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Giant genomes are rare across the plant kingdom and their study has focused almost exclusively on angiosperms and gymnosperms. The scarce genetic data that are available for ferns, however, indicate differences in their genome organization and a lower dynamism compared to other plant groups. Tmesipteris is a small genus of mainly epiphytic ferns that occur in Oceania and several Pacific Islands. So far, only two species with giant genomes have been reported in the genus, T. tannensis (1C = 73.19 Gbp) and T. obliqua (1C = 147.29 Gbp). Low-coverage genome skimming sequence data were generated in these two species and analyzed using the RepeatExplorer2 pipeline to identify and quantify the repetitive DNA fraction of these genomes. We found that both species share a similar genomic composition, with high repeat diversity compared to taxa with small (1C < 10 Gbp) genomes. We also found that, in general, characterized repetitive elements have relatively high heterogeneity scores, indicating ancient diverging evolutionary trajectories. Our results suggest that a whole genome multiplication event, accumulation of repetitive elements, and recent activation of those repeats have all played a role in shaping these genomes. It will be informative to compare these data in the future with data from the giant genome of the angiosperm Paris japonica, to determine if the structures observed here are an emergent property of massive genomic inflation or derived from lineage specific processes., This research was funded by the Spanish Research Council (Ref: PID2019-108173GA-I00) funded by MCIN/AEI/ 10.13039/501100011033. J.P. benefited from a Ramón y Cajal grant (Ref: RYC-2017-2274) funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”. P.F. benefited from a FPU Fellowship (Ref: FPU21/03564) funded by Spanish Ministry of Universities., Introduction Results Discussion Materials and Methods Conclusions Supplementary Materials Author Contributions Funding Institutional Review Board Statement Informed Consent Statement Data Availability Statement Acknowledgments Conflicts of Interest References

Asteraceae, the daisy family, are one of the most diverse families of angiosperms and are predominant in many ecosystems, including grasslands, deserts, savannas and high-elevation mountains. They are characterized by a peculiar inflorescence, the capitulum, which mimics a flower, but is actually made up of many tightly grouped florets. The capitulum is considered a key character underpinning the impressive evolutionary success of the family, and it plays a pivotal role in the economic importance of the family, given that many species are cultivated for their capitulum for agricultural and horticultural purposes. However, to date, there is still no comprehensive understanding of the extent of the morphological diversity of capitula across lineages of Asteraceae. This is mainly due to a lack of appropriate tools for describing such a complex and condensed structure. To address the problem, we present a protocol for characterizing the full diversity of capitula from any lineage of Asteraceae. This involves making a whole dissection of a capitulum from fresh material; it is simple and cost-effective and requires relatively easy-to-transport equipment meaning that it can be done during fieldwork., This research was supported through a research grant by the Winton (Harding) Alpine Plant Conservation & Research Programme (WHAPCRP, https://www.winton.com/philanthropy), by the Lautaret Garden-UMS 3370 (University of Grenoble Alpes, CNRS, SAJF, 38000 Grenoble, France), member of AnaEE-France (ANR-11-INBS-0001AnaEE-Services, Investissements d’Avenir frame) and of the eLTER-Europe network (University of Grenoble Alpes, CNRS, LTSER Zone Atelier Alpes, 38000 Grenoble, France), the project ‘COMPOSITAE’ reference PID2020-116480GB-I00 funded by MCIN/AEI/10.13039/501100011033 and the ‘Ajuts a Grups de Recerca Consolidats’ (2017/SGR/1116) from the Generalitat de Catalunya. OH benefited from a Marie Skłodowska-Curie Action Individual Fellowship (grant agreement number 657918). JP benefited from a Ramón y Cajal grant reference number RYC-2017-2274 funded by MCIN/AEI/10.13039/501100011033 and by ‘ESF Investing in your future’., INTRODUCTION MATERIAL AND METHODS NOTES SUPPORTING INFORMATION ACKNOWLEDGEMENTS REFERENCES Supplementary data

