Sequencing of Camelina neglecta,a diploid progenitor of the hexaploid oilseed Camelina sativa

Camelina neglecta is a diploid species from the genus Camelina, which includes the versatile oilseed Camelina sativa. These species are closely related to Arabidopsis thaliana and the economically important Brassica crop species, making this genus a useful platform to dissect traits of agronomic importance while providing a tool to study the evolution of polyploids. A highly contiguous chromosome-level genome sequence of C. neglecta with an N50 size of 29.1 Mb was generated utilizing Pacific Biosciences (PacBio, Menlo Park, CA) long-read sequencing followed by chromosome conformation phasing. Comparison of the genome with that of C. sativa shows remarkable coincidence with subgenome 1 of the hexaploid, with only one major chromosomal rearrangement separating the two. Synonymous substitution rate analysis of the predicted 34 061 genes suggested subgenome 1 of C. sativa directly descended from C. neglecta around 1.2 mya. Higher functional divergence of genes in the hexaploid as evidenced by the greater number of unique orthogroups, and differential composition of resistant gene analogs, might suggest an immediate adaptation strategy after genome merger. The absence of genome bias in gene fractionation among the subgenomes of C. sativa in comparison with C. neglecta, and the complete lack of fractionation of meiosis-specific genes attests to the neopolyploid status of C. sativa. The assembled genome will provide a tool to further study genome evolution processes in the Camelina genus and potentially allow for the identification and exploitation of novel variation for Camelina crop improvement.     

Shrinking genomes? Evidence from genome size variation in Crepis (Compositae)

Large-scale surveys of genome size evolution in angiosperms show that the ancestral genome was most likely small, with a tendency towards an increase in DNA content during evolution. Due to polyploidisation and self-replicating DNA elements, angiosperm genomes were considered to have a 'one-way ticket to obesity' (Bennetzen & Kellogg 1997). New findings on how organisms can lose DNA challenged the hypotheses of unidirectional evolution of genome size. The present study is based on the classical work of Babcock (1947a) on karyotype evolution within Crepis and analyses karyotypic diversification within the genus in a phylogenetic context. Genome size of 21 Crepis species was estimated using flow cytometry. Additional data of 17 further species were taken from the literature. Within 30 diploid Crepis species there is a striking trend towards genome contraction. The direction of genome size evolution was analysed by reconstructing ancestral character states on a molecular phylogeny based on ITS sequence data. DNA content is correlated to distributional aspects as well as life form. Genome size is significantly higher in perennials than in annuals. Within sampled species, very small genomes are only present in Mediterranean or European species, whereas their Central and East Asian relatives have larger 1C values.     

Detection of a 17 kb unique sequence (T-DNA) in plant chromosomes by in situ hybridization

An approach is described for the detection of a unique sequence, the T-DNA region of the Agrobacterium rhizogenes root-inducing (Ri) plasmid, in plant chromosomes by in situ hybridization. This sequence was introduced into the Crepis capillaris genome (2n=6) by infecting Crepis stem segments with A. rhizogenes. Roots growing from the infection site contain T-DNA and synthesize mannopine, which can be used as a convenient biochemical marker for T-DNA transformation. Southern analysis of DNA isolated from one transformed Crepis root line verified the presence of a single copy of T-DNA (approximate size 17 kb) per diploid Crepis genome. To localize T-DNA, both DNA and RNA probes, labelled with either tritium or biotin, were hybridized to Crepis chromosomes prepared from transformed root tips by a novel spreading method. Biotinylated probes were visualized using reflection-contrast microscopy. In the hybridization experiments described, T-DNA was detected in one homologue of chromosome 3, where it could be assigned to a paracentromeric position in the neighbourhood of the nucleolar organizing region. These results demonstrate that it is possible to localize unique sequences in plant chromosomes by in situ hybridization.     

