Effect of Different Types of Sequence Data on Palaeognath Phylogeny.

Palaeognathae consists of five groups of extant species: flighted tinamous (1) and four flightless groups: kiwi (2), cassowaries and emu (3), rheas (4), and ostriches (5). Molecular studies supported the groupings of extinct moas with tinamous and elephant birds with kiwi as well as ostriches as the group that diverged first among the five groups. However, phylogenetic relationships among the five groups are still controversial. Previous studies showed extensive heterogeneity in estimated gene tree topologies from conserved nonexonic elements, introns, and ultraconserved elements. Using the noncoding loci together with protein-coding loci, this study investigated the factors that affected gene tree estimation error and the relationships among the five groups. Using closely related ostrich rather than distantly related chicken as the outgroup, concatenated and gene tree-based approaches supported rheas as the group that diverged first among groups (1)-(4). Whereas gene tree estimation error increased using loci with low sequence divergence and short length, topological bias in estimated trees occurred using loci with high sequence divergence and/or nucleotide composition bias and heterogeneity, which more occurred in trees estimated from coding loci than noncoding loci. Regarding the relationships of (1)-(4), the site patterns by parsimony criterion appeared less susceptible to the bias than tree construction assuming stationary time-homogeneous model and suggested the clustering of kiwi and cassowaries and emu the most likely with ∼40% support rather than the clustering of kiwi and rheas and that of kiwi and tinamous with 30% support each.

Resolving the Early Divergence Pattern of Teleost Fish Using Genome-Scale Data.

Regarding the phylogenetic relationship of the three primary groups of teleost fishes, Osteoglossomorpha (bonytongues and others), Elopomorpha (eels and relatives), Clupeocephala (the remaining teleost fish), early morphological studies hypothesized the first divergence of Osteoglossomorpha, whereas the recent prevailing view is the first divergence of Elopomorpha. Molecular studies supported all the possible relationships of the three primary groups. This study analyzed genome-scale data from four previous studies: 1) 412 genes from 12 species, 2) 772 genes from 15 species, 3) 1,062 genes from 30 species, and 4) 491 UCE loci from 27 species. The effects of the species, loci, and models used on the constructed tree topologies were investigated. In the analyses of the data sets (1)-(3), although the first divergence of Clupeocephala that left the other two groups in a sister relationship was supported by concatenated sequences and gene trees of all the species and genes, the first divergence of Elopomorpha among the three groups was supported using species and/or genes with low divergence of sequence and amino-acid frequencies. This result corresponded to that of the UCE data set (4), whose sequence divergence was low, which supported the first divergence of Elopomorpha with high statistical significance. The increase in accuracy of the phylogenetic construction by using species and genes with low sequence divergence was predicted by a phylogenetic informativeness approach and confirmed by computer simulation. These results supported that Elopomorpha was the first basal group of teleost fish to have diverged, consistent with the prevailing view of recent morphological studies.

Global Rate Variation in Bony Vertebrates.

This study investigated long-term substitution rate differences using three calibration points, divergences between lobe-finned vertebrates and ray-finned fish, between mammals and sauropsids, and between holosteans (gar and bowfin) and teleost fish with amino acid sequence data of 625 genes for 25 bony vertebrates. The result showed that the substitution rate was two to three times higher in the stem branches of lobe-finned vertebrates before the mammal-sauropsid divergence than in amniotes. The rate in the stem branch of ray-finned fish before the holostean-teleost fish divergence was also a few times higher than the holostean rate, whereas it was similar to or somewhat slower than the teleost fish rate. The phylogenetic relationship of coelacanth and lungfish with tetrapod was difficult to determine because of the short interval of the divergences. Considering the high rate in the stem branches, the divergences of coelacanth and lungfish from the stem branch were estimated as 408-427 Ma and 399-414 Ma, respectively, with the interval of 9-13 Myr. With the external calibration of the mammal-sauropsid split, the estimated times for ordinal divergences within eutherian mammals tend to be smaller than those in previous studies that used the calibration points within the lineage, with deeper divergences before the Cretaceous-Paleogene boundary and shallower ones after the boundary. In contrast the estimated times within birds were larger than those of previous studies, with the divergence between Galliformes and Anseriformes ∼80 Ma and that between Galloanserae and Neoaves 110 Ma.

