Crossing the divide: gene flow produces intergeneric hybrid in feral transgenic creeping bentgrass population

Gene flow is the most frequently expressed public concern related to the deregulation of transgenic events ( Snow 2002 ; Ellstrand 2003 ). However, assessing the potential for transgene escape is complex because it depends on the opportunities for unintended gene flow, and establishment and persistence of the transgene in the environment ( Warwick et al. 2008 ). Creeping bentgrass (Agrostis stolonifera L.), a turfgrass species widely used on golf courses, has been genetically engineered to be resistant to glyphosate, a nonselective herbicide. Outcrossing species, such as creeping bentgrass (CB), which have several compatible species, have greater chances for gene escape and spontaneous hybridization (i.e. natural, unassisted sexual reproduction between taxa in the field), which challenges transgene containment. Several authors have emphasized the need for evidence of spontaneous hybridization to infer the potential for gene flow ( Armstrong et al. 2005 ). Here we report that a transgenic intergeneric hybrid has been produced as result of spontaneous hybridization of a feral‐regulated transgenic pollen receptor (CB) and a nontransgenic pollen donor (rabbitfoot grass, RF, Polypogon monspeliensis (L.) Desf.). We identified an off‐type transgenic seedling and confirmed it to be CB × RF intergeneric hybrid. This first report of a transgenic intergeneric hybrid produced in situ with a regulated transgenic event demonstrates the importance of considering all possible avenues for transgene spread at the landscape level before planting a regulated transgenic crop in the field. Spontaneous hybridization adds a level of complexity to transgene monitoring, containment, mitigation and remediation programmes.     

Wetland and riparian plant communities at risk of invasion by transgenic herbicide-resistant <Emphasis Type="Italic">Agrostis</Emphasis> spp. in central Oregon

Creeping bentgrass (Agrostis stolonifera) and redtop (A. gigantea) are introduced turfgrasses that are naturalized throughout the northern U.S. Interest in creeping bentgrass has risen following the 2003 escape of a genetically modified (GM), herbicide-resistant cultivar near Madras, Oregon. The objectives of this study were to characterize the floristic attributes of the plant communities associated with naturalized Agrostis populations in the Madras area, and to identify plant communities at risk of invasion by transgenic Agrostis. Vegetation data collected from 62 stratified random vegetation plots with and without A. stolonifera and A. gigantea identified 11 distinct plant communities. Community composition was strongly correlated with an indirect soil moisture index based on the wetland status of individual species. Results indicate that wetland plant communities are at the highest risk of invasion by transgenic A. stolonifera. Also, inter-specific gene flow to A. gigantea could affect additional habitats and plant communities where A. stolonifera is not found. Both A. stolonifera and A. gigantea were invasive in wetland and riparian settings in the Madras study area, and introducing glyphosate (e.g., Roundup^®, Rodeo^®) herbicide tolerance into these populations would eliminate the primary means of control for these species.     

Stasipatric speciation: resurrecting a system to bury a hypothesis?

The process of speciation remains a fundamental topic in evolutionary biology. Numerous models of speciation have been proposed and they are as diverse and colourful as the scientists who conceived them ( Coyne & Orr 2004 ). One of the more controversial theories has been the ‘stasipatric speciation’ model, proposed by the pioneering and influential cytogeneticist Michael White and his co‐workers ( White 1968 ; White et al. 1967 ). This is one of a number of speciation models whereby chromosomal rearrangements drive the speciation process. The inspiration for the theory of stasipatric speciation came from White’s karyotypic analyses of a group of Australian grasshoppers of the genus Vandiemenella ( White et al. 1967 ) ( Fig. 1 ). It has been exactly three decades since the last scientific publication on this group of grasshoppers, over which time the molecular revolution dramatically altered the landscape of evolutionary genetics. Kawakami and colleagues have successfully resurrected the Vandiemenella system ( Kawakami et al. 2009a, 2007 ) and in this issue they have applied modern molecular‐based techniques to reassess the validity of the stasipatric speciation model for this historically important group ( Kawakami et al. 2009b ).

Figure 1 Open in figure viewer PowerPoint The grasshopper (Vandiemenella viatica) that inspired Michael White to develop the stasipatric speciation model (photograph by Remko Leijs).     

Distance from forest edge affects bee pollinators in oilseed rape fields

Wild pollinators have been shown to enhance the pollination of Brassica napus (oilseed rape) and thus increase its market value. Several studies have previously shown that pollination services are greater in crops adjoining forest patches or other seminatural habitats than in crops completely surrounded by other crops. In this study, we investigated the specific importance of forest edges in providing potential pollinators in B. napus fields in two areas in France. Bees were caught with yellow pan traps at increasing distances from both warm and cold forest edges into B. napus fields during the blooming period. A total of 4594 individual bees, representing six families and 83 taxa, were collected. We found that both bee abundance and taxa richness were negatively affected by the distance from forest edge. However, responses varied between bee groups and edge orientations. The ITD (Inter‐Tegular distance) of the species, a good proxy for bee foraging range, seems to limit how far the bees can travel from the forest edge. We found a greater abundance of cuckoo bees (Nomada spp.) of Andrena spp. and Andrena spp. males at forest edges, which we assume indicate suitable nesting sites, or at least mating sites, for some abundant Andrena species and their parasites (Fig.  1 ). Synthesis and Applications. This study provides one of the first examples in temperate ecosystems of how forest edges may actually act as a reservoir of potential pollinators and directly benefit agricultural crops by providing nesting or mating sites for important early spring pollinators. Policy‐makers and land managers should take forest edges into account and encourage their protection in the agricultural matrix to promote wild bees and their pollination services.

