QTL analysis of heterostyly in Primula sieboldii and its application for morph identification in wild populations.

BACKGROUND AND AIMS: Primula sieboldii is a perennial clonal herb that is distributed around the Sea of Japan and is endangered in Japan. Its breeding system is characterized by heteromorphic self-incompatibility, and the morph ratio within a population is very important for reproductive success. The aims of this study were to construct a linkage map, map the S locus as a qualitative trait and quantitative trait loci (QTLs) for floral morphological traits related to heterostyly, and predict the morph type in wild populations by using molecular markers for devising a conservation strategy. METHODS: A linkage map was constructed with 126 markers. The QTLs for four floral traits and the S locus were mapped. Using the genotypes of loci that were located near both the S locus and the QTLs with large effects, morphs of 59 wild genets were predicted. KEY RESULTS: The linkage map consisted of 14 linkage groups (LGs). The S locus was mapped to LG 7. Major QTLs for stigma and anther heights were detected in the same region as the S locus. These QTLs exhibited high logarithm of the odds scores and explained a high percentage of the phenotypic variance (>85 %). By analysing these two traits within each morph, additional QTLs for each trait were detected. Using the four loci linked to the S locus, the morphs of 43 genets in three wild populations could be predicted. CONCLUSIONS: This is the first report of a linkage map and QTL analysis for floral morphology related to heterostyly in P. sieboldii. Floral morphologies related to heterostyly are controlled by the S locus in LG 7 and by several QTLs in other LGs. Additionally, this study showed that molecular markers are effective tools for investigating morph ratios in a population containing the non-flowering individuals or during the non-flowering seasons.

Do Small Canopy Gaps Created by Japanese Black Bears Facilitate Fruiting of Fleshy-Fruited Plants?

Japanese black bears often break branches when climbing trees and feeding on fruit in canopies, thereby creating small canopy gaps. However, the role of black bear-created canopy gaps has not been evaluated in the context of multiple forest dynamics. Our hypothesis was that small canopy gaps created by black bears improve light conditions, which facilitates fruiting of adult fleshy-fruited plants located beneath the gaps, and also that this chain interaction depends on interactions among the size of gaps, improved light conditions, forest layers, and life form of plants. The rPPFD, size of black bear-created canopy gaps, and fruiting/non-fruiting of fleshy-fruited plants were investigated in five forest layers beneath black-bear-created canopy gaps and closed canopies of Mongolian oak (Quercus crispula). We found that light conditions improved beneath black bear-disturbed trees with canopy gaps of large size, and the effect of improvement of light conditions was reduced with descending forest layers. Fruiting of fleshy-fruited plants, especially woody lianas and trees, was facilitated by the improvement of light conditions accompanied by an increase in the size of black-bear-created gaps. Data from this study revealed that canopy disturbance by black bears was key for improving light conditions and accelerating fruiting of fleshy-fruited trees and woody lianas in the canopy layers in particular. Therefore, our hypothesis was mostly supported. Our results provide evidence that Japanese black bears have high potential as ecosystem engineers that increase the availability of resources (light and fruit in this study) to other species by causing physical state changes in biotic materials (branches of Q. crispula in this study).

Field fate of Amaranthus patulus seeds subjected to leaf-canopy inhibition of germination.

The germination of seeds of Amaranthus patulus Bertol., is known to be sensitive to leaf-transmitted light. Seeds were enclosed in transparent polyester-mesh envelopes and placed horizontally in 10-cm deep soil or on the soil surface, beneath a closed vegetation cover in the field. Changes in the numbers of firm intact seeds and of germinable seeds were traced for up to 3 years by periodical retrievals and germination tests. Rapid loss of germinable seeds, mainly due to germination, was observed in the buried seed population, in which only 20% of seeds maintained their germinability after 1 year, and a negligible number after 3 years. In contrast, the seeds placed on the soil surface maintained germinability relatively well: over 80% of seeds remained germinable after 1 year and a low percentage still preserved their germinability after 3 years. Assuming exponential decay in germinability, the decay rates on and in the soil were calculated from the data of the 1-year experiment to be 0.21 and 0.84 year-1 respectively. The fate of seeds that were exposed to canopy light on the soil for a month and then buried was shown to be almost the same as that of the seeds which had been continuously in 10-cm deep soil. Correspondingly, the possibility of the induction of secondary (induced) dormancy by exposure to canopy light was excluded in a laboratory experiment, in which it was found that the imbibed seeds suffering leaf-canopy inhibition of germination exuded some diffusible germination inhibitor responsible for apparent dormancy. Estimation of numbers of A. patulus in the seed bank of an early successional field showed that 3,500 seeds/m2 remained in the soil to the depth of 10 cm after 3 years' exclusion of the species following the production of 700,000 seeds/m2, by a population explosively established after experimental induction of secondary succession.

