Received: 22 March 2017
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Revised: 26 May 2017
Accepted: 30 May 2017
DOI: 10.1002/ece3.3206
ORIGINAL RESEARCH
Growth form evolution and hybridization in Senecio
(Asteraceae) from the high equatorial Andes
Eva Dušková1 | Petr Sklenář1
| Filip Kolář1,2 | Diana L. A. Vásquez1 |
Katya Romoleroux3 | Tomáš Fér1 | Karol Marhold1,4
Department of Botany, Faculty of
Science, Charles University, Prague, Czech
Republic
1
Abstract
Changes in growth forms frequently accompany plant adaptive radiations, including
National Centre for Biosystematics, Natural
History Museum, University of Oslo, Oslo,
Norway
páramo–a high-elevation treeless habitat type of the northern Andes. We tested
3
such growth form changes. We also investigated the role of Andean geography and
2
Escuela de Ciencias Biológicas, Pontificia
Universidad Católica del Ecuador, Quito,
Ecuador
4
Institute of Botany, Slovak Academy of
Sciences, Bratislava, Slovak Republic
Correspondence
Petr Sklenář, Department of Botany, Faculty
of Science, Charles University, Prague, Czech
Republic.
Email: petr.sklenar@natur.cuni.cz
Funding information
Grantová Agentura České Republiky, Grant/
Award Number: 206/07/0273; Grant Agency
of the Charles University, Grant/Award
Number: GAUK 261211
whether diverse group of Senecio inhabiting montane forests and páramo represented
environment in structuring genetic variation of this group. We sampled 108 populations and 28 species of Senecio (focusing on species from former genera Lasiocephalus
and Culcitium) and analyzed their genetic relationships and patterns of intraspecific
variation using DNA fingerprinting (AFLPs) and nuclear DNA sequences (ITS). We partitioned genetic variation into environmental and geographical components. ITS-based
phylogeny supported monophyly of a Lasiocephalus-Culcitium clade. A grade of herbaceous alpine Senecio species subtended the Lasiocephalus-Culcitium clade suggesting a
change from the herbaceous to the woody growth form. Both ITS sequences and the
AFLPs separated a group composed of the majority of páramo subshrubs from other
group(s) comprising both forest and páramo species of various growth forms. These
morphologically variable group(s) further split into clades encompassing both the
páramo subshrubs and forest lianas, indicating independent switches among the
growth forms and habitats. The finest AFLP genetic structure corresponded to morphologically delimited species except in two independent cases in which patterns of
genetic variation instead reflected geography. Several morphologically variable species were genetically admixed, which suggests possible hybrid origins. Latitude and
longitude accounted for 5%–8% of genetic variation in each of three AFLP groups,
while the proportion of variation attributed to environment varied between 8% and
31% among them. A change from the herbaceous to the woody growth form is suggested for species of high-elevation Andean Senecio. Independent switches between
habitats and growth forms likely occurred within the group. Hybridization likely played
an important role in species diversification.
KEYWORDS
adaptive radiation, Andes, Culcitium, growth forms, hybridization, Lasiocephalus, Neotropical
montane forest, páramo, Senecio
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2017 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.
Ecology and Evolution. 2017;7:6455–6468.
www.ecolevol.org
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1 | INTRODUCTION
The uplift of Andean cordilleras played a major role in promoting di-
DUŠKOVÁ et al.
Phylogenetic molecular studies of the tribe Senecioneae suggest
that the traditionally recognized Andean genera Lasiocephalus and
Culcitium Bonpl. (scapose herbs forming basal leaf rosettes) belong
versification of the Neotropical flora (Antonelli, Nylander, Persson, &
to Senecio (Pelser, Nordenstam, Kadereit, & Watson, 2007; Pelser
Sanmartin, 2009; Hoorn et al., 2010; Hughes, Pennington, & Antonelli,
et al., 2010). Our previous study of 13 Senecio species from the
2013). The equatorial Andes in particular host a very diverse flora
former Lasiocephalus, which all were diploid, based on nuclear DNA
spanning a great variety of habitats between the montane forest and
sequences (ITS region) and nuclear genome size data (Dušková et al.,
the alpine páramo belts (Churchill, Balslev, Forero, & Luteyn, 1995;
2010), identified two major clades that largely correspond to the two
Luteyn, 1999; Myers, Mittermaier, Mittermaier, da Fonseca, & Kent,
habitat types, that is, montane forest and páramo. The results also sug-
2000). Páramo habitats became available by the end of the north-
gested that Senecio (Culcitium) nivalis (Kunth) Cuatrec. (Figure 1f) was
Andean orogeny ca. 3–5 Ma (van der Hammen & Cleef, 1986). In spite
closer to species of former Lasiocephalus than to other taxa of former
of its relative youth, the páramo flora is especially rich in groups which
Culcitium.
underwent radiations (Madriñán, Cortés, & Richardson, 2013; Sklenář,
Given its likely origin within ca. the last 2 Myr (Pelser et al.,
Dušková, & Balslev, 2011). Elevational shifts of vegetation belts during
2010) and occurrence in the montane-alpine habitats, the former
the Pleistocene, which repeatedly fragmented and reunited plant pop-
Lasiocephalus exemplifies recent plant radiation in the (sub)tropi-
ulations, were coupled with the final uplift of the Andes, which cre-
cal Andes. Based on extensive population sampling throughout the
ated new ecological opportunities and promoted diversification of the
northern Andes and using an extended sample of ITS sequences
flora (Hughes & Eastwood, 2006; Jabaily & Sytsma, 2012; Luebert &
complemented with highly variable AFLP (amplified fragment length
Weigend, 2014; Madriñán et al., 2013).
