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1.  Assignment of SNP allelic configuration in polyploids using competitive allele-specific PCR: application to citrus triploid progeny 
Annals of Botany  2013;111(4):731-742.
Background
Polyploidy is a major component of eukaryote evolution. Estimation of allele copy numbers for molecular markers has long been considered a challenge for polyploid species, while this process is essential for most genetic research. With the increasing availability and whole-genome coverage of single nucleotide polymorphism (SNP) markers, it is essential to implement a versatile SNP genotyping method to assign allelic configuration efficiently in polyploids.
Scope
This work evaluates the usefulness of the KASPar method, based on competitive allele-specific PCR, for the assignment of SNP allelic configuration. Citrus was chosen as a model because of its economic importance, the ongoing worldwide polyploidy manipulation projects for cultivar and rootstock breeding, and the increasing availability of SNP markers.
Conclusions
Fifteen SNP markers were successfully designed that produced clear allele signals that were in agreement with previous genotyping results at the diploid level. The analysis of DNA mixes between two haploid lines (Clementine and pummelo) at 13 different ratios revealed a very high correlation (average = 0·9796; s.d. = 0·0094) between the allele ratio and two parameters [θ angle = tan−1 (y/x) and y′ = y/(x + y)] derived from the two normalized allele signals (x and y) provided by KASPar. Separated cluster analysis and analysis of variance (ANOVA) from mixed DNA simulating triploid and tetraploid hybrids provided 99·71 % correct allelic configuration. Moreover, triploid populations arising from 2n gametes and interploid crosses were easily genotyped and provided useful genetic information. This work demonstrates that the KASPar SNP genotyping technique is an efficient way to assign heterozygous allelic configurations within polyploid populations. This method is accurate, simple and cost-effective. Moreover, it may be useful for quantitative studies, such as relative allele-specific expression analysis and bulk segregant analysis.
doi:10.1093/aob/mct032
PMCID: PMC3605964  PMID: 23422023
SNP genotyping; competitive allele-specific PCR; KASPar; allele dosage; Citrus clementina; C. maxima; C. reticulata; polyploid; triploid; tetraploid
2.  trnL-F is a powerful marker for DNA identification of field vittarioid gametophytes (Pteridaceae) 
Annals of Botany  2013;111(4):663-673.
Background and Aims
The gametophyte phase of ferns plays an important role in habitat selection, dispersal, adaptation and evolution. However, ecological studies on fern gametophytes have been impeded due to the difficulty of species identification of free-living gametophytes. DNA barcoding provides an alternative approach to identifying fern gametophytes but is rarely applied to field studies. In this study, an example of field vittarioid gametophyte identification using DNA barcoding, which has not been done before, is given.
Methods
A combination of distance-based and tree-based approaches was performed to evaluate the discriminating power of three candidate barcodes (matK, rbcL and trnL-F) on 16 vittarioid sporophytes. Sequences of the trnL-F region were generated from 15 fern gametophyte populations by tissue-direct PCR and were compared against the sporophyte dataset, using BLAST.
Key Results trnL-F
earns highest primer universality and discriminatory ability scores, whereas PCR success rates were very low for matK and rbcL regions (10·8 % and 41·3 %, respectively). BLAST analyses showed that all the sampled field gametophytes could be successfully identified to species level. Three gametophyte populations were also discovered to be living beyond the known occurrence of their sporophyte counterparts.
Conclusions
This study demonstrates that DNA barcoding (i.e. reference databasing, tissue-direct PCR and molecular analysis), especially the trnL-F region, is an efficient tool to identify field gametophytes, and has considerable potential in exploring the ecology of fern gametophytes.
doi:10.1093/aob/mct004
PMCID: PMC3605945  PMID: 23380240
DNA barcoding; field gametophyte; independent gametophyte; vittarioid fern
3.  Extreme divergence in floral scent among woodland star species (Lithophragma spp.) pollinated by floral parasites 
Annals of Botany  2013;111(4):539-550.
Backgrounds and Aims
A current challenge in coevolutionary biology is to understand how suites of traits vary as coevolving lineages diverge. Floral scent is often a complex, variable trait that attracts a suite of generalized pollinators, but may be highly specific in plants specialized on attracting coevolved pollinating floral parasites. In this study, floral scent variation was investigated in four species of woodland stars (Lithophragma spp.) that share the same major pollinator (the moth Greya politella, a floral parasite). Three specific hypotheses were tested: (1) sharing the same specific major pollinator favours conservation of floral scent among close relatives; (2) selection favours ‘private channels’ of rare compounds particularly aimed at the specialist pollinator; or (3) selection from rare, less-specialized co-pollinators mitigates the conservation of floral scent and occurrence of private channels.
Methods
Dynamic headspace sampling and solid-phase microextraction were applied to greenhouse-grown plants from a common garden as well as to field samples from natural populations in a series of experiments aiming to disentangle the genetic and environmental basis of floral scent variation.
Key Results
Striking floral scent divergence was discovered among species. Only one of 69 compounds was shared among all four species. Scent variation was largely genetically based, because it was consistent across field and greenhouse treatments, and was not affected by visits from the pollinating floral parasite.
Conclusions
The strong divergence in floral scents among Lithophragma species contrasts with the pattern of conserved floral scent composition found in other plant genera involved in mutualisms with pollinating floral parasites. Unlike some of these other obligate pollination mutualisms, Lithophragma plants in some populations are occasionally visited by generalist pollinators from other insect taxa. This additional complexity may contribute to the diversification in floral scent found among the Lithophragma species pollinated by Greya moths.
doi:10.1093/aob/mct007
PMCID: PMC3605946  PMID: 23365407
Lithophragma affine; L. cymbalaria; L. heterophyllum; L. parviflorum; Saxifragaceae; Prodoxidae; coevolution; obligate mutualism; pollinating floral parasite; private channel; phenotypic plasticity; plant–insect interactions
4.  Ecological importance of sedges: a survey of the Australasian Cyperaceae genus Lepidosperma 
Annals of Botany  2013;111(4):499-529.
