Genome size evolution of the extant lycophytes and ferns

Ferns and lycophytes have remarkably large genomes. However, little is known about how their genome size evolved in fern lineages. To explore the origins and evolution of chromosome numbers and genome size in ferns, we used flow cytometry to measure the genomes of 240 species (255 samples) of extant ferns and lycophytes comprising 27 families and 72 genera, of which 228 species (242 samples) represent new reports. We analyzed correlations among genome size, spore size, chromosomal features, phylogeny, and habitat type preference within a phylogenetic framework. We also applied ANOVA and multinomial logistic regression analysis to preference of habitat type and genome size. Using the phylogeny, we conducted ancestral character reconstruction for habitat types and tested whether genome size changes simultaneously with shifts in habitat preference. We found that 2C values had weak phylogenetic signal, whereas the base number of chromosomes (x) had a strong phylogenetic signal. Furthermore, our analyses revealed a positive correlation between genome size and chromosome traits, indicating that the base number of chromosomes (x), chromosome size, and polyploidization may be primary contributors to genome expansion in ferns and lycophytes. Genome sizes in different habitat types varied significantly and were significantly correlated with habitat types; specifically, multinomial logistic regression indicated that species with larger 2C values were more likely to be epiphytes. Terrestrial habitat is inferred to be ancestral for both extant ferns and lycophytes, whereas transitions to other habitat types occurred as the major clades emerged. Shifts in habitat types appear be followed by periods of genomic stability. Based on these results, we inferred that habitat type changes and multiple whole-genome duplications have contributed to the formation of large genomes of ferns and their allies during their evolutionary history.     

Phylogenetic relationships and the maternal donor of Roegneria (Triticeae: Poaceae) based on three nuclear DNA sequences (ITS,Acc1, and Pgk1) and one chloroplast region (trnL-F)

Roegneria is a polyploid perennial genus in the tribe Triticeae. Some species of Roegneria are morphologically similar to genus Elymus and have been classified in Elymus. To investigate the delimitation and phylogenetic relationships of Roegneria, nuclear (ITS, Acc1, and Pgk1) and chloroplast (trnL–trnF) DNA regions were sequenced for 38 allopolyploid species and 32 diploid species of Triticeae. Phylogenetic analyses of nuclear DNA revealed that all Roegneria species were included in the St and Y genome clades, and that the Y genome was closely related to the V and Xp genomes. The chloroplast DNA dataset showed that Roegneria species were grouped with Pseudoroegneria species. The Pseudoroegneria species from the Middle East (P. libanotica and P. tauri) and Central Asia (P. strigosa) were more closely related to Roegneria species. The results suggested that: (i) the species containing the St and Y genomes should be segregated from Elymus and treated as a distinct genus, Roegneria, based on the genomic constitution; (ii) P. libanotica, P. tauri, and/or P. strigosa potentially served as the maternal donor of the St genome in Roegneria; (iii) The Y genome of Roegneria originated from a diploid Y genome species, and the V and Xp genomes may have contributed to Y genome formation; (iv) among Roegneria species of previously uncertain genomic constitution, R. seriotina was tetraploid and possessed the StY genomes, E. calcicolus was hexaploid with the StYH genomic constitution and should be classified in Campeiostachys, R. glaucifolia possessed the StStY genomes, and R. tschimganica had the genomic constitution St₁St₂Y.     

Nuclear and plastid phylogenomic analyses provide insights into the reticulate evolution,species delimitation,and biogeography of the Sino-Japanese disjunctive Diabelia (Caprifoliaceae)

Understanding biological diversity and the mechanisms of the Sino-Japanese disjunctions are major challenges in eastern Asia biogeography. The Sino-Japanese flora has been broadly studied as an ideal model for plant phylogeography. Diabelia Landrein (Caprifoliaceae) is an East Asian genus, with a disjunctive distribution across the Sino-Japanese region. However, relationships within Diabelia remain elusive. In this study, we reconstructed the phylogeny of Diabelia and inferred historical biogeography and evolutionary patterns based on nuclear and plastid sequences from target enrichment and genome skimming approaches, respectively. We found that the main clades within Diabelia were discordant between nuclear and plastid trees. Both nuclear and plastid phylogenetic analyses supported five main clades: Diabelia serrata (Siebold & Zucc.) Landrein, Diabelia tetrasepala (Koidz.) Landrein, Diabelia sanguinea (Makino) Landrein, Diabelia stenophylla (Honda) Landrein, and Diabelia spathulata (Siebold & Zucc.) Landrein. Species network analyses revealed that Diabelia tetrasepala is likely the result of a hybridization event. Divergence time estimation and ancestral area reconstructions showed that Diabelia originated in Japan during the early Miocene, with subsequent vicariance and dispersal events between Japan and Korea, and between Japan and China. Overall, our results support the division of Diabelia into five main clades and the recognition of five species in the genus. This research provides new insights into the species delimitation and speciation processes of taxonomically complex lineages such as Diabelia.     

