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© 2017 The Linnean Society of London, Botanical Journal of the Linnean Society, 2017, XX, 1–19 1
Botanical Journal of the Linnean Society, 2017, XX, 1–19. With 8 figures.
Comparative cypsela morphology in Disynaphiinae
and implications for their systematics and evolution
(Eupatorieae: Asteraceae)
TAYNARA D. G. SILVA1, JULIANA MARZINEK1, ERIC K. O. HATTORI2,
JIMI N. NAKAJIMA1 and ORLANDO C. DE-PAULA1*
1Universidade Federal de Uberlândia – Instituto de Biologia, Uberlândia 38400-902, Minas Gerais,
Brazil
2Universidade Federal dos Vales do Jequitinhonha e Mucuri – Instituto de Ciências Agrárias, Unaí
38610-000, Minas Gerais, Brazil
Received 23 August 2016; revised 18 September 2017; accepted for publication 16 October 2017
In Asteraceae, subtribe Disynaphiinae comprise c. 50 species distributed among six genera, including the mono-
typic Acanthostyles, Campovassouria (two species), Disynaphia (16 species), Grazielia (ten species), Raulinoreitzia
(three species) and Symphyopappus (14 species). In a recent phylogenetic analysis, the subtribe were shown to be
non-monophyletic; to achieve monophyly, D. praeficta would need to be excluded, and Neocabreria malacophylla,
N. serrulata (Critoiinae) and Radlkoferotoma cistifolium (Ageratinae) would need to be included. However, there
is no strong morphological feature to justify the exclusion of D. praeficta from the subtribe. All other Disynaphia
spp. form a clade with Campovassouria. Symphyopappus is also polyphyletic, but all species remain in the subtribe.
Thus, the circumscription of the genera traditionally based on morphological features became unclear, and the diag-
nostic potential of features like the structure of the fruit (called a cypsela) now needs to be re-evaluated in testing
the new relationships suggested by molecular data and finding morphological features supporting the exclusion of
D. praeficta. The structure of cypselae of all genera of the subtribe was studied using a combination of light and scan-
ning electron microscopy to evaluate its potential for systematics. Our study shows the phytomelanin layer external
to the vascular bundles and vascularized pappus were shared by among all species of the subtribe, but including
D. praeficta. Features of the cypselae of Disynaphiinae such as the carpopodium, floral disc, pappus outer mesocarp,
sclerenchyma, phytomelanin layer, ribs and trichomes are valuable at both generic and specific levels. In addition,
the outer mesocarp was an important feature supporting the new relationship in two of four clades proposed in the
current phylogenetic framework. Moreover, our results revealed the presence of a multiplicative pericarp only in a
few Symphyopappus spp., a rare feature in Asteraceae, which probably evolved independently in the family. Cypsela
structure also supports the exclusion of D. praeficta from the subtribe, since the species has several different features
when compared with other representatives of Disynaphiinae.
ADDITIONAL KEYWORDS: anatomy – carpopodium – cypsela – multiplicative pericarp – pappus – phytomelanin.
INTRODUCTION
Disynaphiinae are one of the 19 subtribes of
Eupatorieae (Asteraceae) and comprise six gen-
era and c. 50 species occurring exclusively in South
America (King & Robinson, 1987; Robinson, Schilling
& Panero, 2009; Rivera et al., 2016; Rivera, Ferreira
& Panero, 2016). Of these six genera, Acanthostyles
R.M.King & H.Rob. is monotypic (Grossi et al.,
2011) and Campovassouria R.M.King & H.Rob.
and Raulinoreitzia R.M.King & H.Rob. have two
and three species, respectively. Disynaphia Hook.
& Arn. comprises 16 species, Grazielia R.M.King &
H.Rob. has ten species and Symphyopappus Turcz.
has 14 species (King & Robinson, 1987). The cur-
rent circumscription of Disynaphiinae was based
on morphological features, and its members can
be recognized by having persistent subimbricate
*Corresponding author. E-mail: orlandocavalari@gmail.com
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2 T. D. G. SILVA ET AL.
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Figure 1. Phylogenetic tree of Disynaphiinae, showing the relationship among their genera (modified from Rivera et al.,
2016). This analysis included Disynaphia praeficta which was closely related to some Gyptidinae.
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CYPSELAE AND DISYNAPHIINAE SYSTEMATICS 3
© 2017 The Linnean Society of London, Botanical Journal of the Linnean Society, 2017, XX, 1–19
involucral bracts, a corymbose or pyramidal inflor-
escence of short- pedicellate heads with five flow-
ers (Robinson et al., 2009). Their cypselae have five
glabrous ribs with micropunctations aligned in
dense transverse bands, an obsolete to annuliform
or short cylindrical carpopodium with thin or some-
times slightly thicker cell walls and a uniseriate
pappus with many bristles, sometimes with rounded
apical cells (King & Robinson, 1987). However, in a
recent phylogenetic analysis, Disynaphiinae were
shown to be non-monophyletic, and to achieve mono-
phyly, it would be necessary to exclude Disynaphia
praeficta (B.L.Rob.) R.M.King & H.Rob., and to
include Neocabreria malacophylla (Klatt) R.M.King
& H.Rob., N. serrulata (DC.) R.M.King & H.Rob.
(Critoiinae) and Radlkoferotoma cistifolium (Less.)
Kuntze (Ageratinae). All other Disynaphia spp. form
a clade with Campovassouria. Symphyopappus is
polyphyletic since S. itatiayensis (Hieron.) R.M.King
& H.Rob. is nested in a clade with all Raulinoreitzia
spp., whereas S. compressus (Gardner) B.L.Rob.
is nested in a clade with all Grazielia spp. plus
N. serrulata (Rivera et al., 2016) (Fig. 1).
Morphological and anatomical features of cypselae of
Asteraceae have a high systematic value (Roth, 1977;
Pandey & Singh, 1982b; Haque & Godward, 1984;
Table 1. Voucher information for studied taxa of Disynaphiinae
Genus Species Voucher
Acanthostyles R.M.King &
H.Rob.
