Acta Palaeobotanica 56(2): 223–245, 2016
DOI: 10.1515/acpa-2016-0017
Pollen morphology of Nothofagus (Nothofagaceae,
Fagales) and its phylogenetic signiicance
DAMIÁN ANDRÉS FERNÁNDEZ1,*, PATRICIO EMMANUEL SANTAMARINA1,*,
MARÍA CRISTINA TELLERÍA2,*, LUIS PALAZZESI 1,* and VIVIANA DORA BARREDA1,*
1
2
Sección Paleopalinología, MACN “B. Rivadavia”, Ángel Gallardo 470 (C1405DJR) C.A.B.A.;
e-mails: dafernandez@macn.gov.ar; santamarinape@gmail.com; lpalazzesi@macn.gov.ar;
vbarreda@macn.gov.ar
Laboratorio de Sistemática y Biología Evolutiva (LASBE), Museo de La Plata, UNLP, Paseo del Bosque s/n°
(B1900FWA) La Plata; e-mail: mariatelleria@fcnym.unlp.edu.ar
*
Consejo Nacional de Investigaciones Cientíicas y Técnicas (CONICET), Buenos Aires, Argentina
Received 31 August 2016, accepted for publication 10 November 2016
ABSTRACT. Nothofagaceae (southern beeches) are a relatively small lowering plant family of trees conined
to the Southern Hemisphere. The fossil record of the family is abundant and it has been widely used as a test
case for the classic hypothesis that Antarctica, Patagonia, Australia and New Zealand were once joined together.
Although the phylogenetic relationships in Nothofagus appear to be well supported, the evolution of some pollen
morphological traits remains elusive, largely because of the lack of ultrastructural analyses. Here we describe
the pollen morphology of all extant South American species of Nothofagus, using scanning electron microscopy
(SEM), transmission electron microscopy (TEM) and light microscopy (LM), and reconstruct ancestral character
states using a well-supported phylogenetic tree of the family. Our results indicate that the main differences
between pollen of subgenera Fuscospora (pollen type fusca a) and Nothofagus (pollen type fusca b) are related to
the size of microspines (distinguishable or not in optical section), and the thickening of colpi margins (thickened
inwards, or thickened both inwards and outwards). In particular, Nothofagus alessandrii, the only extant South
American species of subgenus Fuscospora, presents distinctive pollen features that have not been observed in
any other species of the genus (i.e. a large granular infratectum and spongy apertural endexine). Species of
subgenus Lophozonia are characterized by having the largest pollen grains, with polygonal outline in polar
view, microspines distinguishable in optical section, long and non-thickened colpi, and a thin endexine. The
reconstruction of character states for the node corresponding to the common ancestor to genus Nothofagus leads
us to conclude that the ancestral form of Nothofagaceae should have had: equatorial diameter < 40 μm, circular
outline in polar view, microspines distinguishable in optical section, short colpi thickened inwards, and a thin
endexine. These features are fully consistent with those present in Nothofagidites senectus Dettmann & Playford, the oldest fossil species of Nothofagaceae recorded in Campanian-Maastrichtian sediments of Gondwana.
KEYWORDS: Nothofagus, South America, pollen morphology, exine structure, character evolution
INTRODUCTION
Nothofagus Blume, the only member of
the family Nothofagaceae (Kuprianova 1962),
comprises ca 42 species of prominent trees of
the Southern Hemisphere. It grows in forests
of southeastern Australia including Tasmania,
New Caledonia, New Guinea, New Zealand,
and southwestern South America (Romero
1977). It comprises four subgenera, Lophozonia, Fuscospora, Nothofagus and, Brassospora
recognized on the basis of cupule morphology, leaf architecture and cuticular morphology (Hill & Read 1991). Nothofagus has been
a test case for the hypothesis that Gondwanan
vicariance can explain major biogeographic
patterns; for that reason its evolutionary history has motivated major studies based on its
pollen morphology, fossil record and cladistic
biogeography (Sauquet et al. 2012).
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Phylogenetic relationships of genus Nothofagus have been extensively explored on the
basis of morphological features (Hill & Jordan 1993, Heenan & Smissen 2013) and DNA
sequences (Martin & Dowd 1993, Setoguchi
et al. 1997, Manos 1997, Acosta & Premoli
2010, Premoli et al. 2012, Sauquet et al. 2012).
Morphological features of Nothofagus pollen
have been studied in some detail. For example,
Praglowski (1980, 1982) examined the pollen
of seven species of Nothofagus and deined
three types named fusca, menziesii and brassi,
which differ mainly in features of the colpi.