An analysis of over 10 000 plant genome sizes (GSs) indicates that most species have smaller genomes than expected given the incidence of polyploidy in their ancestries, suggesting selection for genome downsizing. However, comparing ancestral GS with the incidence of ancestral polyploidy suggests that the rate of DNA loss following polyploidy is likely to have been very low (4-70 Mb/million years, 4-482 bp/generation). This poses a problem. How might such small DNA losses be visible to selection, overcome the power of genetic drift and drive genome downsizing? Here we explore that problem, focussing on the role that double-strand break (DSB) repair pathways (non-homologous end joining and homologous recombination) may have played. We also explore two hypotheses that could explain how selection might favour genome downsizing following polyploidy: to reduce (i) nitrogen (N) and phosphate (P) costs associated with nucleic acid synthesis in the nucleus and the transcriptome and (ii) the impact of scaling effects of GS on cell size, which influences CO2 uptake and water loss. We explore the hypothesis that losses of DNA must be fastest in early polyploid generations. Alternatively, if DNA loss is a more continuous process over evolutionary time, then we propose it is a byproduct of selection elsewhere, such as limiting the damaging activity of repetitive DNA. If so, then the impact of GS on photosynthesis, water use efficiency and/or nutrient costs at the nucleus level may be emergent properties, which have advantages, but not ones that could have been selected for over generational timescales., XW is funded by the China Scholarship Council (CSC, Grant 201908370151) from the Ministry of Education of the P.R. China. JP is supported by a Ramón y Cajal Fellowship (2017-RYC-2274) funded by the Ministry of Science and Technology (Government of Spain)., INTRODUCTION IMPACT OF WHOLE-GENOME DUPLICATIONS ON GENOME SIZE DIVERSITY IN ANGIOSPERMS MECHANISMS UNDERPINNING GENOME SIZE DIVERGENCE PREDICTING RATES OF GENOME SIZE CHANGE FOLLOWING WHOLE-GENOME DUPLICATIONS POTENTIAL SOURCES OF SELECTION ACTING ON GENOME SIZE FOLLOWING WHOLE-GENOME DUPLICATIONS Selection for small genomes generated by the nutrient costs of nucleic acids Selection for small genomes to maximise photosynthesis and/or water use efficiency HOW MIGHT GENOME DOWNSIZING ARISE? Genome downsizing arises through selection against genome upsizing Genome downsizing is selected for in early-generation polyploids Genome downsizing is a byproduct of high recombination frequencies in polyploids Genome downsizing is a byproduct of selection for a nutrient-efficient transcriptome CONCLUDING REMARKS AND FUTURE PERSPECTIVES ACKNOWLEDGEMENTS CONFLICT OF INTEREST

Simple telomeric repeats composed of six to seven iterating nucleotide units are important sequences typically found at the ends of chromosomes. Here we analyzed their abundance and homogeneity in 42 gymnosperm (29 newly sequenced), 29 angiosperm (one newly sequenced), and eight bryophytes using bioinformatics, conventional cytogenetic and molecular biology approaches to explore their diversity across land plants. We found more than 10 000-fold variation in the amounts of telomeric repeats among the investigated taxa. Repeat abundance was positively correlated with increasing intragenomic sequence heterogeneity and occurrence at non-telomeric positions, but there was no correlation with genome size. The highest abundance/heterogeneity was found in the gymnosperm genus Cycas (Cycadaceae), in which megabase-sized blocks of telomeric repeats (i.e., billions of copies) were identified. Fluorescent in situ hybridization experiments using variant-specific probes revealed canonical Arabidopsis-type telomeric TTTAGGG repeats at chromosome ends, while pericentromeric blocks comprised at least four major telomeric variants with decreasing abundance: TTTAGGG>TTCAGGG >TTTAAGG>TTCAAGG. Such a diversity of repeats was not found in the sister cycad family Zamiaceae or in any other species analyzed. Using immunocytochemistry, we showed that the pericentromeric blocks of telomeric repeats overlapped with histone H3 serine 10 phosphorylation signals. We show that species of Cycas have amplified their telomeric repeats in centromeric and telomeric positions on telocentric chromosomes to extraordinary high levels. The ancestral chromosome number reconstruction suggests their occurrence is unlikely to be the product of ancient Robertsonian chromosome fusions. We speculate as to how the observed chromosome dynamics may be associated with the diversification of cycads., This project was supported by the Czech Academy of Science, Czech Science Foundation (22-16826S), Czech National Infrastructure for Biological data (ELIXIR CZ, LM2018131), NERC and China Scholarship Council (CSC). JP benefited from a Ramón y Cajal grant Ref: RYC-2017-2274 funded by MCIN/AEI/, INTRODUCTION RESULTS Identification and quantification of telomeric repeats in high-throughput reads In silico identification of telomeric repeat variants Southern blot hybridization analysis of telomeric variants Identification of cycad centromeres by immunostaining of chromatin FISH analysis of telomeric variants Evolution of chromosome numbers and genome sizes across cycads DISCUSSION Variable abundance of telomeric repeats in plant genomes Origin of telomeric repeat variants in cycad genomes Epigenetic modification of telomeric repeats Chromosome evolution in cycads CONCLUSION EXPERIMENTAL PROCEDURES Plant material DNA isolation and Illumina sequencing Estimation of telomeric repeats abundance and diversity from high-throughput sequencing data Ancestral chromosome and genome size reconstruction Southern blot hybridization and DNA methylation analysis DNA probe preparation for FISH and southern blotting FISH Immunohistochemical staining of chromosomes ACKNOWLEDGEMENTS Author CONTRIBUTIONS

The entangled relationship of chromosome number and genome size with species distribution has been the subject of study for almost a century, but remains an open question due to previous ecological and phylogenetic knowledge constraints. To better address this subject, we used the clade Maxillariinae, a widely distributed and karyotypically known orchid group, as a model system to infer such relationships in a robust methodological framework.Based on the literature and new data, we gathered the chromosome number and genome size for 93 and 64 species, respectively. We built a phylogenetic hypothesis and assessed the best macroevolutionary model for both genomic traits. Additionally, we collected together ecological data (preferences for bioclimatic variables, elevation and habit) used as explanatory variables in multivariate phylogenetic models explaining genomic traits. Finally, the impact of polyploidy was estimated by running the analyses with and without polyploids in the sample.The association between genomic and ecological data varied depending on whether polyploids were considered or not. Without polyploids, chromosome number failed to present consistent associations with ecological variables. With polyploids, there was a tendency to waive epiphytism and colonize new habitats outside humid forests. The genome size showed association with ecological variables: without polyploids, genome increase was associated with flexible habits, with higher elevation and with drier summers; with polyploids, genome size increase was associated with colonizing drier environments.The chromosome number and genome size variations, essential but neglected traits in the ecological niche, are shaped in the Maxillariinae by both neutral and adaptive evolution. Both genomic traits are partially correlated to bioclimatic variables and elevation, even when controlling for phylogenetic constraints. While polyploidy was associated with shifts in the environmental niche, the genome size emerges as a central trait in orchid evolution by the association between small genome size and epiphytism, a key innovation to Neotropical orchid diversification.

