Entangling ancient allotetraploidization in Asian Mitella: an integrated approach for multilocus combinations

The reconstruction of an ancient polyploidization history is often challenging, although it is a crucial step in clarifying the mechanisms underlying the contemporary success and diversity of polyploids. Phylogenetic relationships of duplicated gene pairs of polyploids, with respect to their orthologs in related diploids, have been used to address this problem, but they often result in conflicting topologies among different genes. Asimitellaria is an East Asian endemic tetraploid lineage of perennials (genus Mitella; Saxifragaceae) that has diversified in riparian habitats. Phylogenetic analyses of four nuclear-encoded, single-copy (per haploid) genes GBSSI-A, GBSSI-B, GS-II, and PepCK all supported a single allopolyploid origin of Asimitellaria, but they did not lead to a consensus about which diploid lineage gave rise to each of the Asimitellaria subgenomes. To address this issue, we used an integrated approach, whereby the four gene data sets and an additional nuclear ribosomal external transcribed spacer and internal transcribed spacer (including a 5.8S ribosomal DNA) data set were concatenated in all possible combinations, and the most probable data combination was determined together with the phylogenetic inference. This resulted in relatively robust support for the two closely related North American diploid species as the ancestral lineages of the Asimitellaria subgenomes, suggesting ancient intercontinental migration of the diploid or tetraploid lineages and subsequent tetraploid diversification in the Japanese Archipelago. The present approach enabled sorting out the duplicated genes into their original combinations in their preduplication ancestors under a maximum-likelihood framework, and its extension toward genome sequencing data may help in the reconstruction of ancestral, preduplicated, whole-genome structures.     

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.     

Polyploid and hybrid evolution in roses east of the Rocky Mountains

This study investigates the impact of hybridization and polyploidy in the evolution of eastern North American roses. We explore these processes in the Rosa carolina complex (section Cinnamomeae), which consists of five diploid and three tetraploid species. To clarify the status and origins of polyploids, a haplotype network (statistical parsimony) of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) nuclear gene was estimated for polyploids of the complex and for diploids of section Cinnamomeae in North America. A genealogical approach helped to decipher the evolutionary history of polyploids from noise created by hybridization, incomplete lineage sorting, and allelic segregation. At the diploid level, species west of the Rocky Mountains are distinct from eastern species. In the east, two groups of diploids were found: one consists of R. blanda and R. woodsii and the other of R. foliolosa, R. nitida, and R. palustris. Only eastern diploids are involved in the origins of the polyploids. Rosa arkansana is derived from the blanda-woodsii group, R. virginiana originated from the foliolosa-nitida-palustris group, and R. carolina is derived from a hybrid between the two diploid groups. The distinct origins of these polyploid taxa support the hypothesis that the three polyploids are separate species.     

Evolution of biogeographic patterns, ploidy levels, and breeding systems in a diploid-polyploid species complex of Primula

Primula sect. Aleuritia subsect. Aleuritia (Aleuritia) includes diploid, self-incompatible heterostyles and polyploid, self-compatible homostyles, the latter generally occurring at higher latitudes than the former. This study develops a phylogenetic hypothesis for Aleuritia to elucidate the interactions between Pleistocene glacial cycles, biogeographic patterns, ploidy levels and breeding systems. Sequences from five chloroplast DNA loci were analyzed with parsimony to reconstruct a phylogeny, haplotype network, and ancestral states for ploidy levels and breeding systems.The results supported the monophyly of Aleuritia and four major biogeographic lineages: an amphi-Pacific, a South American, an amphi-Atlantic and a European/North American lineage. At least four independent switches to homostyly and five to polyploidy were inferred. An Asian ancestor probably gave origin to an amphi-Pacific clade and to a lineage that diversified on the European and American continents. Switches to homostyly occurred exclusively in polyploid lineages, which mainly occupy previously glaciated areas. The higher success of the autogamous polyploid species at recolonizing habitats freed by glacial retreat might be explained in terms of selection for reproductive assurance.     

