Yoshihisa Suyama

Glacial Survival of Boreal Trees in Northern Scandinavia

Tree Refugia Ideas of how and when boreal plants spread to the formerly glaciated parts of the world following the retreat of the glaciers 9000 years ago are long debated. Models of the postglacial spread of boreal plants argue for dispersal from southern refugia; however, Parducci et al. (p. 1083) have shown that both spruce and pine were present in small ice-free regions of Scandinavia much earlier than thought. DNA haplotyping confirmed that a remnant mitochondrial type of spruce, once unique to Scandinavia, now lives alongside the more common spruce originating from Eastern Europe. Evidence from lake cores collected from central and northern Norway indicated the survival of conifers as early as 22,000 years before the present, when apart from ice-free pockets, most of Scandinavia was covered by ice. DNA from modern and ancient spruce and pine indicate that both survived in ice-free areas during the last glaciations. It is commonly believed that trees were absent in Scandinavia during the last glaciation and first recolonized the Scandinavian Peninsula with the retreat of its ice sheet some 9000 years ago. Here, we show the presence of a rare mitochondrial DNA haplotype of spruce that appears unique to Scandinavia and with its highest frequency to the west—an area believed to sustain ice-free refugia during most of the last ice age. We further show the survival of DNA from this haplotype in lake sediments and pollen of Trøndelag in central Norway dating back ~10,300 years and chloroplast DNA of pine and spruce in lake sediments adjacent to the ice-free Andøya refugium in northwestern Norway as early as ~22,000 and 17,700 years ago, respectively. Our findings imply that conifer trees survived in ice-free refugia of Scandinavia during the last glaciation, challenging current views on survival and spread of trees as a response to climate changes.

Clonal structure in a dwarf bamboo (Sasa senanensis) population inferred from amplified fragment length polymorphism (AFLP) fingerprints.

Amplified fragment length polymorphism (AFLP) fingerprints were used to reveal clonal structure of a dense population of dwarf bamboo, Sasa senanensis, in a 10‐ha study plot at Sugadaira Montane Research Center, University of Tsukuba, Nagano, Japan. We generated AFLP fingerprints for 51 leaf samples, collected at 50 m intervals, using three selective primer pairs. A total of 135–166 fragments were detected per sample, and 22 different fingerprints were identified based on 24–83 differing fragments. Our results demonstrate that the S. senanensis population in our plot consists of at least 22 clones and that the largest single clone occurs over a distance of about 300 m. Furthermore, the clone distribution pattern implies a relationship between site quality and clonal structure.

Ancient DNA from pollen: a genetic record of population history in Scots pine

Assessments of plant population dynamics in space and time have depended on dated records of fossil pollen synthesized on a subcontinental scale. Genetic analyses of extant populations have revealed spatial relationships that are indicative of past spatial dynamics, but lack an explicit timescale. Synthesis of these data requires genetic analyses from abundant dated fossil material, and this has hitherto been lacking. Fossil pollen is the most abundant material with which to fill this data gap. Here we report genetic analyses of fossil pollen retrieved from Holtjärnen postglacial lake sediment in Sweden and show that plastid DNA is recoverable from Scots Pine and Norway spruce pollen grains that are 100 and 10 000 years old. By sequencing clones from two short plastid PCR products and by using multiple controls we show that the ancient sequences were endogenous to the fossil grains. Comparison of ancient sequences and those obtained from an extant population of Scots pine establishes the first genetic link between extant and fossil samples in this species, providing genetic continuity through time. The finding of one common haplotype present in modern, 100‐year old and 10 000‐year old samples suggests that it may have persisted near Holtjärnen throughout the postglacial period. This retrieval of ancient DNA from pollen has major implications for plant palaeoecology in conifer species by allowing direct estimates of population dynamics in space and time.