Given the 2,400-fold range of genome sizes (0.06–148.9 Gbp (gigabase pair)) of seed plants (angiosperms and gymnosperms) with a broadly similar gene content (amounting to approximately 0.03 Gbp), the repeat-sequence content of the genome might be expected to increase with genome size, resulting in the largest genomes consisting almost entirely of repetitive sequences. Here we test this prediction, using the same bioinformatic approach for 101 species to ensure consistency in what constitutes a repeat. We reveal a fundamental change in repeat turnover in genomes above around 10 Gbp, such that species with the largest genomes are only about 55% repetitive. Given that genome size influences many plant traits, habits and life strategies, this fundamental shift in repeat dynamics is likely to affect the evolutionary trajectory of species lineages., We thank Natural Environment Research Council (NE/G020256/1), the Czech Academy of Sciences (RVO:60077344) and Ramón y Cajal Fellowship (RYC-2017-2274) funded by the Ministerio de Ciencia y Tecnología (Gobierno de España) for support. We also thank Natural Environment Research Council for funding a studentship to S.D. and the China Scholarship Council for funding W.W., Main Methods Data availability Code availability References Acknowledgements Author information Ethics declarations Additional information Extended data Supplementary information Rights and permissions About this article Further reading

With c. 24700 species (10% of all flowering plants), Asteraceae are one of the largest and most phenotypically diverse angiosperm families, with considerable economic and ecological importance. Asteraceae are distributed worldwide, from nearly polar latitudes all the way to the tropics, and occur across a diverse range of habitats from extreme deserts to swamps and from lowland rainforests to alpine tundra. Altogether, these characteristics make this family an outstanding model system to address a broad range of eco-evolutionary questions. In this review, we summarize recent progress in our understanding of Asteraceae on the basis of joint efforts by specialists in the fields of palaeobotany, cytogenetics, comparative genomics and phylogenomics. We will highlight how these developments are opening up new possibilities for integrating fields and better comprehending evolution beyond Asteraceae., This research was supported through the project ‘COMPOSITAE’ reference number PID2020-116480GB-I00 funded by MCIN/AEI/10.13039/501100011033, the ‘Ajuts a Grups de Recerca Consolidats’ (2017/SGR/1116) from the Generalitat de Catalunya, and by Agencia Nacional de Promoción Científica y Tecnológica (PICT 2019-03011). J.P. benefited from a Ramón y Cajal grant reference number RYC-2017-2274 funded by MCIN/AEI/10.13039/501100011033 and by ‘ESF Investing in your future’., INTRODUCTION UNCOVERING THE ORIGIN OF ASTERACEAE: EVIDENCE FROM THE FOSSIL RECORD EVOLUTIONARY FRAMEWORK OF ASTERACEAE IN THE AGE OF HIGH-THROUGHPUT SEQUENCING TECHNOLOGIES FROM MODEL SPECIES TO FAMILY WIDE APPROACHES: POLYPLOIDY, GENOME SIZE AND REPETITIVE DNA CONTENT IN ASTERACEAE RESOURCES AVAILABLE FOR FUNCTIONAL GENOMIC STUDIES IN ASTERACEAE SUPPORTING INFORMATION ACKNOWLEDGEMENTS REFERENCES Author notes Supplementary data

Genome size varies 2400-fold across plants, influencing their evolution through changes in cell size and cell division rates which impact plants' environmental stress tolerance. Repetitive element expansion explains much genome size diversity, and the processes structuring repeat "communities" are analogous to those structuring ecological communities. However, which environmental stressors influence repeat community dynamics has not yet been examined from an ecological perspective. We measured genome size and leveraged climatic data for 91% of genera within the ecologically diverse palm family (Arecaceae). We then generated genomic repeat profiles for 141 palm species, and analysed repeats using phylogenetically informed linear models to explore relationships between repeat dynamics and environmental factors. We show that palm genome size and repeat "community" composition are best explained by aridity. Specifically, Ty3-gypsy and TIR elements were more abundant in palm species from wetter environments, which generally had larger genomes, suggesting amplification. By contrast, Ty1-copia and LINE elements were more abundant in drier environments. Our results suggest that water stress inhibits repeat expansion through selection on upper genome size limits. However, elements that may associate with stress-response genes (e.g. Ty1-copia) have amplified in arid-adapted palm species. Overall, we provide novel evidence of climate influencing the assembly of repeat "communities"., JP was supported by a Ramón y Cajal Fellowship (RYC-2017-2274) funded by MCIN/AEI/10.13039/501100011033 and by ‘ESF Investing in your future’. SB was funded by a Garfield Weston Foundation postdoctoral fellowship. PN and JM were supported by the ELIXIR CZ Research Infrastructure Project (Czech Ministry of Education, Youth and Sports; grant no. LM2018131)., IntroductionMaterials and Methods Plant material collection and genome size measurement Phylogenetic, environmental and genomic data collection Modelling relationships between genome size and environmental variables DNA repeat profiling Assessing repeat dynamics in palm genomesResults Palm genome size variation Aridity preferences of palm species help explain genome size variation Ecological metrics of palm repeat ‘communities’ vary with genome size Repeat abundances correlate with genome size Aridity preferences of palm species explain abundances of certain repeat lineagesDiscussion Palm genome size variation Aridity thresholds best explain palm genome size diversity The ‘community ecology’ of repeats correlates with genome size Repeat dynamics may be modulated by aridityConclusionsAcknowledgementsAuthor contributions

