CAM evolution is associated with gene family expansion in an explosive bromeliad radiation.

The subgenus Tillandsia (Bromeliaceae) belongs to one of the fastest radiating clades in the plant kingdom and is characterised by the repeated evolution of Crassulacean acid metabolism (CAM). Despite its complex genetic basis, this water-conserving trait has evolved independently across many plant families and is regarded as a key innovation trait and driver of ecological diversification in Bromeliaceae. By producing high-quality genome assemblies of a Tillandsia species pair displaying divergent photosynthetic phenotypes, and combining genome-wide investigations of synteny, transposable element (TE) dynamics, sequence evolution, gene family evolution and temporal differential expression, we were able to pinpoint the genomic drivers of CAM evolution in Tillandsia. Several large-scale rearrangements associated with karyotype changes between the two genomes and a highly dynamic TE landscape shaped the genomes of Tillandsia. However, our analyses show that rewiring of photosynthetic metabolism is mainly obtained through regulatory evolution rather than coding sequence evolution, as CAM-related genes are differentially expressed across a 24-hour cycle between the two species but are not candidates of positive selection. Gene orthology analyses reveal that CAM-related gene families manifesting differential expression underwent accelerated gene family expansion in the constitutive CAM species, further supporting the view of gene family evolution as a driver of CAM evolution.

Does the evolution of micromorphology accompany chromosomal changes on dysploid and polyploid levels in the Barnardia japonica complex (Hyacinthaceae)?

BACKGROUND: Chromosome number and genome size changes via dysploidy and polyploidy accompany plant diversification and speciation. Such changes often impact also morphological characters. An excellent system to address the questions of how extensive and structured chromosomal changes within one species complex affect the phenotype is the monocot species complex of Barnardia japonica. This taxon contains two well established and distinct diploid cytotypes differing in base chromosome numbers (AA: x = 8, BB: x = 9) and their allopolyploid derivatives on several ploidy levels (from 3x to 6x). This extensive and structured genomic variation, however, is not mirrored by gross morphological differentiation. RESULTS: The current study aims to analyze the correlations between the changes of chromosome numbers and genome sizes with palynological and leaf micromorphological characters in diploids and selected allopolyploids of the B. japonica complex. The chromosome numbers varied from 2n = 16 and 18 (2n = 25 with the presence of supernumerary B chromosomes), and from 2n = 26 to 51 in polyploids on four different ploidy levels (3x, 4x, 5x, and 6x). Despite additive chromosome numbers compared to diploid parental cytotypes, all polyploid cytotypes have experienced genome downsizing. Analyses of leaf micromorphological characters did not reveal any diagnostic traits that could be specifically assigned to individual cytotypes. The variation of pollen grain sizes correlated positively with ploidy levels. CONCLUSIONS: This study clearly demonstrates that karyotype and genome size differentiation does not have to be correlated with morphological differentiation of cytotypes.

Formamide-Free Genomic In Situ Hybridization (ff-GISH).

Fluorescence in situ hybridization allows for the mapping of various sequence types in the genomes and is thus widely used in structural, functional, and evolutionary studies. One particular type of in situ hybridization that specifically allows to map whole parental genomes in diploid and polyploid hybrids is genomic in situ hybridization (GISH). The efficiency of GISH, i.e., the specificity of hybridization of genomic DNA probes to the parental subgenomes in hybrids depends, among others, on the age of the polyploids and the similarity of the parental genomes, specifically their repetitive DNA fractions. Typically, high levels of overall repeat similarity between the parental genomes result in lower efficiency of GISH. Here, we present the formamide-free GISH (ff-GISH) protocol that can be applied to diploid and polyploid hybrids of both monocots and dicots. ff-GISH allows higher efficiency of the labeling of the putative parental genomes compared to the standard GISH protocol and allows discrimination of parental chromosome sets that share up to 80-90% repeat similarity. This modified method is nontoxic, is simple, and lends itself to modifications. It can also be used for standard FISH and mapping of individual sequence types in chromosomes/genomes.

Repeat Dynamics across Timescales: A Perspective from Sibling Allotetraploid Marsh Orchids (Dactylorhiza majalis s.l.).