Dettmann et al. (1990) studied extant and
fossil pollen of Nothofagus and found larger
diversity in fossil pollen grains, distinguishing
four subtypes in addition to those deined by
Praglowski (1982). These subtypes were circumscribed on the basis of characters of the colpi, outline of the pollen, and the ratio between length
of the colpi and equatorial diameter. Wang et al.
(2000) conirmed the division into three pollen
types proposed by Praglowski (1982) from study
of pollen of 26 species using LM, SEM and MET.
The correspondence between the Nothofagus subgenera and their pollen morphology
has been explored using a widely accepted
phylogeny of the family (Manos 1997). However, an ultrastructural analysis of the pollen
grains has never been taken into account. The
main objectives of the present study are to
(1) provide more extensive knowledge of pollen morphology of South American species of
Nothofagus using light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM), including species of subgenus Lophozonia which have never
been observed under SEM and TEM and (2)
sketch the evolutionary trends of pollen features on the maximum likelihood tree provided
by Sauquet et al. (2012).
MATERIAL AND METHODS
Pollen grains from all South American species
of Nothofagus (N. alessandrii, N. alpina, N. glauca,
N. obliqua, N. antarctica, N. betuloides, N. dombeyi,
N. pumilio and N. nitida) and Betula platyphylla were
removed from anthers of herbarium specimens from
CONC, LP and SI (herbarium acronyms follow Index
Herbariorum http://sciweb.nybg.org/science2/IndexHerbariorum.asp).
Specimens studied:
– Betula platyphylla var. japonica (Miq.) H. Hara:
H. Ohashi and Y. Tateishi (SI 187723).
–
Nothofagus alessandrii Espinosa: NN (CONC
155232).
– Nothofagus alpina (Poepp. and Endl.) Oerst.: Schajavskoy (LP).
– Nothofagus antarctica (G. Forst.) Oerst.: Delucchi
591 (LP).
– Nothofagus betuloides (Mirb.) Oerst.: Guerrido
et al. 626 (SI).
– Nothofagus dombeyi (Mirb.) Oerst.: Delucchi 607
(LP).
– Nothofagus glauca (Phil.) Krasser: Picca 160 (SI).
– Nothofagus nitida (Phil.) Krasser: Picca 220 (SI).
– Nothofagus obliqua (Mirb.) Oerst.: Schajavskoy
(LP).
– Nothofagus pumilio (Poepp. and Endl.) Krasser:
Gentili (LP).
For light microscopy (LM), pollen was acetolysed
according to Erdtman (1960); the slides were prepared
by mounting the pollen in glycerol jelly and sealing
with parafin. For SEM, acetolysed and non-acetolysed pollen grains were suspended in 90% ethanol,
mounted on stubs and examined using a Philips XL30
TMP SEM at the Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”. For TEM, the fresh pollen grains were ixed in 1.5% glutaraldehyde and then
buffered in 2% OsO4 for 2 hours at room temperature;
they were washed for 30 minutes in distilled water,
dehydrated in an ethanol series and inally embedded in acetone-Spurr 3:1 for 6 h and twice in Spurr
for 24 h. Ultrathin sections were cut using a diamond
knife itted into a Sorvall Porter-Blum MT2-B ultramicrotome. Sections were mounted in single grids and
stained with lead citrate (1 min) and uranyl acetate
(10 min). The observations were made with a Jeol
JEM 1200 EX II transmission electron microscope
from the Servicio Central de Microscopía Electrónica
of the Facultad de Ciencias Veterinarias, Universidad
Nacional de La Plata.
The classical taxonomy of genus Nothofagus
(Nothofagaceae=Nothofagus) is followed in this contribution (Hill & Read 1991). Terminology used for pollen description follows Punt et al. (2007). We selected
six characters that were found to be variable among
Nothofagus taxa: equatorial diameter, outline in polar
view, aperture length, microspines (distinguishable
or not in optical section), endexine thickening at the
colpi level, and ratio of endexine/ectexine thickness.
We avoided using the polar and equatorial diameter
ratio because Nothofagus pollen grains are oblate to
per-oblate and are usually observed only in polar view.
We also avoided using the character “shape of aperture ends” of Dettmann (1990); we instead used the
character “endexine thickenings at the apertures”. In
fact, if the endexine is thickened at the apertural level
the ends of the colpi are well delimited, whereas if the
endexine is not thickened at the apertural level, the
ends of the colpi appear as issures.
The deinition and states of each character together
with the data matrix are given in Table 1. Equatorial diameter, outline in polar view, aperture length,
microspines and endexine thickening at aperture level
were measured on 25 pollen grains under LM (Appendix A); and the ratio of endexine/ectexine thickness
was measured on 10 grains under TEM.
