Phylogeny and circumscription of Dasyphyllum (Asteraceae: Barnadesioideae) based on molecular data with the recognition of a new genus, Archidasyphyllum

Abstract

Dasyphyllum Kunth is the most diverse genus of the South American subfamily Barnadesioideae (Asteraceae), comprising 33 species that occur in tropical Andes, Atlantic Forest, Caatinga, Cerrado, and Chaco. Based on distribution, variation in anther apical appendages, and leaf venation pattern, it has traditionally been divided into two subgenera, namely, Archidasyphyllum and Dasyphyllum. Further, based on involucre size and capitula arrangement, two sections have been recognized within subgenus Dasyphyllum: Macrocephala and Microcephala (=Dasyphyllum). Here, we report a phylogenetic analysis performed to test the monophyly of Dasyphyllum and its infrageneric classification based on molecular data from three non-coding regions (trnL-trnF, psbA-trnH, and ITS), using a broad taxonomic sampling of Dasyphyllum and representatives of all nine genera of Barnadesioideae. Moreover, we used a phylogenetic framework to investigate the evolution of the morphological characters traditionally used to recognize its infrageneric groups. Our results show that neither Dasyphyllum nor its infrageneric classification are currently monophyletic. Based on phylogenetic, morphological, and biogeographical evidence, we propose a new circumscription for Dasyphyllum, elevating subgenus Archidasyphyllum to generic rank and doing away with the infrageneric classification. Ancestral states reconstruction shows that the ancestor of Dasyphyllum probably had acrodromous leaf venation, bifid anther apical appendages, involucres up to 18 mm in length, and capitula arranged in synflorescence.

Full text

Phylogeny and circumscription of
Dasyphyllum (Asteraceae: Barnadesioideae)
based on molecular data with the
recognition of a new genus,
Archidasyphyllum
Paola de Lima Ferreira
1
, Mariana Machado Saavedra
2
and
Milton Groppo
1
1Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto,
Universidade de São Paulo, Ribeirão Preto, São Paulo, Brasil
2Departamento de Botânica, Instituto de Biologia, Universidade Federal do Rio de Janeiro, Rio de
Janeiro, Rio de Janeiro, Brazil
ABSTRACT
Dasyphyllum Kunth is the most diverse genus of the South American subfamily
Barnadesioideae (Asteraceae), comprising 33 species that occur in tropical Andes,
Atlantic Forest, Caatinga, Cerrado, and Chaco. Based on distribution, variation
in anther apical appendages, and leaf venation pattern, it has traditionally
been divided into two subgenera, namely, Archidasyphyllum and Dasyphyllum.
Further, based on involucre size and capitula arrangement, two sections have been
recognized within subgenus Dasyphyllum:Macrocephala and Microcephala
(=Dasyphyllum). Here, we report a phylogenetic analysis performed to test the
monophyly of Dasyphyllum and its infrageneric classification based on molecular
data from three non-coding regions (trnL-trnF, psbA-trnH, and ITS), using a broad
taxonomic sampling of Dasyphyllum and representatives of all nine genera of
Barnadesioideae. Moreover, we used a phylogenetic framework to investigate
the evolution of the morphological characters traditionally used to recognize its
infrageneric groups. Our results show that neither Dasyphyllum nor its infrageneric
classification are currently monophyletic. Based on phylogenetic, morphological,
and biogeographical evidence, we propose a new circumscription for Dasyphyllum,
elevating subgenus Archidasyphyllum to generic rank and doing away with the
infrageneric classification. Ancestral states reconstruction shows that the ancestor of
Dasyphyllum probably had acrodromous leaf venation, bifid anther apical
appendages, involucres up to 18 mm in length, and capitula arranged in
synflorescence.
Subjects Biodiversity, Evolutionary Studies, Molecular Biology, Plant Science, Taxonomy
Keywords Asterids, Compositae, Character Evolution, South America, Systematics, Taxonomy
INTRODUCTION
Systematics of Asteraceae (Composite) has undergone major change over the last four
decades, mainly due to the insights provided by molecular data. One of the pioneering
How to cite this article Ferreira PdL, Saavedra MM, Groppo M. 2019. Phylogeny and circumscription of Dasyphyllum (Asteraceae:
Barnadesioideae) based on molecular data with the recognition of a new genus, Archidasyphyllum.PeerJ 7:e6475 DOI 10.7717 /peerj.6475
Submitted 11 October 2018
Accepted 17 January 2019
Published 27 February 2019
Corresponding author
Paola de Lima Ferreira,
paolaferreira@usp.br
Academic editor
Richard Cowling
Additional Information and
Declarations can be found on
page 15
DOI 10.7717/peerj.6475
Copyright
2019 Ferreira et al.
Distributed under
Creative Commons CC-BY 4.0

molecular studies demonstrated an inversion of 22 kb in the chloroplast genome of
all Asteraceae, except for the members of subtribe Barnadesiinae, tribe Mutiseae (Jansen &
Palmer, 1987). Subsequent phylogenetic studies indicated that Barnadesiinae is the
sister group to the rest of the family (Bremer, 1987;Jansen et al., 1992); therefore, the
subtribe was elevated to the rank of subfamily as Barnadesioideae (Bremer & Jansen, 1992).
