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1 The causes and molecular consequences of polyploidy in flowering plants 2 3 Short title: Plant polyploidy 4 5 Authors: Gaurav D. Moghe1,2, Shin-Han Shiu1,2,3,* 6 7 Affiliations: 8 1,2 9 State University, East Lansing, MI 48824, USA 10 Programs in Genetics and Quantitative Biology, 3 Department of Plant Biology, Michigan * Corresponding author ([email protected]) 11 12 Correspondence address: 13 2265 Molecular Plant Sciences Building., Dept. of Plant Biology, Michigan State University, 14 East Lansing, MI, 48824 15 Phone #: 517-353-7196 16 17 Keywords: 18 Whole genome duplication, plants, adaptation, expression divergence, fractionation, molecular 19 consequences of polyploidy 20 21 22 1 1 Abstract 2 Polyploidy is an important force shaping plant genomes. All flowering plants are descendants of 3 an ancestral polyploid species, and up to 70% of extant vascular plant species are believed to be 4 recent polyploids. Over the past century, a significant body of knowledge has accumulated 5 regarding the prevalence and ecology of polyploid plants. In this review, we summarize our 6 current understanding of the causes and molecular consequences of polyploidization in 7 angiosperms. We also provide a discussion on the relationships between polyploidy and 8 adaptation and suggest areas where further research may provide a better understanding of 9 polyploidy. 10 2 1 Introduction 2 Polyploidization results in multiplication of the genome and an increase in gene content 3 that frequently leads to morphological and physiological differences between polyploids and 4 their diploid progenitors1. Polyploidy is widespread among flowering plants2,3 and has been 5 postulated as an answer to "Darwin's abominable mystery" regarding the causes behind the rapid 6 acceleration in the diversification of angiosperms in earth's evolutionary history4,5. It is also a 7 major route for origination of new genes via gene duplication and subsequent diversification6,7. 8 Although we have a fairly good idea about the extent of polyploidy in eukaryotes 8–10 and the 9 modes of diversification of duplicate genes derived from polyploidy11, there is still a 10 considerable debate about whether polyploidy indeed confers an evolutionary advantage to the 11 organism and if yes, does it contribute to speciation? In addition, although the primary pathways 12 of polyploid generation have been known for some time12, only recently have we begun to 13 identify the molecular consequences of polyploidization. 14 In this review, we first focus on the genetic and environmental factors that influence the 15 rates of polyploidization. Second, we discuss the impact of polyploidization at the molecular 16 level. Third, we summarize recent studies on the impact of polyploidy on morphology, 17 physiology, and stress biology. Finally, we discuss current evidence on how polyploidy 18 contributes to adaptation and speciation. Our goal in this review is to present a brief overview of 19 our current state of understanding regarding a few different aspects of polyploidy. For additional 20 information, we refer the reader to several excellent resources2,3,13–17 that have covered these 21 topics in greater detail. 22 Causes of polyploidization in flowering plants 23 Cytological pathways leading to polyploidization 24 Diploids mostly propagate by producing haploid gametes, which combine to produce 25 diploid progeny (Figure 1A). In rare cases, polyploids can arise via the somatic doubling of 26 chromosomes in the zygote (Figure 1B) or through the production of unreduced gametes (Figure 27 1C-E). The primary mechanism for polyploid generation is thought to be via the latter12,18. 28 Theoretical models considering unreduced gamete formation and fertilities of plants with 29 different ploidy levels have been used to predict equilibrium ploidy frequencies19–21. Notably, to 30 fit the existing data on ploidy frequency observed in multiple autopolyploid species, the 3 1 unreduced gamete frequency was estimated to be 0.89%21. This high rate of unreduced gamete 2 production is consistent with its involvement in angiosperm polyploidy. 3 Unreduced gametes can be formed in three different ways: 1) pre-meiotic genome 4 doubling due to endoreplication mechanisms including endocycling (alternating periods of S 5 phase, where DNA is replicated, and gap phase, without cell division), endomitosis (mitosis 6 without the final cell division)22, or nuclear fusion, 2) via impairments in meiosis, which can 7 affect either the first or the second meiotic divisions and 3) via post-meiotic genome doubling12. 8 The unreduced gametes from diploids (2X, with X being the base chromosome number of the 9 species) can lead to a tetraploid (4X) in one step by hybridization between unreduced male and 10 female gametes (Type I pathway, Figure 1C,D) or through the creation of an intermediate 11 triploid (Type II pathway, Figure 1E)19. Given that unreduced gametes can be produced at an 12 appreciable frequency21, it is conceivable that unreduced gametes generated in two individuals, 13 or from the same individual (if selfing is feasible), may hybridize and generate polyploids 14 through the Type I pathway. 15 In the Type II pathway, an unreduced gamete hybridizes with a normal gamete to 16 produce triploid plants (3X). The triploid produces mostly aneuploid gametes, which are 17 generally not viable, and a small percentage of viable X, 2X or 3X gametes. These gametes can 18 then hybridize with other X, 2X or 3X gametes to generate plants of higher ploidy levels (Figure 19 1E). Hence, triploid plants are regarded as a "bridge" towards polyploidy (triploid bridge), rather 20 than a dead end (triploid block)12. Currently, it remains unclear which pathway is more 21 prevalent13. Although the production of 3X gametes required in the Type II pathway would be 22 rare in nature, unreduced gametes in artificially generated hybrids of multiple Brassica species 23 are produced at a much higher frequency than in their parents. These gametes have a size 24 distribution corresponding to >2X genome complement, and they are more viable than reduced 25 gametes in the Brassica hybrids. These results support the hypothesis that the triploid bridge 26 scenario may be more prevalent for polyploids generated from inter-specific hybridridization12. 27 Genetic components contributing to polyploidization 28 Several A. thaliana genes that can influence the frequency of unreduced gamete 29 formation have been identified23,24. For example, ~60% of the seeds produced from a mutated 30 version of the SWI1/DYAD protein are triploid25. SWI1/DYAD is required for a proper meiosis 31 I in both male and female germ cells. In the swi1/dyad mutant, cells skip the reduction division 4 1 in meiosis I and directly advance to the equatorial division in meiosis II, producing 2 predominantly unreduced gametes26,27. Mutations in several other genes lead to the production of 3 unreduced gametes by affecting different meiotic and mitotic steps. For example, mutations in 4 the GLUCAN SYNTHASE LIKE 8 protein, which lays down the glucan chains at cell plates 5 during cell division, lead to a flower containing both diploid and polyploid somatic cells. The 6 polyploid cells then go on to produce unreduced gametes28. 