Effects of the Tanaka Line on the genetic structure of Bombax ceiba (Malvaceae) in dry‐hot valley areas of southwest China

Southwest China is an important biodiversity hotspot. The interactions among the complex topography, climate change, and ecological factors in the dry‐hot valley areas in southwest China may have profoundly affected the genetic structure of plant species in this region. In this study, we determined the effects of the Tanaka Line on genetic variation in the wild Bombax ceiba tree in southwest China. We sampled 224 individuals from 17 populations throughout the dry‐hot valley regions. Six polymorphic expressed sequence tag–simple sequence repeat primers were employed to sequence the PCR products using the first‐generation Sanger technique. The analysis based on population genetics suggested that B. ceiba exhibited a high level of gene diversity (H_E: 0.2377–0.4775; I: 0.3997–0.7848). The 17 populations were divided into two groups by cluster analysis, which corresponded to geographic characters on each side of the Tanaka Line. In addition, a Mantel test indicated that the phylogeographic structure among the populations could be fitted to the isolation‐by‐distance model (r² = .2553, p < .001). A barrier test indicated that there were obstacles among populations and between the two groups due to complex terrain isolation and geographic heterogeneity. We inferred that the Tanaka Line might have promoted the intraspecific phylogeographic subdivision and divergence of B. ceiba. These results provide new insights into the effects of the Tanaka Line on genetic isolation and population differentiation of plant species in southwest China.     

A comparative plastomics approach reveals available molecular markers for the phylogeographic study of Dendrobium huoshanense,an endangered orchid with extremely small populations

Comparative plastomics approaches have been used to identify available molecular markers for different taxonomic level studies of orchid species. However, the adoption of such methods has been largely limited in phylogeographic studies. Therefore, in this study, Dendrobium huoshanense, an endangered species with extremely small populations, was used as a model system to test whether the comparative plastomic approaches could screen available molecular markers for the phylogeographic study. We sequenced two more plastomes of D. huoshanense and compared them with our previously published one. A total of 27 mutational hotspot regions and six polymorphic cpSSRs have been screened for the phylogeographic studies of D. huoshanense. The cpDNA haplotype data revealed that the existence of haplotype distribution center was located in Dabieshan Mts. (Huoshan). The genetic diversity and phylogenetic analyses showed that the populations of D. huoshanense have been isolated and evolved independently for long period. On the contrary, based on cpSSR data, the genetic structure analysis revealed a mixed structure among the populations in Anhui and Jiangxi province, which suggested that the hybridization or introgression events have occurred among the populations of D. huoshanense. These results indicated that human activities have played key roles in shaping the genetic diversity and distributional patterns of D. huoshanense. According to our results, both two markers showed a high resolution for the phylogeographic studies of D. huoshanense. Therefore, we put forth that comparative plastomic approaches could revealed available molecular markers for phylogeographic study, especially for the species with extremely small populations.     

Small but attractive: female‐biased nectar production and floral visitors in a dimorphic shrub

• In sexually dimorphic species, hermaphrodite flowers in gynodioecious species or male flowers in dioecious species are often larger and produce more nectar than their conspecific female flowers. As a consequence, hermaphrodite or male flowers frequently receive more pollinator visits.
• Sex ratio, flower size, floral display, nectar production and floral visits were evaluated in two natural populations of Fuchsia thymifolia, a morphologically gynodioecious but functionally subdioecious insect‐pollinated shrub.
• Sex ratio did not differ from the expected 1:1 in the two studied populations. As expected, hermaphrodite flowers were larger than female flowers, but in contrast to the general pattern, hermaphrodite flowers did not produce nectar or produced much less than female flowers. Flower visitors were flies (68%) and bumblebees (24%), both of which showed a preference for female flowers. No sex difference was detected in either flower longevity or floral display across the flowering season.
• Higher nectar production by females may attract more pollinators, and may be a strategy to enhance female reproductive success in this species. Finally, floral dimorphism and insect preferences did not seem to hamper the maintenance of sub‐dioecy or prevent the evolution of dioecy in F. thymifolia.

