Phylogenetic systematics of Vigna sensu stricto in the context of Physostigma and allies.

PREMISE: Vigna includes economically vital crops and wild species. Molecular systematic studies of Vigna species resulted in generic segregates of many New World (NW) species. However, limited Old World (OW) sampling left questions regarding inter- and intraspecific relationships in Vigna s.s. METHODS: African species, including the putative sister genus Physostigma, were comprehensively sampled within the context of NW relatives. Maximum likelihood and Bayesian inference analyses of the chloroplast matK-trnK and nuclear ribosomal ITS/5.8 S (ITS) DNA regions were undertaken to resolve OW Vigna taxonomic questions. Divergence dates were estimated using BEAST to date key nodes in the phylogeny. RESULTS: Analyses of matK and ITS data supported five clades of Vigna s.s.: subg. Lasiospron, a reduced subg. Vigna, subg. Haydonia, subg. Ceratotropis, an enlarged subg. Plectrotropis, and a clade including V. kirkii and V. stenophylla. Genome size estimates of 601 Mb for V. kirkii are near the overall mean of the genus, whereas V. stenophylla had a larger genome (810 Mb), similar to some Vigna subg. Ceratotropis or Plectrotropis species. CONCLUSIONS: Former subg. Vigna is reduced to yellow- and blue-flowered species and subg. Plectrotropis is enlarged to mostly all white-, pink-, and purple-flowered species. The age of the split between NW and OW Vigna lineages is ~6-7 Myr. Genome size estimates cannot rule out a polyploid or hybrid origin for V. stenophylla, potentially involving extinct lineage ancestors of Vigna subg. Ceratotropis or Plectrotropis, as indicated by network and phylogenetic analyses. Taxonomic revisions are suggested based on these results.

Annual-perennial lifespan variation in Chaenactis douglasii suggests a drought escape strategy in warm-arid environments.

PREMISE: Intraspecific variation in drought resistance traits, such as drought escape, appear to be frequent within wild, ruderal forb species. Understanding how these traits are arrayed across the landscape, particularly in association with climate, is critical to developing forbs for wildland restoration programs. Use of forbs is requisite for maintaining biological diversity and ecological services. METHODS: Using 6074 greenhouse-grown Chaenactis douglasii seedlings from 95 wild, seed-sourced populations across the western United States, we recorded bolting phenology and estimated genome size using flow cytometry. Mixed-effects regression models were used to assess whether climate of seed origin was predictive for bolting phenology and genome size. RESULTS: Variation in bolting, reflecting an annual vs. perennial lifespan in this species, was observed in 8.7% of the plants, with bolting plants disproportionately occurring in locations with warm, arid climates. Populations with increasing heat and aridity were positively correlated with observed bolting (r = 0.61, p < 0.0001). About one-third (22%) of the total (61%) lifespan variation was attributed to seed source climate and annual heat moisture index, a measure of aridity. Genome size had no significant effect on bolting. Projected climate modeling for mid-century (2041-2070) supports an increasing occurrence of annual lifespan. CONCLUSIONS: Our analyses support a drought escape, bet-hedging strategy in C. douglasii. Populations exposed to greater aridity exhibited a higher proportion of individuals with an annual lifespan. Drought escape leading to an annual lifespan can affect how seeds are propagated and deployed for climate-informed restoration.

The smallest angiosperm genomes may be the price for effective traps of bladderworts.