Figure 1 Open in figure viewer PowerPoint Left, a Nomada sp male; right, an Andrena sp male. Caption Left, a Nomada sp male; right, an Andrena sp male.

Introduction

Pollinators play an important functional role in most terrestrial ecosystems and provide a key ecosystem service (Ashman et al. 2004 ). Insects, particularly bees, are the primary pollinators for the majority of the world's angiosperms (Ollerton et al. 2012 ). Without this service, many interconnected species and processes functioning within both wild and agricultural ecosystems could collapse (Kearns et al. 1998 ). Brassica napus (oilseed rape, OSR) represents the most widespread entomophilous crop in France with almost 1.5 Mha in 2010 (FAOSTAT August 10th, 2012). Results differ between varieties, but even though it seems that OSR produces 70% of its fruits through self‐pollination (Downey et al. 1970 in Mesquida and Renard 1981 ), native bees are also known to contribute to its pollination (Morandin and Winston 2005 ; Jauker et al. 2012 ). Bee pollination leads to improved yields (Steffan‐Dewenter 2003b ; Sabbahi et al. 2005 ) and to a shorter blooming period (Sabbahi et al. 2006 ), thus increasing the crop's market value (Bommarco et al. 2012 ). The most widely used species in crop pollination is the honeybee (Apis mellifera L) which is sometimes assumed to be sufficient for worldwide crop pollination (Aebi and Neumann 2011 ). However, this assertion has been questioned by different authors (Ollerton et al. 2012 ), and several studies show that many wild bees are also efficient pollinators of crops (Klein et al. 2007 ; Winfree et al. 2008 ; Breeze et al. 2011 ). Recently, Garibaldi et al. ( 2013 ) found positive associations of fruit set with wild‐insect visits to flowers in 41 crop systems worldwide. They demonstrate that honeybees do not maximize pollination, nor can they fully replace the contributions of diverse, wild‐insect assemblages to fruit set for a broad range of crops and agricultural practices on all continents with farmland. Unfortunately, not only are honey bees declining due to a variety of different causes (vanEngelsdorp et al. 2009 ), wild bee populations are also dwindling (Potts et al. 2010 ). Their decline has been documented in two Western European countries (Britain and the Netherlands) by comparing data obtained before and after 1980 (Biesmeijer et al. 2006 ). These losses have mostly been attributed to the use of agrochemicals, the increase in monocultures, the loss of seminatural habitat and deforestation (Steffan‐Dewenter et al. 2002 ; Steffan‐Dewenter and Westphal 2008 ; Brittain and Potts 2011 ). Several studies have shown the importance of natural or seminatural habitats in sustaining pollinator populations or pollination services close to fruit crops (Steffan‐Dewenter 2003a ; Kremen et al. 2004 ; Greenleaf and Kremen 2006a ; Carvalheiro et al. 2010 ). Morandin and Winston ( 2006 ) presented a cost–benefit model that estimates profit in OSR agroecosystems with different proportions of uncultivated land. They calculated that yield and profit could be maximized with 30% of the land left uncultivated within 750 m of field edges. Other studies have demonstrated a negative impact of the distance from forests on pollination services or bee abundance and richness both in tropical ecosystems (De Marco and Coelho 2004 ; Blanche et al. 2006 ; Chacoff and Aizen 2006 ) and in temperate ecosystems (Hawkins 1965 ; Taki et al. 2007 ; Arthur et al. 2010 ; Watson et al. 2011 ). These studies all suggest that natural or seminatural habitats are important sources of pollinators, probably because they provide “partial habitats” (Westrich 1996 ) such as complementary mating, foraging, nesting, and nesting materials sites that bees need to complete their life cycle. In this study, we focused on the effect of distance to forest edge on bee assemblages in OSR ecosystems. Forest edges could provide one or more important partial habitats for different bee species in agricultural landscapes, in particular when associated with a mass‐flowering crop such as OSR (Le Feon et al. 2011 ). For example, the availability of untilled soil and dead branches might provide ground‐nesting and cavity‐nesting bee species with numerous nesting sites. Moreover, during spring at least, the understory and the forest edge can provide cover containing flowering plants and wild trees such as Prunus spp, Castanea sativa, or Salix spp and thereby allow bees to find alternative floral resources. During spring 2010 and 2011, in two areas in France, we examined wild bee abundance and taxa richness both along forest edges and inside OSR fields at different distances from the forest. Like other taxa, bees respond to environmental variables according to their biologic traits that determine access and requirements for nesting, mating, and forage resources, species mobility or physiological tolerance. Specifically, we hypothesized that (1) bee abundance, species richness, and composition of bee communities within the crop field are dependent on the distance from the forest edge (where complementary floral resources, nesting sites, shelters, etc. can be found) and on the orientation of the forest edge; (2) the identity of bees in the crop is related to their foraging range which we measured with the ITD (Inter‐Tegular distance); (3) the forest edge may be the nesting or mating sites for cavity‐nesting or ground‐nesting bees such as Osmia spp or Andrena spp which are important groups of potential early spring pollinators for OSR.     

