S ELF STERILITY IN TWO C SPECIES (L , P TO 1 - Moodle UFSC

123

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

The two principal mechanisms that induce self-sterility in plants are self-incompatibility (SI) and early-acting inbreeding depression (ID). Self-incompatibility involves a series of physi-ological mechanisms that prevent reproduction after self-polli-nation or between genetically closely related individuals. This mechanism is not only one of the most important means of side-stepping autogamy and of promoting the generation of new genotypes among plants, but also it may contribute to angio-sperm diversity and success ( de Nettancourt, 2001 ).

In the SI process, the ovules are not fertilized because of an active rejection of the male gametophytes that carry the same S alleles of the sporophyte ( de Nettancourt, 2001 ). There are basi-cally two kinds of prezygotic SI (i.e., prior-to-fertilization): het-eromorphic and homomorphic. In heteromorphic or diallelic SI, fl owers differ in morphology (i.e., they are heterostylous), whereas in homomorphic SI, fl owers are not morphologically differentiated.

Homomorphic SI is much more common ( Richards, 1986 ) and can be divided into sporophytic and gametophytic. Sporo-phytic SI is controlled by the genotype of the pollen-producing plant, and inhibition usually takes place on the stigma surface ( de Nettancourt, 2001 ). This type of SI has been recorded in families such as Brassicaceae, Asteraceae, Betulaceae, Po-

lemoniaceae, Convolvulaceae, Caryophyllaceae, and Malva-ceae ( Hiscock and Mclnnis, 2003 ), but at a molecular level the phenomenon has been very rarely investigated (e.g., Brassica ; see revision in Charlesworth et al., 2005 ).

In gametophytic SI, the pollen is controlled by its own geno-type, and rejection can occur either on the style, more fre-quently, or on the stigma. Gametophytic SI is more widespread than sporophytic SI and is found in families such as Legumino-sae, Scrophulariaceae, Rosaceae, Solanaceae, Papaveraceae, and Poaceae. At a molecular level, however, the phenomenon has been studied only in Solanaceae, Papaver , Antirrhinum , and Rosaceae (Silva and Goring, 2001; Franklin-Tong and Franklin, 2003 ; Charlesworth et al., 2005 ).

A third kind of SI, late-acting or ovarian SI ( Seavey and Bawa, 1986 ), occurs in two large groups of species. In the fi rst group of species, pollen tubes reach the ovary after self-pollina-tion and can even penetrate the ovules, but fertilization does not take place. This type of rejection is consequently prezygotic. In the second group of species, self-fertilization takes place with subsequent postzygotic rejection ( Seavey and Bawa, 1986 ; Gibbs and Bianchi, 1993 ). Because the expression “ late-acting SI ” assumes that rejection is under genetic control similar to 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., 1994 ) or pistillate sorting ( Bertin et al., 1989 ). The latter has been used to describe those cases in which not only the time of occurrence of syngamy is unknown, but also whether abortion takes place before or after syngamy. The expression involves no presumption about the control mechanisms of those events (maternal, paternal, zygotic, or some combination of these) ( Sage et al., 1994 ). Nevertheless, Lipow and Wyatt (2000) demonstrated in Asclepias exaltata the existence of a single lo-cus with polyallelic control of late-acting SI.

Inbreeding depression (ID) is defi ned as fi tness loss as a re-sult of self-fertilization or fertilization between similar or

1 Manuscript received 1 October 2008; revision accepted 23 September 2009.

The authors thank Dr. M. Gonz á lez (University of Extremadura) for statistical assistance. Suggestions and comments by two anonymous reviewers and the associate editor greatly improved the manuscript. This work was fi nanced by the Ministry of Education and Science of Spain (projects BOS2002-00703 and CGL2005-00783/BOS, both co-fi nanced by ERDF). A predoctoral grant from that Ministry to F.J.V. (BES-2003-2187) is greatly appreciated.

4 Author for correspondence ([email protected])

doi:10.3732/ajb.0800332

SELF-STERILITY IN TWO CYTISUS SPECIES (LEGUMINOSAE, PAPILIONOIDEAE) DUE TO

EARLY-ACTING INBREEDING DEPRESSION 1

Francisco J. Valtue ñ a 2 , Tom á s Rodr í guez-Ria ñ o 2 , Francisco Espinosa 3 , 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.

124 American Journal of Botany [Vol. 97

sis and prepared to receive the pollen tube ( Rodr í guez-Ria ñ o et al., 2006 ); their mature seeds have an aril with a funicular origin ( Rodr í guez-Ria ñ o et al., 2006 ). Cytisus multifl orus presents two fl oral morphs, LF (with large fl owers) and SF (with small fl owers), with SF having the greater reproductive success of the two ( Rodr í guez-Ria ñ o et al., 2004 ). In this work, we shall only study the more widespread SF. Both species are clearly xenoga-mous and pollinated by bees. Self-sterility in these two species is characterized by virtually no seed production and high rates of abscission of fruits that contain ovule-seeds of varying sizes ( Rodr í guez-Ria ñ o et al., 1999 , 2004 ). Variability in the timing of fruit abscission and seed abortion indicates that ID may be operating in both species ( Rodr í guez-Ria ñ o et al., 1999 , 2004 ). In addition, prezygotic SI also contributes to self-sterility in C. striatus as indicated by low rates of ovule penetration ( Rodr í guez-Ria ñ o et al., 1999 ). However, verifi cation of ID or late-acting SI as the cause of self-sterility remains to be demon-strated. The main objective of this study is to assess seed devel-opment after cross- and self-pollination to determine whether postzygotic self-sterility in C. multifl orus and C. striatus is due to ID or late-acting SI. Previously published data ( Rodr í guez-Ria ñ o et al., 1999 , 2004 ) and unpublished data for the years 2004 and 2005 will be considered in the Discussion.

