American Journal of Botany 97(1): 123–135. 2010.

S ELF-STERILITY IN TWO CYTISUS SPECIES (LEGUMINOSAE, PAPILIONOIDEAE) DUE TO EARLY-ACTING INBREEDING DEPRESSION 1

Francisco J. Valtue ñ a2 , Tom á s Rodr í guez-Ria ñ o 2 , Francisco Espinosa3 , and Ana Ortega-Olivencia 2,4

2 Á rea de Bot á nica, Facultad de Ciencias, Universidad de Extremadura, Avda. de Elvas, s.n. 06071-Badajoz, Spain; and 3 Á rea de Fisiolog í a Vegetal, Facultad de Ciencias, Universidad de Extremadura, Avda. de Elvas, s.n. 06071-Badajoz, Spain

In most angiosperms, the endosperm develops before the embryo, but with harmony between the two structures until fi nal seed formation. In an embryological study, we show that inbreeding depression causes disharmony in development of the two structures in two Leguminosae shrubs, Cytisus multifl orus and C. striatus. Our main objective was to test the causes of self-sterility in the two species by comparing the embryological development of the self seeds with that of cross seeds. In developing selfed seeds of C. multifl orus , the embryo reaches at most the globular stage and never forms mature seeds, while in C. striatus a few mature selfed seeds are formed. In both species, the main cause of abortion of developing selfed seeds is diminished endosperm develop- ment (low values of the ratio of endosperm to embryo), which triggers collapse of the endosperm and embryo. The results indicate that self-sterility in C. striatus is postzygotic because of strong, early inbreeding depression, while in C. multifl orus there exists a mixed pre- and postzygotic mechanism; the prezygotic mechanism causes rejection of some self-pollen tubes in the style/ovary, and the early inbreeding depression triggers abortion of fertilized ovules that escaped that action.

Key words: abortion; Cytisus multifl orus ; Cytisus striatus; embryogeny failures; inbreeding depression; late-acting self-in- compatibility; Leguminosae; Mediterranean region; self-sterility.