7 As discussed earlier, one important step in the pathway to polyploidy is the formation of 8 triploid intermediates. The major challenge upon forming this intermediate is the “triploid 9 block”, originally described as the difficulty in generating viable triploids through diploid- 10 tetraploid crosses29, which can lead to reproductive isolation of the newly formed polyploid due 11 to minority cytotype exclusion30. A recent study demonstrated that one genetic component of the 12 triploid block in A. thaliana is a paternally expressed gene ADMETOS31. The authors of this 13 study carried out a mutant screen in a jason mutant background, which produces unreduced 14 gametes at a high frequency but 30% of the triploid seeds produced are aborted. The admetos-1 15 jason double mutant, on the other hand, has only 2% aborted triploid seeds. The admetos-1 16 mutant is a gain-of-function mutant with elevated expression of the ADMETOS gene31. These 17 results suggest that there is genetic control over triploid formation. While such genetic control 18 may exist to create a post-zygotic reproductive barrier for gene flow between species, naturally 19 occurring variation in such control mechanisms may provide an opportunity for polyploids to be 20 generated. 21 Together with the finding that unreduced gamete formation is a trait with high heritability 22 (e.g. 0.40 to 0.60 in alfalfa and clover32,33), at least in domesticated crops experiencing artificial 23 selection, polyploid formation through unreduced gamete and triploid bridge formation is 24 expected to have a significant genetic component. Although these genetic studies are highly 25 informative, it remains unclear whether these newly identified genes are involved in increasing 26 or decreasing the rate of polyploid formation in nature. If these genes are the targets of selection 27 for polyploidization frequency, they may display substantial variation between plant species 28 and/or populations that have variable relative abundances of individuals with different ploidy 29 levels. 5 1 Relationship between environment and polyploidy 2 Nearly 80 years ago, it was demonstrated that a correlation exists between polyploidy and 3 latitudinal cline34, suggesting potential habitat differentiation between plants with different 4 ploidy levels due to the differences in latitudinal environment. Additionally, it was shown in 5 1920 that hot water treated Pisum root tips have increased frequency of tetraploidy in somatic 6 cells35. In 1932, Randolf demonstrated that high temperature (47-48°C) results in an increased 7 frequency of tetraploid embryos in maize36. Subsequently, a number of studies have established 8 that environmental stress, including temperature stress, herbivory, pathogen attack, nutritional 9 stress and water stress, lead to an elevated rate of unreduced gamete production12. 10 In addition to observations linking environmental stress, polyploidy and unreduced 11 gamete production, a large number of studies have focused on how environment influences 12 chromosome behavior and unreduced gamete production18. Although unreduced gametes can be 13 produced due to pre-meiotic, meiotic, or post-meiotic aberrations, recent studies have shown that 14 environmentally induced production of unreduced pollen is mainly due to meiotic irregularities, 15 particularly during telophase II. In Rosa species, the proportion of unreduced pollen produced 16 due to elevated temperature (36°C) differs greatly at different microspore stages37. The elevated 17 temperature led to formation of normal rose pollen tetrads as well as abnormal dyads, triads, and 18 polyads as a result of mis-orientation of meiotic spindles. 19 Although similarly affected during telophase II, the formation of A. thaliana unreduced 20 pollen due to cold shock (4-5°C) is not due to defects in spindle fibers attached to the 21 chromosomes but mainly due to abnormalities in equatorial cell plate formation as a result of 22 misplaced microtubules28. Because the aberration in cell plate formation occurs during telophase 23 II, the cell plate separating homologous chromosomes is in place but the one separating sister 24 chromatids is defective. Therefore, instead of forming pollen tetrads (1X), 2X dyads are formed, 25 each containing two sets of exactly identical chromosomes. It is not clear if the differences in 26 the mechanistic details (spindles vs. cell plate formation) are due to the differences in the type of 27 stress applied or due to differences between species. 28 In this section, we reviewed studies focused on finding the genetic and environmental 29 causes of polyploidization, most of which act by affecting mitosis or meiosis thus producing 30 unreduced gametes. A polyploid, once created, has to establish itself, and the process of 31 neopolyploid establishment continues in the backdrop of molecular and physiological changes 6 1 occurring due to genome duplication (and merging two different genomes in allopolyploids) that 2 a neopolyploid has to go through1. In the remainder of the review, we focus on the impact of 3 polyploidy on genome content, on gene expression, on morphology and finally on adaptation. 4 Impact of polyploidy on genome content 5 Changes in genome organization 6 Polyploids have a tendency to return to a diploidized state over time, experiencing 7 changes in chromosomes organization, gene order, expression, epigenetic modification, and 8 biological network topology (Figures 2-4), a phenomenon known as diploidization38. 9 Diploidization may begin with large scale changes in the genome of neopolyploid plants such as 10 abnormal chromosome segregation, rearrangement, and breakage39,40 and may occur in a 11 haphazard manner in the initial few decades after polyploidization41 (Figure 2A,B). For example, 12 in synthetic allotetraploids between double haploid Brassica oleracea (C genome) and B. rapa 13 (A genome), chromosomal segregation aberrations lead to extensive aneuploidy as early as the 14 first generation, when the aneuploidy rate is 24%. This rate rises to 95% in the 11th generation42. 15 Despite the high rate of aneuploidy, the number of homeologs for a particular chromosome is 16 frequently maintained at four copies; i.e. the loss of chromosome 1 from the A genome is usually 17 associated with gain of the same chromosome from the C genome and vice versa. This 18 compensating aneuploidy suggests a dosage balance requirement at least in the early generations. 19 Compensating aneuploidy also occurs in the naturally occurring allotetraploid Tragopogon 20 miscellus, wherein 85% of aneuploid plants were found to have the expected chromosome 21 number43. Chromosomal losses in early generations have also been reported for synthetic 22 allohexaploids (Triticum aestivum44, Brassica carinata x B. rapa44, A. thaliana x A. suecica45) as 23 well as in autopolyploid potato46, alfalfa and corn47. Recently, the cause of such chromosomal 24 losses, which occur due to meiotic instabilities, was tracked down to a single quantitative trait 25 locus called BOY NAMED SUE (BYS) in synthetic allopolyploids of A. thaliana x A. arenosa48. 26 The authors speculate that the BYS locus may play a role in A. suecica, which is a naturally 27 occurring allopolyploid of A. thaliana x A. arenosa, in ensuring that homeologous chromosomes 28 don't pair with each other, a process that may lead to chromosomal dosage irregularities in the 29 progeny48. 7 1 In addition to changes in chromosome numbers, newly generated polyploids display an 2 elevated rate of genome rearrangements leading to loss of chromosomal fragments (Figure 2A). 3 By tracking a limited number of markers, synthetic autotetraploids of Paspalum notatum49 and 4 Elymus elongatus50 were shown to lose ~10% of genome sequence in the first generation. In 5 Phlox drumondii, up to 25% reduction in parental DNA content was observed as early as the 6 third generation50. On the contrary, studies in synthetic A. thaliana autopolyploids reveal little to 7 no loss51. These observations suggest that genome rearrangement can be prominent in allo- and 8 some auto-polyploids. 