Evaluating the genetic diversity in two tropical leguminous trees, Dalbergia cochinchinensis and D. nigrescens, in lowland forests in Cambodia and Thailand using MIG-seq.

It is vital to measure the levels of genetic diversity and differentiation between populations in a species to understand the current genetic structure and evolution of the species. Here, MIG-seq (multiplexed inter-simple sequence repeat genotyping by sequencing) was employed to assess the genetic variation in two tropical leguminous tree species, Dalbergia cochinchinensis and D. nigrescens, in Cambodia and Thailand. Sequence data for 255-618 loci, each with an approximate length of 100 bp, were obtained, and the nucleotide diversity, Tajima's D and FST were computed. The estimates calculated from the data obtained by MIG-seq were compared to those obtained by Sanger sequencing of nine nuclear coding genes in D. cochinchinensis in our previous study. The nucleotide diversity at the MIG-seq loci was slightly higher than that at silent sites in the coding loci, whereas the FST values at the MIG-seq loci were generally lower than those at the coding loci, although the differences were not significant. Moreover, nucleotide diversities within populations of the two species were similar to each other, at approximately 0.005. Three and four population clusters were genetically recognized in D. cochinchinensis and D. nigrescens, respectively. Although the populations were differentiated from each other, the levels of differentiation among them, as measured by FST, were higher in D. cochinchinensis than in D. nigrescens. This indicates higher levels of gene flow between the populations in the latter species. We recommend using MIG-seq for quick surveys of genetic variation because it is cost-effective and results in smaller variance in the estimates of population genetic parameters.

Inferring the demographic history of Japanese cedar, Cryptomeria japonica, using amplicon sequencing.

The evolution of a species depends on multiple forces, such as demography and natural selection. To understand the trajectory and driving forces of evolution of a target species, it is first necessary to uncover that species' population history, such as past and present population sizes, subdivision and gene flow, by using appropriate genetic markers. Cryptomeria japonica is a long-lived monoecious conifer species that is distributed in Japan. There are two main lines (omote-sugi and ura-sugi), which are distinguished by apparent differences in morphological traits that may have contributed to their local adaptation. The evolution of these morphological traits seems to be related to past climatic changes in East Asia, but no precise estimate is available for the divergence time of these two lines and the subsequent population dynamics in this species. Here, we analyzed the nucleotide variations at 120 nuclear genes in 94 individuals by using amplicon sequencing in combination with high-throughput sequencing technologies. Our analysis indicated that the population on Yakushima Island, the southern distribution limit of C. japonica in Japan, diverged from the other populations 0.85 million years ago (MYA). The divergence time of the other populations on mainland Japan was estimated to be 0.32 MYA suggesting that the divergence of omote-sugi and ura-sugi might have occurred before the last glacial maximum. Although we found modest levels of gene flow between the present populations, the long-term isolation and environmental heterogeneity caused by climatic changes might have contributed to the differentiation of the lines and their local adaptation.

Effects of cyclic changes in population size on neutral genetic diversity.

Recurrent changes in population size are often observed in nature, influencing the efficiency of selection and consequently affecting organismal evolution. Thus, it is important to know whether such changes occurred in the past history of a focal population of evolutionary interests. Here, we focused on cyclic changes in population size and investigated the distributional properties of Tajima's D and its power to distinguish a cyclic change model compared with the standard neutral model, changing the frequency and magnitude of the cyclic change. With very low or very high frequencies of the cycle, the distribution of Tajima's D was similar to that in a constant size population, as demonstrated by previous theoretical works. Otherwise, its mean was negative or positive, and its variance was smaller or larger depending on the time of sampling. The detection rate of the cyclic change against the constancy in size by Tajima's D depended on the sample size, the number of loci, and the time of sampling in addition to the frequency and amplitude of the cycle. Using sequence data of several tens of loci, the detection rate was fairly high if the frequency was intermediate and the sampling was made when population size was large; otherwise, the detection rate was not high. We also found that cyclic change could be discriminated from simple expansion or shrinkage of a population by Tajima's D only if the frequency was in a limited range and the sampling was made when the population was large.

Horizontal Transmission of Symbiotic Green Algae Between Hydra Strains.

