Variable Patterns of Seed Maturation and Abortion in Alliaria Petiolata (Brassicaceae)

1677

Variable patterns of seed maturation and abortion in Alliaria petiolata (Brassicaceae)

David J. Susko and Lesley Lovett-Doust

Abstract: We investigated variation in ovule development within and among fruits in garlic mustard, Alliaria petiolata (M. Bieb.) Cavara & Grande. Individuals were sampled at 14 sites in North America. On average, 94% of ovules showed evidence of fertilization and development. The majority of ovules (mean 68%; range 53.2–82.5%) reached seed maturity. Patterns of seed maturation and abortion varied nonrandomly within and among fruits. In sites in Tennessee, Kentucky, and Ontario, resource limitation seemed to be an important determinant of seed production, as fruits initiated nearest the base of an infructescence were significantly more likely to produce mature seeds than distally located fruits. Also the probability of seed maturation within individual fruits decreased significantly from the pedicellar to the stylar ends. In contrast, for individuals from Ohio sites, the probability of maturing seeds was greatest for centrally located fruits within an infructescence as well as for centrally located ovules within fruits, indicating a greater influence of pollen limitation in addition to resource limitation. We conclude that it could be misleading to infer traits of a species as a whole based on observed patterns of seed maturation in a single site or region. Rather such patterns may reflect prevailing conditions and selection pressures at a local, or regional, scale.

Key words: Alliaria petiolata, Brassicaceae, garlic mustard, ovule abortion, seed maturation, patterns. Résumé : Les auteurs ont examiné la variation du développement de l’ovule à l’intérieur et entre les fruits de l’Alliaria petiolata (M. Bieb.) Cavara & Grande (ail-moutarde). Ils ont échantillonné des individus provenant de 14 sites en Amérique du Nord. Globalement, 94% des ovules montraient des signes de fécondation et de développement. La majorité des ovules ont atteint (en moyenne 68%; écart 53,2 à 82,5%) la maturité. Les patrons de maturation des graines et d’avortement varient de façon non-aléatoire à l’intérieur et entre les fruits. Sur les sites du Tennessee, du Kentucky et de l’Ontario, la limitation en ressources semble être un important facteur déterminant pour la production des graines, puisque les fruits se formant tout près de la base de l’infructescence sont significativement plus enclins à produire des graines mûres que les fruits se formant en position distale. De plus, la probabilité de maturation des graines à l’intérieur du fruit individuel diminue significativement de l’extrémité pédicellaire en allant vers celle du style. Au contraire, chez les individus provenant des sites de l’Ohio, la probabilité que les graines mûrissent est plus élevée pour les fruits localisés au milieu, à l’intérieur de l’infructescence, aussi bien que pour les ovules localisées centralement à l’intérieur du fruit, ce qui indique une plus grande influence d’une pollinisation limitée en plus de la limitation en ressources. Les auteurs concluent qu’il pourrait être trompeur de déduire les caractères d’une espèce dans son ensemble en ne se basant sur les patrons de maturation des graines, à partir d’une seule station ou région. De tels patrons peuvent au contraire refléter les conditions et les pressions sélective prévalant à l’échelle locale ou régionale. Mots clés : Alliaria petiolata, Brassicaceae, ail-moutarde, avortement ovulaire, maturation des graines, patrons. [Traduit par la Rédaction] Susko and Lovett-Doust 1686

tions is the period from ovule fertilization to seed maturation. Ovules may or may not be fertilized, and not all fertil- Plant population studies typically trace individual fates ized ovules reach maturity as viable, fully developed seeds. over time from seed germination and the emergence of seed- The period of seed ontogeny between fertilization and seed lings to reproductive maturity and the eventual death of the maturity is likely to be important in determining the even- individual. Mating systems and methods of seed dispersal, tual reproductive output of a plant and the vigour of its off- as agents driving ecological dynamics, have been studied in- spring. tensively (Howe and Smallwood 1982; Marshall and Folsom 1991; Venable and Brown 1993). However, a significant Several studies have shown that early initiated fruits life-history stage often neglected in demographic investiga- and seeds have a greater probability of reaching maturity than later initiated ones (Lovett-Doust and Eaton 1982; Nakamura 1986; Guitian 1994; Emms 1996). Early fertiliza- Received December 19, 1997. Accepted June 12, 1998. tion may provide a temporal advantage in subsequent inter- and intra-ovary competition for maternal resources. In addi- D.J. Susko and L. Lovett-Doust.1 Department of Biological tion to the timing of fertilization, the spatial arrangement of Sciences, University of Windsor, 401 Sunset Avenue, ovaries and ovules within a plant may also affect the proba- Windsor, ON N9B 3P4, Canada. bility of fruit and seed maturation. Seed mass, fruit mass, 1Author to whom all correspondence should be addressed. and the probability of maturing fruits and seeds have been e-mail: [email protected] shown to be greater in fruits located nearest the main rachis