MATERIALS AND METHODS

Plant material — Populations of both Cytisus species ( C. multifl orus and C. striatus ) are located at the El Hito estate, in the municipal district of Alburquer-que (Badajoz, SW Spain). The population (39 ° 13 ′ N, 6 ° 57 ′ W) is situated be-tween 390 and 395 m a.s.l. on a granitic substrate and subject to a typical Mediterranean climate characterized by winters that are rainy and more or less cold, and summers that are dry and hot. The annual mean temperature is 15.5 ° C, 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 a lesser extent of C. striatus , with a very few individuals of Quercus rotundifo-lia , Crataegus monogyna , Pyrus bourgaeana , and Rubus ulmifolius as the main tree or shrub elements.

Pollination treatments — Pollination experiments were carried out during the years 2003 and 2004. In both species, hand self pollination (HSP) and hand cross pollination (HCP) were performed on separate individuals. Unless other-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-nated at the time of pollination as well as at the collection of samples to avoid the allocation of resources to those fl owers. The gynoecia of the pollinated fl owers were collected, stored, and sectioned following the method of Rodr í guez-Ria ñ o et al. (2006) . Because of the linear arrangement (typical of legumes) and number of ovules in the ovary (8 – 10 ovules/ovary), the ovary was 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 pollination (DAP) in C. multifl orus and 20 DAP in C. striatus, every piece contained only two ovule-seeds of the central zone of the ovary — the zone most likely to form seeds — and in those of very advanced phases only one. In these last ovaries, we only prepared those developing seeds that were in good condi-tion, discarding those that were completely aborted because their advanced degradation did not allow anything to be observed.

Sections were stained either with periodic acid Schiff ’ s reagent (PAS) and contrasted with Gill no. 3 h æ matoxylin solution for starch and general visual-ization of tissues (I. Casimiro, University of Extremadura, Spain, personal com-munication) or sometimes with ruthenium red contrasted with toluidine blue O for visualization of tissues ( Crivellato et al., 1990 , modifi ed). Permanent prepa-rations were mounted in Eukitt. Sections were studied with a light microscope.

The number of analyzed individuals, fl owers, and ovules-seeds in the differ-ent sections of this work (quantifi cation of ovule penetration and double fertil-ization and, seed development after cross, self, or no pollination) are represented in supplemental tables (Appendices S1 and S2, see Supplemental Data with the online version of this article).

closely related genotypes. This phenomenon is one of the most signifi cant factors infl uencing the evolution of plant reproduc-tive systems ( Lloyd, 1980 ; Charlesworth and Charlesworth, 1987 ). There is a tendency for ID to be particularly intense be-tween fertilization and the mature seed phase (early ID) when numerous essential genes are expressed for the fi rst time ( Meinke, 1991 ; Seavey and Carter, 1994 ; Husband and Schem-ske, 1996 ).

As Seavey and Bawa (1986) observed, it is very diffi cult to distinguish which effects are attributable to late-acting SI and which to ID. The following two principal criteria have been suggested for differentiating between the two biological phe-nomena ( Seavey and Bawa, 1986 ; Sage et al., 1994 ; Nic Lughadha, 1998 ; Lipow and Wyatt, 2000 ): (1) Timing of abortion. A uniform failure at a single developmental stage would be interpreted as late-acting SI, whereas a series of fail-ures throughout embryogeny would be seen as the result of ID. Nonetheless, there have been relatively few studies inves-tigating embryological processes to confi rm that fertilization has occurred before the abortion of the developing seeds (but see Gibbs and Bianchi, 1993 ; Seavey and Carter, 1996 ; Gibbs and Sassaki, 1998 ; Nic Lughadha, 1998 ; Bittencourt et al., 2003 ; Pound et al., 2003 ; Sage and Sampson, 2003 ). (2) The amount of variability in selfed seed set among individuals of a population. Null or almost null seed production after self-pollination in all the individuals of a population would indi-cate late-acting SI, whereas variations among individuals in the population would be seen as the result of ID. Nevertheless, genotypes with a high load of lethal genes could also induce complete or almost complete self-sterility ( Seavey and Bawa, 1986 ; Waser and Price, 1991 ). Other criteria include (1) re-sponse of embryos to rescue in tissue culture, (2) dependence of abortion on the paternal vs. the progeny genotype, and (3) induced mutations (see Seavey and Bawa, 1986 ; Klekowski, 1988 ; Sage et al., 1994 ; Lipow and Wyatt, 2000 ; Bittencourt and Semir, 2005 ).