Some hydra strains belonging to the vulgaris group show a symbiotic relationship with green algae Chlorococcum sp. The symbiotic green algae can escape from the host polyps and can form swimming zoospores (which have two flagella) in culture solution. We observed that co-culture with the symbiotic polyps caused horizontal transmission of the symbionts into some non-symbiotic hydra strains that have no symbionts in nature and that belong not only to the vulgaris group but also to other hydra species groups. Although most of the horizontal transmission has ended in transient symbioses, a newly formed symbiosis between the symbiotic Chlorococcum sp. and strain 105 of Hydra vulgaris (Hydra magnipapillata) has been sustained for more than five years and has caused morphological and behavioral changes in the host polyps. We named this strain 105G. The asexual proliferation rate by budding increased under light conditions, although the feeding activity decreased and the polyp size was reduced in strain 105G. This new symbiosis between Chlorococcum sp. and strain 105G of H. vulgaris provides us with an intriguing research system for investigating the origin of symbiosis.

Understanding and monitoring the consequences of human impacts on intraspecific variation.

Intraspecific variation is a major component of biodiversity, yet it has received relatively little attention from governmental and nongovernmental organizations, especially with regard to conservation plans and the management of wild species. This omission is ill-advised because phenotypic and genetic variations within and among populations can have dramatic effects on ecological and evolutionary processes, including responses to environmental change, the maintenance of species diversity, and ecological stability and resilience. At the same time, environmental changes associated with many human activities, such as land use and climate change, have dramatic and often negative impacts on intraspecific variation. We argue for the need for local, regional, and global programs to monitor intraspecific genetic variation. We suggest that such monitoring should include two main strategies: (i) intensive monitoring of multiple types of genetic variation in selected species and (ii) broad-brush modeling for representative species for predicting changes in variation as a function of changes in population size and range extent. Overall, we call for collaborative efforts to initiate the urgently needed monitoring of intraspecific variation.

Inferences of population structure and demographic history for Taxodium distichum, a coniferous tree in North America, based on amplicon sequencing analysis.

PREMISE OF THE STUDY: Studies of natural genetic variation can elucidate the genetic basis of phenotypic variation and the past population structure of species. Our study species, Taxodium distichum, is a unique conifer that inhabits the flood plains and swamps of North America. Morphological and ecological differences in two varieties, T. distichum var. distichum (bald cypress) and T. distichum var. imbricarium (pond cypress), are well known, but little is known about the level of genetic differentiation between the varieties and the demographic history of local populations. METHODS: We analyzed nucleotide polymorphisms at 47 nuclear loci from 96 individuals collected from the Mississippi River Alluvial Valley (MRAV), and Gulf Coastal populations in Texas, Louisiana, and Florida using high-throughput DNA sequencing. Standard population genetic statistics were calculated, and demographic parameters were estimated using a composite-likelihood approach. KEY RESULTS: Taxodium distichum in North America can be divided into at least three genetic groups, bald cypress in the MRAV and Texas, bald cypress in Florida, and pond cypress in Florida. The levels of genetic differentiation among the groups were low but significant. Several loci showed the signatures of positive selection, which might be responsible for local adaptation or varietal differentiation. CONCLUSIONS: Bald cypress was genetically differentiated into two geographical groups, and the boundary was located between the MRAV and Florida. This differentiation could be explained by population expansion from east to west. Despite the overlap of the two varieties' ranges, they were genetically differentiated in Florida. The estimated demographic parameters suggested that pond cypress split from bald cypress during the late Miocene.

Evolutionary rate variation in two conifer species, Taxodium distichum (L.) Rich. var. distichum (baldcypress) and Cryptomeria japonica (Thunb. ex L.f.) D. Don (Sugi, Japanese cedar).

