ORIGINAL ARTICLE
The Iberian endemic species Ranunculus cabrerensis Rothm.:
an intricate history in the Ranunculus parnassiifolius L. polyploid
complex
Eduardo Cires • Jose´ Antonio Ferna´ndez Prieto
Received: 5 October 2010 / Accepted: 19 August 2011 / Published online: 10 September 2011
 Springer-Verlag 2011
Abstract The orophilous plant, Ranunculus parnassiifo-
lius L., is a polyploid complex that is widespread
throughout the southern European mountains, where at
least five taxa have traditionally been recognized. The aim
of this study was to test whether R. parnassiifolius subsp.
cabrerensis should be treated as an independent species of
the R. parnassiifolius polyploid complex and constitutes,
therefore, an evolutionary line in itself. To disentangle its
evolutionary history and taxonomy, we used genome size
estimation based on flow cytometric measurements (FCM),
multivariate morphometric analyses, polymerase chain
reaction-restriction fragment length polymorphisms (PCR–
RFLPs) and subsequent sequencing of cpDNA regions
(trnS, trnR-atpA, atpH-atpI). This study provides molec-
ular and morphological evidence for the recognition of
R. cabrerensis at the species level, rather than as an
intraspecific taxon of R. parnassiifolius. Furthermore, it is
concluded that those plants previously known as R. par-
nassiifolius subsp. muniellensis should be systematized at
the subspecies level as R. cabrerensis subsp. muniellensis.
This contribution highlights the benefit of combining
diverse approaches to obtain knowledge about relict pop-
ulations and for the implementation of suitable conserva-
tion measures.
Keywords cpDNA network  Genome size 
Iberian Peninsula  Polyploidy  Ranunculaceae 
Taxonomy
Introduction
Phylogeography is the phylogenetic analysis of geograph-
ically contextualized genetic data for the testing of
hypotheses regarding the causal relationships among geo-
graphical phenomena, species distributions and the mech-
anisms driving speciation (Avise 2000, 2009). Molecular
phylogeographical studies of plant species from European
mountains have increased in number in recent years (e.g.,
reviewed in Comes and Kadereit 2003; Tribsch and
Scho¨nswetter 2003; Scho¨nswetter et al. 2005, 2009; Kropf
et al. 2008). Most of these studies are focused on the Alps
and neighboring high mountain ranges, such as the Pyre-
nees or the Carpathians, whereas molecular analyses in the
Southwestern European mountains are centred on the
Mediterranean Basin (e.g., Vargas 2003; Kropf et al. 2006,
2008). However, phylogeographical studies considering the
Northwest Iberian Peninsula, and particularly the Canta-
brian Mountains and surroundings (e.g., ‘‘Montes de
Leo´n’’), are still limited (but see Kropf et al. 2003; Vargas
2003; Dixon et al. 2007, 2008; Terrab et al. 2008; Peredo
et al. 2009)—this although, according to Schmitt et al.
(2010), the Cantabrian Mountains represent an important
mountain area for the survival of old lineages that can be
interpreted as relics of the cold stages of a more distant
past.
Species groups with complicated phylogenetic rela-
tionships, evolutionary histories and intraspecific variations
are not rare among vascular plants (see review Hickerson
et al. 2009). A good example is the alpine buttercup
Electronic supplementary material The online version of this
article (doi:10.1007/s00606-011-0529-9) contains supplementary
material, which is available to authorized users.
E. Cires (&)  J. A. F. Prieto
Departamento de Biologı´a de Organismos y Sistemas, A´rea de
Bota´nica. Universidad de Oviedo, Catedra´tico Rodrigo Urı´a s/n,
33071 Oviedo, Spain
e-mail: cireseduardo@gmail.com
123
Plant Syst Evol (2012) 298:121–138
DOI 10.1007/s00606-011-0529-9

Ranunculus parnassiifolius L., a polyploid complex wide-
spread throughout the Southern European mountains with
five subspecies recognized (Ku¨pfer 1974; Bueno Sa´nchez
et al. 1992): R. parnassiifolius subsp. parnassiifolius: dip-
loid, silicicole, endemic in the Eastern Pyrenees; R. par-
nassiifolius subsp. cabrerensis Rothm.: diploid, silicicole,
endemic in the northwestern mountains of Spain; R. par-
nassiifolius subsp. muniellensis Bueno, Fern. Casado &
Fern. Prieto: diploid, silicicole, endemic in the Western
Cantabrian Mountains (‘‘Reserva de la Biosfera de Mun-
iellos’’) with a single population; R. parnassiifolius subsp.
