Phylogenetic relationships in the
southern African genus Drosanthemum
(Ruschioideae, Aizoaceae)
Sigrid Liede-Schumann1 , Guido W. Grimm2 , Nicolai M. Nürk1 ,
Alastair J. Potts3 , Ulrich Meve1 and Heidrun E.K. Hartmann4 ,†
1
Department of Plant Systematics, University of Bayreuth, Bayreuth, Germany
Unaffiliated, Orléans, France
3
African Centre for Coastal Palaeoscience, Nelson Mandela University, Port Elizabeth, Eastern Cape,
South Africa
4
Department of Systematics and Evolution of Plants, University of Hamburg, Hamburg, Germany
†
Deceased.
2
ABSTRACT
Submitted 8 November 2019
Accepted 26 March 2020
Published 8 May 2020
Corresponding author
Sigrid Liede-Schumann,
sigrid.liede@uni-bayreuth.de
Academic editor
Gabriele Casazza
Additional Information and
Declarations can be found on
page 22
Background. Drosanthemum, the only genus of the tribe Drosanthemeae, is widespread
over the Greater Cape Floristic Region in southern Africa. With 114 recognized species,
Drosanthemum, together with the highly succulent and species-rich tribe Ruschieae,
constitute the ‘core ruschioids’ in Aizoaceae. Within Drosanthemum, nine subgenera
have been described based on flower and fruit morphology. Their phylogenetic
relationships, however, have not yet been investigated, hampering understanding of
monophyletic entities and patterns of geographic distribution.
Methods. Using chloroplast and nuclear DNA sequence data, we performed networkand tree-based phylogenetic analyses of 73 species of Drosanthemum with multiple
accessions for widespread species. A well-curated, geo-referenced occurrence dataset
comprising the 134 genetically analysed and 863 further accessions was used to describe
the distributional ranges of intrageneric lineages and the genus as a whole.
Results. Phylogenetic inference supports nine clades within Drosanthemum, seven of
which group in two major clades, while the remaining two show ambiguous affinities.
The nine clades are generally congruent to previously described subgenera within
Drosanthemum, with exceptions such as cryptic species. In-depth analyses of sequence
patterns in each gene region were used to reveal phylogenetic affinities inside the
retrieved clades in more detail. We observe a complex distribution pattern including
widespread, species-rich clades expanding into arid habitats of the interior (subgenera
Drosanthemum p.p., Vespertina, Xamera) that are genetically and morphologically
diverse. In contrast, less species-rich, genetically less divergent, and morphologically
unique lineages are restricted to the central Cape region and more mesic conditions
(Decidua, Necopina, Ossicula, Quastea, Quadrata, Speciosa). Our results suggest that the
main lineages arose from an initial rapid radiation, with subsequent diversification in
some clades.
DOI 10.7717/peerj.8999
Copyright
2020 Liede-Schumann et al.
Subjects Biodiversity, Evolutionary Studies, Plant Science
Distributed under
Creative Commons CC-BY 4.0
Likelihood, MJ Network, Outgroup placement, Phylogeny, SP Network, Subgeneric Classification
Keywords Distribution, Genetic diversity, Greater Cape Floristic Region, Haplotyping, Maximum
OPEN ACCESS
How to cite this article Liede-Schumann S, Grimm GW, Nürk NM, Potts AJ, Meve U, Hartmann HEK. 2020. Phylogenetic relationships
in the southern African genus Drosanthemum (Ruschioideae, Aizoaceae). PeerJ 8:e8999 http://doi.org/10.7717/peerj.8999
INTRODUCTION
In the south-western corner of Africa, the iconic leaf-succulent Aizoaceae (ice plant family,
including Lithops, ‘living stones’; Caryophyllales) is one of the most species-rich families
in the biodiversity hot-spot of the Greater Cape Floristic Region (GCFR; Born, Linder
& Desmet, 2007; Mittermeier et al., 1998; Mittermeier et al., 2004; Mittermeier et al., 2011),
ranking second in the number of endemic genera and fifth in the number of species
(Manning & Goldblatt, 2012). Although Aizoaceae species have received much attention
both in terms of their ecology and evolution (e.g., Klak, Reeves & Hedderson, 2004; Valente
et al., 2014; Ellis, Weis & Gaut, 2007; Hartmann, 2006; Schmiedel & Jürgens, 2004; Powell et
al., 2019), information on phylogenetic relationships within major clades (or subfamilies)
is still far from complete. Here, we aim at filling some of the knowledge-gaps by: (1)
providing a review of the current classification of the family, and origin and distribution
of major clades (in the Introduction), and (2) a study of phylogenetic relationships in the
enigmatic and hitherto, phylogenetically, almost neglected genus Drosanthemum.
Subfamilies of Aizoaceae: relationship of major clades
Aizoaceae currently comprises ca. 1800 species (Hartmann, 2017a; Klak, Hanáček & Bruyns,
2017a) classified in 145 genera and five subfamilies (Klak, Hanáček & Bruyns, 2017a). The
first three subfamilies—Sesuvioideae, Aizooideae, Acrosanthoideae—are successive sisters
to Mesembryanthemoideae + Ruschioideae (Klak et al., 2003; Klak, Reeves & Hedderson,
2004; Thiede, 2004; Klak, Hanáček & Bruyns, 2017b; for authors and species numbers see
Table 1). Species of Mesembryanthemoideae and Ruschioideae, commonly referred to as
‘mesembs’ (Mesembryanthema; Hartmann, 1991), were found in molecular phylogenetic
studies to be reciprocally monophyletic (e.g., Klak et al., 2003; Thiede, 2004; Klak, Hanáček
& Bruyns, 2017b).
Mesembryanthemoideae and Ruschioideae, as well as their sister-group relationship,
are supported by morphological characters. Mesembryanthemoideae + Ruschioideae can
be distinguished from the remaining Aizoaceae by raphid bundles of calcium oxalate
(in contrast to calcium oxalate druses), the presence of petals of staminodial origin,
half-inferior or inferior ovary and a base chromosome number of x = 9 (Bittrich &
Struck, 1989). The conspicuous loculicidal hygrochastic fruit capsules of ca. 98% of the
species (Ihlenfeldt, 1971; Parolin, 2001; Parolin, 2006) are lacking in Acrosanthoideae, for
which xerochastic, parchment-like capsules are apomorphic, but are also predominant
in subfamily Aizooideae (Bittrich, 1990; Klak, Hanáček & Bruyns, 2017a). In Aizooideae,
however, valve wings of the capsules are either absent or very narrow, while they are well
developed in Mesembryanthemoideae + Ruschioideae (Bittrich & Struck, 1989).
The capsules of Mesembryanthemoideae and Ruschioideae differ in the structure of their
expanding keels. The expanding keels are of purely septal origin in Mesembryanthemoideae,
and mainly of valvar origin in Ruschioideae (Hartmann, 1991). In floral structure,
Ruschioideae are characterized almost always by crest-shaped (lophomorphic) nectaries
and a parietal placentation (Hartmann & Niesler, 2009), while Mesembryanthemoideae
possess plain shell-shaped (coilomorphic) nectaries and a central placentation.
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Table 1 Infrafamilial classification of Aizoaceae.
Subfamilies of Aizoaceae: origin and distribution of major clades
Subfamily Sesuvioideae, sister to the rest of Aizoaceae, originated in Africa/Arabia
suggesting an African origin for the entire family (Bohley et al., 2015). While Sesuvioideae
and Aizooideae dispersed as far as Australia and the Americas (Bohley et al., 2015; Klak,
Hanáček & Bruyns, 2017b), Acrosanthoideae, Mesembryanthemoideae and Ruschioideae
are most diverse in southern Africa. Only a small number of Ruschioideae species are found
outside of this area. Delosperma N.E.Br. is native to Madagascar and Réunion and expands
with less than ten species along the East African mountains into the south-eastern part of
the Arabian Peninsula (Hartmann, 2016; Liede-Schumann & Newton, 2018). Additionally,
in Ruschioideae there are nine halophytic species endemic to Australia (Prescott & Venning,
1984; Hartmann, 2017a; Hartmann, 2017b), and possibly one species to Chile (Hartmann,
2017a).
