Deciphering and prediction of transcriptome dynamics under fluctuating field conditions.

Determining the drivers of gene expression patterns is more straightforward in laboratory conditions than in the complex fluctuating environments where organisms typically live. We gathered transcriptome data from the leaves of rice plants in a paddy field along with the corresponding meteorological data and used them to develop statistical models for the endogenous and external influences on gene expression. Our results indicate that the transcriptome dynamics are predominantly governed by endogenous diurnal rhythms, ambient temperature, plant age, and solar radiation. The data revealed diurnal gates for environmental stimuli to influence transcription and pointed to relative influences exerted by circadian and environmental factors on different metabolic genes. The model also generated predictions for the influence of changing temperatures on transcriptome dynamics. We anticipate that our models will help translate the knowledge amassed in laboratories to problems in agriculture and that our approach to deciphering the transcriptome fluctuations in complex environments will be applicable to other organisms.

A transcriptional program underlying the circannual rhythms of gonadal development in medaka.

To cope with seasonal environmental changes, organisms have evolved approximately 1-y endogenous circannual clocks. These circannual clocks regulate various physiological properties and behaviors such as reproduction, hibernation, migration, and molting, thus providing organisms with adaptive advantages. Although several hypotheses have been proposed, the genes that regulate circannual rhythms and the underlying mechanisms controlling long-term circannual clocks remain unknown in any organism. Here, we show a transcriptional program underlying the circannual clock in medaka fish (Oryzias latipes). We monitored the seasonal reproductive rhythms of medaka kept under natural outdoor conditions for 2 y. Linear regression analysis suggested that seasonal changes in reproductive activity were predominantly determined by an endogenous program. Medaka hypothalamic and pituitary transcriptomes were obtained monthly over 2 y and daily on all equinoxes and solstices. Analysis identified 3,341 seasonally oscillating genes and 1,381 daily oscillating genes. We then examined the existence of circannual rhythms in medaka via maintaining them under constant photoperiodic conditions. Medaka exhibited approximately 6-mo free-running circannual rhythms under constant conditions, and monthly transcriptomes under constant conditions identified 518 circannual genes. Gene ontology analysis of circannual genes highlighted the enrichment of genes related to cell proliferation and differentiation. Altogether, our findings support the "histogenesis hypothesis" that postulates the involvement of tissue remodeling in circannual time-keeping.

The Arabidopsis katamari2 Mutant Exhibits a Hypersensitive Seedling Arrest Response at the Phase Transition from Heterotrophic to Autotrophic Growth.

Young seedlings use nutrients stored in the seeds to grow and acquire photosynthetic potential. This process, called seedling establishment, involves a developmental phase transition from heterotrophic to autotrophic growth. Some membrane-trafficking mutants of Arabidopsis (Arabidopsis thaliana), such as the katamari2 (kam2) mutant, exhibit growth arrest during seedling development, with a portion of individuals failing to develop true leaves on sucrose-free solid medium. However, the reason for this seedling arrest is unclear. In this study, we show that seedling arrest is a temporal growth arrest response that occurs not only in kam2 but also in wild-type (WT) Arabidopsis; however, the threshold for this response is lower in kam2 than in the WT. A subset of the arrested kam2 seedlings resumed growth after transfer to fresh sucrose-free medium. Growth arrest in kam2 on sucrose-free medium was restored by increasing the gel concentration of the medium or covering the surface of the medium with a perforated plastic sheet. WT Arabidopsis seedlings were also arrested when the gel concentration of sucrose-free medium was reduced. RNA sequencing revealed that transcriptomic changes associated with the rate of seedling establishment were observed as early as 4 d after sowing. Our results suggest that the growth arrest of both kam2 and WT seedlings is an adaptive stress response and is not simply caused by the lack of a carbon source in the medium. This study provides a new perspective on an environmental stress response under unfavorable conditions during the phase transition from heterotrophic to autotrophic growth in Arabidopsis.

