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Climate change and the resultant extreme weather events have disrupted agricultural ecosystems. In South Taiwan, serious droughts and unpredictable, heavy rainfall have caused slope sliding at lowland areas and led to significant agricultural and economic loss. A team of researchers led by Professor Tzen-Yuh Chiang, that includes Dr Tsai-Wen Hsu and Professor Yu-Chung Chiang, and is based at the University Social Responsibility, National Cheng Kung University, Taiwan, wants to drive change in farming practices in the Dong Shan district, Taiwan. In work supported by the Ministry of Science and Technology in Taiwan, the team is introducing a combination of natural and microbial farming practices. As such, the researchers have isolated and reformulated microbial fertilisers in the lab and applied them to enforce the health of crop roots. They have found that many insects and birds are now back on the farm, acting as biological control, which removes the need for pesticides. The overall goal of the project is to rebuild the vegetation and natural ecosystem along the slopes of lowlands. Key to this is stopping slope exploration, along with using microbial farming practices to enforce and ensure the health of crops. The project's success also relies on the use of microbial technology to culture microbes with sterile medium, which can then be used as fertilisers. This important work will help to integrate knowledge and policy about the agricultural environment and the agricultural economy.

The 2,3,7,8-tetrachlorodibenzodioxin (TCDD), a contaminant in Agent Orange released during the US-Vietnam War, led to a severe environmental crisis. Approximately, 50 years have passed since the end of this war, and vegetation has gradually recovered from the pollution. Soil bacterial communities were investigated by 16S metagenomics in habitats with different vegetation physiognomies in Central Vietnam, namely, forests (S0), barren land (S1), grassland (S2), and developing woods (S3). Vegetation complexity was negatively associated with TCDD concentrations, revealing the reasoning behind the utilization of vegetation physiognomy as an indicator for ecological succession along the gradient of pollutants. Stark changes in bacterial composition were detected between S0 and S1, with an increase in Firmicutes and a decrease in Acidobacteria and Bacteroidetes. Notably, dioxin digesters Arthrobacter, Rhodococcus, Comamonadaceae, and Bacialles were detected in highly contaminated soil (S1). Along the TCDD gradients, following the dioxin decay from S1 to S2, the abundance of Firmicutes and Actinobacteria decreased, while that of Acidobacteria increased; slight changes occurred at the phylum level from S2 to S3. Although metagenomics analyses disclosed a trend toward bacterial communities before contamination with vegetation recovery, non-metric multidimensional scaling analysis unveiled a new trajectory deviating from the native state. Recovery of the bacterial community may have been hindered, as indicated by lower bacterial diversity in S3 compared to S0 due to a significant loss of bacterial taxa and recruitment of fewer colonizers. The results indicate that dioxins significantly altered the soil microbiomes into a state of disorder with a deviating trajectory in restoration.

Microbial farming for habitat recovery in the Taiwanese mountains Professor Tzen-Yuh Chiang's team at National Cheng Kung University looks toward microbial farming in the mountains slopes of South Taiwan to aid habitat recovery, as low-land slopes have been affected by climate change. The team have taken soil structures and underground water into account when helping these slope habitats recover, using sustainable engineering methodologies to stabilize the slopes with vegetation, allowing new species to grow strong roots to increase the adhesive forces in soil. Their other methodologies of accelerating habitat recovery for slope farms include through the revegetation of native plants from neighboring forests, and understanding the importance of soil health with a high diversity of microbes that can interact with plant roots.

Conspecific male animals fight for resources such as food and mating opportunities but typically stop fighting after assessing their relative fighting abilities to avoid serious injuries. Physiologically, how the fighting behavior is controlled remains unknown. Using the fighting fish Betta splendens, we studied behavioral and brain-transcriptomic changes during the fight between the two opponents. At the behavioral level, surface-breathing, and biting/striking occurred only during intervals between mouth-locking. Eventually, the behaviors of the two opponents became synchronized, with each pair showing a unique behavioral pattern. At the physiological level, we examined the expression patterns of 23,306 brain transcripts using RNA-sequencing data from brains of fighting pairs after a 20-min (D20) and a 60-min (D60) fight. The two opponents in each D60 fighting pair showed a strong gene expression correlation, whereas those in D20 fighting pairs showed a weak correlation. Moreover, each fighting pair in the D60 group showed pair-specific gene expression patterns in a grade of membership analysis (GoM) and were grouped as a pair in the heatmap clustering. The observed pair-specific individualization in brain-transcriptomic synchronization (PIBS) suggested that this synchronization provides a physiological basis for the behavioral synchronization. An analysis using the synchronized genes in fighting pairs of the D60 group found genes enriched for ion transport, synaptic function, and learning and memory. Brain-transcriptomic synchronization could be a general phenomenon and may provide a new cornerstone with which to investigate coordinating and sustaining social interactions between two interacting partners of vertebrates., Author summary Agonistic encounters induce changes in the brain and behavior, but their underlying molecular mechanisms remain poorly understood. The fighting fish Betta splendens are small freshwater fish that are well known for their aggressiveness and are widely used to study aggression. Here, by measuring aggressive behavior displays (bite/strike/surface-breathing) between two opponents during fighting, we demonstrate that the two opponents in each fighting pair showed similar fighting configurations by influencing each other. In addition, we compared brain gene expression between opponents and showed synchronization of gene expression within a fighting pair, leading to pair-specific synchronization in genes associated with ion transport, synapse function, and learning and memory. This study presents the possibility that similar behaviors in pairs of animals under similar conditions may trigger synchronizing waves of transcription between the individuals, providing a hint to support the idea that fighting behaviors contain cooperative aspects at the molecular level.