In southern Africa, most species of Acrosanthoideae, Mesembryanthemoideae and
Ruschioideae are native to the Winter Rainfall Region (Verboom et al., 2009; Valente et al.,
2014) in the GCFR. Acrosanthoideae with only six species is endemic to mesic fynbos,
whereas Mesembryanthemoideae and Ruschioideae are speciose in more arid Succulent
Karoo vegetation (Klak, Hanáček & Bruyns, 2017a).
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Within Ruschioideae: relationships of major clades
Ruschioideae constitute the largest clade of Aizoaceae with estimated species richness of
ca. 1,600 (Stevens, 2001, onwards; Klak, Bruyns & Hanáček, 2013). Within Ruschioideae
three tribes, Apatesieae, Dorotheantheae, and Ruschieae s.l., have been distinguished based
on unique combinations of nectary and capsule characters (Chesselet, Smith & Van Wyk,
2002). These three tribes form well supported clades in phylogenetic analyses (Klak et
al., 2003; Thiede, 2004; Valente et al., 2014). Ruschieae s.l. are further characterized by the
possession of wideband tracheids (Landrum, 2001), endoscopic peripheral vascular bundles
in the leaves (Melo-de Pinna et al., 2014), smooth and crested mero- and holonectaries,
well-developed valvar expanding tissue in the capsules (Hartmann & Niesler, 2009), the
loss of the rpoC1 intron in the chloroplast DNA (cpDNA; Thiede, Schmidt & Rudolph,
2007), and the possession of two ARP (Asymmetric Leaves1/Rough Sheath 2/Phantastica)
orthologues in the nuclear DNA (Illing et al., 2009); the duplication most likely took
place after the divergence of the Ruschioideae from the Mesembryanthemoideae, with the
subsequent loss of one paralogue in Apatesieae and Dorotheantheae (Illing et al., 2009).
Within Ruschieae s.l. (‘core ruschioids’ sensu Klak, Reeves & Hedderson, 2004), Klak
et al. (2003) additionally revealed two clades with strong support, Ruschieae s.str. and a
clade consisting only of members of Drosanthemum Schwantes. Species of Delosperma,
considered closely related to Drosanthemum due to a papillate epidermis, often broad, flat
mesophytic leaves, relatively simple hygrochastic fruits and a meronectarium have been
described with Drosanthemum in tribe Delospermeae Chess., Gideon F.Sm. & A.E.van Wyk
(Chesselet, Smith & Van Wyk, 2002). In phylogenetic studies, however, Delosperma species
are nested in Ruschieae s.str. (except for a few species, e.g., Drosanthemum asperulum and
D. longipes, which have been assigned in turn to either Delosperma or Drosanthemum).
Consequently, Chesselet, Smith & Van Wyk (2004) included Delosperma in Ruschieae s.str.
and coined the monogeneric Drosanthemeae Chess., Gideon F. Sm. & A.E. van Wyk as a
distinct tribe sister to Ruschieae s.str. (in the following Drosanthemeae + Ruschieae = core
ruschioids; Table 1).
While Ruschieae are characterized by fused leaf bases (Chesselet, Smith & Van Wyk,
2002), an apomorphic trait is less obvious for its sister tribe Drosanthemeae. Hartmann
& Bruckmann (2000) suggested capsules with a bipartite pedicel, of which the lower part
appears darker due to an inner corky layer, and the upper part often thinner and agreeing
in surface and colour with the capsule base. More generally, species of Drosanthemeae
are considered mesomorphic, compared to the highly succulent, xeromorphic Ruschieae
(Klak, Bruyns & Hanáček, 2013).
Core ruschioids: relationships of lineages
A sister-group relationship of Drosanthemeae and Ruschieae has been revealed by
molecular phylogenetic analyses (Klak, Bruyns & Hanáček, 2013). Whether both groups are
reciprocally monophyletic (and in which circumscription) is less clear (e.g., Klak, Hanáček
& Bruyns, 2017b). For example, molecular phylogenies identified two species erroneously
included in Drosanthemeae. One of these, Drosanthemum diversifolium L.Bolus, was first
transferred to Knersia H.E.K.Hartmann & Liede, a monotypic genus placed in Ruschieae
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(Hartmann & Liede-Schumann, 2013), and later to Drosanthemopsis Rauschert (Ruschieae)
by Klak, Hanáček & Bruyns (2018). The second species, Drosanthemum pulverulentum
(Haw.) Schwantes, with a xeromorphic epidermis untypical for Drosanthemeae, was
retrieved as member of the highly succulent clade ‘‘L1’’ in Ruschieae (Klak, Bruyns &
Hanáček, 2013; not yet formally transferred). With these corrections, Drosanthemeae
comprise a single genus, Drosanthemum, with 114 species presently recognized and a
wide distribution in the GCFR with the centre of diversity in the Cape Floristic Region
(Hartmann, 2017a; Van Jaarsveld, 2015; Van Jaarsveld, 2018; Liede-Schumann, Meve &
Grimm, 2019).
Drosanthemum (Drosanthemeae) systematics
Within Drosanthemum, five floral types have been distinguished, differing mainly
in number, position and relative length of petaloid staminodes (Rust, Bruckmann &
Hartmann, 2002; Fig. 1). Also, ten types of capsules have been described, differing in size
and shape of the capsule base and the capsule membrane, and the presence or absence
of a closing body (Hartmann & Bruckmann, 2000). Based on a combination of these
flower and fruit types, Hartmann (2007) proposed a subdivision of Drosanthemum in eight
subgenera. Later, Hartmann & Liede-Schumann (2014) proposed two more subgenera
based on additional vegetative morphology, and also suggested the union of two of the
previously described subgenera. This reflects an unusually broad variation in flower and
capsule types encountered in the genus compared with other Aizoaceae genera.
Despite this extraordinary morphological diversity, molecular phylogenetic studies
of Drosanthemum have hitherto been restricted to few species: nine species studied for
ten cpDNA regions in Klak, Bruyns & Hanáček (2013) and 16 species studied for two
cpDNA regions and the nuclear-encoded internal transcribed spacer (ITS) region of the
35S ribosomal DNA cistron in Hartmann & Liede-Schumann (2013). Obtaining increased
species coverage representative for the phenotypic and taxonomic diversity present in
Drosanthemum is challenging partly due to ambiguous species assignment to either
Drosanthemum or Delosperma (Hartmann & Liede-Schumann, 2014), but mainly due
to challenges in attributing specimens to published species names in Drosanthemum.
Ambiguous and/or overlapping diagnostic characters are common among closely related
species and also present between subgenera or genera. Specimens of species flocks and
cryptic species (Liede-Schumann, Meve & Grimm, 2019) are often hard to identify with
certainty, a fact that might have hampered investigation of the genetic differentiation
among Drosanthemum species. In this study, we build on Heidrun Hartmann’s huge field
collections of identified specimens of Drosanthemum. The present study would not have
been possible without her enduring commitment to collect, diagnose, and formally name
species in the Aizoaceae.
We present a phylogenetic study of Drosanthemum covering more than 64% of the
species richness (73 of 114 recognized species) representing all subgenera. We analyse
chloroplast and nuclear DNA sequence variation using phylogenetic tree and network
approaches and assemble a taxonomically-verified occurrence dataset. Specifically, we
test whether: (1) Drosanthemum is a monophyletic lineage sister to Ruschieae; (2) the
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Figure 1 Floral diversity in Drosanthemum. (A) Drosanthemum lique (subg. Vespertina, clade IIIa; HH
34800); (B) Drosanthemum nordenstamii (subg. Drosanthemum, clade Ia; HH 31525); (C) Drosanthemum
papillatum (subg. Quastea, clade VI; HH 32425); (D) Drosanthemum cereale (subg. Speciosa, clade Vb; HH
34489)—note the absence of black staminodes; (E) Drosanthemum hallii (subg. Speciosa; clade Vb; HH
34610)—note the black staminodes; (F) Drosanthemum zygophylloides. Photos (A–E): H.E.K. Hartmann;
F: L. Mucina.
