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The international grain trade is a major pathway for the introduction of alien plants because grain commodities can be contaminated with various weed seeds. To evaluate how alien weed seeds derived from imported grain commodities affect local vegetation in international trading ports, we conducted a vegetation survey on a set of grain landing ports and non-grain landing ports in nine regions throughout Japan to compare the vegetation between these two types of ports. We also surveyed weed seed contamination of wheat imported into Japan, and the contamination rate was calculated for each species based on our survey and previous studies on weed seed contamination. The vegetation clearly differed between the grain landing ports and the non-grain ports. In the grain landing ports, alien species were more abundant than in non-grain landing ports. All indicator species of the grain landing ports were reported to be contaminants in grain commodities. Furthermore, there was a tendency for the more abundant species at the grain landing ports to show higher contamination rates in grain commodities. These results indicate that contaminant seeds spill from imported grain in grain landing ports, and highly contaminated species are likely to become established. We clearly show that weed seed contamination in grain commodities plays an important role in propagule pressure. Gathering information about the prevalence of weeds in grain-exporting countries and monitoring the weed species composition in imported grain commodities is becoming increasingly important for predicting the unintentional introduction of troublesome weeds and identifying effective weed management options.

Stable production of crops depends on pollination services provided by animals. Although crop production is more stable in pollinator-independent crops than in pollinator-dependent crops at the global scale, the situation at the local scale is unknown. Here, we investigated the relationship between pollinator dependence and production stability of 40 crops in Japan and show that the global relationship does not apply at the local scale, i.e., most pollinator-dependent crops showed stable production and most pollinator-independent showed instable production possibly because of local policies and agricultural practices. Examining relationships at the local scale will help establish effective local management of pollination services and crop production.

The European honeybee, Apis mellifera L. (Hymenoptera: Apidae), is the most important crop pollinator, and there is an urgent need for a sustained supply of honeybee colonies. Understanding the availability of pollen resources around apiaries throughout the brood-rearing season is crucial to increasing the number of colonies. However, detailed information on the floral resources used by honeybees is limited due to a scarcity of efficient methods for identifying pollen species composition. Therefore, we developed a DNA barcoding method for identifying the species of each pollen pellet and for quantifying the species composition by summing the weights of the pellets for each species. To establish the molecular biological protocol, we analyzed 1008 pellets collected between late July and early September 2016 from five hives placed in a forest/agricultural landscape of Hokkaido, northern Japan. Pollen was classified into 31 plant taxa, of which 29 were identified with satisfactory discrimination (25 species and 4 genera) using trnL-trnF and ITS2 as DNA barcoding regions together with available floral and phenological information. The remaining two taxa were classified to the species level using other DNA barcoding regions. Of the 1008 pollen pellets tested, 1005 (99.7%) were successfully identified. As an example of the use of this method, we demonstrated the change in species composition of pollen pellets collected each week for 9 weeks from the same hive. Electronic supplementary material The online version of this article (10.1007/s13355-018-0565-9) contains supplementary material, which is available to authorized users.

Surveys in the native range of Japanese honeysuckle Lonicera japonica in Honshu, Japan, revealed a natural enemy biota attacking the plant that was rich in potential biocontrol agents for New Zealand (NZ), including 11 herbivorous insect species and one fungal pathogen. This contrasts with previously published surveys in NZ that found only generalist arthropod herbivores and only two weakly pathogenic specialist pathogens. The Japanese insect herbivores included leaf-mining and stem-boring feeding guilds that were absent in NZ. Among the insect herbivores, a nymphalid butterfly Limenitis glorifica, a cerambycid beetle Oberea shirahatai and a cosmopterigid moth Allotalanta sp. were prioritised as potential biocontrol agents. Their larvae cause potentially complementary damage to leaves, woody stems and shoot tips, respectively. Host-range testing of Lim. glorifica and O. shirahatai indicated that both species are sufficiently host-specific to be released in NZ and formal approval to release these species has been obtained. Lim. glorifica was released into the field in 2014 and has become established. O. shirahatai is due to be released in late 2016. Preliminary testing indicates that the fundamental host range for Allotalanta sp. larval feeding and development is narrowly oligophagous. Allotalanta sp. is therefore likely to have potential for use as a biocontrol agent in NZ if the first two agents provide insufficient control, although further host-range testing would be desirable to more accurately predict the likely field host range of this species.