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Table 1. Data matrix of exine characters
TAXA
Outgroups
Fagus grandifolia
Betula platyphylla
Nothofagaceae
Nothofagus
N. antarctica
N. pumilio
N. betuloides
N. nitida
N. dombeyi
Fuscospora
N. truncata
N. cliffortioides
N. fusca
N. solandri
N. gunnii
N. alessandri
Brassospora
N. grandis
N. carrii
N. perryi
N. brassi
N. resinosa
N. discoidea
N. balansae
N. baumanniae
N. codonandra
N. aequilateralis
Lophozonia
N. alpina
N. glauca
N. obliqua
N. cunninghamii
N. moorei
N. menziesii
1
2
3
4
5
6
0
0
1
1
2
0
NA
0
1
0
0
0
0
0
0
0
0
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
2
2
2
2
2
2
2
2
2
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
?
1
?
1
?
?
1
1
1
1
1
1
1
0
1
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
0
0
0
0
0
0
Exine characters used in this analysis.
1 – Equatorial diameter < 40 μm (0), > 40 μm (1).
2 – Outline in polar view (amb) polygonal (0), circular (1), star-like (2).
3 – Aperture length < 11 μm (0), 14–17 μm (1), 25–29 μm (2).
4 – Microspines not distinguishable in optical section (0), distinguishable in optical section (1).
5 – Endexine thickening at aperture level present inwards and outwards (0), present inwards (1), absent (2).
6 – Endexine/ectexine thickness ratio ca 1:10 (0), ca 1:2 (1).
We reconstructed ancestral character states of
Nothofagus pollen using Mesquite 3.10 (Maddison
& Maddison 2016) functions Likelihood Ancestral
States (ML), under the Mk1 model of evolution, and
Parsimony Ancestral States (MP) on the published
phylogenetic tree of Sauquet et al. (2012).
Data for species of subgenus Brassospora were
taken from Praglowski (1980, 1982) and the Australasian Pollen and Spore Atlas (APSA Members 2007).
We use the outgroups selected by Sauquet et al. (2012),
Betula platyphylla Roth. and Fagus grandilora Ehrh.
Betula platyphylla (ultrastructure only) data were
taken from El-Ghazaly (1999), El-Ghazaly and Huysmans (2001) and Blackmore et al. (2003), and Fagus
grandilora data from Praglowski (1982) and Heenan
and Smissen (2013).
RESULTS AND DISCUSSION
Nothofagus pollen grains appear to be
similar among species, but they show some
variation in size, shape, apertural features,
ornamentation and exine structure. These
characters are distinguishable under LM,
which is useful when comparing with fossil
pollen grains. Additionally, details of exine
structure revealed under TEM reinforce
the characterization of the features optimized in the phylogenetic tree of Sauquet
et al. (2012).
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Fig. 1. Diagrammatic interpretations of the pollen wall of subgenus Lophozonia (menziessi type) (a – Nothofagus alpina; b –
N. glauca; c – N. obliqua); d – subgenus Fuscospora (fusca a type, N. alessandrii); e – subgenus Nothofagus (fusca b type).
Colors used to indicate the different layers were taken from Punt et al. (2007). All diagrams are at the same scale. The thickness of the layers is an average of the measures of all the grains studied
GENERAL POLLEN MORPHOLOGY
LM
Pollen grains are isopolar, radially symmetrical, oblate to peroblate, subcircular to elliptic
in equatorial view (Pl. 1, ig. 9), with convex or
nearly straight mesocolpia in polar view (Pl. 1,
igs 1–8). The equatorial diameter range of
the analysed species is 26–57 μm; Nothofagus
alessandrii is the smallest and N. alpina the
largest (Appendix A). The apertures are 4–7
stephanocolpate, colpi are narrow and marginate with rounded ends and parallel edges, or
have a issurate aspect (as a subtle interruption of the exine) (Pl. 1, igs 1–3). Pollen grains
with marginate colpi have the exine thickened
inwards at the aperture level (Pl. 1, ig. 4) or
have both inwards- and outwards-thickened
margins (i.e. protruding above the surface)
(Pl. 1, igs 5–9). The exine is microechinate and
the surface is psilate between microspines in
all species. Sexine and nexine are not distinguishable under LM.
SEM
The sculpture consists of minute and
uneven-sized microspines (Pl. 2–4, igs 2,
6, 10) which are mostly conical. In Nothofagus glauca and N. alessandrii, the microspines appear to be connected with delicate
strands of sporopollenin (Pl. 2, ig. 6; Pl. 3,
ig. 2). In N. alessandrii the microspines are
conspicuous (Pl. 1, ig. 4).