As an ancient seed plant, cycads are one of the few gymnosperms that develop a root symbiosis with cyanobacteria, which has allowed cycads to cope with harsh geologic and climatic conditions during the evolutionary process. However, the endophytic microbes in cycad roots remain poorly identified. In this study, using next-generation sequencing techniques, we investigated the microbial diversity and composition of both the coralloid and regular roots of Cycas bifida (Dyer) K.D. Hill. Highly diverse endophytic communities were observed in both the coralloid and regular roots. Of the associated bacteria, the top five families were the Nostocaceae, Sinobacteraceae, Bradyrhizobiaceae, Bacillaceae, and Hyphomicrobiaceae. The Nectriaceae, Trichocomaceae, and Incertae sedis were the predominant fungal families in all root samples. A significant difference in the endophytic bacterial community was detected between coralloid roots and regular roots, but no difference was observed between the fungal communities in the two root types. Cyanobacteria were more dominant in coralloid roots than in regular roots. The divergence of cycad root structures and the modified physiological processes may have contributed to the abundance of cyanobionts in coralloid roots. Consequently, the colonization of cyanobacteria inhibits the assemblage of other endophytes. Our results contribute to an understanding of the species diversity and composition of the cycad-endophyte microbiome and provide an abbreviated list of potential ecological roles of the core microbes present.

Conspecific male animals fight for resources such as food and mating opportunities but typically stop fighting after assessing their relative fighting abilities to avoid serious injuries. Physiologically, how the fighting behavior is controlled remains unknown. Using the fighting fish Betta splendens, we studied behavioral and neurotranscriptomic changes during the fight between the two opponents. At the behavioral level, surface-breathing and biting/striking occurred only during intervals between mouth-lockings. Eventually, the behaviors of the two opponents became synchronized, with each pair showing a unique behavioral pattern. At the physiological level, we examined the expression patterns of 23,306 brain transcripts using RNA-sequencing data from brains of fighting pairs after a 20-min (D20) and a 60-min (D60) fight. The two opponents in each D60 fighting pairs showed a strong gene expression correlation, whereas those D20 fighting pair showed a weak correlation. Moreover, each fighting pair in the D60 group showed pair-specific gene expression patterns in the grade of membership analysis (GoM) and were grouped as a pair in the heatmap clustering. The observed pair-specific individualization in neurotranscriptomic synchronization (PINS) suggested that this synchronization provides physiological bases for the behavioral synchronization. An analysis using the synchronized genes in fighting pairs of the D60 found genes enriched for ion transport, synaptic function, and learning and memory. PINS could be a general phenomenon and may provide a new cornerstone to investigate coordinating and sustaining social interactions between two interacting partners of higher animals.

Miscanthus in Taiwan occupies a cline along altitude and adapts to diverse environments, e.g., habitats of high salinity and volcanoes. Rhizospheric and endophytic bacteria may help Miscanthus acclimate to those stresses. The relative contributions of rhizosphere vs. endosphere compartments to the adaptation remain unknown. Here, we used targeted metagenomics to compare the microbial communities in the rhizosphere and endosphere among ecotypes of M. sinensis that dwell habitats under different stresses. Proteobacteria and Actinobacteria predominated in the endosphere. Diverse phyla constituted the rhizosphere microbiome, including a core microbiome found consistently across habitats. In endosphere, the predominance of the bacteria colonizing from the surrounding soil suggests that soil recruitment must have subsequently determined the endophytic microbiome in Miscanthus roots. In endosphere, the bacterial diversity decreased with the altitude, likely corresponding to rising limitation to microorganisms according to the species-energy theory. Specific endophytes were associated with different environmental stresses, e.g., Pseudomonas spp. for alpine and Agrobacterium spp. for coastal habitats. This suggests Miscanthus actively recruits an endosphere microbiome from the rhizosphere it influences.

