Botanical Journal of the Linnean Society, 2020, XX, 1–16. With 3 figures.

Phylogenetic relationships based on nuclear and

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plastid DNA sequences reveal recent diversification and
discordant patterns of morphological evolution of the
Chilean genera of Gilliesieae (Amaryllidaceae: Allioideae)
INELIA  ESCOBAR1, EDUARDO RUIZ-PONCE1,*, PAULA J. RUDALL2, ,
MICHAEL F. FAY2,3, OSCAR TORO-NÚÑEZ1, HEIDY M. VILLALOBOS-BARRANTES4,5 and
CARLOS M. BAEZA1
1
Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas, Universidad de
Concepción, Casilla 160-C, Concepción, Chile
2
Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK
3
School of Plant Biology, University of Western Australia, Crawley, WA 6009, Australia
4
Centro de Investigación en Biología Celular y Molecular, Universidad de Costa Rica, San José, Costa
Rica
5
Escuela de Química, Universidad de Costa Rica, San José, Costa Rica

Received 3 July 2019; revised 16 December 2019; accepted for publication 6 May 2020

Gilliesieae are a South American tribe of Amaryllidaceae characterized by high floral diversity. Given different
taxonomic interpretations and proposals for generic and specific relationships, a representative phylogenetic analysis
is required to clarify the systematics of this group. The present study provides a framework for understanding
phylogenetic relationships and contributing to the development of an appropriate taxonomic treatment of Gilliesieae.
Molecular analyses, based on nuclear (ITS) and plastid DNA sequences (trnL-F and rbcL), resolve with strong
support the monophyly of the tribe and the differentiation of two major clades. Clade I comprises the genera Gilliesia,
Gethyum and Solaria and Clade II includes Miersia and Speea. These well-supported clades are mostly congruent with
vegetative and karyotype characters rather than, e.g., floral symmetry. At the generic level, all molecular analyses
reveal the paraphyly of Gilliesia and Miersia. Gethyum was found to be paraphyletic, resulting in the confirmation
of Ancrumia as a distinct genus. Several instances of incongruent phylogenetic signals were found among data sets.
The calibrated tree suggests a recent diversification of the tribe (Pliocene–Pleistocene), a contemporary process of
speciation in which instances of hybridization and incomplete lineage sorting could explain patterns of paraphyly
and incongruence of floral morphology.

ADDITIONAL KEYWORDS:  Ancrumia – Chilean hotspot – Gethyum – Gilliesia – phylogeny – paraphyly –

Solaria – Speea.

INTRODUCTION and Nectaroscordum Lindl.); (2) Tulbaghieae Endl.

ex Meisn. with the sole genus Tulbaghia L. and (3)
Amaryllidaceae consist of three subfamilies:
Gilliesieae Baker with 13 genera, Ancrumia Harv.
Amaryllidoideae, Agapanthoideae and Allioideae
ex Baker, Erinna Phil., Gethyum Phil., Gilliesia
(Chase, Reveal & Fay, 2009). According to these
Lindl. Ipheion Raf., Leucocoryne Lindl., Miersia
authors, Allioideae consist of three tribes: (1) Allieae
Lindl., Nothoscordum Kunth, Schickendantziella
Dumort., including only the type genus Allium
(Speg.) Speg., Solaria Phil., Speea Loes., Trichlora
L. s.l. (including Caloscordum Herb., Milula Prain
Baker and Tristagma Poepp. (Chase et al., 2009).
In Gilliesieae, phylogenetic studies have revealed
two well-supported lineages (Fay & Chase, 1996;
*Corresponding author. E-mail: eruiz@udec.cl

