Molecular cytogenetic analysis of recently evolved Tragopogon (Asteraceae) allopolyploids reveal a karyotype that is additive of the diploid progenitors

Tragopogon mirus and T. miscellus (both 2n = 4x = 24) are recent allotetraploids derived from T. dubius × T. porrifolius and T. dubius × T. pratensis (each 2n = 2x = 12), respectively. The genome sizes of T. mirus are additive of those of its diploid parents, but at least some populations of T. miscellus have undergone genome downsizing. To survey for genomic rearrangements in the allopolyploids, four repetitive sequences were physically mapped. TPRMBO (unit size 160 base pairs [bp]) and TGP7 (532 bp) are tandemly organized satellite sequences isolated from T. pratensis and T. porrifolius, respectively. Fluorescent in situ hybridization to the diploids showed that TPRMBO is a predominantly centromeric repeat on all 12 chromosomes, while TGP7 is a subtelomeric sequence on most chromosome arms. The distribution of tandem repetitive DNA loci (TPRMBO, TGP7, 18S-5.8S-26S rDNA, and 5S rDNA) gave unique molecular karyotypes for the three diploid species, permitting the identification of the parental chromosomes in the polyploids. The location and number of these loci were inherited without apparent changes in the allotetraploids. There was no evidence for major genomic rearrangements in Tragopogon allopolyploids that have arisen multiple times in North America within the last 80 yr.     

Rapid concerted evolution of nuclear ribosomal DNA in two Tragopogon allopolyploids of recent and recurrent origin

We investigated concerted evolution of rRNA genes in multiple populations of Tragopogon mirus and T. miscellus, two allotetraploids that formed recurrently within the last 80 years following the introduction of three diploids (T. dubius, T. pratensis, and T. porrifolius) from Europe to North America. Using the earliest herbarium specimens of the allotetraploids (1949 and 1953) to represent the genomic condition near the time of polyploidization, we found that the parental rDNA repeats were inherited in roughly equal numbers. In contrast, in most present-day populations of both tetraploids, the rDNA of T. dubius origin is reduced and may occupy as little as 5% of total rDNA in some individuals. However, in two populations of T. mirus the repeats of T. dubius origin outnumber the repeats of the second diploid parent (T. porrifolius), indicating bidirectional concerted evolution within a single species. In plants of T. miscellus having a low rDNA contribution from T. dubius, the rDNA of T. dubius was nonetheless expressed. We have apparently caught homogenization of rDNA repeats (concerted evolution) in the act, although it has not proceeded to completion in any allopolyploid population yet examined.     

Comparative proteomics of the recently and recurrently formed natural allopolyploid Tragopogon mirus (Asteraceae) and its parents

? We examined the proteomes of the recently formed natural allopolyploid Tragopogon mirus and its diploid parents (T.?dubius, T.?porrifolius), as well as a diploid F(1) hybrid and synthetic T.?mirus. ? Analyses using iTRAQ LC-MS/MS technology identified 476 proteins produced by all three species. Of these, 408 proteins showed quantitative additivity of the two parental profiles in T.?mirus (both natural and synthetic); 68 proteins were quantitatively differentially expressed. ? Comparison of F(1) hybrid, and synthetic and natural polyploid T.?mirus with the parental diploid species revealed 32 protein expression changes associated with hybridization, 22 with genome doubling and 14 that had occurred since the origin of T.?mirus c. 80?yr ago. We found six proteins with novel expression; this phenomenon appears to start in the F(1) hybrid and results from post-translational modifications. ? Our results indicate that the impact of hybridization on the proteome is more important than is polyploidization. Furthermore, two cases of homeolog-specific expression in T.?mirus suggest that silencing in T.?mirus was not associated with hybridization itself, but occurred subsequent to both hybridization and polyploidization. This study has shown the utility of proteomics in the analysis of the evolutionary consequences of polyploidy.     