Miscanthus ×ogiformis is a hybrid between Miscanthus sacchariflorus and Miscanthus sinensis and its several synonyms indicate the insufficient understanding of the morphological variation of its awn on the spikelet, which is a key characteristic for identifying Miscanthus species. In the present study, all the Miscanthus specimens in 19 herbaria were examined in order to find new localities for Miscanthus ×ogiformis. Thirty-nine Miscanthus ×ogiformis specimens appeared to be naturally occurring hybrids, as their callus hair length, awn length, and callus-to-spikelet ratio differed from those found in the parental species. Therefore, new Miscanthus ×ogiformis individuals with morphological variations are likely to be discovered and these strains might be useful as new genetic resources for this biomass crop.

Summary Compared with natural seed dispersal, human-mediated seed dispersal could spread herbicide resistance genes on a much larger scale. Herbicide-resistant weed seeds have been reported as contaminants in commercial grain. We investigated the contamination of seeds of Lolium species with target-site mutations conferring resistance to acetolactate synthase (ALS)-inhibiting herbicides in wheat imported from the USA, Canada and Australia into Japan. We also investigated the establishment of ALS-inhibiting herbicide-resistant Lolium species in 12 seaports in Japan that are major entry points for international commodities. We found herbicide-resistant Lolium spp. seeds from all classes of wheat samples. Resistant individuals became established at six of eight ports where more than 50 kt of imported wheat is unloaded every year. The establishment of resistant Lolium spp. individuals was common at major grain landing ports. Monitoring over 3 years at one port revealed that the frequency of resistant individuals did not fluctuate between years. Many resistant individuals were distributed in front of the entrance of a fodder company, but a few resistant individuals were found in areas 2 km away from the port. The results indicate that gene flow is rare through pollen or seed movement from resistant plants to peripheral populations. Further extensive and long-term monitoring is necessary to perform a comprehensive risk assessment of herbicide-resistant plants entering Japan through major commercial ports.

We examined fruiting behaviors of 24 dipterocarp species in a lowland rain forest of Peninsular Malaysia during two consecutive episodes of general flowering (GF). The first GF episode (GF2001) occurred from August 2001 to February 2002, and the second GF episode (GF2002) followed immediately, from March to September 2002. The magnitude of GF2002 was greater than that of GF2001 at the community level. Significant positive size dependence of fruiting behavior at the community level was found in both GF2001 and GF2002, but there was no significant association between the fruiting behaviors in GF2001 and GF2002 except for one species. These results imply that tree size was one of the explanatory factors for fruiting behavior of dipterocarp species, but there was no evidence that adjacent reproduction caused the absence of reproduction and decreased fecundity in the subsequent fruiting event. In contrast, strong spatial aggregation of fruiting trees was found in GF2001, suggesting that external factors may affect fruiting behavior of dipterocarps in a minor GF episode. Among the 12 study species, there were large variations in fruiting behavior, but growth type (e.g., fast-growing or slow-growing) did not simply explain the inter-specific pattern of fruiting behavior. Thus, tree size may account for fruiting behavior of dipterocarps during the consecutive GF episodes through species-specific differences in phonological responses to internal and external conditions.