Background
Sedges (Cyperaceae) form an important ecological component of many ecosystems around the world. Sword and rapier sedges (genus Lepidosperma) are common and widespread components of the southern Australian and New Zealand floras, also occurring in New Caledonia, West Papua, Borneo, Malaysia and southern China. Sedge ecology is seldom studied and no comprehensive review of sedge ecology exists. Lepidosperma is unusual in the Cyperaceae with the majority of species occurring in dryland habitats.
Scope
Extensive review of ecological literature and field observations shows Lepidosperma species to be important components of many ecosystems, often dominating understorey and sedge-rich communities. For the first time, a detailed ecological review of a Cyperaceae genus is presented.
Conclusions Lepidosperma
species are long-lived perennials with significant abundance and persistence in the landscape. Speciation patterns in the genus are of considerable interest due to complex biogeographical patterns and a high degree of habitat specificity. Potential benefits exist for medicinal products identified from several Lepidosperma species. Over 178 organisms, including 26 mammals, 42 birds, six reptiles, five amphibians, eight arachnids, 75 insects, three crustaceans and 13 fungi, are found to be dependent on, or making use of, Lepidosperma species. A significant relationship exists between Lepidosperma species and the moth genus Elachista. Implications for the conservation and ecology of both sedges and associated species are discussed.
doi:10.1093/aob/mct008
PMCID: PMC3605947  PMID: 23378523
Lepidosperma; Cyperaceae; Schoeneae; sedge; ecology; fire; mammals; birds; reptiles; insects; spiders; fungi
5.  Differences in membrane selectivity drive phloem transport to the apoplast from which maize florets develop 
Annals of Botany  2013;111(4):551-562.
Background and Aims
Floral development depends on photosynthetic products delivered by the phloem. Previous work suggested the path to the flower involved either the apoplast or the symplast. The objective of the present work was to determine the path and its mechanism of operation.
Methods
Maize (Zea mays) plants were grown until pollination. For simplicity, florets were harvested before fertilization to ensure that all tissues were of maternal origin. Because sucrose from phloem is hydrolysed to glucose on its way to the floret, the tissues were imaged and analysed for glucose using an enzyme-based assay. Also, carboxyfluorescein diacetate was fed to the stems and similarly imaged and analysed.
Key Results
The images of live sections revealed that phloem contents were released to the pedicel apoplast below the nucellus of the florets. Glucose or carboxyfluorescein were detected and could be washed out. For carboxyfluorescein, the plasma membranes of the phloem parenchyma appeared to control the release. After release, the nucellus absorbed apoplast glucose selectively, rejecting carboxyfluorescein.
Conclusions
Despite the absence of an embryo, the apoplast below the nucellus was a depot for phloem contents, and the strictly symplast path is rejected. Because glucose and carboxyfluorescein were released non-selectively, the path to the floret resembled the one later when an embryo is present. The non-selective release indicates that turgor at phloem termini cannot balance the full osmotic potential of the phloem contents and would create a downward pressure gradient driving bulk flow toward the sink. Such a gradient was previously measured by Fisher and Cash-Clark in wheat. At the same time, selective absorption from the apoplast by the nucellar membranes would support full turgor in this tissue, isolating the embryo sac from the maternal plant. The isolation should continue later when an embryo develops.
doi:10.1093/aob/mct012
PMCID: PMC3605948  PMID: 23388879
Apoplast; carboxyfluorescein; floret; glucose; membrane transport; phloem; reflection coefficient; sucrose; symplast; turgor pressure; Zea mays
6.  Breeding systems, hybridization and continuing evolution in Avon Gorge Sorbus 
Annals of Botany  2013;111(4):563-575.
Background and Aims
Interspecific hybridization and polyploidy are key processes in plant evolution and are responsible for ongoing genetic diversification in the genus Sorbus (Rosaceae). The Avon Gorge, Bristol, UK, is a world ‘hotspot’ for Sorbus diversity and home to diploid sexual species and polyploid apomictic species. This research investigated how mating system variation, hybridization and polyploidy interact to generate this biological diversity.
Methods
Mating systems of diploid, triploid and tetraploid Sorbus taxa were analysed using pollen tube growth and seed set assays from controlled pollinations, and parent–offspring genotyping of progeny from open and manual pollinations.
Key Results
Diploid Sorbus are outcrossing and self-incompatible (SI). Triploid taxa are pseudogamous apomicts and genetically invariable, but because they also display self-incompatibility, apomictic seed set requires pollen from other Sorbus taxa – a phenomenon which offers direct opportunities for hybridization. In contrast tetraploid taxa are pseudogamous but self-compatible, so do not have the same obligate requirement for intertaxon pollination.
Conclusions
The mating inter-relationships among Avon Gorge Sorbus taxa are complex and are the driving force for hybridization and ongoing genetic diversification. In particular, the presence of self-incompatibility in triploid pseudogamous apomicts imposes a requirement for interspecific cross-pollination, thereby facilitating continuing diversification and evolution through rare sexual hybridization events. This is the first report of naturally occurring pseudogamous apomictic SI plant populations, and we suggest that interspecific pollination, in combination with a relaxed endosperm balance requirement, is the most likely route to the persistence of these populations. We propose that Avon Gorge Sorbus represents a model system for studying the establishment and persistence of SI apomicts in natural populations.
doi:10.1093/aob/mct013
PMCID: PMC3605949  PMID: 23408832
Hybridization; evolution; polyploidy; apomixis; pseudogamy; self-incompatibility; Sorbus
7.  Visualization of embolism formation in the xylem of liana stems using neutron radiography 
Annals of Botany  2013;111(4):723-730.