TEM
The ectexine is 0.5–1 μm thick; it consists
of a compact tectum 0.2–0.4 μm thick, a thin
granular infratectum and a continuous foot
layer as thick as the tectum. The microspines
are compact and appear to be deep-rooted in
the endexine (Pl. 2, ig. 7).
The endexine is 0.1–0.2 μm thick (Pl. 2–4,
igs 3, 7, 11). In the species of subgenus
Nothofagus it is thickened both inwards and
outwards at the colpi level, forming a margin
(Pl. 3, igs 8, 12; Pl. 4, igs 4, 8, 12).
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R e m a r k s: in the species of subgenus Nothofagus the intine is notably thickened beneath
the aperture, forming an oncus (Pl. 3, igs 8, 12;
Pl. 4, ig. 4). In Nothofagus glauca, N. antarctica, N. dombeyi and N. nitida a lamellate
layer is situated between the endexine and the
intine (Pl. 2, ig. 8; Pl. 3, igs 8, 12; Pl. 4, ig. 4).
This layer is comparable with the MGL (membranous granular layer) deined by El-Ghazaly
and Huysmans (2001).
DIAGNOSTIC FEATURES OF SUBGENERA
Subgenus Lophozonia (menziesii type)
Pl. 1, igs 1–3; Pl. 2
The pollen has straight or more or less
convex mesocolpia; the outline in polar view
is circular to polygonal (Pl. 1, igs 1–3). The
pollen grains are the largest within the genus
(Appendix A). Colpi (4–8) are long, reaching
Fig. 2. Equatorial diameter (a) and outline in polar view (b), of pollen grains of Fagus grandifolia, Betula platyphylla and
Nothofagus sp. div., optimized onto the ML tree of Sauquet et al. (2012) using the Mesquite program, version 3.10 (Maddison
& Maddison 2016). Circles at the tips represent the observed character states in each taxon, while the circles of the nodes are
pie diagrams that indicate the likelihood of each state. The likelihood reconstruction inds the state that maximizes the probability of arriving at the observed states in the terminal taxa, given the Mk1 model of evolution, and considering all possible
assignments to the other ancestral states
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15 µm, and have a issurate appearance. The
exine is thin (1 μm thick or less). Microspines
are usually uneven-sized and distinguishable
in optical section. The endexine/ectexine thickness ratio is ca 1:10 – 1:5 at the mesocolpia
(equatorial section) (Pl. 2, igs 3, 7, 11). The
granular infratectum is ca 0.1 μm thick.
R e m a r k s a n d c o m p a r i s o n s. N. alpina
(Pl. 1, ig. 1; Pl. 2, igs 1–4): the foot layer is
lacking at colpi margins and the endexine is
disrupted deining a concave surface (Pl. 2,
ig. 4; Fig. 1a).
N. glauca (Pl. 1, ig. 2; Pl. 2, igs 5–8): the
colpi appear as an abrupt gap at the ectexine
level. The colpi margins are poorly deined.
The microspines have rounded tips and are
connected by delicate strands; the tectum surface between microspines is scabrate (Pl. 2,
ig. 6). The endexine is slightly thickened at
the colpi margins. A lamellate layer is situated
between the endexine and the intine (Pl. 2,
ig. 8; Fig. 1b). This layer is comparable with
the MGL.
N. obliqua (Pl. 1, ig. 3; Pl. 2, igs 9–12):
an abrupt gap in the ectexine is observed at
the aperture level. The endexine persists discontinuously in the aperture (Pl. 2, ig. 12;
Fig. 1c). The intine is 1.4 µm thick, with two
layers, the external (ca 0.7 μm thick) denser
and darker than the internal. Both layers have
lamellar texture (Pl. 2, ig. 12; Fig. 1c).
