Annals of Botany 81 : 735–740, 1998

Reproductive Biology of Dalbergia miscolobium Benth. (Leguminosae-

Papilionoideae) in SE Brazil : The Effects of Pistillate Sorting on Fruit-set

PETER GIBBS* and REGINA SASSAKI†

*Plant Science Laboratories, The Uni�ersity, St Andrews KY16 9AL, UK and † Instituto de BotaW nica,

CxP 4005, Sah o Paulo 01051, Brazil

Received 17 April 1997 Returned for revision: 30 June 1997 Accepted 18 February 1998

The breeding system of Dalbergia miscolobium Benth. was investigated by means of controlled pollinations andstudies of post-pollination events in pistils. This species does not have homomorphic self-incompatibility, but crossedflowers always have higher fruit set than selfs, despite the fact that embryo development may be initiated in selfedpistils. It is suggested that deleterious recessive alleles, which lead to abortion of most selfed seeds, together with somematernal resource limitation, result in low fruiting success. # 1998 Annals of Botany Company

Key words : Dalbergia miscolobium Benth., Leguminosae, breeding system, pistillate sorting, fruiting success, seed set.

INTRODUCTION

The genus Dalbergia comprises some 100 species of treesand lianes with a pantropical but mainly neotropicaldistribution. Many species are valuable timber trees withdurable, dark-grained wood used for furniture and musicalinstruments. D. miscolobium Benth. (frequently referred toas D. �iolacea (Vogel) Malme in older literature) is one ofthe most widespread of the Brazilian species, occurring inseasonal woodlands from Piauı! state in the NE to Parana! inthe south.

In common with many neotropical trees, Dalbergiamiscolobium has mass flowering but matures relatively fewfruits. Low fruit}flower ratios have been variously attributedto poor pollination success (Bierzychudek, 1981; Lee, 1988;Burd, 1994) or to maternal resource limitation (Stephenson,1979; Lee, 1988). However, independently of pollinatorservice and any maternal resource limitation, other intrinsicfactors may cause abortion of fertilized ovules. Such post-zygotic factors have received relatively little attention intree species but the importance of such ‘pistillate sorting’phenomena (Sage, Bertin and Williams, 1994) has beendemonstrated in some herbaceous species (e.g. Marshall andEllstrand, 1988; Waser and Price, 1991; Seavey and Carter,1996). Here we report a study of fruiting success in a treespecies with this kind of breeding system based on fieldobservations and pollination experiments over a 2 yearperiod, together with analyses of post-pollination eventsand seed number.

MATERIAL AND METHODS

Dalbergia miscolobium is a tree with dense racemose orpaniculate inflorescences of small, approx. 15 mm, typicallypapilionoid flowers, with violet standard and wing petals,and a whitish keel. The single carpel, which contains twoovules, is surrounded by ten diadelphous stamens, and at

anthesis the stigma projects slightly beyond the longestwhorl of anthers. Most fruits contain a single seed.

Studies on trees of D. miscolobium were carried out in anarea of cerrado vegetation about 15 km from Itirapira (Sa4 oPaulo state) in 1992 and 1993. Each year, during theflowering period in January, some trees (two in 1992, threein 1993) were monitored for natural fruiting success bymeans of (1) whole inflorescences with counted buds (in1992 only) and (2) individually tagged flowers (both years).Pistils and newly aborted young fruit were fixed in 70%FAA (formalin-acetic-alcohol).

Other trees (four in 1992, five in 1993) were used forpollination experiments. Inflorescences in bud were enclosedin lightweight nylon gauze bags, and hand pollinations werecarried out on the morning of the day flowers opened (i.e.the standard petal became erect). Cross- and self-pollin-ations were effected by forcing open the tips of the keelpetals to expose the stigma, and pollen applied from donorflowers collected the same morning and used with 2 h. Crossdonor flowers came from trees located about 1 km away.Emasculations were not attempted with hand-pollinatedflowers so as to avoid further damage to the keel petals. In1992, 99 (1993: 49) flowers were cross-pollinated and 83(1993: 88) were selfed. In 1992, 95 (1993: 60) flowers wereleft bagged but untreated to check for automatic self-pollination. In addition, in 1993 28 flowers were selfed bybeing gently squeezed across the keel petals to simulate theweight of a visiting insect, and bagged with no furthertreatment, and 18 flowers were emasculated and leftunpollinated as a check for apomixis.