Progenitor-derivative relationships of Hordeum polyploids (Poaceae, Triticeae) inferred from sequences of TOPO6, a nuclear low-copy gene region

Polyploidization is a major mechanism of speciation in plants. Within the barley genus Hordeum, approximately half of the taxa are polyploids. While for diploid species a good hypothesis of phylogenetic relationships exists, there is little information available for the polyploids (4×, 6×) of Hordeum. Relationships among all 33 diploid and polyploid Hordeum species were analyzed with the low-copy nuclear marker region TOPO6 for 341 Hordeum individuals and eight outgroup species. PCR products were either directly sequenced or cloned and on average 12 clones per individual were included in phylogenetic analyses. In most diploid Hordeum species TOPO6 is probably a single-copy locus. Most sequences found in polyploid individuals phylogenetically cluster together with sequences derived from diploid species and thus allow the identification of parental taxa of polyploids. Four groups of sequences occurring only in polyploid taxa are interpreted as footprints of extinct diploid taxa, which contributed to allopolyploid evolution. Our analysis identifies three key species involved in the evolution of the American polyploids of the genus. (i) All but one of the American tetraploids have a TOPO6 copy originating from the Central Asian diploid H. roshevitzii, the second copy clustering with different American diploid species. (ii) All hexaploid species from the New World have a copy of an extinct close relative of H. californicum and (iii) possess the TOPO6 sequence pattern of tetraploid H. jubatum, each with an additional copy derived from different American diploids. Tetraploid H. bulbosum is an autopolyploid, while the assumed autopolyploid H. brevisubulatum (4×, 6×) was identified as allopolyploid throughout most of its distribution area. The use of a proof-reading DNA polymerase in PCR reduced the proportion of chimerical sequences in polyploids in comparison to Taq polymerase.     

Polyploid origins in a circumpolar complex in Draba (Brassicaceae) inferred from cloned nuclear DNA sequences and fingerprints

Polyploid evolution has been of major importance in the arctic flora, but rarely addressed on the full circumpolar scale. Herein we study the allopolyploid Draba lactea and its close allies, which form a taxonomically intricate arctic-alpine complex including diploids, tetraploids, and hexaploids. Based on samples from the entire circumpolar area, we inferred the origins of polyploids in this complex using cloned DNA sequences from two nuclear regions (one intron from a gene encoding a second largest subunit in the RNA polymerase family, RPD2, and the ribosomal internal transcribed spacer region, ITS) and DNA fingerprints (random amplified polymorphic DNAs, RAPDs). Although D. lactea and all other polyploids examined in Draba are genetic alloploids showing fixed heterozygosity, the data obtained in the present study suggest that each of the polyploids analyzed here may have originated from a single diploid lineage: hexaploid D. lactea via tetraploid D. lactea from the D. palanderiana lineage (not from the D. fladnizensis and D. nivalis lineages as previously hypothesized), the tetraploid D. turczaninovii from the D. fladnizensis lineage, the tetraploid D. porsildii from the D. lonchocarpa lineage, and a tetraploid here named Draba spB from the D. nivalis lineage. Draba lactea has probably originated several times in the Beringian area, and it is not necessary to invoke complex origins based on a combination of different species lineages as previously suggested.     

SPECIATION BY POLYPLOIDY IN TREEFROGS: MULTIPLE ORIGINS OF THE TETRAPLOID,HYLA VERSICOLOR

Speciation by polyploidy is rare in animals, yet, in vertebrates, there is a disproportionate concentration of polyploid species in anuran amphibians. Sequences from the cytochrome b gene of the mitochondrial DNA (mtDNA) were used to determine phylogenetic relationships among 37 populations of the diploid-tetraploid species pair of gray treefrogs, Hyla chrysoscelis and Hyla versicolor. The diploid species, H. chrysoscelis, consists of an eastern and a western lineage that have 2.3% sequence divergence between them. The tetraploid species, H. versicolor, had at least three separate, independent origins. Two of the tetraploid lineages are more closely related to one or the other of the diploid lineages (0.18%–1.4% sequence divergence) than they are to each other (1.9%–3.4% sequence divergence). The maternal ancestor of the third tetraploid lineage is unknown. The phylogenetic relationships between the two species and among lineages within each species support the hypothesis of multiple origins of the tetraploid lineages.     