Full-size DOI: 10.7717/peerj.8999/fig-1
morphologically delineated subgenera are monophyletic, in particular, whether the most
species-rich subgenus Drosanthemum is monophyletic or, alternatively, a ‘‘dustbin’’ for
species that cannot be assigned to other subgenera based on morphology; (3) all accessions
within currently recognized species are indeed each other’s closest relatives; and (4) the
clades detected in this study have distinct geographic distributions in the GCFR.
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MATERIAL AND METHODS
Taxon sampling
We established a collection of georeferenced and identified Drosanthemum samples; each
sample was only included if sufficient material was available to identify key characteristics.
The full collection (‘core collection’; n = 997 samples) represents the most comprehensive
sampling of the currently recognized Drosanthemum species, covering 85 species in total,
with each species represented by up to 30 georeferenced samples (range: 1–30; mean: 5
samples per species). This core collection consists of 590 samples identified to subgenus,
plus 407 identified to species. The subgeneric classification follows Hartmann (2007) and
Hartmann & Liede-Schumann (2014).
A subset of the core collection was used to generate the molecular dataset; this subset
comprised 134 accessions of Drosanthemum, covering 73 of the recognized species, with the
more widespread and morphologically variable species represented by up to 5 accessions.
To cover the full distribution in the larger subgenera, the molecular dataset comprises
21 accessions identified to subgenus, several of which most likely represent hitherto
undescribed species: subg. Drosanthemum (10 accessions), subg. Vespertina (8 accessions),
subg. Xamera (2 accessions), and subg. Ossicula (1 accession). Geographic distributions
of the subgenera that were corroborated with phylogenetic inference in this study (i.e.,
inferred clades, see Results) were plotted on a map using the elevation above sea level
data from the WorldClim climate layers (Hijmans et al., 2005), with a spatial resolution
of 30′ using the raster library v2.8-19 (Hijmans, 2019) in R v3.5.3 (R Core Team, 2019).
Geographic references for the core collection are available at the Dryad digital repository
(Liede-Schumann et al., 2019).
For outgroup comparison we selected a broad spectrum of species representing the three
remaining tribes of Ruschioideae: Apatesieae (two accessions representing one species),
Dorotheantheae (three species), and Ruschieae (49 accessions representing 47 species
and 42 genera). We used the cpDNA dataset of Klak, Bruyns & Hanáček (2013) pruned
to include one to several accessions of each Ruschieae clade (depending on clade size)
with an additional nine species sequenced in previous studies of the present authors.
Nuclear ITS sequences were downloaded from GenBank for accessions identical to the
cpDNA dataset; in five cases different accessions of the same species had to be used:
Dorotheanthus bellidiformis (Burm.f.) N.E.Br., Cheiridopsis excavata L.Bolus, Corpuscularia
lehmannii (Eckl. & Zeyh.) Schwantes, Jacobsenia kolbei (L.Bolus) L.Bolus & Schwantes, and
Prepodesma orpenii (N.E.Br.) N.E.Br. A species shown by Klak, Bruyns & Hanáček (2013),
to belong in Ruschieae clade L1, Drosanthemum pulverulentum (Haw.) Schwantes, was
regarded as part of the outgroup.
PCR and sequencing
We targeted four cpDNA markers and the nuclear rDNA ITS region. These included two
cpDNA markers, the trnS-trn G intergenic spacer region and the rpl 16 intron, that were
found to have the highest intra-generic divergence amongst the seven Drosanthemum
accessions used by Klak, Bruyns & Hanáček (2013); these regions were amplified using the
primers and protocols provided in the original paper. In addition, two cpDNA intergenic
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spacers, trnQ–5′ rps16 and 3′ rpS16 –5′ trnK, were amplified with primers trnQ(UUG) and
rpS16x1 and with primers rpS16x2F2 and trnK(UUU) , respectively (Shaw et al., 2007). The
nuclear ITS region was amplified as detailed in Hassan, Thiede & Liede-Schumann (2005).
Total genomic DNA was extracted from seedlings or from herbarium specimens
using the DNeasy Plant MiniKit (Qiagen, Hilden, Germany), following the protocol
of the manufacturer. For sequencing, the PCR products were sent to Entelechon
(Regensburg, Germany) or Eurofins (Ebersberg, Germany) resulting in 473 new sequences
of Drosanthemum species produced in this study. Forward and reverse sequences were
aligned with CodonCode Aligner, v.3.0.3 (CodonCode Corp., Dedham, Massachusetts,
USA). Sequence data of individual marker regions were aligned with OPAL (Wheeler &
Kececioglu, 2007) and checked visually using Mesquite v.3.51 (Maddison & Maddison,
2018). All sequences newly generated in this study have been submitted to ENA (for
accession numbers see Supplemental Information 1).
Phylogenetic analyses
Phylogenetic tree inference
We used maximum likelihood (ML) and non-parametric bootstrapping (BS) analysis on a
concatenated cpDNA dataset (comprising all four regions) including only Drosanthemum
species (‘Drosanthemum’ dataset: 134 accessions), and on a dataset also including outgroup
species (‘Ruschioideae’ dataset: 188 accessions; see Taxon sampling ) to infer the placement
of the Drosanthemum species in relation to the other Ruschioideae lineages. Note that
prior to this concatenated cpDNA analysis, each marker was analysed individually and in
various division schemes (several data matrices were tested: partitioned and unpartitioned,
also including or excluding the most-divergent and length-polymorphic rps16-trnQ spacer
region, and including/excluding an ITS partition; raw data, code and results are available
at Dryad, Liede-Schumann et al., 2019). No supported topological discordances were
present; thus we used a concatenated four-markers cpDNA dataset. ML tree inference
and BS analysis relied on RAxML v. 8.0.20 (Stamatakis, 2014), partitioned and set to
allow for site-specific variation modelled using the ‘per-site rate’ model approximation
of the Gamma distribution (Stamatakis, 2006). Duplicate sequences were reduced to a
single sequence resulting in 131 accessions in the ML cpDNA tree of Drosanthemum. The
same RAxML settings were used for the ‘Ruschioideae’ dataset. To obtain probability
estimates for the most likely Drosanthemum (ingroup) root, we used the evolutionary
placement algorithm (EPA; Berger, Krompass & Stamatakis, 2011) implemented in RAxML
and following the analytical set-up of Hubert et al. (2014) and Grímsson, Grimm & Zetter
(2018). EPA provides probability estimates (Berger, Krompass & Stamatakis, 2011) for
placing a query sequence (here: outgroup taxa representing the Ruschieae) within a
given topology (here: ML Drosanthemum cpDNA tree) offering identifying a consensus
outgroup-based root while minimising potential biases (e.g., long-branch attraction,
LBA; Bergsten, 2005). To do so, we queried a set of 47 Ruschieae species and calculated
a probability estimate (pR ) by averaging the likelihood weight ratios of query taxa per
inferred rooting scenario over all queried taxa.
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Phylogenetic network inference
We investigated competing support patterns within Drosanthemum by means of BS
consensus networks (Holland & Moulton, 2003; Grimm et al., 2006; Schliep et al., 2017)
using SplitsTree v. 4.1.13 (Huson & Bryant, 2006) and up to 1000 BS (pseudo-) replicate
RAxML trees (see paragraph above). The number of necessary BS replicates was determined
using the extended majority bootstrap criterion (Pattengale et al., 2009). Additionally, we
investigated within-lineage differentiation of subclades within Drosanthemum (‘subclade’
refers here to the nine clades within the genus Drosanthemum defined in the Results section)
using median-joining (MJ; Bandelt, Forster & Röhl, 1999) networks for the cpDNA dataset
and statistical parsimony (SP; Templeton, Crandall & Sing, 1992) networks for the ITS
data. MJ networks were computed with Network v.5.0.0.3 (Fluxus; available online
http://www.fluxus-engineering.com/sharenet.htm) with default settings and no character
weighting and SP networks with pegas v0.11 (Paradis, 2010) in R. In the MJ network
analyses, we used reduced sequence alignments differentiating four sequence patterns
at the intra-subclade level: (i) single-nucleotide polymorphisms (SNPs); (ii) insertions,
duplications and deletions (indels), represented by a single character because gaps are
treated as 5th base by Network by default; (iii) length-polymorphic sequence motifs (LP,
such as multi-A motifs, which were only considered when including mutations additional
to length variation; this category also includes more complex length-polymorphic patterns
such as length-polymorphic AT-dominated sequence regions); and (iv) oligo-nucleotide
motifs (ONM), short motifs with apparently linked mutations that can slightly differ in
length, which were treated as a single mutational event; inversions, like the ones found in
the pseudo-hairpin structure of the trnK-rps16 spacer, are a special form of ONMs. The
highly divergent, length-polymorphic ‘high-div’ region characterising the 5′ end of the rps
16-trnQ intergenic spacer, was generally excluded from the analysis but included in the
haplotype documentation (see Liede-Schumann et al., 2019: file Haplotyping.xlsx).