© 2020 The Linnean Society of London, Botanical Journal of the Linnean Society, 2020, XX, 1–16 1
2  I. ESCOBAR ET AL.
Fay, Rudall & Chase, 2006), which have been subject
to different classification proposals, reflecting diverse
opinions about the taxonomy of this group. As tribes,
Gilliesieae and ‘Ipheieae’ (informally described), with
zygomorphic and actinomorphic flowers, respectively,
have been placed in subfamily Gilliesioideae (Fay &
Chase, 1996; Rudall et al., 2002; Fay et al., 2006). Using
a wider circumscription, species from both lineages
have been also considered as a single tribe, Gilliesieae,
belonging to Allioideae (Amaryllidaceae; Chase et al.,
2009). Subsequently, Sassone, Arroyo-Leuenberger
& Giussani (2014) offered a new circumscription of
tribe Leucocoryneae, recognizing Beauverdia Herter
(a genus resurrected for Amaryllidaceae by Sassone
et al., 2014), Ipheion, Leucocoryne, Nothoscordum, the
recently revalidated Latace Phil. (as Zoellnerallium
Crosa; Sassone, Belgrano & Guaglianone, 2015)
and Tristagma from Gilliesieae, thus recognizing
four tribes in Allioideae: Allieae, Tulbaghieae,
Gilliesieae and Leucocoryneae. Different phylogenetic
studies have retrieved Leucocoryneae as a strongly
supported monophyletic group (Souza et al., 2016;
Sassone & Giussani, 2018). In this way, Gilliesieae
remain, comprising Ancrumia, Gethyum, Gilliesia,
Miersia, Solaria and Speea, mainly distributed in the
Mediterranean zone of Chile, plus Schickendantziella
from Argentina and Bolivia and Trichlora from
Peru. However, a recent phylogenetic study has
considered Gilliesieae and Leucocoryneae as subtribes
(Gilliesiinae and Leucocoryninae, respectively),
belonging to Gilliesieae (Pellicer et al., 2017).
In contrast to Leucocoryneae, which have been
extensively studied from aspects of systematics and
taxonomy, including morphology (Sassone, Giussani
& Guaglianone, 2013, 2014; Sassone et al., 2014),
karyology (Crosa 1972, 1975, 1981, 1988; Souza et al.,
2009, 2016; Souza, Crosa, & Guerra, 2010), cytogenetics
(Sassone et al., 2018) and molecular phylogenetics
(Fay & Chase 1996; Fay et al., 2006, Souza et al.,
2016; Pellicer et al., 2017; Sassone & Giussiani, 2018),
dedicated studies on Gilliesieae are comparatively few
(e.g. Ravenna, 2000b; Rudall et al., 2002, Escobar, 2012).
This relative paucity of publications can be explained
by the difficulty of obtaining appropriate material
with which to conduct thorough systematic studies.
Few specimens are available in herbarium collections
(Escobar, unpubl. data), and local populations are
difficult to locate due to the short flowering periods
(a few weeks at the end of winter; Hoffman, 1989;
Zöellner & Arriagada, 1998) and their rare presence
in their known localities. As a result, most of the key
elements employed in the clarification of the taxonomy
of Gilliesieae remain elusive and confusing, given that
extant taxonomic treatments are based mainly on
floral characters of difficult interpretation (Escobar,
© 2020 The Linnean Society of London, Botanical Journal of the Linnean Society, 2020, XX, 1–16
2012). Different numbers of genera (six to nine) and
species (six to 24) have been historically recognized
in Gilliesieae (Engler 1887; Baker 1879; Reiche 1893;
Krause 1930; Hutchinson 1939; Traub 1976, 1982;
Ravenna 1978, 2000a, b, c, d, 2005a, b; Marticorena &
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Quezada 1985; Rahn 1998).
Several phylogenetic studies have indirectly covered
aspects of the systematics of Gilliesieae, particularly
supporting the monophyletic status relative to
other tribes of Allioideae (Fay & Chase, 1996, Fay
et al., 2006, Souza et al., 2016, Pellicer et al., 2017).
Nonetheless, studies with a more complete and
comprehensive sampling remain necessary to evaluate
infratribal relationships, an important step towards
the formulation of a more robust taxonomic treatment
of genera and species. To date, only cytological studies
have been conducted with a comprehensive sampling
of Gilliesieae (Escobar, Ruiz, & Baeza, 2012), without
considering a phylogenetic framework for taxonomic
evaluation. In this case, three different karyotypes
were revealed: one present in Gethyum, Gilliesia
and Solaria with 2n  = 14 (four metacentric, four
submetacentric and six acrocentric chromosomes);
one present in Miersia and Speea with 2n = 12 (ten
metacentric and two acrocentric chromosomes) and a
unique karyotype present in Miersia chilensis Lindl.
with 2n  = 20 (two metacentric and 18 acrocentric
chromosomes) (Escobar et al., 2012).
Given the need for a more robust taxonomy of
Gilliesieae, a notable native group in the Chilean flora
and the Chilean Valdivian Rainforest biodiversity
hotspot (Myers et al., 2000), we undertook a phylogenetic
study aimed at: (1) providing a phylogenetic hypothesis
for infratribal relationships; (2) eliciting possible
patterns of diversification and (3) clarifying the
phylogenetic congruence from potentially informative
diagnostic characters. Phylogenetic analyses were
conducted using a combination of nuclear (ITS) and
plastid (trnL-F and rbcL) DNA sequences, with the
subsequent evaluation of proposed morphological and
cytological data of taxonomic value.
MATERIAL AND METHODS
Sampling
Following Chase et al. (2009) and the recent tribal
classification of Sassone et al. (2014), 16 taxa were
sampled; 13 corresponded to previously recognized
species in the group (Ravenna, 2000b, c, d, 2005a, b)
and three are of unknown circumscription (Gilliesia
sp. 35, 41, 129, Gilliesia sp. 146, Miersia sp. 84).
Material was collected from 54 localities in the
Mediterranean zone of central Chile (see the studied
material in Supporting Information, Supplementary

Data 1). Outgroup taxa were chosen given their sister
clade status to Gilliesieae [Tristagma bivalve (Hook. ex
Lindl.) Traub, T. uniflorum (Lindl.) Traub, Beauverdia
hirtella (Kunth) Herter, Nothoscordum gracile (Aiton)
Stearn and Leucocoryne pauciflora Phil.; Fay et al.,
2006; Souza et al., 2016; Sassone & Giussani, 2018];
one species of Tulbaghieae (Tulbaghia capensis L.; Fay
& Chase, 1996; Meerow et al., 1999; Fay et al., 2006;
Souza et al., 2016) was included as a final outgroup.
Outgroup sequences were obtained directly from
herbarium specimens and/or previously published
data in Allioideae (Fay & Chase, 1996, Meerow et al.,
1999). All sequences were deposited in GenBank
(see accession numbers in Supporting Information,
Supplementary Data 2).
Molecular data
Genomic DNA was extracted from leaves desiccated
in silica gel, following a modified 2× CTAB protocol
(Doyle & Doyle, 1987). Due to the old age of available
herbarium specimens, it was not possible to extract
DNA from Schickendantziella and Trichlora. For
phylogenetic analysis, the ITS region and the
plastid rbcL and trnL-F were sequenced. The
plastid DNA sequences were selected based on
their demonstrated phylogenetic utility in previous
studies of Amaryllidaceae and closely related
monocotyledons (Fay & Chase, 1996; Meerow et al.,
1999; Fay et al., 2006; Kwembeya et al., 2007; Devey
et al., 2008; Peterson, Levichev, & Peterson, 2008;
Zarrei et al., 2009; Snijman & Meerow, 2010). Target
DNA regions were amplified using the primer
combinations 1F, 724R, 636F and 1352 (rbcL; Chase
et al., 1995; Meerow et al., 1999), c and f (trnL-F;
Taberlet et al., 1991) and ITS4 and ITS5 (ITS; White
et al., 1990).
Amplification and sequencing reactions of plastid
regions were conducted using a combination of 12.5 µl
GoTaq Green Master Mix, 2.5 mM MgCl2 (Promega),
0.5 µl 0.4% BSA (bovine serum albumin), 0.5 µl of each
primer (10 µM), 1–2 µl template DNA and deionized
water to a total reaction volume of 25 µl. PCR reactions
with ITS regions used the same reagents and volumes,
but adding 0.5 μl DMSO (4%) instead of BSA. The
PCR program for ITS comprised an initial stage of
denaturation at 94 °C for 4 min, followed for 28 cycles
of (94 °C for 1 min, 48 °C for 1 min, 72 °C for 1 min) and
a final extension of 72 °C for 7 min. PCR reactions for
plastid regions followed the same protocol, but with 30
cycles and an annealing temperature of 50 °C for 30 s
in each cycle. Sequencing was performed at the Jodrell
Laboratory (Royal Botanic Gardens, Kew, UK) and by
the Molecular Biology Laboratory at the Pontificia
Universidad Católica (Santiago, Chile). Sequences
were assembled in Geneious v.11.0.5 (http://www.
© 2020 The Linnean Society of London, Botanical Journal of the Linnean Society, 2020, XX, 1–16
PHYLOGENETICS OF TRIBE GILLIESIEAE  3
geneious.com, Kearse et al., 2012) and aligned with
MAFFT v.1.3.7 (Katoh & Standley, 2013). Data sets
were visually corrected to align sequences with their
initial ORF (for rbcL data sets) and minimize apparent
character state changes.
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Phylogenetic analyses
Phylogenetic analyses were conducted on plastid
(rbcL + trnL-F), and nuclear ribosomal genomes (ITS)
independently. In both cases, maximum parsimony
(MP) and Bayesian inference (BI) were employed
to compare results among different criteria of
phylogenetic reconstruction. Given that preliminary
analyses showed no effect of the use of the codon model
and there was no evidence of nucleotide saturation
and substitution bias (unpublished results), no codon
scheme was adopted for the rbcL data set (data not
shown). Indels (insertion and deletion events) from
ITS and trnL-F data sets were also included in the
analyses, given their capacity to resolve and increase
support in phylogenetic analyses (e.g. Kelchner,
2000; Simmons & Ocheterena, 2000; Müller, 2006).
Since these characters are usually inferred under a
subjective scheme of alignment, optimal indels were
obtained with RELINDEL server (guidance.tau.ac.il/
RELINDEL/), which detects and ranks indel reliability
based on an alternative multiple sequence alignment
approach (Ashkenazy et al., 2014). All alignment and
indel data sets are available in the Treebase repository
(N 26352). Data sets were permuted through 1000
bootstrap replicates, and subsequently aligned with
MAFFT, retaining every indels with a score of 1 (most
reliable). All employed indels were scored using the
simple indel-coding scheme (Simmons & Ocheterena,
2000) and added as a separate partition in all
phylogenetic analyses.
Maximum parsimony (MP)
The tree search was conducted using a Wagner tree
and TBR swap with 5000 replicates and holding
ten trees per iteration. Unsupported branches were
collapsed, and multiple searches were conducted until
no other most-parsimonious trees were obtained.
Levels of support for branches were assessed with
1000 bootstrap replicates (PB; Felsenstein, 1985).
Clades with support values 0.50–0.75 were considered
as weakly supported, > 0.75 moderately supported
and > 0.90 as strongly supported. Additionally, an
analysis using a concatenated data set including all
molecular and indels data sets was also conducted
to determine levels of support and resolution among
clades not retrieved using individual data sets.
All MP analyses were conducted using TNT v.1.5
(Goloboff & Catalano, 2016).
4  I. ESCOBAR ET AL.