Can. J. Bot. 76: 1677–1686 (1998) © 1998 NRC Canada 1678 Can. J. Bot. Vol. 76, 1998 compared to those situated at more distal positions within a (siliques) are fully mature by early to middle July, at which time plant for a number of species (Lee and Bazzaz 1986; dehiscence begins. Cavers et al. (1979) determined that this species Matthies 1990; Guitian 1994). It is well established that was self-compatible and self-pollinated. plant fecundity is dependent on the ability to acquire poten- tially limiting resources. Thus, fruits and seeds that develop Stages of ovule development closer to maternal resources should have a spatial advantage Like most mustards, garlic mustard has two-loculed, bicar- in inter- and (or) intra-ovary competition (Solomon 1988; pellate, linear fruits. Individual siliques contain 8–24 ovules, gen- Siemens 1994; Navarro 1996). Two physiological models erally arranged alternately on the two sides of the sinus. A useful have been proposed to explain the success of early initiated feature of these fruits for the purpose of this study is that the and proximally located fruits and seeds (see Lee 1988 for re- ovules retain a persistent connection to the wall of the ovary. Thus, view). The source–sink model suggests that early or proxi- if fruits are collected with appropriate care prior to dehiscence, the mal fruits act as stronger sinks for attracting resources. An relative position of every ovule or developing seed within a fruit alternative model suggests that developing early or proximal can be recorded. Four major stages of ovule development could be identified readily. Some ovules showed no apparent signs of devel- fruits actively inhibit later or distal fruits through the pro- opment whatsoever; these ovules were small, shrivelled, and pre- duction of phytohormones. sumably unfertilized (though they could have been fertilized and Variation in ovule fates associated with ovule positions then aborted at such an early stage that they showed no visible within and between infructescences have been observed in a morphological signs of fertilization). Three subsequent stages were number of species (Wyatt 1981; Bawa and Webb 1984; recognized. Some ovules were expanded in size and had begun Rocha and Stephenson 1990). Although the existence of po- some development of the embryo but had not formed a seed coat. sitional effects on ovule development has been recognized These were termed early aborted ovules. Ovules that had pro- for a long time, few studies have addressed whether these ceeded through further expansion and embryo development but patterns of seed maturation vary among individuals, sites, or had not matured were called late-aborted ovules. These did have a seed coat but were characterized by malformations and were still geographical regions (but see Hossaert and Valero 1988). much smaller than a fully mature seed. Mature seeds were recog- Rather, it is implied that an observed pattern for a given site nized as the fourth and final stage of ovule development. These is representative of the species as a whole, regardless of ge- seeds were brown-black in colour and plump, with a completely netic differentiation or differing abiotic site conditions formed seed coat. Mean mass (± SE) of a mature seed was 1.723 ± among sites. The objective of this study was to examine spa- 0.005 mg and ranged from 0.466 to 3.652 mg (D.J. Susko, unpub- tial patterns in ovule development within and among fruits lished data). of garlic mustard, Alliaria petiolata (M. Bieb.) Cavara & Grande (Brassicaceae), which possesses relatively linear Sampling procedure and ovule standardization multiseeded fruits. To test for variation in the probability of Fourteen sites containing garlic mustard were sampled over a 3- seed maturation and abortion, we compared positional pat- week period from July 8 to July 29, 1994 (Table 1). Sites were se- terns both among plants and among sites across a large por- lected from four sampling regions across a >700 km latitudinal tion of this introduced species’ range in North America. transect of the species’ distribution in North America (Table 1). Factors that may contribute to the observed pattern of seed Annual precipitation, temperature, and the number of frost-free maturation and abortion are then discussed. days varied clinally among the regions (Table 1). Each site was located in a closed woodland or forest. Nearly all were associated with a river or creek, suggesting that garlic mustard seed dispersal via waterways may contribute to gene flow and the invasion of previously uncolonized communities. Garlic mustard was a domi- Study species nant understorey herb at all sites. Garlic mustard is a biennial herb native to northern Europe. The All mature fruits (i.e., fruits containing at least a single mature species was probably first introduced in North America in the lat- seed) on 10 randomly selected plants were harvested from each ter half of the 19th century (Cavers et al. 1979). Its current distri- site. All individuals sampled had only one flowering stem bearing bution in North America extends primarily from southern Ontario one terminal infructescence and no lateral infructescences. These to Tennessee (Nuzzo 1993). The species is concentrated predomi- plants were chosen because they represented the predominant ar- nantly in the northeastern United States from the east coast to Illi- chitectural form (D.J. Susko, unpublished data) and to uncouple nois (Nuzzo 1993) but does exist in isolated sites as far west as potential effects of architectural complexity and plant size as Oregon and British Columbia (Cavers et al. 1979). Typically, garlic covariates, since size has been shown to be highly correlated with mustard invades wet forested areas and floodplains but may also reproductive effort in a number of species (Samson and Werk be found in ditches, waste areas, open fields, and along roadsides 1986; Aarssen and Taylor 1992). All fruits were collected sepa- (Cavers et al.1979). rately and identified as to the fruit’s relative position within an In the first growing season, garlic mustard plants exist as ro- infructescence. settes of dark green leaves that persist through the winter. Mature The position and fate (i.e., developmental stage) of each ovule second-year plants bolt in early April and flower in late April – was recorded, proceeding from the (proximal) basal end to the early May in southwestern Ontario. All second-year plants flower (distal) stylar end or tip of a fruit. Since the total number of ovules regardless of size or site conditions, so this species appears to be an varied among fruits, fruits were “standardized” to enable compari- obligate, rather than a facultative biennial (Byers 1988; D.J. Susko, son of the effects of ovule position among fruits of different sizes. personal observation). Most plants produce only a single flowering Fruits were standardized according to the formula developed by stem, but up to six stems may be present on some individuals. Hossaert and Valero (1988) for legume seed pods. This effectively Flowers are arranged in racemes within which flower opening pro- adjusted each fruit examined to a standardized fruit containing the ceeds acropetally. Most plants produce a single terminal raceme, same mean number of ovules. The mean (±SE) number of ovules but one or two additional lateral racemes are not uncommon. Fruits within mature fruits of garlic mustard was 15.7 ± 0.10. Therefore,

