Navigating the Future of 3D Laparoscopic Liver Surgeries: Visualization of Internal Anatomy on Laparoscopic Images With Augmented Reality.

INTRODUCTION: Liver tumor resection requires precise localization of tumors and blood vessels. Despite advancements in 3-dimensional (3D) visualization for laparoscopic surgeries, challenges persist. We developed and evaluated an augmented reality (AR) system that overlays preoperative 3D models onto laparoscopic images, offering crucial support for 3D visualization during laparoscopic liver surgeries. METHODS: Anatomic liver structures from preoperative computed tomography scans were segmented using open-source software including 3D Slicer and Maya 2022 for 3D model editing. A registration system was created with 3D visualization software utilizing a stereo registration input system to overlay the virtual liver onto laparoscopic images during surgical procedures. A controller was customized using a modified keyboard to facilitate manual alignment of the virtual liver with the laparoscopic image. The AR system was evaluated by 3 experienced surgeons who performed manual registration for a total of 27 images from 7 clinical cases. The evaluation criteria included registration time; measured in minutes, and accuracy; measured using the Dice similarity coefficient. RESULTS: The overall mean registration time was 2.4±1.7 minutes (range: 0.3 to 9.5 min), and the overall mean registration accuracy was 93.8%±4.9% (range: 80.9% to 99.7%). CONCLUSION: Our validated AR system has the potential to effectively enable the prediction of internal hepatic anatomic structures during 3D laparoscopic liver resection, and may enhance 3D visualization for select laparoscopic liver surgeries.

Effect of Self-Training Using Virtual Reality Head-Mounted Display Simulator on the Acquisition of Holmium Laser Enucleation of the Prostate Surgical Skills.

PURPOSE: We aimed to evaluate the effect of self-training using a virtual reality head-mounted display simulator on the acquisition of surgical skills for holmium laser enucleation surgery. METHODS: Thirteen medical students without surgical skills for holmium laser enucleation of the prostate were trained using multimedia to learn the technique via simulator manipulation. Thereafter, participants performed the technique on a virtual benign prostatic hyperplasia model A (test A). After a 1-week wash-out period, they underwent self-training using a simulator and performed the technique on model B (test B). Subsequently, participants were asked to respond to Training Satisfaction Questions. Video footage of hand movements and endoscope view were recorded during tests A and B for later review by 2 expert surgeons. A 20-step Assessment Checklist, 6-domain Global Rating Scale, and a Pass Rating were used to compare performance on tests A and B. RESULTS: Thirteen participants completed both tests A and B. The 20-step Assessment Checklist and 6-domain Global Rating Scale evaluation results showed significantly improved scores in test B than in test A (P<0.05). No evaluator rated participants as passed after test A, but 11 participants (84.6%) passed after test B. Ten participants (76.9%) indicated that the simulator was helpful in acquiring surgical skills for holmium laser enucleation of the prostate. CONCLUSION: The virtual reality head-mounted display holmium laser enucleation of the prostate simulator was effective for surgical skill training. This simulator may help to shorten the learning curve of this technique in real clinical practice in the future.

Thliphthisasapphus (Rubiaceae, Rubieae), a new species from Lefkada (Ionian Islands, Greece) and its ecological position.

The new species, Thliphthisasapphussp. nov. (Rubiaceae, Rubieae), a narrow endemic of the white cliffs of Lefkátas on the southwest coast of Lefkada (Greece) is described and illustrated and an IUCN assessment is presented. Vegetation relevés were performed at the single known locality, limestone cliffs facing the sea and revealed a new association, the Thliphthisasapphus-Lomelosietumdallaportae. The chromosome number of Thliphthisasapphus was determined as 2n = 4x = 44, being the single tetraploid species in the genus to date. The species also differs markedly morphologically from its morphologically closest relatives, two Greek steno-endemic oreophytes, Th.baenitzii and Th.muscosa by the following characters: densely setose mericarps and corolla, tetraploidy and by its distribution. An identification key for the Greek species of Thliphthisa is provided. Th.sapphus constitutes the westernmost outpost of a group of Greek steno-endemics, highlighting the importance of coastal habitats and their protection as refugia for poorly competitive chamaephytes.

