The ATP-dependent carboxylate-amine/thiol ligase superfamily is known to contain enzymes catalyzing the formation of various types of peptide, such as D-alanyl-D-alanine, polyglutamate, and ␥-peptide, but, curiously, no enzyme synthesizing ␣-dipeptides of L-amino acids is known. We attempted to find such an enzyme. By in silico screening based on the consensus sequence of the superfamily followed by an in vitro assay with purified enzyme to avoid the degradation of the peptide(s) synthesized, ywfE of Bacillus subtilis was found to code for the activity forming L-alanyl-L-glutamine from L-alanine and L-glutamine with hydrolysis of ATP to ADP. No AMP was formed, supporting the idea that the enzyme belongs to the superfamily. Surprisingly, the enzyme accepted a wide variety of L-amino acids. Among 231 combinations of L-amino acids tested, reaction products were obtained for 111 combinations and 44 kinds of ␣-dipeptides were confirmed by high-performance liquid chromatography analyses, while no tripeptide or longer peptide was detected and the D-amino acids were inert. From these results, we propose that ywfE encodes a new member of the superfamily, L-amino acid ligase.

Molecular phylogeographic studies have revealed the genetic patterns and glacial-interglacial history of many plant and animal species. To infer the Quaternary history of alpine plants in the Japanese archipelago, which is poorly known, we investigated 203 individuals of Potentilla matsumurae and its varieties from 22 populations. We found 11 haplotypes based on approximately 1400 bp of two intergenetic spacers in chloroplast DNA (trnT-L and rpl20-rps20). The distribution of these haplotypes was geographically structured, which was supported by haplotype composition, principal component analysis, and unweighted pair group method with arithmetic mean (UPGMA), and N(ST) (0.71) was significantly greater than G(ST) (0.68). In addition to the positive correlation between genetic and geographic distance (Mantel test, r = 0.497, P < 0.001), an abrupt genetic change was detected between mountains in central Honshu and the Tohoku region. This genetic boundary was further supported by analysis of molecular variance (AMOVA), and high variation (54.0%) was explained by differences on either side of this boundary. Moreover, haplotypes in central Honshu were thought to have diverged, based on an outgroup comparison. These results suggest that mountains in central Honshu served as refugia during the Quaternary climatic oscillation, although the results could not reveal the history of most mountains in the Tohoku region and Hokkaido. Nevertheless, following floristic studies, our results indicate that alpine plants in Japan experienced a history different from that in Europe; i.e. they retreated into refugia during warm periods to avoid forest development, rather than glaciers.

Aim This study aims to elucidate the phylogeography of the Japanese endemic alpine plant, Phyllodoce nipponica Makino (Ericaceae) and to infer the location of refugia of alpine plants in Japan during climatic oscillations.Location Alpine zone in the Japanese archipelago.Methods We determined the chloroplast (cp) DNA haplotypes of 155 individuals (22 populations) based on sequence data from the trnL-F and trnT-L intergenic spacers and the trnL intron, whose phylogenetic relationships were analysed using the program tcs. To examine the genetic structure, analysis of molecular variance (amova) was carried out and the population differentiation was shown by the parameters G ST and N ST . ResultsThe haplotype composition and the results of amova showed that populations in the Japanese Central Mountain Region (JCMR) and in the westernmost region were highly divergent (18.8%). The diversity within populations was very high in the JCMR (h S ¼ 0.421); less variation was found within populations located in other regions at lower elevations.Main conclusions Phyllodoce nipponica survived climatic changes during the Quaternary in the JCMR and the westernmost region. Most of the distribution range was colonized during only one range expansion. The source location from which the range expansion occurred was unclear.

A new flask sampling system (Automatic Air Sampling Equipment (ASE)) was used to conduct atmospheric CO 2 measurements at about 10km altitude over the western Pacific between Australia and Japan using a Japan Airlines (JAL) airliner from December 2005 to April 2007. The observed CO 2 from the ASE agreed well with that determined by the in-situ CO 2 measuring system (Continuous CO 2 Measuring Equipment (CME)) during the same flights. The data from the ASE and CME exhibited similar mean CO 2 for 12 latitudinal bands at an interval of 5° between 30°N and 30°S, although the discrete ASE data are sparse compared to the frequent measurements by the CME. The CO 2 standard scales of the Meteorological Research Institute and the National Institute for Environmental Studies were compared to provide a consistent data set for the first and second phases of the JAL project. The new ASE data clearly depicted a seasonal cycle and increasing trend, which were reproduced by the extrapolated variations deduced from the climatology of previous observations for the past 12 years. In particular, latitudinal changes of detrended seasonal cycles obtained from the new observation were quite similar to those derived from the climatology in the previous observation. These results indicated the consistency in the continuity of the CO 2 record extended by the new JAL observations.

Molecular phylogeography has inferred the history of differentiation between regions and/or among populations following the Pleistocene climatic oscillations, mostly based on the genetic structure of organelle DNA. However, such genetic structure only reflects the history of a single gene, and studies based on single-copy genes of nuclear DNA (nDNA) are required for phylogeography, although their efficiency remains unclear. To examine the utility of nDNA loci, the genetic structures of three genes from Cardamine nipponica, which is closely related to the model species Arabidopsis thaliana, were elucidated: the nDNA genes DET1, PHYA, PHYE, as well as chloroplast DNA (cpDNA). In 279 individuals collected from throughout the range of the species, strong genetic differentiation between northern and central Japan was found for all loci. This result suggested that populations in central Japan experienced a different history from those in northern Japan during the Pleistocene climatic oscillations. In addition, the evidence of refugia at the edges of the distribution, where the genetic structure was less influenced by colonization following range expansion, was shown for several loci. The specific genetic structure within the southernmost populations of northern Japan suggested that this region was also isolated during range expansion. Hence, the consistent history among loci and a more detailed history from several loci indicated that cpDNA can represent the history of vicariance and demonstrated the efficiency of single-copy nuclear genes in phylogeography.