Two cyprinid fishes, Spinibarbus hollandi and Onychostoma alticorpus, are endemic to southeastern Taiwan. This study examined the phylogeography of these two codistributed primary freshwater fishes using mitochondrial DNA cytochrome b sequences (1140 bp) to search for general patterns in the effect of historical changes in southeastern Taiwan. In total, 135 specimens belonging to these two species were collected from five populations. These two codistributed species revealed similar genetic variation patterns. The genetic variation in both species was very low, and the geographical distribution of the genetic variation corresponded neither to the drainage structure nor to the geographical distances between the samples. The results of a statistical dispersal-vicariance analysis suggested that the ancestral populations of these two species were distributed in southern Taiwan before their dispersal. Our study suggests that the initial colonization occurred in the Kaoping River followed by eastern and northward dispersal. Our results also indicate that the Central Range in Taiwan did not act as a barrier to the dispersal of S. hollandi or O. alticorpus.

Megacodon stylophorus (Clarke) Smith is a perennial alpine herb endemic to the species-rich eastern Himalayan region. Its populations are locally scattered as isolated patches throughout this region. Genetic variation within and among six populations of this species was assessed using ISSR fingerprinting with 13 primers. High levels of genetic diversity exist within species (P = 69.83%, HT = 0.1949 and Hsp = 0.3047), while the within-population diversity is low (P = 11.21%, HE = 0.0532 and Hpop = 0.0792). Extraordinarily high levels of genetic differentiation were detected among populations based on various statistics, including Nei’s genetic diversity analysis (72.7%), Shannon’s diversity index (74.01%) and AMOVA (80.70%). That is, populations shared low levels of genetic identity (I = 0.8203 ± 0.0430). This genetic structure was probably due to severe genetic drift of the small-sized patchy populations resulting from postglacial habitat fragmentations. The observed genetic structure of the populations implies that as many populations as possible should be considered for any in situ and ex situ conservation practice on this species.

Juglone (5-hydroxy-1,4-naphthoquinone) is an important allelochemical in walnut trees (Juglans nigra L.). Its allelopathic potential has been reported in different plant species. We investigated the phytotoxic effects of the allelochemical juglone and the protective role of proline in tobacco seedlings. Juglone inhibited the growth of tobacco seedlings and increased reactive oxygen species content in tobacco roots. Moreover, juglone stress increased proline concentration. The expression of two proline synthesis genes, pyrroline-5-carboxylate synthetase and ornithine aminotransferase, was upregulated and that of a proline catabolism gene, proline dehydrogenase, was downregulated with juglone treatment in tobacco roots. Furthermore, plants pretreated with proline and then exposed to juglone showed attenuated toxic effects in roots. Proline was able to modulate allelochemical juglone-induced stress in tobacco. In summary, this study suggested that increased proline content in the tobacco seedlings treated wi...

Young incipient species provide ideal materials for untangling the process of ecological speciation in the presence of gene flow. The Miscanthus floridulus/sinensis complex exhibits diverse phenotypic and ecological differences despite recent divergence (approximately 1.59 million years ago). To elucidate the process of genetic differentiation during early stages of ecological speciation, we analyzed genomic divergence in the Miscanthus complex using 72 randomly selected genes from a newly assembled transcriptome. In this study, rampant gene flow was detected between species, estimated as M = 3.36 × 10(-9) to 1.20 × 10(-6) , resulting in contradicting phylogenies across loci. Nevertheless, beast analyses revealed the species identity and the effects of extrinsic cohesive forces that counteracted the non-stop introgression. As expected, early in speciation with gene flow, only 3-13 loci were highly diverged; two to five outliers (approximately 2.78-6.94% of the genome) were characterized by strong linkage disequilibrium, and asymmetrically distributed among ecotypes, indicating footprints of diversifying selection. In conclusion, ecological speciation of incipient species of Miscanthus probably followed the parapatric model, whereas allopatric speciation cannot be completely ruled out, especially between the geographically isolated northern and southern M. sinensis, for which no significant gene flow across oceanic barriers was detected. Divergence between local ecotypes in early-stage speciation began at a few genomic regions under the influence of natural selection and divergence hitchhiking that overcame gene flow.

Islands isolated by oceans that act as a geographical barrier for plant migration often possess high species endemism and have been deemed as a natural laboratory for studying species divergence. Fatsia Decne. & Planch. (Araliaceae), with three species, is one of the few plant genera absent in continents while exclusively spanning continental and oceanic islands. The nuclear ribosomal internal transcribed spacer (nrITS) phylogeny uncovered a pattern with reciprocal monophyly of Fatsia oligocarpella Koidz. (Bonin) and Fatsia polycarpa Hayata (Taiwan) vs. paraphyly of Fatsia japonica (Thunb.) Decne. & Planch. (Japan and Ryukyus), suggesting ancestry of the species in Japan and a likely temperate origin; whereas, lack of monophyly of all three allopatrically distributed species at chloroplast DNA (cpDNA) trnL–trnF spacer likely resulted from lineage sorting. In spite of the limited habitats for F. oligocarpella, unexpectedly high genetic variations in this species of oceanic islands were likely attributable to multiple colonizations and recurrent gene introgression. Biogeographical analyses suggested that Fatsia likely diverged via southward colonization in Bonin Islands and Taiwan during the late Pliocene to Pleistocene. Besides, Fatsia species with an allopatric distribution provide a perfect model for testing speciation modes of insular endemics. Nonzero gene flow between species was detected based on MIGRATE and STRUCTURE analyses of DNA sequences and microsatellite fingerprints, suggesting that allopatric speciation is less likely.

