Pirani & al. • Molecular phylogeny of Acanthophyllum
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Molecular phylogeny of Acanthophyllum (Caryophyllaceae:
Caryophylleae), with emphasis on infrageneric classification
Atefeh Pirani,1,2 Shahin Zarre,1 Bernard E. Pfeil,2 Yann J.K. Bertrand,2 Mostafa Assadi3 & Bengt Oxelman2
1 Department of Plant Biology, and Center of Excellence in Phylogeny of Living Organisms, School of Biology, College of Science,
University of Tehran, P.O. Box 14155-6455 Tehran, Iran
2 Department of Biological and Environmental Sciences, University of Gothenburg, Box 461, 40530 Göteborg, Sweden
3 Research Institute of Forests and Rangelands, P.O. Box 13185-116, Tehran, Iran
Authors for correspondence: Atefeh Pirani, pirani@khayam.ut.ac.ir; Shahin Zarre, zarre@khayam.ut.ac.ir
DOI http://dx.doi.org/10.12705/633.39
Abstract Despite being one of the larger genera of Caryophyllaceae with about 60 cushion-forming subshrubby species,
Acanthophyllum is represented poorly in previous molecular phylogenetic studies. The genus is an important component of
the subalpine steppe flora in Central to Southwest Asia. Although the placement of Acanthophyllum in the tribe Caryophylleae
and a close relationship to Allochrusa has already been suggested, the monophyly of the genus and its infrageneric taxa, as
well as its relation to other closely related genera, have not been addressed. We have assembled datasets of nuclear ribosomal
internal transcribed spacer (ITS) sequences and intron sequences of the chloroplast gene rps16 for 47 Acanthophyllum species
and 63 species of 11 additional genera from Caryophylleae. Phylogenetic analyses were performed using maximum parsimony,
maximum likelihood and Bayesian methods. Our analysis suggests that Allochrusa, Diaphanoptera, Ochotonophila and Scler
anthopsis are nested within Acanthophyllum but that the traditionally recognized sections of Acanthophyllum are monophyletic
after reassignment of a few species. Emarginate petals may be a synapomorphy for one of the two basal clades of Acanthophyl
lum. Moreover, non-monophyly of the genera Gypsophila and Diaphanoptera is suggested by the present study. The age of the
crown clade of Acanthophyllum s.l. is estimated to be 11.1 Ma by *BEAST species tree analysis.
Keywords Acanthophyllum; Caryophyllaceae; Irano-Turanian; ITS; molecular phylogeny; rps16; species tree
INTRODUCTION
Acanthophyllum C.A.Mey., with ca. 60 species, is a mainly
Irano-Turanian genus (Bittrich, 1993; Ghaffari, 2004) that inhabits areas between Syria and western China (Ghaffari, 2002).
The diversity center of the genus is the Khorassan-Kopet dagh
floristic province in NE Iran and neighboring areas in Afghanistan and Turkmenistan (Ghaffari, 1989, 2004; MahmoudiShamsabad & al., 2012).
Acanthophyllum species are small, shrubby, cushionforming perennials with spiny leaves. They grow in exposed
habitats on sandy or stony hills and rocky slopes (Fig. 1A–B).
They are important components of the steppe (Zohary, 1973)
and mountain vegetation in Central and Southwest Asia.
Several biologically active triterpene saponins have been
reported from different species of Acanthophyllum (Gaidi & al.,
2000, 2004). The genus could be medicinally interesting due
to the highly cytotoxic properties of many saponins. It has
been shown that cytotoxic compounds have potential antitumor
activity (Sparg & al., 2004). Saponins of some Acanthophyl
lum species have been suggested as substitutes for synthetic
surfactants in shampoo (Aghel & al., 2007).
Although Acanthophyllum is one of the larger genera in
the Caryophyllaceae, it has been subjected to few systematic
and phylogenetic studies and no inclusive monograph of the
genus is available. The number of species was estimated to 56
in Bittrich (1993), based on Flora Iranica (Schiman-Czeika,
1988), but 14 species should be added based on Flora of the
U.S.S.R. (Schischkin, 1936), Flora of Uzbekistan (Vvedensky,
1953) and Flora of Tajikistan (Ovchinnikov, 1968). With a few
recently described species (Aytaç, 2001; Mahmoudi-Shamsabad
& al., 2012; Pirani & al., 2013), the species number exceeds 70.
On the other hand, several synonymies have been suggested
by regional studies in Iran and Pakistan (Ghazanfar & Nasir,
1986; Basiri-Esfahani & al., 2011). Clearly, the taxonomy of
Acanthophyllum is in need of revision.
Boissier (1867) recognized five groups within Acantho
phyllum, delimited mainly on the basis of inflorescence and
floral features. The basic framework of his classification was
followed in the next three major taxonomic treatments of the
genus by Golenkin (1893), Schischkin (1936) and SchimanCzeika (1988). Golenkin (1893) recognized 19 species, including the genus Allochrusa Bunge as a sixth section. Schischkin
(1936) classified the genus into two subgenera (Euacantho
phyllum and Allochrusa), recognizing two sections in subg.
Allochrusa (Bunge) Schischk. Schiman-Czeika (1988) transferred sect. Pseudacanthophyllum (Boiss.) Rech.f. from Gyp
sophila L. to Acanthophyllum and a new section, Scapiflora
Received: 2 Jul 2013 | returned for first revision: 6 Sep 2013 | revision received: 11 Feb 2014 | accepted: 13 Feb 2014 | not published online ahead of
inclusion in print and online issues || © International Association for Plant Taxonomy (IAPT) 2014
592
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Pirani & al. • Molecular phylogeny of Acanthophyllum
Schiman-Czeika, was introduced. Acanthophyllum sect. Scapi
flora includes four species, among which two were transferred
from Gypsophila and Saponaria L. A comparison of the four
major infrageneric classifications of Acanthophyllum (with
type for each section) is given in Table 1.
Hitherto used characters in infrageneric classifications of
Acanthophyllum are mostly related to inflorescence, shape and
texture of bracts, and number of ovules. The general inflorescence type in Acanthophyllum is dichasial cymes arranged in
terminal heads and/or axillary verticillasters. The exception is
Fig. 1 (in two parts). A–B, Natural habitats of Acanthophyllum; C–E, A. speciosum (sect. Oligosperma); F–G, A. gracile (sect. Macrostegia);
H, A. bracteatum (sect. Macrostegia); I–K, A. crassifolium (sect. Acanthophyllum); L–M, A. glandulosum (sect. Pleiosperma); N, A. spinosum
(sect. Pleiosperma); O–P, A. caespitosum (sect. Oligosperma). — Photos by Hamid Moazzeni & Atefeh Pirani.
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Pirani & al. • Molecular phylogeny of Acanthophyllum
sect. Macrodonta Boiss. that includes species with 1-flowered
(rarely up to 3-flowered) terminal inflorescences. Among inflorescence characters applied in sectional classification are (1)
1-flowered versus multi-flowered inflorescences, (2) terminal
versus elongated inflorescences and (3) richly branched versus
poorly branched inflorescences (richly branched inflorescences
are composed of several partial dichasial units distributed
along the stem or confined to several apical nodes of the stem,
whereas poorly branched inflorescences are composed of one,
or rarely up to three condensed dichasial unit(s) confined to
only one terminal node of the stem; Fig. 2A–B). Inflorescence
594
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variation in A. sect. Acanthophyllum, sect. Macrostegia Boiss.,
sect. Oligosperma Schischk. and sect. Pleiosperma Boiss. is
shown in Fig. 1C–P.
As with many other genera in the family (Kurtto, 2001;
Oxelman & al., 2001; Fior & al., 2006), the limits of Acantho
phyllum in relation to closely related genera are controversial.
There have been several transfers of species among Acantho
phyllum, Gypsophila, Saponaria and Dianthus L. by different
authors. Moreover, the relationships to the small genera, Scler
anthopsis Rech.f., Allochrusa, Ochotonophila Gilli, Diapha
noptera Rech.f., and Kuhitangia Ovczinn. need to be elucidated.
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Pirani & al. • Molecular phylogeny of Acanthophyllum
Acanthophyllum has been included with few samples in
a small number of molecular studies of Caryophyllaceae. In
a phylogenetic analysis based on the chloroplast DNA matK
region, Allochrusa versicolor Boiss. is sister to Acanthophyl
lum sordidum Bunge ex Boiss. (Fior & al., 2006), but as only
one Acanthophyllum species was included, it is not possible to
judge whether Allochrusa should be retained or not. Greenberg
& Donoghue (2011) showed that All. versicolor, A. sordidum and
A. paniculatum Regel & Herder make a monophyletic clade,
reinforcing the idea that the two genera are closely related. Allo
chrusa was sister to A. sordidum, thus rendering Acanthophyl
lum paraphyletic. The latter work is the most comprehensive
molecular study of Caryophylleae to date.
In the few divergence time studies that included Acantho
phyllum, the approximate age of the Acanthophyllum-Allochrusa
clade was estimated to be 2.88 Ma (Frajman & al., 2009) or
between 3 and 9 Ma (Valente & al., 2010). Since only one species each of Allochrusa and Acanthophyllum were sampled in
these studies, there is a need for a focused study using a wider
sampling.