Comparative microscopic investigations of leaf epidermis in four Ajuga species from Korea.

The genus Ajuga is widely distributed in temperate to subtropical regions, and four species are currently recognized in Korea (A. decumbens, A. multiflora, A. nipponensis, and A. spectabilis), but epidermal anatomical differences across these species have never been described. A comparative study of the leaf micromorphological characteristics of Korean Ajuga species was performed using light microscopy (LM) and scanning electron microscopy (SEM) to elucidate their taxonomic usefulness and to assess leaf micromorphological diversity. Considerable diversity in epidermal and stomatal anatomy was observed across Korean Ajuga species. Species had both hypostomatic or amphistomatic leaves, with anomocytic, anisocytic, diactyic, or actinocytic stomatal complexes. Guard cell length across species ranged from 17.66 ± 0.57 µm to 32.50 ± 2.38 µm and correlated with genome size. Abnormal stomata were frequently observed in three species (A. decumbens, A. multiflora, and A. nipponensis) but not in A. spectabilis. Three types of glandular trichomes were found: peltate in all species, short-stalked in all species, and long-stalked glandular trichomes in A. multiflora. Among the investigated leaf micromophological characters, trichome type, epidermal cell shape, and stomatal morphology were all taxonomically informative traits at a species level. RESEARCH HIGHLIGHTS: A comprehensive micromorphological description of the leaf surface is provided for Korean Ajuga species using scanning electron microscopic (SEM) and light microscopic (LM) analyses. The diverse range of stomatal development and the occurrence of polymorphic stomatal types are documented for the first time in Korean Ajuga species. The great diversity in stomatal and trichome morphology in Korean Ajuga species are taxonomically useful traits for species identification.

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.

Disruption of the standard kinetochore in holocentric Cuscuta species.

The segregation of chromosomes depends on the centromere. Most species are monocentric, with the centromere restricted to a single region per chromosome. In some organisms, the monocentric organization changed to holocentric, in which the centromere activity is distributed over the entire chromosome length. However, the causes and consequences of this transition are poorly understood. Here, we show that the transition in the genus Cuscuta was associated with dramatic changes in the kinetochore, a protein complex that mediates the attachment of chromosomes to microtubules. We found that in holocentric Cuscuta species, the KNL2 genes were lost; the CENP-C, KNL1, and ZWINT1 genes were truncated; the centromeric localization of CENH3, CENP-C, KNL1, MIS12, and NDC80 proteins was disrupted; and the spindle assembly checkpoint (SAC) degenerated. Our results demonstrate that holocentric Cuscuta species lost the ability to form a standard kinetochore and do not employ SAC to control the attachment of microtubules to chromosomes.

Comparative Analyses of Complete Chloroplast Genomes and Karyotypes of Allotetraploid Iris koreana and Its Putative Diploid Parental Species (Iris Series Chinenses, Iridaceae).

