Annals of Botany 88: 89±99, 2001
doi:10.1006/anbo.2001.1435, available online at http://www.idealibrary.com on
Self-incompatibility, Inbreeding Depression and Crossing Potential in Five Brazilian
Pleurothallis (Orchidaceae) Species
E D U A R D O L . B O R B A * , J O AÄ O S E M I R and G E O R G E J . S H E P H E R D
Departamento de BotaÃnica, Instituto de Biologia, Universidade Estadual de Campinas, Caixa Postal 6109, Campinas,
SaÄo Paulo, 13083-970, Brazil
Received: 3 November 2000
Returned for revision: 12 February 2001 Accepted: 23 March 2001
Published electronically: 23 May 2001
Intra- and interspeci®c experimental pollinations were made to determine the mating systems and the interspeci®c
crossing potential in individuals from 24 populations of ®ve Pleurothallis species. Pleurothallis johannensis, P. ochreata
and P. teres show weak or partial self-incompatibility while P. adamantinensis and P. fabiobarrosii are more strictly
self-incompatible. We found no di€erences in fruit set between intra- and interpopulation intraspeci®c crosses, and
there was no correlation between fruit set and genetic variability or inbreeding in these species. All species are
interfertile and showed no correlation between genetic similarity and crossing potential. We scored fruits for seed
viability and observed a strong inbreeding depression in all populations; however, there was no di€erence in seed
viability among intrapopulation, interpopulation intraspeci®c and interspeci®c cross-pollinations. These species are
pollinated by ¯ies with a behavioural pattern that facilitates self-pollination. Self-incompatibility and inbreeding
depression are apparently important in the maintenance of the unusually high levels of genetic variability found in
these species. As generally observed in other orchid species, barriers to hybridization between these Pleurothallis
# 2001 Annals of Botany Company
species are at the level of pollination.
Key words: Pleurothallis, Orchidaceae, mating system, self-incompatibility, inbreeding depression, fruit set, seed set,
polyembryony, interspeci®c compatibility.
I N T RO D U C T I O N
Mating systems are one of the most important factors
determining genetic variability in plant species (Hamrick
et al., 1979; Hamrick, 1982, 1989; Hamrick and Godt,
1990). Orchids are typically self-compatible, and barriers to
autogamy generally occur before pollination (Pijl and
Dodson, 1966; Dressler, 1981, 1993; Borba and Semir,
1999). However, genetic barriers (i.e. self-incompatibility)
have been found in some orchid species. Genetic barriers are
usually associated with species pollinated by insects that
often remain on the ¯owers and in¯orescences for a long
time (e.g. ¯ies); this behaviour may promote geitonogamy
(Christensen, 1992; Pedersen, 1995). Barriers to hybridization between orchid species are also generally mechanical
(i.e. pre-pollination), and related species are potentially
interfertile (Dodson, 1962; Pijl and Dodson, 1966; Dressler,
1968, 1981, 1993; Borba and Semir, 1998a, 1999; Borba
et al., 1999).
We have studied the ¯oral biology (Borba and Semir,
2001) and genetic variability (Borba et al., 2001b) in ®ve
rupicolous Brazilian Pleurothallis species occurring on
rocky outcrops in areas of `campo rupestre' vegetation
from the east of Brazil. These species ¯ower synchronously,
and some of them are sympatric. Species with overlapping
distributions do not share pollinator species (Borba et al.,
2000; Borba and Semir, 2001). Populations of these species
* For correspondence at: Departamento de CieÃncias BioloÂgicas,
Universidade Estadual de Feira de Santana, Av. UniversitaÂria s/n,
Feira de Santana-BA, 44031±060, Brazil. E-mail [email protected]
0305-7364/01/070089+11 $35.00/00
show an unusually high genetic variability (Borba et al.,
2001b) despite being pollinated by ¯ies, whose behaviour
generally increases the likelihood of geitonogamy.
This paper describes the breeding systems of individuals
from 24 natural populations of P. johannensis Barb. Rodr.,
P. teres Lindl., P. fabiobarrosii Borba and Semir, P. ochreata
Lindl., and P. adamantinensis Lindl.Ðspecies which occur
in Brazilian campo rupestre vegetation. Our objective was to
investigate whether: (1) there are genetic mechanisms
preventing sel®ng, thus contributing to the maintenance of
the high levels of genetic variability previously reported; (2)
there is any correlation between fruit set following
intrapopulation cross-pollination and genetic variability or
inbreeding in the populations; (3) there are genetic barriers
preventing hybridization; and (4) there is a correlation
between genetic similarity and crossing potential among
di€erent species.
