Low-frequency somatic mutations are heritable in tropical trees Dicorynia guianensis and Sextonia rubra.

Somatic mutations potentially play a role in plant evolution, but common expectations pertaining to plant somatic mutations remain insufficiently tested. Unlike in most animals, the plant germline is assumed to be set aside late in development, leading to the expectation that plants accumulate somatic mutations along growth. Therefore, several predictions were made on the fate of somatic mutations: mutations have generally low frequency in plant tissues; mutations at high frequency have a higher chance of intergenerational transmission; branching topology of the tree dictates mutation distribution; and exposure to UV (ultraviolet) radiation increases mutagenesis. To provide insights into mutation accumulation and transmission in plants, we produced two high-quality reference genomes and a unique dataset of 60 high-coverage whole-genome sequences of two tropical tree species, Dicorynia guianensis (Fabaceae) and Sextonia rubra (Lauraceae). We identified 15,066 de novo somatic mutations in D. guianensis and 3,208 in S. rubra, surprisingly almost all found at low frequency. We demonstrate that 1) low-frequency mutations can be transmitted to the next generation; 2) mutation phylogenies deviate from the branching topology of the tree; and 3) mutation rates and mutation spectra are not demonstrably affected by differences in UV exposure. Altogether, our results suggest far more complex links between plant growth, aging, UV exposure, and mutation rates than commonly thought.

Polyploid goldback and silverback ferns (Pentagramma) occupy a wider, colder, and wetter bioclimatic niche than diploid counterparts.

PREMISE: The western North American fern genus Pentagramma (Pteridaceae) is characterized by complex patterns of ploidy variation, an understanding of which is critical to comprehending both the evolutionary processes within the genus and its current diversity. METHODS: We undertook a cytogeographic study across the range of the genus, using a combination of chromosome counts and flow cytometry to infer ploidy level. Bioclimatic variables and elevation were used to compare niches. RESULTS: We found that diploids and tetraploids are common and widespread, and triploids are rare and sporadic; in contrast with genome size inferences in earlier studies, no hexaploids were found. Diploids and tetraploids show different geographic ranges: only tetraploids were found in the northernmost portion of the range (Washington, Oregon, and British Columbia) and only diploids were found in the Sierra Nevada of California. Diploid, triploid, and tetraploid cytotypes were found to co-occur in relatively few localities: in the southern (San Diego County, California) and desert Southwest (Arizona) parts of the range, and along the Pacific Coast of California. CONCLUSIONS: Tetraploids occupy a wider bioclimatic niche than diploids both within P. triangularis and at the genus-wide scale. It is unknown whether the wider niche of tetraploids is due to their expansion upon the diploid niche, if diploids have contracted their niche due to competition or changing abiotic conditions, or if this wider niche occupancy is due to multiple origins of tetraploids.

Best practices for instrument settings and raw data analysis in plant flow cytometry.

Flow cytometry (FCM) is now the most widely used method to determine ploidy levels and genome size of plants. To get reliable estimates and allow reproducibility of measurements, the methodology should be standardized and follow the best practices in the field. In this article, we discuss instrument calibration and quality control and various instrument and acquisition settings (parameters, flow rate, number of events, scales, use of discriminators, peak positions). These settings must be decided before measurements because they determine the amount and quality of the data and thus influence all downstream analyses. We describe the two main approaches to raw data analysis (gating and histogram modeling), and we discuss their advantages and disadvantages. Finally, we provide a summary of best practice recommendations for data acquisition and raw data analysis in plant FCM.

Hybridization and introgression are prevalent in Southern European Erysimum (Brassicaceae) species.

