Concerted and Independent Evolution of Control Regions 1 and 2 of Water Monitor Lizards (Varanus salvator macromaculatus) and Different Phylogenetic Informative Markers.

Duplicate control regions (CRs) have been observed in the mitochondrial genomes (mitogenomes) of most varanids. Duplicate CRs have evolved in either concerted or independent evolution in vertebrates, but whether an evolutionary pattern exists in varanids remains unknown. Therefore, we conducted this study to analyze the evolutionary patterns and phylogenetic utilities of duplicate CRs in 72 individuals of Varanus salvator macromaculatus and other varanids. Sequence analyses and phylogenetic relationships revealed that divergence between orthologous copies from different individuals was lower than in paralogous copies from the same individual, suggesting an independent evolution of the two CRs. Distinct trees and recombination testing derived from CR1 and CR2 suggested that recombination events occurred between CRs during the evolutionary process. A comparison of substitution saturation showed the potential of CR2 as a phylogenetic marker. By contrast, duplicate CRs of the four examined varanids had similar sequences within species, suggesting typical characteristics of concerted evolution. The results provide a better understanding of the molecular evolutionary processes related to the mitogenomes of the varanid lineage.

Characterization of five complete Cyrtodactylus mitogenome structures reveals low structural diversity and conservation of repeated sequences in the lineage.

Mitochondrial genomes (mitogenomes) of five Cyrtodactylus were determined. Their compositions and structures were similar to most of the available gecko lizard mitogenomes as 13 protein-coding, two rRNA and 22 tRNA genes. The non-coding control region (CR) of almost all Cyrtodactylus mitogenome structures contained a repeated sequence named the 75-bp box family, except for C. auribalteatus which contained the 225-bp box. Sequence similarities indicated that the 225-bp box resulted from the duplication event of 75-bp boxes, followed by homogenization and fixation in C. auribalteatus. The 75-bp box family was found in most gecko lizards with high conservation (55-75% similarities) and could form secondary structures, suggesting that this repeated sequence family played an important role under selective pressure and might involve mitogenome replication and the likelihood of rearrangements in CR. The 75-bp box family was acquired in the common ancestral genome of the gecko lizard, evolving gradually through each lineage by independent nucleotide mutation. Comparison of gecko lizard mitogenomes revealed low structural diversity with at least six types of mitochondrial gene rearrangements. Cyrtodactylus mitogenome structure showed the same gene rearrangement as found in most gecko lizards. Advanced mitogenome information will enable a better understanding of structure evolution mechanisms.

Karyological characterization and identification of four repetitive element groups (the 18S - 28S rRNA gene, telomeric sequences, microsatellite repeat motifs, Rex retroelements) of the Asian swamp eel (Monopterus albus).

Among teleost fishes, Asian swamp eel (Monopterus albus Zuiew, 1793) possesses the lowest chromosome number, 2n = 24. To characterize the chromosome constitution and investigate the genome organization of repetitive sequences in M. albus, karyotyping and chromosome mapping were performed with the 18S - 28S rRNA gene, telomeric repeats, microsatellite repeat motifs, and Rex retroelements. The 18S - 28S rRNA genes were observed to the pericentromeric region of chromosome 4 at the same position with large propidium iodide and C-positive bands, suggesting that the molecular structure of the pericentromeric regions of chromosome 4 has evolved in a concerted manner with amplification of the 18S - 28S rRNA genes. (TTAGGG)n sequences were found at the telomeric ends of all chromosomes. Eight of 19 microsatellite repeat motifs were dispersedly mapped on different chromosomes suggesting the independent amplification of microsatellite repeat motifs in M. albus. Monopterus albus Rex1 (MALRex1) was observed at interstitial sites of all chromosomes and in the pericentromeric regions of most chromosomes whereas MALRex3 was scattered and localized to all chromosomes and MALRex6 to several chromosomes. This suggests that these retroelements were independently amplified or lost in M. albus. Among MALRexs (MALRex1, MALRex3, and MALRex6), MALRex6 showed higher interspecific sequence divergences from other teleost species in comparison. This suggests that the divergence of Rex6 sequences of M. albus might have occurred a relatively long time ago.

High genetic diversity and demographic history of captive Siamese and Saltwater crocodiles suggest the first step toward the establishment of a breeding and reintroduction program in Thailand.

The Siamese crocodile (Crocodylus siamensis) and Saltwater crocodile (C. porosus) are two of the most endangered animals in Thailand. Their numbers have been reduced severely by hunting and habitat fragmentation. A reintroduction plan involving captive-bred populations that are used commercially is important and necessary as a conservation strategy to aid in the recovery of wild populations. Here, the genetic diversity and population structure of 69 individual crocodiles, mostly members of captive populations, were analyzed using both mitochondrial D-loop DNA and microsatellite markers. The overall haplotype diversity was 0.924-0.971 and the mean expected heterozygosity across 22 microsatellite loci was 0.578-0.701 for the two species. This agreed with the star-like shaped topology of the haplotype network, which suggests a high level of genetic diversity. The mean ratio of the number of alleles to the allelic range (M ratio) for the populations of both species was considerably lower than the threshold of 0.68, which was interpreted as indicative of a historical genetic bottleneck. Microsatellite markers provided evidence of introgression for three individual crocodiles, which suggest that hybridization might have occurred between C. siamensis and C. porosus. D-loop sequence analysis detected bi-directional hybridization between male and female individuals of the parent species. Therefore, identification of genetically non-hybrid and hybrid individuals is important for long-term conservation management. Relatedness values were low within the captive populations, which supported their genetic integrity and the viability of a breeding and reintroduction management plan. This work constitutes the first step in establishing an appropriate source population from a scientifically managed perspective for an in situ/ex situ conservation program and reintroduction of crocodile individuals to the wild in Thailand.

