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Contribution to the taxonomy and phylogeny
of Sarcocapnos DC. (Fumariaceae)
M. J. Salinas
1
, A. T. Romero
1
, G. Blanca
1
, R. de la Herra
´n
2
, M. Garrido-Ramos
2
,
C. Ruı
´z-Rejo
´n
2
, C. Morales
1
, M. Ruı
´z-Rejo
´n
2
,and V. Sua
´rez
1
1
Departamento de Bota
´nica, Facultad de Ciencias, Universidad de Granada, Granada, Spain
2
Departamento de Gene
´tica, Facultad de Ciencias, Universidad de Granada, Granada, Spain
Received July 16, 2002; accepted December 11, 2002
Published online: March 31, 2003
Springer-Verlag 2003
Abstract. To solve problems concerning the status
of the taxa described in the genus Sarcocapnos,we
have conducted a study using morphological,
pollen morphology (light microscopy), cytogenetic
and molecular techniques. Focusing on the last
technique, we have sequenced ITS-1 and ITS-2
of nuclear rDNA. The species differ basically
according to 5 morphological traits (leaf shape,
flower spur, corolla colour, corolla size, and crest
of the stigmatic surface). The cytogenetic analyses
indicated n ¼16 to be the standard chromosome
number. The ITS analyses showed that the genus
is monophyletic, defining two main well-supported
clades, one containing S. saetabensis and S.
enneaphylla, and one containing the rest of the
species. In this second clade, S. speciosa,
S. pulcherrima, and S. baetica subsp. ardalii are
related, as are S. integrifolia,S. crassifolia subsp.
crassifolia, and S. crassifolia subsp. atlantis;
S. baetica subsp. baetica forms a trichotomy with
the foregoing groups. S. speciosa is shown to be
a species separate from S. crassifolia subsp.
crassifolia, as in the case of S. baetica with respect
to S. integrifolia. Palynologically, the parameters
used enabled us to establish clear differences
between the taxa, often corroborating the
macromorphological and genetic data. The flower
spur has been reduced several times in different
groups of the genus, for which the classifications
established on the basis of this trait are paraphy-
letic.
Key words: Fumariaceae, Sarcocapnos, taxo-
nomy, phylogeny, morphology, chromosome
number, ITS-1 and ITS-2 sequences, pollen
morphology.
Introduction
Within Papaveraceae sensu lato (Kadereit et al.
1994), De Candolle (1821) created the genus
Sarcocapnos, which is distributed in the west-
ern Mediterranean area. He recognised two
species, S. crassifolia (Fumaria crassifolia
Desf.) and S. enneaphylla (F. enneaphylla L.).
Boissier (1844) described Aplectrocapnos bae-
tica and A. integrifolia, which later were
included by Nyman (1878) and Cuatrecasas
(1929), respectively, in the genus Sarcocapnos.
Subsequently, Boissier (1854) described
S. speciosa from the southeastern Iberian
Peninsula. Emberger & Maire (in Maire
1932) described S. baetica var. atlantis, which
Lide
´n (1986a) considered a subspecies of
S. crassifolia. After a review of this genus by
Plant Syst. Evol. 237: 153–164 (2003)
DOI 10.1007/s00606-002-0260-7
the latter author (1986a, b), three further taxa
have been described: S. saetabensis (Mateo and
Figuerola 1987), S. pulcherrima (Morales and
Romero 1991) and S. baetica subsp. ardalii
(Lo
´pez-Ve
´lez 1991).
A few previous cytogenetical analyses have
been conducted in this group of plants, with
most chromosome counts giving n ¼16
(Ryberg 1960, Lide
´n 1986a), although some
discordant data (n ¼12) have been found
(Humphries et al. 1978, Lo
´pez-Ve
´lez 1991).
However, uncertainty persists regarding
the status of several taxa as well as the
phylogenetic relationships within this genus
and between it and the rest of the genera in
Papaveraceae sensu lato (Lide
´n 1986a, Lide
´n
et al. 1997).
In the present study, we perform multidis-
ciplinary analyses of all 9 taxa of the genus
Sarcocapnos. Specifically, we study the macro-
morphology and pollen morphology, and we
use sequences of the internal transcribed spac-
ers of the nuclear ribosomal genes (ITS-1 and
ITS-2 sequences) for phylogenetic analyses. All
this information is used to resolve the taxo-
nomic controversies of a group that requires a
detailed study of as many markers as possible.
Rapidly evolving sequences as the spacers for
the ribosomal genes are useful markers for
disclosing phylogenetic relationships between
closely related species. Thus, the ITS sequences
have been successfully used to disclose the
relationships between species of different
families (for review, see Baldwin et al. 1995)
and, specifically, of the family Papaveraceae
(Lide
´n et al. 1995). We have also used the data
available on the relative content of alkaloids
related to isoquinoline in some of the taxa
within the genus (Blanco et al. 1991, Lo
´pez et al.
1991, Tojo et al. 1991).
Material and methods
Cytogenetics and molecular analyses, as well as
morphometric measurements, were performed with
plants from wild populations, including all 9 taxa
of the genus Sarcocapnos (Table 1).
Macromorphology. Morphometric analyses
were carried out with 10 flowering plants of the
species and populations mentioned above. In each
individual, 20 measurements were made for each
character considered.
Pollen morphological analysis. Samples of 20
anthers from different specimens of each popula-
tion (see Table 1) were acetolysed using the method
of Erdtman (1960) as modified by Hideux (1972),
and then mounted in glycerogelatine for light
microscopy. The terminology used is based on that
of Erdtman (1971) and Faegri and Iversen (1975).