The genus Marshallia is made up by seven to ten species of perennial herbs growing mainly in open habitats, whereas the genus Balduina is represented by three sympatric species; two perennial herbs and one annual, growing in open pine forest habitats. Both genera belong to the family Asteraceae, tribe Helenieae, and are endemic to the southeast United States, in North America. Cytogenetic studies concerning these two genera are scarce and genome size data is lacking for both. The main goals of this study were to (i) generate novel insights into the evolution of the genome size and (ii), contribute to filling existing gaps on our knowledge of the Asteraceae family from this point of view. Nuclear DNA contents range from 11.42 pg/2C in Marshallia trinervia to 31.58 pg/2C in Marshallia mohrii. The combination of genome size with chromosome data (and inferred cytotypes) suggests the existence of multiple cytotypes, and provides interesting insights into the potential impact of polyploidy in the evolution of these genera in general, and the shaping of genome size diversity, in particular., This work was supported by the Catalan Government under Grant 2017SGR1116. J.P. benefited from a Ramón y Cajal fellowship supported by the Ministerio de Ciencia y Tecnología (RYC-2017-2274), INTRODUCTION MATERIALS AND METHODS Plant material Nuclear DNA content assessments Chromosome counts Phylogenetic tree and data mapping RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

While a number of studies have suggested that temperate plants typically have larger genome sizes (GS) than tropical ones, recent analyses have not convincingly confirmed this. We have selected the widely distributed Caesalpinia Group (Leguminosae) to test this hypothesis. We used flow cytometry to estimate the amount of DNA in the haploid genome (C-values) of 40 species of the Caesalpinia Group, aiming to explore the relationship between GS and latitude as well as a range of climatic variables. These comparisons were made using Phylogenetic Comparative Methods (PCM) in a spatio-temporal context. 2C-values varied 7.73-fold, ranging from 0.92 pg (in Cenostigma bracteosum) to 7.11 pg (in Pomaria lactea). By analyzing the data within a statistical framework that took into account phylogenetic relationships, we observed a positive correlation between GS and latitude as well as additional correlations with various temperature variables. While the genetic mechanism(s) underpinning the increase or decrease of genome size in a latitudinal gradient is unclear, we hypothesize that after the origin of the group (c. 55.9 Mya), fragmentation of the Succulent Biome led to the formation of small populations (island-like) which were subject to genetic drift. This in turn led to drastic and rapid changes in the repetitive DNA fractions of the genome correlated with temperature variables. Thus, we suggest that the environment has played a role in contributing to the diversity of genome sizes reported here for the Caesalpinia Group.

Premise Phylogenetic studies in the Compositae are challenging due to the sheer size of the family and the challenges they pose for molecular tools, ranging from the genomic impact of polyploid events to their very conserved plastid genomes. The search for better molecular tools for phylogenetic studies led to the development of the family-specific Compositae1061 probe set, as well as the universal Angiosperms353 probe set designed for all flowering plants. In this study, we evaluate the extent to which data generated using the family-specific kit and those obtained with the universal kit can be merged for downstream analyses. Methods We used comparative methods to verify the presence of shared loci between probe sets. Using two sets of eight samples sequenced with Compositae1061 and Angiosperms353, we ran phylogenetic analyses with and without loci flagged as paralogs, a gene tree discordance analysis, and a complementary phylogenetic analysis mixing samples from both sample sets. Results Our results show that the Compositae1061 kit provides an average of 721 loci, with 9¿46% of them presenting paralogs, while the Angiosperms353 set yields an average of 287 loci, which are less affected by paralogy. Analyses mixing samples from both sets showed that the presence of 30 shared loci in the probe sets allows the combination of data generated in different ways. Discussion Combining data generated using different probe sets opens up the possibility of collaborative efforts and shared data within the synantherological community., Funding came from various sources, including the National Science Foundation Division of Environmental Biology (DEB-1745197), and grants from the Calleva Foundation and the Sackler Trust to the Plant and Fungal Trees of Life (PAFTOL) project at the Royal Botanic Gardens, Kew, METHODS Identification of shared loci Taxon selection, plant material, DNA extraction, library preparation, and sequencing Sequence assembly and data analysis RESULTS Identification of shared loci Recovered loci and paralogy Phylogenetic relationships and gene tree discordance DISCUSSION Acknowledgments AUTHOR CONTRIBUTIONS

