Our investigation of the dioecious plant R. nivalis revealed that both female and male sporophytic tissue and female- and male-determining microgametophytes could be reliably distinguished using flow cytometry. This enabled us to determine whether bias was evident in the ratios of the two pollen types produced by male plants. Our earlier work on this species demonstrated female-biased sex ratios during various stages of the sporophytic life cycle (Stehlik & Barrett, 2005, 2006). We found that, on average, pollen production was significantly biased towards female-determining gametophytes, with males from 18 of 22 half-sib families producing more ‘female’ pollen. Additionally, we detected significant between-family variation in the ratios of female- and male-determining pollen, suggesting a genetic component to the production of biased pollen sex ratios.
Diagnosing sex using flow cytometry
Using flow cytometry, we were able to clearly distinguish female from male sporophytic nuclei in R. nivalis (Fig. 3b). This was undoubtedly because of the low ratio of autosomes to heterogametic sex chromosomes, the comparatively large size of the sex chromosomes, and the possession of two Y-chromosomes instead of one in males (Wagenitz, 1981; Navajas-Pérez et al., 2005). These features of the genetic system of R. nivalis resulted in male nuclei containing 9.5% more DNA than female nuclei at the diploid level, with DNA contents of 6.74 pg/2C for females and 7.41 pg/2C for males. These values are comparable to flow cytometric estimates for the related R. acetosa, also in subgenus Acetosa (Błocka-Wandas et al., in press), although the reported difference (7.0 pg/2C for females; 7.5 pg/2C for males) is smaller than for our species. The difference in DNA content between the sexes of R. nivalis is similar to an estimate from Humulus japonicus (9.8%; Grabowska-Joachimiak et al., 2006), an unrelated dioecious species also with heteromorphic sex chromosomes. Doležel & Göhde (1995) also distinguished female and male nuclei peaks with differences of 3.9% and 4.5% in two Melandrium species. Therefore, gender assessment in species with sex chromosomes by flow cytometry can provide a reliable alternative to polymerase chain reaction (PCR)-based approaches using sex-specific sequence characterized amplified region (SCAR) markers (Stehlik & Blattner, 2004).
In addition to 2C nuclei, we also identified a high frequency of 4C and 8C nuclei in leaves of R. nivalis, indicating the occurrence of endopolyploid tissue in somatic cells (Fig. 3a; only 2C and 4C peaks shown). Cell polyploidization results from endoreduplication, which consists of one or more rounds of DNA synthesis in the absence of mitosis (Joubès & Chevalier, 2000). Endoreduplicated cells are commonly found in many tissues, especially those undergoing differentiation or expansion (Galbraith et al., 1991), such as in our case with the leaves of R. nivalis. Thus, R. nivalis can be added to the growing list of plant species in which endoreduplication has been detected (Joubès & Chevalier, 2000). The occurrence of somatic endopolyploidization in R. nivalis posed no problem for distinguishing female and male plants (Fig. 3b), nor did it affect our ability to distinguish female from male microgametophytic nuclei.
In addition to diagnosing gender in sporophytes of R. nivalis, we also detected distinct fluorescence peaks for female- and male-determining microgametophytes (Fig. 1). In accord with our cytological observations of stained pollen grains, female- and male-determining pollen each produced one main fluorescence peak (Fig. 1). This was expected because all pollen grains we examined were trinucleate (Fig. 2) and all three nuclei are expected to be haploid and to have the same DNA content (Bino et al., 1990). Using flow cytometry, Błocka-Wandas et al. (in press) were also able to distinguish female- from male-determining pollen in R. acetosa, indicating that this approach for ‘sexing pollen grains’ may have general applicability in Rumex species with the XX/XY1Y2 genetic system. However, the differences in DNA content in other angiosperm families with heteromorphic sex chromosomes may often be too small for reliable diagnosis of sex differences between pollen grains (Parker, 1990; Ainsworth, 2000).
Flow cytometry studies of pollen nuclei, with few exceptions, usually identify small secondary peaks at fluorescence intensities corresponding to approximately twice the DNA content of the main peaks (reviewed in Suda et al., 2007). Such secondary peaks are often interpreted, with varying degrees of support, as diploidized pollen nuclei resulting from a failure in reduction division of the chromosomes during meiosis (e.g. Bino et al., 1990), or the postmeiotic failure of mitosis in pollen grains (Pan et al., 2004; Błocka-Wandas et al., in press). In our study, we detected three small secondary peaks (Fig. 1). Because the positions of these peaks correspond well to the predicted positions of the three possible doublet types, we interpret them as pollen aggregations of two female-determining, two male-determining, and one female-determining plus one male-determining pollen grain (2F, 2M, and FM, respectively; Fig. 1). This interpretation is further corroborated by the fact that when we used the finer 10-µm filter, which generally yielded stronger and clearer histograms, the smaller 2C peaks were substantially reduced in size. Finally, if substantial numbers of polyploid nuclei were present in addition to doublets, the first and third of these small peaks (2F and 2M in Fig. 1) should be larger than the central peak (FM, Fig. 1). However, we did not observe this pattern in our study. Clearly, caution is necessary when interpreting multiple peaks in histogram profiles of pollen using flow cytometry. Future work on ‘pollen sex ratios’ should pay particular attention to filter size because of the differences we observed between 10- and 30-µm filters. We emphasize, however, that interpreting these 2C peaks as either polyploid nuclei or undivided generative nuclei did not qualitatively affect our results.
