Genome Size of Alexandrium catenella and Gracilariopsis lemaneiformis Estimated by Flow Cytometry

Flow cytometry(FCM) technique has been widely applied to estimating the genome size of various higher plants. However, there is few report about its application in algae. In this study, an optimized procedure of FCM was exploited to estimate the genome size of two eukaryotic algae. For analyzing Alexandrium catenella, an important red tide species, the whole cell instead of isolated nucleus was studied, and chicken erythrocytes were used as an internal reference. The genome size of A. catenella was estimated to be 56.48 ± 4.14 Gb(1C), approximately nineteen times larger than that of human genome. For analyzing Gracilariopsis lemaneiformis, an important economical red alga, the purified nucleus was employed, and Arabidopsis thaliana and Chondrus crispus were used as internal references, respectively. The genome size of Gp. lemaneiformis was 97.35 ± 2.58 Mb(1C) and 112.73 ± 14.00 Mb(1C), respectively, depending on the different internal references. The results of this research will promote the related studies on the genomics and evolution of these two species.

Genome size of 14 species of fireflies (Insecta, Coleoptera, Lampyridae)

Eukaryotic genome size data are important both as the basis for comparative research into genome evolution and as estimators of the cost and difficulty of genome sequencing programs for non-model organisms. In this study, the genome size of 14 species of fireflies （Lampyridae） （two genera in Lampyrinae, three genera in Luciolinae, and one genus in subfamily incertae sedis） were estimated by propidium iodide （PI）-based flow cytometry. The haploid genome sizes of Lampyridae ranged from 0.42 to 1.31 pg, a 3.1-fold span. Genome sizes of the fireflies varied within the tested subfamilies and genera. Lamprigera and Pyrocoelia species had large and small genome sizes, respectively. No correlation was found between genome size and morphological traits such as body length, body width, eye width, and antennal length. Our data provide additional information on genome size estimation of the firefly family Lampyridae. Furthermore, this study will help clarify the cost and difficulty of genome sequencing programs for non-model organisms and will help promote studies on firefly genome evolution.

Genome size evolution of the extant lycophytes and ferns

Ferns and lycophytes have remarkably large genomes.However,little is known about how their genome size evolved in fern lineages.To explore the origins and evolution of chromosome numbers and genome size in ferns,we used flow cytometry to measure the genomes of 240 species(255 samples)of extant ferns and lycophytes comprising 27 families and 72 genera,of which 228 species(242 samples)represent new reports.We analyzed correlations among genome size,spore size,chromosomal features,phylogeny,and habitat type preference within a phylogenetic framework.We also applied ANOVA and multinomial logistic regression analysis to preference of habitat type and genome size.Using the phylogeny,we conducted ancestral character reconstruction for habitat types and tested whether genome size changes simultaneously with shifts in habitat preference.We found that 2 C values had weak phylogenetic signal,whereas the base number of chromosomes(x)had a strong phylogenetic signal.Furthermore,our analyses revealed a positive correlation between genome size and chromosome traits,indicating that the base number of chromosomes(x),chromosome size,and polyploidization may be primary contributors to genome expansion in ferns and lycophytes.Genome sizes in different habitat types varied significantly and were significantly correlated with habitat types;specifically,multinomial logistic regression indicated that species with larger 2 C values were more likely to be epiphytes.Terrestrial habitat is inferred to be ancestral for both extant ferns and lycophytes,whereas transitions to other habitat types occurred as the major clades emerged.Shifts in habitat types appear be followed by periods of genomic stability.Based on these results,we inferred that habitat type changes and multiple whole-genome duplications have contributed to the formation of large genomes of ferns and their allies during their evolutionary history.

The effects of fresh and rapid desiccated tissue on estimates of Ophiopogoneae genome size

Fresh plant material is usually used for genome size estimation by flow cytometry(FCM). Lack of fresh material is cited as one of the main reasons for the dearth of studies on plants from remote locations.Genome sizes in fresh versus desiccated tissue of 16 Ophiopogoneae species and five model plant species were estimated. Our results indicated that desiccated tissue was suitable for genome size estimation; this method enables broader geographic sampling of plants when fresh tissue collection is not feasible. To be useful, after dessication the Ophiopogoneae sample should be green without brown or yellow markings;it should be stored in deep freezer at à80C, and the storage time should be no more than 6 months.