Barnadesioideae comprises nine genera and approximately 85 species, and is
restricted to South America (Bremer, 1987,1994;Jansen et al., 1992;Panero & Funk, 2002;
Funk et al., 2005,2009;Panero et al., 2014;Panero & Crozier, 2016;Saavedra et al., 2018).
Its members are characterized by the presence of axillary spines arranged at the
nodes, in pairs or in fascicles, and by the presence of unbranched three-celled hairs
called “barnadesioid trichomes”on the corollas, cypselae, and pappus (Cabrera, 1959;
Ezcurra, 1985;Bremer & Jansen, 1992;Bremer, 1994;Urtubey, 1999;Erbar & Leins, 2000;
Ulloa, Jørgensen & Dillon, 2002;Stuessy, Urtubey & Gruenstaeudl, 2009).
Dasyphyllum is the largest genus in Barnadesioideae, comprising 33 species
(Saavedra, 2011;Saavedra et al., 2018;Fig. 1) distributed from Venezuela to Northwestern
Argentina, but absent in the Amazon region (Cabrera, 1959;Saavedra, 2011;Saavedra,
Monge & Guimarães, 2014). The genus is morphologically diverse and can be
distinguished from the other genera of Barnadesioideae by including trees, shrubs,
and woody vines with pairs of straight, curved, or fasciculate spines, together with simple,
alternate leaves; monoecious or gynodioecious capitula, comprising discoid heads
with many types of corolla (Stuessy & Urtubey, 2006), and anthers with apical appendages
that are either bifid or undivided (Cabrera, 1959;Stuessy, Urtubey & Gruenstaeudl, 2009;
Saavedra, 2011).
Cabrera (1959) proposed the first infrageneric classification of Dasyphyllum,
recognizing 36 species in two subgenera distinguished by several morphological
characters and disjunct distributions. Subgenus Archidasyphyllum Cabrera comprised
two tree-species and was characterized by the presence of leaves with pinnate venation and
emarginate or obtuse anther apical appendages. Both species are restricted to the
Nothofagus forests of central Chile and Argentina. In contrast, subgenus Dasyphyllum
Cabrera comprised 34 tree or shrubs species, with acrodromous leaf venation and
bifid anther apical appendages, distributed from the Andes eastward into tropical
Argentina, Brazil, and Paraguay. Within subgenus Dasyphyllum, two sections are currently
recognized: section Microcephala Cabrera (23 species) and section Macrocephala
Cabrera (11 species). The two sections are distinguished by involucre size and capitula
arrangement with section Macrocephala having involucre longer than 20 mm in length
and arranged in a solitary or small group of heads (Figs. 1A and 1B) and section
Microcephala having heads arranged in synflorescence (corymbiform cymes) smaller than
18 mm in length (Figs. 1C–1F).
Nonetheless, the treatment by Cabrera (1959) often relied on a single and narrow
morphological concept to define the species. Due to the great morphological
variation, floristic studies undertaken in Brazil have shown that many characteristics
overlap; thus casting doubt on species delimitation (Roque & Pirani, 1997;
Saavedra et al., 2018).
Ferreira et al. (2019), PeerJ, DOI 10.7717/peerj.6475 2/19

In this context, Saavedra (2011) and Saavedra et al. (2018) updated the taxonomy
of Dasyphyllum, recognizing 33 species. Thirty of them were classified in
two sections using the same morphological definition for sections provided by
Cabrera (1959),thatis,Dasyphyllum Cabrera with 24 species, and Macrocephala
Baker ex Saavedra with six species; and the remaining three species
Figure 1 Photos of some Dasyphyllum species. (A) Dasyphyllum reticulatum (DC.) Cabrera.
(B) Dasyphyllum sprengelianum (Gardner) Cabrera. (C) Dasyphyllum brasiliense (Spreng.) Cabrera.
(D) Dasyphyllum leptacanthum (Gardner) Cabrera. (E) Dasyphyllum diamantinense Saavedra & M.Monge.
(F) Dasyphyllum flagellare (Casar.) Cabrera. Photo credits: Photographs by Cláudio N. Fraga, except A (by
Mariana M. Saavedra) and B (by Paola L. Ferreira). Full-size
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DOI: 10.7717/peerj.6475/fig-1
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(D. diacanthoides, D. excelsum belonging to D. subgenus Archidasyphyllum,and
D. hystrix) were placed as incertae sedis.
Several phylogenetic studies aiming to clarify the phylogenetic relationships
within Barnadesioideae have included species of Dasyphyllum (Bremer, 1994;Stuessy,
Sang & DeVore, 1996;Gustafsson et al., 2001;Urtubey & Stuessy, 2001;Gruenstaeudl et al.,
2009) but none of them representative of taxon sampling from each genus. Furthermore,
these phylogenetic results proposed conflicting hypotheses for the relationships
within the subfamily, especially regarding the monophyly of Dasyphyllum and its
infrageneric classification.