Genome-wide association study of leaf photosynthesis using a high-throughput gas exchange system in rice.

Enhancing leaf photosynthetic capacity is essential for improving the yield of rice (Oryza sativa L.). Although the exploitation of natural genetic resources is considered a promising approach to enhance photosynthetic capacity, genomic factors related to the genetic diversity of leaf photosynthetic capacity have yet to be fully elucidated due to the limitation of measurement efficiency. In this study, we aimed to identify novel genomic regions for the net CO2 assimilation rate (A) by combining genome-wide association study (GWAS) and the newly developed rapid closed gas exchange system MIC-100. Using three MIC-100 systems in the field at the vegetative stage, we measured A of 168 temperate japonica rice varieties with six replicates for three years. We found that the modern varieties exhibited higher A than the landraces, while there was no significant relationship between the release year and A among the modern varieties. Our GWAS scan revealed two major peaks located on chromosomes 4 and 8, which were repeatedly detected in the different experiments and in the generalized linear modelling approach. We suggest that high-throughput gas exchange measurements combined with GWAS is a reliable approach for understanding the genetic mechanisms underlying photosynthetic diversities in crop species.

Enhanced decreases in rice evapotranspiration in response to elevated atmospheric carbon dioxide under warmer environments.

A short period of exposure to elevated CO2 is known to decrease evapotranspiration via stomatal closure. Based on theoretical evaluation of a canopy transpiration model, we hypothesized that this decrease in the evapotranspiration of rice under elevated CO2 was greater under higher temperature conditions due to an increased sensitivity of transpiration to changes in CO2 induced by the greater vapour pressure deficit. In a temperature gradient chamber-based experiment, a 200 ppm increase in CO2 concentration led to 0.4 mm (-7%) and 1.5 mm (-15%) decreases in 12 h evapotranspiration under ambient temperature and high temperature (+3.7°C) conditions, respectively. Model simulations revealed that the greater vapour pressure deficit under higher temperature conditions explained the variations in the reduction of evapotranspiration observed under elevated CO2 levels between the temperature treatments. Our study suggests the utility of a simple modelling framework for mechanistic understanding of evapotranspiration and crop energy balance system under changing environmental conditions.

Distribution and Population Genetic Structure of the Hau Giang Medaka, Oryzias haugiangensis, Along the East Coast of the Indochinese Peninsula.

The east coast of the Indochinese Peninsula is a well-known transition zone from subtropical to tropical systems, yet only a small number of studies have been conducted on the biogeography and phylogeography of aquatic organisms in this region. The Hau Giang medaka, Oryzias haugiangensis, was originally described from the Mekong Delta in southern Vietnam, and later reported also from southeastern Thailand, west of the Mekong Delta region. However, the species' full geographic range and population genetic structures remain unknown. Field surveys showed a widespread distribution of this species along the east coast of the Indochinese Peninsula, as far as northern Vietnam. A mitochondrial gene phylogeny and population genetic structure analysis using genome-wide single nucleotide polymorphisms revealed that the populations of O. haugiangensis are highly structuralized along the east coast of Vietnam, with the southernmost Mekong Delta population clearly separated from three populations north of central Vietnam. Further field collections are necessary to determine the boundary between the southern and northern populations, and the presence or absence of a hybrid zone.

The basic leucine zipper transcription factor OsbZIP83 and the glutaredoxins OsGRX6 and OsGRX9 facilitate rice iron utilization under the control of OsHRZ ubiquitin ligases.

