Plant Syst Evol (2011) 291:159–172
DOI 10.1007/s00606-010-0374-2
ORIGINAL ARTICLE
Seed morphology and endosperm structure of selected species
of Primulaceae, Myrsinaceae, and Theophrastaceae and their
systematic importance
Maria Morozowska • Aneta Czarna •
Marcin Kujawa • Andrzej M. Jagodzinski
Received: 4 February 2010 / Accepted: 4 October 2010 / Published online: 11 November 2010
Ó The Author(s) 2010. This article is published with open access at Springerlink.com
A. M. Jagodzinski
Department of Forest Protection,
Poznan University of Life Sciences,
Wojska Polskiego 71c, 60-625 Poznan, Poland
e-mail: amj@man.poznan.pl
shape types, the length/width ratio of the seeds examined
was constant, while their hilum length/width ratio was a
highly variable feature. Three types of seed surface patterns
were observed: (1) reticulate, (2) tuberculate with secondary striations, and (3) poroid-alveolate with the presence of
a spongy outer layer. For seeds of Anagallis arvensis,
A. minima, Cortusa matthioli, Lysimachia nemorum, and
Soldanella carpatica, secondary seed sculpture was
described. The seed coats of all species examined were twolayered, and great differences in testa thickness were found
(9.9–128.6 lm). In general, seeds of the Myrsinaceae species were more often characterized by thick testa. Different
proportions in thickness of the inner and outer testa layers
were observed. In seeds with reticulate seed patterns, the
inner testa layer was twice to several times thicker than the
outer layer, while the opposite proportions were observed in
seeds with the tuberculate or poroid-alveolate seed sculpture pattern. In seeds of all species examined, oxalate
crystals were present on the surface of the inner testa layer.
Thus, the presence or absence of oxalate crystals in testa is
not a feature distinguishing species with angular or subglobose seeds. Four types of endosperm structure were
identified according to the thickness of the endosperm cell
walls and the relief of their inner surface: (1) with evenly
thickened and smooth cell walls, (2) with evenly thickened
cell walls and circular or helical thickenings on their inside
surfaces, (3) with very thick, but not evenly thickened cell
walls with constrictions (‘‘pitted’’), and (4) with very thin
papery and undulate cell walls. There is no rule concerning
the seed shape, type of the seed sculpture, testa thickness, or
endosperm structure that corresponds to the family affiliation of the species examined.
A. M. Jagodzinski
Institute of Dendrology, Polish Academy of Sciences,
Parkowa 5, 62-035 Kornik, Poland
Keywords Seed morphology Seed sculpture Testa
Endosperm structure
Abstract Seed size and shape, seed coat surface pattern,
seed coat thickness, and endosperm structure were investigated in Androsace septentrionalis, Cortusa matthioli,
Hottonia palustris, Primula elatior, Soldanella carpatica
(Primulaceae), Anagallis arvensis, A. minima, Cyclamen
purpurascens, Glaux maritima, Lysimachia nemorum,
L. vulgaris, Trientalis europaea (Myrsinaceae), and Samolus valerandi (Theophrastaceae). Three seed size categories
were distinguished on the basis of biometric measurements.
Almost all seeds examined were found to be small with an
angular shape classified as sectoroid or polyhedral. A new
type of seed shape, suboval, was identified for H. palustris.
Cyclamen purpurascens seeds differed from seeds of all
other species examined because of their large size, subglobose shape, and concave hilar area. Despite the different
M. Morozowska (&) A. Czarna
Department of Botany, Poznan University of Life Sciences,
Wojska Polskiego 71c, 60-625 Poznan, Poland
e-mail: mariamor@up.poznan.pl
A. Czarna
e-mail: czarna@up.poznan.pl
M. Kujawa
Laboratory of Electron and Confocal Microscope,
Faculty of Biology, Adam Mickiewicz University,
Umultowska 89, 61-614 Poznan, Poland
e-mail: wpmek@amu.edu.pl
123
160
Introduction
The phylogenetic relationships within the group of three
families––Primulacaeae, Myrsinaceae, and Theophrastaceae––based either on DNA sequences from plastid (atpB,
ndhF, rbcL, and trnL-F), nuclear (ITS), and mitochondrial
(atp1 and matR) genes or morphological data have been
analyzed in many papers (Anderberg and Ståhl 1995;
Anderberg et al. 1998, 2000, 2002; Källersjö et al. 2000;
Caris et al. 2000; Hao and Hu 2001; Mast et al. 2001;
Källersjö and Ståhl 2003; Martins et al. 2003; Hao et al.
2004; Ståhl and Anderberg 2004; Manns and Anderberg
2005, 2007; Oh et al. 2008). As a result of some of these
studies, strong support for a sister group relationship
between Myrsinaceae s.str. and the tribe Lysimachieae of
Primulaceae was found. Källersjö et al. (2000) recognized
four families among the ‘‘primuloid’’ families of the
Ericales s.l., namely Primulaceae, Myrsinaceae, Theophrastaceae, and Maesaceae. In their opinion the recognition
of four families was the best way of preserving taxonomic
stability but required some generic realignments. These
realignments concerned Samolus, which was transferred to
Theophrastaceae; Maesa, which was raised to family level;
and Lysimachia, Anagallis, Trientalis, Glaux, Asterolinon,
Pelletiera, Coris, Ardisiandra, and Cyclamen, which were
included in Myrsinaceae. In other works on molecular
phylogeny of the Primulales s.l., many close or distant
relationships of certain taxa were described. Martins et al.
(2003) found that Anagallis minima is rather distantly
related to A. arvensis, which, along with Asterolinon and
Pelletiera, is closely related to Lysimachia sect. Lerouxia.
