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Keywords:
Abstract
comparative analysis;
evo-devo;
microsporogenesis;
morphospace;
pollen aperture pattern.
Although much attention has been paid to the role of stabilizing selection,
empirical analyses testing the role of developmental constraints in evolutionary stasis remain rare, particularly for plants. This topic is studied here with a
focus on the evolution of a pollen ontogenetic feature, the last points of callose
deposition (LPCD) pattern, involved in the determination of an adaptive
morphological pollen character (aperture pattern). The LPCD pattern exhibits
a low level of evolution in eudicots, as compared to the evolution observed in
monocots. Stasis in this pattern might be explained by developmental
constraints expressed during male meiosis (microsporogenesis) or by selective
pressures expressed through the adaptive role of the aperture pattern. Here,
we demonstrate that the LPCD pattern is conserved in Euphorbiaceae s.s. and
that this conservatism is primarily due to selective pressures. A phylogenetic
association was found between the putative removal of selective pressures on
pollen morphology after the origin of inaperturate pollen, and the appearance
of variation in microsporogenesis and in the resulting LPCD pattern,
suggesting that stasis was due to these selective pressures. However, even in
a neutral context, variation in microsporogenesis was biased. This should
therefore favour the appearance of some developmental and morphological
phenotypes rather than others.
Introduction
Given the Darwinian process of descent with modification, evolutionary change is to be expected as a common
occurrence. Although such evolutionary change occurs in
many cases, several examples of lineages exhibiting little
morphological change for an individual character, for
character complexes or for the whole organism are known
(Futuyma, 2010). It may be inferred that a character
exhibits little morphological change in a lineage when the
rate of change of the character is slower than it theoretically could be (see Bradshaw, 1991; Williams, 1992;
Futuyma, 2010 for examples). The amount of morphological variation of a character can be visualized by
determining the theoretical morphospace of all the posCorrespondence: Alexis Matamoro-Vidal, Department of Biological Science,
Florida State University, Tallahassee, FL 32310, USA.
Tel.: +1 850 645 8521; fax: +1 850 645 8447; e-mail: amv@bio.fsu.edu
sible states that can exist for the character and then
looking at how the morphospace has been filled during the
course of evolution (Raup, 1966; McGhee, 2007). Morphospaces are delineated by the fact that all the members
of a lineage have inherited a limited number of developmental modules (Breuker et al., 2006; Muller, 2007;
Wagner et al., 2007) and that the morphological variation
allowed by these modules is finite (Lauder, 1981; Wake &
Larson, 1987; Raff, 1996; Salazar-Ciudad, 2006; Schlosser,
2007). Therefore, the evolution of a character may only
occur within the limits allowed by these modules unless
new modules are acquired and added to the previous ones.
The utilization of morphospace allows testing for the
occurrence of stasis or phylogenetic conservatism between
related lineages that share common developmental modules. If it is found that, for a given character, a lineage
explored a smaller subset of the morphospace than
another lineage, it may be stated that this character
exhibits stasis in the former relative to the latter.
1077
1078
A. MATAMORO-VIDAL ET AL.
1079
(a) Tetragonal
4 cleavage planes
View
1
Centripetal according to the
cleavage plane = CpP
2
Centripetal according to the
tetrad = CpT
Whole tetrad
1
4
Section
(b) Rhomboidal
5 cleavage planes
Whole tetrad
1
2
5
4
Section
(c) Tetrahedral
6 cleavage planes
Whole tetrad
5
Section
(planes 4, 5, 6 only)
Fig. 1 Tetrad forms and modes of callose deposition accounting for six different patterns for the number and position of the last points of
callose deposition (LPCD). Only centripetal callose deposits via simultaneous cytokinesis are represented, for tetragonal (a), rhomboidal (b)
and tetrahedral (c) tetrads. These three tetrad configurations differ in the number of cleavage planes of callose that are synthesized to separate
the microspores during microsporogenesis (46 planes, respectively, for tetragonal, rhomboidal and tetrahedral tetrads). Numbering of planes
is arbitrary for tetragonal and rhomboidal tetrads. For tetrahedral tetrads, planes numbered 13 are those that are roughly parallel to the image
plane, and planes numbered 46 are those that are orthogonal to the image plane. For each tetrad form, the formation of the cleavage planes is
illustrated according to the mode of callose deposition. 1 Callose deposition centripetal according to the cleavage plane (abbreviated CpP):
callose begins to be deposited at the border of each plane and progresses towards the planes centre. 2 Callose deposition centripetal according
to the tetrad (abbreviated CpT): callose begins to be deposited at the border of the tetrad and progresses towards the tetrads centre. For
each tetrad form mode of callose deposition combination, the formation of the cleavage planes is illustrated with a view of the whole tetrad
and with a view of a section of the tetrad, to help with the interpretation of photographs in Figs 46. The section represented is in the plane
delimited by the red points (seen as dark grey points in print edition) that figure on the view of the whole tetrad. Callose is represented in blue
(seen as grey surfaces in print edition). Arrows on the view of the section indicate the direction of callose progression. White areas on whole
tetrads are the places where callose was not yet deposited at the stage represented in this scheme. For each LPCD pattern, a pollen grain (grey
sphere) with the corresponding aperture pattern is illustrated. According to Ressayre et al. (2002a), an aperture (coloured black) is expected to
occur in each area of the pollen adjoining the areas where callose is deposited last.