A. buniifolius (Hook. ex Arn.) R.M.King &
H.Rob.
Covas, G. 18516 (CEN)
Campovassouria R.M.King
& H.Rob.
C. barbosae H.Rob. Silva, J.M. et al. 6064 (MBM)
C. cruciata (Vell.) R.M.King & H.Rob. Barbosa, A.A.A. 1225 (HUFU)
Disynaphia Hook. & Arn. ex
DC.
D. halimifolia (DC.) R.M.King & H.Rob. Romero, R. 2258 (HUFU);
Romero, R. et al. 4960 (HUFU)
D. littoralis (Cabrera) R.M.King & H.Rob. Hattori, E.K.O. et al. 1122 (BHCB)
D. multicrenulata (Sch. Bip. ex Baker)
R.M.King & H.Rob.
Ribas, O.S. 2622 (HUFU)
D. praeficta (B.L.Rob.) R.M.King & H.Rob. Contro, F.L. & Hattori, E.K.O. s.n. (HUFU)
D. senecionidea (Baker) R.M.King & H.Rob. Barbosa, A.A. 3375 (HUFU)
Grazielia R.M.King & H.Rob. G. dimorpholepis (Baker) R.M.King &
H.Rob.
Amorim, E.H. 541 (HUFU)
G. gaudichaudeana (DC.) R.M.King &
H.Rob.
Hattori, E.K.O. et al. 1335 (BHCB);
Hattori, E.K.O. et al. 1117 (BHCB)
G. intermedia (DC.) R.M.King & H.Rob. Hattori, E.K.O. et al. 1331 (BHCB);
Hattori, E.K.O. et al. 1116 (BHCB)
G. mollicoma (B.L. Rob.) R.M.King &
H.Rob.
Hattori, E.K.O. 1224 (HUFU)
G. multifida (DC.) R.M.King & H.Rob. Camilo, S.B. s.n. (HUFU)
G. nummularia (Hook. & Arn.) R.M.King &
H.Rob.
Hattori, E.K.O. 1103 (BHCB)
G. serrata (Spreng.) R.M.King & H.Rob. Hattori, E.K.O. et al. 1097 (BHCB)
Raulinoreitzia R.M.King &
H.Rob.
R. crenulata (Spreng.) R.M.King & H.Rob. Hattori, E.K.O. 1230 (HUFU)
R. tremula (Hook. & Arn.) R.M.King &
H.Rob.
Pires, A. 336 (HUFU)
Symphyopappus Turcz. S. angustifolius Cabrera Hattori, E.K.O. 1066 (HUFU)
S. apurimacensis H.Rob. Hattori, E.K.O. 1402
S. brasiliensis (Gardner) R.M.King &
H.Rob.
Hattori, E.K.O. 1488 (BHCB)
S. compressus (Gardner) B.L.Rob. Duarte, A.P. 7607 (HUFU)
S. cuneatus (DC.) Sch.Bip. ex Baker Fernandes, A.C. 1025 (BHCB)
S. decussatus Turcz. Mendonça, C.V. 1303 (HUFU)
S. itatiayensis (Hieron.) R.M.King & H.Rob. Sakane, M. 538 (HUFU)
S. lymansmithii B.L.Rob. Meireles, L.D. 3005 (HUFU)
S. myricifolius B.L.Rob. Leoni, L.S. 3980 (HUFU)
S. polystachyus Baker Carvalho, M.G. 1362 (HUFU)
S. reitzii (Cabrera) R.M.King & H.Rob. Hattori, E.K.O. s.n. (HUFU)
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CYPSELAE AND DISYNAPHIINAE SYSTEMATICS 5
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Källersjö, 1985; Bruhl & Quinn, 1990; Bean, 2001; Pak,
Park & Whang, 2001; Hood & Semple, 2003; Marzinek,
De-Paula & Oliveira, 2010; Marzinek & Oliveira, 2010;
Pandey, Stuessy & Mathur, 2014; Tadesse & Crawford,
2014; Franca et al., 2015; Freitas et al., 2015). In the
Disynaphiinae, morphological features of the carpopo-
dium and pappus in cypselae have been used for the sep-
aration of genera and species (King & Robinson, 1987).
However, the recent phylogenetic analysis of Rivera
et al. (2016) suggests that morphological features used
by King & Robinson (1987) may be homoplastic and
that the boundaries between (or circumscriptions of)
the genera are not as clear as previously thought and
need to be re-evaluated to test the new relationships
proposed by molecular data. In addition, knowledge
of cypselae anatomy in Disynaphiinae is restricted to
date to Symphyopappus reticulatus Baker (Marzinek
& Oliveira, 2010).
In this study, we compared the morphology and the
anatomy of the 28 species of six genera in the subtribe
sensu King & Robinson (1987) to re-evaluate the poten-
tial value of the external (morphological) and internal
(anatomical) structure of the cypsela for the circum-
scription of the subtribe and the resolution of their
systematic affinities with other members of Eupatorieae.
We also aimed at re-evaluating the monophyly of each
genus, paraphyly of Symphyopappus and potential
exclusion of D. praeficta with a morphological and
anatomical approach.
MATERIAL AND METHODS
Cypselae were obtained from specimens deposited as
the BHCB, CEN, HUFU and MBM herbaria (Table 1).