     

Role of the gibberellin receptors GID1 during fruit‐set in Arabidopsis

Gibberellins (GAs) play a critical role in fruit‐set and fruit growth. Gibberellin is perceived by its nuclear receptors GA INSENSITIVE DWARF1s (GID1s), which then trigger degradation of downstream repressors DELLAs. To understand the role of the three GA receptor genes (GID1A, GID1B and GID1C) in Arabidopsis during fruit initiation, we have examined their temporal and spatial localization, in combination with analysis of mutant phenotypes. Distinct expression patterns are revealed for each GID1: GID1A is expressed throughout the whole pistil, while GID1B is expressed in ovules, and GID1C is expressed in valves. Functional study of gid1 mutant combinations confirms that GID1A plays a major role during fruit‐set and growth, whereas GID1B and GID1C have specific roles in seed development and pod elongation, respectively. Therefore, in ovules, GA perception is mediated by GID1A and GID1B, while GID1A and GID1C are involved in GA perception in valves. To identify tissue‐specific interactions between GID1s and DELLAs, we analyzed spatial expression patterns of four DELLA genes that have a role in fruit initiation (GAI, RGA, RGL1 and RGL2). Our data suggest that GID1A can interact with RGA and GAI in all tissues, whereas GID1C–RGL1 and GID1B–RGL2 interactions only occur in valves and ovules, respectively. These results uncover specific functions of each GID1–DELLA in the different GA‐dependent processes that occur upon fruit‐set. In addition, the distribution of GA receptors in valves along with lack of expression of GA biosynthesis genes in this tissue, strongly suggests transport of GAs from the developing seeds to promote fruit growth.     

Seed dispersal in the mycoheterotrophic orchid Yoania japonica: Further evidence for endozoochory by camel crickets

• Although orthopterans are rarely considered to be effective seed dispersal agents, the large flightless crickets known as ‘weta’ have been suggested to function as ecological replacements for small mammals in New Zealand, where such mammals are absent. In addition, a recent study reported that camel crickets mediate seed dispersal of several heterotrophic plants, including Yoania amagiensis in Japan.
• I investigated the seed dispersal mechanism of Yoania japonica because the fruit morphology is similar to Y. amagiensis. Specifically, I aimed to determine whether Y. japonica fruits are consumed by camel crickets and, if so, whether the seeds defecated by camel crickets remains intact, by checking seed viability with TTC staining, and whether germination rate is different between seeds collected directly from fruits and defecated seeds by comparing in situ seed germinability.
• The present study provides evidence that camel crickets function as seed dispersal agents of Y. japonica. Camel crickets were important consumers of Y. japonica fruits, and a substantial portion of the consumed seeds remained viable after passing through the digestive tract. In situ seed germination experiments revealed that the seeds defecated by camel crickets actually germinated in the field. In addition, the germination rate of defecated seeds was even higher than that of intact seeds, although the difference was not significant.
• Taken together with recent reports of insect‐mediated endozoochory, such a seed dispersal system may be common in plants with fleshy indehiscent fruits and small seeds, even in locations where other seed dispersal agents are present.

     

A novel and fully scalable Agrobacterium spray‐based process for manufacturing cellulases and other cost‐sensitive proteins in plants

Transient transfection of plants by vacuum infiltration of Agrobacterium vectors represents the state of the art in plant‐based protein manufacturing; however, the complexity and cost of this approach restrict it to pharmaceutical proteins. We demonstrated that simple spraying of Nicotiana plants with Agrobacterium vectors in the presence of a surfactant can substitute for vacuum inoculation. When the T‐DNA of Agrobacterium encodes viral replicons capable of cell‐to‐cell movement, up to 90% of the leaf cells can be transfected and express a recombinant protein at levels up to 50% of total soluble protein. This simple, fast and indefinitely scalable process was successfully applied to produce cellulases, one of the most volume‐ and cost‐sensitive biotechnology products. We demonstrate here for the first time that representatives of all hydrolase classes necessary for cellulosic biomass decomposition can be expressed at high levels, stored as silage without significant loss of activity and then used directly as enzyme additives. This process enables production of cellulases, and other potential high‐volume products such as noncaloric sweetener thaumatin and antiviral protein griffithsin, at commodity agricultural prices and could find broad applicability in the large‐scale production of many other cost‐sensitive proteins.     