GENETIC VARIATION IN TRAGOPOGON SPECIES: ADDITIONAL ORIGINS OF THE ALLOTETRAPLOIDS T. MIRUS AND T. MISCELLUS (COMPOSITAE)

Genetic diversity in the introduced diploids Tragopogon dubius, T. porrifolius, and T. pratensis and their neoallotetraploid derivatives T. mirus and T. miscellus was estimated to assess the numbers of recurrent, independent origins of the two tetraploid species in the Palouse region of eastern Washington and adjacent Idaho. These tetraploid species arose in this region, probably within the past 50–60 yr, and provide one of the best models for the study of polyploidy in plants. The parental species of both T. mirus and T. miscellus have been well documented, and each tetraploid species has apparently formed multiple times. However, a recent survey of the distributions of these allotetraploids revealed that both tetraploid species have expanded their ranges considerably during the past 50 yr, and several new populations of each species were discovered. Therefore, to evaluate the possibility that these recently discovered populations are of recent independent origin, a broad analysis of genetic diversity in T. mirus, T. miscellus, and their diploid progenitors was conducted. Analyses of allozymic and DNA restriction site variation in all known populations of T. mirus and T. miscellus in the Palouse and several populations of each parental diploid species revealed several distinct genotypes in each tetraploid species. Four isozymic multilocus genotypes were observed in T. mirus, and seven were detected in T. miscellus. Tragopogon mirus possesses a single chloroplast genome, that of T. porrifolius, and two distinct repeat types of the 18S-26S ribosomal RNA genes. Populations of T. miscellus from Pullman, Washington, have the chloroplast genome of T. dubius; all other populations of T. miscellus have the chloroplast DNA of T. pratensis. All populations of T. miscellus combine the ribosomal RNA repeat types of T. dubius and T. pratensis, as demonstrated previously. When all current and previously published data are considered, both T. mirus and T. miscellus appear to have formed numerous times even within the small geographic confines of the Palouse, with estimates of five to nine and two to 21 independent origins, respectively. Such recurrent polyploidization appears to characterize most polyploid plant species investigated to date (although this number is small) and may contribute to the genetic diversity and ultimate success of polyploid species.     

ALLOPOLYPLOID SPECIATION IN TRAGOPOGON: INSIGHTS FROM CHLOROPLAST DNA

Tragopogon mirus and T. miscellus are classic examples of recent allopolyploid speciation. Previous studies documented that the diploid parents of T. mirus are T. dubius and T. porrifolius and those of T. miscellus are T. dubius and T. pratensis. Restriction fragment analysis of chloroplast DNA (cpDNA) provided additional evolutionary information regarding the origin of the allotetraploids. We analyzed 39 populations of the three diploid and two allotetraploid species with 18 restriction endonucleases. Six restriction site mutations and three length mutations were identified; these unambiguously differentiated the parental diploids. Previous morphological, cytological, and electrophoretic analyses indicated that T. mirus arose independently at least three times. Chloroplast DNA data suggest that T. porrifolius has consistently been the maternal parent of T. mirus. Chloroplast DNA data also document a minimum of two independent origins of T. miscellus: 1) populations from Pullman, Washington, have T. dubius as the maternal parent; 2) all other populations have T. pratensis as the maternal parent. Two restriction site mutations implicate certain populations of T. dubius in the formation of the Pullman populations of T. miscellus. The two rare diploid species, T. porrifolius and T. pratensis, typically appear as maternal parents of the allotetraploids; the widespread and common T. dubius is the maternal parent only for two populations of T. miscellus. These data suggest that pollen load may be an important factor in determining the male and female parents of allopolyploid angiosperms.     