The main goals of this study are: (1) to test the monophyly
of Acanthophyllum and its infrageneric taxa using DNA sequence data; (2) to evaluate the infrageneric classification of
Acanthophyllum in light of molecular data; (3) to check the
evolution of morphological characters on the background of
the molecular phylogeny; (4) to estimate the divergence time
of the crown node of Acanthophyllum; (5) to investigate the
phylogenetic position of Acanthophyllum within tribe Caryophylleae.
Table 1. Major infrageneric classifications of Acanthophyllum including types for sectional taxa.
Schischkin (1936)
Subg.
Euacanthophyllum
(Boiss.) Schischk.a
Subg.
Allochrusa
(Bunge) Schischk.
Boissier (1867)
Golenkin (1893)
Schiman-Czeika (1988)
Type
Sect.
Macrodonta
Sect.
Macrodonta
—
—
Sect.
Macrodonta
Boiss.
A. grandiflorum
Stocks
Sect.
Macrostegia
Sect.
Macrostegia
Boiss.
Sect.
Macrostegia
Boiss.
—
Sect.
Macrostegia
Boiss.
A. bracteatum
Boiss.
Sect.
Turbinaria
Sect.
Turbinaria Boiss.
Sect.
Turbinaria
Boiss.
—
Sect.
Acanthophyllum
A. mucronatum
C.A.Mey.
Sect.
Pleiosperma
Sect.
Pleiosperma
Boiss.
Sect.
Pleiosperma
Boiss.
—
Sect.
Pleiosperma
Boiss.
A. spinosum
(Desf.) C.A.Mey.
Sect.
Euacanthophylla
Sect.
Euacanthophylla
Sect.
Oligosperma
Schischk.
—
Sect.
Oligosperma
Schischk.
A. squarrosum
Boiss.
—
—
—
—
Sect. Pseudacanthophyllum A. laxiflorum
(Boiss.) Rech.f.
Boiss.
Syn.: Gypsophila sect.
Pseudacanthophyllum Boiss.
—
—
—
—
Sect.
Scapiflora
Schiman-Czeika
A. scapiflorum
(Akhtar) Schiman-Czeika
—
—
—
Sect.
Versicoloria
Schischk.
—
A. versicolor
Fisch. & C.A.Mey.
—
Sect.
Paniculata
Golenk.
—
Sect.
Paniculata
Golenk.
—
A. paniculatum
Regel & Herder
a
Acanthophyllum when described by Meyer (1831) included only one species which should automatically serve as the type of the genus. Boissier
(1867) made the first infrageneric classification of the genus including five sections (indicated by “§”). He was not aware of typification rules
and assigned the type to the genus in sect. Turbinaria. Most of earlier taxonomists ignored to put the type of the genus in section Acanthophyllum
(or sect. Euacanthophylla). Schiman-Czeika (1988) recognized the problem and defined section Acanthophyllum with A. mucronatum as the type
and including formerly described sect. Turbinaria.
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Pirani & al. • Molecular phylogeny of Acanthophyllum
MATERIALS AND METHODS
Sampling strategy and plant material. — All genera from
Caryophylleae represented in former phylogenetic studies plus
four small genera allied to Acanthophyllum were included. The
genus Silene L. was chosen as outgroup (Sileneae is well established as sister group to Caryophylleae; Harbaugh & al., 2010;
Greenberg & Donoghue, 2011).
New sequences were obtained from specimens deposited at
FUMH, GAZI, GB, M, MSB, TARI, TMRC and TUH; a total
of 108 sequences representing 47 species of Acanthophyllum,
4 of Diaphanoptera, 2 of Allochrusa, 1 of Ochotonophila, 1 of
Scleranthopsis and 1 of Gypsophila. All sections of Acantho
phyllum recognized by Schiman-Czeika (1988) and Schischkin
(1936), covering the whole morphological variation within the
genus, have been included. Amplification of the rps16 region for
A. lilacinum Schischk. and D. afghanica Podlech failed. There
was no material of A. paniculatum available, therefore only ITS
sequence for this species could be obtained from GenBank.
Other ITS and rps16 sequences representing 21 Gypsophila, 3
Saponaria, 23 Dianthus, 4 Petrorhagia (Ser.) Link, 1 Velezia L.,
1 Psammosilene W.C.Wu & C.Y.Wu, 1 Vaccaria Medik., 1 Bolbo
saponaria Bondarenko and 17 Silene species were obtained from
GenBank. In several cases only the ITS or the rps16 sequence
was available for a species, so the ITS and rps16 datasets do
not strictly match.
The final dataset for ITS contained 117 sequences representing 117 taxa and for rps16 72 sequences representing 72
taxa. Voucher information is listed in Appendix 1.
DNA extraction, PCR, and sequencing. — Genomic
DNA was extracted from herbarium specimens using the
E.Z.N.A. SP Plant DNA Mini Kit (Omega Bio-Tek, Norcross,
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Georgia, U.S.A.) according to the manufacturer’s protocol, or
using a modified Carlson/Yoon method (Oxelman & Lidén,
1995; Rautenberg & al., 2010). The nuclear ribosomal internal
transcribed spacer (ITS) region was amplified using primer
pairs P17/26S-82R (Popp & Oxelman, 2001; Kool & al., 2012).
The complete intron of the plastid rps16 gene was amplified
using primer pairs rpsF/rpsR2R (Oxelman & al., 1997; Petri
& Oxelman, 2011; Kool & al., 2012) or rpsF/rpsR3R. Multiscreen PCR (Millipore, Billerica, Massachusetts, U.S.A.) was
used to purify amplification products, according to the manufacturer’s instructions. The ITS region was sequenced using
primer pairs P16/ITS4 (Eggens & al., 2007; Popp & Oxelman,
2007), whereas the rps16 region was sequenced using primer
pairs rpsF2a/rpsR3R (Popp & al., 2005). Sequencing was performed by Macrogen (Amsterdam, Netherlands).
Sequence assembly, alignment and analysis. — Sequence
editing, contig assembly and alignments (see TreeBase, http://
purl.org/phylo/treebase/phylows/study/TB2:S15296) were performed using Geneious v.5.5.8. For multiple alignments, we
used the MUSCLE plug-in with default settings. The alignments were checked and adjusted manually. Indels were coded
using SeqState v.1.4.1 (Müller, 2005), under the simple indelcoding option (Simmons & Ochotorena, 2000). The best substitution model for each alignment was selected using jModelTest v.0.1.1 (Posada, 2008), under the Bayesian information
criterion (BIC). The GTR + G model was determined as the
best-fit model for both nuclear and chloroplast markers. The
binary data model (Lewis, 2001), as implemented in MrBayes,
was used for indel characters. Bayesian inference (BI) of the
individual gene analyses was performed using MrBayes v.3.1.2
(Huelsenbeck & Ronquist, 2001), with default prior settings,
for ten million MCMC generations in four parallel runs, each
Fig. 2. A, richly branched inflorescence; B, poorly branched
inflorescence; C, dentate petal;
D, emarginate petal; E, bifid petal;
F, entire petal; G, fimbriate petal;
H, sinuate petal. — A–B after
Schiman-Czeika, 1988; C–H after
Laínz & Garmendia (1990).
596
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Clade E
Clade G
0.81
0.74
Ovules ≥ 8 0.82
0.83
0.63
0.98
Clade H
Clade C
Membranous bracts
0.7
0.92
0.97
Clade F
0.54
Clade K
0.66
Clade B
Clade L
0.93
0.61
0.96
Clade J
CLADE I
0.46
0.77
0.94
Solitary flowers
0.99
0.56
Clade D
0.51
0.88
Clade A
0.96
Poorly branched inflorescence 0.91
0.99
CLADE II
0.71
0.5
0.99
Emarginate petals
12.5
10
7.5
MIOCENE
5
0.76
0
2.5
PLIOCENE
QUATERNARY
597
Fig. 3. *BEAST species tree of Acanthophyllum with node bars representing 95% HPD intervals for the root, Clade I and Clade II nodes. Bayesian posterior probabilities are indicated above
branches. Values below 50% are not shown.