Background and Aims
Cold neutron radiography was applied to directly observe embolism in conduits of liana stems with the aim to evaluate the suitability of this method for studying embolism formation and repair. Potential advantages of this method are a principally non-invasive imaging approach with low energy dose compared with synchrotron X-ray radiation, a good spatial and temporal resolution, and the possibility to observe the entire volume of stem portions with a length of several centimetres at one time.
Methods
Complete and cut stems of Adenia lobata, Aristolochia macrophylla and Parthenocissus tricuspidata were radiographed at the neutron imaging facility CONRAD at the Helmholtz-Zentrum Berlin für Materialien und Energie, with each measurement cycle lasting several hours. Low attenuation gas spaces were separated from the high attenuation (water-containing) plant tissue using image processing.
Key results
Severe cuts into the stem were necessary to induce embolism. The formation and temporal course of an embolism event could then be successfully observed in individual conduits. It was found that complete emptying of a vessel with a diameter of 100 µm required a time interval of 4 min. Furthermore, dehydration of the whole stem section could be monitored via decreasing attenuation of the neutrons.
Conclusions
The results suggest that cold neutron radiography represents a useful tool for studying water relations in plant stems that has the potential to complement other non-invasive methods.
doi:10.1093/aob/mct014
PMCID: PMC3605950  PMID: 23393096
Xylem; plant water transport; embolism; neutron radiography; Adenia lobata; Aristolochia macrophylla; Parthenocissus tricuspidata.
8.  Stand variation in Pinus radiata and its relationship with allometric scaling and critical buckling height 
Annals of Botany  2013;111(4):675-680.
Background and Aims
Allometric relationships and the determination of critical buckling heights have been examined for Pinus radiata in the past. However, how they relate to more mature Pinus radiata exhibiting a wide range of stem diameters, slenderness and modulus of elasticity (E) at operationally used stand densities is largely unknown. The aim of this study was to examine the relationship between Pinus radiata stand structure variables and allometric scaling and critical buckling height.
Methods
Utilizing a Pinus radiata Nelder trial with stand density and genetic breed as variables, critical buckling height was calculated whilst reduced major axis regression was used to determine scaling exponents between critical height (Hcrit), actual height (H), ground line diameter (D), slenderness (S), density-specific stiffness (E/ρ) and modulus of elasticity (E).
Key Results
Critical buckling height was highly responsive to decreasing diameter and increasing slenderness. Safety factors in this study were typically considerably lower than previously reported margins in other species. As density-specific stiffness scaled negatively with diameter, the exponent of 0·55 between critical height and diameter did not meet the assumed value of 0·67 under constant density-specific stiffness. E scaled positively with stem slenderness to the power of 0·78.
Conclusions
The findings suggest that within species density-specific stiffness variation may influence critical height and the scaling exponent between critical height and diameter, which is considered so important in assumptions regarding allometric relationships.
doi:10.1093/aob/mct015
PMCID: PMC3605951  PMID: 23388878
Allometric scaling; critical buckling height; Euler buckling; modulus of elasticity; Nelder; Pinus radiata; safety factor; stem slenderness
9.  Significant involvement of PEP-CK in carbon assimilation of C4 eudicots 
Annals of Botany  2013;111(4):577-589.
Background and Aims
C4 eudicot species are classified into biochemical sub-types of C4 photosynthesis based on the principal decarboxylating enzyme. Two sub-types are known, NADP-malic enzyme (ME) and NAD-ME; however, evidence for the occurrence or involvement of the third sub-type (phosphoenolpyruvate carboxykinase; PEP-CK) is emerging. In this study, the presence and activity of PEP-CK in C4 eudicot species of Trianthema and Zaleya (Sesuvioideae, Aizoaceae) is clarified through analysis of key anatomical features and C4 photosynthetic enzymes.
Methods
Three C4 species (T. portulacastrum, T. sheilae and Z. pentandra) were examined with light and transmission electron microscopy for leaf structural properties. Activities and immunolocalizations of C4 enzymes were measured for biochemical characteristics.
Key Results
Leaves of each species possess atriplicoid-type Kranz anatomy, but differ in ultrastructural features. Bundle sheath organelles are centripetal in T. portulacastrum and Z. pentandra, and centrifugal in T. sheilae. Bundle sheath chloroplasts in T. portulacastrum are almost agranal, whereas mesophyll counterparts have grana. Both T. sheilae and Z. pentandra are similar, where bundle sheath chloroplasts contain well-developed grana while mesophyll chloroplasts are grana deficient. Cell wall thickness is significantly greater in T. sheilae than in the other species. Biochemically, T. portulacastrum is NADP-ME, while T. sheilae and Z. pentandra are NAD-ME. Both T. portulacastrum and Z. pentandra exhibit considerable PEP-CK activity, and immunolocalization studies show dense and specific compartmentation of PEP-CK in these species, consistent with high PEP-CK enzyme activity.