OWNBEY'S TRAGOPOGONS: 40 YEARS LATER

Two of the classic examples of recent allopolyploid speciation are Tragopogon mirus and T. miscellus. Previous studies have documented that both allotetraploids originated within the past 50–60 years; the diploid parents of T. minis are T. dubius and T. porrifolius; those of T. miscellus are T. dubius and T. pratensis. It has now been 40 years since these allotetraploids were first described by Ownbey in 1950. To assess whether population size (the absolute number of plants) and population number of these species have changed during the past 40 years, we determined their distribution and numbers in the Palouse region of eastern Washington and northern Idaho in the spring of 1990. We visited 90 localities, 60 in Washington and 30 in Idaho. Tragopogon minis is found in nine locations, with eight of these sites located in eastern Whitman County, Washington. Tragopogon miscellus is much more widespread, occurring in 38 of 90 locations. The latter species is now one of the most common weeds in the vicinity of Spokane, Washington. Comparison of the current distributions of these species with historical records indicates that both allotetraploids, especially T. miscellus, have increased substantially in both geographic range and numbers. These Tragopogon species offer an excellent model system for testing questions concerning the ecology and population biology of recently derived allotetraploid species and their diploid progenitors.     

Plant–soil interactions in the expansion and native range of a poleward shifting plant species

Climate warming causes range shifts of many species toward higher latitudes and altitudes. However, range shifts of host species do not necessarily proceed at the same rates as those of their enemies and symbionts. Here, we examined how a range shifting plant species performs in soil from its original range in comparison with soil from the expansion range. Tragopogon dubius is currently expanding from southern into north-western Europe and we examined how this plant species responds to soil communities from its original and expansion ranges. We compared the performance of T. dubius with that of the closely related Tragopogon pratensis , which has a natural occurrence along the entire latitudinal gradient. Inoculation with the rhizosphere soil from T. dubius populations of the original range had a more negative effect on plant biomass production than inoculation with rhizosphere soil from the expansion range. Interestingly, the nonrange expander T. pratensis experienced a net negative soil effect throughout this entire range. The effects observed in this species pair may be due to release from soil born enemies or accumulation of beneficial soil born organisms. If this phenomenon applies broadly to other species, then range expansion may enable plants species to show enhanced performance.     

Polyploids with different origins and ancestors form a single sexual polyploid species

Polyploidization is one of the few mechanisms that can produce instantaneous speciation. Multiple origins of tetraploid lineages from the same two diploid progenitors are common, but here we report the first known instance of a single tetraploid species that originated repeatedly from at least three diploid ancestors. Parallel evolution of advertisement calls in tetraploid lineages of gray tree frogs has allowed these lineages to interbreed, resulting in a single sexually interacting polyploid species despite the separate origins of polyploids from different diploids. Speciation by polyploidization in these frogs has been the source of considerable debate, but the various published hypotheses have assumed that polyploids arose through either autopolyploidy or allopolyploidy of extant diploid species. We utilized molecular markers and advertisement calls to infer the origins of tetraploid gray tree frogs. Previous hypotheses did not sufficiently account for the observed data. Instead, we found that tetraploids originated multiple times from extant diploid gray tree frogs and two other, apparently extinct, lineages of tree frogs. Tetraploid lineages then merged through interbreeding to result in a single species. Thus, polyploid species may have complex origins, especially in systems in which isolating mechanisms (such as advertisement calls) are affected directly through hybridization and polyploidy.     

Suppression of Homoeologous Pairing by <Emphasis Type="Italic">B</Emphasis> Chromosomes in a <Emphasis Type="Italic">Lolium</Emphasis> Species Hybrid

INTERSPECIFIC hybridization together with polyploidy has been an important force in the evolution of many of our graminaceous crop plants. Both wheat (Triticum aestivum) and oats (Avena sativa), for example, are natural allohexaploids derived in each case from the hybridization of three separate but related diploid species. The efforts of plant breeders to synthesize stable and fertile polyploids of this kind have, on the whole, been unsuccessful. The main reason for this is that whereas meiosis in natural allopolyploids such as wheat is extremely regular this is not the case with “synthetic” polyploids. In wheat precise control over pairing at meiosis is achieved by a gene or a cluster of genes on chromosome SB. The gene acts by restricting the pairing to homologous chromosomes with the result that only bivalents are formed, disjunction is regular and inheritance is completely disomic^1,2. In the artificial polyploids at pachytene there is pairing between both homologous chromosomes (from the same species) and “corresponding” homoeologous chromosomes (from different species). The result is an extremely irregular metaphase 1 comprising multivalents and univalents as well as bivalents. Segregation is irregular and a certain degree of infertility is inevitable.     