9 Gene duplicate loss and retention 10 Polyploidization initially results in multiplication of gene content, however, the 11 predominant fate of gene duplicates is loss52. Studies of newly sequenced genomes shed light on 12 the extent of gene loss in species which underwent polyploidization events several million years 13 ago (MYA). In the A. thaliana genome, only 17% of duplicates were retained after a 14 paleopolyplodization (β) event took place ~50 MYA53. In the paleopolyploid Glycine max, two 15 rounds of whole genome duplications took place ~59 and ~13 MYA54. In the homologous genes 16 from the more recent duplication event, 56.6% of duplicates are no longer detectable, compared 17 to 74.1% genes lost after the older Glycine polyploidization. Thus the rates of gene loss are 4.4% 18 and 1.3% per million years for the younger and the older duplication event, respectively, 19 indicating that gene loss rate is high initially but slows down over time54. In Brassica rapa and 20 Raphanus raphanistrum, which experienced genome triplication ~25 MYA55–57, assuming the 21 ancestral gene number prior to triplication was similar to that in A. thaliana (~30,000), the 22 number of extant B. rapa genes (~41,000) and R. raphanistrum genes (~38,000) indicates that as 23 many as 55% of the genes derived from genome triplication were lost58,59. 24 The process of loss of polyploidy-derived genes is referred to as “fractionation”, a 25 collection of mutational mechanisms leading to the removal of duplicates derived from 26 polyploidization60,61 (Figure 2B). Studies of gene collinearity between duplicate regions in A. 27 thaliana62, Z. mays63 and B. rapa64 suggest a bias in the genes lost from certain parental 28 genomes. In B. rapa, one of the three subgenomes experienced significantly fewer gene losses 29 than the others58,65. This phenomenon is also reflected at the expression level, where genes 30 located on one subgenome tend to have higher expression than others, indicating genome 31 dominance66. The duplicated gene copy producing the most RNA molecules appears to be the 8 1 one retained63. It has recently been suggested that transposon silencing due to small RNAs may 2 contribute to the phenomenon of genome dominance, with the parental genome having the 3 lowest proportion of transposons being the more dominant66. Fractionation of genes also leads to 4 preferential gene retention, which has been reviewed recently61. Retained duplicates derived 5 from polyploidization have a number of distinguishing characteristics compared to genes that 6 remain single copy, including biased gene function67,68, higher gene complexity (number of 7 exons and protein domains)69,70, higher levels of gene expression71, significant parental genome 8 dominance63,72 and higher network connectivity62. Duplicate genes playing a role in stress 9 response, development, signaling and transcriptional regulation tend to be retained, a feature 10 consistent across multiple polyploidization events and time scales59,70. 11 Why are duplicates with these types of characteristics retained? Retained duplicates may 12 experience a brief period of complete functional redundancy, but eventually obtain new 13 functions6, and/or partition ancestral functions leading to subfunctionalization73. In addition to 14 these mechanisms, the retention of duplicate genes may be due to balanced gene drive/gene 15 balance74,75, functional buffering69, dosage selection76 and escape from adaptive conflict77 16 (reviewed by Innan and Kondrashov11 and Edger and Pires78). Among the mechanisms 17 explaining duplicate retention, some imply adaptive evolution (e.g. neofunctionalization, dosage 18 selection) while the others require the need for purifying selection to maintain the ancestral 19 functions (e.g. subfunctionalization, dosage balance). Examples of adaptive duplication are 20 accumulating but it remains unclear what fraction of gene duplicates was fixed due to adaptive 21 evolution79. 22 Mutation and transposable element activities 23 Because more than one gene copy is present, increased ploidy can mask the effect of 24 deleterious mutations80. Meanwhile, the newly formed polyploid species has a very small 25 effective population size, assuming postzygotic isolation from its parental diploids. In this 26 situation, genetic drift is expected to play a more dominant role in polyploid evolution. Therefore 27 the selective pressure against any mutation in polyploid genomes would be more relaxed; leading 28 to increased frequencies of otherwise deleterious alleles. Although it is not clear whether the 29 spontaneous mutation rate is higher in polyploids compared to diploids, there is generally a 30 higher mutation density in mutagenized polyploids compared to diploids81. Because this pattern 31 is similar between natural and synthetic polyploids, and because the mutations tend to exist in 9 1 heterozygous states, the elevated mutation density is likely a consequence of masking recessive 2 deleterious mutations81. In a comparison between wheat Triticum aestivum, which experienced 3 recent whole genome duplication, and three other non-duplicated grass species, there are more 4 non-synonymous substitutions, gene structural rearrangements, and alternative splice forms of 5 genes in wheat82, suggesting relaxed selection on deleterious alleles. 6 The elevated mutation rate in polyploids can also be due to elevated transposable element 7 activities83,84. The proliferation of transposons in polyploids is expected due to reduced 8 population size, masked deleterious transposon insertion, and/or conflict in transposition 9 repressors due to genome merger84 (Figure 2C). Despite this expectation, current studies present 10 conflicting results regarding whether proliferation of transposons is correlated with 11 polyploidy83,84. For example, the numbers of transposons in the Au short interspersed nuclear 12 element (SINE) family in natural polyploid wheat species are significantly higher than those in 13 diploids85, although it is difficult to ascertain whether the difference observed is due to 14 polyploidy, hybridization, and/or lineage divergence. Additionally, one of three synthetic 15 allopolyploids has higher number of Au SINEs by the fourth generation85. In this case, it remains 16 unclear whether genome doubling or hybridization contributes to the higher number of Au SINEs 17 observed. 18 Studies on the activities of transposons in autopolyploids are also contradictory. In A. 19 thaliana synthetic autotetraploids, activation of Sunfish, a DNA transposon, was observed39. On 20 the other hand, a study assaying naturally occurring autotetraploid A. arenosa accessions found 21 evidence for purifying selection against expansion of Ac-III transposon family86. In addition to 22 the possibility of transposon proliferation, transposons may be involved in recombination events 23 leading to sequence losses in polyploid genomes87–89. For example, illegitimate recombination 24 mediated by TE elements was shown to underlie the variation observed between diploid and 25 polyploid wheat species in the Hardness locus90. Such transposon-mediated recombination can 26 also contribute to differential expansion and contraction of subgenomes as shown in maize91. 27 10 1 Impact of polyploidy on gene expression and biological network 2 Expression divergence between homeologs 3 In addition to experiencing widespread changes at the DNA level, polyploids have 4 considerable differences in gene expression compared to diploids, and this has been reviewed 5 extensively92–96. Divergence in the expression states of duplicated genes may lead to the 6 following outcomes: they may gain new expression states (neo-functionalization6), partition their 7 ancestral functions (sub-functionalization73) or lose their expression state completely leading to 8 eventual pseudogenization52. In A. thaliana, 57% and 75% of the duplicates derived from the 9 more recent α and the older β polyploidization events were found to have diverged in 10 expression67. It was also found that whole genome duplicates tend to diverge in expression at a 11 slower rate than tandem duplicates, presumably because entire intergenic regions are duplicated 12 during polyploidization, but only a fraction might end up being duplicated during tandem 13 duplication due to the random nature of DNA breakage and recombination97. 