Conclusions
Involvement of PEP-CK in C4 NADP-ME T. portulacastrum and NAD-ME Z. petandra occurs irrespective of biochemical sub-type, or the position of bundle sheath chloroplasts. Ultrastructural traits, including numbers of bundle sheath peroxisomes and mesophyll chloroplasts, and degree of grana development in bundle sheath chloroplasts, coincide more directly with PEP-CK recruitment. Discovery of high PEP-CK activity in C4 Sesuvioideae species offers a unique opportunity for evaluating PEP-CK expression and suggests the possibility that PEP-CK recruitment may exist elsewhere in C4 eudicots.
doi:10.1093/aob/mct017
PMCID: PMC3605952  PMID: 23388881
Aizoaceae; C4 photosynthesis; granal development; Kranz anatomy; NADP-ME sub-type; NAD-ME sub-type; PEP-CK sub-type; Sesuvioideae; thylakoid; Trianthema portulacastrum; Trianthema sheilae; Zaleya pentandra
10.  A new insight into arable weed adaptive evolution: mutations endowing herbicide resistance also affect germination dynamics and seedling emergence 
Annals of Botany  2013;111(4):681-691.
Background and Aims
Selective pressures exerted by agriculture on populations of arable weeds foster the evolution of adaptive traits. Germination and emergence dynamics and herbicide resistance are key adaptive traits. Herbicide resistance alleles can have pleiotropic effects on a weed's life cycle. This study investigated the pleiotropic effects of three acetyl-coenzyme A carboxylase (ACCase) alleles endowing herbicide resistance on the seed-to-plant part of the life cycle of the grass weed Alopecurus myosuroides.
Methods
In each of two series of experiments, A. myosuroides populations with homogenized genetic backgrounds and segregating for Leu1781, Asn2041 or Gly2078 ACCase mutations which arose independently were used to compare germination dynamics, survival in the soil and seedling pre-emergence growth among seeds containing wild-type, heterozygous and homozygous mutant ACCase embryos.
Key Results
Asn2041 ACCase caused no significant effects. Gly2078 ACCase major effects were a co-dominant acceleration in seed germination (1·25- and 1·10-fold decrease in the time to reach 50 % germination (T50) for homozygous and heterozygous mutant embryos, respectively). Segregation distortion against homozygous mutant embryos or a co-dominant increase in fatal germination was observed in one series of experiments. Leu1781 ACCase major effects were a co-dominant delay in seed germination (1·41- and 1·22-fold increase in T50 for homozygous and heterozygous mutant embryos, respectively) associated with a substantial co-dominant decrease in fatal germination.
Conclusions
Under current agricultural systems, plants carrying Leu1781 or Gly2078 ACCase have a fitness advantage conferred by herbicide resistance that is enhanced or counterbalanced, respectively, by direct pleiotropic effects on the plant phenology. Pleiotropic effects associated with mutations endowing herbicide resistance undoubtedly play a significant role in the evolutionary dynamics of herbicide resistance in weed populations. Mutant ACCase alleles should also prove useful to investigate the role played by seed storage lipids in the control of seed dormancy and germination.
doi:10.1093/aob/mct018
PMCID: PMC3605953  PMID: 23393095
ACCase (acetyl-coenzyme A carboxylase); adaptation; agricultural ecosystem; Alopecurus myosuroides; fitness; grass weed; herbicide resistance; lipid biosynthesis; pleiotropic effect; seed dormancy; survival analysis
11.  Patterns of plastid and nuclear variation among apomictic polyploids of Hieracium: evolutionary processes and taxonomic implications 
Annals of Botany  2013;111(4):591-609.
Background and Aims
Apomictic species (with asexual seed production) make up for 20–50 % of all taxonomically recognized species in northern Europe, but the phylogenetic relationships of apomictic species and the mode of evolution and speciation remain largely unknown and their taxonomy is consequently disputed.
Methods
In the present study, plastid psbD-trnT sequences (349 accessions) and 12 nuclear microsatellite loci (478 accessions) were used to create an overview of the molecular variation in (mainly) northern European members of the most species-rich of all plant genera, Hieracium s.s. The results are discussed and interpreted in the context of morphological and cytological data on the same species.
Key Results and Conclusions
The complete psbD-trnT alignment was 1243 bp and 50 polymorphisms defined 40 haplotypes. All haplotypes found in the sections of the genus distributed in the northern European lowlands fell into one of two main groups, group H and group V, mutually separated by seven or eight polymorphisms. All accessions belonging to H. sects. Foliosa, Hieracioides (viz. H. umbellatum) and Tridentata and all but one accession of triploid species of H. sects. Oradea and Vulgata showed haplotypes of group V. Haplotypes of group H were found in all accessions of H. sects. Bifida and Hieracium and in all tetraploid representatives of H. sects. Oreadea and Vulgata. Additional haplotypes were found in accessions of the genus Pilosella and in southern European and Alpine sections of Hieracium. In contrast, the distribution of individual haplotypes in the two major groups appeared uncorrelated with morphology and current taxonomy, but polymorphisms within species were only rarely encountered. In total, 160 microsatellite alleles were identified. Levels of variation were generally high with only nine pairs of accessions being identical at all loci (in all cases representing accessions of the same species). In the neighbor-joining analysis based on the microsatellite data, accessions of the same species generally clustered together and some smaller groups of species congruent with morphology and/or current taxonomy were recovered but, except for H. sect. Oreadea, most larger groups were not correlated with morphology. Although the plastid DNA sequences show too little variation and the nuclear microsatellites are too variable to resolve relationships successfully among species or to fully understand processes of evolution, it is concluded that both species and sections as defined by morphology are largely congruent with the molecular data, that gene flow between the sections is rare or non-existent and that the tetraploid species may constitute the key to understanding evolution and speciation in this genus.
doi:10.1093/aob/mct019
PMCID: PMC3605954  PMID: 23393094
Apomixis; polyploidy; phylogeny; speciation; taxonomy; chloroplast haplotypes; microsatellites; Hieracium; Hieracium sect. Hieracium; Hieracium sect. Vulgata; Hieracium sect. Bifida; Hieracium sect. Oreadea
12.  Extensive contemporary pollen-mediated gene flow in two herb species, Ranunculus bulbosus and Trifolium montanum, along an altitudinal gradient in a meadow landscape 
Annals of Botany  2013;111(4):611-621.