Overexpression of the leucine‐rich receptor‐like kinase gene LRK2 increases drought tolerance and tiller number in rice

Drought represents a key limiting factor of global crop distribution. Receptor‐like kinases play major roles in plant development and defence responses against stresses such as drought. In this study, LRK2, which encodes a leucine‐rich receptor‐like kinase, was cloned and characterized and found to be localized on the plasma membrane in rice. Promoter–GUS analysis revealed strong expression in tiller buds, roots, nodes and anthers. Transgenic plants overexpressing LRK2 exhibited enhanced tolerance to drought stress due to an increased number of lateral roots compared with the wild type at the vegetative stage. Moreover, ectopic expression of LRK2 seedlings resulted in increased tiller development. Yeast two‐hybrid screening and bimolecular fluorescence complementation (BiFC) indicated a possible interaction between LRK2 and elongation factor 1 alpha (OsEF1A) in vitro. These results suggest that LRK2 functions as a positive regulator of the drought stress response and tiller development via increased branch development in rice. These findings will aid our understanding of branch regulation in other grasses and support improvements in rice genetics.     

Precision genome editing in plants via gene targeting and piggyBac‐mediated marker excision

Precise genome engineering via homologous recombination (HR)‐mediated gene targeting (GT) has become an essential tool in molecular breeding as well as in basic plant science. As HR‐mediated GT is an extremely rare event, positive–negative selection has been used extensively in flowering plants to isolate cells in which GT has occurred. In order to utilize GT as a methodology for precision mutagenesis, the positive selectable marker gene should be completely eliminated from the GT locus. Here, we introduce targeted point mutations conferring resistance to herbicide into the rice acetolactate synthase (ALS) gene via GT with subsequent marker excision by piggyBac transposition. Almost all regenerated plants expressing piggyBac transposase contained exclusively targeted point mutations without concomitant re‐integration of the transposon, resulting in these progeny showing a herbicide bispyribac sodium (BS)‐tolerant phenotype. This approach was also applied successfully to the editing of a microRNA targeting site in the rice cleistogamy 1 gene. Therefore, our approach provides a general strategy for the targeted modification of endogenous genes in plants.     

Global distribution,diversity hot spots and niche transitions of an astaxanthin‐producing eukaryotic microbe

Microbes establish very diverse but still poorly understood associations with other microscopic or macroscopic organisms that do not follow the more conventional modes of competition or mutualism. Phaffia rhodozyma, an orange‐coloured yeast that produces the biotechnologically relevant carotenoid astaxanthin, exhibits a Holarctic association with birch trees in temperate forests that contrasts with the more recent finding of a South American population associated with Nothofagus (southern beech) and with stromata of its biotrophic fungal parasite Cyttaria spp. We investigated whether the association of Phaffia with Nothofagus–Cyttaria could be expanded to Australasia, the other region of the world where Nothofagus are endemic, studied the genetic structure of populations representing the known worldwide distribution of Phaffia and analysed the evolution of the association with tree hosts. The phylogenetic analysis revealed that Phaffia diversity in Australasia is much higher than in other regions of the globe and that two endemic and markedly divergent lineages seem to represent new species. The observed genetic diversity correlates with host tree genera rather than with geography, which suggests that adaptation to the different niches is driving population structure in this yeast. The high genetic diversity and endemism in Australasia indicate that the genus evolved in this region and that the association with Nothofagus is the ancestral tree association. Estimates of the divergence times of Phaffia lineages point to splits that are much more recent than the break‐up of Gondwana, supporting that long‐distance dispersal rather than vicariance is responsible for observed distribution of P. rhodozyma.     