With the advance of sequencing technologies, large-scale data of expressed sequence tags and full-length cDNA sequences have been reported for several conifer species. Comparative analyses of evolutionary rates among diverse taxa provide insights into taxon-specific molecular evolutionary features and into the origin of variation in evolutionary rates within genomes and between species. Here, we estimated evolutionary rates in two conifer species, Taxodium distichum and Cryptomeria japonica, to illuminate the molecular evolutionary features of these species, using hundreds of genes and employing Chamaecyparis obtusa as an outgroup. Our results show that the mutation rates based on synonymous substitution rates (dS) of T. distichum and C. japonica are approximately 0.67 × 10(-9) and 0.59 × 10(-9)/site/year, respectively, which are 15-25 times lower than those of annual angiosperms. We found a significant positive correlation between dS and GC3. This implies that a local mutation bias, such as context dependency of the mutation bias, exists within the genomes of T. distichum and C. japonica, and/or that selection acts on synonymous sites in these species. In addition, the means of the ratios of synonymous to nonsynonymous substitution rate in the two species are almost the same, suggesting that the average intensity of functional constraint is constant between the lineages. Finally, we tested the possibility of positive selection based on the site model, and detected one candidate gene for positive selection.

Difference in flowering time can initiate speciation of nocturnally flowering species.

Isolation mechanisms that prevent gene flow between populations prezygotically play important roles in achieving speciation. In flowering plants, the nighttime flowering system provides a mechanism for isolation from diurnally flowering species. Although this system has long been of interest in evolutionary biology, the evolutionary process leading to this system has yet to be elucidated because of the lack of good model species. However, the genetic mechanisms underlying the differences in flowering times and the traits that attract pollinators between a pair of diurnally and nocturnally flowering species have recently been identified in a few cases. This identification enables us to build a realistic model for theoretically studying the evolution of a nocturnally flowering species. In this study, based on previous experimental data, we assumed a model in which two loci control the flowering time and one locus determines a trait that attracts pollinators. Using this model, we evaluated the possibility of the evolution of a nocturnally flowering species from a diurnally flowering ancestor through simulations. We found that a newly emerging nighttime flowering flower exhibited a sufficiently high fitness, and the evolution of a nocturnally flowering species from a diurnally flowering species could be achieved when hybrid viability was intermediate to low, even in a completely sympatric situation. Our results suggest that the difference in flowering time can act as a magic trait that induces both natural selection and assortative mating and would play an important role in speciation between diurnally and nocturnally flowering species pairs.

High prevalence of non-synonymous substitutions in mtDNA of cichlid fishes from Lake Victoria.

When a population size is reduced, genetic drift may fix slightly deleterious mutations, and an increase in nonsynonymous substitution is expected. It has been suggested that past aridity has seriously affected and decreased the populations of cichlid fishes in Lake Victoria, while geographical studies have shown that the water levels in Lake Tanganyika and Lake Malawi have remained fairly constant. The comparably stable environments in the latter two lakes might have kept the populations of cichlid fishes large enough to remove slightly deleterious mutations. The difference in the stability of cichlid fish population sizes between Lake Victoria and the Lakes Tanganyika and Malawi is expected to have caused differences in the nonsynonymous/synonymous ratio, ω (=dN/dS), of the evolutionary rate. Here, we estimated ω and compared it between the cichlids of the three lakes for 13 mitochondrial protein-coding genes using maximum likelihood methods. We found that the lineages of the cichlids in Lake Victoria had a significantly higher ω for several mitochondrial loci. Moreover, positive selection was indicated for several codons in the mtDNA of the Lake Victoria cichlid lineage. Our results indicate that both adaptive and slightly deleterious molecular evolution has taken place in the Lake Victoria cichlids' mtDNA genes, whose nonsynonymous sites are generally conserved.

Genetic structure of pelagic and littoral cichlid fishes from Lake Victoria.

The approximately 700 species of cichlids found in Lake Victoria in East Africa are thought to have evolved over a short period of time, and they represent one of the largest known examples of adaptive radiation. To understand the processes that are driving this spectacular radiation, we must determine the present genetic structure of these species and elucidate how this structure relates to the ecological conditions that caused their adaptation. We analyzed the genetic structure of two pelagic and seven littoral species sampled from the southeast area of Lake Victoria using sequences from the mtDNA control region and 12 microsatellite loci as markers. Using a Bayesian model-based clustering method to analyze the microsatellite data, we separated these nine species into four groups: one group composed of pelagic species and another three groups composed mainly of rocky-shore species. Furthermore, we found significant levels of genetic variation between species within each group at both marker loci using analysis of molecular variance (AMOVA), although the nine species often shared mtDNA haplotypes. We also found significant levels of genetic variation between populations within species. These results suggest that initial groupings, some of which appear to have been related to habitat differences, as well as divergence between species within groups took place among the cichlid species of Lake Victoria.