BACKGROUND AND AIMS: Hybridization is a common and important force in plant evolution. One of its outcomes is introgression - the transfer of small genomic regions from one taxon to another by hybridization and repeated backcrossing. This process is believed to be common in glacial refugia, where range expansions and contractions can lead to cycles of sympatry and isolation, creating conditions for extensive hybridization and introgression. Polyploidization is another genome-wide process with a major influence on plant evolution. Both hybridization and polyploidization can have complex effects on plant evolution. However, these effects are often difficult to understand in recently evolved species complexes. METHODS: We combined flow cytometry, analyses of transcriptomic sequences and pollen tube growth assays to investigate the consequences of polyploidization, hybridization and introgression on the recent evolution of several Erysimum (Brassicaceae) species from the South of the Iberian Peninsula, a well-known glacial refugium. This species complex differentiated in the last 2 million years, and its evolution has been hypothesized to be determined mainly by polyploidization, interspecific hybridization and introgression. KEY RESULTS: Our results support a scenario of widespread hybridization involving both extant and 'ghost' taxa. Several taxa studied here, most notably those with purple corollas, are polyploids, probably of allopolyploid origin. Moreover, hybridization in this group might be an ongoing phenomenon, as pre-zygotic barriers appeared weak in many cases. CONCLUSIONS: The evolution of Erysimum spp. has been determined by hybridization to a large extent. Species with purple (polyploids) and yellow flowers (mostly diploid) exhibit a strong signature of introgression in their genomes, indicating that hybridization occurred regardless of colour and across ploidy levels. Although the adaptive value of such genomic exchanges remains unclear, our results demonstrate the significance of hybridization for plant diversification, which should be taken into account when studying plant evolution.

Ploidy effects on the relationship between floral phenotype, reproductive investment, and fitness in an autogamous species complex.

PREMISE: The relationships between reproductive investment, phenotype, and fitness have been broadly studied in cross-pollinated plants in contrast to selfing species, which are considered less interesting in this area because they are supposed to be a dead end in any evolutionary pathway. Still, selfing plants are unique systems to study these questions since the position of reproductive structures and traits related to flower size play an important role in female and male pollination success. METHODS: Erysimum incanum s.l. is a selfing species complex that has three levels of ploidy (diploids, tetraploids, and hexaploids) and traits that are typically associated with the selfing syndrome. Here, we used 1609 plants belonging to these three ploidies to characterize the floral phenotype and spatial configuration of reproductive structures, reproductive investment (pollen and ovule production), and plant fitness. Then, we used structural equation modelling to analyze the relationship between all these variables across ploidy levels. RESULTS: An increase in ploidy level leads to bigger flowers with anthers exserted farther and more pollen and ovules. In addition, hexaploid plants had higher absolute values for herkogamy, which is positively correlated with fitness. Ovule production significantly mediated the natural selection acting on different phenotypic traits and pollen production, a pattern that is maintained across ploidies. CONCLUSIONS: The changes in floral phenotypes, reproductive investment, and fitness with ploidy level suggest that genome duplication can be a driver for transitions in reproductive strategy by modifying the investment in pollen and ovules and linking them with plant phenotype and fitness.

Plant material selection, collection, preservation, and storage for nuclear DNA content estimation.

In theory, any plant tissue providing intact nuclei in sufficient quantity is suitable for nuclear DNA content estimation using flow cytometry (FCM). While this certainly opens a wide variety of possible applications of FCM, especially when compared to classical karyological techniques restricted to tissues with active cell division, tissue selection and quality may directly affect the precision (and sometimes even reliability) of FCM measurements. It is usually convenient to first consider the goals of the study to either aim for the highest possible accuracy of estimates (e.g., for inferring genome size, detecting homoploid intraspecific genome size variation, aneuploidy, among others), or to decide that histograms of reasonable resolution provide sufficient information (e.g., ploidy level screening within a single model species). Here, a set of best practices guidelines for selecting the optimal plant tissue for FCM analysis, sampling of material, and material preservation and storage are provided. In addition, factors potentially compromising the quality of FCM estimates of nuclear DNA content and data interpretation are discussed.

Application-based guidelines for best practices in plant flow cytometry.

Flow cytometry (FCM) is currently the most widely-used method to establish nuclear DNA content in plants. Since simple, 1-3-parameter, flow cytometers, which are sufficient for most plant applications, are commercially available at a reasonable price, the number of laboratories equipped with these instruments, and consequently new FCM users, has greatly increased over the last decade. This paper meets an urgent need for comprehensive recommendations for best practices in FCM for different plant science applications. We discuss advantages and limitations of establishing plant ploidy, genome size, DNA base composition, cell cycle activity, and level of endoreduplication. Applications of such measurements in plant systematics, ecology, molecular biology research, reproduction biology, tissue cultures, plant breeding, and seed sciences are described. Advice is included on how to obtain accurate and reliable results, as well as how to manage troubleshooting that may occur during sample preparation, cytometric measurements, and data handling. Each section is followed by best practice recommendations; tips as to what specific information should be provided in FCM papers are also provided.

