Flora 208 (2013) 412–419

Contents lists available at ScienceDirect

Flora

j ournal homepage: www.elsevier.com/locate/flora

Pollination and self-interference in

a,∗ a,b

Cristian Daniel Torres , Javier Guido Puntieri

a

Instituto Nacional de Investigaciones en Biodiversidad y Medioambiente (Universidad Nacional del Comahue-CONICET), Quintral 1250, 8400 San Carlos de Bariloche, Argentina

b

Universidad Nacional de Río Negro, Sede Andina, San Carlos de Bariloche, Argentina

a r t i c l e i n f o a b s t r a c t

Article history: Interference between male and female functions within a monoecious may hinder crossing and

Received 16 January 2013

decrease seed set. We assessed the probability of self-pollination and the effect of self-pollination on

Accepted 1 June 2013

cross-pollination for two self-incompatible species: and N. nervosa. The probability of

Available online 27 July 2013

self-pollination was studied by tracking the phenologies of staminate and pistillate flowers, including an

analysis of stigmatic receptivity. Pure and mixed pollinations were performed in order to evaluate the

Keywords:

effect of self-pollination upon cross-pollination. Phenological observations suggest that self-pollination

Nothofagus

is highly likely in both species. Compared to pure cross-pollination, the application of self-pollination

Phenology

prior to cross-pollination resulted in lower numbers of germinated pollen grains for both species, and

Pollen release

also in a lower production of viable seeds in N. nervosa. The low proportion of viable seeds often observed

Stigma receptivity

Self-interference in natural populations of N. obliqua and N. nervosa may be related to self-pollination.

Anemophily © 2013 Elsevier GmbH. All rights reserved.

Introduction to identify since it is determined by secretions promoting pollen

hydration and germination, and pollen-tube growth (Herrero and

In monoecious (with both sexes in each individual), Hormaza, 1996; Sanzol et al., 2003; Waites and Ågren, 2006).

self-pollination within the same individual may interfere with In some species, the period of stigmatic receptivity is evidenced

the crossing with conspecific individuals (Barrett, 2002). Since through morphological changes affecting the stigmas (Oddie and

most monoecious species are self-incompatible (Allen and Hiscock, McComb, 1998; Tal, 2011; Yi et al., 2006). In many other species this

2008), self-pollination may impose physical or chemical barri- period is not externally apparent, so that indirect techniques based

ers to cross-pollination hindering the production of viable seeds on the detection of enzymatic activity related to receptivity are

(Barrett, 2002; Cesaro et al., 2004; Dai and Galloway, 2011). It has often applied (Dafni and Maués, 1998; Jones, 2002). A reliable way

been argued that the intra-individual interference between male of evaluating stigmatic receptivity is the application of sequential

and female functions (self-interference) is an important selective hand-pollinations followed by the assessment of viable-seed pro-

force influencing plant breeding. Both the spatial and temporal duction and/or pollen-grain germination on the stigmas (Oddie and

intra-individual separations of male and female functions have McComb, 1998; Ofosu-Anim et al., 2006; Page et al., 2006; Sanzol

been interpreted to evolve as mechanisms that tend to reduce et al., 2003). The description of flower phenology and the degree

self-interference (Barrett, 2002). Accurate information about the of temporal overlapping between male and female functions for a

phenologies of staminate and pistillate flowers and the mechanism given species contribute to the reproduction of plants with com-

of self-incompatibility are necessary in order to assess the degree mercial and/or conservation value (Tooke and Battey, 2010).

of self-interference in each species. Nothofagus species are dominant in temperate forests of the

Knowledge about the degree of temporal overlapping between Southern Hemisphere (e.g. Veblen et al., 1996). Their diaspores

pollen release and pistillate flowers receptivity is essential in the are one-seeded, indehiscent (achenes). High quantitative

study of self-interference. The ripening of staminate flowers is often and qualitative inter-annual variations in seed production are

evidenced by changes in the morphology and color of anthers, as commonly observed in natural populations of these species

well as by pollen release. Stigmatic receptivity is more difficult (Donoso et al., 2006a,b; Marchelli and Gallo, 1999). The production

of empty seeds was found to be high in many populations of

Nothofagus species (Bahamonde et al., 2011; Marchelli and Gallo,

1999; Martínez-Pastur et al., 1994). Although the amount of

∗

Corresponding author. Tel.: +54 2944 423374/428505x280;

empty seeds is often related to damage by insects (Carrillo and

fax: +54 294 4422111.