The Iris series Chinenses in Korea comprises four species (I. minutoaurea, I. odaesanensis, I. koreana, and I. rossii), and the group includes some endangered species, owing to their high ornamental, economic, and conservation values. Among them, the putative allotetraploid, Iris koreana (2n = 4x = 50), is hypothesized to have originated from the hybridization of the diploids I. minutoaurea (2n = 2x = 22) and I. odaesanensis (2n = 2x = 28) based on morphological characters, chromosome numbers, and genome size additivity. Despite extensive morphological and molecular phylogenetical studies on the genus Iris, little is known about Korean irises in terms of their complete chloroplast (cp) genomes and molecular cytogenetics that involve rDNA loci evolution based on fluorescence in situ hybridization (FISH). This study reports comparative analyses of the karyotypes of the three Iris species (I. koreana, I. odaesanensis, and I. minutoaurea), with an emphasis on the 5S and 35S rDNA loci number and localization using FISH together with the genome size and chromosome number. Moreover, the cp genomes of the same individuals were sequenced and assembled for comparative analysis. The rDNA loci numbers, which were localized consistently at the same position in all species, and the chromosome numbers and genome size values of tetraploid Iris koreana (four 5S and 35S loci; 2n = 50; 1C = 7.35 pg) were additively compared to its putative diploid progenitors, I. minutoaurea (two 5S and 35S loci; 2n = 22; 1C = 3.71 pg) and I. odaesanensis (two 5S and 35S loci; 2n = 28; 1C = 3.68 pg). The chloroplast genomes were 152,259-155,145 bp in length, and exhibited a conserved quadripartite structure. The Iris cp genomes were highly conserved and similar to other Iridaceae cp genomes. Nucleotide diversity analysis indicated that all three species had similar levels of genetic variation, but the cp genomes of I. koreana and I. minutoaurea were more similar to each other than to I. odaesanensis. Positive selection was inferred for psbK and ycf2 genes of the three Iris species. Phylogenetic analyses consistently recovered I. odaesanensis as a sister to a clade containing I. koreana and I. minutoaurea. Although the phylogenetic relationship, rDNA loci number, and localization, together with the genome size and chromosome number of the three species, allowed for the inference of I. minutoaurea as a putative maternal taxon and I. odaesanensis as a paternal taxon, further analyses involving species-specific molecular cytogenetic markers and genomic in situ hybridization are required to interpret the mechanisms involved in the origin of the chromosomal variation in Iris series Chinenses. This study contributes towards the genomic and chromosomal evolution of the genus Iris.

Comparative analysis of two Korean irises (Iris ruthenica and I. uniflora, Iridaceae) based on plastome sequencing and micromorphology.

Iris ruthenica Ker Gawl. and I. uniflora Pall. ex Link, which are rare and endangered species in Korea, possess considerable horticultural and medicinal value among Korean irises. However, discrimination of the species is hindered by extensive morphological similarity. Thus, the aim of the present study was to identify discriminating features by comparing the species' complete plastid genome (i.e., plastome) sequences and micromorphological features, including leaf margins, stomatal complex distribution (hypostomatic vs. amphistomatic leaves), anther stomata density, and tepal epidermal cell patterns. Plastome comparison revealed slightly divergent regions within intergenic spacer regions, and the most variable sequences, which were distributed in non-coding regions, could be used as molecular markers for the discrimination of I. ruthenica and I. uniflora. Phylogenetic analysis of the Iris species revealed that I. ruthenica and I. uniflora formed a well-supported clade. The comparison of plastomes and micromorphological features performed in this study provides useful information for elucidating taxonomic, phylogenetic, and evolutionary relationships in Iridaceae. Further studies, including those based on molecular cytogenetic approaches using species specific markers, will offer insights into species delimitation of the two closely related Iris species.

Implications of foliar epidermal micromorphology using light and scanning electron microscopy: A useful tool in taxonomy of Korean irises.

The genus Iris L., comprising approximately 210 species, is one of the most species-rich genera in the family Iridaceae. In this study, the first comprehensive leaf micromorphological characters of Korean irises were studied using light and scanning electron microscopy. Our objective was to evaluate the foliar micromorphological characteristics (namely epidermal cells, stomata types, and guard cell size) of Korean Iris taxa in a systematic context. All the investigated Korean Iris taxa had amphistomatic or hypostomatic leaves with anomocytic stomatal complexes. Guard cell length varied among species, ranging from 24.8 µm (I. rossii) to 56.0 µm (I. domestica). Although the presence of papillae on the outer periclinal wall is not of taxonomic significance, leaf margin pattern, guard cell size, and sunken stomata type were useful for species-level identification of Korean Iris species. The occurrence of polymorphic stomatal types was reported here for the first time, and the correlation between genome size and epidermal guard cell length was discussed.

Cytological Study of Cypripedium japonicum Thunb. (Orchidaceae Juss.): An Endangered Species from Korea.