Fig. 3. Pollen grains of Fagus grandifolia, Betula platyphylla and Nothofagus sp. div. Aperture length (a) and microspines
(distinguishable or not in optical section) (b), optimized onto the ML tree of Sauquet et al. (2012) using Mesquite program
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Fig. 4. Pollen grains of Fagus grandifolia, Betula platyphylla and Nothofagus sp. div. Endexine thickening at aperture level
(a) and endexine/ectexine thickness ratio (b) optimized onto the ML tree of Sauquet et al. (2012) using Mesquite program
Subgenus Fuscospora (fusca a type)
Pl. 1, ig. 4; Pl. 3, igs 1–4
The pollen has slightly convex or sometimes
straight mesocolpia; the outline in polar view
is circular to subcircular (Pl. 1, ig. 4). The pollen grains are the smallest within the genus
(Appendix A). The colpi (5–7) are short, with
rounded ends, with inwards-thickened colpi
margins (Pl. 1, ig. 4; Pl. 3, ig. 4; Fig. 1d). The
microspines are conspicuous, connected by
delicate sinuous strands distinguishable under
SEM (Pl. 3, ig. 2). The exine is thicker than
that in the menziesii type (Fig. 1d; Appendix A). The endexine/ectexine thickness ratio
is ca 1:10 at the mesocolpia (equatorial section)
(Appendix A). The granular infratectum is ca
0.2 µm thick, with lower density of granules
than that observed in subgenera Nothofagus
and Lophozonia (Pl. 3, igs 3, 4). The endexine
has a spongy appearance at the level of colpi
margins (Pl. 3, ig. 4).
Subgenus Nothofagus (fusca b type)
Pl. 1, igs 5–9; Pl. 3, igs 5–12; Pl. 4, igs 1–12
The pollen has slightly convex mesocolpia;
the outline in polar view is subcircular. The
equatorial diameter range is 26–40 μm. The
colpi (4–7) are short, with rounded ends and
parallel edges, and marginate (Pl. 1, igs 5–9).
The margins are formed by both inwards- and
outwards-thickened endexine (Pl. 3, igs 5, 8, 9,
12; Pl. 4, igs 1, 4, 5, 8, 9, 12; Fig. 1e). The microspines are not distinguishable in optical section
(Pl. 1, igs 5–9). The exine is thicker than that
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observed in the menziesii type (Appendix A;
Pl. 3, igs 7, 11; Pl. 4, igs 3, 7, 11; Fig. 1e). The
endexine/ectexine thickness ratio is ca 1:10 –
1:5 at the equatorial mesocolpia (Appendix A).
The granular infratectum is ca 0.1 μm thick
(Pl. 3, igs 7, 11; Pl. 4, igs 3, 7, 11; Fig. 1e). The
endexine at colpi level is more compact than
that in the fusca type (a), and 1–1.5 μm thick
(Pl. 3, igs 8, 12; Pl. 4, igs 4, 8, 12; Fig. 1e).
A p e r t u r e l e n g t h (Fig. 3a): the external
aperture of Fagus is a relatively long colpus
(l = 25–29 μm). Betula presents a pore (diameter = 1–3 μm). A short aperture (l = 1–11 μm) is
reconstructed as an ancestral character state
shared by Betula, Fuscospora, Nothofagus and
Brassopora (p = 0.70). Medium-sized colpus
(l = 14–17 μm) is a synapomorphy of subgenus
Lophozonia.
R e m a r k s a n d c o m p a r i s o n s. N. antarctica (Pl. 1, ig. 5; Pl. 3, igs 5–8), N. dombeyi
(Pl. 1, ig. 6; Pl. 3, igs 9–12) and N. nitida (Pl. 1,
ig. 7; Pl. 4, igs 1–4) show a lamellate layer between the endexine and the intine comparable
with the MGL (Pl. 3, igs 8, 12; Pl. 4, ig. 4).
In particular, N. nitida has an exine less than
1 μm thick (Appendix A).
N. pumilio (Pl. 1, ig. 8; Pl. 4, igs 5–8): exine
less than 1 μm thick (Appendix A).
N. betuloides (Pl. 1, ig. 9; Pl. 4, igs 9–12):
with microspines stronger than in the other
species of the subgenus Nothofagus. The
endexine is sometimes barely distinguishable
from the intine (Pl. 4, ig. 11).
M i c r o s p i n e s (Fig. 3b): microspines distinguishable in optical section is the inferred
ancestral state in the genus (p = 0.97). Only
subgenus Nothofagus has microspines not
distinguishable in optical section, which is
derived within genus Nothofagus and homoplastic with the same state in Betula. Fagus is
psilate; using SEM the surface is vermicularrugulose (Praglowski 1982).
PHYLOGENETIC SIGNIFICANCE
OF EXINE CHARACTERS
The results of optimizing selected pollen features onto the ML tree of Sauquet et al. (2012)
(Figs 2–4) allow us to formulate hypotheses on
their evolution across the family. It is worth
noting that the results obtained with the ML
method agree with those obtained using MP
(see supplementary igures).