Chemical composition of Crepis foetida L. and C. rubra L. volatile constituents and evaluation of the in vitro anti-inflammatory activity of salicylaldehyde rich volatile fraction

The genus Crepis L. (Asteraceae) comprises more than 200 currently recognized species. Several studies have been conducted on non-volatile phytochemicals of Crepis spp., featuring mainly sesquiterpene lactones and phenolic derivatives. Nevertheless, no report has been made on assessing the volatile constituents of the genus. Therefore, the present study is the first report to the chemical composition of the volatile constituents of two odoriferous Crepis spp., namely C. foetida L. and C. rubra L. Flowers and stems with leaves volatiles were analysed separately by gas chromatography coupled with mass spectrometry. In total, 37 volatile compounds were detected. Salicylaldehyde, carvacrol and aliphatic hydrocarbons are the main components of both C. foetida fractions, however C. rubra flowers are characterized by the abundance of β-sitosterol and eicosanoic acid, while the stems-leaves volatiles revealed to be more complex with hydrocarbons as main constituents.In addition, we investigated the salicylaldehyde rich volatile fraction for its in vitro activity on TNF-α induced ICAM-1 expression.     

Chromosome-level genome assembly of an agricultural pest,the rice leaffolder Cnaphalocrocis exigua (Crambidae,Lepidoptera)

The rice leaffolder Cnaphalocrocis exigua (Crambidae, Lepidoptera) is an important agricultural pest that damages rice crops and other members of related grass families. C. exigua exhibits a very similar morphological phenotype and feeding behaviour to C. medinalis, another species of rice leaffolder whose genome was recently reported. However, genomic information for C. exigua remains extremely limited. Here, we used a hybrid strategy combining different sequencing technologies, including Illumina, PacBio, 10× Genomics, and Hi – C scaffolding, to generate a high-quality chromosome-level genome assembly of C. exigua. We initially obtained a 798.8 Mb assembly with a contig N50 size of 2.9 Mb, and the N50 size was subsequently increased to 25.7 Mb using Hi – C technology to anchor 1413 scaffolds to 32 chromosomes. We detected a total of 97.7% Benchmarking Universal Single-Copy Orthologues (BUSCO) in the genome assembly, which was comprised of ~52% repetitive sequence and annotated 14,922 protein-coding genes. Of note, the Z and W sex chromosomes were assembled and identified. A comparative genomic analysis demonstrated that despite the high synteny observed between the two rice leaffolders, the species have distinct genomic features associated with expansion and contraction of gene families and selection pressure. In summary, our chromosome-level genome assembly and comparative genomic analysis of C. exigua provide novel insights into the evolution and ecology of this rice insect pests and offer useful information for pest control.     

Restoring Forbs for Sage Grouse Habitat: Fire, Microsites, and Establishment Methods

Abstract The decline and range reduction of sage grouse populations are primarily due to permanent loss and degradation of sagebrush–grassland habitat. Several studies have shown that sage grouse productivity may be limited by the availability of certain preferred highly nutritious forb species that have also declined within sagebrush ecosystems of the Intermountain West, U.S.A. The purpose of this study was to determine the suitability of three species of forbs for revegetation projects where improving sage grouse habitat is a goal. Species suitability was determined by evaluating the emergence, survival, and reproduction of Crepis modocensis, C. occidentalis, and Astragalus purshii in response to method of establishment (seeding or transplanting), site preparation treatment (burned or unburned), and microsite (mound or interspace) in an Artemisia tridentata ssp. wyomingensis vegetation association in south central Oregon. For seeded plants A. purshii had the lowest emergence (8%) of all three species. Both seeded Crepis species had similar overall emergence (38%). Significantly more Crepis seedlings emerged from shrub mounds in unburned areas (50%) than in any other fire‐by‐microsite treatment (33 to 36%). Approximately 10% more Crepis seedlings survived in mounds compared with interspaces. Nearly twice as many emerging Crepis seedlings survived in the burned areas as opposed to unburned areas (p < 0.01). This resulted in more plant establishment in burned mounds despite higher emergence in unburned mounds. Astragalus purshii seedlings also survived better in burned areas (p = 0.06) but had no differential response to microsite. Fire enhanced survival of both Crepis and A. purshii transplants (p = 0.08 and p = 0.001). We believe additional research is needed to improve A. purshii emergence before it will become an effective plant for restoring sage grouse habitat. Conversely, we conclude that these Crepis species provide a viable revegetation option for improving sage grouse habitat in south central Oregon.     

