Improvement of alfalfa forage quality and management through the down‐regulation of MsFTa1

Alfalfa (Medicago sativa L.) is one of the most important forage crops worldwide. As a perennial, alfalfa is cut several times each year. Farmers face a dilemma: if cut earlier, forage nutritive value is much higher but regrowth is affected and the longevity of the stand is severely compromised. On the other hand, if alfalfa is cut later at full flower, stands persist longer and more biomass may be harvested, but the nutritive value diminishes. Alfalfa is a strict long‐day plant. We reasoned that by manipulating the response to photoperiod, we could delay flowering to improve forage quality and widen each harvesting window, facilitating management. With this aim, we functionally characterized the FLOWERING LOCUS T family of genes, represented by five members: MsFTa1, MsFTa2, MsFTb1, MsFTb2 and MsFTc. The expression of MsFTa1 correlated with photoperiodic flowering and its down‐regulation led to severe delayed flowering. Altogether, with late flowering, low expression of MsFTa1 led to changes in plant architecture resulting in increased leaf to stem biomass ratios and forage digestibility. By manipulating photoperiodic flowering, we were able to improve the quality of alfalfa forage and management, which may allow farmers to cut alfalfa of high nutritive value without compromising stand persistence.     

Phytoavailability of Heavy Metals and Metalloids in Soils Irrigated with Wastewater,Akaki, Ethiopia: A Greenhouse Study

Irrigation with untreated wastewater from several industrial, commercial, and domestic discharges for decades caused accumulation of various heavy metals and metalloids in soils along the Akaki River in Ethiopia. Assessment of environmental threats and the potential phytoremediation of the soils require understanding of the toxic elements’ uptake and distribution in plant parts. Hence, a greenhouse study was performed to examine the phytoavailability and distribution of Cr, Ni, Co, Cu, Zn, Cd, Pb, Hg, Se, V, and As in forage grasses: Oat (Avena sativa), Rhodes grass (Chloris gayana), Setaria (Setaria sphacelata), and the legumes Alfalfa (Medicago sativa) and Desmodium (Desmodium unicinatum). The average contents of Cr, Ni, Co, Cu, Zn, Pb, Hg, Se, and V in the plants were generally higher than the background levels for forage grasses/legumes, and some of these elements were in the phytotoxic range. Root bioconcentration factor (BCF = root to soil concentration ratio) > 1 was observed for Cu (Oat, Rhodes, Desmodium, and Setaria: Fluvisol), Zn (Setaria: Fluvisol), Cd (Rhodes: Fluvisol; Setaria from both soils) and Hg (Oat and Alfalfa: Fluvisol). Alfalfa and Desmodium displayed translocation factor > 1 (TF = shoot to root concentration ratio) for most heavy metals. Most heavy metals/metalloids may pose a health threat to humans and stock via introduction to the food chain. The plant factors (species and plant part), soil factors (soil type, soil fractions, pH, and CEC), and their interactions significantly (p < 0.05) influenced plant heavy metal and metalloid levels. However, the role of plant part and species emerged as the most important on heavy metal uptake, translocation, sequestration, and ultimately transfer to the food chain. Accordingly, the uptake and distribution of heavy metals/metalloids in the plants reflect the potential environmental and health hazards attributable to the use of fodder grasses, legumes, and cultivation of vegetables in soils with polymetallic and metalloid contamination.     

Genetic diversity and population structure identify the potential source of the invasive red clover casebearer moth, <Emphasis Type="Italic">Coleophora deauratella</Emphasis>, in North America

The red clover casebearer, Coleophora deauratella, is an invasive pest of red clover grown for seed in North America. In 2006, an outbreak in Alberta, Canada was discovered that resulted in significant seed losses, while further invasion threatens the world’s largest red clover forage seed production region in Oregon, USA. Prior to the recent outbreak, C. deauratella was thought to be restricted to eastern North America in its invasive range. We sequenced a 615-bp fragment of the mitochondrial cytochrome c oxidase subunit 1 gene, and developed three microsatellite markers to assess the genetic diversity and population structure of C. deauratella in North America and its native range in Europe. We observed signatures of a founder effect in North American populations and a further loss of genetic diversity within Alberta populations. Most genetic differentiation was found between continents, with no evidence of isolation-by-distance within each continent. From the limited number of European populations sampled, a single introduction from Switzerland is the most probable source of North American populations based on similar mitochondrial diversity and lack of population differentiation. Within North America, based on increased genetic diversity compared to the rest of the continent, the first North American record from Ithaca, NY, and the first documented outbreak in southern Ontario in 1989, the initial C. deauratella invasion most likely occurred in southern Ontario, Canada or adjacent states in the USA, followed by transport throughout the continent. This study provides insight into the phylogeographic history of C. deauratella in North America and Europe and may help to identify a regional source of future classical biological control agents.     