Increase in multiple paternity across the reproductive lifespan in a sperm‐storing,hermaphroditic freshwater snail

Polyandry is a common phenomenon and challenges the traditional view of stronger sexual selection in males than in females. In simultaneous hermaphrodites, the physical proximity of both sex functions was long thought to preclude the operation of sexual selection. Laboratory studies suggest that multiple mating and polyandry in hermaphrodites may actually be common, but data from natural populations are sparse. We therefore estimated the rate of multiple paternity and its seasonal variability in the annual, sperm‐storing, simultaneously hermaphroditic freshwater snail Radix balthica for the entire duration of the reproductive lifespan. We also tested whether multiple paternity was associated with clutch size or embryonic development. To obtain these data, we measured and genotyped 60 field‐collected egg clutches using nine highly polymorphic microsatellite markers. Overall, 50% of the clutches had multiple fathers, and both the frequency (20–93% of clutches) and magnitude of multiple paternity (mean 1.3–3.8 fathers per clutch) substantially increased over time, probably because of extensive sperm storage. Most multiply sired clutches (83%) had a dominant father, but neither clutch size nor the proportion of developed embryos per clutch was associated with levels of multiple paternity. Both the evident promiscuity and the frequent skew of paternity shares suggest that sexual selection may be an important evolutionary force in the study population.     

Evolution of life‐history traits and mating strategy in males: a case study on two populations of a Drosophila parasitoid

Abiotic and biotic factors affect life‐history traits and lead populations to exhibit different behavioural strategies. Due to the direct link between their behaviour and fitness, parasitoid females have often been used to test the theories explaining these differences. In male parasitoids, however, such investigations are vastly understudied, although their mating strategy directly determines their fitness. In this study, we compared the pattern of life history traits and the mating strategy of males in two populations of the Drosophila parasitoid Asobara tabida, exposed to different biotic and abiotic conditions, with the major difference being that one of them was recently exposed to strong competition with the dominant competitor Leptopilina boulardi after recent climate change, the other population being settled in a location where L. boulardi has not been recorded. The results showed that individuals of both populations have a different reproductive strategy: in one population, females produced a more female‐biased sex ratio, while males accumulated more lipids during their larval development, invested more energy in reproduction and decreased their locomotor activity, suggesting a higher proportion of matings on their emergence patch, all of these factors being possibly linked to the new competition pressure. In both populations, mating without sperm transfer may persist for several days after males become sperm‐depleted, and may be more frequent than mating with sperm transfer over their whole lifespan. This point is discussed from an evolutionary point of view.     

Cones structure and seed traits of four species of large‐seeded pines: Adaptation to animal‐mediated dispersal

Seed dispersal selection pressures may cause morphological differences in cone structure and seed traits of large‐seeded pine trees. We investigated the cone, seed, and scale traits of four species of animal‐dispersed pine trees to explore the adaptations of morphological structures to different dispersers. The four focal pines analyzed in this study were Chinese white pine (Pinus armandi), Korean pine (P. koraiensis), Siberian dwarf pine (P. pumila), and Dabieshan white pine (P. dabeshanensis). There are significant differences in the traits of the cones and seeds of these four animal‐dispersed pines. The scales of Korean pine and Siberian dwarf pine are somewhat opened after cone maturity, the seeds are closely combined with scales, and the seed coat and scales are thick. The cones of Chinese white pine and Dabieshan white pine are open after ripening, the seeds fall easily from the cones, and the seed coat and seed scales are relatively thin. The results showed that the cone structure of Chinese white pine is similar to that of Dabieshan white pine, whereas Korean pine and Siberian dwarf pine are significantly different from the other two pines and vary significantly from each other. This suggests that species with similar seed dispersal strategies exhibit similar morphological adaptions. Accordingly, we predicted three possible seed dispersal paradigms for animal‐dispersed pines: the first, as represented by Chinese white pine and Dabieshan white pine, relies upon small forest rodents for seed dispersal; the second, represented by Korean pine, relies primarily on birds and squirrels to disperse the seeds; and the third, represented by Siberian dwarf pine, relies primarily on birds for seed dispersal. Our study highlights the significance of animal seed dispersal in shaping cone morphology, and our predictions provide a theoretical framework for research investigating the coevolution of large‐seeded pines and their seed dispersers.     

