Plant Syst Evol (2009) 280:219–227

DOI 10.1007/s00606-009-0184-6

ORIGINAL ARTICLE

Cytotaxonomy of diploid and polyploid Aristolochia

(Aristolochiaceae) species based on the distribution of CMA/DAPI
bands and 5S and 45S rDNA sites
Regina Berjano Æ Fernando Roa Æ Salvador Talavera Æ
Marcelo Guerra

Received: 20 January 2009 / Accepted: 30 March 2009 / Published online: 25 April 2009
Ó Springer-Verlag 2009

Abstract Aristolochia is the largest genus of the family Introduction

Aristolochiaceae and the only one with large chromosome
number variation. A combination of fluorochrome banding The genus Aristolochia L. ‘‘sensu lato’’ (Aristolochiaceae,
and in situ hybridization of 5S and 45S rDNA probes was Aristolochioideae) is the largest and most diverse genus of
used to evaluate the structural karyotype variability of the family, consisting of about 500 species characterized by
representatives of two subgenera: Siphisia, which seems to their peculiar, zygomorphic tubular flowers that exhibit a
have a single chromosome number (2n = 32), probably particular fly-trap mechanism of pollination (Sakai 2002).
derived from an old polyploidization event, and Aristolo- Species of the genus are generally perennial herbs, shrubs,
chia, including the Old World section Diplolobus and the or lianas, and are distributed mainly in tropical regions,
New World Gymnolobus. Based on chromosome mor- although they are also found in subtropical or temperate
phology and on the degree of diploidization of rDNA sites, habitats (De Groot et al. 2006). However, the circum-
A. serpentaria (Siphisia) was identified as an old hexaploid, scription of this genus is unclear and several authors have
whereas A. paucinervis (Diplolobus) seemed to be a recent proposed up to 15 segregates from Aristolochia s.l. (Wanke
hexaploid (2n = 34). The karyotypes of the five analyzed et al. 2006). Among the different taxonomic frameworks
species of section Gymnolobus were structurally more suggested for the genus Aristolochia s.l., it is noteworthy
stable than those from Diplolobus, which varied consider- the recognition of three subgenera: Siphisia, Pararistolo-
ably in the type of heterochromatin, chromosome number, chia, and Aristolochia (González and Stevenson 2000). The
and morphology. These data indicate that fluorochrome latter is the largest and comprehends the section Diplolo-
banding and rDNA localization may substantially improve bus, composed of ca. 120 species from Europe, N. and
the cytotaxonomical analysis of this genus. E. Africa, Asia, and N. Australia, and section Gymnolobus,
with ca. 210 species from the New World. More recent
Keywords Aristolochia
Cytotaxonomy
studies propose that the genus Aristolochia s.l. consists of
Heterochromatin
rDNA two major lineages, divided into four genera: Endodeca
and Isotrema within the subtribe Isotrematinae (previously
treated as subgenus Siphisia) and Pararistolochia and
Aristolochia ‘‘sensu stricto’’ within Aristolochiinae (Kelly
and González 2003; Neinhuis et al. 2005). Despite the
different taxonomic ranks, these systems are highly similar.
Studies on chromosome numbers have helped to char-
R. Berjano
S. Talavera
Department of Plant Biology and Ecology, University of Seville, acterize clades in Aristolochia s.l., with most of them
41080, Seville, Spain characterized by a single chromosome number (Sugawara
et al. 2001). Thus, within the subgenus Aristolochia,
F. Roa
M. Guerra (&)
2n = 12 is predominant in the subsection Podanthemum,
Laboratory of Plant Cytogenetics, Department of Botany,
Federal University of Pernambuco, Recife, PE 50670-420, Brazil 2n = 16 in the series Thyrsicae, and 2n = 14 in the sub-
e-mail: msfguerra@hotmail.com sections Aristolochia and Pentandrae. The other two