Support for Lungfish as the Closest Relative of Tetrapods by Using Slowly Evolving Ray-Finned Fish as the Outgroup.

In a previous analysis of the phylogenetic relationships of coelacanths, lungfishes and tetrapods, using cartilaginous fish (CF) as the outgroup, the sister relationship of lungfishes and tetrapods was constructed with high statistical support. However, using as the outgroup ray-finned fish (RF), which are more taxonomically closely related to the three lineages than CF, the sister relationship of coelacanths and tetrapods was most often constructed depending on the methods and the data sets, but the statistical support was generally low except in the cases in which the data set including a small number of species was analyzed. In this study, instead of the fast evolving ray-finned fish, teleost fish (TF), in the previous data sets, by using two slowly evolving RF, gar and bowfin, as the outgroup, we showed that the sister relationship of lungfishes and tetrapods was reconstructed with high statistical support. In our analysis the evolutionary rates of gar and bowfin were similar to each other and one third to one half of TF. The difference of the amino acid frequencies of the two species with other lineages was larger than those of TF. This study provides a strong support for lungfishes as the closest relative of tetrapods and indicates the importance of using an appropriate outgroup with small divergence in phylogenetic construction.

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.

POPTREEW: web version of POPTREE for constructing population trees from allele frequency data and computing some other quantities.

POPTREE software, including the command line (POPTREE) and the Windows (POPTREE2) versions, is available to perform evolutionary analyses of allele frequency data, computing distance measures for constructing population trees and average heterozygosity (H) (measure of genetic diversity within populations) and G(ST) (measure of genetic differentiation among subdivided populations). We have now developed a web version POPTREEW (http://www.med.kagawa-u.ac.jp/∼genomelb/takezaki/poptreew/) to provide cross-platform access to all POPTREE functions including interactive tree editing. Furthermore, new POPTREE software (POPTREE, POPTREE2, and POPTREEW) computes standardized G(ST) and Jost's D, which may be appropriate for data with high variability, and accepts genotype data in GENEPOP format as an input.

POPTREE2: Software for constructing population trees from allele frequency data and computing other population statistics with Windows interface.

Currently, there is a demand for software to analyze polymorphism data such as microsatellite DNA and single nucleotide polymorphism with easily accessible interface in many fields of research. In this article, we would like to make an announcement of POPTREE2, a computer program package, that can perform evolutionary analyses of allele frequency data. The original version (POPTREE) was a command-line program that runs on the Command Prompt of Windows and Unix. In POPTREE2 genetic distances (measures of the extent of genetic differentiation between populations) for constructing phylogenetic trees, average heterozygosities (H) (a measure of genetic variation within populations) and G(ST) (a measure of genetic differentiation of subdivided populations) are computed through a simple and intuitive Windows interface. It will facilitate statistical analyses of polymorphism data for researchers in many different fields. POPTREE2 is available at http://www.med.kagawa-u.ac.jp/ approximately genomelb/takezaki/poptree2/index.html.

Genomic drift and evolution of microsatellite DNAs in human populations.

In recent years, copy number variation (CNV) of DNA segments has become a hot topic in the study of genetic variation, and a large amount of CNVs has been uncovered in human populations. The CNVs involving the smallest units of DNA segments are microsatellite DNAs, and the evolutionary change of microsatellite DNAs is believed to occur mostly by the increase or decrease of one repeat unit at a time in a more or less neutral fashion. If we note that eukaryotic genomes contain millions of microsatellite loci, this pattern of nucleotide change is expected to generate random changes of genome size, that is, genomic drift, and will provide a neutral model of CNV evolution. We therefore investigated the amount of variation of the total number of repeats (TNR) per individual concerned with 145 microsatellite loci in three human populations, Africans, Europeans, and Asians. It was shown that the TNR follows the normal distribution in all three populations and that the extent of variation of TNR is more than 50% greater in Africans than in Europeans and Asians as expected from the hypothesis of African origin of modern humans. If we consider all microsatellite loci in the human genome and compute the variation of the total number of nucleotides involved (TNN), it is possible to study the contribution of microsatellite loci to the genome size variation. This study has shown that the genome sizes of human individuals are affected considerably by genomic drift of microsatellite DNA alone. This pattern of evolution is similar to that of olfactory receptor (OR) genes previously studied in human populations and support the idea that the number of OR genes has evolved in a more or less neutral fashion. However, this conclusion does not necessarily apply to the genomewide CNVs of various DNA segments, and it appears that long variant DNA fragments are deleterious and under purifying selection.