Bayesian inference (BI)
A Bayesian phylogenetic reconstruction approach

0.96
0.96
0.94

0.94
RI
was employed applying a general likelihood mixed
model approach to gene sequence evolution with
the program BayesPhylogenies v.1.1 (Pagel &

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Meade, 2004). This program implements a mixture

0.81
0.82
0.87

0.87
model, which accommodates more than one model

CI
of sequence evolution without partitioning data sets
(Pagel & Meade, 2004). By employing reversible-jump
Markov chain Monte Carlo (rjMCMC; Green, 1995),

length
Tree
this approach can efficiently explore and sample

693
708
906

923
complex parameters space finding the best model that
summarizes observed sequence evolution in data sets
(Pagel & Meade, 2006). An rjMCMC analysis was run

Potentially parsimoniously Number of

for 20 000 000 generations, retaining a sample every
10 000 trees to assure appropriate independence.

1
1
21290

19070
Table 1.  Results obtained with MP using different individual data sets. CI = consistency index, RI = retention index.

trees
Indels were analysed with a non-time-reversible model
of change (M2P; Pagel & Meade, 2004). Convergence
was evaluated in all models and tree parameters
sampling the effecting sampling size (ESS > 300),

informative characters
after discarding 10% of all samples as burn-in, using
TRACER v.1.5 (Rambaut & Drummond, 2009). All
retrieved trees were summarized in a maximum clade
credibility tree (MCCT) with the BEAST co-distributed

202 (9.7%)

206 (9.8%)
257 (35%)
265 (36%)
package TreeAnnotator v.1.10 (Suchard et al., 2018),
considering clades with a posterior probability (PP) >
0.95 as optimally supported.

Divergence times among lineages

non-informative
Parsimoniously

Divergence times with ITS sequences were also

2085 (90.3%)

2093 (90.2%)
458 (65%)
465 (64%)
explored to contextualize trends of diversification
characters

of taxa of Gilliesieae. A Bayesian estimation of

node ages was conducted using similar settings to
those proposed by Sassone & Giussani (2018). First,
additional ITS sequences published from other
members of Leucocoryneae, Tulbaghieae and Allieae
2287 (1324 + 963)

and Lycoris radiata (L’Hér.) Herb and L. sprengeri

Comes ex Baker (Amaryllidaceae) were included to
characters

provide robust outgroup sampling (Table 1) (Sassone &

Giussani, 2018). Following previous studies (Sassone
Total

2299

& Giussani, 2018), we include a substitution rate of

715
730

5.09 × 10–9 substitutions/site/year and the addition of

calibration constraints at the Allium and Leucocoryne
nodes, although with slight modifications to make
Number
of taxa

improvements in MCMC searches. For the node age of

Allium, the estimate of Li et al. (2016) was employed
80
80
80

80

with a mean of 34.26 My and a normal standard

distribution of 1.0. For the node age estimate for
(trnL-F + rbcL)

Leucocoryne, we preferred to use a mean of 11.4 My

plastid DNA +

and a standard distribution of 1.7 (Jara-Arancio et al.,

plastid DNA
ITS + indels

2014). A model of molecular substitution (GTR+G+I)