The characters measured were P (polar axis), E
(equatorial axis), A (apocolpium length), M (meso-
colpium length), Ee (thickness of the equatorial
exine), Ep (thickness of the polar exine), C (colpus
width) and L (colpus length; Fig. 1).
In each sample, 30 pollen grains were mea-
sured. To compare the differences or similarities in
pollen size between taxa, a simple analysis of
variance was performed on P and E parameters.
The data were normalised by the expression
x¢¼ln(x + 1). The statistical analyses were per-
formed with the program SYSTAT, version 7.0,
1997, SPSS INC.
Cytogenetic analysis. To determine the chro-
mosome number of each species, meiotic PMC’s
(Pollen Mother Cells) were analysed by staining
squashed young anthers in aceto-carmine (see, for
example, Ruiz-Rejo
´n et al. 1990).
ITS sequences. Total DNA from nine Sarco-
capnos taxa, Dicentra spectabilis (L.) Pers., Fuma-
ria officinalis L., and Platycapnos spicata (L.)
Bernh. (Table 1) was isolated from fresh tissue,
following Doyle and Doyle (1987). In a few cases
DNA was isolated from herbarium materials
(Sarcocapnos crassifolia subsp. atlantis and S.
enneaphylla). For the remaining species, the se-
quences were taken from the GenBank database
[Corydalis decumbens (Thunb.) Pers., C. tauricola
(Cullen & Davis) Lide
´n, C. wilsonii N. E. Br., and
Hypecoum imberbe Sm.]. Outgroup taxa were
chosen to represent the various genera of Fumari-
aceae considered as closely allied to Sarcocapnos
(e.g. Heywood 1964; Lide
´n 1986a, b). For each
species, the complete region between the 3¢end of
the 18S ribosomal gene and the 5¢end of the 28S
ribosomal gene (Internal transcribed spacer
1 -ITS1-, 5.8S ribosomal gene and Internal
transcribed spacer 2 -ITS2-) was obtained by
PCR amplification using the universal primers
154 M. J. Salinas et al.: Taxonomy and phylogeny of Sarcocapnos (Fumariaceae)
Table 1. Populations studied and analyses performed in each of the populations. Mmorphological analyses; Ppalynological studies; Ccyto-
genetical studies; IITS analyses. Voucher specimens are deposited in the herbarium of the University of Granada, GDAC. MA: Herbarium of the
Royal Botanic Garden of Madrid
Code Taxa Accession number
(GenBank Database)
Population Voucher Analyses
ITS-1 ITS-2
SB-1 S. baetica (Boiss. & Reut.)
Nyman subsp. baetica
AJ250623 AJ429199 Molino de los Batanes (Albacete, Spain) 44353 M, P, C, I
SB-2 Sierra de Cazorla (Jae
´n, Spain) 44354 M, P, C
SBA S. baetica subsp. ardalii Lo
´pez Ve
´lez AJ250624 AJ429200 Embalse de la Fuensanta (Albacete, Spain) 44355 M, I
SC-1 S. crassifolia (Desf.) DC.
subsp. crassifolia
AJ429195 AJ429196 Col du Zad (Morocco) 44364 M, P, I
SC-2 Ifrane (Morocco) 44525 M, P
SC-3 Gorges du Dades, Haut Atlas (Morocco) 44750 M
SC-4 S. crassifolia (Desf.) DC.
subsp. atlantis (Emb. & Maire) Lide
´n
AJ429197 AJ429198 El Kelaa des Mgouna, Amesker (Morocco) MA434149 M, I
SE-1 S. enneaphylla (L.) DC. AJ493442 AJ493443 Ve
´lez de Benaudalla (Granada, Spain) 44351 M,P,C,I
SE-2 O
´rgiva (Granada, Spain) 22864 M,P,C
SE-3 Cabo de Gata (Almerı
´a, Spain) 44356 M,P,C
SI S. integrifolia (Boiss.) Cuatrec. AJ250621 AJ429201 La Cerradura (Jae
´n, Spain) 44352 M,P,C,I
SP-1 S. pulcherrima C. Morales
& Romero Garcı
´a
AJ250627 AJ429204 Moclı
´n (Granada, Spain) 22850 M,P,C,I
SP-2 Bermejales (Granada, Spain) 44362 M,P,C
SP-3 Sierra de Ma
´gina (Jae
´n, Spain) 4508 M,C
SP-4 Fuensanta de Martos (Jae
´n, Spain) 28030 M,C
SS-1 S.saetabensis Mateo & Figuerola AJ250625 AJ429202 Sierra de Benicadell (Valencia, Spain) 44361 M,P,C,I
SS-2 Sierra de Marı
´a (Almerı
´a, Spain) 44359 M,C
SS-3 Ja
´tiva (Valencia, Spain) 44360 M,P,C
SSP-1 S. speciosa Boiss. AJ250626 AJ429203 La Ragua, Sierra Nevada
(Granada, Spain)
44358 M,P,C,I
SSP-2 Jeres, Sierra Nevada (Granada, Spain) 44344 M,P
Dicentra spectabilis (L.) Pers. AJ493444 AJ493445 Cultivated at Department of Botany
(Granada, Spain)
–I
Fumaria officinalis L. AJ493446 AJ493447 Acequia de Perliz, Go
´jar
(Granada, Spain)
–I
Platycapnos spicata (L.) Bernh. AJ493448 AJ493449 Albolote (Granada, Spain) 32052 I
M. J. Salinas et al.: Taxonomy and phylogeny of Sarcocapnos (Fumariaceae) 155
ITS-1 and ITS-4 described in White et al. (1990).