The impact of habitat loss on pollination services for a threatened dune endemic plant.

Habitat loss is currently a major threat to biodiversity, affecting species interactions, such as plant-pollinator interactions. This is particularly important in self-incompatible plants relying on pollinators to reproduce and sustain their populations. Here, we evaluated how habitat loss affects the pollination system, plant individual-pollinator species interaction network, and plant reproductive fitness of the self-incompatible Jasione maritima var. sabularia, a threatened taxon from dune systems. This plant is a pollinator generalist, visited by 108 species from distinct taxonomic groups. Results suggest that increasing habitat loss led to a significant decline in pollinator richness, increased pollen limitation, and a decrease in reproductive fitness of J. maritima var. sabularia. Visitation rate per individual did not significantly change with available area, indicating that the quality of pollen differed across populations. The topology of the network between J. maritima var. sabularia individuals and its pollinator species did not change, which may be attributed to the stability in the core of pollinator species. This suggests that the lower fitness of plants with increasing habitat degradation may be explained not only by the lower richness of peripheral pollinators but also by the genetic structure of the plant populations, as there is a possible higher transference of less quality pollen by pollinators, ultimately compromising the persistence of plant populations. Our study highlights the need of future studies to integrate the fine details provided by individual-level networks, which will increase our understanding of the pattern of species interactions and its consequences for the fitness of threatened plant populations.

Carcass traits and meat characteristics of grazing Nellore cattle submitted to different supplementation strategies in the tropics.

This study explored the effects of different supplementation strategies during the dry and rainy seasons in the tropics on the carcass traits and meat quality of Nellore cattle produced under grazing conditions. Additionally, a cost assessment of the supplementation strategies was conducted to define the most suitable ones from an economic standpoint. Twenty-eight non-castrated male animals (18 months) with an initial body weight of 327.9 ± 4.2 kg were used. The animals were equitably distributed in a randomized complete design thorough four supplementation strategies as follows: (i) mineral supplementation (MS) in both dry and rainy seasons (MS/MS), (ii) MS during the dry season and concentrate supplementation (CS) during the rainy season (MS/CS), (iii) CS during the dry season and MS during the rainy season (CS/MS), and (iv) CS in both dry and rainy seasons (CS/CS). Thereafter, carcass traits, primary carcass cut yields, meat quality traits, and chemical composition of the meat of cattle produced across different supplementation strategies were determined. Data revealed that animals under CS/CS showed the greatest (P < 0.01) hot carcass weights among the other supplementation strategies evaluated. Conversely, supplementation strategy did not affect (P > 0.10) the carcass traits (the ribeye area, final pH, and forequarter), meat quality traits (shear force, myofibrillar fragment index, sarcomere length, and color), and meat chemical composition (crude protein, fat, and moisture) of the animals. A cost assessment of the supplementation strategies revealed that CS/CS had the highest production costs. Nevertheless, CS/CS had the greatest income and profit, while MS/MS had the lowest ones. In conclusion, data suggest that cattle grazing on tropical forage under CS during at least one season (i.e., dry or rainy) produce similar meat quality traits and chemical composition of meat to those observed for animals under CS in both seasons. Additionally, the last supplementation strategy revealed the greatest profit indicators among the other explored.

The use of flow cytometry for fungal nuclear DNA quantification.

Genome size information is sparse across fungi, with information being available for less than 2000 species. So far, most records have been obtained using static, microscope-based cytometry methods or derived from genome sequencing projects. Flow cytometry is now considered the state-of-the-art method for obtaining genome size measurements, and appropriate methods and DNA standards are available, enabling the analysis of most genome size ranges in a rapid, robust and inexpensive way. The average fungal genome size is 60 Mbp, but sizes vary across phylogeny, ranging from 2.2 (Encephalitozoon romaleae) to 3706 Mbp (Jafnea semitosta). In several fungal clades, genome size expansion seems to accompany evolution either to plant mutualism or to plant parasitism (particularly biotrophy), and fungi that interact with plants seem to have larger genomes than saprobes and those that interact with animals. Whereas flow cytometry for nuclear DNA quantification is routinely employed in plant sciences for genome size and ploidy studies, its use in fungal biology is still infrequent. Appropriate standards, methods and best practices are described here, with the aim of stimulating a more generalized and widespread use of flow cytometry for fungal genome size measurement.