For morphological observations, cypselae were mounted
directly on aluminium stubs with double-sided adhesive
carbon tape, coated with gold in a sputter coater (Leica
EM SCD050, Heidelberg, Germany), examined using
a scanning electron microscope (Zeiss EVO MA 100,
Jena, Germany) and the most representative regions
captured digitally. To determine the divergence angle
and surface of the bristles of pappus, we followed the
terminology adapted from Hickey (1979) and Barthlott
et al. (1998), respectively. Three divergence angles
were recognized (i.e. narrow acute <45°; moderately
acute 46°–65°; wide acute 66°–89°), and surfaces were
described as being either smooth or striated.
For anatomical observations, the dried cypselae were
initially rehydrated in a solution of 5 M NaOH for
36 h (Anderson, 1963), subsequently dehydrated in an
ascending ethanol series and embedded in historesin
(Leica Microsystems, Heidelberg, Germany). The sam-
ples were sectioned with a rotary microtome in trans-
verse and longitudinal series 10 μm thick. The sections
were stained with 0.05% toluidine blue in acetate buffer,
pH 4.7 (O’Brien, Feder & McCully, 1964 modified)
and mounted with synthetic resin (Entellan, Merck,
Darmstadt, Germany). The slides were examined
under a light microscope (Olympus BX51, Olympus,
Southall, UK), and the most representative regions were
photographed with a digital camera (Olympus DP70,
Olympus, Southall, UK). The results were described
using the pericarp sensu lato proposed by Roth (1977), in
which the exocarp develops from the outer epidermis of
the ovary, the endocarp from the inner epidermis of the
ovary and the mesocarp from the region supplied by the
vascular bundles. The terminology used to describe the
trichomes was based on Castro, Leitão-Filho & Monteiro
(1997). The images were organized and brightness and
contrast were adjusted in Photoshop image-editing
software (Redwood City, CA, USA). In some SEM images,
the background was blackened.
RESULTS
Morphology
All cypselae are slightly curved with a prismatic shape
and conspicuous ribs (Fig. 2A–L; Table 2). The carpo-
podia are distinct in most species studied (Fig. 2M–R),
except in D. multicrenulata (Sch.Bip. ex Baker)
R.M.King & H.Rob. (Fig. 2E), D. senecionidea (Baker)
R.M.King & H.Rob., G. mollicoma (B.L.Rob.) R.M.King
& H.Rob. and G. multifida (DC.) R.M.King & H.Rob.,
in which they are indistinct. Only D. praeficta has an
asymmetric carpopodium (Fig. 2O; Table 2).
The floral disc is constricted in most species
(Fig. 2S–W), except in D. praeficta, G. multifida,
Figure 2. SEM of fruit in Disynaphiinae. A–L, general view. A, Acanthostyles buniifolius. B, Campovassouria barbosae. C,
Campovassouria cruciata. D, Disynaphia halimifolia. E, Disynaphia multicrenulata. F, Grazielia dimorpholepis. G, Grazielia
serrata. H, Raulinoreitzia crenulata. I, Symphyopappus decussatus. J, Symphyopappus itatiayensis. K, Symphyopappus
myricifolius. L, Symphyopappus reitzii. M–R, carpopodium. M, Acanthostyles buniifolius. N, Campovassouria cruciata.
O, Disynaphia praeficta. P, Grazielia intermedia. Q, Raulinoreitzia crenulata. R, Symphyopappus lymansmithii. S–X,
detail of the apical region of the fruit showing the floral disc (some with reticulate ornamentation). S, Acanthostyles
buniifolius. T, Campovassouria cruciata. U, Disynaphia halimifolia. V, Grazielia dimorpholepis. W. Raulinoreitzia crenulata.
X, Symphyopappus polystachyus. Arrow, floral disc; arrowhead, rib; ca, carpopodium; pa, pappus. Scale bars: A–L, U–W,
300 μm; N, T, 200 μm; M, O–S, X, 100 μm.
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Table 2. Summary of the morphological features in Disynaphiinae cypsela
Genera Species Cypsela
slightly
curved
Carpopodium Floral disc Pappus
Constricted Reticulated Series Connate at
the basis
Bristle angle Apical cells Surface
Acanthostyles A. buniifolius x Distinct x x 1 Narrow acute Acuminate Smooth
Campovassouria C. barbosae x Distinct x 1 Narrow acute Acuminate Smooth
C. cruciata x Distinct x x 1 Narrow acute Acuminate Smooth
Disynaphia D. halimifolia x Distinct x 1 Moderately acute Rounded Smooth
D. littoralis x Distinct x 1 Wide acute Rounded Smooth
D. multicrenulata x Indistinct x x 1 x Narrow acute Rounded Smooth
D. praeficta x Asymmetric 1 Moderately acute Rounded Smooth
D. senecionidea x Indistinct x 1 Narrow acute Acuminate Smooth
Grazielia G. dimorpholepis x Distinct x x 1 x Moderately acute Rounded Smooth
G. gaudichaudeana x Distinct x x 1 Moderately acute Rounded Smooth
G. intermedia x Distinct x x 1 Moderately acute Acuminate Smooth
G. mollicoma x Indistinct x x 1 Moderately acute Acuminate Smooth
G. multifida x Indistinct 1 Moderately acute Acuminate Smooth
G. nummularia x Distinct x x 1 x Moderately acute Acuminate Smooth
G. serrata x Distinct x x 1 x Moderately acute Acuminate Smooth
Raulinoreitzia R. crenulata x Distinct x x 1 Narrow acute Rounded Striated
R. tremula x Distinct x x 1 x Narrow acute Rounded Striated
Symphyopappus S. angustifolius x Distinct x 2 Narrow acute Rounded Smooth
S. apurimacensis x Distinct x 1 Narrow acute Acuminate Smooth
S. brasiliensis x Distinct 2 x Narrow acute Rounded Smooth
S. compressus x Distinct x x 2 Moderately acute Acuminate Smooth
S. cuneatus x Distinct x x 1 x Moderately acute Rounded Smooth
S. decussatus x Distinct x 2 x Moderately acute Rounded Smooth
S. itatiayensis x Distinct x x 1 Narrow acute Acuminate Smooth
S. lymansmithii x Distinct x x 1 Moderately acute Acuminate Smooth
S. myricifolius x Distinct x x 1 Moderately acute Acuminate Smooth
S. polystachyus x Distinct 1 x Moderately acute Acuminate Smooth
S. reitzii x Distinct x x 2 x Moderately acute Rounded Smooth
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CYPSELAE AND DISYNAPHIINAE SYSTEMATICS 7
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S. brasiliensis (Gardner) R.M.King & H.Rob.,
S. decussatus Turcz. and S. polystachyus Baker. It also
has a reticulated surface in A. buniifolius (Hook. ex
Arn.) R.M.King & H.Rob. (Fig. 2S), C. cruciata (Vell.)