Sequence-tagged-sites (STSs) of cDNA clones in Cryptomeria japonica and their evaluation as molecular markers in conifers

Abstract We have generated 66 sequence-tagged-site (STS) markers from cDNA clones of Cryptomeria japonica, and 60% of them have already been mapped into C. japonica linkage groups. All of the STS markers showed a single fragment following polymerase chain reaction (PCR) amplification. We investigated by polymorphism of these STS markers in a mapped F2 population and 15 plus trees by means of a restriction endonuclease analysis. Polymorphism levels were 10.6% and 22.7% in the F2 population and the 15 plus trees, respectively. PCR amplification levels of the 66 STS markers in 14 conifer species varied depending on their genetic relationship with C. japonica. Taxodium, which is closely related to C. japonica, had the most amplifications (31.82%), followed by Sequoiadendron giganteum, which is of the same family. The average proportion of PCR amplifications in each family gradually declined in the following order: from Taxodiaceae to Cuppresaceae, Sciadopityaceae, Pinaceae, and Taxaceae. These results are in general agreement with a molecular phylogenetic relationship based on chloroplast DNA. The 66 STS markers will be useful as on anchor point for genome mapping and population genetics, and some of them will also be useful when studying other conifers.

Selfing and inbreeding depression in seeds and seedlings of Neobalanocarpus heimii (Dipterocarpaceae).

We evaluated the degree of selfing and inbreeding depression at the seed and seedling stages of a threatened tropical canopy tree, Neobalanocarpus heimii, using microsatellite markers. Selection resulted in an overall decrease in the level of surviving selfed progeny from seeds to established seedlings, indicating inbreeding depression during seedling establishment. Mean seed mass of selfed progeny was lower than that of outcrossed progeny. Since the smaller seeds suffered a fitness disadvantage at germination in N. heimii, the reduced seed mass of selfed progeny would be one of the determinants of the observed inbreeding depression during seedling establishment. High selfing rates in some mother trees could be attributed to low local densities of reproductive individuals, thus maintenance of a sufficiently high density of mature N. heimii should facilitate regeneration and conservation of the species.

Defoliation effects on the community structure of arbuscular mycorrhizal fungi based on 18S rDNA sequences

The effects of defoliation on arbuscular mycorrhizal (AM) associations in the field were investigated in terms of the community structure of AM fungi colonizing roots of grassland plants; the carbohydrate balance of the host plants was also determined. We focused on two plant species dominating Japanese native grasslands: the grazing-intolerant species Miscanthus sinensis and the grazing-tolerant species Zoysia japonica. Community structures of AM fungi were determined from 18S rRNA gene sequences. The dominant fungal group in both plant species was the Glomus clade, which was classified into several phylogenetic groups based on genetic distances and topology. In Miscanthus roots, the Glomus-Ab, Glomus-Ac, and Glomus-Ad groups were detected almost equally. In Zoysia roots, the Glomus-Ab group was dominant. Defoliation effects on the community structure of AM fungi differed between the plant species. In Miscanthus roots, the percentage of root length colonized (%RLC) by the Glomus-Ac and Glomus-Ad groups was significantly reduced by defoliation treatment. On the other hand, AM fungal group composition in Zoysia roots was unaffected by defoliation except on the last sampling date. Decreased %RLC by Glomus-Ac and Glomus-Ad coincided with decreased non-structural carbohydrate (NSC) levels in host plants; also, significant positive correlations were found between the %RLC and some NSC levels. On the other hand, the %RLC by Glomus-Ab in both plant species was unaffected by the NSC level. These results suggest that AM fungal groups have different carbohydrate requirements from host plants.

Differentiation of mitochondrial DNA polymorphisms in populations of five Japanese Abies species