The two principal mechanisms that induce self-sterility in lemoniaceae, Convolvulaceae, Caryophyllaceae, and Malva- plants are self-incompatibility (SI) and early-acting inbreeding ceae (Hiscock and Mclnnis, 2003), but at a molecular level the depression (ID). Self-incompatibility involves a series of physi- phenomenon has been very rarely investigated (e.g., Brassica ; ological mechanisms that prevent reproduction after self-polli- see revision in Charlesworth et al., 2005 ). nation or between genetically closely related individuals. This In gametophytic SI, the pollen is controlled by its own geno- mechanism is not only one of the most important means of side- type, and rejection can occur either on the style, more fre- stepping autogamy and of promoting the generation of new quently, or on the stigma. Gametophytic SI is more widespread genotypes among plants, but also it may contribute to angio- than sporophytic SI and is found in families such as Legumino- sperm diversity and success ( de Nettancourt, 2001 ). sae, Scrophulariaceae, Rosaceae, Solanaceae, Papaveraceae, In the SI process, the ovules are not fertilized because of an and Poaceae. At a molecular level, however, the phenomenon active rejection of the male gametophytes that carry the same S has been studied only in Solanaceae, Papaver , Antirrhinum , alleles of the sporophyte (de Nettancourt, 2001). There are basi- and Rosaceae (Silva and Goring, 2001; Franklin-Tong and cally two kinds of prezygotic SI (i.e., prior-to-fertilization): het- Franklin, 2003 ; Charlesworth et al., 2005 ). eromorphic and homomorphic. In heteromorphic or diallelic SI, A third kind of SI, late-acting or ovarian SI (Seavey and fl owers differ in morphology (i.e., they are heterostylous), Bawa, 1986 ), occurs in two large groups of species. In the fi rst whereas in homomorphic SI, fl owers are not morphologically group of species, pollen tubes reach the ovary after self-pollina- differentiated. tion and can even penetrate the ovules, but fertilization does not Homomorphic SI is much more common ( Richards, 1986 ) take place. This type of rejection is consequently prezygotic. In and can be divided into sporophytic and gametophytic. Sporo- the second group of species, self-fertilization takes place with phytic SI is controlled by the genotype of the pollen-producing subsequent postzygotic rejection (Seavey and Bawa, 1986; plant, and inhibition usually takes place on the stigma surface Gibbs and Bianchi, 1993). Because the expression “ late-acting ( de Nettancourt, 2001 ). This type of SI has been recorded in SI” assumes that rejection is under genetic control similar to families such as Brassicaceae, Asteraceae, Betulaceae, Po- typical mechanisms of prezygotic SI and because these mecha- nisms have not yet been fully determined, some researchers prefer to use other terms, such as ovarian sterility ( Sage et al., 1 Manuscript received 1 October 2008; revision accepted 23 September 1994 ) or pistillate sorting ( Bertin et al., 1989 ). The latter has 2009. been used to describe those cases in which not only the time of The authors thank Dr. M. Gonzá lez (University of Extremadura) for occurrence of syngamy is unknown, but also whether abortion statistical assistance. Suggestions and comments by two anonymous takes place before or after syngamy. The expression involves reviewers and the associate editor greatly improved the manuscript. This no presumption about the control mechanisms of those events work was fi nanced by the Ministry of Education and Science of Spain (maternal, paternal, zygotic, or some combination of these) (projects BOS2002-00703 and CGL2005-00783/BOS, both co-fi nanced by ERDF). A predoctoral grant from that Ministry to F.J.V. (BES-2003- (Sage et al., 1994). Nevertheless, Lipow and Wyatt (2000) 2187) is greatly appreciated. demonstrated in Asclepias exaltata the existence of a single lo- 4 Author for correspondence ([email protected]) cus with polyallelic control of late-acting SI. Inbreeding depression (ID) is defi ned as fi tness loss as a re- doi:10.3732/ajb.0800332 sult of self-fertilization or fertilization between similar or 123 124 American Journal of Botany [Vol. 97 closely related genotypes. This phenomenon is one of the most sis and prepared to receive the pollen tube ( Rodr í guez-Ria ñ o et signifi cant factors infl uencing the evolution of plant reproduc- al., 2006 ); their mature seeds have an aril with a funicular origin tive systems (Lloyd, 1980; Charlesworth and Charlesworth, ( Rodr í guez-Ria ñ o et al., 2006 ). Cytisus multifl orus presents 1987). There is a tendency for ID to be particularly intense be- two fl oral morphs, LF (with large fl owers) and SF (with small tween fertilization and the mature seed phase (early ID) when fl owers), with SF having the greater reproductive success of the numerous essential genes are expressed for the fi rst time two (Rodr í guez-Ria ñ o et al., 2004). In this work, we shall only (Meinke, 1991; Seavey and Carter, 1994; Husband and Schem- study the more widespread SF. Both species are clearly xenoga- ske, 1996 ). mous and pollinated by bees. Self-sterility in these two species As Seavey and Bawa (1986) observed, it is very diffi cult to is characterized by virtually no seed production and high rates distinguish which effects are attributable to late-acting SI and of abscission of fruits that contain ovule-seeds of varying sizes which to ID. The following two principal criteria have been ( Rodr í guez-Ria ñ o et al., 1999 , 2004 ). Variability in the timing suggested for differentiating between the two biological phe- of fruit abscission and seed abortion indicates that ID may be nomena (Seavey and Bawa, 1986; Sage et al., 1994; Nic operating in both species (Rodr í guez-Ria ñ o et al., 1999, 2004 ). Lughadha, 1998; Lipow and Wyatt, 2000): (1) Timing of In addition, prezygotic SI also contributes to self-sterility in C. abortion. A uniform failure at a single developmental stage striatus as indicated by low rates of ovule penetration would be interpreted as late-acting SI, whereas a series of fail- ( Rodr í guez-Ria ñ o et al., 1999 ). However, verifi cation of ID or ures throughout embryogeny would be seen as the result of late-acting SI as the cause of