14 In allopolyploids, the combination of gene sets in two species is expected to create a 15 “transcriptome shock”, defined as abrupt and rapid changes in patterns of parental gene 16 expression in the polyploid98. Through a number of intriguing studies in the past decade or so, 17 several basic features have emerged regarding homeolog expression in allopolyploids. The 18 transcriptome shock contributes to significant differences in expression levels between 19 homeologs in allopolyploids, albeit to varying extents94, and the corresponding genes in diploid 20 parents have been documented in Arabidopsis99, B. napus100, and cotton101. But a recent study 21 also shows that in wheat synthetic polyploids fewer than 1% of genes show non-additive 22 expression102. In addition, concerns on how non-additivity is defined in polyploids have been 23 raised103. Nonetheless, analysis of non-additive patterns of expression in allopolyploid 24 homeologs has lead to the discovery of two related phenomena, “homeolog expression bias" and 25 "expression level dominance"104 (Figure 3A). 26 Homeolog expression bias takes place when one homeolog tends to have significantly 27 reduced level of expression or is silenced altogether in the allopolyploid105. For example, in the 28 first RNA-sequencing study examining expression bias in cotton polyploids, 59-62% and 48% of 29 genes are differentially expressed when comparing diploids against the natural and synthetic 30 polyploids, respectively106. It was found that genes from a particular subgenome were expressed 11 1 in allopolyploids, and the nature of the subgenome differed in the natural alopolyploid vs. 2 synthetic allopolyploid106. 3 distinguishing homeolog origins, only 0.71-0.75% of genes have no detectable expression in 4 synthetic polyploids. Given that the cotton allopolyploidization event took place 1-2 MYA107, 5 there may have been insufficient time for gene loss and/or the expression of pseudogenized 6 copies are still detectable. Related to homeolog expression bias, expression level dominance 7 (originally called genomic dominance) describes a situation where the sum of expression levels 8 of a homeologous gene pair tends to be more similar to that in one parent, regardless of the 9 expression level of the gene in the parent in question104,108. These phenomena have been well- 10 documented in multiple natural and synthetic allopolyploids. The cause of expression biases may 11 be partially attributed to cis and trans regulatory differences between the hybridized genomes109 12 and epigenetic regulation, which is discussed in a later section. 13 Contribution of hybridization and genome doubling to transcriptome shock Interestingly, among over 25,000 cotton genes containing SNPs 14 In addition to homeolog expression bias and expression level dominance, another focus is 15 on the relative contribution of genome doubling and hybridization to non-additive expression in 16 allopolyploids. Through comparisons of hybrid diploids, synthetic allopolyploids, and 17 neopolyploids (particularly Senecio and Tragopogon species, which formed in the past 100 18 years110,111), the effect of hybridization as well as genome doubling on gene expression changes 19 has been quantified, and these studies suggest that hybridization likely plays a more dominant 20 role. For example, only 88 genes are differentially expressed between A. thaliana diploid and 21 synthetic isogenic autotetraploids, compared to >1,700 genes with significantly different 22 expression levels between synthetic allotetraploid A. thaliana x A. arenosa and the average of 23 two parents112. In a study of diploid, autotetraploid, and autohexaploid Helianthus decapetalus, 24 ploidy level does not contribute significantly to expression differences113. Similarly in Senecio, 25 twice as many genes in S. x baxteri, a triploid hybrid between S. vulgaris (2n=4x) and S. 26 squalidus (2n=2x), are differentially expressed compared to the parental species than in a 27 synthetic allohexaploid derived from the triploid S. x baxteri98. Interestingly, genome doubling in 28 fact ameliorates the effect of hybridization in Senecio98, a finding that is also reported in 29 neopolyploid Spartina114. Thus the non-additive gene expression changes in these allopolyploids 30 is likely due to inter-specific hybridization and not simply genome doubling. 12 1 Although hybridization seems to play a dominant role, studies in cotton, Spartina, and 2 domesticated rice subspecies suggest the contribution of genome doubling may be important. In 3 the natural polyploid cotton, among assayed genes with expression bias toward one parental 4 diploid, only 25% are biased in the same direction as the diploid hybrid101, suggesting the 5 expression bias in the remaining 75% is due to genome doubling. However, allopolyploid cotton 6 was established ~1-2 MYA. Thus one cannot rule of the possibility that the expression bias is 7 due to regulatory variation that accumulated in the past 1-2 million years. In Spartina, the 8 allopolyploid tends to have a higher number of transgressively over-expressed genes compared 9 to species hybrid114, again suggesting a prominent role of genome doubling. Nonetheless, given 10 that the parental Spartina species are hexaploids, it remains unclear if the findings are applicable 11 to comparisons between lower ploidy levels. On the other extreme, genome doubling seems to 12 have a more prominent role in transcriptome shock than hybridization in a comparison between a 13 hybrid of Oryza sativa subsp. indica and japonica and synthetic tetraploid rice109. But given that 14 there has been repeated hybridization between japonica and indica115, it remains unclear how 15 hybridization may have impacted the findings. 16 Involvement of epigenetic modifications and small RNAs in transcriptome shock 17 Transcriptome shock results in non-additive gene expression and silencing of homeologs. 18 In allopolyploids, trans factors and cis regulatory components from two genetic backgrounds can 19 interact due to the hybridization between two genomes, contributing to changes in gene 20 expression between polyploids and their diploid progenitors116,117. In addition, epigenetic factors 21 including DNA methylation, histone modifications, and small RNA have been implicated in 22 modulating gene expression in allpolyploids66,116,118–122. Extensive DNA methylation changes 23 have been reported between allopolyploids and their parents in Brassica napus123, wheat124, 24 Spartina anglica88,125 and Arabidopsis126 (Figure 3B). Inconsistent with the above observations, 25 the synthetic allopolyploid Cucumis hystrix x sativus has approximately the same methylation 26 density compared to the parents or the F1 hybrids127. 27 The direct involvement of DNA methylation in expression changes between homeologs 28 has also been demonstrated. In the natural allotetraploid A. suecica, the transcription factor TCP3 29 was silenced when chemical inhibitors of DNA methyltransferases were applied105, suggesting 30 methylation is important for proper TCP3 expression. Another line of evidence comes from 31 methyltransferase 1 (MET1) RNA interference (RNAi) lines in the A. suecica background128. 