Explanations for female-biased sex ratios
We detected a significant overall bias in the production of female- vs male-determining pollen in R. nivalis using flow cytometry. The degree of bias was sensitive to filter size and several assumptions that we made concerning the interpretation of peaks in our histograms. However, 18 of the 22 families that we examined produced pollen with a prevalence of female-determining microgametophytes (Fig. 5) and an ANOVA detecting between-family variation provided evidence of a genetic component to biased pollen sex ratios. We are therefore confident that our finding of female-biased pollen sex ratios in R. nivalis, although involving a relatively small numerical difference, represents a real biological phenomenon. Błocka-Wandas et al. (in press) also investigated pollen sex ratios using flow cytometry in an unspecified number of R. acetosa plants from a meadow in Poland. They also demonstrated female bias (0.55, a value slightly higher than we found in R. nivalis). These results are significant because of earlier reports of sex ratio bias in populations of Rumex species as these also involve female bias. These findings raise the question of what mechanism(s) might account for the pattern of sex ratio bias in male gametophytes.
During microgametophyte development in R. nivalis, diploid microspore mother cells located in anthers should contain the full complement of XY1Y2 sex chromosomes, as in all other somatic cells. At meiosis, it would be expected that microspore mother cells divide to form two classes of microspores containing either X or Y1Y2 sex chromosomes. As anther development proceeds, these microspores mature to become gametophytes and equal ratios of female- and male-determining pollen are generally expected, although this has never been verified directly.
Our results suggest that unknown mechanisms cause deviations from the expected 1 : 1 ratio of female- and male-determining pollen. The number of female-determining pollen could be inflated if meiotic disruptions generate microspores with single Y-chromosomes that are indistinguishable from those with X-chromosomes. However, cytological studies of R. acetosa provide no evidence for this phenomenon (Błocka-Wandas et al., in press) and we consider this explanation unlikely. If such an effect were to occur, and if pollen with single Y-chromosomes were inviable, a smaller female bias would still be present, with meiotic disruption being one mechanism contributing to the loss of male pollen. Stehlik & Barrett (2005, 2006) review several additional hypotheses that might influence the quantity and quality of pollen in Rumex and affect sex ratio bias. These ideas, summarized in the following paragraph, are functionally associated with the particular type of chromosomal sex determination that occurs in Rumex section Acetosa.
Female-biased pollen sex ratios are most easily explained by the mortality of microspores or male-determining pollen during early gametophyte development. This could result from trivalent formation and nondisjunction of the sex chromosomes during meiosis as a result of the difference in chromosome numbers between the sexes. However, a study of R. acetosa provided no evidence of disturbance during meiosis (e.g. anaphase bridges or delayed chromosomes) and the presence of irregular nuclei or micronuclei was not observed (Błocka-Wandas et al., in press). In their study, ~2% of pollen grains were considered inviable based on Alexander's test. Rumex acetosa and R. nivalis (both subgenus Acetosa) share the same XY1Y2 chromosomal system and similar female biases in sporophytic and gametophytic life stages (Zarzycki & Rychlewski, 1972; Rychlewski & Zarzycki, 1975, 1986; Błocka-Wandas et al., in press). It is thus possible that similar mechanisms causing female bias operate in both species. Assuming that all inviable pollen is male-determining, the estimate of inviable pollen in R. acetosa is similar to our mean bias toward female-determining pollen. Future studies in R. nivalis are required to confirm that meiosis is regular and to establish levels of pollen viability. In particular, it would be important to determine if variation in pollen viability is correlated with the degree of female bias in families.