Cerda, 1987; Marchelli and Gallo, 1999), non-damaged empty

E-mail addresses: [email protected], [email protected]

(C.D. Torres), [email protected] (J.G. Puntieri). seeds are common, which could be related to pollen limitation,

0367-2530/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.flora.2013.07.002

C.D. Torres, J.G. Puntieri / Flora 208 (2013) 412–419 413

self-pollination and/or self-interference. Nothofagus species are

anemophilous, so the small size of stigmas and the sessile nature

of the pistillate flowers may limit pollen capture. Moreover, the

development of both flower types on the same shoots (Puntieri

et al., 2009; Torres et al., 2012) would promote the highly inef-

fective self-pollination (Riveros et al., 1995b). However, as the

period of stigmatic receptivity has not been investigated, a possible

overlapping between the periods of pollen release and stigmatic

receptivity within an individual has not been confirmed. Moreover,

the effect of self-pollination on viable seed production has not

been tested experimentally in Nothofagus so far.

In the present work we selected two Nothofagus species in

order to assess (A) the time of stigmatic receptivity through the

application of sequential pollinations and the evaluation of pollen

germination, (B) the intra-individual overlapping between the

periods of pollen release and stigmatic receptivity, and (C) the

degrees of self-incompatibility and self-interference. The selected

species, Nothofagus obliqua (Mirb.) Oerst. and Nothofagus nervosa

(Phil.) Krasser (=N. alpina (Poepp. et Endl.) Oerst.) are two of the

economically most valued Nothofagus species due to their fast

Fig. 1. Semi-schematic representation of a trimerous pistillate inflorescence of

growth and high wood quality (Donoso et al., 2006a,b; Tuley, 1980).

Nothofagus obliqua at anthesis.

Expanding the current knowledge about the reproductive biology

of these species is necessary in order to understand their natural

regeneration dynamics and support domestication programs. and avoid fungal growth and pollen clumping. Previous studies

◦

indicate that pollen grains of N. obliqua stored at 5 C maintain

their viability for up to three months (Báez et al., 2002).

Materials and methods

Pollination was applied to the pistillate flowers of one branch

per tree at one of the following times: at the time of anthesis

Flowering trees of Nothofagus obliqua and N. nervosa with acces-

(0 DAA), three days after anthesis (3 DAA), six days after anthesis

sible flower-bearing branches (at less than 5 m on the trunk) are not

(6 DAA), ten days after anthesis (10 DAA), and 15 days after anthesis

easy to find. Moreover, inter-annual variation in flower production

(15 DAA). Pistillate flowers of Nothofagus species have a rudimen-

within each tree complicates the selection of suitable trees. Due

tary perigonium, so that we considered that the anthesis of these

to these difficulties, this study was carried out in three stages on

flowers corresponds with the unfolding of the subtending and

different trees. In such stages we addressed: (A) the period of stig-

their stipules, which makes the stigmas accessible to pollen (Fig. 1).

matic receptivity, (B) flowering phenology of reproductive shoots

Only the most proximal pistillate inflorescence in each shoot was

and (C) interference in cross-pollination and seed production due

pollinated since pistillate inflorescences on different nodes of a

to self-pollination.