13 1 Notably, only 200 genes were found to be differentially expressed between A. suecica wildtype 2 plants and MET1 RNAi lines and only 34 of these 200 genes overlap with the ~1400 genes with 3 expression changes in synthetic tetraploids between A. thaliana and A. arenosa (the presumed 4 diploid parents of A. suecica)112. In addition, 33 of the 200 differentially expressed genes in 5 MET1 RNAi lines are pseudogenes or transposons128. Thus, MET1 mediated DNA methylation 6 differences between diploids and polyploids appear to be more relevant to controlling 7 heterochromatic states than to contributing to transcriptome shock. Nonetheless, the DNA 8 methylation machinery is complex129 and more loss-of-function studies will be necessary for a 9 more complete picture of the influence of DNA methylation on transcriptome shock. 10 Similar to DNA methylation, there are indications that histone modifications likely play a 11 significant role in transcriptome shock. Using diploids derived from transgenic autotetraploid A. 12 thaliana containing epialleles that either silence or allow the expression of a resistance gene 13 marker130, a screen for mutants releasing the silencing effect of the epiallele resulted in 14 identification of loss-of-function alleles in DECREASE IN DNA METHYLATION1 (DDM1) and 15 HOMOLOGOUS GENE SILENCING1 (HOG1)131. Global changes in both DNA and histone 16 methylation in the mutant background likely contribute to silencing release. Although the 17 epiallele action appears to be tied to the ploidy level130, it remains to be determined whether the 18 mutations in DDM1 and/or HOG1 contribute to similar phenomenon in a polyploidy 19 background. In addition to histone methylation, histone acetylation is implicated in transcriptome 20 shock based on studies of a histone deacetylase (ATHD1/ATHDA19) mutant132. Genes 21 differentially regulated between wildtype and athd1/athda19 mutants overlap significantly with 22 genes differentially expressed between synthetic allopolyploid Arabidopsis and the diploid 23 parents. 24 In addition to DNA methylation and histone modifications, the expression profiles of 25 small RNAs including both micro RNA and small interfering (both repeat-associated and trans- 26 acting) RNAs are affected post-polyploidization (Figure 3C). Such changes have been 27 documented in resynthesized allotetraploids of A. thaliana x arenosa133 and Ae. tauschii x 28 Triticum turgidum134. As with DNA methylation and histone modification, mutants in genes 29 controlling small RNA biogenesis have been used to study the impact of small RNA on 30 transcription and other phenotypic changes associated with polyploidy. RNAi lines interfering 31 with the expression of DICER LIKE-1 and ARGONAUTE 1, both involved in small RNA 14 1 biogenesis, have been created135. Similar to the DNA methylation machinery, the complexity of 2 epigenetic regulatory machineries in plants is well appreciated136. Additional loss-of-function 3 studies in polyploids will be highly informative. In addition to their effect on transcription, as 4 discussed earlier, small RNAs may contribute to the phenomenon of genome dominance66. Aside 5 from transcriptional regulatory mechanisms, a recent study examining ribosome associated 6 transcripts in the recently formed (∼100,000 years ago) allotetraploid G. dolichocarpa indicated 7 that transcripts subject to translational regulation tend to be retained homeologs from an ancient 8 whole genome duplication event137. It will be interesting to perform similar experiments in the 9 diploid parents and F1s to assess the impact of genome doubling and genome merger on 10 translational regulation. 11 Perturbations of biological networks due to polyploidy 12 Preferential retention and loss of genes, coupled with extensive transcriptional and 13 genomic changes post-polyploidy, necessarily leads to alterations in the molecular networks 14 operating in the organism (Figure 4). The network level changes in turn are expected to affect the 15 phenotype of the organism. Such phenotypic impact has been reviewed recently using flowering 16 time as an example120. While most retention/loss events may be random, there is evidence for 17 preferential retention/loss of certain types of genes. According to the "gene balance hypothesis", 18 the random loss of genes can result in a perturbation of stoichiometric relationships between 19 gene products, leading to genomic imbalance in a newly created polyploid and loss of fitness138. 20 Hence, neopolyploid lineages which happen to retain certain types of genes may be able to better 21 establish themselves. 22 There is a growing body of literature discussing the functional evolution of gene 23 duplicates derived from whole genome duplication76,138,139. For example, studies in B. rapa show 24 that genes involved in circadian rhythm such as CIRCADIAN CLOCK ASSOCIATED 1 and 25 LATE ELONGATED HYPOCOTYL were preferentially retained post polyploidization, leading to 26 an altered flowering time pathway140. Such preferential retention was also found in A. thaliana 27 genes duplicated in the α polyploidization event 50-65 MYA related to specific metabolic 28 pathways141 as well as MADS-box genes involved in various aspects of plant development142. In 29 poplar, two duplicate 30 paleopolyploidization event are expressed at different times of the year and have distinct roles in 31 influencing flowering genes time FLOWERING and vegetative LOCUS T1 growth143. and T2 derived from Sub-functionalization of a two 15 1 polyploidization derived phytochrome (PHY) genes in maize – PHY B1 and B2 - where the two 2 proteins have both overlapping and unique functions in seedlings and adult plants, is also 3 known144. 4 Most of our understanding of the impact of polyploidy on biological network evolution 5 comes from budding yeast, Saccharomyces cerevisiae, which underwent a polyploidization event 6 ~100 MYA145. This polyploidization event was associated with a large scale rewiring of the 7 transcriptional network via changes in cis-regulatory motif usage, which led to the evolution of 8 facultatively anaerobic post-polyploidization species whereas several pre-polyploidization 9 species are aerobic146. It was also found that despite 100 million years of evolution, genetic 10 redundancy may still exist between several pairs of duplicated genes, although there is evidence 11 of significant partitioning of ancestral functions and gain of new functions between these 12 pairs147. 13 Impact of polyploidy on morphology and physiology 14 Morphological alterations 15 Some fundamental commonalities in morphology and physiology are found among 16 polyploid species, however, the specific outcomes of a particular polyploidization event can vary 17 widely between taxa (reviewed in Levin, 2002148). Anatomically, polyploids have larger cell 18 volumes, stomatal guard cells, pollen, and seeds compared to diploids. Polyploids also have 19 broader and thicker leaves with fewer stems per plant (reviewed in Otto and Whitton, 2000149). 20 Such changes may affect processes such as water relations, gas exchange, cold tolerance and 21 shade tolerance of polyploid plants1. Some phenotypic modifications may occur due to increased 22 DNA content in the nucleus and a need to maintain a particular nuclear-to-cytoplasmic volume 23 ratio, while other changes may be induced by genome restructuring and regulatory and network 24 level modifications. For example, in a synthetic autotetraploid line of A. thaliana, larger aerial 25 organs compared to the diploid are a result of faster expansion rates and a longer expansion 26 duration during cell division. Such behavior is due to up-regulation of cyclin dependent kinases 27 in tetraploids compared to diploids150. 28 Polyploidization is also expected to impact the morphology of the reproductive system by 29 affecting the size of the flower, relative sizes of petals, the spatial relationships between different 30 floral organs as well as flowering time of the plant151. These changes can influence pollinator 16 1 preferences. In addition to morphological changes in reproductive structure, polyploidy may lead 2 to the breakdown of self-incompatibility. In the Solanaceae family, polyploid species are 3 significantly more likely to be self-compatible than diploid species152. A broader study of 235 4 angiosperm species also found a significant association between polyploidy and selfing153. 