BACKGROUND: Species of the carnivorous family Lentibulariaceae exhibit the smallest genomes in flowering plants. We explored the hypothesis that their minute genomes result from the unique mitochondrial cytochrome c oxidase (COX) mutation. The mutation may boost mitochondrial efficiency, which is especially useful for suction-bladder traps of Utricularia, but also increase DNA-damaging reactive oxygen species, leading to genome shrinkage through deletion-biased DNA repair. We aimed to explore this mutation's impact on genome size, providing insights into genetic mutation roles in plant genome evolution under environmental pressures. METHODS: We compiled and measured genome and mean chromosome sizes for 127 and 67 species, respectively, representing all three genera (Genlisea, Pinguicula, and Utricularia) of Lentibulariaceae. We also isolated and analyzed COX sequences to detect the mutation. Through phylogenetic regressions and Ornstein-Uhlenbeck models of trait evolution, we assessed the impact of the COX mutation on the genome and chromosome sizes across the family. RESULTS: Our findings reveal significant correlations between the COX mutations and smaller genome and chromosome sizes. Specifically, species carrying the ancestral COX sequence exhibited larger genomes and chromosomes than those with the mutation. This evidence supports the notion that the COX mutation contributes to genome downsizing, with statistical analyses confirming a directional evolution towards smaller genomes in species harboring these mutations. CONCLUSIONS: Our study confirms that the COX mutation in Lentibulariaceae is associated with genome downsizing, likely driven by increased reactive oxygen species production and subsequent DNA damage requiring deletion-biased repair mechanisms. While boosting mitochondrial energy output, this genetic mutation compromises genome integrity and may potentially affect recombination rates, illustrating a complex trade-off between evolutionary advantages and disadvantages. Our results highlight the intricate processes by which genetic mutations and environmental pressures shape genome size evolution in carnivorous plants.

Expansions and contractions of repetitive DNA elements reveal contrasting evolutionary responses to the polyploid genome shock hypothesis in Brachypodium model grasses.

Brachypodium grass species have been selected as model plants for functional genomics of grass crops, and to elucidate the origins of allopolyploidy and perenniality in monocots, due to their small genome sizes and feasibility of cultivation. However, genome sizes differ greatly between diploid or polyploid Brachypodium lineages. We have used genome skimming sequencing data to uncover the composition, abundance, and phylogenetic value of repetitive elements in 44 representatives of the major Brachypodium lineages and cytotypes. We also aimed to test the possible mechanisms and consequences of the "polyploid genome shock hypothesis" (PGSH) under three different evolutionary scenarios of variation in repeats and genome sizes of Brachypodium allopolyploids. Our data indicated that the proportion of the genome covered by the repeatome in the Brachypodium species showed a 3.3-fold difference between the highest content of B. mexicanum-4x (67.97%) and the lowest of B. stacei-2x (20.77%), and that changes in the sizes of their genomes were a consequence of gains or losses in their repeat elements. LTR-Retand and Tekay retrotransposons were the most frequent repeat elements in the Brachypodium genomes, while Ogre retrotransposons were found exclusively in B. mexicanum. The repeatome phylogenetic network showed a high topological congruence with plastome and nuclear rDNA and transcriptome trees, differentiating the ancestral outcore lineages from the recently evolved core-perennial lineages. The 5S rDNA graph topologies had a strong match with the ploidy levels and nature of the subgenomes of the Brachypodium polyploids. The core-perennial B. sylvaticum presents a large repeatome and characteristics of a potential post-polyploid diploidized origin. Our study evidenced that expansions and contractions in the repeatome were responsible for the three contrasting responses to the PGSH. The exacerbated genome expansion of the ancestral allotetraploid B. mexicanum was a consequence of chromosome-wide proliferation of TEs and not of WGD, the additive repeatome pattern of young allotetraploid B. hybridum of stabilized post-WGD genome evolution, and the genomecontraction of recent core-perennials polyploids (B. pinnatum, B. phoenicoides) of repeat losses through recombination of these highly hybridizing lineages. Our analyses have contributed to unraveling the evolution of the repeatome and the genome size variation in model Brachypodium grasses.

Repetitive element expansions contribute to genome size gigantism in Pamphagidae: A comparative study (Orthoptera, Acridoidea).

Pamphagidae is a family of Acridoidea that inhabits the desert steppes of Eurasia and Africa. This study employed flow cytometry to estimate the genome size of eight species in the Pamphagidae. The results indicate that the genome size of the eight species ranged from 13.88 pg to 14.66 pg, with an average of 14.26 pg. This is the largest average genome size recorded for the Orthoptera families, as well as for the entire Insecta. Furthermore, the study explored the role of repetitive sequences in the genome, including their evolutionary dynamics and activity, using low-coverage next-generation sequencing data. The genome is composed of 14 different types of repetitive sequences, which collectively make up between 59.9% and 68.17% of the total genome. The Pamphagidae family displays high levels of transposable element (TE) activity, with the number of TEs increasing and accumulating since the family's emergence. The study found that the types of repetitive sequences contributing to the TE outburst events are similar across species. Additionally, the study identified unique repetitive elements for each species. The differences in repetitive sequences among the eight Pamphagidae species correspond to their phylogenetic relationships. The study sheds new light on genome gigantism in the Pamphagidae and provides insight into the correlation between genome size and repetitive sequences within the family.