Numerous herbaceous and, above all, shrub species have late-acting SI (see e.g., Bawa and Beach, 1983 ; Owens, 1985 ; Kenrick et al., 1986 ; Seavey and Bawa, 1986 ; Gibbs and Bi-anchi, 1993 , 1999 ; Sage et al., 1994 , 1999 , 2006 ; Lipow and Wyatt, 2000; Sage and Sampson, 2003 ; Bittencourt and Semir, 2006 ). A postzygotic mechanism of ID inducing self-sterility has been shown in Epilobium obcordatum ( Seavey and Carter, 1994 ), Gomidesia ( Nic Lughadha, 1998 ), Dalbergia miscolo-bium ( Gibbs and Sassaki, 1998 ), Calluna vulgaris ( Mahy and Jacquemart, 1999 ), several species of Vaccinium ( Guillaume and Jacquemart, 1999 ; Hokanson and Hancock, 2000 ), and Bul-bine bulbosa ( Owen and Vaughton, 2003 ) among others. Nev-ertheless, species are also known with mixed prezygotic SI and postzygotic mechanisms explaining self-sterility, as happens in Medicago sativa ( Cooper, 1940 ), Eucalyptus spathulata ( Ellis and Sedgley, 1992 ), Dombeya acutangula ( Gigord et al., 1998 ), and Clintonia borealis ( Dorken and Husband, 1999 ). Neither can one rule out the probable existence of both postzygotic mechanisms (late-acting SI and ID) in a given species (e.g., Rhododendron prinophyllum, Padrutt et al., 1992 ) or even both together with prezygotic SI (e.g., Dipterocarpus tempehes, Kenta et al., 2002 ).

The present research is centered on two shrub species of Le-guminosae, Cytisus multifl orus (L ’ H é r.) Sweet and C. striatus (Hill) Rothm., endemics from the western Mediterranean re-gion. Both have a monosporic Polygonum type of embryo sac development, with all ovules per ovary mature at fl ower anthe-

125January 2010] Valtue ñ a et al. — Inbreeding depression in CYTISUS

F 1,39 = 8.02, P < 0.01, R 2 = 0.84, see the solid and dashed lines in Fig. 1A ), but not in C. striatus (ANCOVA, F 1,48 = 0.02, P > 0.05, R 2 = 0.61, see the solid and dashed lines in Fig. 1B ). In both treatments, there was a trend toward delayed penetration and double fertilization in C. multifl orus with respect to C. stri-atus , although this was not tested statistically.

Ovule and seed development after cross, self, and no polli-nation — All results refer to both species, except where differ-ences between them are explicitly indicated. This section begins with a description of normal seed development. We will then show by comparing the two treatments (self vs. cross) that the selfed embryo had either a similar or an accelerated develop-ment, although not signifi cant, relative to the crossed embryo, while the development of its endosperm was signifi cantly de-layed. This lag was refl ected in very low values of the En/E ra-tio compared to those obtained for the crossed seeds. This disharmony in the development between these two structures (embryo and endosperm) would be ultimately responsible for the abortion of the selfed seeds due to delayed development of the endosperm, which subsequently caused the collapse of the developing embryo.

Seed development after HCP was considered to be normal development. The pollen tube penetrates the ovule through a zig-zag micropyle and then penetrates the embryo sac through the fi liform apparatus. Prior to penetration of the ovule, one of the synergids degenerates. After fertilization, the second syner-gid degenerates. The embryo development is delayed with re-spect to endosperm development and passes through the following phases: proembryo, globular, heart, torpedo, and cot-yledon ( Fig. 2 ). Both species present a nuclear endosperm that develops through the consecutive divisions of the primary en-dosperm nucleus ( Rodr í guez-Ria ñ o et al., 2006 ). Later, in the late globular – early heart phase, the endosperm becomes cellu-lar except for the chalazal area, where cell wall formation never occurs. As far as the fi rst developmental stages of the embryo are concerned, both species follow a variant pattern of the On-agrad type, according to the classifi cation of Johansen (1950) , a pattern well known in Leguminosae ( Prakash, 1987 ; Batygina, 2006 ). Although in this family the endosperm development is very uniform, embryo development patterns vary considerably (Asterad, Caryophyllad, Onagrad, and Solanad types; see e.g., Cooper, 1938 ; Prakash, 1987 ; Lim and Prakash, 1994 ; Lersten, 2004 ; Batygina, 2006 ; Rodr í guez-Pontes, 2007 ).

Embryo, endosperm, and endosperm to embryo ratio — In both species, the development of the endosperm and the en-dosperm to embryo ratio (En/E ratio) were dependent on the treatment (cross vs. self), but the development of the embryo was not.

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-sion of the process in C. multifl orus (in a single ovule). That is, the zygote began its development before the primary endosperm nucleus.