Phylogenomic and comparative analyses of Rheum (Polygonaceae,Polygonoideae)

Species of Rheum have high medicinal value, with the center of diversity in the Qinghai–Tibet Plateau (QTP) and adjacent regions. However, phylogenetic relationships of Rheum are still unclear due to fragment markers providing insufficient informative loci. Here, we sequenced and annotated plastomes of nine Rheum species, and compared the genome structure among the novel nine species along with three published species. Comparative analyses revealed that plastomes of Rheum share a relatively conserved structure. Five highly divergent regions (accD, ccsA, matK, ndhF, and ndhH) can be used as valuable molecular markers for further species delimitation and population genetic studies. Twenty-two accessions representing 17 species were used for phylogenetic analysis, which generated a robust phylogenetic tree and revealed two major clades within Rheum. Phylogenetic results showed that glasshouse structures and cushions of Rheum are results of parallel evolution during adaptation to similar environments. Inconsistent tree topology between concatenated and coalescent methods was detected, implying that incomplete lineage sorting and hybridization may have occurred in the evolutionary history of Rheum. Divergence time estimation based on two fossil calibrations and three secondary calibrations revealed a Miocene to middle Oligocene origin of Rheum. Our study provides valuable genomic resources for the medicinally important genus Rheum, while gaining helpful insights into its systematics and evolution.     

Endo-allopolyploidy of autopolyploids and recurrent hybridization—A possible mechanism to explain the unresolved Y-genome donor in polyploid Elymus species (Triticeae: Poaceae)

The genus Elymus L. in the tribe Triticeae (Poaceae) includes economically and ecologically important forage grasses. The genus contains the pivotal St genome from Pseudoroegneria in combination with other genomes in the tribe. Many Elymus species are tetraploids containing the StY genomes. It is thought that polyploidization characterizes the speciation of the genus in which the Y is considered as another key genome. Based on data from cytological, genome in situ hybridization, and molecular studies, we hypothesized an endo-allopolyploidy origin of the StY-genome species from the autotetraploid Pseudoroegneria species. To test this hypothesis, we amplified, cloned, and sequenced five single-copy nuclear genes (i.e., alcohol dehydrogenase 1–3, Adh1–Adh3, RNA polymerase II, Rpb2; and Waxy) from Elymus, Pseudoroegneria, and Hordeum species. The phylogenetic trees constructed based on the sequencing analyses of all genes indicated that diploid and autotetraploid Pseudoroegneria species were closely related, although with considerable genetic variation in tetraploids. In addition, the StY-genome Elymus species tended to have a close relationship with the diploid and autotetraploid Pseudoroegneria species, although different phylogenetic relationships among the gene trees were detected. These results indicated that the StY-genome species may have an autotetraploid origin and experienced recurrent hybridization. The complex St genomes in Pseudoroegneria in the polyploid state may gain more opportunities for within-species differentiation and recurrent hybridization. As a result, series modified versions of St genomes evolved into the StY genomes in some Elymus species.     

Genome size variation and species diversity in salamanders

Salamanders (Urodela) have among the largest vertebrate genomes, ranging in size from 10 to 120 pg. Although changes in genome size often occur randomly and in the absence of selection pressure, nonrandom patterns of genome size variation are evident among specific vertebrate lineages. Several reports suggest a relationship between species richness and genome size, but the exact nature of that relationship remains unclear both within and across different taxonomic groups. Here, we report (a) a negative relationship between haploid genome size (C‐value) and species richness at the family taxonomic level in salamander clades; (b) a correlation of C‐value and species richness with clade crown age but not with diversification rates; (c) strong associations between C‐value and both geographic area and climatic‐niche rate. Finally, we report a relationship between C‐value diversity and species diversity at both the family‐ and genus‐level clades in urodeles.     