As herbicide-resistant weeds have spread in the agricultural fields of grain-exporting countries, their seeds could be introduced into other countries as contaminants in imported grain. The spread of resistance genes through seed and pollen can cause significant economic loss. In order to assess the extent of the problem, we investigated the contamination by herbicide-resistant annual ryegrass (Lolium rigidum) of wheat imported from Western Australia into Japan. Annual ryegrass seeds were recovered from wheat shipments and seed bioassays were conducted to identify resistance to the herbicides that are commonly used in Australia: diclofop-methyl, sethoxydim, chlorsulfuron, and glyphosate. Nearly 4500 ryegrass seeds were detected in 20 kg of wheat that was imported in both 2006 and 2007. About 35% and 15% of the seeds were resistant to diclofop-methyl, 5% and 6% were resistant to sethoxydim, and 56% and 60% were resistant to chlorsulfuron in 2006 and 2007, respectively. None was resistant to glyphosate in either year. As the contamination of crops by herbicide-resistant weeds is probably a common phenomenon, the monitoring of incoming grain shipments is necessary to stem the further spread of herbicide-resistant weeds into importing countries.

†Background and Aims Knowledge of pollen dispersal patterns and variation of fecundity is essential to under-standing plant evolutionary processes and to formulating strategies to conserve forest genetic resources.Nevertheless, the pollen dispersal pattern of dipterocarp, main canopy tree species in palaeo-tropical forestremains unclear, and flowering intensity variation in the field suggests heterogeneity of fecundity.†Methods Pollen dispersal patterns and male fecundity variation of Shorea leprosula and Shorea parvifolia ssp.parvifolia on Peninsular Malaysian were investigated during two general flowering seasons (2001 and 2002),using a neighbourhood model modified by including terms accounting for variation in male fecundity amongindividual trees to express heterogeneity in flowering.†Key Results The pollen dispersal patterns of the two dipterocarp species were affected by differences in con-specific tree flowering density, and reductions in conspecific tree flowering density led to an increased selfingrate. Active pollen dispersal and a larger number of effective paternal parents were observed for both speciesin the season of greater magnitude of general flowering (2002).†Conclusions The magnitude of general flowering, male fecundity variation, and distance between pollen donorsand mother trees should be taken into account when attempting to predict the effects of management practices onthe self-fertilization and genetic structure of key tree species in tropical forest, and also the sustainability of poss-ible management strategies, especially selective logging regimes.Key words: Dipterocarp, general flowering, male fecundity variation, microsatellite marker, paternity analysis,pollen dispersal, Shorea, tropical forest.

Summary International trade is a major route by which non-indigenous organisms are introduced into new habitats. Various kinds of weed seeds have been introduced through grain trade. The objectives of this study were to understand the factors that affect the initial assemblage of plant species introduced by the international grain trade and to extract their general attributes. We surveyed weed seed contamination of spring wheat imported from Canada to Japan and analysed the effects of the field abundance of each weed and of harvesting and cleaning on the quantity of weed seed included in the imported wheat. The field abundance was positively correlated with the weed seed quantity. Seeds of short weeds and seeds with a pappus were eliminated from the wheat by the harvesting or cleaning process. Many other crop plants contaminated the wheat. Because various transportation vehicles, temporary storage sites and port elevators are used commonly with all exported crops and it is difficult to remove all residues from them, other crops might be carried over into the wheat commodity. These relationships also apply to other grains.

BACKGROUND AND AIMS: Flower shapes are important visual cues for pollinators. However, the ability of pollinators to discriminate between flower shapes under natural conditions is poorly understood. This study focused on the diversity of flower shape in Primula sieboldii and investigated the ability of bumblebees to discriminate between flowers by combining computer graphics with a traditional behavioural experiment. METHODS: Elliptic Fourier descriptors described shapes by transforming coordinate information for the contours into coefficients, and principal components analysis summarized these coefficients. Using these methods, artificial flowers were created based on the natural diversity of petal shape in P. sieboldii. Dual-choice tests were then performed to investigate the ability of the bumblebees to detect differences in the aspect ratio of petals and the depth of their head notch. KEY RESULTS: The insects showed no significant ability to detect differences in the aspect ratio of the petals under natural conditions unless the morphological distance increased to an unrealistic level. These results suggest the existence of a perception threshold for distances in this parameter. The bumblebees showed a significant preference for narrow petals even after training using flowers with wide petals. The bumblebees showed a significant ability to discriminate based on the depth of the petal head notch after training using artificial flowers with a deep head notch. However, they showed no discrimination in tests with training using extreme distances between flowers in this parameter. CONCLUSIONS: A new type of behavioural experiment was demonstrated using real variation in flower corolla shape in P. sieboldii. If the range in aspect ratios of petals expands much further, bumblebees may learn to exhibit selective behaviour. However, because discrimination by bumblebees under natural conditions was low, there may be no strong selective behaviour based on innate or learned preferences under natural conditions.