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subgenera, Siphisia and Pararistolochia, have rather 1976; Guerra 2000). FISH with rDNA probes reveals
invariable chromosome numbers of 2n = 32 and 2n = 12, valuable additional markers for chromosome and genome
respectively (Ohi-Toma et al. 2006). According to Grant characterization. Tandemly repetitive 45S rRNA genes are
(1982), the basic number in Aristolochia should be x = 7, usually located on the secondary constriction and adjacent
but this assumption is now barely sustainable. The varia- heterochromatin, both of which constitute the nucleolar
tion in chromosome number currently known is much organizer region (NOR). These sites are commonly
higher than before (2n = 6, 8, 10, 12, 14, 24, 26, 28, 32, detected with fluorochromes such as CMA?/DAPI- bands
and 36) and the distribution of these numbers in the phylo- (Cabral et al. 2006; Fregonezi et al. 2006), but FISH with
genetic tree is rather complex (Ohi-Toma et al. 2006). 45S rDNA probes provides a more reliable recognition of
Furthermore, the number 2n = 28, which supported the nucleolar organizer regions (NORs), especially the
Grant’s assumption of x = 7 and which was reported ear- minor and inactive sites (Brasileiro-Vidal et al. 2007). On
lier for several Aristolochia species, is probably a miscount the other hand, 5S rDNA sites are exclusively detected by
(Sugawara et al. 2001). The base number of a genus should FISH, as they do not generate chromosome constrictions
parsimoniously explain the chromosome variability of that and are rarely visible as heterochromatin blocks (Cabral
group and have a clear relationship with the closest related et al. 2006).
groups, as reviewed by Guerra (2008). However, Thottea, In the present work, a combination of CMA/DAPI
the sister group of the genus Aristolochia, has x = 13 fluorochrome staining and FISH with 5S and 45S rDNA
(Morawetz 1985), a number found only in a single species probes was used to characterize the karyotypes of ten
of the section Diplolobus. species of Aristolochia s.l. belonging to the subgenera
According to Sugawara et al. (2001), species of Aris- Siphisia and Aristolochia, aiming to understand the
tolochia have, in general, symmetrical karyotypes with karyotype evolution and the taxonomic relationships
small (0.5–2 lm) meta or submetacentric chromosomes, so among some species of this complex group.
that karyotype differentiation would be practically restric-
ted to chromosome number variation. Techniques such as
chromosome banding or fluorescent in situ hybridization Materials and methods
(FISH) have proved to be useful tools for additional
karyotype differentiation, helping to clarify evolutionary Plant material
and phylogenetic relationships among species (Vaio et al.
2005; Almeida et al. 2007; de Moraes et al. 2007), but, till Ten species of Aristolochia, one of them belonging to subgenus
now, they have never been applied to Aristolochia species. Siphisia (A. serpentaria) and nine from the subgenus Aristol-
Base-specific fluorochromes are generally used to ochia, were analyzed. Tubers or seeds of Aristolochia species
identify heterochromatic regions, which, in general, consist were collected and maintained in cultivation at the greenhouses
of non-coding tandemly repetitive DNA sequences. The of the University of Seville, Spain. Most vouchers were
most used fluorochromes in plant cytogenetics are deposited at the SEV herbarium, University of Seville, Spain,
chromomycin A3 (CMA), which binds preferentially to with the exception of A. birostris, which is at the EAN
GC-rich DNA, and 40 ,6-diamidino-2-phenylindole (DAPI), herbarium, Federal University of Paraı́ba, Areia, Brazil. For
which preferentially stains AT-rich regions (Schweizer collection localities and voucher numbers, see Table 1.

Table 1 Species of Aristolochia analyzed, with their respective provenance, voucher code, and chromosome number
Species Provenance Voucher Chromosome number (2n)

A. baetica L. Hinojos, Huelva, Spain SEV 218049 14

A. birostris Duch. Serra Negra de Bezerros, Pernambuco, Brazil EAN 12216 14
A. brasiliensis Mart. & Zucc. Gravatá, Pernambuco, Brazil SEV 218054 14
A. clematitis L. Ferreries, Menorca, Spain SEV 218053 14
A. cymbifera Mart. & Zucc. Conceição do Mato Dentro, Minas Gerais, Brazil SEV 217014 14
A. gigantea Hook. Uberlândia, Minas Gerais, Brazil SEV 217015 14
A. loefgrenii Hoehne Uberlândia, Minas Gerais, Brazil SEV 217000 14
A. paucinervis Pomel Hinojos, Huelva, Spain SEV 218051 34
A. rotunda L. Es Mercandal, Menorca, Spain SEV 217315 12
A. serpentaria L. Gainesville, California, USA SEV 218056 32

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Cytotaxonomy of diploid and polyploid Aristolochia (Aristolochiaceae) species 221