Background and Aims
Genetic connectivity between plant populations allows for exchange and dispersal of adaptive genes, which can facilitate plant population persistence particularly in rapidly changing environments.
Methods
Patterns of historic gene flow, flowering phenology and contemporary pollen flow were investigated in two common herbs, Ranunculus bulbosus and Trifolium montanum, along an altitudinal gradient of 1200–1800 m a.s.l. over a distance of 1 km among five alpine meadows in Switzerland.
Key Results
Historic gene flow was extensive, as revealed by Fst values of 0·01 and 0·007 in R. bulbosus and T. montanum, respectively, by similar levels of allelic richness among meadows and by the grouping of all individuals into one genetic cluster. Our data suggest contemporary pollen flow is not limited across altitudes in either species but is more pronounced in T. montanum, as indicated by the differential decay of among-sibships correlated paternity with increasing spatial distance. Flowering phenology among meadows was not a barrier to pollen flow in T. montanum, as the large overlap between meadow pairs was consistent with the extensive pollen flow. The smaller flowering overlap among R. bulbosus meadows might explain the slightly more limited pollen flow detected.
Conclusions
High levels of pollen flow among altitudes in both R. bulbosus and T. montanum should facilitate exchange of genes which may enhance adaptive responses to rapid climate change.
doi:10.1093/aob/mct021
PMCID: PMC3605955  PMID: 23408831
Elevation; flowering phenology; gene flow; herbs; managed meadows; microsatellites; pollen flow; pollen-pool analysis; Ranunculus bulbosus; Trifolium montanum
13.  Chromosome evolution in marginal populations of Aegilops speltoides: causes and consequences 
Annals of Botany  2013;111(4):531-538.
Background
Genome restructuring is an ongoing process in natural plant populations. The influence of environmental changes on the genome is crucial, especially during periods of extreme climatic fluctuations. Interactions between the environment and the organism manifest to the greatest extent at the limits of the species' ecological niche. Thus, marginal populations are expected to exhibit lower genetic diversity and higher genetic differentiation than central populations, and some models assume that marginal populations play an important role in the maintenance and generation of biological diversity.
Scope
In this review, long-term data on the cytogenetic characteristics of diploid Aegilops speltoides Tauch populations are summarized and discussed. This species is distributed in and around the Fertile Crescent and is proposed to be the wild progenitor of a number of diploid and polyploid wheat species. In marginal populations of Ae. speltoides, numerical chromosomal aberrations, spontaneous aneuploidy, B-chromosomes, rDNA cluster repatterning and reduction in the species-specific and tribe-specific tandem repeats have been detected. Significant changes were observed and occurred in parallel with changes in plant morphology and physiology.
Conclusions
Considerable genomic variation at the chromosomal level was found in the marginal populations of Ae. speltoides. It is likely that a specific combination of gene mutations and chromosomal repatterning has produced the evolutionary trend in each specific case, i.e. for a particular species or group of related species in a given period of time and in a certain habitat. The appearance of a new chromosomal pattern is considered an important factor in promoting the emergence of interbreeding barriers.
doi:10.1093/aob/mct023
PMCID: PMC3605956  PMID: 23393097
Aegilops speltoides; wheat; marginal populations; chromosomes; evolution; speciation
14.  The impact of extensive clonal growth on fine-scale mating patterns: a full paternity analysis of a lily-of-the-valley population (Convallaria majalis) 
Annals of Botany  2013;111(4):623-628.
Background and Aims
The combination of clonality and a mating system promoting outcrossing is considered advantageous because outcrossing avoids the fitness costs of selfing within clones (geitonogamy) while clonality assures local persistence and increases floral display. The spatial spread of genetically identical plants (ramets) may, however, also decrease paternal diversity (the number of sires fertilizing a given dam) and fertility, particularly towards the centre of large clumped clones. This study aimed to quantify the impact of extensive clonal growth on fine-scale paternity patterns in a population of the allogamous Convallaria majalis.
Methods
A full analysis of paternity was performed by genotyping all flowering individuals and all viable seeds produced during a single season using AFLP. Mating patterns were examined and the spatial position of ramets was related to the extent of multiple paternity, fruiting success and seed production.
Key Results
The overall outcrossing rate was high (91 %) and pollen flow into the population was considerable (27 %). Despite extensive clonal growth, multiple paternity was relatively common (the fraction of siblings sharing the same father was 0·53 within ramets). The diversity of offspring collected from reproductive ramets surrounded by genetically identical inflorescences was as high as among offspring collected from ramets surrounded by distinct genets. There was no significant relationship between the similarity of the pollen load received by two ramets and the distance between them. Neither the distance of ramets with respect to distinct genets nor the distance to the genet centre significantly affected fruiting success or seed production.
Conclusions
Random mating and considerable pollen inflow most probably implied that pollen dispersal distances were sufficiently high to mitigate local mate scarcity despite extensive clonal spread. The data provide no evidence for the intrusion of clonal growth on fine-scale plant mating patterns.
doi:10.1093/aob/mct024
PMCID: PMC3605957  PMID: 23439847
Paternity analysis; mate diversity; outcrossing; mating opportunities; AFLP; reproductive success; seed set; ramet; lily-of-the-valley; Convallaria majalis
15.  Ecological significance of seed desiccation sensitivity in Quercus ilex 
Annals of Botany  2013;111(4):693-701.
