Taizo Hogetsu

An improved technique for isolating codominant compound microsatellite markers

An approach for developing codominant polymorphic markers (compound microsatellite (SSR) markers), with substantial time and cost savings, is introduced in this paper. In this technique, fragments flanked by a compound SSR sequence at one end were amplified from the constructed DNA library using compound SSR primer (AC)6(AG)5 or (TC)6(AC)5 and an adaptor primer for the suppression-PCR. A locus-specific primer was designed from the sequence flanking the compound SSR. The primer pairs of the locus-specific and compound SSR primers were used as a compound SSR marker. Because only one locus-specific primer was needed for design of each marker and only a common compound SSR primer was needed as the fluorescence-labeled primer for analyzing all the compound SSR markers, this approach substantially reduced the cost of developing codominant markers and analyzing their polymorphism. We have demonstrated this technique for Dendropanax trifidus and easily developed 11 codominant markers with high polymorphism for D. trifidus. Use of the technique for successful isolation of codominant compound SSR markers for several other plant species is currently in progress.

ECTOMYCORRHIZAL FUNGI ON ESTABLISHED SHRUBS FACILITATE SUBSEQUENT SEEDLING ESTABLISHMENT OF SUCCESSIONAL PLANT SPECIES

Ectomycorrhizal (ECM) fungi are symbiotic microorganisms that can significantly improve the water and nutrient uptake of host plants. With reference to ECM fungi, we studied the effects of established shrubs on the subsequent seedling establishment of three woody plant species in a volcanic desert on Mt. Fuji, Japan, during early primary succession. Salix reinii, an alpine dwarf willow, is the pioneer ECM plant at this research site, and many ECM fungi have colonized established willow shrubs. ECM formation was found to be high on S. reinii seedlings that had been transplanted near established willow shrubs. The growth and nitrogen content of the seedlings increased significantly with the numbers of associated ECM fungal species and ECM root tips. ECM formation on transplanted seedlings of Betula ermanii and Larix kaempferi, two subsequent successional tree species, was also higher near the established willow shrubs. Molecular identification showed that almost all of the ECM fungi on transplanted seedlings of the three plant species were of the same species as those observed on the established willow shrubs. These results indicate that ECM fungi associated with established willow shrubs are essential in facilitating seedling establishment of successional plant species in the early successional volcanic desert.

Outcrossing and paternity analysis of Pinus densiflora (Japanese red pine) by microsatellite polymorphism.

This study employed microsatellite loci to analyse outcrossing rate and pollen dispersal in Japanese red pine (Pinus densiflora) in an isolated stand. The average offspring outcrossing rate for 29 cones was 0.955. Significant differences in outcrossing rates between offspring groups on individual branches that extended in different directions at different heights were not detected. Male parents of 874 offspring collected from the maternal tree were assessed by exclusion using polymorphisms at three microsatellite loci. Paternity analysis indicated that at least 31% of the offspring were fertilized by pollen from trees outside the stand. The average distance of pollen migration within the study stand was 68 m, with a maximum value of 325 m. There was excess mating with nearby P. densiflora trees, of which only a few were predominant pollen donors. In addition, a weakly directional bias in P. densiflora pollination was also detected in the study stand, suggesting that female strobili on a branch of the maternal tree were more easily fertilized by pollen from trees in that direction.

Genetic structure and reproduction dynamics of Salix reinii during primary succession on Mount Fuji, as revealed by nuclear and chloroplast microsatellite analysis

The early stage of volcanic desert succession is underway on the southeastern slope of Mount Fuji. We used markers of nuclear microsatellites (simple sequence repeats; SSR) and chloroplast microsatellites (cpSSR) to investigate the population genetic structure and reproduction dynamics of Salix reinii, one of the dominant pioneer shrubs in this area. The number of S. reinii genets in a patch and the area of the largest genet within the patch increased with patch area, suggesting that both clonal growth and seedling recruitment are involved in the reproduction dynamics of S. reinii. Five polymorphic cpSSR markers were developed for S. reinii by sequencing the noncoding regions between universal sequences in the chloroplast genome. Nineteen different cpSSR haplotypes were identified, indicating that S. reinii pioneer genets were created by the long‐distance dispersal of seeds originating from different mother genets around the study site, where all vegetation was destroyed during the last eruption. Furthermore, the clustered distributions of different haplotypes within each patch or plot suggested that newly colonized genets tended to be generated from seeds dispersed near the initially established mother genets. These results revealed that the establishment of the S. reinii population on the southeastern slope of Mount Fuji involved two sequential modes of seed dispersal: long‐distance dispersal followed by short‐distance dispersal.

MICROSATELLITE MARKERS REVEAL POPULATION GENETIC STRUCTURE OF THE TOXIC DINOFLAGELLATE ALEXANDRIUM TAMARENSE (DINOPHYCEAE) IN JAPANESE COASTAL WATERS1

This is the first report to explore the fine‐scale diversity, population genetic structure, and biogeography of a typical planktonic microbe in Japanese and Korean coastal waters and also to try to detect the impact of natural and human‐assisted dispersals on the genetic structure and gene flow in a toxic dinoflagellate species. Here we present the genetic analysis of Alexandrium tamarense (Lebour) Balech populations from 10 sites along the Japanese and Korean coasts. We used nine microsatellite loci, which varied widely in number of alleles and gene diversity across populations. The analysis revealed that Neis genetic distance correlated significantly with geographic distance in pair‐wise comparisons, and that there was genetic differentiation in about half of 45 pair‐wise populations. These results clearly indicate genetic isolation among populations according to geographic distance and restricted gene flow via natural dispersal through tidal currents among the populations. On the other hand, high P‐values in Fishers combined test were detected in five pair‐wise populations, suggesting similar genetic structure and a close genetic relationship between the populations. These findings suggest that the genetic structure of Japanese A. tamarense populations has been disturbed, possibly by human‐assisted dispersal, which has resulted in gene flow between geographically separated populations.