polymorphism) markers, here we present deeper insights into the re-
Adaptation to newly emerging supra-forest habitats has been an
lationships among the Andean species of Senecio formerly classified
important source of functional diversity in the páramo flora (Sklenář
in Lasiocephalus. Specifically, we examine a hypothesis put forward by
et al., 2011). DNA-based phylogenetic studies indicate that the north-
Dušková et al. (2010) that independent transitions between the mon-
Andean genus Espeletia Mutis ex Bonpl. (Asteraceae) evolved dis-
tane forest and páramo habitats occurred that were accompanied by
tinct growth forms upon colonizing the páramo (Cuatrecasas, 1986;
growth form changes. We further examine patterns of genetic diver-
Panero, Jansen, & Clevinger, 1999; Rauscher, 2002). In Aragoa Kunth
sity within the group, and particularly their correlation with environ-
(Plantaginaceae), another genus endemic to the northern Andes, ar-
mental factors and Andean geography.
borescent plants of the montane forest evolved into páramo shrubs
(Bello, Chase, Olmstead, Rønsted, & Albach, 2002; Fernández-Alonso,
1995), and growth form changes have been found in other Andean
genera such as Lupinus L., Hinterhubera Sch. Bip. ex Wedd., Laestadia
Kunth ex Less., and Westoniella Cuatrec. (Hughes & Eastwood, 2006;
2 | MATERIALS AND METHODS
2.1 | Plant material
Karaman-Castro & Urbatsch, 2009). However, deep insights into
Samples of species from the former Lasiocephalus and former
growth form evolution among north-Andean plant groups, based upon
Culcitium, along with co-occurring species of Senecio, were collected
genetic markers with sufficiently detailed resolution have been rare
during 2006–2010 in Bolivia, Ecuador, Venezuela, and Colombia
(Jabaily & Sytsma, 2012; Uribe-Convers, Settles, & Tank, 2016).
(Appendix S1). Due to the sampling gap in the central Andes, we
Species of the genus Senecio L. (Asteraceae), which were tradition-
lacked the single Peruvian species of former Lasiocephalus, a broad-
ally placed in the genus Lasiocephalus Willd. ex Schltdl. (Cuatrecasas,
leaved liana S. loeseneri Hieron. This species is, nevertheless, some-
1978), comprise a morphologically and ecologically diverse plant
times considered conspecific with S. campanulatus Sch. Bip. ex Klatt
group in the northern and central Andes. About 25 species are distrib-
from Bolivia (Calvo & Freire, 2016), which was included in our study.
uted from Venezuela to Bolivia, with the highest richness in Ecuador
Multiple populations were sampled for most of the species through-
(Calvo & Freire, 2016; Cuatrecasas, 1978). Two main growth forms
out their distribution ranges (Figure 2b). At each locality, geographical
are recognized. Broad-leaved lianas (Figure 1 g,h) inhabit montane
coordinates and elevation were recorded. Young, intact leaves were
forests and secondary thickets usually between 2,800 and 3,800 m,
collected and desiccated in silica gel; vouchers were deposited in COL,
although some species also occur in the forest–páramo shrubby eco-
PRC, QCA, QCNE, and VEN.
tone called subpáramo (usually at 3,800 m). The other growth form is
ascending or erect, narrow-leaved subshrub (Figure 1a–c,e) that occur
in the páramo dominated by tussock grasses (3,800–4,300 m) and in
2.2 | AFLP fingerprinting and DNA sequencing
the uppermost belt of patchy vegetation called superpáramo (up to
In total, 356 accessions of 18 Senecio species formerly classified
4,800–5,000 m). One species, Senecio mojandensis Hieron. (Figure 1d),
as Lasiocephalus and 18 accessions of Senecio nivalis were geno-
a basal rosette herb of wet páramo habitats cannot be satisfactorily
typed using AFLP fingerprinting (Vos et al., 1995) (see Appendix S2
placed in either of these categories. Most species are morphologically
for details on the protocol). Fragments were manually scored with
distinct and readily identifiable, although some are variable in leaf size
genemarKer
and shape, such as S. otophorus Wedd.
in the range of 60-500 bp. were scored, regardless of their intensity
version 1.80 (SoftGenetics). Only unambiguous fragments
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DUŠKOVÁ et al.
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F I G U R E 1 Growth form variation
among the investigated Senecio species
from the high Andes: (a) S. lingulatus,
Ecuador, páramo; (b) S. longepenicillatus,
Venezuela, superpáramo; (c) highelevation form of S. otophorus, Colombia,
superpáramo; (d) S. mojandensis, Ecuador,
páramo; (e) S. superandinus, Ecuador,
superpáramo; (f) Senecio nivalis, Ecuador,
superpáramo, (g) S. pindilicensis, Ecuador,
montane forest; (h) S. patens, Ecuador,
montane forest. Symbols are colored
according to species assignment to the
main strUctUre clusters (see Figure 2);
symbol shape indicates the growth form,
that is, square–basal rosette herb, circle–
narrow-leaved subshrub, triangle–broadleaved liana
(Tribsch, Schönswetter, & Stuessy, 2002). For 5% of the samples, the
runs (Nordborg et al., 2005) and delta K (Evanno, Regnaut, & Goudet,
whole AFLP protocol was repeated from the isolated DNA onwards
2005), both calculated using the R-script Structure-sum-2009 (Ehrich,
to test the reproducibility of the method (Bonin et al., 2004). Internal
2006). The Ks with consistent results over ten repeats were consid-
transcribed spacer (ITS) regions were directly sequenced using the
ered to be plausible and further examined. As the analysis of the entire
primers ITS4 and ITS5 (White, Bruns, Lee, & Taylor, 1990) for 50 in-
dataset showed that only runs with K = 3 converged to a consistent
dividuals of Andean Senecio (i.e., 44 of former Lasiocephalus, two of
solution in ten repeats, subsequent, separate
former Culcitium, four other members of Senecio). We selected the
each of these three partitions (hereafter named clusters A, B, and C)
individuals in order to representatively cover all species of the former
were conducted using the same parameters. Only individuals assigned
Lasiocephalus as well as all clusters and subgroups identified by AFLPs.