The Amentotaxus argotaenia (Taxaceae) species complex is comprised of four relict and endangered gymnosperms. Amentotaxus argotaenia and A. yunnanensis are scattered throughout South and Central China, and A. formosana, and A. poilanei are restricted to Taiwan and Vietnam. In the present study, we examined genetic divergence and biogeographical patterns in the species complex based on chloroplast DNA atpI-atpH and rpS16-trnK intergenic spacers, the mitochondrial DNA nad1 intron, and microsatellite markers. Genealogical analyses revealed systematic inconsistencies between organellar DNA markers, with paraphyletic species inferred based on cpDNA, versus a monophyletic A. formosana, and clustering of A. poilanei within A. yunnanensis at mtDNA. AMOVA revealed that most organellar DNA variants resided among species and populations, a pattern also supported by clustering with STRUCTURE analyses on microsatellites. Higher levels of genetic diversity in the Yongxin population of A. argotaenia reflected features of a refugium. Most speciation events were rather recent, with divergence no earlier than 2.50 MYA, although many ancient lineages derived 4.12-13.84 MYA were maintained within species. Significant genetic structuring within A. argotaenia was detected among three geographical regions-a phylogeographical pattern likely attributable to past fragmentations. In contrast to paraphyletic A. argotaenia based on the maintenance of ancestral polymorphisms, interspecific hybridization likely contributed to polyphyly in A. yunnanensis. Furthermore, despite the low likelihood of recurrent gene flow due to geographical isolation, IM analyses revealed continued interspecific gene flow after species divergence, reflecting a parapatric speciation model.

Identifying the species composition of a microbial ecosystem is often hampered by difficulties in culturing the organisms and in the low sequencing depth of traditional DNA barcoding. Metagenomic analysis, a huge-scale nucleotide-sequence-based tool, can overcome such difficulties. In this study, Sanger sequencing of 500 nrITS clones uncovered 29 taxa of 19 fungal genera, whereas metagenomics with next-generation sequencing identified 512 operational taxonomic units (OTUs) for ITS1/2 and 364 for ITS3/4. Nevertheless, high throughput sequencing of PCR amplicons of ITS1/2, ITS3/4, nrLSU-LR, nrLSU-U, mtLSU, and mtATP6, all with at least 1,300× coverage and about 21 million reads in total, yielded a very diverse fungal composition. The fact that 74 % of the OTUs were exclusively uncovered with single barcodes indicated that each marker provided its own insights into the fungal flora. To deal with the high heterogeneity in the data and to integrate the information on species composition across barcodes, a rank-scoring strategy was developed. Accordingly, 205 genera among 64 orders of fungi were identified in healthy Phalaenopsis roots. Of the barcodes utilized, ITS1/2, ITS3/4, and nrLSU-U were the most competent in uncovering the fungal diversity. These barcodes, though detecting different compositions likely due to primer preference, provided complementary and comprehensive power in deciphering the microbial diversity, especially in revealing rare species.

Primers for eight microsatellites were developed; they successfully amplified DNA from 20 domesticated Formosan Sambar deer (Cervus unicolor swinhoei). All loci were polymorphic, with 10-19 alleles per locus. The average observed heterozygosity across loci and samples was 0.310, ranging from 0 to 0.750 at each locus. All loci but one, CU18, deviated from Hardy-Weinberg equilibrium due to excessive homozygosity in these domesticated broodstocks, reflecting inbreeding. These microsatellite loci will be useful, not only for assessment of population structure and genetic variability, but also for conservation of wild deer populations in Taiwan.

Little has been known about the impacts of past vicariance events on the phylogeography and population structure of freshwater fishes in East Asia.The aims of this study are to assess the genetic variability with extensive sampling throughout the range of Chinese spiny loach, Cobitis sinensis, andto infer the genetic structure and evolutionary history of populations. Cobitis sinensis in China may have initiated from two ancestral populations,namely Yangtze and Pearl Rivers, which diverged about 7.24 MYA likely due to drainage systems alteration. In the phylogroup I, a southward dis-persal event occurred from East China (Yangtze River) to south ZheMin and Hainan subregions, followed by eastward dispersal from ZheMin to southTaiwan. In the phylogroup II, eastward colonization took place from Pearl River to north Taiwan in the late Pliocene, coupled with loss of geneticdiversity in the island populations. This study showed that Cenozoic tectonic movements and climatic and sea-level fluctuations may have shaped thegenetic structure of C. sinensis in concert. Highly diverged mtDNA sequences suggest existence of cryptic species in morphospecies C. sinensis.Key words: Cobitis sinensis – coalescence – cryptic species – tectonic movements – phylogeography