self-sterility remains to be demon- ID. Nonetheless, there have been relatively few studies inves- strated. The main objective of this study is to assess seed devel- tigating embryological processes to confi rm that fertilization opment after cross- and self-pollination to determine whether has occurred before the abortion of the developing seeds (but postzygotic self-sterility in C. multifl orus and C. striatus is due see Gibbs and Bianchi, 1993; Seavey and Carter, 1996; Gibbs to ID or late-acting SI. Previously published data (Rodr í guez- and Sassaki, 1998; Nic Lughadha, 1998; Bittencourt et al., Ria ñ o et al., 1999 , 2004 ) and unpublished data for the years 2003 ; Pound et al., 2003 ; Sage and Sampson, 2003 ). (2) The 2004 and 2005 will be considered in the Discussion. amount of variability in selfed seed set among individuals of a population. Null or almost null seed production after self- MATERIALS AND METHODS pollination in all the individuals of a population would indi- cate late-acting SI, whereas variations among individuals in Plant material— Populations of both Cytisus species (C. multifl orus and C. the population would be seen as the result of ID. Nevertheless, striatus) are located at the El Hito estate, in the municipal district of Alburquer- genotypes with a high load of lethal genes could also induce que (Badajoz, SW Spain). The population (39 ° 13 ′ N, 6 ° 57 ′ W) is situated be- complete or almost complete self-sterility (Seavey and Bawa, tween 390 and 395 m a.s.l. on a granitic substrate and subject to a typical 1986; Waser and Price, 1991). Other criteria include (1) re- Mediterranean climate characterized by winters that are rainy and more or less sponse of embryos to rescue in tissue culture, (2) dependence cold, and summers that are dry and hot. The annual mean temperature is 15.5 ° C, of abortion on the paternal vs. the progeny genotype, and (3) and the mean annual rainfall is 642 mm. The current vegetation is broom scru- bland due to range-farming activities. It consists mostly of C. multifl orus and to induced mutations (see Seavey and Bawa, 1986 ; Klekowski, a lesser extent of C. striatus, with a very few individuals of Quercus rotundifo- 1988; Sage et al., 1994; Lipow and Wyatt, 2000; Bittencourt lia , Crataegus monogyna , Pyrus bourgaeana, and Rubus ulmifolius as the main and Semir, 2005). tree or shrub elements. Numerous herbaceous and, above all, shrub species have late-acting SI (see e.g., Bawa and Beach, 1983 ; Owens, 1985 ; Pollination treatments— Pollination experiments were carried out during Kenrick et al., 1986 ; Seavey and Bawa, 1986 ; Gibbs and Bi- the years 2003 and 2004. In both species, hand self pollination (HSP) and hand anchi, 1993 , 1999 ; Sage et al., 1994 , 1999 , 2006 ; Lipow and cross pollination (HCP) were performed on separate individuals. Unless other- Wyatt, 2000; Sage and Sampson, 2003; Bittencourt and Semir, wise stated, all hand pollinations were carried out following the method of Rodr í guez-Ria ñ o et al. (1999 , 2004 ). The unmanipulated fl owers were elimi- 2006 ). A postzygotic mechanism of ID inducing self-sterility nated at the time of pollination as well as at the collection of samples to avoid has been shown in Epilobium obcordatum (Seavey and Carter, the allocation of resources to those fl owers. The gynoecia of the pollinated 1994 ), Gomidesia (Nic Lughadha, 1998), Dalbergia miscolo- fl owers were collected, stored, and sectioned following the method of bium (Gibbs and Sassaki, 1998), Calluna vulgaris ( Mahy and Rodr í guez-Ria ñ o et al. (2006) . Because of the linear arrangement (typical of Jacquemart, 1999), several species of Vaccinium ( Guillaume legumes) and number of ovules in the ovary (8 – 10 ovules/ovary), the ovary was and Jacquemart, 1999; Hokanson and Hancock, 2000), and Bul- transversally sectioned to avoid pieces that were too large and to facilitate in- clusion of resin and the use of the microtome. In ovaries more than 13 d after bine bulbosa (Owen and Vaughton, 2003) among others. Nev- pollination (DAP) in C. multifl orus and 20 DAP in C. striatus, every piece ertheless, species are also known with mixed prezygotic SI and contained only two ovule-seeds of the central zone of the ovary — the zone most postzygotic mechanisms explaining self-sterility, as happens in likely to form seeds — and in those of very advanced phases only one. In these Medicago sativa ( Cooper, 1940 ), Eucalyptus spathulata ( Ellis last ovaries, we only prepared those developing seeds that were in good condi- and Sedgley, 1992), Dombeya acutangula (Gigord et al., 1998), tion, discarding those that were completely aborted because their advanced and Clintonia borealis ( Dorken and Husband, 1999 ). Neither degradation did not allow anything to be observed. Sections were stained either with periodic acid Schiff ’ s reagent (PAS) and can one rule out the probable existence of both postzygotic contrasted with Gill no. 3 hæ matoxylin solution for starch and general visual- mechanisms (late-acting SI and ID) in a given species (e.g., ization of tissues (I. Casimiro, University of Extremadura, Spain, personal com- Rhododendron prinophyllum, Padrutt et al., 1992) or even both munication) or sometimes with ruthenium red contrasted with toluidine blue O together with prezygotic SI (e.g., Dipterocarpus tempehes, for visualization of tissues (Crivellato et al., 1990, modifi ed). Permanent prepa- Kenta et al., 2002 ). rations were mounted in Eukitt. Sections were studied with a light The present research is centered on two shrub species of Le- microscope. The number of analyzed individuals, fl owers, and ovules-seeds in the differ- guminosae, Cytisus multifl orus (L ’ H é r.) Sweet and C. striatus ent sections of this work (quantifi cation of ovule penetration and double fertil- (Hill) Rothm., endemics from the western Mediterranean re- ization and, seed development after cross, self, or no pollination) are represented gion. Both have a monosporic Polygonum type of embryo sac in supplemental tables (Appendices S1 and S2, see Supplemental Data with the development, with all ovules per ovary mature at fl ower anthe- online version of this article). January 2010] Valtue ñ a et al. — Inbreeding depression in C YTISUS 125