R.M.King & H.Rob. (Fig. 2T), D. multicrenulata,
G. dimorpholepis (Baker) R.M.King & H.Rob. (Fig. 2V),
G. gaudichaudeana (DC.) R.M.King & H.Rob., G. inter-
media (DC.) R.M.King & H.Rob., G. mollicoma,
G. nummularia (Hook. & Arn.) R.M.King & H.Rob.,
G. serrata (Spreng.) R.M.King & H.Rob., R. crenulata
(Spreng.) R.M.King & H.Rob. (Fig. 2W), S. compressus
(Gardner) B.L.Rob., S. cuneatus (DC.) Sch.Bip. ex Baker,
S. decussatus, S. itatiayensis (Hieron.) R.M.King &
H.Rob., S. lymansmithii B.L.Rob., S. myricifolius B.L.Rob.
and S. reitzii (Cabrera) R.M.King & H.Rob. (Table 2).
The pappus is uniseriate (organized in a sin-
gle whorl), except in S. angustifolius Cabrera,
S. brasiliensis, S. compressus, S. decussatus and
S. reitzii, in which it is biseriate (organized in two
whorls) (Table 2). It is free in most species (Fig. 2A–L,
S–X) and connate at the base in D. multicrenulata,
G. dimorpholepis, G. nummularia, G. serrata, R.
tremula (Hook. & Arn.) R.M.King & H.Rob., S. bra-
siliensis, S. cuneatus, S. decussatus, S. polystachyus
(Fig. 2X) and S. reitzii (Table 2). In all species, the
pappus is flattened at the base (Fig. 3A–R; Table 2),
and becomes rounded towards the apex (Fig. 3A′–R′).
However, the projections of the bristles have a narrow
acute angle in A. buniifolius (Fig. 3A), Campovassouria
(Fig. 3C, D), D. multicrenulata (Fig. 3E), D. senecionidea
(Fig. 3G), Raulinoreitzia (Fig. 3K), S. angustifolius
(Fig. 3L), S. apurimacensis H.Rob. (Fig. 3M) ,
S. brasiliensis (Fig. 3N) and S. itatiayensis. The angle
is moderately acute in D. halimifolia (DC.) R.M.King &
H.Rob., Grazielia (Fig. 3H, I), S. compressus (Fig. 3O), S.
cuneatus, S. decussatus, S. lymansmithii (Fig. 3P), S. myric-
ifolius (Fig. 3Q), S. polystachyus and S. reitzii (Fig. 3R).
A wider acute angle was only observed in Disynaphia lit-
toralis (Cabrera) R.M.King & H.Rob. (Fig. 3D; Table 2). In
addition, the apex of the pappus is round in most species
(Fig. 3B, D′, F′, K′, L′, N′, R ′; Table 2), and acuminate only in
A. buniifolius (Fig. 3A′), C. cruciata ( Fig. 3C′),
D. senecionidea ( Fig. 3G′), G. intermedia,
G. mollicoma (Fig. 3H′), G. multifida (Fig. 3I′), G. num-
mularia, G. serrata (Fig. 3J′), S. apurimacensis
(Fig. 3M′), S. compressus (Fig. 3O′), S. itatiayensis,
S. lymansmithii (Fig. 3P′), S. myricifolius (Fig. 3Q′) and
S. polystachyus (Table 2). The surface of the bristles is
striated in Raulinoreitzia, but smooth in all other species
and genera (Fig. 3Kd; Table 2).
AnAtoMy
In all species studied here, the cypselae are polygo-
nal in cross-section and have a uniseriate exocarp
(Figs 4–8), with isodiametrical and juxtaposed cells,
except in A. buniifolius (Fig. 4B, C), D. senecionidea,
G. gaudichaudeana, G. multifida, S. compressus and
S. itatiayensis, in which they are periclinally flattened.
In S. brasiliensis (Fig. 6E, F) and S. reitzii (Fig. 6N, O),
the exocarp is bulkier and has thicker outer walls. In
S. brasiliensis (Fig. 6E, F) and S. decussatus (Fig. 6H,
I), the exocarp is lignified (Table 3).
The outer mesocarp has two or three layers of bulky
cells in A. buniifolius (Fig. 4B), D. praeficta (Fig. 4K),
Raulinoreitzia (Fig. 5K) and S. itatiayensis and three
or four layers in Campovassouria (Fig. 4E), all other
Disynaphia (Fig. 5B, E, H), S. apurimacensis (Fig. 6B)
and S. lymansmithii (Table 3). In all other species of
Symphyopappus, the mesocarp has five or six layers
(Fig. 6E, H, K, N; Table 3), formed by secondary cellu-
lar divisions of the ovary mesophyll during the cypsela
development (Fig. 6P).