Effects of Life Histories on Genome Size Variation in Squamata

Genome size changes significantly among taxonomic levels,and this variation is often related to the patterns shaped by the phylogeny,life histories and ecological factors.However,there are mixed evidences on the main factors affecting molecular evolution in animals.In this study,we used phylogenetic comparative analysis to investigate the evolutionary rate of genome size and the relationships between genome size and life histories(i.e.,hatchling mass,clutch size,clutches per year,age at sexual maturity,lifespan and body mass)among 199 squamata species.Our results showed that the evolutionary rate of genome size in Lacertilia was significantly faster than Serpentes.Moreover,we also found that larger species showed larger hatchling mass,more clutches per year and clutch size and longer lifespan.However,genome size was negatively associated with clutch size and clutches per year,but not associated with body mass we looked at.The findings suggest that larger species do not possess the evolution of large genomes in squamata.

Ecological selection of bacterial taxa with larger genome sizes in response to polycyclic aromatic hydrocarbons stress

Polycyclic aromatic hydrocarbons(PAHs)are ubiquitous priority pollutants that cause great damage to the natural environment and health.Average genome size in a community is critical for shedding light on microbiome's functional response to pollution stress within an environment.Here,microcosms under different concentrations were performed to evaluate the selection of PAHs stress on the average genome size in a community.We found the distinct communities of significantly larger genome size with the increase of PAHs concentration gradients in soils,and consistent trends were discovered in soils at different latitudes.The abundance of Proteobacteria and Deinococcus-Thermus with relatively larger genomes increased along with PAHs stress and well adapted to polluted environments.In contrast,the abundance of Patescibacteria with a highly streamlined and smaller genome decreased,implying complex interactions between environmental selection and functional fitness resulted in bacteria with larger genomes becoming more abundant.Moreover,we confirmed the increased capacity for horizontal transfer of degrading genes between communities by showing an increased connection number per node positively related to the nid A gene along the concentration gradients in the co-occurrence network.Our findings suggest PAHs tend to select bacterial taxa with larger genome sizes,with significant consequences for community stability and potential biodegradation strategies.

Genome Size Evolution Mediated by Gypsy Retrotransposons in Brassicaceae

The dynamic activity of transposable elements(TEs)contributes to the vast diversity of genome size and architecture among plants.Here,we examined the genomic distribution and transposition activity of long terminal repeat retrotransposons(LTR-RTs)in Arabidopsis thaliana(Ath)and three of its relatives,Arabidopsis lyrata(Aly),Eutrema salsugineum(Esa),and Schrenkiella parvula(Spa),in Brassicaceae.Our analyses revealed the distinct evolutionary dynamics of Gypsy retrotransposons,which reflects the different patterns of genome size changes of the four species over the past million years.The rate of Gypsy transposition in Aly is approximately five times more rapid than that of Ath and Esa,suggesting an expanding Aly genome.Gypsy insertions in Esa are strictly confined to pericentromeric heterochromatin and associated with dramatic centromere expansion.In contrast,Gypsy insertions in Spa have been largely suppressed over the last million years,likely as a result of a combination of an inherent molecular mechanism of preferential DNA removal and purifying selection at Gypsy elements.Additionally,species-specific clades of Gypsy elements shaped the distinct genome architectures of Aly and Esa.

Genome sizes of four important medicinal species in Kadsura by flow cytometry

Objective:Dianjixueteng is a geoherb in Yunnan Province,the source plant of which is Kadsura interior.However,the formation of this geoherb is not clear in genetic mechanism,in which genome size is the first step that should be known on the genomic level.In this study we aimed to estimate the genome sizes of source plants of K.interior and three related herbs K.heteroclita,K.longipedunculata,and K.coccinea by flow cytometry(FCM)and make a comparison.Methods:The genome sizes of K.interior,K.heteroclita,K.longipedunculata and K.coccinea,i.e.,the source plants of Dianjixueteng and its relative medicinal materials,were estimated by FCM.The nuclei of K.interior were isolated using modified LB01 buffer,for the rest species,by the Galbraith’s buffer.Results:The genome sizes of K.interior,K.heteroclita,K.longipedunculata,and K.coccinea were 7.36,7.12,7.01,and 5.15 pg/1 C,respectively.Genome size of K.interior had no significant variation with those of K.heteroclita and K.longipedunculata(P=0.296),which was significantly larger than that of K.coccinea.Conclusion:Genome size can not distinguish K.interior from K.heteroclita and K.longipedunculata,but could distinguish them from K.coccinea,which lays the foundation for future studies on genetic mechanism of the geoherb formation.