M AT E R I A L S A N D M E T H O D S
Plants of the ®ve Pleurothallis species used for the crossing
experiments were sampled from 24 natural populations at
16 localities in the states of Minas Gerais (MG), Bahia (BA)
and Pernambuco (PE) in southeastern and northeastern
Brazil: P. johannensis Barb. Rodr. (seven populations),
P. teres Lindl. (ten populations), P. ochreata Lindl. ( four
populations), P. fabiobarrosii Borba & Semir (one population), and P. adamantinensis Brade (two populations)
[Table 1; see Borba and Semir (2001) for the distribution of
these species and characterization of the areas in which they
# 2001 Annals of Botany Company
90
Borba et al.ÐMating Systems and Interspeci®c Compatibility in Pleurothallis (Orchidaceae)
T A B L E 1. Populations of Pleurothallis johannensis, P. teres, P. ochreata, P. fabiobarrosii and P. adamantinensis studied,
and number of individuals (N) used in experimental crossings
Species/population
Name
N
Location
Voucher
P. johannensis
Carrancas-MG ( pop. 1)
Carrancas-MG ( pop. 2)
Carrancas-MG ( pop. 3)
Itutinga-MG
Santa R. Ibitipoca-MG
SaÄo JoaÄo Del Rei-MG
Nazareno-MG
C1
C2
C3
IT
IB
SJ
NZ
24
13
17
17
28
29
18
218300 2800 S; 448360 0000 W
218280 0300 S; 448320 5600 W
218280 2500 S; 448370 0000 W
218170 5200 S; 448420 4500 W
218360 S; 438550 W
218080 2200 W; 448170 2800 W
218180 3800 S; 448350 2700 W
Borba
Borba
Borba
Borba
Borba
Borba
Borba
P. teres
Serra do Rola MocËa-MG
Serra do CipoÂ-MG ( pop. 1)
Serra do CipoÂ-MG ( pop. 2)
Serra do CipoÂ-MG ( pop. 3)
Serra do CipoÂ-MG ( pop. 4)
Serra do CipoÂ-MG ( pop. 5)
Serra do CipoÂ-MG ( pop. 6)
Ouro Preto-MG
CaeteÂ-MG
Diamantina-MG
RM
CA
CC
CVG
CVL
CLC
CJ
OP
CT
DI
05
13
05
02
05
01
02
20
27
06
208030 3700 S; 448010 5800 W
198170 3200 S; 438350 3700 W
198190 3000 S; 438330 5000 W
198140 5000 S; 438300 4000 W
198130 S; 438350 W
198080 S; 438410 W
198150 S; 438330 W
208240 S; 438290 W
198480 5000 S; 438400 3100 W
188140 3600 S; 438380 0500 W
Borba 520
Semir & Vitta s.n.
Borba 521
Borba 502
Semir & Vitta s.n.
Semir & Vitta s.n.
Semir & Vitta s.n.
Borba 522
Borba & Lucca 509
Borba 523
P. ochreata
GraÄo Mogol-MG
Morro do ChapeÂu-BA
Jacobina-BA
Camocim de SaÄo FeÂlix-PE
GM
MC
JC
CS
15
12
06
06
168330 S; 428540 W
118330 S; 418090 W
118400 2300 S; 408400 1700 W
88210 S; 358450 W
Borba 505
Solferini & Vaccarelli s.n.
Romero et al. 5657
Semir s.n.
P. fabiobarrosii
GraÄo Mogol-MG
GM
13
168330 S; 428540 W
Borba & Felix 512
P. adamantinensis
GraÄo Mogol-MG
Diamantina-MG
GM
DI
10
07
168330 S; 428540 W
188140 S; 438380 W
Borba & Felix 550
Borba 551
516
517
518
507
511
& Lucca 504
519
Vouchers are deposited at UEC.
occur]. Great care was taken to collect individuals from
di€erent clones, since these species can reproduce vegetatively. We used a total of 301 individuals of the ®ve species.
The individuals were maintained in a glasshouse at
Universidade Estadual de Campinas, SaÄo Paulo state,
Brazil (228490 S, 478060 W) for at least 6 months before
beginning experiments. The climate experienced by wild
populations of these species is similar to that of Campinas,
Cwb in KoÈeppen's (1948) classi®cation. Vouchers are
deposited in the herbarium of the Universidade Estadual
de Campinas (UEC; Table 1).
Seven (six for P. teres and P. fabiobarrosii) experimental
hand-pollination treatments were carried out for each
species as the pollen receptor: self-pollination; intraspeci®c
intrapopulation cross-pollination; intraspeci®c interpopulation cross-pollination; and interspeci®c cross-pollination
with each of the other four species. Interspeci®c crosses were
not carried out between P. teres and P. fabiobarrosii due to
the lack of sucient ¯owers in the latter species. Only
recently opened ¯owers were used. The order in which the
treatments were applied in each in¯orescence was random,
and we did not perform more than six pollinations in a single
in¯orescence (the highest number of fruits in a single
in¯orescence in natural populations). The number of crosses
varied among treatments and species, depending on the
availability of ¯owers. Fruit initiation and development was
monitored until fruits were mature (3±5 months depending
on the species). We tested for di€erences in mean fruit set
among the three intraspeci®c treatments within each species
(self-pollination, intrapopulation and interpopulation crosspollination), and the same intraspeci®c treatment among
di€erent species. Data were arcsin transformed, and oneway ANOVA was performed with further Tukey multiple
comparisons (Zar, 1999). Besides hand-pollination experiments, more than 50 unpollinated, non-emasculated ¯owers
per population were monitored for the occurrence of
apomixy and spontaneous self-pollination.