Quantifi cation of ovule penetration and double fertilization 2 — Ovule pen- F1,39 = 8.02, P < 0.01, R = 0.84, see the solid and dashed lines etration and fertilization were studied on collected ovaries during the fi rst 10 in Fig. 1A ), but not in C. striatus (ANCOVA, F 1,48 = 0.02, P > DAP after sectioning and staining and, in some phases, the sample size was R2 increased with the analysis of ovules after aniline blue staining and their obser- 0.05, = 0.61, see the solid and dashed lines in Fig. 1B ). In vation under fl uorescence microscopy ( Rodr í guez-Ria ñ o et al., 1999 ). Ovule both treatments, there was a trend toward delayed penetration penetration was verifi ed by the presence of the pollen tube through the micro- and double fertilization in C. multifl orus with respect to C. stri- pyle, and fertilization was verifi ed by the presence of (1) a degenerated and atus , although this was not tested statistically. strongly stained synergid with the formation of a dense cytoplasmic loop be- tween egg cell and central cell (which seems to be related to the transfer of the Ovule and seed development after cross, self, and no polli- sperm cell nuclei; see Bittencourt et al., 2003), (2) one or more endosperm nu- nation— All results refer to both species, except where differ- clei, and (3) a zygote or embryo. In previous work (Rodr í guez-Ria ñ o et al., 1999, 2004 ), the percentage of ovule penetration had already been determined ences between them are explicitly indicated. This section begins but only on ovaries at 5 DAP, and they had been viewed with fl uorescence mi- with a description of normal seed development. We will then croscopy (C. striatus : 90.6% vs. 72.5%; C. multifl orus : 56.8% vs. 54.9%; show by comparing the two treatments (self vs. cross) that the crossed vs. selfed, respectively). This last method does not resolve whether the selfed embryo had either a similar or an accelerated develop- self-penetration is delayed with respect to cross-penetration or how the penetra- ment, although not signifi cant, relative to the crossed embryo, tion increases over time. We therefore needed to evaluate the percentage of while the development of its endosperm was signifi cantly de- ovule penetration again, but in a different way to try to respond to those two questions. Ovule penetration and double fertilization following HSP was deter- layed. This lag was refl ected in very low values of the En/E ra- mined on 183 and 164 ovules of C. multifl orus and C. striatus, respectively, and tio compared to those obtained for the crossed seeds. This after HCP on 201 and 126 ovules (online Appendices S1 and S2). Because ev- disharmony in the development between these two structures ery penetrated ovule was observed to be fertilized, we did not consider differ- (embryo and endosperm) would be ultimately responsible for ences between the penetrated and fertilized ovules. the abortion of the selfed seeds due to delayed development of the endosperm, which subsequently caused the collapse of the Ovule and seed development after cross, self, and no pollination — Ovule developing embryo. and seed development were studied in sectioned and stained ovules and devel- Seed development after HCP was considered to be normal oping seeds. We tested several features. (1) Anatomical observation: the seed development was determined after cross- and self-pollination, and in nonpene- development. The pollen tube penetrates the ovule through a trated/nonfertilized ovules. (2) Ovule-seed length: the median longitudinal sec- zig-zag micropyle and then penetrates the embryo sac through tion of ovules and developing seeds was measured in all phases of development the fi liform apparatus. Prior to penetration of the ovule, one of studied as the greatest distance between the chalazal-most and the micropylar- the synergids degenerates. After fertilization, the second syner- most points. (3) Number of endosperm nuclei (En) and number of embryo cells gid degenerates. The embryo development is delayed with re- (E) by counting the number of endosperm nuclei and embryo cells, respec- spect to endosperm development and passes through the tively, on all the sections obtained per developing seed, followed by calculation of the ratio En/E. These estimates of endosperm nuclei and embryo cells were following phases: proembryo, globular, heart, torpedo, and cot- only made until 20 DAP in C. multifl orus and 14 DAP in C. striatus (hence- yledon ( Fig. 2 ). Both species present a nuclear endosperm that forth, period I) because of the diffi culty of counting them at later phases (period develops through the consecutive divisions of the primary en- II). Morphological features, En/E ratios and ovule-seed length following HSP dosperm nucleus (Rodr í guez-Ria ñ o et al., 2006). Later, in the was determined on a maximum of 332 and 277 ovules-seeds of C. multifl orus late globular – early heart phase, the endosperm becomes cellu- and C. striatus , respectively, and after HCP on a maximum of 300 and 215 lar except for the chalazal area, where cell wall formation never ovules-seeds (For more details, see online Appendices S1 and S2.). occurs. As far as the fi rst developmental stages of the embryo are concerned, both species follow a variant pattern of the On- Statistical analyses— The SPSS statistical package, version 15.0.1, was used for all statistical analyses. The comparison between treatments (self and agrad type, according to the classifi cation of Johansen (1950), a cross pollination) in all the variables studied (percentage of penetrated ovules, pattern well known in Leguminosae ( Prakash, 1987 ; Batygina, length of ovule or developing seed, number of cells of the embryo [E], number 2006). Although in this family the endosperm development is of endosperm nuclei [En], ratio En/E, and percentage of aborted developing very uniform, embryo development patterns vary considerably seeds) was performed using ANCOVA, considering the treatment as the princi- (Asterad, Caryophyllad, Onagrad, and Solanad types; see e.g., pal factor and time as the covariable in the linear model studied, as well as the Cooper, 1938 ; Prakash, 1987 ; Lim and Prakash, 1994 ; Lersten, effect of the interaction of the two. Where the effect of this interaction was not signifi cant, the model was refi tted without it. Prior to all analyses, the data were 2004 ; Batygina, 2006 ; Rodr í guez-Pontes, 2007 ). checked for the preconditions of normality and homoscedasticity. Note that to perform the ANCOVA analyses we used only the data from those ovules or Embryo, endosperm, and endosperm to embryo ratio — In developing seeds that showed no or only slight signs of abortion. A Mann – both species, the development of the endosperm and the en- Whitney test was used specifi cally to determine any differences in the ratio dosperm to embryo ratio (En/E ratio) were dependent on the En/E at the end of period I (day 14 in C. striatus and day 20 in C. multifl orus ) treatment (cross vs. self), but the development of the embryo after cross- and self-pollination. This nonparametric test was applied because at the end of that period, the data did not satisfy the conditions of normality and was not. homoscedasticity. The fi rst zygotic division takes place at about 5 DAP, when the endosperm presents 4 – 16 nuclei in C. multifl orus and 8 – 16 nuclei in C. striatus . After self-pollination, there was an inver- RESULTS sion of the process in C. multifl orus (in a single ovule). That is, the zygote began its development before the primary endosperm Quantifi cation of ovule penetration and double fertiliza- nucleus. tion— In neither species was there a delay in the self-pollen The model of embryo growth as measured by number of cells tubes reaching the ovules relative to the cross-pollen tubes. The did not differ signifi cantly between treatments in either species 2 fi rst pollen tubes to reach the ovules were observed at 3 and 4 (ANCOVA C. multifl orus , F1,64 = 2.93, P > 0.05, R = 0.74; 2 DAP in C. striatus and C. multifl orus , respectively, regardless ANCOVA C. striatus , F1,50 = 0.08, P > 0.05, R = 0.54; Figs. of whether they were self or cross. However, the percentage 3A and 4A). However, in C. multifl orus, we observed a trend penetration of ovules was signifi cantly greater after cross pol- toward more cells after self-pollination than after cross-pollina- lination than after self pollination in C. multifl orus (ANCOVA, tion during embryo development (Fig. 3A). The development 126 American Journal of Botany [Vol. 97