Underlying the outer mesocarp, one sclereid layer was
observed in A. buniifolius (Fig. 4B), Campovassouria
(Fig. 4E), Disynaphia (Fig. 4H, K), in most Grazielia
(Fig. 5B, H), Raulinoreitzia (Fig. 5K), S. compressus
and S. lymansmithii; and two or three layers in
G. mollicoma (Fig. 5E), S. angustifolius, S. apurimacensis
(Fig. 6B), S. brasiliensis (Fig. 6E), S. cuneatus,
S. decussatus (Fig. 6H), S. itatiayensis, S. myrici-
folius (Fig. 6K), S. polystachyus and S. reitzii (Fig. 6N;
Table 3).
The inner mesocarp is partially consumed in
D. praeficta (Fig. 5K, L), G. mollicoma (Fig. 5E),
S. angustifolius, S. apurimacensis (Fig. 6B) and
S. polystachyus (Table 3). Only in D. praeficta,
structures shaped like secretory ducts following
the vascular bundles were observed (Fig. 5J, L). In
the other species, that layer and the endocarp are
completely consumed (Figs 4B, E, H, 5B, H, K, 6E, H,
K, N; Table 3).
Phytomelanin is deposited in the schizogenous
space between the outer mesocarp and sclereids
(Figs 4A–C, 5G–L, 6G–O) and forms a continuous
layer external to the vascular bundles in all species
of Disynaphiinae (Figs 4A–L, 5A–L, 6A–P; Table 3).
Disynaphia multicrenulata and G. mollicoma (Fig.
5D) have three vascular bundles. Acanthostyles bunii-
folius (Fig. 4A), Campovassouria (Fig. 4D), D. hal-
imifolia (Fig. 4G), D. littoralis, D. praeficta (Fig. 4J),
D. senecionidea, G. gaudichaudeana, G. intermedia,
G. multifida, G. serrata (Fig. 5G), Raulinoreitzia
(Fig. 5J) and Symphyopappus (Fig. 6A, D, G, J, M)
have five vascular bundles. Grazielia dimorpholepis
(Fig. 5A) and G. nummularia have six vascular
bundles (Table 3). In all species, the vascular bundles
are associated with the ribs except in D. praeficta
(Fig. 4J), in which some ribs have no vascular
bundles (Table 3).
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The carpodia is lignified only in S. brasiliensis,
S. itatiayensis, S. lymansmithii (Fig. 7I) and S. reitzii
(Table 3). The floral disc is similar to the regions
previously described for the pericarp, but the outer
mesocarp and sclerenchyma have more layers
(Fig. 7C–H; Table 3). The outer mesocarp cells also
tend to be more elongated radially when compared
with those of the rest of the cypsela (Fig. 7C–H). The
pappus is composed of lignified cells and supplied by a
central vascular bundle (Fig. 7I; Table 3).
Seven types of trichomes were observed on cypselae
of Disynaphiinae (Fig. 8; Table 3). Acanthostyles
buniifolius has only biseriate non-glandular trichomes
(Fig. 8A; Table 3). Campovassouria has biseriate
non-glandular, capitate uniseriate glandular and
capitate biseriate glandular trichomes (Table 3).
Disynaphia has biseriate non-glandular, capitate
uniseriate glandular (Fig. 8B), filamentous uniseriate
glandular, globular biseriate and stalked capitate
biseriate glandular trichomes (Fig. 8F; Table 3).
Grazielia has biseriate non-glandular, filamentous
uniseriate glandular and capitate biseriate glandular
trichomes (Table 3). Raulinoreitzia had capitate
uniseriate glandular and capitate filamentous uniseri-
ate glandular trichomes (Table 3). Symphyopappus has
biseriate non-glandular, capitate uniseriate glandular,
filamentous uniseriate glandular (Fig. 8C), capitate
filamentous uniseriate glandular (Fig. 8D), globular
biseriate (Fig. 8E) and capitate biseriate glandular
trichomes (Fig. 8F; Table 3).
DISCUSSION
Morphology
All current representatives of Disynaphiinae shared
the slightly curved cypsela. However, this is also
present in D. praeficta, which was recently shown
to be more closely related to some Gyptidinae than
to other species of the genus and other members of
Disynaphiinae (Rivera et al. 2016), and it is thus a
weak feature to support the monophyly of the subtribe.
The carpopodium is the abscission zone of the cypsela,
which aids in the detachment of the cypsela and its
dispersion (Robinson & King, 1977). Its morphology
may be different between tribes (Mukherjee &
Nordenstam, 2004), and has been used in Asteraceae
to segragate some genera (Haque & Godward, 1984).
However, there is a divergence of interpretation of their
states in our study and in those of King & Robinson
(1987) and those of Rivera et al. (2016). In King &
Robinson (1987), the carpopodia of Disynaphia are
described as being indistinct and those of Grazielia as
being distinct, whereas Rivera et al. (2016) described
the carpopodia of all Disynaphiinae as being distinct.
However, our pictures clearly show there is a dis-
tinct carpopodium in D. halimifolia, D. littoralis and
D. praeficta, but not in G. mollicoma and G. multifida.
In addition, our results suggest that the carpopodium
is symmetrical in all Disynaphiinae except D. praeficta,
and this provides good support for its exclusion from
the tribe as suggested by molecular phylogenetic
analyses.
The floral disc and pappus were heterogeneous
and did not form any pattern for the subtribe or
clades identified in phylogenetic analyses or in the
genera. These features can be used in species seg-
regation. The main feature of the pappus reported
in Disynaphiinae is the frequent detachment of
the floral disc with the pappus in a unit (King &
Robinson, 1987; Robinson, 2006; Robinson et al.,
2009; Marzinek & Oliveira, 2010). However, in
the studied species, this feature was only seen in
A. buniifolius, S. decussatus and S. polystachyus.
Furthermore, there is ornamentation in the pappus
surface of Raulinoreitzia that was absent in the
other taxa studied, and in S. reticulatus, in the study
of Marzinek & Oliveira (2010).