Pirani & al. • Molecular phylogeny of Acanthophyllum
Ochotonophila allochrusoides
A. pleiostegium
Sect. Acanthophyllum
A. schugnanicum
A. glandulosum
Sect. Pleiosperma
6x; ovules 8–12(–16)
A. crassinodum
A. spinosum
A. longicalyx
Sect. Macrodonta
Diaphanoptera lindbergii
Diaphanoptera stenocalycina
A. kandaharicum
A. gracile
A. bracteatum
2x; ovules 4
Sect. Macrostegia
A. pachycephalum
A. leucostegium
A. andarabicum
A. heratense
A. pachystegium
A. borsczowii
A. squarrosum
A. ejtehadii
A. diezianum
A. speciosum
A. heterophyllum
Sect. Oligosperma
2x; ovules 4
A. laxiusculum
A. yasamin-nassehiae
A. stocksianum
A. andersenii
A. subglabrum
A. kabulicum
A. adenophorum
A. korshinskyi
Scleranthopsis aphanantha
A. macrodon
A. grandiflorum
Sect. Macrodonta
A. xanthoporphyranthum
A. anisocladum
Sect. Scapiflora
A. raphiophyllum
A. stewartii
Sect. Pseudacanthophyllum
A. laxiflorum
A. scapiflorum
Sect. Scapiflora
Diaphanoptera ekbergii
A. sordidum
Sect. Pleiosperma
4x; ovules (6–)8(–10)
Allochrusa versicolor
Allochrusa bungei
A. acerosum
A. verticillatum
A. mucronatum
A. microcephalum
Sect. Acanthophyllum 4x; ovules 4–8
A. kurdicum
A. crassifolium
A. oppositiflorum
A. caespitosum
Sect. Oligosperma
2x; ovules 4
0.84
Pirani & al. • Molecular phylogeny of Acanthophyllum
with four parallel chains for rps16, and four runs each with
eight chains under increased temperature of t = 0.1 for the ITS
dataset. Tracer v.1.5 (Rambaut & Drummond, 2009) was used
to check convergence of runs and diagnose MCMC chains.
Trees were summarized in TreeAnnotator v.1.6.1 (Drummond
& Rambaut, 2007) with the 25% first generations discarded as
burn-in. Maximum likelihood (ML) analyses for individual nuclear and chloroplast datasets were conducted using RAxMLHPC2 v.7.3.2 on the Cipres Science Gateway (Miller & al.,
2010) under the GTRCAT model with 1000 bootstrap replicates.
Bootstrap proportions on the ML tree were summarized using Sumtrees from the Python Dendropy library (Sukumaran
& Holder, 2010). Maximum parsimony (MP) analyses were
performed on both datasets after excluding outgroup taxa, using PAUP* v.4.0b10 (Swofford, 2003), which was also used to
calculate consistency and retention indices, and tree length.
Parameters for the parsimony heuristic searches included 100
random addition replicates, tree bisection-reconnection branch
swapping and multrees off. Trees were visualized using FigTree
v.1.3.1 (Rambaut, 2009).
Species tree inference and dating. — The *BEAST method
as implemented in BEAST v.1.7.3 (Drummond & al., 2006;
Drummond & Rambaut, 2007) was used to estimate the species tree for 52 species (covering those species common to both
plastid and nuclear datasets except for Gypsophila cerastioi
des D.Don). The data matrix was composed of two unlinked
partitions: (1) ITS sequences, (2) rps16 sequences, using the
GTR + G as substitution model for both partitions, and an uncorrelated relaxed lognormal clock. One MCMC chain was run
for 20 million generations, with tree sampling every 1000 steps.
Since there is no known fossil record for the study group, we
used published ITS and chloroplast substitution rates. The prior
probability of the clock rate was set to a truncated normal distribution with a mean of 2.15 × 10−3, ranging from 0.38 × 10−3 to
7.83 × 10−3 substitutions per site per Ma for ITS and a truncated
normal distribution with a mean of 0.67 × 10−3, ranging from
0.5 × 10−4 to 2.6 × 10−3 substitutions per site per Ma for rps16.
ITS substitution rates were set according to previous estimates
for perennial woody plants (Kay & al., 2006). The rps16 rates
could have been independently estimated, but in order to speed
up convergence (particularly by excluding highly suboptimal
starting rates) we instead provided rate priors where the lower
and upper rates of ITS were divided by three (substitution rates
in the chloroplast genome are typically slower by similar magnitudes compared to rates in the nuclear genome in plants; Wolfe
& al., 1987; Gaut & al., 1996; Yue & al., 2010). The rps16 prior
standard deviation was set to 0.7 × 10−3 that fits in the truncated
normal distribution. The value of standard deviation is larger
than the mean and unlikely to bias its posterior estimate. Then
the estimated rates were checked and modified to the values that
allowed the rps16 posteriors to be able to fluctuate within these
priors. The ploidy level for rps16 was set to haploid.
Tracer was used to check the convergence and mixing of
MCMC chains and the effective sample sizes for all parameters
to be above 200. Trees were summarized in TreeAnnotator with
the 10% first generations discarded as burn-in, and visualized
using FigTree.
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TAXON 63 (3) • June 2014: 592–607
RESULTS
Species tree inference and dating. — Two major clades
are retrieved within Acanthophyllum in a wide sense (see below) by the *BEAST species tree analysis (Fig. 3). These clades
are referred to as Clade I and Clade II hereafter. Two clades
are recognized within Clade I (PP 0.94): (1) clade A (PP 0.91),
including A. sect. Pseudacanthophyllum and sect. Scapiflora,
with Diaphanoptera ekbergii Hedge & Wendelbo as sister
to these sections; (2) clade B (PP 0.93), including A. sect.
Macrodonta, sect. Macrostegia, sect. Pleiosperma and sect.
Oligosperma as well as Ochotonophila allochrusoides Gilli,
Scleranthopsis aphanantha (Rech.f.) Rech.f., Diaphanoptera
stenocalycina Rech.f. & Schiman-Czeika and D. lindbergii
Hedge & Wendelbo. A single species of A. sect. Acantho
phyllum (A. pleiostegium Schiman-Czeika) also nests within
clade B. Clade II (PP 0.99) largely corresponds to A. sect.
Acanthophyllum. Allochrusa bungei Boiss. and All. versi
color form a subclade within Clade II with strong support
(PP 0.99). Acanthophyllum sordidum of A. sect. Pleiosperma
and A. caespitosum Boiss. of A. sect. Oligosperma also resolve
within Clade II.
This analysis estimates the age of the Acanthophyllum
clade (crown node) to be 11.1 Ma (3.8–32.4 Ma, 95% HPD interval). Clades I and II are estimated to be 8.2 Ma (3.2–22.2 Ma,
95% HPD interval) and 3.9 Ma (1.7–11.1 Ma, 95% HPD interval),
respectively.
Phylogenetic analyses. — BI, ML and MP analyses of
individual nuclear and plastid markers yielded mostly congruent trees within each marker, with no strongly supported
differences. Therefore, the results of BI are presented and
discussed in detail, whereas those of ML and MP analyses
including tree length, invariable characters, number of informative characters, number of indels, consistency and retention
indices, and likelihood bootstraps are summarized in Table 2
and Figs. 4–5.
Table 2. Models chosen by ModelTest, sequence and parsimony statistics.
ITS
rps16
Number of sequences (number of ingroup taxa)
56
53
BIC model choice
GTR + G GTR + G
Sequence length [bp]
674
839
Number of coded indels
63
40
Invariable characters [bp]
446
742
Variable sites [bp]
228
97
Variable sites [%]
33.8
11.6
Parsimony-informative characters [bp]
140
43
Parsimony-informative characters [%]
20.7
5.1
Consistency index, excl. uninformative characters
0.57
0.78
Retention index
0.8
0.94
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Pirani & al. • Molecular phylogeny of Acanthophyllum
Two major well-supported clades are recognized within
Acanthophyllum in both the ITS (Fig. 4) and the rps16 (Fig. 5)
trees. These clades are congruent with the Clades I and II obtained by *BEAST species tree analysis. The genera Ochotono
phila, Scleranthopsis, Diaphanoptera (pro majora parte) and
one species of Gypsophila are nested within Acanthophyllum.
However, Allochrusa is sister to Acanthophyllum in the ITS
phylogeny (PP 0.86; Fig. 4), whereas it nests within Acantho
phyllum Clade II in the rps16 phylogeny (PP 1.0; Fig. 5). Clade I
is composed of A. sect. Oligosperma, sect. Pleiosperma, sect.
Scapiflora, sect. Pseudacanthophyllum, sect. Macrostegia and
sect. Macrodonta. Clade I also contains representatives of the
genera Diaphanoptera, Ochotonophila, Scleranthopsis and
Gypsophila. Moreover, monophyly of A. sect. Oligosperma,
Silene
1
Vaccaria hispanica
Diaphanoptera afghanica
Bolbosaponaria bucharica
1
99
1
98
Gypsophila
1
100
Gypsophila
Dianthus
1
100
Velezia rigida
1
100
1
Petrorhagia
Gypsophila muralis
Gypsophila cerastioides
Diaphanoptera ekbergii
1
86
Ochotonophila allochrusoides
1
1
1
1
99
1
1
68
0.86
Clade I
1
1
100
Acanthophyllum s.l.