shoot do not reach anthesis simultaneously. Pollination was applied

once for each treatment with a thin painting brush. In all, 278 pistil-

Stigma receptivity late flowers from 93 inflorescences were pollinated. were

fixed in FAA 24 h after pollination and stored for 90 days. Fixed

The analysis of stigmatic receptivity was made on flowers were softened in 1.5 M NaOH for 24 h, then gently washed

hydroponically-maintained flowering branches in order to with distilled water, stained for 24 h in a 0.1% (w/v) solution of ani-

simplify the periodic observation of flowers and their isolation in line blue in 0.1 M K2POH4, mounted on glycerine and squashed. For

prevention of unwanted pollinations. Preliminary tests showed each flower, germinated grains were counted with a fluorescence

that shoots of N. obliqua placed in hydroponic culture present microscope (Olympus BX51) equipped with a HBO 50 W lamp, and

normal bud-break and flowering. In September 2009, in an area a filter for UV excitation (BP 330–385 nm, BA 420 nm; Fig. 2). The

of 80 ha close to Lácar Lake (Lanín National Park, Argentina), three flowers of each inflorescence were considered individually.

four N. obliqua trees with flowering buds on accessible branches

were identified. Nothofagus nervosa was not included since trees Phenology of flowering shoots

with high flower production could not be found in that year.

Before budbreak, five 17–50 cm long flowering branches of each In order to describe the phenology of flowering shoots, N. obliqua

selected N. obliqua tree were cut and placed in water immediately and N. nervosa trees were selected at the “Unidad de Mejoramiento

afterwards. An additional N. obliqua tree located at San Carlos de Ecológico, Genético y Forestal”, EEA INTA Bariloche, situated about

Bariloche (brought from another natural population of this species 70 and 100 km south of the nearest natural population of N. obliqua

in Argentina) was used as pollen donor. Branches were taken to and N. nervosa respectively. Based on bibliographic information,

a polycarbonate greenhouse and with external ventilation. Each mean temperature and precipitations at the nursery are similar to

3

branch was placed individually in 500 cm bottles filled with those registered in natural populations of N. nervosa and N. obliqua

running water. Temperatures in the greenhouse ranged between 8 at Lanín National Park (Conti, 1998). In spring 2011, three N. obli-

◦

and 25 C during the experiment. Every 48 h, bottles were refilled qua and two N. nervosa trees were chosen based on the presence of

with water and 1 cm of the proximal end of each branch stem flowering buds. Because of the scarcity of trees with flowers in the

×

was cut while keeping it under water. After bud-break, all shoots studied season, three reproductive N. nervosa N. obliqua hybrid

of these branches were emasculated. Staminate flowers from the trees were also included. For each tree, three shoots extended one

tree used as pollen donor were cut short before pollen release year before sampling (2010–2011), hereafter referred to as parent

◦

and placed in open petri dishes at room temperature (15–20 C). shoots, were chosen among those with reproductive buds. Phe-

◦

Once anthers opened, pollen was placed in small jars at 5 C nological observations were made on all annual shoots derived

with silica-gel in order to maintain a fresh and dry environment, from these parent shoots. Altogether, 98, 64 and 77 annual shoots

414 C.D. Torres, J.G. Puntieri / Flora 208 (2013) 412–419

fruits were separated by the technique of flotation in ethanol 96%;

fruits with embryo sink immediately, in contrast to empty fruits.

Filled fruits were immediately washed with distilled water and

◦

stored at 5 C. To test seed germination, fruits were washed with

sodium hypochlorite 10%, then with distilled water, and stratified

◦ ®

at 4 C for 90 days in cotton soaked with the antifungal Vitavax 2%.

After stratification, fruits were placed in a germination chamber

◦

at 22 C. A seed was considered as germinated when the emerging

radicle was visible. The number of germinated seeds per treatment

was counted.