Table 1. List of garlic mustard sites surveyed across a latitudinal gradient of the species’ distribution in North America. Annual Mean annual Number of Latitude (N) and Elevation Region precipitation (mm) temperature (°C) frost-free days Site Location longitude (W) (m) Tennessee 1143 15.6 224 TN-1 Wartrace 35°32′, 86°20′ 170 TN-2 Cumberland County 36°00′, 85°00′ 270 Kentucky 1106 13.7 190 KY-3 Lexington 38°03′, 84°30′ 300 KY-4 Kleber Wildlife Area 38°11′, 85°51′ 270 KY-5 Frankfort 38°12′, 84°52′ 270 KY-6 Franklin County 38°13′, 84°53′ 270 Ohio 924 11.1 161 OH-7 Scioto River near Delaware 40°18′, 83°04′ 250 OH-8 Delaware 40°18′, 83°04′ 250 OH-9 Delaware Wildlife Area 40°18′, 83°03′ 250 OH-10 Alum State Park 40°18′, 83°04′ 250 Ontario 836 9 173 ON-11 Point Pelee National Park 42°00′, 82°33′ 190 ON-12 Kent County 42°05′, 82°32′ 200 ON-13 LaSalle 42°18′, 83°00′ 180 ON-14 Windsor Airport 42°18′, 82°58′ 190 Note: Climatological data (NOAA 1974; Matthews and Morrow 1985) are taken from the nearest weather station within each region (central Tennessee, Nashville; northern Kentucky, Frankfort; central Ohio, Delaware; southwestern Ontario, Windsor). to approximate that value, all fruits were transformed to a standardized size of 16 ovule positions. The formula used was Geographic variation in ovule fate Fruits differed significantly among regions (Table 2) in [1] P = 16(f )/N terms of the numbers of undeveloped ovules (df = 3, 1114, F = 150.8, p < 0.001), early aborted ovules (df = 3, 1114, F = where P is the new (i.e., standardized) ovule position, f is the for- 56.2, p < 0.001), late-aborted ovules (df = 3, 1114, F = 18.9, mer (i.e., observed) ovule position, and N is the total number of p < 0.001), mature seeds (df = 3, 1114, F = 57.0, p < 0.001), ovules in a given fruit. and the total number of ovules (df = 3, 1114, F = 6.6, p < To examine fruit- and ovule-position effects on ovule fates, data 0.001). Fruits from the regions of Kentucky and Ontario did were assigned to the following fruit and ovule classes. Three not differ significantly from each other in their numbers of classes of fruit position within an infructescence were identified: undeveloped or early aborted ovules. Undeveloped ovules basal, middle, and distal (nearest the apex) fruits. Fruits were as- were rare (fewer than 6%) in fruits from all sites outside the signed to these fruit-position classes by dividing the number of four Ohio sites (Fig. 1), which had significantly greater mature fruits in the infructescence into thirds. If there was one numbers of both undeveloped and early aborted ovules than fewer or one more fruit than necessary for equal division, then that missing or extra fruit was assigned to the middle class. Standard- any other region (Table 2). Tennessee fruits had the fewest ized ovules were assigned to one of four ovule-position classes as of both these early stages. Significantly fewer late-aborted follows: 1 (the quarter of the total ovules nearest the pedicel), 2 ovules were observed in fruits from Ohio and Ontario than and 3 (both in the middle quarters, with 2 being closer to the low- in those from Tennessee and Kentucky (Table 2). Overall, est position class), and 4 (the quarter nearest to the stigma). Each about 68% of all ovules matured to fully developed seeds ovule-position class consisted of four standardized ovules. (Table 2). The numbers of mature seeds in fruits from Tennessee and Ontario did not differ significantly (Table 2), but fruits from these regions had significantly more mature Statistical analyses seeds than fruits from Kentucky, which in turn had signifi- Variables were transformed as necessary to enhance normality cantly more than those from Ohio. The majority of ovules and meet the assumptions of parametric statistical analysis. To within fruits from all sites (minimum of 53.2% in OH-10; compare the mean number of ovules in each stage of ovule development across geographical regions, sites were grouped by region maximum of 82.5% in ON-11) became mature seeds (Table 1). Regional differences were analysed by a general linear (Fig. 1). Overall, the total number of ovules was greatest in model (GLM) analysis of variance (ANOVA), and a Tukey– fruits from Ontario sites (Table 2). Tennessee, Kentucky, and Kramer test where ANOVA indicated significant effects (SYSTAT Ohio fruits did not differ significantly from each other with Inc. 1992). Differences among sites, individual plants, and fruit- respect to total number of ovules (Table 2). position classes were analysed by a three-level nested ANOVA (SAS Institute Inc. 1985). Next we determined the effect of fruit- Fruit-position effects on ovule fate position class and site on ovule fates using two-way ANOVA with The number of ovules at each stage of ovule development, SYSTAT GLM (Systat Inc. 1992). A two-way ANOVA was also used to test for differences among ovule-position classes and as well as the total number of ovules per fruit, differed sig- among sites. The effect of ovule position on ovule fates was ana- nificantly among sites and among fruit-position classes lysed by a three-level nested ANOVA on all four ovule-position (Table 3). Furthermore, the numbers of undeveloped ovules, classes to test for differences among sites, individuals, and fruit- early aborted ovules, and total ovules differed significantly position classes (SAS Institute Inc. 1985). among plants within sites (Table 3). However, the numbers