A shade tolerance index for common understory species of northeastern North America

Since Baker’s [Baker, F.S., 1949. A revised tolerance table. J. For. 47, 179–181] classic contribution, shade tolerance indices have not been much modified for North American plant species. While many common tree and shrub species are included in the shade tolerance index, much less is known about this characteristic for the abundant and rich understory vascular and nonvascular plant layers. The classification of the shade tolerance is widely used to compare relative growth and survival among plant species under closed canopies and is also fundamental to an understanding of stand development following small and large scale disturbances. Although qualitative, it is frequently used both in research and management implications. Here we provide a significant revision to Baker’s shade tolerance table to include the most common forest understory plant species found in northeastern North America forests. Our index is based on: (1) the compilation of the opinions of five experts, (2) a comparison with Ellenberg’s index from Europe as well as, (3) information from current literature. For most of the 347 plant species investigated, a consistent and robust shade tolerance index, ranging between 1 (very tolerant) and 9 (very intolerant), was found. Here we present revised shade indices for 71 tree and shrub species, 185 herbaceous species, and 91 bryophyte and lichen species.     

Alfalfa Carbon and Nitrogen Sequestration Patterns and Effects of Temperature and Precipitation in Three Agro-Pastoral Ecotones of Northern China

Alfalfa (Medicago sativa L.) is a primary and widely cultivated forage crop in China. As a perennial leguminous grass, continuous planted alfalfa may influence carbon and nitrogen sequestration in soils. We evaluated the effect of alfalfa, planted for different lengths of time, and temperature and precipitation on soil organic carbon (SOC) and total nitrogen (TN) contents, and estimated soil SOC and TN inventories from 0–60 cm in three agro-pastoral ecotones of northern China. Alfalfa SOC and TN storage patterns were significantly different with increasing soil depths between the three regions of northern China. Continuous alfalfa grassland planted had a positive effect on accumulation of both SOC and TN in the Northwest region, whereas SOC storage peaked 6–7 years after planting in the Northeast and North region. Moreover, relatively higher TN storage appeared 7 years after planting in the Northeast and North regions. This study controlled as many factors as possible, but we caution that such temporal inferences could be artifacts of site selection. The regression analysis indicated that SOC and TN accumulation was mainly dependent on temperature (≥10°C of effective total accumulated temperature) in the North region. Precipitation in the growing season was the main limiting factor for SOC storage in the Northwest region and TN accumulation in the North regions. Therefore, the different climate factors affecting SOC and TN sequestration in alfalfa occurred at a regional scale.     

Genetic diversity of rhizobia nodulating Trifolium ambiguum in North America

Kura clover (Trifolium ambiguum M.B.) is a persistent rhizomatous forage legume, whose use in the U.S.A. is limited by establishment difficulties in part attributable to nodulation problems. In this study, soil was collected from established stands of Kura clover growing in 9 diverse North American environments. Rhizobia were plant-trapped using Kura clover cv. Endura as host, then rhizobia from nodules fingerprinted using BOX-PCR. The diversity of isolates from North America was then contrasted to that of rhizobia from a single Caucasian environment (Russia), the center of origin for this species. Populations were characterized using clustering methods, and genetic diversity estimated using the Shannon-Weaver diversity index. The genetic diversity of the North American populations was extremely limited, all isolates being closely related to two of the strains found in a locally available commercial inoculant. In contrast, Russian isolates formed a distinct cluster with significant internal genetic diversity. Genetic diversity indices for the North American and Russian populations were 3.5 and 10.76, respectively. The implication of this and other studies is that Kura clover is highly specific in Rhizobium requirement. If the performance of this legume in the U.S.A. is to be improved, either by modifying current establishment practices or plant breeding, it is essential that these studies be paralleled by more collections and evaluation of rhizobia from its center of origin, given the extremely limited diversity of rhizobia found in North America.     