We carried out a correlation analysis between genetic
variability and fruit set in intrapopulation cross-pollinations, and between Fis (inbreeding coecient) and fruit set
in intrapopulation cross-pollinations in P. johannensis,
P. teres, and P. ochreata, and for all populations of all
®ve species pooled. Populations of P. adamantinensis and
P. fabiobarrosii were not analysed separately as we only had
data for one population of each. We used observed mean
heterozygosity (Ho) from allozyme data as a measure of
genetic variability [data from Borba et al., (2001b) in which
the same individuals of these populations were used]. The
inbreeding coecient, which is a measure of the reduction
in heterozygosity due to non-random mating within a
population (Wright, 1978), was calculated as (He ÿ Ho)/
He , where He is the expected mean heterozygosity (data
Borba et al.ÐMating Systems and Interspeci®c Compatibility in Pleurothallis (Orchidaceae)
from Borba et al., 2001b). All data were arcsin transformed
before calculating the correlation coecient; its signi®cance
was tested using table B.17 in Zar (1999).
Bidirectional interspeci®c crosses between each pair of
species were pooled to construct a matrix that was
compared to a matrix of genetic similarity of these species
( from Borba et al., 2001b) using the Mantel test in PCOrd
4.10 (McCune and Me€ord, 1999). Data were randomized
with a Monte Carlo test. The estimated fruit set following
cross-pollination in each species was calculated by pooling
results for intra- and interpopulation crosses.
We carried out a preliminary seed viability test, placing
fresh seeds in a 1 % solution of 2,3,5-triphenyltetrazolium
chloride. All well-developed embryos stained red, indicating
viable seeds; none of the rudimentary embryos were stained.
Given these resuts, seeds from mature fruits were removed
after the start of dehiscence and ®xed in 50 % FAA. A
sample of 200 seeds per fruit was scored for viability on a
morphological basis only: seeds with well-developed
embryos were considered viable, and seeds with no embryo
or a rudimentary embryo were considered inviable.
Mature fruits, aborted fruits, and ¯owers that did not
begin fruit development were ®xed in 50 % FAA. These
were softened in 8N NaOH at 60 8C for 60 min ( fruits) or
30 min ( ¯owers), washed in distilled water and then stained
with aniline blue, squashed and observed by epi¯uorescence
microscopy (modi®ed from Martin, 1959) to examine
pollen tube growth and ovule/seed morphology.
R E S U LT S
Fruit set
Intraspeci®c pollinations. Fruits were not produced from
spontaneous sel®ng or apomixy in any individual of any of
the ®ve species studied; thus pollinator visits are essential
for fruit formation. We carried out a total of 2741
experimental crossings, with 1907 intraspeci®c and 834
interspeci®c pollinations. Flowers that did not begin fruit
development withered and fell about 7±10 d after pollination. Fruits ripened after about 3 (P. johannensis and
P. adamantinensis), 4 (P. ochreata and P. teres), or 5
(P. fabiobarrosii) months. A few fruits did not ripen, and
aborted during development. Fruit set following experimental pollinations is summarized in Table 2.
Fruit set ranged from 0 to 38.5 % per population and
from 4.2 to 12.6 % per species following self-pollination,
from 21.1 to 80.8 % per population and 39.4 to 80.8 % per
species following intrapopulation cross-pollination and
from 33.3 to 82.7 % per population and 44.43 to 77.8 %
per species after interpopulation intraspeci®c crosspollination (Table 2). Considerable variation was observed
among di€erent populations of the same species. Populations with the highest fruit set following self-pollination did
not necessarily have the highest fruit set following
intraspeci®c cross-pollination. Fruit set was higher following intraspeci®c cross-pollination (both intra- and interpopulation crosses) than following self-pollination in all
populations: it was 3.2 to 5.3 times higher in P. johannensis,
P. teres and P. ochreata, and 14.8 to 19.2 times higher in
91
P. adamantinensis and P. fabiobarrosii. These values are all
statistically signi®cant (all values of P 5 0.01, except
intrapopulation crosses for P. ochreata with P ˆ 0.021;
Table 2). Signi®cant di€erences between intra- and interpopulation intraspeci®c cross-pollination did not occur in
any of the species (all values of P 4 0.99). The three
intraspeci®c treatments did not di€er signi®cantly among
di€erent species (all values of P 4 0.38).
There were no signi®cant correlations between genetic
variability and fruit set in intrapopulation cross-pollinations in P. johannensis (r ˆ 0.174, d.f. ˆ 5, P 4 0.05),
P. teres (r ˆ 0.443, d.f. ˆ 4, P 4 0.05), and P. ochreata
(r ˆ ±0.738, d.f. ˆ 2, P 4 0.05), or in pooled populations
of all ®ve species (r ˆ 0.002, d.f. ˆ 17, P 4 0.05; Fig. 1A).