Therefore, the main purposes of this work were to: (1) infer the intergeneric
relationships of Dasyphyllum based on three molecular markers (plastid trnL-trnF and
psbA-trnH, and nuclear ITS) using a broad taxonomic sampling of Barnadesioideae;
(2) test the current circumscription of Dasyphyllum and its infrageneric classification
according to Saavedra (2011) and Saavedra et al. (2018), and update the taxonomy; and
(3) investigate the character evolution of Dasyphyllum.
MATERIALS AND METHODS
Taxon sampling
A total of 60 out of the 85 species of Barnadesioideae, representing all nine genera, were
sampled in this study. This included 27 of the 33 species (82%) from all sections of
Dasyphyllum (Saavedra, 2011;Saavedra et al., 2018), covering most of its morphological
diversity and geographical distribution. The six species missing in our analysis
were not included due to unsuccessful DNA extractions or because we could not
obtain voucher materials on loan for DNA extraction. A total of 61 accessions were
newly sequenced and deposited in GenBank (Table S1); additionally, 125 accessions were
obtained from previous studies (Gustafsson et al., 2001;Gruenstaeudl et al., 2009;
Katinas et al., 2008;Funk & Roque, 2011;Funk et al., 2014;Table S2). Two species of
Mutisia (Asteraceae: Mutisioideae) and one species of Calycera (Calyceraceae) were used
as outgroups. All phylogenetic trees were rooted against to Calyceraceae, the sister
family of Asteraceae (Barker et al., 2016;Panero & Crozier, 2016).
Molecular analysis
Total genomic DNA was extracted from three to five mg of silica-gel dried leaves using the
Qiagen DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) according to the instructions
by the manufacturer. We selected and amplified three regions previously used to infer
the phylogenetic relationships in Barnadesioideae: trnL-trnF using primers “c”and “f”
(Taberlet et al., 1991); psbA-trnH using primers “psbAF”and “trnHR”(Sang, Crawford &
Stuessy, 1997); and ITS using primers 18s F and 26s R (Gruenstaeudl et al., 2009).
PCR reaction mixtures and purification were carried out after as per Bruniera, Kallunki &
Groppo (2015). Thermal cycling for plastid amplification was performed using initial
denaturation at 94 C (8 min), followed by 30 cycles at 94 C (1 min), 54 C (1 min), 72 C,
(2 min), ending with an elongation at 72 C (3 min). Nuclear thermal cycling was
performed according to Barfuss et al. (2005), except for the annealing temperature of 62 C
Ferreira et al. (2019), PeerJ, DOI 10.7717/peerj.6475 4/19

(used in this study). Sequencing of the amplified DNA regions was performed at CREBIO
(Jaboticabal, São Paulo, Brazil) with the same primers used for PCR amplification.
Sequences were assembled and edited using the Biological Sequence Alignment Editor
(BioEdit), version 7.2.5 (Hall, 1999). We performed sequence alignments using
MAFFT version 7 (Katoh & Standley, 2013) with default parameters, followed by manual
adjustments with Mesquite version 3.51 (Maddison & Maddison, 2018). All data
matrices generated are included in Data S1.
Phylogenetic trees for each molecular region and the combined datasets were constructed
under parsimony (PA), maximum likelihood (ML), and Bayesianinference (BI). PA analyses
were performed in PAUPversion 4.0b10 (Swofford, 2002). Heuristics searches were
performed with 10,000 random addition sequence replicates holding 10 trees at each step,
tree-bisection-reconnection (TBR) branch swapping, with the “steepest descent”and
“multrees”options off. All characters were unordered and equally weighted. Bootstrapping
was implemented with 1,000 pseudoreplicates, 10,000 random taxon addition, and TBR
branch-swapping algorithm. Bootstrap (BP) support values in the following ranges were
considered strong (>88%), moderate (76–87%), weak (63–75%), and ambiguous (<63%)
following Bruniera, Kallunki & Groppo (2015).
Maximum likelihood and BI analyses were performed on the CIPRES Science Gateway
(Miller, Pfeiffer & Schwartz, 2010). The most appropriate model of sequence evolution
for each matrix was selected using the Akaike information criterion (Akaike, 1973)
in jModelTest version 2.1.9 (Posada, 2008;Darriba et al., 2012). Selected models were
GTR + I + G for ITS and GTR + G for both psbA-trnH and trnL-trnF.
Maximum likelihood analyses were performed using RaxML version 8 (Stamatakis,
2014) associated with a rapid BP analysis of 1,000 replicates under the GTRCAT model.
ML BP were interpreted as in the PA analyses.
Bayesian inference analyses were performed in MrBayes version 3.2.6 (Ronquist et al.,
2012) using two independent runs, each run with four simultaneous Markov chains
(three heated chains and one cold chain) started from random trees. Analyses were run for
20 million generations, and values were sampled every 1,000 generations. The stationarity
and convergence of runs, as the effective sample size 200 were ascertained using
Tracer version 1.6 (Rambaut et al., 2013). The first 25% of the sample trees were
discarded as burn-in and a 50% majority-rule consensus tree was calculated from the
remaining trees using the sumt option. Posterior probabilities (PP) above 0.95 were
considered as strong support.