The model of embryo growth as measured by number of cells did not differ signifi cantly between treatments in either species (ANCOVA C. multifl orus , F 1,64 = 2.93, P > 0.05, R 2 = 0.74; ANCOVA C. striatus , F 1,50 = 0.08, P > 0.05, R 2 = 0.54; Figs. 3A and 4A ). However, in C. multifl orus , we observed a trend toward more cells after self-pollination than after cross-pollina-tion during embryo development ( Fig. 3A ). The development

Quantifi cation of ovule penetration and double fertilization — Ovule pen-etration and fertilization were studied on collected ovaries during the fi rst 10 DAP after sectioning and staining and, in some phases, the sample size was increased with the analysis of ovules after aniline blue staining and their obser-vation under fl uorescence microscopy ( Rodr í guez-Ria ñ o et al., 1999 ). Ovule penetration was verifi ed by the presence of the pollen tube through the micro-pyle, and fertilization was verifi ed by the presence of (1) a degenerated and 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 sperm cell nuclei; see Bittencourt et al., 2003 ), (2) one or more endosperm nu-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 but only on ovaries at 5 DAP, and they had been viewed with fl uorescence mi-croscopy ( C. striatus : 90.6% vs. 72.5%; C. multifl orus : 56.8% vs. 54.9%; crossed vs. selfed, respectively). This last method does not resolve whether the self-penetration is delayed with respect to cross-penetration or how the penetra-tion increases over time. We therefore needed to evaluate the percentage of 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-mined on 183 and 164 ovules of C. multifl orus and C. striatus , respectively, and after HCP on 201 and 126 ovules (online Appendices S1 and S2). Because ev-ery penetrated ovule was observed to be fertilized, we did not consider differ-ences between the penetrated and fertilized ovules.

Ovule and seed development after cross, self, and no pollination — Ovule and seed development were studied in sectioned and stained ovules and devel-oping seeds. We tested several features. (1) Anatomical observation: the seed development was determined after cross- and self-pollination, and in nonpene-trated/nonfertilized ovules. (2) Ovule-seed length: the median longitudinal sec-tion of ovules and developing seeds was measured in all phases of development studied as the greatest distance between the chalazal-most and the micropylar-most points. (3) Number of endosperm nuclei (En) and number of embryo cells (E) by counting the number of endosperm nuclei and embryo cells, respec-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 only made until 20 DAP in C. multifl orus and 14 DAP in C. striatus (hence-forth, period I) because of the diffi culty of counting them at later phases (period II). Morphological features, En/E ratios and ovule-seed length following HSP was determined on a maximum of 332 and 277 ovules-seeds of C. multifl orus and C. striatus , respectively, and after HCP on a maximum of 300 and 215 ovules-seeds (For more details, see online Appendices S1 and S2.).

Statistical analyses — The SPSS statistical package, version 15.0.1, was used for all statistical analyses. The comparison between treatments (self and cross pollination) in all the variables studied (percentage of penetrated ovules, length of ovule or developing seed, number of cells of the embryo [E], number of endosperm nuclei [En], ratio En/E, and percentage of aborted developing seeds) was performed using ANCOVA, considering the treatment as the princi-pal factor and time as the covariable in the linear model studied, as well as the 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 checked for the preconditions of normality and homoscedasticity. Note that to perform the ANCOVA analyses we used only the data from those ovules or developing seeds that showed no or only slight signs of abortion. A Mann – Whitney test was used specifi cally to determine any differences in the ratio En/E at the end of period I (day 14 in C. striatus and day 20 in C. multifl orus ) 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 homoscedasticity.

RESULTS

Quantifi cation of ovule penetration and double fertiliza-tion — In neither species was there a delay in the self-pollen tubes reaching the ovules relative to the cross-pollen tubes. The fi rst pollen tubes to reach the ovules were observed at 3 and 4 DAP in C. striatus and C. multifl orus , respectively, regardless of whether they were self or cross. However, the percentage penetration of ovules was signifi cantly greater after cross pol-lination than after self pollination in C. multifl orus (ANCOVA,


fertilized, were either similar in size to virgin ovules or only slightly larger ( Figs. 3D and 4D ). The ANCOVA showed sig-nifi cant differences among the three types of ovule (cross- and self-fertilized, and nonfertilized ovules) (ANCOVA C. multi-fl orus , F 2,155 = 197.12, P < 0.001, R 2 = 0.95; ANCOVA C. stria-tus , F 2,66 = 33.44, P < 0.001, R 2 = 0.85). These differences were due solely to the smaller size of the nonfertilized relative to the fertilized ovules (cross and self), but there were no differences between the latter two treatments (cross vs. self; Figs. 3D and 4D ).

On the contrary, in later phases (period II; Figs. 3E and 4E ), the behavior after HSP of the two species was very different. In C. striatus , most of the few healthy selfed seeds remaining in the fruits (6.14%) followed a development similar to that of the crossed seeds ( Fig. 2 ) but with one difference: they were gener-ally larger (ANCOVA, F 1,55 = 7.50, P < 0.01, R 2 = 0.56) be-cause in each fruit there was usually just one developing seed compared with up to seven seeds/fruit in the crossed fruits. This similar development of selfed and crossed seeds made it possi-ble for there to exist seeds with normal development after HSP up to the occurrence of embryos that had differentiated radicle and cotyledons and fi nally the production of mature seeds. In C. multifl orus , the development of the selfed seeds followed the same pattern as the crossed seeds (ANCOVA, F 1,33 = 3.52, P > 0.05, R 2 = 0.82), although they were somewhat smaller in size ( Fig. 3E ).

