Developing DNA barcodes for species identification in Podophylloideae (Berberidaceae)

Species of Podophyllum, Dysosma, Sinopodophyllum, and Diphylleia, genera from Podophylloideae of Berberidaceae, have long been used in traditional herbal medicine in East Asia and/or North America. Accurate identification of the species of these four genera is crucial to their medicinal uses. In this study, we tested the utility of nine barcodes (matK, rbcL, atpH-atpI, rpl32-trnL^UAG, rps18-clpp, trnL-trnF, trnL-ndhJ, trnS-trnfM, and internal transcribed spacer (ITS)) to discriminate different species of Podophylloideae. Thirty-six individuals representing 12 species of Podophylloideae were collected from different locations in China, Japan, and North America. We assessed the feasibility of amplification and sequencing of all markers, examined the levels of the barcoding gap based on DNA sequence divergence between ranges of intra- and interspecific variation using pairwise distances, and further evaluated successful identifications using each barcode by similarity-based and tree-based methods. Results showed that nine barcodes, except rps18-clpp, have a high level of primer universality and sequencing success. As a single barcode, ITS has the most variable sites, greater intra- and interspecific divergences, and the highest species discrimination rate (83%), followed by matKwhich has moderate variation and also high species discrimination rates. However, these species can also be discriminated by ITS alone, except Dysosma versipellis (Hance) M. Cheng ex T. S. Ying and D. pleiantha (Hance) Woodson. The combination of ITS + matK did not improve species resolution over ITS alone. Thus, we propose that ITS may be used as a sole region for identification of most species in Podophylloideae. The failure of ITS to distinguish D. versipellis and D. pleiantha is likely attributed to incomplete lineage sorting due to recent divergence of the two species.     

Chemical Analysis of Melanin Pigments in Feather Germs of Japanese Quail Bh (Black at Hatch) Mutants

Bh (black at hatch) is a mutation of Japanese quails which causes darkening or lightening of the plumage in heterozygotes or homozygotes, respectively. We chemically analyzed melanin pigments in feather germs of Bh mutant embryos and in feathers of adult animals. Dark brown dorsal feathers of wild-type adult animals had white barrings, but heterozygous ones lacked clear barrings. The feathers of wild-type and heterozygote animals contained both eumelanins and pheomelanins, the latter being more pheomelanic. On the dorsal skin of 10-day old wild-type embryos, longitudinal stripes from black and yellow rows of feather germs developed; two or three longitudinal rows of black feather germs and then two or three rows of yellow feather germs next to the short central feather germs. Heterozygous embryos appeared black in plumage pigmentation, due to the presence of‘gray’feather germs in rows of dorsal feather germs that corresponded to yellow rows in wild-type embryos. Homozygous dorsal feather germs did not develop the black and yellow longitudinal stripes, but were brown. Chemical analysis showed that embryos of each genotype contained both eumelanins and pheomelanins in the feather germs; however, the eumelanin content in ho-mozygous feather germs was very low. These results suggest that the Bh mutation causes pheomelanic changes in feathers of quails.     

Influence of Temperature on Lactate Utilization by Round Spermatids from Rat Testes

Rotenone-sensitive ¹⁴CO₂ formation from [¹⁴C]lactate and oxygen consumption by round spermatids were found to be greater at elevated temperatures than at 34°C. More than 96% of the total radioactivity of the metabolized [¹⁴C]lactate was recovered in the released CO₂ and the acid soluble fraction of the cells. There was practically no incorporation of [¹⁴C]latctate into the lipid, nucleic acid, and protein fractions. Intracellular level of ATP in spermatids was enhanced in the presence of lactate (20 mM) at 34°C (scrotal temperature), whereas it was decrease at 37°C (body temperature). However, this was reversible when the cells were transferred from the elevated temperature to 34°C. It was also found that oxygen consumption and CO₂ production were increased at 34°C by 2, 4-dinitrophenol (DNP), but decreased by oligomycin. On the other hand, oligomycin and DNP had no effect on oxygen consumption and ¹⁴CO₂ formation at the elevated temperature.
These findings provide evidence that lactate utilization by spermatids is coupled with oxidative phosphorylation at scrotal temperature, but becomes uncoupled at elevated temperature, although more lactate is consumed.     

Faunal make-up, host range and infestation rate of weevils and tephritid flies associated with flower heads of the thistle Cirsium (Cardueae: Astaraceae) in Japan

From 1988 to 1998, we collected flower heads of 39 thistle taxa (35 taxa of Cirsium, one species each of Breea, Synurus, Saussurea and Arctium; Cardueae; Astaraceae) in Japan, mainly from Hokuriku and other parts of central Honshu, and kept them in the laboratory to breed weevils and tephritid flies, the core fauna. We report the faunal make‐up, host plants, geographic distribution and the attack levels of the insects. Results indicated that (i) three Larinus species (Curculionidae) and three species of tephritid flies (Tephritis, Urophora and Xyphosia) comprised the core fauna; (ii) two insect species belonging to the same taxonomic group (either Curculionidae (Larinus) or Tephritidae) tended to use different host plant species; (iii) two sympatric Larinus species (L. latissimus and L. meleagris) segregated the host plants seasonally in central Honshu (Cirsium blooming in spring and autumn, respectively); and (iv) two tephritid fly species, Xyphosia punctigera and Urophora sachalinensis, segregated geographically (the former on the Japan Sea side and the latter on the Pacific Ocean side). In comparison with their European counterparts, the weevils and tephritids of the Japanese Cirsium are characterized by a lower species richness and a lower degree of specialization in usage of the thistle flower heads, with gall‐formers being distinctly under‐represented, and callus tissue‐feeders being absent. This reflects the fact that Japanese thistles are so closely related that hybridization frequently occurs, and also that the thistles have had a short history of interaction with the insects since the thistles’ arrival in Japan.     

ROLE OF SULFUR METABOLISM IN COPPER-RESISTANCE OF YEAST

Cells of a normal yeast strain and of its copper-resistant substrainwere more severely injured by copper treatment under sulfurdeficiency than under nitrogen or carbon deficiency. Even underthese starved conditions, the resistant substrain gave higherviable counts than the parent strain after the copper treatment.Several hours were needed for the sulfur-starved cells to recovertheir copper-resistance when resupplied with sulfur. The observed effect of the amount of sulfur supply on the cellyield was not contradictory to the idea that resistant cellsuse for their resistance mechanism a part of sulfur they absorb.Methionine made cells more sensitive to copper, and this effectwas antagonized by ethionine to some extent. The importance of sulfur metabolism in copper resistance isdiscussed. ¹This work was supported by a Grant in Aid for Fundamental ScientificResearch, Ministry of Education, Japan. ²Present address: Konan University, Kobe, Japan. (Received October 8, 1959; )