We estimated the required sample sizes for estimating large-scale soil respiration (for areas from 1 to 2 ha) in four ecosystems (primary and secondary forests, and oil palm and rubber plantations) in Malaysia. The soil respiration rates were 769 ± 329 mg CO2 m−2 h−1 in the primary forest (2 ha, 50 sample points), 708 ± 300 mg CO2 m−2 h−1 in the secondary forest (2 ha, 50 points), 815 ± 363 mg CO2 m−2 h−1 in the oil palm plantation (1 ha, 25 points), and 450 ± 178 mg CO2 m−2 h−1 in the rubber plantation (1 ha, 25 points). According to our sample size analysis, the number of measurement points required to determine the mean soil respiration rate at each site with an error in the mean of no more than 10% ranged from 67 to 85 at the 95% probability level. These results suggest that evaluating the spatial heterogeneity of soil respiration rates in the tropics may require more measurement points than in temperate forests.

We investigated the fine-scale genetic structure of three tropical-rainforest trees, Hopea dryobalanoides, Shorea parvifolia and S. acuminata (Dipterocarpaceae), in Peninsular Malaysia, all of which cooccurred within a 6-ha plot in Pasoh Forest Reserve. A significant genetic structure was found in H. dryobalanoides, weaker (but still significant) genetic structure in S. parvifolia and nonsignificant structure in S. acuminata. Seeds of all three species are wind dispersed, and their flowers are thought to be insect pollinated. The most obvious difference among these species is their height: S. parvifolia and S. acuminata are canopy species, whereas H. dryobalanoides is a subcanopy species. Clear differences were also found among these species in their range of seed dispersal, which depends on the height of the release point; so taller trees disperse their seed more extensively. The estimates of seed dispersal area were consistent with the degree of genetic structure found in the three species. Therefore, tree height probably had a strong influence on the fine-scale genetic structure of the three species.

The spatial genetic structure of Polygonum thunbergii was investigated by DNA fingerprinting, using an oligonucleotide probe (GACA)4 and restriction enzyme Dra I. The genetic similarity of DNA fingerprints among 15 plants collected over a distance of 230 m continuously decreased with increasing distance. This significant negative correlation between similarity of DNA fingerprint and distance means that there is a spatial genetic structure within the population of P. thunbergii because similarity of DNA fingerprint and genetic relatedness are strongly correlated. The genetic structure would be caused by the strictly limited seed dispersal in this species, which has cleistogamous and chasmogamous flowers with no specialized seed dispersal mechanism.

Pollen flow and population genetic structure among 30 potentially flowering individuals of Neobalanocarpus heimii , a tropical emergent tree, were investigated in a lowland tropical rainforest of Malaysia using microsatellite polymorphism. The 248 offspring in the vicinity of five reproductive trees of the 30 potentially flowering trees were used in paternity analysis for pollen-flow study. Four primer pairs, developed in different species of dipterocarps, were adopted to detect microsatellite polymorphism. Based upon microsatellite polymorphism, pollen flow and seed migration were detected. Pollen-flow events of more than 400 m were observed directly, based on paternity analysis in the study plot. The estimated average mating distance of the five reproductive trees was 524 m. This result suggests that reproduction of this species is mediated by a long-distance pollinator. The haplotypes of some offspring were not compatible with the nearest reproductive tree. Thus, the results suggest that some seeds are dispersed by a seed dispersal vector. Investigation of genetic structure showed significant and negative correlation of genetic relatedness and spatial distances between the 30 potentially flowering trees, but this correlation was weak. We suggest that long-distance gene flow and seed migration are responsible for the poorly developed genetic structure of this species.