The incongruence length difference test (ILD; Farris et al., 1995) was performed to
test the congruence between the plastid marker datasets (psbA-trnH and trnL-trnF)
and the combined marker datasets generated in this study (psbA-trnH, trnL-trnF, and
ITS). The ILD test was performed using PAUPversion 4.0b10 (Swofford, 2002) with
1,000 replicates and the same parameters used for PA searches.
Taxonomy
The electronic version of this article in portable document format will represent a
published work according to the international code of nomenclature for algae, fungi, and
Ferreira et al. (2019), PeerJ, DOI 10.7717/peerj.6475 5/19

plants (ICN), and hence the new names contained in the electronic version are effectively
published under that Code from the electronic edition alone. In addition, new names
contained in this work which have been issued with identifiers by IPNI will eventually be
made available to the global names index. The IPNI LSIDs can be resolved and the
associated information viewed through any standard web browser by appending the LSID
contained in this publication to the prefix“http://ipni.org/”. The online version of this
work is archived and available from the following digital repositories: PeerJ, PubMed
Central, and CLOCKSS.
Ancestral state reconstruction
In order to understand how the morphological features traditionally used to recognize the
infrageneric groups have evolved in Dasyphyllum, we reconstructed ancestral
character traits using the Bayesian majority-rule consensus tree based on the combined
datasets (trnL-trnF, psbA-trnH, and ITS) and further ultrametrized using the
chronopl function with default parameters in the R package “ape”(Paradis, Claude &
Strimmer, 2004). Ancestral state reconstructions were estimated from 1,000 iterations of
Bayesian stochastic character mapping (Bollback, 2006) using the function make.simmap
in the R package phytools (Revell, 2012). Coding of morphological characters was
extracted from the literature (Cabrera, 1959;Stuessy, Urtubey & Gruenstaeudl, 2009;
Funk & Roque, 2011;Saavedra, 2011;Saavedra, Monge & Guimarães, 2014;Saavedra et al.,
2018) and from examination of specimens from the following herbaria: ALCB, B,
BAF, BHCB, BM, BOTU, BR, CEN, CEPEC, CESJ, CONC, CVRD, EAC, ESA, GFJP,
GOET, GUYN, HB, HEPH, HPBR, HRCB, HST, HUEFS, HUFU, IBGE, ICN, IPA, JBP, K,
LP, M, MBM, MBML, MO, MOSS, NY, OUPR, P, PACA, PEUFR, QCA, R, RB, S, SI,
SP, SPF, SPFR, UB, UEC, UFG, UFMT, UFP, UFRN, UPCB, US, VIC (herbaria acronyms
follow Thiers, 2018). A list of morphological characters and their character state coding
used for the ancestral state reconstruction is detailed in Table 1.
Scanning electron microscopy was used to examine anther apical appendages
in two species of Dasyphyllum. Dried florets were rehydrated with hot water and
stored in 70% ethanol; then, anthers were critically point dried, sputter coated with gold
and analyzed using an EVO 50 scanning electron microscope (Carl Zeiss,
Cambridge, UK).
RESULTS
Phylogenetic analyses
The ILD test did not indicate incongruences between the plastid and combined datasets
(P> 0.05), thus allowing both to be used for further phylogenetic analyses.
Moreover, based on the results of BP and PP (>80), we did not find any evidence of
significant incongruence among the relationships that differed between the trees
(Fig. 2;Figs. S1–S4). Therefore, we decided to discuss our results based on the combined
analysis of the three regions as it includes the largest number of taxa (Fig. 2).
Our combined alignment consisted of 2,414 bp (trnl-trnF = 912 bp; psbA-trnH = 537;
ITS = 965 bp) for 63 taxa (see summary statistics for each dataset in Table 2).
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In all phylogenetic hypotheses, Dasyphyllum was found to be non-monophyletic due
to the highly supported position of D. diacanthoides and D. excelsum (formely
subgenus Archidasyphyllum) as sister clade to Fulcaldea and Arnaldoa (Fig. 2, Node 1,
PA BP 99%, ML BP 100%, PP 1).
Dasyphyllum sensu stricto,defined here by excluding D. diacanthoides and D. excelsum,
was recovered as monophyletic with moderate or strong support (Fig. 2; Node 2;
Table 1 Diagnostic feature coding used to infer the Bayesian stochastic character mapping analyses.