Where are they now? The fates of two genetic lineages in an introduced Hawaiian reef fish

Humans, both wittingly and unwittingly, have been transporting marine organisms beyond their native ranges for centuries ( Ruiz et al. 1997 ). A central challenge of invasion biology is to identify the factors that determine whether introduced species fail to become established, become benign members of a community, or spread so far and reach such densities as to be considered invasive. Organismal features such as physiological tolerance, niche breadth and fecundity are critical, but by themselves are inaccurate predictors of the fates of introduced species ( Sakai et al. 2001 ). The size, age distribution, and genetic makeup of founder populations are also important, but because they are usually unknown they are most often viewed as sources of uncertainty. For marine species with planktonic larvae, the challenge is even greater because the consequences of a planktonic phase for dispersal and population viability are not well understood. In this issue, Gaither et al. (2010a) present a remarkable account of the introduction of a reef fish for which the number and genetic makeup of the founders are known. Between 1956 and 1961, the Division of Fish and Game for the Territory of Hawaii introduced 12 non‐indigenous fish species into Hawaiian waters to establish commercial and sport fisheries. The introduction of Lutjanus kasmira, the bluestriped snapper, was the most successful ( Fig. 1 ). There were two releases of fish from French Polynesia. In 1958, 2431 fish from the Marquesas Islands were released on Oahu, followed in 1961 with an additional 728 fish from the Society Islands. The blue striped snapper rapidly spread to the other Hawaiian Islands, reaching the northwestern end of the archipelago by 1992. The choice of the Marquesas as one of two sources for the introduction was fortuitous. Gaither et al. (2010b) found that the Marquesas population is genetically distinct from all other Indo‐Pacific populations of L. kasmira. Mitochondrial cytochrome b sequences of fish from the Marquesas belong to a separate lineage that diverged from others in the species roughly half a Ma. Allele frequencies for several nuclear loci are also distinct. This provided Gaither et al. (2010a) with an extraordinary opportunity to examine what became of the mixed genetic heritage of Hawaiian blue striped snappers after 50 years.

Figure 1 Open in figure viewer PowerPoint The bluestriped snapper, Lutjanus kasmira, introduced to Hawaii 50 years ago and now an abundant reef fish expanded from a small founder population with minimal changes in the diversity or frequencies of mitochondrial and nuclear genetic markers.     

Determining parentage and relatedness from genetic markers sheds light on patterns of marine larval dispersal

Population connectivity, the extent to which geographically separated subpopulations exchange individuals and are demographically linked, is important to the scientific management of marine living resources. In theory, the design of a marine protected area, for example, depends on an explicit understanding of how dispersal of planktonic larvae affects metapopulation structure and dynamics ( Botsford et al. 2001 ). In practice, for most marine metazoans with planktonic larvae, the mean and variance of the distances that larvae disperse are unobservable quantities, owing to the small sizes of larvae and the very large volumes through which they are distributed. Simulation of dispersal kernels with models that incorporate oceanography and limited aspects of larval biology and behaviour, coupled with field studies of larval distribution, abundance, and settlement, have provided the best available approaches to date for understanding connectivity of marine populations ( Cowen et al. 2006 ). On the other hand, marine population connectivity has often been judged by spatial variation in the frequencies of alleles and genotypes, although the inherent limitations of this indirect approach to measuring larval dispersal have often been overlooked ( Hedgecock et al. 2007 ). More recently, researchers have turned to genetic methods and highly polymorphic markers that can provide direct evidence of population connectivity in the form of parentage or relatedness of recruits (e.g. Jones et al. 2005 ). In this issue, Christie et al. (2010) provide a particularly elegant example, in which both indirect and novel direct genetic methods are used to determine the major ecological processes shaping dispersal patterns of larval bicolour damselfish Stegastes partitus, a common and widespread reef fish species in the Caribbean Basin ( Fig. 1 ).

Figure 1 Open in figure viewer PowerPoint The bicolour damselfish Stegastes partitus shows substantial self‐recruitment of juveniles to their natal coral reef habitat. Below, a male guarding an artificial nest made from PVC pipe; differential reproductive success of parents or differential survival of egg clutches or the larvae that hatch from them may account for signals of sweepstakes reproductive success in this species (photo credits: top, Bill Harward; bottom, Darren Johnson).     

Massive parallel MHC genotyping: titanium that shines

The power of population genetic analyses is often limited by sample size resulting from constraints in financial resources and time to genotype large numbers of individuals. This particularly applies to nonmodel species where detailed genomic knowledge is lacking. Next‐generation sequencing technology using primers ‘tagged’ with an individual barcode of a few nucleotides offers the opportunity to genotype hundreds of individuals at several loci in parallel ( Binladen et al. 2007 ; Meyer et al. 2008 ). The large number of sequence reads can also be used to identify artefacts by frequency distribution thresholds intrinsically determined for each run and data set. In Babik et al. (2009 ), next‐generation deep sequencing was used to genotype several major histocompatibility complex (MHC) class IIB loci of the European bank vole ( Fig. 1 ). Their approach can be useful for many researchers working with complex multiallelic templates and large sample sizes.

Figure 1 Open in figure viewer PowerPoint Hypothetical example of parallel genotyping of two individuals using individually bar‐coded primers. Polymerase chain reactions (PCRs) are performed separately for each individual using a forward primer with a unique Tag‐sequence of four nucleotides. After sequencing of pooled PCR products, sequences can be sorted by their forward primer Tag (Tag‐sorting error rate was estimated < 0.1%). Rare sequences most likely represent artefacts and due to the large amount of sequences obtained (up to 10⁶) the artefact threshold can be determined intrinsically for each data set and was estimated to be around 3% in the case of bank vole MHC class IIB genes ( Babik et al. 2009 ). Photos by Gabriela Bydlon.     