SummaryGenome size (GS) is a key trait related to morphology, life history, and evolvability. Although GS is, by definition, affected by presence/absence variants (PAVs), which are ubiquitous in population sequencing studies, GS is often treated as an intrinsic property of a species. Here, we studied intra- and interspecific GS variation in taxonomically complex British eyebrights (Euphrasia).We generated GS data for 192 individuals of diploid and tetraploidEuphrasiaand analysed GS variation in relation to ploidy, taxonomy, population affiliation, and geography. We further compared the genomic repeat content of 30 samples.We found considerable genuine intraspecific GS variation, and observed isolation-by-distance for GS in outcrossing diploids. TetraploidEuphrasiashowed contrasting patterns, with GS increasing with latitude in outcrossingEuphrasia arctica, but little GS variation in the highly selfingEuphrasia micrantha. Interspecific differences in GS genomic repeat percentages were small.We show the utility of treating GS as the outcome of polygenic variation. Like other types of genetic variation, such as single nucleotide polymorphisms, GS variation may be increased through hybridisation and population subdivision. In addition to selection on associated traits, GS is predicted to be affected indirectly by selection due to pleiotropy of the underlying PAVs.

[Background and Aims] Extant plant groups with a long fossil history are key elements in understanding vascular plant evolution. Horsetails (Equisetum, Equisetaceae) have a nearly continuous fossil record dating back to the Carboniferous, but their phylogenetic and biogeographic patterns are still poorly understood. We use here the most extensive phylogenetic analysis to date as a framework to evaluate their age, biogeography and genome size evolution. [Methods] DNA sequences of four plastid loci were used to estimate divergence times and investigate the biogeographic history of all extant species of Equisetum. Flow cytometry was used to study genome size evolution against the framework of phylogenetic relationships in Equisetum. [Key Results] On a well-supported phylogenetic tree including all extant Equisetum species, a molecular clock calibrated with multiple fossils places the node at which the outgroup and Equisetum diverged at 343 Mya (Early Carboniferous), with the first major split among extant species occurring 170 Mya (Middle Jurassic). These dates are older than those reported in some other recent molecular clock studies but are largely in agreement with a timeline established by fossil appearance in the geological record. Representatives of evergreen subgenus Hippochaete have much larger genome sizes than those of deciduous subgenus Equisetum, despite their shared conserved chromosome number. Subgenus Paramochaete has an intermediate genome size and maintains the same number of chromosomes. [Conclusions] The first divergences among extant members of the genus coincided with the break-up of Pangaea and the resulting more humid, warmer climate. Subsequent tectonic activity most likely involved vicariance events that led to species divergences combined with some more recent, long-distance dispersal events. We hypothesize that differences in genome size between subgenera may be related to the number of sperm flagellae., INTRODUCTION MATERIALS AND METHODS Taxonomic sampling and DNA sequencing Phylogenetic analyses Divergence time estimates using fossil calibrations Ancestral range reconstruction analyses Genome size estimation using flow cytometry RESULTS Phylogenetic analyses Molecular clock analysis using fossil calibrations Ancestral range reconstruction analyses Genome size analysis of Equisetum DISCUSSION Origin, age and diversification of Equisetum Biogeography of Equisetum Genome size diversity and evolution in Equisetum Conclusions SUPPLEMENTARY DATA ACKNOWLEDGEMENTS LITERATURE CITED Supplementary data

Background and aims: Genome size varies considerably across the diversity of plant life. Although genome size is, by definition, affected by genetic presence/absence variants, which are ubiquitous in population sequencing studies, genome size is often treated as an intrinsic property of a species. Here, we studied intra- and interspecific genome size variation in taxonomically complex British eyebrights (Euphrasia, Orobanchaceae). Our aim is to document genome size diversity and investigate underlying evolutionary processes shaping variation between individuals, populations and species. Methods: We generated genome size data for 192 individuals of diploid and tetraploid Euphrasia and analysed genome size variation in relation to ploidy, taxonomy, population affiliation and geography. We further compared the genomic repeat content of 30 samples. Key results: We found considerable intraspecific genome size variation, and observed isolation-by-distance for genome size in outcrossing diploids. Tetraploid Euphrasia showed contrasting patterns, with genome size increasing with latitude in outcrossing Euphrasia arctica, but with little genome size variation in the highly selfing Euphrasia micrantha. Interspecific differences in genome size and the genomic proportions of repeat sequences were small. Conclusions: We show the utility of treating genome size as the outcome of polygenic variation. Like other types of genetic variation, such as single nucleotide polymorphisms, genome size variation may be affected by ongoing hybridization and the extent of population subdivision. In addition to selection on associated traits, genome size is predicted to be affected indirectly by selection due to pleiotropy of the underlying presence/absence variants., A.D.T. is supported by NERC research grants NE/L011336/1 and NE/N006739/1. The Royal Botanic Garden Edinburgh (RBGE) is supported by the Scottish Government’s Rural and Environment Science and Analytical Services Division. J.P. is supported by a Ramón y Cajal Fellowship (RYC-2017–2274) funded by the Ministerio de Ciencia y Tecnología (Gobierno de España)., Issue Cover Volume 128Issue 5 8 October 2021 Article Contents Abstract INTRODUCTION METHODS The study system Population and species-level genome size variation Population sampling Genome size measurements Repeat content variation Sequence data generation Repeat content Statistical analyses Data availability RESULTS Population and species-level genome size variation Variation in genomic repeat content DISCUSSION Genome size variation mirrors population genetic patterns Genome size differences and genomic repeats Evolution of genome size variation SUPPLEMENTARY DATA ACKNOWLEDGEMENTS FUNDING LITERATURE CITED Supplementary data

