DISCUSSION

2n gamete formation in the potato and genetic consequences: A number of meiotic mutations related to spindle formation and cytokinesis have been associated with 2n gamete production. In the potato, the main mechanism of 2n pollen formation is parallel spindle (ps; Watanabe and Peloquin 1993). In normal meiosis during microsporogenesis the metaphase II spindles are oriented so that their poles define a tetrahedron, and following cytokinesis, a tetrad of four 1n microspores is produced. When ps is present, the spindles are parallel, and a dyad of two 2n microspores is formed at the end of meiosis. Mok and Peloquin (1975) demonstrated that this mutation is controlled by a single recessive gene. Parallel spindle is genetically equivalent to a first-division restitution (FDR) mechanism. All loci from the centromere to the first crossover that are heterozygous in the parent will be heterozygous in the gametes, and half the heterozygous parental loci beyond the first crossover will be heterozygous in the gametes (due to small chromosome size, there is normally only one crossover per chromosome arm).

Several mutations leading to 2n egg formation have also been found (Werner and Peloquin 1991a). Among them, the most common is the omission of the second meiotic division (os). In normal megasporogenesis, the cell plate forms after the first and second divisions, and four 1n megaspores are formed. When os is present, after the first division there is no second division, and the chromatids just fall apart. Of the resulting two 2n megaspores, one gives rise to the 2n female gametophyte and the other degenerates. Omission of the second division is also a simply inherited Mendelian recessive (Werner and Peloquin 1990) that has been detected in a large number of Solanum species (Den Nijs and Peloquin 1977; Stelly and Peloquin 1985; Werner and Peloquin 1991a). Omission of the second division is genetically equivalent to a second-division restitution (SDR) mechanism. All the loci from the centromere to the first crossover will be homozygous in the gametes, and all loci past the first crossover, which are heterozygous in the parent, will be heterozygous in the gametes. On the basis of chromosome structure, position of the centromere, and chiasmata frequencies, it has been estimated that FDR 2n gametes transmit ∼80% of heterozygosity and large fractions of epistasis from the parent to the offspring (Peloquin 1983). On the other hand, SDR 2n gametes transmit a smaller amount of heterozygosity (∼40%) and of epistatic interactions.

The frequency at which 2n gametes are formed is also very important, in that it may affect the rate of new polyploid formation. Ramsey and Schemske (1998) suggested that the high frequency of polyploid species in harsh environments can be related to the impact of environmental conditions on 2n gamete production. The frequency of 2n gametes may be highly variable both between and within species. Watanabe and Peloquin (1993) found that 2n pollen frequency in diploid, tetraploid, and hexaploid Solanum species ranged from 1.9 to 36.3%. A significant variation in the frequency of 2n eggs in Solanum species (from 4.9 to 22.6%) has been also reported by Werner and Peloquin (1991a). The variability found in Solanum is consistent with that reported in other species like Dactylis glomerata, Trifolium nigrescens, Manihot, and Medicago (see Bretagnolle and Thompson 1995) and has been attributed mainly to the incomplete penetrance and variable expressivity of the mutant alleles responsible for 2n gamete production, as well as to environmental factors. Generally, available data indicate that the range of 2n gamete frequency is between 1 and 40%. This suggests that plants tend to hold high frequencies of n gametes, probably to maintain their diploid level while allowing polyploidization.

Solanum species and the EBN: Potato species form a polyploid series with a basic chromosome number (x) of 12 and a minimal chromosome differentiation among taxa. Most of them (∼70%) occur as diploids (2n = 24), and the others are triploids (2n = 36), tetraploids (2n = 48), pentaploids (2n = 60), and hexaploids (2n = 72). The main cultivated potato is a tetrasomic tetraploid originating from South America. S. tuberosum Group Andigena is cultivated mainly under short days and is now restricted to South America. S. tuberosum Group Tuberosum is the potato cultivated worldwide under long-day conditions. Also, some diploids, triploids, and pentaploids are still cultivated in South America, as well as a large number of wild tuber-bearing species (∼160). They grow in a wide range of habitats from the southern part of the United States through Mexico and Central America down to southern Chile.