Analysis of phylogenetic relationships of Potamogeton species in China based on chloroplast trnT-trnF sequences

Sequences of the chloroplast trnT-trnF region were analyzed for species of the genus Potamogeton distributed in China to reconstruct phylogenetic relationships. The phylogenetic analyses showed that the genus Potamogeton could be divided into two clades. Eighteen species formed a monophyletic clade while the remaining four formed a second, distinguishable one, supporting the conventional treatment that the genus Potamogeton contains the submerged linear-leaved group and the submerged broad-leaved group. The first clade, which represented the subgenus Potamogeton, could be further divided into two subclades. The second clade, which represented the subgenus Coleogeton, displayed a close phylogenetic relationship with the subgenus Potamogeton and occupied a unique position within the genus Potamogeton. This finding suggested that the treatment of the subgenus Coleogeton, which was once regarded as the genus Stuckenia Börner, may need to be reconsidered. Furthermore, identification of maternal donors of some hybrids was successfully applied based on sequence of maternally inherited chloroplast genome. The female parents of three putative hybrids, P. × malainoides, P. × anguillanus, and P. × orientalis, were proved to be accordant with previous morphological conclusions.     

Polyploidy does not control all: Lineage‐specific average chromosome length constrains genome size evolution in ferns

Recent studies investigating the evolution of genome size diversity in ferns have shown that they have a distinctive genome profile compared with other land plants. Ferns are typically characterized by possessing medium‐sized genomes, although a few lineages have evolved very large genomes. Ferns are different from other vascular plant lineages as they are the only group to show evidence for a correlation between genome size and chromosome number. In this study, we aim to explore whether the evolution of fern genome sizes is not only shaped by chromosome number changes arising from polyploidy but also by constraints on the average amount of DNA per chromosome. We selected the genus Asplenium L. as a model genus to study the question because of the unique combination of a highly conserved base chromosome number and a high frequency of polyploidy. New genome size data for Asplenium taxa were combined with existing data and analyzed within a phylogenetic framework. Genome size varied substantially between diploid species, resulting in overlapping genome sizes among diploid and tetraploid spleenworts. The observed additive pattern indicates the absence of genome downsizing following polyploidy. The genome size of diploids varied non‐randomly and we found evidence for clade‐specific trends towards larger or smaller genomes. The 578‐fold range of fern genome sizes have arisen not only from repeated cycles of polyploidy but also through clade‐specific constraints governing accumulation and/or elimination of DNA.     

Sauropod dinosaurs evolved moderately sized genomes unrelated to body size

Sauropodomorph dinosaurs include the largest land animals to have ever lived, some reaching up to 10 times the mass of an African elephant. Despite their status defining the upper range for body size in land animals, it remains unknown whether sauropodomorphs evolved larger-sized genomes than non-avian theropods, their sister taxon, or whether a relationship exists between genome size and body size in dinosaurs, two questions critical for understanding broad patterns of genome evolution in dinosaurs. Here we report inferences of genome size for 10 sauropodomorph taxa. The estimates are derived from a Bayesian phylogenetic generalized least squares approach that generates posterior distributions of regression models relating genome size to osteocyte lacunae volume in extant tetrapods. We estimate that the average genome size of sauropodomorphs was 2.02 pg (range of species means: 1.77–2.21 pg), a value in the upper range of extant birds (mean = 1.42 pg, range: 0.97–2.16 pg) and near the average for extant non-avian reptiles (mean = 2.24 pg, range: 1.05–5.44 pg). The results suggest that the variation in size and architecture of genomes in extinct dinosaurs was lower than the variation found in mammals. A substantial difference in genome size separates the two major clades within dinosaurs, Ornithischia (large genomes) and Saurischia (moderate to small genomes). We find no relationship between body size and estimated genome size in extinct dinosaurs, which suggests that neutral forces did not dominate the evolution of genome size in this group.     