Background and Aims
Several widespread tree species of temperate forests, such as species of the genus Quercus, produce recalcitrant (desiccation-sensitive) seeds. However, the ecological significance of seed desiccation sensitivity in temperate regions is largely unknown. Do seeds of such species suffer from drying during the period when they remain on the soil, between shedding in autumn and the return of conditions required for germination in spring?
Methods
To test this hypothesis, the Mediterranean holm oak (Quercus ilex) forest was used as a model system. The relationships between the climate in winter, the characteristics of microhabitats, acorn morphological traits, and the water status and viability of seeds after winter were then investigated in 42 woodlands sampled over the entire French distribution of the species.
Key Results
The percentages of germination and normal seedling development were tightly linked to the water content of seeds after the winter period, revealing that in situ desiccation is a major cause of mortality. The homogeneity of seed response to drying suggests that neither intraspecific genetic variation nor environmental conditions had a significant impact on the level of desiccation sensitivity of seeds. In contrast, the water and viability status of seeds at the time of collection were dramatically influenced by cumulative rainfall and maximum temperatures during winter. A significant effect of shade and of the type of soil cover was also evidenced.
Conclusions
The findings establish that seed desiccation sensitivity is a key functional trait which may influence the success of recruitment in temperate recalcitrant seed species. Considering that most models of climate change predict changes in rainfall and temperature in the Mediterranean basin, the present work could help foresee changes in the distribution of Q. ilex and other oak species, and hence plant community alterations.
doi:10.1093/aob/mct025
PMCID: PMC3605958  PMID: 23388882
Quercus ilex; acorn; desiccation sensitivity; holm oak; drought; ecological filtering; germination; Mediterranean climate; recalcitrance; winter rainfall
16.  Linking ascorbic acid production in Ribes nigrum with fruit development and changes in sources and sinks 
Annals of Botany  2013;111(4):703-712.
Background and Aims
Understanding the synthesis of ascorbic acid (l-AsA) in green tissues in model species has advanced considerably; here we focus on its production and accumulation in fruit. In particular, our aim is to understand the links between organs which may be sources of l-AsA (leaves) and those which accumulate it (fruits). The work presented here tests the idea that changes in leaf and fruit number influence the accumulation of l-AsA. The aim was to understand the importance of leaf tissue in the production of l-AsA and to determine how this might provide routes for the manipulation of fruit tissue l-AsA.
Methods
The experiments used Ribes nigrum (blackcurrant), predominantly in field experiments, where the source–sink relationship was manipulated to alter potential leaf l-AsA production and fruit growth and accumulation of l-AsA. These manipulations included reductions in reproductive capacity, by raceme removal, and the availability of assimilates by leaf removal and branch phloem girdling. Natural variation in fruit growth and fruit abscission is also described as this influences subsequent experimental design and the interpretation of l-AsA data.
Key Results
Results show that fruit l-AsA concentration is conserved but total yield of l-AsA per plant is dependent on a number of innate factors many of which relate to raceme attributes. Leaf removal and phloem girdling reduced fruit weight, and a combination of both reduced fruit yields further. It appears that around 50 % of assimilates utilized for fruit growth came from apical leaves, while between 20 and 30 % came from raceme leaves, with the remainder from ‘storage’.
Conclusions
Despite being able to manipulate leaf area and therefore assimilate availability and stored carbohydrates, along with fruit yields, rarely were effects on fruit l-AsA concentration seen, indicating fruit l-AsA production in Ribes was not directly coupled to assimilate supply. There was no supporting evidence that l-AsA production occurred predominantly in green leaf tissue followed by its transfer to developing fruits. It is concluded that l-AsA production occurs predominantly in the fruit of Ribes nigrum.
doi:10.1093/aob/mct026
PMCID: PMC3605959  PMID: 23419248
l-Ascorbic acid; blackcurrant; fruit; Ribes nigrum; source sinks; vitamin C
17.  Modelling temperature, photoperiod and vernalization responses of Brunonia australis (Goodeniaceae) and Calandrinia sp. (Portulacaceae) to predict flowering time 
Annals of Botany  2013;111(4):629-639.
Background and Aims
Crop models for herbaceous ornamental species typically include functions for temperature and photoperiod responses, but very few incorporate vernalization, which is a requirement of many traditional crops. This study investigated the development of floriculture crop models, which describe temperature responses, plus photoperiod or vernalization requirements, using Australian native ephemerals Brunonia australis and Calandrinia sp.
Methods
A novel approach involved the use of a field crop modelling tool, DEVEL2. This optimization program estimates the parameters of selected functions within the development rate models using an iterative process that minimizes sum of squares residual between estimated and observed days for the phenological event. Parameter profiling and jack-knifing are included in DEVEL2 to remove bias from parameter estimates and introduce rigour into the parameter selection process.
Key Results
Development rate of B. australis from planting to first visible floral bud (VFB) was predicted using a multiplicative approach with a curvilinear function to describe temperature responses and a broken linear function to explain photoperiod responses. A similar model was used to describe the development rate of Calandrinia sp., except the photoperiod function was replaced with an exponential vernalization function, which explained a facultative cold requirement and included a coefficient for determining the vernalization ceiling temperature. Temperature was the main environmental factor influencing development rate for VFB to anthesis of both species and was predicted using a linear model.