1080
A. MATAMORO-VIDAL ET AL.
2002; Sagun et al., 2006). For the study of microsporogenesis, a data set of microsporogenesis features was
obtained using information from the literature for 16
species and our own observations for 17 additional
species (Table 1). Data on microsporogenesis were
obtained for aperturate species representing all the major
clades of the family. For inaperturate species, data were
obtained only for species of the inaperturate crotonoids.
Data were not obtained for species of tribe Plukenetieae
(clade A8) because these were not available from the
living collections.
For the description of microsporogenesis, fresh material was obtained from the living collections of botanical
gardens or from plants cultivated in a greenhouse at the
Laboratoire Ecologie, Systematique, Evolution, Orsay,
France (Table 1). Anthers were dissected from unopened
buds of various sizes to find the ontogenetic stages
corresponding to callose deposition and pollen assembled
in mature tetrads. Anthers were squashed on a slide and
mounted with aniline blue solution (aniline blue, 1 g L)1;
K3PO4, 21.2 g L)1; glycerol 187.4 g L)1), according to a
protocol modified from the study of Arens (1949). Aniline
blue staining causes fluorescence of the intersporal
callose walls when illuminated with UV light. Slides
were observed under a Zeiss Axiophot fluorescence
microscope (DAPI filter, excitation at 345 nm, emission
at 425-nm-long pass). For each species, pollen morphology, tetrad form, cytokinesis type and the mode of callose
deposition during intersporal wall formation were recorded. When more than one tetrad configuration was
observed for a given species, a survey of the proportion of
each tetrad form was carried out by counting a number of
tetrads ranging from c. 200 to c. 300 per plant. Chi-square
tests were performed using R (R Development Core Team,
2008) to test (i) whether each tetrad form was produced in
similar proportions within plants and (ii) whether the
distribution of the proportions of tetrad forms was equivalent among the heteromorphic species (i.e. species for
which individuals produce more than one form of tetrad).
Then, a phylogenetic framework was used to study the
evolution of microsporogenesis in relation to aperture
pattern.
Phylogenetic analysis
Phylogenetic relationships of the Euphorbiaceae s.s.
species for which we acquired data on pollen morphology
and microsporogenesis were obtained using DNA
sequences of rbcL and trnL-F genes (Wurdack et al.,
2005; Tokuoka, 2007; Sagun, 2008) (Table S1). Although
molecular data were not available for some of our
investigated species, the resulting species sampling is
sufficient to yield statistically significant results with the
test of Pagel (1994). We used the alignment matrix of the
combined data for rbcL and trnL-F in Wurdack et al.
(2005). Taxon sampling was modified by removing taxa
that lacked morphological data and by adding new taxa
Reference
Kapil (1960)
Kapil (1960)
Mukherjee (1964)
Johri & Kapil (1953)
Thathachar (1952)
Mukherjee (1964)
Albert et al. (2009)
Lyon (1898)
Mukherjee (1960)
Kapil (1961) and Murgia et al. (1986)
Mukherjee (1960)
Mukherjee (1965)
Rao & Devi (1974)
Rao (1964)
Liu et al. (2007)
Rao (1962)
1081
1082
A. MATAMORO-VIDAL ET AL.
Table 2 Records of tetrad form, mode of callose deposition and cytokinesis type in species of Euphorbiaceae s.s. investigated, according to their
aperture pattern.