Table 2. Population means and regional means (±SE) for different stages of ovule development in fruits of garlic mustard collected across the species’ range in North America. Population No. of fruits No. of undeveloped No. of early No. of late-aborted No. of mature Total no. of or region examined ovules aborted ovules ovules seeds ovules TN-1 71 0.30±0.12 1.90±0.20 2.14±0.35 12.73±0.66 17.08±0.45 TN-2 74 0.04±0.02 0.86±0.12 2.20±0.30 11.44±0.44 14.54±0.24 KY-3 105 0.90±0.16 2.85±0.26 3.87±0.43 9.34±0.51 16.93±0.36 KY-4 107 0.45±0.16 2.18±0.21 0.92±0.19 12.65±0.43 16.19±0.26 KY-5 68 0.48±0.11 2.91±0.23 1.94±0.22 7.51±0.29 12.85±0.33 KY-6 74 0.36±0.10 1.35±0.13 1.55±0.23 12.56±0.37 15.91±0.20 OH-7 76 3.01±0.32 3.18±0.22 0.55±0.11 8.62±0.39 15.37±0.41 OH-8 89 2.58±0.22 3.86±0.25 0.22±0.08 9.28±0.32 15.93±0.31 OH-9 79 1.91±0.23 3.35±0.29 0.84±0.17 7.82±0.43 13.91±0.42 OH-10 89 1.46±0.23 4.33±0.28 2.03±0.26 8.91±0.38 16.73±0.39 ON-11 78 0.12±0.05 1.42±0.16 1.22±0.25 13.03±0.47 15.75±0.28 ON-12 71 0.21±0.06 3.12±0.23 1.28±0.26 12.49±0.48 17.10±0.23 ON-13 72 0.45±0.09 2.45±0.20 0.83±0.17 12.95±0.40 16.67±0.27 ON-14 65 0.20±0.05 1.76±0.18 2.21±0.35 11.87±0.56 16.04±0.39 Tennessee 145 0.17±0.06c 1.39±0.13c 2.17±0.23a 12.10±0.40a 15.83±0.28b Kentucky 354 0.52±0.06b 2.27±0.11b 1.98±0.14a 10.46±0.23b 15.25±0.17b Ohio 333 2.22±0.13a 3.71±0.13a 0.93±0.09b 8.68±0.19c 15.54±0.20b Ontario 286 0.25±0.03b 2.21±0.11b 1.37±0.13b 12.58±0.24a 16.41±0.15a Total 1118 0.91±0.05 2.57±0.06 1.54±0.07 10.69±0.13 15.71±0.10 Note: Values among regions (within columns) followed by different letters are significantly different at p < 0.05 (Tukey–Kramer test).