INCIPIENT POLYPLOID SPECIATION IN THE MAIDENHAIR FERN (ADIANTUM PEDATUM; ADIANTACEAE)?

Despite the widespread occurrence of polyploids in plant taxa and the many advantages attributed to polyploidy, very little is known about the specific processes that lead to the establishment of polyploids in nature. Classical models suggested that polyploids arise following somatic chromosome doubling in hybrids. However, the production of polyploids from unreduced meiotic products has been receiving greater attention. During an enzyme electrophoretic study of a local population of Adiantum pedatum, a mutant producing viable unreduced spores along with abortive spores was discovered. Studies of sporogenesis showed that a synaptic mutation caused the paired chromosomes to disassociate, with mostly univalents remaining at metaphase I. In such aberrant spore mother cells, one of two pathways was followed in the remaining stages of meiosis. Cells attempting both meiotic divisions formed abortive spores. However, in spore mother cells that bypassed meiosis I and formed a restitution nucleus, meiosis II and subsequent stages of sporogenesis proceeded normally. Unreduced diploid spores resulted from this second pathway. When sown on either agar or sterile soil, these diplospores germinated and produced diploid gametophytes. Tetraploid sporophytes were produced by the gametophytes growing on sterile soil. The discovery of diploid sporophytes producing unreduced spores provided the opportunity to characterize the first step of one possible route to polyploid formation. Continued observations of the natural population may provide insights into the earliest stages of natural polyploid formation.     

Origin and evolution of North American polyploid Silene (Caryophyllaceae)

Nuclear DNA sequences from introns of the low-copy nuclear gene family encoding the second largest subunit of RNA polymerases and the ribosomal internal transcribed spacer (ITS) regions, combined with the psbE-petL spacer and the rps16 intron from the chloroplast genome were used to infer origins and phylogenetic relationships of North American polyploid Silene species and their closest relatives. Although the vast majority of North American Silene species are polyploid, which contrasts to the diploid condition dominating in other parts of the world, the phylogenetic analyses rejected a single origin of the North American polyploids. One lineage consists of tetraploid Silene menziesii and its diploid allies. A second lineage, Physolychnis s.l., consists of Arctic, European, Asian, and South American taxa in addition to the majority of the North American polyploids. The hexaploid S. hookeri is derived from an allopolyploidization between these two lineages. The tetraploid S. nivea does not belong to any of these lineages, but is closely related to the European diploid S. baccifera. The poor resolution within Physolychnis s.l. may be attributed to rapid radiation, recombination among homoeologues, homoplasy, or any combination of these factors. No extant diploid donors could be identified in Physolychnis s.l.     

Evidence of 2n microspore production in a natural diploid population of Turnera sidoides subsp. carnea and its relevance in the evolution of the T. sidoides (Turneraceae) autopolyploid complex

Turnera sidoides is a complex of distylous perennial rhizomatous herbs that includes five subspecies. Since polyploidy has played a prominent role within this species (x?=?7), ongoing studies in T. sidoides are focused on the understanding of the mechanisms involved in the origin and the establishment of polyploids. Therefore, aiming to contribute to the understanding of the mode of polyploid formation, in this study we investigated the frequency of unreduced microspores in a natural diploid population of T. sidoides subsp. carnea by analyzing the size range of pollen and the constitution of the sporads. The results showed that some of the individuals studied produced 2n and 4n microspores, both in short- and long-styled floral morphs. The analysis performed documents the production of unreduced microspores in T. sidoides subsp. carnea and provides evidence that support the hypothesis of sexual polyploidization as one of the most probable mechanisms involved in the origin of polyploids within this species complex. The role of unreduced pollen in the establishment and persistence of newly formed polyploids in diploid populations of T. sidoides is also discussed.     