Taxon Leaf
venation
Anther apical
appendage
Involucre
size
Capitula
arrangement
Arnaldoa macbrideana 00 0 0
Arnaldoa weberbaueri 00 0 0
Dasyphyllum argenteum 01 1 1
Dasyphyllum armatum 01 1 1
Dasyphyllum brasiliense 01 1 1
Dasyphyllum brevispinum 01 1 1
Dasyphyllum colombianum 01 1 1
Dasyphyllum diacanthoides 12 1 0
Dasyphyllum diamantinense 01 1 1
Dasyphyllum donianum 01 0 0
Dasyphyllum excelsum 12 1 1
Dasyphyllum ferox 01 1 1
Dasyphyllum flagellare 01 1 1
Dasyphyllum floribundum 01 1 1
Dasyphyllum fodinarum 01 0 0
Dasyphyllum hystrix 01 1 0
Dasyphyllum inerme 01 1 1
Dasyphyllum lanceolatum 01 1 1
Dasyphyllum leptacanthum 01 1 0
Dasyphyllum popayanense 01 1 1
Dasyphyllum reticulatum 01 0 0
Dasyphyllum spinescens 01 1 1
Dasyphyllum sprengelianum 01 0 0
Dasyphyllum trichophyllum 01 0 0
Dasyphyllum vagans 01 1 1
Dasyphyllum sp. nov. (1) 0 1 0 0
Dasyphyllum sp. nov. (2) 0 1 1 1
Dasyphyllum sp. nov. (3) 0 1 1 1
Dasyphyllum sp. nov. (4) 0 1 1 1
Fulcaldea laurifolia 00 1 1
Fulcaldea stuessy 00 1 1
Note:
Leaf venation: (0) Acrodomous, (1) Pinnate. Anther apical appendage: (0) Acute, (1) Bifid, (2) Obtuse. Involucre size:
(0) 20 mm, (1) 18 mm. Capitula arrangement: (0) Solitary or few capitula (1) Capitula arranged in synflorescences
(corymbiform cymes).
Ferreira et al. (2019), PeerJ, DOI 10.7717/peerj.6475 7/19

PA BP 76%, ML BP 98%, PP 1). However, at the intrageneric level, both currently-accepted
sections (Dasyphyllum and Macrocephala) were found to be non-monophyletic.
Members of Dasyphyllum sensu stricto are divided into four main lineages: (1) lineage “A”
is composed only of D. hystrix and is sister to the rest of the genus (PA BP 76%, ML BP
98%, PP 1); (2) lineage “B”comprises seven species classified in section. Dasyphyllum
of Saavedra (2011) and is only supported in the Bayesian analysis (PP 0.97); (3) lineage “C”
is composed of 11 species, including approximately 46% of the species currently classified
in sect. Dasyphyllum of Saavedra (2011), with no strong support in any analysis;
(4) lineage “D”is composed of five of the six species positioned in sect. Macrocephala of
Saavedra et al. (2018), plus one undescribed Brazilian species (Dasyphyllum sp. nov. 1)
Figure 2 Phylogenetic relationships of Dasyphyllum based on combined datasets inferred from
Bayesian inference. Support values are indicated above the branches in the order of parsimony, max-
imum likelihood, and Bayesian analyses. Support values less than 63% are indicated by a dash (–). Capital
letters on internal clades of Dasyphyllum are discussed in the article.
Full-size
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previously positioned in sect. Dasyphyllum of Saavedra (2011), and it is only strongly
supported in the Bayesian analysis (PP 0.97).
The phylogenetic analyses of individual (Figs. S1 and S2) and combined (Fig. S3) plastid
marker datasets do not have good resolutions or supports and do not clarify the
relationships of Dasyphyllum sensu stricto and the rest of the subfamily. On the other hand,
in the ITS (Fig. S4) and combined phylogenies (Fig. 2), Dasyphyllum is placed as
sister to the clade comprising Arnaldoa,Fulcaldea, D. diacanthoides, and D. excelsum
((PA BP 98%, ML BP 100%, PP1) support values for ITS; PA BP 99%, ML BP 100%, PP 1
support values for combined).
Ancestral state reconstruction analyses
Bayesian stochastic character mapping demonstrated that the ancestral condition in
Dasyphyllum sensu stricto is acrodromous leaf venation (PP = 0.99; Fig. 3A), bifid anther
apical appendages (PP = 0.96; Fig. 3B), and small involucres (PP = 0.99; Fig. 3C) with
capitula arranged into an synflorescence (PP = 0.66; Fig. 3D). Pinnate venation (Fig. 3A)
and obtuse anther apical appendages (Fig. 3B) evolved in the ancestor of the clade
comprising D. diacanthoides and D. excelsum (PP 0.95 and PP 0.82, respectively). The
larger involucre larger (20 mm) is inferred to have evolved twice, since it appears in the
ancestor of lineage “D”(PP 0.98), and in the Arnaldoa clade (PP 0.95). Regarding capitula
arrangement, solitary, or arranged in few inflorescences (2–4) is a derived state and
appears at least five times over the evolutionary history of the group.
DISCUSSION
Previous molecular phylogenetic hypotheses aimed to clarify the intergeneric relationships
within Barnadesioideae, but they only included a limited taxonomic sampling from
each genus (Gustafsson et al., 2001;Gruenstaeudl et al., 2009). Our combined phylogeny
greatly improves the taxonomic coverage by including almost 82% of the species
recognized as belonging to Dasyphyllum. The results obtained here allowed us to review
the generic taxonomy and to discuss the morphological features used to recognize the
infrageneric groups within this genus.