Considering separately the individuals studied of the two species, we found that after both treatments (cross and self pol-lination) their behavior did not vary signifi cantly between indi-viduals (Appendix S3, see online Supplemental Data) in all the parameters considered (embryo and endosperm development and En/E ratio). In all individuals, the development of the en-dosperm after self pollination was slower than after cross pol-lination, while the development of the embryo was similar. As discussed, these patterns imply a somewhat different behavior of the species in their En/E ratio. Thus, in C. multifl orus , the slower development of the endosperm after self pollination was enhanced by faster development of the embryo (En/E ratios very far from the normal). In contrast, in C. striatus , the slow

of the endosperm depended signifi cantly on the type of treat-ment (ANCOVA C. multifl orus , F 1,63 = 57.71, P < 0.001, R 2 = 0.78; ANCOVA C. striatus , F 1,49 = 35.46, P < 0.001, R 2 = 0.80; Figs. 3B and 4B ). This difference was refl ected in far slower development of the endosperm after self-pollination than after cross-pollination, as measured by the number of nuclei ( Figs. 3B and 4B ). Similarly, the pollination treatment (self vs. cross) signifi cantly infl uenced the ratio En/E (ANCOVA C. multifl o-rus , F 1,63 = 66.84, P < 0.001, R 2 = 0.79; ANCOVA C. striatus , F 1,48 = 6.87, P < 0.05, R 2 = 0.52; Figs. 3C and 4C ).

Considering the En/E ratio after cross pollination to repre-sent ideal development of the seed (i.e., developmental har-mony between the embryo and the endosperm), we must remark that, in both species, after self-pollination (similar or high em-bryo development and low endosperm development), this ratio was lower in value (i.e., there was disharmony in the develop-ment of the two structures). These tendencies were due to (1) a marked increase in the cross ratio ( Figs. 3C and 4C ) caused by a surge of endosperm development in period I ( Figs. 3B and 4B ), and (2) a slight increase in the self ratio ( Figs. 3C and 4C ) from the rapid growth undergone by the embryo, accompanied by weak growth of the endosperm ( Figs. 3B and 4B ). Neverthe-less, in Fig. 4C, we observed that in C. striatus the cross and self ratios tended to approach each other at ~14 DAP (see in Fig. 4C, the plus sign represents the crossed data and the open circles the selfed data). This convergence was due to the decline in the cross ratio caused by the greater growth of the embryo ( Fig. 4A ) relative to the endosperm ( Fig. 4B ), causing a pro-gressive decrease in that ratio ( Fig. 4C ). The result was the ab-sence of signifi cant differences between the cross and the self ratios at the end of period I ( U 14DAP = 10.00, P > 0.05). The convergence of the two ratios in this species could be translated into a reharmonization of the selfed embryo and endosperm de-velopment, albeit with a slower development than after HCP.

Ovule size — During period I, the growth of the ovule was determined by the existence or absence of fertilization (fertil-ized vs. nonfertilized), but not by the type of treatment (cross vs. self). The nonpenetrated ovules, or penetrated but not yet

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.


low in both species (c. 16% of the pollinated fl owers in C. stria-tus and 6% in C. multifl orus ). These fruits tended to be small, pale-green or yellowish, with no turgidity, and with already aborted or aborting seeds. All individuals of C. multifl orus ini-tially had more or less massive fruit-drop that slowly continued, but differed as to the timing of the dropping (e.g., one individ-ual maintained most of its selfed fruits until approximately 35 – 40 DAP). In contrast, in C. striatus fruit-drop took place gradually, and the individuals behaved more uniformly than in the case of C. multifl orus .

DISCUSSION

The most important contribution of this work is the demon-stration of disharmony in endosperm/embryo development as one of the main causes of seed abortion after selfi ng in Cytisus multifl orus and C. striatus , thus representing a conceptual ad-vance in the study of inbreeding depression (ID). Although ID is the main cause of self-sterility in both species, in the case of C. multifl orus , to this postzygotic mechanism we must add an-other mechanism, prezygotic rejection — a phenomenon that has rarely been documented in the literature.

Prezygotic mechanism of SI in C. multifl orus — The lower percentage of penetrated ovules after self-pollination as com-pared to cross-pollination in C. multifl orus clearly supports a partial prezygotic SI mechanism acting at the level of the style/ovary. The low penetration percentage in the selfed ovules of C. multifl orus is not attributable to their immaturity at anthesis be-cause morphologically the ovules are completely formed and mature at that time ( Rodr í guez-Ria ñ o et al., 2006 ). Sage et al. (1999 , 2006 ) indicated the presence of long-distance signaling in which the presence of self-pollen or self-pollen tubes nega-tively infl uences ovule development (e.g., Narcissus triandrus and Ipomopsis aggregata ). In the species studied, there was no evidence of this long-distance signaling in the self ovules; we observed no morphological or structural changes compared with the ovules of unpollinated fl owers or with unpenetrated ovules of crossed fl owers. The only structural changes observed in unpenetrated ovules were those related with their progressive deterioration. In Dipterocarpus tempehes , Kenta et al. (2002) blame a massive fall of fl owers in the early stages after fertiliza-tion on the possible action of late-acting SI. In our case, most of the selfed fl owers collected during the fi rst DAP had ovaries with a large proportion of unfertilized ovules, with which late-acting SI (as a postzygotic phenomenon) does not seem consistent.