A novel genome‐wide microsatellite resource for species of Eucalyptus with linkage‐to‐physical correspondence on the reference genome sequence

Keystone species in their native ranges, eucalypts, are ecologically and genetically very diverse, growing naturally along extensive latitudinal and altitudinal ranges and variable environments. Besides their ecological importance, eucalypts are also the most widely planted trees for sustainable forestry in the world. We report the development of a novel collection of 535 microsatellites for species of Eucalyptus, 494 designed from ESTs and 41 from genomic libraries. A selected subset of 223 was evaluated for individual identification, parentage testing, and ancestral information content in the two most extensively studied species, Eucalyptus grandis and Eucalyptus globulus. Microsatellites showed high transferability and overlapping allele size range, suggesting they have arisen still in their common ancestor and confirming the extensive genome conservation between these two species. A consensus linkage map with 437 microsatellites, the most comprehensive microsatellite‐only genetic map for Eucalyptus, was built by assembling segregation data from three mapping populations and anchored to the Eucalyptus genome. An overall colinearity between recombination‐based and physical positioning of 84% of the mapped microsatellites was observed, with some ordering discrepancies and sporadic locus duplications, consistent with the recently described whole genome duplication events in Eucalyptus. The linkage map covered 95.2% of the 605.8‐Mbp assembled genome sequence, placing one microsatellite every 1.55 Mbp on average, and an overall estimate of physical to recombination distance of 618 kbp/cM. The genetic parameters estimates together with linkage and physical position data for this large set of microsatellites should assist marker choice for genome‐wide population genetics and comparative mapping in Eucalyptus.     

A rare case of plastid protein‐coding gene duplication in the chloroplast genome of Euglena archaeoplastidiata (Euglenophyta)

Gene duplication is an important evolutionary process that allows duplicate functions to diverge, or, in some cases, allows for new functional gains. However, in contrast to the nuclear genome, gene duplications within the chloroplast are extremely rare. Here, we present the chloroplast genome of the photosynthetic protist Euglena archaeoplastidiata. Upon annotation, it was found that the chloroplast genome contained a novel tandem direct duplication that encoded a portion of RuBisCO large subunit (rbcL) followed by a complete copy of ribosomal protein L32 (rpl32), as well as the associated intergenic sequences. Analyses of the duplicated rpl32 were inconclusive regarding selective pressures, although it was found that substitutions in the duplicated region, all non‐synonymous, likely had a neutral functional effect. The duplicated region did not exhibit patterns consistent with previously described mechanisms for tandem direct duplications, and demonstrated an unknown mechanism of duplication. In addition, a comparison of this chloroplast genome to other previously characterized chloroplast genomes from the same family revealed characteristics that indicated E. archaeoplastidiata was probably more closely related to taxa in the genera Monomorphina, Cryptoglena, and Euglenaria than it was to other Euglena taxa. Taken together, the chloroplast genome of E. archaeoplastidiata demonstrated multiple characteristics unique to the euglenoid world, and has justified the longstanding curiosity regarding this enigmatic taxon.     

Identification structure of the mating‐type locus and distribution of Kabatiella zeae in China

The 51 isolates, the causing agents of maize eyespot, were identified as Kabatiella zeae with morphological and molecular methods. The structure of the MAT locus in K. zeae JLMHK‐9 strain contains MAT1‐1 and MAT1‐2 genes which are transcribed in opposite directions, DNA lyase gene (APN2) which is adjacent to the 3′ flanking region of MAT1‐2‐1 gene and a pleckstrin homology domain (PH) which is adjacent to the 3′ flanking region of MAT1‐1‐1 gene. The specific primers are used to identify the mating types of K. zeae isolates collected from six provinces in China, and our findings speculate that K. zeae is a homothallic species.     