These results also supported the monophyly of a group
consisting of the four genera Hottonia, Soldanella, Bryocarpum, and Omphalogramma; provided evidence against
monophyly of the large genera Primula, Androsace, and
Lysimachia; and showed that Cyclamen is not a member of
the Myrsinaceae-Lysimachieae clade such that its position
remains unclear. Hao et al. (2004) found only Glaux to be
placed within Lysimachia, and Anderberg et al. (2007)
suggested that in addition to Glaux, such genera as Anagallis, Asterolinon, and Pelletiera should also be nested
within Lysimachia. Recent work by Oh et al. (2008) concerning seed morphology of some species from the family
Myrsinaceae, especially the genus Lysimachia and a few
related taxa, showed that mapping of seed characters onto a
previously obtained phylogenetic tree may provide potentially synapomorphic character states for those subclades of
Lysimachia that were examined. The general conclusion of
these authors was that seed morphology may provide some
important evidence for understanding the phylogeny of the
taxa being examined.
Because the phylogenetic interrelationships within the
primuloid families are still not completely resolved, we
123
M. Morozowska et al.
have investigated seed structure of some species of Primulaceae, Myrsinaceae, and Theophrastaceae. The aims of
our study were (1) to examine and describe seed morphology and endosperm structure of some genera from
Primulaceae, Myrsinaceae, and Theophrastaceae, (2) to
find seed characters of systematic importance, and (3) to
evaluate their usefulness in confirming or constraining a
new systematic classification of the genera studied.
Materials and methods
Biometric measurements
Morphological and anatomical seed characters of Androsace septentrionalis, Cortusa matthioli, Hottonia palustris,
Primula elatior, Soldanella carpatica (Primulaceae), Anagallis arvensis, A. minima, Cyclamen purpurascens, Glaux
maritima, Lysimachia nemorum, L. vulgaris, Trientalis
europaea (Myrsinaceae), and Samolus valerandi (Theophrastaceae) were studied using seeds collected during the
years 2005–2007 from living plants in natural localities and
from herbarium collections (Table 1). In natural localities,
plants grew in habitats optimal for them. In each locality,
seeds were collected from 15 individuals, and the representative seed sample was used for biometric measurements. Herbarium specimens were used to obtain seeds of
two species; for Anagallis arvensis, a representative seed
sample was composed of seeds from 20 individuals, and
for Cyclamen purpurascens, seeds were obtained from the
few individuals available. Measurements of seed and hilum
size were carried out with light microscopy (LM) on 30
seeds of each species. Seed coat thickness was measured on
five seeds of each species with the help of scanning electron microscopy (SEM) pictures. Seed length was measured along the longest axis in the plane of the hilum and
microphyle, seed width along the longest axis at a 90°
angle to the plane of the hilum and microphyle, and seed
thickness along the dorsiventral axis. Length and width of
the hilum were also measured, and length/width ratio for
both seed and hilum was calculated. For testa thickness, the
inner (IL) and outer (OL) layers were measured on the
longitudinal seed sections.
Seed preparations for SEM
Before cutting, seeds were fixed in a mixture of ethyl
alcohol and acetic acid in a 3:1 proportion for 12 h. After
rinsing in distilled water (two times for 10 min), they were
sectioned at -15°C in a cryostat LEICA CM 1850, washed
several times to remove the freezing medium, and dried
using an acetone sequence in the following concentrations:
30, 50, 70, 90, and 100%, three times for 6 min in each.
Seed morphology and endosperm structure of Primulaceae, Myrsinaceae and Theophrastaceae
Table 1 List of species
analyzed in the study
161
Family
Species
Specimen collection data
Figures
Myrsinaceae
Anagallis minima L.
N 53°350
4f–m
E 18°350
leg. Aneta Czarna
Myrsinaceae
Cyclamen purpurascens Mill.
TRN Herbarium, PL
3r–v
leg. Andrzej Zielski
Myrsinaceae
Glaux maritima L.
N 52°100
3m–q
E 17°410
leg. Aneta Czarna
Myrsinaceae
Lysimachia nemorum L.
N 49°080
4n–r
E 22°340
leg. Aneta Czarna
Myrsinaceae
Lysimachia vulgaris L.
N 52°210
3a–d
E 15°520
leg. Maria Morozowska
Myrsinaceae
Trientalis europaea L.
Myrsinaceae
Anagallis arvensis L.
N 53°290
E 22°420
3e–l
leg. Aneta Czarna
POZNB Herbarium, PL
4a–e
leg. Aneta Czarna
Primulaceae
Androsace septentrionalis L.
N 53°460
5a–d
E 20°280
leg. Aneta Czarna
Primulaceae
Cortusa matthioli L.
N 49°170
6f–i
E 19°560
leg. Aneta Czarna
Primulaceae
Hottonia palustris L.
N 52°060
6j–n
0
E 17°04
leg. Aneta Czarna
Primulaceae
Primula elatior (L.) Hill
N 49°080
5e–l
E 22°340
Primulaceae
Soldanella carpatica Vierh.
leg. Aneta Czarna
N 49°170
6a–e
0
E 19°56
leg. Aneta Czarna
Theophrastaceae
Samolus valerandi L.
N 52°100
6o–r
E 17°410
leg. Aneta Czarna
For SEM, seeds and seed cuttings were covered with gold
and examined with a Zeiss EVO 40 electron microscope at
8–15 kV depending on the species.
Statistical analysis
The biometric data were analyzed statistically. For each
seed trait, one-factor analysis of variance (ANOVA) was
used to examine differences in means among species
studied. If critical differences were noted, multiple
comparisons were carried out based on Tukey’s test for
equal sample sizes. To show similarities and differences
among taxa studied, Ward’s hierarchical clustering method
was used to compute cluster groups of species based on the
seed characters analyzed. Statistical analyses were performed using JMP 8.0 (SAS Institute, Cary, NC, USA;
http://www.sas.com/).
The specimens and the seed material are deposited in the
herbarium of the Botany Department (POZNB), Poznan
University of Life Sciences, Poland.
