Ocean Currents Drove Genetic Structure of Seven Dominant Mangrove Species Along the Coastlines of Southern China.

Mangrove forest ecosystems, which provide important ecological services for marine environments and human activities, are being destroyed worldwide at an alarming rate. The objective of our study was to use molecular data and analytical techniques to separate the effects of historical and contemporary processes on the distribution of mangroves and patterns of population genetic differentiation. Seven mangrove species (Acanthus ilicifolius, Aegiceras corniculatum, Avicennia marina, Bruguiera gymnorrhiza, Kandelia obovata, Lumnitzera racemosa, and Rhizophora stylosa), which are predominant along the coastlines of South China, were genotyped at nuclear (nSSR) and chloroplast (cpSSR) microsatellite markers. We estimated historical and contemporary gene flow, the genetic diversity and population structure of seven mangrove species in China. All of these seven species exhibited few haplotypes, low levels of genetic diversity (H E = 0.160-0.361, with the exception of K. obovata) and high levels of inbreeding (F IS = 0.104-0.637), which may be due to their marginal geographical distribution, human-driven and natural stressors on habitat loss and fragmentation. The distribution patterns of haplotypes and population genetic structures of seven mangrove species in China suggest historical connectivity between populations over a large geographic area. In contrast, significant genetic differentiation [F ST = 0.165-0.629 (nSSR); G ST = 0.173-0.923 (cpSSR)] indicates that populations of mangroves are isolated from one another with low levels of contemporary gene flow among populations. Our results suggest that populations of mangroves were historically more widely inter-connected and have recently been isolated, likely through a combination of ocean currents and human activities. In addition, genetic admixture in Beibu Gulf populations and populations surrounding Hainan Island and southern mainland China were attributed to asymmetric gene flow along prevailing oceanic currents in China in historical times. Even ocean currents promote genetic exchanges among mangrove populations, which are still unable to offset the effects of natural and anthropogenic fragmentation. The recent isolation and lack of gene flow among populations of mangroves may affect their long-term survival along the coastlines of South China. Our study enhances the understanding of oceanic currents contributing to population connectivity, and the effects of anthropogenic and natural habitat fragmentation on mangroves, thereby informing future conservation efforts and seascape genetics toward mangroves.

Pathway and sink activity for photosynthate translocation in Pisolithus extraradical mycelium of ectomycorrhizal Pinus thunbergii seedlings.

The purpose of this study was to identify the pathway and sink activity of photosynthate translocation in the extraradical mycelium (ERM) of a Pisolithus isolate. We labelled ectomycorrhizal (ECM) Pinus thunbergii seedlings with (14)CO2 and followed (14)C distribution within the ERM by autoradiography. (14)C photosynthate translocation in the ERM resulted in (14)C distribution in rhizomorphs throughout the ERM, with (14)C accumulation at the front. When most radial mycelial connections between ECM root tips and the ERM front were cut, the whole allocation of (14)C photosynthates to the ERM was reduced. However, the overall pattern of (14)C distribution in the ERM was maintained even in regions immediately above and below the cut, with no local (14)C depletion or accumulation. We inferred from this result that every portion in the ERM has a significant sink activity and a definite sink capacity for photosynthates and that photosynthates detour the cut and reach throughout the ERM by translocation in every direction. Next, we prepared paired ECM seedlings, ERMs of which had been connected with each other by hyphal fusion, alongside, labelled the left seedling with (14)CO2, and shaded none, one or both of them. (14)C photosynthates were acropetally and basipetally translocated from the left ERM to ECM root tips of the right seedling through rhizomorphs in the left and right ERMs, respectively. With the left seedling illuminated, (14)C translocation from the left to the right ERM increased by shading the right seedling. This result suggests that reduced photosynthate transfer from the host to its ERM increased sink activity of the ERM.

Genet dynamics and ecological functions of the pioneer ectomycorrhizal fungi Laccaria amethystina and Laccaria laccata in a volcanic desert on Mount Fuji.