To provide insights into the fate of transposable elements (TEs) across timescales in a post-polyploidization context, we comparatively investigate five sibling Dactylorhiza allotetraploids (Orchidaceae) formed independently and sequentially between 500 and 100K generations ago by unidirectional hybridization between diploids D. fuchsii and D. incarnata. Our results first reveal that the paternal D. incarnata genome shows a marked increased content of LTR retrotransposons compared to the maternal species, reflected in its larger genome size and consistent with a previously hypothesized bottleneck. With regard to the allopolyploids, in the youngest D. purpurella both genome size and TE composition appear to be largely additive with respect to parents, whereas for polyploids of intermediate ages we uncover rampant genome expansion on a magnitude of multiple entire genomes of some plants such as Arabidopsis. The oldest allopolyploids in the series are not larger than the intermediate ones. A putative tandem repeat, potentially derived from a non-autonomous miniature inverted-repeat TE (MITE) drives much of the genome dynamics in the allopolyploids. The highly dynamic MITE-like element is found in higher proportions in the maternal diploid, D. fuchsii, but is observed to increase in copy number in both subgenomes of the allopolyploids. Altogether, the fate of repeats appears strongly regulated and therefore predictable across multiple independent allopolyploidization events in this system. Apart from the MITE-like element, we consistently document a mild genomic shock following the allopolyploidizations investigated here, which may be linked to their relatively large genome sizes, possibly associated with strong selection against further genome expansions.

Descending Dysploidy and Bidirectional Changes in Genome Size Accompanied Crepis (Asteraceae) Evolution.

The evolution of the karyotype and genome size was examined in species of Crepis sensu lato. The phylogenetic relationships, inferred from the plastid and nrITS DNA sequences, were used as a framework to infer the patterns of karyotype evolution. Five different base chromosome numbers (x = 3, 4, 5, 6, and 11) were observed. A phylogenetic analysis of the evolution of the chromosome numbers allowed the inference of x = 6 as the ancestral state and the descending dysploidy as the major direction of the chromosome base number evolution. The derived base chromosome numbers (x = 5, 4, and 3) were found to have originated independently and recurrently in the different lineages of the genus. A few independent events of increases in karyotype asymmetry were inferred to have accompanied the karyotype evolution in Crepis. The genome sizes of 33 Crepis species differed seven-fold and the ancestral genome size was reconstructed to be 1C = 3.44 pg. Both decreases and increases in the genome size were inferred to have occurred within and between the lineages. The data suggest that, in addition to dysploidy, the amplification/elimination of various repetitive DNAs was likely involved in the genome and taxa differentiation in the genus.

Spatial and Ecological Drivers of Genetic Structure in Greek Populations of Alkanna tinctoria (Boraginaceae), a Polyploid Medicinal Herb.

Background and Aims: Quantifying genetic variation is fundamental to understand a species' demographic trajectory and its ability to adapt to future changes. In comparison with diploids, however, genetic variation and factors fostering genetic divergence remain poorly studied in polyploids due to analytical challenges. Here, by employing a ploidy-aware framework, we investigated the genetic structure and its determinants in polyploid Alkanna tinctoria (Boraginaceae), an ancient medicinal herb that is the source of bioactive compounds known as alkannin and shikonin (A/S). From a practical perspective, such investigation can inform biodiversity management strategies. Methods: We collected 14 populations of A. tinctoria within its main distribution range in Greece and genotyped them using restriction site-associated DNA sequencing. In addition, we included two populations of A. sieberi. By using a ploidy-aware genotype calling based on likelihoods, we generated a dataset of 16,107 high-quality SNPs. Classical and model-based analysis was done to characterize the genetic structure within and between the sampled populations, complemented by genome size measurements and chromosomal counts. Finally, to reveal the drivers of genetic structure, we searched for associations between allele frequencies and spatial and climatic variables. Key Results: We found support for a marked regional structure in A. tinctoria along a latitudinal gradient in line with phytogeographic divisions. Several analyses identified interspecific admixture affecting both mainland and island populations. Modeling of spatial and climatic variables further demonstrated a larger contribution of neutral processes and a lesser albeit significant role of selection in shaping the observed genetic structure in A. tinctoria. Conclusion: Current findings provide evidence of strong genetic structure in A. tinctoria mainly driven by neutral processes. The revealed natural genomic variation in Greek Alkanna can be used to further predict variation in A/S production, whereas our bioinformatics approach should prove useful for the study of other non-model polyploid species.

Genome Size and Chromosome Number Evolution in Korean Iris L. Species (Iridaceae Juss.).