123
162
Results
Seed shape and size
To describe seed shape for the species examined, we have
adopted two of the three major shape types proposed by Oh
et al. (2008). These were the sectoroid seed shape, which was
observed for seeds of Androsace septentrionalis, Cortusa
matthioli, Soldanella carpatica (Primulaceae), Anagallis
arvensis, A. minima, G. maritima, Lysimachia nemorum, L.
vulgaris, and T. europaea (Myrsinaceae), and the polyhedral
seed shape, which was observed for seeds of P. elatior
(Primulaceae) and Samolus valerandi (Theophrastaceae)
(Table 2). We have described two other seed shape types:
suboval and subglobose with a concave hilar area (Fig. 1).
The suboval shape corresponds to the lateral view of the seed
and is typical for H. palustris. Seeds are laterally flattened,
and the lateral faces are slanted towards the ventral margin.
The hilum is located along the ridge on the top side (Fig. 6j, k).
Subglobose seeds are typical for Cyclamen purpurascens.
Their shape is similar to rugose seeds described by Oh et al.
(2008) for Asterolinon linum-stellatum, but their surface is
not rugose. In dorsal view they are elliptic, and on the ventral
side, they have a concave hilar area (Fig. 3r, s). Three
seed size categories were distinguished: small, medium,
and large. Most of the species examined have small seeds
with the following length, width, and thickness ratios:
0.5–1.6 9 0.3–1.2 9 0.4–0.8 mm. T. europaea has medium
size seeds (1.6 9 1.4 9 0.6 mm), and seeds of Cyclamen
purpurascens are substantially larger than all other species
(2.8 9 2.4 9 1.3 mm) (Table 2).
Statistical analyses showed significant differences
(P \ 0.0001) among species studied for all features analyzed (Tables 3, 4). Ward’s agglomerative grouping
method distinguished three main groups of taxa (Fig. 2).
Two of them comprise species representing both Primulaceae and Myrsinaceae, and Cyclamen purpurascens is
isolated in the third one. In the first group, the closest
similarities were found for species representing the same
family, while in the second group, the closest similarities
were observed for species representing different families
and different shape types. Multiple comparisons based on
Tukey’s test showed the presence of two uniform groups of
species based on mean values of the length/width ratio. The
first group included all species of Myrsinaceae examined
and only one species of Primulceae, i.e., P. elatior, while
the second group included all other species of Primulaceae
(except Soldanella carpatica) and Samolus valerandi of
Theophrastaceae (Table 3). Despite the different sizes and
shape types of seeds, length/width proportions remained
fairly similar within particular families (CV = 23.4%). In
contrast, the hilum length/width ratio was found to be
a highly variable feature (CV = 53.7%) (Table 4).
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M. Morozowska et al.
The species examined were characterized by either narrow,
narrowly elliptic, or elliptic hilum shapes (ratios from
longest axis to shortest axis ranged from 8:1 to 5:1, 4.5:1 to
2.5:1, and 2:1 to 1.5:1, respectively).
Most of the examined species of Primulaceae and
Myrsinaceae have narrowly elliptic or elliptic hila more or
less flush with the surface. Soldanella carpatica, Cortusa
matthioli, and Samolus valerandi have narrow hilum
(Fig. 6a, b, f, o), and Cyclamen purpurascens has extremely different, elliptic immersed concave hila (Fig. 3s;
Table 2). Seeds of Cyclamen purpurascens were also significantly different from seeds of Myrsinaceae, Primulaceae, and Theophrastaceae in all other biometric features
(Tables 3, 4). In turn, seeds of Samolus valerandi were
found to be very similar to seeds of Anagallis minima
according to testa thickness, and no significant differences
were noted between these species for seed length, width, or
thickness.
Seed surface and testa thickness
Three types of the seed sculpture were observed among all
species examined. The most common was reticulate ornamentation of the seed surface, observed for G. maritima
(Fig. 3n), Cyclamen purpurascens (Fig. 3t), Androsace
septentrionalis (Fig. 5b), Soldanella carpatica (Fig. 6c),
Cortusa matthioli (Fig. 6h), H. palustris (Fig. 6l), and
Samolus valerandi (Fig. 6q). The reticulate and partly
vesiculose seed pattern of Cortusa matthioli and the
reticulate seed sculpture of Soldanella carpatica were also
characterized by secondary sculpture; in Cortusa matthioli
the surface of the outer cell walls exhibited a striate microornamentation (Fig. 6h) and in Soldanella carpatica small
secondary protrusions were observed (Fig. 6d). A tuberculate seed surface pattern characterized P. elatior seeds,
and tuberculate sculpture with secondary striations was
observed for Anagallis arvensis, A. minima, and Lysimachia nemorum (Fig. 4d, i, p). Two species, Lysimachia
vulgaris and T. europaea, were characterized by the presence of a sponge-like outer layer with a poroid-alveolate
surface pattern. On L. vulgaris seeds, this outer layer has a
columnar look (Fig. 3b) that was previously described by
Oh et al. (2008). The observed thickness of the outer layer
was about 80–110 lm (Table 2). The sponge-like outer