We used 100 ng of DNA from each species as a
template for PCR amplification. Thermal param-
eters were 40 cycles of 94
o
C for 90 seconds, 50
o
C
for 90 seconds and 72
o
C for 90 seconds. The
amplifications were done in 10 mM Tris-HCl, pH
8.3; 5 mM NH
4
Cl; 50 mM KCl; 2 mM MgCl
2
;
0.2 mM of each dNTP and 1.25 units Taq DNA
polymerase (Boehringer). The amplified products
were used as templates for sequencing by the
dideoxynucleotide chain terminator method of
Sanger et al. (1977). Sequencing products were
analysed by an automated laser fluorescent DNA
sequencer (Pharmacia). Three individuals of each
analysed taxon have been sequenced and we found
no differences among them. ITS-1 and ITS-2
sequences used in this paper are deposited at the
EMBL sequence database under the accession
numbers indicated in Table 1. The sequences of
ITS-1 and ITS-2 from each species were linked,
and this combined sequence was used as input for
the phylogenetic analysis. The sequences of all the
species were then aligned by the use of the
CLUSTALX 1.7 program (Thompson et al.
1997) and corrected manually.
Phylogenetic and molecular evolutionary anal-
yses were conducted using MEGA version 2.1
(Kumar et al. 2001). The maximum parsimony
method was applied, and the search was made by
the heuristic method with a search factor of 4. For
all analyses of sequence data, gaps were treated as
missing data. The number of replicates of the
bootstrap analysis was 1000.
Results
Macromorphology. The morphometric traits
used for distinguishing the species of Sarco-
capnos appear in Table 2. The genus can be
separated into 3 groups of species. The first
includes S. enneaphylla and S. saetabensis,
which can be distinguished from the other
species by having a cristate stigma and leaves
with a greater number of leaflets. The second
comprises S. baetica and S. integrifolia, which
have very small flowers without a spur.
S. crassifolia subsp. atlantis lacks a spur also
and has larger flowers, traits that relate it with
the third group, which includes in addition
S. crassifolia subsp. crassifolia,S. pulcherrima
and S. speciosa, with spurred corollas. S. bae-
tica subsp. ardalii does not differ significantly
from S. baetica in morphological characters;
the only notable feature is the presence of
abundant hairs on the stems and leaves of the
former, although, in the classical site of the
subsp. ardalii, only 1% of the individuals have
such hairs. S. pulcherrima and S. saetabensis
are distinguished from the other members of
their group by having flowers of greater size
and pinkish colour.
Pollen morphology. The largest pollen
grains were observed in S. enneaphylla (SE-1,
P¼55.7 ± 3.1 lm; E ¼52.2 ± 5.3 lm), and
the smallest ones in S. saetabensis (SS-3,
P¼34.2 ± 2.9 lm; E ¼32.2 ± 2.4 lm, Ta-
ble 3). S. baetica has larger pollen grains than
S. integrifolia (for P, F ¼11.0, p < 0.001; for
E, F ¼27.6, p < 0.001), and S. crassifolia
subsp. crassifolia has smaller pollen than S.
pulcherrima (for P, F ¼164.0, p < 0.001; for
E, F ¼128.0, p < 0.001). This was also ob-
served between S. crassifolia subsp. crassifolia
and S. speciosa (for P, F ¼216.1, p < 0.001;
for E, F ¼171.9, p < 0.001). S. speciosa and
S. pulcherrima showed no significant differenc-
es in any of the parameters studied. This is
similar to what happened between S. crassifo-
lia subsp. crassifolia and S. integrifolia.
S. enneaphylla has larger pollen grains than
S. saetabensis (for P, F ¼148.7, p < 0.001; for
E, F ¼182.2, p < 0.001).
Fig. 1. Schematic drawing of a pollen grain in polar
view (A) and in equatorial view (B) illustrating the
parameters studied. P(polar axis), E(equatorial
axis), A(apocolpium length), M(mesocolpium
length), Ee (thickness of the equatorial exine),
Ep (thickness of the polar exine), C(colpus width)
and L(colpus length)
156 M. J. Salinas et al.: Taxonomy and phylogeny of Sarcocapnos (Fumariaceae)
Table 2. Main morphological features of the taxa studied. Absent character (–)
Traits S. saetabensis S. enneaphylla S. pulcherrima S. crassifolia
subsp.
crassifolia
S. crassifolia
subsp.
atlantis
S. speciosa S. baetica S. baetica
subsp.
