Phylogenetic relationships of Sakhalin taimen Parahucho perryi inferred from PCR-RFLP analysis of mitochondrial DNA

RFLP analysis of three amplified mtDNA fragments (Cytb/D-loop, ND1/ND2, and ND3/ND4L/ND4) was performed in the following taxa: Parahucho perryi, Hucho taimen, Brachymystax lenok, B. tumensis, Salmo salar, Salvelinus leucomaenis, and S. levanidovi. For mtDNA of P. perryi, a substantial decrease in the haplotype and nucleotide diversity was observed as a result of random genetic drift, caused by a reduction in the effective population size. Nucleotide divergence estimates between the mtDNA haplotypes were determined. Sakhalin taimen P. perryi was found to be approximately equally diverged from S. salar and from the charrs of the genus Salvelinus, by 11.0 and 10.0%, respectively. The divergence between P. perryi and H. taimen constituted 14.6%, between P. perryi and lenoks of the genus Brachymystax, 14.2%, and between H. taimen and Brachymystax, 7.7%. The analysis of possible phylogenetic relationships of the mtDNA from P. perryi among the group of taxa examined confirmed validity of the genus Parahucho. Phylogenetic reconstructions performed showed that robustness of the trees constructed for the complex of phylogenetically informative characters over three mtDNA fragments was considerably higher than that of the trees constructed for individual genes.     

Spawning behaviour of Sakhalin taimen, <Emphasis Type="Italic">Parahucho perryi</Emphasis>, from northern Hokkaido,Japan

A video camera mounted in an underwater housing and remotely operated was used to monitor the behaviour of five different Sakhalin taimen (Parahucho perryi), females and attendant males spawning in three coastal tributary streams in Northern Hokkaido, Japan. Based on three complete and two incomplete spawnings, we describe in detail for the first time the complete spawning behavioural repertoire of this species. The Sakhalin taimen was originally placed within Hucho, then removed from that genus based on morphological, life history and molecular data. Our study supports that removal—none of the behavioural traits we recorded clustered Parahucho with Hucho uniquely. Similarities between the two genera were all plesiomorphic traits that are widespread throughout the salmonines. The immediate behaviour right after spawning was found to be a major difference between Hucho and Parahucho. Like female Oncorhynchus and Salmo, Sakhalin taimen females cover their eggs by beats of their tails immediately after spawning. This is different from the “rest, then cover” behaviour shown by Siberian taimen (Hucho taimen) as well as lenok (Brachymystax lenok), supporting again that the Sakhalin taimen be removed from Hucho and placed in its own genus.     

Migration of Sakhalin taimen (<Emphasis Type="Italic">Parahucho perryi</Emphasis>): evidence of freshwater resident life history types

Sakhalin taimen (Parahucho perryi) range from the Russian Far East mainland along the Sea of Japan coast, and Sakhalin, Kuril, and Hokkaido Islands and are considered to primarily be an anadromous species. We used otolith strontium-to-calcium ratios (Sr/Ca) to determine the chronology of migration between freshwater and saltwater and identify migratory contingents of taimen collected from the Koppi River, Russia. In addition, we examined taimen from the Sarufutsu River, Japan and Tumnin River, Russia that were captured in marine waters. Transects of otolith Sr/Ca for the Sarufutsu River fish were consistent with patterns observed in anadromous salmonids. Two fish from the Tumnin River appeared to be recent migrants to saltwater and one fish was characterized by an otolith Sr/Ca transect consistent with marine migration. Using these transects as benchmarks, all Koppi River taimen were classified as freshwater residents. These findings suggest more work is needed to assess life history variability among locations and the role of freshwater productivity in controlling migratory behavior in taimen.     

Phylogenetic analysis of <Emphasis Type="Italic">Antrodia</Emphasis> species and <Emphasis Type="Italic">Antrodia camphorata</Emphasis> inferred from internal transcribed spacer region

The species of Antrodia are one of the difficult-to-classify and obscure groups of poroid Aphyllophorales based on morphological appearance. However, it is becoming increasingly important to reliably identify the entire suite of Antrodia camphorata strains and Antrodia species due to the potential pharmaceutical value of their biologically active ingredients. In this study, the internal transcribed spacer (ITS) region of the ribosomal RNA gene (rDNA) was sequenced and phylogenetically analyzed in a number of Antrodia fungal species and strains. ITS amplicons from the Antrodia species tested ranged in size from 543 to 610 bp; the size of the ITS of A. camphorata strains ranged from 592 to 596 bp. The overall sizes of ITS2 and 5.8S ribosomal RNA gene of all A. camphorata strains tested in this study were shown to be 217 and 158 bp, respectively. A phylogenetic analysis of ITS data generated, which included sequences of 11 A. camphorata strains and nine other Antrodia species, showed three clearly distinct groups. Group 1 includes A. camphorata, Antrodia salmonea, and Antrodia carbinca strains. Within Group 2, Antrodia sinuosa and Antrodia xantha were clustered together. Group 3 contained Antrodia albida, A. heteromorpha, A. serialis, and A. malicola. The observed sequence diversity among ITS alleles provided an effective tool for differentiating strains of A. camphorata, A. salmonea, A. xantha, A. sinuosa, or A. serialis. Polymorphisms arising within the ITS1-5.8S-ITS2 region can provide practical markers for establishing a foundation for the further expansion of an ITS sequence database of medically important fungi.     