Cytophotometric evidence of variation in genome size of desmognathine salamanders

The amount of DNA per haploid genome, the C-value, is often directly correlated with nuclear and cell volume, but inversely correlated with cell replication rate. Also, rates of cellular growth sometimes appear to be correlated with organismal developmental rates and life history patterns. Among vertebrates, salamanders exhibit the greatest variation in genome size. In the present study we have examined interspecific and intraspecific variation in blood cell DNA levels in the genus Desmognathus, which shows greater variation in life history traits than any other salamander genus. Specimens of Desmognathus quadramaculatus, D. monticola, D. ochrophaeus and D. wrighti were collected from nature at two localities in the southern Appalachian Mountains. Estimates of genome size in pg of DNA were obtained from blood smears by DNA-Feulgen cytophotometry, using erythrocyte nuclei of Xenopus laevis as an internal reference standard of 6.35 pg DNA per cell. C-values of Desmognathus are the smallest in the order Caudata. Although significant variation in DNA levels was found among the four species, the differences were small, and do not support previously proposed relationships between C-value and life-history variation.     

Phylogenomic relationships and species identification of the olive genus Olea (Oleaceae)

The olive genus Olea includes c. 30–40 taxa in three subgenera (Olea, Tetrapilus, and Paniculatae) within the family Oleaceae. Historically, the Olea genus was classified into four groups that were overall well supported by reconstructed phylogenies, despite incomplete sampling of subgenus Tetrapilus and poor resolution within clades. These analyses also showed that the genus was not monophyletic. Reliable identification of Olea species is important for both their conservation and utilization of this economically important genus. In this study, we used phylogenomic data from genome skimming to resolve relationships within Olea and to identify molecular markers for species identification. We assembled the complete plastomes, and nrDNA of 26 individuals representing 13 species using next-generation sequencing and added 18 publicly available accessions of Olea. We also developed nuclear SNPs using the genome skimming data to infer the phylogenetic relationships of Olea. Large-scale phylogenomic analyses of 138 samples of tribe Oleeae supported the polyphyly of Olea, with Olea caudatilimba and Olea subgenus Tetrapilus not sharing their most recent common ancestor with the main Olea clade (subgenus Paniculatae and subgenus Olea). The interspecific phylogenetic resolution was poor owing to a possible rapid radiation. By comparing with the plastome data, we identified the markers ycf1b and psbE-petL as the best Olea-specific chloroplast DNA barcodes. Compared with universal barcodes, specific DNA barcodes and super-barcode exhibited higher discriminatory power. Our results demonstrated the power of phylogenomics to improve phylogenetic relationships of intricate groups and provided new insights into barcodes that allow for accurate identification of Olea species.     

Chloroplast genomes of key species shed light on the evolution of the ancient genus Isoetes

Although phylogenetic studies have revealed major clades, the deepest relationships in Isoetes remain unresolved. The use of next-generation sequencing provides enormous amounts of gene sequences, which allows not only clarification of the basal relationships but also rapid radiations. Plastomes of six key Isoetes species were annotated, revealing a total of 129 or 130 genes, depending on the species. Our phylogenomic analyses comprising representatives of all major clades yielded well-supported nodes and identical topologies using maximum likelihood and Bayesian inference. The phylogenetic reconstructions detangled the deep relationships in Isoetes and illuminated the more recent radiations in the genus. A basal dichotomy was found that grouped Isoetes spp. from Brazil and South Africa into a clade sister to the remaining Isoetes groups. Interestingly, I. andicola was found to be sister to the North American species complex. Genomic trait mapping analysis showed that the missing introns in the atpF and clpP genes were well conserved in two major clades. The absence of trnK-UUU was observed in the Brazilian tropical species and in I. velata. Among lycophytes, the gene trnR-CCG was missing only in I. eludens. In general, genomic traits such as the presence or absence of internal stop codons, a tRNA, and an intron were revealed to be conserved within groups, suggesting that these genomic traits might reveal vital information about the evolution of the genus. This study will contribute to understanding the diversification of Isoetes and the establishment of a better framework to address the evolutionary history of the genus.     

Genome size and chromosome number in the New Zealand species of Schoenus (Cyperaceae)

Schoenus (Cyperaceae) has holocentric chromosomes. Chromosome numbers were counted and nuclear DNA amounts were measured for all the New Zealand species of the genus. Chromosome numbers ranged from 2n = 8 to c. 2n = 90. Two chromosome races, with 2n = 28 and 2n = 56, were found in S. pauciflorus. Flow cytometry using propidium iodide‐stained nuclei was used to measure genome size. A 14.8‐fold variation in 2C DNA content was found, with values ranging from 1.33 to 19.71 pg/2C nucleus. Phylogenetic trees based on sequence variation in the internal transcribed spacer (ITS) region of the 45S ribosomal DNA locus were constructed using several phylogenetic models to reveal possible evolutionary relationships among the New Zealand Schoenus spp. and a sample of Australian Schoenus spp. Analysis revealed heterogeneity of chromosome number, size and DNA C value within clades. Meiosis in four species showed only bivalent formation at metaphase I. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 169 , 555–564.     