Besides having different ploidy levels, Solanum species also differ in the EBN (Johnstonet al. 1980). The EBN represents their “effective ploidy” and is not always in accordance with the actual chromosome number. It varies from 1 to 4: diploid species can be either 1 or 2EBN, triploids 2EBN, tetraploids 2 or 4EBN, pentaploids and hexaploids 4EBN (Hanneman 1994). The cultivated S. tuberosum is 4EBN. The EBN deserves special attention in the context of the evolution of tuber-bearing Solanum species. It acts as an effective mechanism to regulate endosperm development after interspecific/interploidy crosses and thus to determine their success or failure. According to the model hypothesized, normal endosperm development occurs only when there is a 2:1 maternal-to-paternal EBN ratio in the hybrid endosperm. In all the other cases the endosperm degenerates. Clearly, EBN differences between parents prevent hybridization between different ploidies within a species and between species. For this reason, for example, tetraploid S. acaule (2EBN) cannot be crossed with S. tuberosum, which is also tetraploid but has an EBN of 4. On the other hand, S. acaule hybridizes with diploid (2EBN) species. A number of studies have demonstrated that the EBN is under the control of genes with additive effects. Ehlenfeldt and Hanneman (1988) and Camadro and Masuelli (1995) reported that three and two genes were involved in the determination of the EBN of Solanum species, respectively. Evidence for EBN-like systems has been reported in several other species. In Impatiens, Arisumi (1982) assigned EBNs to several species according to their crossability with 2EBN testers. Parrott and Smith (1986) provided evidence that EBN is operating in Trifolium and assigned EBN values from 2 to 8. Ehlenfeldt and Hanneman (1992) classified the species belonging to the Lycopersicon esculentum crossability group as 2EBN, whereas higher EBN values were assigned to the L. peruvianum complex. Katsiosis et al. (1995) grouped oat species into four EBN groups from 1 to 4. For a review of the EBN, its genetic control, and its usefulness, see Carputo et al. (1999).

Origin of the cultivated potato: According to the evolutionary pathway proposed by Hawkes (1990), tuber-bearing wild species evolved first in Mexico and then spread throughout Central and South America. The point of origin of the cultivated potato was somewhere between central Peru and central Bolivia, but the exact nature of the original wild parent is still uncertain. The most primitive form of the cultivated tetraploid potato is the diploid S. tuberosum group Stenotomum from which all the cultivated forms have arisen either directly or indirectly. Chloroplast DNA analysis confirmed very close relationships among 2x cultivated potatoes and thus the idea of the common origin of the cultivated 2x species from S. tuberosum group Stenotomum (Hosaka and Hanneman 1988). S. tuberosum group Phureja is believed to have originated by mutation and selection from S. tuberosum group Stenotomum after the Andean farmers had taken it to lower and warmer valleys. Among cultivated triploids, S. tuberosum group Chaucha is made up of a series of natural triploids between group Stenotomum and group Andigena. S. × juzepczukii is a natural hybrid between S. acaule and group Stenotomum. The pentaploid S. × curtilobum was formed following hybridization between S. × juzepczukii and group Andigena. Hawkes (1990) also proposed that the cultivated S. tuberosum group Andigena was derived from group Stenotomum and the diploid weedy species S. sparsipilum through polyploidization. On the other hand, Matsubayashi (1991) hypothesized that it is mainly a group Stenotomum-group Phureja derivative and that S. sparsipilum had only a secondary role in the origin of group Andigena. The potato that is cultivated worldwide, S. tuberosum group Tuberosum, derived from group Andigena as a result of artificial selection for tuberization under long-day conditions.

In this complex pattern of species, hybrids, and chromosome numbers, polyploidization has been a major mechanism of speciation. But how did polyploidization occur?

Sexual vs. asexual polyploidization: The two main mechanisms of polyploid formation are somatic doubling of chromosomes in meristem tissues of sporophytes, zygotes, or embryo (somatic polyploidization) and meiotic nuclear restitution leading to 2n gamete production (sexual polyploidization). In somatic polyploidization chromosome restitution takes place during mitosis and all the chromosomes of a somatic cell are included in one daughter nucleus, giving rise to a cell with a doubled chromosome number. The most common example of somatic doubling is Primula kewensis, a disomic polyploid, which arose by somatic doubling of some flowering branches in the sterile diploid hybrid P. floribunda × P. verticillata (Newton and Pellew 1929). Somatic doubling is also known to occur in Solanum (Jorgensen 1928) and Mimulus (Hieseyet al. 1971). Little is known about either the natural frequency of somatic doubling in plants or the effects of interspecific hybridization on its occurrence. From the literature available, Harlan and De Wet (1975) concluded that somatic doubling is a relatively unimportant phenomenon in the production of polyploids.