Fig. 1. Relationship between ovule penetration percentage and days after hand self pollination (HSP) and hand cross pollination (HCP). Lines show penetration models estimated by ANCOVA. (A) Cytisus multifl orus . (B) C. striatus . Each data point shows the mean value ± SD/2 per individual and per day after pollination (DAP). Only the upper (HCP) or lower (HSP) SD bar is shown for clarity. of the endosperm depended signifi cantly on the type of treat- fertilized, were either similar in size to virgin ovules or only 2 ment (ANCOVA C. multifl orus , F1,63 = 57.71, P < 0.001, R = slightly larger ( Figs. 3D and 4D ). The ANCOVA showed sig- 2 0.78; ANCOVA C. striatus , F1,49 = 35.46, P < 0.001, R = 0.80; nifi cant differences among the three types of ovule (cross- and Figs. 3B and 4B ). This difference was refl ected in far slower self-fertilized, and nonfertilized ovules) (ANCOVA C. multi- 2 development of the endosperm after self-pollination than after fl orus , F2,155 = 197.12, P < 0.001, R = 0.95; ANCOVA C. stria- 2 cross-pollination, as measured by the number of nuclei (Figs. tus , F2,66 = 33.44, P < 0.001, R = 0.85). These differences were 3B and 4B). Similarly, the pollination treatment (self vs. cross) due solely to the smaller size of the nonfertilized relative to the signifi cantly infl uenced the ratio En/E (ANCOVA C. multifl o- fertilized ovules (cross and self), but there were no differences 2 rus , F 1,63 = 66.84, P < 0.001, R = 0.79; ANCOVA C. striatus , between the latter two treatments (cross vs. self; Figs. 3D and 2 F1,48 = 6.87, P < 0.05, R = 0.52; Figs. 3C and 4C ). 4D ). Considering the En/E ratio after cross pollination to repre- On the contrary, in later phases (period II; Figs. 3E and 4E ), sent ideal development of the seed (i.e., developmental har- the behavior after HSP of the two species was very different. In mony between the embryo and the endosperm), we must remark C. striatus , most of the few healthy selfed seeds remaining in that, in both species, after self-pollination (similar or high em- the fruits (6.14%) followed a development similar to that of the bryo development and low endosperm development), this ratio crossed seeds ( Fig. 2 ) but with one difference: they were gener- 2 was lower in value (i.e., there was disharmony in the develop- ally larger (ANCOVA, F 1,55 = 7.50, P < 0.01, R = 0.56) be- ment of the two structures). These tendencies were due to (1) a cause in each fruit there was usually just one developing seed marked increase in the cross ratio ( Figs. 3C and 4C ) caused by compared with up to seven seeds/fruit in the crossed fruits. This a surge of endosperm development in period I ( Figs. 3B and similar development of selfed and crossed seeds made it possi- 4B ), and (2) a slight increase in the self ratio ( Figs. 3C and 4C ) ble for there to exist seeds with normal development after HSP from the rapid growth undergone by the embryo, accompanied up to the occurrence of embryos that had differentiated radicle by weak growth of the endosperm (Figs. 3B and 4B). Neverthe- and cotyledons and fi nally the production of mature seeds. In C. less, in Fig. 4C, we observed that in C. striatus the cross and multifl orus , the development of the selfed seeds followed the self ratios tended to approach each other at ~14 DAP (see in same pattern as the crossed seeds (ANCOVA, F 1,33 = 3.52, P > Fig. 4C, the plus sign represents the crossed data and the open 0.05, R 2 = 0.82), although they were somewhat smaller in size circles the selfed data). This convergence was due to the decline ( Fig. 3E ). in the cross ratio caused by the greater growth of the embryo Considering separately the individuals studied of the two ( Fig. 4A ) relative to the endosperm ( Fig. 4B ), causing a pro- species, we found that after both treatments (cross and self pol- gressive decrease in that ratio (Fig. 4C). The result was the ab- lination) their behavior did not vary signifi cantly between indi- sence of signifi cant differences between the cross and the self viduals (Appendix S3, see online Supplemental Data) in all the ratios at the end of period I (U 14DAP = 10.00, P > 0.05). The parameters considered (embryo and endosperm development convergence of the two ratios in this species could be translated and En/E ratio). In all individuals, the development of the en- into a reharmonization of the selfed embryo and endosperm de- dosperm after self pollination was slower than after cross pol- velopment, albeit with a slower development than after HCP. lination, while the development of the embryo was similar. As discussed, these patterns imply a somewhat different behavior Ovule size— During period I, the growth of the ovule was of the species in their En/E ratio. Thus, in C. multifl orus , the determined by the existence or absence of fertilization (fertil- slower development of the endosperm after self pollination was ized vs. nonfertilized), but not by the type of treatment (cross enhanced by faster development of the embryo (En/E ratios vs. self). The nonpenetrated ovules, or penetrated but not yet very far from the normal). In contrast, in C. striatus , the slow January 2010] Valtue ñ a et al. — Inbreeding depression in C YTISUS 127 development of the endosperm was mitigated by a somewhat low in both species (c. 16% of the pollinated fl owers in C. stria- slower development of the embryo, leading to En/E ratios that tus and 6% in C. multifl orus). These fruits tended to be small, in some cases were similar to those obtained after cross pale-green or yellowish, with no turgidity, and with already pollination. aborted or aborting seeds. All individuals of C. multifl orus ini- tially had more or less massive fruit-drop that slowly continued, Developing seed abortion— Very few fertilized selfed ovules but differed as to the timing of the dropping (e.g., one individ- developed normally. Indeed, fertilized selfed ovules were ob- ual maintained most of its selfed fruits until approximately served aborting on every DAP (Figs. 3F, 4F, and 5) . This abor- 35– 40 DAP). In contrast, in C. striatus fruit-drop took place tion occurred continuously throughout the development of the gradually, and the individuals behaved more uniformly than in seed, although its greatest incidence was during the proembryo the case of C. multifl orus . phase (until about 32 DAP in C. multifl orus and until 18– 20 DAP in C. striatus , Fig. 5A – D ). Abortion also occurred in the globular phase of C. multifl orus , and in the globular ( Fig. 5E ), DISCUSSION heart, and torpedo (Fig. 5F) phases in C. striatus. The embryo never surpassed the globular phase in C. multifl orus , but reached The most important contribution of this work is the demon- the mature seed phase in C. striatus. The percentage of aborted stration of disharmony in endosperm/embryo development as developing seeds during the proembryo phase in C. multifl orus one of the main causes of seed abortion after selfi ng in Cytisus and proembryo – globular phase in C. striatus differed signifi - multifl orus and C. striatus, thus representing a conceptual ad- cantly between treatments and between DAP (ANCOVA C. vance in the study of inbreeding depression (ID). Although ID 2 multifl orus , F1,89 = 5.25, P < 0.05, R = 0.32; ANCOVA C. stri- is the main cause of self-sterility in both species, in the case of 2 atus , F 1,82 = 17.67, P < 0.001, R = 0.74; Figs. 3F and 4F, re- C. multifl orus, to this postzygotic mechanism we must add an- spectively). Abortion was identifi ed by some structural features; other mechanism, prezygotic rejection — a phenomenon that the most important and valuable were the presence of delayed has rarely been documented in the literature. and/or densely staining endosperm (Fig. 5), aborting embryos (with vacuolated cells, Fig. 5C ), or aborted embryos with Prezygotic mechanism of SI in C. multifl orus— The lower strongly stained and irregular cells (Fig. 5A, B, and D). In these percentage of penetrated ovules after self-pollination as com- aborting selfed seeds, if the abortion process was incipient, we pared to cross-pollination in C. multifl orus clearly supports a observed problems in the endosperm and embryo not accompa- partial prezygotic SI mechanism acting at the level of the style/ nied by changes in the other tissues (for example, amount of ovary. The low penetration percentage in the selfed ovules of C. starch grains, degree of staining of ovular tissue cells). How- multifl orus is not attributable to their immaturity at anthesis be- ever, when abortion was well advanced, a number of character- cause morphologically the ovules are completely formed and istics in the nonembryonic tissues were observed that indicated mature at that time ( Rodr í guez-Ria ñ o et al., 2006 ). Sage et al. deterioration (e.g., they appeared more lightly stained, and the (1999 , 2006 ) indicated the presence of long-distance signaling amount of starch grains had declined [ Fig. 5C] or even disap- in which the presence of self-pollen or self-pollen tubes nega- peared completely). tively infl uences ovule development (e.g., Narcissus triandrus In developing selfed seeds, occasional abnormalities of en- and Ipomopsis aggregata). In the species studied, there was no dosperm development were observed related to karyokinetic evidence of this long-distance signaling in the self ovules; we failures. They involved the presence of abnormally large nuclei observed no morphological or structural changes compared in which one or more nucleoli could be distinguished. They with the ovules of unpollinated fl owers or with unpenetrated were observed in early stages of endosperm development, i.e., ovules of crossed fl owers. The only structural changes observed the primary endosperm nucleus ( Fig. 6A, B ) and less frequently in unpenetrated ovules were those related with their progressive in the binucleate endosperm phase (micropylar and chalazal nu- deterioration. In Dipterocarpus tempehes , Kenta et al. (2002) clei), in which case, they may affect only the chalazal en- blame a massive fall of fl owers in the early stages after fertiliza- dosperm nucleus ( Fig. 6C ) or both endosperm nuclei ( Fig. 6D ). tion on the possible action of late-acting SI. In our case, most of The absence of normal development of endosperm inhibits deg- the selfed fl owers collected during the fi rst DAP had ovaries radation of the starch grains surrounding the central cell. Con- with a large proportion of unfertilized ovules, with which late- sequently, starch grains persist around the primary endosperm acting SI (as a postzygotic phenomenon) does not seem nucleus ( Fig. 6A ). consistent. Both problems (low En/E ratios and endosperm abnormali- ties during period II) were also observed after HCP, but with Postzygotic mechanism of self-sterility in both species— To different relevance. After cross pollination, the existence of low distinguish between late-acting SI and early-acting ID, one of values of this ratio was infrequent (1.98% in C. multifl orus and the principal criteria is the time until the developing seed aborts almost 0% in C. striatus ), while the existence of endosperm (e.g., Seavey and Bawa, 1986 ; Sage et al., 1994 ; Nic Lughadha, abnormalities ( Fig. 6 ) was similar in incidence to that of the 1998; Lipow and Wyatt, 2000). We have reported in previous selfed seeds (cross = 4.5%, self = 4.6% in C. multifl orus ; cross work ( Rodr í guez-Ria ñ o et al., 1999 , 2004 ) that in both Cytisus = 4.3%, self = 6.5% in C. striatus ). species most of the self-penetrated ovules were probably self- Another type of failure, that generally was more frequent af- fertilized because of the presence of a remaining aril. This fea- ter HSP, was the vacuolization of the tissues surrounding the ture — self-fertilization of ovules — has been suffi ciently embryo sac (Fig. 6E). This failure was always greater after HSP demonstrated in the present work, and their subsequent abor- ( C. multifl orus = 4.29%; C. striatus = 6.04%) than after HCP tion indicates the existence of postzygotic rejection. After ovule ( C. multifl orus = 3.55%; C. striatus = 1.83%). self-penetration, almost all structural developmental features Finally, it has to be noted that, in most individuals, the num- (fertilization percentage, developing seed size, ovule tissue de- ber of surviving fruits 25 d after self-pollination was extremely velopment, etc.) were practically identical to those after cross- 128 American Journal of Botany [Vol. 97