Table 2 Summary statistics of the datasets used in this study.
trnL-trnF psbA-trnH ITS Plastid
dataset
Combined
dataset
Number of taxa included 53 49 60 53 63
Aligned length (BP) 912 537 965 1,449 2,414
Number of constant characters (%) 807 (88.49) 386 (71.88) 499 (51.71) 1,139 (78.61) 1,692 (70.09)
Number of variable characters (%) 105 (11.51) 151 (28.12) 466 (48.29) 310 (21.39) 722 (29.91)
Number of parsimony informative characters (%) 53 (5.81) 61 (11.36) 346 (35.85) 114 (7.87) 460 (19.06)
Tree length of best parsimony tree (steps) 120 222 1,375 348 1,743
Number of most parsimonious trees 20.251 3.120 309 11.337 3,475
Consistency index (CI) 0.9083 0.8018 0.4611 0.1753 0.4102
Retention index (RI) 0.9722 0.8739 0.4412 0.9181 0.8314
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Re-circumscription of Dasyphyllum
All phylogenetic analyses show that, as traditionally circumscribed, Dasyphyllum is
non-monophyletic due to the well-supported placement of D. diacanthoides and
D. excelsum, which belong to Dasyphyllum subg. Archidasyphyllum, sensu Cabrera (1959),
in a clade sister to Arnaldoa and Fulcaldea (Fig. 2;Figs. S1–S4), a finding that
confirms previous studies based on molecular data (Gustafsson et al., 2001;Gruenstaeudl
et al., 2009;Funk & Roque, 2011;Padin, Calviño & Ezcurra, 2015). Despite their
shared Andean distribution, the clade comprising Arnaldoa,Fulcaldea,D. diacanthoides,
and D. excelsum is morphologically diverse and well-defined into distinct genera:
Fulcaldea comprises two species of shrubs or small trees found in southern Ecuador,
northern Peru, and Brazil; the species of this genus are distinguished by having
single-flowered capitula, a style with subapical swelling, and villose pappus with red
or pink bristles (Gustafsson et al., 2001;Stuessy, Urtubey & Gruenstaeudl, 2009;Funk &
Roque, 2011). On the other hand, Arnaldoa comprises three shrubs species distributed
in Ecuador and northern Peru; they are distinguished by their large and solitary
Figure 3 History of the morphological characters traditionally used to circumscribe infrageneric
groups of Dasyphyllum.(A) Leaf venation. (B) Anther apical appendage. (C) Involucre size. (D) Capi-
tula arrangement. Squares to the right and left of the phylogeny are color-coded according to each
character state. Pie charts at nodes represent posterior probabilities of ancestral states using Bayesian
inference. Full-size
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capitula with sub-bilabiate, white, orange, or purple corollas (Stuessy & Sagástegui, 1993;
Ulloa, Jørgensen & Dillon, 2002). In contrast, D. diacanthoides and D. excelsum are
restricted to the relict Nothofagus forests of central Chile and adjacent areas of Argentina
(Cabrera, 1959;Gustafsson et al., 2001;Gruenstaeudl et al., 2009;Stuessy, Urtubey &
Gruenstaeudl, 2009) and are easily distinguished from Fulcaldea and Arnaldoa because
D. diacanthoides and D. excelsum are tall trees (up to 30 m) with leaves showing pinnate
venation (Figs. 3A,4A and 4B), solitary or spiciform (Fig. 3D), gynodioecious or
monoecious capitula with more than one flower, and emarginated or obtuse anther apical
Figure 4 Diaphanized leaves showing the differences in venation. (A and B) show the pinnate
venation of Dasyphyllum subgenus Archidasyphyllum. (C and D) show the acrodomous venation of
Dasyphyllum sensu stricto. Photos: (A) Dasyphyllum excelsum. (B) Dasyphyllum diacanthoides.
(C) Dasyphyllum argenteum. (D) Dasyphyllum brasiliense. All photographs were extracted from Saavedra
(2011).Full-size
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DOI: 10.7717/peerj.6475/fig-4
Ferreira et al. (2019), PeerJ, DOI 10.7717/peerj.6475 11/19

appendages (Figs. 3B and 5A;Cabrera, 1959;Saavedra, 2011). Due to the great
morphological diversity, classifying Arnaldoa,Fulcaldea, and Dasyphyllum subg.
Archidasyphyllum together in one single unit would result in several undesirable
taxonomic changes and create a drastically broader genus concept with no obvious
morphological support.
Instead, we propose a new circumscription of Dasyphyllum by elevating subg.
Archidasyphyllum to the generic rank, Archidasyphyllum. This proposal is phylogenetically
well-supported and consistent with leaf venation pattern (Fig. 4), anther apical appendage
shape (Fig. 5), and distributional data (Stuessy, Sang & DeVore, 1996;Gruenstaeudl
et al., 2009;Saavedra, 2011). New combinations and a key for this genus, as well as other
commentaries about the distribution and phenology of the species, are presented at the
end of the manuscript.