Sect. Acanthophyllum
A. pleiostegium
A. longicalyx
Sect. Macrodonta
A.
schugnanicum
1
1 91
A. crassinodum
Sect. Pleiosperma
0.66
A. glandulosum
72
A. spinosum
Diaphanoptera lindbergii
1
91
Diaphanoptera stenocalycina
A. pachycephalum
A. bracteatum
A. leucostegium
Sect. Macrostegia
1
A. kandaharicum
100
A. gracile
1
A. stocksianum
89
A. subglabrum
A. andersenii
A. squarrosum
A. heterophyllum
A. borsczowii
A. pachystegium
0.94
A. lilacinum
A. heratense
A. speciosum
Sect. Oligosperma
A. diezianum
1
A. yasamin-nassehiae
98
A. kabulicum
1
A. laxiusculum
53
A. ejtehadii
A. korshinskyi
A. adenophorum
A. andarabicum
Sect. Paniculata
A. paniculatum
Scleranthopsis aphanantha
A. xanthoporphyranthum
1
A. macrodon
Sect. Macrodonta
100
A. grandiflorum
A.
scapiflorum
1
Sect. Scapiflora
87
A. anisocladum
A. laxiflorum
A. stewartii
A. raphiophyllum
A. oppositiflorum
A. acerosum
A. crassifolium
A. verticillatum
A. mucronatum
A. caespitosum
A. kurdicum
A. microcephalum
A. sordidum
Allochrusa versicolor
Allochrusa bungei
71
1
100
Clade II
1
84
1
89
1
100
1
Psammosilene tunicoides
1
100
Sect. Pseudacanthophyllum
Sect. Acanthophyllum
Sect. Oligosperma
Sect. Acanthophyllum
Sect. Pleiosperma
Saponaria
Fig. 4. Majority-rule consensus tree inferred from Bayesian analysis of ITS data. Posterior probability values are indicated above branches, ML
bootstrap values below. Values below 50% are not shown.
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Pirani & al. • Molecular phylogeny of Acanthophyllum
TAXON 63 (3) • June 2014: 592–607
sect. Macrodonta and sect. Macrostegia is not obtained in the
rps16 tree. Clade II includes all examined species of A. sect.
Acanthophyllum (except for A. pleiostegium) as well as A. sor
didum (sect. Pleiosperma) and A. caespitosum (sect. Oligo
sperma). The Allochrusa species are included in this clade in
the rps16 tree. The resolution within Clade II is low in both
datasets.
DISCUSSION
Phylogenetic position of Acanthophyllum within tribe
Caryophylleae. — Acanthophyllum has gained little attention
in previous phylogenetic analyses of tribe Caryophylleae. The
most comprehensive molecular study included only two species (Fior & al., 2006; Harbaugh & al., 2010; Valente & al.,
Silene
1
97
1
100
Dianthus
Petrorhagia saxifraga
0.85
0.94
1
79
1
100 0.98
64
1
93
1
100
1
93
1
95
Gypsophila cerastioides
Ochotonophila allochrusoides
A. pleiostegium
Sect. Acanthophyllum
A. spinosum
A. schugnanicum
Sect. Pleiosperma
A. crassinodum
A. glandulosum
Diaphanoptera lindbergii
Diaphanoptera stenocalycina
A. longicalxy
Sect. Macrodonta
A. gracile
A. kandaharicum
A. pachycephalum
A. bracteatum
Sect. Macrostegia
A.leucostegium
A. heratense
A. squarrosum
A. kabulicum
A. pachystegium
A. heterophyllum
A. subglabrum
A. korshinskyi
A. yasamin-nassehiae
A. laxiusculum
1
79
A. borsczowii
A. ejtehadii
1
90
A. speciosum
A. andersenii
A. adenophorum
A. stocksianum
A. diezianum
A. andarabicum
Scleranthopsis aphanantha
A. macrodon
0.97
A. grandiflorum
66
A. xanthoporphyranthum
1
100
Acanthophyllum s.l.
1
100
Clade I
Sect. Oligosperma
Sect. Macrodonta
A. raphiophyllum Sect. Pseudacanthophyllum
0.99 A. stewartii
60
0.9
A. anisocladum
63
1
A. laxiflorum
87
0.68
54
Diaphanoptera ekbergii
Fig. 5. Majority-rule consensus tree inferred from Bayesian
analysis of rps16 data. Posterior
probability values are indicated
above branches, ML bootstrap
values below. Values below 50%
are not shown.
A. oppositiflorum
A. crassifolium
A. kurdicum
A. acerosum
1
86
Sect. Acanthophyllum
0.94
1
100
Clade II
1
93
600
A. scapiflorum
Sect. Scapiflora
Sect. Pseudacanthophyllum
Sect. Scapiflora
A. verticillatum
A. mucronatum
A. microcephalum
0.96
Allochrusa versicolor
72 1
95
Allochrusa bungei
A. sordidum
A. caespitosum
Sect. Pleiosperma
Sect. Oligosperma
Gypsophila paniculata
1
100
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TAXON 63 (3) • June 2014: 592–607
Pirani & al. • Molecular phylogeny of Acanthophyllum
2010; Greenberg & Donoghue, 2011) and the small genera allied
to Acanthophyllum, i.e., Scleranthopsis, Ochotonophila and
Diaphanoptera, were ignored. In our trees these small genera
are embedded in Acanthophyllum s.l. (Figs. 4–5). Cushionforming subshrubby to somewhat woody habit is a character
that distinguishes the Acanthophyllum s.l. clade (Acanthophyl
lum, Scleranthopsis, Allochrusa, Ochotonophila, Diaphanop
tera p.p.) from the rest of tribe Caryophylleae. We conclude
that the morphological characters used for delimiting Acan
thophyllum s.str., Allochrusa, Scleranthopsis, Ochotonophila
and Diaphanoptera are either not congruent with the phylogenetic patterns or need to be reassessed. Therefore, we suggest a
new classification for this group of taxa based on more reliable
characters and the evidence from molecular phylogenetics (see
below under Circumscription of Acanthophyllum).
The analyses of nuclear and plastid loci show that the Acan
thophyllum s.l. clade (including Allochrusa, Ochotonophila,
Diaphanoptera p.p. and Scleranthopsis), (PP 1.0; Figs. 4–5) is
sister to a clade holding Dianthus and Petrorhagia (and Vele
zia only in ITS tree) (PP 1.0; Figs. 4–5). This is in agreement
with previous studies (Fior & al., 2006; Harbaugh & al., 2010;
Valente & al., 2010; Greenberg & Donoghue, 2011). Gypsophila
cerastioides nests within Acanthophyllum s.l.
The placement of G. muralis L. as sister to the Dianthus
Petrorhagia clade in the present ITS tree is consistent with the
results of Greenberg & Donoghue (2011).
Circumscription of Acanthophyllum. — This study reveals
that Acanthophyllum as currently circumscribed (SchimanCzeika, 1988; Bittrich, 1993; Takhtajan, 2009) is not monophyletic. The circumscription of Acanthophyllum is discussed in
relation to its closely related genera residing within Acantho
phyllum s.l. clade.
Scleranthopsis. — The monospecific Scleranthopsis is
distributed in SW, E and central Afghanistan. The position of
this genus has been uncertain and was discussed by Rechinger
(1957, 1967). It was first described as Acanthophyllum aphanan
thum Rech.f. and classified in the monotypic sect. Aphanantha
Rech.f. (Rechinger, 1957). It was later raised to generic level,
Scleranthopsis, based on the following characters: non-clawed
petals, short petals enclosed within the calyx, and unequal
calyx teeth (Rechinger, 1967).
Our study supports returning Scleranthopsis to Acantho
phyllum and re-establishing the sect. Aphanantha.
Allochrusa. — Allochrusa has approximately seven species
distributed in Armenia, Turkey, NW Iran, NE Afghanistan,
Tajikistan and Turkmenistan. It was first proposed as distinct
from Acanthophyllum (Boissier, 1867) based on non-spiny
leaves and enclosed stamens but was included in Acanthophyl
lum by Golenkin (1893) and Schischkin (1936). Golenkin made
Allochrusa a section, whereas Schischkin (1936) treated it as a
subgenus. He divided subg. Allochrusa into two sections, sect.
Versicoloria Schischk. and sect. Paniculata Golenk. (Table 1).
The species of the latter section have a short calyx and petals,
as well as loose inflorescences. Barkoudah (1962) followed
Schischkin. However, Allochrusa has later been accepted as
a separate genus (Yukhananov, 1974; Schiman-Czeika, 1987;
Bittrich, 1993; Takhtajan, 2009).
Allochrusa versicolor and All. bungei form a strongly
supported clade within Acanthophyllum s.l. (Figs. 3–5). The
Bayesian analysis of the plastid marker as well as the results of
the *BEAST species tree analyses are congruent in resolving
this clade within Clade II. However, Bayesian analysis of ITS
suggests that the Allochrusa clade is sister group to Acantho
phyllum (PP 1.0, but note PP only 0.86 for monophyly of the
latter). We cannot resolve the reason for this incongruence at
the moment. Emarginate petals is a possible synapomorphy
for Clade II, including Allochrusa bungei and All. versicolor
(= Allochrusa s.str.). Our ITS dataset also included Acantophyl
lum paniculatum, which was classified in subg. Allochrusa sect.
Paniculata by Schischkin (1936) and in the genus Allochrusa by
Schiman-Czeika (1988). Acanthophyllum paniculatum which
has entire petals, is placed in Clade I in the ITS tree, together
with other species with entire petals.
Interestingly, in Clade II, All. versicolor and bungei that
are distributed in NW Iran and Armenia come together with
Acanthophyllum species with a mainly western distribution
pattern, i.e., Syria, Turkey, Iraq and W Iran, whereas the more
eastern A. paniculatum (Kazakhstan, Kyrgyzstan, Tajikistan,
Turkmenistan, Uzbekistan) is placed in Clade I, which includes
taxa with an eastern distribution. Our study supports the inclusion of Allochrusa in the Acanthophyllum s.l. clade.