Fig. 1. Mean frequency of each stage of ovule development within fruits from 14 populations of garlic mustard in North America. .Ovule fates are shown as (ٗ) undeveloped ovules, ( ) early aborted ovules, ( ) late-aborted ovules, (᭿) mature seeds 100

Frequency (%) 0 TN-1 TN-2 KY-3 KY-6 KY-5 KY-4 OH-9 OH-8 OH-7 ON-11 ON-12 OH-10 ON-13 ON-14 Site of late-aborted ovules and mature seeds did not differ signif- site and fruit-position class (Table 4). Similar patterns of icantly among plants within sites. Differences among plants ovule development were observed consistently among fruit- accounted for very little (0–7.9%) of the variation observed position classes in plants from all sites in Tennessee, Ken- in the numbers of ovules in different developmental stages tucky, and Ontario (Fig. 2). The frequency of undeveloped (Table 3). Differences within fruit-position classes ac- ovules in fruits from these sites remained very low (0.3– counted for the majority (37.6–63.0%) of the observed vari- 7.6%) regardless of fruit position within an infructescence. ation in numbers of ovules in various developmental stages. Fruits nearest the base of an infructescence had the fewest Most of the remaining variation in the numbers of undevel- early aborted and late-aborted ovules, and consequently had oped ovules (33.0%) and early aborted ovules (20.0%) oc- the greatest number of mature seeds. The occurrence of early curred among sites. Differences among fruit-position classes aborted and late-aborted ovules increased from the base to accounted for most of the remaining variation in late-aborted the tip of an infructescence; correspondingly, the frequency ovules (33.3%), mature seeds (40.3%), and total ovule num- of mature seeds decreased in distally located fruits. ber (20.8%) (Table 3). Patterns of ovule development among fruit-position The probability of seed maturation depended significantly classes in plants from Ohio sites differed significantly from upon fruit-position class, site, and the interactive effect of those observed for the other sites (Table 4, Fig. 2). In Ohio

Table 3. Results of three-level nested analysis of variance testing for effects of populations, plants, and fruit-position classes on the number of ovules in each stage of ovule development.

Undeveloped Early aborted Late-aborted Mature Total no. Source of variation ovules ovules ovules seeds of ovules

Among populations F(13, 126) 19.972*** 11.794*** 10.563*** 13.878*** 6.852*** (32.98) (19.96) (13.04) (22.14) (14.12)

Among plants within populations F(126, 280) 1.851*** 1.676*** 0.772ns 0.958ns 1.775*** (7.95) (7.43) (0) (0) (10.47)

Among fruit-position classes within plants F(280, 698) 1.522*** 1.401*** 2.628*** 3.822*** 2.000*** (9.80) (9.62) (33.27) (40.31) (20.79) Within fruit-position classes (error) –49.28 –62.99 –53.69 –37.55 –54.62 Note: Values in parentheses are the percentages of variation that are, in each instance, explained by that variable. Significance of F values is shown as follows: ***, p < 0.001; ns, not statistically significant.

Table 4. F values of two-way analyses of variance of the effects on ovule fates of site and either the position of fruits within the infructescence or the position of ovules within fruits. Undeveloped Early aborted Late-aborted Mature df ovules ovules ovules seeds Fruit-position effects Site 13, 1076 44.781*** 23.748*** 18.610*** 35.344*** Fruit-position class 2, 1076 3.278* 13.187*** 132.894*** 199.718*** Site × fruit-position class 26, 1076 3.902*** 3.515*** 5.020*** 5.079*** Ovule-position effects Site 13, 4416 97.047*** 44.696*** 46.795*** 79.029*** Ovule-position class 3, 4416 57.075*** 107.579*** 110.460*** 337.007*** Site × ovule-position class 39, 4416 10.394*** 3.507*** 3.867*** 4.812*** Note: Significance of F values is shown as follows: *, p < 0.05; ***, p < 0.001. plants the frequency of undeveloped ovules was signifi- sites in Tennessee, Kentucky, and Ontario (Fig. 4). As with cantly higher (4.8–26.7%) and decreased from the base to ovule fates among fruits in an infructescence, the patterns of the tip of an infructescence. As in the other sites, more late- ovule development among ovule-position classes in plants aborted ovules were present in distal fruit positions than from Ohio differed significantly from those of the three proximal ones. However, the frequency of early aborted other regions (Fig. 4). Undeveloped ovules were either ovules did not exhibit any consistent pattern among fruit po- found most frequently at the tip and base of individual fruits sitions. Also, in these Ohio plants, fruits located in the mid- (OH-7 and OH-8) or increased in frequency towards the tip dle of an infructescence matured more seeds than either of fruits (OH-9 and OH-10). The converse pattern was ob- basal or distal fruits. served for mature seeds. Mature seeds either occurred most often in the middle of fruits (OH-7 and OH-8) or decreased in frequency towards the tips of fruits (OH-9 and OH-10). Ovule-position effects on ovule fate The frequency of early aborted ovules increased towards the The probability of seed maturation depended significantly tips of fruits, while the frequency of late-aborted ovules ei- upon ovule-position class and site (Table 4). Significant in- ther increased towards the tips of fruits (OH-8, OH-9, and teraction terms showed that the patterns of ovule fate in OH-10) or remained constant throughout the length of the ovule-position classes varied among sites (Table 4). There fruit (OH-7). were highly significant differences among sites in the numbers of ovules at each stage of development for each ovule- position class (Table 5). Nearly all differences in terms of frequencies of the four stages of ovule development for each ovule-position class were highly significant among fruit- Geographic variation in ovule fate position classes but not significant among individual plants Patterns of seed maturation and abortion in garlic mustard within populations (Table 5). The probability of seed matu- differed significantly among sites. Overall, regional trends ration was significantly dependent on ovule position within were also apparent. Sites in Tennessee, Kentucky, and a fruit (Fig. 3). More seeds were matured near the base of a Ontario showed similar, high levels of seed maturation; the fruit, while more early aborted and late-aborted ovules were probability of seed maturation was approximately 76% in found near the tip. The frequency of undeveloped ovules fruits grouped from sites in these three regions. The proba- was lowest in the middle of fruits and increased slightly at bility of seed maturation in the Ohio region was consistently both the base and the tip. This pattern of ovule development lower at about 55%. We cannot attribute the significantly was observed among ovule-position classes in plants from lower levels of seed maturation in Ohio sites to any differ-