Dasyphyllum sensu stricto—intergeneric relationships and
infrageneric classification
The phylogenetic relationships of Dasyphyllum with genera in Barnadesioideae remains
unresolved. Our phylogenetic hypotheses are consistent with the placement of Dasyphyllum
as a sister clade to the clade comprising Arnaldoa,Fulcaldea,andArchidasyphyllum (Fig. 2;
Fig. S4). This relationship was also supported by previous molecular phylogenetic
analyses (Gustafsson et al., 2001;Gruenstaeudl et al., 2009;Funk & Roque, 2011).
As stated in the introduction, Dasyphyllum sensu stricto (D. subgenus Dasyphyllum,
sensu Cabrera, 1959) has been traditionally divided into two sections based on involucre
size and capitula arrangement. Our results indicated that neither section is monophyletic
(Fig. 2). Section Macrocephala comprises six species found in adjacent areas of Bolivia
Figure 5 Scanning electron microscopy images showing the differences in anther apical appendages.
(A) apical appendages obtuse of Dasyphyllum diacanthoides (Dasyphyllum subgenus Archidasyphyllum).
(B) apical appendages bifidofDasyphyllum trichophyllum (Baker) Cabrera (Dasyphyllum sensu stricto).
Full-size
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DOI: 10.7717/peerj.6475/fig-5
Ferreira et al. (2019), PeerJ, DOI 10.7717/peerj.6475 12/19

and Paraguay (Saavedra et al., 2018) that share the presence of few large capitula, solitary
or in small groups of heads (Figs. 1A and 1B), and it can be recognized as a monophyletic
group by inclusion of Dasyphyllum. sp. nov. (1). Although these morphological features
have evolved more than once over evolutionary history (Figs. 3C and 3D), they are useful
to define this clade. Moreover, our Bayesian stochastic mapping analyses showed that
the character states previously used to define section Dasyphyllum (involucre up to 18 mm
in length and capitula arranged in synflorescences; Figs. 3C and 3D) are plesiomorphic,
and therefore cannot be used to delimitate infrageneric groups as previously
proposed by Cabrera (1959) and Saavedra (2011).
Based on our taxonomic sampling, species of Dasyphyllum sensu stricto fall into four
heterogeneous and poorly supported lineages (Fig. 2; lineages A–D). Therefore, the results
of this work do not corroborate the subdivision of Dasyphyllum into sections and they
should be abandoned.
Taxonomic treatment
Archidasyphyllum (Cabrera) P.L.Ferreira, Saavedra & Groppo, stat. nov. hDasyphyllum
subgenus Archidasyphyllum Cabrera, Revista Mus. de La Plata,Secc. Bot., 9(38):
44. 1959. Type: Archidasyphyllum diacanthoides (Less.) P.L.Ferreira, Saavedra & Groppo.
Etymology. Archi (Greek) = First, Primitive; Dasyphyllum = genus that belongs to
Barnadesioideae. Cabrera (1959) suggested that Dasyphyllum subgenus Archidasyphyllum
is the earliest diverging group of the subfamily Barnadesioideae.
Key to species of Archidasyphyllum
1. Capitula solitary on the branches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. diacanthoides
1. Capitula arranged in spiciform synflorescences . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. excelsum
New combinations:
Archidasyphyllum diacanthoides (Less.) P.L.Ferreira, Saavedra & Groppo comb. nov. h
Flotovia diacanthoides Less, Syn. Gen. Compos.: 95. 1832. hPiptocarpha diacanthoides
(Less.) Hook. & Arn., Comp. Bot. Mag. 1: 110. 1835. hDasyphyllum diacanthoides
(Less.) Cabrera, Revista Mus. La Plata, Secc. Bot., 9(38): 44. 1959. - Type: Chile, Antuco,
E.F. Poeppig [Coll. pl. Chil. III, Syn. pl. Amer. austr. msc., Diar. 793], XII.1828 (Lectotypus
hic designatus: P! [P00703408]; Isolectotypi:B†[photo F! [F0BN015834]], BM!
[BM001010220], BR! [BR541864], M! [M-0030607], NY! [00169364, 00169365]).
Distribution and Habitat—Archidasyphyllum diacanthoides is distributed in southern
Chile and adjacent areas of Argentina between 38and 43S. This species is found in
forested areas ranging from 400 to 1,200 m in elevation.
Phenology—Flowering from November to April.
Note—Flotovia diacanthoides was described by Lessing (1832) based on the material
“Chuquiraga leucoxilon Pöpp. mss. n. 793”(nomen nudum) collected by Poeppig.
According to Stafleu (1969), the plants collected by Poeppig in Chile were distributed by
Kunze under the designation “Coll. pl. Chi.”. Although all the type materials assigned to
Flotovia diacanthoides are indicated with the phrase “Coll. pl. Chl.”, we designated the
Ferreira et al. (2019), PeerJ, DOI 10.7717/peerj.6475 13/19

sheet deposited at P herbarium as the lectotype because it is the only material which also
bears a handwritten label “N. 793 Chuquiraga leucoxilon”.