Species
Aperture
pattern
Acalypha alnifolia
Acalypha brachystachya
Acalypha ciliata
Acalypha indica
Acalypha lanceolata
Acalypha malabaria
Bocquillonia spicata
Caperonia serrata
Dalechampia roezliana
Euphorbia corollata
Euphorbia dracunculoides
Euphorbia dulcis
Equatorial
Equatorial
Equatorial
Equatorial
Equatorial
Equatorial
Equatorial
Equatorial
Equatorial
Equatorial
Equatorial
Equatorial
Td
Td
Td
Td
Td
Td
Td
Td
Td
Td
Td
Td
Euphorbia microphylla
Euphorbia peltata
Euphorbia vermiculata
Hevea brasiliensis
Hura crepitans
Macaranga tanarius
Mercurialis annua
Mercurialis perennis
Micrococca mercurialis
Pedilanthus tithymaloides
subsp. tithymaloides
Ricinus communis
Baloghia inophylla
Codiaeum variegatum
Croton californicus
Eremocarpus setigerus
Jatropha curcas
Jatropha gossypiifolia
Jatropha integerrima
Jatropha multifida
Jatropha podagrica
Manihot esculenta
Equatorial
Equatorial
Equatorial
Equatorial
Equatorial
Equatorial
Equatorial
Equatorial
Equatorial
Equatorial
Td ) Tg*
Td
Td
Td
Td
Td
Td
Td
Td ) Dc*
Td
Equatorial
Inaperturate
Inaperturate
Inaperturate
Inaperturate
Inaperturate
Inaperturate
Inaperturate
Inaperturate
Inaperturate
Global
Td
Td
Td
Td
Td
Td
Td
Td
Td
Td
Td
) Tg ) Dc*
) Tg ) Dc*
) Tg ) Dc*
) Tg*
) Tg*
) Tg ) Dc
(70 %) ) Rh (25 %)
) Rh ) Tg
(77 %) ) Rh (19 %)
(51 %) ) Rh (29 %)
) Rh
(89 %) ) Rh (10 %)
(61 %) ) Rh (32 %)
(72 %) ) Rh (24 %)
(83 %) ) Rh (15 %)
(80%) ) Rh (20%)
) Tg (5 %)
) Tg (4 %)
) Tg (19 %)
)
)
)
)
Tg
Tg
Tg
Tg
(1
(7
(4
(2
%)
%)
%)
%)
Callose
deposition
Cytokinesis
Reference Figure
?
?
CpP
Centripetal
Centripetal
Centripetal
CpP
CpP
CpP
?
Centripetal
?
Simultaneous
Simultaneous
Simultaneous
Simultaneous
Simultaneous
Simultaneous
Simultaneous
Simultaneous
Simultaneous
Simultaneous
Simultaneous
?
Centripetal
?
?
Centripetal
CpP
CpP
CpP
CpP
?
CpP
Simultaneous
?
Simultaneous
Simultaneous
Simultaneous
Simultaneous
Simultaneous
Simultaneous
Simultaneous
Simultaneous
Kapil (1960)
Kapil (1960)
Mukherjee (1964)
Johri & Kapil (1953)
Thathachar (1952)
Mukherjee (1964)
Fig. 4
Fig. 4
Fig. 4
Lyon (1898)
Mukherjee (1960)
Kapil (1961); Murgia
et al. (1986)
Mukherjee (1960)
Mukherjee (1965)
Rao & Devi (1974)
Rao (1964)
Fig. 4
Fig. 4
Fig. 4
Fig. 4
Rao (1962)
Fig. 4
CpP
CpP CpT
CpP CpT CfT
CpP
CpP CpT
Centripetal
CpP CpT
CpP CpT
CpP
CpP CpT
CpP CpT
Simultaneous
Simultaneous
Heteromorphic
Simultaneous
Simultaneous
Simultaneous
Simultaneous
Simultaneous
Simultaneous
Simultaneous
Simultaneous
Fig. 4
Fig. 6
Albert et al. (2009)
Fig. 6
Fig. 6
Liu et al. (2007)
Fig. 5
Fig. 5
Fig. 5
Fig. 5
Fig. 6
Td, tetrahedral; Tg, tetragonal; Rh, rhomboidal; Dc, decussate; CpP, centripetal according to the cleavage plane; CpT, centripetal according
to the tetrad; CfT, centrifugal according to the tetrad. * denotes the literature reports on tetrads that have been reinterpreted (see Results).