Difference in flowering time as an isolating barrier.

Although many theoretical studies have reported strong effects of different flowering times on reproductive isolation, such studies have all focused on the different flowering time within a season, and the subsequently developed models are difficult to apply to the cases of diurnal- and nocturnal-flowering species pairs. The different flowering times within a day differ from those within a season because of the simultaneous opening and closing of the flowers for each species and the carry-over of the pollen from early to later times. In this study, we consider pollinator-mediated, diurnal- and nocturnal-flowering plants and build a new model to study the effects of the different flowering times within a day on reproductive isolation. We assume two loci, each with two alleles, which determine the opening and closing times of flowers, respectively. We numerically calculate the changes in the frequencies of the gametes in a model incorporating the reductions in hybrid viability, flowering costs, recombination rate and degree of dominance at each locus. We found that the early-opening flowers had a much higher fitness than the late-opening flowers and that the maintenance of the two species was difficult even if their flowering times were not overlapping. Therefore, some other mechanisms, such as pollinator preference, may be required to explain the coexistence of closely related diurnal and nocturnal flowers.

Symbiosis between hydra and chlorella: molecular phylogenetic analysis and experimental study provide insight into its origin and evolution.

Although many physiological studies have been reported on the symbiosis between hydra and green algae, very little information from a molecular phylogenetic aspect of symbiosis is available. In order to understand the origin and evolution of symbiosis between the two organisms, we compared the phylogenetic relationships among symbiotic green algae with the phylogenetic relationships among host hydra strains. To do so, we reconstructed molecular phylogenetic trees of several strains of symbiotic chlorella harbored in the endodermal epithelial cells of viridissima group hydra strains and investigated their congruence with the molecular phylogenetic trees of the host hydra strains. To examine the species specificity between the host and the symbiont with respect to the genetic distance, we also tried to introduce chlorella strains into two aposymbiotic strains of viridissima group hydra in which symbiotic chlorella had been eliminated in advance. We discussed the origin and history of symbiosis between hydra and green algae based on the analysis.

Speciation history of three closely related oak gall wasps, Andricus mukaigawae, A. kashiwaphilus, and A. pseudoflos (Hymenoptera: Cynipidae) inferred from nuclear and mitochondrial DNA sequences.

The Andricus mukaigawae complex of oak gall wasps is composed of cyclically parthenogenetic species: A. mukaigawae and Andricus kashiwaphilus, and a parthenogenetic species, Andricus pseudoflos. The component species differ in life history, host plant, karyotype, and asexual gall shape, although little difference is found in the external morphology of asexual adults. To understand the speciation history of this species complex, DNA sequences of one mitochondrial region and nine nuclear gene regions were investigated. The genetic relationship among the species suggested that a loss of sex occurred after host shift. Unexpectedly, two or three distinct groups in the parthenogenetic species, A. pseudoflos, were revealed by both mitochondrial and nuclear DNA data. Gene flow in nuclear genes from the species not infected by Wolbachia (A. kashiwaphilus) to the species infected by it (A. mukaigawae) was suggested by a method based on coalescent simulations. On the other hand, gene flow in mitochondrial genes was suggested to be in the opposite direction. These findings indicate possible involvement of Wolbachia infection in the speciation process of the A. mukaigawae complex.

Linkage disequilibrium in a population undergoing periodic fragmentation and admixture.