Multiplex CRISPR/Cas9-mediated mutagenesis of alfalfa FLOWERING LOCUS Ta1 (MsFTa1) leads to delayed flowering time with improved forage biomass yield and quality

Alfalfa (Medicago sativa L.) is a perennial flowering plant in the legume family that is widely cultivated as a forage crop for its high yield, forage quality and related agricultural and economic benefits. Alfalfa is a photoperiod sensitive long-day (LD) plant that can accomplish its vegetative and reproductive phases in a short period of time. However, rapid flowering can compromise forage biomass yield and quality. Here, we attempted to delay flowering in alfalfa using multiplex CRISPR/Cas9-mediated mutagenesis of FLOWERING LOCUS Ta1 (MsFTa1), a key floral integrator and activator gene. Four guide RNAs (gRNAs) were designed and clustered in a polycistronic tRNA–gRNA system and introduced into alfalfa by Agrobacterium-mediated transformation. Ninety-six putative mutant lines were identified by gene sequencing and characterized for delayed flowering time and related desirable agronomic traits. Phenotype assessment of flowering time under LD conditions identified 22 independent mutant lines with delayed flowering compared to the control. Six independent Msfta1 lines containing mutations in all four copies of MsFTa1 accumulated significantly higher forage biomass yield, with increases of up to 78% in fresh weight and 76% in dry weight compared to controls. Depending on the harvesting schemes, many of these lines also had reduced lignin, acid detergent fibre (ADF) and neutral detergent fibre (NDF) content and significantly higher crude protein (CP) and mineral contents compared to control plants, especially in the stems. These CRISPR/Cas9-edited Msfta1 mutants could be introduced in alfalfa breeding programmes to generate elite transgene-free alfalfa cultivars with improved forage biomass yield and quality.     

Comparative proteomic analysis of male and female plants in Jojoba (<Emphasis Type="Italic">Simmondsia chinensis</Emphasis>) leaves revealed changes in proteins involved in photosynthesis,metabolism, energy,and biotic and abiotic stresses

Jojoba is a dioecious shrub with female and male flowers in separated individuals. The plant native to North and Central American deserts, it’s cultivated in many other places worldwide for its valuable liquid wax. The male-biased ratio in cultivated jojoba affects the yield. To develop protein molecular markers for early gender differentiation, comparative proteomic study been conducted on male and female leaves. Using gel-based proteomic, 45 proteins were identified representing 19 different proteins with 18 known functions. The identified proteins were involved in photosynthesis, energy, metabolism and the respond to biotic and abiotic stress. Ribulose-1,5-bisphosphate carboxylase (Rubisco) and ATP synthase were the most abundant proteins in both male and female of jojoba leaves, both were upregulated in male compared to female. Both proteins have the potential to serve as protein biomarkers for early differentiation between male and female in jojoba plant. These results could help in better understanding the molecular mechanism of gender differentiation in jojoba.     

Talus fragmentation mitigates the effects of pikas, Ochotona princeps, on high alpine meadows

North American pikas (Ochotona princeps) are central-place foragers that are restricted to talus slopes in the mountains of western North America. They graze meadows surrounding talus producing a gradient of grazing pressure extending out from the talus edge. We asked: 1) how does the physical structure of the talus slope affect pika foraging behavior? and 2) how do differences in foraging behavior affect the relative abundance of different plant functional groups?
Pikas at three talus-meadow interfaces differed in the distances they traveled for forage, depending upon the structure of the talus slope. Pikas fed preferentially on vegetation within the talus, ventured into the surrounding meadow when intra-talus vegetation was rare, and ranged farther when haying than when grazing. They ventured into the surrounding meadow less when talus fields were more fragmented and intra-talus vegetation was more abundant.
We tested the effects of pikas on meadow vegetation by excluding pikas from small plots located at various distances from talus edges. When habitat structure forced pikas to venture out from the talus to forage, they decreased total vegetative biomass by as much as 80% and increased the relative importance of cushion plants and sedges. These effects were undetectable at distances beyond one meter. These patterns appear to result from a change in the relative performance of different plant functional groups caused by herbivory.     