Fig. 2. Effect of fruit position within an infructescence on ovule mustard. The latter group also determined that, for plants fates for each population grouped by geographical region. Fruit- from sites in Iowa, self-pollination only occurred in the ab- position classes were as follows: B, basal fruits; M, fruits in the sence of cross-pollination. The conflicting results of these -middle; T, fruits near the tip. Ovule fates are shown as (ٗ) two studies suggest that pollination and the effective breed undeveloped ovules, ( ) early aborted ovules, ( ) late-aborted ing system may vary due to phenotypic differences in floral ovules, (᭿) mature seeds. biology among sites (Anderson et al. 1996) or among geo- TN-1 TN-2 graphic regions. Schoen (1982) demonstrated that average 100 outcrossing rates of Gilia achilleifolia Benth. could vary 80 widely from about 0.15–0.96, with autogamy being most 60 pronounced in sites in the northern portion of the species’ 40 distribution. He concluded that selection acting on variation in the degree of protandry may have been responsible for 20 differences in breeding systems among sites of this species. 0 We suspect that the elevated frequencies of undeveloped and BMT BMT early aborted ovules and the lower seed set we observed in KY-3 KY-4 KY-5 KY-6 garlic mustard plants from Ohio sites may be the result of 100 having individuals with higher outcrossing rates. An exami- 80 nation of the floral biology (e.g., degree of protandry, stigma 60 exsertion, flower size, etc.) of garlic mustard is needed to test for differentiation of floral traits among sites and among 40 geographic regions. Indeed, if plants in Ohio sites act more 20 as facultative outcrossers (as do plants in Iowa, according to 0 Cruden et al. (1996)), then the amount of pollen deposited BMT BMT BMT BMT on stigmas and pollinator activity are more likely to limit OH-7OH-8 OH-9 OH-10 ovule fertilization. For example, cross-pollination will be in- 100 fluenced by local availability and visitation rates of insect

Frequency (%) 80 pollinators, and cumulative pollinator activity over the sea- 60 son is likely to be lower in this region that has only 161 40 frost-free days. Field data from Ohio on the activity of 20 pollinators, such as syrphid flies and honeybees, which are common visitors throughout the flowering period in Ontario 0 BMT BMT BMT BMT sites (D.J. Susko, personal observation), are needed to test this hypothesis. The fact that seed set was lowest in Ohio ON-11ON-12 ON-13 ON-14 plants is interesting, because based on Schoen’s (1982) work 100 on G. achilleifolia, one might have predicted that a shorter 80 pollinator season at a more northerly (or in this case, a more 60 continental) site would favour autogamy. 40 The proportion of ovules aborting at either early or late 20 stages of development varied significantly among sites of 0 garlic mustard but not among individuals within those sites. BMT BMT BMT BMT In particular, high frequencies of early aborted ovules were observed in Ohio sites. Most studies have attributed ovule Fruit-position class abortions either to lethal allele combinations (Wiens 1984) or to limited resources (Stephenson 1981). In an experiment ences in temperature, rainfall, or elevation among sites in with the legume, Cassia fasciculata Michx., Martin and Lee the four geographic regions (Table 1). Only the number of (1993) demonstrated that both pollen donor effects and frost-free days was positively correlated with the probability resource availability operated together to influence ovule of maturation, and Ohio sites had the lowest value for both abortion. When they limited resources by manipulating photo- parameters. In this indeterminately flowering plant, a shorter period and numbers of fruits, they found that ovule abortion growing season could promote earlier senescence and trun- increased in some self-pollinated plants and not in others. cate late-season seed production in Ohio plants. However, We believe that, as in C. fasciculata, ovule abortion in garlic this seems an unlikely explanation for a spring-flowering mustard is a function of both intrinsic genetic factors and species like garlic mustard, which typically finishes matur- extrinsic environmental conditions. Unfortunately, abiotic ing seeds by late summer. soil conditions were not assessed in the present study. We In a controlled pollination experiment on garlic mustard did visually note that resource status varied among the sites in Illinois, Anderson et al. (1996) demonstrated that self- surveyed. Plants that were harvested in each site grew in pollination occurred before the flowers had even opened, similar shaded, forested habitats, but soil moisture and soil and concluded that autogamy was the most likely breeding type differed among sites. Shade intensity, soil nutrient and system for the sites they studied. However, in another recent moisture content, and soil type have been shown to influ- study, Cruden et al. (1996) noted that only small amounts of ence seed mass and (or) number in a variety of species self-pollen were visible on stigmas prior to anthesis in garlic (Westoby et al. 1992).