Data analyses

Stigma receptivity

The proportion of flowers with germinated pollen grains was

2

compared among pollination times by means of tests. The

Fig. 2. Pollen grains of Nothofagus obliqua germinated on the three stigmatic sur-

number of germinated grains was compared among pollination

faces of a pistillate flower (dark areas) observed with a fluorescence microscope.

times, taking into account only those flowers that presented at

Arrows indicate (a) a pollen grain, (b) a pollen tube and (c) stigmas.

least one germinated pollen grain. Due to data non-normality,

such comparisons were made by means of non-parametrical

corresponding respectively to N. obliqua, N. nervosa and hybrid trees Kruskal–Wallis tests (Sokal and Rohlf, 1981); pairwise comparisons

were observed. Every two to three days starting at the day of bud- were applied whenever significant differences were found with the

break, the number of unfolded , the number and phenological Kruskal–Wallis test.

stage of the staminate inflorescences (from the initiation of their

expansion in budbreak to pollen release) and the number of visi-

ble pistillate inflorescences (i.e. in anthesis or post-anthesis) were Phenology of flowering shoots

registered for each flowering shoot. During the period of observa- Phenophases of staminate flowers were identified from about

tion air temperature and relative humidity was registered with a the time of budbreak, when flower extension allows their distinc-

data logger (Lascar EL-USB-2; data not shown but available from tion from other organs of the growing shoot, to anther dehiscence.

the authors on request). For each entity we observed the variation over time in the number

of staminate flowers at each phenophase, the number of pistillate

inflorescences at anthesis and post-anthesis and the deployment

Self-interference

of green leaves.

During spring 2010, three N. obliqua and four N. nervosa trees

were selected by identifying flowering buds. Two of the N. obli- Self-interference

qua trees are located at San Martín de los Andes town and

The number of pollen grains germinated on the stigmas was

the other one belongs to a natural population close to Lácar

compared among pollination treatments (I–V) by means of analy-

Lake (Lanín National Park). Nothofagus nervosa trees are located

sis of variance (ANOVA) including each tree as a block. Square-root

close to Lolog Lake (Lanín National Park). Shortly after budbreak,

data transformation was applied so as to fulfill the normality and

flowering branches of each tree were selected and all of their

variance homogeneity requirements for ANOVA. The proportion

flowering shoots were emasculated. When pistillate flowers were

of filled and empty seeds was compared among pollination treat-

visible, the following pollinations were manually applied with 2

ments by means of tests. For filled seeds of each treatment, the

a thin painting brush: (I) cross-pollination, (II) self-pollination,

percentage of germination was calculated, although the application

(III) cross-pollination followed by self-pollination 24 h later, (IV)

of statistical analyses was not possible in this case due to the low

self-pollination followed by cross-pollination 24 h later, and (V)

number of seeds with embryo in some treatments.

simultaneous cross-pollination and self-pollination. For each tree,

all pollinations were performed within a lapse of 1 h. Pollinations

corresponding to treatments I, II and V were applied twice so that Results

all flowers received similar quantities of pollen. The pollen applied

was obtained from shoots placed in hydroponics in warm and dry Stigma receptivity

conditions which allowed pollen to be obtained prior to natural

pollen release. Pollen for cross-pollination derived, in each case, Pistillate flowers with at least one germinated pollen grain were

from another of the trees of the same species selected for this study. detected throughout the period of time considered (from anthe-

◦ 3

Pollen obtained was stored at 5 C in 1.5 cm air-tight jars with sis to 15 DAA; Fig. 3A). The percentage of flowers with germinated

silica-gel. grains (Fig. 2) was similar among pollination times 0, 3, 6 and

The results of each pollination were assessed in three steps: (1) 10 DAA (about 50–60%), whereas the proportion of pistillate flow-

germination of pollen grains on the stigmas, (2) production of filled ers with germinated grains corresponding to pollination 15 DAA

2

fruits (i.e. with an embryo in the seed), and (3) seed germination. was significantly lower ( = 18.3; p = 0.001; Fig. 3A). The num-