Flowers tagged for natural fruit-set or involved inexperimental pollinations were monitored initially every2 d, subsequently at 10, then at 20 d intervals up to 118 dwhen surviving fruits were mature. The percentage ofsurviving fruit was calculated for each census and anarcsine transformation was carried out on the percentagedata to permit s.e. to be calculated.

0305-7364}98}060735­06 $25.00}0 bo980623 # 1998 Annals of Botany Company

736 Gibbs and Sassaki—The Effects of Pistillate Sorting on Fruit-set

Subsamples of the hand-pollinated flowers were fixed in70% FAA at intervals (24 h–6 d) for studies of pollen tubegrowth using fluorescence microscopy and leuco-anilineblue staining (Martin, 1959) or for histological observations.For the latter, pistils were embedded in paraffin waxfollowing the tertiary butanol (2-methylpropan-2-ol) sched-ule of Johansen (1940) and sectioned at 14 µm, or embeddedin resin (LKB historesin kit schedule) and semi-thin sectionscut at 3 µm. All sections were cut using a LKB microtomeand stained with either Delafield’s haematoxylin or safraninand fast green (Sass, 1951).

RESULTS

Natural fruiting success

Trees of D. miscolobium did not flower each year.Individuals which flowered in 1992 did not flower orproduced very few flowers in 1993, whilst those whichflowered in 1993 had not flowered the previous year.

In 1992, only 0±9% (n¯ 538) and 9±3% (n¯ 233) of budsin tagged inflorescences matured fruits on trees 1 and 2respectively, whilst of the individually tagged flowers, 1±4%(n¯ 122) and 19±7% (n¯ 70) formed fruits (Fig. 1A). In

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F. 1. Fruit-set following natural pollination in Dalbergia miscolobium.A, 1992 census. Buds in inflorescences : tree 1 (E) tree 2 (+) ; taggedflowers : tree 1 (D) tree 2 (*). B, 1993 census. Tagged flowers : tree 3

(^) tree 4 (V) tree 5 (x).

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F. 2. Fruit-set in D. miscolobium following hand-pollinations in 1992(A) and 1993 (B). Crosses (D) ; selfs (*) ; bagged controls (^) ;squeezed-keel selfs (V) ; emasculated controls (x). Points with the

same letter are not significantly different.

1993 tagged flowers on trees 3, 4 and 5 produced 0% (n¯68), 5±1% (n¯ 140) and 7±1% (n¯ 59) fruits, respectively(Fig. 1B). In both years the majority of flowers monitoredfor natural pollination were abscised during the first weekafter anthesis. However, fruit survival at day 7 (31±5% in1993—84 from 267 individually tagged flowers) was notmarkedly different from that of hand-pollinated selfedflowers (see below).

Hand pollination treatments

In 1992, 30±3% (1993: 20±5%) of crossed flowersdeveloped mature fruits compared to 3±6% (1993: 3±4%) ofselfed flowers ; flowers selfed by simply squeezing the keelpetals matured 10±7% fruits (Fig. 2A and B). Unfortunately,the number of squeezed-keel flowers (total 28 between twotrees) and fruits matured (3) did not permit a statisticalcomparison with crossed flowers. However, in 1993 hand-crossed flowers produced significantly more fruits (10}49)than hand-selfs plus squeezed-keel selfs (6}116) ; χ#¯ 9±13,d.f.¯ 1, P! 0±01.

Gibbs and Sassaki—The Effects of Pistillate Sorting on Fruit-set 737

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F. 3. Fruit enlargement in selfed �s. crossed flowers. Points with thesame letter are not significantly different.

In both years about half of the hand cross- and hand self-pollinated flowers}pistils abscised within 1 week of pol-lination. Thus, in 1993, 47% of cross- and 49% of selfedpistils remained on day 7, compared with 11±7% baggedcontrol flowers left for automatic self-pollination, and byday 10, 33% of cross- and 30% of selfed pistils remained,compared with 1±7% bagged controls. Both crosses andselfs continued to lose more young fruit over subsequentweeks, but such losses were much more marked in selfedpistils : in 1993, crossed fruit survival declined from 33% onday 10 to 20% on day 118, whereas selfed fruits declinedfrom 30% to 3% over this period. The cohort of crossedfruits showed a faster rate of elongation than selfs for thefirst 20 d after pollination, after which time there were nosignificant differences in size between surviving selfed andcrossed fruits (Fig. 3)

Fluorescence microscopy studies showed that self-pollentubes, as with crosses, grew through the style to the ovarywith no obvious inhibition (Fig. 4A and B). It was not easyto observe ovules penetrated by pollen tubes within theovary in either selfs or crosses, but of 31 pistils (youngfruits) observed up to 6 d after cross-pollination, a total often ovules with pollen ‘tails ’ at the micropyle were scored.In 26 selfed pistils fixed over 6 d only two ovules wereobserved to be penetrated.

