Masato Nikaido

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 of Major Cetacean Lineages: The Monophyly of Toothed Whales and the Paraphyly Of River Dolphins

SINE (short interspersed element) insertion analysis elucidates contentious aspects in the phylogeny of toothed whales and dolphins (Odontoceti), especially river dolphins. Here, we characterize 25 informative SINEs inserted into unique genomic loci during evolution of odontocetes to construct a cladogram, and determine a total of 2.8 kb per taxon of the flanking sequences of these SINE loci to estimate divergence times among lineages. We demonstrate that: (i) Odontocetes are monophyletic; (ii) Ganges River dolphins, beaked whales, and ocean dolphins diverged (in this order) after sperm whales; (iii) three other river dolphin taxa, namely the Amazon, La Plata, and Yangtze river dolphins, form a monophyletic group with Yangtze River dolphins being the most basal; and (iv) the rapid radiation of extant cetacean lineages occurred some 28–33 million years B.P., in strong accord with the fossil record. The combination of SINE and flanking sequence analysis suggests a topology and set of divergence times for odontocete relationships, offering alternative explanations for several long-standing problems in cetacean evolution.

Mitochondrial Phylogenetics and Evolution of Mysticete Whales

The phylogenetic relationships among baleen whales (Order: Cetacea) remain uncertain despite extensive research in cetacean molecular phylogenetics and a potential morphological sample size of over 2 million animals harvested. Questions remain regarding the number of species and the monophyly of genera, as well as higher order relationships. Here, we approach mysticete phylogeny with complete mitochondrial genome sequence analysis. We determined complete mtDNA sequences of 10 extant Mysticeti species, inferred their phylogenetic relationships, and estimated node divergence times. The mtDNA sequence analysis concurs with previous molecular studies in the ordering of the principal branches, with Balaenidae (right whales) as sister to all other mysticetes base, followed by Neobalaenidae (pygmy right whale), Eschrichtiidae (gray whale), and finally Balaenopteridae (rorquals + humpback whale). The mtDNA analysis further suggests that four lineages exist within the clade of Eschrichtiidae + Balaenopteridae, including a sister relationship between the humpback and fin whales, and a monophyletic group formed by the blue, sei, and Brydes whales, each of which represents a newly recognized phylogenetic relationship in Mysticeti. We also estimated the divergence times of all extant mysticete species, accounting for evolutionary rate heterogeneity among lineages. When the mtDNA divergence estimates are compared with the mysticete fossil record, several lineages have molecular divergence estimates strikingly older than indicated by paleontological data. We suggest this discrepancy reflects both a large amount of ancestral polymorphism and long generation times of ancestral baleen whale populations.

Maximum likelihood analysis of the complete mitochondrial genomes of eutherians and a reevaluation of the phylogeny of bats and insectivores.

Abstract. The complete mitochondrial genomes of two microbats, the horseshoe bat Rhinolophus pumilus, and the Japanese pipistrelle Pipistrellus abramus, and that of an insectivore, the long-clawed shrew Sorex unguiculatus, were sequenced and analyzed phylogenetically by a maximum likelihood method in an effort to enhance our understanding of mammalian evolution. Our analysis suggested that (1) a sister relationship exists between moles and shrews, which form an eulipotyphlan clade; (2) chiropterans have a sister-relationship with eulipotyphlans; and (3) the Eulipotyphla/Chiroptera clade is closely related to fereuungulates (Cetartiodactyla, Perissodactyla and Carnivora). Divergence times on the mammalian tree were estimated from consideration of a relaxed molecular clock, the amino acid sequences of 12 concatenated mitochondrial proteins and multiple reference criteria. Moles and shrews were estimated to have diverged approximately 48 MyrBP, and bats and eulipotyphlans to have diverged 68 MyrBP. Recent phylogenetic controversy over the polyphyly of microbats, the monophyly of rodents, and the position of hedgehogs is also examined.

Interordinal relationships and timescale of eutherian evolution as inferred from mitochondrial genome data

Extensive phylogenetic analyses of the updated sequence data of mammalian mitochondrial genomes were carried out using the maximum likelihood method in order to resolve deep branchings in eutherian evolution. The divergence times in the mammalian tree were estimated by a relaxed molecular clock of the mitochondrial proteins calibrated with multiple references. A Chiroptera/Eulipotyphla (i.e. bat/mole) clade and a close relationship of this clade to Fereuungulata (Carnivora+Perissodactyla+Cetartiodactyla) were reconfirmed with high statistical significance. However, a support for a monophyly of Fereuungulata relative to the Chiroptera/Eulipotyphla clade was fragile, and we suggest that the three branchings among Carnivora, Perissodactyla, Cetartiodactyla and Chiroptera/Eulipotyphla occurred successively in a short time period, estimated to be approximately 77Myr BP. The Chiroptera/Eulipotyphla divergence was estimated to roughly coincide with the Cretaceous-Tertiary boundary (65Myr BP). The monophyly of Rodentia, the Lagomorpha/Rodentia clade (traditionally called Glires), and the Afrotheria/Xenarthra clade were preferred over alternative relationships, but the supports of these clades were not strong enough to exclude other possibilities. Although several super-order taxa of eutherians were strongly supported by the analyses of the mitochondrial genome data, the branching order in the deepest part of the eutherian tree remained ambiguous from the data presently available.