Pollinator‐mediated assemblage processes in California wildflowers

Community assembly is the result of multiple ecological and evolutionary forces that influence species coexistence. For flowering plants, pollinators are often essential for plant reproduction and establishment, and pollinator‐mediated interactions may influence plant community composition. Here, we use null models and community phylogenetic analyses of co‐occurrence patterns to determine the role of pollinator‐mediated processes in structuring plant communities dominated by congeners. We surveyed three species‐rich genera (Limnanthes, Mimulus and Clarkia) with centres of diversity in the Sierra Nevada of California. Each genus contains species that co‐flower and share pollinators, and each has a robust phylogeny. Within each genus, we surveyed 44–48 communities at three spatial scales, measured floral and vegetative traits and tested for segregation or aggregation of: (i) species, (ii) floral traits (which are likely to be influenced by pollinators), and (iii) vegetative traits (which are likely affected by other environmental factors). We detected both aggregation and segregation of floral traits that were uncorrelated with vegetative trait patterns; we infer that pollinators have shaped the community assembly although the mechanisms may be varied (competition, facilitation, or filtering). We also found that mating system differences may play an important role in allowing species co‐occurrence. Together, it appears that pollinators influence community assemblage in these three clades.     

Genetic control of α‐farnesene production in apple fruit and its role in fungal pathogenesis

Terpenes are important compounds in plant trophic interactions. A meta‐analysis of GC‐MS data from a diverse range of apple (Malus × domestica) genotypes revealed that apple fruit produces a range of terpene volatiles, with the predominant terpene being the acyclic branched sesquiterpene (E,E)‐α‐farnesene. Four quantitative trait loci (QTLs) for α‐farnesene production in ripe fruit were identified in a segregating ‘Royal Gala’ (RG) × ‘Granny Smith’ (GS) population with one major QTL on linkage group 10 co‐locating with the MdAFS1 (α‐farnesene synthase‐1) gene. Three of the four QTLs were derived from the GS parent, which was consistent with GC‐MS analysis of headspace and solvent‐extracted terpenes showing that cold‐treated GS apples produced higher levels of (E,E)‐α‐farnesene than RG. Transgenic RG fruit downregulated for MdAFS1 expression produced significantly lower levels of (E,E)‐α‐farnesene. To evaluate the role of (E,E)‐α‐farnesene in fungal pathogenesis, MdAFS1 RNA interference transgenic fruit and RG controls were inoculated with three important apple post‐harvest pathogens [Colletotrichum acutatum, Penicillium expansum and Neofabraea alba (synonym Phlyctema vagabunda)]. From results obtained over four seasons, we demonstrate that reduced (E,E)‐α‐farnesene is associated with decreased disease initiation rates of all three pathogens. In each case, the infection rate was significantly reduced 7 days post‐inoculation, although the size of successful lesions was comparable with infections on control fruit. These results indicate that (E,E)‐α‐farnesene production is likely to be an important factor involved in fungal pathogenesis in apple fruit.     

Genetic variation in Tertiary relics: The case of eastern‐Mediterranean Abies (Pinaceae)

The eastern‐Mediterranean Abies taxa, which include both widely distributed species and taxa with minuscule ranges, represent a good model to study the impacts of range size and fragmentation on the levels of genetic diversity and differentiation. To assess the patterns of genetic diversity and phylogenetic relationships among eastern‐Mediterranean Abies taxa, genetic variation was assessed by eight nuclear microsatellite loci in 52 populations of Abies taxa with a focus on those distributed in Turkey and the Caucasus. Both at the population and the taxon level, the subspecies or regional populations of Abies nordmanniana s.l. exhibited generally higher allelic richness, private allelic richness, and expected heterozygosity compared with Abies cilicica s.l. Results of both the Structure analysis and distance‐based approaches showed a strong differentiation of the two A. cilicica subspecies from the rest as well as from each other, whereas the subspecies of A. nordmanniana were distinct but less differentiated. ABC simulations were run for a set of scenarios of phylogeny and past demographic changes. For A. ×olcayana, the simulation gave a poor support for the hypothesis of being a taxon resulting from a past hybridization, the same is true for Abies equi‐trojani: both they represent evolutionary branches of Abies bornmuelleriana.