In sectioned ovules at 4–6 d post-pollination, a globularembryo with endosperm nuclei was present in both crossedand selfed pistils and at this stage it was impossible todistinguish between selfed �s. crossed embryos (Fig. 4C andD). Due to problems with sectioned material we were notable to positively identify embryos in either ovule for anumber of pistils, but 28±6% of ovules in crossed- �s. 21% ofovules in selfed pistils were observed with embryos. If ovuleswhich were scored to have either a pollen ‘tail ’ at themicropyle (in squash preparations with fluorescence mi-croscopy) or an embryo (in sectioned pistils) are all treatedas ‘fertilized ovules ’, then there is no significant differencebetween self- �s. cross-pollinated pistils in this respect(cross : 22 fertilized}total 104 ovules ; self : 10 fertilized}total90 ovules ; χ#¯ 3±31, d.f.¯ 1, P" 0±05).

DISCUSSION

Trees ofD.miscolobium in the study area flowered biennially.In addition, natural fruit-set was low, ranging from 0–20%in the five trees monitored over a 2 year period. Mostflowers}young fruits were lost during the first 4–10 d afteranthesis. This suggests that low fruiting success is not due tolack of flower visitation and pollination, and this view issupported by the fact that the majority of pistils}youngfruits which were sampled and observed using fluorescencemicroscopy had pollen tubes in the styles and usually atleast one penetrated ovule.

Fruit-set following hand cross-pollinations ranged from20–30% which, despite the problem of manipulation ofsmall papilionoid flowers (see below), was higher than thatfound in naturally pollinated flowers, and markedly higherthan the less than 4% of hand-selfed flowers. Clearly,crossed flowers set more fruit than selfs, and althoughbiennial flowering and considerable loss of crossed flowerssuggests that some maternal resource constraints are alsolikely to be involved, poor fruit-set in D. miscolobium islikely to be depressed to a significant extent by the incidenceof self- (intrafloral or geitonogamous) pollinations withinthe canopies of these mass-flowering trees.

Although less than 4% of hand self-pollinated flowersformed fruits, flowers selfed by simply squeezing the keelpetals matured 10% fruits. Since flowers which were simplyleft bagged never formed fruits it seems likely that somekind of physical ‘ tripping’ of the keel petals is necessary forself-pollen to germinate. It is possible that the position ofthe stigma projecting slightly beyond the anthers at anthesisis sufficient to protect it from self-pollen deposition in anuntripped flower. The difference in selfing success betweenhand-pollinated �s. ‘ squeezed’}triggered flowers also sug-gests that the floral manipulation needed to effect handpollinations resulted in some loss of flowers, but thispresumably applied equally to cross- and selfed flowers.

It seems unlikely thatD.miscolobium is a self-incompatible(SI) species manifesting homomorphic, gametophytic SI,with prezygotic rejection of self- pollen tubes, as found insome other Leguminosae (Williams, 1951; Shivanna andOwens, 1989). None of the selfed pistils which were studiedby fluorescence microscopy showed any indication of a siteof pollen tube rejection in the style, as in a species with‘ leaky’ gametophytic SI in which occasional self pollentubes manage to grow to the ovary. And although thenumbers of ovules observed to be penetrated with a pollentube (in crosses and selfs) were not numerous, overall therewas no significant difference between crosses and selfs in theincidence of penetrated ovules together with ovules observedwith an embryo. Furthermore, in each year, over 45% ofhand selfed flowers survived for at least 7 d, parallelingsurvival of crossed flowers, whereas only 11±7% baggedcontrol flowers (left for possible automatic self-pollinationbut probably mostly unpollinated) survived for this period.This is not the pattern of predominant pollen tube rejectionin the style and rapid loss of selfed flowers which one wouldexpect to find in a species with ‘ leaky’ homomorphic SI.Rather, selfing in D. miscolobium seems to be controlled bya post-zygotic mechanism.