Nine polymorphic microsatellite markers in Crepis sancta (Asteraceae)

We describe nine new variable di‐ and trinucleotide microsatellites in the plant Crepis sancta, as well as conditions for multiplexing and simultaneous genotyping sets of loci. Their variability was assessed in four populations. Four to 33 alleles were detected per locus; two to 23 alleles per population. Significant deviations from Hardy–Weinberg equilibrium could be attributed either to a mating system involving partial selfing or to null alleles. Genetic differentiation over all loci was significant among the four populations studied. These markers will constitute an efficient tool for investigating dispersal patterns in fragmented landscapes. Cross‐species amplification provided few loci variability in three other Crepis species (C. pulcra, C. foetida and C. rubra).     

Inferring climatic controls of rice stem borers’ spatial distributions using maximum entropy modelling

Accurate assessment of pest potential distributions is needed to identify their establishment risks that play a key role in pest management in agricultural ecosystems. We used a correlative niche modelling method (Maxent) to predict and map the spatial distributions of two important rice stem borers, Chilo suppressalis and Sesamia cretica, in paddy fields of Iran. In total, 195 presence occurrence records (101 records for C. suppressalis and 94 records for S. cretica) were compiled. A set of environmental and topographic variables, with the highest effects on the species distributions and the lowest correlations among themselves, were used. The results showed that mainly the northern parts of Iran were the most suitable areas for C. suppressalis, and north, north‐east and south‐west of Iran as the most suitable areas for S. cretica. Both models performed well, with an area under the receiver operating characteristic curve (AUC) of 0.983 and 0.786 for C. suppressalis and S. cretica, respectively. The Maxent models showed higher accuracy for predicting the distribution of the specialist pest with the small range sizes compared to the generalist species. Assessing the importance of environmental variables, which were derived from the jackknife test, showed the precipitation as the variable with the highest contribution (66%) in explaining the spatial distribution of C. suppressalis compared to the other variables. The distribution of S. cretica was influenced by a set of variables derived from both the precipitation and temperature. The Maxent predictions were useful to map the geographical distributions of the risk for both rice stem borers that is needed to develop effective management strategies.     

Evolution of the African slippery frogs (Anura: Conraua), including the world’s largest living frog

Although Conraua goliath is well known as the largest living frog species, the diversity and evolution of the genus Conraua across sub-Saharan Africa remain poorly understood. We present multilocus phylogenetic analyses of the six currently recognized species that provide insights into divergence times, biogeography, body size evolution and undescribed species. An analysis of divergence times demonstrates that crown-group Conraua arose some time during the latest Oligocene to mid-Miocene followed by divergence into major lineages in the mid-Miocene that may reflect the fragmentation of widespread tropical forests in Africa that began at this time. We find three pairs of sister species, C. crassipes + C. beccarii, C. alleni + C. derooi and C. goliath + C. robusta, each of which diverged during the Miocene. These relationships reject phylogenetic hypotheses based solely on biogeography as the geographically peripheral C. beccarii from north-eastern Africa is nested within western African species and the Central African species do not form a clade. Our species delimitation analyses provide support for undescribed species in C. alleni, C. beccarii and C. derooi, and possibly C. crassipes, suggesting that the current taxonomy substantially underestimates species diversity. There is no clear directional trend of either increasing or decreasing body size in Conraua and the three largest species do not form a clade. With a robust phylogenetic hypothesis in hand, further field-based studies are needed to understand the evolution of morphology and life history in this charismatic African anuran clade.     