ardalii
S. integrifolia
Pilosity
(leaves and stem)
Glabrous Glabrous
or hairy
Glabrous Glabrous Glabrous Glabrous Glabrous Glabrous
or hairy
Glabrous
Leaves
shape ternate ternate ternate ternate ternate ternate ternate ternate simple
nleaflets 3–9 (15) (3) 9–20 (28) 3–6 3–6 3 3–7 3–6 (7) 3–6 1
Sepal length (mm) 1.5–2 1–1.6 2.5–3.5 1–2.1 1–1.2 1.2–2 1.5–2 2 1.8
Corolla
colour pinkish white pinkish white white white white white white
length (mm,
spur included)
15–18 13–16 20–23 14–15 8–8.5 18–20 7.2–7.5 6.2–6.5 5.5
Lower petal
apex truncated to
weakly
emarginated
truncated emarginated emargi-
nated
emarginated emargi-
nated
weakly
emarginated
truncated to
weakly
emarginated
truncated
total length
(mm)
11–14 9–11 18–19 12–13 8–8.5 13–15 7.2–7.5 6.5 5
limb width
(mm)
9–12 3.5–5.2 11–13 7.5–8.5 5–6 7–11 6–8 6.3 6
limb length
(mm)
6–8 3.5–5.2 10–12 5.5–6 4–5 6–8 4.5–5 4.5 2–5
claw length
(mm)
5–5.5 5–6 7–8 6–7 3.5 7–8 2.5 2 2.5
Spur length
(mm)
4.5–5.5 4.5–6.5 3.8–4.5 3–3.5 – 3.5–4.2 – – –
Stigma shape cristate cristate non-cristate non-cristate non-cristate non-cristate non-cristate non-cristate non-cristate
Fruit length
(mm)
3.8–5.0 3–4.9 4.0–6.8 3.2–4.8 2.8 3.4–4.8 2.9–4.2 2.5–4.2 2.8–3.2
M. J. Salinas et al.: Taxonomy and phylogeny of Sarcocapnos (Fumariaceae) 157
Table 3. Variation in pollen characters (in lm) in different populations of Sarcocapnos species, showing mean values (x), standard deviations
(SD), and range. Ppolar axis; Eequatorial axis; Mmesocolpium length; Aapocolpium length; Lcolpus length; Ccolpus width; Ee thickness of
the equatorial exine; Ep thickness of the polar exine
Species Population P E M A L C Ee Ep
x SD range x SD range x SD range x SD range x SD range x SD range x SD range x SD range
S. baetica SB-1 50.3 6.8 38.4–66.6 47.7 7.9 38.4–66.56 31.0 7.0 19.2–51.2 6.2 2.1 3.8–12.8 29.0 4.8 19.2–38.4 2.9 1.4 1.3–6.4 2.3 0.6 1.3–3.2 3.3 0.6 2.6–4.5
SB-2 48.6 6.8 41.0–69.1 49.2 6.4 41.0–62.7 37.8 6.9 28.2–49.9 5.7 1.5 3.8–10.2 34.3 4.5 28.2–44.8 2.2 0.9 1.3–3.8 2.0 0.4 1.2–2.4 2.6 0.4 1.9–3.8
SBA 34.4 3.4 30.7–47.4 33.7 2.8 29.4–39.7 20.5 3.1 15.4–26.9 5.8 1.3 3.8–7.7 19.8 2.9 15.4–29.4 1.9 0.6 1.3–2.6 1.6 0.4 1.3–2.6 2.2 0.4 1.3–3.2
S. crassifolia SC-1 37.9 3.3 30.7–44.8 37.5 3.2 33.3–47.4 23.6 3.3 17.9–30.7 6.5 1.5 3.8–10.2 19.2 3.6 14.1–28.2 1.5 0.5 1.3–2.6 2.1 0.5 1.3–2.6 2.5 0.5 1.3–3.2
SC-2 34.6 2.3 29.4–38.4 33.7 4.0 29.4–44.8 23.1 4.0 15.4–30.7 7.6 1.3 5.1–10.2 17.7 2.7 14.1–23.0 1.7 0.8 1.3–3.8 1.9 0.6 1.3–2.6 2.4 0.4 1.3–3.2
S. enneaphylla SE-1 55.7 3.1 47.3–62.7 52.2 5.3 43.5–64.0 31.9 4.8 19.2–41.0 6.3 1.5 3.8–9.0 29.0 5.5 17.9–38.4 2.6 1.2 1.3–5.1 1.7 0.5 1.3–2.6 3.1 0.6 1.9–4.5
SE-2 44.0 4.3 35.8–49.9 43.3 3.6 35.8–48.6 30.5 3.2 24.3–35.8 6.9 1.4 3.8–9.0 23.0 3.2 17.9–28.2 1.6 0.4 1.3–2.6 1.9 0.5 1.3–2.6 2.6 0.6 1.9–3.8
SE-3 53.5 8.4 38.4–76.8 51.5 7.5 38.4–64.0 28.7 6.9 17.9–43.5 5.9 2.1 2.6–10.2 30.1 5.4 16.6–38.4 2.9 1.5 1.3–7.7 2.1 0.6 1.3–3.8 3.2 0.7 2.6–5.1
S. integrifolia SI 38.4 4.3 28.2–48.6 34.5 5.6 25.6–47.4 24.1 4.6 16.6–32.0 6.6 1.8 2.6–9.0 20.4 5.2 11.5–33.3 2.1 1.0 1.3–5.1 1.6 0.4 1.3–2.6 2.8 0.6 1.3–3.8
S. saetabensis SS-1 41.9 3.1 35.8–48.6 39.5 3.7 32.0–46.1 26.8 5.8 16.6–35.8 6.4 1.8 3.8–10.2 21.3 3.4 14.1–25.6 2.0 0.8 1.3–3.8 1.5 0.4 1.3–2.6 2.3 0.5 1.3–3.2
SS-3 34.2 2.9 28.2–38.4 32.2 2.4 26.9–37.1 25.3 3.3 19.2–30.7 6.9 1.9 3.8–10.2 17.5 2.5 12.8–23.0 1.7 0.7 1.3–3.8 1.3 0.2 0.6–1.9 2.1 0.5 1.3–2.6
S. speciosa SSP-1 45.4 4.7 38.4–61.4 43.1 3.9 35.8–51.2 28.0 4.2 19.2–34.6 6.6 2.1 3.8–12.8 23.6 4.5 15.4–30.7 1.8 0.8 1.3–3.8 1.4 0.2 1.3–1.9 2.5 0.6 1.3–3.8
SSP-2 48.9 6.3 38.4–64.0 47.2 5.7 35.8–61.4 29.8 5.1 17.9–38.4 7.2 2.1 3.8–12.8 24.9 4.9 15.4–33.3 1.9 0.8 0.6–3.8 1.5 0.4 1.3–2.6 2.6 0.6 1.3–3.8