Sexual polyploidization involves the function of 2n gametes. The union of two 2n gametes, as well as of 1n and 2n gametes, produces polyploid embryos, which can give rise to a new polyploid population, depending on the genotypes involved and the environmental conditions. Compared to somatic polyploidization, more information is available on the genetic and environmental factors influencing the frequency of 2n gamete production. There is also stronger circumstantial evidence that 2n gametes have contributed to polyploid evolution. However, further quantitative assessments of the frequencies of somatic doubling and 2n gametes in natural populations will allow better evaluation of the contribution of somatic and sexual polyploidization in plants. The availability of genetic markers and a detailed knowledge of mechanisms involved in 2n gamete production will be extremely useful for this purpose.

Although both sexual and somatic polyploidization result in chromosome doubling, the genetic consequences of the two modes of polyploidization are very different. Three features of 2n gametes and sexual polyploidization should be emphasized in this context.

1. It is well known that heterozygosity is very important in a polysomic polyploid like the potato in that maximum heterosis for polygenic traits is expected with maximum heterozygosity (Werner and Peloquin 1991b). Heterozygosity refers to the possibility of more than two alleles per locus, and diversity at tetra-allelic loci (A₁A₂A₃A₄) gives maximum heterozygosity with six first-order, four second-order, and one third-order interaction. 2n gametes are the result of modified meiosis and, as already pointed out, they transmit high levels of parental heterozygosity and epistasis, circumventing the disruptive effects of meiosis on intra- and interlocus interactions. Somatic doubling transmits all the parental heterozygosity, but if a heterozygous individual A₁A₂ is doubled, a balanced diallelic tetraploid is formed, which has only one first-order interaction as its progenitor and high levels of inbreeding.

2. The genetic determination of 2n gamete formation ensures repeated events of sexual polyploidization and the incorporation of genetic diversity of more than one diploid species. This “progressive introgression,” besides having implications for the taxonomy of polyploids, constitutes the basis for especially high levels of heterozygosity. Newly arisen polyploids may thus possess a wide range of novel phenotypic, ecological, and physiological characteristics upon which natural selection can act. In recent years, use of various molecular techniques allowed detailed analyses of the genomic or DNA sequence constitution of several polyploids, including angiosperms, pteridophytes, and bryophytes (Leitch and Bennett 1997; Soltis and Soltis 2000). All these studies confirmed that nearly all polyploids were polyphyletic and that polyploidy is a dynamic and reversible process. Although the authors recognized this as a significant source of genetic diversity, none of them discussed sexual polyploidization as the cause of the phenomenon.

3. The genetic determination of 2n gametes also allows the reciprocal origin of polyploids when diploid progenitors produce both 2n pollen and 2n eggs. This can result in differing morphological, biochemical, and physiological characteristics, as well as differing nuclear-cytoplasmic gene interactions. As a result, further genetic variability is created.

Computer simulation has been performed to compare the genetic consequences of sexual and somatic polyploidization (Watanabeet al. 1991). The results confirmed that regardless of the position of a locus in relation to the centromere, sexual polyploidization generally produces less inbreeding, more genotypic diversity, and a higher frequency of tri- and tetra-allelic genotypes. As the number of alleles at a locus increases, these results become even more prominent. Only when two alleles are possible at a locus can somatic polyploidization be equal to sexual polyploidization. SDR × FDR proved the most stable mechanisms for sexual polyploidization against extreme recombination events. Table 1 reports coefficients of inbreeding and the number of genotypes at a locus in tetraploid populations deriving from somatic doubling and an SDR × FDR crossing scheme.

In brief, there is genetic evidence that while somatic doubling does not change the genotype in any way except that all alleles become redundant and doubled plants become inbred, sexual polyploidization is a creative phenomenon providing new allelic diversity and gene combinations while avoiding inbreeding. It would be extremely interesting to know the rate of fitness of sexual polyploids vs. diploid progenitors in natural habitats and the specific genetic mechanisms that may influence fitness. These studies would allow a better understanding of the role of polyploidization in adaptation. Recently, Otto and Whitton (2000) suggested that sexual and asexual polyploids can adapt faster than diploids, especially if beneficial mutations appear in relatively small populations and have a partially dominant effect on fitness.