Empirical tests of the reliability of phylogenetic trees constructed with microsatellite DNA.

Microsatellite DNA loci or short tandem repeats (STRs) are abundant in eukaryotic genomes and are often used for constructing phylogenetic trees of closely related populations or species. These phylogenetic trees are usually constructed by using some genetic distance measure based on allele frequency data, and there are many distance measures that have been proposed for this purpose. In the past the efficiencies of these distance measures in constructing phylogenetic trees have been studied mathematically or by computer simulations. Recently, however, allele frequencies of 783 STR loci have been compiled from various human populations. We have therefore used these empirical data to investigate the relative efficiencies of different distance measures in constructing phylogenetic trees. The results showed that (1) the probability of obtaining the correct branching pattern of a tree (PC) is generally highest for DA distance; (2) FST*, standard genetic distance (DS), and FST/(1-FST) give similar PC-values, FST* being slightly better than the other two; and (3) (deltamu)2 shows PC-values much lower than the other distance measures. To have reasonably high PC-values for trees similar to ours, at least 30 loci with a minimum of 15 individuals are required when DA distance is used.

The N-acylethanolamine-hydrolyzing acid amidase (NAAA).

Bioactive N-acylethanolamines, including the endocannabinoid anandamide and anti-inflammatory N-palmitoylethanolamine, are hydrolyzed to fatty acids and ethanolamine in animal tissues by the catalysis of fatty acid amide hydrolase (FAAH). We recently cloned cDNA of N-acylethanolamine-hydrolyzing acid amidase (NAAA), another enzyme catalyzing the same reaction, from human, rat, and mouse. NAAA reveals no sequence homology with FAAH and belongs to the choloylglycine hydrolase family. The most striking catalytic property of NAAA is pH optimum at 4.5-5, which is consistent with its immunocytochemical localization in lysosomes. In rat, NAAA is highly expressed in lung, spleen, thymus, and intestine. Notably, the expression level of NAAA is exceptionally high in rat alveolar macrophages. The primary structure of NAAA exhibits 33-35% amino acid identity to that of acid ceramidase, a lysosomal enzyme hydrolyzing ceramide to fatty acid and sphingosine. NAAA actually showed a low, but detectable ceramide-hydrolyzing activity, while acid ceramidase hydrolyzed N-lauroylethanolamine. Thus, NAAA is a novel lysosomal hydrolase, which is structurally and functionally similar to acid ceramidase. These results suggest a unique role of NAAA in the degradation of N-acylethanolamines.

Mhc class I genes of the cichlid fish Oreochromis niloticus.

In terms of number of species, perciform (perch-like) fishes are one of the most diversified groups of modern vertebrates. Within this group, the family Cichlidae is best known for its spectacular adaptive radiation in the great lakes of East Africa. The molecular tool kit used in the study of this radiation includes the major histocompatibility complex (Mhc) genes. To refine this tool, information about the organization of the Mhc regions is badly needed. In this study, the first step was taken toward providing such information for the Mhc class one regions of Oreochromis niloticus, a representative species of the tilapiine branch of the Cichlidae, for which good bacterial artificial chromosome library is available. Screening of the library with class I gene probes led to the identification and isolation of 31 class-I-positive clones. Sequencing of one of these clones and partial characterization of the remaining clones for the presence of class I exons resulted in the construction of two contigs representing the class I region of this species as well as identification of seven additional class-I-positive singleton clones. The O. niloticus genome was shown to contain at least 28 class I genes or gene fragments. The shorter of the two contigs was approximately 330 kb long and contained eight class I genes/gene fragments; the longer contig encompassed 1,200 kb of sequence and contained minimally 17 class I genes/gene fragments; three additional class I genes were found to be borne by a clone that might be part of the shorter contig.

The phylogenetic relationship of tetrapod, coelacanth, and lungfish revealed by the sequences of forty-four nuclear genes.