to a particular cluster with posterior probability >0.9 in the initial
strUctUre
analyzes of
analysis were included in these subsequent analyzes. Major trends in
2.3 | Clustering of AFLP data
Genetic structure was inferred using a Bayesian clustering method
implemented in
the AFLP variation were visualized using principal coordinate analyzes
(PCoA) based on Jaccard interindividual distances computed using
famD
1.31 (Schlüter & Harris, 2006).
2.2.3 (Falush, Stephens, & Pritchard, 2007)
We further investigated the relationships among the major clus-
employing a recessive allele model with admixture, assuming inde-
ters based on a reduced subset of 266 individuals that were identi-
strUctUre
pendent allele frequencies with 1,100,000 MCMC (Markov chain
fied as nonadmixed (i.e., with posterior probabilities of membership to
Monte Carlo) generations, and discarding the first 100,000 genera-
both major clusters and subgroups >0.9) in the strUctUre analyzes. We
tions as burn-in. We limited the number of clusters (K) to 1–10, each
reconstructed phylogenetic relationships using a likelihood model for
K was replicated with ten runs, and we further assessed stability of
binary restriction site data implemented in mrBayes v3.2.5 (Ronquist
the results by calculating similarity coefficients between the replicate
& Huelsenbeck, 2003). This model approximates the gain and loss of
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DUŠKOVÁ et al.
(a)
(b)
(c)
F I G U R E 2 (a) Assignment of 374
individuals (entire dataset) of highelevation Andean Senecio into three main
AFLP clusters inferred in strUctUre; (b)
Geographical locations of populations
with growth form and strUctUre cluster
assignment indicated; (c) Ordination of
AFLP phenotypes by use of principal
coordinate analysis (PCoA) based on
Jaccard distances. The symbol coloration
reflects the assignment of the individuals to
the main strUctUre clusters (white–admixed
individuals with assignment probability
below 0.5); symbol shape indicates the
growth form, that is, square–basal rosette
herb, circle–narrow-leaved subshrub,
triangle–broad-leaved liana
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DUŠKOVÁ et al.
fragments by setting a condition that the characters that are absent
6459
fitting model. Association of growth forms and phylogeny was tested
(i.e., 0) in all individuals cannot be observed. We performed two inde-
by computing Pagel’s lambda (Freckleton, Harvey, & Pagel, 2002)
pendent runs of 5,000,000 generations each using the default prior
using the function fitDiscrete in the package geiger (Pennell et al.,
settings, setting the restriction site model (lset nst = 1 coding = noab-
2014) in R (Ihaka & Gentleman, 1996). Statistical significance of es-
sencesites) and discarding the first 25% generations as burn-in.
timated lambda was tested by computing likelihood ratio test (LRT)
against lambda = 0 model.
2.4 | DNA sequence analyzes
Sequences of the ITS region were aligned by mafft 7 (Katoh &
2.6 | Geographical analyzes of AFLP data
Standley, 2013) and edited using aliView (Larsson, 2014). In addition,
Geographical correlates of the genetic (AFLP) variation were examined
we included in the final matrix previously published ITS sequences
after the admixed (i.e., posterior probability <.9), and Bolivian samples
of: (1) 11 directly sequenced accessions of other Andean Senecio
were excluded to avoid bias due to unclear cluster assignment and sam-
(Pelser et al., 2007) and (2) 26 cloned individuals from the former
pling gap, respectively. We tested for a significant correlation between
Lasiocephalus (10–12 colonies per accessions were cloned; putative
matrices of genetic and geographical distances among populations (iso-
PCR errors and potential chimaeric sequences were removed previ-
lation by distance) using a Mantel test in adegenet. Among-population
ously by Dušková et al., 2010; no excessively long branches indicating
genetic chord distances derived from AFLP fragment frequencies were
nonfunctional copies were found). To reduce the number of phyloge-
inferred using a Bayesian method with nonuniform priors (Zhivotovsky,
netically uninformative tip branches (and number of pseudoreplicates
1999) as implemented in famD 1.31 (Schlüter & Harris, 2006).
for trait mapping analyzes), we collapsed the cloned sequences from
Climatic data describing mean annual temperature, daily and an-
each individual to a consensus and intra-individual polymorphisms
nual temperature ranges, annual rainfall, and its inter-annual variation
were coded using IUPAC (International Union of Pure and Applied
expressed as coefficient of variation for each collection site were ex-
Chemistry) ambiguity codes in cases where clones from single species
tracted from the WorldClim database (Hijmans, Cameron, Parra, Jones,
formed a monophyletic cluster or fell within an unresolved polytomy.