Ochotonophila. — Ochotonophila comprises two species inhabiting central and E Afghanistan (Gilli, 1956; Hedge
& Wendelbo, 1963; Schiman-Czeika, 1988). They are lowgrowing multi-stemmed perennials with a woody base. In the
original description by Gilli (1956) the genus (with the single
species O. allochrusoides) was considered intermediate between Acanthophyllum and Gypsophila. Hedge & Wendelbo
(1963) described O. eglandulosa Hedge & Wendelbo, again emphasizing similarities to Acanthophyllum and Gypsophila. Later
work by Schiman-Czeika (1988) suggested that Ochotonophila
is more closely related to Acanthophyllum, Scleranthopsis and
Allochrusa. The tubular calyx and the presence of bracteoles are
features shared by Ochotonophila, Allochrusa, Scleranthopsis
and Acanthophyllum but not Gypsophila. Ochotonophila differs
from Acanthophyllum by its non-spiny leaves, enclosed stamens
and, non-appressed bracteoles. Ochotonophila allochrusoides
is placed close to A. sect. Pleiosperma, A. pleiostegium (A. sect.
Acanthophyllum) and A. longicalyx Hedge & Wendelbo (A. sect.
Macrodonta) in our analyses (Figs. 3–5). An important feature
shared by Ochotonophila and A. sect. Pleiosperma is the high
number of ovules (10–21). We suggest reducing Ochotonophila
to a section under Acanthopyllum s.l.
Diaphanoptera. — Diaphanoptera has six species and
is distributed in NE Iran, Turkmenistan and Afghanistan
(Schiman-Czeika, 1988). They are perennial multi-stemmed
plants with a woody base. The genus was described by
Rechinger (1940) with the single species D. khorassanica
Rech.f., distinguished from Acanthophyllum by diaphanous
wings on the calyx (Rechinger, 1940; Hedge & Wendelbo,
1963). However, the other five currently accepted species of
the genus lack diaphanous wings. Lack of bracteoles and a
non-tubular calyx also separate Diaphanoptera from Acan
thophyllum (Schiman-Czeika, 1988). Diaphanoptera afghanica
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Pirani & al. • Molecular phylogeny of Acanthophyllum
(included only in the ITS dataset) is sister to Bolbosaponaria, a
genus closely related to Gypsophila s.str., whereas Diaphanop
tera stenocalycina, D. lindbergii and D. ekbergii, nest inside
Acanthophyllum Clade I in all trees (Figs. 3–5). Diaphanop
tera lindbergii and D. stenocalycina have 10–12 ovules in each
ovary, in accordance with its placement in clade E (PP 0.82;
Fig. 3) that contains taxa with more than eight ovules. However, the support for clade E is not strong and is due only to
rps16 (PP 0.98; Fig. 5). The placement of D. ekbergii as sister
to A. sects. Pseudacanthophyllum and Scapiflora is likewise
poorly supported in the species tree (PP 0.91; Fig. 3) and the
rps16 tree (PP 0.68; Fig. 5) and not at all in the ITS tree (Fig. 4),
where it is resolved as sister to Gypsophila cerastioides. We
were not successful in generating high-quality sequences for
D. khorassanica, the type of the genus. Therefore, the delimitation of Diaphanoptera from Acanthophyllum, Gypsophila
and Ochotonophila cannot be ascertained, but it is clear that
Diaphanoptera as currently circumscribed contains unrelated
elements.
Gypsophila. — Gypsophila has about 150 species and is
distributed in temperate Asia and Europe, Egypt, Australia,
North America and China (Amini & al., 2011). The delimitation
of Acanthophyllum from Gypsophila has long been controversial. General habit, calyx shape, petal shape in terms of developing a claw, leaf texture, duration of bracts and bracteoles,
calyx shape and mode of capsule dehiscence (Barkoudah, 1962;
Gilli, 1964; Schiman-Czeika, 1987) are the most commonly
used characters for discriminating the two genera. Emphasis
on different characters has led to several transfers of species.
Acanthophyllum sect. Pseudacanthophyllum is one of the most
controversial groups transferred from Gypsophila to Acantho
phyllum (Oliver, 1859; Bentham & Hooker, 1862; Boissier, 1867;
Williams, 1889; Barkoudah, 1962; Schiman-Czeika, 1988). The
examined species of this section nest within Acanthophyllum
in our analyses (Figs. 3–5).
In both analyses of ITS and rps16 (Figs. 4–5) G. cerasti
oides falls within Acanthophyullum s.l. Gypsophila muralis
resolves as sister to the Dianthus-Petrorhagia clade in the ITS
tree (consistent with Greenberg & Donoghue, 2011), and the
remaining species of Gypsophila are found in two additional
clades. This shows that Gypsophila is non-monophyletic. A
broad molecular study (Zarre & al., in prep.) shows that apart
from G. cerastioides no other species of Gypsophila appear
to belong to the Acanthophyllum clade. Therefore, the poor
sampling of Gypsophila is not likely to affect our taxonomic
conclusions.
Infrageneric classification of Acanthophyllum s.str. — The
sections in individual ITS and rps16 trees do not fully match.
Acanthophyllum sect. Pseudacanthophyllum and sect. Scapi
flora are intermingled in the plastid tree, whereas they are sister
sections in the nuclear tree. Acanthophyllum sect. Macrostegia
appears monophyletic only in the ITS tree. Acanthophyllum
sect. Pleiosperma is monophyletic only in rps16 tree, except
for one species. The large section Oligosperma forms a clade
in both nuclear and plastid trees, except for one and two species, respectively. Acanthophyllum sect. Macrodonta is monophyletic in both gene trees if A. longicalyx is excluded. Most
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TAXON 63 (3) • June 2014: 592–607
species of A. sect. Acanthophyllum form a clade in the ITS tree;
only one species is excluded from this clade while one species
of A. sect. Oligosperma is nested within it. In rps16, A. sect.
Acanthophyllum is also monophyletic except for two species.
The differences between the rps16 and ITS trees can be
explained by different numbers of informative characters in
plastid and nuclear datasets. It can also be attributed to stochastic effects, hybridizations, or incomplete lineage sorting.
The implementation of the multispecies coalescent model in
*BEAST takes the latter into account (Heled & Drummond,
2010), and by using the information from all datasets simultaneously, the stochastic effects can be expected to decrease.
Therefore, we will from now on discuss the phylogenetic status
of the taxa in relation to the *BEAST species tree (Fig. 3).
Emarginate petals appear to be a synapomorphy for Clade
II, whereas Clade I holds taxa with mainly entire petals (some
other petal types, e.g., dentate-sinuate in A. pleiostegium and
deeply bifid in O. allochrusoides are rarely seen in Clade I).
The common petal shapes in outgroups are entire, bifid, emarginate, fimbriate and sinuate. Some common petal shapes in
Caryophylloideae are shown in Fig. 2C–H.
Clade I. — Acanthophyllum sect. Scapiflora, sect. Pseud
acanthophyllum, sect. Macrodonta, sect. Pleiosperma, sect.
Macrostegia and sect. Oligosperma together form Clade I (PP
0.94; Fig. 3). Acanthophyllum sects. Scapiflora and Macrodonta
are not resolved as monophyletic. A single species of A. sect.
Acanthophyllum, A. pleiostegium, also nests within Clade
I. Acanthophyllum sect. Scapiflora, sect. Pseudacanthophyl
lum, sect. Macrodonta, sect. Pleiosperma, sect. Macrostegia
and sect. Oligosperma correspond to the subclades A, D, G, H
and K, respectively. Clade A and clade B, the latter including
clades D, E and H, have posterior probabilities from 0.82 to
0.99, and are in the following discussed in relation to putative
synapomorphies. The other clades have low support and are
not discussed.
Clade A. – Clade A (PP 0.91; Fig. 3) includes A. sect. Pseud
acanthophyllum and sect. Scapiflora and D. ekbergii. A putative synapomorphy for Acanthophyllum sections in this clade
is poorly branched inflorescence (Fig. 2B) of one (rarely up to
three in A. laxiflorum Boiss.) condensed unit(s) confined to
only one terminal node of the stem and composed of multiflowered partial cymes (with more than seven flowers). These
sections are almost confined to Afghanistan.
Acanthophyllum anisocladum Schiman-Czeika and A. sca
piflorum (Akhtar) Schiman-Czeika of sect. Scapiflora and
A. laxiflorum, A. raphiophyllum (Rech.f.) Barkoudah and
A. stewartii (Thoms. ex Edgew & Hook.f.) of A. sect. Pseud
acanthophyllum constitute clade A. The two sections do not
form separate clades in the *BEAST tree (Fig. 3), despite morphological differences that make them easily recognizable.
Species of A. sect. Scapiflora have elongated, leafless peduncles whereas species of A. sect. Pseudacanthophyllum have a
condensed habit with the inflorescence set close to the leaves.