Under low iron availability, plants induce the expression of various genes for iron uptake and translocation. The rice (Oryza sativa) ubiquitin ligases OsHRZ1 and OsHRZ2 cause overall repression of these iron-related genes at the transcript level, but their protein-level regulation is unclear. We conducted a proteome analysis to identify key regulators whose abundance was regulated by OsHRZs at the protein level. In response to iron deficiency or OsHRZ knockdown, many genes showed differential regulation between the transcript and protein levels, including the TGA-type basic leucine zipper transcription factor OsbZIP83. We also identified two glutaredoxins, OsGRX6 and OsGRX9, as OsHRZ-interacting proteins in yeast and plant cells. OsGRX6 also interacted with OsbZIP83. Our in vitro degradation assay suggested that OsbZIP83, OsGRX6 and OsGRX9 proteins are subjected to 26S proteasome- and OsHRZ-dependent degradation. Proteome analysis and our in vitro degradation assay also suggested that OsbZIP83 protein was preferentially degraded under iron-deficient conditions in rice roots. Transgenic rice lines overexpressing OsGRX9 and OsbZIP83 showed improved tolerance to iron deficiency. Expression of iron-related genes was affected in the OsGRX9 and OsGRX6 knockdown lines, suggesting disturbed iron utilization and signaling. OsbZIP83 overexpression lines showed enhanced expression of OsYSL2 and OsNAS3, which are involved in internal iron translocation, in addition to OsGRX9 and genes related to phytoalexin biosynthesis and the salicylic acid pathway. The results suggest that OsbZIP83, OsGRX6 and OsGRX9 facilitate iron utilization downstream of the OsHRZ pathway.

Single-Cell RNA Sequencing of Arabidopsis Leaf Tissues Identifies Multiple Specialized Cell Types: Idioblast Myrosin Cells and Potential Glucosinolate-Producing Cells.

The glucosinolate-myrosinase defense system (GMDS), characteristic of Brassicales, is involved in plant defense. Previous single-cell transcriptomic analyses have reported the expression profiles of multiple GMDS-related cell types (i.e. myrosinase-rich myrosin idioblasts and multiple types of potential glucosinolate synthetic cells as well as a candidate S-cell for glucosinolate accumulation). However, differences in plant stages and cell-type annotation methods have hindered comparisons among studies. Here, we used the single-cell transcriptome profiles of extended Arabidopsis leaves and verified the distribution of previously used markers to refine the expression profiles of GMDS-associated cell types. Moreover, we performed beta-glucuronidase promoter assays to confirm the histological expression patterns of newly obtained markers for GMDS-associated candidates. As a result, we found a set of new specific reporters for myrosin cells and potential glucosinolate-producing cells.

Apoplast-Localized ß-Glucosidase Elevates Isoflavone Accumulation in the Soybean Rhizosphere.

Plant specialized metabolites (PSMs) are often stored as glycosides within cells and released from the roots with some chemical modifications. While isoflavones are known to function as symbiotic signals with rhizobia and to modulate the soybean rhizosphere microbiome, the underlying mechanisms of root-to-soil delivery are poorly understood. In addition to transporter-mediated secretion, the hydrolysis of isoflavone glycosides in the apoplast by an isoflavone conjugate-hydrolyzing ß-glucosidase (ICHG) has been proposed but not yet verified. To clarify the role of ICHG in isoflavone supply to the rhizosphere, we have isolated two independent mutants defective in ICHG activity from a soybean high-density mutant library. In the root apoplastic fraction of ichg mutants, the isoflavone glycoside contents were significantly increased, while isoflavone aglycone contents were decreased, indicating that ICHG hydrolyzes isoflavone glycosides into aglycones in the root apoplast. When grown in a field, the lack of ICHG activity considerably reduced isoflavone aglycone contents in roots and the rhizosphere soil, although the transcriptomes showed no distinct differences between the ichg mutants and wild-types (WTs). Despite the change in isoflavone contents and composition of the root and rhizosphere of the mutants, root and rhizosphere bacterial communities were not distinctive from those of the WTs. Root bacterial communities and nodulation capacities of the ichg mutants did not differ from the WTs under nitrogen-deficient conditions either. Taken together, these results indicate that ICHG elevates the accumulation of isoflavones in the soybean rhizosphere but is not essential for isoflavone-mediated plant-microbe interactions.