For the first analysis two to six pistillate inflorescences per shoot ber of germinated pollen grains varied significantly among flowers

were fixed in FAA for about three months and pollen germination pollinated at different times (Kruskal–Wallis H = 38.7; p < 0.001;

was assessed (see protocol used for the receptivity experiment Fig. 3B). Flowers pollinated 6 DAA had significantly more ger-

described above). The remaining flowers were left undisturbed minated grains than flowers pollinated at the other times. The

to allow development. Ripe fruits were harvested in late numbers of germinated grains of flowers pollinated 0, 3 and 10 DAA

February to evaluate the presence of embryos. Fruits derived from were intermediate between those pollinated 6 DAA and those pol-

open pollination were also taken from each tree. Filled and empty linated 15 DAA.

C.D. Torres, J.G. Puntieri / Flora 208 (2013) 412–419 415

flower primordia are no longer hidden by the cataphylls of the

flowering bud, and flowers begin their expansion; stamens remain

covered by the perigonium; (II) the perigonium has opened but the

stamens are tightly packed and their filaments remain short; (III)

filaments have extended so that stamens loosen and protrude, but

their anthers remain closed; (IV) anthers are open and pollen is

released.

For flowering shoots of all trees, phenophase I of the staminate

flowers began in early spring, together with the initiation of the

deployment of the proximal green leaves of the reproductive shoot

(Fig. 5A–C). Phenophase duration decreased from phenophase I to

III (Fig. 5A–C). Since phenophase III was markedly shorter than

phenophases I and II, a change from phenophase II to phenophase

IV was registered for several staminate flowers (Fig. 5). Phenophase

III was less evident for staminate flowers of hybrid trees than for

flowers of N. obliqua and N. nervosa, and several staminate flow-

ers did not exhibit those characteristics associated with this phase

before pollen release (Fig. 5C). For all trees, pollen release began

about 25 days after the initiation of phenophase I. In broad terms,

most of the pistillate inflorescences of the selected reproductive

shoots had reached anthesis before the time of pollen release from

the same shoots (Fig. 5A–C).

Self-interference

The mean number of germinated pollen grains per pistillate

flower varied significantly (p < 0.001) depending on the pollina-

tion treatment for both species, more notably so for N. nervosa

(F = 23.8; n = 130) than for N. obliqua (F = 4.8; n = 122; Fig. 6). Inter-

individual variation (block effect) was significant both for N. obliqua

(F = 12.1; p < 0.001) and N. nervosa (F = 7.0; p = 0.002). The mean

number of germinated grains was lowest for self-pollinated flow-

Fig. 3. (A) Percentage of receptive pistillate flowers and (B) number of pollen grains

germinated on the stigmas corresponding with pollination at 0, 3, 6, 10 and 15 ers in both species (treatment II; Fig. 6A and B). In the case of

days after anthesis (DAA) in Nothofagus obliqua. Different letters indicate significant

N. obliqua, the number of germinated grains observed for self-

2

differences regarding the proportion of receptive flowers ( test) and the number

pollinated flowers was lower than for flowers corresponding to

of germinated grains between treatments (Kruskal–Wallis test).

treatments I (cross-pollination), III (cross-pollination followed by

self-pollination 24 h later) and V (simultaneous cross- and self-

Phenology of reproductive shoots

pollination), whereas flowers of treatment IV (self-pollination

The development of the staminate flowers, from budbreak to followed by cross-pollination 24 h later) presented intermedi-

pollen release can be described by four phenophases (Fig. 4): (I) ate values (Fig. 6A). Regarding N. nervosa, the mean number of

Fig. 4. Phenophases of staminate flowers in Nothofagus: I: staminate flowers detached from other primordia of the breaking bud with perigonium still attached to the

stamens; II: perigonium unfolded, stamens short and tightly packed; III: stamens lengthened and loosen; IV: anthers split open, pollen release.