Data set

was used with an uncorrelated lognormal relaxed clock

indels

for the model tree and a Yule process as speciation

ITS

model. This analysis was performed mostly with one

© 2020 The Linnean Society of London, Botanical Journal of the Linnean Society, 2020, XX, 1–16

sequence per OTU, as lack of convergence and mixing
were detected with the use of multiple intraspecific
entries. All analyses were conducted with BEAST
10.0, employing four independent MCMC searches of
20 000 000 iterations and sampling every 2000 replicate
states. All outputs were scrutinized with TRACER
v.1.7 to determine appropriate convergence and mixing
(> 200 ESS in target parameters like tree likelihood,
topology, model selection and the most common recent
ancestor for node constraints) after discarding a
burn-in of 20% of sampled states. Also, we explored the
convergence of tree splits among replicates with the
R package RWTY (Warren, Geneva, & Lanfear, 2017).
Finally, all runs were combined with LOGCOMBINER
v.1.10 and all trees were summarized in a MCCT using
the mean height per node with TreeAnotator v.1.10
(Rambaut & Drummond, 2009). All MP and BI trees
were visualized and annotated using FigTree v.1.3.1
(Rambaut, 2009) and Dendroscope v.3.5.9 (Huson &
Scornavacca, 2012).
Phenotypes
Morphological data were recorded to evaluate the
correspondence of relevant taxonomic characters
with the phylogenetic reconstruction obtained at the
genus level. Twenty-four characters (19 reproductive,
three vegetative and two karyological) were chosen
from herbarium and field collected specimens (based
on Escobar, 2012; see morphological state matrix in
Supporting Information, Supplementary Data 3).
Initially, all characters were evaluated to determine
patterns of variation for the major clades detected
in Gilliesieae. For this purpose, characters were
optimized using a parsimony criterion with Mesquite
v.3.51 (Maddison & Maddison, 2018) on a consensus
tree inferred with a reduced data set (21 OTUs) and
concatenated molecular regions (inferred with TNT
following same search settings than the full data
set and no indels included). From them, characters
were selected based on their synapomorphy (or
symplesiomorphy) of their character states and their
documented taxonomic use, to determine the error of
association on a phylogenetic tree rather than expected
by chance (Parker, Rambaut & Pybus, 2008). This
analysis was conducted with the last 100 posterior sets
of MCMC trees obtained from an analysis employing
the same reduced data set with concatenated molecular
regions (no indels) and following the same search
and model settings as in our earlier BI analyses. The
association index (AI; Wang et al., 2001), the parsimony
score (PS; Fitch 1971) and the maximum exclusive
single-state clade size score (Salemi et al., 2005) were
employed to evaluate association of each character
as implemented in the program BaTS (Parker et al.,
© 2020 The Linnean Society of London, Botanical Journal of the Linnean Society, 2020, XX, 1–16
PHYLOGENETICS OF TRIBE GILLIESIEAE  5
2008). The evaluation was expressed to test the null
hypothesis that no significant differences (P > 0.05)
are found from the phylogenetic-trait association in
evaluated clades than a random expectation.
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RESULTS
Sequence data
Obtained sequences yielded data sets of 715 bp for
ITS and 2287 bp for plastid DNA, of which 257 (35%)
and 202 (9.7%) were potentially parsimoniously
informative, respectively. These numbers were not
significantly altered with the inclusion of indels,
which added 15 and 12 additional characters for ITS
and plastid DNA data sets, respectively (Table 1).
Analysis based on MP of the ITS data sets yielded a
single most-parsimonious tree of 693 steps (708 steps
including indels), whereas the plastid DNA data sets
yielded 21 290 trees of 906 steps (19 070 trees, 923
steps including indels). Analyses of both data sets
(including indels) showed no significant differences
in the main groups retrieved in MP and BI analyses,
except for the BI analyses of plastid DNA data sets
for which significantly improved support and clade
resolution were obtained when indels were added to
the analyses. Therefore, all results will be shown in
the context of BI analyses of data sets including indels
(Fig. 1). All individual phylogenetic trees and their
statistics are available in Supporting Information,
Supplementary Data 4.
Phylogenetic analyses
The BI analyses using both data sets independently
revealed the monophyly of Gilliesieae (PP = 1 with both
data sets; Fig. 1), which contained two consistent and
highly supported clades. Clade I comprised Gethyum
cuspidatum (Harv. ex Baker) Muñoz-Schick, Gethyum
atropurpureum Phil., Solaria miersioides Phil. and all
Gilliesia spp. (PP = 1 with both data sets). Clade II
comprised Speea humilis (Phil.) Loes. and all Miersia
spp. (PP = 1 with both data sets; Fig. 1). In both clades,
the major genera (Gilliesia and Miersia) were retrieved
as paraphyletic and different patterns of association
and levels of support were obtained, depending on the
analysed data sets.
In Clade I, both data sets retrieved Gethyum
cuspidatum as monophyletic and sister to other
species of Clade I (PP = 1 with both data sets; Fig. 1).
Likewise, a well-supported clade combined Gethyum
atropurpureum with Gilliesia isopetala Ravenna
(PPITS = 0.97; PPplastidDNA = 0.98) and both species were
retrieved as monophyletic (PPITS = 0.98; PPplastidDNA = 0.99
and PPITS = 1; PPplastidDNA = 0.94, respectively; Fig. 1).
6  I. ESCOBAR ET AL.

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Figure 1.  MCC trees inferred with BI criterion. Left: MCCT inferred with ITS + indels; Right: MCCT inferred with plastid
DNA + indels. Monotypic genera (Ancrumia, Gethyum, Speea and Solaria) are delimited in grey boxes. Morphologically
divergent individual (Gilliesia sp. 86) is delimited in a black box. Numbers above branches represent posterior probabilities
for significant (PP > 0.95) or nearly significant (PP > 0.90) supported clades. Figures depict the following species: A, Gethyum
cuspidatum; B, Gethyum atropurpureum; C, Gilliesia sp.; D, Gilliesia dimera; E, Gilliesia montana; F, Solaria miersioides;
G, Gilliesia graminea: H, Miersia chilensis; I, Miersia tenuiseta; J, Miersia minor; K, Speea humilis; L, Miersia leporine and
M, Miersia cornuta.