ps and evolution: Most genetic studies of 2n gametes concern 2n pollen, and results obtained in Solanum provide useful information to extend our understanding of the evolution of polyploids. The genetic determination of 2n pollen formation and the large number of diploid Solanum species in which 2n pollen has been found (Den Nijs and Peloquin 1977; Watanabe and Peloquin 1993) represent the first clear proof that 2n pollen was involved in the evolution of polyploid potatoes. Significantly, 2n pollen has not been recovered in species belonging to taxonomic series (e.g., Circaeifolia, Morelliformia) that are entirely diploid, whereas it is largely present in series (e.g., Tuberosa) where a wide range of ploidy levels is found. Studies by Camadro and Peloquin (1980) and Watanabe and Peloquin (1989) demonstrated that the ps allele was widely distributed in natural populations of diploid species and that a high frequency of recessive homozygous and heterozygous diploid wild genotypes can be found at the ps locus. Table 2 summarizes the results of these researches and gives the estimated gene frequency for a number of wild species from Peru, Chile, and Argentina. It should be pointed out that these values are probably underestimated, given that ps, like most meiotic mutations, is characterized by variable penetrance and expressivity, so that plants that do not produce 2n pollen may have the psps genotype. It is also remarkable that Iwanaga and Peloquin (1982), in screening several tetraploid cultivars for ps gene frequency, found that most of them were simplex (Pspspsps). The high ps gene frequency in wild diploid species and related tetraploids is further convincing evidence that sexual polyploidization through genetically determined 2n pollen is an important evolutionary pathway in Solanum species.

Comparison between ps gene frequency in polyploid species and their original diploid ancestors provides further proof of the major role played by parallel spindle in polyploid evolution. Gene frequency for 2n pollen in the tetraploids should be higher if sexual polyploidization through 2n pollen is involved in the origin of polyploid potato than if polyploidization occurred through somatic doubling. In fact, in the latter case, the same gene frequency in the tetraploids as in their diploid progenitors is expected. But if polyploids originate from sexual polyploidization, a higher gene frequency for ps can be expected in tetraploids because (1) both ps alleles are transmitted from the male diploid parent to the tetraploid population that originated through sexual polyploidization and (2) the ps gene frequency in 2n eggs is independent of the position of the ps locus in respect to the centromere (Watanabe and Peloquin 1989). As a result, the ps gene frequency in new tetraploids is increased from that in the original diploid populations.

Genetic analysis by Camadro and Peloquin (1980), Watanabe and Peloquin (1989), and Iwanaga and Peloquin (1982) supported this hypothesis (Table 2). The ps gene frequency in possible progenitors of tetraploid S. tuberosum group Andigena and group Tuberosum was 0.39 for S. sparsipilum, 0.42 for S. tuberosum group Stenotomum, and 0.47 for S. tuberosum group Phureja. By contrast, the ps gene frequency was much higher in S. tuberosum group Andigena (0.85) and group Tuberosum (0.69). Similarly, the ps gene frequency of diploid S. gourlayi and S. infundibuliforme, possible progenitors of tetraploid S. gourlayi, was 0.46 and 0.37, respectively. Again, in tetraploid S. gourlayi it was much higher (0.80). In all these studies the ps gene frequencies in tetraploid species were almost twice those of the diploid progenitors. Without doubt this indicates that the tetraploids originated from sexual polyploidization.

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Figure 1.

—Complementary role of endosperm and 2n gametes in maintaining and breaking the sexual isolation of Solanum species and in determining the success of interspecific hybridization during the evolution of tuber-bearing Solanums. The EBN acts as a screen for either 1n or 2n gametes, allowing the maintenance of the ploidy integrity of the diploid parental species through 2x(2EBN) × 2x(2EBN) crosses, while permitting polyploidization through interploidy inter-EBN crosses. In the case of 3x(2EBN) × 2x(2EBN) crosses to obtain tetraploids, the functional gametes of the 3x parent have a 24-chromosome complement and an EBN of 2.

Although the os gene frequency has not been estimated, data on 2n egg production support the hypothesis that 2n gametes were involved in the origin of tetraploids. Werner and Peloquin (1991a) screened diploid species and group Tuberosum haploids for the frequency of plants producing 2n eggs through omission of the second meiotic division. About 60% of the group Tuberosum haploids produced 2n eggs vs. only 24% of wild species.