Flow cytometric analysis of pollen and spores: An overview of applications and methodology.

Pollen grains are the male gametophytes in a seed-plant life cycle. Their small, particulate nature and crucial role in plant reproduction have made them an attractive object of study using flow cytometry (FCM), with a wide range of applications existing in the literature. While methodological considerations for many of these overlap with those for other tissue types (e.g., general considerations for the measurement of nuclear DNA content), the relative complexity of pollen compared to single cells presents some unique challenges. We consider these here in the context of both the identification and isolation of pollen and its subunits, and the types of research applications. While the discussion here mostly concerns pollen, the general principles described here can be extended to apply to spores in ferns, lycophytes, and bryophytes. In addition to recommendations provided in more general studies, some recurring and notable issues related specifically to pollen and spores are highlighted.

Isolation of plant nuclei for estimation of nuclear DNA content: Overview and best practices.

A critical aspect for obtaining accurate, reliable, and high-resolution estimates of nuclear DNA content is the release of nuclei from the cytoplasm in sufficient amounts, while maintaining their integrity throughout the analysis, protecting their DNA from degradation by endonucleases, and enabling stoichiometric DNA staining. In embryophytes, the most common method consists of chopping the plant material with a sharp razor blade to release nuclei into an isolation buffer, filtering the homogenate, and staining the nuclei in buffered suspension with a fluorochrome of choice. Despite the recent description of alternative approaches to isolate nuclei, the chopping procedure remains the most widely adopted method, due to its simplicity, rapidity, and effectiveness. In this review article, we discuss the specifics of nuclei isolation buffers and the distorting effects that secondary metabolites may have in nuclear suspensions and how to test them. We also present alternatives to the chopping procedure, options for filtering and fluorochromes, and discuss the applications of these varied approaches. A summary of the best practices regarding the isolation of plant nuclei for the estimation of nuclear DNA content is also provided.

Genetic similarities versus morphological resemblance: Unraveling a polyploid complex in a Mediterranean biodiversity hotspot.

The Balkan Peninsula is recognized as one of the hotspots of biodiversity in Europe. This area has shown since the Last Glacial Maximum appropriate conditions for species diversification and hybridization, which has led to the existence of numerous taxonomically unresolved entities. Here, we focus on the Western Balkans and explore the genetic structure and relationships among species belonging to the V. austriaca - V. orbiculata diploid-polyploid complex, including populations showing intermediate morphologies. A combination of nuclear markers (microsatellites), plastid DNA regions (trnH-psbA, ycf6-psbM) and ploidy level estimations using flow cytometry are employed to assess the genetic structure and evolutionary dynamics of this polyploid complex. To reconstruct the evolutionary history, an approximate Bayesian computation approach is combined with projections of the species distribution models onto the climatic scenarios of the Mid-Holocene (6 ka BP) and Last Glacial Maximum (22 ka BP). Four main groups were found: one well-established entity within the diploid level, V. dalmatica, a second diploid-tetraploid group which corresponds to V. orbiculata, a hexaploid cluster harboring V. austriaca subsp. jacquinii individuals, and an enigmatic tetraploid group. According to the molecular data obtained, this latter cluster represents an allopolyploid cryptic lineage −with V. orbiculata and V. dalmatica as putative parents− morphologically similar to V. orbiculata, but genetically more related to V. austriaca subsp. jacquinii. Veronica dalmatica and this "uncertain tetraploid" group are involved in the formation of the hexaploid taxon V. austriaca subsp. jacquinii, with the possibility of recent gene flow among different cytotypes. The present study supports a scenario of diversification from a diploid common ancestor leading to two different but interrelated lineages. The first one would correspond with the diploid V. orbiculata plus tetraploid individuals of this species arising through allo- and autopolyploidization, and the second one would involve all ploidy levels with allopolyploidization being prevalent.

Correction to: Assessing genotype-phenotype associations in three dorsal colour morphs in the meadow spittlebug Philaenus spumarius (L.) (Hemiptera: Aphrophoridae) using genomic and transcriptomic resources.

Following publication of the original article [1], it has been brought to the authors' attention that in their paper (Rodrigues et al. 2016) they reported the genome size based on 2C values (diploid genome) when it is more common to present it as 1C value.