favargeri P. Ku¨pfer: diploid, calcicole, endemic in the
Cantabrian Mountains and the Western Pyrenees; and
finally, R. parnassiifolius subsp. heterocarpus P. Ku¨pfer:
tetraploid, calcicole, spread throughout the Cantabrian
Mountains, the Central Pyrenees and the Alps. Addition-
ally, R. parnassiifolius behaves like an apomictic taxa (i.e.,
asexual reproduction via seeds) with geographical parthe-
nogenesis (Ho¨randl et al. 2008; Ho¨randl 2009, 2011; Cires
et al. 2010) where the only taxon reaching the Alps,
R. parnassiifolius subsp. heterocarpus, is a tetraploid
apomict and therefore most likely a derivative of the dip-
loid sexual subspecies of R. parnassiifolius, endemic to the
Iberian Peninsula. Geographical parthenogenesis is often
described as an infraspecific phenomenon, e.g., the wide-
spread occurrence of asexual populations compared to
sexual populations. In some apomictic plant complexes,
however, the apomicts may include hundreds or thousands
of agamospecies that differ from the sexual counterparts
and from each other by ploidy level, morphological and
ecological features. In the specific case of the genus
Ranunculus, at least three independent origins of apomixis
(R. auricomus, R. kuepferi, R. parnassiifolius) have been
described, each of them showing a pattern of geographical
parthenogenesis (alpine apomictic plants reviewed by
Ho¨randl 2011).
According to the published phylogenies of Ranunculus
(Ho¨randl et al. 2005; Paun et al. 2005), R. parnassiifolius
appears in the clade ‘white-flowered European alpines’
(clades IX and X in the former, II and III in the latter
study), which are situated on rather basal branches of the
respective trees. However, in previous papers we have
proved that this classification is not entirely clear (Cires
et al. 2009, 2010), and it has therefore become essential
to gain deeper insight into the evolutionary history of
R. parnassiifolius group and to elucidate its taxonomy. We
consider there are numerous arguments to support the
separation of the R. parnassiifolius subsp. cabrerensis from
the R. parnassiifolius s.l. polyploid complex, and that it
should consequently be treated as an independent species
(R. cabrerensis vs. R. parnassiifolius) and that in fact it
constitutes an evolutionary line in itself. This treatment
was adopted by Rothmaler (1934) when he described the
taxon ‘‘Ranunculus cabrerensis Rothmaler nov. sp. (…) Ex
affinitate R. parnassifolii L., a quo differt habitu robustiore,
foliis utrinque lanatis, petiolis lanatis, rostro carpelli lon-
giore spiram formante’’ (l.c. pp. 148). However, later in the
same paper Rothmaler (1934) concluded that there was
insufficient reason to describe the taxon as a species, and
it was therefore included as a geographical race of the
R. parnassiifolius complex: ‘‘Est forma geographica et
melius R. parnassifolius L. ssp. R. cabrerensis Rothm.
nominanda’’ (l.c. pp. 148). Additionally, as was indicated
by Bueno Sa´nchez et al. (1992), R. parnassiifolius subsp.
muniellensis has stronger similarities (morphological,
ecological, etc.) with R. parnassiifolius subsp. cabrerensis,
which leads us to hypothesize that it could be a lineage
within of R. cabrerensis rather than of R. parnassiifolius.
To explore this main hypothesis, we supported our study
by the use of morphological data, flow cytometry (FCM)
analysis and plastid genome (cpDNA) data. In fact, the use
of morphological data together with genome size variation
to resolve complex groups (e.g., Suda et al. 2007) has been
frequently used. The estimation of nuclear DNA content in
absolute terms or in relative units as an indicator of ploidy
level is by far the dominant use of FCM: it enables cyto-
type surveys over large spatial scales and involves large
population samples. It has consequently revolutionized
many fields of polyploid plant biology and changed the
perception of the magnitude of ploidy variation and its
dynamics in natural conditions (Dolezˇel et al. 2007).
Similarly, the use of cpDNA regions that are structurally
stable, haploid, non-recombinant and maternally inherited
in the majority of angiosperms studied has proved valuable
in resolving closely related taxa (e.g., Mort et al. 2007;
Hufbauer and Sforza 2008).
Within this framework, we addressed the following
objectives: (1) to resolve taxonomic uncertainties among
the orophilous taxa R. parnassiifolius subsp. cabrerensis
and R. parnassiifolius subsp. muniellensis versus the
R. parnassiifolius s.l. polyploid complex—that is to say, to
determine whether they might be considered subspecies
of the same taxon or otherwise have their own identity at
the species level; and (2) to infer, for the first time,
the phylogeography and population structure within the
R. parnassiifolius group. Finally, taking into account the
results achieved, a renewed taxonomic treatment is presented.
Materials and methods
Study area
As a rule, 15–20 plants of R. parnassiifolius polyploid
complex were collected from each sample site throughout
the whole distribution range between 2006 and 2008
122 E. Cires, J. A. F. Prieto
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