Multiplexed ISSR Genotyping by Sequencing (MIG-Seq).

Multiplexed inter-simple sequence repeat (ISSR) genotyping by sequencing (MIG-seq) is a simple, rapid, and inexpensive method for detecting single-nucleotide polymorphisms (SNPs) using next-generation sequencing (NGS). The advantages of MIG-seq include easy application to various species without prior genetic information. In addition, this method opens the door to genome-wide nucleotide sequence analyses of low-quality and trace-level deoxyribonucleic acid (DNA) samples, which have previously been difficult to analyze. Another advantage is that the procedure is simple, time-saving, and inexpensive. Recently, MIG-seq has been applied to wild and cultivated plants and has produced novel results. Using invisible DNA information, questions related to gene flow through pollination and seed dispersal, the genetic structure and diversity of populations, clonality, and the hybridization of wild and cultivated plants are being rapidly answered. In this chapter, I present the results of plant research based on MIG-seq and describe the procedure for this method as a user of MIG-seq.

Effects of El Niño drought on seedling dynamics in a seasonally dry tropical forest in Northern Thailand.

As El Niño is predicted to become stronger and more frequent in the future, it is crucial to understand how El Niño-induced droughts will affect tropical forests. Although many studies have focused on tropical rainforests, there is a paucity of studies on seasonally dry tropical forests (SDTFs), particularly in Asia, and few studies have focused on seedling dynamics, which are expected to be strongly affected by drought. Seedlings in SDTFs are generally more drought-tolerant than those in the rainforests, and the effects of El Niño-induced droughts may differ between SDTF and tropical rainforests. In this study, we explored the impact of El Niño-induced drought at an SDTF in northern Thailand by monitoring the seedling dynamics at monthly intervals for 7 years, including a period of strong El Niño. The effects were compared between two forest types in an SDTF: a deciduous dipterocarp forest (DDF), dominated by deciduous species, and an adjacent lower montane forest (LMF) with more evergreen species. El Niño-induced drought increased seedling mortality in both the forest types. The effect of drought was stronger in evergreen than in the deciduous species, resulting in higher mortality in the LMF during El Niño. However, El Niño increased seedling recruitment only in the DDF, mainly because of the massive recruitment of the deciduous oak, Quercus brandisiana (Fagaceae), which compensated for the mortality of seedlings in the DDF. As a result, El Niño increased seedling density in the DDF and decreased it in the LMF. This is the first long-term study to identify the differences in the impacts of El Niño on seedlings between the two forest types, and two leaf habits, evergreen and deciduous, in Southeast Asia. Our findings suggest that future climate change may alter the species composition and spatial distribution of seedlings in Asian SDTFs.

Community phylogeny and spatial scale affect phylogenetic diversity metrics in a species-rich rainforest in Borneo.

Community phylogenetic analysis is an effective approach to understanding the process of community formation. The phylogenetic tree of the species pool is reconstructed in the first step, and the phylogenetic tree obtained in the second step is used to analyze phylogenetic diversity. Sythetic trees have often been used in the construction of phylogenentic trees; however, in tropical rainforests with many closely related species, synthetic trees contain many unresolved nodes, which may affect the results of phylogenetic structure analysis. Here, we constructed a phylogenetic tree using DNA barcode sequences (rbcL, matK, trnH-psbA) for 737 tree species from the rainforests of Borneo, which have a high-species diversity and many closely related species. The phylogenetic tree had fewer polytomies and more branch length variations than the Phylocom synthetic trees. Comparison of community phylogenetic analyses indicated that values of the standardized effect size of mean pairwise distance (SES-MPD) were highly correlated between Phylocom and DNA barcode trees, but less so for the standardized effect size of mean nearest taxon distance (SES-MNTD), suggesting that caution is needed when using synthetic trees for communities containing many congeneric species, especially when using SES-MNTD. Simulation analysis suggested that spatial dependence on phylogenetic diversity is related to the phylogenetic signal of the species' habitat niche and the spatial structure of habitat, indicating the importance of detailed phylogeny in understanding community assembly processes.