416 C.D. Torres, J.G. Puntieri / Flora 208 (2013) 412–419

Fig. 6. Mean (±standard error) of germinated pollen grains on the stigmas of N.

obliqua (A) and N. nervosa (B) after (I) cross-pollination, (II) self-pollination, (III)

cross-pollination followed by self-pollination 24 h later, (IV) self-pollination fol-

lowed by cross-pollination 24 h later and (V) simultaneous cross-pollination and

self-pollination. Different letters indicate significant differences between treat-

ments in the number of germinated grains (Tukey’s test).

Fig. 5. Numbers of unfolded green leaves, staminate flowers at phenophases I to IV,

and pistillate flowers as related to observation date for N. obliqua (A), N. nervosa (B)

and N. nervosa × N. obliqua hybrid trees (C).

germinated grains was similar for treatments II and IV and notably

lower than the number observed for the other treatments (Fig. 6B).

In all three N. obliqua trees, significant differences among polli-

nation treatments were detected regarding the proportion of filled

2

fruits ( = 482.3; p < 0.001; Fig. 7A). In this species, the proportion

of filled fruits was similar after treatments I, III, IV and V (between

60 and 80%) and notably lower for treatment II (self-pollination)

and open pollination (less than 15%; Fig. 7A). For N. nervosa the

proportion of filled fruits also varied significantly among polli-

2

nation treatments ( = 83.2; p < 0.001; Fig. 7B), and the lowest

proportion of filled fruits was obtained by self-pollination. The

proportion of filled fruits obtained by self-pollination followed by

cross-pollination (treatment IV) was also low for N. nervosa (less

than 20%; Fig. 7B). Between 40 and 60% of filled fruits were observed

Table 1

Percentage of germination of seeds with embryo obtained after the application of

different pollination treatments (I–V) and after open pollination. Treatments codes

as in Fig. 6. n: number of seeds.

Treatment N. obliqua N. nervosa

% Germination n % Germination n

I 94 89 95 116

Fig. 7. Percentage of filled fruits obtained from (A) three N. obliqua and (B)

II 89 9 100 4

three N. nervosa trees after (I) cross-pollination, (II) self-pollination, (III) cross-

III 95 99 88 19

pollination followed by self-pollination 24 h later, (IV) self-pollination followed by

IV 100 61 85 13

cross-pollination 24 h later, (V) simultaneous cross- and self-pollination and open

V 100 80 100 31

pollination. The total numbers of fruits observed are indicated at the top of the bars.

Open 85 168 79 202

C.D. Torres, J.G. Puntieri / Flora 208 (2013) 412–419 417

for treatments I, III and V and for open pollination (Fig. 7B). A low release and stigma receptivity, considering the period of stigma

variation was observed regarding the germination of seeds with receptivity observed for N. obliqua and the frequent occurrence

embryo among treatments in either species (Table 1). of several pistillate inflorescences in each shoot (Puntieri et al.,

2009). A study on Nothofagus species from New Zealand, although

Discussion lacking methodological specifications, arrived at similar conclu-

sions (Poole, 1950). The similarities observed between N. obliqua

Flowering phenology in Nothofagus and probability of and N. nervosa regarding the anthesis dynamics of staminate and

self-pollination pistillate flowers at an intra-individual scale, suggest that the latter

species would also exhibit high probabilities of self-pollination.

For Nothofagus species the period of stigmatic receptivity had

not been investigated until now. In cases like Nothofagus spp., in Self-incompatibility and self-interference

which stigmatic receptivity is not externally evident, the assess-

ment of the stigma’s capacity to promote pollen hydration and The present study confirmed the high degree of self-

germination is a reliable way to determine the period of stigmatic incompatibility previously mentioned for Nothofagus (Riveros et al.,

receptivity (Ofosu-Anim et al., 2006; Sanzol et al., 2003). By apply- 1995b), a feature that is shared with other related species (Clausen,

ing such technique we found that pistillate flowers of N. obliqua are 1966; Steiner and Gregorius, 1999; Yacine and Bouras, 1997). In the

receptive at about the time of unfolding of their subtending leaf (a present work, self-incompatibility was evidenced not only by a low

stage that may then be referred to as anthesis). The early start of production of filled fruits after self-pollination but also by a low

stigmatic receptivity observed here is consistent with observations germination of pollen grains on pistillate flowers of the plant from

in the related genus Quercus (Boavida et al., 1999; Ducousso et al., which pollen was obtained. This assessment of self-compatibility