Eq u a t o r i a l d i a m e t e r (Fig. 2a): small size
(less than 40 μm) is the ancestral (plesiomorphic) character state (p = 0.82) present in the
outgroups (Betula and Fagus) and retained in
subgenera Fuscospora, Nothofagus and Brassospora. Equatorial diameter larger than 40 μm
is a synapomorphy of subgenus Lophozonia.
O u t l i n e i n p o l a r v i e w (amb) (Fig. 2b):
circular outline is reconstructed as the ancestral character state shared by Betula, Fagus,
basal species of subgenus Fuscospora (Nothofagus alessandrii and N. gunnii), and Nothofagus
(p = 0.57). Polygonal outline is a derived state
that appears as parallelisms in Lophozonia (with
a reversal to circular in N. glauca) and in Fuscospora (N. truncata, N. fusca and N. solandri).
Subgenus Brassospora has a derived star-like
outline that is a synapomorphy of this clade.
Endexine thickenings at the apert u r e s (Fig. 4a): endexine thickenings (of two
types) are shared by Betula, Fagus and all the
ingroup with the exception of subgenus Lophozonia (p = 0.59), which seems to have lost them
during the early evolution of the family. This
loss appears to be a synapomorphy of Lophozonia. The apertural endexine is thickened
inwards in Fagus, Fuscospora and Brassospora,
while Betula and subgenus Nothofagus shared,
as a parallelism, apertural endexine thickened
outwards, protruding above the surface.
Endexine/ectexine thickness ratio
(Fig. 4b): a thin endexine (endexine/ectexine
thickness ratio ca 1:10) is reconstructed as an
ancestral character state present in the outgroups and retained in subgenera Fuscospora,
Nothofagus and Lophozonia (p = 0.99). A thicker
endexine (endexine/ectenxine thickness ratio
ca 1:2) is a synapomorphy of subgenus Brassospora, this may be functionally or developmentally related to the characteristic star-like, concave polygonal pollen grains of this subgenus.
CONCLUSION
Our study of the pollen morphology of species of Nothofagus – some of them previously
poorly studied, such as N. alpina, N. glauca
and N. nitida – under LM, SEM and TEM, and
the optimization of pollen characters on a well
supported phylogenetic tree, provide a better understanding of their evolution within
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the genus, and also further evidence that the
South American species cannot be recognized
to a lower taxonomic level than subgenus on
the basis of pollen morphology. In general, the
morphology is in line with that reported in previous contributions, such as Praglowski (1980,
1982), Zheng et al. (1999) and Wang et al.
(2000). Equatorial diameter, outline in polar
view, ornamentation and apertural morphology
are the most distinctive features. Concerning
the pollen of subgenera Fuscospora and Nothofagus, regarded as indistinguishable by Manos
(1997), our examination showed that pollen of
Fuscospora have microspines distinguishable in
optical section and endexine thickened inwards
at colpi level, whereas pollen of species of subgenus Nothofagus has microspines not distinguishable in optical section and the endexine
thickened both inwards and, outwards at colpi
level. N. alessandrii, the only extant South
American species of subgenus Fuscospora, presents distinctive ultrastructural features of the
exine that have not been observed in any other
species of the genus (e.g. thick granular infratectum, spongy apertural endexine) (Appendix A; Pl. 1, ig. 4; Pl. 3, igs 1–4).
Morphological differences between the pollen grains of the subgenus Lophozonia and the
rest of the family are noticeable. This subgenus
has larger pollen grains, a polygonal outline
in polar view, microspines distinguishable in
optical section, colpi long, and without endexine thickening and a thin endexine (Appendix A; Pl. 1, igs 1–3; Pl. 2, igs 1–12).
Reconstruction of character states for the
node corresponding to the common ancestor of
genus Nothofagus leads us to conclude that it
had a small equatorial diameter, circular amb,
microspines distinguishable in optical section,
short apertures thickened inwards and a thin
endexine. These features are fully consistent
with those recorded in Nothofagidites senectus
Dettmann & Playford, the oldest fossil species
of the family, recorded from the Campanian
and Maastrichtian over wide areas of southern Gondwana (Dettmann et al. 1990). This
congruence increases conidence in both the
phylogeny and the fossils as reliable records of
diversiication of the genus.
Future work on fossils of extinct pollen
types of Nothofagus, including SEM and TEM
analyses, would provide further evidence to
explore the evolutionary signiicance of the
here analysed pollen characters and to test
palaeobiogeographic hypotheses related to the
presence of the subgenus Brassospora in Cenozoic sediments from Patagonia (Dettmann
et al. 1990).