We aimed to analyze the phytotoxic effect of the allelopathic volatile myrcene on rice seedlings, particularly root growth, reactive oxygen species (ROS) generation, and activity of antioxidant enzymes and defense-related genes. Myrcene inhibited the growth of rice seedlings. The activity of ROS and lipoxygenase (LOX) was significantly increased with increasing myrcene concentration in roots and that of antioxidant enzymes was altered dose-dependently. The activity of superoxide dismutase and peroxidase was changed at 24 h after myrcene treatment in rice roots. Furthermore, the mRNA expression of three mitogen-activated protein kinase genes (OsMPK2, 3, and 4), WRKY transcription factor gene (OsWRKY71), LOX gene (OsLOX3), pathogenesis-related protein 1b gene (OsPR1b) and cyclin-dependent protein kinase inhibitor (OsCKI) was upregulated and that of cyclin genes OsCycA1;1, OsCycB1;1 and OsCycD1;1 was downregulated with myrcene treatment in rice roots. These results may provide new insights into the molecular basis of the allelopathic volatile response in plants.

Gene flow between species may last a long time in plants. Reticulation inevitably causes difficulties in phylogenetic reconstruction. In this study, we looked into the genetic divergence and phylogeny of 20 Lilium species based on multilocus analyses of 8 genes of chloroplast DNA (cpDNA), the internally transcribed nuclear ribosomal DNA (nrITS) spacer and 20 loci extracted from the expressed sequence tag (EST) libraries of L. longiflorum Thunb. and L. formosanum Wallace. The phylogeny based on the combined data of the maternally inherited cpDNA and nrITS was largely consistent with the taxonomy of Lilium sections. This phylogeny was deemed the hypothetical species tree and uncovered three groups, i.e., Cluster A consisting of 4 taxa from the sections Pseudolirium and Liriotypus, Cluster B consisting of the 4 taxa from the sections Leucolirion, Archelirion and Daurolirion, and Cluster C comprising 10 taxa mostly from the sections Martagon and Sinomartagon. In contrast, systematic inconsistency occurred across the EST loci, with up to 19 genes (95%) displaying tree topologies deviating from the hypothetical species tree. The phylogenetic incongruence was likely attributable to the frequent genetic exchanges between species/sections, as indicated by the high levels of genetic recombination and the IMa analyses with the EST loci. Nevertheless, multilocus analysis could provide complementary information among the loci on the species split and the extent of gene flow between the species. In conclusion, this study not only detected frequent gene flow among Lilium sections that resulted in phylogenetic incongruence but also reconstructed a hypothetical species tree that gave insights into the nature of the complex relationships among Lilium species.

Postglacial climate changes alter geographical distributions and diversity of species. Such ongoing changes often force species to migrate along the latitude/altitude. Altitudinal gradients represent assemblage of environmental, especially climatic, variable factors that influence the plant distributions. Global warming that triggered upward migrations has therefore impacted the alpine plants on an island. In this study, we examined the genetic structure of Juniperus morrisonicola, a dominant alpine species in Taiwan, and inferred historical, demographic dynamics based on multilocus analyses. Lower levels of genetic diversity in north indicated that populations at higher latitudes were vulnerable to climate change, possibly related to historical alpine glaciers. Neither organellar DNA nor nuclear genes displayed geographical subdivisions, indicating that populations were likely interconnected before migrating upward to isolated mountain peaks, providing low possibilities of seed/pollen dispersal across mountain ranges. Bayesian skyline plots suggested steady population growth of J. morrisonicola followed by recent demographic contraction. In contrast, most lower-elevation plants experienced recent demographic expansion as a result of global warming. The endemic alpine conifer may have experienced dramatic climate changes over the alternation of glacial and interglacial periods, as indicated by a trend showing decreasing genetic diversity with the altitudinal gradient, plus a fact of upward migration.

Summary Asian rice, Oryza sativa, consists of two major subspecies, indica and japonica, which are physiologically differentiated and adapted to different latitudes. Genes for photoperiod sensitivity are likely targets of selection along latitude. We examined the footprints of natural and artificial selections for four major genes of the photoperiod pathway, namely PHYTOCHROME B (PhyB), HEADING DATE 1 (Hd1), HEADING DATE 3a (Hd3a), and EARLY HEADING DATE 1 (Ehd1), by investigation of the patterns of nucleotide polymorphisms in cultivated and wild rice. Geographical subdivision between tropical and subtropical O. rufipogon was found for all of the photoperiod genes in plants divided by the Tropic of Cancer (TOC). All of these genes, except for PhyB, were characterized by the existence of clades that split a long time ago and that corresponded to latitudinal subdivisions, and revealed a likely diversifying selection. Ssp. indica showed close affinity to tropical O. rufipogon for all genes, while ssp. japonica, which has a much wider range of distribution, displayed complex patterns of differentiation from O. rufipogon, which reflected various agricultural needs in relation to crop yield. In japonica, all genes, except Hd3a, were genetically differentiated at the TOC, while geographical subdivision occurred at 31°N in Hd3a, probably the result of varying photoperiods. Many other features of the photoperiod genes revealed domestication signatures, which included high linkage disequilibrium (LD) within genes, the occurrence of frequent and recurrent non-functional Hd1 mutants in cultivated rice, crossovers between subtropical and tropical alleles of Hd1, and significant LD between Hd1 and Hd3a in japonica and indica.