Genetic variability in anadromous fishes,chum salmon <Emphasis Type="Italic">Oncorhynchus keta</Emphasis> (Walbaum, 1792), and Sakhalin taimen <Emphasis Type="Italic">Parahucho perryi</Emphasis> (Brevoort, 1856) from the Northwestern Pacific as a reflection of paleoclimate oscilations

The genetic variability distribution of two mtDNA segments of chum salmon (Oncorhynchus keta) (Walbaum, 1792) and Sakhalin taimen (Parahucho perryi) (Brevoort, 1856) was examined in populations of the Sea of Japan and the Sea of Okhotsk. The values of haplotype and nucleotide variability in these species are, in general, of the same level. The dating of the divergence time of species haplotypes revealed four evolutionary periods in Sakhalin taimen and three in chum salmon. In the taimen, the first divergence time occurred approximately 430 thousand years (kyr) ago, the second 220 kyr ago, and the third 70 kyr ago. In the chum salmon, the first divergence time corresponds to 220 kyr; the second is approximately 100 kyr ago. In both species, the main portion of presently revealed haplotypes evolved over the past 50–10 kyr. Certain glacioeustatic sea level fluctuations influenced each stage of evolution history of species, contributing to their geographic isolation. Demographic population history research found that the initial stage of population growth in the taimen occurred at the time period of approximately 12 kyr ago and was apparently associated with the end of the Last Glacial Maximum. In the chum salmon, this period began somewhat earlier, 30–35 kyr ago; it has accelerated in the past 10–15 kyr. The last glaciation to a lesser extent impacted the demographics of chum salmon, probably due to the greater eurythermity and to the larger range of this species.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Sectional relationships in the genus <Emphasis Type="Italic">Musa</Emphasis> L. inferred from the PCR-RFLP of organelle DNA sequences

The objective of this study was to construct a molecular phylogeny of the genus Musa using restriction-site polymorphisms of the chloroplast (cpDNA) and mitochondrial DNA (mtDNA). Six cpDNA and two mtDNA sequences were amplified individually in polymerase chain reaction (PCR) experiments in 13 species representing the four sections of Musa. Ensete ventricosum (W.) Ch. was used as the outgroup. The amplified products were digested with ten restriction endonucleases. A total of 79 restriction-site changes were scored in the sample. Wagner parsimony using the branch and bound option defined two lines of evolution in Musa. One lineage comprised species of the sections Australimusa and Callimusa which have a basic number of x = 10 chromosomes, while most species of sections Eumusa and Rhodochlamys (x = 11) formed the other lineage. Musa laterita Cheesman (Rhodochlamys) had identical organellar genome patterns as some subspecies of the Musa acuminata Colla complex. The progenitors of the cultivated bananas, M. acuminata and Musa balbisiana Colla, were evolutionarily distinct from each other. Musa balbisiana occupied a basal position in the cladogram indicating an evolutionarily primitive status. The close phylogenetic relationship between M. laterita and M. acuminata suggests that species of the section Rhodochlamys may constitute a secondary genepool for the improvement of cultivated bananas.Communicated by H.F. Linskens     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Genetic population structure of <Emphasis Type="Italic">Lefua echigonia</Emphasis> inferred from allozymic and mitochondrial cytochrome <Emphasis Type="Italic">b</Emphasis> variations

We examined the genetic population structure of Lefua echigonia (Japanese name, hotoke-dojo) using polymerase chain reaction restriction-fragment length polymorphisms (PCR-RFLPs) of the mitochondrial cytochrome b gene and two allozymic loci. The phylogenetic relationships of L. echigonia and those among L. echigonia, Lefua sp. (nagare hotoke-dojo), and L. nikkonis (ezo hotoke-dojo) were also investigated based on the nucleotide sequences of the cytochrome b gene. PCR-RFLP analysis revealed 18 mitotypes in L. echigonia, 2 in Lefua sp., and 1 in L. nikkonis. Phylogenetic trees based on the cytochrome b sequences indicated that the 18 mitotypes in L. echigonia were divided into five distinct groups (South-Kanto, North-Kanto, Tohoku, Echigo, and Tokai-Kinki clades) that differed by 8.5–15.3%, reflecting region-specific geographic distributions. The distributions of alleles in two allozymic loci roughly corresponded to those of the mitotype groups. The divergence times of the five groups were estimated to be about 3.4–7.7 million years ago by applying a general rate for mitochondrial DNA, suggesting that the divergence among them might have occurred in the late Tertiary. It can be inferred that the regional differentiation of each group was mainly due to geographic isolation and that this has been maintained, because the boundaries among the groups corresponded to geological features. The trees also supported the existence of three taxa, L. echigonia, Lefua sp., and L. nikkonis. We concluded that Lefua sp. was distinguished from other species in Lefua by morphological and ecological characters and also by genetic divergences of the cytochrome b gene. Our study also demonstrated the superior efficacy and simplicity of PCR-RFLP analysis as a method for detecting genetic variation in L. echigonia.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Phylogenetic relationships of <Emphasis Type="Italic">Citrus</Emphasis> and its relatives based on <Emphasis Type="Italic">rbcL</Emphasis> gene sequences