ACKNOWLEDGEMENTS
We thank: James A. Doyle and an anonymous
reviewer for their very helpful comments; Isabel
Farías, Fernanda Faisal and Fabián G. Tricárico for
their technical assistance; Alicia Marticorena, and the
authorities of CONC, LP and SI for allowing the use of
herbarium material for this study and Pablo Picca for
providing N. glauca material; the Chair of Sistemática
Teórica (FCEyN, UBA) for their helpful assistance and
comments. Financial support was provided by Agencia
Nacional de Promoción Cientíica y Tecnológica (PICT
0911) and Facultad de Ciencias Naturales y Museo
(UNLP) (Beca de Experiencia Laboral, Resolución del
HCA Nº 07/11).
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PLATES
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Plate 1
Pollen of genus Nothofagus under LM. Most grains are in polar view
1. Nothofagus alpina (Schajavskoy, LP)
2. Nothofagus glauca (Picca 160, SI)
3. Nothofagus obliqua (Schajavskoy, LP)
4. Nothofagus alessandrii (NN, CONC 155232)
5. Nothofagus antarctica (Delucchi 591, LP)
6. Nothofagus dombeyi (Delucchi 607, LP)
7. Nothofagus nitida (Picca 220, SI)
8. Nothofagus pumilio (Gentili, LP)
9. Nothofagus betuloides (Guerrido et al. 626, SI). Subequatorial view
Scale bar: 10 μm
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Plate 2
Pollen of subgenus Lophozonia
1–4. Nothofagus alpina (Schajavskoy, LP)
1. SEM, polar view showing apertures (arrows)
2. SEM, detail of microspines
3. TEM, exine section of mesocolpium showing the thin granular infratectum and the microspines deeprooted in the endexine
4. SEM, aperture showing the disrupted endexine (arrows)
5–8. Nothofagus glauca (Picca 160, SI)
5. SEM, subequatorial view showing apertures (arrows)
6. SEM, microspines are connected by delicate strands (arrows)
7. TEM, exine section of mesocolpium showing the thin granular infratectum and the microspines deeprooted in the endexine
8. SEM, aperture showing the lamellate layer (arrows)
9–12. Nothofagus obliqua (Schajavskoy, LP)
9. SEM, polar view
10. SEM, detail of microspines
11. TEM, exine section of mesocolpium showing the thin granular infratectum and the microspines deeprooted in the endexine
12. SEM, aperture showing endexine remains (arrows)
Abbreviations: In – intine; E – endexine; F – foot layer; G – granular infratectum; T – tectum; Ms – microspines.
Scale bars of 1, 5 and 9: 10 µm; of 2–4, 6–8 and 10–12: 2 µm
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Plate 3
Pollen of subgenus Fuscospora and Nothofagus
1–4. Nothofagus alessandrii (NN, CONC 155232)
1. SEM, subequatorial view
2. SEM, microspines are connected by delicate strands (arrows)
3. TEM, exine section of mesocolpium showing the relatively thick granular infratectum. Section is not perpendicular to the surface
4. SEM, aperture showing the endexine with spongy appearance (arrows)
5–8. Nothofagus antarctica (Delucchi 591, LP)
5. SEM, subequatorial view
6. SEM, detail of microspines
7. TEM, exine section of mesocolpium
8. SEM, aperture showing the endexine thickenings and the lamellate layer between the endexine and the
intine (arrows)
9–12. Nothofagus dombeyi (Delucchi 607, LP)
9. SEM, subpolar view
10. SEM, detail of microspines
11. TEM, exine section of mesocolpium
12. SEM, aperture showing the endexine thickenings and the lamellate layer between the endexine and the
intine (arrows)
Abbreviations: In – intine; E – endexine; F – foot layer; G – granular infratectum; T – tectum; Ms – microspines.
Scale bars of 1, 5 and 9: 5 µm; of 2, 6 and 10: 2 µm; of 3, 4, 7, 8, 11 and 12: 1 µm
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Plate 4
Pollen of subgenus Nothofagus
1–4. Nothofagus nitida (Picca 220, SI)
1. SEM, polar view
2. SEM, detail of microspines
3. TEM, exine section of mesocolpium
4. SEM, aperture showing the endexine thickenings and the lamellate layer between the endexine and the
intine (arrows)
5–8. Nothofagus pumilio (Gentili, LP)
5. SEM, subpolar view
6. SEM, detail of microspines
7. TEM, exine section of mesocolpium
8. SEM, aperture showing the endexine thickenings (arrows)
9–12. Nothofagus betuloides (Guerrido et al. 626, SI)
9. SEM, subequatorial view
10. SEM, detail of microspines
11. TEM, exine section of mesocolpium showing the endexine barely distinguishable from the intine (arrows)
12. SEM, aperture showing the endexine thickenings
Abbreviations: In – intine; E – endexine; F – foot layer; G – granular infratectum; T – tectum; Ms – microspines.