Influence of polyploidy on morphology and distribution of the Cypress Spurge (Euphorbia cyparissias, Euphorbiaceae).

Polyploidy can cause differences in phenotypic and physiological traits among different cytotypes of the same species. Polyploids may have larger organs or occupy different ecological niches than their diploid counterparts, therefore they are hypothesized to have larger distributions or prosper in stressful environments, such as higher elevations. The Cypress spurge (Euphorbia cyparissias L.; Euphorbiaceae) is a widespread European heteroploid species including di- (2x), tetra- (4x) and hexaploid (6x) cytotypes. We tested the hypotheses that polyploids are more widespread and more abundant at higher elevations and have larger organs than their diploid ancestors in the case of E. cyparissias. We also analysed whether genome downsizing had occurred after polyploidisation. We conducted a comprehensive geographic sampling of 617 populations of E. cyparissias throughout Europe. We estimated their relative genome size using flow cytometry and inferred ploidy level of each population. We scored 13 morphological traits of vegetative and seed characters and performed statistical analyses. The study indicates that polyploidisation facilitated colonisation of new areas in E. cyparissias, where the tetraploids are most widespread, whereas the diploids are limited to putative Pleistocene refugia, mostly in southern Europe. On the other hand, the three ploidies do not differ in their elevational distribution. Although some quantitative morphological traits exhibited an increasing trend with increasing ploidy, most traits did not differ significantly among the three ploidies, and there was no overall phenotypic differentiation among them. Given that individuals of different ploidies thrive in similar habitats across the same elevations, we suggest that ecological segregation following polyploidisation is a more important trigger for morphological differentiation than polyploidisation itself in autopolyploid plants. The study demonstrates that polyploidisation can be crucial for the colonisation of new areas and for range expansion, but it does not necessarily influence elevational distribution nor confer a different phenotype.

Deciphering Pteronia's evolution in the Cape Floristic Region: A comprehensive study disputes polyploid deficiency and affirms diploid radiation.

The Greater Cape Floristic Region (GCFR) is renowned for its exceptional biodiversity, accommodating over 11 000 plant species, notable degree of endemism, and substantial diversification within limited plant lineages, a phenomenon ascribed to historical radiation events. While both abiotic and biotic factors contribute to this diversification, comprehensive genomic alterations, recognized as pivotal in the diversification of angiosperms, are perceived as uncommon. This investigation focuses on the genus Pteronia, a prominent representative of the Asteraceae family in the GCFR. Employing NGS-based HybSeq and RADSeq methodologies, flow cytometry, karyology, and ecological modeling, we scrutinize the intricacies of its polyploid evolution. Phylogenetic reconstructions using 951 low-copy nuclear genes confirm Pteronia as a well-supported, distinct clade within the tribe Astereae. The ingroup displays a structure indicative of rapid radiation likely antedating polyploid establishment, with the two main groups demarcated by their presence or absence in the fynbos biome. Genome size analysis encompasses 1293 individuals across 347 populations, elucidating significant variation ranging from 6.1 to 34.2 pg (2C-value). Pteronia demonstrates substantially large genome sizes within Astereae and phanerophytes. Polyploidy is identified in 31% of the studied species, with four discerned ploidy levels (2x, 4x, 6x, 8x). Cytotypes exhibit marked distinctions in environmental traits, influencing their distribution across biomes and augmenting their niche differentiation. These revelations challenge the presumed scarcity of polyploidy in the Cape flora, underscoring the imperative need for detailed population studies. The intricate evolutionary history of Pteronia, characterized by recent polyploidy and genome size variation, contributes substantially to the comprehension of diversification patterns within the GCFR biodiversity hotspot.

Synthetic lethality and the minimal genome size problem.