Ancestry of American polyploid Hordeum species with the I genome inferred from 5S and 18S-25S rDNA

* BACKGROUND AND AIMS: The genus Hordeum exists at three ploidy levels (2x, 4x and 6x) and presents excellent material for investigating the patterns of polyploid evolution in plants. Here the aim was to clarify the ancestry of American polyploid species with the I genome. * METHODS: Chromosomal locations of 5S and 18S-25S ribosomal RNA genes were determined by fluorescence in situ hybridization (FISH). In both polyploid and diploid species, variation in 18S-25S rDNA repeated sequences was analysed by the RFLP technique. * KEY RESULTS: Six American tetraploid species were divided into two types that differed in the number of rDNA sites and RFLP profiles. Four hexaploid species were similar in number and location of both types of rDNA sites, but the RFLP profiles of 18S-25S rDNA revealed one species, H. arizonicum, with a different ancestry. * CONCLUSIONS: Five American perennial tetraploid species appear to be alloploids having the genomes of an Asian diploid H. roshevitzii and an American diploid species. The North American annual tetraploid H. depressum is probably a segmental alloploid combining the two closely related genomes of American diploid species. A hexaploid species, H. arizonicum, involves a diploid species, H. pusillum, in its ancestry; both species share the annual growth habit and are distributed in North America. Polymorphisms of rDNA sites detected by FISH and RFLP analyses provide useful information to infer the phylogenetic relationships of I-genome Hordeum species because of their highly conserved nature during polyploid evolution.     

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.     

Molecular phylogeny and reticulate origins of the polyploid Bromus species from section Genea (Poaceae)

The origin of polyploid Bromus species of section Genea was investigated using molecular data. This group of annual species native from the Old-World is composed of three diploids, two tetraploids, one hexaploid, and one octoploid. Molecular cloning, sequencing, and phylogenetic analyses were performed on several accessions per species. We used the low copy nuclear gene Waxy, repeated rDNA spacers ITS1 and ITS2 and chloroplast spacers trnT-trnL and trnL-trnF. Our analyses revealed four different lineages involved in the parentage of the polyploids and confirmed their reticulate origin. Three of these lineages are closely related to the diploid species B. sterilis, B. tectorum, and B. fasciculatus. The fourth lineage could not be related to any diploid according to the available data. Our data gave insights on the origin of all the polyploids of section Genea, and chloroplast data allowed us to identify the maternal lineages. The Waxy gene was the most informative regarding origin of the polyploids. The Waxy copies duplicated by polyploidy appear selectively maintained in the polyploid species. No sequence heterogeneity was encountered in the ITS region, where concerted evolution seems to have occurred toward either maternal or paternal repeats. These results provide new information about the origin and molecular evolution of these polyploids and will allow a more accurate taxonomic treatment of the concerned species, based on their evolutionary history.     

Molecular evidence for hybrid origins of the invasive polyploids Carthamus creticus and C. turkestanicus (Cardueae, Asteraceae)

The hexaploids Carthamus creticus and C. turkestanicus are noxious weeds with wide but non-overlapping Mediterranean distributions, and C. creticus, together with another polyploid, C. lanatus, have also invaded similar climatic regions in North and South America, South Africa and Australia. Here we infer their origins using sequences of the plastid intergenic spacer trnH-psbA and the intron trnK and three introns of nuclear low-copy genes of the RNA Polymerase family (RPD2 and the duplicated RPC2), as well as RAPD markers (random amplified polymorphic DNA). Phylogenetic analyses of the nuclear introns and additivity analysis of the RAPD markers support the hypotheses that the two hexaploids are allopolyploids sharing a tetraploid progenitor lineage represented by the broadly Mediterranean C. lanatus, combined with different diploid progenitor lineages consistent with the different geographic distributions of the hexaploids. Whereas C. leucocaulos from the south-eastern Greek Islands represents the diploid progenitor lineage of the western C. creticus, the Irano-Turanian C. glaucus represents the diploid progenitor lineage of the eastern C. turkestanicus. The plastid data suggest that the diploid lineages served as the maternal progenitors of the hexaploids.     

The genetic legacy of polyploid Bolivian Daphnia: the tropical Andes as a source for the North and South American D. pulicaria complex

We investigated genetic variation in asexual polyploid members of the water flea Daphnia pulex complex from a set of 12 Bolivian high-altitude lakes. We used nuclear microsatellite markers to study genetic relationships among all encountered multilocus genotypes, and combined this with a phylogenetic approach using DNA sequence data of three mitochondrial genes. Analyses of mitochondrial gene sequence divergence showed the presence of three very distinct clades that likely represent cryptic undescribed species. Our phylogenetic results suggest that the Daphnia pulicaria group, a complex of predominantly North American species that has diversified rapidly since the Pleistocene, has its origin in South America, as specific tests of topology indicated that all three South American lineages are ancestral to the North American members of this species group. A comparison between variation of nuclear and mitochondrial markers revealed that closely related polyploid nuclear genotypes sometimes belonged to very divergent mitochondrial lineages, while distantly related nuclear genotypes often belonged to the same mitochondrial lineage. This discrepancy suggests that these South American water fleas originated through reciprocal hybridization between different endemic, sexually reproducing parental lineages. It is also likely that polyploidy of the investigated lineages resulted from this hybridization. Nevertheless, no putative diploid parental lineages were found in the studied region.     

