timing of expression differed. On the other hand, LjEngrailedC transcripts were in the pharyngeal ectoderm and the ventral ectoderm of the body wall. In addition, LjEngrailedA was expressed in the ventral side of the epibranchial muscle precursors. LjEngrailedD transcripts were seen in the mesodermal cells of the mandibular arch and later in a group of cells responsible for the formation of the upper lip, lower lip, and velum. Our results provide clues to the evolution of these structures as well as a possible scenario for duplication events of Engrailed genes. Developmental Dynamics 237:1581–1589, 2008.

Retroposons: Genetic Footprints on the Evolutionary Paths of Life

Retroposons such as short interspersed elements (SINEs) and long interspersed elements are abundant transposable elements in eukaryote genomes. Recent large-scale comparative genome analyses have revealed that retroposons are a major component of genomes, wherein they provide structural diversity between species and uniqueness to each species. SINEs have been used as powerful markers in phylogenetic analyses of various species. This approach, which has been termed the SINE insertion method, infers phylogenetic relationships based on the presence/absence of SINEs among lineages. However, the method is not yet used extensively among biologists, especially molecular phylogenetists, because it is based on an understanding of the molecular mechanisms of retroposition, which may be unfamiliar to many researchers. Moreover, the method may require a large amount of bench work to characterize a new SINE family and to screen genomic libraries of the species of interest. In this chapter, we present the basic theory and detailed technical steps involved in a SINE insertion analysis. Furthermore, we explain the isolation and characterization of a new SINE family from the genome of a species of interest using as an example a known SINE family in mammals.

Characterization and evolutionary landscape of AmnSINE1 in Amniota genomes

Discovery of a large number of conserved non-coding elements (CNEs) in vertebrate genomes provides a cornerstone to elucidate molecular mechanisms of macroevolution. Extensive comparative genomics has proven that transposons such as short interspersed elements (SINEs) were an important source of CNEs. We recently characterized AmnSINE1, a SINE family in Amniota genomes, some of which are present in CNEs, and demonstrated that two AmnSINE1 loci play an important role in mammalian-specific brain development by functioning as an enhancer (Sasaki et al. Proc. Natl. Acad. Sci. USA 2008). To get more information about AmnSINE1s, we here performed a multi-species search for AmnSINE1, and revealed the distribution and evolutionary history of these SINEs in amniote genomes. The number of AmnSINE1 regions in amniotes ranged from 160 to 1200; the number in the eutherians were under 500 and the largest was that in chicken. Phylogenetic analysis established that each AmnSINE1 locus has evolved uniquely, primarily since the divergence of mammals from reptiles. These results support the notion that AmnSINE1s were amplified as an ancient retroposon in a common ancestor of Amniota and subsequently have survived for 300 Myr because of functions acquired by mutation-coupled exaptation prior mammalian radiation. On the basis of sequence homology and conserved synteny, we detected the orthologs of AmnSINE1 for candidates of further enhancer analysis, which are more conserved than two loci that were shown to have been involved in mammalian brain development. The present work provides a comprehensive data set to test the role of AmnSINE1s, many of which were exapted and contributed to mammalian macroevolution.

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.

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.

MetaSINEs: Broad Distribution of a Novel SINE Superfamily in Animals

SINEs (short interspersed elements) are transposable elements that typically originate independently in each taxonomic clade (order/family). However, some SINE families share a highly similar central sequence and are thus categorized as a SINE superfamily. Although only four SINE superfamilies (CORE-SINEs, V-SINEs, DeuSINEs, and Ceph-SINEs) have been reported so far, it is expected that new SINE superfamilies would be discovered by deep exploration of new SINEs in metazoan genomes. Here we describe 15 SINEs, among which 13 are novel, that have a similar 66-bp central region and therefore constitute a new SINE superfamily, MetaSINEs. MetaSINEs are distributed from fish to cnidarians, suggesting their common evolutionary origin at least 640 Ma. Because the 3′ tails of MetaSINEs are variable, these SINEs most likely survived by changing their partner long interspersed elements for retrotransposition during evolution. Furthermore, we examined the presence of members of other SINE superfamilies in bivalve genomes and characterized eight new SINEs belonging to the CORE-SINEs, V-SINEs, and DeuSINEs, in addition to the MetaSINEs. The broad distribution of bivalve SINEs suggests that at least three SINEs originated in the common ancestor of Bivalvia. Our comparative analysis of the central domains of the SINEs revealed that, in each superfamily, only a restricted region is shared among all of its members. Because the functions of the central domains of the SINE superfamilies remain unknown, such structural information of SINE superfamilies will be useful for future experimental and comparative analyses to reveal why they have been retained in metazoan genomes during evolution.

Resolving the Phylogenetic Position of Coelacanth: The Closest Relative Is Not Always the Most Appropriate Outgroup

Determining the phylogenetic relationship of two extant lineages of lobe-finned fish, coelacanths and lungfishes, and tetrapods is important for understanding the origin of tetrapods. We analyzed data sets from two previous studies along with a newly collected data set, each of which had varying numbers of species and genes and varying extent of missing sites. We found that in all the data sets the sister relationship of lungfish and tetrapods was constructed with the use of cartilaginous fish as the outgroup with a high degree of statistical support. In contrast, when ray-finned fish were used as the outgroup, which is taxonomically an immediate outgroup of lobe-finned fish and tetrapods, the sister relationship of coelacanth and tetrapods was supported most strongly, although the statistical support was weaker. Even though it is generally accepted that the closest relative is an appropriate outgroup, our analysis suggested that the large divergence of the ray-finned fish as indicated by their long branch lengths and different amino acid frequencies made them less suitable as an outgroup than cartilaginous fish.