Stomatal dynamics are regulated by leaf hydraulic traits and guard cell anatomy in nine true mangrove species

Stomatal regulation is critical for mangroves to survive in the hyper-saline intertidal zone where water stress is severe and water availability is highly fluctuant.However,very little is known about the stomatal sensitivity to vapour pressure deficit(VPD)in mangroves,and its co-ordination with stomatal morphology and leaf hydraulic traits.We measured the stomatal response to a step increase in VPD in situ,stomatal anatomy,leaf hydraulic vulnerability and pressure-volume traits in nine true mangrove species of five families and collected the data of genome size.We aimed to answer two questions:(1)Does stomatal morphology influence stomatal dynamics in response to a high VPD in mangroves?with a consideration of possible influence of genome size on stomatal morphology;and(2)do leaf hydraulic traits influence stomatal sensitivity to VPD in mangroves?We found that the stomata of mangrove plants were highly sensitive to a step rise in VPD and the stomatal responses were directly affected by stomatal anatomy and hydraulic traits.Smaller,denser stomata was correlated with faster stomatal closure at high VPD across the species of Rhizophoraceae,and stomata size negatively and vein density positively correlated with genome size.Less negative leaf osmotic pressure at the full turgor(πo)was related to higher operating steady-state stomatal conductance(gs);and a higher leaf capacitance(Cleaf)and more embolism resistant leaf xylem were associated with slower stomatal responses to an increase in VPD.In addition,stomatal responsiveness to VPD was indirectly affected by leaf morphological traits,which were affected by site salinity and consequently leaf water status.Our results demonstrate that mangroves display a unique relationship between genome size,stomatal size and vein packing,and that stomatal responsiveness to VPD is regulated by leaf hydraulic traits and stomatal morphology.Our work provides a quantitative framework to better understand of stomatal regulation in mangroves in an environment with high salinity and dynamic water availability.

Relaxation of Selective Constraint on the Ultra-Large Mitochondrial Genomes of Arcidae (Mollusca: Bivalvia)

The mitochondrial genomes(mitogenomes)are purportedly under selection for smaller size to improve their replica-tion and translation efficiency.However,the mitogenomes of Arcidae species are larger than those of other bivalves,and are among the largest metazoan mitogenomes reported to date.In order to explore the differences of base composition and selective constraints between the large and small mitogenomes,we compared the mitogenomes of 9 large arcid mitogenomes and 77 small bivalves mitogenomes.Base composition analyses indicated that Arcidae mitogenomes have significantly greater GC skews in both their whole genomes and coding sequences.This result suggests that the replication of the large mitogenomes in Arcidae may be slower than those in other bivalves,exposing the parental strand to deamination for a longer time.Selection pressure analyses showed that the mitochondrial protein-coding genes of Arcidae species have significantly higher Ka/Ks ratios than other bivalves,suggesting that they have accumulated more nonsynonymous nucleotide substitutions.Seven protein-coding genes(atp6,cox1-3,nad1,nad4 and nad5)show significant difference for Ka/Ks ratios between the Arcidae and non-Arcidae groups.However,these divergences are not observed in the nuclear gene within histone H3.From these observations,we concluded that the large mitoge-nomes of Arcidae species experienced more relaxed selective constraints.As some Arcidae species are more tolerant to hypoxia that can lead to low metabolic rate,the relaxed selective constraints of mitogenomes may be energy-related.This study provides new insights into the evolution of Arcidae mitogenomes.

Flow cytometric analysis of DNA content for four commercially important crabs in China

The genome size（C-value） of an organism is referring to the DNA content of its non-replicated haploid chromosome complement,generally deduced from measuring somatic diploid nuclei.We presented genome size（C-value） data obtained by flow cytometry for four commercially important crabs（Portunus trituberculatus,Charybdis japonica,Scylla paramamosain,and Eriocheir sinensis） common in the coast of China.Gallus domesticus（2C=2.5 pg） was used as the internal standard.The results showed that the C-value for P.trituberculatus,C.japonica,S.paramamosain,and E.sinensis were（2.31±0.01） pg,（2.33±0.03） pg,（1.64±0.02） pg,and（2.29±0.03） pg,respectively.The C-value of P.trituberculatus,C.japonica and S.paramamosain were reported for the first time.The data represented by the four species indicated that they had lower DNA contents than average DNA values in crustaceans（（4.99±0.48） pg）,and three of the four values were very similar if not identical.The results provide useful data for future studies in the fields of biodiversity,species conservation,and phylogeny of these commercial crabs.They will also be helpful in instructing the hybridization breeding program and estimating the cost of the whole genome sequencing project.