The root tips of A. brasiliensis were obtained from Results

seeds germinating in Petri dishes, whereas for the other
species, they were obtained from plants growing in pots. Most species (A. baetica, A. brasiliensis, A. loefgrenii,
They were pretreated with 2 mM 8-hydroxyquinoline for A. cymbifera, A. clematitis, A. birostris, and A. gigantea)
3.5 h at room temperature, fixed overnight in 3:1 etha- had 2n = 14 and exhibited more or less symmetrical
nol–acetic acid (v/v) at room temperature, and stored at karyotypes. Nevertheless, chromosome number 1 was
-20°C. conspicuously larger than the others in A. clematitis,
A. birostris, and A. gigantea (Fig. 1). Among the remain-
Fluorochrome staining ing species, A. serpentaria (2n = 32) and A. paucinervis
(2n = 34) were polyploids with symmetrical karyotypes
For chromosome preparations, fixed root tips were washed (Figs. 1 and 2a, g), while A. rotunda had a reduced
in distilled water, digested in 2% (w/v) cellulase Onozuka chromosome number (2n = 12) and a rather asymmetri-
R-10 (Serva) and 20% (v/v) pectinase (Sigma), and squa- cal karyotype (Figs. 1 and 3a), with remarkable chro-
shed in 45% acetic acid. Coverslips were removed by mosome size variation. The smallest chromosome on
freezing in liquid nitrogen and the slides were air-dried. each karyotype was about 1.5–2.2 lm in all of the
The best slides were selected after a brief staining with a analyzed species, whereas the largest reached 2.5–
DAPI (2 mg/ml):glycerol mixture (1:1, v/v), destained in 3.9 lm. Chromosome morphology as well as the posi-
3:1 ethanol–acetic acid for 30 min, and dehydrated in tions of 5S and 45S rDNA sites and band patterns
100% ethanol for at least 2 h. obtained with CMA and DAPI fluorochromes were
For CMA/DAPI staining, the slides were aged for summarized in the ideograms of Fig. 1. Known phylo-
3 days and stained with 0.5 mg/ml CMA (Sigma) for 1 h genetic relationships among these species are shown by
and with 2 lg/ml DAPI (Sigma) for 30 min. The slides the adapted cladograms on the right side of this figure.
were mounted in McIlvaine’s pH 7 buffer:glycerol (1:1, Only in A. serpentaria and A. clematitis was the chro-
v/v). Images were acquired with a Leica DM LB micro- mosome morphology unclear, so that the centromere
scope equipped with a Cohu CCD camera and Leica position was not indicated. Likewise, in A. rotunda, the
QFISH software. The images were optimized for best number and position of 5S rDNA sites were not reliably
contrast and brightness with Adobe Photoshop CS3 (Adobe identified.
Systems, Inc.). The slides were destained again and stored After CMA/DAPI double-staining, some chromosome
at -20°C until use for FISH. regions were CMA?/DAPI- (i.e., brighter with CMA and
duller with DAPI), whereas others were CMA-/DAPI?,
Fluorescent in situ hybridization and sometimes neutral for one of the fluorochromes (CMA0
or DAPI0). Figure 2a shows the secondary constriction of
The FISH procedure was based on Moscone et al. (1996) A. serpentaria, which is barely visualized with CMA,
with minor modifications. Two rDNA probes, R2 and D2, whereas in A. baetica (Fig. 2c), the large secondary con-
were used to locate the 45S and 5S rDNA sites, respec- striction is CMA0. The number and position of bands
tively. R2 is a 6.5-kb fragment containing an 18S–5.8S– varied among species, with CMA? bands predominantly
25S rDNA repeat unit from Arabidopsis thaliana, and D2 located at the centromeric or pericentromeric region in
is a 500-bp fragment of the 5S rDNA repeat unit from species of the section Gymnolobus and also possibly in
Lotus japonicus (see Almeida et al. 2007). They were A. clematitis (Figs. 2f, 3c, d, f, g, j).
labeled using a nick translation kit (Gibco) with digoxi- On the other hand, the number of 5S and 45S rDNA sites
genin-11-dUTP (Roche) and biotin-11-dUTP (Sigma), was remarkably stable, with only one pair of sites of each
respectively. The 5S rDNA was detected with mouse anti- rDNA family. The only exception was the polyploid
biotin (Roche) and the signals were amplified with rabbit A. paucinervis, which displayed two pairs of 5S rDNA and
anti-mouse TRITC conjugate (Dako). The 45S rDNA was three pairs of 45S rDNA sites (Fig. 2g, h). The location
detected with sheep anti-digoxigenin FITC conjugate of the 45S rDNA site always coincided with one of the
(Roche) and amplified with rabbit anti-sheep FITC conju- terminal CMA?/DAPI- bands, except in A. serpentaria,
gate (Dako). All preparations were counterstained with where the 45S rDNA site seemed to be colocalized with a
DAPI (2 lg/mL) and mounted in Vectashield (Vector). non-terminal CMA?/DAPI- band (Fig. 2a, b). The 45S
The cell images were acquired as above. Ideograms were rDNA site was located on the largest chromosome of all
constructed based on the analysis of at least five well- species, except for A. baetica and A. paucinervis. On the
spread metaphases using Adobe Photoshop CS3 software. contrary, the 5S rDNA sites were always located in
Chromosomes were ordered from the largest one to the euchromatic CMA0/DAPI0 regions, almost always on
shortest. proximal or interstitial positions.