1993). In this study a high proportion of N. obliqua pistillate flow- in a stage previous to embryo development is useful to disentangle

ers were receptive for 10 days after anthesis, and some degree of the mechanisms associated with self-incompatibility in Nothofa-

receptivity could be detected 15 days after anthesis. This period of gus. The obtained results suggest pre-zygotic anti-selfing barriers

stigmatic receptivity seems to be longer than that of Quercus (about acting on the stigmatic surface. When self-pollen is rejected on

5 days; Boavida et al., 1999; Ducousso et al., 1993). A long period the stigmatic surface, that is, pollen germination is inhibited, the

of receptivity is helpful whenever the probabilities of pollination incompatibility of the pollen grain is identified by the diploid

per time unit are low (Arroyo et al., 1985; Waites and Ågren, 2006). genome of the parental plant (sporophytic self-incompatibility;

Considering the axillary position and the small size of the pistillate Allen and Hiscock, 2008). Sporophytic self-incompatibility has been

flowers of N. obliqua, a long period of stigma receptivity could be previously observed for Betulaceae (Germain, 1994; Hampson and

helpful to increase the probability of pollination. Azarenko, 1993), but for Nothofagaceae, as well as for Fagaceae,

Our data indicate that stigmatic receptivity in N. obliqua has a self-incompatibility mechanisms have been mentioned to take

normal distribution with a distinct peak about six days after anthe- place in the ovary, prior to fertilization (Allen and Hiscock, 2008;

sis. Similarly, in Eucalyptus species, a peak in receptivity has been Yacine and Bouras, 1997). The present research indicates that

observed by applying sequential pollinations (Oddie and McComb, the low proportion of filled fruits obtained after self-pollination

1998). Variations in the degree of receptivity could be linked with in N. obliqua and N. nervosa would be due to a low probabil-

changes in the proportion of the stigmatic surface that is recep- ity of pollen germination on the stigmas, suggesting sporophytic

tive at a given moment, as observed in Kunzea pomifera by means self-incompatibility. These results indicate that more investigation

of peroxidase activity (Page et al., 2006). Since more pollen grains about pre-zygotic anti-selfing barriers in Nothofagaceae is needed.

germinate within the receptivity peak, pollination at this time No evidence of post-zygotic barriers was found since the percent-

could determine competition among pollen grains deposited on age of filled fruits that germinate was similar among cross- and

the stigmas and, therefore, be beneficial in terms of pollen selection self-pollinated flowers. Nevertheless, the low number of filled fruits

(Herrero and Hormaza, 1996). This argument would be meaningful obtained by self-pollination prevents us from being conclusive

if pollen tube growth speed is slow relative to the duration of the regarding the significance of post-zygotic barriers in these species.

phase of maximum receptivity. In many species with reduced self-compatibility, self-

It has been presumed for some time that self-pollination in pollination could result in low seed production or low germination

Nothofagus could be prevented by the more proximal position rates even in the presence of compatible pollen (Cesaro et al.,

in the flowering shoots of the staminate flowers compared to 2004; Dai and Galloway, 2011). For the Nothofagus species studied

the pistillate flowers (e.g., Donoso et al., 2006a,b; Riveros et al., here, the high probabilities of self-pollination, suggested by the

1995a). Only one previous study provides accurate information in phenological observations, highlight the importance of assessing

this regards: “female flowering started 10 to 15 days after male self-interference. Both pollen germination on the stigmas and