Nor were correlations between the inbreeding coecient
and fruit set in intrapopulation cross-pollinations in
P. johannensis (r ˆ 0.056, d.f. ˆ 5, P 4 0.05), P. teres
(r ˆ 0.588, d.f. ˆ 4, P 4 0.05), P. ochreata (r ˆ ÿ0.037,
d.f. ˆ 2, P 4 0.05) or pooled populations of all ®ve species
(r ˆ 0.014, d.f. ˆ 17, P 4 0.05; Fig. 1B) signi®cant.
Interspeci®c pollinations. Fruit set following interspeci®c
pollination was variable among di€erent pairs of species and
di€erent populations of a single species, as noted for intraspeci®c crosses (Table 2). In all species, fruit set following
interspeci®c crosses was always higher than that following self-pollination and was usually lower than that
following intraspeci®c cross-pollinations, except in
P. adamantinensis P. johannensis, P. fabiobarrosii P. adamantinensis, and in the majority of the crosses in
which P. ochreata was the pollen receptor. The di€erence in
fruit set between reciprocal crosses was high in ®ve of the
nine pairs of species crossed; these crosses always involved
P. adamantinensis or P. ochreata. However, some of the
lowest di€erences were found in reciprocal crosses between
these two species (Table 2).
A matrix of the pooled bidirectional interspeci®c crosses
is presented in Table 3, and a matrix of genetic similarity
based on allozyme data is given in Table 4 ( from Borba
et al., 2001b). The Mantel test shows no signi®cant
correlation between genetic similarity and fruit set in
interspeci®c crosses in these species (P ˆ 0.261).
Seed viability
We scored 1014 fruits (689 from intraspeci®c crosses and
325 from interspeci®c crosses) for seed viability, with a total
of 202 800 seeds. Fruits from intra- and interpopulation
intraspeci®c cross-pollinations had a high percentage of
viable seedsÐover 80 % on average (Fig. 2A). There was
little di€erence between these two treatments or among
species. The percentage of viable seeds in fruits produced as
a result of self-pollination was much lower (usually less
than 30 %) than that observed following intraspeci®c crosspollinations; variation among fruits was also greater. The
majority of seeds in self-pollinated fruits had rudimentary
or partially developed embryos or no embryo at all
(Fig. 3A). Seed viability in fruits from interspeci®c crosses
was similar to that of intraspeci®c cross-pollinations
(Fig. 2).
92
Borba et al.ÐMating Systems and Interspeci®c Compatibility in Pleurothallis (Orchidaceae)
T A B L E 2. Percentage fruit set in experimental crosses in Pleurothallis johannensis, P. teres, P. ochreata, P. adamantinensis,
and P. fabiobarrosii
Species/population
self
intrapop
interpop
X joh
X ter
X och
X ada
18.8 (16)
59.1 (22)
30.0 (20)
6.1 (33)
45.0 (20)
28.6 (21)
79.2 (24)
56.1 (41)
37.6 (133)
40.0 (40)
44.8 (105)
53.8 (13)
53.8 (13)
P. johannensis
C1
C2
C3
IB
IT
NZ
SJ
Mean
5.9
8.3
13.6
19.1
10.3
4.5
19.4
12.4
(34)
(24)
(22)
(47)
(29)
(22)
(31)
(209)Aa
56.7
63.3
50.0
46.0
62.9
56.0
81.3
59.0
(30)
(30)
(20)
(50)
(35)
(25)
(32)
(222)Ab
80.6
82.7
75.0
48.1
77.2
50.0
55.6
66.0
P. teres
CA
CC
CT
DI
OP
RM
Mean
20.0
38.5
12.9
17.6
4.3
0.0
12.6
(20)
(13)
(31)
(17)
(47)
(10)
(159)Aa
71.4
58.8
46.4
45.5
21.1
70.0
44.3
(21)
(17)
(28)
(22)
(57)
(10)
(167)Ab
82.4 (51)
33.3 (9)
42.9
60.6
41.2
33.3
52.9
(49)
(33)
(85)
(18)
(240)Ab
47.6 (21)
33.3 (42)
11.8 (17)
41.7 (84)
12.9 (31)
25.0 (20)
41.4 (133)
23.1 (78)
18.9 (37)
P. ochreata
CS
GM
JC
MC
Mean
35.0
9.7
0.0
0.0
12.4
(20)
(31)
(13)
(20)
(89)Aa
57.1
36.8
26.1
40.0
39.4
(21)
(38)
(23)
(50)
(132)Ab
45.5
48.0
37.5
42.4
44.3
(44)
(50)
(16)
(66)
(176)Ab
P. adamantinensis
DI
GM
Mean
5.0 (20)
4.2 (24)
80.8 (26)
80.8 (26)
P. fabiobarrosii
GM
Mean
5.3 (19)
5.3 (19)
78.6 (14)
78.6 (14)
(36)
(52)
(20)
(108)
(123)
(46)
(27)
(412)Ab
77.8 (9)
77.8 (9)
77.8 (18)
X fab
45.5 (11)
41.7 (12)
82.6 (86)
59.5 (42)
40.5 (42)
66.7 (9)
63.2 (19)
79.0 (105)
55.6 (27)
58.0 (69)
40.5 (42)
66.7 (9)
90.9 (11)
90.9 (11)
35.0 (20)
38.1 (21)
45.0 (20)
39.1 (23)
36.4 (11)
36.4 (11)
55.6 (9)
55.6 (9)
36.4 (11)
36.4 (11)
100.0 (12)
100.0 (12)
Self, self-pollination; intrapop, intrapopulation cross-pollination; interpop, interpopulation intraspeci®c cross-pollination; X joh, X ter, X och,
X ada, X fab, interspeci®c pollination with P. johannensis, P. teres, P. ochreata, P. adamantinensis and P. fabiobarrosii as pollen donors,
respectively. See Table 1 for abbreviations of the populations. Sample size in parentheses. For populations in which less than ®ve crosses were
made, percentage fruit set is not given, but these crosses were used in the calculation of the overall mean of the species. Values followed by the
same capital letter in a column or lowercase letter in a row are not signi®cantly di€erent (ANOVA one-way with Tukey test; P 5 0.05; only
intraspeci®c crosses within and among species were tested).