? denotes missing data.
(simultaneous and successive no taxa were monomorphic for successive); (ii) mode of callose deposition:
centripetal according to the cleavage plane or heteromorphic (centripetal according to the cleavage plane and
centripetal according to the tetrad); and (iii) tetrad form:
tetrahedral or heteromorphic (tetrahedral, rhomboidal
and tetragonal). The resulting data matrix is shown in
Table S1.
Correlation between aperture pattern and
microsporogenesis
To test for correlated evolution between aperture pattern
and microsporogenesis, the aperture pattern was binarycoded (apertures equatorial vs. global absent), as correlation tests can only be performed on binary traits. The
Results
Phylogenetic analysis
Maximum-likelihood analyses resulted in a single tree
with a score of log-likelihood = )19082.20 (Fig. 2). The
phylogeny obtained with our analysis recognized all
the subclades (Adenoclineae s.l.; Articulated crotonoids;
C12; Pseudanthial; H12; Alchorneoids; A18) found in
the two previous studies on Euphorbiaceae s.s. (Wurdack
et al., 2005; Tokuoka, 2007). The three major clades in
the previous studies (Crotonoideae s.l.; Euphorbioideae;
and Acalyphoideae s.s.) also coincided with those of our
analysis except that our analysis suggests the monophyly
of the Crotonoideae s.l., whereas the other two exclude
the Adenoclineae from this group. Crotonogynopsis groups
with the Alchorneoids instead of expected clade A3 (i.e.
Wurdack et al., 2005; Tokuoka, 2007), but this difference
does not affect our conclusions. A substantial difference is
in the relationships between the major clades. The
Wurdack et al. (2005) analysis grouped Euphorbioideae
1083
77
56
100
73
87
67
100
96
80
98
100
99
98
50
66
76
99
98
100
100
72
52
100
Strophioblachia fimbricalyx
Codiaeum variegatum
Sagotia racemosa
Sandwithia guyanensis
Joannesia princeps
Jatropha integerrima
Jatropha curcas
Jatropha gossypiifolia
Jatropha podagrica
Beyeria leschenaultii
Baloghia inophylla
Tannodia cordifolia
Domohinea perrieri
Ostodes paniculata
Dodecastigma amazonicum
Ricinodendron heudelotii
Baliosperm motanum
Plagiostyles africana
Nealchornea yapurensis
Dichostemma glaucescens
Neoguillauminia cleopatra
100
Aleurites moluccana
Garcia nutans
Cavacoa aurea
53
99
Outgroups
Cheilosoideae
Endospermum moluccanum
Omphalea diandra
Ditta myricoides
Suregada africana
Elateriospermum tapos
Glycydendron amazonicum
Hevea brasiliensis
97
Manihot esculenta
Manihot grahami
100
Clutia pulchella
0.03
C2
C1
84
Acalypha brachystachya
Acalypha lanceolata
Acalypha ciliata
Tragia fallax
A3
A2
A4
A8
A7
A6
A5
A1
Pseudanthial
Inaperturate crotonoids
Articulated crotonoids
Gelonieae
Adenoclineae s.l.
