Pollination, fertilization and seed maturation
Successful sexual reproduction in plants involves a series of events beginning with flower development and ending with the recruitment of progeny into the adult population, and is impaired here at several stages between flower development and seed maturation (Table 1). The lower flower production in monomorphic populations combined with their smaller population size may attract fewer pollinators than in trimorphic populations. Lower flower density may also result in low pollen loads on individual pollinators, further reducing the deposition of conspecific pollen. Data on pollinator abundance and visitation in monomorphic vs. trimorphic populations are needed to evaluate these possibilities (e.g. Sih & Baltus 1987; Jennersten 1988).
Reduced pollen deposition in monomorphic populations was, in turn, associated with fewer pollen tubes in styles and ovules penetrated. Some of the difference in pollen tube numbers could be due to higher levels of self-pollination because natural pollinations will involve mostly self-pollen in single-genotype monomorphic populations, compared with a mixture of self- and cross-pollen in trimorphic populations. Because pollen tube growth in this species is faster for cross- than self-pollen, and this is manifested in greater numbers of tubes from cross-pollen during the first 24 hours after pollination (Eckert & Allen 1997), we might expect fewer pollen tubes in styles from monomorphic than trimorphic populations shortly after pollination. However, self-tubes eventually reach the ovary with the same frequency as cross-tubes and, in natural populations of D. verticillatus, almost all pollen deposition occurs within the first 12 hours after anthesis (C.G. Eckert, G.P. Grabas and C. Münch, unpublished data), so that the difference in tube numbers disappears by 36 hours after pollination (Eckert & Allen 1997). We collected flowers 48 hours after anthesis. Slower growth of self-tubes is therefore unlikely to explain much of the large difference in pollen tube numbers.
Ultimately, low pollination and pollen tube growth in monomorphic populations was associated with extremely scant seed production. In fact, plants sampled from seven of 18 monomorphic populations produced no seed at all. There is very little difference in seed production by D. verticillatus after self-compared with cross-pollination (Eckert & Barrett 1994a), suggesting that early acting inbreeding depression following self-pollination accounts for little of the striking reduction in seed production in monomorphic populations.
Sex is impaired at several stages
If sexual reproduction was being impaired at only one critical developmental stage, the average proportional difference between monomorphic and trimorphic populations should remain more or less constant throughout later stages. Our results, however, show a consistent increase from 41% for pollen deposition, to 77% for pollen tube number, 95% for ovules penetrated by a pollen tube, and 99% for seeds per flower. More detailed analysis (not shown) revealed that, although the relative reproductive performance of each monomorphic population tends to decrease incrementally at each stage, populations differ in terms of which stage is most severely impaired. For example, low seed production in populations ME-M5 and ME-M14 was due to the production of very few flowers whereas ME-M13 produced abundant flowers but few were pollinated, and as much pollen was deposited in ME-M3 and ME-M15 as in trimorphic populations but pollen tube growth and ovule penetration were severely impaired. Reduced sexual reproduction is probably due to the impairment of almost all stages, including pollination, fertilization and seed formation, to varying degrees in individual populations, rather than the failure of any single stage.
The association between seed fertility and genotypic diversity is seen among monomorphic populations as well as very clearly between monomorphic and trimorphic populations. All of the seven monomorphic populations that failed to produce any seed consisted of single genotypes. Of the 11 populations that produced some seed, three, including two with the highest seed production, contained more than one genotype. Low seed production therefore seems to limit the recruitment of sexually produced progeny in monomorphic populations. Too few seeds were produced to test whether sexual recruitment is further limited by germination and seedling establishment, but even if this does not occur the difference in seed fertility between monomorphic and trimorphic populations (more than two orders of magnitude) is sufficient to cause very large differences in sexual recruitment.