Gilliesia dimera Ravenna was also strongly supported as
monophyletic (PPITS = 0.98; PPplastidDNA = 0.99). Different
patterns of relationships were found for Solaria
miersioides. Using ITS, S. miersioides is closely related
to Gilliesia graminea Lindl. (PPITS = 0.95) and with
plastid DNA it is closely related to Gilliesia montana
Poepp. (PPplastidDNA = 1). With both data sets, Gilliesia
montana and Gilliesia sp. (35, 41, 129, probably a comb.
nov.; Escobar, in prep.), were retrieved as paraphyletic. In
Clade II, more incongruent phylogenetic patterns were
found. With ITS, Miersia chilensis, M. tenuiseta Ravenna
and Speea humilis were retrieved as monophyletic, but
only S. humilis was strongly supported (PPITS = 0.99).
With plastid DNA, only Miersia cornuta Phil. was
retrieved as monophyletic and strongly supported
(PPplastidDNA = 0.99; Fig. 1).

© 2020 The Linnean Society of London, Botanical Journal of the Linnean Society, 2020, XX, 1–16

The MP analyses are congruent with BI analysis in
retrieving Gethyum cuspidatum, Gilliesia isopetala,
Gethyum atropurpureum and Gilliesia dimera as
monophyletic in Clade I with both data sets and
Miersia tenuiseta and Speea humilis as monophyletic
and strongly supported in Clade II with only ITS.
Both MP and BI were congruent with each data set
analysed independently (see phylogenetic trees in
Supporting Information, Supplementary Data 4).
Although MP analyses with the full concatenated data
set gave similar results to those with the individual
data sets, the discussion was based on the individual
data sets as resolution is lost in several well-supported
clades retrieved with individual data set analyses (see
consensus tree of concatenated data set in Supporting
Information, Supplementary Data 4).
Divergence time analyses
Most divergence times from our analyses were
congruent with those from earlier studies (Sassone
& Giussani, 2018), but our tree topology differed
from those reported from previous studies, adding
discordance for some node ages at tribal levels (Fig. 2).
This result is explained by the lack of congruence and
mixing found in MCMC searches, following proposed
priors and node age constraints. Exploratory analyses
suggest that the use of the highly divergent ITS
sequences of Allium leads to a consistent retention of
phylogenetic trees with an unclear root for Allioideae,
precluding an adequate fit in targeted node age and
model estimates (results not shown). Although this
effect seemed unavoidable given the divergent nature
of all ITS sequences for Allium, consistent results were
still retrieved in node ages of Gilliesieae, independently
of the analysis settings employed. Therefore, we
decided to conduct this analysis by forcing the
monophyly of Allioideae (Tulbaghieae + Leucocoryneae
+ Alliae + Gilliesieae) and leaving Amaryllioideae and
Agapanthoideae as outgroup. Hence, we focused our
discussion on the results obtained for Gilliesieae and
the closely allied tribe Leucocoryneae.
Our results indicate that Gilliesieae originated
during the Oligocene/Miocene (95% HPD: 10.52–
43.23 Mya), with a range of divergence close to that
for Leucocoryneae (95% HPD: 21.23–56.35 Mya;
Fig. 2). In Gilliesieae, node ages in the Miocene/
Pleistocene were detected for Clade I (95% HPD:
3.05–19.99 Mya) and Clade II (95% HPD: 2.05–
17.33 Mya). The remaining taxa in both clades in
our analyses mostly had origins in the Pliocene/
Pleistocene, except for the divergence of Gethyum
cuspidatum during the Miocene (95% HPD: 3.05–
19.99 Mya). In Clade I, the node ages suggest recent
periods of diversification, particularly for the clades
G. graminea + S. miersioides (95% HPD: 0.003–2.49
© 2020 The Linnean Society of London, Botanical Journal of the Linnean Society, 2020, XX, 1–16
PHYLOGENETICS OF TRIBE GILLIESIEAE  7
My), G. isopetala + Gethyum atropurpureum + Gilliesia
sp. 146 (95% HPD: 0.24–4.1 My) and G. dimera +
G. montana + Gilliesia sp. 129 (95% HPD: 0.03–2.89
My). In Clade II, node ages were particularly recent
in M. leporina Ravenna + M. cornuta (95% HPD:
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0.0001–1.65 My) and the remaining Miersia spp. +
S. humilis (95% HPD: 0.42–5.45 My). Our estimated
node ages in Leucocoryneae were similar to those
previously reported by Sassone & Giussani (2018).
In particular, the Latace + Leucocoryne clade (95%
HPD: 12.64–35.93 My), Nothoscordum + Beauverdia
clade (95% HPD: 8.50–29.28 My) and the Tristagma
clade (95% HPD: 8.51–29.44 My) revealed node ages
in the Eocene/Miocene (Fig. 2).
Phenotypic character state reconstruction
and TIP association analyses
Results of character state optimization analyses of
all characters are shown in Supporting Information,
Supplementary Data 5. Fifteen out of 24 morphological
characters were selected for the character association
analysis: five vegetative, eight reproductive and two
karyological characters. (Fig. 3, Table 2; only congruent
characters are shown). Nine character states were
found to be synapomorphic for all OTUs of Clade
I (Fig. 3). The exceptions were leaf shape in Solaria
miersioides (with navicular leaves) and number of
stamens in G. cuspidatum (with two stamens). Similar
results were found in Clade II, with eight characters
retrieved as symplesiomorphic (shared with outgroup)
and one as synapomorphic (Fig. 3). Statistical support
for this phylogenetic association was significant in
most of the vegetative characters analysed (P < 0.05;
Table 2). Most character states revealed significant
phylogenetic association (P < 0.05) in almost all
of the tests, except for leaf shape (navicular),
karyotype asymmetry (predominantly acrocentric),
floral symmetry (radial flowers), tepal arrangement
(tepals fused to a level above the ovary) and number
of stamens (six stamens; see detailed statistics for
phenotypic association in Supporting Information,
Supplementary Data 6).
DISCUSSION
Phylogenetic and divergence time analyses
All analyses with molecular data sets showed
Gilliesieae to be monophyletic in Allioideae, consisting
of two clades that separate the two major genera and
their allies: Gilliesia (plus Gethyum and Solaria) and
Miersia (plus Speea) (Fig. 1). In the molecular analyses
(ITS), most of the species are retrieved as monophyletic,
providing a phylogenetic framework in which to
delimit entities taxonomically and revealing insights
8  I. ESCOBAR ET AL.

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Figure 2.  Time calibrated tree inferred with ITS in BEAST. Date tips are shown above branches and bars depict the 95%
credible intervals.