Postzygotic mechanism of self-sterility in both species — To distinguish between late-acting SI and early-acting ID, one of the principal criteria is the time until the developing seed aborts (e.g., Seavey and Bawa, 1986 ; Sage et al., 1994 ; Nic Lughadha, 1998 ; Lipow and Wyatt, 2000 ). We have reported in previous work ( Rodr í guez-Ria ñ o et al., 1999 , 2004 ) that in both Cytisus species most of the self-penetrated ovules were probably self-fertilized because of the presence of a remaining aril. This fea-ture — self-fertilization of ovules — has been suffi ciently demonstrated in the present work, and their subsequent abor-tion indicates the existence of postzygotic rejection. After ovule self-penetration, almost all structural developmental features (fertilization percentage, developing seed size, ovule tissue de-velopment, etc.) were practically identical to those after cross-

development of the endosperm was mitigated by a somewhat slower development of the embryo, leading to En/E ratios that in some cases were similar to those obtained after cross pollination.

Developing seed abortion — Very few fertilized selfed ovules developed normally. Indeed, fertilized selfed ovules were ob-served aborting on every DAP ( Figs. 3F, 4F, and 5 ) . This abor-tion occurred continuously throughout the development of the seed, although its greatest incidence was during the proembryo 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 ), heart, and torpedo ( Fig. 5F ) phases in C. striatus . The embryo never surpassed the globular phase in C. multifl orus , but reached the mature seed phase in C. striatus . The percentage of aborted developing seeds during the proembryo phase in C. multifl orus and proembryo – globular phase in C. striatus differed signifi -cantly between treatments and between DAP (ANCOVA C. multifl orus , F 1,89 = 5.25, P < 0.05, R 2 = 0.32; ANCOVA C. stri-atus , F 1,82 = 17.67, P < 0.001, R 2 = 0.74; Figs. 3F and 4F , re-spectively). Abortion was identifi ed by some structural features; the most important and valuable were the presence of delayed and/or densely staining endosperm ( Fig. 5 ), aborting embryos (with vacuolated cells, Fig. 5C ), or aborted embryos with strongly stained and irregular cells ( Fig. 5A, B, and D ). In these aborting selfed seeds, if the abortion process was incipient, we observed problems in the endosperm and embryo not accompa-nied by changes in the other tissues (for example, amount of starch grains, degree of staining of ovular tissue cells). How-ever, when abortion was well advanced, a number of character-istics in the nonembryonic tissues were observed that indicated deterioration (e.g., they appeared more lightly stained, and the amount of starch grains had declined [ Fig. 5C] or even disap-peared completely).

In developing selfed seeds, occasional abnormalities of en-dosperm development were observed related to karyokinetic failures. They involved the presence of abnormally large nuclei in which one or more nucleoli could be distinguished. They were observed in early stages of endosperm development, i.e., the primary endosperm nucleus ( Fig. 6A, B ) and less frequently in the binucleate endosperm phase (micropylar and chalazal nu-clei), in which case, they may affect only the chalazal en-dosperm nucleus ( Fig. 6C ) or both endosperm nuclei ( Fig. 6D ). The absence of normal development of endosperm inhibits deg-radation of the starch grains surrounding the central cell. Con-sequently, starch grains persist around the primary endosperm nucleus ( Fig. 6A ).

Both problems (low En/E ratios and endosperm abnormali-ties during period II) were also observed after HCP, but with different relevance. After cross pollination, the existence of low values of this ratio was infrequent (1.98% in C. multifl orus and almost 0% in C. striatus ), while the existence of endosperm abnormalities ( Fig. 6 ) was similar in incidence to that of the selfed seeds (cross = 4.5%, self = 4.6% in C. multifl orus ; cross = 4.3%, self = 6.5% in C. striatus ).

Another type of failure, that generally was more frequent af-ter HSP, was the vacuolization of the tissues surrounding the embryo sac ( Fig. 6E ). This failure was always greater after HSP ( C. multifl orus = 4.29%; C. striatus = 6.04%) than after HCP ( C. multifl orus = 3.55%; C. striatus = 1.83%).

Finally, it has to be noted that, in most individuals, the num-ber of surviving fruits 25 d after self-pollination was extremely


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.


Time of selfed pistils or young fruits fall after self-pollina-tion — Late-acting SI is usually manifest in a massive fall of selfed pistils/young fruits in a very short period of time after self-pollination (e.g., between 3 – 8 DAP; Chorisia chodatii , C. speciosa , Tabebuia caraiba , T. ochracea : Gibbs and Bianchi, 1993 ; Dolichandra cynanchoides and Tabebuia nodosa : Gibbs and Bianchi, 1999 ; Spathodea campanulata : Bittencourt et al., 2003 ; Jacaranda racemosa : Bittencourt and Semir, 2006 ). In a study of Hymenaea stigonocarpa , abscission of most of the selfed fl owers occurred at 7 – 8 d, but two pistils persisted for 6 – 7 mo, indicating the existence of postzygotic rejection ( Gibbs et al., 1999 ) without discriminating between late-acting SI or ID. In taxa with ID, the selfed-pistils/young fruits usually ab-scised over weeks to months ( Gibbs and Sassaki, 1998 ; Nic Lughadha, 1998 ). Thus, in Dalbergia miscolobium ( Gibbs and Sassaki, 1998 ), half of the fl owers whether crossed or selfed had fallen by 1 wk after pollination, but the abscission contin-ued for nearly 4 mo. This gradual fall over time implies that the developing seeds abort at different stages of development.