Genetic analysis of pungency deficiency in Japanese chili pepper 'Shishito' (Capsicum annuum) revealed its unique heredity and brought the discovery of two genetic loci involved with the reduction of pungency.

The sensation of pungency generated by capsaicinoids is a characteristic trait of chili peppers (Capsicum spp.), and the presence or absence of pungency is central in determining its usage as a spice or a vegetable. In the present study, we aimed to clarify the heredity and genetic factors involved in the deficiency of pungency (quite low pungency) that is uniquely observed in the Japanese chili pepper 'Shishito' (Capsicum annuum). First, the F2 population ('Shishito' × pungent variety 'Takanotsume') was used for segregation analysis, and pungency level was investigated using capsaicinoid quantification with high-performance liquid chromatography. Also, restriction site associated DNA sequencing of the F2 population was performed, and genetic map construction and quantitative trait locus (QTL) mapping were implemented. The results indicated that the F2 population showed varying capsaicinoid content and two major QTLs were detected, Shql3 and Shql7, which explained 39.8 and 19.7% of the genetic variance, respectively. According to these results, the quite low pungency of 'Shishito' was a quantitative trait that involved at least the two loci. Further, this trait was completely separate from general non-pungent traits controlled by individual recessive genes, as described in previous studies. The present study is the first report to investigate the genetic mechanism of pungency deficiency in Japanese chili peppers, and our results provide new insights into the genetic regulation of pungency in chili pepper.

Genomic landscape of introgression from the ghost lineage in a gobiid fish uncovers the generality of forces shaping hybrid genomes.

Extinct lineages can leave legacies in the genomes of extant lineages through ancient introgressive hybridization. The patterns of genomic survival of these extinct lineages provide insight into the role of extinct lineages in current biodiversity. However, our understanding on the genomic landscape of introgression from extinct lineages remains limited due to challenges associated with locating the traces of unsampled 'ghost' extinct lineages without ancient genomes. Herein, we conducted population genomic analyses on the East China Sea (ECS) lineage of Chaenogobius annularis, which was suspected to have originated from ghost introgression, with the aim of elucidating its genomic origins and characterizing its landscape of introgression. By combining phylogeographic analysis and demographic modelling, we demonstrated that the ECS lineage originated from ancient hybridization with an extinct ghost lineage. Forward simulations based on the estimated demography indicated that the statistic γ of the HyDe analysis can be used to distinguish the differences in local introgression rates in our data. Consistent with introgression between extant organisms, we found reduced introgression from extinct lineage in regions with low recombination rates and with functional importance, thereby suggesting a role of linked selection that has eliminated the extinct lineage in shaping the hybrid genome. Moreover, we identified enrichment of repetitive elements in regions associated with ghost introgression, which was hitherto little known but was also observed in the re-analysis of published data on introgression between extant organisms. Overall, our findings underscore the unexpected similarities in the characteristics of introgression landscapes across different taxa, even in cases of ghost introgression.

Transcriptome dynamics of rice in natura: Response of above and below ground organs to microclimate.

The long-term dynamics of the transcriptome under natural field conditions remain unclear. We conducted comprehensive gene expression analyses of rice leaves and roots grown under natural field conditions for a long period, from the tillering stage to the ripening stage. In this experiment, changes in the transcriptome were captured in relation to microclimatic parameters, particularly potential evaporation (Ep), which is a multiple meteorological factor and acts as an indicator of transpirational demand. The results indicated  that many genes were regulated by changes in temperature and Ep in both leaves and roots. Furthermore, the correlation between gene expression and meteorological factors differed significantly between the vegetative and reproductive stages. Since Ep triggers transpiration, we analyzed aquaporin gene expression, which is responsible for water transport, and found that many aquaporin genes in leaves were positively correlated with Ep throughout the growth period, whereas in roots, two plasma membrane intrinsic aquaporins, PIP2;4 and PIP2;5 were strongly correlated with Ep during reproductive growth. Other genes closely related to productivity, such as those involved in nutrient absorption and photosynthesis, exhibited different responses to meteorological factors at different growth stages. The stage-dependent shift in the microclimate response provides an important perspective on crop physiology in light of climate change.