Pedilanthus tithymaloides
Euphorbia pulcherrima
Croton californicus
Eremocarpus setigerus
Maprounea guianensis
Homalanthus populneus
100
H1
Gymnanthes lucida
64
Dalembertia poplifolia
94
Pachystroma longifolium
100
Hura crepitans
Colliguaja integerrima
66
H2
Hippomane mancinella
100
99
Grimmeodendron eglandulosum
Bonania cubana
Pseudagrostistachys ugandensis
Cyttaranthus congolensis
84
100
Discoglypremma caloneura
Amyrea sambiranensis
Mareyopsis longifolia
81 70
Alchorneopsis floribunda
Alchorneoids
100 Gavarretia terminalis
Crotonogynopsis usambarica
100
Aparisthmium cordatum
85
100
Alchornea laxiflora
Alchornea cordifolia
100
Macaranga tanarius
98
Trewia nudiflora
Mallotus japonicus
100
Seidelia triandra
Leidesia procumbens
68
Chiropetalum tricoccum
99
Caperonia palustris
Philyra brasiliensis
100
100
Lasiocroton bahamensis
Adelia ricinella
98
Caryodendron orinocense
Adenophaedra grandifolia
83
Plukenetia volubilis
73
82
Dalechampia spathulata
Astrococcus cornutus
Cnesmone javanica
70
97
Platygyna hexandra
Monotaxis grandiflora
Adriana tomentosa
Ricinus communis
Discocleidion rufescens
94
Koilodepas bantamense
Cephalocroton mollis
Chrozophora tinctoria
99
Sumbaviopsis albicans
Melanolepis multiglandulosa
Mercurialis perennis
100
Lobanilia bakeriana
100
69
Micrococca capensis
Claoxylon australe
Homonoia riparia
100
Pycnocoma macrophylla
Argomuellera macrophylla
Mareya micrantha
82
100
91
63
86
96
63
55
76
58
87
84
71
100
Pera bicolor
Neoscortechinia kingii
Klaineanthus gaboniae
Acalyphoideae s.s.
Euphorbioideae
Crotonoideae s.l.
Fig. 2 Relationships of the species of Euphorbiaceae investigated in this study. The tree was obtained from a combined analysis of the markers rbcL and trnL-F using a maximum-likelihood
reconstruction method (log-likelihood = )19 082.20). ML-estimated branch lengths are shown. Numbers next to branches are the maximum-likelihood bootstrap values (only values
> 50 are shown). The names of major clades follow the study of Wurdack et al. (2005).
Euphorbiaceae s.s.
88
Vantanea guianensis
1084
A. MATAMORO-VIDAL ET AL.
(a)
(b)
Gelonieae
Crotonoideae s.l.
1085
Articulated crotonoids
Euphorbiaceae s.s.
Inaperturate
crotonoids
Euphorbioideae
Vantanea guianensis
Pera bicolor
Clutia pulchella
Neoscortechinia kingii
Klaineanthus gaboniae
Endospermum moluccanum
Omphalea diandra
Ditta myricoides
Suregada africana
Elateriospermum tapos
Glycydendron amazonicum
Hevea brasiliensis
Manihot esculenta
Manihot grahami
Croton californicus
Eremocarpus setigerus
Sagotia racemosa
Sandwithia guyanensis
Joannesia princeps
Jatropha integerrima
Jatropha curcas
Jatropha gossypifolia
Jatropha podagrica
Garcia nutans
Cavacoa aurea
Strophioblachia fimbricalyx
Codiaeum variegatum
Aleurites moluccana
Beyeria leschenaultii
Baloghia inophylla
Tannodia cordifolia
Domohinea perrieri
Ostodes paniculata
Dodecastigma amazonicum
Ricinodendron heudelotii
Baliospermum motanum
Plagiostyles africana
Nealchornea yapurensis
Dichostemma glaucescens
Neoguillauminia cleopatra
Pedilanthus tithymaloides
Euphorbia pulcherrima
Maprounea guianensis
Homalanthus populneus
Gymnanthes lucida
Dalembertia populifolia
Pachystroma longifolium
Hura crepitans
Colliguaja integerrima
Hippomane mancinella
Grimmeodendron eglandulosum
Bonania cubana
Pseudagrostistachys ugandensis
Acalyphoideae s.s.