We sequenced the rbcL genes of 64 accessions from 24 genera of Citrus relatives and analyzed them by neighbor-joining and maximum parsimony methods. Both trees supported Swingle and Reece’s (1967) treatment of the subfamily Aurantioideae as monophyletic. However, the trees did not support Swingle and Reece’s treatment of tribes and subtribes. The subgenera Citrus and Papeda were not clustered clearly. The analysis associated the Fortunella group with mandarin, Poncirus with Citrus ichangensis, Severinia buxifolia with Atalantia ceylanica, Microcitrus with Eremocitrus and Citrus micrantha, and Hesperethusa crenulata with Citropsis. Furthermore, Atalantia species showed polytomy. The classification of Swingle and Reece should be reviewed.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

A PCR-RFLP method on faecal samples to distinguish <Emphasis Type="Italic">Martes martes</Emphasis>, <Emphasis Type="Italic">Martes foina</Emphasis>, <Emphasis Type="Italic">Mustela putorius</Emphasis> and <Emphasis Type="Italic">Vulpes vulpes</Emphasis>

A non-invasive genetic approach has been recently employed in the population genetics of wild species, using faeces that could be easily collected, particularly of elusive or endangered species. However, faeces of pine (Martes martes) and beech marten (M. foina) can be morphologically similar and could be confused with those of polecat (Mustela putorius) and red fox (Vulpes vulpes). On this basis, a rapid, simple and inexpensive RFLP protocol using a cytochrome b (Cyt b) gene fragment of mtDNA, isolated from faecal samples, was performed to distinguish the above-mentioned species.     

Prevalence,intensity and associated factor analysis of <Emphasis Type="Italic">Tropilaelaps</Emphasis><Emphasis Type="Italic">mercedesae</Emphasis> infesting <Emphasis Type="Italic">Apis</Emphasis><Emphasis Type="Italic">mellifera</Emphasis> in China

Tropilaelaps mercedesae is a serious ectoparasite of Apis mellifera in China. The aim of this study was to investigate the infestation rates and intensity of T. mercedesae in A. mellifera in China, and to explore the relative importance of climate, district, management practices and beekeeper characteristics that are assumed to be associated with the intensity of T. mercedesae. Of the 410 participating apiaries, 379 apiaries were included in analyses of seasonal infestation rates and 352 apiaries were included in multivariable regression analysis. The highest infestation rate (86.3%) of T. mercedesae was encountered in autumn, followed by summer (66.5%), spring (17.2%) and winter (14.8%). In autumn, 28.9% (93) of the infested apiaries were in the north (including the northeast and northwest of China), 71.1% (229) were in the central and south (including east, southeast and southwest China), and 306 apiaries (82.9%) were co-infested by both T. mercedesae and Varroa. Multivariable regression analysis showed that geographical location, season, royal jelly collection and Varroa infestation were the factors that influence the intensity of T. mercedesae. The influence of beekeeper’s education, time of beekeeping, operation size, and hive migration on the intensity of T. mercedesa was not statistically significant. This study provided information about the establishment of the linkage of the environment and the parasite and could lead to better timing and methods of control.     

Phylogenetic analysis of <Emphasis Type="Italic">IRIS</Emphasis> L. from China on chloroplast <Emphasis Type="Italic">TRN</Emphasis>L-F sequences

Phylogenetic relationships among the six Iris subgenera were reconstructed by chloroplast trnL-F sequences data using maximum likelihood. The entire matrix of aligned bases analyzed includes 1043 characters and the length of all sequences varied from 747 bp to 893 bp, and mutation sites accounted for 18.79% of the total length. The cluster analysis results accorded well with the subgeneric classification of Chinese Iris species. Results suggested rhizomes and sepals lacking ornament are ancestral characters, and subg. Xyridion and subg. Limniris are more primordial than another four subgenera. Subgenus Nepalensis and subg. Xyridion were resolved as monophyletic.     

Phylogenetic Studies of <Emphasis Type="Italic">Gordonia</Emphasis> Species Based on <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">secA1</Emphasis> Gene Analyses

Phylogenetic relations within the genus Gordonia were analyzed using partial gyrB and secA1 gene sequences of 23 type species in comparison with those of 16S rRNA gene. The gyrB and secA1 phylogenies showed agreement with that constructed using 16S rRNA gene sequences. The degrees of divergence of the gyrB and secA1 genes were approximately 3.4 and 1.7 times greater, respectively, than that of 16S rRNA gene. The gyrB gene showed more discriminatory power than either the secA1 or 16S rRNA gene, facilitating clear differentiation of any two Gordonia species using gyrB gene analysis. Our data indicate that gyrB and secA1 gene sequences are useful as markers for phylogenetic study and identification at the species level of the genus Gordonia.