Fig. 2. Seed development in Cytisus multifl orus after hand cross pollination (embryo and endosperm). (A) Proembryo phase and nuclear endosperm (28 days after pollination [DAP]). (B) Early globular phase and nuclear endosperm (42 DAP). (C) Late globular phase and cellular endosperm (44 DAP). (D) Late heart phase and cellular endosperm (57 DAP). (E) Torpedo phase and cellular endosperm (62 DAP). (F) Cotyledon phase and cellular endosperm, except for the (not shown) chalazal area (70 DAP). Abbreviations : ce, cellular endosperm; co, cotyledon; e, embryo; es, embryo sac; ii, inner integument; ne, nuclear endosperm; pl, palisade layer; pr, proembryo; r, radicle; s, suspensor; vb, vascular bundle; *, micropylar area. Stain: A – C and E, PAS + h æ ma- toxylin; D and F, toluidine blue + ruthenium red. Bars: A – C = 100 µ m; D = 150 µ m; E = 250 µ m; F = 500 µ m. January 2010] Valtue ñ a et al. — Inbreeding depression in C YTISUS 129 pollination. The only different character between the two (cross Time of selfed pistils or young fruits fall after self-pollina- vs. self) was the endosperm development and, consequently, tion— Late-acting SI is usually manifest in a massive fall of the endosperm to embryo (En/E) ratio. Endosperm develop- selfed pistils/young fruits in a very short period of time after ment, measured as number of nuclei, could be a factor involved self-pollination (e.g., between 3 – 8 DAP; Chorisia chodatii , C. in abortion of the developing seed. However, rather than merely speciosa , Tabebuia caraiba , T. ochracea: Gibbs and Bianchi, the poorer development of the endosperm, the disharmony that 1993 ; Dolichandra cynanchoides and Tabebuia nodosa : Gibbs arises between the development of the embryo and of the en- and Bianchi, 1999; Spathodea campanulata: Bittencourt et al., dosperm, i.e., the En/E ratio, is really responsible. 2003 ; Jacaranda racemosa: Bittencourt and Semir, 2006). In a Early ID is believed to be due to the expression of recessive study of Hymenaea stigonocarpa , abscission of most of the alleles during embryo development and/or endosperm forma- selfed fl owers occurred at 7 – 8 d, but two pistils persisted for tion. In both Cytisus species, abortion of the developing seed 6 – 7 mo, indicating the existence of postzygotic rejection ( Gibbs appears to be linked to the relationship between embryo and et al., 1999 ) without discriminating between late-acting SI or endosperm development (En/E ratio). Thus, while selfed seeds ID. In taxa with ID, the selfed-pistils/young fruits usually ab- may have delayed endosperm development with respect to scised over weeks to months (Gibbs and Sassaki, 1998; Nic crossed seeds, if the endosperm’ s relative growth is not suffi - Lughadha, 1998). Thus, in Dalbergia miscolobium ( Gibbs and ciently out of synchrony with that of the embryo, abortion Sassaki, 1998 ), half of the fl owers whether crossed or selfed would not occur. Therefore, the convergence observed of the had fallen by 1 wk after pollination, but the abscission contin- En/E cross and self ratios at the end of period I in C. striatus ued for nearly 4 mo. This gradual fall over time implies that the would be translated into survival of the selfed seeds that had developing seeds abort at different stages of development. managed to surpass this period. Nonetheless, because there is Apart from the massive fall of selfed pistils in C. multifl orus no such convergence in C. multifl orus, the developing selfed at a few days after pollination, previously attributed to nonpen- seeds always abort. The genetic load probably acts by causing a etration of the ovules, in both species, the selfed fruits gradually disharmonization in embryo and endosperm development, be- fell over time. Therefore, with respect to the period of abscis- ing more severe in C. multifl orus as a consequence of the ex- sion of selfed fl owers, the two Cytisus species share more char- pression of more lethal recessive alleles in the different acteristics with species that are subject to ID rather than those developing seeds, as proposed by K ä rkk ä inen and Savolainen that present late-acting SI, so the hypothesis of the existence of (1993) for most conifers. This different action of ID would lead ID seems more plausible in both. to a great variability in the size of the aborted seeds because abortion would occur when the En/E ratio reaches a minimum, Size variability or uniformity of the aborted seeds— It is as- from which point survival would no longer be possible. sumed that late-acting SI provokes a uniform failure of self- Abortion occurred continuously throughout the development fertilized ovules, while early ID triggers failure at various stages of the seed, with its greatest incidence at the proembryo phase. of development (see Seavey and Bawa, 1986 ; Sage et al., 1994 ; But because this phase covers a very long time span (i.e., until Nic Lughadha, 1998 ; Lipow and Wyatt, 2000 ). In previous pa- about 32 DAP in C. multifl orus and until 18– 20 DAP in C. stri- pers ( Rodr í guez-Ria ñ o et al., 1999 , 2004 ), the presence of a atus ), changes in the development of the embryo and/or en- gradient of fertilized ovule sizes and aborted fruits in both Cy- dosperm cannot be considered as a single stage of development. tisus species was described. Our embryological results indicate Thus, abortion within one phase of development is considered that these aborted seeds are at different phases of development, to be due to a continuing failure at different stages within that with selfed embryos reaching the late proembryo – early globu- phase. The observed sequence of faults usually began with a lar stage in Cytisus multifl orus and far more advanced phases in delay in the development of the endosperm, causing the abor- C. striatus (i.e., heart, torpedo, and cotyledon phases). The high tion of the embryo at some point in its development (whenever proportion of aborted seeds of widely varying sizes in selfed an aborting seed was observed, the endosperm was delayed fruits and their presence in crossed fruits but at a much lower and/or degraded). Only when the collapse of the endosperm proportion lends support to the ID hypothesis in both species. and/or embryo was in advanced phases did we observe effects Thus, as proposed by Hufford and Hamrick (2003), the mater- on the other, nonembryonic tissues of the developing seed (e.g., nal embryonic genetic load may be expressed in both selfed and decrease in integument starch grains). These observations in the outcrossed progeny, with the degree of infertility related to how aborted or aborting seeds support the hypothesis set out in the many deleterious alleles are combined. previous paragraph of the action of lethal recessive alleles on the development of the endosperm and embryo, so that embry- Amount of variability in selfed seed set among individuals of onic failure is the cause of abortion and not its fi nal consequence a population— Variability among self-pollinated individuals is (Cooper et al., 1937; Cooper, 1940). In contrast, Sage and Web- thought to be due to ID. This criterion is fully satisfi ed in C. ster (1990) observed that in Phaseolus vulgaris abortion of the striatus because some selfed individuals produced fruits or seeds was a consequence of changes in the maternal tissue (i.e., seeds, showing a variable range of fruit set (0 – 15%) and seed nonembryonic tissue, such as integument and nucellus cells, set (0– 3.1%) (T. Rodrí guez-Ria ñ o, F. J. Valtueñ a, and A. Or- starch depletion) causing abortion of embryonic tissues (embryo tega-Olivencia, unpublished data). In contrast, this criterion is and endosperm). In this last case, abortion occurred due to the not satisfi ed in C. multifl orus . If one considers fruiting to in- diversion of assimilates to nonaborting seeds, i.e., it was unre- volve the formation of mature fruit with at least one mature lated to the action of lethal recessive alleles. Similar conclusions seed, the selfed fruit set was always absolutely null (Rodr í guez- had been reached by Briggs et al. (1987) for Pisum sativum . Ria ñ o et al., 1999 , 2004 ), because inside the few fruits formed ( < 1%, T. Rodr í guez-Ria ñ o, F. J. Valtue ñ a, and A. Ortega-Ol- Other features supporting early ID— In both species, sev- ivencia, unpublished data), there was generally only one shriv- eral different features confi rm or support the presence of early eled seed. Nonetheless, mature fruits but with no fertile seeds ID as a postzygotic mechanism of self-sterility. were also produced in some individuals of C. striatus ( < 3%). 130 American Journal of Botany [Vol. 97