Few observations have been made on temporal changes in the siring success of flowers in the male stage. In this study, we estimated both male and female contributions to fitness for 21 plants of protandrous andromonoeciosHeracleum lanatum with differing dates of first flowering. The results of multiple regression analysis showed that total male fitness significantly increases with the advance of the first-flowering date but does not depend upon plant size, whereas female fitness increases with plant size but does not depend upon the first-flowering date. We also showed that the earlier-flowering plants have more late-blooming male flowers in their secondary umbels. Based on these results, we suggest that polymorphism of the early- and late-bloomers may be maintained by frequency-dependent selection through temporally changing male reproductive success.

Six microsatellite loci were characterized in Dioscorea tokoro, a wild yam species in East Asia. All six loci were polymorphic in a sample of 23 individuals from natural populations in Japan. The microsatellite loci displayed many alleles (6.2 alleles per locus on average), and the observed heterozygosity (Ho = 0.54) as well as expected heterozygosity (He = 0.68) were high. The heterozygosities were far more than that previously detected by allozyme analysis of D. tokoro (Ho = 0.23, He = 0.28). Five microsatellite loci were sufficient to provide a paternity exclusion rate (Q) of Q = 0.98, which enables monitoring of the pollen-mediated gene flow between plants in a population. Microsatellite loci are abundant and highly polymorphic in D. tokoro and other plants and are therefore ideal markers for plant population genetic studies.Key words: microsatellite, simple sequence repeat, population genetics, Dioscorea tokoro.

We monitored the reproductive status of all trees with diameters at breast height (dbh) >30 cm in a 40-ha plot at Pasoh, west Malaysia, and investigated the individual fecundity of 15 Shorea acuminata Dyer (Dipterocarpaceae) trees using seed-trapping methods during two consecutive general flowering periods in 2001 (GF2001) and 2002 (GF2002). The proportion of flowering trees was higher, and not dependent on size, in GF2002 (84.2%), than in GF2001 (54.5%), when flowering mainly occurred in trees with a dbh ≤70 cm. Fecundity parameters of individual trees per event varied widely (221,000–35,200,000 flowers, 0–139,000 mature seeds, and 1.04–177 kg total dry matter mass of fruit (TDM) per tree). Monotonic increases with increasing tree size were observed for flower production and TDM amongst trees up to 90 cm in dbh, but not for mature seed production or for any of these parameters amongst larger trees. The pattern of reproductive investment during the two consecutive reproductive events clearly differed between medium-sized and large trees; the former concentrated their reproductive investment in one of the reproductive events whereas the latter allocated their investment more evenly to both reproductive events. Our results suggest size-related differences in the resource allocation pattern for reproduction.

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.

Tropical rain forests are becoming fragmented into small patches and simplified in structure and composition due to their exploitation for timber. Consequently, genetic variation may be decreased and the genetic differentiation between the patches may rise. Developing sustainable forestry regimes that retain genetic diversity is one of the major goals in tropical forestry today. Tropical tree species have predominantly allogamous mating systems, mediated by many kinds of pollinators, so the gene flow through pollen and seeds is one of the most important factors influencing genetic diversity in tropical forests. Recently, molecular markers have been used to determine the genetic diversity and differentiation of forest tree populations. In particular, microsatellite DNA markers, which are highly polymorphic, have been used to evaluate the gene flow through pollen and seeds within forests. We have also developed microsatellite DNA markers in dipterocarp species, and used these markers to analyze mating systems, genetic structures and gene flow in several dipterocarp species at ecological parmanent plots such as those in Pasoh Forest Reserve (Pasoh FR). We discuss, here, characteristics of the pollination and breeding systems in dipterocarps that have been revealed by microsatellite analysis, and the factors that influence them. Finally, the potential value and limitations of using molecular approaches to develop genetic criteria for gene conservation in sustainable forestry are discussed.