Intra- and interfamily relationships of Vespertilionidae inferred by various molecular markers including SINE insertion data

The family Vespertilionidae comprises the largest number of species in the order Chiroptera, and its members are distributed over most of the world. We collected sequences of the mitochondrial NDI (NADH dehydrogenase subunit 1) gene from 38 species as well as 20 sequences of exon28 of the vWF (von Willebrand Factor) gene and analyzed phylogenetic relationships in Vespertilionidae using maximum-likelihood analyses and SINE (short interspersed elements) insertions. Our data strongly suggest the following: (1) Murininae and Myotis, each of which is classified into different subfamilies, are monophyletic; (2) the monophyly of Murininae, Myotis, Pipistrellini, and Plecotini is supported by our analyses of the NDI and nuclear vWF as well as data from SINE insertions, while Miniopterinae was not included in this group; and (3) Rhinolophoidea (microchiroptera) is separated from all the other microchiropteran species. Our study represents the most comprehensive phylogenetic model of Vespertilionidae to date.

Monophyletic Origin of the Order Chiroptera and Its Phylogenetic Position Among Mammalia, as Inferred from the Complete Sequence of the Mitochondrial DNA of a Japanese Megabat, the Ryukyu Flying Fox (Pteropus dasymallus)

Abstract. Complete sequences of mitochondrial DNA (mtDNA) are useful for the reconstruction of phylogenetic trees of mammals and, in particular, for inferring higher-order relationships in mammals. In this study, we determined the complete sequence (16,705 bp) of the mtDNA of a Japanese megabat, the Ryukyu flying fox (Pteropus dasymallus). We analyzed this sequence phylogenetically by comparing it with the complete sequence of mtDNAs of 35 mammals in an effort to reevaluate the enigmatic relationship between Megachiroptera and Microchiroptera and the relationships between them and other mammals. Maximum-likelihood analysis of 12 concatenated mitochondrial proteins from 36 mammals strongly suggested the monophyly of the order Chiroptera and its close relationship to Fereuungulata (Carnivora + Perissodactyla + Cetartiodactyla). We estimated that megabats and microbats diverged approximately 58 MyrBP and discussed the origin and early evolution of Chiroptera based on our findings.

Retroposon Mapping in Molecular Systematics

Advances in genome sciences are demonstrating the dynamic nature of noncoding DNA regions, which are comprised largely of repetitive elements with no apparent function. Retroposons are one class of mobile genetic elements that amplify and move about the genome via a copy-and-paste mechanism that employs an RNA intermediate. Short and long interspersed elements (SINEs and LINEs, respectively) are types of retroposons of particular interest because of their active role in shaping the architecture of genomes and their diagnostic value as evolutionary markers for studies of phylogeny and population biology. Although the use of SINEs and LINEs for molecular systematic studies is proliferating, a comprehensive laboratory protocol that explicitly outlines how to isolate and characterize retroposons for systematic studies in a detailed, step-by-step fashion has been lacking. The present chapter addresses this gap in the literature by focusing on the strategy for isolating new SINEs from a genomic library, the screening process, the sequencing and characterization of clones into subfamilies, quantification of copy number in host taxa, and the critical diagnosis of phylogenetically informative SINE and LINE insertion patterns. Practical limits to the method are discussed in relation to sampling design, systematic character theory, and the empirical distribution of elements observed in eukaryotic lineages. Major steps in the experimental process are illustrated with case examples from a diversity of taxonomic groups and by published results in the molecular biology and systematics literature.

The status of the Japanese and East Asian bats of the genus Myotis (Vespertilionidae) based on mitochondrial sequences

The genus Myotis includes the largest number of species in the family Vespertilionidae (Chiroptera), and its members are distributed throughout most of the world. To re-evaluate the phylogenetic position of East Asian Myotis species with respect to Myotis species worldwide, we analyzed mitochondrial gene sequences of NADH dehydrogenase subunit 1 and cytochrome b from 24 East Asian individuals as well as 42 vespertilionid bats determined previously. The results suggest that: (1) some individuals having the same species name in Europe and Japan do not form a monophyletic clade, indicating that some bat species exhibit morphological convergence, (2) Japanese Myotis mystacinus forms a sister relationship with Myotis brandtii (Palaearctic), and both species are included in the American clade implying that an ancestor of these species originated in North America, and (3) the Black whiskered bat, Myotis pruinosus, is endemic to Japan and forms sister relationships with Myotis yanbarensis and Myotis montivagus collected from Okinawa (Japan) and Selangor (Malaysia), respectively, implying that M. pruinosus originated from the south. The systematics of Japanese and East Asian Myotis bats were revisited by considering their phylogenetic relationships. Our study provides the first extensive phylogenetic hypothesis of the genus Myotis that includes East Asian and Japanese species.

Mitochondrial phylogeny of hedgehogs and monophyly of Eulipotyphla.

We sequenced the complete mitochondrial (mt) genomes of three insectivores: the long-eared hedgehog Hemiechinus auritus, the Japanese mole Mogera wogura, and the greater Japanese shrew-mole Urotrichus talpoides. These mtDNA data together with other previously sequenced mtDNAs were analyzed using a maximum likelihood method to infer their phylogenetic relationships among eutherians. Previous mitochondrial protein analyses used a simple model that did not consider site-heterogeneity, and Erinaceoidea (hedgehogs and moonrats) was placed at the basal eutherian position that is separated from Soricoidea (shrews) and Talpoidea (moles), suggesting the exclusion of the Erinaceoidea-Eulipotyphla tree. By including the new mtDNA sequences and introducing site-heterogeneity into the model, the Erinaceoidea-Eulipotyphla tree emerges as the best tree or as a tree with a log-likelihood score indistinguishable from that of the best tree. However, this conclusion depends on species sampling in Erinaceoidea, demonstrating the importance of both species sampling and use of an appropriate substitution model when inferring phylogenetic relationships.