Gene knockout studies suggest that ~300 genes in a bacterial genome and ~1,100 genes in a yeast genome cannot be deleted without loss of viability. These single-gene knockout experiments do not account for negative genetic interactions, when two or more genes can each be deleted without effect, but their joint deletion is lethal. Thus, large-scale single-gene deletion studies underestimate the size of a minimal gene set compatible with cell survival. In yeast Saccharomyces cerevisiae, the viability of all possible deletions of gene pairs (2-tuples), and of some deletions of gene triplets (3-tuples), has been experimentally tested. To estimate the size of a yeast minimal genome from that data, we first established that finding the size of a minimal gene set is equivalent to finding the minimum vertex cover in the lethality (hyper)graph, where the vertices are genes and (hyper)edges connect k-tuples of genes whose joint deletion is lethal. Using the Lovász-Johnson-Chvatal greedy approximation algorithm, we computed the minimum vertex cover of the synthetic-lethal 2-tuples graph to be 1,723 genes. We next simulated the genetic interactions in 3-tuples, extrapolating from the existing triplet sample, and again estimated minimum vertex covers. The size of a minimal gene set in yeast rapidly approaches the size of the entire genome even when considering only synthetic lethalities in k-tuples with small k. In contrast, several studies reported successful experimental reductions of yeast and bacterial genomes by simultaneous deletions of hundreds of genes, without eliciting synthetic lethality. We discuss possible reasons for this apparent contradiction.IMPORTANCEHow can we estimate the smallest number of genes sufficient for a unicellular organism to survive on a rich medium? One approach is to remove genes one at a time and count how many of such deletion strains are unable to grow. However, the single-gene knockout data are insufficient, because joint gene deletions may result in negative genetic interactions, also known as synthetic lethality. We used a technique from graph theory to estimate the size of minimal yeast genome from partial data on synthetic lethality. The number of potential synthetic lethal interactions grows very fast when multiple genes are deleted, revealing a paradoxical contrast with the experimental reductions of yeast genome by ~100 genes, and of bacterial genomes by several hundreds of genes.

Genome size and chromosome number are critical metrics for accurate genome assembly assessment in Eukaryota.

The number of genome assemblies has rapidly increased in recent history, with NCBI databases reaching over 41,000 eukaryotic genome assemblies across about 2,300 species. Increases in read length and improvements in assembly algorithms have led to increased contiguity and larger genome assemblies. While this number of assemblies is impressive, only about a third of these assemblies have corresponding genome size estimations for their respective species on publicly available databases. In this paper, genome assemblies are assessed regarding their total size compared to their respective publicly available genome size estimations. These deviations in size are assessed related to genome size, kingdom, sequencing platform, and standard assembly metrics, such as N50 and BUSCO values. A large proportion of assemblies deviate from their estimated genome size by more than 10%, with increasing deviations in size with increased genome size, suggesting nonprotein coding and structural DNA may be to blame. Modest differences in performance of sequencing platforms are noted as well. While standard metrics of genome assessment are more likely to indicate an assembly approaching the estimated genome size, much of the variation in this deviation in size is not explained with these raw metrics. A new, proportional N50 metric is proposed, in which N50 values are made relative to the average chromosome size of each species. This new metric has a stronger relationship with complete genome assemblies and, due to its proportional nature, allows for a more direct comparison across assemblies for genomes with variation in sizes and architectures.

Extreme overall mushroom genome expansion in Mycena s.s. irrespective of plant hosts or substrate specializations.

Mycena s.s. is a ubiquitous mushroom genus whose members degrade multiple dead plant substrates and opportunistically invade living plant roots. Having sequenced the nuclear genomes of 24 Mycena species, we find them to defy the expected patterns for fungi based on both their traditionally perceived saprotrophic ecology and substrate specializations. Mycena displayed massive genome expansions overall affecting all gene families, driven by novel gene family emergence, gene duplications, enlarged secretomes encoding polysaccharide degradation enzymes, transposable element (TE) proliferation, and horizontal gene transfers. Mainly due to TE proliferation, Arctic Mycena species display genomes of up to 502 Mbp (2-8× the temperate Mycena), the largest among mushroom-forming Agaricomycetes, indicating a possible evolutionary convergence to genomic expansions sometimes seen in Arctic plants. Overall, Mycena show highly unusual, varied mosaic-like genomic structures adaptable to multiple lifestyles, providing genomic illustration for the growing realization that fungal niche adaptations can be far more fluid than traditionally believed.