The two sections are, however, resolved as monophyletic in
the ITS tree (Fig. 4). It will require sampling of other nuclear
loci to resolve whether the incongruence should be attributed
to stochasticity or needs a biological explanation.
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Pirani & al. • Molecular phylogeny of Acanthophyllum
Clade B. – Clade B (PP 0.93; Fig. 3) includes A. sect. Macro
donta (clade D), sect. Pleiosperma (clade G), sect. Macrostegia
(clade H) and sect. Oligosperma (clade K), a single species of
A. sect. Acanthophyllum, as well as Diaphanoptera, Ochotono
phila and Scleranthopsis. No morphological synapomorphy for
this clade could be found.
Clade D. – Within clade B, subclade D is strongly supported (PP 0.99; Fig. 3) and holds all examined species of
A. sect. Macrodonta except A. longicalyx. Clade D is characterized by solitary flowers and four ovules. Acanthophyllum
longicalyx has one to three terminal flowers and eight ovules
and resolves within clade E. This species also possesses the
longest calyx and the shortest calyx teeth among the taxa assigned to sect. Macrodonta (Hedge & Wendelbo 1963; Schiman-Czeika 1988).
Clade E. – Clade E (PP 0.82; Fig. 3) holds Ochotonophila
allochrusoides, A. sect. Pleiosperma (except A. sordidum),
A. pleiostegium (sect. Acanthophyllum), A. longicalyx (sect.
Macrodonta) and Diaphanoptera lindbergii and D. stenoca
lycina. Apart from A. pleiostegium with 4–6 ovules in each
ovary, the rest of the taxa in this clade produce eight or more
ovules (up to 21 ovules in O. allochrusoides).
Clade H. – Clade H (PP 0.92; Fig. 3) corresponds to A. sect.
Macrostegia. Broadly ovate to lanceolate membranous bracts
and bracteoles constitute a synapomorphy for this clade. It is
widespread in Iran, Iraq, Turkmenistan, Tajikistan, Kyrgyzstan, Uzbekistan, Kazakhstan, Afghanistan and Pakistan.
Acanthophyllum sect. Oligosperma. — Clade K (PP 0.66;
Fig. 3) has poor support but is more or less identical to the large
and taxonomically complicated A. sect. Oligosperma (except
for A. caespitosum which is resolved as member of Clade II
in all analyses). However, without A. andarabicum Podl. ex
Schiman-Czeika, the rest of the clade (clade L) has strong support in all trees. Only the ITS phylogeny (Fig. 4) has strong
support (PP 1.0) for clade K.
Schischkin (1936) divided A. sect. Oligosperma into five
series. Series Elatiora Schischk. (including A. elatius Bunge
and A. borsczowii Litv.) was later elevated to sectional rank
(Zakirov & Musaeva, 1981). However, Schiman-Czeika (1988)
included A. elatius and A. borsczowii within sect. Oligosperma.
We can confirm the latter opinion, as A. borsczowii falls within
the core group of A. sect. Oligosperma in the species (PP 0.96;
Fig. 3), ITS (PP 1.0; Fig. 4) and rps16 (PP 1.0; Fig. 5) trees.
Clade II. — Clade II (PP 0.99; Fig. 3) is characterized
by emarginate petals. A large part of this clade corresponds
to A. sect. Acanthophyllum which is recognised by branched
elongated inflorescences of white flowers (bright pink flowers are rarely seen in some populations of A. acerosum Sosn.).
However, the section is not resolved as monophyletic, with
A. oppositiflorum Aytaç falling outside this group including
A. acerosum, A. verticillatum Hand.-Mazz., A. mucronatum
C.A.Mey., A. microcephalum Boiss., A. kurdicum Boiss. &
Hausskn. ex Boiss. and A. crassifolium Boiss. Acantophyllum
oppositiflorum possesses elongated inflorescences with only
two opposite flowers at each node, whereas the other species
of A. sect. Acantophyllum have inflorescences with multiflowered verticillasters.
Acanthophyllum sect. Acanthophyllum shows a mainly
western distribution and inhabits Syria, Iraq, Turkey, Armenia, Azerbaijan, Iran and Turkmenistan. The Afghan species
A. pleiostegium was assigned to sect. Acanthophyllum by Schiman-Czeika (1988), but belongs to clade E (see above).
Acanthophyllum caespitosum (sect. Oligosperma) and
A. sordidum (sect. Pleiosperma) also nest in Clade II. They
are the only species of their sections that have emarginate petals. Acanthophyllum caespitosum is an odd member of A. sect.
Oligosperma also in its procumbent densely branched habit,
extremely reduced stems and imbricate leaves. The other taxa
of A. sect. Oligosperma have tall stems and non-imbricate
leaves. Further, A. caespitosum is the only known species of
Acantophyllum with a haploid base chromosome number of
x = 14 instead of x = 15 (Ghaffari, 2004). Ghaffari discussed
these morphological and base chromosome number differences
and suggested that A. caespitosum should form a section of its
own. Exclusion of A. caespitosum from A. sect. Oligosperma
is corroborated by our analyses and it is placed together with
the species of A. sect. Acanthophyllum (Figs. 3–5). However, A. caespitosum differs from A. sect. Acanthophyllum in
4-ovulate ovaries, reduced stems and reduced inflorescences.
Furthermore, all examined representatives of sect. Acantho
phyllum are tetraploids (Ghaffari, 2004), whereas A. caespi
tosum is diploid. Taking these complexities into account, it is
not possible to decisively judge the taxonomic placement of
A. caespitosum in A. sect. Acanthophyllum.
Acanthophyllum sordidum has been assigned to A. sect.
Pleiosperma (Boissier, 1867; Schischkin, 1936; Golenkin, 1893;
Schiman-Czeika, 1988; Basiri-Esfahani & al., 2011), but our
analysis shows that it is remotely related to the rest of the section, which resides within Clade I (Figs. 3–5). The non-clawed
linear emarginate petals of A. sordidum are not seen in any
other taxa of A. sect. Pleiosperma. The ploidy level reported
for A. sordidum is tetraploid (Ghaffari 2004), the same as for
A. sect. Acanthophyllum, whereas the rest of the examined species of A. sect. Pleiosperma are hexaploid (Ghaffari, 2004).
However, non-elongated inflorescences and non-clawed petals
of A. sordidum separate it from A. sect. Acanthophyllum. We
conclude that A. sordidum should be excluded from A. sect.
Pleiosperma.
Some conclusions with regard to the circumscription and
infrageneric classification of Acanthophyllum s.l. and the geographical distribution of its infrageneric taxa can be derived
from our study: (1) The majority of the morphological characters used in infrageneric classifications of Acanthophyllum
show low levels of homoplasy, and are thus useful in interpreting phylogenetic patterns within the genus. We conclude
that the value of petal morphology has been underestimated.
Emarginate petals, for instance, characterizes one of the two
major clades. However, the state of “clawed petal” which is
frequent among the studied group, is lost in several places in
the trees to form the “non-clawed petals” in A. aphananthum
(see above under Scleranthopsis), O. allochrusoides (see above
under Ochotonophila), A. pachycephalum Schiman-Czeika,
A. pleiostegium, A. sordidum and A. oppositiflorum; (2) Increase in ploidy level is accompanied by increase in the number
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Pirani & al. • Molecular phylogeny of Acanthophyllum
of ovules (see Fig. 3). Acanthophyllum sect. Macrostegia and
sect. Oligosperma, which have diploid chromosome numbers,
always produce four ovules per ovary, whereas tetraploid
A. sect. Acanthophyllum produces 4–8 ovules, and hexaploid
A. sect. Pleiosperma produces 8–16 ovules; (3) The number
of Acanthophyllum species is estimated to amount to about
80–90. Acanthophyllum sect. Acanthophyllum constitutes the
westernmost limit of the genus. The diversity center of this section is in NW Iran and neighboring areas in Turkey and Iraq.
The examined species of this section reside within Clade II in
our trees. The easternmost limit is set by A. pungens Boiss.
(sect. Oligosperma), which inhabits Central Asia to W China.
Acanthophyllum sect. Oligosperma is otherwise most diverse in
NE Iran and adjacent areas of Afghanistan and Turkmenistan,
corresponding to the diversity center of the genus as a whole.
Acanthophyllum sect. Oligosperma falls into Clade I in our
analyses. Generally, most Acanthophyllum species are in Clade
I, found in NE Iran or further east. Afghanistan harbors the
highest number of Acanthophyllum sections, including three
endemic sections (A. sect. Scapiflora, sect. Ochotonophila and
sect. Aphanantha; see below).
Divergence time of Acanthophyllum. — Valente & al. (2010)
obtained an approximate age of 5 Ma (3–9 Ma, 95% HPD interval) for the AcanthophyllumAllochrusa clade. BEAST analysis
of matK data by Frajman & al. (2009) estimated the age to 2.88
Ma. These studies included only one species each of Acan
thophyllum and Allochrusa: A. sordidum and All. versicolor.