Evolution and polyploid origins in North American Arctic Puccinellia (Poaceae) based on nuclear ribosomal spacer and chloroplast DNA sequences

The proportion of polyploid plant species increases at higher latitudes, and it has been suggested that original postglacial Arctic immigrants of some large groups, including grasses, were polyploid. We analyzed noncoding nuclear and chloroplast DNA of all North American diploid Puccinellia (Poaceae) and a subset of arctic polyploids to hypothesize evolutionary relationships among diploids and to evaluate the parentage of polyploids. Diploids formed three lineages: one uniting arctic species P. arctica and P. banksiensis; a second comprising arctic species P. tenella, P. alaskana, P. vahliana, and P. wrightii; and a third uniting the two temperate species P. lemmonii and P. parishii. The arctic species P. angustata (hexaploid) and P. andersonii (primarily octoploid) apparently derive from the P. arctica-P. banksiensis lineage based on ITS and chloroplast sequences, and share an ancestor with arctic triploid/tetraploid P. phryganodes based on nrDNA sequences. Sequence comparisons also suggest tetraploid P. bruggemannii evolved from two arctic lineages: P. vahliana-P. wrightii and P. arctica-P. banksiensis. These patterns and the predominance of arctic rather than temperate diploid species support the idea that diploid Puccinellia recolonized the Arctic from northern glacial refugia like Beringia, and also formed stabilized polyploid hybrids during these refugial events or subsequently during postglacial colonization.     

Regulation of toxocariasis in mice selectively reared for high and low immune responses to Nematospiroides dubius

Test mice have been selectively reared for high (H) or low (L) immune responses to Nematospiroides dubius. After secondary infection with N. dubius, the L mice voided ten times as many eggs in their faeces as the H mice, and at necropsy, 71% versus 20% of the inoculum of N. dubius were recovered as adult worms from the L and H mice respectively. Furthermore, N. dubius were more fecund in the L than in H mice. High or low immune responsiveness was not restricted to N. dubius infection in these mice but was also observed during Toxocara canis infection. The migration of T. canis larvae from gut via the liver to skeletal muscle and CNS was inhibited in H versus L mice. Many more larvae were recovered from the livers of H compared with L mice which was indicative of greater immunity in the H mice. The protective immune response in H compared with L mice to both N. dubius and T. canis included pronounced eosinophilia and elevated antiparasite antibody titres.     

A genetic appraisal of a new synthetic Nicotiana tabacum (Solanaceae) and the Kostoff synthetic tobacco

Polyploids have significantly influenced angiosperm evolution. Understanding the genetic consequences of polyploidy is advanced by studies on synthetic allopolyploids that mimic natural species. In Nicotiana, Burk (1973) and Kostoff (1938) generated synthetic tobacco (N. tabacum) using the parents ♀N. sylvestris × ♂N. tomentosiformis. We previously reported rapid genetic changes in the Burk material. Kostoff's material has 24 chromosomes of N. sylvestris origin (S-genome), 24 of N. tomentosiformis origin (T-genome), and a large intergenomic translocation, but not an additive distribution of ribosomal DNA (rDNA) families as expected from the parental contribution. Our new synthetic tobacco lines TR1 and TR2 are chromosomally balanced with no intergenomic translocations and are either sterile or have highly reduced fertility, supporting the nuclear cytoplasmic hypothesis that allopolyploid fertility is enhanced by intergenomic translocations. Two plants of TR1 (TR1-A, TR1-B) have the expected number, structure, and chromosomal distribution of rDNA families, in contrast to Burk's and Kostoff's synthetic tobaccos and to synthetic polyploids of Arabidopsis. Perhaps allopolyploids must pass through meiosis before genetic changes involving rDNA become apparent, or the genetic changes may occur stochastically in different synthetic allopolyploids. The lack of fertility in the first generation of our synthetic tobacco lines may have uses in biopharmacy.