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222 R. Berjano et al.

Fig. 1 Ideograms of the

Aristolochia species analyzed.
Species were organized in
subgenera according to
Ohi-Toma et al. (2006).
Cladograms (right) were
adapted from: (1) Ohi-Toma
et al. 2006; (2) Wanke et al.
2006; (3) De Groot et al. 2006

Figures 1–3 illustrate a few other noteworthy details DAPI staining, two other DAPI-brilliant chromosome
besides the CMA/DAPI bands and rDNA sites. In several regions were observed, which could be confused with
species, the secondary constriction was very distended DAPI-positive heterochromatin. One such region was
(Fig. 2a), and in some of them, the labeling with 45S rDNA observed in the prophase or prometaphase chromosomes of
was typically diffuse and spread over a large area in the A. baetica and A. paucinervis, which display a sharp dif-
center of the nucleus. Figure 2g illustrates the diffuse ferentiation between more condensed and less condensed
labeling found in every cell from interphase to prometa- chromatin (Fig. 2d, g). However, in completely condensed
phase and most metaphase cells of A. paucinervis. metaphases, such differentiation was never observed,
Although all secondary constrictions were CMA?, when indicating that it is not a kind of heterochromatin but,
distended, they often became CMA0/DAPI- (arrows in rather, an early condensed chromatin. Some other DAPI-
Fig. 2c, d). brilliant regions were observed in several species only after
DAPI? bands were only observed in A. rotunda and in situ hybridization. In A. brasiliensis, A. loefgrenii, and
A. clematitis, which also display relatively asymmetric A. cymbifera, the centromeric and pericentromeric regions,
karyotypes. Besides the DAPI? bands observed with CMA/ which were DAPI- after CMA/DAPI staining, became

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Cytotaxonomy of diploid and polyploid Aristolochia (Aristolochiaceae) species 223

Fig. 2 Distribution of heterochromatin and rDNA sites in the green or red images from FISH. Observe in a and b the distended
chromosome complements of species of subgenus Siphisia (a, b) secondary constriction between two chromosome parts (arrows). The
and subgenus Aristolochia, section Diplolobus (c–h). a, b A. arrows in c point to secondary constrictions. The arrows in b, h and
serpentaria. c, d A. baetica. e, f A. clematitis. g, h A. paucinervis. the inserts of d–f show 5S or 45S rDNA sites. The bar in h represents
CMA (yellow), DAPI (blue), 5S rDNA (red), 45S rDNA (green). The 10 lm
grey color in the inserts of d–f shows DAPI pseudocolor merged with

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224 R. Berjano et al.

Fig. 3 Distribution of heterochromatin and rDNA sites in the color in the inserts of c, f show DAPI contrast merged with green or
chromosome complements of subgenus Aristolochia sections Diplol- red images from FISH. Compare the centromeric heterochromatin
obus (a, b) and Gymnolobus (c–l). a, b A. rotunda. c A. birostris. d, e observed as CMA? bands in d, g, j, DAPI- bands in h, k, and deeply
A. brasiliensis. f A. gigantea. g–i A. cymbifera. j–l A. loefgrenii. CMA stained regions after FISH denaturing in e, i, l (arrows). The bar in l
(yellow), DAPI (blue), 5S rDNA (red), 45S rDNA (green). The grey represents 10 lm

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Cytotaxonomy of diploid and polyploid Aristolochia (Aristolochiaceae) species 225