AnAtoMy
The anatomy of the cypselae has great value in the
systematics in tribes of Asteraceae (Freitas et al.,
2015). A pericarp with a one-layered exocarp, a
parenchymatous outer mesocarp, a sclerenchymatous
median mesocarp, a consumed inner mesocarp and
endocarp, a schizogenous space formed between the
outer mesocarp and sclereids filled with phytome lanin,
as found in all species of Disynaphiinae studied here,
is also a common feature of cypselae in Eupatorieae
(Pandey & Singh, 1983; Pandey, Wilcox & Stuessy,
1989; Pandey & Singh, 1994; Marzinek & Oliveira,
2010; De-Paula et al., 2013; Batista, 2014; Franca
Figure 3. SEM of pappus in Disynaphiinae. A–R, base. A′–R′, apex. A, Acanthostyles buniifolius. B, Campovassouria
barbosae. C, Campovassouria cruciata. D, Disynaphia littoralis. E, Disynaphia multicrenulata. F, Disynaphia praeficta. G,
Disynaphia senecionidea. H, Grazielia mollicoma. I, Grazielia multifida. J, Grazielia serrata. K, Raulinoreitzia crenulata.
d, note the wall ornamentation. L, Symphyopappus angustifolius. M, Symphyopappus apurimacensis. N, Symphyopappus
brasiliensis. O, Symphyopappus compressus. P, Symphyopappus lymansmithii. Q, Symphyopappus myricifolius. R,
Symphyopappus reitzii. Scale bars: R, 100 μm; B, C–K, K′–Q′, R′, 50 μm; A–A′, B′, 25 μm; Kd, 10 μm.
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et al., 2015), reinforcing its value in systematics of
Asteraceae. A similar pattern without a schizogenous
space is reported for Coreopsideae (Pandey &
Singh 1982a; Julio & Oliveira, 2009; Batista 2014) ,
Heliantheae (Misra, 1972; Stuessy & Liu, 1983; Pandey
& Singh, 1994; Batista, 2014), Madieae (Pandey &
Singh, 1994), Millerieae (Stuessy & Liu, 1983; Batista,
2014; Frangiote-Pallone & Souza, 2014), Neurolaeneae
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(Batista, 2014) and Tageteae (Misra, 1964; Pandey,
1998; Frangiote-Pallone & Souza, 2014). All tribes
listed above, including Disynaphiinae, as currently
circumscribed, form a monophyletic group called
the Heliantheae alliance by Anderberg et al. (2007).
Another deposition pattern in the family is found in
Heterocoma DC. currently nested in Cichorioideae–
Vernonieae, in which the phytomelanin is deposited
internally to the sclerenchyma (Freitas et al., 2015).
In addition to the deposition mode, the position of
the phytomelanin layer in relation to the vascular
bundles is an important feature in Disynaphiinae. In
all species studied, the phytomelanin layer is external
to the vascular bundle. The only concern is that this
layer was also observed in D. praeficta. Possibly, this
feature can be (1) notable among the subtribes or (2)
independently appear in Eupatorieae as a homoplastic
character state.
In lower ranks of the classification, the pericarp
structure is poorly explored. In Disynaphiinae, some
Symphyopappus spp. differ from other species in the
subtribe and tribe by having the outer mesocarp with
a larger number of layers formed by divisions of the
ovary mesophyll during the cypsela development.
Multiplicative pericarp as found in this genus is a
rare feature in Asteraceae. In some cases, this mer-
istematic tissue layer is responsible for forming an
aerenchyma as in the cypsela of Eclipta prostrata
(L.) L. and Heliopsis helianthoides Britton, Sterns
& Poggenb. var. scabra (Dunal) T.R.Fisher (Loose,
1891) or a periderm-like tissue in Helianthus annuus
L. (Hanausek, 1902), all currently circumscribed in
Heliantheae (Funk et al., 2009).
The number of layers of the outer mesocarp
appears as an important evolutionary feature in
the phylogenetic hypothesis proposed by Rivera
et al. (2016). Acanthostyles buniifolius and the
Raulinoreitzia–S. itatiayensis clade (Fig. 1) have
two to three layers. As A. buniifolius is sister to all
other Disynaphiinae, two to three layers of outer
mesocarp appear as a potential ancestral state in the
subtribe. The Campovassouria–Disynaphia (exclud-
ing D. praeficta) clade (Fig. 1) has three to four
layers as the apomorphic character state. However,
the Grazielia–Symphyopappus clade (except
S. itatiayensis), proposed by Rivera et al. (2016), is not
supported by the outer mesocarp layers. All Grazielia
spp., S. apurimacensis and S. lymansmithii have two
to four layers, whereas most Symphyopappus spp.
have five to six layers of the outer mesocarp, includ-
ing S. compressus which appears to be more closely
related to Grazielia than to other Symphyopappus
spp. Our results also suggest the outer mesocarp
with five to six layers evolved independently in
Symphyopappus s.s. and S. compressus (Fig. 1).
The number of sclerenchyma layers varies in
Disynaphiinae. Most taxa have one layer, but two or
more layers are more common in Symphyopappus,
including S. itatiayensis currently associated with
Raulinoreitzia (Rivera et al., 2016). The character state
of two or more layers possibly appeared independently
in the subtribe. According to Robinson et al. (2009), the
cypsela wall has phytomelanin perforations, with cells
connected to each other being arranged in horizon-
tal bands in all Disynaphiinae. This feature was also
used by King & Robinson (1987) to circumscribe the
subtribe and was also reported in S. apurimacensis,
S. decemflorus (Robinson, 2006) and S. brasiliensis
(Hattori, 2013). In this work, this feature was only
observed in A. buniifolius and Raulinoreitzia, which
have fewer cell layers in the outer mesocarp. The
micropunctations observed by Robinson et al. (2009),
King & Robinson (1987), Robinson (2006) and Hattori
(2013) are the projections of the sclereids becoming
visible from dehydration of the outer mesocarp by the
herborization process and can be an unreliable taxo-
nomic feature.