& Jarvis, 2005). Those data together with elevation formed a group
The single exception with highly divergent haplotypes was 88_Pi, in
of environmental variables, while site latitude and longitude repre-
which the two divergent haplotypes were retained as separate acces-
sented geographical variables. Variance of the AFLP data matrix was
sions when constructing the tree. We performed phylogenetic analysis
partitioned into environmental and geographical components (and
on the resulting matrix of 630 characters and 87 individuals using both
their interaction) by a series of redundancy analyzes (RDA) and par-
maximum parsimony (in
tial RDA ordinations (Borcard, Legendre, & Drapeau, 1992) employing
paUp
v4.0b10; Swofford, 2002; treating gaps
as characters) and Bayesian analyzes (in mrBayes v3.2.2; Ronquist &
canOcO
fOr
winDOws 4.5 (ter Braak & Šmilauer, 1998). As the RDA
Huelsenbeck, 2003). The most parsimonious trees were searched
employs Euclidean distance to measure dissimilarity between pairs of
heuristically with 1,000 replicates of random sequence addition, tree
samples (Šmilauer & Lepš, 2014), this makes it analogous to analysis of
bisection reconnection swapping and MulTrees on and the data set
molecular variance (AMOVA; Excoffier, Smouse, & Quattro, 1992) but
were bootstrapped using 1,000 replicates. In Bayesian analyzes, we
provides an opportunity to make partial tests to discriminate between
applied the generalized time reversible (GTR) substitution model
pure effects of explanatory variables and their interaction.
(as selected by the Bayesian Information Criterion in JModeltest 2;
Darriba, Taboada, Doallo, & Posada, 2012) with gamma distribution of
rate heterogeneity and simultaneously ran two MCMCMC runs with
four chains each for 2,000,000 generations, sampling every 1000th
generation using the default priors. The posterior probability of the
3 | RESULTS
3.1 | AFLP fingerprinting
phylogeny and its branches were determined from the combined set
AFLP analysis of 374 accessions resulted in 269 reliable fragments, of
of trees, discarding the first 25% of trees as burn-in.
which 264 (98%) were polymorphic. The overall reproducibility of the
dataset was 95%.
2.5 | Growth form evolution
Character state reconstructions of the growth forms were performed
3.1.1 | Main grouping within the entire dataset
employing a maximum likelihood approach implemented in the func-
Bayesian clustering of the entire dataset yielded the highest values
tion rayDISC, part of the package corHMM (Beaulieu, O’Meara, &
of ΔK and among-replicate similarity (1.0) for partition into three
Donoghue, 2013) in R (Ihaka & Gentleman, 1996). This method al-
clusters A, B, and C (Figure 2a, Appendix S3A). Cluster A contained
lows for reconstructions of multistate characters, unresolved nodes,
mostly lianas of montane forest and forest–páramo ecotone, one of
and ambiguities (polymorphic taxa or missing data). Three models of
which, however, also formed a morphologically distinct subshrub-like
character evolution were evaluated as follows: equal rates (ER), sym-
high-elevation population (pop. 51). Cluster B was morphologically
metrical (SYM), and all rates different (ARD); an Akaike information
variable, encompassing montane forest lianas, narrow-leaved páramo
criterion corrected for sample size (AICc) was used to select the best
subshrubs, and a basal rosette herb. Cluster C contained exclusively
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DUŠKOVÁ et al.
narrow-leaved (super)páramo subshrubs (Figure 2b, Table 1). Four
accessions were admixed, while most accessions of S. otophorus (ex-
species comprised mostly individuals that were admixed either be-
cluding those from southern Ecuador) fell into the second subgroup
tween all three clusters (S. josei Sklenář, S. iscoensis Hieron.), between
(Appendix S3B). With K = 3, two subgroups partly corresponded
clusters A and C (S. aff. quitensis), or between clusters B and C (S. sub-
to species limits, namely (1) a subgroup of Colombian and north
involucratus Cuatrec.), although certain admixture was also found in
Ecuadorian populations of S. involucratus (Kunth) DC. (along with two
some individuals of S. patens (Kunth) DC., S. mojandensis, S. pindilicen-
S. aff. quitensis Cuatrec. accessions) and (2) a subgroup of Colombian
sis Hieron., S. longepenicillatus Sch. Bip. ex Sandwith, S. imbaburensis
to central Ecuadorian accessions of S. otophorus. The third subgroup
Sklenář & Marhold, S. lingulatus (Schltdl.) Cuatrec., and S. superandinus
comprised all populations from southern Ecuador disregarding spe-
Cuatrec.. The Bayesian clustering was also reflected in PCoA ordina-
cies identity (S. involucratus, S. cuencanus, and S. otophorus) along
tion, separating the three clusters along the first (cluster A vs. C) and
with populations of S. involucratus from northern and central Ecuador
second (cluster B vs. A + C) axes (Figure 2c).
(Figure 3a,b). The PCoA of cluster A confirmed the Bayesian grouping,
separating Colombian to central Ecuadorian populations of S. otophorus along the first axis and Colombian to north Ecuadorian populations
3.1.2 | Finer structure within the main clusters
of S. involucratus along the second axis (Figure 3c).
Separate Bayesian clustering of the accessions assigned to cluster
Separate analysis of cluster B yielded the same similarity co-
A revealed that K = 2 and K = 3 exhibited high similarity among in-
efficient, 1.0, for K = 2, 3, 4, and 5, although K = 3 had the highest
dependent runs (>0.998 in both partitions, although the former had
ΔK (Appendix S3A). The finest partitioning (K = 5) separated all five
higher ΔK; Appendix S3A), and the finer structuring was plotted
species with almost no admixture (Figure 4a), whereas S. patens and
onto the map (Figure 3b). With K = 2, S. involucratus and S. cuencanus
S. pindilicensis merged at K = 4, and S. campanulatus and S. longepen-
Hieron. were classified in the first subgroup, although most of their
icillatus joined this subgroup at K = 3 and K = 2, respectively, leaving
Senecio nivalis apart from all other species at K = 2 (Appendix S3C).