Correlated evolution of genome size and seed mass

Previous investigators have identified strong positive relationships between genome size and seed mass within species, and across species from the same genus and family. Here, we make the first broad-scale quantification of this relationship, using data for 1222 species, from 139 families and 48 orders. We analyzed the relationship between genome size and seed mass using a statistical framework that included four different tests. A quadratic relationship between genome size and seed mass appeared to be driven by the large genome/seed mass gymnosperms and the many small genome size/large seed mass angiosperms. Very small seeds were never associated with very large genomes, possibly indicating a developmental constraint. Independent contrast results showed that divergences in genome size were positively correlated with divergences in seed mass. Divergences in seed mass have been more closely correlated with divergences in genome size than with divergences in other morphological and ecological variables. Plant growth form is the only variable examined thus far that explains a greater proportion of variation in seed mass than does genome size.     

Patterns of genome size in the copepoda

Adult somatic nuclear DNA contents are reported for eleven cyclopoid species (Megacyclops latipes, Mesocyclops edax, M. longisetus, M. ruttneri, M. leuckarti, M. woutersi, Macrocyclops albidus, Cyclops strenuus, Acanthocyclops robustus, Diothona oculata, Thermocyclops crassus) and for the harpacticoid Tigriopus californicus and range from 0.50 to 4.1 pg DNA per nucleus. These diploid genome sizes are consistent with previously published values for four Cyclops species (0.28–1.8 pg DNA per nucleus), but are strikingly smaller than those reported for marine calanoids (4.32–24.92 pg DNA per nucleus). We discuss three explanations, none of them exclusive of another, to account for the smaller size and range of cyclopoid genome sizes relative to calanoid genome sizes: (1) higher prevalence of chromatin diminution in the Cyclopoida, (2) phylogenetic structure or older age of the Calanoida relative to Cyclopoida and (3) nucleotypic selection that may influence life history variation and fitness. Measurements of genome size were made on Feulgen stained, somatic cell nuclei, using scanning microdensitometry which is well suited to the sparse and heterogeneous populations of copepod nuclei. The importance of measuring large numbers of nuclei per specimen, possible sources of variation associated with cytophotometric measurements, and appropriate use of internal reference standards and stoichiometry of the Feulgen stained nuclei are discussed.     

Plastid genome structure and loss of photosynthetic ability in the parasitic genus Cuscuta

The genus Cuscuta (dodder) is composed of parasitic plants, some species of which appear to be losing the ability to photosynthesize. A molecular phylogeny was constructed using 15 species of Cuscuta in order to assess whether changes in photosynthetic ability and alterations in structure of the plastid genome relate to phylogenetic position within the genus. The molecular phylogeny provides evidence for four major clades within Cuscuta. Although DNA blot analysis showed that Cuscuta species have smaller plastid genomes than tobacco, and that plastome size varied significantly even within one Cuscuta clade, dot blot analysis indicated that the dodders possess homologous sequence to 101 genes from the tobacco plastome. Evidence is provided for significant rates of DNA transfer from plastid to nucleus in Cuscuta. Size and structure of Cuscuta plastid genomes, as well as photosynthetic ability, appear to vary independently of position within the phylogeny, thus supporting the hypothesis that within Cuscuta photosynthetic ability and organization of the plastid genome are changing in an unco-ordinated manner.     

Evolution of genome size in the angiosperms

Genome size varies extensively across the flowering plants, which has stimulated speculation regarding the ancestral genome size of these plants and trends in genome evolution. We investigated the evolution of C-values across the angiosperms using a molecular phylogenetic framework and C-values not previously available for crucial basal angiosperms, including Amborella, Illiciaceae, and Austrobaileya. Reconstructions of genome size across the angiosperms and extant gymnosperms indicate that the ancestral genome size for angiosperms is very small (1C ≤ 1.4 pg), in agreement with an earlier analysis of Leitch et al. (1998). Furthermore, a very small genome size (1C ≤ 1.4 pg) is ancestral not only for the angiosperms in general, but also for most major clades of flowering plants, including the monocots and the eudicots. The ancestral genome of core eudicots may also have been very small given that very low 1C-values appear to be ancestral for major clades of core eudicots, such as Caryophyllales, Saxifragales, and asterids. Very large genomes occur in clades that occupy derived positions within the monocots and Santalales.     