Over the years, the amount of DNA in a nucleus (genome size) has been estimated using a variety of methods, but increasingly, flow cytometry (FCM) has become the method of choice. The popularity of this technique lies in the ease of sample preparation and in the large number of particles (i.e., nuclei) that can be analyzed in a very short period of time. This chapter presents a step-by-step guide to estimating the nuclear DNA content of plant nuclei using FCM. Attempting to serve as a tool for daily laboratory practice, we list, in detail, the equipment required, specific reagents and buffers needed, as well as the most frequently used protocols to carry out nuclei isolation. In addition, solutions to the most common problems that users may encounter when working with plant material and troubleshooting advice are provided. Finally, information about the correct terminology to use and the importance of obtaining chromosome counts to avoid cytological misinterpretations of the FCM data are discussed.

Over the years, the amount of DNA in a nucleus (genome size) has been estimated using a variety of methods, but increasingly, flow cytometry (FCM) has become the method of choice. The popularity of this technique lies in the ease of sample preparation and in the large number of particles (i.e., nuclei) that can be analyzed in a very short period of time. This chapter presents a step-by-step guide to estimating the nuclear DNA content of plant nuclei using FCM. Attempting to serve as a tool for daily laboratory practice, we list, in detail, the equipment required, specific reagents and buffers needed, as well as the most frequently used protocols to carry out nuclei isolation. In addition, solutions to the most common problems that users may encounter when working with plant material and troubleshooting advice are provided. Finally, information about the correct terminology to use and the importance of obtaining chromosome counts to avoid cytological misinterpretations of the FCM data are discussed.

Artículo de 12 páginas con mapa de especificación geográfica, con gráficos de detalle de especies y tablas de datos., Asexual reproduction has often been regarded as an evolutionary dead end, but asexual lineages (most notably those that are apomictic) are present in several angiosperm families and often comprise a large number of taxa, both widespread and endemic. Investigating correlations between genetic, environmental and taxonomic factors and the incidence of apomixis has represented a challenge for many years, with previous analyses frequently omitting one or more of these variables. Here, flow cytometric seed screening, cytological data and ecological variables have been integrated in a phylogenetic framework to create a comprehensive dataset for 229 of Asteraceae from the European Alps. Data were analysed using phylogenetically informed generalized linear mixed models (pMCMCglmm) where elevation, ploidy and phenology were assessed for their potential correlation with asexual reproduction and apomixis type. Although apomixis is not dominant among the species studied, our results confirm that an odd ploidy (e.g. 3x) and, to a lesser extent, an even polyploid level (i.e. 4x) significantly increase its probability, most probably due to chromosome misalignments during meiosis. The distribution of apomictic species does not correlate with elevation, and there is a weak correlation between early flowering initiation and aposporous apomixis. Although current and future changes in climate may severely impact the survival of the flora of the European Alps, asexual reproduction and polyploidization may prove to be, at least temporarily, lifelines for the survival of a species under the novel climatic conditions. Therefore, uncovering how apomicts and polyploids evolve and persist will be essential for understanding the ecology of the European Alps and hence informing future conservation strategies., This research was funded through a research grant by the Winton (Harding) Alpine Plant Conservation & Research Programme (https://www.winton.com/philanthropy) and a predoctoral contract awarded to Luca Pegoraro. Oriane Hidalgo benefited from a Marie Skłodowska-Curie Action Individual Fellowship (European Commission - Ref: 657918) and a Ramón y Cajal fellowship (Ministerio de Ciencia e Innovación, Gobierno de España - Ref: RYC-2016-21176). Jaume Pellicer benefited from Ramón y Cajal fellowship (Ministerio de Ciencia e Innovación, Gobierno de España - Ref: RYC-2017-2274)., INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION CONCLUSIONS SUPPORTING INFORMATION ACKNOWLEDGEMENTS REFERENCES Supplementary data

While polyploidization is recognized as a major evolutionary driver for ferns and angiosperms, little is known about its impact in gymnosperms, where polyploidy is much less frequent. We explore Ephedra to evaluate (i) the extent of genome size diversity in the genus and the influence polyploidy has had on the evolution of nuclear DNA contents, and (ii) identify where shifts in genome size and polyploidy have occurred both temporally and spatially. A phylogenetic framework of all Ephedra species together with genome sizes and karyotypes for 87% and 67% of them respectively, were used to explore ploidy evolution and its global distribution patterns. Polyploidy was shown to be extremely common, with 41 species (83%) being polyploid (up to 8×) or having polyploid cytotypes – the highest frequency and level reported for any gymnosperm. Genome size was also diverse, with values ranging ~5-fold (8.09–38.34 pg/1C) – the largest range for any gymnosperm family – and increasing in proportion to ploidy level (i.e. no genome downsizing). Our findings provide novel data which support the view that gymnosperms have a more conserved mode of genomic evolution compared with angiosperms., The work was partially supported by a DAAD Research Fellowship to SIB for a research stay in the Renner Lab at LMU, Munich, Germany. OH and JP benefited from Ramón y Cajal fellowships (RYC-2016-21176 and RYC-2017-22742, respectively)., Highlights Abstract Graphical abstract Keywords 1. Introduction 2. Materials and methods 3. Results 4. Discussion Author contributions Declaration of Competing Interest Acknowledgements Appendix A. Supplementary material References