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 PHYLOGENETICS OF TRIBE GILLIESIEAE  9

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Figure 3.  Character tip association test. MCC tree (cladogram enforced) inferred with the combination of ITS and plastid
data on Gilliesieae and outgroups, for vegetative (above) and floral (below) characters.

into the relationships and evolution of morphological
characters in Gilliesieae.
In previous treatments, most of the studied taxa
(ingroup) have been placed in Gilliesieae and grouped
at different hierarchical levels (Lindley, 1826; Baker,
1879; Engler, 1887; Reiche, 1893; Hutchinson, 1939;
Traub, 1963), apart for the exclusion of Erinna
gilliesioides Phil. (synonymized under Leucocoryne
by Ravenna, 2000a). Previous phylogenetic studies
have considered only two species for the preliminary

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10  I. ESCOBAR ET AL.

Table 2.  Summary with P values on character association with the association index (AI), parsimony score (PS) and the
maximum exclusive single-state clade size score (MC) tests. Bold letters in grey cells: non-significant values.

AI PS MC (state 0) MC (state 1) MC (state 2) MC (state 3) MC (state 4)

Bulb shape (A) 0.000 0.000 0.030 0.010

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N leaves per bulb (B) 0.000 0.000 0.023 0.002
N floral scapes per bulb (C) 0.000 0.002 0.008 0.004
Leaf shape (D) 0.000 0.000 0.023 0.002 1.000
Capsule shape (E) 0 0 0.014 0.006 0.001
Karyotype asymmetry (F) 0.000 0.000 0.004 0.005 0.138
Chromosome number (G) 0.010 0.000 0.530 0.001 1.000 1.000 1.000
Floral symmetry (H) 0.000 0.000 0.747 0.001 0.005
Number of tepals (I) 0.461 1.000 0.347 1.000 1.000
Perigonium shape (J) 0.087 0.004 0.070 1.000 0.003
Tepals arrangement (K) 0.005 0.001 0.021 0.002 0.073
Presence of staminoids (L) 0 0 0.014 0.001
Number of stamens (M) 0.001 0.013 0.009 0.036 1.000
Length of floral scapes (N) 0.012 0.002 0.007 0.007
Degree of fusion in filaments (O) 0.000 0.000 0.255 0.002 0.001 1.000

recognition of the monophyly of this subtribe: Gilliesia
graminea and Gethyum atropurpureum, which
have always been retrieved as well-supported sister
species despite their striking differences in floral
morphology and cytology (Fay & Chase, 1996; Meerow
et al., 1999; Rudall et al., 2002; Fay et al., 2006). This
preliminary hypothesis of the monophyly of Gilliesieae
is congruent with segregation from other tribes of
Allioideae (Sassone et al., 2014). This hypothesis was
also confirmed by phylogenetic analyses including
more species of Gilliesieae (Souza et al., 2016; Sassone
& Giussani, 2018).
An important finding is that both main genera of
Gilliesieae (Miersia and Gilliesia) are retrieved as
paraphyletic (Fig. 1). This pattern of molecular evolution
could be explained by the result of recent process of
diversification, as most of the studied species diverged
during the Pliocene–Pleistocene (except Gethyum
cuspidatum; Fig. 2). Although speciation requires some
degree of reproductive isolation that affects a group
of populations within a species, other populations
could remain genetically cohesive, producing typical
patterns of paraphyly (Hörandl, 2006). Studies have
documented the presence of non-monophyletic groups
with high levels of morphological divergence attributed
to processes of recent and contemporary radiation
(Ramdhani, Barker & Baijnath, 2009; Kellner et al.,
2010; Rymer et al., 2010; Ramdhani, Barker, & Cowling,
2011). Recent diversification would explain how some
taxa have experienced a contemporary process of
speciation and adaptation, resulting in the presence
of non-monophyletic groups with highly diverse
floral morphology, constant chromosome numbers
and restricted geographical distribution. In the same
context, hybridization and incomplete lineage sorting
are also well-known sources of discordant patterns of
phylogenetic relationships among gene trees (Doyle
1992; Maddison, 1997), as rapid and recent divergent
events reduce the possibility of sorting lineages
before cladogenesis is complete (Syring et al., 2007,
Ramdhani et al., 2009). As such, this hypothesis should
not be discarded for Gilliesieae and other allied tribes,
as recent diversification and reticulated phylogenetic
relationships have also played an important role in
patterns of recent diversification in Allioideae (e.g.
Sassone & Giussani, 2018).
Patterns of phenotypic and karyotypic
variation in Gilliesieae
Based on our results, Gilliesieae are a group with
variable trends in character state variation. Most
character state and tip association showed more
positive correlation with vegetative and karyotypic
than floral characters (Fig. 3), following a congruent
characterization for Clades I and II rather than genera
(Fig. 3). This pattern suggests contradictory trends
in morphological variation of flowers that do not fit
existing taxonomic proposals, and instead indicates
several instances of homoplasy among the analysed
characters (e.g. floral symmetry; Fig. 3 and Supporting
Information, Supplementary Data 6). Character
optimization showed some characters that exhibited
evolutionary trends congruent to delimit Gilliesieae
and the infratribal major clades. At the tribal level,
Gilliesieae display a trend of change to zygomorphic
flowers, with an open (or close to campanulate)
perigonium, different degrees of fusion in staminal

© 2020 The Linnean Society of London, Botanical Journal of the Linnean Society, 2020, XX, 1–16