2n gametes and endosperm: The endosperm, together with 2n gametes, has been a major factor in the origin and evolution of polyploids. The endosperm dosage system regulates the within-species and between-species congruity in that endosperm development is dependent on the relative proportion of maternal and paternal contributions. Due to endosperm imbalance, for example, a strong triploid block exists in the majority of angiosperms. The development of the EBN system in Solanum has shed new light on the role of the endosperm in polyploid evolution. In fact, the EBN in combination with 2n gametes maintains the ploidy integrity of diploid ancestral species, while providing the flexibility for either unilateral or bilateral sexual polyploidization. Den Nijs and Peloquin (1977) proposed an evolution pathway in which 1n and 2n gametes link all ploidy levels of Solanum species, thus overcoming the ploidy barriers and giving opportunity for gene flow throughout sympatric species. In this evolutionary scheme the endosperm, through the EBN incompatibility system, strongly complements the role of 2n gametes. It acts as a powerful screen for either 1n or 2n gametes, depending on the EBN and chromosome number of parents. This guarantees the maintenance of the ploidy level integrity of the two diploid parental species in crossing with other diploids, while allowing polyploidization.

Sexual polyploidization can occur bilaterally or unilaterally (Harlan and De Wet 1975; Bretagnolle and Thompson 1995). In the former, both parents produce 2n gametes and have the same EBN value. This may lead to the direct synthesis of a tetraploid following 2x × 2x crosses. In the latter, one parent produces 2n gametes and the ploidy level of the offspring will depend on the EBN of the parents. In a 4x(2EBN) × 2x(2EBN) cross, the required 2:1 ratio between female and male parental EBNs favors 1n gametes of the male parent and an intermediate 3x progeny is generated. By contrast, a 4x(4EBN) × 2x(2EBN) cross will favor 2n gametes of the male parent and a tetraploid offspring is produced. Tetraploid potatoes most likely originated from bilateral sexual polyploidization via fusion of 2n gametes of diploids with the same EBN. It is unlikely that diploid ancestors had different EBNs since that would have caused endosperm degeneration. After the emergence of tetraploids, chances for “progressive introgression” between diploids and newly arisen tetraploids through unilateral sexual polyploidization (4x × 2x crosses) were then possible. Under the complementary hypothesis of 2n gametes and endosperm, 4x(4EBN) S. tuberosum group Andigena may have arisen from the fusion of 2n gametes of 2x(2EBN) S. stenotomum and S. sparsipilum (Hawkes 1990).

Strong evidence of EBN-like systems exists for several species (Avena, Impatiens, Medicago, and Trifolium) in which 2n gametes and ploidy series have also been found. Thus, a combined role of 2n gametes and endosperm can be hypothesized to explain the polyploid evolution of these species as well. It should be pointed out that in the Lycopersicon system 2n gametes have not been detected, ploidy series do not exist, but the EBN does operate (Ehlenfeldt and Hanneman 1992). In this case it acts as an isolating mechanism between the “esculentum group” species and the “peruvianum group” species and has probably evolved through genic changes within a diploid system rather than through polyploidization events, as in Solanum.

Recently, Ramsey and Schemske (1998) reviewed the role of triploids in the evolution of polyploids, their fertility and meiotic behavior, and the chromosome number of progeny deriving from triploid parents. Their results indicated that triploids are often semifertile, producing 1x, 2x, and 3x gametes, and thus contribute significantly to the formation of both polysomic and disomic polyploids. These findings are consistent with the 2n gamete-EBN-based evolution pathway of the potato (Figure 1). In 2x(2EBN) × 2x(1EBN) crosses, the required 2:1 ratio between female and male in the endosperm favors 2n gametes of the male parent, resulting in the formation of a viable tetraploid endosperm associated with a triploid embryo. An example is provided by S. chacoense and S. commersonii, two sympatric diploid species from Argentina. S. chacoense is 2x(2EBN) while S. commersonii is 2x(1EBN), and the two species do not normally intercross. However, if S. commersonii produces 2n gametes, triploids will result. Alternatively, triploids can also be produced following 2x(2EBN) × 4x(4EBN) crosses if endomitosis in the polar nuclei of the 2x parent occurs (Carputoet al. 1999). Newly arisen triploids can function as parents in 3x(2EBN) × 2x(2EBN) crosses to obtain tetraploids. In this case they produce functional gametes with a 24-chromosome complement and have an EBN of 2. They can also self or by sexual polyploidization via 2n gametes from both parents form hexaploids with 4EBN.

Conclusions: Polyploidization is a major factor in the evolution of plants. Somatic doubling and sexual polyploidization are two of the possible origins for polyploids. In-depth analyses of the Solanum polyploid series suggest that 2n gametes and EBN are the mechanisms leading to the formation of polyploids. This process of sexual polyploidization maximizes heterosis and genetic diversity, characteristics of polyploids, yet allows interploidy gene flow. The same evolution path may account for polyploid origin and evolution in other plant families.