Effects of SAG12-ipt expression on cytokinin production,growth and senescence of creeping bentgrass (<Emphasis Type="Italic">Agrostis stolonifera</Emphasis> L.) under heat stress

The study was conducted to determine the effects of expression of a transgene encoding adenine isopentenyl transferase (ipt), which controls cytokinin synthesis, on growth and leaf senescence of creeping bentgrass (Agrostis stolonifera L.), subjected to heat stress. Creeping bentgrass (cv. Penncross) was transformed with ipt ligated to a senescence-activated promoter (SAG12). Eight SAG12-ipt transgenic lines exhibiting desirable turf quality and a transgenic control line (transformed with the empty vector) were evaluated for morphological and physiological changes under normal growth temperature (20°C) and after 14 days of heat stress (35°C) in growth chambers. Six of the SAG12-ipt lines developed more tillers than the control line during establishment under normal growth temperature of 20°C. Following 14 days of heat stress, four of the SAG12-ipt lines had increased 65–83% of roots and for all six SAG12-ipt lines root elongation continued, whereas root production ceased and total root length decreased for the control line. Root isopentenyl adenine (iPA) content increased 2.5–3.5 times in five of the SAG12-ipt lines, whereas in the control line iPA decreased 20% after 14 days at 35°C. Total zeatin riboside (ZR) content was maintained at the original level or increased in five of the SAG12-ipt lines, whereas in the control line ZR decreased under heat stress. Our results suggest expression of SAG12-ipt in creeping bentgrass stimulated tiller formation and root production, and delayed leaf senescence under heat stress, suggesting a role for cytokinins in regulating cool-season grass tolerance to heat stress.     

Beyond splitting hares and rabbiting on about major histocompatibility complex complexity

The genes of the major histocompatibility complex (MHC) have become the target of choice for studies wishing to examine adaptively important genetic diversity in natural populations. Within Molecular Ecology alone, there have been 71 papers on aspects of MHC evolution over the past few years, with an increasing year on year trend. This focus on the MHC is partly driven by the hypothesized links between MHC gene dynamics and ecologically interesting and relevant traits, such as mate choice and host–parasite interactions. However, an ability to pin down the evolutionary causes and ecological consequences of MHC variation in natural populations has proven challenging and has been hampered by the very issue that is attractive about MHC genes – their high levels of diversity. Linking high levels of MHC diversity to ecological factors in inherently complex natural populations requires a level of experimental design and analytical rigour that is extremely difficult to achieve owing to a plethora of potentially confounding and interacting variables. In this issue of Molecular Ecology, Smith et al. (2010) elegantly overcome the challenge of detecting complex interactions in complex systems by using an intricate analytical approach to demonstrate a role for MHC in the reproductive ability of a natural population of the European hare Lepus europaeus ( Fig. 1 ). Also in this issue, Oppelt et al. (2010) demonstrate a role for MHC variation in determining levels of hepatic coccidian infection in the European rabbit Oryctolagus cuniculus ( Fig. 2 ).

Figure 1 Open in figure viewer PowerPoint The European hare (Lepus europaeus).     

Cultivated and weedy rice interactions and the domestication process

Examining the targets of selection in crop species and their wild and weedy relatives sheds light on the evolutionary processes underlying differentiation of cultivars from progenitor lineages. On one hand, human‐mediated directional selection in crops favours traits associated with the streamlining of controllable and predictable monoculture practices alongside selection for desired trait values. On the other hand, natural selection in wild and especially weedy relatives presumably favours trait values that increase the probability of escaping eradication. Gene flow between crops and wild species may also counter human‐mediated selection, promoting the evolution and persistence of weedy forms. In this issue, two studies from a group of collaborators examine diversity and divergence patterns of genes underlying two traits associated with red rice (Oryza sp.), the conspecific relative of cultivated rice (Oryza sativa) that is a non‐native weed (see Fig. 1 ). In the first study by Gross et al. (2010) , genetic variation in the major gene underlying the hallmark red pigmentation characterizing most weedy rice (Rc) is found to have a pattern consistent with non‐reversion from U.S. cultivated rice (i.e. the cultivar did not ‘go feral’). This suggests that U.S. weedy rice is not an escaped lineage derived from U.S. cultivated rice populations; weedy rice likely differentiated prior to the selective sweep occurred in this gene within cultivated rice populations. Using the major seed shattering locus sh4 gene and the neighbouring genomic region, Thurber et al. (2010) track the molecular evolutionary history of the high shattering phenotype, a trait contributing dramatically to the success of crop selection in cultivated rice as well as the persistence and expansion of weedy red rice. In this study, the shared fixation of a sh4 mutation in both cultivated rice and weedy rice indicates that weedy rice arose subsequent to the strong selective sweep leading to significant reduction in seed shattering in cultivated rice.

Figure 1 Open in figure viewer PowerPoint A weedy, brown hulled red rice individual with long awns surrounded by a field of cultivated rice (photo by A. Lawton‐Rauh).     

Transgenic creeping bentgrass overexpressing Osa‐miR393a exhibits altered plant development and improved multiple stress tolerance

MicroRNA393 (miR393) has been implicated in plant growth, development and multiple stress responses in annual species such as Arabidopsis and rice. However, the role of miR393 in perennial grasses remains unexplored. Creeping bentgrass (Agrostis stolonifera L.) is an environmentally and economically important C3 cool‐season perennial turfgrass. Understanding how miR393 functions in this representative turf species would allow the development of novel strategies in genetically engineering grass species for improved abiotic stress tolerance. We have generated and characterized transgenic creeping bentgrass plants overexpressing rice pri‐miR393a (Osa‐miR393a). We found that Osa‐miR393a transgenics had fewer, but longer tillers, enhanced drought stress tolerance associated with reduced stomata density and denser cuticles, improved salt stress tolerance associated with increased uptake of potassium and enhanced heat stress tolerance associated with induced expression of small heat‐shock protein in comparison with wild‐type controls. We also identified two targets of miR393, AsAFB2 and AsTIR1, whose expression is repressed in transgenics. Taken together, our results revealed the distinctive roles of miR393/target module in plant development and stress responses between creeping bentgrass and other annual species, suggesting that miR393 would be a promising candidate for generating superior crop cultivars with enhanced multiple stress tolerance, thus contributing to agricultural productivity.     