Glacial and interglacial cycles are considered to have caused the fragmentation and admixture of populations in many organisms. A simple model incorporating such periodic changes of the population structure is analysed in order to investigate the behaviour of neutral genetic variation at one and two loci. The equilibrium is reached very quickly in terms of cycles if the length of a cycle is long, as would be expected of the glaciation cycles. Heterozygosity and linkage disequilibrium are shown to depend on the length of time of the fragmented and admixed phases, population sizes, and number (n) of subpopulations in the fragmented phase. If the population size is small in the fragmented phase and its duration is long, the squared correlation coefficient of two loci (a measure of linkage disequilibrium) just after the admixture is approximated by 1/(n-1) for n > 1. After admixture, the correlation decays at a rate of approximately twice the recombination rate. Therefore, if post-glaciation admixture created linkage disequilibrium, we expect to observe linkage disequilibrium even between moderately linked loci, and its decay pattern along the chromosome is very different from that in a random mating population at equilibrium. This is especially true in organisms with long generation times such as trees.

Correlation between nuptial colors and visual sensitivities tuned by opsins leads to species richness in sympatric Lake Victoria cichlid fishes.

Reproductive isolation that prevents interspecific hybridization between closely related coexisting species maintains sympatric species diversity. One of the reproductive isolations is mate choice based on color signals (breeding color perceived by color vision). This is well known in several animal taxa, yet little is known about its genetic and molecular mechanism. Lake Victoria cichlid fishes are thought to be an example of sympatric species diversity. In the species inhabiting different light environments in rocky shore, speciation by sensory drive through color signals has been proposed by analyses of the long wavelength-sensitive (LWS) opsin gene and the male nuptial coloration. However, the genetic and molecular mechanism of how diversity of sympatric species occurring in the same habitat is maintained remains unknown. To address this issue, we determined nucleotide sequences of eight opsins of six sympatric species collected from a sandy-muddy shore--an ideal model system for studying sympatric species. Among eight opsins, the LWS and RH1 alleles were diversified and one particular allele is dominant or fixed in each species, and we propose that this is due to natural selection. The functions of their LWS alleles were also diversified as shown by absorption measurements of reconstituted visual pigments. To analyze the relationship between nuptial coloration and the absorption of LWS pigments, we systematically evaluated and defined nuptial coloration. We showed that the coloration was species specific with respect to hue and significantly differentiated by the index values of hue (dominant wavelength: λ(d)). The λ(d) value of the male nuptial coloration correlated with the absorption of LWS pigments from all the species, suggesting that reproductive isolation through mate choice using color signals may prevent sympatric interspecific hybridization, thereby maintaining the species diversity in sympatric species in Lake Victoria.

Extended linkage disequilibrium in noncoding regions in a conifer, Cryptomeria japonica.

We measured linkage disequilibrium in mostly noncoding regions of Cryptomeria japonica, a conifer belonging to Cupressaceae. Linkage disequilibrium was extensive and did not decay even at a distance of 100 kb. The average estimate of the population recombination rate per base pair was 1.55 × 10(-5) and was <1/70 of that in the coding regions. We discuss the impact of low recombination rates in a large part of the genome on association studies.

Evolution of the number of LRRs in plant disease resistance genes.

The largest group of plant resistance (R) genes contain the regions that encode the nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains (NBS-LRR genes). To gain new resistance, amino acid substitutions and changes in number of the LRRs that recognize the presence of pathogens are considered important. In this study, we focus on the evolution of the number of LRRs and analyze the genome data of five plant species, Arabidopsis thaliana, Oryza sativa, Medicago truncatula, Lotus japonicus and Populus trichocarpa. We first categorized the NBS-LRR genes in each species into groups and subgroups based on the phylogenetic relationships of their NBS domain sequences. Then we estimated the evolutionary rate of the number of LRRs relative to the synonymous divergence in the NBS domain sequences by a maximum likelihood method assuming the single stepwise mutation model. The estimates ranged from 4.5 to 600 and differed between groups in the same species or between species. This indicated different roles played by different groups of the NBS-LRR genes within a species or the effects of various life history characteristics, such as generation time, of the species. We also tested the fit of the model to the data using the variance of number of LRRs in each subgroup. In some subgroups in some plants (16 out of 174 subgroups), the results of simulation using the estimated rates significantly deviated from the observed data. Those subgroups may have undergone different modes of selection from the other subgroups.

Molecular phylogenetic study in genus Hydra.