The red-spot prawn, Metapenaeopsis barbata, is a commercially important, widely distributed demersal species in the Indo-West Pacific Ocean. Overfishing has made its populations decline in the past decade. To study conservation genetics, eight polymorphic microsatellite loci were isolated. Genetic characteristics of the SSR (simple sequence repeat) fingerprints were estimated in 61 individuals from adjacent seas of Taiwan and China. The number of alleles, ranging from 2 to 4, as well as observed and expected heterozygosities in populations, ranging from 0.048 to 0.538, and 0.048 and 0.654, respectively, were detected. No deviation from Hardy–Weinberg expectations was detected at either locus. No significant linkage disequilibrium was detected in locus pairs. The polymorphic microsatellite loci will be useful for investigations of the genetic variation, population structure, and conservation genetics of this species.

In the present work, a series of simulation tools were used to determine structure-activity relationships for the endomorphins (EMs) and derive μ-pharmacophore models for these peptides. Potential lowest energy conformations were determined in vacuo by systematically varying the torsional angles of the Tyr(1)-Pro(2) (ω(1)) and Pro(2)-Trp(3)/Phe(3) (ω(2)) as tuning parameters in AM1 calculations. These initial models were then exposed to aqueous conditions via molecular dynamics simulations. In aqueous solution, the simulations suggest that endomorphin conformers strongly favor the trans/trans pair of the ω(1)/ω(2) amide bonds. From two-dimensional probability distributions of the ring-to-ring distances with respect to the pharmacophoric angles for EMs, a selectivity range of μ(1) is ca. 8.3 ~ 10.5 Å for endomorphin-2 and selectivity range of μ(2) is ca. 10.5 ~ 13.0 Å for endomorphin-1 were determined. Four-component μ-pharmacophore models are proposed for EMs and are compared to the previously published δ- and κ-pharmacophore models.

Nine polymorphic microsatellite makers were isolated and characterized in 20 individuals of Parapenaeopsis hardwickii collected from the fishing ground near Ilan, Taiwan. Number of alleles per locus ranged from 3 to 8, observed and expected heterozygosities ranged from 0.5263 to 0.7632, and from 0.4225 to 0.8044, respectively. Only one locus showed a Hardy–Weinberg equilibrium deviation. No linkage disequilibrium was found among these studied loci. This set of microsatellites provides a suitable tool for population genetic structure and fishery management of this species in Asian coastal waters.

Molecular dynamics simulations of the biphalin molecule, (Tyr-D-Ala-Gly-Phe-NH)2, and the active tetrapeptide hydrazide, Tyr-D-Ala-Gly-Phe-NH-NH2 were performed to investigate the cause of the increased μ and δ receptor binding affinities of the former over the latter. The simulation results demonstrate that the acylation of the two equal tetrapeptide fragments of biphalin produces the constrained hydrazide bridges \( {\hbox{C}}_4^{\alpha } - {{\hbox{C}}_4}\prime - {{\hbox{N}}_9} - {{\hbox{N}}_{{10}}} \) and \( {{\hbox{N}}_9} - {{\hbox{N}}_{{10}}} - {{\hbox{C}}_5}\prime - {\hbox{C}}_5^{\alpha } \), which in turn increase the opportunity of conformations for binding to μ or δ receptors. Meanwhile, the connection of the two active tetrapeptide fragments of biphalin also results in the constrained side chain torsion angle χ2 at one of the two residues Phe. This constrained side chain torsion angle not only significantly increases the δ receptor binding affinity but also makes most of the δ receptor binding conformations of biphalin bind to the δ receptor through the fragment containing the mentioned residue Phe.

A. halleri is a psuedometallophyte with a patchy distribution in Europe and is often spread by human activity. To determine the population history and whether this history is consistent with potential human effects, we surveyed nucleotide variation using 24 loci from 12 individuals in a large A. halleri population. The means of total and silent nucleotide variation (theta(W)) are within the range expected for the species. The population genetic neutrality tests Tajima's D and Wall's B had significant composite results rejecting panmixia, and Approximate Bayesian Computation analysis revealed that a subdivision model better explained the variation than the standard neutral model, refugia (or admixture), bottleneck or change of population size models. A categorical regression analysis further supports the subdivision model, and under the subdivision model, the neutrality tests are no longer significant. The best support was for two source populations, a situation consistent with the mixing of two populations possibly mediated by human activity. This scenario might limit the genetic diversity and adaptive potential of the population. The non-neutral population variation described here should be considered in bioinformatic searches for adaptation.