Genomic Gigantism is not Associated with Reduced Selection Efficiency in Neotropical Salamanders.

Genome size variation in eukaryotes has myriad effects on organismal biology from the genomic to whole-organism level. Large genome size may be associated with lower selection efficiency because lower effective population sizes allow fixation of deleterious mutations via genetic drift, increasing genome size and decreasing selection efficiency. Because of a hypothesized negative relationship between genome size and recombination rate per base pair, increased genome size could also increase the effect of linked selection in the genome, decreasing the efficiency with which natural selection can fix or remove mutations. We used a transcriptomic dataset of 15 and a subset of six Neotropical salamander species ranging in genome size from 12 to 87 pg to study the relationship between genome size and efficiency of selection. We estimated dN/dS of salamanders with small and large genomes and tested for relaxation of selection in the larger genomes. Contrary to our expectations, we did not find a significant relationship between genome size and selection efficiency or strong evidence for higher dN/dS values in species with larger genomes for either species set. We also found little evidence for relaxation of selection in species with larger genomes. A positive correlation between genome size and range size (a proxy of population size) in this group disagrees with predictions of stronger drift in species with larger genomes. Our results highlight the complex interactions between the many forces shaping genomic variation in organisms with genomic gigantism.

Corrigendum: LocoGSE, a sequence-based genome size estimator for plants.

[This corrects the article DOI: 10.3389/fpls.2024.1328966.].

Comparative mitochondrial genomics in Nematoda reveal astonishing variation in compositional biases and substitution rates indicative of multi-level selection.

BACKGROUND: Nematodes are the most abundant and diverse metazoans on Earth, and are known to significantly affect ecosystem functioning. A better understanding of their biology and ecology, including potential adaptations to diverse habitats and lifestyles, is key to understanding their response to global change scenarios. Mitochondrial genomes offer high species level characterization, low cost of sequencing, and an ease of data handling that can provide insights into nematode evolutionary pressures. RESULTS: Generally, nematode mitochondrial genomes exhibited similar structural characteristics (e.g., gene size and GC content), but displayed remarkable variability around these general patterns. Compositional strand biases showed strong codon position specific G skews and relationships with nematode life traits (especially parasitic feeding habits) equal to or greater than with predicted phylogeny. On average, nematode mitochondrial genomes showed low non-synonymous substitution rates, but also high clade specific deviations from these means. Despite the presence of significant mutational saturation, non-synonymous (dN) and synonymous (dS) substitution rates could still be significantly explained by feeding habit and/or habitat. Low ratios of dN:dS rates, particularly associated with the parasitic lifestyles, suggested the presence of strong purifying selection. CONCLUSIONS: Nematode mitochondrial genomes demonstrated a capacity to accumulate diversity in composition, structure, and content while still maintaining functional genes. Moreover, they demonstrated a capacity for rapid evolutionary change pointing to a potential interaction between multi-level selection pressures and rapid evolution. In conclusion, this study helps establish a background for our understanding of the potential evolutionary pressures shaping nematode mitochondrial genomes, while outlining likely routes of future inquiry.

An adventurous journey toward and away from fern apomixis: Insights from genome size and spore abortion patterns.

PREMISE: Apomixis in ferns is relatively common and obligatory. Sterile hybrids may restore fertility via apomixis at a cost of long-term genetic stagnation. In this study, we outlined apomixis as a possible temporary phase leading to sexuality and analyzed factors relating to transitioning to and away from apomixis, such as unreduced and reduced spore formation in apomict and apo-sex hybrid ferns. METHODS: We analyzed the genome size of 15 fern species or hybrids ("taxa") via flow cytometry. The number of reduced and unreduced gametophytes was established as a proxy for viable spore formation of either type. We also calculated the spore abortion ratio (sign of reduced spores) in several taxa, including the apo-sex hybrid Dryopteris × critica and its 16 apomictically formed offspring. RESULTS: Four of 15 sampled taxa yielded offspring variable in genome size. Specifically, each variable taxon formed one viable reduced plant among 12-451 sampled gametophytes per taxon. Thus, haploid spore formation in the studied apomicts was very rare but possible. Spore abortion analyses indicated gradually decreasing abortion (haploid spore formation) over time. In Dryopteris × critica, abortion decreased from 93.8% to mean 89.5% in one generation. CONCLUSIONS: Our results support apomixis as a transitionary phase toward sexuality. Newly formed apomicts hybridize with sexual relatives and continue to form haploid spores early on. Thus, they may get the genomic content necessary for regular meiosis and restore sexuality. If the missing relative goes extinct, the lineage gets locked into apomixis as may be the case with the Dryopteris affinis complex.