Changes in chromosome number and karyotype evolution are important to plant diversification, as they are both major drivers of speciation processes. Herein, chromosome number, karyotype, and genome size of the Korean lady's slipper orchid Cypripedium japonicum Thunb., an endangered species, were investigated in natural populations. Furthermore, all cytological data from this species are reported herein for the first time. The chromosome number of all investigated C. japonicum plants was diploid (2n = 2x = 22), with x = 11 as base chromosome number, whereby the species can now be clearly distinguished from the Japanese lady's slipper orchid. The karyotypes of all studied individuals were of similar length, symmetrical, and rather unimodal. Flow cytometry of the C. japonicum revealed that the genome size ranged from 28.38 to 30.14 pg/1C. Data on chromosome number and karyotypes were largely consistent with previous results indicating that Korean (x = 11) populations of C. japonicum are more closely related to Chinese populations (x = 11) compared to Japanese (x = 10) populations. These comprehensive cytological results will benefit the efforts to discriminate the geographically isolated and endangered Eastern Asian (China, Japan, and Korea) lady's slipper orchid species.

Micromorphological differentiation of Korean Disporum species using light and scanning electron microscopy.

The genus Disporum Salisb. is widely distributed in East Asia, yet phylogenetically relevant morphological traits useful for differentiating many of the small, perennial, herbaceous species remain poorly described. To address this, leaf, floral, pollen, and orbicule micromorphology of four Korean Disporum species was investigated using light and scanning electron microscopy. All Korean Disporum species examined had hypostomatic leaves, with anomocytic stomatal complexes found only on the abaxial epidermis. Guard cell length varied among species, ranging from 44.30 µm in D. viridescens to 53.49 µm in D. uniflorum. The epidermal cells of the investigated Disporum taxa had sinuate anticlinal cell walls on both adaxial and abaxial surfaces. The surface of the guard and subsidiary cells were either smooth with weak striations or had strongly wrinkled striations. The pollen grains of all Korean Disporum taxa were monads, monosulcate with granular aperture membranes, subprolate to prolate in shape with microreticulate or verrucate exine surfaces. The mean size of pollen grains ranged from 46.38 to 49.92 µm in polar length and from 34.39 to 39.58 µm in equatorial diameter across species. Sexine ornamentation was a taxonomically relevant trait for differentiating Korean Disporum taxa. Additionally, the presence of orbicules as well as the orbicular characters (e.g., size, shape, ornamentation, and association pattern) are described for the first time in species from this genus. The present investigation of leaf and floral micromorphology using light and scanning electron microscopy provides valuable information for the taxonomic differentiation and identification of Disporum species in Korea. RESEARCH HIGHLIGHTS: A detailed micromorphological description of leaf, floral characters (tepal, stigma, style), pollen and orbicule is provided for Korean Disporum species using scanning electron microscopy (SEM) and light microscopy (LM). The presence of orbicules and their taxonomic implications in Korean Disporum species are described for the first time. Phylogenetically informative pollen and orbicule micromorphological characters are described, improving understanding the systematic relationships of Korean species in the genus Disproum.

Impact of parasitic lifestyle and different types of centromere organization on chromosome and genome evolution in the plant genus Cuscuta.

The parasitic genus Cuscuta (Convolvulaceae) is exceptional among plants with respect to centromere organization, including both monocentric and holocentric chromosomes, and substantial variation in genome size and chromosome number. We investigated 12 species representing the diversity of the genus in a phylogenetic context to reveal the molecular and evolutionary processes leading to diversification of their genomes. We measured genome sizes and investigated karyotypes and centromere organization using molecular cytogenetic techniques. We also performed low-pass whole genome sequencing and comparative analysis of repetitive DNA composition. A remarkable 102-fold variation in genome sizes (342-34 734 Mbp/1C) was detected for monocentric Cuscuta species, while genomes of holocentric species were of moderate sizes (533-1545 Mbp/1C). The genome size variation was primarily driven by the differential accumulation of LTR-retrotransposons and satellite DNA. The transition to holocentric chromosomes in the subgenus Cuscuta was associated with loss of histone H2A phosphorylation and elimination of centromeric retrotransposons. In addition, basic chromosome number of holocentric species (x = 7) was smaller than in monocentrics (x = 15 or 16). We demonstrated that the transition to holocentricity in Cuscuta was accompanied by significant changes in epigenetic marks, chromosome number and the repetitive DNA sequence composition.