S. pulcherrima SP-1 43.4 3.5 34.6–51.2 42.2 3.0 35.8–49.9 29.6 3.1 23.0–34.6 7.0 1.6 2.6–10.2 23.3 3.3 16.6–29.4 1.9 0.8 1.3–3.8 1.5 0.3 1.3–2.6 2.8 0.5 1.9–3.8
SP-2 48.1 5.1 38.4–58.9 45.2 4.2 38.4–58.9 30.0 4.5 16.6–37.1 7.5 1.2 5.1–10.2 23.3 3.9 16.6–32.0 2.2 0.9 1.3–3.8 1.6 0.5 1.3–2.6 2.5 0.7 1.3–3.8
158 M. J. Salinas et al.: Taxonomy and phylogeny of Sarcocapnos (Fumariaceae)
Chromosome counts. Our analyses indicate
that n ¼16 is the standard chromosome num-
ber of this genus (Table 4). Only S. crassifolia
subsp. atlantis has not been counted.
Molecular analyses. The complete se-
quence of the ITS1 region of the Sarcocapnos
species had a length varying between 153 and
156 bp while the complete sequence for the
ITS-2 varied between 226 and 230 bp. This is
an exceptionally short length for the ITS-1
region among the species of the Papaverales
for which ITS-1 sequences are on deposit
in databases (about 250 bp). This is not
observed for the case of the ITS-2 sequence,
which is about 220 bp in length for the
species of the Papaverales for which these
sequences are on deposit in databases.
Although the length of the ITS-1 region of
the species used as an outgroup far exceeded
that of the Sarcocapnos species, the sites
which were homologous to these species were
clearly identifiable and the inserted/deleted
parts of the ITSs not present in the ITSs of
Sarcocapnos could be identified and removed
for this alignment.
The total aligned length was 497 bp, with
233 (46.9%) variable sites and 123 (24.7%)
parsimony-informative sites. Parsimony anal-
ysis of the ITS-1 and ITS-2 sequence data
yielded 9 equally most parsimonious trees of
395 steps. Consistency Index was CI ¼0.83,
and Retention Index was RI ¼0.77. One of the
most parsimonious trees is shown in Fig. 2,
and the strict consensus tree is shown in Fig. 3.
Hypecoum,Dicentra,Corydalis,Fumaria,
and Platycapnos form successively closer
outgroups to Sarcocapnos, all supported with
relatively high bootstrap values.
Table 4. Chromosome numbers of the taxa studied. See Table 1 for codes of the populations
Species and codes n Previous counts
S. baetica 2n = 32: Lide
´n (1986a)
SB-1 16
SB-2 16
S. baetica subsp. ardalii – 2n = 24: Lo
´pez Ve
´lez (1991)
S. crassifolia n = 12: Humphries et al. (1978)
SC-1 – 2n = 32: Lide
´n (1986a)
SC-2 –
S. enneaphylla 2n = c. 32: Ryberg (1960)
SE-1 16 2n = 32: Lide
´n (1986a)
SE-2 16
SE-3 16
S. integrifolia
SI 16
S. pulcherrima 2n = 32: Lide
´n (1986a)
sub S. crassifolia subsp. speciosa
SP-1 16
SP-2 16
SP-3 16
SP-4 16
S.saetabensis
SS-1 16
SS-2 16
SS-3 16
S. speciosa 2n = c. 32: Ryberg (1960)
sub S. crassifolia
SSP 16
M. J. Salinas et al.: Taxonomy and phylogeny of Sarcocapnos (Fumariaceae) 159
Within the genus Sarcocapnos, bootstrap
values of 100% support two main clades: one
with S. enneaphylla and S. saetabensis, and one
with the other species (Fig. 3). Within this
second clade, low sequence divergence resulted
in poor resolution and relatively low bootstrap
levels, but some conclusions emerge. Notably,
there are the two strongly supported group-
ings: S. crassifolia subsp. crassifolia and
S. crassifolia subsp. atlantis, and S. speciosa
and S. pulcherrima. With a considerably lower
bootstrap value (66%), S. integrifolia is
grouped as a sister species to the group of
S. crassifolia. With the same bootstrap value,
S. baetica subsp. ardalii is placed in the group
made up by S. pulcherrima and S. speciosa.In
the original tree (Fig. 2), S. baetica subsp.
baetica was placed together with S. baetica
subsp. ardalii, but in the strict consensus tree
the former species makes a trichotomy with the
two aforementioned groups.
Discussion
Morphological analysis. We distinguish 7 spe-
cies within the genus Sarcocapnos, identifiable
by the key included at the end of this section.