End-to-end chromosome attachments in mitotic interphase and their possible significance to meiotic chromosome pairing

Cytological studies on telophase and early prophase in roottip cells of several plant species (Allium cepa, 2n=16; four Crepis species, including Crepis capillaris, 2n=6; Callitriche hermaphroditica, 2n=6; Nigella arvensis, 2n=12; Secale cereale, 2n=14) revealed that chromosome ends are attached two by two forming chains of chromosomes (interphase associations). In these chains homologous chromosomes are presumably located adjacent to each other. In Crepis capillaris it was observed that the two nucleolar chromosomes form a separate ring one end attached to the ring of the four remaining chromosomes and the other end attached to the nucleolus. It is proposed that these end-to-end attachments have significance for chromosome pairing in meiosis. The adjacent location of homologous chromosomes in the interphase associations would facilitate rapid and regular synapsis.     

Inter‐ and intraspecific variation in body‐ and genome size in calanoid copepods from temperate and arctic waters

The tendency of ectotherms to get larger in the cold (Bergmann clines) has potentially great implications for individual performance and food web dynamics. The mechanistic drivers of this trend are not well understood, however. One fundamental question is to which extent variation in body size is attributed to variation in cell size, which again is related to genome size. In this study, we analyzed body and genome size in four species of marine calanoid copepods, Calanus finmarchicus, C. glacialis, C. hyperboreus and Paraeuchaeta norvegica, with populations from both south Norwegian fjords and the High Arctic. The Calanus species showed typical interspecific Bergmann clines, and we assessed whether they also displayed similar intraspecific variations—and if correlation between genome size and body size differed between species. There were considerable inter‐ as well as intraspecific variations in body size and genome size, with the northernmost populations having the largest values of both variables within each species. Positive intraspecific relationships suggest a functional link between body and genome size, although its adaptiveness has not been settled. Impact of additional drivers like phylogeny or specific adaptations, however, was suggested by striking divergences in body size – genome size ratios among species. Thus, C. glacialis and C. hyperboreus, had fairly similar genome size despite very different body size, while P. norvegica, of similar body size as C. hyperboreus, had the largest genome sizes ever recorded from copepods. The inter‐ and intraspecific latitudinal body size clines suggest that climate change may have major impact on body size composition of keystone species in marine planktonic food webs.     

Simple sequence repeats reveal uneven distribution of genetic diversity in chloroplast genomes of <Emphasis Type="Italic">Brassica oleracea</Emphasis> L. and (<Emphasis Type="Italic">n </Emphasis>= 9) wild relatives

Diversity in the chloroplast genome of 171 accessions representing the Brassica ‘C’ (n = 9) genome, including domesticated and wild B. oleracea and nine inter-fertile related wild species, was investigated using six chloroplast SSR (microsatellite) markers. The lack of diversity detected among 105 cultivated and wild accessions of B. oleracea contrasted starkly with that found within its wild relatives. The vast majority of B. oleracea accessions shared a single haplotype, whereas as many as six haplotypes were detected in two wild species, B. villosa Biv. and B. cretica Lam.. The SSRs proved to be highly polymorphic across haplotypes, with calculated genetic diversity values (H) of 0.23–0.87. In total, 23 different haplotypes were detected in C genome species, with an additional five haplotypes detected in B. rapa L. (A genome n = 10) and another in B. nigra L. (B genome, n = 8). The low chloroplast diversity of B. oleracea is not suggestive of multiple domestication events. The predominant B. oleracea haplotype was also common in B. incana Ten. and present in low frequencies in B. villosa, B. macrocarpa Guss, B. rupestris Raf. and B. cretica. The chloroplast SSRs reveal a wealth of diversity within wild Brassica species that will facilitate further evolutionary and phylogeographic studies of this important crop genus. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Island species radiation and karyotypic stasis in <Emphasis Type="Italic">Pachycladon</Emphasis>allopolyploids

Background  

Pachycladon (Brassicaceae, tribe Camelineae) is a monophyletic genus of ten morphologically and ecogeographically differentiated, and presumably allopolyploid species occurring in the South Island of New Zealand and in Tasmania. All Pachycladon species possess ten chromosome pairs (2n = 20). The feasibility of comparative chromosome painting (CCP) in crucifer species allows the origin and genome evolution in this genus to be elucidated. We focus on the origin and genome evolution of Pachycladon as well as on its genomic relationship to other crucifer species, particularly to the allopolyploid Australian Camelineae taxa. As species radiation on islands is usually characterized by chromosomal stasis, i.e. uniformity of chromosome numbers/ploidy levels, the role of major karyotypic reshuffling during the island adaptive and species radiation in Pachycladon is investigated through whole-genome CCP analysis.     