Fruit set (%)
90
A
B
60
30
0
0
0.1
0.2
0.3
0.4
Observed mean heterozygosity (Ho)
0.5
0
0.1
0.2
0.3
0.4
0.5
Inbreeding coefficient (Fis)
F I G . 1. Relationship between fruit set and (A) genetic variability and (B) inbreeding coecient in 17 populations of Pleurothallis johannensis (s),
P. teres (j), P. ochreata (n), P. adamantinensis (), and P. fabiobarrosii (e). Two populations of P. johannensis are superposed in A.
Borba et al.ÐMating Systems and Interspeci®c Compatibility in Pleurothallis (Orchidaceae)
93
T A B L E 3. Matrix of percentage fruit set in experimental crossings among ®ve Pleurothallis species
Species
P.
P.
P.
P.
P.
P. johanensis
P. teres
P. ochreata
P. adamantinensis
P. fabiobarrosii
63.6
39.5
61.9
70.8
39.1
49.4
39.5
25.9
Ð
42.2
40.0
61.1
79.6
69.6
78.6
johanensis
teres
ochreata
adamantinensis
fabiobarrosii
Bidirectional crosses between species pairs were pooled. Values on the diagonal represent pooled intrapopulation and interpopulation results.
T A B L E 4. Matrix of mean genetic similarity among ®ve Pleurothallis species (Nei's, 1978, unbiased genetic identity) using
allozyme data from Borba et al. (2001a)
Species
P.
P.
P.
P.
P.
P. johanensis
P. teres
0.98
0.77
0.65
0.50
0.57
johanensis
teres
ochreata
adamantinensis
fabiobarrosii
P. ochreata
0.89
0.58
0.52
0.60
P. adamantinensis
0.92
0.50
0.66
P. fabiobarrosii
0.97
0.73
0.90
Values on the diagonal represent mean similarity between populations of each species.
A
1.0
1
19
11
1
A
Ax AxA F
7
18 103 251 10
43
62
11
41 111
Ox OxO T
Tx TxT
B
2 10
4
7
9
2
4
5
2 39 45 13
6 76 35 5
43 18
Proportion of viable seeds
0.8
0.6
0.4
0.2
0.0
Fx
J
Jx JxJ
O
Intraspecific crossings
AxF AxJ AxO AxT FxA FxJ FxO JxA JxF JxO JxT OxA OxF OxJ OxT TxA TxJ TxO
Interspecific crossings
F I G . 2. Box-plots of seed viability per fruit in intraspeci®c (A) and interspeci®c (B) experimental crosses involving Pleurothallis johannensis (J),
P. teres (T), P. ochreata (O), P. adamantinensis (A), and P. fabiobarrosii (F). A, F, J, O, T, self-pollination; Ax, Fx, Jx, Ox, Tx, intrapopulation
cross-pollination; AxA, FxF, JxJ, OxO, TxT, interpopulation intraspeci®c cross-pollination. The second letter indicates the pollen donor in
interspeci®c crosses. The number of fruits sampled is shown on the upper axis. The box comprises 50 % of the data and the central line marks the
median. Inner and outer fences are de®ned by interquartile ranges. Asterisks are outlying values and circles indicate extreme outliers (SYSTAT,
1992).
Seeds with two embryos (three embryos in one case)
were occasionally observed in the majority of treatments
(Table 5). These embryos were usually less developed
than normal embryos but were apparently viable. These
polyembryo seeds rarely exceed 2 % (up to 6 %) of
the total seeds in a fruit. Two-embryo seeds were
practically absent in self-pollinated fruits (in which
seeds rarely ever had any embryo) or following interspeci®c crosses in which P. fabiobarrosii was the pollen
donor, but were frequent when this species was the pollen
receptor (including intraspeci®c crosses). No pattern could
be detected among di€erent treatments or among species
in relation to the percentage of fruits with two-embryo
seeds.