T A B L E 1 Summary of the associations between growth forms
(uppercase letters) and genetic relationships reconstructed by the
two sets of molecular markers (AFLPs, ITS sequences) in highelevation north Andean Senecio (see Figures 2–5 for AFLP (sub)
groups and Figure 6a for ITS (sub)clades delimitation). B–basal
rosette herb, mostly from lower páramo; L–broad-leaved liana, from
montane forest and forest-páramo ecotone; N–narrow-leaved
subshrub, from páramo to superpáramo. Others refer to admixed
samples with equivocal assignment to AFLP (sub)groups and
accessions not genotyped by AFLPs (see Appendix S1)
Páramo ITS
clade
Forest ITS clade
p1
f1
p2
f2
f3
separated S. campanulatus and S. pindilicensis, respectively.
Analysis of cluster C showed the highest ΔK and similarity coefficient (0.995) to be yielded by K = 5 (Appendix S3A). The five subgroups
comprised as follows: (1) S. puracensis (Cuatrec.) Cuatrec. + S. gargantanus (Cuatrec.) Cuatrec.; (2) S. imbaburensis; (3) S. lingulatus
(except for southern Ecuador) + S. subinvolucratus; (4) S. superandinus (except for southern Ecuador) + S. superparamensis Sklenář, (5)
southern Ecuadorian populations of S. superandinus and S. lingulatus
(Figure 5a,b). Senecio aff. quitensis was highly admixed and was scattered among three subgroups. The PCoA ordination diagram showed
f4
f5
f6
incomplete discrimination of the species (Figure 5c), but its first and
second axes, respectively, suggested separation of the S. garganta-
AFLP cluster A
A1
L
A2
L
A3
L
N, L
AFLP cluster B
B1
The PCoA of cluster B (Figure 4c) separated S. nivalis and S. patens
along the first two ordination axes, whereas the third and fourth axes
N
nus + S. puracensis group and supported the distinct position of the
southern Ecuadorian populations of S. superandinus and S. lingulatus.
3.2 | ITS and AFLP phylogeny
Bayesian analysis of ITS sequences showed monophyly of a clade
B2
comprising all accessions of the former Lasiocephalus and former
N
B3
L
Culcitium (together with Senecio chionogeton), although it did not sup-
B4
L
port separation of the two former genera (Figure 6a). Instead, along
B5
with several unresolved former Culcitium accessions, we identified
L
two clades, corresponding to “páramo” and “forest” clades of Dušková
AFLP cluster C
C1
N
C2
N
C3
N
N
C4
N
N
C5
Others
et al. (2010), which with a few exceptions corresponded to major AFLP
N
clusters C and A + B, respectively (Figure 6a,b, Table 1). The “forest
clade” further split into several well supported subclades (with uncertain relationships among them) which mostly corresponded to AFLP
subgroups (namely B2, B3 + B4, B5, A1 + A3, A2 + A3 subgroups).
N
B
N, L
B
L
N, L
While the “páramo clade” comprised narrow-leaved subshrubs and
N
one basal rosette herb, the “forest clade” contained representatives
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DUŠKOVÁ et al.
6461
(a)
(b)
(c)
F I G U R E 3 Genetic structure and geographical distribution of 149 individuals of high-elevation Andean Senecio from cluster A. (a) Posterior
probabilities for membership of each individual in the three resulting subgroups (designated by different colors) as identified in a separate
strUctUre analysis of cluster A members. (b) Geographical distribution of the analyzed populations. (c) Ordination of AFLP phenotypes (PCoA);
symbol color refers to the strUctUre subgroups (>0.5 posterior probability), symbol shape indicates species
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DUŠKOVÁ et al.
(a)
(b)
(c)
F I G U R E 4 Genetic structure and geographical distribution of 47 individuals of high-elevation Andean Senecio from cluster B. (a) Posterior
probabilities for membership of each individual in the five resulting subgroups (designated by different colors) as identified in a separate
strUctUre analysis of cluster B members. (b) Geographical distribution of the analyzed populations. (c) Ordination of AFLP phenotypes (PCoA);
symbol color refers to the strUctUre subgroups (>0.5 posterior probability), symbol shape indicates species
of all three growth forms. Broad-leaved liana is reconstructed as the
a supported lineage (p1) within the “páramo” clade (Figure 6a). AFLP
ancestral state within the “forest clade,” which contains one subclade
cluster C1 was split into both major ITS clades, with the Colombian and
(f4) formed by lianas only, three subclades (f1, f5, f6) comprising a
Ecuadorian accessions being parts of the “forest” and “páramo” clades,
liana and a narrow-leaved subshrub, one subclade (f3) with a narrow-
respectively. ITS clones isolated from a single accession of S. aff. quiten-
leaved subshrub (S. longepenicillatus), and one subclade (f2) comprising
sis (pop. 88_Pi) fell into both major ITS clades. Finally, S. puracensis from
a narrow-leaved subshrub and a basal rosette herb (Table 1).
“páramo” cluster C appears nested within the ITS “forest clade.”
There were several remarkable incongruences among ITS and AFLP
Bayesian phylogenetic analysis of AFLP phenotypes of nonad-
data. In particular, Senecio nivalis (cluster B1) shared the same ITS hap-
mixed individuals confirmed monophyly (>90% posterior probability)
lotypes with S. superparamensis (cluster C4) and both species formed
of most of the AFLP subgroups. However, it failed to provide support
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DUŠKOVÁ et al.