layer on T. europaea seeds consists of rectangular cells
(RC) with a roof-like upper wall and longitudinal thickenings on the inner cell wall surface (Fig. 3j). The thickness of the outer layer for this species was about 30–62 lm
(Table 2). The surface of the sponge-like outer layer on
T. europaea sometimes showed a reticulate pattern with
secondary striations (Fig. 3g). Testa structure of all species
examined was characterized by two layers, an inner layer
(IL) and outer layer (OL) of different thicknesses, which
Species
Anagallis
arvensis
Seed shape
Sectoroid
Anagallis minima Sectoroid
Androsace
septentrionalis
Sectoroid
Cortusa matthioli Sectoroid (±
angled)
Spongy Surface
outer
layer
Hilum
shape
Edges
Testa thickness (lm)
Endosperm cell walls
Testa
Outer
layer
Inner
layer
Type of
thickness
Inner surface
Absent Tuberculate with
Narrow
secondary striations
Not winged or
keeled; weakly or
strongly winged
71.6
49.9
21.7
Evenly thickened
Smooth or with little
protrusions
Absent Tuberculate with
Narrowly elliptic
secondary striations
Not winged, rarely
weakly keeled
18.6
12.5
6.1
Very thin, paperlike
Strongly undulate,
partly with circular
thickenings
Absent Reticulate
Narrow
Not winged or
keeled
19.0
4.0
15.0
Evenly thickened
or ‘‘pitted’’
If pitted, then with
‘‘hollows’’
Absent Reticulate/vesiculose Narrow
with secondary
striations
Not winged or
keeled
31.5
6.4
25.1
Evenly thickened
Smooth
Cyclamen
purpurascens
Subglobose
Absent Strongly reticulate
Narrowly elliptic
immersed
concave hilum
–
45.8
32.3
13.5
Very thick, not
evenly
thickened,
‘‘pitted’’
With ‘‘hollows’’
Glaux maritima
Sectoroid
Absent Reticulate
Narrowly elliptic
Weakly keeled
93.1
34.2
58.9
Evenly thickened
or very thin,
paper-like
Smooth, or if thin,
then with circular
thickenings
Absent Weakly reticulate
Elliptic
–
10.5
3.0
7.5
Thin, paper-like
63.2
57.7
Evenly thickened
Smooth, slightly
undulate
With helical
thickenings
Hottonia palustris Suboval (±
angled)
Lysimachia
Sectoroid,
nemorum
polyhedral
Absent Tuberculate with
Elliptic
secondary striations
Not winged or
keeled
121.9
101.1–128.6 82.5–110.0 18.6
Sectoroid
Present Poroid-alveolate
Narrowly elliptic
Not winged or
keeled
Primula elatior
Polyhedral
Absent Tuberculate
Elliptic
Not winged or
keeled
87.0
69.0
Samolus
valerandi
Polyhedral
Absent Reticulate to weakly Narrow
reticulate
Not or very weekly
keeled or winged
10.7
Soldanella
carpatica
Sectoroid (±
angled)
Absent Weakly reticulate
with secondary
protrusions
Narrow
Not winged or
keeled
9.9
Trientalis
europaea
Sectoroid/
polyhedral
Present Poroid-alveolate (±
reticulate, with
secondary
striations)
Narrowly elliptic
to elliptic
Not winged or
keeled
66.6–96.1
Thin, paper-like
Smooth or slightly
undulate
18.0
Evenly thickened
and rarely
‘‘pitted’’ or very
thin
Smooth, slightly
undulate, if pitted,
then with
‘‘hollows’’
3.6
7.1
Evenly thickened
Smooth
4.6
5.3
Thin, paper-like
Smooth, strongly
undulate
34.1
Evenly thickened
or very thin,
paper-like
Strongly undulate
with circular
thickenings
32.5–62.0
163
123
Lysimachia
vulgaris
Seed morphology and endosperm structure of Primulaceae, Myrsinaceae and Theophrastaceae
Table 2 Seed morphological and anatomical characters of the species examined from Primulaceae, Myrsinaceae, and Theophrastaceae
164
Fig. 1 Schematic illustration of seed shapes of a Hottonia palustris
(suboval laterally flattened, side faces, cavernous, and slanted towards
the ventral margin) and b Cyclamen purpurascens (subglobose with
concave hilar area). Seed shape in ventral view (left column), lateral
view (middle column), and transverse section (right column)
closely adhere to each other. Primulaceae seeds had a
smaller range of testa thickness (9.9–87.0 lm) than seeds
of Myrsinaceae (18.6–128.6 lm). Seeds with especially
thin testa were found for Soldanella carpatica, H. palustris,
and Samolus valerandi for which testa thickness ranged
from 9.9–10.7 lm (Table 2). For all species examined with
reticulate seed coat patterns, the inner testa layer was
thicker than the outer testa layer (ratios of inner layer to
outer layer ranged from 1.7:1 to 3.9:1). The opposite pattern was found for seeds with tuberculate seed sculpture
and seeds with presence of the sponge-like outer layer. For
example, in seeds with rather thick testa (71.6–96.1 lm),
such as P. elatior (Fig. 5h, i, j) (Primulaceae), Anagallis
arvensis (Fig. 4e), and T. europaea (Fig. 3j) (Myrsinaceae), the ratio of the thickness of the inner/outer testa
layers was 1:3.8, 1:2.3, and 1:1.8, respectively. In seeds of
Lysimachia nemorum (Fig. 4q) and L. vulgaris (Fig. 3a, b),
in which the testa was very thick (121.9–128.6 lm), the
same ratios were 1.1:1 and 5.9:1, respectively (Table 2).
The feature common for all species examined was the
presence of oxalate crystals on the surface of the inner testa
layer (Figs. 3j, o, u; 4e, m; 5h, l; 6m). For Androsace
septentrionalis (Fig. 5b), Soldanella carpatica, and Cortusa matthioli (Fig. 6c, h), outlines of crystals were visible
on the ventral view of the seed surface, under the cuticle
cell layer.
Endosperm structure
Endosperm structure differed strongly among species
examined. We have defined two endosperm characters: cell
wall thickness and the relief of the inside cell wall surfaces.