Table 5. Results of three-level nested analysis of variance testing for effects of populations, plants, and fruit-position classes on the number of ovules in each stage of ovule development and each ovule-position class.

Ovule-position Undeveloped Early aborted Late-aborted Mature Source of variation class ovules ovules ovules seeds

Among populations F(13, 126) 1234 23.571*** 6.197*** 3.018** 16.477*** 2 6.329*** 9.311*** 3.129*** 10.647*** 3 5.115*** 8.589*** 5.514*** 15.248*** 4 9.292*** 6.951*** 8.855*** 11.616***

Among plants within populations F(126, 280) 1234 1.079ns 1.417** 0.876ns 1.155ns 2 1.152ns 1.103ns 0.745ns 0.718ns 3 1.572*** 1.156ns 0.471ns 0.482ns 4 2.224*** 1.415** 0.657ns 0.675ns

Among fruit-position classes within plants F(280, 698) 1234 1.394*** 1.027*** 1.686*** 1.132*** 2 1.508*** 1.081ns 1.870*** 1.980*** 3 1.076*** 1.322*** 2.294*** 2.202*** 4 0.878ns 1.224*** 2.679*** 2.472***

Note: Ovule-position classes are numbered 1–4 from pedicellar to stigmatic ends of fruits. Significance of F values is indicated as follows: ***, p < 0.001; **, p < 0.01; ns, not statistically significant.

Fig. 3. Mean frequency of each stage of ovule development by standardized ovule position within fruits of garlic mustard. Ovule fates .are shown as (ٗ) undeveloped ovules, ( ) early aborted ovules, ( ) late-aborted ovules, (᭿) mature seeds base tip 100

Frequency (%) 20

0 12345 6 7 8 9 10 11 12 13 14 15 16 Standardized ovule position within fruit

Fruit-position effects on ovule fate to the base of a garlic mustard plant have larger vascular Within garlic mustard plants the probability of early and connections than distal fruits (D.J. Susko, personal observa- late ovule abortions increased from the base to the tip of the tion). Such differential vascularization has been reported in infructescence in most of the sites surveyed from Tennessee, Lupinus luteus L. by Van Steveninck (1957); the observation Kentucky, and Ontario. A similar pattern of abortion has cited here was based on a limited pilot study, and we are un- been found in the crucifer Brassica napus L., where seeds in able to speculate yet about the generality of this observation. apical pods were more likely to abort than those in basal Inflorescences of garlic mustard plants only rarely produce pods (Pechan and Morgan 1985). We interpret this pattern as leaves between the developing flowers and fruits. Hence, a consequence of competition for limited resources among basal fruits are located nearer to the few subtending cauline fruits of garlic mustard where the sink strength of the earlier leaves. These leaves are probably responsible for much of formed basal fruits is stronger than that of more distal ones. the current photosynthesis in the plant, since the older, ro- Fruits nearest the base of a raceme are physically closer to sette leaves appear withered at, or before, the onset of flow- photosynthate, nutrients, and water as these resources move ering (D.J. Susko, personal observation). In addition to up from the roots, which serve as storage sites for carbohy- being spatially closer to plant resources, basal fruits of garlic drates accumulated during the first year of this biennial mustard were also initiated earlier than distal fruits. Flower plant’s life. Also, light microscopy shows that fruits nearest opening and subsequent fruit initiation then proceed acro-