Conclusions
The phenology models for B. australis and Calandrinia sp. described development rate from planting to VFB and from VFB to anthesis in response to temperature and photoperiod or vernalization and may assist modelling efforts of other herbaceous ornamental plants. In addition to crop management, the vernalization function could be used to identify plant communities most at risk from predicted increases in temperature due to global warming.
doi:10.1093/aob/mct028
PMCID: PMC3605960  PMID: 23404991
Brunonia australis; Calandrinia sp.; modelling; flowering; phenology; temperature; photoperiod; vernalization
18.  Morphological and molecular identification to secure cultivar maintenance and management of self-sterile Rubus arcticus 
Annals of Botany  2013;111(4):713-721.
Background and Aims
Preservation of cultivar purity creates a particular challenge for plants that are self-incompatible, require insects for cross-pollination, and have easily germinating seeds and vigorously spreading rhizomes. As the fields must be planted with mixed populations, and a balance must be maintained between the cultivars to achieve effective pollination, methods for field monitoring of the relative density of different cultivars must be practical. Furthermore, a DNA-based method is needed for cultivar verification in the collections and outside of the growing season. The aim of this study was to develop both types of methods for Rubus arcticus (arctic bramble).
Methods
Morphological parameters were measured from six cultivars grown on three farms. Observations from the flowers and fruits included: petal and sepal number, flower diameter, arrangement of petals, size of calyx in relation to corolla, fruit weight, yield and soluble sugars. Observations from the leaves included: width and height of middle leaflet, shape of the base of terminal leaflet, shape of terminal leaflet, leaf margin serration and fingertip touch. The applicability of simple sequence repeat (SSR) or microsatellite DNA markers developed for red raspberry was tested on eight arctic bramble cultivars.
Key Results and Conclusions
Morphological and molecular identification methods were developed for R. arcticus. The best morphological characteristics were the length-to-width ratio of the middle leaflet and leaf margin serration. A particular characteristic, fingertip touch, was shown by electron microscopy to be related to the density and quality of the leaf hairs. Red raspberry SSR marker no. 126 proved to be applicable for differentiation of the eight arctic bramble cultivars tested. These identification methods are critical to secure the maintenance and management of R. arcticus. However, the challenges faced and approaches taken are equally applicable to other species with similar biology.
doi:10.1093/aob/mct029
PMCID: PMC3605961  PMID: 23456688
Arctic bramble; authenticity; cultivar purity; identification; morphological markers; Rubus arcticus ssp. arcticus; self-incompatibility; SSR markers; UPOV; preservation; cultivar maintenance; genetic resources conservation
19.  High ploidy diversity and distinct patterns of cytotype distribution in a widespread species of Oxalis in the Greater Cape Floristic Region 
Annals of Botany  2013;111(4):641-649.
Background and Aims
Genome duplication is widely acknowledged as a major force in the evolution of angiosperms, although the incidence of polyploidy in different floras may differ dramatically. The Greater Cape Floristic Region of southern Africa is one of the world's biodiversity hotspots and is considered depauperate in polyploids. To test this assumption, ploidy variation was assessed in a widespread member of the largest geophytic genus in the Cape flora: Oxalis obtusa.
Methods
DNA flow cytometry complemented by confirmatory chromosome counts was used to determine ploidy levels in 355 populations of O. obtusa (1014 individuals) across its entire distribution range. Ecological differentiation among cytotypes was tested by comparing sets of vegetation and climatic variables extracted for each locality.
Key Results
Three majority (2x, 4x, 6x) and three minority (3x, 5x, 8x) cytotypes were detected in situ, in addition to a heptaploid individual originating from a botanical garden. While single-cytotype populations predominate, 12 mixed-ploidy populations were also found. The overall pattern of ploidy level distribution is quite complex, but some ecological segregation was observed. Hexaploids are the most common cytotype and prevail in the Fynbos biome. In contrast, tetraploids dominate in the Succulent Karoo biome. Precipitation parameters were identified as the most important climatic variables associated with cytotype distribution.
Conclusions
Although it would be premature to make generalizations regarding the role of genome duplication in the genesis of hyperdiversity of the Cape flora, the substantial and unexpected ploidy diversity in Oxalis obtusa is unparalleled in comparison with any other cytologically known native Cape plant species. The results suggest that ploidy variation in the Greater Cape Floristic Region may be much greater than currently assumed, which, given the documented role of polyploidy in speciation, has direct implications for radiation hypotheses in this biodiversity hotspot.
doi:10.1093/aob/mct030
PMCID: PMC3605962  PMID: 23425783
Cape Floristic Region; cytogeography; flow cytometry; Fynbos; Oxalis obtusa; polyploidy; Succulent Karoo; vegetation
20.  Differences in dichogamy and herkogamy contribute to higher selfing in contrasting environments in the annual Blackstonia perfoliata (Gentianaceae) 
Annals of Botany  2013;111(4):651-661.
Background and Aims
The establishment of plant populations in novel environments may generate pronounced shifts in floral traits and plant mating systems, particularly when pollinators are scarce. In this study, floral morphology and mating system functioning are compared between recently established and older populations of the annual plant Blackstonia perfoliata that occur in different pollinator environments.
Methods
Hand-pollination and emasculation experiments were conducted to assess the extent of pollinator-mediated pollen deposition and pollen limitation, and the contribution of autonomous selfing to total seed production. Detailed measurements of key floral traits were performed to compare the flower morphology and mating system functioning between plants from both pollination environments.