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(a)
(b)
(c)
F I G U R E 5 Genetic structure and geographical distribution of 116 individuals of high-elevation Andean Senecio from cluster C. (a) Posterior
probabilities for membership of each individual in the five resulting subgroups (designated by different colors) as identified in a separate
strUctUre analysis of cluster C members. (b) Geographical distribution of the analyzed populations. (c) Ordination of AFLP phenotypes (PCoA);
symbol color refers to the strUctUre subgroups (>0.5 posterior probability), symbol shape indicates species
6464
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DUŠKOVÁ et al.
F I G U R E 6 (a) Phylogenetic reconstruction of 87 accessions of northern Andean Senecio based on sequences of ITS region of ribosomal
DNA. Bayesian 50% majority rule consensus tree with posterior probabilities >0.90 and bootstrap values >50% inferred with maximum
parsimony are indicated, respectively, before and after the slash above each supported branch. Supported subclades of the “forest” and
“páramo” clades are marked as f1–f6 and p1–p2, respectively. Growth form of each accession is marked by a symbol, membership in the AFLP
subgroups (if applicable) is denoted by corresponding letters (A1–C5), accessions with ambiguous strUctUre assignment are marked “MIX.” The
presence of highly divergent ITS sequences in the same individual of S. aff. quitensis is marked by an arrow. Senecio doryphyllus, S. decipiens,
and S. alatopetiolatus, although belonging to the former Lasiocephalus, were not analyzed using the AFLPs. Reconstruction of the growth form
evolution according to the equal rates (ER) model has been superimposed onto the ITS tree (see Appendix S3E for original). (b) Relationships
among AFLP phenotypes of 266 nonadmixed (see Section 2) individuals of former Lasiocephalus and Senecio nivalis reconstructed in Bayesian
framework. Cluster codes correspond with Figures 3–5; branches with posterior probabilities >0.95 are marked with dots
for relationships among the AFLP subgroups, except for supported
clade” except for S. cocuyanus (basal rosette herb) and S. aff. quitensis
monophyly of cluster A (Figure 6b, Appendix S3D).
(varying between broad-leaved liana and narrow-leaved subshrub).
The growth form was strongly associated with phylogeny (Pagel’s
3.3 | Growth form evolution
lambda = 0.89, ln(lambda) = −75.63, ln(lambda = 0) = −150.38, LRT
p-value < .001).
Equal rates (ER) model had the lowest AICc (94.24), compared to SYM
(97.77) and ARD (107.13) models. The ER model reconstructed the
basal rosette herb as the ancestral growth form for the LasiocephalusCulcitium species group (Figure 6a, Appendix S3E). The herbs switched
3.4 | Geographical and environmental analyzes of
AFLP data
to broad-leaved lianas in the “forest clade,” with further changes
Senecio populations as a whole and members of cluster A showed a
to narrow-leaved subshrub (S. longepenicillatus, S. puracensis) and
very weak correlation between genetic and geographical distances
a reversal to a basal rosette herb (S. mojandensis). The growth form
(isolation by distance, IBD), whereas this relationship was nonsig-
of narrow-leaved subshrub is present in all species of the “páramo
nificant in the other two clusters (Table 2). Subgroups with sufficient
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DUŠKOVÁ et al.
6465
numbers of populations were available only in cluster A; here we
ITS “forest clade” (except for S. puracensis) (Figure 6, Table 1); incon-
observed significant correlations in both Ecuadorian-Colombian sub-
gruences will be discussed below.
groups A1 and A2 but a lack of correlation in the southern Ecuadorian
subgroup A2.
Less than 10% of total variance in the entire AFLP dataset was
accounted for by the effects of either environmental (rainfall, tem-
4.2 | Growth form changes and habitat shifts
Species of different growth forms and preferences for páramo or mon-
perature, elevation; altogether 6% of variability) geographical (latitude,
tane forest fell within several different AFLP (sub)groups and ITS (sub)
longitude; 2% of variability) components or their interaction (1.5% of
clades, suggesting that independent shifts in ecology were accompa-
variability) (Table 2). When the three AFLP clusters were analyzed sep-
nied by changes in morphology. Both AFLP and ITS data indicate that
arately, the geographical component accounted for a similar (5%–8%)
at least two distinct genetic entities occur in the páramo, representa-
proportion of the total variation, whereas the environmental compo-
tives of which demonstrate convergence in such traits as growth form,
nent accounted for almost a third of variation in cluster B but only
size and number of capitula, and leaf morphology (Figure 1). The first
8%–9% in clusters A and C. Moreover, there was a distinct interaction
entity is the páramo-dwelling AFLP cluster C (largely corresponding
(5%) between the two sets of variables in cluster A, whereas the inter-
to the ITS “páramo clade”), species of which occur throughout most
action was very low or lacking in the two other clusters.
of Ecuador and southern Colombia. The second entity is represented
by Venezuelan S. longepenicillatus (AFLP cluster B, f3 subclade within
the ITS “forest clade”), which is a narrow-leaved páramo subshrub but
4 | DISCUSSION
sporadically also appears in a broad-leaved form at the tree-line ecotone. In addition, S. otophorus (AFLP cluster A, f6 subclade within the
4.1 | Lasiocephalus-Culcitium species group
ITS “forest clade”), which grows as a slender liana twining in subpáramo
Pelser et al. (2007, 2010) and Dušková et al. (2010) pointed to
thickets, also occurs as a narrow-leaved subshrub in the superpáramo of
close relationships between the former genera of Lasiocephalus and
Colombian Cordillera Oriental (Figure 1c), and similar habit variation is
Culcitium. The present study, using ITS sequences and an extended
demonstrated by montane forest and superpáramo plants of S. doryphyl-
list of species, suggests monophyly of the Lasiocephalus-Culcitium spe-
lus Cuatrec. from Sierra Nevada de Santa Marta (northern Colombia).
cies group but with neither of the two former genera monophyletic.