Multiple colonizations and hybridization of a freshwater fish group on a satellite island of Sulawesi.

Repeated colonizations and resultant hybridization may increase lineage diversity on an island if introgression occurs only in a portion of the indigenous island lineage. Therefore, to precisely understand how island biodiversity was shaped, it is essential to reconstruct the history of secondary colonization and resultant hybridization both in time and space. In this study, we reconstructed the history of multiple colonizations of the Oryzias woworae species group, a freshwater fish group of the family Adrianichthyidae, from Sulawesi Island to its southeast satellite island, Muna Island. Phylogenetic and species tree analyses using genome-wide single-nucleotide polymorphisms revealed that all local populations on Muna Island were monophyletic, but that there were several genetically distinct lineages within the island. Population structure and phylogenetic network analyses demonstrated that colonization of this island occurred more than once, and that secondary colonization and resultant introgressive hybridization occurred only in one local population on the island. The spatially heterogeneous introgression induced by the multiple colonizations were also supported by differential admixture analyses. In addition, the differential admixture analyses detected reverse colonization from Muna Island to the Sulawesi mainland. Coalescence-based demographic inference estimated that these mutual colonizations occurred during the middle to late Quaternary period, during which sea level repeatedly declined; this indicates that the colonizations occurred via land bridges. We conclude that these mutual colonizations between Muna Island and the Sulawesi mainland, and the resultant spatially heterogeneous introgression shaped the current biodiversity of this species group in this area.

Ghost introgression in ricefishes of the genus Adrianichthys in an ancient Wallacean lake.

Because speciation might have been promoted by ancient introgression from an extinct lineage, it is important to detect the existence of 'ghost introgression' in focal taxa and examine its contribution to their diversification. In this study, we examined possible ghost introgression and its contributions to the diversification of ricefishes of the genus Adrianichthys in Lake Poso, an ancient lake on Sulawesi Island, in which some extinctions are known to have occurred. Population-genomic analysis revealed that two extant Adrianichthys species, A. oophorus and A. poptae are reproductively isolated from each other. Comparisons of demographic models demonstrated that introgression from a ghost population, which diverged from the common ancestor of A. oophorus and A. poptae, is essential for reconstructing the demographic history of Adrianichthys. The best model estimated that the divergence of the ghost population greatly predated the divergence between A. oophorus and A. poptae, and that the ghost population secondarily contacted the two extant species within Lake Poso more recently. Genome scans and simulations detected a greatly divergent locus, which cannot be explained without ghost introgression. This locus was also completely segregated between A. oophorus and A. poptae. These findings suggest that variants that came from a ghost population have contributed to the divergence between A. oophorus and A. poptae, but the large time-lag between their divergence and ghost introgression indicates that the contribution of introgression may be restricted.

Genetic differentiation and evolution of broad-leaved evergreen shrub and tree varieties of Daphniphyllum macropodum (Daphniphyllaceae).

Tree form evolution is an important ecological specialization for woody species, but its evolutionary process with adaptation is poorly understood, especially on the microevolutionary scale. Daphniphyllum macropodum comprises two varieties: a tree variety growing in a warm temperate climate with light snowfall and a shrub variety growing in a cool temperate climate with heavy snowfall in Japan. Chloroplast DNA variations and genome-wide single-nucleotide polymorphisms across D. macropodum populations and D. teijsmannii as an outgroup were used to reveal the evolutionary process of the shrub variety. Population genetic analysis indicated that the two varieties diverged but were weakly differentiated. Approximate Bayesian computation analysis supported a scenario that assumed migration between the tree variety and the southern populations of the shrub variety. We found migration between the two varieties where the distributions of the two varieties are in contact, and it is concordant with higher tree height in the southern populations of the shrub variety than the northern populations. The genetic divergence between the two varieties was associated with snowfall. The heavy snowfall climate is considered to have developed since the middle Quaternary in this region. The estimated divergence time between the two varieties suggests that the evolution of the two varieties may be concordant with such paleoclimatic change.