1

flowering ” (Riveros et al., 1995a). Whether this result complies the production of filled fruits were similar after cross-pollination

or not with the present study depends on the definition of “male followed by self-pollination 24 h later, simultaneous self- and

flowering”. If we consider the onset of male flowering to be denoted cross-pollination, and pure cross-pollination. Therefore, no inter-

by the initiation of the distinction of staminate flowers from the ference seems to occur if self-pollination is simultaneous or follows

breaking bud (phenophase I in this study), then our results would cross-pollination. On the other hand, self-pollination prior to cross

agree with those of the cited work. However, according to our pollination in N. nervosa resulted in a notably lower number of

results, staminate flowers of N. obliqua, N. nervosa and their hybrids germinated pollen grains and lower production of filled fruits with

would require a period of about 25 days between the phase of respect to pure cross-pollination. This was not observed for N.

flower distinction from the breaking bud to pollen release. There- obliqua. The results indicate that N. nervosa is more susceptible to

fore pistillate inflorescences would reach anthesis before pollen self-interference than N. obliqua and that such interference would

release within the same individual. Our results suggest a high occur only if self-pollination precedes cross-pollination. It would

degree of intra-individual overlapping between the times of pollen be interesting to assess whether time intervals between self- and

cross-pollination longer than that tested here (24 h) would render

self-interference significant in N. obliqua.

1 Another aspect to be considered is the quantitative balance

In the original: “la floración femenina se inició 10 a 15 días más tarde que la

masculina”. between self-pollen and cross-pollen. For example, it has been

418 C.D. Torres, J.G. Puntieri / Flora 208 (2013) 412–419

observed for Turnera ulmifolia (Turneraceae) that self-interference Arroyo, M.T.K., Armesto, J.J., Primack, R.B., 1985. Community studies in pollination

ecology in the High Temperate Andes of central Chile. Plant Syst. Evol. 149,

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Báez, P., Riveros, M., Lehnebach, C., 2002. Viability and longevity of pollen of Nothofa-

dence of post-zygotic effects of self-interference was found here for gus species in south Chile. New Zeal. J. Bot. 40, 671–678.

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(Quercus suber L.). I. The progamic phase. Sex. Plant Reprod. 11, 347–353.

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and pistillate flowers in flowering shoots of the latter species. It Cesaro, A.C., Barrett, S.C.H., Maurice, S., Vaissiere, B.E., Thompson, J.D., 2004. An

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Clausen, K.E., 1966. Studies of compatibility in Betula. In: Joint Proc. 2nd Genet.

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USDA Forest Serv. Res. Pap. NC-6, pp. 48–52.

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Conti, H.A., 1998. Características climáticas de la Patagonia. In: Correa, M.N. (Ed.),

The negative effect of self-interference on seed production of N.

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et al., 2004; Lin et al., 2012; Yacine and Bouras, 1997; Zhang et al.,

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2011), and could explain, at least partially, the high proportions

ence in a self-incompatible species? Funct. Ecol. 25, 271–278.

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de los bosques templados de Chile y Argentina. Marisa Cuneo Ediciones, Valdivia,

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Donoso, P., Donoso, C., Gallo, L., Azpilicueta, M.M., Baldini, A., Escobar, B., 2006b.

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Knapp, E.E., Goedde, M.A., Rice, K.J., 2001. Pollen-limited reproduction in blue oak:

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Marchelli, P., Gallo, L., 1999. Annual and geographic variation in seed traits of Argen-

The present work showed, for N. obliqua and N. nervosa, a high tinean populations of southern beech Nothofagus nervosa (Phil.) Dim. et Mil. For.

Ecol. Manage. 121, 239–250.

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Martínez-Pastur, C., Arena, M.E., Fernández, C., 1994. Nota sobre la inferencia del

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For these species, stigmas act as a first line of prevention of self- gus betuloides (Mirb.) Oerst. Invest. Agrar. Sist. Recur. For. 3, 83–89.

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Oddie, R.L.A., McComb, J.A., 1998. Stigma receptivity in Eucalyptus camaldulensis

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