These findings suggest that convergent growth form evolved indepen-
Although relationships within the group are only partly resolved in
dently in various parts of the northern Andes, although the variation in
both the ITS and AFLP datasets, suggesting a recent diversification
S. otophorus and S. doryphyllus may only represent phenotypic plasticity.
(Turner et al., 2013), there is a partial congruence between the two
Transitions in growth form associated with habitats at different
markers, as AFLP cluster C corresponds to the ITS “páramo clade” (ex-
elevations have been presented for various Andean plant groups.
cept for Senecio nivalis) and AFLP clusters A and B correspond to the
Whereas Lobeliaceae (Knox, Muasya, & Nuchhaala, 2008), Huperzia
Bernh. (Wilkström, Kenrick, & Chase, 1999), Chusquea Kunth (Fisher
et al., 2009), and Disterigma (Klotzsch) Nied. (Pedraza-Peñalosa, 2009)
T A B L E 2 Eco-geographical covariates of genetic variation of
north-Andean Senecio. Variance partitioning (by means of RDA) of
AFLP genetic variation into environmental (rainfall, temperature,
elevation) and geographical (latitude, longitude) components and
correlation between genetic and geographical distances (by means of
Mantel test and quantified by correlation coefficient rM). The
partitioning was based on characteristics of the original collection
sites after genetically admixed individuals, and spatially remote
Bolivian samples were excluded; all RDA ordinations were significant
at p = .002 under 499 Monte Carlo permutations
Chaetanthera Ruiz & Pav. and Puya Molina migration was suggested in
the opposite or both directions, respectively (Hershkovitz, Arroyo, Bell,
& Hinojosa, 2006; Jabaily & Sytsma, 2012). As a grade of herbaceous
Senecio species from alpine habitats subtends (although the support is
weak) the Lasiocephalus-Culcitium clade (Figure 6a), the ITS phylogeny
is consistent with an alpine-to-forest transition for the evolution of
the Lasiocephalus-Culcitium group as a whole. If such a relationship is
confirmed, a change from the herbaceous (basal leaf rosette) to the
woody (liana, ascending subshrub, shrub) state is implied, similar to, for
Entire
dataset
Cluster
A
Cluster
B
Cluster
C
Environment
5.8%
7.8%
31.2%
8.8%
sette herbs does not permit interpretation of the growth form transi-
Environmenta
geography
1.5%
5.2%
0%
1.1%
tions within the Lasiocephalus-Culcitium clade to evaluate Cuatrecasas’
Geography
2.2%
6%
7.7%
5.2%
cies evolved from the growth form of their montane forest ancestor(s).
Residual
90.5%
81%
61.1%
84.9%
However, Cuatrecasas’ view could be valid for some páramo subshrubs
Isolation by distance
(Mantel test)
rM = .12,
p = .04
rM = .12,
p = .04a
n.s.
n.s.
found within the “forest clade” (e.g., S. longepenicillatus). Moreover, the
Variance partitioning (RDA)
example, Andean Valeriana L., Gentianella Moench, and Loricaria Wedd.
(Kolář, Dušková, & Sklenář, 2016; Sklenář et al., 2011). The polytomy
consisting of the “páramo clade,” “forest clade,” and several basal ro-
Subgroups, A1: rM = .44, p = .02; A2: n.s.; A3: rM = .31, p = .03.
a
apparently colonized alpine habitats from the montane forest, for
(1978) idea that the páramo growth form of former Lasiocephalus spe-
ITS phylogeny suggests another transition in this clade, that is, to a
basal rosette herb in S. mojandensis.
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4.3 | Role of hybridization
Gene flow is known to occur among closely related alpine spe-
DUŠKOVÁ et al.
morphologically (Figure 1a,e) and are genetically distinct throughout
most of Ecuador. Their populations in southern Ecuador, however,
merge and form another distinct genetic subgroup (Figure 5a,b).
cies (Vargas, 2003; Wagstaff & Garnock-Jones, 2000; Winkworth,
Similarly, two morphologically distinct species from the montane
Wagstaff, Glenny, & Lockhart, 2005), yet the role of hybridization
forest-páramo ecotone, S. involucratus and S. otophorus (cluster A),
in the evolution of the Andean flora has not been documented, ex-
appear as distinct AFLP entities in northern-central Ecuador and
cept in the cases of Polylepis Rioz & Pav. (Schmidt-Lebuhn, Kessler,
Colombia, but the markers fail to discriminate between them in south-
& Kumar, 2006), Hypochaeris (Tremetsberger et al., 2006), and Puya
ern Ecuador (Figure 3a,b). There, the species form a separate sub-
(Jabaily & Sytsma, 2012). In agreement with frequent hybridization in
group together with another morphologically distinct montane forest
the Senecioneae (Hodálová & Marhold, 1996; Kirk, Máčel, Klinkhamer,
liana, S. cuencanus.