5 However, a study on self-sterility data from 1266 angiosperm species contrasting self- 6 compatible, self-incompatible, and mix-mating groups found that polyploid species did not tend 7 to be more self-compatible compared to diploids154. If the results of the latter study are true, 8 there may be a short-term breakdown of self-incompatability in the neopolyploids enabling it to 9 establish its population154, after which self-compatibility gets established again. Alternatively, 10 other features associated with the mating system such as inflorescence size, floral display, 11 pollinator behaviour may minimize the effect of reproductive isolation due to polyploidization in 12 neopolyploids. Nonetheless, one issue with the 1266 species study is that phylogenetic 13 relationships are not considered154, although there is non-random association between phylogeny 14 and mating system. In addition, the selfing rate was binned into three broad categories. On the 15 other hand, the 235 species study considered species phylogeny and modeled selfing rate as a 16 continuous variable despite a smaller sample size153. Thus the lack of correlation between 17 polyploidy and selfing rate in the 1266 species study can be due to confounding factors of 18 phylogenetic relations and selfing rate binning. 19 Physiological changes and stress tolerance 20 Polyploidy significantly influences photosynthesis (reviewed in Warner and Edwards, 21 1993155), and this effect is particularly obvious under stress conditions. In greenhouse grown 22 Betula papyrifer, water stress treatment leads to a complete cessation of photosynthesis in 23 diploids but not in penta- and hexapolyploids. Such behavior can be partly attributed to earlier 24 stomata closure in diploids156. But it remains unclear if the photosynthetic activities under water 25 deficit in polyploid is advantageous because it would contribute to carbon fixation even under 26 stress or whether it is detrimental due to water loss. Under excess light, the capacity for photo- 27 protection and non-photosynthetic electron transport are higher in the natural allotetraploid G. 28 dolichocarpa than in its diploid progenitors157. This enhanced photo-protection appears 29 beneficial because the allotetraploid is reported to photobleach later than its diploid progenitors. 30 Because of the prevalence of polyploid plants and the perceived broader ecological 31 tolerance (reviewed in Otto and Whitton 2000149), one potential physiological consequence of 17 1 polyploidy is its increased tolerance to environmental stress. However, the degree of 2 environmental stress tolerance does not necessarily correlate with cytotypes (see Maherali et al, 3 2009158 and references therein). Taking drought tolerance as an example, in a comparison 4 between the fireweed Chamerion angustifolium diploid and tetraploids (both natural and 5 synthetic) in a controlled environment, the natural tetraploids took 20-30% longer to wilt 6 compared to both diploid and synthetic tetraploids158, consistent with the higher xylem hydraulic 7 conductivity observed in natural tetraploids. However, the vulnerability of stems to drought- 8 induced cavitation was similar among C. angustifolium cytotypes. In field grown tetraploid and 9 hexaploid Atriplex canescens159, the leaf-specific hydraulic conductivity as well as susceptibility 10 to cavitation was lower in plants with higher ploidy levels. The inconsistency between earlier 11 studies and the two recent ones highlighted above can be attributed to, for example, differences 12 in whether the studies were conducted in controlled or natural environments, how physiological 13 measurements were taken, and how and when the polyploids were established. 14 Polyploids are also hypothesized to be more resistant to pathogens160. Mathematical 15 models of interactions between pathogens and either diploids or neopolyploids have shown that 16 newly formed polyploid populations of hosts are expected to be more resistant161. However, 17 similar to studies of the relationships between polyploidy and abiotic stress tolerance, the few 18 empirical studies conducted so far have generated mixed results162,163. For plant-insect 19 interactions, the autotetrapolid gooseberry-leaf alumroot Heuchera grossulariifolia is more likely 20 to be attacked by the specialist moth herbivore Greya politella than diploids164. In another field 21 study of the resistance of H. grossulariifolia to three moth species G. politella, G. piperella, and 22 Eupithecia misturata was tested165. Interestingly, G. piperella tends to attack and lay eggs on 23 diploids, suggesting herbivore species may provide selective pressure differently due to 24 differences in ploidy levels. Nonetheless, it remains unclear whether there are additional genetic 25 differences independent from polyploidy that contribute to the difference. 26 27 Polyploidy and adaptation 28 Survival in adverse environments 29 The question of whether polyploidization contributes to the long-term evolutionary 30 success of plant species has been raised repeatedly since Stebbins166. There are already a number 18 1 of excellent reviews on this topic17,149,167,168, thus, in this section, we focus on providing a 2 summary of earlier findings and a discussion of some recent results. 3 Polyploids are more frequent at higher elevations, higher latitudes and may be more 4 tolerant to dry conditions38,158,169, suggesting a fitness advantage for polyploids under those 5 environments. The timing of multiple polyploidization events in angiosperms coincides with the 6 timing of the creation of the Cretaceous-Tertiary boundary170. This coincidence has led to the 7 hypothesis that species with genome doubling could adapt better to the changing environment 8 than their diploid relatives during the mass extinction event. However, considering the influence 9 of environmental factors on unreduced gamete formation, it is also possible that the intense 10 climatic changes during the mass extinction event may have increased the frequency of 11 unreduced gamete formation, creating polyploids at a faster rate than normal. In addition, 12 polyploids were found to be more successful in colonizing the Arctic after deglaciation than 13 diploids169. A study sampling 640 endangered and 81 invasive species worldwide has led to the 14 conclusion that endangered species tend to be diploids while invasive species tend to be 15 polyploids, suggesting that polyploidization may increase tolerance to diverse ecological 16 conditions171. It has also been shown that polyploid A. thaliana accessions are more tolerant to 17 and have better reproductive success under high salinity compared to diploid cytotypes172. It will 18 be particularly interesting to determine if such high salinity adaptation can also be observed 19 under field conditions. 20 Not all findings support the notion that polyploids tend to survive better in adverse 21 environments. A 1940 study of 100 polyploid and diploid plant species found no correlation 22 between polyploidy and winter hardiness173. More recently, in a study of two diploid and one 23 polyploid species from each of 144 North American plant genera, no association was found 24 between ploidy level and species range area, minimum/maximum temperature, precipitation, or 25 latitudes174. Furthermore, as discussed in the previous section, physiological changes associated 26 with polyploidy do not necessarily confer superior tolerance and/or resistance to abiotic and 27 biotic factors. These observations suggest that the relationship between polyploidy and 28 adaptation is quite complex and may depend on not only the species undergoing polyploidization 29 but also the environment. 