A8
Aperture pattern
Max. parsimony
Max. likelihood
Cyttaranthus congolensis
Discoglypremma caloneura
Amyrea sambiranensis
Mareyopsis longifolia
Alchorneopsis floribunda
Gavarretia terminalis
Crotonogynopsis usambarica
Aparisthmium cordatum
Alchornea laxiflora
Alchornea cordifolia
Macaranga tanarius
Trewia nudiflora
Mallotus japonicus
Seidelia triandra
Leidesia procumbens
Chiropetalum tricoccum
Caperonia palustris
Philyra brasiliensis
Lasiocroton bahamensis
Adelia ricinella
Caryodendron orinocense
Adenophaedra grandifolia
Plukenetia volubilis
Dalechampia spathulata
Astrococcus cornutus
Cnesmone javanica
Tragia fallax
Platygyna hexandra
Monotaxis grandiflora
Adriana tomentosa
Ricinus communis
Discocleidion rufescens
Koilodepas bantamense
Cephalocroton mollis
Chrozophora tinctoria
Sumbaviopsis albicans
Melanolepis multiglandulosa
Mercurialis perennis
Lobanilia bakeriana
Micrococca capensis
Claoxylon australe
Homonoia riparia
Pycnocoma macrophylla
Argomuellera macrophylla
Mareya micrantha
Acalypha brachystachya
Acalypha lanceolata
Acalypha ciliata
Tetrad form
Max. parsimony
Max. likelihood
Equatorial
Tetrahedral
Global
Heteromorphic
Inaperturate
Fig. 3 Mirror tree of Euphorbiaceae s.s. comparing optimizations of the aperture pattern (a) and tetrad form (b). Colour of the boxes near
species names indicates the state of the corresponding character (box absence indicates data absence). Branch colours indicate the ancestral
states obtained using MP optimization. Pie charts on the nodes indicate the ancestral states obtained using ML. Pie chart colours represent the
probability that this node is at a particular state. Analyses were conducted on the phylogram shown in Fig. 2 but are shown here on a
cladogram for clarity. The names of major clades follow the study of Wurdack et al. (2005).
1086
A. MATAMORO-VIDAL ET AL.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
(m)
(n)
(o)
Fig. 4 Pollen morphology and microsporogenesis for nine Euphorbiaceae species producing pollen with equatorial apertures. (a)
(c) Dalechampia roezliana. (a) Triaperturate
pollen (equatorial section). (b) Tetrahedral
tetrad with apertures arranged in Fischers
configuration (i.e. apertures are grouped in
pairs in the tetrad arrows). (c) Planes 13 of
a tetrahedral tetrad during callose wall formation. Callose deposition follows the CpP
mode (Fig. 1c-1, whole tetrad). Callose (ca)
begins to be deposited at the border of each
plane and progresses towards the centre of
the plane (asterisks). At the stage represented here, the centre of each plane remains
empty of callose. (d)(f) Ricinus communis. (d)
Triaperturate pollen (polar view). (e) Tetrahedral tetrad (same configuration as in b). (f)
Planes 46 of a tetrahedral tetrad during callose wall formation. Callose deposition follows the CpP mode (Fig. 1c-1, section).
Callose begins to be deposited at the border
of each plane (arrows the central border is
common to the three planes) and progresses
towards the centre of the plane. (g)(i) Pedilanthus tithymaloides subsp. tithymaloides. (g)
Triaperturate pollen (longitudinal section).
(h) Tetrahedral tetrad (same configuration as
in b). (i) Planes 13 of a tetrahedral tetrad
during callose wall formation. The callose
deposition follows the CpP mode (same
configuration as in c). (j)(l) Mercurialis
annua. (j) Triaperturate pollen (equatorial
section). (k) Tetrahedral tetrad (same configuration as in b). (l) Planes 46 of a
tetrahedral tetrad during callose wall formation (same configuration as in f). (m)(o)
Mercurialis perennis. (m) Triaperturate grain
(polar view). (n) Tetrahedral tetrad. Apertures are grouped in pairs (arrows) in accordance with Fischers configuration.
(o) Planes 46 of a tetrahedral tetrad during
callose wall formation (same configuration as
in f). (p)(r) Bocquillonia spicata. (p) Triaperturate pollen (equatorial section). (q) Tetrahedral tetrad with apertures (asterisk) in
Fishers configuration. (r) Planes 46 of a
tetrahedral tetrad during callose wall formation (same configuration as in f). (s)(u)
Hura crepitans. (s) Triaperturate pollen
(equatorial section). (t) Tetrahedral tetrad.
Apertures are grouped in pairs (arrows) in
Fig. 4 (Continued)
accordance with Fischers configuration.
(u) Planes 46 of a tetrahedral tetrad during
callose wall formation (same configuration
as in f). (v)(x) Macaranga tanarius. (v)
Triaperturate pollen (polar view). (w) Tetrahedral tetrad. (x) Planes 46 of a tetrahedral
tetrad during callose wall formation (same
configuration as in f). (y)(a) Caperonia serrata. (y) Hexa-aperturate grain (polar view).
(z) Tetrahedral tetrad. Apertures are grouped
in pairs (arrows) in accordance with Fischers
configuration. (a) Planes 46 of a tetrahedral
tetrad during callose wall formation (same
configuration as in f). All photographs are
taken using fluorescence microscopy except
(e) and (j), which are with light microscopy.