Chromosome numbers, karyotypes, and genome sizes of 14 Iris L. (Iridaceae Juss.) species in Korea and their closely related taxon, Sisyrinchium rosulatum, are presented and analyzed in a phylogenetic framework. To date, understanding the chromosomal evolution of Korean irises has been hampered by their high chromosome numbers. Here, we report analyses of chromosome numbers and karyotypes obtained via classic Feulgen staining and genome sizes measured using flow cytometry in Korean irises. More than a two-fold variation in chromosome numbers (2n = 22 to 2n = 50) and over a three-fold genome size variation (2.39 pg to 7.86 pg/1 C) suggest the putative polyploid and/or dysploid origin of some taxa. Our study demonstrates that the patterns of genome size variation and chromosome number changes in Korean irises do not correlate with the phylogenetic relationships and could have been affected by different evolutionary processes involving polyploidy or dysploidy. This study presents the first comprehensive chromosomal and genome size data for Korean Iris species. Further studies involving molecular cytogenetic and phylogenomic analyses are needed to interpret the mechanisms involved in the origin of chromosomal variation in the Iris.

Euchromatic Supernumerary Chromosomal Segments-Remnants of Ongoing Karyotype Restructuring in the Prospero autumnale Complex?

Supernumerary chromosomal segments (SCSs) represent additional chromosomal material that, unlike B chromosomes, is attached to the standard chromosome complement. The Prospero autumnale complex (Hyacinthaceae) is polymorphic for euchromatic large terminal SCSs located on the short arm of chromosome 1 in diploid cytotypes AA and B7B7, and tetraploid AAB7B7 and B6B6B7B7, in addition to on the short arm of chromosome 4 in polyploid B7B7B7B7 and B7B7B7B7B7B7 cytotypes. The genomic composition and evolutionary relationships among these SCSs have been assessed using fluorescence in situ hybridisation (FISH) with 5S and 35S ribosomal DNAs (rDNAs), satellite DNA PaB6, and a vertebrate-type telomeric repeat TTAGGG. Neither of the rDNA repeats were detected in SCSs, but most contained PaB6 and telomeric repeats, although these never spanned whole SCSs. Genomic in situ hybridisation (GISH) using A, B6, and B7 diploid genomic parental DNAs as probes revealed the consistently higher genomic affinity of SCSs in diploid hybrid B6B7 and allopolyploids AAB7B7 and B6B6B7B7 to genomic DNA of the B7 diploid cytotype. GISH results suggest a possible early origin of SCSs, especially that on chromosome 1, as by-products of the extensive genome restructuring within a putative ancestral P. autumnale B7 genome, predating the complex diversification at the diploid level and perhaps linked to B-chromosome evolution.

Organization and evolution of two repetitive sequences, 18-24J and 12-13P, in the genome of Chenopodium (Amaranthaceae).

The abundance and chromosomal organization of two repetitive sequences named 12-13P and 18-24J were analyzed in 24 diploid and nine polyploid species of Chenopodium s.l., with special attention to Chenopodium s.s. Both sequences were predominantly present in species of Chenopodium s.s.; however, differences in the amplification levels were observed among the species. The 12-13P repeat was highly amplified in all of the analyzed Eurasian species, whereas the American diploids showed a marked variation in the amplification levels. The 12-13P repeat contains a tandemly arranged 40 bp minisatellite element forming a large proportion of the genome of Chenopodium (up to 3.5%). FISH revealed its localization to the pericentromeric regions of the chromosomes. The chromosomal distribution of 12-13P delivered additional chromosomal marker for B-genome diploids. The 18-24J repeat showed a dispersed organization in all of the chromosomes of the analyzed diploid species and the Eurasian tetraploids. In the American allotetraploids (C. quinoa, C. berlandieri) and Eurasian allohexaploids (e.g., C. album) very intense hybridization signals of 18-24J were observed only on 18 chromosomes that belong to the B subgenome of these polyploids. Combined cytogenetic and molecular analyses suggests that reorganization of these two repeats accompanied the diversification and speciation of diploid (especially A genome) and polyploid species of Chenopodium s.s.

Multiple Origins and Nested Cycles of Hybridization Result in High Tetraploid Diversity in the Monocot Prospero.