The carpopodial exocarp is lignified in the clade
formed by S. itatiayensis and Raulinoreitzia, in
S. brasiliensis nested in Symphyopappus s.s. and in
S. lymansmithii and S. reitzii which were not
included in the phylogenetic analysis of Rivera et al.
(2016). This character has probably thus evolved
independently in Symphyopappus, and a larger
anatomical and phylogenetic sample is necessary to
study if this character state evolved more than twice
in Dysinaphiinae.
The ribs, floral disc and pappus are heterogeneous
and do not form any pattern for the subtribe or
clades in phylogenetic analyses or in the genera.
These features can, however, be used for species
segregation. Concerning the ribs, Dysinaphiinae
are characterized by having cypselae with five ribs
(King & Robinson, 1987), but our results show a
Figure 4. Transversal sections of the pericarp in Acanthostyles, Campovassouria and Disynaphia. A–C, Acanthostyles
buniifolius. A, general view of the pericarp. B, intercostal region. C, costal region. D–F, Campovassouria cruciata. D, general
view of the pericarp. E, intercostal region. F, costal region. G–I, Disynaphia halimifolia. G, general view of the pericarp. H,
intercostal region. I, costal region. J–L, Disynaphia praeficta. J, general view of the pericarp. K, intercostal region. L, costal
region. Arrow, rib with a vascular bundle; arrowhead, phytomelanin; asterisk, rib without a vascular bundle; ex, exocarp;
om, outer mesocarp; pe, pericarp; sc, seminal chamber; sd, secretory duct; sl, sclerenchyma; tr, trichome; vb, vascular bundle.
Scale bars: A, D, G, J, 200 μm; I, 50 μm; E, F, H, K, L, 25 μm; B, 10 μm.
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wide variation in the number of ribs. Disynaphia
praeficta and G. mollicoma have ribs without vas-
cular bundles, supporting the observations by
Marzinek et al. (2010), who stated that some ribs
can be formed by the compression exerted by the
cypsela on others and that this feature should be
carefully used in the identification keys.
The vascularized pappus, as found in all cypselae
of Disynaphiinae studied, also occurs in S. reticulatus
(Marzinek & Oliveira, 2010) and is commonly found
in Eupatorieae [Helogyne Nutt. (Alomiinae), King &
Robinson, 1987; Campuloclinium macrocephalum
DC. (Gyptidinae), Chromolaena stachyophylla
(Spreng.) R.M.King & H.Rob. (Praxelinae), Mikania
micrantha Kunth (Mikaniinae), Praxelis pauciflora
(Kunth) R.M.King & H.Rob. (Praxelinae) and Vittetia
orbiculata (DC.) R.M.King & H.Rob. (Gyptidinae),
Marzinek & Oliveira, 2010], but also occurs in
Tageteae [Porophyllum ruderale (Jacq.) Cass.] and
Millerieae (Tridax procumbens L.) (Frangiote-Pallone
& Souza, 2014). Although underestimated, the vascu-
larized pappus appears have evolved independently in
Asteraceae.
Trichomes have had great value in the delimita-
tion of Asteraceae since the study of Hess (1938),
who described biseriate trichomes as being unique
to the family and not being found in any other
angiosperm lineage. These trichomes, as found in
cypselae of Disynaphiinae, are called ‘twin hairs’
or Zwillingshaare, due to them having a single ini-
tial cell (Hanausek, 1910; Hess, 1938; Roth, 1977).
Different functions are attributed to them, but it
has been demonstrated that they act as protection
against herbivores in vegetative organs (Appezzato-
da-Glória et al., 2012). They also help more specifi-
cally in the fixation and water absorption by cypselae
(De-Paula et al., 2015). Besides the twin hairs, the
family has several other types of trichomes used at
different taxonomic levels (Castro et al., 1997). In
lower ranks of the classification, trichomes can be
used for the delimitation of genera, or combined with
other species-delimiting features as initially pro-
posed by King & Robinson (1970). Although type IV
and V trichomes are restricted to Symphyopappus,
only type V trichomes support the Symphyopappus
s.s. clade, because type IV trichomes also occur in S.
compressus, which falls in a clade with Grazielia in
the study of Rivera et al. (2016).
CONCLUSIONS AND PERSPECTIVES
The micropunctuations found in all Disyphiineae
are also found in other members of Eupatorieae,
including D. praeficta, and thus cannot be a synapo-
morphy of the subtribe as proposed earlier by King
& Robinson (1987). These micropunctations become
visible due to dehydration of the pericarp and can be
more or less visible due to the herborization method
and especially the number of layers of the outer
mesocarp. The phytomelanin layer external to the
vascular bundles and vascularized pappus were pre-
sent in all species including D. praeficta. The phy-
tomelanin position in relation to vascular supplying
should be studied in other subtribes to test if (1)
they represent a synapomorphy for Disynaphiinae
and related subtribes or (2) they evolved indepen-
dently in Eupatorieae. Our study revealed that the
number of layers of the outer mesocarp is an impor-
tant feature supporting the relationship among two
of four clades studied here and suggests that a two-
or three-celled thick mesocarp may be the ancestral
state for the subtribe. Only type V trichomes sup-
ported Symphyopappus as a clade (excluding S. com-
pressus and S. itatiayensis). Even the multiplicative
pericarp, a rare feature in Asteraceae, has evolved
in Symphyopappus s.s. and S. compressus indepen-
dently. Our results also supported the exclusion of
D. praeficta from the subtribe, previously suggested
only by molecular data. Disynaphia praeficta has ribs
lacking a vascular bundle and an asymmetric carpo-
podium and secretory ducts in the pericarp, which
are absent in all other members of the subtribe. As
a perspective for future research, the knowledge
of the cypsela structure in Neocabreria R.M.King
& H.Rob., Radlkoferotoma Kuntze, Urolepis (DC.)