& Vrieling, 2004; Lowe & Abbott, 2000; Osborne, Chapman, Nevado,
As morphologically intermediate plants between S. superandinus
& Filatov, 2016), our molecular data, morphological observations, and
and S. lingulatus occur in southern Ecuador, gene flow due to hybrid-
previously published genome size values (Dušková et al., 2010) indi-
ization might have generated the observed pattern. In contrast, we did
cate that homoploid hybridization likely occurred among multiple spe-
not observe any putative hybrids between S. involucratus and S. oto-
cies of former Lasiocephalus.
phorus, nor did we find any consistent morphological distinction in
Traces of hybridization are indicated by consistently admixed AFLP
plants of either species from southern Ecuador. Therefore, we hypoth-
profiles across multiple populations of several species (Figure 2a). This
esize that southern Ecuador represents an ancestral area of cluster
is especially apparent for Senecio aff. quitensis, a taxon with morphol-
A where high levels of ancestral polymorphisms have been retained,
ogy varying between subshrubs and lianas and whose accessions vari-
preventing the genetic discrimination of species by means of the
ously combine AFLP profiles of clusters A (forest lianas) and C (páramo
AFLPs. Both species might have independently migrated northwards,
subshrubs). Furthermore, ITS sequences of this species (including di-
leaving a footprint of gradual genetic differentiation which is docu-
vergent ITS copy types from a single individual) are placed in the diver-
mented by a significant isolation by distance relationship observed in
gent “páramo” and “forest” clades, and its genome size is intermediate
A1 and A3 subgroups. Such northward migration would be consistent
between them (Dušková et al., 2010).
with the biogeographical reconstructions of Andean plant groups such
Incongruence between the AFLP and ITS datasets suggests that
as Azorella, Oreobolus R. Br., and Puya (Andersson, Kocsis, & Eriksson,
hybridization might also have been involved in the origin of other
2006; Chacón, Madriñán, & Bruhl, 2006; Jabaily & Sytsma, 2012).
Andean Senecio species. This was particularly documented for Senecio
The northern and central Ecuadorian Andes experienced a different
superparamensis, a species of intermediate morphology and genome
Quaternary history from the south of the country, namely in having
size (Dušková et al., 2010; as “L. sp. 4” there) between S. superand-
volcanism and glaciation (Jørgensen & Ulloa, 1994). Glaciation events
inus, with which it was assigned to AFLP subgroup C4, and S. niva-
and volcanism may have structured the genetic patterns of the species
lis, with which it shares ITS sequences (Figure 6a). Such conflict may
through the effects of repeated bottlenecks and founder events (Luo
be explained by (past) gene flow of S. nivalis ITS haplotypes that was
et al., 2016; Vásquez, Balslev, Hansen, Sklenář, & Romoleroux, 2016).
followed by rapid homogenization of the ITS sequences (Alvarez
The genetic structure of species from the montane forest-páramo
& Wendel, 2003) toward S. nivalis-like paralogues. A similar process
ecotone (cluster A) and páramo (cluster C) showed little association
might have led to “deeper” incongruences in other species (S. nivalis
with environmental variables, which we acknowledge might be at least
and S. puracensis; Figure 6a) that also exhibit conflicts among the three
partly due to the lack of precision of extrapolated climatic variables
major AFLP clusters versus the two main ITS clades. Such indications
for high mountains (Hijmans et al., 2005; Kirchheimer et al., 2016).
of past hybridization events, however, should be interpreted with cau-
However, in cluster B, the high proportion of genetic variation as-
tion as AFLP data do not allow distinguishing admixture from incom-
sociated with environmental factors is consistent with the variety of
plete lineage sorting.
habitats occupied by its species. The ecological differentiation coupled with high AFLP and morphological diversification may suggest a
4.4 | Geographical and ecological correlates of
genetic variation
Geographical barriers along with ecological differentiation promote
relatively long divergence time and/or efficient isolation (Kolář et al.,
2016). The very small or entirely lacking association between environment and geography in clusters C and B, respectively, suggests that
two distinct and (largely) independent signals are involved. However,
species diversification in mountains (Kolář et al., 2016; Luo et al.,
the stronger association in cluster A suggests that migration along the
2016). As a geographical signal was comparably strong in the three
cordilleras was coupled with a shift in species ecology, such as the
main AFLP clusters, geography may structure genetic variation in a
entry of S. otophorus in the superpáramo in Colombia.
similar way in both the Andean montane forest and the páramo. In
support of this, the AFLP data reveal two strikingly similar cases of genetic separation corresponding to geography which are incongruent
AC KNOW LEDG M ENTS
with morphology-based species limits. Two páramo species, Senecio
We thank F. Ávila, L. Flašková, E. Gámez, J. Krejčíková, M. Lučanová,
lingulatus and S. superandinus (cluster C), are readily distinguished
P. Macek, R. Sanchez, D. Stančík, J. Suda, P. Ubiergo, O. Vargas, and E.
DUŠKOVÁ et al.
Záveská for help with the field and laboratory work and acknowledge
comments of three anonymous reviewers on an earlier draft of the
manuscript. Computational resources were provided by the CESNET
LM2015042 and the CERIT Scientific Cloud LM2015085, provided
under the programme “Projects of Large Research, Development,
and Innovations Infrastructures.” The research was supported by the
Czech Science Foundation (project 206/07/0273), and by Charles
University (project GAUK 261211).
CO NFLI CT OF I NTERE ST
None Declared.
AUT HOR CONTRI BUTI O NS
All co-authors participated in the field work and writing. Specifically,
E.D. did most of the molecular work. F.K. lead analyzes of molecular
data. P.S. conceived the ideas. D.V. participated in the molecular work.
T.F. did the character state reconstruction. K.R. arranged for research
permits in Ecuador. K.M. supervised the research.
Editor: Şerban Procheş
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SUP P O RT I NG I NFO R M AT I O N
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How to cite this article: Dušková E, Sklenář P, Kolář F, et al.
Growth form evolution and hybridization in Senecio
(Asteraceae) from the high equatorial Andes. Ecol Evol.
2017;7:6455–6468. https://doi.org/10.1002/ece3.3206