Four main types of endosperm were distinguished: (1) with
evenly thickened cell walls and smooth inside cell wall
123
M. Morozowska et al.
surfaces, (2) with evenly thickened cell walls and circular
or helical thickenings on their inside surfaces, (3) with very
thick, unevenly thickened cell walls with constrictions
(‘‘pitted’’) and with dark deep hollows on the inside cell
wall surfaces, (4) with very thin, paper-like cell walls
slightly or strongly undulate. The first type of endosperm
was found in seeds of Anagallis arvensis, for which little
protrusions were also observed on the inner cell wall surfaces (Fig. 4e). The same type of endosperm, but without
protrusions, was typical for G. maritima, Cortusa matthioli,
and Samolus valerandi seeds (Figs. 3p; 6i, r). The endosperm structure of T. europaea and G. maritima seeds was
very variable, as two different types of endosperm structure
were present in them (Fig. 3k, l, p, q). The second endosperm type was found in Lysimachia nemorum seeds
(Fig. 4r). The third type was found in seeds of Cyclamen
purpurascens, Androsace septentrionalis, and P. elatior
(Figs. 3v; 5d, i, j, k). It is worth mentioning that in seeds of
P. elatior, the endosperm structure was not uniform, as its
cells located on the end of the longest axis were characterized by papery thin cell walls (Fig. 5j). The fourth
endosperm type was observed in seeds of G. maritima
(Fig. 3q), Anagallis minima (Fig. 4k, l), and Soldanella
carpatica (Fig. 6e), consisting of cells with very strongly
undulate walls. The same type of endosperm was found in
seeds of Lysimachia vulgaris, T. europaea (Fig. 3d, k), and
H. palustris (Fig. 6m, n), in which the cell walls were only
slightly undulate.
Discussion
Recent molecular and morphological phylogenetic studies
on the relationships among taxa of ‘‘primuloid’’ families of
the Ericales s.l. (Primulaceae, Myrsinaceae, and Theophrastaceae) have shown that, despite molecular evidence for
close relationships of certain taxa, the addition of morphological information may lead to a slight increase, or
decrease, in support for phylogenetic relationships of some
groups (Anderberg et al. 2007; Oh et al. 2008). Based on
our observations, the testa thickness, presence or absence
of oxalate crystals in the outer testa layer, and the endosperm structure are very important features that may be
potentially informative characters of systematic importance. According to the general rule (Anderberg and Ståhl
1995), the subglobose seeds of the former MyrsinaceaeMyrsinoideae have ‘‘pitted’’ endosperm cell walls and their
testa are devoid of crystals, while the angular seeds of the
former Primulaceae have evenly thickened, smooth endosperm cell walls and rather thick, usually distinctly twolayered testa, generally with rhomboid crystals. However,
among species we examined for this study, we found
angular seeds of the former Primulaceae with either thin or
Species
Seeds
(n)
Seed length (mm)
Seed width (mm)
Length/width ratio
Min. Max. Mean (±SE)
CV
(%)
Min. Max. Mean (±SE)
Anagallis arvensis
30
0.91 1.56
1.235 (0.024)g
10.7
0.68 1.10
0.944 (0.021)ef 12.5
1.028 1.794 1.318 (0.023)d
9.6
0.52 0.94
0.668 (0.020)cd
16.4
Anagallis minima
30
0.41 0.62
0.551 (0.009)j
9.1
0.36 0.73
0.424 (0.012)hi 16.0
0.562 1.590 1.325 (0.034)d
14.1
0.31 0.56
0.356 (0.009)hi
13.9
Androsace
septentrionalis
30
0.91 1.25
1.046 (0.018)h
9.3
0.47 0.78
0.621 (0.016)g
1.269 2.383 1.715 (0.050)b
15.8
0.41 0.73
0.560 (0.017)efg
17.0
CV
(%)
14.1
Min.
Seed thickness (mm)
Max. Mean (±SE)
CV
(%)
Min. Max. Mean (±SE)
CV
(%)
Cortusa matthioli
30
1.07 2.08
1.636 (0.038)bc 12.6
0.57 1.43
1.001 (0.039)de 21.5
1.068 2.754 1.698 (0.070)b
22.6
0.44 0.78
0.635 (0.017)cde
14.7
Cyclamen
purpurascens
30
1.82 3.42
2.803 (0.069)a
1.79 2.85
2.373 (0.049)a
1.015 1.465 1.184 (0.022)d
10.0
0.95 1.75
1.320 (0.034)a
14.1
Galux maritima
30
1.09 1.38
1.237 (0.014)fg
6.2
0.73 1.07
0.903 (0.019)ef 11.3
1.101 1.781 1.384 (0.027)cd 10.6
0.39 0.73
0.505 (0.014)g
15.0
Hottonia palustris
30
0.57 0.99
0.759 (0.014)i
10.3
0.34 0.62
0.486 (0.010)h
11.4
1.036 2.294 1.586 (0.047)bc 16.2
0.31 0.47
0.378 (0.007)h
10.0
Lysimachia nemorum 30
1.17 1.53
1.398 (0.018)de
7.1
0.86 1.38
1.095 (0.018)cd
9.0
1.026 1.571 1.284 (0.022)d
0.39 0.70
0.552 (0.016)fg
15.6
Lysimachia vulgaris
30
1.22 1.82
1.523 (0.029)cd 10.5
0.91 1.38
1.200 (0.025)c
11.6
1.047 1.824 1.285 (0.037)d
15.7
0.62 0.83
0.697 (0.010)c
7.9
Primula elatior
30
1.10 1.72
1.372 (0.030)ef 11.9
0.48 1.43
1.095 (0.037)cd 18.3
0.846 2.542 1.317 (0.073)d
30.3
0.57 1.00
0.817 (0.017)b
11.4
Samolus valerandi
30
0.39 0.57
0.481 (0.008)j
9.2
0.23 0.42
0.314 (0.010)i
16.9
1.000 2.130 1.576 (0.054)bc 18.8
0.23 0.34
0.282 (0.005)i
10.4
Soldanella carpatica
30
1.38 1.87
1.684 (0.021)b
7.0
0.70 0.91
0.834 (0.012)f
7.8
1.516 2.414 2.035 (0.043)a
11.7
0.39 0.88
0.619 (0.023)cdef 20.2
Trientalis europaea
30
1.30 1.82
1.583 (0.025)bc
8.5
0.91 1.51
1.358 (0.025)b
10.1
1.013 1.857 1.177 (0.031)d
14.3
0.39 0.73
0.611 (0.016)def
ANOVA
df
F
P
F
P
F
P
F
P
Species
12
428.6
\0.0001
427.6
\0.0001
32.7
\0.0001
222.4
\0.0001
df error
378
13.6
11.3
9.2
14.5
Seed morphology and endosperm structure of Primulaceae, Myrsinaceae and Theophrastaceae
Table 3 Ranges (minimum–maximum), mean values (±SE), and coefficients of variation (CV) of seed morphological features of the species studied (e.g., seed length, seed width, length/width
ratio, and seed thickness)
One way ANOVAs were performed separately for each of the seed features to determine the differences among species studied. Same letters indicate a lack of statistically significant differences
between analyzed species according to Tukey’s a posteriori test (P \ 0.05)
165
123
166
123
Table 4 Ranges (minimum–maximum), mean values (±SE), and coefficients of variation of seed morphological features of the species studied (e.g., hilum length, hilum width, and length/
width ratio)
Species
Seeds (n)
Hilum length (mm)
Hilum width (mm)
Min.