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.

Ancient hybridization and repetitive element proliferation in the evolutionary history of the monocot genus Amomum (Zingiberaceae).

Genome size variation is a crucial aspect of plant evolution, influenced by a complex interplay of factors. Repetitive elements, which are fundamental components of genomic architecture, often play a role in genome expansion by selectively amplifying specific repeat motifs. This study focuses on Amomum, a genus in the ginger family (Zingiberaceae), known for its 4.4-fold variation in genome size. Using a robust methodology involving PhyloNet reconstruction, RepeatExplorer clustering, and repeat similarity-based phylogenetic network construction, we investigated the repeatome composition, analyzed repeat dynamics, and identified potential hybridization events within the genus. Our analysis confirmed the presence of four major infrageneric clades (A-D) within Amomum, with clades A-C exclusively comprising diploid species (2n = 48) and clade D encompassing both diploid and tetraploid species (2n = 48 and 96). We observed an increase in the repeat content within the genus, ranging from 84% to 89%, compared to outgroup species with 75% of the repeatome. The SIRE lineage of the Ty1-Copia repeat superfamily was prevalent in most analyzed ingroup genomes. We identified significant difference in repeatome structure between the basal Amomum clades (A, B, C) and the most diverged clade D. Our investigation revealed evidence of ancient hybridization events within Amomum, coinciding with a substantial proliferation of multiple repeat groups. This finding supports the hypothesis that ancient hybridization is a driving force in the genomic evolution of Amomum. Furthermore, we contextualize our findings within the broader context of genome size variations and repeatome dynamics observed across major monocot lineages. This study enhances our understanding of evolutionary processes within monocots by highlighting the crucial roles of repetitive elements in shaping genome size and suggesting the mechanisms that drive these changes.

Distinction of chia varieties in vivo and in vitro based on the flow cytometry and rosmarinic acid production.

Flow cytometry has made a significant contribution to the study of several complex fundamental mechanisms in plant cytogenetics, becoming a useful analytical tool to understand several mechanisms and processes underlying plant growth, development, and function. In this study, the genome size, DNA ploidy level, and A-T/G-C ratio were measured for the first time for two genotypes of chia, Salvia hispanica, an herbaceous plant commonly used in phytotherapy and nutrition. This study also evaluated, for the first time by flow cytometry, the capacity to produce organic acids of tissues stained with LysoTracker Deep Red after elicitation with either yeast extract or cadmium chloride. Rosmarinic acid content differed between the two chia varieties treated with different elicitor concentrations, compared with non-elicited plant material. Elicited tissues of both varieties contained a higher content of rosmarinic acid compared with non-elicited cultures, and cadmium chloride at 500 µM was much better than that at 1000 µM, which led to plant death. For both genotypes, a dose-response was observed with yeast extract, as the higher the concentration of elicitor used, the higher rosmarinic acid content, resulting also in better results and a higher content of rosmarinic acid compared with cadmium chloride. This study demonstrates that flow cytometry may be used as a taxonomy tool, to distinguish among very close genotypses of a given species and, for the first time in plants, that this approach can also be put to profit for a characterization of the cytoplasmic acid phase and the concomitant production of secondary metabolites of interest in vitro, with or without elicitation. KEY POINTS: • Genome size, ploidy level, A-T/G-C ratio, and cytoplasm acid phase of S. hispanica • Cytometry study of cytoplasm acid phase of LysoTracker Deep Red-stained plant cells • Yeast extract or cadmium chloride elicited rosmarinic acid production of chia tissues.

Genome size variation in Cape schoenoid sedges (Schoeneae) and its ecophysiological consequences.