Are patterns of fine-scale spatial genetic structure consistent between sites within tropical tree species?

Documenting the scale and intensity of fine-scale spatial genetic structure (FSGS), and the processes that shape it, is relevant to the sustainable management of genetic resources in timber tree species, particularly where logging or fragmentation might disrupt gene flow. In this study we assessed patterns of FSGS in three species of Dipterocarpaceae (Parashorea tomentella, Shorea leprosula and Shorea parvifolia) across four different tropical rain forests in Malaysia using nuclear microsatellite markers. Topographic heterogeneity varied across the sites. We hypothesised that forests with high topographic heterogeneity would display increased FSGS among the adult populations driven by habitat associations. This hypothesis was not supported for S. leprosula and S. parvifolia which displayed little variation in the intensity and scale of FSGS between sites despite substantial variation in topographic heterogeneity. Conversely, the intensity of FSGS for P. tomentella was greater at a more topographically heterogeneous than a homogeneous site, and a significant difference in the overall pattern of FSGS was detected between sites for this species. These results suggest that local patterns of FSGS may in some species be shaped by habitat heterogeneity in addition to limited gene flow by pollen and seed dispersal. Site factors can therefore contribute to the development of FSGS. Confirming consistency in species' FSGS amongst sites is an important step in managing timber tree genetic diversity as it provides confidence that species specific management recommendations based on species reproductive traits can be applied across a species' range. Forest managers should take into account the interaction between reproductive traits and site characteristics, its consequences for maintaining forest genetic resources and how this might influence natural regeneration across species if management is to be sustainable.

Dioecious plants are more precocious than cosexual plants: A comparative study of relative sizes at the onset of sexual reproduction in woody species.

The reproductive capacities of dioecious plant species may be limited by severe pollen limitation and narrow seed shadows for the two reasons. First, they are unable to self-pollinate, and seed production occurs only with pollinator movement from males to females. Second, only 50% of the individuals in populations contribute to seed production. Despite these handicaps, dioecious plants maintain their populations in plant communities with cooccurring cosexual plants, and no substantial difference in population growth rates has been found between dioecious and cosexual plants. Hence, dioecious plants are thought to mitigate these disadvantages by adopting ecological traits, such as insect pollination, animal-dispersed fleshy fruits, and precocious flowering. We studied the relationship between flowering and plant size in 30 woody species with different sex expressions, leaf habits, fruit types, and maximum plant sizes. The study site was located in an evergreen broad-leaved forest on the island of Honshu, Japan. A phylogenetic linear regression model showed that dioecious species tended to mature at smaller sizes than did cosexual taxa. At the population level, given equal plant densities and reproductive efforts, the precocity of dioecious plants could serve as one of the factors that mitigate the limitations of pollen and seed-shadow handicaps by increasing the density of reproductive individuals in the population. At the individual level, smaller size of onset of flowering may play a role in enhancing reproductive success over a lifetime by increasing reproductive opportunities. We discussed the possible effect of the relationship between precocity and some ecological traits of dioecious plants, such as small flowers pollinated by unspecialized insects, fleshy fruit dispersed by animals, and their preferential occurrence in the tropics and in island habitats. The universality of precocity among dioecious plants should be investigated in diverse plant communities. Such studies will increase our understanding of the evolution of plant breeding systems.

Geographical variation in soil bacterial community structure in tropical forests in Southeast Asia and temperate forests in Japan based on pyrosequencing analysis of 16S rRNA.