738 Gibbs and Sassaki—The Effects of Pistillate Sorting on Fruit-set

F. 4. For legend see facing page.

Gibbs and Sassaki—The Effects of Pistillate Sorting on Fruit-set 739

Such a situation has been variously referred to as ‘ late-acting self-incompatibility ’ (LSI) (Seavey and Bawa, 1986),‘cryptic self-compatibility ’ (Bertin and Sullivan, 1988) or,perhaps the best generic term to date, ‘pistillate sorting’(Bertin, Barnes and Guttman, 1989; Sage et al., 1994). Theterm late-acting self-incompatibility is perhaps unfortunatesince, as employed by Seavey and Bawa (1986), it en-compassed literally ‘ late ’ (i.e. within the ovary or ovule) butstill pre-zygotic self-pollen tube rejection, e.g. as in Acaciaretinodes (Kenrick, Kaul and Williams, 1986) and alsospecies with (probably diverse) post-zygotic self-rejectionphenomena.

Bawa and Webb (1984) studied fruit and seed abortion inseven neotropical tree species which included Dalbergiaretusa. They concluded that the excess of flowers to fruits inthese species was due to sexual selection effects rather thanpollinator or resource limitation. D. retusa was reported tobe self-incompatible. However, Seavey and Bawa (1986)subsequently listed D. retusa, together with two otherleguminous trees, Dipteryx panamensis and Myrospermumfrutescens, as species with a post-fertilization self rejectionmechanism. All three species were cited from unpublishedstudies by Bawa and no further details were provided. Ourstudy indicates that in D. miscolobium apparent self-incompatibility is due to a post-zygotic rejection, andtogether with Bawa’s observations suggests that such amechanism occurs more widely in the genus Dalbergia. Thiskind of effectively ‘pistillate sorting’ control of selfing hasalso been reported in several herbaceous papilionoidlegumes, e.g. Lotus corniculatus (Bubar, 1958) and Medicagosati�a (Brink and Cooper, 1938). Indeed, reports for ‘ late-acting’ or ‘pistillate sorting’ type control of selfing inlegume species outnumber those detailing conventionalhomomorphic SI in this family.

Exactly what causes massive rejection of selfed flowers inspecies where the self pollen tubes reach the ovules withequal facility to crossed ones, and fertilization occurs, is stillconjectural. On the one hand, some authors have doubtedwhether flowering plant species are likely to have sufficientembryonic lethals to virtually mimic multiallelic self-incompatibility. Waser and Price (1991) emphasized that atleast nine to ten lethals are necessary to produce over 90%self-sterility. And in cases where selfed flowers are uniformlyrejected at a stage when only a resting zygote is present, i.eno embryonic divisions have occurred as in Chorisia andTabebuia (Gibbs and Bianchi, 1993), it is difficult to see howsuch lethals may operate to curtail development. On theother hand, Klekowski (1988) and other workers (see reviewin Sage et al., 1994) have argued that the number ofembryonic lethals in woody species may well reach levelssufficient to simulate self-incompatibility.

In the case of D. miscolobium, the following factors mustbe taken into account: (1) there seems to be some differencesin selfing ability between individual trees, a situation whichwould be expected under the recessive lethal model ; (2) early

F. 4. Pollen tube growth and embryo development in hand pollinated D. miscolobium pistils}fruits. A, Self-pollen tubes in stigma and upperstyle ; B, self-pollen tubes entering ovary (multicellular glands which cover ovary wall very evident) ; C and D, globular embryo with endospermnuclei (arrowed) in crossed �s. selfed pistils at 6 d post-pollination; C, wax-embedded section, 14 µm; D, resin-embedded section, 3 µm. Bar¯

100 µm (A, B), 25 µm (C, D).

embryo development, rather than a resting zygote situation,occurs in this species which would give the opportunity forthe action of deleterious lethals ; and (3) although themajority of selfed carpels}young fruits fall within 5–10 d (asdo many crossed fruits), selfed fruits continue to absciseover a period of 30–60 d or more. Again, a protracted lossof selfed fruits would be expected under the inbreedingmodel.

These aspects indicate that the most likely explanation forthe poor fruit-set following selfing found in D. miscolobiumis deleterious recessive alleles taking effect at various stagesin selfed embryo development (Seavey and Carter, 1994,1996). And, since geitonogamous selfing is likely to befrequent, this would have a marked impact on overallfruiting success. However, since the majority of hand-crossed flowers also failed to set fruit (although thedetrimental role of floral manipulation is unknown here)and biennial flowering also seems to be the rule, it is likelythat this species has additional maternal resource constraintswhich also limit the fruiting success of most flowers.

ACKNOWLEDGEMENTS

This work was part of a study of the reproductive biologyand developmental physiology of cerrado plants supportedby the European Union ISC contract CI1}0620. RS wasalso funded by the Brazilian CNPq.

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