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Allopolyploidization accounts for a significant fraction of speciation events in many eukaryotic lineages. However, existing phylogenetic and dating methods require tree-like topologies and are unable to handle the network-like phylogenetic relationships of lineages containing allopolyploids. No explicit framework has so far been established for evaluating competing network topologies, and few attempts have been made to date phylogenetic networks. We used a four-step approach to generate a dated polyploid species network for the cosmopolitan angiosperm genus Viola L. (Violaceae Batch.). The genus contains ca 600 species and both recent (neo-) and more ancient (meso-) polyploid lineages distributed over 16 sections. First, we obtained DNA sequences of three low-copy nuclear genes and one chloroplast region, from 42 species representing all 16 sections. Second, we obtained fossil-calibrated chronograms for each nuclear gene marker. Third, we determined the most parsimonious multilabeled genome tree and its corresponding network, resolved at the section (not the species) level. Reconstructing the "correct" network for a set of polyploids depends on recovering all homoeologs, i.e., all subgenomes, in these polyploids. Assuming the presence of Viola subgenome lineages that were not detected by the nuclear gene phylogenies ("ghost subgenome lineages") significantly reduced the number of inferred polyploidization events. We identified the most parsimonious network topology from a set of five competing scenarios differing in the interpretation of homoeolog extinctions and lineage sorting, based on (i) fewest possible ghost subgenome lineages, (ii) fewest possible polyploidization events, and (iii) least possible deviation from expected ploidy as inferred from available chromosome counts of the involved polyploid taxa. Finally, we estimated the homoploid and polyploid speciation times of the most parsimonious network. Homoploid speciation times were estimated by coalescent analysis of gene tree node ages. Polyploid speciation times were estimated by comparing branch lengths and speciation rates of lineages with and without ploidy shifts. Our analyses recognize Viola as an old genus (crown age 31 Ma) whose evolutionary history has been profoundly affected by allopolyploidy. Between 16 and 21 allopolyploidizations are necessary to explain the diversification of the 16 major lineages (sections) of Viola, suggesting that allopolyploidy has accounted for a high percentage-between 67% and 88%-of the speciation events at this level. The theoretical and methodological approaches presented here for (i) constructing networks and (ii) dating speciation events within a network, have general applicability for phylogenetic studies of groups where allopolyploidization has occurred. They make explicit use of a hitherto underexplored source of ploidy information from chromosome counts to help resolve phylogenetic cases where incomplete sequence data hampers network inference. Importantly, the coalescent-based method used herein circumvents the assumption of tree-like evolution required by most techniques for dating speciation events., (© The Author(s) 2014. Published by Oxford University Press, on behalf of the Society of Systematic Biologists.)

The phylogenies of allopolyploids take the shape of networks and cannot be adequately represented as bifurcating trees. Especially for high polyploids (i.e., organisms with more than six sets of nuclear chromosomes), the signatures of gene homoeolog loss, deep coalescence, and polyploidy may become confounded, with the result that gene trees may be congruent with more than one species network. Herein, we obtained the most parsimonious species network by objective comparison of competing scenarios involving polyploidization and homoeolog loss in a high-polyploid lineage of violets (Viola, Violaceae) mostly or entirely restricted to North America, Central America, or Hawaii. We amplified homoeologs of the low-copy nuclear gene, glucose-6-phosphate isomerase (GPI), by single-molecule polymerase chain reaction (PCR) and the chloroplast trnL-F region by conventional PCR for 51 species and subspecies. Topological incongruence among GPI homoeolog subclades, owing to deep coalescence and two instances of putative loss (or lack of detection) of homoeologs, were reconciled by applying the maximum tree topology for each subclade. The most parsimonious species network and the fossil-based calibration of the homoeolog tree favored monophyly of the high polyploids, which has resulted from allodecaploidization 9-14 Ma, involving sympatric ancestors from the extant Viola sections Chamaemelanium (diploid), Plagiostigma (paleotetraploid), and Viola (paleotetraploid). Although two of the high-polyploid lineages (Boreali-Americanae, Pedatae) remained decaploid, recurrent polyploidization with tetraploids of section Plagiostigma within the last 5 Ma has resulted in two 14-ploid lineages (Mexicanae, Nosphinium) and one 18-ploid lineage (Langsdorffianae). This implies a more complex phylogenetic and biogeographic origin of the Hawaiian violets (Nosphinium) than that previously inferred from rDNA data and illustrates the necessity of considering polyploidy in phylogenetic and biogeographic reconstruction.

Background: The genomes of higher plants are, on the majority, polyploid, and hybridisation is more frequent in plants than in animals. Both polyploidisation and hybridisation contribute to increased variability within species, and may transfer adaptations between species in a changing environment. Studying these aspects of evolution within a diversified species complex could help to clarify overall spatial and temporal patterns of plant speciation. The Arabidopsis lyrata complex, which is closely related to the model plant Arabidopsis thaliana, is a perennial, outcrossing, herbaceous species complex with a circumpolar distribution in the Northern Hemisphere as well as a disjunct Central European distribution in relictual habitats. This species complex comprises three species and four subspecies, mainly diploids but also several tetraploids, including one natural hybrid. The complex is ecologically, but not fully geographically, separated from members of the closely related species complex of Arabidopsis halleri, and the evolutionary histories of both species compexes have largely been influenced by Pleistocene climate oscillations., Results: Using DNA sequence data from the nuclear encoded cytosolic phosphoglucoisomerase and Internal Transcribed Spacers 1 and 2 of the ribosomal DNA, as well as the trnL/F region from the chloroplast genome, we unravelled the phylogeography of the various taxonomic units of the A. lyrata complex. We demonstrate the existence of two major gene pools in Central Europe and Northern America. These two major gene pools are constructed from different taxonomic units. We also confirmed that A. kamchatica is the allotetraploid hybrid between A. lyrata and A. halleri, occupying the amphi-Beringian area in Eastern Asia and Northern America. This species closes the large distribution gap of the various other A. lyrata segregates. Furthermore, we revealed a threefold independent allopolyploid origin of this hybrid species in Japan, China, and Kamchatka., Conclusions: Unglaciated parts of the Eastern Austrian Alps and arctic Eurasia, including Beringia, served as major glacial refugia of the Eurasian A. lyrata lineage, whereas A. halleri and its various subspecies probably survived in refuges in Central Europe and Eastern Asia with a large distribution gap in between. The North American A. lyrata lineage probably survived the glaciation in the southeast of North America. The dramatic climatic changes during glaciation and deglaciation cycles promoted not only secondary contact and formation of the allopolyploid hybrid A. kamchatica, but also provided the environment that allowed this species to fill a large geographic gap separating the two genetically different A. lyrata lineages from Eurasia and North America. With our example focusing on the evolutionary history of the A. lyrata species complex, we add substantial information to a broad evolutionary framework for future investigations within this emerging model system in molecular and evolutionary biology.

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.

Polyploidy, the duplication of entire genomes, plays a major role in plant evolution. In allopolyploids, genome duplication is associated with hybridization between two or more divergent genomes. Successive hybridization and polyploidization events can build up species complexes of allopolyploids with complicated network-like histories, and the evolutionary history of many plant groups cannot be adequately represented by phylogenetic trees because of such reticulate events. The history of complex genome mergings within a high-polyploid species complex in the genus Cerastium (Caryophyllaceae) is here untangled by the use of a network algorithm and noncoding sequences of a low-copy number gene. The resulting network illustrates how hybridization and polyploidization have acted as key evolutionary processes in creating a plant group where high-level allopolyploids clearly outnumber extant parental genomes.