Polyploidy is a major driving force in angiosperm evolution, but our understanding of establishment and early diversification processes following allo- vs. auto-polyploidy is limited. An excellent system to address such questions is the monocot plant Prospero autumnale, as it comprises several genomically and chromosomally distinct diploid cytotypes and their auto- and allotetraploid derivatives. To infer origins and evolutionary trajectories of the tetraploids, we use genome size data, in situ hybridization with parental genomic DNAs and specific probes (satDNA, rDNAs), as well as molecular-phylogenetic analyses. Thus, we demonstrate that an astounding range of allotetraploid lineages has been formed recurrently by chromosomal re-patterning, interactions of chromosomally variable parental genomes and nested cycles of extensive hybridization, whereas autotetraploids have originated at least twice and are cytologically stable. During the recurrent formation and establishment across wide geographic areas hybridization in some populations could have inhibited lineage diversification and nascent speciation of such a hybrid swarm. However, cytotypes that became fixed in populations enhanced the potential for species diversification, possibly exploiting the extended allelic base, and fixed heterozygosity that polyploidy confers. The time required for polyploid cytotype fixation may in part reflect the lag phase reported for polyploids between their formation and species diversification.

Dating the Species Network: Allopolyploidy and Repetitive DNA Evolution in American Daisies (Melampodium sect. Melampodium, Asteraceae).

Allopolyploidy has played an important role in the evolution of the flowering plants. Genome mergers are often accompanied by significant and rapid alterations of genome size and structure via chromosomal rearrangements and altered dynamics of tandem and dispersed repetitive DNA families. Recent developments in sequencing technologies and bioinformatic methods allow for a comprehensive investigation of the repetitive component of plant genomes. Interpretation of evolutionary dynamics following allopolyploidization requires both the knowledge of parentage and the age of origin of an allopolyploid. Whereas parentage is typically inferred from cytogenetic and phylogenetic data, age inference is hampered by the reticulate nature of the phylogenetic relationships. Treating subgenomes of allopolyploids as if they belonged to different species (i.e., no recombination among subgenomes) and applying cross-bracing (i.e., putting a constraint on the age difference of nodes pertaining to the same event), we can infer the age of allopolyploids within the framework of the multispecies coalescent within BEAST2. Together with a comprehensive characterization of the repetitive DNA fraction using the RepeatExplorer pipeline, we apply the dating approach in a group of closely related allopolyploids and their progenitor species in the plant genus Melampodium (Asteraceae). We dated the origin of both the allotetraploid, Melampodium strigosum, and its two allohexaploid derivatives, Melampodium pringlei and Melampodium sericeum, which share both parentage and the direction of the cross, to the Pleistocene ($<$1.4 Ma). Thus, Pleistocene climatic fluctuations may have triggered formation of allopolyploids possibly in short intervals, contributing to difficulties in inferring the precise temporal order of allopolyploid species divergence of M. sericeum and M. pringlei. The relatively recent origin of the allopolyploids likely played a role in the near-absence of major changes in the repetitive fraction of the polyploids' genomes. The repetitive elements most affected by the postpolyploidization changes represented retrotransposons of the Ty1-copia lineage Maximus and, to a lesser extent, also Athila elements of Ty3-gypsy family.

Origin and Diversification of South American Polyploid Silene Sect. Physolychnis (Caryophyllaceae) in the Andes and Patagonia.

The Andes are an important biogeographic region in South America extending for about 8000 km from Venezuela to Argentina. They are - along with the Patagonian steppes - the main distribution area of ca. 18 polyploid species of Silene sect. Physolychnis. Using nuclear ITS and plastid psbE-petG and matK sequences, flow cytometric ploidy level estimations and chromosome counts, and including 13 South American species, we explored the origin and diversification of this group. Our data suggest a single, late Pliocene or early Pleistocene migration of the North American S. verecunda lineage to South America, which was followed by dispersal and diversification of this tetraploid lineage in the Andes, other Argentinian mountain ranges and the Patagonian steppes. Later in the Pleistocene South American populations hybridized with the S. uralensis lineage, which led to allopolyploidisation and origin of decaploid S. chilensis and S. echegarayi occurring at high elevations. Additionally, we show that the morphological differentiation in leaf shape correlated with divergent habitats (high elevation Andes vs. lower elevation Patagonian steppes) is also supported phylogenetically, especially in the ITS tree. Lastly, the species boundaries among the narrow-leaved Patagonian steppe species are poorly resolved and need more thorough taxonomic revision.

Genome-wide repeat dynamics reflect phylogenetic distance in closely related allotetraploid Nicotiana (Solanaceae).