Evolutionary importance of the relationship between cytogeography and climate:New insights on creosote bushes from North and South America

Relationships between genome size and environmental variables suggest that DNA content might be adaptive and of evolutionary importance in plants.The genus Larrea provides an interesting system to test this hypothesis,since it shows both intra-and interspecific variation in genome size.Larrea has an amphitropical distribution in North and South American deserts,where it is most speciose.Larrea tridentata in North America shows a gradient of increasing autopolyploidy;while three of the four studied South American species are diploids,Larrea divaricata,Larrea nitida,Larrea ameghinoi,and the fourth is an allopolyploid,Larrea cuneifolia.We downloaded available focal species’georeferenced records from seven data reservoirs.We used these records to extract biologically relevant environmental variables from WorldClim at 30 arc seconds scale,to have a broad characterization of the variable climatic conditions of both regions,and a climatic envelope for each species.We estimated relative DNA content index and relative monoploid genome values,by flow cytometry,of four most abundant Larrea species throughout their respective ranges.Then we winnow the bioclimatic dataset down to uncorrelated variables and sampled locales,to analyse the degree of association between both intra-and interspecific relative DNA content and climatic variables that are functionally relevant in arid environments using Pearson correlations,general linear and mixed effects models.Within the genus Larrea,relative DNA content increases with rising temperature and decreases with rising precipitation.At the intraspecific level,all four species show relative DNA content variation across climatic conditions.Larrea is a genus that shows genome size variation correlated with climate.Our results are also consistent with the hypothesis that extreme environmental pressures may have facilitated repeated whole genome duplication events in North America,while in South America,reticulate evolution,as allopolyploidization,and speciation might have been climate-dependent since the Oligocene.

Adaptive Bird-like Genome Miniaturization During the Evolution of Scallop Swimming Lifestyle

Genome miniaturization drives key evolutionary innovations of adaptive traits in vertebrates,such as the flight evolution of birds.However,whether similar evolutionary processes exist in invertebrates remains poorly understood.Derived from the second-largest animal phylum,scallops are a special group of bivalve molluscs and acquire the evolutionary novelty of the swimming lifestyle,providing excellent models for investigating the coordinated genome and lifestyle evolution.Here,we show for the first time that genome sizes of scallops exhibit a generally negative correlation with locomotion activity.To elucidate the co-evolution of genome size and swimming lifestyle,we focus on the Asian moon scallop(Amusium pleuronectes)that possesses the smallest known scallop genome while being among scallops with the highest swimming activity.Whole-genome sequencing of A.pleuronectes reveals highly conserved chromosomal macrosynteny and microsynteny,suggestive of a highly contracted but not degenerated genome.Genome reduction of A.pleuronectes is facilitated by significant inactivation of transposable elements,leading to reduced gene length,elevated expression of genes involved in energy-producing pathways,and decreased copy numbers and expression levels of biomineralization-related genes.Similar evolutionary changes of relevant pathways are also observed for bird genome reduction with flight evolution.The striking mimicry of genome miniaturization underlying the evolution of bird flight and scallop swimming unveils the potentially common,pivotal role of genome size fluctuation in the evolution of novel lifestyles in the animal kingdom.

