NOTE

Cryptic seed abortion and the estimation of ovule fertilization

ROBERT R. NAKAMURA AND MAUREEN L. STANTON Department of Botany, University of California, Davis, CA, U.S.A. 95616

Received February 17, 1987

NAKAMURA, R. R., and STANTON, M. L. 1987. Cryptic seed abortion and the estimation of ovule fertilization. Can. J. Bot. 65: 2463 -2465.

In a natural population of Raphanus sativus L. 41 % of all fertilized ovules aborted before seed maturation. However, in mature fruits over 75 % of the abortion was undetected. The cryptic abortion of developing seeds was likely due to the early time of abortion and the corky nature of the pericarp. The results are important for the accurate measurement of ovule fertiliza- tion and seed abortion in ecological studies of plant reproduction.

NAKAMURA, R. R., et STANTON, M. L. 1987. Cryptic seed abortion and the estimation of ovule fertilization. Can. J . Bot. 65 : 2463 - 2465.

Chez une population naturelle du Raphanus sativus L., 41 % de tous les ovules fCcondCs ont avortC avant la maturation des graines. Cependant, chez les fruits mQrs, plus de 75 % des avortements sont passCs inaper~us. L'avortement cachC des graines a dCveloppement incomplet Ctait probablement dB a un avortement prCcoce et la nature subireuse du pCricarpe. Les rksultats sont importants pour un mesurage precis de la fecondation de l'ovule et de l'avortement sCminal dans les Ctudes Ccologiques de la reproduction chez les vCgCtaux.

[Traduit par la revue]

Introduction The comparison between number of ovules and the number

of ovules fertilized measures one contribution to the disparity between the potential and the realized fecundity of maternal plants. Understanding this difference and subsequent seed abortion is important to research in several areas of plant repro- ductive ecology. In the demography of plant populations new individuals are conceived at ovule fertilization, and mortality through seed abortion can significantly reduce population size (Harper 1977). In pollination research a low rate of ovule fertilization is evidence for pollen limitation of reproduction (Rathcke 1983), while seed abortion may be due to restricted maternal resources (Lee 1987). For genetic studies the forma- tion of embryos is the first propagation of genes into the next sporophytic generation, and the selective death of embryos would be one factor influencing total fitness (Solbrig 1980).

Most ecologists have estimated ovule fertilization or seed abortion from the examination of mature, or nearly mature, fruits (e.g., Aker 1982; Wolf et al. 1986). In these studies the numbers of mature and aborted seeds were counted along with any unfertilized ovules. However, if ovules or seeds break down during fruit development so that no traces remain, collecting only mature fruits will underestimate the number of ovules and the mortality of embryos. ,One alternative is to examine fruits very early in development (e.g., Guth and Weller 1986); this method allows more precise estimates of the total number of ovules and fertilized ovules. However, the subsequent abortion of fertilized ovules would prevent an early measure of ovule fertilization from predicting the number of mature seeds per fruit.

Full understanding of this cryptic phase in the plant life cycle requires measurement of ovule fertilization, seed abortion, and tissue degradation in fruits at different ages from a natural population. Here we describe such a study of wild radish, Raphanus sativus L. We addressed three questions in the study. (i) Is seed abortion a major source of mortality in Printed in Canada 1 lmprimt au Canada

R. sativus populations? (ii) Do the actual numbers of ovules, fertilized ovules, and aborted seeds differ from estimates made only from mature fruits? (iii) What is the relationship of ovule fertilization and seed abortion to the growth patterns of developing fruits and seeds?

Materials and methods

Wild radish, Raphanus sativus L., is an annual weedy crucifer. Plants are self-incompatible and grow in disturbed habitats. Usually R. sativus fecundity is not pollen limited (Stanton 1987). The study population was in a field with annual grasses and ruderal forbs at the Sacramento Metro Airport (Sacramento County, CA, U.S.A.). M. L. Stanton and R. E. Preston (unpublished) name this location the South Airport site. On 3 April 1985 we randomly chose 20 wild radish plants from the population. For each plant five newly opened flowers were tagged and left open to natural pollination. To establish a time- table for fruit and seed development we collected one tagged fruit from each plant 5, 10, 15, and 21 days after tagging and at fruit maturation. The mature fruits were collected on 22 May 1985. We dissected the fruits on the day of collection to note the developmental stage and length of the ovules and seeds. In newly opened flowers unfertilized ovules were always 5 0 . 5 rnm in length. We scored all larger ovules as either developing or aborting seeds (i.e., fertilized ovules). Counting all expanded ovules as fertilized could have over- estimated fertilization because of possible pollen-induced hormonal stimulation of the growth of unfertilized ovules. Conversely, because some embryos could have aborted very soon after fertilization and only expanded ovules were counted as fertilized, the number of abort- ing seeds may have been underestimated. Aborting seeds were deflated and yellow or brown in color. Developing seeds were full and green before maturation. The dry mass of the entire fruit and the dry mass of one randomly chosen developing seed from each fruit completed the measurements. We used the GLM procedure (SAS Institute Inc. 1985) for analysis of variance of the transformed data. Counts of seeds and ovules per fruit were ranked, and measurements of mass were transformed to natural logarithms.