Max.
Mean (±SE)
CV (%)
Min.
Max.
Length/width ratio
Mean (±SE)
CV (%)
Min.
Max.
Mean (±SE)
CV (%)
Anagallis arvensis
30
0.39
0.78
0.511 (0.017)cd
18.2
0.08
0.18
0.114 (0.005)c
25.1
3.056
6.125
4.616 (0.138)de
16.3
Anagallis minima
30
0.06
0.14
0.094 (0.004)h
21.1
0.05
0.09
0.066 (0.002)e
19.2
0.875
2.000
1.454 (0.063)i
23.6
Androsace septentrionalis
30
0.30
0.43
0.378 (0.006)ef
8.5
0.04
0.12
0.074 (0.004)de
27.3
2.917
9.000
5.442 (0.271)bcd
27.3
Cortusa matthioli
Cyclamen purpurascens
30
30
0.39
0.38
0.91
1.60
0.626 (0.027)b
1.045 (0.056)a
23.6
29.6
0.05
0.19
0.08
0.61
0.078 (0.001)de
0.371 (0.017)a
9.8
25.8
4.875
1.000
11.400
7.000
8.102 (0.362)a
3.028 (0.228)g
24.5
41.3
Galux maritima
30
0.23
0.78
0.411 (0.020)ef
27.1
0.08
0.13
0.100 (0.004)cd
19.3
2.300
6.000
4.176 (0.177)ef
23.2
Hottonia palustris
30
0.10
0.18
0.121 (0.004)gh
20.0
0.02
0.13
0.075 (0.005)de
38.6
0.769
6.500
1.987 (0.225)hi
62.1
Lysimachia nemorum
30
0.34
0.57
0.429 (0.011)de
13.5
0.16
0.26
0.195 (0.005)b
12.7
1.619
3.167
2.226 (0.063)ghi
15.6
Lysimachia vulgaris
30
0.31
0.52
0.420 (0.010)def
13.0
0.05
0.16
0.093 (0.005)cde
27.8
2.750
8.800
4.885 (0.311)cde
34.9
Primula elatior
30
0.26
0.44
0.323 (0.010)f
16.6
0.05
0.16
0.110 (0.005)c
25.7
1.625
7.800
3.173 (0.221)fg
38.2
Samolus valerandi
30
0.13
0.26
0.194 (0.005)g
15.0
0.03
0.03
0.030 (0.000)f
0.0
4.333
8.667
6.478 (0.178)b
15.0
Soldanella carpatica
30
0.31
0.80
0.576 (0.023)bc
22.0
0.08
0.13
0.100 (0.002)cd
10.2
3.100
8.500
5.848 (0.281)bc
26.3
Trientalis europaea
30
0.26
0.60
0.458 (0.015)de
18.0
0.13
0.23
0.175 (0.005)b
14.4
1.714
3.563
2.648 (0.088)gh
18.2
ANOVA
df
F
P
F
P
F
P
Species
12
135.8
\0.0001
199.7
\0.0001
78.0
\0.0001
df error
378
One-way ANOVAs were performed separately for each seed feature to determine the differences among species studied. Same letters indicate a lack of statistically significant differences
between analyzed species according to Tukey’s a posteriori test (P \ 0.05)
M. Morozowska et al.
Seed morphology and endosperm structure of Primulaceae, Myrsinaceae and Theophrastaceae
Anagallis arvensis
Lysimachia vulgaris
Glaux maritima
Primula elatior
Lysimachia nemorum
Trientalis europaea
Cortusa matthioli
Soldanella carpatica
Androsace septentrionalis
Samolus valerandi
Anagallis minima
Hottonia palustris
Cyclamen purpurascens
Fig. 2 Dendrogram of cluster groupings of the species studied on the
basis of seed morphological features
thick testa. After the reclassification of that family and
transferring such species as Lysimachia, Anagallis, Trientalis, Glaux, and Cyclamen to Myrsinaceae (Källersjö et al.
2000), only P. elatior (among those species examined) was
characterized by thick testa. This is in agreement with the
results of our previous study in which the presence of
rather thick testa was also described for P. veris seeds
(Klimko et al. 2001). In this study we observed endosperm
with irregularly thickened cell walls with constrictions in
seeds of not only Cyclamen purpurascens (which fits well
with the description of Myrsinaceae), but also in small
angular seeds of P. elatior and Androsace septentrionalis.
Anderberg and Kelso (1996) in their earlier investigations
on phylogenetic implications of endosperm cell wall morphology in Douglasia, Androsace, and Vitaliana (Primulaceae), noted that the endosperm of Primula and
Androsace is characterized by evenly thickened cell walls
without constrictions. According to the same authors, the
endosperm cell walls with irregular thickening and narrow
constrictions characterizes all Douglasia and Vitaliana
species and supports the monophyly of these genera.
However, a few years later Ståhl and Anderberg (2004)
found that in seeds of some Androsace, the endosperm
consists of cells with unevenly thickened cell walls with
narrow constrictions. We have found Androsace septentrionalis to be another example of an Androsace species
with ‘‘pitted’’ endosperm cell walls. According to our
observations, the feature of the irregularly thickened
endosperm cell walls is due to the presence of the deep
dark hollows on the inner surface of the cell walls, and
such hollows were observed by us in seeds of P. elatior and
Androsace septentrionalis.