The reasoning for the use of MJ and SP networks is because there were few consistent
mutations at the intrageneric level within subclades—this results in a flat likelihood surface
of the tree space and, in this situation, parsimony can be more informative than probabilistic
approaches (Felsenstein, 2004). In contrast to phylogenetic trees, MJ networks include all
equally parsimonious solutions to a dataset and produce n-dimensional splits graphs that
can include topological alternatives. Also, MJ and SP haplotype networks directly depict
ancestor-descendant relationships, and hence, can assist in deciding whether inferred
clades in the tree are monophyletic in a strict sense, i.e., groups of inclusive common
origin (Hennig, 1950; see also Felsenstein, 2004, chapter 10). Because the MJ networks can
easily become diffuse or complex, especially when analysing interspecific relations, we
summarized the inferred haplotypes into haplotype groups for visualizing and interpreting
MJ networks.
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Table 2 Alignment and analysis parameters for the targeted sequence regions.
Gene region
Matrix dimension
(OTU × characters)
NLD
PUC
DAP
NBS
Approx.
model
ITS
112 × 440a
trnS-trnG
121× 1157
27
2.7%
130
500
aabcde
34
30.8%
305
800
rpl16 intron
112× 1193
aabbba
36
22.0%
201
450
aabaaa
trnK-rps16
rps16-trnQ
122× 768
31
20.6%
241
450
aabccc
127 × 788b
19
21.8%
245
550
aababa
Notes.
a
Only ITS1 and ITS2, flanking rRNA and 5.8S rRNA genes not included.
b
After exclusion of the ‘high-div’ region.
NLD, number of literally duplicate (identical) sequences; PUC, proportion of undetermined matrix cells (gappyness); DAP,
number of distinct alignment patterns; NBS, number of necessary BS pseudoreplicates; Approx. model, approximate of the
DNA substitution model optimized by RAxML for each gene region (in alphabetical order: A ↔ C, A ↔ G, A ↔ T, C ↔ G, C
↔ T, G ↔ T).
RESULTS
Patterns of DNA sequence diversity
We targeted the most variable cpDNA gene regions currently known for Aizoaceae, which
provided a relatively high number of distinct alignment patterns (Table 2), although each
cpDNA marker on its own provides low topological resolution (single plastid gene-region
ML trees and BS consensus networks are provided in Liede-Schumann et al. (2019). Lengthpolymorphism was common, hence, the high proportion of gaps (undetermined cells) in
the alignments, but often restricted to duplications or deletions, rarely insertions, and
explicitly alignable. An exception was the rps16-trnQ intergenic spacer, which includes
regions with extreme length-polymorphism and highly complex sequence patterns that
are only alignable amongst closely related species. A notable feature is a ‘pseudo-hairpin’
sequence found in the trnK-rps16 intergenic spacer, which includes a partly clade-diagnostic
strictly complementary upstream-downstream sequence pattern composed of duplications
of two short sequence motifs and subsequent deletions and a ‘‘terminal’’ inversion (shown
in the coding example in Supplemental Information 2, Fig. S2A; for more details see
Liede-Schumann et al., 2019: Haplotype.xlsx).
In general, cpDNA sequence patterns in Drosanthemum are highly diagnostic at and
below the level of major clades, in most cases allowing identification of haplotypes or
clade-unique substitution pattern. This includes a few, potentially synapomorphic (sensu
Hennig, 1950: uniquely shared derived traits) single-base mutations in generally lengthhomogenous sequence portions (see Liede-Schumann et al., 2019). Indel patterns appear to
be largely homoplastic, but sometimes diagnostic at the species level or for species flocks. In
contrast, mutation patterns in the length-homogeneous (SNPs) and length-polymorphic
regions (LP, indels, ONMs) are largely congruent, with few conflicting signals, for taxon
splits.
The nuclear-encoded ITS region has low divergence and contains little signal for tree
discrimination, which is typical for the Aizoaceae (e.g., Klak, Bruyns & Hanáček, 2013),
and was not included for defining major clades or testing their coherence with the earlier
proposed subgenera. Still, the genetic diversity present (Table 2) allows for the identification
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of more ancestral vs. more derived genotypes (Supplemental Information 3), which were
mapped onto the cpDNA tree (Fig. 2).
Phylogenetic inference and potential Drosanthemum roots
The ML tree of Drosanthemum (based on the concatenated cpDNA dataset) indicates
nine moderately (>65% BS support) to well supported (>90% BS support) clades (Fig. 2).
Seven of these group in two major clades, with high support for the clade I+II+III+IV
(98% BS support; addressed informally as ‘Drosanthemum core clade’, Fig. 3) and low
support for the second clade V+VI+VII (58% BS support). Clades VIII and IX have
ambiguous affinities (results not shown; for full documentation see Liede-Schumann et al.,
2019). Two species, D. longipes (sister to clade VII) and D. zygophylloides (sister to VIII)
are not included in the nine described clades (see Discussion –Phylogenetic inference reflects
taxonomic classification). Notably, the nine clades overall group into six lineages (clades
I–IV, V+VI, VII+ D. longipes, VIII, XI, and D. zygophylloides), but relationships among the
six lineages were weakly supported. Specifically, the earliest branching events in the ML
tree are ambiguously resolved (Fig. 2)
The ML tree of Ruschioideae (based on the ‘Ruschioideae’ dataset) inferred
Drosanthemum a monophyletic sister to Ruschieae (100% BS support, Fig. S4.1), supporting
the ‘core ruschioids’’ hypothesis (Table 1; for details see Liede-Schumann et al., 2019). In
this tree, the topology within Drosanthemum, however, differs in parts (clade VIII and
IX successive sister to the rest; not supported) from the tree inferred by the analysis of
the ‘Drosanthemum’ dataset (Fig. 2; both datasets comprise the same four concatenated
cpDNA regions). Taken together, phylogenetic inference is consistent with a rapid initial
diversification within Drosanthemum that was potentially too fast to leave a signal in
cpDNA sequence variation in the studied markers.
Placement of the 49 queried outgroup taxa indicates eleven potential Drosanthemum root
positions (Fig. 3). However, six of these positions are unlikely considering the probability
estimates pR (an order of magnitude lower), number of supporting queries (0 to 2), and
phylogenetic evidence (Fig. S4). The remaining five root positions are summarized as
follows: scenario 1, clade I–IV sister to clade V–IX, supported by 33 queries and pR =
0.26 (Fig. 2; Fig. S4.2); scenario 2, clade IX sister to the rest, eight queries and pR = 0.23
(Fig. S4.3); scenario 3, clade VIII + D. zygophylloides sister to the rest, one query and pR
= 0.14 (Fig. S4.4); scenario 4, cladeV–VII + D. longipes sister to clade I–IV + VIII + D.
zygophylloides + IX, four queries and pR = 0.14 (Fig. S4.5); scenario 5, clade I–IV + VIII
+ D. zygophylloides sister to clade V–IIV + D. longipes + IX, supported by zero queries and
pR = 0.14 (Fig. S4.6). Because the outgroup samples are notably distant in the targeted
plastid gene regions to Drosanthemum favouring attraction of most distinct accessions,
scenarios 3–5 may be artefacts generated by outgroup-ingroup (long) branch attraction.