Development of an efficient plant regeneration system for Russian wildrye (<Emphasis Type="Italic">Psathyrostachys juncea</Emphasis>)

Russian wildrye [Psathyrostachys juncea (Fisch.) Nevski] is a cool-season forage grass with a broad adaptation to semi-arid regions of North America. In order to explore the potential of biotechnology for genetic improvement of this important forage species, we developed an efficient tissue culture system. Embryogenic calli were induced from mature embryos with an induction frequency in the range of 2-7%. The selected highly embryogenic calli allowed the regeneration of dozens of plants from a single callus. Individual embryogenic calli were then used to establish single genotype-derived suspension cultures. Eighteen embryogenic cell suspension lines were established from three cultivars (Bozoisky-Select, Sawki and Tetracan). A relatively high green plant regeneration frequency, up to 70%, was achieved from plated cell clusters of the established suspension cultures. The regenerated plants were fertile after two winters of vernalization in the field. This efficient plant regeneration system provides a solid basis for generating transgenic plants.     

Reassessment of phylogeographical structure in an eastern North American tree using Monmonier’s algorithm and ecological niche modelling

Aim Patterns of phylogeographical diversity in eastern North America have been well documented, with suggestions of Pleistocene refugia in both coastal and interior regions. However, most studies to date have assessed these patterns only qualitatively, largely through visual observation of haplotype networks. Furthermore, many plant studies use only one or two individuals per locality, which probably limits the recovery of haplotype diversity. The aim of this study is to address the issues of sampling strategy and quantitative assessment of phylogeographical patterns in an eastern North American tree, Fagus grandifolia (American beech). Location Eastern North America. Methods Comparing two sampling strategies (more localities with lower sample size within localities versus fewer localities with increased sample size within localities), we analysed chloroplast DNA sequence data from more than 230 individuals across 130 localities using statistical parsimony, maximum parsimony, maximum likelihood and Bayesian analyses. We then assessed support for inferred phylogenetic relationships using Monmonier’s algorithm and analysis of molecular variance. As an additional test of biogeographic hypotheses, we employed ecological niche models (ENMs), which are used to predict the geographic range of a species from occurrence data and environmental records. Here we predict both present and palaeodistributions. Results More haplotypes were recovered when more localities were sampled, but novel haplotypes and haplotype distributions were recovered using both strategies. Phylogeographical patterns suggest possible Pleistocene refugia along the Gulf and Atlantic coasts according to ENMs, as well as a more interior refugium according to Monmonier’s algorithm. Main conclusions Monmonier’s algorithm supports previous findings of an interior refugium in the Lower Mississippi River Valley/Upper Midwest, while ENMs indicate that the Gulf and Atlantic coasts may have provided the most suitable habitat for F. grandifolia during the Pleistocene. Our findings allow us to highlight the strengths and weaknesses of the two approaches. We propose that future phylogeographical studies should follow a step‐wise sampling strategy, balancing cost and expected outcomes.     

Fodder resources and feeding of semifree bison (<Emphasis Type="Italic">Bison bison</Emphasis>) on a steppe pasture in the Western Manych valley

In 2012–2013, the species composition, aboveground phytomass, and nutrition of freely ranging bison were investigated on a natural steppe pasture of the Western Manych valley. The aboveground mass of plants on the investigated pasture varied from 16.1 to 19.4 c/ha. The composition of the plants consumed by bison and their share in the bison’s diet were determined by microscopy of feces. In the snowless season, graminoids was the basis of the bison’s diet (79–87%). The use of forbs varied from 4.7 to 17.5%. Sedges were present in the forage in summer; they amounted to 6.5–15.4%. The quantitative assessment of the food selectivity was determined using the ratio of the share of a plant species in the diet to its portion in the plant community of the pasture. The most selected were Festuca valesiaca (the selectivity is 12.0), Agropyron sp. sp. (1.7), Polygonum patulum (1.2), and Carex sp. sp. (1.1). In other forage plants (Poa bulbojsa, Tanacetum achileifolium, and other forbs), the selectivity was 0.01–0.3. The nutritional value of fodder vegetation and food indexes (daily intake, digestibility of forage, consumption of metabolizable energy) were estimated. The digestibility was determined by the ratio of inert (undigestible) components (silicon, lignin) in the diet to their amount in the feces. The daily intake of forage was calculated based on the mass of released feces and digestibility of forage. In the snowless season, bison consumed from 6.3–7.0 to 9.2–9.9 kg/ind. (dry weight) per day. The maximum consumption (9.9 kg/ind.) was observed in summer. The digestibility of forage changed from 45 to 53% (the average is 50%). The consumption of metabolizable energy in different seasons varied from 0.60 to 0.91 MJ/kg^0.75/day, which corresponded to maintenance energy.     