The presence of endosperm with very thin, paper-like
cell walls and slight or strong undulations, which was
observed in seeds of Anagallis minima, G. maritima, and
T. europaea, the three species transferred recently to
Myrsinaceae (Källersjö et al. 2000), as well as in seeds of
167
Lysimachia vulgaris, may suggest that this type of endosperm structure is common among Myrsinaceae species.
However, in seeds of Lysimachia nemorum, although the
endosperm cell walls were rather thin and undulate, the
relief of their inside wall surfaces was rather different,
characterized by the presence of very distinct helical
thickenings.
Results of this study show that rhomboid crystals can be
present in testae of subglobose and angular seeds, so that
this feature is not exclusive to small angular seeds, and lack
of crystal presence in subglobose seeds of Cyclamen purpurascens before now does not support the transfer of that
species to Myrsinaceae. Our results concerning seed
structure of other species of the present Myrsinaceae, such
as Anagallis arvensis and A. minima, for which there are
many different reports concerning their molecular and
morphological similarity (Martins et al. 2003; Manns and
Anderberg 2005), showed on one hand very close similarity of their tuberculate seed sculpturing with secondary
striations, while on the other hand, the results on other
features examined did not confirm that similarity. For
example, seeds of these two species differed in the presence or absence of winged and keeled seed edges,
respectively. The testa was more than three times thicker in
seeds of Anagallis arvensis than in seeds of A. minima, and
striking differences in endosperm structure of these two
species were observed. In Anagallis arvensis, seed endosperm consisted of cells with evenly thickened smooth cell
walls, while in seeds of A. minima, the endosperm cell
walls were papery, thin, and strongly undulate. Differences
in the seed structure observed in these two species are in
agreement with the results of molecular studies showing
their distant phylogenetic relationships (Martins et al.
2003).
Our results concerning description and comparison of
the seed structure of Anagallis arvensis and Lysimachia
nemorum, for which there is strong molecular evidence of a
very close relationship (Källersjö et al. 2000; Manns and
Anderberg 2007; Oh et al. 2008), add some more morphological support for the reclassification of Anagallis
from Primulaceae to Myrsinaceae. Similarities in the seed
shape, seed sculpture, presence of oxalate crystals as well
as thickness of endosperm cell walls within these two
species were observed. However, we found one obvious
difference between them: the relief of the inside surface of
endosperm cell walls, which in L. nemorum is characterized by helical thickenings. One other difference concerns
the testa thickness, which was more than 1.5 times thicker
in seeds of L. nemorum than in seeds of A. arvensis. To
resolve the differentiation between these two species, more
taxa from both genera should be examined.
Some of our results showed similarities in the seed
structure of H. palustris and Soldanella carpatica. These
123
168
M. Morozowska et al.
Fig. 3 Seeds of a–
d Lysimachia vulgaris,
e–l Trientalis europaea,
m–q Glaux maritima, and
r–v Cyclamen purpurascens. m,
s Ventral view showing
narrowly elliptic (m) hilum
(H) and concave hilar area (s).
e, r Dorsal view showing seed
outline and the reticulate surface
pattern. c, g, h, i Surface of the
spongy outer layer of
Lysimachia vulgaris (c) and
Trientalis europaea seeds (g, h,
i). n, t Reticulate (n) and
strongly reticulate (t) pattern of
the outer layer (OL). f Partly
removed spongy outer layer
showing presence of oxalate
crystals (OxC; arrow). a, b, j, o,
u Longitudinal sections (L.S.)
showing testa (T) thickness and
rhomboid crystals (arrows) on
the surface of the inner layer
(IL). b, j L.S. showing columnar
cells (CC) and rectangular cells
(RC) of spongy outer layers of
Lysimachia vulgaris (b) and
Trientalis europaea (j) seeds.
d, k L.S. showing very thin,
papery walls of endosperm
(End). l L.S. showing
endosperm cells with evenly
thickened, undulate walls with
circular thickenings on the inner
cell walls surface. p, q L.S.
showing Glaux maritima
endosperm cells with smooth
evenly thickened (p) and papery
thin, with circular thickenings
on the inner cell wall surface
(q). v L.S. showing ‘‘pitted’’
endosperm structure with
unevenly thickened endosperm
cell walls with dark hollows on
the inside cell wall surfaces
(arrows)
included the reticulate seed pattern, very thin testae, presence of rhomboid crystals, and very thin, papery, and
smooth endosperm cell walls. According to some earlier
molecular studies (Trift et al. 2002; Martins et al. 2003),
there is some support for the assumption of common
ancestry of Hottonia and Soldanella. Similarities in the
123
seed structure of these two species we describe here adds
support for that hypothesis.
The inclusion of Samolus valerandi in the family Theophrastaceae is the subject of much discussion, as there is
very weak morphological support for its transfer to that
family (Källersjö and Ståhl 2003; Martins et al. 2003; Caris
Seed morphology and endosperm structure of Primulaceae, Myrsinaceae and Theophrastaceae
169
Fig. 4 Seeds of a–e Anagallis
arvensis, f–m A. minima, and
n–r Lysimachia nemorum. a, f,
o Ventral view showing
narrowly elliptic (a, f) and
elliptic (o) hilum (H) shape. b,
g, n Dorsal view showing lack
of winged or keeled edges of
dorsal faces. c, h Lateral view
showing strongly winged edges
of dorsal face (c) and lack of
winged or keeled dorsal face
(h). d, i, p Seed surface showing
tuberculate pattern with
secondary striations. j Seed
surface with partly removed
outer layer (OL) and crystal
visible on the surface of the
inner layer (IL). e, m,
q Longitudinal section (L.S.)