19 1 Local adaptation 2 In addition to the meta-analyses discussed above, multiple studies provide specific 3 examples of adaptation that are potentially due to polyploidy. In H. grossulariifolia, pollinators 4 visit tetraploid individuals more often than diploid ones175. Similarly in the fireweed C. 5 angustifolium, where populations ranges of polyploid and diploid varieties overlap, tetraploids 6 have a disproportionately higher number of bee visits and a greater pollen siring advantage 7 compared to diploids176,177. The invasiveness of allotetraploid cordgrass Spartina anglica may 8 indicate a fitness increase due to polyploidy, however, this is more likely a consequence of 9 heterosis than genome doubling178. 10 In wild yarrow Achillea borealis, which has hexaploid and tetraploid populations 11 occupying nearby but different environments, there is clear evidence of local adaptation, and 12 polyploidization is likely the initial trigger for diversification and adaptation to a new habitat179. 13 Consistent with this notion, the evolvability of the neo-autotetraploid C. angustifolium is higher 14 than both diploid and established autotetraploids, suggesting that genome doubling, without 15 hybridization, may initially alter evolutionary rate and contribute to adaptive evolution180. In 16 addition, reciprocal transplant experiments demonstrated that C. angustifolium diploids and 17 tetraploids survived best at their native elevations181. Overall, these observations suggest that 18 polyploidization may have positive fitness consequences and can lead to adaptation to local or 19 regional environments. 20 Species richness 21 Polyploidy leads to instantaneous reproductive isolation of polyploid individuals through 22 the phenomenon of minority cytotype exclusion1,30, but such individuals also possess a greater 23 capacity for functional innovation. If polyploids in general have better fitness compared to 24 diploids, the speciation rate of polyploids may be higher than that of diploids. Multiple 25 observations suggest a positive association between species richness and percent polyploid 26 species in different plant clades7,149,182. Additionally, a large number of species in major plant 27 families such as Poaceae183, Asteraceae184, Brassicaceae57 and the subfamily Papilionoideae185 28 have descended from a polyploid ancestor, suggesting a possible increase in diversification rates 29 post-polyploidization. Overall, it has been estimated that a significant proportion - ~15% of 30 angiosperm and ~31% of fern speciation events - may have been accompanied by ploidy 31 increase186. On the contrary, some other studies provide conflicting results. One study arrived at 20 1 the conclusion that polyploids tend to have lower diversification rates than diploids and have a 2 greater chance of extinction187. It has also been estimated that only 2-4% of polyploidization 3 events in angiosperms have actually resulted in speciation149. In addition, analyses of genome 4 sequences of flowering plants suggests that the difference in the estimated timing between 5 successive detectable paleopolyploidization events is ~10-30 million years in flowering plant 6 lineages5. Thus, considering the abundance of polyploids among flowering plants, most of the 7 polyploidization events were not recorded in the genomes of extant species. The inference is that 8 most of these polyploids have gone extinct. 9 These observations suggest two possibilities - either a majority of the polyploid lineages 10 indeed go extinct, making them "evolutionary dead ends"166, or they hybridize with their parental 11 species, creating lineages of mixed ploidy. It seems that both of these scenarios might occur in 12 nature. Hybridization between polyploids and their diploid ancestors has been reported in natural 13 populations of multiple species such as C. angustifolium188, species of the genus Epidendrum189 14 and Jacobaea190 and several others191, in regions known as hybrid zones191. Mathematical 15 simulations on whether a triploid hybrid between an autotetraploid and a diploid can help a 16 tetraploid population to establish suggest that even partially fit triploids can assist in long-term 17 tetraploid fixation19. Thus, polyploidization may not necessarily be an "evolutionary dead end" 18 but may create interesting possibilities for further innovation in the lineage. 19 20 Conclusions 21 In this article, we highlight the findings that the formation of a polyploid is associated 22 with extensive changes at the genomic, epigenetic, transcriptional and network levels. These 23 genomic, transcriptomic, and other omic changes must have contributed to morphological, 24 physiological, and ecological “phenotype” differences between polyploids and their diploid 25 progenitors. However, the exact molecular changes responsible for the phenotypic differences 26 between cytotypes remain unclear in most cases. Also, we have a relatively better understanding 27 of the molecular and phenotypic consequences of allopolyploidy than autopolyploidy, and 28 studies comparing and contrasting mechanisms of molecular evolution in these two forms of 29 polyploidy are lacking. For example, it is not clear whether the rate of neo- and sub- 30 functionalization and pseudogenization differ between auto and allopolyploids, given the 31 homeologs in allopolyploids are already slightly divergent from each other. Also, given the 21 1 extent of functional redundancy is higher in autopolyploids, do mutations have a stronger 2 deleterious effects in allopolyploids than autopolyploids? Additional research would be needed 3 to address these questions. 4 In a review by Soltis et al13, a number of intriguing questions are raised regarding what 5 we still don’t know about polyploidy. To expand on the long list, one challenge lies in 6 establishing the genetic basis in cases where polyploids are shown to be successful. Among the 7 unknowns, one particularly challenging question is whether polyploids are more successful than 8 their diploid progenitors13. A related question is under what ecological situations is polyploidy 9 adaptive. Theoretical considerations as well as empirical evidence have provided contradictory 10 answers to these two questions so far17,149,166–168. Nevertheless, the second question is more 11 tractable as it does not require any generalizations and can be examined experimentally on a 12 species-by-species basis. Polyploidy is an extreme form of duplication and can be seen as a 13 mutation mechanism. Considering the nearly neutral theory of molecular evolution 192, the null 14 hypothesis is that the effect of polyploidy is neutral or nearly neutral. In this framework, we can 15 test under what situations, e.g. different abiotic/biotic environments, the null hypothesis can be 16 rejected. 17 Taking C. angustifolium as an example, diploid and tetraploid varieties show significant 18 differences in drought tolerance158. Given the understanding of drought tolerance in model 19 plants193,194, a targeted survey of candidate gene transcription in field conditions can potentially 20 be informative. The optimal timing for the assay is not trivial to determine and molecular 21 changes other than transcription can be more important. In addition, drought tolerance may not 22 be the main environmental factor, highlighting the need to assess potentially relevant 23 abiotic/biotic factors in controlled environments in addition to the field. Nonetheless, a candidate 24 gene approach is a reasonable starting point. In case the candidate gene approach does not bear 25 fruit, because sampling the "omes" – genomes, transcriptomes, proteomes, or metabolomes – of 26 “non-model” species is no longer a rate limiting step, a global study of molecular changes may 27 provide viable hypotheses for further testing. These considerations are not unique to C. 28 angustifolium but other polyploid study systems as well. We surmise that good experimental 29 designs incorporating both molecular and ecological considerations of polyploids have the 30 potential to make the most impact in the near future. 