The origin of tetrapods is a major outstanding issue in vertebrate phylogeny. Each of the three possible principal hypotheses (coelacanth, lungfish, or neither being the sister group of tetrapods) has found support in different sets of data. In an attempt to resolve the controversy, sequences of 44 nuclear genes encoding amino acid residues at 10,404 positions were obtained and analyzed. However, this large set of sequences did not support conclusively one of the three hypotheses. Apparently, the coelacanth, lungfish, and tetrapod lineages diverged within such a short time interval that at this level of analysis, their relationships appear to be an irresolvable trichotomy.

Phylogenetic relationships among East African haplochromine fish as revealed by short interspersed elements (SINEs).

Genomic DNA libraries were prepared from two endemic species of Lake Victoria haplochromine (cichlid) fish and used to isolate and characterize a set of short interspersed elements (SINEs). The distribution and sequences of the SINEs were used to infer phylogenetic relationships among East African haplochromines. The SINE-based classification divides the fish into four groups, which, in order of their divergence from a stem lineage, are the endemic Lake Tanganyika flock (group 1); fish of the nonendemic, monotypic, widely distributed genus Astatoreochromis (group 2); the endemic Lake Malawi flock (group 3); and group 4, which contains fish from widely dispersed East African localities including Lakes Victoria, Edward, George, Albert, and Rukwa, as well as many rivers. The group 4 haplochromines are characterized by a subset of polymorphic SINEs, each of which is present in some individuals and absent in others of the same population at a given locality, the same morphologically defined species, and the same mtDNA-defined haplogroup. SINE-defined group 4 contains six of the seven previously described mtDNA haplogroups. One of the polymorphic SINEs appears to be fixed in the endemic Lake Victoria flock; four others display the presence-or-absence polymorphism within the species of this flock. These findings have implications for the origin of Lake Victoria cichlids and for their founding population sizes.

Origin and speciation of haplochromine fishes in East African crater lakes investigated by the analysis of their mtDNA, Mhc genes, and SINEs.

The Western Branch of the East African Great Rift Valley is pocketed with craters of extinct or dormant volcanoes. Many of the craters are filled with water, and the lakes are inhabited by fishes. The objective of the present study was to determine the amount and nature of genetic variation in haplochromine fishes inhabiting two of these crater lakes, Lake Lutoto and Lake Nshere, and to use this information to infer the origin and history of the two populations. To this end, sequences of mitochondrial (mt) DNA control region, exon 2 of major histocompatibility complex (Mhc) class II B genes, and short interspersed elements (SINEs) were analyzed. The results indicate that the Lake Nshere and Lake Lutoto fishes originated from different but related large founding populations derived from the Kazinga Channel, which connects Lake Edward and Lake George. Some of the genetic polymorphism that existed in the ancestral populations was lost in the populations of the two lakes. The polymorphism that has been retained has persisted for some 50000 generations (years). During this time, new mutations arose and became fixed in each of the two populations in the mtDNA, giving rise to sets of diagnostic substitutions. Each population evolved in isolation after the colonization of the lakes less than 50000 years ago. There appears to be no population structure within the crater lake fishes, and their present effective population sizes are in the order of 104 to 105 individuals. Comparisons with the endemic haplochromine species of Lake Victoria reveal interesting parallels, as well as differences, which may help to understand the nature of the speciation process.

Identification and characterization of a TAP-family gene in the lamprey.

An expressed sequence tag obtained from a sea lamprey ( Petromyzon marinus) cDNA library was used to obtain a full-length coding sequence showing significant similarity to ABCB transporter proteins. The sequence is closely related to the mammalian ABCB9 protein and the TAP1 and TAP2 proteins that transport peptides for loading onto nascent Mhc class I molecules. The Pema-ABCB9 gene has an exon-intron organization similar to that of the mammalian TAP genes, with the exception of exon 2, which in the lamprey is split into two by a 949-bp long intron. The gene probably occurs in a single copy in the haploid lamprey genome. The ABCB9 genes appear to be evolving four-to-ten times slower than the TAP1 and TAP2 genes. Six putative transmembrane helices and the nucleotide-binding domain of the lamprey ABCB9 protein show high sequence similarity with the TAP1 and TAP2 molecules. The lamprey protein also contains sequence stretches that resemble the putative peptide interacting parts of the TAP1 and TAP2 molecules, but are peppered with ABCB9-specific residues.

Macrophage migration inhibitory factor (MIF) of jawed and jawless fishes: implications for its evolutionary origin.