Key Results
Pollinator-mediated pollen deposition was about twice as low in the recently colonized and pollinator-poor environment compared with the old and pollinator-rich sites, but total seed set was little affected by any type of pollen limitation. The contribution of autonomous selfing to total seed production was higher in the pollinator-poor sites than in the pollinator-rich sites (index of reproductive assurance = 0·56 and 0·17, respectively), and seed production was only poorly affected by selfing, whereas in the pollinator-rich populations selfing reduced total reproductive output by about 40 % compared with outcross pollination. Plants originating from pollinator-poor environments produced smaller flowers that showed significantly lower levels of dichogamy (i.e. protogyny) and herkogamy. These reductions resulted in a 2-fold higher capacity for autonomous selfing under pollinator-free conditions (index of autonomous selfing = 0·81 and 0·41 in plants originating from the pollinator-poor and pollinator-rich environment, respectively).
Conclusions
The results illustrate that plant populations colonizing novel environments can differ markedly in floral morphology and mating system functioning. Due to a temporal shift in the male phase, the breeding system of B. perfoliata shifted from delayed selfing under pollinator-rich conditions towards competing selfing in recently established populations, providing greater reproductive assurance when pollinators and/or reproductive partners are limited.
doi:10.1093/aob/mct031
PMCID: PMC3605963  PMID: 23408833
Autogamy; competing selfing; delayed selfing; dichogamy; herkogamy; pollen limitation; pollen quality; reproductive assurance; Blackstonia perfoliata; Gentianaceae
21.  Plant Cuttings 
Annals of Botany  2013;111(4):iv-vi.
doi:10.1093/aob/mct066
PMCID: PMC3605965
22.  ContentSnapshots 
Annals of Botany  2013;111(4):i-iv.
doi:10.1093/aob/mct068
PMCID: PMC3605966
23.  The trafficking pathway of a wheat storage protein in transgenic rice endosperm 
Annals of Botany  2014;113(5):807-815.
Background and Aims
The trafficking of proteins in the endoplasmic reticulum (ER) of plant cells is a topic of considerable interest since this organelle serves as an entry point for proteins destined for other organelles, as well as for the ER itself. In the current work, transgenic rice was used to study the pattern and pathway of deposition of the wheat high molecular weight (HMW) glutenin sub-unit (GS) 1Dx5 within the rice endosperm using specific antibodies to determine whether it is deposited in the same or different protein bodies from the rice storage proteins, and whether it is located in the same or separate phases within these.
Methods
The protein distribution and the expression pattern of HMW sub-unit 1Dx5 in transgenic rice endosperm at different stages of development were determined using light and electron microscopy after labelling with antibodies.
Key results
The use of HMW-GS-specific antibodies showed that sub-unit 1Dx5 was expressed mainly in the sub-aleurone cells of the endosperm and that it was deposited in both types of protein body present in the rice endosperm: derived from the ER and containing prolamins, and derived from the vacuole and containing glutelins. In addition, new types of protein bodies were also formed within the endosperm cells.
Conclusions
The results suggest that the HMW 1Dx5 protein could be trafficked by either the ER or vacuolar pathway, possibly depending on the stage of development, and that its accumulation in the rice endosperm could compromise the structural integrity of protein bodies and their segregation into two distinct populations in the mature endosperm.
doi:10.1093/aob/mcu008
PMCID: PMC3962248  PMID: 24603605
HMW-GS; immunolocalization; protein trafficking; protein bodies; cereal endosperm; transgenic rice; Oryza sativa; wheat; Triticum aestivum
24.  Congratulations, you have been carefully chosen to represent an important developmental regulator! 
Annals of Botany  2012;111(3):329-333.
Background
Studying a process in a new species often relies on focusing our attention to a candidate gene, encoding a protein similar to one with a known function. Not all the choices seem to be prudent.
Scope
This Viewpoint includes an overview of issues that are encountered during research of candidate genes. Defining a match for a gene of interest, deciding whether variation in ESTs or RNAseq data for a certain transcript, represent more than one gene. The problem of incorrect annotation of genes due to incorrect in-silico splicing, is also mentioned. The author's humble opinion on how to deal with these issues is provided.
Conclusions
The vast amount of new sequence data provides us with great possibilities for giant leaps in our understanding. Still, we cannot afford to skip over the tedious steps required to confirm that we are indeed studying the correct gene, and try to be sure that the complex expression pattern we observe is not a composite of several genes.
doi:10.1093/aob/mcs161
PMCID: PMC3579433  PMID: 22811510
Homology; orthology; MADS box; FLOWERING LOCUS T; FLC; candidate gene; citrus; Citrus clementina; apple; Malus domestica
25.  Current methods for detecting ethylene in plants 
Annals of Botany  2012;111(3):347-360.
Background
In view of ethylene's critical developmental and physiological roles the gaseous hormone remains an active research topic for plant biologists. Progress has been made to understand the ethylene biosynthesis pathway and the mechanisms of perception and action. Still numerous questions need to be answered and findings to be validated. Monitoring gas production will very often complete the picture of any ethylene research topic. Therefore the search for suitable ethylene measuring methods for various plant samples either in the field, greenhouses, laboratories or storage facilities is strongly motivated.
Scope
This review presents an update of the current methods for ethylene monitoring in plants. It focuses on the three most-used methods – gas chromatography detection, electrochemical sensing and optical detection – and compares them in terms of sensitivity, selectivity, time response and price. Guidelines are provided for proper selection and application of the described sensor methodologies and some specific applications are illustrated of laser-based detector for monitoring ethylene given off by Arabidopsis thaliana upon various nutritional treatments.
Conclusions
Each method has its advantages and limitations. The choice for the suitable ethylene sensor needs careful consideration and is driven by the requirements for a specific application.
doi:10.1093/aob/mcs259
PMCID: PMC3579434  PMID: 23243188
Ethylene; Arabidopsis thaliana; gas sampling; gas chromatography; electrochemical sensing; laser-based detector

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