Scenario 1 is identified as the most likely root position and has additionally the highest
probability estimate and number of supporting queries by the distribution of ITS genotypes,
indicating underived variants in clade I and V and both un- and derived ITS variants in
the smaller clades outside ‘Drosanthemum core clade’ (Fig. 2; see also Results, Identification
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Nuclear ITS genotypes
(not included during tree inference)
D. hispidum HH 33866
D. hispidum HH 34262
D. floribundum HH 34403
D. candens HH 31897
D. candens HH 25014
D. spec. HH 30568
D. spec. HH 32382
D. candens HH 30541
D. dejagerae HH 31812
[Xamera]
D. spec. HH 33182
D. tuberculiferum HH 32398
D. prostratum HH 34592
D. prostratum aff HH 34316
D. archeri HH 31788
D. archeri HH 31774
D. glabrescens HH 32256
D. muirii HH 32209
D. ambiguum HH 30388
D. opacum HH 32212
D. spec. HH 25365
D. spec. HH 34496
D. spec. HH 32392neu
D. spec. Mucina 161005_14
Species with genus-consensus ITS
Species with ITS deviating by a single
mutation from the genus-consensus
Ic
Least derived V-type ITS, one consistent
transversion to genus consensus
Ia
Bootstrap support
75 — 89.9
69
I
90 — 100
(Ib)
II
77
1 CU, 1 Sh
III
subg.
Drosanthemum
D. intermedium HH 32200
D. intermedium HH 32439
D. spec. HH 34472
D. spec. HH 25376
D. framesii HH 32339
D. muirii HH 30321
D. subplanum HH 32259
D. oculatum HH 31710
D. schoenlandianum Bruyns 7172
D. oculatum HH 31680
D. schoenlandianum HH 25751
D. latipetalum HH 31568
D. latipetalum HH 31605
D. nordenstamii HH 31533
D. latipetalum HH 32217
D. spec. HH 32218
D. marinum HH 32205
D. fourcadei HH 33900
D. fourcadei HH 34404
D. dipageae HH 34409
D. spec. HH 34424
D. spec. HH 34586
D. delicatulum HH 30782
[Drosanthemum]
D. delicatulum HH 32462
D. curtophyllum HH 32673
D. subclausum HH 25737
[Drosanthemum]
D. praecultum HH 31824
D. obibense HH 25979
D. obibense HH 25972
D. luederitzii HH 26095
D. nollothense HH 31547, HH31569*
IIIb
75
IV
99
2–3 CU, 2 Sh
Va
V
subg.
Vespertina
D. micans LeRoux 83_2
D. micans HH 34597
D. boerharvii HH 34596
D. hallii HH 34610
D. boerharvii HH 34478
D. speciosum HH 34619
D. pulchrum HH 34712
D. speciosum Bruyns 9006
D. chrysum HH 34631
D. austricola HH 34695 [Ossicula] D. boerharvii HH 34697*, D. lavisii HH 34693*
D. flavum HH 34706
D. cereale HH 34492
D. cereale HH 34491
D. cereale HH 34490
D. flammeum HH 34460
D. brakfonteinense HH 34688
D. edwardsiae HH 34648
D. edwardsiaeHH 34651
D. uniondalense HH 34813
D. boerhavii HH 34480
[Vespertina]
3 Sh
VII 97
IX 98
2 Sh
VIII
0.001
99
1 CU, 1 Sh
D. attenuatum HH 11826
D. attenuatum aff HH 34650
D. pallens Bayer 7454
D. striatum HH 29013
D. striatum HH 34698
D. spec. HH 34699
3 (–4) CU, 1 Sh
91
subg.
Speciosa
D. hispifolium HH 34587
D. hispifolium HH 32206
Vb
VI
Drosanthemum
(IIIa)
D. gracillimum Bruyns 7170
82
Xamera
subg.
D. brevifolium HH 32242
D. ramosissimum US110492
D. cymiferum HH 25811
[Quastea]
D. cymiferum HH 31686
D. acutifolium HH 32406
D. erigeriflorum HH 26196
D. parvifolium HH 30399
D. spec. HH 25424
D. spec. HH 33490
D. spec. HH 31925
D. spec. HH 31059
D. spec. HH 26170a
D. crassum HH 25896
D. spec. HH 32404
D. albiflorum HH 32383
D. lique HH 34447
D. spec. HH 34804
D. spec. HH 32378
subg.
reduced by factor 2
subg.
Ossicula
D. striatum HH 34720
subg.
D. acuminatum HH 34608
D. ecclesianum HH 34814
D. ecclesianum HH 34815
D. semiglobosum HH 34593
D. thudichumii HH 34714
D. bicolor HH 32459
D. papillatum HH 34614
D. papillatum HH 34607
D. papillatum HH 34624
D. papillatum HH 34629
D. calycinum HH 32211
D. papillatum HH 34456
D. expersum HH 34598
D. expersum HH 34590
D. cymiferum HH 32250
D. longipes Bruyns 6037
[Decidua]
D. longipes van Jaarsveld s.n.
D. deciduum HH 32241
D. deciduum Klak 1638
D. anemophilum van Jaarsveld s.n.
D. inornatum HH 32654
D. inornatum Bruyns 10066
Necopina
subg.
Quastea
subg.
D. longipes
Decidua
subg.
D. asperulum Bruyns 9005
D. tetramerum HH 34488
D. asperulum HH 34502
D. quadratum Bruyns 7812
D. quadratum HH 34503
Quadrata
D. zygophylloides Mucina 130216_4
D. zygophylloides Klak 830
D. zygophylloides
Figure 2 Phylogeny of Drosanthemum. ML tree inferred by partitioned analysis of the cpDNA sequence
data. Edge lengths are scaled on expected number of substitutions. The nine main clades are annotated by
roman numbers I–IX and coloured branches, with ML bootstrap support indicated by edge width (values given for the nine main clades). Bars and names to the right indicate subgeneric classification sensu
Hartmann, 2007. An asterisk after tip names indicate accessions with literally duplicate sequences. CU,
clade unique ITS mutation pattern(s); Sh, shared ITS mutation pattern found occasionally also in other
clades. Rooting is according to the most likely position inferred by outgroup-EPA (scenario 1; outgroups
removed).
Full-size DOI: 10.7717/peerj.8999/fig-2
Liede-Schumann et al. (2020), PeerJ, DOI 10.7717/peerj.8999
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D. no
ll
othe
n
se
D. brevifolium
D. ramosissimum
dei
e
D. fourca
agea
D.
cy
c.
D. spe
sii
3
me
H
ii H
30
IV
iae
dw
ar
ds
80
erh
av
VIII
um
IX
m
liu
es
D. long
ip
ifo
uum
nuatu
m
isp
H
9
um
69
34
D. deci
d
D. cereale
h
D.
.H
ec
sp
D.
D. inornatum
rum
D. hallii
D. att
e
tum
ram
e
D. micans
D. austricola , D. boerhavii HH 34697, D. lavisii
D. chrysum
D
D. . bra
fla kfo
mm nt
eu eine
m ns
e
D. st
oko
ei
D. striatum
D. te
t
34
D. pulchrum
D. speciosum
ria
D. s
p
b
D.
st
D.
m
ac
D.
su
bo
glo
a
D.
n
em
ii
mi
op
r
D.
thu
d
ich
um
tum
ina
D. calycinum
D. papillatum
um
ian
s
ccle
D. e
m
rsu
xpe rsum
D. e expe
D.
0
3225
m HH
miferu
D. cy
456
um HH 34
D. papillat
BSML = 100
um
hil
m
D. striatum
er
illimu
D. b
icolo
ec.
VII
HH
D. speciosum
Vb
se
D.
sp
D. a
ulum
e
ns
ale
nd
nio
rh
oe
u
D.
D. flavum
D. gra
c
V
VI
s
drat
ua
D. q
D.
e
44
D. spec.
HH 32378
oide
ii
8
47 rhav
e
34
H . bo
ii H D
av
Va
yll
goph
D. zy
6
59
D. spec.
HH 34804
III
D. spec.
Mucina 161005-14
ir
mu
D.
D. spec. HH 25365
D. spec. HH 34496
HH
25424
H3
I
Ia
HH
323
92n
eu
D. in
term
ediu
m
ra
D. f
21
pR <<0.1 0.1 0.2 0.3
334
D.
bo
D. spec.
HH 34472
76
253
H
c. H
HH
D. albiflorum
D. subp
e
D. sp
90
p
D. s
D. lique
II
Ic
lanum
Outgroup-inferred
root possition
ec.
ium m
fol oru
uti rifl m
ac ge oliu
D. D. eri arvif
p
D.