showing testa thickness and
presence of oxalate crystals
(OxC; arrows) on the surface of
the inner layer (IL). e, k, l,
r L.S. showing endosperm
(End) structure with evenly
thickened cell walls with little
protrusions on the inner cell
wall surfaces (e); very thin,
papery cell walls (k, l); evenly
thickened cell walls with helical
thickening on the inner cell wall
surfaces (r)
and Smets 2004). We found that seeds of Samolus valerandi were very similar to seeds of H. palustris and
Soldanella carpatica (Primulaceae) in size proportions,
faintly reticulate seed ornamentation, presence of oxalate
crystals, and very thin testae. Some similarity (due to the
presence of very thin testae) was observed between seeds
of S. valerandi and seeds of Androsace septentrionalis
(Primulaceae) and Anagallis minima (Myrsinaceae). The
structure of very thin testa in seeds of S. valerandi was
described in our earlier study (Morozowska and Czarna
2006). On the other hand, thickness of the endosperm cell
walls in seeds of S. valerandi differed from the endosperm
structure in seeds of the species mentioned above; seeds of
A. septentrionalis had endosperm with ‘‘pitted’’ cell walls
123
170
M. Morozowska et al.
Fig. 5 Seeds of a–d Androsace
septentrionalis and e–l Primula
elatior. a, f Ventral view
showing linear (a) and elliptic
(f) hilum (H) shape. e Dorsal
view showing lack of winged or
keeled edges of dorsal face.
b Outer layer (OL) surface with
reticulate seed pattern and
oxalate crystals (OxC) visible
under cuticle cells (arrows).
g Tuberculate seed pattern. c, h,
l Longitudinal section (L.S.)
showing testa (T) thickness and
presence of oxalate crystals (h,
l; arrows) on the surface of the
inner layer (IL). c, d, i, j, k L.S.
showing endosperm (End)
structure with irregular
thickenings of the cell walls and
dark hollows on the inner cell
wall surfaces (arrows)
while the other species were characterized by papery thin
endosperm cell walls. In general, the features of S. valerandi seeds we described compare well with the description of Theophrastaceae seeds given by Ståhl and
Anderberg (2004), according to whom seeds of Theophrastaceae are characterized by faintly reticulate seed ornamentation and endosperm with smooth or irregularly
thickened cell walls. In our opinion, the best way to find
more, stronger morphological support for transferring
Samolus to Theophrastaceae would be to examine the seed
structure of the large number of species of that genus.
With regard to Cyclamen purpurascens, whose placement within Myrsinaceae is still under discussion (Martins
et al. 2003), we found that, although the genus Cyclamen is
characterized by capsular fruits, its seed shape and hilum,
rather thin testae and ‘‘pitted’’ endosperm cell walls bear a
good resemblance to the description of seeds typical for
drupaceous species from Myrsinaceae (Ståhl and Anderberg 2004). The only feature that contradicts that description was the presence of oxalate crystals observed on the
surface of the inner testa layer.
123
In conclusion, our study of the seed structure of some
species of Primulaceae, Myrsinaceae, and Theophrastaceae
provided some important new data concerning seed shape
and size, seed coat pattern, presence of oxalate crystals on
the surface of the outer testa layer, thickness of testa, and
endosperm structure. In general, seeds, as generative
organs that are only slightly influenced by environmental
conditions, are important diagnostic features (but see Janyszek et al. 2008; Janyszek and Jagodzinski 2009). Their
high structural diversity provides the most valuable criteria
for classification at species and family levels. Thus, a
detailed analysis of the morphological and anatomical
structure of seeds greatly increases our knowledge of
individual species and may be helpful in better understanding the phylogeny of the taxa examined. According to
our observations, the relief of the secondary wall thickenings forming the secondary seed sculpture on some of the
seeds examined is of high systematic importance. In any
case, for purposes of comparative systematics, it is necessary to define these secondary sculptures structurally. Their
nature can often be revealed by TEM examinations of thin
Seed morphology and endosperm structure of Primulaceae, Myrsinaceae and Theophrastaceae
171
Fig. 6 Seeds of a–e Soldanella
carpatica, f–i Cortusa matthioli,
j–n Hottonia palustris, and
o–r Samolus valerandi. a, f, j,
o Ventral view showing narrow
(a, f) and narrowly elliptic (j,
o) hilum (H) shape. j, k Ventral
(j) and lateral (k) view showing
suboval seed shape with
cavernous lateral faces. p Dorsal
view showing very weakly
keeled edges between dorsal
and lateral faces. c, d Outer
surface layer with reticulate
seed pattern with secondary
protrusions. g, h Reticulate/
vesiculose seed pattern with
secondary striations and oxalate
crystals (OxC) visible under
cuticle cells (h) (arrows). l,
q Weakly reticulate seed pattern
with oxalate crystals visible
under cuticle cells. c, i, m,
r Longitudinal sections showing
testa (T) thickness and presence
of oxalate crystals (arrows) on
the surface of the inner layer.
e, i, m, n, r Longitudinal
sections showing endosperm
(End) structure with evenly
thickened cell walls (i, r) and
very thin, papery cell walls
slightly (m, n) or very strongly
(e) undulate
sections (Barthlott 1990). We also conclude that the presence of oxalate crystals in testae, which were observed in
seeds of all species examined, should not be used as the
feature distinguishing species with angular or subglobose
seeds. The other character that in our opinion is of high
systematic interest is the endosperm structure, especially
the thickenings of the cell walls and their inner surface
relief. In analyzing those features, we have shown some
similarities in endosperm structure between species from
different genera and families not reported earlier. However,
as a single source of characters, endosperm structure cannot be expected to resolve all unclear relationships among
123
172
the families examined. Such studies should be continued,
based on a broader sampling of taxa, and including both
molecular and morphological evidence.
Acknowledgments We kindly thank Dr. Lee E. Frelich (University
of Minnesota, USA) for linguistic support and valuable comments on
the early draft of the manuscript. The authors would like to thank two
anonymous reviewers for their suggestions and comments made on an
earlier version of the manuscript.
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
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