Nouelia insignis, an endangered species, is distributed in the Jinsha and Nanpan drainage areas in southwestern China. In this study, we examined the genetic diversity and population structure based on the sequences of the cpDNA rpL 16 intron. Low levels of genetic variation were detected within all populations of the endemic species. A gene genealogy of 11 haplotypes recovered two major lineages I and II, with haplotypes H1 and H6 nested as interior nodes, respectively. Haplotype H1 was widespread in all populations, while haplotype H6 was restricted to populations southern of the Jinsha River. Low levels of genetic differentiation were detected, as most F (st) values between populations were zero. This result, however, contradicts previous studies based on allozymes and fingerprinting. Genetic analyses suggested that coancestry due to low evolutionary rates resulted in the lack of geographical subdivision. Molecular dating estimated that the two lineages split about 3.224 MYA (95% CI 1.070-6.089 MYA). Maintenance of ancestral polymorphisms was possibly attributable to a long-standing large effective population size until recently. Postglacial demographic expansion was supported by a unimodal mismatch distribution and star-like phylogenies.

Formosan lily (Lilium formosanum), a species endemic in Taiwan, is characterized by showy and fragrant flowers. To understand the gene expression at its reproductive phase, we constructed a suppression subtractive cDNA library of immature flower buds, from which 1,324 expressed sequence tags (ESTs) were randomly selected and sequenced. These EST sequences were clustered into 974 nonredundant sequences. Based on BLAST searching, functions of 376 sequences (39%) were determined, and 80 sequences showed high similarities to genes encoding hypothetical proteins without known functions. Another 518 sequences did not show significant homology to any known sequences and were therefore classified as novel sequences. Further analyses of the 376 ESTs sequences revealed high abundance of stress-related and flower-development genes. The highly expressing stress-related transcripts include 39 with high similarities to lipid transfer proteins, five to ascorbate peroxidases, and five to heat shock proteins 70. Using real-time quantitative RT-PCR analysis, we further revealed the expression of these three genes in the immature flower buds and in the pistils or stamens of the blooming flower of Formosan lily collected from alpine regions. These results suggest that the flower of L. formosanum possesses a significantly elevated level of stress genes in response to alpine environment and the ESTs analyzed here represent a valuable resource for studying a resistance mechanism of the reproductive organs of Formosan lily.

Lin, C.-J., Lin, H.-D., Wang, J.-P., Chao, S.-C. & Chiang, T.-Y. (2007). Phylogeography of Hemibarbus labeo (Cyprinidae): secondary contact of ancient lineages of mtDNA. —Zoologica Scripta, 39, 23–35. Nucleotide sequences of the D-loop control region of mtDNA were used to assess the genetic structure and phylogeography of Hemibarbus labeo in Mainland China and Taiwan. A hierarchical analysis of molecular variance of populations in 11 major streams from three geographical regions revealed significant structuring among populations and geographical regions. High levels of nucleotide diversity (π = 1.88%) and haplotype diversity (h = 96 ± 0.009%) suggest a large effective population size. A maximum likelihood tree based on mtDNA variation identified two ancient mtDNA lineages, which split approximately 3.39 million years ago. Overlapping distribution of the major lineages displayed low correspondence with geographical regions and reflects a scenario of secondary mergence after long isolation. Gene genealogy further revealed a unidirectional migration. Nevertheless, there existed a phylogeographical structure that mostly agreed with a biogeographical hypothesis. That is, within each lineage, a close phylogeny between populations of the River-Campagna and East-Pacific regions was supported by the mtDNA gene genealogy, although monophyly of each geographical region was not supported. The degree of genetic differentiation was correlated with geographical distances between populations, displaying a pattern of ‘isolation by distance’. Gene genealogy of mtDNA revealed that Yangtzejiang population may act as a divergence centre of H. labeo. In addition, Taiwan population was colonized via a recent a founder event, likely from population Yangtzejiang River about 131 000 years before present. Low haplotype number and genetic variability also suggested possible bottleneck events in the Rongjiang and Dazhangjiang populations.

Ludwigia, a genus with rampant interspecific hybridization, is an ideal model for examining the impact of gene introgression and polyploidization on species diversification. We examined the evolution of the Ludwigia sect. Isnardia, which is a polyploid complex (x = 8) of five species distributed in North America. The phylogeny of L. sect. Isnardia was reconstructed based on sequences of cpDNA atpB-rbcL intergenic spacer and nrITS (internal transcribed spacer). Most L. sect. Isnardia species are polyphyletic at both loci, indicating possible recurrent hybridization, based on maximum likelihood trees rooted at L. sect. Microcarpium. A minimum spanning network was also constructed to examine genealogical relationships among haplotypes. In the cpDNA network, haplotypes of tetraploid L. spathulata are nested at the most interior nodes, suggesting a parental genome origin of an extinct diploid or an unsampled extant diploid with a DD cytotype. Allopolyploid L. arcuata contributed its cpDNA to hexaploid L. repens and L. brevipes. ITS haplotypes of diploid L. palustris were clustered with L. spathulata, suggesting L. palustris as the paternal genome donor. Ludwigia brevipes may have arisen via bi-directional hybridization between L. palustris and L. arcuata followed by polyploidy. Genetic and phylogenetic analyses using molecular markers with different inheritance modes provided sufficient insights into the reticulate evolution of the species of L. sect. Isnardia. Interspecific hybridization and polyploidy play a key role in the species evolution in L. sect. Isnardia.