One size does not fit all: no evidence for an optimal body size on islands

Aim Optimal body size theories predict that large clades have a single, optimal, body size that serves as an evolutionary attractor, with the full body size spectrum of a clade resulting from interspecific competition. Because interspecific competition is believed to be reduced on islands, such theories predict that insular animals should be closer to the optimal size than mainland animals. We test the resulting prediction that insular clade members should therefore have narrower body size ranges than their mainland relatives. Location World‐wide. Methods We used body sizes and a phylogenetic tree of 4004 mammal species, including more than 200 species that went extinct since the last ice age. We tested, in a phylogenetically explicit framework, whether insular taxa converge on an optimal size and whether insular clades have narrow size ranges. Results We found no support for any of the predictions of the optimal size theory. No specific size serves as an evolutionary attractor. We did find consistent evidence that large (> 10 kg) mammals grow smaller on islands. Smaller species, however, show no consistent tendency to either dwarf or grow larger on islands. Size ranges of insular taxa are not narrower than expected by chance given the number of species in their clades, nor are they narrower than the size ranges of their mainland sister clades – despite insular clade members showing strong phylogenetic clustering. Main conclusions The concept of a single optimal body size is not supported by the data that were thought most likely to show it. We reject the notion that inclusive clades evolve towards a body‐plan‐specific optimum.     

Phylogenomic study of Amorphophallus (Alismatales; Araceae): When plastid DNA gene sequences help to resolve the backbone subgeneric delineation

Encompassing ca. 200 species distributed in paleotropical Africa and Asia, Amorphophallus is one of the largest genera of Araceae. In spite of the great economic interest in its glucomannan production, only a few studies have attempted to grasp the evolutionary history of this genus. In the current state of knowledge, four main clades, mostly linked to biogeographical delineation, have been identified from phylogenies based on a few genes. However, relationships among and within these clades still remain unclear, due to the rapid radiation that occurred during the early evolutionary history of the genus. Here, we generated genome skimming libraries for 43 specimens from 36 species distributed across the 4 clades, which allowed us to produce a phylogenetic matrix for a set of 71 plastid genes. Our phylogenies confirm the monophyly of these clades but show a new and well-resolved arrangement among these clades. Our analyses therefore provide a new scenario and timeline for the evolution of the main Amorphophallus clades, consistent with the morphological characteristics of the clades. The inferred scenario is also in agreement with climate dynamics and the onset of long-distance dispersal by the earliest migratory birds near the Oligocene/Miocene transition around 23 million years ago. Our study provides an up-to-date baseline to understand biogeographic and ecological processes that shaped the current diversity and distribution of Amorphophallus, paving the way for larger-scale phylogenomic studies based on plastid and nuclear genomes.     

Phylogenomics and rapid diversification of the genus Eutrema on the Qinghai–Tibet Plateau and adjacent regions

Both geographic isolation and polyploidization are assumed to play an important role in driving species diversification. However, this is rarely illustrated through phylogenomic analyses. The genus Eutrema (Brassicaceae), which also includes the salt-resistant species, are distributed mainly in Asia with extensive species diversification in the Qinghai–Tibet Plateau (QTP) and adjacent regions. In this study, we revealed almost fully resolved backbone relationships of the genus with genome re-sequencing data for genomes of 168 individuals from 28 species. Phylogenetic analyses of both plastomes and single-copy nuclear genes from the whole genome recovered six well-supported clades with almost consistent relationships. The first two clades are mainly distributed in central China and central Asia, while the other four in the QTP and adjacent regions. All of them diversified within 12 million years. Within each clade, we recovered numerous conflicts in the interspecific relationships between nuclear and plastome phylogenies, likely suggesting hybridization and incomplete lineage sorting during species diversification. Our estimation of genome size and comparison of the number of the single-copy nuclear genes demonstrated frequent occurrences of polyploids in the genus. Except for an establishment of the backbone phylogeny, our phylogenomic analyses suggest that in addition to strong geographic isolation, polyploidization may have played an important role in species diversification of this genus.