Geographical variation in soil bacterial community structure in 26 tropical forests in Southeast Asia (Malaysia, Indonesia and Singapore) and two temperate forests in Japan was investigated to elucidate the environmental factors and mechanisms that influence biogeography of soil bacterial diversity and composition. Despite substantial environmental differences, bacterial phyla were represented in similar proportions, with Acidobacteria and Proteobacteria the dominant phyla in all forests except one mangrove forest in Sarawak, although highly significant heterogeneity in frequency of individual phyla was detected among forests. In contrast, species diversity (α-diversity) differed to a much greater extent, being nearly six-fold higher in the mangrove forest (Chao1 index = 6,862) than in forests in Singapore and Sarawak (~1,250). In addition, natural mixed dipterocarp forests had lower species diversity than acacia and oil palm plantations, indicating that aboveground tree composition does not influence soil bacterial diversity. Shannon and Chao1 indices were correlated positively, implying that skewed operational taxonomic unit (OTU) distribution was associated with the abundance of overall and rare (singleton) OTUs. No OTUs were represented in all 28 forests, and forest-specific OTUs accounted for over 70% of all detected OTUs. Forests that were geographically adjacent and/or of the same forest type had similar bacterial species composition, and a positive correlation was detected between species divergence (ß-diversity) and direct distance between forests. Both α- and ß-diversities were correlated with soil pH. These results suggest that soil bacterial communities in different forests evolve largely independently of each other and that soil bacterial communities adapt to their local environment, modulated by bacterial dispersal (distance effect) and forest type. Therefore, we conclude that the biogeography of soil bacteria communities described here is non-random, reflecting the influences of contemporary environmental factors and evolutionary history.

Isolation and characterization of microsatellite loci for the large-seeded tree Protorhus deflexa (Anacardiaceae).

PREMISE OF THE STUDY: Protorhus deflexa is an endemic large-seeded tree in Madagascar that depends heavily on insects for cross-pollination and on large-bodied frugivores for seed dispersal. Because such mutualistic relationships are vulnerable to human disturbance, the development of microsatellite markers will enhance analyses of gene flow in this tree species in degraded forests. • METHODS AND RESULTS: Nineteen microsatellite markers were developed for P. deflexa using 454 pyrosequencing. The number of alleles ranged from two to nine, and the ranges of observed and expected heterozygosities were 0.200-0.800 and 0.303-0.821, respectively. The parentage exclusion probability by the 19 loci reached 0.98583 for the first parent and 0.99971 for the second parent. • CONCLUSIONS: These markers will be useful for studying gene flow via pollination and seed dispersal by animals and the genetic structure of P. deflexa in protected and degraded forests in Madagascar.

Sex change towards female in dying Acer rufinerve trees.

BACKGROUND AND AIMS: Sex changes within the genus Acer (Aceraceae) may occur because of associations of sex expression and plant health. In this study, a natural population of Acer rufinerve was monitored to clarify the sex change patterns, the relationship between sex expression and plant health, and the causal environmental conditions that precede sex changes. METHODS: Sex expression, growth rate and mortality of A. rufinerve trees in a natural population were monitored from 1992 to 1997. KEY RESULTS: Three types of sex expression were observed among A. rufinerve: male, female and bisexual. Among the three types of sex expression, sex changes occurred in all directions. In the growing season of 1994, precipitation was reduced. Stem growth rate decreased and mortality was high in 1994. In the spring of 1995, a drastic sex change from male to female or to bisexual occurred. As a result, the sex ratio became female-biased in 1995, although it had been male-biased from 1992 to 1994. In 1996 and 1997, the proportion of males in the population increased, partly as a result of female mortality and partly as a result of female-to-male sex changes. Sex expression of A. rufinerve was associated with their growth rate and mortality. The growth rate decreased for trees whose sex changed from male to female or to bisexual, and increased for trees whose sex changed from female to male or to bisexual. Dead trees reproduced as females before they died, except for those that died as males in 1994. CONCLUSIONS: One explanation for the sex change towards increasing femaleness for this A. rufinerve population in 1995 was the deterioration of plant health in the previous growing season, because of reduced precipitation. Sex changes of unhealthy and dying A. rufinerve towards femaleness may facilitate re-occupancy by offspring in gaps created by the death of A. rufinerve trees.