Nicotiana sect. Repandae is a group of four allotetraploid species originating from a single allopolyploidisation event approximately 5 million years ago. Previous phylogenetic analyses support the hypothesis of N. nudicaulis as sister to the other three species. This is concordant with changes in genome size, separating those with genome downsizing (N. nudicaulis) from those with genome upsizing (N. repanda, N. nesophila, N. stocktonii). However, a recent analysis reflecting genome dynamics of different transposable element families reconstructed greater similarity between N. nudicaulis and the Revillagigedo Island taxa (N. nesophila and N. stocktonii), thereby placing N. repanda as sister to the rest of the group. This could reflect a different phylogenetic hypothesis or the unique evolutionary history of these particular elements. Here we re-examine relationships in this group and investigate genome-wide patterns in repetitive DNA, utilising high-throughput sequencing and a genome skimming approach. Repetitive DNA clusters provide support for N. nudicaulis as sister to the rest of the section, with N. repanda sister to the two Revillagigedo Island species. Clade-specific patterns in the occurrence and abundance of particular repeats confirm the original (N. nudicaulis (N. repanda (N. nesophila + N. stocktonii))) hypothesis. Furthermore, overall repeat dynamics in the island species N. nesophila and N. stocktonii confirm their similarity to N. repanda and the distinctive patterns between these three species and N. nudicaulis. Together these results suggest that broad-scale repeat dynamics do in fact reflect evolutionary history and could be predicted based on phylogenetic distance.

rDNA Loci Evolution in the Genus Glechoma (Lamiaceae).

Glechoma L. (Lamiaceae) is distributed in eastern Asia and Europe. Understanding chromosome evolution in Glechoma has been strongly hampered by its small chromosomes, constant karyotype and polyploidy. Here phylogenetic patterns and chromosomal variation in Glechoma species are considered, using genome sizes, chromosome mapping of 5S and 35S rDNAs by fluorescence in situ hybridisation (FISH), and phylogenetic analyses of internal transcribed spacers (nrITS) of 35S rDNA and 5S rDNA NTS sequences. Species and populations of Glechoma are tetraploid (2n = 36) with base chromosome number of x = 9. Four chromosomes carry pericentric 5S rDNA sites in their short arms in all the species. Two to four of these chromosomes also carry 35S rDNA in subterminal regions of the same arms. Two to four other chromosomes have 35S rDNA sites, all located subterminally within short arms; one individual possessed additional weak pericentric 35S rDNA signals on three other chromosomes. Five types of rDNA locus distribution have been defined on the basis of 35S rDNA variation, but none is species-specific, and most species have more than one type. Glechoma hederacea has four types. Genome size in Glechoma ranges from 0.80 to 0.94 pg (1C), with low levels of intrapopulational variation in all species. Phylogenetic analyses of ITS and NTS sequences distinguish three main clades coinciding with geographical distribution: European (G. hederacea-G. hirsuta), Chinese and Korean (G. longituba), and Japanese (G. grandis). The paper presents the first comparative cytogenetic analyses of Glechoma species including karyotype structure, rDNA location and number, and genome size interpreted in a phylogenetic context. The observed variation suggests that the genus is still in genomic flux. Genome size, but not rDNA loci number and distribution, provides a character for species delimitation which allows better inferences of interspecific relationships to be made, in the absence of well-defined morphological differentiation.

The Impact of Reconstruction Methods, Phylogenetic Uncertainty and Branch Lengths on Inference of Chromosome Number Evolution in American Daisies (Melampodium, Asteraceae).

Chromosome number change (polyploidy and dysploidy) plays an important role in plant diversification and speciation. Investigating chromosome number evolution commonly entails ancestral state reconstruction performed within a phylogenetic framework, which is, however, prone to uncertainty, whose effects on evolutionary inferences are insufficiently understood. Using the chromosomally diverse plant genus Melampodium (Asteraceae) as model group, we assess the impact of reconstruction method (maximum parsimony, maximum likelihood, Bayesian methods), branch length model (phylograms versus chronograms) and phylogenetic uncertainty (topological and branch length uncertainty) on the inference of chromosome number evolution. We also address the suitability of the maximum clade credibility (MCC) tree as single representative topology for chromosome number reconstruction. Each of the listed factors causes considerable incongruence among chromosome number reconstructions. Discrepancies between inferences on the MCC tree from those made by integrating over a set of trees are moderate for ancestral chromosome numbers, but severe for the difference of chromosome gains and losses, a measure of the directionality of dysploidy. Therefore, reliance on single trees, such as the MCC tree, is strongly discouraged and model averaging, taking both phylogenetic and model uncertainty into account, is recommended. For studying chromosome number evolution, dedicated models implemented in the program ChromEvol and ordered maximum parsimony may be most appropriate. Chromosome number evolution in Melampodium follows a pattern of bidirectional dysploidy (starting from x = 11 to x = 9 and x = 14, respectively) with no prevailing direction.