The role of multiple reproductive barriers: strong post-pollination interactions govern cytotype isolation in a tetraploid-octoploid contact zone.

BACKGROUND AND AIMS: Polyploidy is an important contributor to sympatric speciation and assortative mating is a key mechanism driving cytotype interactions in contact zones. While strong reproductive barriers can mediate the coexistence of different cytotypes in sympatry, positive frequency-dependent mating disadvantage ultimately drives the transition to single-ploidy populations. However, comprehensive estimates of reproductive isolation among cytotypes and across multiple barriers are rare. We quantify the strength of isolation across multiple reproductive stages in a tetraploid-octoploid contact zone to understand the potential for coexistence. METHODS: Assortative mating due to flowering asynchrony, pollinator behaviour, morphological overlap, self-fertilization and gametic competition between tetraploid and octoploid Gladiolus communis in a contact zone in the Western Iberian Peninsula were assessed in natural and experimental populations to quantify reproductive isolation (RI) between cytotypes. KEY RESULTS: Tetraploids and octoploids have a high degree of overlap in flowering time and similar floral morphology, and are visited by generalist insects without cytotype foraging preferences, resulting in weak pre-pollination RI (from 0.00 to 0.21). In contrast, post-pollination isolation resulting from gametic selection was a strong barrier to inter-cytotype mating, with ploidy composition in stigmatic pollen loads determining the levels of RI (from 0.54 to 1.00). Between-cytotype cross-incompatibility was relatively high (RI from 0.54 to 0.63) as was isolation acquired through self-pollination (RI of 0.59 in tetraploids and 0.39 in octoploids). CONCLUSIONS: Total RI was high for both tetraploids (from 0.90 to 1.00) and octoploids (from 0.78 to 0.98). Such high rates of assortative mating will enable cytotype coexistence in mixed-ploidy populations by weakening the impacts of minority cytotype exclusion. This study reveals the key role of gametic selection in cytotype siring success and highlights the importance of comprehensive estimates across multiple reproductive barriers to understand cytotype interactions at contact zones.

Environmental differences are correlated with the distribution pattern of cytotypes in Veronica subsection Pentasepalae at a broad scale.

BACKGROUND AND AIMS: The distribution of cytotypes and its potential correlation with environmental variables represent a cornerstone to understanding the origin and maintenance of polyploid lineages. Although many studies have addressed this question in single species at a regional scale, only a few have attempted to decipher this enigma in groups of closely related species at a broad intercontinental geographical scale. Here, we consider approx. 20 species of a diploid-polyploid complex (Veronica subsect. Pentasepalae) of recent and rapid diversification represented in Europe and North Africa to study the frequency and distribution of cytotypes and their relationship to environmental variables. METHODS: A total of 680 individuals (207 populations) were sampled. Ploidy levels were determined using flow cytometry. Ecological differentiation among cytotypes was tested using climatic and environmental variables related to temperature, precipitation, vegetation and biogeographical region, among others, and by performing univariate and multivariate (constrained principal coordinates analysis) analyses. KEY RESULTS: Four ploidy levels (2x, 4x, 6x and 8x) were found and genome downsizing was observed to occur within the group. Plants of different ploidy level are ecologically differentiated, with hexaploids and octoploids occurring in wetter and colder habitats with a higher seasonality than diploids. A south to north distribution pattern was found, with diploids occupying southern refugial areas and octoploids being more frequent in northern regions of Europe above the permafrost boundary. CONCLUSIONS: The distribution of cytotypes can be explained by ecological differentiation, the geographical position of refuge areas during the Quaternary climatic oscillations as well as by ice and permafrost retreat patterns. The Balkan Peninsula constitutes the most important contact zone between cytotypes. This work provides the first comprehensive ploidy screening within V. subsect. Pentasepalae at a broad scale and indicates that polyploidy and genome downsizing might have contributed to the colonization of new habitats in a recently diverged polyploid complex.

Is selfing a reproductive assurance promoting polyploid establishment? Reduced fitness, leaky self-incompatibility and lower inbreeding depression in neotetraploids.