This taxonomic scheme agrees with that
established by Lide
´n (1986a, b), except for
the choice of rank. This author recognized
only 3 species (S. enneaphylla,S. crassifolia
and S. baetica), classifying S. speciosa and
S. integrifolia as subspecies of S. crassifolia
and S. baetica, respectively. Similar combina-
tions had long before been proposed by Rouy
(1884). The two other species, S. saetabensis
and S. pulcherrima, were described more
recently than Lide
´n’s work.
Fig. 2. One of the 9 most parsimonious trees derived from cladistic analysis of ITS-1 and ITS-2 sequence data.
Number above branches represents branch lengths
160 M. J. Salinas et al.: Taxonomy and phylogeny of Sarcocapnos (Fumariaceae)
S. saetabensis can be distinguished from
S. enneaphylla on the basis of floral colour
(pinkish), corolla, sepals, lower petal, fruit size
(Table 2), and pollen size (Table 3). In addi-
tion, Blanco et al. (1991), Lo
´pez et al. (1991)
and Tojo et al. (1991) found significant differ-
ences in the alkaloid contents: S. enneaphylla
presented a higher percentage of protopines
(23.6%) than did S. saetabensis (11.0%), and
the presence of ribasines (0.9%), which were
absent in the latter species. On the other hand,
aporphines, benzophenanthridines and mor-
phinandienones proved more abundant in
S. saetabensis (4.8%, 21.2%, 1.8%, respective-
ly) than in S. enneaphylla (2.6%, 14.8%, 0.4%,
respectively) and benzylisoquinolines have not
been found in S. enneaphylla. These differences
suggested that the rate of evolution in these
characters was higher than the mutation rate
of ITS.
S. pulcherrima can be easily distinguished
from S. speciosa by its pinkish corolla, by its
longer sepals, and by its lower petals being
longer and its fruits larger (Table 2), in
addition to its ecology and geographic distri-
bution (Morales and Romero 1991). While
S. pulcherrima lives on limestone walls and
is distributed over a large part of the eastern
Betic sierras, at altitudes of 800–1200 m a. s. l.,
S. speciosa is located in micaschists of the Sierra
Nevada massif (Granada and Almerı
´a), at
generally higher altitudes (1800–2000 m a. s. l.).
S. baetica subsp. baetica and S. baetica
subsp. ardalii present no morphological differ-
ences (Table 2). One of the two traits pointed
out by Lo
´pez-Ve
´lez (1991) to distinguish the
subspecies ardalii is pilosity on the leaves;
nevertheless, in the classic site (Sierra del
Ardal, Embalse de la Fuensanta, Albacete,
Spain), glabrous specimens are the more
abundant, while those with pilosity are scarce.
Glabrous and pilose individuals also cohabit in
S. enneaphylla and S. pulcherrima populations
in which pilosity lacks sufficient taxonomic
importance to separate subspecies. Lo
´pez-
Ve
´lez (1991) also separated subsp. ardalii on
the basis of chromosome number 2n ¼24,
while subsp. baetica presents 2n ¼32 (Table 4).
The chromosome number 2n ¼24 is doubtful
in the genus Sarcocapnos. Humphries et al.
(1978) detected this chromosome number in a
North African population of S. crassifolia
Fig. 3. Strict consensus of 9 shortest trees derived from cladistic analysis of ITS-1 and ITS-2 sequence data.
Number above branches represents bootstrap percentages
M. J. Salinas et al.: Taxonomy and phylogeny of Sarcocapnos (Fumariaceae) 161
subsp. crassifolia, the same in which Lide
´n
(1986a) later found 2n ¼32, in agreement also
with our results for North African populations
(see Table 4).
Monophyly of Sarcocapnos.The phyloge-
netic analysis of the sequence data from the
ITS-1 and ITS-2 suggests that Sarcocapnos is
monophyletic. The size of the ITS-1, consider-
ably shorter than in the other species of
Papaverales compared and shorter than in
most plant species (Baldwin et al. 1995), would
represent a synapomorphy in the genus, which
seems to have evolved by one (or more)
deletion events, as in other angiosperms (Mo
¨l-
ler and Cronk 1997).
Congruence between ITS-based phylogeny
and traditional classification. The phylogenetic
relationships between the outgroups used
herein and Sarcocapnos are in accordance with
the tribes and subtribes established by Lide
´n
(1986a) within the Fumariaceae family.
Within the genus, the presence of cristate
stigma and also leaves with a greater number
of leaflets in S. enneaphylla and S. saetabensis
are morphologic traits that clearly separate
these species from the rest. The two groups
thus formed are phylogenetically consistent
according to the ITS analysis. Lide
´n (1986a)
noted, on the basis of morphological features,
that S. enneaphylla stands isolated in the
genus.
The presence or absence of a floral spur, a
conspicuous trait usually used in traditional
classification, provides a paraphyletic classifi-
cation according to the ITS analysis. Heywood
(1964) established two sections: Section Sarco-
capnos, for the species with a spur, and
Aplectocapnos for those without a spur.
Among the species without a spur, S. integri-
folia was considered a subspecies of S. baetica,
when the ITS analysis separated the two into
different clades. On the other hand, S. crassi-
folia subsp. atlantis was differentiated from
S. crassifolia subsp. crassifolia fundamentally
for lacking a floral spur (Lide
´n 1986a,
Table 2), but the two taxa are genetically very
close (Figs. 2 and 3).
Traditionally, S. speciosa (and by extension
S. pulcherrima, previously included in the
former species) has been considered a subspe-
cies of S. crassifolia. However, the morpho-
logical data and the ITS analysis indicate
considerable divergence between the two for-
mer species and S. crassifolia, which is suffi-
cient to consider these independent species.