Differentiation of spring emerging and autumn emerging ecotypes in Galium spurium L. var. echinospermon.

Ecotypes of Galium spurium L. var. echinospermon with distinct germination phenologies were found to occur in two adjacent plots of a grassland nature reserve with different management histories: a spring-germinating population is present in a winter-burnt plot, and an autumn-germinating type in an unburnt plot. These ecotypes share common flowering and fruiting phenologies, and disperse their seeds in early summer. Markedly contrasting thermal dormancy/germination characteristics were demonstrated for their seeds in systematic laboratory tests performed after several types of seed storage including storage in the field. The primary dormancy of seeds of the spring germinator was removed by moist-chilling or field winter-chilling, while that of the autumn germinator was removed by moist storage at 25°C or field summer temperatures. Biseasonal seedling emergence of the species appears to be due to a local differentiation of distinct ecotypes.

Effects of light and soil water availability on leaf photosynthesis and growth of Arisaema heterophyllum, a riparian forest understorey plant.

The effects of soil-water availability on leaf light acclimation and whole-plant carbon gain were examined in Arisaema heterophyllum Blume, a riparian deciduous forest understorey plant. Photosynthesis, above-ground morphology and ramet biomass accumulation (relative growth rate: RGR of a corm for a full leaf life-span) were measured on plants raised under three light treatments combined with two soil water conditions. The two higher light treatments during growth (high: max. 550 micro mol photons m(-2) s(-1); medium: 150 micro mol photons m(-2) s(-1)) resulted in a twofold increase in RGRs, 30% higher photosynthetic capacities and 20% less photosynthetic low-light use efficiency than those under a low light condition (50 micro mol photons m(-2) s(-1)). Leaf area was the smallest and leaf mass area ratio was the largest under the high light treatment. Water stress decreased both photosynthetic rate and leaf area and, hence, RGR in all the light regimes. However, water stress did not alter the general patterns of physiological and morphological responses to different light regimes. We estimated that higher photosynthetic low-light use efficiency and larger leaf area in the low light leaf would lead to a threefold carbon gain as compared with the high light leaf under simulated low light conditions. Both experimental and simulation results suggest that the physiological and morphological acclimations tend to be beneficial to carbon gain when light availability is low, whereas they favor increased water use efficiency when light availability is sufficiently high.

Comparison of generalist predators in winter-flooded and conventionally managed rice paddies and identification of their limiting factors.

Winter-flooding of rice paddies without the application of agricultural chemicals is attracting attention as a new agricultural method for enhancing the habitat conditions of wintering waterfowl in rice paddy ecosystems throughout Japan and east Asia. Conditions in these paddies are expected to result in restoration of not only the winter habitats of waterfowl but also those of other taxonomic groups during the rice growing season. In this study, we tested whether the diversity of summer spiders--ubiquitous predators in rice paddies--was higher in the winter-flooded paddies than in the conventional ones by conducting field measurements in 31 winter-flooded and 7 conventional paddies. Limiting factors of spiders in the winter-flooded paddies were then examined. Results revealed that both the density and species richness of spiders were significantly higher in the winter-flooded paddies than in the conventional ones both before and after the insecticide application against pecky rice bug Stenotus rubrovittatus (Matsumura)(Hemiptera: Miridae) to conventional paddies. In addition, spider density and species richness in the winter-flooded paddies correlated with the availability of two prey groups--chironomids and other nematocera. These findings suggest that in the winter-flooded paddies the diversity of generalist predators is higher than in the conventional ones during the rice-growing season and that the combination of management at both the landscape and field level is likely more effective for increasing spider abundance in winter-flooded paddies.