94
Borba et al.ÐMating Systems and Interspeci®c Compatibility in Pleurothallis (Orchidaceae)
F I G . 3. A, Seeds with aborted rudimentary embryo (black arrow), seeds with small aborted embryo (white arrow), seeds with two embryos
(arrowhead), and seeds with a well-developed embryo (P. johannensis; interpopulation intraspeci®c cross). B and C, Anomalous pollen tube
growth in the stylar canal of P. teres (self-pollination). D, Normal pollen tubes in the stylar canal of P. johannensis (intrapopulation crosspollination). E, Normal pollen tubes and normal seeds (arrow) in the ovary of P. johannensis (interpopulation intraspeci®c cross). F, Anomalous
pollen tubes and seeds lacking an embryo in the ovary of P. johannensis (self-pollination). Bar ˆ 1 mm.
Pollen tube growth
Pollen grain germination usually occurred on stigmas
even when no fruit was formed or initiated. The absence of
pollen germination was rare. In all experimental treatments,
morphologically normal pollen tubes grew down the stylar
canal, to the middle of the column. Thereafter, in ¯owers
which did not start fruit development, they became
irregular, ®lled with callose and usually had a dilated tip
which sometimes burst and liberated the cytoplasm; growth
ceased near the base of the column (Fig. 3B, C; Fig. 3D for
a comparison with normal pollen tubes in the stylar canal).
These pollen tubes never reached the ovary. Only placental
primordia were visible in this phase, and ovary expansion
did not occur.
Borba et al.ÐMating Systems and Interspeci®c Compatibility in Pleurothallis (Orchidaceae)
95
T A B L E 5. Percentage of fruits with two-embryo seeds in experimental crossings in Pleurothallis johannensis, P. teres,
P. ochreata, P. adamantinensis and P. fabiobarrosii
self
P.
P.
P.
P.
P.
johanensis
teres
ochreata
adamantinensis
fabiobarrosii
11.1
0
0
0
0
(18)
(11)
(10)
(1)
(1)
intrapop
interpop
26.2
17.1
20.9
26.3
42.9
28.3
28.8
14.5
27.3
(103)
(41)
(43)
(19)
(7)
(251)
(111)
(62)
(19)
X joh
11.4
18.2
50.0
0
(44)
(77)
(10)
(2)
X ter
X och
X ada
X fab
33.3 (45)
43.6 (9)
55.6 (18)
0 (5)
40.0 (5)
7.7 (13)
0 (2)
34.3 (35)
28.6 (7)
25.0 (4)
75.0 (4)
44.4 (9)
0 (6)
0 (2)
Self, self-pollination; intrapop, intrapopulation cross-pollination; interpop, interpopulation intraspeci®c cross-pollination; X joh, X ter, X och,
X ada, X fab, interspeci®c pollination with P. johannensis, P. teres, P. ochreata, P. adamantinensis and P. fabiobarrosii as pollen donors,
respectively. Sample size in parentheses.
Pollen tubes observed in fruits with a high percentage of
viable seeds were normal during the extension process
(Fig. 3E). Pollen tubes persisted and could be stained so
that indicators of fertilization were observable even after
fruit ripening. Even fruits with a high percentage of seeds
lacking embryos showed normal pollen tube growth to the
placenta, at which point many of the tubes became
irregular, ®lled with callose and formed a tangled mass
around the ovules (Fig. 3F). A large number of unfertilized
ovules and aborted seeds were visible. The greater the
proportion of inviable seeds, the greater the proportion of
anomalous pollen tubes, which were more frequent following self-pollination. Pollen tubes in aborted fruits showed
the same morphology as abnormal tubes in fruits with low
seed viability.
DISCUSSION
Intraspeci®c pollinations
Classic self-incompatibility and self-compatibility are
extremes of a continuum between which there is often no
clear di€erence. As relatively few species ®t these extremes
exactly (Schemske and Lande, 1985), many authors have
utilized di€erent indices, generally comparing seed set
between sel®ng and outcrossing, to determine whether a
particular species is self-compatible or self-incompatible
(Bawa, 1974, 1979; Zapata and Arroyo, 1978; Sobrevilla
and Arroyo, 1982; Jaimes and Ramirez, 1999). The
distinction between the two conditions is, however,
arbitrary, and various authors have used di€erent values.
Some populations or species in this study, namely
P. johannensis, P. teres and P. ochreata, could be considered
to be self-incompatible or self-compatible depending on
which criterion is employed. However, all these species
showed pollen tube reactions similar to those observed in
species with homomorphic gametophytic self-incompatibility (de Nettancourt, 1977; Dafni and Calder, 1987; Murfett
et al., 1996; Richards, 1997) and were homogeneous in the
timing of the reaction in the stylar canal. These characteristics lead us to suggest the occurrence of weak or partial
self-incompatibility in these three species. Conversely,
P. adamantinensis and P. fabiobarrosii can be clearly
considered self-incompatible.