To understand the reproduction of the pioneer ectomycorrhizal fungi Laccaria amethystina and Laccaria laccata in a volcanic desert on Mount Fuji, Japan, the in situ genet dynamics of sporocarps were analysed. Sporocarps of the two Laccaria species were sampled at fine and large scales for 3 and 2 consecutive years, respectively, and were genotyped using microsatellite markers. In the fine-scale analysis, we found many small genets, the majority of which appeared and disappeared annually. The high densities and annual renewal of Laccaria genets indicate frequent turnover by sexual reproduction via spores. In the large-scale analysis, we found positive spatial autocorrelations in the shortest distance class. An allele-clustering analysis also showed that several alleles were distributed in only a small, localised region. These results indicate that Laccaria spores contributing to sexual reproduction may be dispersed only short distances from sporocarps that would have themselves been established via rare, long-distance spore dispersal. This combination of rare, long-distance and frequent, short-distance Laccaria spore dispersal is reflected in the establishment pattern of seeds of their host, Salix reinii.

Fine-scale distribution of ectomycorrhizal fungi colonizing Tsuga diversifolia seedlings growing on rocks in a subalpine Abies veitchii forest.

Numerous species of ectomycorrhizal (ECM) fungi coexist under the forest floor. To explore the mechanisms of coexistence, we investigated the fine-scale distribution of ECM fungal species colonizing root tips in the root system of Tsuga diversifolia seedlings in a subalpine forest. ECM root tips of three seedlings growing on the flat top surface of rocks were sampled after recording their positions in the root system. After the root tips were grouped by terminal-restriction fragment length polymorphism (T-RFLP) analysis of ITS rDNA, the fungal species representing each T-RFLP group were identified using DNA sequencing. Based on the fungal species identification, the distribution of root tips colonized by each ECM fungus was mapped. Significant clustering of root tips was estimated for each fungal species by comparing actual and randomly simulated distributions. In total, the three seedlings were colonized by 40 ECM fungal species. The composition of colonizing fungal species was quite different among the seedlings. Twelve of the 15 major ECM fungal species clustered significantly within a few centimeters. Some clusters overlapped or intermingled, while others were unique. Areas with high fungal species diversity were also identified in the root system. In this report, the mechanisms underlying generation of these ECM root tip clusters in the root system are discussed.

Structural and functional interactions between extraradical mycelia of ectomycorrhizal Pisolithus isolates.

Extraradical mycelia from different ectomycorrhizal (ECM) roots coexist and interact under the forest floor. We investigated structural connections of conspecific mycelia and translocation of carbon and phosphorus between the same or different genets. Paired ECM Pinus thunbergii seedlings colonized by the same or different Pisolithus isolates were grown side by side in a rhizobox as their mycelia contacted each other. (14)CO(2) or (33)P-phosphoric acid was fed to leaves or a spot on the mycelium in one of the paired seedlings. Time-course distributions of (14)C and (33)P were visualized using a digital autoradiographic technique with imaging plates. Hyphal connections were observed between mycelia of the same Pisolithus isolate near the contact site, but hyphae did not connect between different isolates. (14)C and (33)P were translocated between mycelia of the same isolate. In (33)P-fed mycelia, accumulation of (33)P from the feeding spot toward the host ECM roots was observed. No (14)C and (33)P translocation occurred between mycelia of different isolates. These results provide direct evidence that contact and hyphal connection between mycelia of the same ECM isolate can cause nutrient translocation. The ecological significance of contact between extraradical mycelia is discussed.

Transfer of 14C-photosynthate to the sporocarp of an ectomycorrhizal fungus Laccaria amethystina.

Sporocarps of ectomycorrhizal fungi are strong carbon sinks for the source in host trees, but the details of carbon transfer from the host to the sporocarp are unknown. In this study, single seedlings of Japanese red pine (Pinus densiflora) colonised by Laccaria amethystina were grown on floral foam plates fitted in rhizoboxes, resulting in fruiting on the substrate. The seedlings were photosynthetically labelled with (14)CO(2); (14)C-labelled photosynthate transfer from leaves to sporocarps was then chased using a time-course autoradiography technique. (14)C was transferred to healthy, fresh sporocarps in a purple colour ranging from primordial to elongate sporocarps, but hardly to senesced ones that had faded to white or grey, or browned. This suggested that C is transferred only to physiologically active sporocarps. Two seedlings associated with a growing sporocarp were labelled again 7 and 16 days after the first labelling, respectively. (14)C accumulation in the sporocarps rose in a stepwise manner after the second labelling, indicating that sporocarps mainly used recently rather than previously photosynthesised C.