Asymmetric dispersal structures a riverine metapopulation of the freshwater pearl mussel Margaritifera laevis.

Unidirectional water flow results in the downstream-biased, asymmetric dispersal of many riverine organisms. However, little is known of how asymmetric dispersal influences riverine population structure and dynamics, limiting our ability to properly manage riverine organisms. A metapopulation of the freshwater pearl mussel Margaritifera laevis may be sensitive to river currents because mussels are repeatedly exposed to downstream drift during floods-a parasitic life stage is the only, limited period (∼40 days) during which larvae (glochidia) can move upstream with the aid of host fish. We hypothesized that water-mediated dispersal would overwhelm upstream dispersal via host fish, and therefore, that upstream subpopulations play a critical role as immigrant sources. To test this hypothesis, we examined the effects of both up- and downstream immigrant sources on the size of target subpopulations in the Shubuto River system, Hokkaido, Japan. We found that target subpopulation size was dependent on the upstream distribution range of reproductive subpopulations and the number of upstream tributaries, which are proxies for the number of potential immigrants moving downstream. In contrast, little influence was observed of downstream immigrant sources (proximity to downstream reproductive subpopulations). These results were consistent even after accounting for local environments and stream size. Our finding suggests that upstream subpopulations can be disproportionately important as immigrant sources when dispersal is strongly asymmetric.

Facilitation of a native pest of rice, Stenotus rubrovittatus (Hemiptera: Miridae), by the non-native Lolium multiflorum (Cyperales: Poaceae) in an agricultural landscape.

Source populations of polyphagous pests often occur on host plants other than the economically damaged crop. We evaluated the contribution of patches of a non-native meadow grass, Lolium multiflorum Lam. (Poaceae), and other weeds growing in fallow fields or meadows as source hosts of an important native pest of rice, Stenotus rubrovittatus (Matsumura) (Hemiptera: Miridae), in an agricultural landscape of northern Japan. Periodical censuses of this mirid bug by using the sweeping method, vegetation surveys, and statistical analysis revealed that L. multiflorum was the only plant species that was positively correlated with the density of adult S. rubrovittatus through two generations and thus may be the most stable and important host of the mirid bug early in the season before the colonization of rice paddies. The risk and cost of such an indirect negative effect on a crop plant through facilitation of a native pest by a non-native plant in the agricultural landscape should not be overlooked.

Contrasting impacts of invasive engineers on freshwater ecosystems: an experiment and meta-analysis.

Invasion by common carp (Cyprinus carpio) and red swamp crayfish (Procambarus clarkii) in shallow lakes have been followed by stable-state changes from a macrophyte-dominated clear water state to a phytoplankton-dominated turbid water state. Both invasive carp and crayfish are, therefore, possible drivers for catastrophic regime shifts. Despite these two species having been introduced into ecosystems world-wide, their relative significance on regime shifts remains largely unexplored. We compared the ecological impacts of carp and crayfish on submerged macrophytes, water quality, phytoplankton, nutrient dynamics, zooplankton and benthic macroinvertebrates by combining an enclosure experiment and a meta-analysis. The experiment was designed to examine how water quality and biological variables responded to increasing carp or crayfish biomass. We found that even at a low biomass, carp had large and positive impacts on suspended solids, phytoplankton and nutrients and negative impacts on benthic macroinvertebrates. In contrast, crayfish had a strong negative impact on submerged macrophytes. The impacts of crayfish on macrophytes were significantly greater than those of carp. The meta-analysis showed that both carp and crayfish have significant effects on submerged macrophytes, phytoplankton, nutrient dynamics and benthic macroinvertebrates, while zooplankton are affected by carp but not crayfish. It also indicated that crayfish have significantly greater impacts on macrophytes relative to carp. Overall, the meta-analysis largely supported the results of the experiment. Taken as a whole, our results show that both carp and crayfish have profound effects on community composition and ecosystem processes through combined consequences of bioturbation, excretion, consumption and non-consumptive destruction. However, key variables (e.g. macrophytes) relating to stable-state changes responded differently to increasing carp or crayfish biomass, indicating that they have differential ecosystem impacts.