more brilliant than the rest of the chromosomes after FISH with other species of section Diplolobus has been demon-
(Fig. 3d, e, h, i, k, l). In these cases, the brilliant blocks are strated (De Groot et al. 2006; Fig. 1). This species is included
heterochromatic but not necessarily AT-rich regions. Even together with A. baetica and A. clematitis in the section
some CMA? bands were observed as DAPI-brilliant blocks Diplolobus, subsection Aristolochia, a monophyletic clade
after in situ hybridization (Fig. 3d, e, g, i, j, l). mainly with 2n = 14 and basic number x = 7 (González
1999; Ohi-Toma et al. 2006). Since 2n = 34 has been
observed only in this subsection and most species of the
Discussion subgenus Aristolochia have 2n = 14, the polyploidy in this
case should be a derived condition of recent origin. Analysis
The variability of chromosome numbers found in Aristol- of the ideogram of A. paucinervis depicted in Fig. 1 reveals
ochia species in the present work is in agreement with that that the chromosomes can be organized by size in groups of
reported by other authors (revised by Sugawara et al. 2001; three (except for the two smallest ones), as would be
Ohi-Toma et al. 2006). Chromosome numbers from most expected for a hexaploid. The presence of three pairs of 45S
species investigated were previously known, except for rDNA and two pairs of 5S rDNA sites suggests that they have
A. birostris and A. loefgrenii, which were reported here been conserved since the polyploidization event, because all
for the first time. Only the diploid numbers found for diploid species exhibited only one site of each rDNA family.
A. serpentaria (2n = 32) and A. paucinervis (2n = 34) In general, old and well established polyploid lineages dis-
were different from that previously registered (2n = 28 play strong reduction in the number of 5S and 45S rDNA
and 2n = 36, respectively) by other authors (Gregory sites as part of the diploidization process, as seems to be the
1956; Nardi 1984). case of A. serpentaria, whereas young polyploids, such as
The chromosome number found here in A. serpentaria is A. paucinervis, tend to conserve the additive number of
in agreement with recent analyses for other species of the rDNA sites (de Melo and Guerra 2003; Clarkson et al. 2005).
subgenus Siphisia. The species of this subgenus were ini- These data suggest that A. paucinervis is most probably a
tially admitted as having a stable chromosome number of hexaploid based on x = 6 which suffered a dysploid reduc-
2n = 28 (Gregory 1956; Ma 1989). However, Sugawara tion from 2n = 36 to 2n = 34 or, alternatively, it could be a
and Murata (1992) and Sugawara et al. (2001) observed the hexaploid based on x = 7, displaying a strong dysploid
number 2n = 32 in 23 species of this subgenus, including reduction.
six of those previously counted as 2n = 28. The present Variation in chromosome size among Aristolochia spe-
finding of 2n = 32 in A. serpentaria, which was previously cies has been previously demonstrated by Ohi-Toma et al.
reported by Gregory (1956) as having 2n = 28, further (2006), who observed that Pararistolochia species have
support the conclusion that 2n = 32 is characteristic to this larger chromosomes than those of the other two subgenera.
group (Sugawara et al. 2001). In general, the chromosomes of Aristolochia species are
The chromosome number 2n = 32 of Siphisia is at least much smaller than those of other Aristolochiaceae genera,
twice as high as those previously found in the other two such as Asarum, Hexastylis, and Thottea (Soltis 1984;
Aristolochia subgenera: Pararistolochia, with 2n = 12, Morawetz 1985). In the present work, we refine the karyo-
and Aristolochia, with 2n = 6, 12, 14, 16 (Ohi-Toma et al. typic analysis and found out that chromosomes of Aristolo-
2006). Therefore, the subgenus is most probably of poly- chia also display a considerable structural variation, both in
ploid origin, although the exact ploidy level is not evident, heterochromatic bands detected by fluorochrome staining
since the base number of the genus is unknown. The and in the position of 5S and 45S rDNA sites.
occurrence of two paralogous copies of the nuclear enco- The five species of section Gymnolobus were relatively
ded genes phytochrome A, APETALA3, and PISTILLATA stable in relation to chromosome size and morphology,
in some Siphisia species and a single homolog of each of banding pattern, and rDNA sites position. The small amount
these genes in diploid species of other subgenera is further of CMA-positive heterochromatin detected varied between
evidence of the tetraploid origin of subgenus Siphisia species of this section, but it is quite possible that most
(Stellari et al. 2004; Jaramillo and Kramer 2004; Ohi-Toma chromosomes have some kind of proximal heterochromatin,
et al. 2006). as observed with DAPI after FISH. It is known that hetero-
The diploid number observed here for A. paucinervis, chromatic blocks only visible after FISH with DAPI should
2n = 34, differed from that reported by Nardi (1984) for the correspond to C-bands but are not necessarily AT-rich (Be-
same species (2n = 36). The different results may be due to sendorfer et al. 2002). Differently from the karyotype vari-
intraspecific variation, species misidentification, or chro- ation found in Aristolochia, specially within section
mosome number miscount. No other species with 2n = 34 Diplolobus, the other two genera of the family investigated
or 2n = 36 is known in the genus (Sugawara et al. 2001; by C-banding, Thottea and Asarum, displayed a fairly stable
Ohi-Toma et al. 2006), but its phylogenetic relationship banding pattern (Morawetz 1985; Na and Kondo 1994).

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The 5S rDNA site was localized adjacent to the 45S Cabral JS, Felix LP, Guerra M (2006) Heterochromatin diversity and
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