Apart from the massive fall of selfed pistils in C. multifl orus at a few days after pollination, previously attributed to nonpen-etration of the ovules, in both species, the selfed fruits gradually fell over time. Therefore, with respect to the period of abscis-sion of selfed fl owers, the two Cytisus species share more char-acteristics with species that are subject to ID rather than those that present late-acting SI, so the hypothesis of the existence of ID seems more plausible in both.

Size variability or uniformity of the aborted seeds — It is as-sumed that late-acting SI provokes a uniform failure of self-fertilized ovules, while early ID triggers failure at various stages of development (see Seavey and Bawa, 1986 ; Sage et al., 1994 ; Nic Lughadha, 1998 ; Lipow and Wyatt, 2000 ). In previous pa-pers ( Rodr í guez-Ria ñ o et al., 1999 , 2004 ), the presence of a gradient of fertilized ovule sizes and aborted fruits in both Cy-tisus species was described. Our embryological results indicate that these aborted seeds are at different phases of development, with selfed embryos reaching the late proembryo – early globu-lar stage in Cytisus multifl orus and far more advanced phases in C. striatus (i.e., heart, torpedo, and cotyledon phases). The high proportion of aborted seeds of widely varying sizes in selfed fruits and their presence in crossed fruits but at a much lower proportion lends support to the ID hypothesis in both species. Thus, as proposed by Hufford and Hamrick (2003) , the mater-nal embryonic genetic load may be expressed in both selfed and outcrossed progeny, with the degree of infertility related to how many deleterious alleles are combined.

Amount of variability in selfed seed set among individuals of a population — Variability among self-pollinated individuals is thought to be due to ID. This criterion is fully satisfi ed in C. striatus because some selfed individuals produced fruits or seeds, showing a variable range of fruit set (0 – 15%) and seed set (0 – 3.1%) (T. Rodr í guez-Ria ñ o, F. J. Valtue ñ a, and A. Or-tega-Olivencia, unpublished data). In contrast, this criterion is not satisfi ed in C. multifl orus . If one considers fruiting to in-volve the formation of mature fruit with at least one mature seed, the selfed fruit set was always absolutely null ( Rodr í guez-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-ivencia, unpublished data), there was generally only one shriv-eled seed. Nonetheless, mature fruits but with no fertile seeds were also produced in some individuals of C. striatus ( < 3%).

pollination. The only different character between the two (cross vs. self) was the endosperm development and, consequently, the endosperm to embryo (En/E) ratio. Endosperm develop-ment, measured as number of nuclei, could be a factor involved in abortion of the developing seed. However, rather than merely the poorer development of the endosperm, the disharmony that arises between the development of the embryo and of the en-dosperm, i.e., the En/E ratio, is really responsible.

Early ID is believed to be due to the expression of recessive alleles during embryo development and/or endosperm forma-tion. In both Cytisus species, abortion of the developing seed appears to be linked to the relationship between embryo and endosperm development (En/E ratio). Thus, while selfed seeds may have delayed endosperm development with respect to crossed seeds, if the endosperm ’ s relative growth is not suffi -ciently out of synchrony with that of the embryo, abortion would not occur. Therefore, the convergence observed of the En/E cross and self ratios at the end of period I in C. striatus would be translated into survival of the selfed seeds that had managed to surpass this period. Nonetheless, because there is no such convergence in C. multifl orus , the developing selfed seeds always abort. The genetic load probably acts by causing a disharmonization in embryo and endosperm development, be-ing more severe in C. multifl orus as a consequence of the ex-pression of more lethal recessive alleles in the different developing seeds, as proposed by K ä rkk ä inen and Savolainen (1993) for most conifers. This different action of ID would lead to a great variability in the size of the aborted seeds because abortion would occur when the En/E ratio reaches a minimum, from which point survival would no longer be possible.