TAXONOMIC REEXAMINATION OF CHARA GLOBULARIS (CHARALES, CHAROPHYCEAE) FROM JAPAN BASED ON OOSPORE MORPHOLOGY AND rbcL GENE SEQUENCES, AND THE DESCRIPTION OF C. LEPTOSPORA SP. NOV.(1).

Chara globularis Thuillier (=f. globularis sensu R. D. Wood) is a widespread species of the genus and inhabits fresh- and brackish-water environments. In an attempt to reexamine the taxonomic status of C. globularis collected from Japan, we reassessed vegetative and oospore morphology of Japanese material and herbarium specimens originating from Europe (including the type specimen) and conducted molecular phylogenetic analyses based on rbcL gene sequences. Although the other vegetative morphologies were consistent with the description of C. globularis f. globularis sensu R. D. Wood, we identified two types of branchlets within the Japanese materials: one has elongate end segments (EL type), and the other has short end segments (SH type) corresponding to the type material. Moreover, the oospore wall of the EL type was different from that in the SH type. The oospores of the EL type were dark brown to reddish brown and had a spongy pattern with the pusticular elevations on the fossa wall, whereas the fossa wall of the SH type was black with a granulate to papillate or fine pusticular pattern. In addition, our sequence data demonstrated that these two types are separated phylogenetically from each other. Therefore, we describe the EL type as a new species, C. leptospora sp. nov.

Characteristics of leaf photosynthesis and simulated individual carbon budget in Primula nutans under contrasting light and temperature conditions.

Primula nutans Georgi is widely distributed in hummock-and-hollow wetlands on the Qinghai-Tibetan Plateau. To assess the ecophysiology of this species in responding to microenvironments, we examined the photosynthetic characteristics and individual carbon gain of plants growing in different microsites from a hummock-and-hollow wetland on the Qinghai-Tibetan Plateau and under laboratory conditions. Plants from wetland hummock microsites showed significantly higher light-saturated photosynthetic CO(2) uptake (A (max)) than those from microsites in hollows at a controlled temperature of 15 degrees C in leaf chamber. Leaf dark respiration rate (R) was only significantly higher in plants from hummocks than hollows at the measuring temperature of 35 degrees C. Optimum temperature for A (max) was 15 degrees C for all plants in the field despite different microsites. In plants growing under laboratory conditions differing in light and temperature, both A (max) and R were significantly higher under higher growth light (photosynthetic photon flux density, PPFD: 800 or 400 micromol m(-2) s(-1)) than low light of 90 micromol m(-2) s(-1). No statistically significant differences in A (max) and R existed in plants differing in growing temperatures. Estimates derived from the photosynthetic parameters of field plants, and microsite environmental measures including PPFD, air temperature and soil temperature showed that the optimum mean daily temperature for net daily carbon gain was around 10 degrees C and the net daily carbon gain was largely limited under lower daily total PPFD. These results suggest that the differences in A (max) and R in P. nutans are strongly affected by growing light regimes but not by temperature regimes.

Phenological changes in rate of respiration and annual carbon balance in a perennial herbaceous plant, Primula sieboldii.

Primula sieboldii E. Morren is a clonal herbaceous species with a short foliar period from spring to early summer. We have studied the temperature-dependence of the rate of respiration at the whole-ramet level throughout the phenological stages of P. sieboldii to reveal its photosynthate-utilization strategy. P. sieboldii ramets were grown in a chamber enabling simulation of seasonal changes in temperature. Rates of respiration were measured at three phenological stages--the foliar period, the before-chilling defoliated (BCD) period, and the after--chilling defoliated (ACD) period. In the foliar period the rate of respiration, on a biomass basis at 20 degrees C (R (20)), of the above-ground plant parts was 2.5 times that of the below-ground parts. The R (20) of the below-ground parts in the foliar period was 6.5 times that in the BCD period and 1.6 times that in the ACD period. Estimation of the ramet carbon balance under these growth conditions showed that ramets respired 87% of total photosynthate production during the experimental period (8.5 months). Respiratory consumption in the foliar period accounted for 70% of the yearly total, whereas 24 and 6% were consumed in the BCD and ACD periods, respectively. An extremely low rate of respiration during the long defoliated period led to a positive net annual carbon balance for P. sieboldii ramets.