Both genetic and ecological factors cause reduced sexual reproduction
Monomorphic populations exhibited much lower fertility than trimorphic populations when compared in a common, and probably more benign, glasshouse environment (Fig. 2, Eckert et al. 1999). Moreover, reduced pollen tube growth and ovule penetration, which contributed substantially to reduced seed production under field conditions, were also observed in the glasshouse. It seems therefore that one or more intrinsic, probably genetic, factors were the primary cause of the reduced sexual fertility of monomorphic populations observed in the field. Detailed genetic analysis involving an infertile population from Ontario suggests that infertility may be the result of specific nuclear sterility mutations (Eckert et al. 1999). Sexual infertility is unlikely to involve the accumulation of generally deleterious mutations in old clones, as recent glasshouse experiments have shown that there is no difference in vegetative growth between monomorphic and trimorphic populations (C.G. Eckert, M.E. Dorken and F. Thompson, unpublished data).
A substantial role for ecological factors in reducing the fertility of monomorphic populations is also indicated because most exhibited higher fertility in the glasshouse than in the field (Fig. 2). For example, monomorphic population ME-M8 produced almost as many seeds per pollination in the glasshouse as trimorphic populations, whereas seed production in the field was well below the range of trimorphic populations. Moreover, this population appears to be fixed for a genotype that is heterozygous at two loci (Table 3), implying that sexual reproduction is rare (see Appendix 1 in the Journal of Ecology archive on the World Wide Web). In contrast, fertility of trimorphic populations differed little between environments. Our comparisons suggest that environmental factors may be responsible for about 20% of the difference in seeds per fruit between monomorphic and trimorphic populations observed in the field, although because most monomorphic populations have extremely low fertility even under benign glasshouse conditions, this may be an underestimate. Reciprocal transplants of inherently fertile genotypes between the locations of trimorphic and monomorphic populations would help clarify this issue.
It is likely that sexual reproduction is limited by one or more biotic and/or abiotic factors that covary with latitude. Both the prevalence of monomorphic populations (Fig. 1 and Eckert & Barrett 1992) and sexual reproduction of our study populations (Table 6) covary strongly with latitude. Although southerly, trimorphic populations are inherently much more fertile than northerly, monomorphic populations, even under glasshouse conditions (Fig. 2), latitude also correlates negatively with fruit and seed production when the analysis is restricted to monomorphic populations (Table 6), and these correlations are not confounded by any relation between latitude and the inherent sexual fertility of monomorphic populations (all P > 0.22).
Vegetative growth and survival may also covary with latitude. Plants in monomorphic populations produced fewer clonal progeny per branch but consisted of more branches than those in trimorphic populations (Table 3): both correlated negatively with latitude, although only branches per plant correlated significantly (Table 6). The propensity for individual branches to produce clonal progeny is largely a function of apical extension, because branches must make contact with wet substrate for rooting to occur, suggesting lower vegetative growth in monomorphic populations. Despite this, plants in monomorphic populations tended to be larger (Table 3) suggesting that the recruitment of any type of progeny (either sexual or clonal) is less frequent. Preliminary results from an ongoing experiment suggest that the difference in vegetative performance between monomorphic and trimorphic populations observed in the field disappears when they are grown in a common glasshouse environment (C.G. Eckert, M.E. Dorken and F. Thompson, unpublished data).
Although we cannot, as yet, tell which of several potential biotic and abiotic factors are critical, temperature may well be a major determinant of sexual reproduction in northern populations of D. verticillatus. First, temperature has been shown to affect many aspects of sexual reproduction in plants (e.g. Herrera 1995; McKee & Richards 1996; Mikesell 1997; Woodward 1997) and is widely thought to shape the geographical distributions of plant species (e.g. Woodward 1990; Beerling 1993; reviewed in Salisbury 1942). Secondly, temperature usually varies with latitude and may therefore explain the common transition from trimorphism to monomorphism observed in D. verticillatus across geologically and ecologically diverse regions such as Ontario, Michigan and New England (Eckert & Barrett 1992). Finally, because both monomorphic and trimorphic populations occur in a wide variety of habitats (lake margins, marshes, swamps, bogs), there is unlikely to be a consistent difference in any other ecological variable that could so drastically affect long-term sexual reproduction across the northern edge of the species’ range.