R.M.King & H.Rob. and Malmeanthus R.M.King &
H.Rob would be useful in a new circumscription of
Disynaphiinae.
Figure 5. Transversal sections of pericarp in Grazielia and Raulinoreitzia. A, D, G, J, visão geral do pericarpo. B, E, H, K,
regiões intercostais e (C, F, I, L) costais. A–C, Grazielia dimorpholepis. A, general view of the pericarp. B, intercostal region.
C, costal region. D–F, Grazielia mollicoma. D, general view of the pericarp. E, intercostal region. F, costal region. Note the
double layer of sclereids in E and F. G–I, Grazielia serrata. G, general view of the pericarp. H, intercostal region I, costal
region. J–L, Raulinoreitzia tremula. J, general view of the pericarp. K, intercostal region. L, costal region. Arrow, rib with a
vascular bundle; arrowhead, phytomelanin; ex, exocarp; im, inner mesocarp; pe, pericarp; om, outer mesocarp; se, seed; sl,
sclerenchyma; tr, trichome; vb, vascular bundle. Scale bars: A, D, G, J, 200 μm; B, C, E, F, H, I, K, L, 25 μm.
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Figure 6. Transversal sections of pericarp in Symphyopappus. A–C, Symphyopappus apurimacensis. A, general view of
the pericarp. B, intercostal region. C, costal region. D–F, Symphyopappus brasiliensis. D, general view of the pericarp. E,
intercostal region. F, costal region. G–I, Symphyopappus decussatus. G, general view of the pericarp. H, intercostal region. I,
costal region. J–L, Symphyopappus myricifolius. J, general view of the pericarp. K, intercostal region. L, costal region. M–O
Symphyopappus reitzii. M, general view of the pericarp. N, intercostal region. O, costal region. P, Symphyopappus polys-
tachys young fruit showing the cell divisions in the pericarp. Arrow, rib with a vascular bundle; arrowhead, phytomelanin;
double arrowhead, cellular division; ex, exocarp; im, inner mesocarp; pe, pericarp; om, outer mesocarp; sl, sclerenchyma; tr,
trichome; vb, vascular bundle. Scale bars: A, D, G, J, M, 200 μm; B, C, E, F, H, I, K, L, N–P, 50 μm.
Figure 7. Transversal sections of a floral disc, pappus and carpopodium in Disynaphiinae. A, B, carpopodium. A,
Campovassouria cruciata. B, Symphyopappus lymansmithii. C–H, floral disc. C, Acanthostyles buniifolius. D, Campovassouria
cruciata. E, Disynaphia senecionidea. F, Grazielia intermedia. G, Raulinoreitzia tremula. H, Symphyopappus itatiayensis. I,
pappus of Symphyopappus cuneatus, note the vascularization of the pappus. Arrowhead, phytomelanin; vb, vascular bundle.
Scale bars: A–H, 100 μm; I, 25 μm.
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Table 3. Summary of the anatomical features in Disynaphiinae cypsela
Genera Species Layers of
the outer
mesocarp
Sclerenchyma
layer(s)
Phytomelanin
layer above
vascular bundles
Ribs Vascular
bundles
Carpopodium Pappus Trichome types (Fig . 8)
Lignified Vascularized Biseriate
non-glandular
Capitate
uniseriate
glandular
Filamentous
uniseriate
glandular
Capitate
filamentous
uniseriate
glandular
Globular
biseriate
Capitate
biseriate
glandular
Stalked
capitate
biseriate
glandular
Acanthostyles A. buniifolius 2–3 1 x 5 5 x x
Campovassouria C. barbosae 3–4 1 x 5 5 x x x
C. cruciata 3–4 1 x 5 5 x x
Disynaphia D. halimifolia 3–4 1 x 5 5 x x x
D. littoralis 3–4 1–2 x 5 5 x x x
D. multicrenulata 3–4 1 x 3 3 x x x
D. praeficta 2–3 1 x 9 5 x x x x x
D. senecionidea 3–4 1 x 5 5 x x x
Grazielia G. dimorpholepis 3–4 1 x 6 6 x x x
G. gaudichaudeana 3–4 1 x 5 5 x
G. intermedia 3–4 1 x 5 5 x x x
G. mollicoma 3–4 2–3 x 3 3 x x x
G. multifida 3–4 1 x 5 5 x x
G. nummularia 3–4 1 x 6 6 x x
G. serrata 3–4 1 x 5 5 x x
Raulinoreitzia R. crenulata 2–3 1 x 5 5 x x
R. tremula 2–3 1 x 5 5 x
Symphyopappus S. angustifolius 5–6 2–3 x 5 5 x x
S. apurimacensis 3–4 2–3 x 5 5 x x x x
S. brasiliensis 5–6 2–3 x 5 5 x x x x
S. compressus 5–6 1 x 5 5 x x
S. cuneatus 5–6 2–3 x 5 5 x x x
S. decussatus 5–6 2–3 x 5 5 x x x
S. itatiayensis 2–3 2–3 x 5 5 x x x x x
S. lymansmithii 3–4 1 x 5 5 x x x x
S. myricifolius 5–6 2–3 x 5 5 x x
S. polystachyus 5–6 2–3 x 5 5 x x x
S. reitzii 5–6 2–3 x 5 5 x x x x
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ACKNOWLEDGEMENTS
The authors would like to thank the BHCB, CEN,
HUFU and MBM herbaria for providing the mater-
ial studied and the Laboratório Multiusuário
de Microscopia Eletrônica of the Faculdade de
Engenharia Química (UFU) for support with SEM.
T.D.G.S. would like to thank CAPES (Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior) for the
scholarship granted. We thank Julien Bachelier and
two anonymous reviewers for their helpful comments
and suggestions.
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