Scale bars 10 lm unless mentioned otherwise.
(p)
(q)
(r)
5 m
5 m
5 m
(s)
(t)
(u)
(v)
(w)
(x)
5 m
(y)
1087
5 m
5 m
(z)
( )
1088
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(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
(m)
(n)
(o)
(p)
Fig. 5 Pollen morphology and microsporogenesis of four inaperturate Jatropha species. (a)(d) Jatropha podagrica. (a) Inaperturate pollen.
(b) Mature tetragonal tetrad. (c) Rhomboidal tetrad during callose wall formation. Callose deposition follows the CpP mode (Fig. 1b-1, whole
tetrad). Callose begins to be deposited at the border of each plane and progresses towards the centre of the plane. (d) Planes 13 of a tetrahedral
tetrad during callose wall formation. Callose deposition follows the CpT mode (Fig. 1c-2, whole tetrad). Callose begins to be deposited at the
border of the tetrad and progresses towards the centre of the tetrad. Note the absence of callose at the centre of the tetrad (arrow), allowing us
to distinguish this configuration from the one where callose deposition is centripetal according to the cleavage planes. (e)(h) Jatropha
integerrima. (e) Inaperturate pollen. (f) Tetragonal tetrad during callose wall formation. Callose deposition follows the CpP mode (Fig. 1a-1,
section). Callose begins to be deposited at the border of each plane (arrows the central border is common to the four planes) and progresses
towards the centre of the plane. At this stage, the centre of each plane (asterisks) remains empty of callose. (g) Mature rhomboidal tetrad. (h)
Planes 46 of a tetrahedral tetrad during callose wall formation. Callose deposition follows the CpT mode (Fig. 1c-2, section). Callose begins to
be deposited at the border of the tetrad and progresses towards the centre of the tetrad (arrow). (i)(l) Jatropha gossypiifolia. (i) Inaperturate
pollen. (j) Tetragonal tetrad during callose wall formation. Callose deposition follows the CpP mode (same configuration as in f). (k) Mature
rhomboidal tetrad. (l) Planes 46 of a tetrahedral tetrad during callose wall formation. Callose deposition follows the CpT mode (same
configuration as in h). The arrow indicates the centre of the tetrad, which remains empty of callose at this stage. (m)(p) Jatropha multifida.
(m) Inaperturate pollen. (n) Mature tetragonal tetrad. (o) Rhomboidal tetrad during callose wall formation. (p) Tetrahedral tetrad during CpP
callose wall formation (Fig. 1c-1, section). All photographs are taken using fluorescence microscopy. Scale bars 10 lm.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
(m)
(n)
(o)
(p)
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Fig. 6 Pollen morphology and microsporogenesis for three inaperturate and one panto-aperturate species of Euphorbiaceae. (a)(d) Croton
californicus. (a) Inaperturate pollen. (b) Mature tetragonal tetrad. (c) Rhomboidal tetrad during callose wall formation. Callose deposition
follows the CpP mode (Fig. 1b-1, section). Callose begins to be deposited at the border of each plane (arrows) and progresses towards the centre
of each plane. (d) Planes 46 of a tetrahedral tetrad during callose wall formation. Callose deposition follows the CpP mode (Fig. 1c-1, section).
(e)(h) Baloghia inophylla. (e) Inaperturate pollen. (f) Mature tetragonal tetrad. (g) Rhomboidal tetrad during callose wall formation. Callose
deposition follows the CpT mode (Fig. 1b-2, whole tetrad). Callose begins to be deposited at the borders of the tetrad (arrows) and progresses
towards the centre of the tetrad (asterisk). At the stage represented here, the centre of the tetrad remains empty of callose. (h) Planes 13 of a
tetrahedral tetrad during callose wall formation. Callose deposition follows the CpP mode (Fig. 1c-1, whole tetrad). Callose (c) begins to be
deposited at the border of each plane and progresses towards the centre of the plane (asterisks). At the stage represented here, the centre
of each plane remains empty of callose. (i)(l) Eremocarpus setigerus. (i) Inaperturate pollen. (j) Tetragonal tetrad during callose wall formation.