The macrophage migration inhibitory factor (MIF) is a cytokine produced by T lymphocytes and macrophages in response to inflammatory stimuli. We sequenced MIF cDNA clones of two jawless fishes, the sea lamprey (Petromyzon marinus) and the North Atlantic hagfish (Myxine glutinosa), as well as of the jawed (cichlid) fish Paralabidochromis chilotes. The fish MIF-encoding genes have the same exon-intron organization as the mammalian MIF genes and are present in one copy per haploid genome. Secondary and tertiary structure predictions suggest that the fish MIF proteins have a topology characteristic of the entire MIF-family of proteins. Phylogenetic analysis separates the known nematode members of the family into two groups, one having a sister group relationship with the mammalian D-dopachrome tautomerase (DDT) proteins and the other being related to vertebrate MIFs. It also reveals a high degree of convergent evolution among the members of the family. Finally, it suggests that the divergence of MIF and DDT occurred before the emergence of nematodes in metazoan evolution.

Molecular phylogeny of early vertebrates: monophyly of the agnathans as revealed by sequences of 35 genes.

Extant vertebrates are divided into three major groups: hagfishes (Hyperotreti, myxinoids), lampreys (Hyperoartia, petromyzontids), and jawed vertebrates (Gnathostomata). The phylogenetic relationships among the groups and within the jawed vertebrates are controversial, for both morphological and molecular studies have rendered themselves to conflicting interpretations. Here, we use the sequences of 35 nuclear protein-encoding genes to provide definitive evidence for the monophyly of the Agnatha (jawless vertebrates, a group encompassing the hagfishes and lampreys). Our analyses also give a strong support for the separation of Chondrichthyes (cartilaginous fishes) before the divergence of Osteichthyes (bony fishes) from the other gnathostomes.

Origin and affinities of indigenous Siberian populations as revealed by HLA class II gene frequencies.

Gene frequencies of eight Siberian populations (Mansi, Tuva, Todja, Tofalar, Buryat, Okhotsk Evenki, Ulchi, and Negidal) were determined for the three most polymorphic HLA class II loci ( DRB1, DQA1, and DQB1) by a combination of single-stranded conformational polymorphism typing and DNA sequencing. The number of alleles per population ranged from 16 to 25, from seven to eight, and from nine to 14 for the DRB1, DQA1, and DQB1 loci, respectively. The alleles at the three loci occurred in 66 different combinations (haplotypes), most of which appeared to be of ancient origin, but some may have arisen within the Siberian populations. Phylogenetic analysis of the frequency data suggests that the HLA genes of Asian and indigenous American populations stem from a single pool distinct from the gene pools of European and African populations. The Asian populations separate into two clusters, one of which encompasses nearly all the Siberian populations and all the indigenous American populations tested, while the other consists of Central, Eastern, and Southeastern Asian populations. The position of the Tuva people appears to be near the node from which the two clusters diverge. The divergence time of the two clusters is estimated to be 21,000-24,000 years BP. Three different branches of the native Siberian peoples seem to have contributed founders for the indigenous American ethnic groups.

Sequencing of amphioxus PSMB5/8 gene and phylogenetic position of agnathan sequences.

Reanalyses of vertebrate PSMB5 and PSMB8 genes were conducted with a newly obtained amphioxus sequence. The phylogenetic analysis indicated that the amphioxus sequence is an outgroup of both vertebrate PSMB5 and PSMB8 genes. The agnathan sequences were previously thought to be closer to PSMB5 than PSMB8 sequences. However, in this study the phylogenetic trees supported the clustering of agnathan sequences with PSMB8 rather than with PSMB5 sequences. In the alignment there are many sites in which the agnathan sequences are more similar to PSMB5 than PSMB8 sequences. Parsimony analysis showed that the amino acid pattern at these sites could be explained by one substitution on the ancestral branch leading to the jawed vertebrate PSMB8 cluster. The number of substitutions required at these sites was the same for different tree topologies with respect to the position of agnathans. The phylogenetic analyses of PSMB6/PSMB9 and PSMB7/PSMB10 genes indicated that all the interferon-gamma-inducible forms have evolved two to three times faster than the housekeeping forms in the jawed vertebrate lineage. Hence although the agnathan sequences cluster with the PSMB8 sequences, they have a slower rate of evolution similar to that of the housekeeping forms, and may be functioning as housekeeping forms.