Results Overall, the wild radish plants abscised 18% of the tagged

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2464 CAN. J. BOT. VOL. 65, 1987

TABLE 1 . Means (SD) for fruit and seed growth in Raphanus sativus at different times after anthesis

Age

5 * lo* 15* 21* Mature ( n = 1 9 ) ( n = 1 5 ) ( n = 1 5 ) ( n = 1 4 ) ( n = 1 5 ) F P

Fruit mass (mg) 2.14d 1 3 . 7 2 ~ 33.886 39.716 7 2 . 4 1 ~ 105.99 0.0001 (0.91) (6.68) (15.20) (22.37) (22.90)

Developing seed mass (mg) - 0 . 4 0 ~ 0.846 1.336 5 . 2 1 ~ 57.25 0.0001 - (0.17) (0.28) (0.56) (2.96)

NOTE: Means followed by the same letter are not significantly different at P 5 0.05 by Tukey's test. *Days after anthesis.

TABLE 2 . Means (SD) for seed development in Raphanus sativus at different times after anthesis

5 * lo* 15* 21* Mature ( n = 1 9 ) ( n = 1 7 ) ( n = 1 7 ) ( n = 1 4 ) ( n = 1 5 ) F P

Unfertilized ovuleslfruit 0 . 1 6 ~ O.Oa O.Oa O.Oa O.Oa 2.96 0.0251 (0.37) (0.0) (0.0) (0 .0) (0 .0)

Aborting seedslfruit 1 . 0 0 6 , ~ 2 . 4 1 ~ 1.82a,b 1.36a,b,c 0 . 4 7 ~ 4.97 0.0013 (1.73) (1.84) (1.59) (1.34) (0.64)

Developing seedslfruit 5.2 la 3 . 4 7 ~ 3 . 8 8 ~ 3 . 7 1 ~ 3 . 7 3 ~ 1.65 0.1704 (2.27) (2.03) (2.50) (1.86) (1.49)

Ovules + seedslfruit 6 . 3 7 ~ 5 . 8 8 ~ 5.71a,b 5.07a,b 4.206 4.66 0.0020 (1.57) (1.4 1 ) (1.40) (1.94) (1.32)

NOTE: Means followed by the same letter are not significantly different at P 5 0.05 by Tukey's test. * Days after anthesis.

flowers before collection; the remainder were gathered. Seventy-eight percent of the tagged flowers produced fruits with at least one normally developing seed. These fruits gained most of their dry mass in the first 21 days after tagging (Table 1). Most growth in mean dry mass for individual seeds occurred after 21 days from anthesis.

Close analysis of fruit development showed that substantial seed abortion occurred within 10 days of anthesis in R. sativus (Table 2). Almost all ovules in collected fruits appeared to be fertilized. On average, only 0.16 of the 6.37 ovules per fruit were unexpanded by day 5. No unfertilized ovules were found in older fruits. The number of aborting seeds detected in- creased sharply between days 5 and 10 and then declined steadily as fruits matured. At 10 days 41 % of all fertilized ovules in a fruit were aborted, but in mature fruits over 75 % of these abortions were not detectable. In contrast, the number of developing seeds per fruit remained unchanged. Most aborted seeds of R. sativus left no visible remains in mature fruits, apparently as a result of early degradation. One would greatly underestimate the frequency of seed abortion by examining only young fruits ( 5 5 days) or fruits close to maturation.

Discussion Our results demonstrate the value of dissecting fruits at dif-

ferent ages to assess accurately ovule number, ovule fertiliza- tion, seed abortion, and the proportion of mature seeds to ovules in a natural R. sativus population. Although fruits of different ages showed little discrepancy in the proportion of ovules fertilized (0.98 to 1.00), cryptic seed abortion did sub- stantially hide the total number of ovules, the number of fertil- ized ovules, and the mortality of embryos when only mature fruits were examined. Seed abortion occurred early in fruit ontogeny, before most of the gain in fruit and seed dry mass.

Therefore the aborted seeds contained little of the maternal plant's allocation to reproduction. One would expect seed and (or) fruit abortion in early stages of fruit growth as this pattern enhances maternal fecundity through resource conservation (Nakamura 1986).

The likelihood of cryptic seed abortion may well depend upon fruit morphology and the time of abortion. In mature R. sativus fruits the corklike pericarp fully envelopes the developing seeds, and in fruit development seeds abort early. In other species the fruit characteristics and the time of abdr- tion make counting the number of ovules and aborted seeds less difficult. The borage Cryptanthaflava, for example, has a fixed number of ovules per ovary (Casper 1983). In the legume Phaseolus vulgaris, because the mature pod interior is largely open and some seed abortion is late in ontogeny (R. R. Naka- mum, personal observation), the remains of undeveloped ovules and aborted seeds are relatively easy to observe. How- ever, in many other plant species, besides wild radish, embryos abort or degenerate at early stages of development (Cooper et al . 1937; Abernethy et al . 1977; Meinke 1982; Levin 1984; Guth and Weller 1986).

We conclude with the suggestion that in quantitative studies of ovule fertilization and seed abortion, investigators should compare the total number of ovules in an ovary of a fresh flower with the total number of ovules estimated from a mature fruit. A difference, if found, provides evidence of cryptic seed abortion, and one should then not rely entirely upon data from mature fruits. Unless this is done, significant genetic and ecol- ogical events during seed ontogeny are likely to be missed.

Acknowledgements We thank T. Sage for suggesting references, and S. Mazer

and A. Snow for reviewing the manuscript. Financial support

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came from National Science Foundation grants DEB-8214508 and BSR-8516333 to M. Stanton.

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