Salinity tolerance of three competing rangeland plant species: Studies in hydroponic culture

Halogeton (Halogeton glomeratus) is an invasive species that displaces Gardner's saltbush (Atriplex gardneri) on saline rangelands, whereas, forage kochia (Bassia prostrata) potentially can rehabilitate these ecosystems. Salinity tolerance has been hypothesized as the predominant factor affecting frequency of these species. This study compared relative salinity tolerance of these species, and tall wheatgrass (Thinopyrum ponticum) and alfalfa (Medicago sativa). Plants were evaluated in hydroponics, eliminating the confounding effects of drought, for 28 days at 0, 150, 200, 300, 400, 600, and 800 mmol/L NaCl. Survival, growth, and ion accumulation were determined. Alfalfa and tall wheatgrass shoot mass were reduced to 32% of the control at 150 mmol/L. Forage kochia survived to 600 mmol/L, but mass was reduced at all salinity levels. Halogeton and Gardner's saltbush increased or maintained shoot mass up to 400 mmol/L. Furthermore, both actively accumulated sodium in shoots, indicating that Na⁺ was the principle ion in osmotic adjustment, whereas, forage kochia exhibited passive (linear) Na⁺ accumulation as salinity increased. This study confirmed the halophytic nature of these three species, but, moreover, discovered that Gardner's saltbush was as saline tolerant as halogeton, whereas, forage kochia was less tolerant. Therefore, factors other than salinity tolerance drive these species’ differential persistence in saline‐desert ecosystems.     

Systematics and management of natural resources: the case of <Emphasis Type="Italic">Spartina</Emphasis> species on European shores

Discrepancies in the identification of some plants and, in consequence on their autochthonous or allochthonous character, can lead to the adoption of inappropriate habit management strategies such as conservation, control, elimination, etc. A clear illustration of this is the case of a plant with an evident expansive behaviour located on the North Atlantic coast of the Iberian Peninsula, which has been considered by some authors as Spartina versicolor, a native of the European coasts. However, other authors have identified this plant as Spartina patens, originally from the North American Atlantic coasts, but introduced on both the Atlantic and Mediterranean coasts of Europe. This species shows an invading behaviour, playing a clear and evident role in the transformation of the habitats that it colonizes. In this work, results based on the use of sequences of the internal transcribed spacer (ITS) region of nrDNA, widely used in taxonomy and molecular phylogeny between closely related species, is reported. These data indicate that the identity of those plants growing on the European littoral is similar to those native to the North American Atlantic coasts.     

Novel microsatellite loci reveal high genetic diversity yet low population structure for alfalfa leafcutting bees in North America

The alfalfa leafcutting bee, Megachile rotundata (ALCB) is an economically important pollinator necessary for seed production of the critical forage crop alfalfa, Medicago sativa. The pollinator was accidentally introduced to North America from Europe approximately 70 years ago, and it is primarily produced in Canada and shipped to the United States annually en masse for seed field pollination. We investigate how the large-scale commercial movement of this bee affects the genetic structure of populations in the North American seed growing system and compare the genetic diversity and structure of introduced North American bees with two native European populations. Using 16 newly developed microsatellite loci, we describe the North American population structure of this bee. ALCBs collected from alfalfa seed farms have a degree of genetic variability similar to one native European population, but lower than the second. Considering that the species was accidentally introduced into North America, we anticipated more signature of a founder effect. Despite the level of genetic variability, we found little, if any, genetic structuring across North America, other than that the North American populations were distinct from the European populations sampled. While we detected some sub-structure in North American populations using Bayesian methods, the structuring was without geographic pattern, and we propose it is the result of the intense human management and movement of these bees. The trade and movement of these bees by humans has created a nearly panmictic M. rotundata population across the continent, which has implications relevant to the preservation and conservation of other bee pollinators.     