filaments (never adnate to the perigonium), absence
of nectaries and presence of osmophores (Supporting
Information, Supplementary Data 5).
Species of Clade I are characterized by elongated
bulbs, a single linear-lanceolate leaf (except for
Solaria miersioides with navicular leaves), and one
floral scape per bulb, mostly erect and tall plants
with length of floral scape up to 1.2 m (except Solaria
miersioides), with three stamens (except Gethyum
cuspidatum with two stamens) basally connate and
one, two or three staminodes, 2n = 14 (x = 7), and a
more asymmetric karyotype (four metacentric, four
submetacentric and six acrocentric chromosomes)
(Fig. 3). On the other hand, one character could be
considered a symplesiomorphy for Clade I: the oblong
capsule. This clade is defined by a tendency to include
species with a reduced number of floral structures
(tepals and stamens) and strong zygomorphy, resulting
from a highly elaborated androecium with prominent
appendages (resembling the body of an insect) typically
found in Gilliesia (Ravenna, 2000b; Rudall et al., 2002,
Escobar, 2012). However, this pattern is contradictory
from a phylogenetic perspective, as Gilliesia is also
found to be non-monophyletic due the inclusion of
species with a weak asymmetry in their perigonium,
such as Gethyum cuspidatum and Solaria miersioides
(and Gilliesia isopetala with similar features; Fig. 1).
A similar pattern of character disassociation was
found in the paraphyly of Gethyum, which reveals
the lack of a single diagnostic character state that
allows a clear circumscription of the genus. Instead,
Gethyum atropurpureum is shown as distinct from
Gethyum cuspidatum by the presence of two instead
of three stamens (Fig. 3). In contrast, species of Clade
II are readily characterized by an ovoid bulb with
two to nine generally linear leaves, one to four floral
scapes per bulb, six stamens, no staminodes, 2n = 12
(x = 6, except for Miersia chilensis, with 2n = 20), a
more symmetric karyotype (ten metacentric and two
acrocentric chromosomes), except for Miersia chilensis
(two metacentric and 18 acrocentric chromosomes),
and larger chromosomes (86.5–104 µm) than Clade
I (56.5–68.5 µm; Escobar et al., 2012). Clade II
comprises all Miersia spp. and Speea humilis (Fig. 1),
a group mainly characterized by the presence of a
constant number of tepals and stamens (six), slightly
bilateral flowers (except M. chilensis) and small plants
(up to 45 cm height).
All analyses support the monophyly of Gilliesieae
with two subgroups (Clade I and II); this topology
helps in contextualizing the information generated
for karyotype variation and evolution. Although
wider karyotype variability is present in Allioideae,
in Gilliesieae a more stable karyotype is present.
The karyotype of the sister tribe Leucocoryneae
comprises variable chromosome numbers (2n and FN)
© 2020 The Linnean Society of London, Botanical Journal of the Linnean Society, 2020, XX, 1–16
PHYLOGENETICS OF TRIBE GILLIESIEAE  11
and karyotype formula (Souza et al., 2016; Sassone
et al., 2018). Nothoscordum has 2n = 10/8 (x = 4 or
5; depending the section) with a variable karyotype
formula (Souza et al., 2010; Sassone et al., 2018) and
includes polyploids (tetraploids and triploids; Souza
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et al., 2009), Leucocoryne has 2n  = 10 with 6M+4A
(Crosa, 1988; Souza et al., 2016), Tristagma has 2n = 8
with 6M+2A and includes tetraploids (Crosa, 1981).
However, higher chromosome numbers and more
asymmetric karyotypes are found in Ipheion and
Latace. Ipheion has a variable chromosome number
and karyotype formula (2n = 12 with 2SM+10A; 14A;
20 with 4SM+16A; 24 with 4SM+20A; Souza et al.,
2010) and Latace has 2n = 24 with 8M+16A (Crosa,
2004, as Zoellnerallium).
In Gilliesieae, three karyotypes are found, all
with FN = 11. One of them is present only in Clade
I (Gethyum, Gilliesia and Solaria; 2n  = 14 with
4M+4SM+6A). A second karyotype is present in Clade
II, comprising most Miersia spp. and Speea humilis
(2n = 12, with 10M+2A). A third karyotype is present
only in Miersia chilensis (2n = 20, 2M+18A; Escobar
et al., 2012). Coincidentally, a more asymmetric
karyotype (2n  = 14) with smaller chromosomes is
present in species of Clade I and a more symmetric
karyotype with larger chromosomes occurs in species
of Clade II, with the exception of an asymmetric
karyotype in Miersia chilensis (TLC = 104 µm; Escobar
et al., 2012).
The change in karyotype symmetry has
been proposed as the result of Robertsonian
rearrangements through fusion of acrocentric
chromosomes to form metacentrics or, alternatively,
fission of metacentrics to form acrocentrics (Goldblatt,
1976). The first alternative was postulated for two
species of Gilliesieae (Miersia chilensis and Gethyum
atropurpureum; Goldblatt 1976). However, ancestral
state reconstruction of the basic chromosome number
for Leucocoryneae, using Gethyum atropurpureum
and Speea humilis as outgroup taxa, indicates that the
ancestral chromosome number was symmetric with
x  = 8, and the change was toward asymmetry with
x  = 7 or x  = 6, present in Gethyum atropurpureum,
Speea humilis and Tulbaghia spp. (Souza et al., 2016).
More recently, Pellicer et al. (2017) estimated the
ancestral chromosome number for tribe Gilliesieae to
be x = 6 (present in Clade II of Gilliesieae, changing to
x = 5 in species of Leucocoryneae). These changes in
Gilliesieae probably resulted from transformation of a
more symmetric karyotype (in Clade II), with 2n = 12
(x = 6), into a more asymmetric karyotype (in Clade
I), with 2n  = 14 (x  = 7), through the broken arm of
a metacentric chromosome forming two acrocentrics.
A similar mechanism could be proposed for the
karyotype of Miersia chilensis, through the broken arms
of four metacentric chromosomes of some Miersia spp.
12  I. ESCOBAR ET AL.
forming nine acrocentric chromosomes. In Gilliesieae,
this type of rapid chromosome change has become an
important factor in karyotype evolution (Crosa, 1988;
Jones, 1998; Pires et al., 2006; Souza et al., 2009, 2010;
Leitch et al., 2010), probably playing a pivotal role
in the evolution and diversification of Gilliesieae. As
such, more specific and detailed studies are needed to
elicit more evidence about these patterns.
Taxonomic and systematic considerations
Based on our results, we identified five broad
taxonomic issues that need to be discussed and/or
clarified. (1) Gilliesieae are monophyletic and clearly
different from other tribes of Allioideae. Our results
support this separation (Figs 1, 3), which is congruent
with other published studies (Fay & Chase, 1996;
Fay et al., 2006; Souza et al., 2016; Pellicer et al.,
2017; Sassone & Giussani, 2018). The karyology and
molecular results of this study support the tribal
classification proposed by Sassone et al. (2014) based
on morphological circumscription: subfamily Allioideae
consisting of four tribes (Allieae, Tulbaghieae,
Gilliesieae and Leucocoryneae). These four tribes in
Allioideae are now recognized in the recent update
of the Angiosperm Phylogeny Website (Stevens, 2001
onwards). (2) Although the two main genera of the tribe
are retrieved as paraphyletic (Gilliesia and Miersia),
two other genera, Speea and Solaria, are shown to be
monophyletic (and monotypic). Based on the presence
of unique character states when compared with closely
related taxa (a campanulate perigonium for Speea
humilis and navicular leaves for Solaria miersioides,
Fig. 3), it is possible to characterize both genera from a
morphological point of view. (3) Although some recent
taxonomists have considered Gethyum to be composed
of G. atropurpureum and G. cuspidatum (Muñoz, 2000;
Zuloaga, Morrone, & Bergrano, 2008; García 2019), other
proposals have suggested that the latter species belongs
to Ancrumia, leaving Gethyum as monotypic. Based
on our results, we favour the suggestion to revalidate
Ancrumia (as a monotypic genus with A. cuspidata
Baker), as this taxon is retrieved as monophyletic and
early diverged (Fig. 1) and morphologically distinct
from Gethyum and any other taxa of Gilliesineae.
Ancrumia is characterized by its highly restricted
distribution, occurring in coastal and inland areas of
the Coquimbo Region (Escobar, 2012), and it is readily
characterized as the only taxon with two stamens in
Gilliesieae (Fig. 3). (4) Further analyses are required to
clarify the taxonomy of Gilliesia, Gethyum, Solaria and
Miersia. The cases of Gilliesia and Solaria are difficult
to resolve, as our results confirm previous observations
of paraphyly and are especially confusing with the
well-supported relationship of Solaria miersioides
and G. graminea (the latter being the type species of
© 2020 The Linnean Society of London, Botanical Journal of the Linnean Society, 2020, XX, 1–16
Gilliesia). Other considerations are also necessary to
explain the strongly supported relationship between
Gethyum atropurpureum and Gilliesia isopetala
(Fig. 1), which could merit a new combination if
patterns of morphological variation (especially of floral
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characters) are well understood. In the case of Miersia,
problems remain in defining monophyletic groups
among its species; this can probably be explained by
the lack of a clear phylogenetic signal, given its recent
age of diversification (Fig. 2). In this context, the
situation of Speea is easier to interpret as a distinctive
taxon from Miersia, which remains as a monophyletic
group with clear diagnostic morphological characters.
(5) An important topic of our analysis is the status
of unassigned specimens of Gilliesieae, as their
delimitation can be determined given the presence
of reticulated patterns of phylogenetic relationships
between analysed data sets (Fig. 1). Especially
intriguing is the case of Gilliesia sp. 146, which
was consistently retrieved in a hard topological
incongruence (sensu Johnson & Soltis, 1998) with the
Gethyum atropurpureum plus Gilliesia isopetala clade
(nuclear data set) and Solaria miersioides plus Gilliesia
montana (plastid data set) (Fig. 1). Field observations
suggest consistent and viable populations of this
taxon (Escobar, unpubl. data), which could suggest
that possible new taxa have resulted from reticulated
speciation events (possible hybrid origin). A different
situation applies to the samples of Gilliesia sp. 35,
41 and 129, which, according to Escobar (2012), could
correspond to a new combination in Gilliesia, despite
not receiving significant support from molecular data
(Fig. 3, Supporting Information, Supplementary Data
4, 5). Similarly, Miersia sp. 84 resolved with no well-
supported relationships; this result could be explained
by the lack of phylogenetic resolution seen in Clade
II (Fig. 2). We consider these three cases as examples
of the rapid and recent diversification currently in
process in Gilliesieae, reflecting intricate patterns
of variation that could not be appropriately captured
through traditional taxonomic treatments. Additional
observations and individuals are required to determine
the origin and integrity of these entities to assess their
merit to be considered as new taxa for the tribe.
Finally, we emphasize that this study represents
the first detailed attempt to understand patterns of
phylogenetic relationships in Gilliesieae. As mentioned
above, additional work will be necessary to clarify the
monophyly of groups that we consider sufficiently robust
to warrant an appropriate taxonomic treatment. The use
of genomic approaches could prove useful for this task,
given their capacity to provide phylogenetic evidence
even in circumstances of recent diversification and
cryptic divergence (e.g. Mort et al., 2015; Shahl-Shavvon
et al., 2017; Fernández-Mazuecos et al., 2018). It is
equally important to add material of Schickendantziella