[Background and Aims] Changes in the amount of repetitive DNA (dispersed and tandem repeats) are considered the main contributors to genome size variation across plant species in the absence of polyploidy. However, the study of repeatome dynamism in groups showing contrasting genomic features and complex evolutionary histories is needed to determine whether other processes underlying genome size variation may have been overlooked. The main aim here was to elucidate which mechanism best explains genome size evolution in Anacyclus (Asteraceae). [Methods] Using data from Illumina sequencing, we analysed the repetitive DNA in all species of Anacyclus, a genus with a reticulate evolutionary history, which displays significant genome size and karyotype diversity albeit presenting a stable chromosome number. [Key Results] By reconstructing ancestral genome size values, we inferred independent episodes of genome size expansions and contractions during the evolution of the genus. However, analysis of the repeatome revealed a similar DNA repeat composition across species, both qualitative and quantitative. Using comparative methods to study repeatome dynamics in the genus, we found no evidence for repeat activity causing genome size variation among species. [Conclusions] Our results, combined with previous cytogenetic data, suggest that genome size differences in Anacyclus are probably related to chromosome rearrangements involving losses or gains of chromosome fragments, possibly associated with homoploid hybridization. These could represent balanced rearrangements that do not disrupt gene dosage in merged genomes, for example via chromosome segment exchanges., This work was supported by the Catalan government (grant number 2017SGR1116) and by the Spanish government [grant numbers CGL2010-18039, CGL2013-49097-C2-1-P, CGL2013-49097-C2-2-P, CGL2016-75694-P (AEI/FEDER, UE), RYC-2014–16608 to S.G. and RYC-2016–21176 to O.H.]

Artículo de 7 páginas sobre secuencación de genomas que incluye tablas de estadísticas, gráficos de correlación e histogramas., Each day, as the amount of genomic data and bioinformatics resources grows, researchers are increasingly challenged with selecting the most appropriate approach to analyze their data. In addition, the opportunity to undertake comparative genomic analyses is growing rapidly. This is especially true for fungi due to their small genome sizes (i.e., mean 1C=44.2Mb). Given these opportunities and aiming to gain novel insights into the evolution of mutualisms, we focus on comparing the quality of whole genome assemblies for fungus-growing ants cultivars (Hymenoptera: Formicidae: Attini) and a free-living relative. Our analyses reveal that currently available methodologies and pipelines for analyzing whole-genome sequence data need refining. By using different genome assemblers, we show that the genome assembly size depends on what software is used. This, in turn, impacts gene number predictions, with higher gene numbers correlating positively with genome assembly size. Furthermore, the majority of fungal genome size data currently available are based on estimates derived from whole-genome assemblies generated from short-read genome data, rather than from the more accurate technique of flow cytometry. Here, we estimated the haploid genome sizes of three ant fungal symbionts by flow cytometry using the fungus Pleurotus ostreatus (Jacq.) P. Kumm. (1871) as a calibration standard. We found that published genome sizes based on genome assemblies are 2.5- to 3-fold larger than our estimates based on flow cytometry. We, therefore, recommend that flow cytometry is used to precalibrate genome assembly pipelines, to avoid incorrect estimates of genome sizes and ensure robust assemblies., P.W.K. received funding from CAPES-PrInt (Grant #88887.468939/2019-00) and J.P. benefited from a Ramon y Cajal Fellowship (RYC-2017-2274) from the government of Spain., Results and Discussion Supplementary Material Acknowledgments Literature Cited Supplementary data

Background and aims Recent tissue-level observations made indirectly via flow cytometry suggest that endoreplication (duplication of the nuclear genome within the nuclear envelope in the absence of subsequent cell division) is widespread within the plant kingdom. Here, we also directly observe ploidy variation among cells within individual petals, relating size of nucleus to cell micromorphology and (more speculatively) to function. Methods We compared the labella (specialized pollinator-attracting petals) of two European orchid genera: Dactylorhiza has a known predisposition to organismal polyploidy, whereas Ophrys exhibits exceptionally complex epidermal patterning that aids pseudocopulatory pollination. Confocal microscopy using multiple staining techniques allowed us to observe directly both the sizes and the internal structures of individual nuclei across each labellum, while flow cytometry was used to test for progressively partial endoreplication. Key results In Dactylorhiza, endoreplication was comparatively infrequent, reached only low levels, and appeared randomly located across the labellum, whereas in Ophrys endoreplication was commonplace, being most frequent in large peripheral trichomes. Endoreplicated nuclei reflected both endomitosis and endocycling, the latter reaching the third round of genome doubling (16C) to generate polytene nuclei. All Ophrys individuals studied exhibited progressively partial endoreplication. Conclusions Comparison of the two genera failed to demonstrate the hypothesized pattern of frequent polyploid speciation in genera showing extensive endoreplication. Endoreplication in Ophrys appears more strongly positively correlated with cell size/complexity than with cell location or secretory role. Epigenetic control of gene overexpression by localized induction of endoreplication within individual plant organs may represent a significant component of a plant's developmental programme, contributing substantially to organ plasticity.