Spatial distribution of Raffaelea quercivora in xylem of naturally infested and inoculated oak trees.

Mass mortality of Japanese oak caused by Raffaelea quercivora due to Japanese oak wilt (JOW) has been tremendous since the late 1980s in Japan. We investigated detailed distribution of R. quercivora hyphae in a naturally infected Quercus serrata tree using fluorescein-conjugated wheat germ agglutinin and also examined spatial relationships between hyphal distribution, water conduction loss, and host reactions in xylem in inoculated Q. crispula saplings. Hyphae of R. quercivora elongated longitudinally in vessels and transversely in ray tissues in both naturally infected and inoculated Quercus trees. Hyphae were confined within a relatively small area near the inoculation site. Vessel dysfunction was also confined and overlapped with hyphal distribution. The reaction zone consisting of two types of fluorescent substance was formed surrounding the outside of the hyphal area in xylem and was always found in non-water-conductive zone. These results suggest that wilting of Quercus trees caused by JOW may not be induced by dysfunction of a small number of vessels, but by that of many vessels, and it requires that R. quercivora hyphae spread from many galleries bored by beetles during mass attacks.

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.

Nuclear and chloroplast microsatellite analysis of Abies sachalinensis regeneration on fallen logs in a subboreal forest in Hokkaido, Japan.

Fallen logs are the main regeneration sites for Abies sachalinensis (Sachalin fir) in subboreal forests in Japan. We surveyed the spatial genetic structure of different demographic stages in a 4.29-ha natural population of A. sachalinensis. Genetic structure was significant at short distances throughout all stages in this wind-dispersed conifer. To evaluate the effects of fallen-log-dependent recruitment on demography and spatial genetic structure, we conducted parentage analysis of offspring with highly polymorphic nuclear simple sequence repeat and chloroplast simple sequence repeat markers, and developed a new hierarchical Bayesian model to estimate the effects of mother trees and fallen logs on seed dispersal and offspring recruitment. Combined application of nuclear and chloroplast simple sequence repeat markers allowed us to unambiguously identify mother trees of most offspring (> 90%). Female reproductive success was extremely skewed; a few adults produced most of the offspring on fallen logs. Limited distance of effective dispersal and recruitment was seen in the parentage analysis and modelling estimation. These recruitment characters of A. sachalinensis, with a fallen-log-dependent recruitment process, may result in significant genetic structure in early demographic stages.

Mating system, pollen and propagule dispersal, and spatial genetic structure in a high-density population of the mangrove tree Kandelia candel.

Mangrove tree species form ecologically and economically important forests along the tropical and subtropical coastlines of the world. Although low intrapopulation genetic diversity and high interpopulation genetic differentiation have been detected in most mangrove tree species, no direct investigation of pollen and propagule dispersal through paternity and/or parentage analysis and spatial genetic structure within populations has been conducted. We surveyed the mating system, pollen and propagule dispersal, and spatial genetic structure in a natural population of Kandelia candel, one of the typical viviparous mangrove tree species, using nuclear and chloroplast microsatellite markers. High diversity and outcrossing rates were observed. Paternity and parentage analysis and modelling estimations revealed the presence of an extremely short-distance component of pollen and propagule dispersal (pollen: 15.2+/-14.9 m (SD) by paternity analysis and 34.4 m by modelling; propagule: 9.4+/-13.8 m (SD) by parentage analysis, and 18.6 m by modelling). Genetic structure was significant at short distances, and a clumped distribution of chloroplast microsatellite genotypes was seen in K. candel adults. We conclude that the K. candel population was initiated by limited propagule founders from outside by long-distance dispersal followed by limited propagule dispersal from the founders, resulting in a half-sib family structure.

Isolation of compound microsatellite loci in the herbaceous perennial Cirsium purpuratum (Maxim.) Matsum.

The herbaceous perennial Cirsium purpuratum is a pioneer on the southeast side of Mount Fuji in Japan. For genetic analysis of reproduction in this species, we developed polymorphic compound microsatellite markers using an adaptor-ligated library method and a simpler method called the intercompound microsatellite method. The latter method was an effective method for developing compound simple sequence repeat markers. In total, 11 polymorphic, codominant microsatellite markers were developed and characterized for this species. These polymorphic markers had three to 20 alleles per locus, a range of observed heterozygosity from 0.25 to 0.90, and were considered effective for genetic analysis.