Key to the taxa of Sarcocapnos
1 Flowers without spur . .............2
1¢Flowers with spur. . . . ............. 4
2 Leaves simple; flowers 5–5.5 mm .......
..................... S. integrifolia
2¢Leaves ternate; flowers 6.2–8.5 mm . . . . 3
3 Sepals 1.5–2 mm; flowers 6.2–7.5 mm. . . .
........................S. baetica
3¢Sepals 1–1.2 mm; flowers 8–8.5 mm . . . . .
........... S. crassifolia subsp. atlantis
4 Stigma cristate . . . . . . .............5
4¢Stigma non-cristate. . . .............6
5 Corolla 13–16 mm, white; lower petal
9–11 mm, with limb 3.5–5.2 mm wide . . .
.....................S. enneaphylla
5¢Corolla 15–18 mm, pinkish; lower petal
11–14 mm, with limb 9–12 mm wide . . . .
..................... S. saetabensis
6 Sepals 2.5–3.5 mm; corolla pinkish, lower
petal 18–19 mm . . . . . ....S. pulcherrima
6¢Sepals 1.0–2.1 mm; corolla white, lower
petal 12–15 mm . . . . . .............7
7 Corolla 14–15 mm; lower petal 12–13 mm;
spur 3–3.5 mm . . . . . . ..............
......... S. crassifolia subsp. crassifolia
7¢Corolla 18–20 mm; lower petal 13–15 mm;
spur 3.5–4.2 mm. . . . . ...... S. speciosa
Conclusions
1) ITS data indicate that the genus Sarcocap-
nos is monophyletic.
2) The presence or absence of a floral spur is
not an adequate character for classification,
as it generates paraphyletic groups.
162 M. J. Salinas et al.: Taxonomy and phylogeny of Sarcocapnos (Fumariaceae)
3) S. enneaphylla and S. saetabensis are closely
related taxa, and clearly separated from the
rest by molecular and morphological data.
4) S. speciosa is well differentiated from
S. crassifolia as initially proposed by Bois-
sier (1854) based only on morphology.
5) S. integrifolia and S. baetica should be
considered different species.
6) Although the molecular data indicate diver-
gence between S. baetica subsp. ardalii and
S. baetica subsp. baetica, the morphological
data do not support the separation of
S. baetica subsp. ardalii, since there are no
diagnostic traits to separate the latter from
S. baetica subsp. baetica. More evidence is
needed to determine the phylogenetic rela-
tionships between these taxa.
7) S. pulcherrima and S. speciosa are very close
together, differentiated by morphological
characters and by geographical distribution,
for which more genetic data are needed to
establish their taxonomic status.
We wish to express our gratitude to Dr.
M. Lide
´n for helpful suggestions on this work,
and anonymous reviewers for their valuable
comments to improve the quality of the manu-
script. We are grateful to David Nesbitt for the
linguistic review of the English manuscript. The
research project was supported by the Direccio
´n
General de Investigacio
´n Cientı
´fica y Te
´cnica
(Spanish Scientific and Technical Research Direc-
torate [DGICYT and DGESIC], PB95-1218 and
PB98-1288).
References
Baldwin B. G., Anderson M. J., Porter J. M.,
Wojciechowski M. F., Campbell C. S., Don-
oghue M. J. (1995) The ITS region of nuclear
ribosomal DNA: a valuable source of evidence
on angiosperm phylogeny. Ann. Missouri Bot.
Gard. 82: 247– 277
Blanco O., Castedo L., Corte
´s D., Villaverde M. C.
(1991) Alkaloids from Sarcocapnos saetabensis.
Phytochem. 30: 2071–2074.
Boissier P. E. (1843-1854) Diagnoses Plantarum
Orientalium Novarum 1. Gene
`ve. Leipzig. Paris.
Como.
Boissier P. E. (1854-1859) Diagnoses Plantarum
Orientalium Novarum 2. Gene
`ve. Leipzig. Paris.
Como.
Cuatrecasas J. (1929) Estudio sobre la flora y
vegetacio
´n del macizo de Ma
´gina. Treb. Mus.
Ci. Nat. Barcelona, Se
´r. Bot. 12: 3–510.
De Candolle A. P. (1821) Regni vegetabilis systema
naturale 2. Treuttel et Wu
¨rtz, Paris.
Doyle J. J., Doyle J. D. (1987) A rapid DNA
isolation procedure for small quantities of fresh
leaf tissue. Phytochem. Bull. 19: 11–15.
Erdtman G. (1960) The acetolysis method – a
revised description. Svensk Bot. Tidskrift 54:
561–564.
Erdtman G. (1971) Pollen morphology and plant
taxonomy (Angiosperms). Hafner Publishing
Company. New York.
Faegri K., Iversen J. (1975) Textbook of pollen
analysis. Munksgaard. Copenhagen.
Heywood V. H. (1964) Sarcocapnos DC. In: Tutin
T. G., Heywood V. H., Burges N. A., Valentine
D. H., Walters S. M., Webb D. A. (eds.) Flora
Europaea, vol. I. Cambridge University Press.
Cambridge, p. 254.
Hideux M. (1972) Techniques d’e
´tude du pollen au
MEB: effects compare
´s des differents traitments
physicochimiques. Micron 3: 1–31.
Humphries C. J., Murray B. G., Bocquet G.,
Vasudevan K. N. (1978) Chromosome numbers
of phanerogams from Morocco and Algeria.