In our *BEAST species tree (Fig. 3) Allochrusa nests within
Acanthophyllum and A. sordidum is resolved as sister to All.
versicolor and All. bungei). Although weakly supported, the
A. sordidum–Allochrusa clade has an approximate age of 2.48
Ma (0.1–7.7 Ma, 95% HPD interval) in our analysis. The species
tree analysis suggests a somewhat younger age for the A. sor
didum–Allochrusa clade, compared to the age estimated by
Valente & al. (2010) using gene trees, although the credibility
intervals of both estimates largely overlap. The age of 2.48 Ma
estimated by the present species tree analysis for the AllochrusaAcanthophyllum clade is very close to the age (2.88 Ma) gained
by the BEAST analysis of matK (Frajman & al., 2009).
The *BEAST analysis estimated the age of the Acantho
phyllum s.l. crown clade as 11.1 Ma (3.8–32.4 Ma, 95% HPD
interval), a time span covering the Oligocene, Miocene and
Pliocene (Fig. 3).
There are some fossils assigned to Caryophyllaceae
(Jordan & Macphail, 2003; Huang & al., 2012), but we did not
use them as the taxa were phylogenetically far from our focal
group, which would either introduce long branches to our tree
or require an extremely increased taxon sampling. Therefore,
we used only clock rate calibration analysis.
TAXONOMIC IMPLICATIONS
Acanthophyllum C.A.Mey., Verz. Pfl. Casp. Meer.: 210. 1831
– Type: A. mucronatum C.A.Mey.
= Ochotonophila Gilli in Feddes Repert. Spec. Nov. Regni Veg.
59: 169. 1956, syn. nov. – Type: O. allochrusoides Gilli.
604
TAXON 63 (3) • June 2014: 592–607
= Allochrusa Bunge in Boissier, Fl. Orient. 1: 559. 1867 ≡
Acanthophyllum subg. Allochrusa (Bunge) Schischk., Fl.
U.S.S.R. 6: 800. 1936 – Type (designated by SchimanCzeika, 1988): All. versicolor (Fisch. & C.A.Mey.) Boiss.
New names and combinations
Acanthophyllum sect. Ochotonophila (Gilli) Pirani, comb.
& stat. nov. ≡ Ochotonophila Gilli in Feddes Repert. Spec.
Nov. Regni Veg. 59: 169. 1956 – Type: A. allochrusoides
(Gilli) Pirani
Perennial low-growing plants, multi-stemmed. Leaves
non-spiny. Bracteoles distant from flowers. Ovary stipitate,
ovules 10–18; capsule many-seeded.
Acanthophyllum allochrusoides (Gilli) Pirani, comb. nov.
≡ Ochotonophila allochrusoides Gilli in Feddes Repert. Spec. Nov. Regni Veg. 59: 169. 1956 – Holotype:
AFGHANISTAN. In montibus calc. NE Bamian, 3000 m,
1949, Gilli 1269 (W [photo!]).
Acanthophyllum eglandulosum (Hedge & Wendelbo) Pirani,
comb. nov. ≡ Ochotonophila eglandulosa Hedge &
Wendelbo in Aarbok Univ. Bergen, Mat.-Naturvitensk.
Ser. 18: 21. 1964 – Holotype: AFGHANISTAN. Kabul,
in decl. orientalibus jugi Shibar, 2750 m, 1962, Hedge &
Wendelbo W-4239 (E n.v.; isotypes: BG n.v., W [photo!]).
Section to be reinstated
Acanthophyllum sect. Aphanantha Rech.f. in Oesterr. Bot. Z. 104:
174. 1957 ≡ Scleranthopsis Rech.f. in Ann. Naturhist. Mus.
Wien 70: 37. 1967 – Type: Acathophyllum aphananthum
Rech.f. in Oesterr. Bot. Z. 104: 174. 1957.
ACKNOWLEDGEMENTS
We thank Magnus Lidén and an anonymous reviewer as well as
the Associate Editor Sigrid Liede-Schumann and the Managing Editor Michael Pirie for valuable suggestions on the manuscript. We are
grateful to Hamid Moazzeni, Anna Petri, Vivian Alden, Claes Persson,
Zeynep Aydin and Zekí Aytaç for their kind help during different
steps of this work. We also wish to thank curators at herbaria B, BM,
FUMH, G, GB, JE, K, KEW, LD, M, MSB, TARI, TMRC, TUH and
WU for the loan and permission to study plant material used in this
study. The herbarium of RBG Edinburgh (E) is thanked for providing
photos of their Acanthophyllum specimens. All analyses (except ML)
were performed on the bioinformatics computer cluster Albiorix at the
Department of Biological and Environmental Sciences, University of
Gothenburg. This paper is a part of Ph.D. thesis conducted by AP at the
University of Tehran. Financial support provided by the University of
Tehran is appreciated. BO and BEP are supported by grants from the
Swedish Research Council, the Royal Swedish Academy of Sciences,
Lars Hiertas Minnesfond, The Royal Physiographic Society in Lund,
Helge Ax:son Johnsons fond and Lundgrenska fonden.
Version of Record (identical to print version).
TAXON 63 (3) • June 2014: 592–607
Pirani & al. • Molecular phylogeny of Acanthophyllum
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Appendix 1. Voucher information: species name, geographical origin, collector(s), voucher (herbarium), GenBank accession numbers for ITS and rps16,
respectively (* indicates sequences new for this study).
Acanthophyllum acerosum Sosn., Iran, W Azarbaijan, Zarre & al. 41900 (TUH), *KF924680, *KF924732; Acanthophyllum adenophorum Freyn, Iran,
Khorassan, Pirani & Moazzeni 1947 (TMRC), *KF924679, *KF924731; Acanthophyllum andarabicum Podl. ex Schiman-Czeika, Afghanistan, Baghlan,
Podlech 10985 (MSB), *KF924678, *KF924730; Acanthophyllum andersenii Rech.f. & Schiman-Czeika, Iran, Khorassan, Anders & Peterson 320 (GB),
*KF924677, *KF924729; Acanthophyllum anisocladum Schiman-Czeika, Afghanistan, Lugar, Ekberg W9180 (GB), *KF924676, *KF924728; Acanthophyllum borsczowii Litv., Iran, Khorassan, Zarre & al. 41034 (TUH), *KF924675, *KF924727; Acanthophyllum bracteatum Boiss., Iran, Kohghiluyeh & Boyerahmad, Pirani & Moazzeni 2104 (TMRC), *KF924674, *KF924726; Acanthophyllum caespitosum Boiss., Iran, Hamadan, Zarre & al. 41903 (TUH),
*KF924673, *KF924725; Acanthophyllum crassifolium Boiss., Iran, Mazandaran, Pirani & Moazzeni 41702 (TUH), *KF924672, *KF924724; Acanthophyllum crassinodum Yukhan. & J.R.Edm., Iran, Kerman, Pirani & Moazzeni 2923 (TMRC), *KF924671, *KF924723; Acanthophyllum diezianum Hand.-Mazz.,
Iran, Khorassan, Zarre & al. 41040 (TUH), *KF924670, *KF924722; Acanthophyllum ejtehadii Mahmoudi & Vaezi, Iran, Khorassan, Pirani & Moazzeni
2181 (TMRC), *KF924669, *KF924721; Acanthophyllum glandulosum Bunge ex Boiss., Iran, Khorassan, Zarre & al. 41037 (TUH), *KF924668, *KF924720;
Acanthophyllum gracile Bunge ex Boiss., Iran, Semnan, Pirani & Moazzeni 2989 (TMRC), *KF924667, *KF924719; Acanthophyllum grandiflorum Stocks,
Afghanistan, Bamian, Podlech 1340 (MSB), *KF924666, *KF924718; Acanthophyllum heratense Schiman-Czeika, Iran, Isfahan, Pirani & Moazzeni 2152
(TMRC), *KF924665, *KF924717; Acanthophyllum heterophyllum Rech.f., Iran, Kerman, Pirani & Moazzeni 2921 (TMRC), *KF924664, *KF924716;
Acanthophyllum kabulicum Schiman-Czeika, Afghanistan, Ghazni, Frey s.n. (GB), *KF924663, *KF924715; Acanthophyllum kandaharicum Gilli, Iran,
Khorassan, Joharchi & Zangouei 36245 (FUMH), *KF924662, *KF924714; Acanthophyllum korshinskyi Schischk., Iran, Khorassan, Pirani & Moazzeni
2123 (TMRC), *KF924661, *KF924713; Acanthophyllum kurdicum Boiss. & Hausskn. ex Boiss., Iran, Ilam, Hamzehee & Lashkarbolooki 1756 (TARI),
*KF924660, *KF924712; Acanthophyllum laxiflorum Boiss., Afghanistan, Lugar, Ekberg W9184 (GB), *KF924659, *KF924711; Acanthophyllum
606
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Appendix 1. Continued.