Recently, considerable discussion has taken place over the
occurrence of inbreeding depression or late-acting selfincompatibility, especially in cases in which post-zygotic
rejection occurs. The high number of seeds with no embryo
or with aborted embryos in these Pleurothallis species may
be due to inbreeding depression. This suggestion is based on
the great variation in the number of viable seeds in selfpollinated fruits, together with di€erences in the size of
aborted embryos, indicating di€erences in the timing of
embryo death. Embryo abortion due to late-acting selfincompatibility is likely to occur at the same time, thus
aborted embryos should be uniform in size. In contrast,
inbreeding depression usually causes abortion at several
stages, as well as a greater variation in seed set (Seavey
and Bawa, 1986; Barrett, 1988; Gibbs et al., 1999; Lewis
and Gibbs, 1999; Sage et al., 1999; but see Mulcahy and
Mulcahy, 1983).
The presence of abnormal pollen tubes in the ovaries of
aborted fruits or in fruits containing a high percentage of
aborted seeds is more dicult to explain. Di€erences in
pollen tube morphology between successful crosses and
incompatible crosses in species exhibiting late-acting selfincompatibility, with rejection after micropylar penetration
or post-zygotic rejection, are not common (James and
Knox, 1993; Humeau et al., 1999; Lipow and Wyatt, 1999;
Sage et al., 1999), and such di€erences would not be
expected with inbreeding depression either. There may be a
third mechanism of rejection in the ovary in these
Pleurothallis species. Some authors have employed the
expression `pistillate sorting' to explain instances in which
inbreeding depression occurs in association with selfincompatibility system(s), making a conclusive analysis
dicult (Bertin et al., 1989; Gibbs and Sassaki, 1998).
Diallelic crosses in progeny arrays are necessary for a more
precise evaluation of the mechanisms involved in fruit
failure and embryo abortion of these Pleurothallis species as
well as in other orchid species presenting complex selfrejection mechanisms (Borba et al., 1999), but they are
unlikely to be carried out due to the long period required
for maturation (about 5±8 years).
The level of inbreeding depression observed in all ®ve
Pleurothallis species is usually found in exclusively allogamous populations, being low as a rule in partially inbreeding
populations, indicating that these Pleurothallis species may
be mainly outcrossing in natural populations (Stebbins,
96
Borba et al.ÐMating Systems and Interspeci®c Compatibility in Pleurothallis (Orchidaceae)
1974; Lande and Schemske, 1985; Schemske and Lande,
1985; Charlesworth and Charlesworth, 1987; Wiens et al.,
1987; Husband and Schemske, 1996; Byers and Waller,
1999; but see Charlesworth and Charlesworth, 1990, and
Charlesworth et al., 1990). Similar inbreeding depression at
early stages of development (embryo abortion) has been
reported for several orchid species (Lock and Pro®ta, 1975;
Stort and Martins, 1980; Catling, 1982; Stort and Galdino,
1984; Matias et al., 1996), and it may be widespread in the
family. Besides being highly expressed during zygote
formation and embryo development, inbreeding depression
in angiosperms continues to be expressed in subsequent life
stages of the plant, mainly growth and reproduction (Seavey
and Bawa, 1986; Husband and Schemske, 1996; Culley et al.,
1999; Daehler, 1999; Kittelson and Maron, 2000). It is likely
that only a few reproductive individuals are ever produced
by self-pollination in these Pleurothallis species under
natural conditions.
Self-incompatibility, inbreeding depression and genetic
variability
The absence of any correlation between heterozygosity
(Ho) or the inbreeding coecient (Fis) and fruit set indicates
that the relatively low fruit set in some populations is not
due to crosses beween close relatives, as might be expected
(Lipow and Wyatt, 1999). The similarities in fruit set and
seed viability between intra- and interpopulation crosses
support this conclusion. It is possible that these crosses
show similar levels of fruit set for di€erent reasons, with
fruit set being low in intrapopulation crosses due to crosses
between relatives and in interpopulation crosses due to
population di€erentiation. This explanation seems unlikely
because of the high genetic similarities among these
populations (Borba et al., 2001b). Variation in self-fertility
among populations was relatively high in some species,
especially in P. teres, and similar results have been found in
other plant species (e. g. Lipow et al., 1999).
Mating systems have been postulated to be one of the
most important factors determining the genetic variability
in plant species (Hamrick et al., 1979; Hamrick, 1982;
Hamrick and Godt, 1990; Maki et al., 1999; Wong and
Sun, 1999; Evans et al., 2000). We believe that both partial
self-incompatibility and inbreeding depression are responsible for the maintenance of the high genetic variability
found in these Pleurothallis species (Borba et al., 2001b)Ð
levels that are much higher than previously reported for
other Orchidaceae (Scacchi and De Angelis, 1989; Schlegel
et al., 1989; Scacchi et al., 1990; Corrias et al., 1991; Klier
et al., 1991; Case, 1994; Wong and Sun, 1999).