Fig. 3. Relationship between different studied parameters and days after hand self pollination (HSP), hand cross pollination (HCP), and no pollination (NP), in Cytisus multifl orus. Lines show models estimated by ANCOVA tests (in formulas in each panel, y = studied parameter and x = days after pollination [DAP]). (A) Number of embryo cells. (B) Number of endosperm nuclei. (C) Endosperm to embryo ratio. (D) Ovule-seed length until 20 DAP. (E) Develop- ing seed length from 22 to 46 DAP. (F) Ovule and seed abortion percentage. Each data point shows the mean value per individual and per DAP.

Although no cases are known in which all the individuals in a greater than 50 m. In the case of C. multifl orus , there is also a population are self-sterile due to genetic load (but see Wiens et greater density of individuals per patch. These individuals may al., 1989 in Dedeckera eurekensis), complete self-sterility has be closely related to one another genetically, especially those indeed been found in individual plants (see reviews in Seavey that are closer than 2 m, because unpublished data indicate and Carter, 1994 ; Gigord et al., 1998 ; Hokanson and Hancock, lower levels of fruit and seed set than for those farther apart. 2000 ) and is therefore indicative of severe ID. Both species are strongly xenogamous and entomophilous, with null levels of spontaneous self pollination. This spontaneous Population structure— The study populations of C. multifl o- self pollination is avoided not by the existence of herkogamy or rus and C. striatus consist of small patches separated from each dichogamy in the fl ower, but because the stigma surface must other by cropland, with an average distance between them be scratched by a pollinator for the pollen to germinate on the January 2010] Valtue ñ a et al. — Inbreeding depression in C YTISUS 131