Karyotype constancy and genome size variation in BulgarianCrepis foetida s. l. (Asteraceae)

Ten populations ofCrepis foetida from Bulgaria belonging to the three subspeciesfoetida, rhoeadifolia, andcommutata were analyzed karyologically using haematoxylin staining, Giemsa C-banding, fluorochrome banding, Ag-NOR staining, Feulgen cytophotometry (scanning densitometry and video-based image analysis), and propidium iodide flow cytometry. The quantitatively-evaluated karyotype structure was similar among all populations, with minor variation in a few intercalary sites only and in the amount of NOR-associated heterochromatin (satellites). In contrast to the karyotypic constancy the genome size ofC. foetida subsp.commutata was about 10% lower than those of the other two subspecies, which had similar genome sizes. The genome size measurements using three different methods resulted in highly correlated data. The genome size difference adds some weight to previous taxonomic opinions treatingC. foetida subsp.commutata at species level, asC. commutata. Prof. Dr. Stefan Kozhuharov (4 January 1933–24 August 1997).     

NUCLEOMORPH KARYOTYPE DIVERSITY IN THE FRESHWATER CRYPTOPHYTE GENUS CRYPTOMONAS1

Cryptophytes are unicellular, biflagellate algae with plastids (chloroplasts) derived from the uptake of a red algal endosymbiont. These organisms are unusual in that the nucleus of the engulfed red alga persists in a highly reduced form called a nucleomorph. Nucleomorph genomes are remarkable in their small size (<1,000 kilobase pairs [kbp]) and high degree of compaction (～1 kbp per gene). Here, we investigated the molecular and karyotypic diversity of nucleomorph genomes in members of the genus Cryptomonas. 18S rDNA genes were amplified, sequenced, and analyzed from C. tetrapyrenoidosa Skuja CCAP979/63, C. erosa Ehrenb. emmend. Hoef‐Emden CCAP979/67, Cryptomonas sp. CCAP979/52, C. lundii Hoef‐Emden et Melkonian CCAP979/69, and C. lucens Skuja CCAP979/35 in the context of a large set of publicly available nucleomorph 18S rDNA sequences. Pulsed‐field gel electrophoresis (PFGE) was used to examine the nucleomorph genome karyotype of each of these strains. Individual chromosomes ranged from ～160 to 280 kbp in size, with total genome sizes estimated to be ～600–655 kbp. Unexpectedly, the nucleomorph karyotype of Cryptomonas sp. CCAP979/52 is significantly different from that of C. tetrapyrenoidosa and C. lucens, despite the fact that their 18S rDNA genes are >99% identical to one another. These results suggest that nucleomorph karyotype similarity is not a reliable indicator of evolutionary affinity and provides a starting point for further investigation of the fine‐scale dynamics of nucleomorph genome evolution within members of the genus Cryptomonas.     

Genome size evolution at the speciation level: The cryptic species complex Brachionus plicatilis (Rotifera)

Background  

Studies on genome size variation in animals are rarely done at lower taxonomic levels, e.g., slightly above/below the species level. Yet, such variation might provide important clues on the tempo and mode of genome size evolution. In this study we used the flow-cytometry method to study the evolution of genome size in the rotifer Brachionus plicatilis, a cryptic species complex consisting of at least 14 closely related species.     