Fig. 4. Effect of ovule position within a fruit on ovule fates for infructescence. It seems possible that these patterns may re- each population, grouped by geographical region. Ovule-position sult from a greater frequency of outcrossing in the Ohio classes were as follows: (1) region nearest the pedicel; plants. The frequency of ovule fertilization increased pro- (2) middle of fruit near pedicel; (3) middle of fruit near stigma; gressively through the reproductive season, indicating that ,region nearest the stigma. Ovule fates are shown as (ٗ) self-pollination may be suppressed early in the year. Thus (4) undeveloped ovules, ( ) early aborted ovules, ( ) late-aborted the development of ovules early in the season may depend ovules, (᭿) mature seeds. more on pollination intensity and quality, rather than on re- TN 1 TN 2 source levels (which are unlikely to be limiting earlier in the 100 season). As flowering proceeds, the degree of selfing may 80 increase to promote seed production in flowers that did not 60 receive outcrossed pollen. Such a strategy would be advanta- geous for monocarpic species such as this and is not uncom- 40 mon in other species (Lee and Bazzaz 1982a, 1982b; 20 Nakamura 1986; Charlesworth et al. 1987; Becker et al. 0 1234 1234 1992). Male gametophytic competition may be reduced with increased selfing. As a consequence, resource availability may become a dominant determinant of seed maturation later KY-3 KY-4 KY-5 KY-6 in the flowering season. The observation that more seeds 100 were matured in centrally located fruits (initiated in the mid- 80 dle of the flowering period) could result from superior pater- 60 nal fertilization success and quality at the peak of the season, supported by enhanced maternal resource allocation. 40 20 Ovule-position effects on ovule fate 0 1234 1234 1234 1234 Patterns of seed maturation and abortion were nonrandom within fruits of garlic mustard For this species, the majority of ovules at all positions within a fruit, except the two ovule OH-7 OH-8 OH-9 OH-10 positions nearest the stigmatic end, reached seed maturity. In 100 Frequency (%) general, the probability of abortion increased from the base 80 of a fruit towards the tip. We believe that these patterns of 60 ovule development are largely the product of competition 40 among ovules for limiting maternal resources. The situation 20 for seed maturation is analogous to that for fruit maturation. Ovules nearest the base of a given fruit are located closer to 0 1234 1234 1234 1234 the vascular tissue of the pedicel, which supplies the developing fruit with photosynthate, nutrients, and water. Some reproductive structures are green and, thereby, capable of ON-11 ON-12 ON-13 ON-14 conducting their own photosynthesis (Bazzaz et al. 1979), 100 but the majority of photoassimilates are probably transported 80 into a fruit from nearby leaves, where these are present (see 60 discussion above on leaf-position effects on fruit develop- 40 ment) or via the vasculature from root reserves. 20 Fruits from Ohio sites displayed two different patterns of ovule development. Fruits from the OH-9 and OH-10 sites 0 1234 1234 1234 1234 had patterns consistent with the resource-limitation hypothesis that applies for the Tennessee, Kentucky, and Ontario Ovule-position class sites. In contrast, fruits from the OH-7 and OH-8 sites differed in that seeds matured more frequently in central ovule- petally within an infructescence. Consequently, we cannot position classes in these fruits. Also, undeveloped ovules rule out the temporal advantages of early resource acquisi- were more frequent at both the base and the tip of a fruit, in- tion on fruit development (see Lovett-Doust and Eaton dicating that resource limitation within these fruits is not the 1982). However, separating the relative effects of time of only limiting factor for ovule development. Differences in production and position of fruits is problematic in garlic pollination and fertilization may also be affecting within- mustard, as in most other species that make terminal fruit seed set. For instance, in legume pods subject to intense racemes, since the two are highly correlated (Nicholls 1987; pollen competition, more seeds are set in the stylar end, be- Guitian 1994; Stocklin and Favre 1994). cause of a fertilization gradient (Bawa and Webb 1984). In Plants from Ohio sites showed markedly different patterns fruits of the self-incompatible wild radish, Raphanus rapha- of ovule development among fruit-position classes. In Ohio, nistrum, Hill and Lord (1986) demonstrated that the order of seeds were more likely to reach maturity in the middle of ovule fertilization was variable. Sometimes ovules near the infructescences than at either end. Also, the frequency of un- stigma were fertilized first, while, in other cases, ovules in developed ovules decreased from the base to the tip of an the middle of fruits were fertilized first. They concluded that

© 1998 NRC Canada Susko and Lovett-Doust 1685 the transmitting tissues in the ovaries of this member of the Cavers, P.B., Heagy, M.I., and Kokron, R.F. 1979. The biology of Brassicaceae allowed growing pollen tubes to bypass ovules Canadian weeds. 35. Alliaria petiolata (M. Bieb.) Cavara and while moving through the septum. Pollen tubes could then Grande. Can. J. Plant Sci. 59: 217–229. exit the septum and fertilize lower ovules. The tendency of Charlesworth, D., Schemske, D.W., and Sork, V.L. 1987. The evo- some pollen tubes to skip over ovules was associated with lution of plant reproductive characters: sexual versus natural se- both the pollen donor and the maternal plant (Hill and Lord lection. In The evolution of sex and its consequences. Edited by 1986). This phenomenon may explain why in fruits of Ulex S.C. Stearns. Birkhauser Verlag, Basel. pp. 317–335. europaeus L. and Ulex gallii, two species of woody le- Cruden, R.W., McClain, A.M., and Shrivastava, G.P. 1996. 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