Scale bars of 1 and 5: 10 µm; of 9: 5 µm; of 2–4, 6, 8, 10 and 12: 2 µm; of 7 and 11: 1 µm
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Character / Species
Fuscospora
(fusca a)
Nothofagus (fusca b)
Subgenus (pollen type)
N. antarctica
N. pumilio N. betuloides
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APPENDIX A
Lophozonia (menziessi)
Outgroup
N. nitida
N. dombeyi
N. alessandri
N. alpina
N. glauca
N. obliqua
Betula platyphylla
LM
26–35
30–40
29–39
31–40
31–37
26–30
44–58
40–57
38–48
23–30
Average E (μm)
30.7
33.3
33.4
36.0
33.4
28.3
52.3
48.2
41.6
25.9
dS (μm)
2.5
2.2
2.3
2.2
1.3
1.3
4.1
4.6
1.9
1.7
4–6 (5)
4–7 (6)
5–7 (6)
5–7 (6)
5–7 (6)
5–7 (6)
4–8 (7)
4–7 (6)
6–7 (6)
3
a
2–3 (2)
1.5–2.5 (2)
2
2
2
(slightly >) 1–2
NA
NA
NA
1.5–2 (2)
b
1–2 (1.5)
2–3 (3)
2–4 (2)
2–3 (2)
1.5–3 (2)
0
NA
NA
NA
NA
c
1–2 (1.5)
2–3 (3)
2–4 (2)
2–3 (2)
1.5–3 (2)
0
NA
NA
NA
NA
Apertural depth (μm)
2.5–3.5 (2.5)
2–4 (3)
2.5–3 (2.5)
2.5–4 (3)
2.5–3 (2.5)
2–3.75 (2.5)
1–19
1–20
1–15
2–4 (3)
Apertural length (μm)
5–7 (5)
3–6 (4)
5–7 (5)
6
5–7 (6)
4–7 (5)
ca 15
ca 15
ca 15
1–3 (2)
Apertural width (µm)
?
1–3 (2)
1–3 (1 y 2)
2
1–3 (2)
1–2 (1)
?
5?
?
2–3 (2)
never
never
never
never
never
always (stronger)
yes
yes
yes
NA
1
<1
1
<1
1
1
<1
1
<1
1
SC
SC
P,SP,C,SC
(SP)
SC
SC/SP
SC
E (μm)
Aperture number
Aperture thickening (μm)*:
Exine thickness (μm)
Outline in polar view
SC
SC
SC
SC
SEM
Ornamentation
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MS
MS
MS
MS
MS
conspicuous MS
MS
MS
MS
MS
Minute processes
present
present
present
present
present
present
present
present
present
present
Strands
absent
absent
absent
absent
absent
present
absent
present
absent
absent
Aperture shape
elliptic
elliptic
elliptic
elliptic
elliptic
elliptic
NA
NA
NA
circular-subcircular
1/10
1/5
1/5
1/10
1/5
1/10
1/10
1/10
1/5
spongy ET
NT
NT
NT
D
PD
PD
PD
TEM
Endexine/ectexine
Apertural morphology
Bacular layer
compact ET
PD
compact ET compact ET compact ET compact ET
PD
PD
PD
PD
Pollen morphological data. Abbreviations used: E – equatorial diameter, dS – standard deviation, P – polygonal, SP – subpolygonal, C – circular, SC – subcircular, MS – microspines,
ET – endexine thickenings, NT – not thickened, PD – poorly developed, D – developed. * sensu Romero (1977).
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MS visible in optical section
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SUPPLEMENTARY FIGURES
Optimization using MP method
SFig. 1. Pollen grains of Fagus grandifolia, Betula platyphylla and Nothofagus sp. div. Equatorial diameter (a) and outline
in polar view (b) optimized onto the ML tree of Sauquet et al. (2012) using Mesquite program
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SFig. 2. Pollen grains of Fagus grandifolia, Betula platyphylla and Nothofagus sp. div. Aperture length (a) and microspines
(distinguishable or not in optical section) (b), optimized onto the ML tree of Sauquet et al. (2012) using Mesquite program
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SFig. 3. Pollen grains of Fagus grandifolia, Betula platyphylla and Nothofagus sp. div. Endexine thickening at aperture level
(a) and endexine/ectexine thickness ratio (b), optimized onto the ML tree of Sauquet et al. (2012) using Mesquite program
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