Archidasyphyllum excelsum (D. Don) P.L.Ferreira, Saavedra & Groppo comb. nov. h
Chuquiraga excelsa D. Don, Phil. Mag. 11: 392. 1832. hPiptocarpha excelsa (D. Don)
Hook. & Arn., Comp. Bot. Mag. 1:110. 1835. hDasyphyllum excelsum (D. Don)
Cabrera, Revista Mus. La Plata, Secc. Bot., 9(38): 46. 1959. Typus: Chile, Valparaiso,
H. Cuming 328, 1832 (Lectotypus hic designatus: K! [K000527920]; Isolectotypi:
BM! [000522369], FI [107436 [image!]], GH [00006351 [image!]], P! [P00703407]).
Distribution and Habitat—Archidasyphyllum excelsum is endemic to central Chile between
32and 34S. This species is found in forested areas ranging from 350 to 900 m in elevation.
Phenology—Flowering from November to April.
Note—According to Stafleu & Cowan (1976–1998), the herbarium of David Don was
donated to the Linnean Society of London and should be conserved at the LINN
herbarium. However, we have been unable to trace this material and we designated the
lectotype in the K herbarium due to the specimen being well-represented in its
reproductive and vegetative forms, besides the high preservation of the material.
CONCLUSIONS
This study comprises the most extensive molecular sampling for Dasyphyllum to date
and provides a sound foundation for the re-circumscription of the genus. In so
doing, it also sheds new light on the evolution of morphological features. Our phylogenetic
analysis demonstrated that as currently circumscribed, Dasyphyllum is not
monophyletic, because of D. diacanthoides and D. excelsum (Dasyphyllum subgenus
Archidasyphyllum) being placed outside the genus, as sister to a clade comprising
Arnaldoa and Fulcaldea. A well-supported phylogeny coupled with morphological and
biogeographical data corroborate our taxonomic decision to elevate Dasyphyllum
subgenus Archidasyphyllum to generic status as Archidasyphyllum. In addition,
both sections of D. sensu stricto were also rejected. However, we prefer not to propose a
new infrageneric classification until new data with unequivocal synapomorphies for the
internal clades are available. Moreover, phylogenetic relationships between Dasyphyllum
and other genera of Barnadesioideae remain to some extent unresolved. We suggest
that future studies including additional characters from phylogenomics might better clarify
the relationships of the internal clades in Dasyphyllum, as well as the relationships within
the whole subfamily Barnadesioideae.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge all herbarium curators for their assistance during visits
and for the loan of materials. Our gratitude to André Simões and Benoit Loeuille for
suggestions on earlier versions of the manuscript. Special thanks to Cíntia Silva-Luz and
Marcelo Monge Egea for the collecting work in the Andean region, Carla Poleselli
Bruniera and Fernando Farache for their support with phylogenetic analyses, Jefferson
Prado for his suggestions regarding botanical nomenclature.
Ferreira et al. (2019), PeerJ, DOI 10.7717/peerj.6475 14/19

ADDITIONAL INFORMATION AND DECLARATIONS
Funding
This study was funded by the Fundação de Amparo à Pesquisa no Estado de São Paulo
(FAPESP, grants 2011/10446-0, 2015/09458-6 and 2016/06260-2). Paola Ferreira
was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES, Finance Code 001). Milton Groppo was funded by the Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq, grants 309994/2012-8 and
309088/2016-0). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Grant Disclosures
The following grant information was disclosed by the authors:
Fundação de Amparo à Pesquisa no Estado de São Paulo: 2011/10446-0, 2015/09458-6 and
2016/06260-2.
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes, Finance Code 001).
Conselho Nacional de Desenvolvimento Científico e Tecnológico: 309994/2012-8 and
309088/2016-0.
Competing Interests
The authors declare that they have no competing interests.
Author Contributions
Paola de Lima Ferreira conceived and designed the experiments, performed the
experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared
figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.
Mariana Machado Saavedra conceived and designed the experiments, analyzed the
data, contributed reagents/materials/analysis tools, authored or reviewed drafts of the
paper, approved the final draft.
Milton Groppo conceived and designed the experiments, analyzed the data, contributed
reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved
the final draft.
Data Availability
The following information was supplied regarding data availability:
The GenBank accession numbers are provided in Table S1 and Table S2. Molecular
matrices are provided in the Supplemental Data S1.
New Species Registration
The following information was supplied regarding the registration of a newly
described species:
Archidasyphyllum (Cabrera) P.L. Ferreira, Saavedra & Groppo LSID: 77194153-1.
Archidasyphyllum diacanthoides (Less.) P.L. Ferreira, Saavedra & Groppo LSID: 77194155-1.
Archidasyphyllum excelsum (D. Don) P.L. Ferreira, Saavedra & Groppo LSID: 77194156-1.
Ferreira et al. (2019), PeerJ, DOI 10.7717/peerj.6475 15/19

Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
peerj.6475#supplemental-information.
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