Lack of satellite DNA species-specific homogenization and relationship to chromosomal rearrangements in monitor lizards (Varanidae, Squamata).

BACKGROUND: Satellite DNAs (stDNAs) are highly repeated sequences that constitute large portions of any genome. The evolutionary dynamics of stDNA (e.g. copy number, nucleotide sequence, location) can, therefore, provide an insight into genome organization and evolution. We investigated the evolutionary origin of VSAREP stDNA in 17 monitor lizards (seven Asian, five Australian, and five African) at molecular and cytogenetic level. RESULTS: Results revealed that VSAREP is conserved in the genome of Asian and Australian varanids, but not in African varanids, suggesting that these sequences are either differentiated or lost in the African varanids. Phylogenetic and arrangement network analyses revealed the existence of at least four VSAREP subfamilies. The similarity of each sequence unit within the same VSAREP subfamily from different species was higher than those of other VSAREP subfamilies belonging to the same species. Additionally, all VSAREP subfamilies isolated from the three Australian species (Varanus rosenbergi, V. gouldii, and V. acanthurus) were co-localized near the centromeric or pericentromeric regions of the macrochromosomes, except for chromosomes 3 and 4 in each Australian varanid. However, their chromosomal arrangements were different among species. CONCLUSIONS: The VSAREP stDNA family lack homogenized species-specific nucleotide positions in varanid lineage. Most VSAREP sequences were shared among varanids within the four VSAREP subfamilies. This suggests that nucleotide substitutions in each varanid species accumulated more slowly than homogenization rates in each VSAREP subfamily, resulting in non-species-specific evolution of stDNA profiles. Moreover, changes in location of VSAREP stDNA in each Australian varanid suggests a correlation with chromosomal rearrangements, leading to karyotypic differences among these species.

Higher-order repeat structure in alpha satellite DNA occurs in New World monkeys and is not confined to hominoids.

Centromeres usually contain large amounts of tandem repeat DNA. Alpha satellite DNA (AS) is the most abundant tandem repeat DNA found in the centromeres of simian primates. The AS of humans contains sequences organized into higher-order repeat (HOR) structures, which are tandem arrays of larger repeat units consisting of multiple basic repeat units. HOR-carrying AS also occurs in other hominoids, but results reported to date for phylogenetically more remote taxa have been negative. Here we show direct evidence for clear HOR structures in AS of the owl monkey and common marmoset. These monkeys are New World monkey species that are located phylogenetically outside of hominoids. It is currently postulated that the presence of HOR structures in AS is unique to hominoids. Our results suggest that this view must be modified. A plausible explanation is that generation of HOR structures is a general event that occurs occasionally or frequently in primate centromeres, and that, in humans, HOR-carrying AS became predominant in the central region of the centromere. It is often difficult to assemble sequence reads of tandem repeat DNAs into accurate contig sequences; our careful sequencing strategy allowed us to overcome this problem.

Role of Chromosome Changes in Crocodylus Evolution and Diversity.

The karyotypes of most species of crocodilians were studied using conventional and molecular cytogenetics. These provided an important contribution of chromosomal rearrangements for the evolutionary processes of Crocodylia and Sauropsida (birds and reptiles). The karyotypic features of crocodilians contain small diploid chromosome numbers (30~42), with little interspecific variation of the chromosome arm number (fundamental number) among crocodiles (56~60). This suggested that centric fusion and/or fission events occurred in the lineage, leading to crocodilian evolution and diversity. The chromosome numbers of Alligator, Caiman, Melanosuchus, Paleosuchus, Gavialis, Tomistoma, Mecistops, and Osteolaemus were stable within each genus, whereas those of Crocodylus (crocodylians) varied within the taxa. This agreed with molecular phylogeny that suggested a highly recent radiation of Crocodylus species. Karyotype analysis also suggests the direction of molecular phylogenetic placement among Crocodylus species and their migration from the Indo-Pacific to Africa and The New World. Crocodylus species originated from an ancestor in the Indo-Pacific around 9~16 million years ago (MYA) in the mid-Miocene, with a rapid radiation and dispersion into Africa 8~12 MYA. This was followed by a trans-Atlantic dispersion to the New World between 4~8 MYA in the Pliocene. The chromosomes provided a better understanding of crocodilian evolution and diversity, which will be useful for further study of the genome evolution in Crocodylia.

Reduction in the structural instability of cloned eukaryotic tandem-repeat DNA by low-temperature culturing of host bacteria.

Summary For accurate analyses of eukaryotic tandem-repeat DNA, it is often required to clone a genomic DNA fragment into a bacterial plasmid. It is, however, a serious problem that tandem-repeat DNA is frequently subjected to structural changes during maintenance or amplification in the host bacteria. Here, we show an example of a clear difference in the instability of tandem-repeat DNA between different culturing temperatures. A fragment of monkey centromeric DNA carried by pUC19 was considerably degraded by culturing bacteria at 37 °C, but the damage was reduced at 25 °C. Thus, culturing temperature is a significant factor for avoiding degradation, in addition to the genotype of the host bacteria.