Another hypothesis that could explain sex ratio biases in Rumex species involves Y-chromosome degeneration and the accumulation of deleterious mutations (Smith, 1963; Lloyd, 1974b; Charlesworth, 2002; Stehlik & Barrett, 2005). Y-chromosomes in section Acetosa appear to be heterochromatic and there is some evidence for chromosome degeneration (Żuk, 1969; Negrutiu et al., 2001; Vyskot & Hobza, 2004; Mosiolek et al., 2005). These effects may be attributable to a lack of recombination and the accumulation of slightly deleterious mutations (Vyskot & Hobza, 2004). This degenerative process is likely to be particularly accelerated on Y-chromosomes because they are present in only one gender and, because of reduced effective population size, are more susceptible to random processes such as genetic drift, Muller's ratchet, the Hill–Robertson effect or genetic hitchhiking (Vyskot & Hobza, 2004). The detrimental effects of Y-chromosome degeneration should be most strongly expressed at the haploid stage and could lead to the abortion of male-determining microgametophytes and the observed bias towards female-determining pollen. In common with the previous explanation based on nondisjunction, evidence for irregular microspores or inviable pollen could provide supporting evidence for the Y-chromosome degeneration hypothesis. Whether the ~2% estimate of pollen inviability in R. acetosa (Błocka-Wandas et al., in press) is associated with Y-chromosome degeneration is not known, but the value is not very different from the average degree of female bias that would occur with this amount of male-determining pollen death.
Another mechanism that could potentially cause biased ratios of female vs male-determining pollen involves a system of sex ratio distorters and restorers of the type reported in dioecious Silene latifolia (Taylor, 1994, 1999). This species shares several similarities with Rumex, including female-biased sex ratios and heteromorphic sex chromosomes, in which females are homogametic and males are heterogametic (Westergaard, 1958). However, the mechanisms responsible for sex ratio bias in these taxa appear to be quite different, particularly with respect to the relative roles of genetics and ecology. Therefore we doubt that the biases reported in Rumex species have a similar mechanistic basis.
In S. latifolia, female bias is caused by segregation distortion during microgametophyte development in one class of males termed ‘driving males’ (Taylor, 1994, 1999; Taylor & Ingvarsson, 2003). Pollen production in driving males diverges strongly from an expected unbiased female- to male-determining ratio as driving males produce little to no male-determining pollen. Sporophytic sex ratios of seeds and adults in S. latifolia depend solely on the bias established by the pollen of driving males involved at fertilization, with no indication of environmentally induced sex-biased mortality among adults (Taylor, 1994, 1999). By contrast, R. nivalis and several other species of Rumex section Acetosa exhibit different seed and adult sex ratios (R. acetosa, Rumex hastatulus and R. thyrsiflorus; Żuk, 1963; Putwain & Harper, 1972; Zarzycki & Rychlewski, 1972; Rychlewski & Zarzycki, 1975; Conn & Blum, 1981; Korpelainen, 1991). For example, in R. nivalis, the degree of bias increases progressively from the gametophytic to the sporophytic generation, and from seed to flowering as a result of gender-based differences in mortality (Fig. 6). Moreover, experimental studies clearly indicate that the amount of pollen captured by stigmas influences the degree of female bias in seeds (Stehlik & Barrett, 2006), indicating another way in which environmental factors play a role in determining sex ratios.
Figure 6. Sex ratio dynamics during the life cycle of Rumex nivalis. Values plotted are the mean proportion of female-determining pollen and the sex ratio at different life-history stages (with standard errors). (1) pollen (this study); (2) open-pollinated seeds from the field; (3) 18-month-old plants in the glasshouse; (4) vegetative plants in the field; (5) flowering plants in the field. (2)–(5) are from Stehlik & Barrett (2005). Sex ratios (4) and (5) were assessed in 18 natural populations from Switzerland. Ratios (2) and (3) are from seed and from an adult glasshouse population grown from seed collected from the same populations. The pollen ratio (1) is from the second offspring generation (open-pollinated) of (3). The dashed line indicates an unbiased ratio.
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Changes in sex ratio during the life cycle and the role of pollination intensity in determining seed sex ratios suggest a strong ecological component to sex ratio-variation in Rumex species of section Acetosa. This pattern would not be predicted if a system of genetic sex ratio distorters were involved. Different mechanisms involved in sex ratio bias in Silene and Rumex may reflect the independent origins and ages of sex chromosomes in these two groups. Sex chromosomes in Rumex section Acetosa are considered to be the most evolutionary advanced and also the most degenerate among the 11 flowering plant families known to possess sex chromosomes (Matsunaga & Kawano, 2001; Vyskot & Hobza, 2004). There are two distinct systems of sex determination in Rumex (XX/XY and XX/XY1Y2) and phylogenetic evidence clearly indicates that the Acetosa type is derived (Navajas-Pérez et al., 2005). It would therefore be valuable to compare the patterns of sex ratio bias in sporophytes and gametophytes of Rumex species with different sex determination systems to determine if the degree of sex-chromosome differentiation is the key factor in causing female bias.