Flavonoids induce germination of basidiospores of the ectomycorrhizal fungus Suillus bovinus.

Under laboratory conditions, spores of ectomycorrhizal fungi usually germinate very poorly or not at all. In a previous study, we showed that spores of the ectomycorrhizal fungus Suillus bovinus germinated through the combination of activated charcoal treatment of media and co-culture with seedlings of Pinus densiflora, which suggested that some substances contained in root exudates induced the germination. Among the compounds reported from root exudates, flavonoids have been elucidated to play various and substantial roles in plant-microbe interactions; we therefore investigated the effects of flavonoids on basidiospore germination of S. bovinus by the diffusion gradient assay on water agar plates pretreated with charcoal powder. Seven out of the 11 flavonoids tested, hesperidin, morin, rutin, quercitrin, naringenin, genistein, and chrysin, had greater effects than controls, whereas flavone, biochanin A, luteolin, and quercetin showed no positive effects. The effective concentration presumably corresponded to several micromolar levels, which was equivalent to those effective for pollen development, nod gene induction, and spore germination of F. solani f. sp. pisi and AM fungi. The results suggest that flavonoids play a role as signaling molecules in symbiotic relationships between woody plants and ectomycorrhizal fungi.

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.

Host effects on ectomycorrhizal fungal communities: insight from eight host species in mixed conifer-broadleaf forests.

To advance our understanding of host effects on the community structure of ectomycorrhizal fungi (EMF), EMF communities were compared among different host species, genera and families in two mixed conifer-broadleaf forests in Japan. Using molecular identification methods we examined EMF root tips of eight coexisting species belonging to six genera (three families): Abies and Tsuga (Pinaceae), Betula and Carpinus (Betulaceae) and Fagus and Quercus (Fagaceae). In total, 205 EMF species were detected, and the total richness was estimated to exceed 300 species using major estimators. Of the 55 EMF species occurring three or more times, eight showed significantly biased host preference. A Mantel test showed a significantly negative correlation between EMF community similarity and host taxonomic distance. Detrended correspondence analysis separated EMF communities mainly by host taxonomic and successional status. Thus, EMF communities are similar on hosts with similar taxonomic and successional status. A significant proportion of EMF exhibited host specificity, which may contribute to the extremely diverse EMF community in conifer-broadleaf forests.

Genetic Structure of Populations of the Pinewood Nematode Bursaphelenchus xylophilus, the Pathogen of Pine Wilt Disease, Between and Within Pine Forests.

ABSTRACT We analyzed the genetic structure of Bursaphelenchus xylophilus populations within individual trees (subpopulations) in three distant pine forests (Tanashi, Tsukuba, and Chiba in Japan) based on the polymorphism of four microsatellite (SSR) markers. Most of the nematodes from subpopulations in Tanashi showed the same genotype over 2 years, indicating that nematodes of that genotype dominated there for years. In contrast, 16 and 15 genotypes were identified in nematode populations from Tsukuba and Chiba, respectively. Despite the high genetic diversity within the Tsukuba and Chiba populations, extremely low genetic diversity was observed within the subpopulations. The genetic difference between the Tsukuba and Chiba populations was significantly smaller than that between Tanashi and either Tsukuba or Chiba. Observed heterozygosity was significantly less than expected based on Hardy-Weinberg equilibrium. These findings are best explained by a founder effect, geographic isolation between populations, explosive nematode multiplication from a small number within individual trees, and the Wahlund effect.

Tricholoma matsutake in a natural Pinus densiflora forest: correspondence between above- and below-ground genets, association with multiple host trees and alteration of existing ectomycorrhizal communities.

Tricholoma matsutake (matsutake) is an ectomycorrhizal (ECM) fungus that produces economically important mushrooms in Japan. Here, we use microsatellite markers to identify genets of matsutake sporocarps and below-ground ECM tips, as well as associated host genotypes of Pinus densiflora. We also studied ECM fungal community structure inside, beneath and outside the matsutake fairy rings, using morphological and internal transcribed spacer (ITS) polymorphism analysis. Based on sporocarp samples, one to four genets were found within each fairy ring, and no genetic differentiation among six sites was detected. Matsutake ECM tips were only found beneath fairy rings and corresponded with the genotypes of the above-ground sporocarps. We detected nine below-ground matsutake genets, all of which colonized multiple pine trees (three to seven trees per genet). The ECM fungal community beneath fairy rings was species-poor and significantly differed from those inside and outside the fairy rings. We conclude that matsutake genets occasionally establish from basidiospores and expand on the root systems of multiple host trees. Although matsutake mycelia suppress other ECM fungi during expansion, most of them may recover after the passage of the fairy rings.