Le Grand Nord et la religion

The Far North, considered as the northern limit of the human ecumene characterized by an arctic climate, extended to contemporaneous temperate zones during glacial periods. Yet humans, an animal originating in intertropical zones, spread further in the Far North over 2 million years, confronting, even during full glacials, exceptional climatic constraints in order to take advantage of the abundant herbivores. Soon after 30,000, humans deeply penetrated within the Arctic Circle, both in Europe and Siberia. It was during the Last Glacial Maxium, in the Far North and Eurasia, that the first archaeological evidence for religious representations as well as significant social and technological innovations appeared. It was also via the Far North that the North American continent was reached.     

Factors influencing forage selection by the North American beaver (Castor canadensis)

Across their range, a large number of biotic and abiotic factors are known to influence the choice of browse plant and the foraging behaviour of the North American beaver (Castor canadensis). We used generalized linear mixed-effects models to investigate sets of variables that may influence the foraging choices of beaver: forage species, distance of forage from water, forage density, and site. Communities across the study sites in central British Columbia, Canada, were dominated by Salix sitchensis, Salix lucida, and Alnus spp. Density had no impact on forage selection, while site, distance from water, and species identity all influenced the foraging decisions of beaver. We postulated that these factors may be ordered hierarchically: large-scale factors, such as site, followed by the medium-scale distance from water, and species of plant at the finest scale. Forage items in some sites had a higher probability of being browsed than in others, while in all sites the probability of being browsed decreased with increasing distance from water. Beaver appeared to be foraging as “picky” generalists; of the 9 plant species examined, 3 species of Salix (S. scouleriana, S. drummondiana and S. sitchensis) were selected by beaver, Salix bebbiana was avoided, and 5 species were neither selected for nor against. Browse selection within the genus Salix implied that beaver were able to differentiate among closely related species. Detailed information on forage selection is a crucial first step in designing and interpreting models that predict large-scale distributional patterns of beaver.     

High migration rates shape the postglacial history of amphi‐Atlantic bryophytes

Paleontological evidence and current patterns of angiosperm species richness suggest that European biota experienced more severe bottlenecks than North American ones during the last glacial maximum. How well this pattern fits other plant species is less clear. Bryophytes offer a unique opportunity to contrast the impact of the last glacial maximum in North America and Europe because about 60% of the European bryoflora is shared with North America. Here, we use population genetic analyses based on approximate Bayesian computation on eight amphi‐Atlantic species to test the hypothesis that North American populations were less impacted by the last glacial maximum, exhibiting higher levels of genetic diversity than European ones and ultimately serving as a refugium for the postglacial recolonization of Europe. In contrast with this hypothesis, the best‐fit demographic model involved similar patterns of population size contractions, comparable levels of genetic diversity and balanced migration rates between European and North American populations. Our results thus suggest that bryophytes have experienced comparable demographic glacial histories on both sides of the Atlantic. Although a weak, but significant genetic structure was systematically recovered between European and North American populations, evidence for migration from and towards both continents suggests that amphi‐Atlantic bryophyte population may function as a metapopulation network. Reconstructing the biogeographic history of either North American or European bryophyte populations therefore requires a large, trans‐Atlantic geographic framework.     

Mechanisms underlying the effects of fall dormancy on the cold acclimation and winter hard- iness of Medicago sativa

As a global planting forage legume, alfalfa (Medicago sativa) is a valuable material to study the evolutionary and ecological mechanisms on plant adaptation to freezing due to their contrasting winter hardiness induced by fall dormancy (FD). This paper reveals that FD is an important growth characteristic that is adaptative to short-day and reducing temperature in late autumn, followed by a higher overwintering rate. Alfalfa cultivars are grouped into 11 FD ratings (numbered from 1 to 11), and this phenomenon is related to the extensive spread and cultivation for thousands of years in the globe. Alfalfa cultivars are under different climate habitats for a long time, leading to FD adaptive evolution, which provides rich genetic resources for human. In general, adaptative process associated with cold acclimation and winter hardiness in alfalfa is affected by FD, thus differences in winter hardiness exist among alfalfa cultivars. So far, regulation of FD by light and temperature and effects of FD on physiological and ecological processes involved in cold acclimation have been reported largely. However, signal transduction and the regulatory network associated with gene expression, especially the molecular mechanisms by which antifreeze proteins function in cold adaptation, are still poorly understood. Several scientific problems that need to be addressed in the future studies are highlighted in this review.