Transgenic tobacco plants expressing grass <Emphasis Type="Italic">AstEXPA1</Emphasis> gene show improved performance to several stresses

Expansins are cell wall-loosening proteins and now widely accepted to associate with the plant resistance against various abiotic stresses. In this study, we cloned an expansin gene of AstEXPA1 from Agrostis stolonifera, a heat-resistant creeping bentgrass cultivar, and transformed it into tobacco plants. Physiological index test showed that the transgenic lines were resistant to various abiotic stresses of drought, heat, cold, and salt in comparison to non-transgenic plants. Comprehensive analysis of four physiological response indices showed that the transgenic plants performed much better resistance to drought, following to heat, cold and salt stress, respectively. Meanwhile soluble sugar content displayed more weight to plant resistance by over-expressing AstEXPA1 gene, followed as proline content, REL, and MDA content. The results here would expand our understanding of the expansin roles and drive better insights into plant molecular breeding against stress.     

Greenhouse evaluation of fitness-related reproductive traits in roundup<Superscript>®</Superscript>-tolerant transgenic creeping bentgrass (<Emphasis Type="Italic">Agrostis Stolonifera</Emphasis> L.)

Summary Creeping bentgrass is a very important turfgrass species used extensively on golf course greens, fairways, and tees. One of the challenges of creeping bentgrass management is the control of grassy weeds, most of which respond to herbicides in a similar manner to that of creeping bentgrass. As part of a weed management program for golf courses, Roundup^?-tolerant creeping bentgrass will be simple to employ and more effective in controlling problem weeds than currently available methods. The goal of this research was to evaluate fitness-related reproductive traits in four transgenic creeping bentgrass events modified to express a Roundup^?-tolerant gene, cp4 epsps, to determine if these creeping bentgrass events had gained an unexpected reproductive fitness advantage. We compared transgenic events ASR 333, ASR801 with their nontransformed tissue culture line, C99056L and transgenic events ASR365, ASR368 with their non-transformed tissue culture line, B99061R. Populations of plants from three conventional cultivars were also included for comparison to determine whether significant variations, if present in transgenic events, were novel to the non-transformed organism, Agrostis stolonifera L. Our results showed that none of the four transgenic events surveyed were significantly different from the respective non-transformed tissue culture line plants for the following characteristics: first heading date, anthesis duration, inflorescence length, number of florets per inflorescence, pollen size, and seed-set capacity through open-pollination. One of the transgenic events, ASR333, needed significantly more days for anthesis initiation than the nontransformed tissue culture line, C99056L; while another transgenic event, ASR801, exhibited significantly shorter pollen longevity than plants of the tissue culture line, C99056L. However, ASR801 was not significantly different from the conventional cultivars ‘Penn A-4’ and ‘Penncross’ for pollen longevity. Plants of both transgenic events ASR365 and ASR368 did not differ significantly from plants of the tissue culture line, B99061R, for all characters measured.     

Retraction Statement: CXCL12/CXCR4 chemokine signaling in spinal glia induces pain hypersensitivity through MAPKs‐mediated neuroinflammation in bone cancer rats

‘CXCL12/CXCR4 chemokine signaling in spinal glia induces pain hypersensitivity through MAPKs‐mediated neuroinflammation in bone cancer rats’ by Hu X.‐M., Liu Y.‐N., Zhang H.‐L., Cao S.‐B., Zhang T., Chen L.‐P. and Shen W. The above article from Journal of Neurochemistry, published online on 26 January 2015 and in volume 132, issue 4, pages 452–463 (available through www.onlinelibrary.wiley.com ), and its subsequent Corrigendum, published online on 5 February 2015 and in volume 132, issue 4, p. 487, have been retracted by agreement between the Journal's Editor‐in‐Chief, Jörg Schulz, corresponding author Wen Shen on behalf of the authors, and John Wiley & Sons Ltd. The retraction has been agreed as the same GFAP immunostaining image was used to represent different experimental conditions in two different publications (Shen et al. [2014] in the Journal of Neuroinflammation and Hu et al. [2015] in the Journal of Neurochemistry), with apparent brightness changes between the images. Shen et al. (2014) show in the outer right panel of Figure 4a, as well as in Fig. 8A for the GFAP/sham condition, a GFAP immunostaining after treatment with TCI + Fluorocitrate. The same image, at a lower intensity, is used in Hu et al. (2015) in the first panel of figure 5b as a sham control. The shape of the tissue margins of the spinal cord section as well as several landmark epitopes that point towards identical images are encircled:

Shen et al. 2014 Figure 4a     

Discovery of keratin function and role in genetic diseases: the year that 1991 was