The evolutionary history of rice azaleas (Rhododendron tschonoskii alliance) involved niche evolution to a montane environment.

PREMISE: The formation of isolated montane geography on islands promotes evolution, speciation, and then radiation if there are ecological changes. Thus, investigating evolutionary histories of montane species and associated ecological changes may help efforts to understand how endemism formed in islands' montane floras. To explore this process, we investigated the evolutionary history of the Rhododendron tschonoskii alliance, which grows in montane environments of the Japanese archipelago and the Korean Peninsula. METHODS: We studied the five species in the R. tschonoskii alliance and 30 outgroup species, using genome-wide single-nucleotide polymorphisms and cpDNA sequences, in association with environmental analyses. RESULTS: The monophyletic R. tschonoskii alliance diverged since the late Miocene. Species in the alliance currently inhabit a cold climatic niche that is largely different from that of the outgroup species. We observed clear genetic and niche differentiations between the taxa of the alliance. CONCLUSIONS: The association of the alliance's evolution with the formation of cooler climates on mountains indicates that it was driven by global cooling since the mid-Miocene and by rapid uplift of mountains since the Pliocene. The combination of geographic and climatic isolation promoted high genetic differentiation between taxa, which has been maintained by climatic oscillations since the Quaternary.

Genetic architectures of postmating isolation and morphology of two highly diverged rockfishes (genus Sebastes).

Postmating isolation is thought to be an important driver of the late stages of speciation. However, relatively little is empirically known about the process compared with other isolating mechanisms that drive the early stages of speciation, especially in non-model organisms. We characterized the genetic architecture of postmating isolation between 2 rockfishes, Sebastes schlegelii and S. trivittatus, whose reproductive isolation is complete. We examined transmission ratio distortion (TRD) patterns of genetic markers in 2 reciprocal backcross populations. Markers showing either of the 2 types of TRD was widespread across the genome, with some of the distorted markers forming extensive clusters around the recombination coldspots. These suggest that the postmating isolation effectively prevents gene flow across the genome and the recombination landscape contributes to the genetic architecture. Comparisons between 2 backcross families and 2 developmental stages showed little similarity in the distorted markers, suggesting asymmetry and stage specificity of the isolation. This may be due to hybrid incompatibility involving maternal factors or extrinsic selection. The lack of sex-ratio distortion in the mapping families suggested that Haldane's rule in terms of hybrid inviability does not hold. Additionally, quantitative trait loci (QTL) mapping detected significant QTLs for sex and the morphological traits relevant to speciation and convergence of rockfishes, including body coloration. Genes in the melanocortin system, including agouti-signaling protein 1 (asip1) and melanocortin 1 receptor (mc1r), might underlie the horizontal and vertical color patterns on the body, respectively. These findings constitute an essential step toward a comprehensive understanding of speciation and morphological diversification of rockfishes.

A Koshihikari X Oryza rufipogon Introgression Line with a High Capacity to Take up Nitrogen to Maintain Growth and Panicle Development under Low Nitrogen Conditions.