31 22 1 2 Competing interests No competing interests exist 3 4 Acknowledgements 5 We would like to thank the two anonymous reviewers as well as the editor for providing us 6 critical feedback on the manuscript. SHS is supported by National Science Foundation grants 7 DEB-0919452 and IOS-1126998. 8 Figure and table legends 9 10 Figure 1: Pathways of tetraploid formation from diploid plants. The symbol X represents the 11 base chromosome number of the species, with 1X corresponding to haploid gametes. (A) The 12 normal pathway wherein a diploid is produced as F1 progeny of two diploid parents. (B) Somatic 13 doubling leading to tetraploid (4X) generation from diploid. (C) Fusion of unreduced gametes 14 can lead to tetraploid generation in one step. (D) A diploid produced in F1 may generate a certain 15 proportion of aneuploid gametes (AP, most of which are not viable) and unreduced gametes that 16 can lead to tetraploid generation in F2. The frequency of unreduced gamete formation can be 17 high if the parents belong to different species and F1 is a hybrid. (E) The triploid bridge scenario 18 where an intermediate triploid produces unreduced gametes leading to generation of tetraploids 19 and individuals of higher ploidy. These pathways of tetraploid formation have been adapted from 20 information presented in Ramsey and Schemske, 1998. For example polypoids formed via each 21 pathway, as well as pathways of formation of polyploids of higher ploidy levels, please refer to 22 the original publication. 23 24 Figure 2: Genomic consequences of polyploidy. (A) P1: Parent 1, P2: Parent 2. Some possible 25 scenarios with respect to genomic rearrangements such as chromosome loss, chromosomal 26 translocation and chromosome fragment loss have been depicted in a simplified manner using 27 only two chromosomes. (B) The process of gene loss in a parent-of-origin manner, termed 28 fractionation. In the depicted scenario, the chromosomal copy from P2 loses most of the genes. 29 (C) Proliferation of transposable elements over time. Such proliferation may lead to changes in 30 gene order, gene function and gene expression. 23 1 2 Figure 3: Effects of polyploidy on gene expression and epigenetic regulation (A) A 3 hypothetical scenario depicted expression divergence upon polyploidization. P1: Parent 1, P2: 4 Parent 2. In “Homeolog expression bias”, the homeologous genes are expressed in a parent-of- 5 origin manner. In “Expression level dominance”, the sum of the expression level of both the 6 genes is similar to that in one parent. (B) A gene which is silenced by DNA methylation in the 7 parent is demethylated upon polyploidization or hybridization, leading to its transcription by 8 RNA polymerase. (C) A gene with a repressor bound to its promoter region is not expressed in 9 the parent but upon polyploidization is expressed as the repressor is removed due to regulation 10 by small RNAs transcribed elsewhere in the genome. 11 12 Figure 4: Effect of polyploidy on gene networks P1: Parent 1, P2: Parent 2. An example of two 13 genes - Gene 1 and Gene 2 - in parents P1 and P2 is shown. Each square represents a genetic or 14 physical interaction of Gene 1 or Gene 2. After polyploidization, over the course of several 15 generations, the network topology begins to evolve. 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After polyploidization, over the course of several generations, the network topology begins to evolve. If both the homeologous copies do not undergo gene loss via deletion or pseudogenization and are retained, divergence between them will lead to eventual sub-functionalization or neofunctionalization between duplicates, changing the network topology and possibly, network function. Figure 2: Molecular consequences of polyploidy. The myriad effects of polyploidy at the molecular level are explained in this schematic. Figure 1 A. Parents 2X 2X Gametes X X B. 2X F1 2X 2X X X 2X 4X D. Parents 2X 2X Gametes X X 2X 2X 2X E. AP X 2X 2X 2X X 3X 2X AP X 2X 3X Gamete 1 X 2X X 2X 3X X 2X 3X X 2X 3X 4X 2X 3X 4X Gamete 2 Gamete 2 Unreduced gamete formation 4X Zygote somatic doubling Gamete 1 F2 2X 2X F1 F1 gametes C. 2X 3X 4X 5X 3X 4X 5X 6X Figure 1: Pathways of tetraploid formation from diploid plants. The symbol X represents the base chromosome number of the species, with 1X corresponding to haploid gametes. (A) The normal pathway wherein a diploid is produced as F1 progeny of two diploid parents. (B) Somatic doubling leading to tetraploid (4X) generation from diploid. (C) Fusion of unreduced gametes can lead to tetraploid generation in one step. (D) A diploid produced in F1 may generate a certain proportion of aneuploid gametes (AP, most of which are not viable) and unreduced gametes that can lead to tetraploid generation in F2. The frequency of unreduced gamete formation can be high if the parents belong to different species and F1 is a hybrid. (E) The triploid bridge scenario where an intermediate triploid produces unreduced gametes leading to generation of tetraploids and individuals of higher ploidy. These pathways of tetraploid formation have been adapted from information presented in Ramsey and Schemske, 1998. For example polypoids formed via each pathway, as well as pathways of formation of polyploids of higher ploidy levels, please refer to the original publication. Figure 2 A. P1 Chromosome loss P2 Chr A Translocation t Chr B Chromosomal fragment loss B. P1 P2 t Homeologous genes Gene losses C. Transposable element P1 P2 Homeologous genes Transcriptional activation or repression t Proliferation Insertion into gene Figure 2: Genomic consequences of polyploidy. (A) P1: Parent 1, P2: Parent 2. Some possible scenarios with respect to genomic rearrangements such as chromosome loss, chromosomal translocation and chromosome fragment loss have been depicted in a simplified manner using only two chromosomes. (B) The process of gene loss in a parent-of-origin manner, termed fractionation. In the depicted scenario, the chromosomal copy from P2 loses most of the genes. (C) Proliferation of transposable elements over time. Such proliferation may lead to changes in gene order, gene function and gene expression. Figure 2: Molecular consequences of polyploidy. The myriad effects of polyploidy at the molecular level are explained in this schematic. Figure 3 Expression level A. Homeologous genes P1 2 1 2 3 4 2 2 3 4 1 1 0 P2 1 0 Genes Genes Polyploidization Divergence Expression level dominance Homeolog expression bias Expression level 2 1 0 1 2 3 4 1 2 3 4 2 1 0 Genes DNA methylation B. Gene Polyploidization Demethylation RNA polymerase Gene Transcribed mRNA C. Repressor Gene RNA polymerase Polyploidization small RNA transcription Gene small RNAs Transcribed mRNA Figure 3: Effects of polyploidy on gene expression and epigenetic regulation (A) A hypothetical scenario showing expression divergence upon polyploidization. P1: Parent 1, P2: Parent 2. In “Homeolog expression bias”, the homeologous genes are expressed in a parent-of-origin manner. In “Expression level dominance”, the sum of the expression level of both the genes is similar to that in one parent. (B) A gene which is silenced by DNA methylation in the parent is demethylated upon polyploidization or hybridization, leading to its transcription by RNA polymerase. (C) A gene with a repressor bound to its promoter region is not expressed in the parent but upon polyploidization is expressed as the repressor is removed due to regulation by small RNAs transcribed elsewhere in the genome.