IIIa
Ib
D. oculatum
D. schoenlandianum
r
de
lue
e
D. bibens
D. o
ii H
c. HH
'Drosanthemum
ii core clade'
itz
D. lue
mi
deritz
fer
ii
um
D. spec. HH 26170a
D. crassum
D. spec. HH
32404
D.
D. s spec. H
pec H 3
. HH 10
319 59
25
ii H
s
H3
D. a
22
mbig
D. m
uum 09
3221 arinum
D. latipet
8
alum H
H32217
D. opac
um
D. latipetalum HH 31568
D . latisepalum HH 31605
ii
D. nordenstam
D. sp
e
IIIb
D. praecu
D. tubercu
res
cen
ltum
liferum
ae
ger
eja 182
D. d HH 33
ri
.
he
pec
arc
D. s
D.
ab
D. d
ip
897
m
atu
str
pro
D.
D. curtophyllum
D. subclausum
D. spec. HH 344
D. spec. HH 345 24
86
H 31
um
nd
D.
gl
D. delicatulum
D. candens HH 25014
ibu
H
dens
or
D.
mu
ir
D. spec. HH
30568
D. canden
D. spec. s HH 30541
HH 32382
n
D. ca
fl
D.
D. hispidum
Figure 3 Bootstrap consensus network of Drosanthemum. Consensus network based on 600 pseudoreplicate samples inferred by partitioned ML analysis of the cpDNA sequence data. Edge lengths are
proportional to the frequency of the phylogenetic split in the pseudoreplicate sample. Branch colours and
labels are as in Fig. 2. Black arrows indicate potential root positions inferred by outgroup-EPA, with arrow
size proportional to the probability estimate pR (Supplemental Information 4, Table S4).
Full-size DOI: 10.7717/peerj.8999/fig-3
of ITS genotypes). Overall, the results obtained by outgroup-EPA are consistent with a fast
radiation generating the main lineages early in the evolution of Drosanthemum.
Inter- and intra-clade differentiation patterns
The haplotype analyses (of each gene region) is in overall congruence with the combined
cpDNA tree (Fig. 2). However, in some genes and/or clades coherent mutational
patterns are shared by several species, which lack uniquely shared sequence patterns
in other gene regions. Thus, detailed haplotype networks (Figs. 4–7) further illuminate
phylogenetic relationships in clades VII–IX (and the two isolated species D. longipes and
D. zygophylloides), and further corroborate subgroups within clades I, III, and V (Figs. 2
and 3).
Clade I is divided into three subclades and the haplotype analysis supports a monophyly
of clades Ia and Ic, but not Ib (Figs. 4A–4D). Members of clade Ib are characterized by
haplotypes either ancestral to those found in clades Ia and Ic (rpl16 intron, trnK-rps16 ) or
unique and strongly divergent from each other (rps16 -trnQ). Clade II haplotypes are more
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Figure 4 Median-joining networks of Drosanthemum clades I–IV. Collapsed Median networks;
collapsed network portions (haplotype groups) represented by by circles; letters in bold refer to LiedeSchumann et al. (2019) (file Haplotyping.xlsx; archive includes full networks). Circle size does not
show haplotype frequency but gives the maximum number of mutations between grouped haplotypes/
connective medians (a group’s dimension); edge length (minimum) number of mutations between
haplotype groups (Grimm, 2019). (A–D) Clade I. Note that subclade Ib is paraphyletic to clades Ia and
Ic according to rpl16 intron, trnK-rps 16 and rps16-trnQ. Filled black circles (medians) denote position
of the consensus sequence of the clade. (E–H). Clades III and IV. Note that grade IIIa (cf. Fig. 2) bridges
between haplotype groups diagnostic for clades IIIb and IV, which could be an indication of paraphyly,
i.e., grade IIIa species originate from a radiation predating the formation and subsequent radiation of
clades IIIb and IV.
Full-size DOI: 10.7717/peerj.8999/fig-4
similar to ancestral haplotypes in clade I than to those in clades III or IV. The haplotypes
in clades III and IV are very similar to each other (Figs. 4E–4H). Clade III is divided into a
more diverse (likely paraphyletic) grade IIIa and a monophyletic clade IIIb (Figs. 2 and 3,
4E–4H). Within clade III, clade IIIb forms an increasingly derived (monophyletic) lineage
(D. luederitzii + D. obibense → D. nollothense → D. brevifolium + D. ramossissimum)
that starts with grade IIIa individuals having D. cymiferum-like morphology but being
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Figure 5 Median-joining networks of Drosanthemum clades V–IX. Collapsed Median networks;
collapsed network portions (haplotype groups) represented by circles; letters in bold refer to (LiedeSchumann et al., 2019) (file Haplotyping.xlsx; archive includes full networks). Circle size does not
show haplotype frequency but gives the maximum number of mutations between grouped haplotypes/
connective medians (a group’s dimension); edge length (minimum) number of mutations between
haplotype groups (Grimm, 2019). A–D. Clades V and VI. Note the central (trnS-trnG) or ancestral
(rps16-trnQ) position of D. gracillimum (no rpl16 and trn K–rps16 data available). E–H. Clades VII–IX.
Note that members of each clade are clearly differentiated but differ in the level of derivation per gene
region.
Full-size DOI: 10.7717/peerj.8999/fig-5
genetically distinct from D. cymiferum. Clade V includes two sequentially coherent and
mutually exclusive (reciprocally monophyletic) clades, Va and Vb (Fig. 3). In general,
haplotypes of clade Vb show more unique shared mutational patterns than those of clade
Va (Figs. 5A–5D). Figures 5A–5D also include the relatively similar haplotypes of the sister
lineage, clade VI, which can be used to root the MJ networks (note that the edge length
reflects the difference in the variable genetic patterns within clade V and does not include
sequence patterns uniquely found in clade VI). Two markers, trnK-rps16 and trnS-trnG,
reflect the potential reciprocal monophyly of both clades. Drosanthemum gracillimum is
not included in clade Va or Vb (Figs. 2, 3). Only two of the considered cpDNA markers are
available for this species, trnS-trnG and rps16-trnQ, with no lineage-diagnostic sequence
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pattern and obviously showing the putative ancestral haplotype within clade V (Figs.
5A–5D).
Whereas haplotypes can be very divergent at the inter- and even intra-clade level (e.g.,
Fig. 4), they are relatively similar to each other in the smaller clades VII–IX (Figs. 5E–5H).
Drosanthemum longipes trnS-trnG and rps16-trnQ haplotypes are highly similar to those
of clade VII. Each gene region has a series of mutational patterns in which D. longipes
and all members of clade VII are distinct from clade VIII and IX. In the lowest-divergent
trnK-rps16 intergenic spacer region, the D. longipes haplotype can directly be derived from
the one of clades VIII and IX (Figs. 5E–5H). Drosanthemum longipes is genetically closer
to the putative Drosanthemum ancestor than to members of clade VII. In contrast, the
haplotypes of D. zygophylloides are visibly unique within the genus (Figs. 5E–5H), which is
also reflected in its long terminal branches in the cpDNA tree (Fig. 2).
Identification of ITS genotypes in Drosanthemum
Analysis of nuclear ITS sequence variation reveals 62 genotypes, for which SP analysis
produces an overall, but highly reticulated, star-shaped network with genotypes linked to
various cpDNA lineages in the centre (Fig. 6; Supplemental Information S3). The least
derived but most common genotypes are found in distantly related clades: genotype 33 in
clade I and genotype 6 clade V (Fig. 6). Genotypes 6 and 33 resemble the consensus
of all ITS genotypes differing only by a single point mutation (note that genotype
33 collects several subtypes differing in an indel pattern that is ignored by the SP
network; Supplemental Information S3; for details see Liede-Schumann et al., 2019: files
Haplotype.xlsx, DataSummary.xlsx). Genotype 3 is shared by members of clade VII and IX
and is central to most other (including the most common 33 and 6; Fig. 6). Clades VII–IX
and the two phylogenetically isolated species, D. longipes, D. zygophylloides, have unique,
derived genotypes. The ITS genotypes of clades II, IIIb and IV can be derived from the
most ancestral ones in clade I and IIIa. The fact that ITS evolution, a stepwise derivation
of a putatively ancestral, consensus sequence into genotypes that are unique within clades
and can be mapped on the cpDNA phylogeny (Fig. 2) indicates that ITS differentiation in
the ‘Drosanthemum core clade’ is in overall congruence with the cpDNA tree.