PREMISE: Newly formed polyploids face significant obstacles to persistence and population establishment because of fitness costs of intercytotype mating. Selfing provides the opportunity to escape mate limitation, enabling production of new individuals and increasing the likelihood of fixation of new polyploid lineages. Still, association between self-compatibility and polyploidy is not always clear. We compared self-incompatibility and inbreeding depression in neotetraploids and their diploid progenitor to explore the direct effects of whole genome duplications on self-incompatibility and the implications of ploidy-driven changes for polyploid establishment. METHODS: Outcross and self-pollinations were performed in diploids and synthetic neotetraploids of Jasione maritima var. maritima, and reproductive success was measured through fruit and seed production and seed germination. Self- and outcross offspring were grown under controlled conditions, and plant performance was measured through several fitness parameters. RESULTS: Neotetraploids showed an overall lower performance than diploids. Reproductive success was negatively affected by selfing in both cytotypes. However, greater variation in the expression of self-incompatibility was observed in neotetraploids; additionally, developmental and physiological parameters were not affected by selfing on neotetraploids, with an overall similar fitness of outcrossed and selfed individuals, resulting in lower inbreeding depression indexes. CONCLUSIONS: Neotetraploids might have benefited from selfing at initial stages after their formation. Genome duplications resulted in leaky self-incompatibility, enabling the production of offspring under minority cytotype disadvantage with similar fitness as outcrossed offspring. Our results support theoretical assumptions that selfing might be important for neopolyploid establishment, although changes in self-incompatibility might not be abrupt.

The challenge of species delimitation in the diploid-polyploid complex Veronica subsection Pentasepalae.

A reliable taxonomic framework and the identification of evolutionary lineages are essential for effective decisions in conservation biodiversity programs. However, phylogenetic reconstruction becomes extremely difficult when polyploidy and hybridization are involved. Veronica subsection Pentasepalae is a diploid-polyploid complex of ca. 20 species with ploidy levels ranging from 2x to 10x. Here, DNA-ploidy level estimations and AFLP fingerprinting were used to determine the evolutionary history, and species boundaries were reviewed in an integrated approach including also previous data (mainly morphology and sequence-based phylogenetic reconstructions). Molecular analyses were performed for 243 individuals from 95 populations, including for the first time all taxa currently recognized within the subsection. Phylogenetic reconstruction identified four main groups corresponding almost completely to the four clusters identified by genetic structure analyses. Multiple autopolyploidization events have occurred in the tetraploid V. satureiifolia giving rise to octoploid entities in central Europe and north of Spain, whereas hybridization is demonstrated to have occurred in several populations from the Balkan Peninsula. Furthermore, our study has established the taxonomic status of taxa, for the most part recovered as monophyletic. Cryptic taxa within the group have been identified, and a new species, Veronica dalmatica, is fully described. This study highlights the implications of polyploidy in species delimitation, and illustrates the importance to conserve polyploid populations as potential sources of diversification due to evolutionary significance of genome duplications in plant evolution.

Experimental insights on Darwin's cross-promotion hypothesis in tristylous purple loosestrife (Lythrum salicaria).

PREMISE OF THE STUDY: Darwin proposed that the reciprocal arrangement of anthers and stigmas in heterostylous plants promotes cross-pollination through pollen segregation on pollinators' bodies. The floral tube in most heterostylous species constrains the feeding posture of pollinators determining the site of contact with sex organs located at different heights within a flower. Here, we evaluate Darwin's hypothesis in tristylous Lythrum salicaria, a species with a partially tubular corolla, and examine the extent to which the location of sex organs within a flower influence compatible and incompatible pollination. We predicted that the proficiency of cross-pollination would increase for more inserted sex organs due to the restrictions imposed by the floral tube on pollinator positioning. METHODS: We used experimental trimorphic and monomorphic arrays and emasculated flowers to quantify intermorph pollen transfer and capture among all sex-organ heights, and estimated the contribution of intraflower self-pollination, geitonogamous self-pollination, and intramorph outcross pollination to total intramorph pollination. KEY RESULTS: As predicted, disassortative pollination varied significantly with sex-organ height and was highest for short-level organs and lowest for long-level organs. In monomorphic arrays, most intramorph pollination resulted from outcross pollination followed by intraflower and geitonogamous self-pollination. CONCLUSIONS: Our results provide evidence for the Darwinian hypothesis. Reciprocal herkogamy promoted varying degrees of disassortative pollination with the magnitude strongly influenced by sex-organ height within a flower.