laxiusculum Schiman-Czeika, Iran, Qom, Pirani & Moazzeni 1941 (TMRC), *KF924658, *KF924710; Acanthophyllum leucostegium Schiman-Czeika, Iran,
Bandar Abbas, Ghahreman & Mozaffarian 5656 (TUH), *KF924657, *KF924709; Acanthophyllum lilacinum Schischk., Afghanistan, Badghis, Podlech &
Jarmal 29855 (MSB), *KF924656, –; Acanthophyllum longicalyx Hedge & Wendelbo, Afghanistan, Jawzjan, Freitag 6580 (MSB), *KF924655, *KF924708;
Acanthophyllum macrodon Edgew., Afghanistan, Kandahar, Hedge & al. W7641 (GB), *KF924654, *KF924707; Acanthophyllum microcephalum Boiss.,
Iran, Tehran, Rajamand & Bazargan 32055 (TARI), *KF924653, *KF924706; Acanthophyllum mucronatum C.A.Mey., Iran, W Azarbaijan, Assadi & Olfat
68668 (TARI), *KF924652, *KF924705; Acanthophyllum oppositiflorum Aytaç, Turkey, Sivas, Aytaç 7476 (GAZI), *KF924651, *KF924704; Acanthophyllum paniculatum Regel & Herder, JN589016, –; Acanthophyllum pachycephalum Schiman-Czeika, Iran, Tehran, Ganjalizadeh 6106 (TUH), *KF924650,
*KF924703; Acanthophyllum pachystegium Rech.f., Afghanistan, Badakhshan, Hedge & Wendelbo W9282 (GB), *KF924649, *KF924702; Acanthophyllum
pleiostegium Schiman-Czeika, Afghanistan, Kataghan, Grey-Wilson & Hewer 1320 (GB), *KF924648, *KF924701; Acanthophyllum raphiophyllum (Rech.f.)
Barkoudah, Afghanistan, Kapisa, Podlech 12548 (MSB), *KF924647, *KF924700; Acanthophyllum scapiflorum (Akhtar) Schiman-Czeika, Afghanistan,
Kabul, Podlech 31232 (MSB), *KF924646, *KF924699; Acanthophyllum schugnanicum Schischk., Afghanistan, Bamian, Wendelbo & Ekberg W9796 (GB),
*KF924645, *KF924698; Acanthophyllum sordidum Bunge ex Boiss., Iran, Isfahan, Pirani & Moazzeni 2147 (TMRC), *KF924644, *KF924697; Acanthophyllum speciosum Rech.f. & Schiman-Czeika, Iran, Khorassan, Pirani & Moazzeni 2186 (TMRC), *KF924643, *KF924733; Acanthophyllum spinosum
C.A.Mey., Iran, Isfahan, Pirani & Moazzeni 2150 (TMRC), *KF924642, *KF924696; Acanthophyllum squarrosum Boiss., Iran, Semnan, Pirani & Moazzeni
2974 (TMRC), *KF924641, *KF924695; Acanthophyllum stewartii (Thoms. ex Edgew. & Hook.f.) Barkoudah, Afghanistan, Khost, Anders 8994 (MSB),
*KF924640, *KF924694; Acanthophyllum stocksianum Boiss., Afghanistan, Kandahar, Toncev s.n. (MSB), *KF924639, *KF924693; Acanthophyllum
subglabrum Schischk., Afghanistan, Nangarhar, Hedge & al. W7483 (GB), *KF924638, *KF924692; Acanthophyllum verticillatum Hand.-Mazz., Iran,
Markazi, Mozaffarian & Sardabi 42175 (TARI), *KF924637, *KF924691; Acanthophyllum xanthoporphyranthum Hedge & Wendelbo, Afghanistan, Herat,
Hedge & al. W8003 (GB), *KF924636, *KF924690; Acanthophyllum yasamin-nassehiae Joharchi & Pirani, Iran, Khorassan, Memariani & Zangouei 41448
(FUMH), *KF924635, *KF924689; Allochrusa bungei Boiss., Iran, E Azarbaijan, Rechinger 43834 (M), *KF924634, *KF924688; Allochrusa versicolor
Boiss., Turkey, Kars, Nydegger 43597b (MSB), *KF924633, *KF924687; Bolbosaponaria bucharica Bondarenko, JN589057, –; Dianthus anatolicus Boiss.,
MA-690057, GU440777, –; Dianthus armeria L., –, FJ404903; Dianthus capitatus J.St.-Hil., GU440792, –; Dianthus carthusianorum L., –, EF674194; Dianthus charidemi Pau, GU440795, –; Dianthus ciliatus Guss., GU440798, –; Dianthus corymbosus Sibth. & Sm., GU440801, –; Dianthus costae Willk.,
GU440802, –; Dianthus crassipes De Roem., GU440803, –; Dianthus crinitus Sm., GU440805, –; Dianthus cyri Fisch. & C.A.Mey., GU440808, –; Dianthus
diffusus Sm., GU440811, –; Dianthus erinaceus Boiss., GU440814, –; Dianthus gracilis Sm., JN589061, –; Dianthus hyssopifolius L., GU440826, –; Dianthus laricifolius Boiss. & Reut., GU440831, –; Dianthus microlepis Boiss., GU440840, –; Dianthus micropetalus Ser., GU440841, –; Dianthus orientalis
Adams, GU440847, –; Dianthus pyrenaicus Bernh. ex Steud., GU440854, –; Dianthus serratifolius Sm., GU440858, –; Dianthus thunbergii S.S.Hooper,
GU440872, –; Dianthus turkestanicus Preobr., GU440876, –; Dianthus versicolor Fisch. ex Link, GU440878, –; Diaphanoptera afghanica Podlech,
Afghanistan, Baghlan, Podlech 21075 (MSB), *KF924632, –; Diaphanoptera ekbergii Hedge & Wendelbo, Afghanistan, Takhar, Podlech 11848 (MSB) &
11760 (MSB), *KF924631, *KF924686; Diaphanoptera lindbergii Hedge & Wendelbo, Afghanistan, Fariab, Hedge & al. W8336 (GB), *KF924630 *KF924685;
Diaphanoptera stenocalycina Rech.f. & Schiman-Czeika, Iran, Golestan, Attar & Mehdigholi 24422 (TUH), *KF924629, *KF924684; Gypsophila arrostii
Guss., JN589043, –; Gypsophila aucheri Boiss., JN589077, –; Gypsophila bicolor Grossh., JN589151, –; Gypsophila capituliflora Rupr., JN589143, –; Gypsophila cephalotes (Schrenk) Raikova, JN589105, –; Gypsophila cerastioides D.Don., Pakistan, Hazar, Ewald & Zetterlund 6227 (GB), *KF924628, *KF924683;
Gypsophila curvifolia Fenzl, JN589159, –; Gypsophila desertorum Fenzl, JN589021, –; Gypsophila elegans M.Bieb., JN589130, –; Gypsophila fastigiata L.,
JN589144, –; Gypsophila heteropoda Freyn, JN589110, –; Gypsophila montserratii Fern.Casas, JN589155, –; Gypsophila muralis L., JN589037, –; Gypsophila
paniculata L., JN589150, FJ404908; Gypsophila patrinii Ser., JN589076, –; Gypsophila pilulifera Boiss. & Heldr, JN589132, –; Gypsophila pinifolia Boiss.
& Hausskn. ex Boiss., JN589050, –; Gypsophila scorzonerifolia Ser., JN589100, –; Gypsophila silenoides Rupr., JN589049, –; Gypsophila steveni Besser,
JN589022, –; Gypsophila violacea Fenzl, JN589068, –; Ochotonophila allochrusoides Gilli, Afghanistan, Bamian, Wendelbo & Ekberg W9801 (GB),
*KF924627, *KF924682; Petrorhagia prolifera (L.) P.W.Ball & Heywood, GU440883, –; Petrorhagia saxifrage Link, –, FJ404930; Petrorhagia thessala
(Boiss.) P.W. Ball & Heywood, GU440885, –; Petrorhagia velutina (Guss.) P.W. Ball & Heywood, AY857974, –; Psammosilene tunicoides W.C.Wu & C.Y.Wu,
JN589122, –; Saponaria ocymoides L., AY936271, FJ404936; Saponaria officinalis L., AY594313, FJ404937; Saponaria pumila Hayek, AY594311, –; Scleranthopsis aphanantha (Rech.f.) Rech.f., Afghanistan, Kabul, Rechinger 31265 (M), *KF924626, *KF924681; Silene ampullata Boiss., EF060223, –; Silene
austroiranica Rech.f., Aellen &Esfand., EF060204, EF061364; Silene campanulata Pers., –, DQ908812; Silene cariensis Boiss., EF060205, EF061365; Silene
conoidea L., FN821101, –; Silene corinthiaca Boiss., EF060206, EF061366; Silene dioica (L.) Clairv., –, FN821276; Silene echinosperma Boiss. & Heldr.,
X86845.1, Z83196; Silene fruticosa L., X86865, Z83188.1; Silene latifolia Poir., –, Z83171; Silene martyi Emb. & Maire, EF060213, EF061373; Silene mentagensis Coss., EF060236, EF061396; Silene nana Kar. & Kir, EF060217, EF061377; Silene nutans L., EF061361; Silene reticulata Desf., EF060216, Ef061376;
Silene vulgaris (Moench) Garcke, –, EF674192; Vaccaria hispanica (Mill.) Rauschert, AY857969, –; Velezia rigida L., GU440888, –.
Version of Record (identical to print version).
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