Geographical clade structure in Drosanthemum
All clades retrieved in the present analysis show their own characteristic distribution
pattern. All clades are present in the south-western Cape, and three clades (VI, VII, VIII)
hardly extend beyond this narrow region. The species-rich clades I, II, and IV cover the
largest areas. Clade V extends along the southern Cape coast and clades III and IX extend
along the West coast (Fig. 7).
DISCUSSION
Genetic differentiation patterns indicate fast radiation initiating
diversification within Drosanthemum
Phylogenetic analysis, in-depth haplotype analyses of cpDNA, and mapping of ITS
evolution on the ML cpDNA tree point towards a rapid initial diversification within
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32
ML cp clade
I
II
III
39
IV
V
VI
VII
VIII
D. zygophylloides
58
IX
35
11
24
54
16
57
9
31
44
42
17
48
5
55
53
59
56
3
34
62
43
1
21
2
14
45
18
8
61
47
4
49
5
28
36
12
9
10
7
29
33
37
6
11
15
23
30
40
20
51
25
60
27
50
22
26
41
52
19
46
38
13
Figure 6 Statistical parsimony network of Drosanthemum ITS genotypes. Network inferred by analysis of the ITS sequence data under an infinite site model. Genotypes are indicated by circles coloured according to clades inferred by cpDNA sequence analysis (see Figs. 3 and 4). Circle size indicate absolute
frequency of genotypes (see legend). Black lines indicate steps in the network, filled black circles missing
genotypes, and dashed grey lines alternative links. Genotypes in the centre of the graph are ancestral, those
in the periphery most derived. Genotype 4 represents the genus consensus sequence found in several accessions of clade I (for details see Supplemental Information 3).
Full-size DOI: 10.7717/peerj.8999/fig-6
the genus Drosanthemum. The best outgroup-EPA inferred rooting position indicates
Drosanthemum species to group in two large clades, with clade I–IV, the ‘Drosanthemum
core clade’, sister to clade V–IX (Fig. 2). The uncertainty in root position (Fig. 3;
Supplemental Information S4) is consistent with a pattern expected in initial radiations
(Graham & Iles, 2009; Saarela et al., 2007). The star-like (but reticulated) structure of
the SP network (nuclear ITS data) suggests an initial bottleneck early in the evolution
of Drosanthemum followed by rapid diversification (Fig. 6, Supplemental Information
3). Similarly, the plastid sequence variation provides sufficient information to resolve
nine well-supported clades within the genus Drosanthemum. However, the ‘backbone’
relationships among the nine clades, or more specifically, the six lineages, are not resolved
(Figs. 2 and 3). Taken together, the difficulties to separate and clarify the exact sequence of
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Figure 7 Distribution of Drosanthemum. (A) Overall distribution of Drosanthemum in Africa. (B–
J). Clade-wise distribution of Drosanthemum species in southern Africa. Filled symbols indicate accessions used in the phylogeny, empty symbols indicate the remaining accessions in the occurrence dataset of
Drosanthemum. D.zygophyl., D.zygophylloides. Maps were created using the elevation above sea level data
from the WorldClim climate layers (Hijmans et al., 2005), with a spatial resolution of 30′ using the raster
library v2.8-19 (Hijmans, 2019) in R v3.5.3 (R Core Team, 2019).
Full-size DOI: 10.7717/peerj.8999/fig-7
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early branching events is a characteristic pattern in rapid evolutionary radiations among
the plant tree of life, and has been found at various phylogenetic levels, for example, in
Saxifragales (Fishbein et al., 2001), within the genus Hypericum (Hypericaceae; Nürk et al.,
2013; Nürk et al., 2015) and in a group of South American Lithospermum (Boraginaceae;
Weigend et al., 2010). It remains to be seen, however, whether analyses of nuclear markers
apart from ITS support the patterns retrieved here.
Phylogenetic inference reflects taxonomic classification
Within Drosanthemum, nine clades are revealed, which generally correspond to the
recognized subgenera (Hartmann, 2017a), although some exceptions exist. The deviations
in morphology-based classification and phylogenetic evidence produced in this study
reveals cryptic species and several new relationships. For example, the species D.
zygophylloides, D. gracillimum, and D. longipes, have either never been included into the
subgeneric classification (D. zygophylloides), or phylogenetic evidence indicates affinities
different from classification (D. gracillimum, D. longipes; Fig. 2). Considering our results,
these species cannot be included in any of the proposed subgenera (Hartmann, 2017a).
Note that both D. longipes and the species in clade IX shed leaves in summer and resprout
with the winter rains.
Subgenus Drosanthemum is revealed as biphyletic, with most of its species in clade I,
sister to clade II (subgenus Xamera; see below). Drosanthemum hispidum, the type species
of Drosanthemum, groups in clade I (subclade Ic; Fig. 2). The rest of the species classified in
subgenus Drosanthemum group within clade III. No morphological diagnostic characters
are obvious to distinguish the clade III species from those in clade I, and thus, clade III is
not yet circumscribed as a tenth subgenus. Likewise, subgenus Drosanthemum species in
clade I group in three subclades Ia, Ib, and Ic, but morphological characters defining these
clades cannot yet be named. Hence, this species-rich subgenus is obviously biphyletic, but
species assigned to it are not distributed all over the tree, i.e., subgenus Drosanthemum does
not appear to be a ‘‘dustbin’’ for species that cannot be assigned based on morphology to
any other subgenera.
The discussed clades I–III, together with clade IV, constitute the informally named
‘Drosanthemum core clade’. Clade IV corresponds to the night-flowering subgenus
Vespertina that is characterized by flowers of the long cone type (Rust, Bruckmann &
Hartmann, 2002). Subgenus Xamera (clade II) is characterized by usually six-locular
capsules and four tiny spinules below the capsule stalk and on older lateral branches
(Hartmann, 2007). Drosanthemum delicatulum and D. subclausum of clade II also show
this character, so that their listing under subgenus Drosanthemum in Hartmann (2017a:
508, 532) is clearly erroneous as is also indicated by the listing of D. subclausum among the
species of Xamera in Hartmann (2017a: p 495). Conversely, D. dejagerae L.Bolus, attributed
to Xamera by Hartmann (2007) and Hartmann (2017a) due to the presence of a six-locular
capsules characteristic for the subgenus, is placed in clade Ic (subgenus Drosanthemum
p.p.).
Of the six remaining subgenera, four—Speciosa (clade Va), Ossicula (clade Vb), Necopina
(clade VI), and Quastea (clade VII)—group in one clade that is, however, not well supported
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(Fig. 2) and also lacks obvious commonly shared, derived morphological characters. In
particular, the stout and often large capsules (to one cm diam.) of subgenus Speciosa
(Hartmann & Bruckmann, 2000) contrast strongly with the tender and smaller capsules
of the other three subgenera. However, bone-shaped closing bodies in the capsules,
considered unique for subgenus Ossicula, have also been found in capsules of Speciosa
species (Hartmann & Le Roux, 2011), reducing their potential as a diagnostic character
for Ossicula. This is illustrated by D. austricola L.Bolus, which is retrieved in subclade Va,
corresponding to subgenus Speciosa, despite its conspicuous bone-shaped closing body, a
character for which it was placed in Ossicula by Hartmann (2008).
While the subgeneric classification of Drosanthemum (Hartmann, 2007; Hartmann &
Liede-Schumann, 2014) is largely confirmed, a few unexpected placements of single species
deserve mentioning. Of the three samples of Drosanthemum cymiferum, attributed to
subgenus Quastea in Hartmann (2007), only one sample was retrieved in the Quastea clade
VII, the other two in clade III (Drosanthemum p.p.). This species was studied in some
more detail in Liede-Schumann, Meve & Grimm (2019), who did not find any consistent
morphological differences between these samples and suggested a case of cryptic speciation
(following the definition of Bickford et al., 2007). A similar case is found in D. muirii L.Bolus,
of which the two samples are retrieved with good support in subclades Ia and Ic, respectively
(Fig. 2).