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.

Micromorphological and cytological comparisons between Youngia japonica and Youngia longiflora using light and scanning electron microscopy.

The genus Youngia Cass. is taxonomically complex and widely distributed in East Asia. Despite the high diversity of species, only two species, Youngia japonica (L.) DC. and Youngia longiflora (Babc. & Stebbins) C.Shih, are currently found in Korea. Among them, Y. longiflora was once recognized as a subspecies of Y. japonica. However, based on chloroplast and nuclear DNA sequences, as well as morphology, Y. longiflora was found to be a distinct species. Here, the two Korean Youngia species were analyzed using scanning electron microscope and light microscope for chromosome number, pollen, cypselae, and leaf micromorphological characters to evaluate if the examined characters are informative for taxonomy. Pollen morphology is described, and the micromorphological characters of Y. longiflora are described for the first time. Cypselae and pollen surface ornamentation, as well as trichome morphology and stomatal size varied between the two species. Y. japonica had longer chromosomes than Y. longiflora, while other chromosomal characters including the localization of the NOR (nucleolar organizer region) were similar. The present micromorphological and cytological results are of great taxonomic value for Youngia species identification in Korea.

Super-Resolution Microscopy Reveals Diversity of Plant Centromere Architecture.

Centromeres are essential for proper chromosome segregation to the daughter cells during mitosis and meiosis. Chromosomes of most eukaryotes studied so far have regional centromeres that form primary constrictions on metaphase chromosomes. These monocentric chromosomes vary from point centromeres to so-called "meta-polycentromeres", with multiple centromere domains in an extended primary constriction, as identified in Pisum and Lathyrus species. However, in various animal and plant lineages centromeres are distributed along almost the entire chromosome length. Therefore, they are called holocentromeres. In holocentric plants, centromere-specific proteins, at which spindle fibers usually attach, are arranged contiguously (line-like), in clusters along the chromosomes or in bands. Here, we summarize findings of ultrastructural investigations using immunolabeling with centromere-specific antibodies and super-resolution microscopy to demonstrate the structural diversity of plant centromeres. A classification of the different centromere types has been suggested based on the distribution of spindle attachment sites. Based on these findings we discuss the possible evolution and advantages of holocentricity, and potential strategies to segregate holocentric chromosomes correctly.

Mitotic Spindle Attachment to the Holocentric Chromosomes of Cuscuta europaea Does Not Correlate With the Distribution of CENH3 Chromatin.

The centromere is the region on a chromosome where the kinetochore assembles and spindle microtubules attach during mitosis and meiosis. In the vast majority of eukaryotes, the centromere position is determined epigenetically by the presence of the centromere-specific histone H3 variant CENH3. In species with monocentric chromosomes, CENH3 is confined to a single chromosomal region corresponding to the primary constriction on metaphase chromosomes. By contrast, in holocentrics, CENH3 (and thus centromere activity) is distributed along the entire chromosome length. Here, we report a unique pattern of CENH3 distribution in the holocentric plant Cuscuta europaea. This species expressed two major variants of CENH3, both of which were deposited into one to three discrete regions per chromosome, whereas the rest of the chromatin appeared to be devoid of CENH3. The two CENH3 variants fully co-localized, and their immunodetection signals overlapped with the positions of DAPI-positive heterochromatic bands containing the highly amplified satellite repeat CUS-TR24. This CENH3 distribution pattern contrasted with the distribution of the mitotic spindle microtubules, which attached at uniform density along the entire chromosome length. This distribution of spindle attachment sites proves the holocentric nature of C. europaea chromosomes and also suggests that, in this species, CENH3 either lost its function or acts in parallel to an additional CENH3-free mechanism of kinetochore positioning.

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.

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.