Among 8000-9000 species of Cnidaria, only several dozens of species of Hydrozoa have been found in the fresh water. Hydra is such a fresh water polyp and has been used as a good material for research in developmental biology, regeneration and pattern formation. Although the genus Hydra has only a few ten species, its distribution is cosmopolitan. The phylogenetic relationship between hydra species is fascinating from the aspect of evolutionary biology and biogeography. However, only a few molecular phylogenetic studies have been reported on hydra. Therefore, we conducted a molecular phylogenetic study of the genus Hydra based on mitochondrial and nuclear nucleotide sequences using a hydra collection that has been kept in the National Institute of Genetics (NIG) of Japan. The results support the idea that four species groups comprise the genus Hydra. Within the viridissima group (green hydra) and braueri group, genetic distances between strains were relatively large. In contrast, genetic distances between strains among the vulgaris and oligactis groups were small irrespective of their geographic distribution. The vulgaris group strains were classified at least (as far as our investigated samples) into three sub-groups, vulgaris sub-group, carnea sub-group, and H. sp. (K5 and K6) sub-group. All of the vulgaris sub-group and H. sp. (K5 and K6) sub-group strains were collected in Eurasia. The carnea sub-group strains in NIG collection were all collected in North America. A few newly collected samples in Japan, however, suggested belonging to the carnea sub-group according to the molecular phylogenic analysis. This suggests a trans-Pacific distribution of the carnea sub-group hydra.

Multilocus patterns of nucleotide polymorphism and demographic change in Taxodium distichum (Cupressaceae) in the lower Mississippi River alluvial valley.

PREMISE OF THE STUDY: Studies of the geographic patterns of genetic variation can give important insights into the past population structure of species. Our study species, Taxodium distichum L. (bald-cypress), prefers riparian and wetland habitats and is widely distributed in southeastern North America and Mexico. We compared the genetic variation of T. distichum with that of its close relative, Cryptomeria japonica, which is endemic to Japan. • METHODS: Nucleotide polymorphisms of T. distichum in the lower Mississippi River alluvial valley, USA, were examined at 10 nuclear loci. • KEY RESULTS: The average nucleotide diversity at silent sites, π(sil), across the 10 loci in T. distichum was higher than that of C. japonica (π(sil) = 0.00732 and 0.00322, respectively). In T. distichum, Tajima's D values were each negative at 9 out of 10 loci, which suggests a recent population expansion. Maximum-likelihood and Bayesian estimations of the exponential population growth rate (g) of T. distichum populations indicated that this species had expanded approximately at the rate of 1.7-1.0 × 10(-6) per year in the past. • CONCLUSIONS: Taxodium distichum had significantly higher nucleotide variation than C. japonica, and its patterns of polymorphism contrasted strikingly with those of the latter, which previously has been inferred to have experienced a reduction in population size.

Genetic variation and demographic history of the Haplochromis laparogramma group of Lake Victoria-An analysis based on SINEs and mitochondrial DNA.

More than 500 endemic haplochromine cichlid species inhabit Lake Victoria. This striking species diversity is a classical example of recent explosive adaptive radiation thought to have happened within the last approximately 15,000 years. In this study, we examined the population structure and historical demography of 3 pelagic haplochromine cichlid species that resemble in morphology and have similar niche, Haplochromis (Yssichromis) laparogramma, Haplochromis (Y.) pyrrhocephalus, and Haplochromis (Y.) sp. "glaucocephalus". We investigated the sequences of the mitochondrial DNA control region and the insertion patterns of short interspersed elements (SINEs) of 759 individuals. We show that sympatric forms are genetically differentiated in 4 of 6 cases, but we also found apparent weakening of the genetic differentiation in areas with turbid water. We estimated the timings of population expansion and species divergence to coincide with the refilling of the lake at the Pleistocene/Holocene boundary. We also found that estimates can be altered significantly by the choice of the shape of the molecular clock. If we employ the nonlinear clock model of evolutionary rates in which the rates are higher towards the recent, the population expansion was dated at around the event of desiccation of the lake ca. 17,000 YBP. Thus, we succeeded in clarifying the species and population structure of closely related Lake Victoria cichlids and in showing the importance of applying appropriate clock calibrations in elucidating recent evolutionary events.