The nocturnal distribution and resource use by Ryukyu flying foxes was studied along 28 transects, covering five types of habitats, on Iriomote Island, Japan, from early June to late September, 2005. Bats were mostly encountered solitarily (66.8%) or in pairs (16.8%), with a mean linear density of 2.5 ± 0.6 bats/km of transect/night. Across the island, however, bat densities were distributed non-randomly among transect-nights, not correlated with transect length, and showed a slightly clumped distribution (variance/mean = 3.3). Outskirt trails contributed higher values to the relative importance of bat abundance, but the highest mean abundances occurred mostly at village sites on the west coast, which on average devoted only a quarter of their land area to agriculture/husbandry compared to those on the east coast. This supports our prediction that higher bat abundances are found in areas with less anthropogenic interference and more forest. Among habitats, the mean total abundance and density were lower in cultivated areas than in villages and inland forests. Bat perches in cultivated areas were also lower, and were in correspondence with lower shrub and canopy heights, and less canopy coverage. Flying fox abundance was correlated moderately with the heterogeneity of the tree composition, and strongly with the density of major fruiting trees. Thirty-nine species of plants and some animal items were used by Ryukyu flying foxes, including at least 31 species of fruits, 13 species of flowers, and leaves of seven species, with 14 species new to the record. Ficus septica and F. variegata were the most frequently encountered and dominant items in both fecal and rejecta/dropped samples, followed mostly by other figs and mulberries in the former, but by larger-seed non- Moraceae plants in dropped samples. Our results suggest that for conservation of flying foxes undisturbed forests providing an adequate resource basis are of major importance.

Caridina gracilipes, an atyid shrimp of Taiwan, inhabits the fresh and brackish waters. Its life history can be classified into amphidromous and landlocked types. Recent human’s transportation has caused concerns of genetic contamination between these long evolved lineages and ecological instabilities. Molecular markers with high sensitivities are required for detecting the possible introgression due to human disturbance. In the study, we described the development of 15 polymorphic microsatellite loci in C. gracilipes for genetic and ecological studies. These new markers were tested in 20 individuals of three populations. The number of alleles ranged from 6 to 17. Expected (H E) and observed (H O) heterozygosities ranged from 0.653 to 0.954, averaged at 0.865, and from 0.000 to 0.4, averaged at 0.146, respectively. All loci are significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency. Significant genetic differentiation among populations was detected based on F ST values, which were estimated from 0.82 to 0.88.

Miscanthus sinensis (Poaceae) is typical of many dominant grasses of East Asia. Due to its characteristics of fast growth and high biomass, Miscanthus, a C4 plant, has been long explored for the potential usage as biofuel. In this study, we described the development of nine microsatellite loci from M. sinensis for genetic studies. These new markers were tested in 25 individuals of five populations in three varieties. The number of alleles ranged from 3 to 8. The expected (H E) and observed (H O) heterozygosities were 0.37–0.78 and 0.05–0.52, respectively. All microsatellite loci are significantly deviated from Hardy–Weinberg expectations likely due to the population structure within samples. Linkage disequilibrium between four loci pairs were observed from the total 36 pairwise comparisons of loci. Cross-species transferability revealed that all loci can be applied to its closely related species, M. floridulus.

Aphyocypris kikuchii is a cyprinid species endemic to northern and eastern Taiwan and is the only primary freshwater fish native east of the Coastal Mountain Range. In total, 92 individuals of A. kikuchii from seven populations in three regions of the island were surveyed for mitochondrial DNA (mtDNA) variation. High haplotype diversity (h= 0·989) and low nucleotide diversity (π= 0·009) of mtDNA were detected. Negative values of Tajima’s D and unimodal mismatch distributions probably reflect a history of recent demographic expansions from small populations. Three major haplotype clusters displayed geographically non-overlapping distributions, indicating a long-term isolation between regions. Hierarchical analysis of molecular variance showed significant genetic structuring among populations (ΦST= 0·66). Significant haplotype heterogeneity was also detected among populations within regions (ΦSC= 0·41, P < 0·001) and among regions (ΦCT= 0·43, P < 0·05). Molecular clock estimates of coalescence in the three major mtDNA lineages indicated coalescence in the most recent common ancestor c. 0·11–0·39 million years ago. Haplotypes of cluster B nested as interior nodes in the haplotype network, indicating that migrations from Shueilian (SL) populations to the northern region (cluster A) and to the eastern region (cluster C) may have occurred independently. Lineages A and B + C should be managed as two distinct evolutionarily significant units, while the northern, SL and southern groups should be managed as separate management units.