In 1991, a set of transgenic mouse studies took the fields of cell biology and dermatology by storm in providing the first credible evidence that keratin intermediate filaments play a unique and essential role in the structural and mechanical support in keratinocytes of the epidermis. Moreover, these studies intimated that mutations altering the primary structure and function of keratin filaments underlie genetic diseases typified by cellular fragility. This Retrospective on how these studies came to be is offered as a means to highlight the 25th anniversary of these discoveries.Although intermediate filaments (IFs) have been characterized at some level for a longer period of time (Oshima, 2007 ), they were officially discovered as such as recently as 1968 by Howard Holtzer and colleagues while studying the developing skeletal muscle (Ishikawa et al., 1968 ). The advent of gene cloning methods and monospecific antibody production in the late 1970s and throughout the 1980s led to an explosion of data and knowledge about IFs that established them as a large family of genes and proteins that are individually regulated in a tight and evolutionarily conserved tissue- and differentiation-specific manner. Researchers also uncovered some of the remarkable properties of IFs as purified elements in vitro and in living systems and recognized that they occur in the nucleus as well as in the cytoplasm. In spite of the fast pace of progress during that period, however, it was not possible to produce evidence that spoke unequivocally about the functional importance of IFs in cells and tissues, let alone their role in disease.Beginning in the mid- to late 1980s, pioneering experimentation along two distinct lines was underway in the laboratory of Elaine Fuchs, then at the University of Chicago. The eventual merger of these approaches yielded the first formal insight into IF function in vivo, as well as into their direct involvement in human disease. In an effort to define structure–function relationships with regard to the assembly and network formation properties of IFs, one such approach was the application of systematic deletion mutagenesis to keratin 14 (K14), a type I IF that is expressed with its type II partner keratin 5 (K5) in the progenitor basal layer of the epidermis and related complex epithelia. These studies demonstrated that deleting sequences from either end of the central α-helical rod domain of the K14 protein was deleterious for filament formation in a dominant manner both in transfected cells (Albers and Fuchs, 1987 , 1989 ; Figure 1) and the setting of IF polymerization assays involving purified proteins in vitro (e.g., Coulombe et al., 1990 ). The second key effort in the Fuchs lab in the late 1980s resulted in the demonstration that the proximal 2.5 kb and distal 700 base pairs corresponding respectively to the 5′ upstream and 3′ downstream regions of the cloned human K14 gene were sufficient to confer tissue-specific, that is, K14-like, regulation in transgenic mice in vivo (Vassar et al., 1989 ; Figure 1). This tour de force paved the way for the production of a human K14 gene promoter–based cassette (e.g., Saitou et al., 1995 ) that could reliably direct the expression of any open reading frame in a K14-like manner in transgenic mice. As an aside, this tool has had a profound effect on epithelial and skin biology research.Open in a separate windowFIGURE 1:Schematic representation of the strategy and outcome of the experiments that led to the discovery of keratin function and role in genetic disease. Original figures are reproduced to give a realistic account of the data. (A) Examples of a disrupted keratin filament network in cultured epithelial cells transfected with and expressing a dominantly acting K14 deletion mutant (arrows). (Reproduced from Albers and Fuchs, 1987 , with permission.) (B) Preferential expression of a substance P-epitope–tagged transgenic human K14 protein in the basal layer of tail skin epidermis in mouse, conveying the tissue- and differentiation-specific behavior of the transgene. (Reproduced from Vassar et al., 1989 , with permission.) (C) The two experimental approaches described in A and B were combined to assess the consequences of tissue-specific expression of dominantly acting K14 mutants in skin tissue in vivo. (D) Newborn mouse littermates. The mouse at the top is transgenic (Tg) and expresses a mutated form of K14 in the epidermis. It is showing severe skin blistering (arrows), particularly in its front paws, which are heavily used by mouse newborns to feed from their mother. The bottom mouse is a nontransgenic control showing no such blistering. (E, F) Hematoxylin-eosin–stained skin tissue sections showing the location of subepidermal cleavage within the epidermis of a K14 mutant–expressing transgenic mouse (opposing arrows in E). Cleavage occurs at the level of the basal layer, where the mutant keratin is expressed. Again, this is never seen in control wild-type (Wt) skin (F). Bar, 100 μm (E, F). (D–F are from Coulombe et al., 1991b , with permission.) (G) Leg skin in a patient suffering from the Dowling–Meara form of epidermolysis bullosa simplex. Characteristic of this severe variant of this disease, several skin blisters are often grouped in a herpetiform manner (Fine et al., 1991 ).Subsequent use of the human K14 promoter–based cassette to direct the expression of epitope-tagged and selected deletion mutants of K14 gave rise to transgenic mouse pups that exhibited extensive blistering of the skin preferentially at sites of frictional trauma (Coulombe et al., 1991b ; Vassar et al., 1991 ; Figure 1). Electron microscopy showed that skin blistering occurred secondary to a loss of the integrity of keratinocytes located in the basal layer of the epidermis, that is, the precise site of mutant K14 protein accumulation. Such blistering did not occur in transgenic mice expressing a full-length version of human K14 modified to carry only an epitope tag at the C-terminus at similar or higher levels (Coulombe et al., 1991b ; Vassar et al., 1991 ). In addition, the severity of skin blistering in mutant K14–expressing transgenic pups could be directly related to the extent to which the mutant protein had been shown to disrupt filament assembly in transfected cell assays and in IF reconstitution assays in vitro. For instance, tissue-specific expression of a K14 mutant that could severely disrupt 10-nm filament assembly was associated with whole-body skin blistering and the untimely death of mouse pups and, from a pathology perspective, with “tonofilament clumping” and a paucity of visible keratin IFs in transgenic basal keratinocytes. By comparison, expression of another K14 mutant with a less deleterious effect on 10-nm IF assembly was compatible with the survival of transgenic mouse pups and resulted in skin blistering largely limited to the front paws in newborn mice together with altered organization of keratin IFs in basal keratinocytes of transgenic epidermis in situ, albeit without tonofilament clumping. This initial set of mouse strains thus revealed the existence of a direct link between the so-called “genotype” (i.e., mutant K14 characteristics) and the skin phenotype (Coulombe et al., 1991b ; Vassar et al., 1991 ; Fuchs and Coulombe, 1992 ). Electrophoretic analyses of protein samples confirmed that the K14 mutant proteins acted dominantly to produce such spectacular phenotypes in transgenic mouse skin. Finally, blistering also occurred in the mutant K14–expressing transgenic mice in other stratified epithelia known both to express K14 and experience trauma, notably in the oral mucosa (Coulombe et al., 1991b ; Vassar et al., 1991 ).It is worth celebrating the 25th anniversary of these pioneering experiments for the following two reasons. First, the study of these mice provided the first formal demonstration that keratin IFs play a fundamentally important role in structural support in surface epithelia such as the epidermis and oral mucosa. Without proper IF support, epidermal keratinocytes are rendered fragile and cannot sustain trivial frictional stress (Coulombe et al., 1991b ; Fuchs and Coulombe, 1992 ). The second reason is the observation that the phenotype of these K14 mutant–expressing mice proved eerily similar to those of individuals afflicted with the disease epidermolysis bullosa simplex (EBS), a rare, dominantly inherited and debilitating skin condition in which the epidermis and oral mucosa undergo blistering after exposure to trivial mechanical trauma. As observed in the mouse model, tissue cleavage had been shown to result from the loss of integrity of keratinocytes located in the basal layer (Fine et al., 1991 ). Further, other researchers had previously reported on anomalies in the organization of keratin IFs in the basal epidermal keratinocytes of EBS patients (Anton-Lamprecht, 1983 ; Ito et al., 1991 ) or in cultures of epidermal keratinocytes established from EBS patients (Kitajima et al., 1989 ). The Fuchs laboratory thus teamed up with Amy Paller, a physician-scientist and pediatric dermatologist with deep expertise in genodermatoses, and mutations were soon discovered in the K14 gene of two independent and sporadic cases of a severe variant of the disease known as Dowling–Meara EBS (Coulombe et al., 1991a ; Figure 1). The two mutations were heterozygous missense alleles that affected the very same codon in K14 (Arg-125) and were correctly predicted at the time to correspond to a mutational hot spot in type I keratin genes. The mutations were shown to dominantly disrupt 10-nm IF assembly in vitro and/or in transfected keratinocytes in culture (Coulombe et al., 1991a ). Soon thereafter, a team led by Ervin Epstein at University of California, San Francisco (San Francisco, CA), reported on the use of classical linkage analysis to uncover a missense mutation in the K14 gene of a small pedigree with Koebner-type EBS, a less severe variant of the disease (Bonifas et al., 1991 ). The next year, Birgit Lane and colleagues (Lane et al., 1992 ) reported on the occurrence of mutations in keratin 5 (K5), the formal type II keratin assembly partner for K14 in vivo, in another instance of Dowling–Meara EBS.In the years since 1991, a role in structural support has been formally demonstrated for all classes of IFs (Coulombe et al., 2009 ), including the nuclear-localized lamins (e.g., Lammerding et al., 2004 ). Moreover, we now know of several hundred independent instances of mutations in either K5 or K14 in the setting of the EBS disease, with the vast majority of those consisting of dominantly acting missense alleles (Szeverenyi et al., 2008 ; Human Intermediate Filament Database, www.interfil.org, maintained at the Centre for Molecular Medicine and Bioinformatics Institute, Singapore). We also learned that, as anticipated, EBS largely represents a loss-of-function phenotype, since K14-null mice (Lloyd et al., 1995 ), K14-null individuals (Chan et al., 1994 ; Rugg et al., 1994 ), and K5-null mice (Peters et al., 2001 ) all exhibit an EBS-like skin-blistering phenotype (Coulombe et al., 2009 ). Mutations such as Arg125Cys in K14 markedly compromise the remarkable mechanical properties of keratin filaments (Ma et al., 2001 ), as well as the steady-state dynamics of keratin filaments in transfected keratinocytes in culture (Werner et al., 2004 ). Finally, mutations affecting the coding sequence of IF genes have been shown to underlie >100 diseases affecting the human population (Omary et al., 2004 ; Szeverenyi et al., 2008 ; www.interfil.org). Consistent with the exquisite tissue- and cell type–specific regulation of IF genes, these diseases collectively affect a myriad of tissues and organs and are relevant to nearly all branches of medicine. These observations attest to the importance and profound effect that the generation and characterization of mutant K14–expressing transgenic mice has had for cell biology, epithelial physiology, dermatology, and medicine.Many thoughts spring to mind when reminiscing about my involvement with this body of work. First, this effort was prescient of the power of team science and, in particular, of the potential effect of close collaborations involving biologists and physician-scientists. I learned a great deal and benefited immensely from working closely with many colleagues on this project, including Bob Vassar, Kathryn Albers, Linda Degenstein, Liz Hutton, Anthony Letai, Amy Paller, and, last but not least, my postdoctoral mentor and the laboratory head, Elaine Fuchs. Second, there is no substitute for elements such as innovation, hard work, perseverance, boldness, accountability, and great leadership. Elaine had the vision and created the exceptional circumstances necessary to make this set of discoveries possible, and, of equal importance, she was an integral part of the day-to-day progress and maturation of the entire project. Finally, as we all know, there is an intangible element of luck involved in discovery research. In this instance, a strong argument can be made that the studies highlighted here may not have had such a deep and defining effect had the effort been devoted to any IF other than the K5–K14 keratin pairing.What are some of the lingering issues regarding this specific topic that preoccupy us still, 25 years later? Two challenges loom particularly large. First, we have yet to achieve a satisfactory understanding of how mutations in keratin proteins can cause disease. This is due in part to the lack of an atomic-level understanding of the core structure of IFs (which has been a tough nut to crack; Lee et al., 2012 ), along with the reality that, for any relevant IF gene, there is a broad variety of disease-associated (mostly missense) mutations that pepper their primary structure (www.interfil.org). Second, we have yet to achieve success toward the treatment of EBS or any IF-based disorder. Disease characteristics such as low incidence, a dominantly inherited character, genetic heterogeneity (e.g., broad mutational landscape), and, in the case of EBS and related conditions, an intrinsically high rate of cell turnover within the main target tissue significantly add to the challenge of devising safe and effective therapeutic strategies (Coulombe et al., 2009 ). Although efforts are still underway to foster progress on these two challenging issues, the field as a whole has made significant progress in uncovering a plethora of noncanonical functions of keratin IFs (Hobbs et al., 2016 ) in addition to understanding their regulation, dynamics, and many remarkable properties.     