A novel method to infer the origin of polyploids from Amplified Fragment Length Polymorphism data reveals that the alpine polyploid complex of Senecio carniolicus (Asteraceae) evolved mainly via autopolyploidy

Despite its evolutionary and ecological relevance, the mode of polyploid origin has been notoriously difficult to be reconstructed from molecular data. Here, we present a method to identify the putative parents of polyploids and thus to infer the mode of their origin (auto‐ vs. allopolyploidy) from Amplified Fragment Length Polymorphism (AFLP) data. To this end, we use Cohen's d of distances between in silico polyploids, generated within a priori defined scenarios of origin from a priori delimited putative parental entities (e.g. taxa, genetic lineages), and natural polyploids. Simulations show that the discriminatory power of the proposed method increases mainly with increasing divergence between the lower‐ploid putative ancestors and less so with increasing delay of polyploidization relative to the time of divergence. We apply the new method to the Senecio carniolicus aggregate, distributed in the European Alps and comprising two diploid, one tetraploid and one hexaploid species. In the eastern part of its distribution, the S. carniolicus aggregate was inferred to comprise an autopolyploid series, whereas for western populations of the tetraploid species, an allopolyploid origin involving the two diploid species was the most likely scenario. Although this suggests that the tetraploid species has two independent origins, other evidence (ribotype distribution, morphology) is consistent with the hypothesis of an autopolyploid origin with subsequent introgression by the second diploid species. Altogether, identifying the best among alternative scenarios using Cohen's d can be straightforward, but particular scenarios, such as allopolyploid origin vs. autopolyploid origin with subsequent introgression, remain difficult to be distinguished.     

Parallel changes in mate-attracting calls and female preferences in autotriploid tree frogs

For polyploid species to persist, they must be reproductively isolated from their diploid parental species, which coexist at the same time and place at least initially. In a complex of biparentally reproducing tetraploid and diploid tree frogs in North America, selective phonotaxis--mediated by differences in the pulse-repetition (pulse rate) of their mate-attracting vocalizations--ensures assortative mating. We show that artificially produced autotriploid females of the diploid species (Hyla chrysoscelis) show a shift in pulse-rate preference in the direction of the pulse rate produced by males of the tetraploid species (Hyla versicolor). The estimated preference function is centred near the mean pulse rate of the calls of artificially produced male autotriploids. Such a parallel shift, which is caused by polyploidy per se and whose magnitude is expected to be greater in autotetraploids, may have facilitated sympatric speciation by promoting reproductive isolation of the initially formed polyploids from their diploid parental forms. This process also helps to explain why tetraploid lineages with different origins have similar advertisement calls and freely interbreed.     

DYNAMICS OF POLYPLOID FORMATION IN TRAGOPOGON (ASTERACEAE): RECURRENT FORMATION,GENE FLOW,AND POPULATION STRUCTURE

Polyploidy is a major feature of angiosperm evolution and diversification. Most polyploid species have formed multiple times, yet we know little about the genetic consequences of recurrent formations. Among the clearest examples of recurrent polyploidy are Tragopogon mirus and T. miscellus (Asteraceae), each of which has formed repeatedly in the last ～80 years from known diploid progenitors in western North America. Here, we apply progenitor‐specific microsatellite markers to examine the genetic contributions to each tetraploid species and to assess gene flow among populations of independent formation. These data provide fine‐scale resolution of independent origins for both polyploid species. Importantly, multiple origins have resulted in considerable genetic variation within both polyploid species; however, the patterns of variation detected in the polyploids contrast with those observed in extant populations of the diploid progenitors. The genotypes detected in the two polyploid species appear to represent a snapshot of historical population structure in the diploid progenitors, rather than modern diploid genotypes. Our data also indicate a lack of gene flow among polyploid plants of independent origin, even when they co‐occur, suggesting potential reproductive barriers among separate lineages in both polyploid species.     

Phylogenetic and biogeographical relationships among some holarctic frog lung flukes (Digenea: Haematoloechidae).