Morphological plasticity of Primula nutans to hummock-and-hollow microsites in an alpine wetland.

Hummock-and-hollow microtopography is common in wetlands of the Qinghai-Tibetan Plateau. The physical environment of hummocks contrasts strongly with that of hollows. To address how Primula nutans Georgi, a herbaceous species broadly distributed on the plateau, can inhabit both hummocks and hollows, we investigated the plasticity of its morphology and biomass growth in relation to the environmental variables during the growing season. The results are as follows: (1) total daily photosynthetic photon flux density, maximum soil temperature, and diurnal soil temperature range were significantly larger on the hummocks than in the hollows; (2) individual ramets had smaller leaves, higher leaf mass per unit area, and shorter petiole and peduncle length on the hummocks, but leaf and root dry weights per ramet differed little between microsites; (3) P. nutans allocated most of its dry mass to peduncles and flowers and the proportion of reproductive mass was significantly higher for plants in the hollows than on the hummocks; (4) the coefficients of variation of most morphological and biometric parameters was higher on hummocks than in hollows. The results suggest that P. nutans favors hummocks but can tolerate hollows through morphological adjustments in that the mass investment to peduncles and flowers is much larger in plants in the hollows than hummocks.

Effect of flowering phenology on pollen flow distance and the consequences for spatial genetic structure within a population of Primula sieboldii (Primulaceae).

To evaluate the effects of flowering phenology on pollen flow distance and spatial genetic structure in a population of a bumblebee-pollinated herb, Primula sieboldii, we investigated the flowering phenology of 1712 flowers of 97 genets in a population in Nagano Prefecture, Japan, and constructed a mating model based on the observed mating pattern, which was revealed by paternity analysis using 11 microsatellite markers. The effects of flowering phenology were inferred by comparing estimated pollen flow distance and the level of heterozygosity in the next generation between two scenarios. In the first scenario, both the intergenet distance and flowering phenology influenced mating opportunity, while in the second scenario only intergenet distance influenced mating opportunity. Although the frequency distribution of pollen flow distance at the population level did not differ significantly between the two scenarios, the mean pollen flow distance of several flowers increased by more than 10 m as a result of variation in flowering phenology. Furthermore, accounting for flowering phenology predicted change in heterozygosity in the next generation from -0.04 to 0.07. The results showed that flowering phenology can affect pollen flow distance and spatial genetic structure.

Gene flow and inbreeding depression inferred from fine-scale genetic structure in an endangered heterostylous perennial, Primula sieboldii.

We estimated the gene dispersal distance and the magnitude of inbreeding depression from the fine-scale genetic structure in the endangered heterostylous perennial Primula sieboldii. We indirectly estimated the neighbourhood size (Nb) and the standard deviation of gene dispersal distance (sigma(g)) from the detected genetic structure by using 10 microsatellite markers. We also estimated the fitness reduction in mating among neighbouring individuals caused by biparental inbreeding according to the genetic structure. We found clear fine-scale genetic structure (a significantly positive kinship coefficient within 42.3 m), and the indirect estimates of sigma(g) and Nb were 15.7 m and 50.9, respectively. These indirect estimates were similar to the direct estimates (18.4 m and 44.0). The slightly larger indirect estimate of Nb may reflect that inbreeding depression and genetic structure or rare long-distance dispersal that were overlooked in the direct estimate have elongated the long-term average of gene dispersal distance. P. sieboldii is also likely to suffer about 19% fitness reduction in progenies from mating among individuals 5 m apart. Our results suggest that biparental inbreeding and genetic structure can affect the range of gene dispersal and seed reproductive success in P. sieboldii.