Abortion occurred continuously throughout the development of the seed, with its greatest incidence at the proembryo phase. But because this phase covers a very long time span (i.e., until about 32 DAP in C. multifl orus and until 18 – 20 DAP in C. stri-atus ), changes in the development of the embryo and/or en-dosperm cannot be considered as a single stage of development. Thus, abortion within one phase of development is considered to be due to a continuing failure at different stages within that phase. The observed sequence of faults usually began with a delay in the development of the endosperm, causing the abor-tion of the embryo at some point in its development (whenever an aborting seed was observed, the endosperm was delayed and/or degraded). Only when the collapse of the endosperm and/or embryo was in advanced phases did we observe effects on the other, nonembryonic tissues of the developing seed (e.g., decrease in integument starch grains). These observations in the aborted or aborting seeds support the hypothesis set out in the previous paragraph of the action of lethal recessive alleles on the development of the endosperm and embryo, so that embry-onic failure is the cause of abortion and not its fi nal consequence ( Cooper et al., 1937 ; Cooper, 1940 ). In contrast, Sage and Web-ster (1990) observed that in Phaseolus vulgaris abortion of the seeds was a consequence of changes in the maternal tissue (i.e., nonembryonic tissue, such as integument and nucellus cells, starch depletion) causing abortion of embryonic tissues (embryo and endosperm). In this last case, abortion occurred due to the diversion of assimilates to nonaborting seeds, i.e., it was unre-lated to the action of lethal recessive alleles. Similar conclusions had been reached by Briggs et al. (1987) for Pisum sativum .

Other features supporting early ID — In both species, sev-eral different features confi rm or support the presence of early ID as a postzygotic mechanism of self-sterility.


greater than 50 m. In the case of C. multifl orus , there is also a greater density of individuals per patch. These individuals may be closely related to one another genetically, especially those that are closer than 2 m, because unpublished data indicate lower levels of fruit and seed set than for those farther apart. Both species are strongly xenogamous and entomophilous, with null levels of spontaneous self pollination. This spontaneous self pollination is avoided not by the existence of herkogamy or dichogamy in the fl ower, but because the stigma surface must be scratched by a pollinator for the pollen to germinate on the

Although no cases are known in which all the individuals in a population are self-sterile due to genetic load (but see Wiens et al., 1989 in Dedeckera eurekensis ), complete self-sterility has indeed been found in individual plants (see reviews in Seavey and Carter, 1994 ; Gigord et al., 1998 ; Hokanson and Hancock, 2000 ) and is therefore indicative of severe ID.

Population structure — The study populations of C. multifl o-rus and C. striatus consist of small patches separated from each other by cropland, with an average distance between them

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.


als ’ level of homozygosity. We would therefore expect a de-crease of the viability of these patches ( Gigord et al., 1998 ).

Conjoint action of prezygotic and postzygotic self-sterility mechanisms — The production of selfed progeny may be re-duced by both pre- and postzygotic mechanisms of self-sterility acting not only in isolation, but also conjointly in a stepwise form in a given species, as has been shown in Medicago sativa ( Cooper, 1940 ), Cribum erubescens ( Manasse and Pinney, 1991 ), Eucalyptus spathulata ( Ellis and Sedgley, 1992 ),

stigma ( Rodr í guez-Ria ñ o et al., 1999 , 2004 ). Nonetheless, this role of the stigma surface does not prevent geitonogamous pol-lination because there is a great quantity of open fl owers per individual. In populations of this type with fragmented patches, the recessive or partially deleterious recessive alleles that would be masked as heterozygotes in large populations are exposed by the increased rate of selfi ng and biparental inbreeding that often occurs in small populations (see references in Gigord et al., 1998 ; Shi et al., 2005 ). Isolation of patches from each other would diminish interpatch gene fl ow, increasing the individu-

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.


tempehes . They suggested the presence of three factors: a pre-zygotic SI and two postzygotic mechanisms — a late-acting SI and an early-acting ID.

In the case of C. striatus , we should be add that there is also the possibility of the involvement of some phenomenon of ma-ternal selection of resources. In the individuals that formed selfed seeds, these tended to be larger than the crossed seeds, perhaps due to a greater availability of resources in the selfed fruits, which usually had only a single developing seed com-pared with a larger number in the crossed fruits. Indeed, in the latter, we would expect not only greater resource competition among the seeds, but also competition for space.

Dombeya acutangula ( Gigord et al., 1998 ), Clintonia borealis ( Dorken and Husband, 1999 ), E. globulus ( Pound et al., 2002a , b ), and Leptosiphon jepsonii ( Goodwillie and Knight, 2006 ). The infl uence of the two phenomena on self-sterility is relative. Thus, in Eucalyptus woodwardii , Sedgley (1989) indicated that the lower seed production was almost exclusively due to prezy-gotic rejection, with a minimal contribution from the postzy-gotic mechanism. In C. multifl orus , the two rejection mechanisms would have a similar infl uence on total self-sterility because about half of the selfed ovules are unpenetrated and the other half are aborted due to postzygotic effects. A more complex situation was proposed by Kenta et al. (2002) in Dipterocarpus

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.


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In sum, in this work we have demonstrated that (1) the al-most total self-sterility in C. striatus is of postzygotic type due to strong early ID and that (2) the total self-sterility in C. multi-fl orus is explained by the existence of both pre- and postzygotic mechanisms. Cytisus multifl orus presents a physiological par-tial prezygotic SI with rejection of part of the self- pollen tubes in the style or ovary and an early ID that causes abortion of those fertilized ovules that escaped the action of prezygotic SI. This postzygotic abortion after selfi ng is due to a disharmony in the development of the endosperm with respect to that of the embryo, refl ected in smaller endosperm to embryo ratios than after cross fertilization.

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S ELF STERILITY IN TWO C SPECIES (L , P TO 1 - Moodle UFSC

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