Mitochondrial DNA polymorphism of 40 populations of five Abies species was investigated using PCR‐amplified coxI and coxIII gene probes. Using four combinations of probe and restriction enzyme, we detected three major haplotypes and 15 total haplotypes. We also found varied levels of gene diversity for the different species: 0.741, 0.604, 0.039, 0.000, and 0.292 for A. firma, A. homolepis, A. veitchii, A. mariesii, and A. sachalinensis, respectively. The marginal and southern populations of A. firma and A. homolepis have unique haplotypes, especially the Kyushu, Shikoku, and Kii Peninsula populations, which inhabit areas coinciding with probable refugia of the last glacial period and possess high levels of mtDNA genetic diversity. The haplotypes in some populations suggested mtDNA capture also occurred between species through introgression/hybridization. The strong mtDNA population differentiation in Abies is most likely due to the maternal inheritance of mitochondria and restricted seed dispersal. A phenetic tree based on the genetic similarity of the mtDNA suggests that some species are polyphyletic. Based on mtDNA variation, the five Abies species could be divided roughly into three groups: (1) A. firma and A. homolepis, (2) A. veitchii and A. sachalinensis, and (3) A. mariesii. However, we found that all these Abies species, except A. mariesii, are genetically very closely related according to an analysis of their cpDNA sequences. This showed that the chloroplast rbcL gene differed by only one base substitutions among the four species. We believe that the mtDNA variation and cpDNA similarity clearly reflect relationships among, and the dissemination processes affecting these Abies species since the last glacial period.

Microsatellite Analysis of Japanese Sea Cucumber, Stichopus (Apostichopus) japonicus, Supports Reproductive Isolation in Color Variants

The genetic relationship among the three color variants (Red, Green, and Black) of the Japanese sea cucumber, S. japonicus, was investigated using 11 microsatellite markers. Genetic differentiation testing among the three sympatric color types showed the strong heterogeneity of Red (p < 0.001), while no significant difference was observed between Green and Black (p = 0.301 to 0.961). UPGMA trees constructed from 10 sample lots from 5 localities showed two distinct clusters, one from the Red types and the other from the Green and Black types. In addition, the sympatric Green and Black formed one subcluster with strong bootstrap support at each locality. These results indicate the separate species status of Red and the other color types, and also support the population identity of sympatric Green and Black.

A linkage map for sugi (Cryptomeria japonica) based on RFLP, RAPD, and isozyme loci

A linkage map for sugi was constructed on the basis of restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), and isozyme loci using a three-generation pedigree prepared for genetic analysis of heartwood color. A total of 128 RFLP (123 cDNA and 5 genomic probes), 33 RAPD, 2 isozyme, and 1 morphological (dwarf) loci segregated in 73 progeny. Of the 164 segregating loci, 145 loci were distributed in 20 linkage groups. Of these loci, 91 with confirmed map positions were assigned to 13 linkage groups, covering a total of 887.3 cM. A clustering of markers with distorted segregation was observed in 6 linkage groups. In the four clusters, distortions with a reduction in the number of homozygotes from one parent only were found.

Molecular‐ and pollen‐based vegetation analysis in lake sediments from central Scandinavia

Plant and animal biodiversity can be studied by obtaining DNA directly from the environment. This new approach in combination with the use of generic barcoding primers (metabarcoding) has been suggested as complementary or alternative to traditional biodiversity monitoring in ancient soil sediments. However, the extent to which metabarcoding truly reflects plant composition remains unclear, as does its power to identify species with no pollen or macrofossil evidence. Here, we compared pollen‐based and metabarcoding approaches to explore the Holocene plant composition around two lakes in central Scandinavia. At one site, we also compared barcoding results with those obtained in earlier studies with species‐specific primers. The pollen analyses revealed a larger number of taxa (46), of which the majority (78%) was not identified by metabarcoding. The metabarcoding identified 14 taxa (MTUs), but allowed identification to a lower taxonomical level. The combined analyses identified 52 taxa. The barcoding primers may favour amplification of certain taxa, as they did not detect taxa previously identified with species‐specific primers. Taphonomy and selectiveness of the primers are likely the major factors influencing these results. We conclude that metabarcoding from lake sediments provides a complementary, but not an alternative, tool to pollen analysis for investigating past flora. In the absence of other fossil evidence, metabarcoding gives a local and important signal from the vegetation, but the resulting assemblages show limited capacity to detect all taxa, regardless of their abundance around the lake. We suggest that metabarcoding is followed by pollen analysis and the use of species‐specific primers to provide the most comprehensive signal from the environment.