Distinct geographic distributions in the Greater Cape Floristic Region
Inside the genus Drosanthemum, six lineages originate from a soft polytomy (precisely,
they root in an unsupported part of the tree; Fig. 2), suggesting a radiation right at the start
of the evolutionary history of Drosanthemum. To which extent this radiation was driven by
ecological or geographical factors remains an open question. Interestingly, several clades
comprising only 3–6 species are distributed over a restricted geographical range: clade VI
Necopina (6 spp), clade VII Quastea (4 spp), and clade VIII Quadrata (3 spp) are restricted
to the western part of the Cape Mountains (Figs. 7F–7I). One species-poor lineage, clade
IX Decidua (3 spp.), extends along the West Coast into Namibia Fig. 7J). Species in clade V,
14 in clade Va Speciosa and 6 in clade Vb Ossicula, are almost restricted to the fynbos of
GCFR (Fig. 7F), whereas the comparatively higher species number in Speciosa might be
the result of more thorough studies in this showy, horticulturally valuable subgenus (e.g.,
Hartmann, 2008; Hartmann & Le Roux, 2011). Notably, these clades are genetically and
morphologically coherent, that is, possess unique and derived sequence patterns as well as
characteristic morphologies.
The more or less narrow distribution pattern of these clades (Figs. 7F–7I) contrasts
to a wide distribution of the ‘Drosanthemum core clade’ (Figs. 7B–7E), harbouring more
widespread lineages with more species potentially indicating broader overall-habitat
preferences: clade IV Vespertina (12 spp), clade II Xamera (8 spp) and the genetically
and morphologically most diverse clade I (Drosanthemum p.p.; ≥ 55 spp; Fig. 7). The
bulk of species diversity has been described in subgenus Drosanthemum, which falls in
three subclades Ia–Ic not previously recognized (Fig. 2). These three subclades show
distinct distribution patterns, with Ia restricted more or less to the fynbos area of GCFR, Ic
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Aizoaceae
Ruschioideae
Sesuvioideae
genera
species
5
65
Aizooideae
Acrosanthoideae
5
116
1
6
Mesembryanthemoideae Apatesieae
1 a / 16 b
105
Dorotheantheae
7
12
Drosanthemeae
1
114
4
14
Ruschieae
106
~1,000 c / 1,600 d
7.0 e / 1.5 f Ma
core ruschioids
14.5 e * / 3.3 f Ma
19.0 e * / 4.5 f Ma
29.0 e / 6.0 f Ma
36.4 e Ma
40.5 e * / 7.9 f Ma
41.5 e Ma
Stem age Aizoaceae: 74.9 g / 51.5 e * / 13.8 f Ma
Figure 8 Phylogeny of Aizoaceae. A summary cladogram indicating recognized subfamilies (sensu Klak,
Hanáček & Bruyns, 2017a) and tribes (sensu Chesselet, Van Wyk & Smith, 2004) detailing the number
of genera and species and estimated node ages. Superscript letters denote reference: a, Klak & Bruyns
(2013); b, Hartmann (2017a, 2017b); c, Stevens (2001, onwards); d, Klak, Bruyns & Hanáček (2013); e, Klak,
Hanáček & Bruyns (2017b); f, Valente et al. (2014); g, Magallón, Gómez-Acevedo & Sánchez-Reyes (2015). A
superscript asterisk denotes ages according to Klak, Hanáček & Bruyns (2017a, Fig. S2).
Full-size DOI: 10.7717/peerj.8999/fig-8
extending far into the east and northeast, while Ib extends north to 28◦ S (Fig. 7B). Clade
III, composed of species hitherto considered to belong to subgenus Drosanthemum, shows
the most diverse distribution of all clades, with a southern group of poorly resolved species,
and a lineage of several species extending to the northernmost locality of Drosanthemum,
the Brandberg in Namibia (Liede-Schumann, Meve & Grimm, 2019; Fig. 7D).
Some more species-rich clades within Drosanthemum have also wide ecological
preferences, with representatives both at lower and higher elevations. Morphological
adaptations to arid habitats are capsules with deep pockets caused by false septa enabling
seed retention (Hartmann & Bruckmann, 2000), which have been evolved in parallel in
clade Ia and IIIb. However, whether the possession of false septa in the capsules is restricted
to species of arid habitats remains an open question.
CONCLUSIONS
In this study, we present a comprehensive phylogenetic investigation of Drosanthemum, a
morphologically diverse genus that has so far been relatively overlooked in evolutionary
studies of Aizoaceae. Our results confirm Drosanthemum (= Drosanthemeae) as sister
lineage to Ruschieae, which is in accord with the ‘core ruschioids’ hypothesis (Klak, Reeves
& Hedderson, 2004; Klak, Bruyns & Hanáček, 2013; Fig. 8). Additionally, our phylogenetic
evidence signifies Drosanthemum as a genetically well-structured but heterogenous lineage
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of mesomorphic plants that is, however, less species-rich than its sister clade; a pattern
of diversity distribution common in the plant tree of life (Donoghue & Sanderson, 2015).
Still, our analysis suggest that Drosanthemum is not simply a depauperate lineage sister to
a radiation, but instead exemplifies a radiation by itself as indicated by complex plastid
and nuclear DNA sequence differentiation patterns (Figs. 2, 3, 6), and the flower and fruit
diversity present in the genus that is unusual for Aizoaceae.
Occurrence patterns among the evolutionary lineages might further indicate geographic
factors playing a role in species diversification in Drosanthemum. While most of the
evolutionary history of the genus seem to have taken place in a relatively mesic environment
in the southwestern parts in the GCFR, several lineages apparently have started to adapt
to more arid and/or winter-cold areas. Genetically relictual species from at least two
early radiations co-exist among rapidly evolving lineages, reflecting species-delimitation
problems in species-rich clades. This is mirrored in the present study that largely supports
the current taxonomic concepts in Drosanthemum with few interesting exceptions, among
others, cryptic species.
ACKNOWLEDGEMENTS
Laco Mucina (Univ. of Western Australia) is thanked for a pleasant field trip and a
sample of D. zygophylloides and Hans-Dieter Ihlenfeldt (Univ. Hamburg) for contributing
several of the outgroup samples. SLS thanks the participants of the MSc Module F1 at the
University of Bayreuth from 2008 to 2015 for their work on Drosanthemum herbarium
specimens. Angelika Täuber and Margit Gebauer (UBT) are thanked for their enduring
and conscientious lab work.
ADDITIONAL INFORMATION AND DECLARATIONS
Funding
This work was supported by two awards of the Mesemb Study Group (M.S.G.) in 2010
and 2013. The publication of this work was funded by the German Research Foundation
(DFG) and the University of Bayreuth in the funding programme Open Access Publishing.
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:
Mesemb Study Group (M.S.G.).
German Research Foundation (DFG).
University of Bayreuth.
Competing Interests
Alastair J. Potts is an Academic Editor for PeerJ.
Liede-Schumann et al. (2020), PeerJ, DOI 10.7717/peerj.8999
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Author Contributions
• Sigrid Liede-Schumann conceived and designed the experiments, performed the
experiments, analyzed the data, authored or reviewed drafts of the paper, and approved
the final draft.
• Guido W. Grimm analyzed the data, prepared figures and/or tables, authored or reviewed
drafts of the paper, and approved the final draft.
• Nicolai M. Nürk prepared figures, authored and reviewed drafts of the paper, and
approved the final draft.
• Alastair J. Potts analyzed the data, authored or reviewed drafts of the paper, and approved
the final draft.
• Ulrich Meve performed the experiments, analyzed the data, authored or reviewed drafts
of the paper, curated herbarium material, and approved the final draft.
• Heidrun E.K. Hartmann conceived and designed the experiments, authored or reviewed
drafts of the paper.
DNA Deposition
The following information was supplied regarding the deposition of DNA sequences:
The sequences are available at ENA: LR030506 to LR030978.
Data Availability
The following information was supplied regarding data availability:
The data is available at Dryad: doi: https://doi.org/10.5061/dryad.n2z34tms2.
Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
peerj.8999#supplemental-information.
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