PREMISE: Increases in genome size in plants-often associated with larger, low-density stomata and greater water-use efficiency (WUE)-could affect plant ecophysiological and hydraulic function. Variation in plant genome size is often due to polyploidy, having occurred repeatedly in the austral sedge genus Schoenus in the Cape Floristic Region (CFR), while species in the other major schoenoid genus in the region, Tetraria, have smaller genomes. Comparing these genera is useful as they co-occur at the landscape level, under broadly similar bioclimatic conditions. We hypothesized that CFR Schoenus have greater WUE, with lower maximum stomatal conductance (gwmax) imposed by larger, less-dense stomata. METHODS: We investigated relationships between genome size and stomatal parameters in a phylogenetic context, reconstructing a phylogeny of CFR-occurring Schoeneae (Cyperaceae). Species' stomatal and functional traits were measured from field-collected and herbarium specimens. Carbon stable isotopes were used as an index of WUE. Genome size was derived from flow-cytometric measurements of leafy shoots. RESULTS: Evolutionary regressions demonstrated that stomatal size and density covary with genome size, positively and negatively, respectively, with genome size explaining 72-75% of the variation in stomatal size. Larger-genomed species had lower gwmax and C:N ratios, particularly in culms. CONCLUSIONS: We interpret differences in vegetative physiology between the genera as evidence of more-conservative strategies in CFR Schoenus compared to the more-acquisitive Tetraria. Because Schoenus have smaller, reduced leaves, they likely rely more on culm photosynthesis than Tetraria. Across the CFR Schoeneae, ecophysiology correlates with genome size, but confounding sources of trait variation limit inferences about causal relationships between traits.

Diversity of genome size and chromosome number in homothallic and heterothallic strains of the Closterium peracerosum-strigosum-littorale complex (Desmidiales, Zygnematophyceae, Streptophyta).

The evolutionary transitions of mating systems between outcrossing and self-fertilization are often suggested to associate with the cytological and genomic changes, but the empirical reports are limited in multicellular organisms. Here we used the unicellular zygnematophycean algae, the Closterium peracerosum-strigosum-littorale (C. psl.) complex, to address whether genomic properties such as genome sizes and chromosome numbers are associated with mating system transitions between homothallism (self-fertility) and heterothallism (self-sterility). Phylogenetic analysis revealed the polyphyly of homothallic strains, suggesting multiple transitions between homothallism and heterothallism in the C. psl. complex. Flow cytometry analysis identified a more than 2-fold genome size variation, ranging from 0.53 to 1.42 Gbp, which was positively correlated with chromosome number variation between strains. Although we did not find consistent trends in genome size change and mating system transitions, the mean chromosome sizes tend to be smaller in homothallic strains than in their relative heterothallic strains. This result suggests that homothallic strains possibly have more fragmented chromosomes, which is consistent with the argument that self-fertilizing populations may tolerate more chromosomal rearrangements.

Remarkably High Repeat Content in the Genomes of Sparrows: The Importance of Genome Assembly Completeness for Transposable Element Discovery.

Transposable elements (TE) play critical roles in shaping genome evolution. Highly repetitive TE sequences are also a major source of assembly gaps making it difficult to fully understand the impact of these elements on host genomes. The increased capacity of long-read sequencing technologies to span highly repetitive regions promises to provide new insights into patterns of TE activity across diverse taxa. Here we report the generation of highly contiguous reference genomes using PacBio long-read and Omni-C technologies for three species of Passerellidae sparrow. We compared these assemblies to three chromosome-level sparrow assemblies and nine other sparrow assemblies generated using a variety of short- and long-read technologies. All long-read based assemblies were longer (range: 1.12 to 1.41 Gb) than short-read assemblies (0.91 to 1.08 Gb) and assembly length was strongly correlated with the amount of repeat content. Repeat content for Bell's sparrow (31.2% of genome) was the highest level ever reported within the order Passeriformes, which comprises over half of avian diversity. The highest levels of repeat content (79.2% to 93.7%) were found on the W chromosome relative to other regions of the genome. Finally, we show that proliferation of different TE classes varied even among species with similar levels of repeat content. These patterns support a dynamic model of TE expansion and contraction even in a clade where TEs were once thought to be fairly depauperate and static. Our work highlights how the resolution of difficult-to-assemble regions of the genome with new sequencing technologies promises to transform our understanding of avian genome evolution.