Callose deposition follows the CpP mode (Fig. 1a-1, whole tetrad). Only two of the four cleavage plane formations are visible on this view
(the two other planes are orthogonal to the picture plane and thus callose deposition cannot be observed). Callose (c) begins to be deposited at
border of the cleavage plane and progresses towards the centre of each plane (asterisk). (k) Rhomboidal tetrad during callose wall formation
stage. Callose deposition follows the CpT mode (Fig. 1b-2, whole tetrad). (l) Planes 46 of a tetrahedral tetrad during callose wall formation.
Callose deposition follows the CpP mode (Fig. 1c-1, section). (m)(p) Manihot esculenta. (m) Panto-aperturate pollen. (n) Rhomboidal tetrad
during callose wall formation (same configuration as in c). (o) Planes 1 and 2 of a tetrahedral tetrad during callose wall formation (same
configuration as in h). (p) Planes 46 of a tetrahedral tetrad during callose wall formation. Callose deposition follows the CpT mode (Fig. 1c-2,
section). Callose begins to be deposited at the border of the tetrad and progresses towards the centre of the tetrad (arrow). All photographs
are taken using fluorescence microscopy except (a), which is with light microscopy. Scale bars 10 lm.
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Discussion
Increasingly, in the literature, an important role is given
to mutational and developmental systems in limiting the
direction that can be taken by a lineage during its
evolution (e.g. Bradshaw, 1991; Lynch, 2007; Stoltzfus &
Yampolsky, 2009; Braendle et al., 2010; Futuyma, 2010).
Other studies are inclined to argue that limitations on
morphological diversity are caused by the action of
natural selection rather than mutational biases or developmental constraints (e.g. Beldade et al., 2002; Eldredge
et al., 2005; Wiens et al., 2006). In this study, we show
that limitations in the evolution of pollen morphology
and in the evolution of its developmental system are
likely to be caused by selective pressures acting on pollen
morphology. However, even in the absence of these
selective pressures, a bias in the variability of the
developmental system remains.
Stasis in pollen morphology and development
The aperture pattern is relatively conserved in the
Euphorbiaceae s.s.: most species in this group (for which
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Conclusion
The pattern observed here indicates that evolutionary
stasis in pollen aperture morphology and development
may be due, in part, to stabilizing selection because
removing the presumed target of selection permits the
appearance of variation. However, not all possible variants occur, indicating that constraints also contribute for
stability. In the debate concerning selection and constraints, it appears that neither of these two factors can
totally account for evolutionary stasis. It is the interaction
between them that may determine the direction of
evolution.
Acknowledgments
The authors thank R. Bateman, J. Becerra, P. Chesselet,
J. Doyle, D. Hinsinger, S. Magallon-Puebla, S. Nadot,
A. Ressayre, H. Sauquet, M. Lopez-Villavicencio and
three anonymous reviewers for helpful comments and
discussion; V. Sagun for sharing his Acalypha sequence
alignments; A. Dubois, M. Geze, C. Raquin, L. Saunois
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Supporting information
Additional Supporting Information may be found in the
online version of this article:
Table S1 Genbank accession numbers (or source) of the
sequences used for the phylogenetic analysis; and matrix
used for the optimization and for the test of correlated
evolution
Table S2 Number of tetrads for each type of tetrad
observed in the heteromorphic species, and results of the
chi-square tests, testing if each type of tetrad is produced
in similar proportions within a plant
Figure S1 Number and position of callose cleavage
planes, relative to the arrangement of the microspores in
the tetrad and to the cytokinesis type
Figure S2 Mirror tree of Euphorbiaceae s.s. comparing
optimizations of the aperture pattern (a) and mode of
callose deposition (b)
Figure S3 Mirror tree of Euphorbiaceae s.s. comparing
optimizations of the aperture pattern (a) and cytokinesis
type (b)
Figure S4 Mirror tree of Euphorbiaceae s.s. comparing
optimizations of the aperture pattern (a) and tetrad form
(b) using the topology of Wurdack et al. (2005)
Figure S5 Mirror tree of Euphorbiaceae s.s. comparing
optimizations of the aperture pattern (a) and mode of
callose deposition (b) using the topology of Wurdack
et al. (2005)
As a service to our authors and readers, this journal
provides supporting information supplied by the authors.
Such materials are peer-reviewed and may be reorganized for online delivery, but are not copy-edited
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addressed to the authors.
Received 2 December 2011; revised 22 February 2012; accepted 24
February 2012