Fig. 4. Relationship between different parameters and days after hand self pollination (HSP), hand cross pollination (HCP), and no pollination (NP) in Cytisus striatus . Lines show models estimated by ANCOVA tests (in formulas in each panel, y = studied parameters and x = days after pollination [DAP]). (A) Number of embryo cells. (B) Number of endosperm nuclei. (C) Endosperm to embryo ratio. (D) Ovule-seed length till 14 DAP. (E) Developing seed length from 16 to 40 DAP. (F) Ovule and seed abortion percentage. Each data point shows the mean value per individual and per DAP. stigma ( Rodr í guez-Ria ñ o et al., 1999 , 2004 ). Nonetheless, this als’ level of homozygosity. We would therefore expect a de- role of the stigma surface does not prevent geitonogamous pol- crease of the viability of these patches ( Gigord et al., 1998 ). lination because there is a great quantity of open fl owers per individual. In populations of this type with fragmented patches, Conjoint action of prezygotic and postzygotic self-sterility the recessive or partially deleterious recessive alleles that would mechanisms— The production of selfed progeny may be re- be masked as heterozygotes in large populations are exposed by duced by both pre- and postzygotic mechanisms of self-sterility the increased rate of selfi ng and biparental inbreeding that often acting not only in isolation, but also conjointly in a stepwise occurs in small populations (see references in Gigord et al., form in a given species, as has been shown in Medicago sativa 1998; Shi et al., 2005). Isolation of patches from each other ( Cooper, 1940 ), Cribum erubescens (Manasse and Pinney, would diminish interpatch gene fl ow, increasing the individu- 1991 ), Eucalyptus spathulata (Ellis and Sedgley, 1992), 132 American Journal of Botany [Vol. 97

Fig. 5. Aborting seeds after hand self-pollination at different phases in both Cytisus species (C. multifl orus , A – C and C. striatus , D – F). (A) Four- celled, collapsed proembryo and aborting endosperm (22 days after pollination [DAP]). (B) Collapsed proembryo with > 14 cells and aborted endosperm (32 DAP). (C) Vacuolated proembryo with > 18 cells and aborting nuclear endosperm (40 DAP). (D) Three-celled, collapsed proembryo with delayed en- dosperm (12 DAP). (E) Aborting embryo in globular phase with suspensor cells collapsing and delayed endosperm (24 DAP). Inset: comparison with well developed globular embryo phase. (F) Torpedo phase with collapsed cellular endosperm (38 DAP). Insets, top: detail of collapsed cellular endosperm; bot- tom: detail of well-developed cellular endosperm. Abbreviations : ane, aborted nuclear endosperm; ap, aborted proembryo; cce, collapsed cellular en- dosperm; e, embryo; es, embryo sac; ii, inner integument; ne, nuclear endosperm; nu, nucellus; pl, palisade layer; pr, proembryo; s, suspensor; black arrowhead, starch grains; *, micropylar area. Stain: A, B, toluidine blue + ruthenium red; C – F, PAS + h æ matoxylin. Bars: A, E = 100 µ m; B – D = 50 µ m; F = 300 µ m.

Dombeya acutangula ( Gigord et al., 1998 ), Clintonia borealis tempehes . They suggested the presence of three factors: a pre- (Dorken and Husband, 1999), E. globulus (Pound et al., 2002a, zygotic SI and two postzygotic mechanisms — a late-acting SI b ), and Leptosiphon jepsonii (Goodwillie and Knight, 2006). and an early-acting ID. The infl uence of the two phenomena on self-sterility is relative. In the case of C. striatus , we should be add that there is also Thus, in Eucalyptus woodwardii , Sedgley (1989) indicated that the possibility of the involvement of some phenomenon of ma- the lower seed production was almost exclusively due to prezy- ternal selection of resources. In the individuals that formed gotic rejection, with a minimal contribution from the postzy- selfed seeds, these tended to be larger than the crossed seeds, gotic mechanism. In C. multifl orus , the two rejection mechanisms perhaps due to a greater availability of resources in the selfed would have a similar infl uence on total self-sterility because fruits, which usually had only a single developing seed com- about half of the selfed ovules are unpenetrated and the other pared with a larger number in the crossed fruits. Indeed, in the half are aborted due to postzygotic effects. A more complex latter, we would expect not only greater resource competition situation was proposed by Kenta et al. (2002) in Dipterocarpus among the seeds, but also competition for space. January 2010] Valtue ñ a et al. — Inbreeding depression in C YTISUS 133

Fig. 6. Abnormalities in developing seed in Cytisus multifl orus (A – C) and C. striatus (D, E) after cross and self pollination. (A, B) Undivided primary endosperm nucleus (it undergoes no karyokinesis) surrounded by large starch grains (A) with one nucleolus, 18 days after pollination (DAP) and (B) with several nucleoli, 12 DAP. (C) Binucleate endosperm phase: two successive sections (top and bottom) showing the micropylar nucleus with normal develop- ment and chalazal nucleus without karyokinesis, 14 DAP. (D) Two nonsuccessive sections (left and right) showing both tetranucleolate nuclei (micropylar and chalazal), 5 DAP. (E) Vacuolated ovule with well-developed embryo, 34 DAP. Abbreviations : ane, aborted nuclear endosperm; es, embryo sac; f, fu- niculus; ii, inner integument; men, micropylar endosperm nucleus; mi, micropyle; n, nucellus; ne, nuclear endosperm; oi, outer integument; pr, proembryo; tcen, tetranucleolate chalazal endosperm nucleus; tmen, tetranucleolate micropylar endosperm nucleus; tpen, tetranucleolate primary endosperm nucleus; ucen, undivided chalazal endosperm nucleus; upen, undivided primary endosperm nucleus; black arrowhead, starch grains; *, micropylar area. Stain: A, B, D, and E, PAS + h æ matoxylin; C, toluidine blue + ruthenium red. Bars: A, E = 100 µ m; B – D = 50 µ m.

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