A Simple Method for Developing Microsatellite Markers using Amplified Fragments of Inter-simple Sequence Repeat (ISSR)

10 A primer IP1 designed from the sequenced region at one end of the microsatellite and for nested PCR another primer IP2 based on the sequence between IP1 and the microsatellite were prepared. These two primers were used to determine the other sequence flanking the microsatellite by a “walking” method. With this approach, we developed several microsatellite markers from Salix reinii, Pinus densiflora and Robinia pseudoacacia, respectively. The absence of enrichment processes and screening procedures makes it easier to develop microsatellite markers, and this approach provides an alternative for the development of microsatellite markers in any organism.

Competition between ectomycorrhizal fungi colonizing Pinus densiflora

Abstract Interactive competition of Pisolithus tinctorius (Pers.) Coker et Couch with an unidentified species Tanashi 01 and Suillus luteus (L.: Fr.) S. F. Gray was investigated using a rhizobox. Pinus densiflora Sieb. et Zucc. was used as the host plant and mycelia were distinguished by hyphal color. The speed of mycelial spread differed between the fungi;P. tinctorius and Tanashi 01 grew faster than S. luteus. A P. tinctorius mycorrhizal seedling and a Tanashi 01 mycorrhizal seedling were transplanted on opposite sides of the rhizobox. The mycelia and mycorrhizae of P. tinctorius were overgrown by Tanashi 01 hyphae and development of P. tinctorius was gradually inhibited. The areas occupied by mycelia and mycorrhiza of P. tinctorius decreased by 52% and 37%, respectively, 154 days after transplantation relative to that at 91 days. In the overlap area of P. tinctorius and Tanashi 01, the latter fungus infected new root tips emerging from P. tinctorius mycorrhiza, which lacked a mantle of P. tinctorius hyphae, and formed a composite mycorrhizal structure. P. tinctorius mycorrhizae were progressively replaced by Tanashi 01 mycorrhizae. Mycelial spread of P. tinctorius and S. luteus were naturally inhibited but there was no interaction in mycorrhizal formation.

Spatial distribution of the subterranean mycelia and ectomycorrhizae of Suillus grevillei genets

Suillus grevillei, and found that one, SG-5, is species-specific to S. grevillei and polymorphic. Using the SG-5 marker, we analyzed for the first time the horizontal and vertical distribution of the subterranean parts of several S. grevillei genets. The spatial distribution of S. grevillei genets in the soil demonstrated that the development of S. grevillei sporocarps is correlated with that of extra-radical mycelia and ectomycorrhizae of the same genet, which are distributed in a narrow area. However, sporocarps are not always centered over the subterranean parts and the amount of subterranean mycelia and mycorrhizae is not always correlated to the number of sporocarps formed on them. No S. grevillei mycelia and mycorrhizae were detected beneath the positions where S. grevillei sporocarps emerged in the previous year. The observation indicates the rapid alteration of the subterranean parts of S. grevillei genets, and suggests that S. grevillei genets change location rather than merely extend their habitat year after year.

Germination and infectivity of ectomycorrhizal fungal spores in relation to their ecological traits during primary succession

The spores of ectomycorrhizal fungi (EMF) play critical roles in the population and community development of EMF. Here, the germination and infectivity of EMF spores are examined with reference to the ecological traits of the EMF species. Spores were collected from 12 EMF species, whose successional patterns have been studied in the volcanic desert on Mount Fuji, Japan. Spore germination experiments were conducted with host plants (Salix reinii), with nonhost plants (Polygonum cuspidatum), and without plants. The mycorrhizal formation ability of spores was also examined in seven EMF using spore inoculation experiments. To determine the effects of the spore preservation period, both experiments were repeated up to 1 yr after spore collection. Spore germination was very low in the absence of host plants. In the presence of hosts, even 30 d after spore collection, spore germination was significantly enhanced in all pioneer EMF (c. 20%) but less so in late-stage EMF (< 5%), except in Hebeloma species. Mycorrhizal formation from spores was also greater in pioneer EMF but was significantly reduced by 1 yr of spore preservation. High spore germination and infectivity of pioneer EMF should enable these species to colonize disturbed and isolated areas in accordance with their ecological traits.

Community structure of arbuscular mycorrhizal fungi in a primary successional volcanic desert on the southeast slope of Mount Fuji

Community structure of arbuscular mycorrhizal fungi (AMF), evaluated as spore samples and mycorrhizal roots of four herbaceous plant species, was investigated at different altitudes in a primary successional volcanic desert on Mount Fuji using molecular methods (fragment and sequence analysis of the large ribosomal subunit RNA gene). In total, 17 different AMF clades were identified, and most were members of the Glomaceae, Acaulosporaceae, and Gigasporaceae. The AMF community structures detected by spore sampling were inconsistent with those from plant roots. Of all AMF clades, six (35.3%) were detected only on the basis of spores, six (35.3%) only in roots, and five corresponded to both spores and roots (29.4%). Although an Acaulospora species was the most dominant among spores (67.1%), it accounted for only 6.8% in root samples. A species analysis of AMF communities at different altitudes demonstrated that AMF species diversity increased as altitude decreased and that the species enrichment at lower altitudes resulted from the addition of new species rather than species replacement. The inconsistencies in the species composition of spore communities with those in roots and the change in species diversity with altitude are discussed.