Ovipositional preferences and larval survival of annual bluegrass weevil,Listronotus maculicollis,on Poa annua and selected bentgrasses (Agrostis spp.)

The annual bluegrass weevil (ABW), Listronotus maculicollis Kirby (Coleoptera: Curculionidae), is a serious and expanding pest of short‐cut turfgrass on golf courses in eastern North America. Increasing problems with the development of insecticide resistance in this pest highlights the need for more sustainable management approaches. Plant resistance is one of the most promising alternative strategies. Bentgrasses are the dominant grass species on golf course fairways, tees, and putting greens in the areas affected by ABW. But Poa annua L. (Poaceae), a highly invasive weed, often constitutes a large percentage of turf stands in short‐mown golf courses and is thought to be particularly susceptible to ABW. We studied resistance to ABW in four cultivars of creeping bentgrass, Agrostis stolonifera L., and two cultivars each of colonial bentgrass, Agrostis capillaris L., and velvet bentgrass, Agrostis canina L. (Poaceae), in comparison with P. annua by addressing the three major components of resistance: antixenosis (adult ovipositional and feeding preferences), antibiosis (larval survival and growth), and grass tolerance (grass damage). Our findings suggest that antixenosis/non‐preference is at least partially involved in bentgrass resistance to ABW. Even though oviposition was observed in all tested grasses, females laid significantly fewer eggs in Agrostis spp. than in P. annua. Compared to P. annua, Agrostis spp. were also less suitable for larval development with lower numbers of ABW immatures recovered and larvae weighing less and being less advanced in development. Resistance levels to ABW larvae varied significantly among Agrostis spp. and cultivars. Agrostis canina was least preferred by females for oviposition and A. stolonifera was the least suitable for larval survival and development. Agrostis spp., especially A. stolonifera, were more tolerant to ABW feeding than P. annua. Our findings suggest that reduction in P. annua and replacement with Agrostis spp., especially A. stolonifera, wherever feasible should be integral to more sustainable approaches to ABW management.     

Heritability not missing—genetic basis of sexual weaponry uncovered

The chase to uncover the genetic underpinnings of quantitative traits of ecological and evolutionary importance has been on for a good while. However, the potential power of genome‐wide association studies (GWAS) as an approach to identify genes of interest in wild animal populations has remained untapped. Setting technical and economic explanations aside, the sobering lack of success in human GWAS might have fed this restraint. Namely, while GWAS have successfully identified genetic variants associated with hundreds of complex traits (e.g. Ku et al. 2010 ), these variants have generally captured only a low percentage of variance in traits known to be highly heritable—an observation came to be known as the ‘missing heritability’ ( Maher 2008 ; Aulchenko et al. 2009 ). Hence, if the vastly resourced human studies have been unsuccessful (but see: Yang et al. 2010 ), why should we expect that less resourced studies of wild animal populations would be able do better? In this issue of Molecular Ecology, Johnston et al. (2011) prove this line of thinking wrong. In an impressive and what may well be the most advanced gene mapping study ever performed in a wild population, they identify a single locus (RXFP2) responsible for explaining horn phenotype in feral domestic sheep from St Kilda ( Fig. 1 ). This same locus is also shown to account for up to 76% of additive genetic variance in horn size in male sheep: this contrasts sharply with most human GWAS where mapped loci explain only a modest proportion of genetic variation in a given trait.

Figure 1 Open in figure viewer PowerPoint The Soay sheep of the St Kilda archipelago are a primitive feral breed of domestic sheep. Pictured are a male with vestigial horns (=‘scurred’; left) and two normal‐horned males (centre and right). Photograph courtesy of Peter Korsten.     

Testing evolutionary hypotheses for phenotypic divergence using landscape genetics

Understanding the evolutionary causes of phenotypic variation among populations has long been a central theme in evolutionary biology. Several factors can influence phenotypic divergence, including geographic isolation, genetic drift, divergent natural or sexual selection, and phenotypic plasticity. But the relative importance of these factors in generating phenotypic divergence in nature is still a tantalizing and unresolved problem in evolutionary biology. The origin and maintenance of phenotypic divergence is also at the root of many ongoing debates in evolutionary biology, such as the extent to which gene flow constrains adaptive divergence ( Garant et al. 2007 ) and the relative importance of genetic drift, natural selection, and sexual selection in initiating reproductive isolation and speciation ( Coyne & Orr 2004 ). In this issue, Wang & Summers (2010) test the causes of one of the most fantastic examples of phenotypic divergence in nature: colour pattern divergence among populations of the strawberry poison frog (Dendrobates pumilio) in Panama and Costa Rica ( Fig. 1 ). This study provides a beautiful example of the use of the emerging field of landscape genetics to differentiate among hypotheses for phenotypic divergence. Using landscape genetic analyses, Wang & Summers were able to reject the hypotheses that colour pattern divergence is due to isolation‐by‐distance (IBD) or landscape resistance. Instead, the hypothesis left standing is that colour divergence is due to divergent selection, in turn driving reproductive isolation among populations with different colour morphs. More generally, this study provides a wonderful example of how the emerging field of landscape genetics, which has primarily been applied to questions in conservation and ecology, now plays an essential role in evolutionary research.

Figure 1 Open in figure viewer PowerPoint Divergent colour morphs observed among populations of the strawberry poison frog, Dendrobates pumilio. Frogs are from San Cristobal (upper left), Cerro Brujo (upper right), Bastimentos (lower right), and Agua (lower left).