Gigantic Genomes Provide Empirical Tests of Transposable Element Dynamics Models

Transposable elements(TEs)are a major determinant of eukaryotic genome size.The collective properties of a genomic TE community reveal the history of TE/host evolutionary dynamics and impact present-day host structure and function,from genome to organism levels.In rare cases,TE community/genome size has greatly expanded in animals,associated with increased cell size and changes to anatomy and physiology.Here,we characterize the TE landscape of the genome and transcriptome in an amphibian with a giant genome—the caecilian Ichthyophis bannanicus,which we show has a genome size of 12.2 Gb.Amphibians are an important model system because the clade includes independent cases of genomic gigantism.The I.bannanicus genome differs compositionally from other giant amphibian genomes,but shares a low rate of ectopic recombination-mediated deletion.We examine TE activity using expression and divergence plots;TEs account for 15%of somatic transcription,and most superfamilies appear active.We quantify TE diversity in the caecilian,as well as other vertebrates with a range of genome sizes,using diversity indices commonly applied in community ecology.We synthesize previous models that integrate TE abundance,diversity,and activity,and test whether the caecilian meets model predictions for genomes with high TE abundance.We propose thorough,consistent characterization of TEs to strengthen future comparative analyses.Such analyses will ultimately be required to reveal whether the divergent TE assemblages found across convergent gigantic genomes reflect fundamental shared features of TE/host genome evolutionary dynamics.

Environmental selection and evolutionary process jointly shape genomic and functional profiles of mangrove rhizosphere microbiomes

Mangrove reforestation with introduced species has been an important strategy to restore mangrove ecosystem functioning.However,how such activities affect microbially driven methane(CH4),nitrogen(N),and sulfur(S)cycling of rhizosphere microbiomes remains unclear.To understand the effect of environmental selection and the evolutionary process on microbially driven biogeochemical cycles in native and introduced mangrove rhizospheres,we analyzed key genomic and functional profiles of rhizosphere microbiomes from native and introduced mangrove species by metagenome sequencing technologies.Compared with the native mangrove(Kandelia obovata,KO),the introduced mangrove(Sonneratia apetala,SA)rhizosphere microbiome had significantly(p<0.05)higher average genome size(AGS)(5.8 vs.5.5 Mb),average 16S ribosomal RNA gene copy number(3.5 vs.3.1),relative abundances of mobile genetic elements,and functional diversity in terms of the Shannon index(7.88 vs.7.84)but lower functional potentials involved in CH4 cycling(e.g.,mcrABCDG and pmoABC),N2 fixation(nifHDK),and inorganic S cycling(dsrAB,dsrC,dsrMKJOP,soxB,sqr,and fccAB).Similar results were also observed from the recovered Proteobacterial metagenome-assembled genomes with a higher AGS and distinct functions in the introduced mangrove rhizosphere.Additionally,salinity and ammonium were identified as the main environmental drivers of functional profiles of mangrove rhizosphere microbiomes through deterministic processes.This study advances our understanding of microbially mediated biogeochemical cycling of CH_(4),N,and S in the mangrove rhizosphere and provides novel insights into the influence of environmental selection and evolutionary processes on ecosystem functions,which has important implications for future mangrove reforestation.

Estimation of Nuclear DNA Content in Tannin-rich Medicinal Plant Cornus officinalis by Flow Cytometry

Objective The amount of nuclear DNA（C-value）is a key biodiversity character that provides strong unifying elements in revealing the phylogenetic regularity and relationship between genome size and functional traits for plant resource.The estimation of C-values could primarily extend our knowledge on the genetic background and genome diversity for medicinal plants,and thereby the variation of pharmacological constituents and phylogenetic mechanism of medicinal plant taxa will be revealed.However,a large number of medicinal plants（e.g.Cornus officinalis）typically contain a series of secondary metabolites,especially tannic acid,which would significantly affect the estimation of DNA content by flow cytometry（FCM）.Methodological discussions and improvement need to be made to solve this problem.Methods Two isolation buffers LB01 and Otto 1 were selected to prepare nuclear suspension with additional treatments of pre-soaking and centrifugation combination of gradient centrifugal force and duration.The best isolation and estimation methods were determined by FCM measurement in C.officinalis.Results The dry leaves were pre-soaked in Otto I buffer for 15 min and the Otto I nuclear suspension was centrifugated at 1.0×103 g for 2 min.The results showed that debris and nuclei were better separated and the scatterplots of good quality were obtained with low coefficient of variation（CV）.Contrarily,the nuclear DNA content of C.officinalis could not be accurately estimated for nuclei extracted by LB01 buffer.Finally,2C-value and genome size of C.officinalis were first estimated as 5.92 pg and 2893 Mbp,respectively.Conclusion The new methods proposed here are able to accurately estimate DNA content of C.officinalis,which provides valuable references for the estimation of genome size in other tannin-rich medicinal plants.