Change in nuclear DNA content and pollen size with polyploidisation in the sweet potato (Ipomoea batatas,Convolvulaceae) complex

• Genome size evolution and its relationship with pollen grain size has been investigated in sweet potato (Ipomoea batatas), an economically important crop which is closely related to diploid and tetraploid species, assessing the nuclear DNA content of 22 accessions from five Ipomoea species, ten sweet potato varieties and two outgroup taxa.
• Nuclear DNA amounts were determined using flow cytometry. Pollen grains were studied using scanning and transmission electron microscopy.
• 2C DNA content of hexaploid I. batatas ranged between 3.12–3.29 pg; the mean monoploid genome size being 0.539 pg (527 Mbp), similar to the related diploid accessions. In tetraploid species I. trifida and I. tabascana, 2C DNA content was, respectively, 2.07 and 2.03 pg. In the diploid species closely related to sweet potato e.g. I. ×leucantha, I. tiliacea, I. trifida and I. triloba, 2C DNA content was 1.01–1.12 pg. However, two diploid outgroup species, I. setosa and I. purpurea, were clearly different from the other diploid species, with 2C of 1.47–1.49 pg; they also have larger chromosomes. The I. batatas genome presents 60.0% AT bases.
• DNA content and ploidy level were positively correlated within this complex. In I. batatas and the more closely related species I. trifida, the genome size and ploidy levels were correlated with pollen size. Our results allow us to propose alternative or complementary hypotheses to that currently proposed for the formation of hexaploid Ipomoea batatas.

     

Differences in gene flow in a twofold secondary contact zone of pond turtles in southern Italy (Testudines: Emydidae: Emys orbicularis galloitalica,E. o. hellenica,E. trinacris)

Using virtually range‐wide sampling for three pond turtle taxa (Emys orbicularis galloitalica, E. o. hellenica, E. trinacris), we analyse gene flow across their southern Italian contact zone. Based on population genetic analyses of 15 highly polymorphic microsatellite loci and a mitochondrial marker, we show that the general genetic pattern matches well with the current taxon delimitation. Yet, single individuals with conflicting genetic identity suggest translocation of turtles by humans. In addition, we identify in south‐western France and the vicinity of Rome populations being heavily impacted by introduced turtles. Cline analyses reveal that the major genetic break between E. o. galloitalica and E. o. hellenica corresponds well with the currently accepted intergradation zone in southern Italy. However, introgression is largely unidirectional from E. o. galloitalica into E. o. hellenica. In the distribution range of the latter subspecies, genetic footprints of E. o. galloitalica are evident along most of the Italian east coast. Our results corroborate that E. o. galloitalica was introduced long ago in Corsica and Sardinia and naturalized there. Gene flow between E. orbicularis and E. trinacris is negligible, with the Strait of Messina matching well with the narrow cline centre between the two species. This contrasts with other Mediterranean freshwater turtle species with extensive transoceanic gene flow. Compared to the two subspecies of E. orbicularis, the Sicilian E. trinacris shows an unexpectedly strong population structuring, a finding also of some relevance for conservation. The differences between the two taxon pairs E. orbicularis/E. trinacris and E. o. galloitalica/E. o. hellenica support their current taxonomic classification and make them attractive objects for follow‐up studies to elucidate the underlying mechanisms of speciation by comparing their properties.     

Correlated evolution of LTR retrotransposons and genome size in the genus <Emphasis Type="Italic">eleocharis</Emphasis>

Background  

Transposable elements (TEs) are considered to be an important source of genome size variation and genetic and phenotypic plasticity in eukaryotes. Most of our knowledge about TEs comes from large genomic projects and studies focused on model organisms. However, TE dynamics among related taxa from natural populations and the role of TEs at the species or supra-species level, where genome size and karyotype evolution are modulated in concert with polyploidy and chromosomal rearrangements, remain poorly understood. We focused on the holokinetic genus Eleocharis (Cyperaceae), which displays large variation in genome size and the occurrence of polyploidy and agmatoploidy/symploidy. We analyzed and quantified the long terminal repeat (LTR) retrotransposons Ty1-copia and Ty3-gypsy in relation to changes in both genome size and karyotype in Eleocharis. We also examined how this relationship is reflected in the phylogeny of Eleocharis.