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.

Genetic structure of Cenococcum geophilum populations in primary successional volcanic deserts on Mount Fuji as revealed by microsatellite markers.

Polymorphic simple sequence repeat (SSR) markers were used to investigate the genetic structure in a Cenococcum geophilum population associated with Salix reinii in an early successional volcanic desert at Gotenba, on the south-eastern slope of Mount Fuji in Japan, and in three other populations associated with the same host at more developed sites on the mountain, one at Fujinomiya and two at Subashiri. The genotype richness of C. geophilum tended to be higher in more developed vegetation patches as well as in more developed sites, suggesting that genotype richness increased with advanced succession because new genotypes might have been introduced into these sites over time. High genotypic similarity was observed between the Gotenba and Fujinomiya populations but not between the Gotenba and Subashiri populations, suggesting that C. geophilum genotypes in Gotenba were introduced from the direction of Fujinomiya. Genotypes in the Gotenba population were clearly distinguishable into two groups. The absence of any intermediate genotype suggests the absence of frequent recombination in this C. geophilum population associated with early successional vegetation.

Root hydraulic conductivity and whole-plant water balance in tropical saplings following a shade-to-sun transfer.

We hypothesized that pioneer and late successional species show different morphological and physiological responses in water use after gap formation. The magnitude of the responses was compared between two pioneer species (Macaranga gigantea and Trema orientalis) and four late successional species (Shorea sp.), in an experiment in which saplings were transferred from shade to sun. Although transpiration demand increased following the transfer, root hydraulic conductivity (Lp(r)) decreased. Lp(r) was sensitive to brief treatments with HgCl(2) (a specific inhibitor of aquaporins). This allows Lp(r) to be divided into two components: cell-to-cell and apoplastic pathways. The Lp(r) of cell-to-cell pathway decreased in all species following the transfer, relating to aquaporin depression in roots. Following the transfer, leaf osmotic potentials at full hydration decreased and both leaf mass per area [leaf mass/leaf area (LMA)] and fine-root surface area/leaf surface area (root SA/leaf SA) increased in almost all species, allowing saplings to compensate for the decrease in Lp(r). Physiologically, pioneer species showed larger decreases in Lp(r) and more effective osmotic adjustment than late successional species, and morphologically, pioneer species showed larger increases in root SA/leaf SA and LMA. Water balance at the whole-plant level should be regulated by coupled responses between the aboveground and the belowground parts. Interspecific differences in responses after gap formation suggest niche differentiation in water use between pioneer and late successional species in accordance with canopy-gap size.

High somatic instability of a microsatellite locus in a clonal tree, Robinia pseudoacacia.

Robinia pseudoacacia L. is a clonal tree species. To investigate a mutation within eight microsatellite loci of R. pseudoacacia, we analyzed DNA samples obtained from different leaf samples within each ramet, leaves from ramets within the genet, and seeds. Of the eight loci, locus Rops15 (AG motif) displayed hypermutability. The mutation rates of Rops15 within each ramet, among ramets within the genet, and offspring were 6.27% (ranging from 0 to 31.1%), 6.11% (from 0 to 25.0%) and 3.78% (from 0 to 10.9%), respectively. The mutation rate increased with allele size (13-71 repeat units). The mutation patterns observed in Rops15 were distinctive in two ways. First, there was a significant bias toward additions over deletions, and both addition and deletion of single repeats were dominant at alleles with lengths less than 232 bp (63 repeats). Second, for the longest allele of 248 bp (71 repeats), the number of losses was higher than the number of gains. These observations suggest that the mutation patterns of microsatellites in R. pseudoacacia may follow a generalized stepwise mutation model, and that the tendency of long alleles to mutate to shorter lengths acts to prevent infinite growth. Finally, the observation of somatic hypermutability at locus Rops15 highlights the need for caution when using highly polymorphic microsatellites for population genetic structure and paternity analysis in tree species.