Nitrogen (N) is an important macronutrient for plant growth and development. Currently, N fertilizers are required for the efficient production of modern crops such as rice due to their limited capacity to take up N when present at low concentrations. Wild rice represents a useful genetic resource for improving crop responses to low nutrient stress. Here, we describe the isolation and characterization of an introgression line, KRIL37, that carries a small region of the Oryza rufipogon genome in the Oryza sativa L. cv Koshihikari (KH) background. This line was found to grow better under low N conditions and have similar or lower C/N ratios in aerial portions compared to those in the parental KH cultivar, suggesting that KRIL37 has a higher capacity to take up and assimilate N when present at low concentrations. KRIL37 performance in the field was also better than that of KH cultivated without N and fertilizer (-F). Transcriptome analyses of 3-week-old seedlings based on RNA-sequencing revealed that KH induced a wider suite of genes than the tolerant line KRIL37 in response to low N conditions. Some ammonium transporters and N assimilation genes were found to be induced under low N in KRIL37, but not in KH. Our findings suggest that the superior growth performance of KRIL37 under limited N conditions could be due to the expression of wild alleles influencing N uptake and assimilation. Our study demonstrates the potential to use wild rice genomes to improve modern crops for low nutrient tolerance.

Ethanol-Mediated Novel Survival Strategy against Drought Stress in Plants.

Water scarcity is a serious agricultural problem causing significant losses to crop yield and product quality. The development of technologies to mitigate the damage caused by drought stress is essential for ensuring a sustainable food supply for the increasing global population. We herein report that the exogenous application of ethanol, an inexpensive and environmentally friendly chemical, significantly enhances drought tolerance in Arabidopsis thaliana, rice and wheat. The transcriptomic analyses of ethanol-treated plants revealed the upregulation of genes related to sucrose and starch metabolism, phenylpropanoids and glucosinolate biosynthesis, while metabolomic analysis showed an increased accumulation of sugars, glucosinolates and drought-tolerance-related amino acids. The phenotyping analysis indicated that drought-induced water loss was delayed in the ethanol-treated plants. Furthermore, ethanol treatment induced stomatal closure, resulting in decreased transpiration rate and increased leaf water contents under drought stress conditions. The ethanol treatment did not enhance drought tolerance in the mutant of ABI1, a negative regulator of abscisic acid (ABA) signaling in Arabidopsis, indicating that ABA signaling contributes to ethanol-mediated drought tolerance. The nuclear magnetic resonance analysis using 13C-labeled ethanol indicated that gluconeogenesis is involved in the accumulation of sugars. The ethanol treatment did not enhance the drought tolerance in the aldehyde dehydrogenase (aldh) triple mutant (aldh2b4/aldh2b7/aldh2c4). These results show that ABA signaling and acetic acid biosynthesis are involved in ethanol-mediated drought tolerance and that chemical priming through ethanol application regulates sugar accumulation and gluconeogenesis, leading to enhanced drought tolerance and sustained plant growth. These findings highlight a new survival strategy for increasing crop production under water-limited conditions.

Microevolution of Pieris butterfly genes involved in host plant adaptation along a host plant community cline.

Herbivorous insects have evolved counteradaptations to overcome the chemical defences of their host plants. Several of these counteradaptations have been elucidated at the molecular level, in particular for insects specialized on cruciferous host plants. While the importance of these counteradaptations for host plant colonization is well established, little is known about their microevolutionary dynamics in the field. In particular, it is not known whether and how host plant diversity shapes diversity in insect counteradaptations. In this study, we examine patterns of host plant use and insect counteradaptation in three Pieris butterfly species across Japan. The larvae of these butterflies express nitrile-specifier protein (NSP) and its paralogue major allergen (MA) in their gut to overcome the highly diversified glucosinolate-myrosinase defence system of their cruciferous host plants. Pieris napi and Pieris melete colonize wild Brassicaceae whereas Pieris rapae typically uses cultivated Brassica as a host, regardless of the local composition of wild crucifers. As expected, NSP and MA diversity was independent of the local composition of wild Brassicaceae in P. rapae. In contrast, NSP diversity correlated with local host plant diversity in both species that preferred wild Brassicaceae. Both P. melete and P. napi revealed two distinct major NSP alleles, which shaped diversity among local populations, albeit with different evolutionary trajectories. In comparison, MA showed no indication for local adaptation. Altogether, MA appeared to be evolutionary more conserved than NSP, suggesting that both genes play different roles in diverting host plant chemical defence.