Ophrys is amongst the best known orchid genera and is an established system for the study of pollinator-mediated floral evolution. Two species, Ophrys fusca s.l. and Ophrys dyris (= O. omegaifera s...

11 p., tablas, mapas -- Post-print del artículo publicado en Plant Systematics and Evolution. Versión revisada y corregida -- La versión online de este artículo contiene material suplementario, disponible en: http://doi.org/10.1007/s00606-017-1387-x, The study of fragmentation processes should be tackled using different approaches, in order to obtain solid and robust evidence that could help in identifying potential barriers and threats for species. In this study, we have evaluated the spatial fragmentation patterns in Artemisia crithmifolia (Asteraceae) along its current distribution along the Atlantic coastlines of Europe, from Portugal to the Netherlands and the UK. Niche modelling analyses considering current and past climatic conditions, combined with plastid markers, have been used to evaluate the disconnected distribution pattern of the species, genetic flow between populations and habitat suitability for present and past populations. Plastid markers showed very low variability, while suggesting that the westernmost populations from the Atlantic coast of the Iberian Peninsula are partially genetically isolated, due to the existence of a potential barrier for gene flow on the northern Spanish coast. However, niche models identify this area as a potential habitat for the species, even during the last glacial maximum (20,000 years ago). The combination of both techniques allows identification of potential refugia for the species, highlighting the most likely recolonisation routes and distribution patterns which resulted in the overall low levels of genetic diversity. Anthropogenic activities (urban sprawl, beach expansions, etc.) are most likely behind local extinctions, thus preventing the establishment and expansion of new populations., The study has been supported by Projects CGL2007-64839-C02-01/-02, CGL2010-22234-C02-01.02/BOS and CGL2013-49097-C2-2-P (Spanish Government) and 2014SGR514 (Catalan Government).

Ecosystems are at increasing risk from the global pollination crisis. Gaining better knowledge about pollinators and their interactions with plants is an urgent need. However, conventional methods of manually recording pollinator activity in the field can be time- and cost-consuming in terms of labour. Field-deployable video recording systems have become more common in ecological studies as they enable the capture of plant-insect interactions in fine detail. Standard video recording can be effective, although there are issues with hardware reliability under field-conditions (e.g. weatherproofing), and reviewing raw video manually is a time-consuming task. Automated video monitoring systems based on motion detection partly overcome these issues by only recording when activity occurs hence reducing the time needed to review footage during post-processing. Another advantage of these systems is that the hardware has relatively low power requirements. A few systems have been tested in the field which permit the collection of large datasets. Compared with other systems, automated monitoring allows vast increases in sampling at broad spatiotemporal scales. Some tools such as post-recording computer vision software and data-import scripts exist, further reducing users’ time spent processing and analysing the data. Integrated computer vision and automated species recognition using machine learning models have great potential to further the study of pollinators in the field. Together, it is predicted that future advances in technology-based field monitoring methods will contribute significantly to understanding the causes underpinning pollinator declines and, hence, developing effective solutions for dealing with this global challenge.

Although it is now widely accepted that speciation can occur in the face of continuous gene flow, with little or no spatial separation, the mechanisms and genomic architectures that permit such divergence are still debated. Here, we examined speciation in the face of gene flow in the Howea palms of Lord Howe Island, Australia. We built a genetic map using a novel method applicable to long-lived tree species, combining it with double digest restriction site-associated DNA sequencing of multiple individuals. Based upon various metrics, we detected 46 highly differentiated regions throughout the genome, four of which contained genes with functions that are particularly relevant to the speciation scenario for Howea, specifically salt and drought tolerance.

The genome evolution of ferns has been considered to be relatively static compared with angiosperms. In this study, we analyse genome size data and chromosome numbers in a phylogenetic framework to explore three hypotheses: the correlation of genome size and chromosome number, the origin of modern ferns from ancestors with high chromosome numbers, and the occurrence of several whole-genome duplications during the evolution of ferns. To achieve this, we generated new genome size data, increasing the percentage of fern species with genome sizes estimated to 2.8% of extant diversity, and ensuring a comprehensive phylogenetic coverage including at least three species from each fern order. Genome size was correlated with chromosome number across all ferns despite some substantial variation in both traits. We observed a trend towards conservation of the amount of DNA per chromosome, although Osmundaceae and Psilotaceae have substantially larger chromosomes. Reconstruction of the ancestral genome traits suggested that the earliest ferns were already characterized by possessing high chromosome numbers and that the earliest divergences in ferns were correlated with substantial karyological changes. Evidence for repeated whole-genome duplications was found across the phylogeny. Fern genomes tend to evolve slowly, albeit genome rearrangements occur in some clades.