A phylogenetic study of 8 North American and European species of frog lung flukes belonging to Haematoloechus was conducted using approximately 850 to 1,000 bases of the intemal transcribed spacer region (ITS 1 + 5.8S + ITS 2) and 1,250 bases of the large subunit (LSU) of the nuclear ribosomal DNA. Adequate phylogenetic resolution could not be obtained from 5.8S or ITS 2 data. Analysis of ITS 1 data produced 2 equally parsimonious trees that differed only in the position of Haematoloechus breviplexus relative to H. medioplexus and H. varioplexus. Single, identical trees were produced by analysis of both LSU sequence data and a data set comprised of all ITS and LSU data. All trees demonstrated 3 distinct evolutionary lineages within the Holarctic Haematoloechus examined. The results confirmed the taxonomic validity of H. abbreviatus and demonstrated that the presence or absence of extracecal uterine loops is not a character meaningful to the recognition of evolutionary lineages or differentiation of genera. Examination of ITS sequence data revealed almost no intraspecific variation within 5 species of Haematoloechus and demonstrated an approximately 150-base indel common to the North American H. longiplexus and the European H. asper. Two of 3 clades revealed by the phylogenetic analyses are comprised of both European and North American species, indicating that lineages of Haematoloechus arose before the breakup of Laurasia and radiated after Eurasia and North America split. Within each of 3 evolutionary lineages, members share similar patterns of arthropod host specificity distinct from patterns found in the other lineages. This suggests that second intermediate host specificity may be a trait that has been conserved through evolutionary time.     

Geographic and phylogenetic evidence for dispersed nuclear introgression in a daphniid with sexual propagules

The role of among-species gene flow in eukaryotic evolution remains controversial. Putative hybrid lineages are common in water fleas, but their ecological success is often associated with polyploidy and the production of asexual propagules. Advanced hybrid lineages with sexual propagules are expected to be geographically restricted because their successful dispersal is contingent on overcoming fertility complications, assimilation by parent taxa, and competition with parent taxa. Here we provide evidence that a diploid lineage of Daphnia has been formed by introgression between distantly related species and attained a broad distribution (Nearctic) despite its requirement for sexual propagules. The evidence is based on geographical discordance, phylogenetic discordance, recombinant genotypes and additive genotypes of the nuclear internal transcribed spacer regions (ITS) and mitochondrial DNA. Additive genotypes also provided evidence of hybridization between introduced European Daphnia and North American Daphnia. We argue that the unique biology of Holarctic lacustrine water fleas and the spatial separation of lineages during Pleistocene glaciation have promoted hybridization and its evolutionary consequences.     

SYSTEMATIC INFERENCES FROM VARIATION IN ISOZYME PROFILES OF ARCTIC AND ALPINE CESPITOSE FESTUCA (POACEAE)

Variation in isozyme patterns was used to assess species boundaries in North American arctic and alpine representatives of the Festuca ovina L. complex. Isozyme profiles, in combination with chromosome number, delimit four discrete entities within the complex: F. brevissima Jurtzev (diploid); F. aggr. auriculata Drobov (diploid); F. brachyphylla Schultes (hexaploid); and tetraploid populations corresponding in morphology to F. baffinensis Polunin (arctic Canada) and F. minutiflora Rydberg (alpine United States). Although no fixed difference was detected between isozyme profiles of the latter two taxa, they are morphologically distinct. Thus variation in isozymes, morphology, and chromosome number delimits five taxa within the F. ovina complex in North America. Some alleles observed in the polyploid taxa were not detected among the diploids, and some observed in F. brachyphylla, the hexaploid taxon, were not detected in either the diploid or the tetraploid species. One possible explanation for these occurrences is that the North American polyploids originated in Eurasia, where many other potential diploid and tetraploid progenitors occur.     

Inferring the history of the polyploid Silene aegaea (Caryophyllaceae) using plastid and homoeologous nuclear DNA sequences

The origin of the rare allotetraploid Silene aegaea was inferred from plastid rps16 intron sequences, homoeologous copies of nuclear ribosomal internal transcribed spacer (ITS) sequences, and an intron from the nuclear gene coding for the second largest subunit of RNA polymerase II (RPB2). The nuclear DNA regions support the S. sedoides and S. pentelica lineages as most closely related to the two S. aegaea paralogues. A few recombinant ITS sequences were found, but as PCR recombination could be demonstrated, no true recombination could be demonstrated. No recombination was found in the RPB2 sequences. Plastid rps16 intron sequences strongly support S. pentelica as the maternal lineage. The strength of the approach of using homoeologous sequences of several loci is demonstrated, and its usefulness for the study of phylogenies of groups including polyploids is emphasized.