Orchids are usually considered self-compatible, with
autogamy prevented mainly by herkogamy and pollinator
behaviour (Lenz and Wimber, 1959; Pijl and Dodson, 1966;
Dressler, 1981, 1993; Gill, 1989; Paulus and Gack, 1990;
Nilsson, 1992) or less frequently by several pre-pollination
mechanisms (Pijl and Dodson, 1966; Adams and Goss,
1976; Stoutamire, 1978; Ackerman and Mesler, 1979;
Catling and Catling, 1991a; Borba and Semir, 1998b,
1999; Singer and Cocucci, 1999). However, exceptions have
been found in some orchid groups, in which genetic barriers
are responsible for low inbreeding (Stoutamire, 1975;
Agnew, 1986; Johansen, 1990; Christensen, 1992; Dressler,
1993; Pedersen, 1995). These genetic and mechanical
barriers are mainly associated with plants pollinated by
insects that often remain on the ¯owers and in¯orescences
for a long time, promoting geitonogamy, as in these
Pleurothallis species (Borba and Semir, 2001) and other
myophilous species (Christensen, 1992; Pedersen, 1995;
Borba and Semir, 1998b, 1999; Singer and Cocucci, 1999).
Unfortunately, we know of no other study reporting genetic
variability in other self-incompatible members of the
Orchidaceae, thus we cannot make comparisons among
di€erent strategies.
Polyembryony
Polyembryony has been reported in the Orchidaceae and
is almost always associated with apomixy, the embryo being
derived from the inner integument (Swamy, 1949; Lenz and
Wimber, 1959; Catling, 1982, 1987; Catling and Catling,
1991b). In all these cases, a higher percentage of multipleembryo seeds was reported (28±95 %) than was found for
the Pleurothallis species in this study. Interspeci®c crosses
usually lead to higher rates of apomictic embryo formation
(O'Neill, 1997); however, we did not ®nd any clear
di€erence in the percentage of polyembryo seeds between
intra- and interspeci®c crosses. PintauÂdi et al. (1990)
reported the presence of a few rare two-embryo seeds in
non-apomictic fruits of Xylobium squalens Lindl. (Orchidaceae). In that case, the second embryo originated from a
division of the zygote. These characteristics, in addition to
the fertilizations we observed, lead us to suppose that the
extra-numerary embryos found in Pleurothallis may have a
similar origin to those of Xylobium, and are unlikely to be
the result of apomixy.
Interspeci®c crossing potential
Some studies have shown a positive correlation between
genetic similarity or phylogeny and interspeci®c crossing
potential (Sanford, 1964, 1967; Johansen, 1990; Scacchi
et al., 1990; Borba et al., 1999), failure in crosses being
caused by evolutionary divergence between species (Hogenboom, 1975). However, we found no correlation between
these variables in our study. We also found no correlation
between genetic reproductive isolation and sympatry, which
has been reported for some species (Coyne, 1992).
The Pleurothallis species studied here ®t the general model
proposed for Orchidaceae in which related species are
usually potentially interfertile and reproductive isolation is
mainly assured by pre-pollination barriers (Lenz and
Wimber, 1959; Dodson, 1962; Pijl and Dodson, 1966;
Dressler, 1968, 1981, 1993; Steiner et al., 1994; Borba and
Semir, 1998a, b, 1999; Silva et al., 1999). The Pleurothallis
species that occur sympatrically attract di€erent groups of
pollinators (Borba and Semir, 2001), and mechanical barriers prevent hybridization in the few cases in which the
pollinator of one species visits another. Although P. ochreata
and P. teres occur in the same mountain formation in Minas
Gerais (the EspinhacËo Range), they have never been found
Borba et al.ÐMating Systems and Interspeci®c Compatibility in Pleurothallis (Orchidaceae)
in sympatric populations. However, hybridization might be
expected to occur if these species come into contact as they
are interfertile, attract the same pollinators, and ¯ower
synchronously (Borba and Semir, 2001).
Interpopulation crossing potential in P. ochreata
The GraÄo Mogol population of P. ochreata is the only
one known in the state of Minas GeraisÐthe species occurs
almost exclusively in the northeastern region of Brazil,
mainly Bahia state (see Fig. 1 of Borba and Semir, 2001).
This population shows some di€erentiation in chemical
(Borba et al., 2001a) and leaf traits, but allozyme data do
not support its segregation as a new species (Borba et al.,
2001b). This view is strengthened by data on ¯oral biology
(Borba and Semir, 2001), together with these results
showing intercompatibility among all the populations of
this species.
AC K N OW L E D GE M E N T S
We thank Julie H. A. Dutilh, Vera N. Solferini, Eliana R.
F. Martins, Mark W. Chase, Je€ Ollerton and Peter E.
Gibbs for improvements to the manuscript, Juliana M.
Felix, Veridiana N. Vaccarelli, MaÂrcio Lucca and Graciela
S. Oliver for help on ®eld trips, Angela B. Martins and
Rosana Romero for providing specimens of P. ochreata,
and Geraldo W. Fernandes for permission to collect
samples on his ranch. ELB received a fellowship from the
FundacËaÄo de Amparo aÁ Pesquisa do Estado de SaÄo Paulo
(FAPESP #97/08800-1). This study was funded by a grant
from FAPESP to JS (#97/08795±8).
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