Molecular and cytogenetic evidence for an allotetraploid origin of Chenopodium quinoa and C. berlandieri (Amaranthaceae).

Most of the cultivated chenopods are polyploids, but their origin and evolutionary history are still poorly understood. Phylogenetic analyses of DNA sequences of four plastid regions, nrITS and nuclear 5S rDNA spacer region (NTS) of two tetraploid chenopods (2n=4x=36), Andean C. quinoa and North American C. berlandieri, and their diploid relatives allowed inferences of their origin. The phylogenetic analyses confirmed allotetraploid origin of both tetraploids involving diploids of two different genomic groups (genomes A and B) and suggested that these two might share very similar parentage. The hypotheses on the origin of the two allopolyploid species were further tested using genomic in situ hybridization (GISH). Several diploid Chenopodium species belonging to the two lineages, genome A and B, suggested by phylogenetic analyses, were tested as putative parental taxa. GISH differentiated two sets of parental chromosomes in both tetraploids and further corroborated their allotetraploid origin. Putative diploid parental taxa have been suggested by GISH for C. quinoa and C. berlandieri. Genome sizes of the analyzed allotetraploids fit nearly perfectly the expected additive values of the putative parental taxa. Directional and uniparental loss of rDNA loci of the maternal A-subgenome was revealed for both C. berlandieri and C. quinoa.

Structural polymorphisms and distinct genomic composition suggest recurrent origin and ongoing evolution of B chromosomes in the Prospero autumnale complex (Hyacinthaceae).

Supernumerary B chromosomes (Bs) are genomic parasitic components, originating from the A complement via chromosomal rearrangements, which follow their own evolutionary trajectories. They often contain repetitive DNAs, some shared with regular chromosomes and some newly evolved. Genomic composition, origin and evolution of Bs have been analysed in the chromosomally variable Prospero autumnale complex. Two rDNAs and a satellite DNA (PaB6) from regular chromosomes were mapped to Bs of 26 plants from three diploid cytotypes, their hybrids and polyploid derivatives. In homoploid diploid hybrids, genomic in situ hybridization (GISH) allowed B painting with the parental DNAs. Bs were structurally variable and highly enriched in 5S rDNA and satDNA PaB6, and rarely in 35S rDNA. Eleven combinations of rDNA and PaB6 localization were observed. The quantities of PaB6 in Bs and regular chromosomes were not correlated, suggesting amplification mechanisms other than recombination. PaB6 and 5S rDNA amounts increased with increasing ploidy level. GISH revealed two independent origins of Bs. The structural variation, repeat content, repeat-type fluctuations and differing genomic affinities of Bs in different cytotypes suggest that they represent young proto-B chromosomes. Bs in P. autumnale probably form recurrently as by-products of the extensive genome restructuring within this chromosomally variable species complex.

Formamide-Free Genomic in situ Hybridization Allows Unambiguous Discrimination of Highly Similar Parental Genomes in Diploid Hybrids and Allopolyploids.

Polyploidy and hybridization play an important role in plant diversification and speciation. The application of genomic in situ hybridization (GISH) allows the identification of parental genomes in hybrids, thus elucidating their origins and allowing for analysis of their genomic evolution. The performance of GISH depends on the similarity of the parental genomes and on the age of hybrids. Here, we present the formamide-free GISH (ff-GISH) protocol applied to diploid and polyploid hybrids of monocots (Prospero, Hyacinthaceae) and dicots (Melampodium, Asteraceae) differing in similarity of the parental genomes and in chromosome and genome sizes. The efficiency of the new protocol is compared to the standard GISH protocol. As a result, ff-GISH allowed efficient labeling and discrimination of the parental chromosome sets in diploid and allopolyploid hybrids in Prospero autumnale species complex. In contrast, the standard GISH protocol failed to differentiate the parental genomes due to high levels of similar repetitive DNA. Likewise, an unambiguous identification of parental genomes in allotetraploid Melampodium nayaritense (Asteraceae) was possible after ff-GISH, whereas the standard GISH hybridization performance was suboptimal. The modified method is simple and non-toxic and allows the discrimination of very similar parental genomes in hybrids. This method lends itself to modifications and improvements and can also be used for FISH.