and Trichlora to the study of Chilean Gilliesieae, as
these potentially early diverging taxa might constitute a
valuable source of information to assess the origins of the
tribe and expand the evaluation of character evolution.
ACKNOWLEDGEMENTS
We thank CONC, CORD and K for providing access
to samples and study specimens of Gilliesieae and
allied taxa. This study was funded with support from
the KLARF program (Kew Latin American Research
Fellowship Program; Royal Botanic Gardens, Kew, UK),
Andes Foundation postgraduate fellowship (project
C-14055/7), CONICYT postgraduate scholarship,
CONCIYT assistance program for ending doctoral
dissertations, MECESUP scholarship (UCO0708),
DIUC (208.111.049-1.0) and the Flora of Chile Project.
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:
Supplementary Data 1. Studied material.
Supplementary Data 2. Sequences of taxa: collector name and number (herbarium acronym) and GenBank

Downloaded from https://academic.oup.com/botlinnean/advance-article-abstract/doi/10.1093/botlinnean/boaa035/5859233 by guest on 18 June 2020

accession number for each sequenced DNA marker of this study.
Supplementary Data 3. Morphological data matrix used for phylogenetic tip association analysis. Character
states are included under each character column. Dashes (-) correspond to missing or inapplicable character states.
Supplementary Figure S1. MCCT obtained with BI criterion from the plastid DNA data set (trnL-F + rbcL, no
indels included). Numbers on branches represent significant (pp > 0.95) or nearly significant (PP > 0.9) posterior
probabilities for each delimited clade.
Supplementary Data 5. Full results obtained from character tip association on phylogenetic data conducted in
Gilliesieae and allied tribes. Bold letters in grey cells: no significant values.
Supplementary Data 6. Parsimonious optimization of phenotypic characters present in Gilliesieae.

© 2020 The Linnean Society of London, Botanical Journal of the Linnean Society, 2020, XX, 1–16