Bot. Not. 131: 391–404.
Kadereit J. W., Blattner F. R., Jork K. B.,
Schwarzbach A. (1994) Phylogenetic analysis
of the Papaveraceae s.l. (incl. Fumariaceae,
Hypecoaceae, and Pteridophyllum) based on
morphological characters. Bot. Jahrb. Syst.
116: 361–390.
Kumar S., Tamura K., Jakobsen I. B., Nei M.
(2001) MEGA2: Molecular Evolutionary Genet-
ics Analysis software. Bioinformatics (in press).
Lide
´n M. (1986a) Synopsis of Fumarioideae
(Papaveraceae) with a monograph of the tribe
Fumarieae. Opera Bot. 88: 1–133.
Lide
´n M. (1986b) Sarcocapnos DC. In: Castroviejo
S., Laı
´nz M., Lo
´pez G., Montserrat P., Mun
˜oz
F., Paiva J., Villar L. (eds.) Flora Iberica, vol. 1.
Real Jardı
´n Bota
´nico-CSIC, Madrid, pp. 433–
438.
Lide
´n M., Fukuhara T., Axberg T. (1995) Phylog-
eny of Corydalis, ITS and morphology. Plant
Syst. Evol. [Suppl.] 9: 183–188.
M. J. Salinas et al.: Taxonomy and phylogeny of Sarcocapnos (Fumariaceae) 163
Lide
´n M., Fukuhara T., Rylander J., Oxelman, B.
(1997) Phylogeny and classification of Fumaria-
ceae, with emphasis on Dicentra s. l. based on the
plastid gene rps16 intron. Plant Syst. Evol. 206:
411–420.
Lo
´pez S., Tojo E., Blanco O., Villaverde M. C.,
Castedo L. (1991) Alkaloids from spanish Sar-
cocapnos species. Phytochem. 30: 1175–1182.
Lo
´pez-Ve
´lez G. (1991) Sarcocapnos baetica (Boiss.
& Reuter) Nyman subsp. ardalii G. Lo
´pez Ve
´lez,
subespecie nueva para la Penı
´nsula Ibe
´rica. Acta
Bot. Malacit. 16: 437–447.
Maire R. (1932) Contributions a
`l’E
´tude de la flore
de l’Afrique du Nord 19(1). Bull. Soc. Hist. Nat.
Afr. Nord 23: 163–222.
Mateo G., Figuerola R. (1987) Flora analı
´tica de la
provincia de Valencia. IAM, Valencia.
Mo
¨ller M., Cronk Q. C. B. (1997) Origin and
relationships of Saintpaulia (Gesneriaceae) based
on ribosomal DNA internal transcribed spacer
(ITS) sequences. Amer. J. Bot. 84: 956–965.
Morales C., Romero A. T. (1991) A new species of
the genus Sarcocapnos (Fumariaceae) from east-
ern Andalusia (Spain). Plant Syst. Evol. 177: 1–
10.
Nyman C. F. (1878–1884) Conspectus florae euro-
paeae 1. Officinae Bohlinianae, O
¨rebro.
Ryberg M. (1960) A morphological study of
Fumariaceae and the taxonomic significance of
the characters examined. Acta Horti. Berg. 19:
121–248.
Rouy G. (1884) Excursions botaniques en Espagne
(Mai-Juin 1883). Denia-Madrid. I. Comptes
rendus des herborisations. I. Denia. Bull. Soc.
Bot. France 31: 52–56.
Ruiz-Rejo
´n C., Blanca G., Cueto M., Lozano R.,
Ruiz-Rejo
´n M. (1990) Asphodelus tenuifolius and
A. fistulosus (Liliaceae) are morphologically,
genetically, and biologically different species.
Plant Syst. Evol. 169: 1–12.
Sanger F., Nicklen S., Coulson A. R. (1977) DNA
sequencing with chain-terminating inhibitors.
Proc. Nat. Acad. Sci. USA 74: 5463–5467.
Thompson J. D., Gibson T. J., Plewniak F.,
Jeanmougin F., Higgins D. G. (1997) The
ClustalX windows interface: flexible strategies
for multiple sequence alignment aided by
quality analysis tools. Nucleic Acids. Res. 24:
4876–4882.
Tojo E., Domı
´nguez D., Castedo L. (1991) Alka-
loids from Sarcocapnos enneaphylla. Phytochem.
30: 1005–1010.
White T. J., Bruns T., Lee S., Taylor J. (1990)
Amplification and direct sequencing of fungal
ribosomal RNA genes for phylogenetics. In:
Innis M., Gelfand D., Sninsky J., White T. (eds.)
PCR protocols: a guide to methods and appli-
cation. Academic Press, San Diego, pp. 315–322.
Addresses of the authors: M. Jacoba Salinas,
Ana T. Romero, Gabriel Blanca, Concepcio
´n
Morales-Torres, and Vı
´ctor Sua
´rez, Departamento
de Bota
´nica, Facultad de Ciencias, Universidad
de Granada, 18071 Granada Spain (e-mail:
maria@ugr.es). Roberto de la Herra
´n, Manuel
Garrido-Ramos, Carmelo Ruiz Rejo
´n and Manuel
Ruiz Rejo
´n, Departamento de Gene
´tica, Facultad
de Ciencias, Universidad de Granada, 18071 Gra-
nada, Spain.
164 M. J. Salinas et al.: Taxonomy and phylogeny of Sarcocapnos (Fumariaceae)