Plant Syst Evol (2010) 288:201–211
DOI 10.1007/s00606-010-0325-y
ORIGINAL ARTICLE
Phylogenetic position of African and Malagasy Pimpinella species
and related genera (Apiaceae, Pimpinelleae)
Anthony Richard Magee • Ben-Erik van Wyk
Patricia M. Tilney • Stephen R. Downie
•
Received: 23 March 2010 / Accepted: 1 July 2010 / Published online: 31 July 2010
Ó Springer-Verlag 2010
Abstract The phylogenetic position of the African and
Malagasy species of Pimpinella is assessed using nrDNA
ITS sequence data and a representative sampling of the
genus, including 16 species from Africa and Madagascar
and 26 species from Eurasia. The results of maximum
parsimony and Bayesian analyses of these data show that
the African and Malagasy species ally with their Eurasian
counterparts in Pimpinelleae. The genus Pimpinella is
rendered paraphyletic by the inclusion of African Cryptotaenia and the small African and Malagasy endemic genera
Frommia and Phellolophium. Within a paraphyletic
Pimpinella, three major clades are recovered, with the
African species occupying two of these clades. The current
sectional classification of the genus, based predominantly
on fruit vestiture, is largely artificial. Chromosome base
number, however, was found to be consistent with the
groupings recovered in the molecular analyses. Those
African and Malagasy Pimpinella species with a chromosome base number of x = 11 and largely glabrous petals
and fruits, form the earliest diverging clade together with
Frommia, which also has a base count of n = 11 and
A. R. Magee B.-E. van Wyk P. M. Tilney
Department of Botany and Plant Biotechnology,
University of Johannesburg, P.O. Box 524,
Auckland Park, Johannesburg 2006, South Africa
A. R. Magee (&)
South African National Biodiversity Institute,
Compton Herbarium, Private Bag X7,
Claremont, Cape Town 7735, South Africa
e-mail: A.Magee@sanbi.org.za
S. R. Downie
Department of Plant Biology,
University of Illinois at Urbana-Champaign,
Urbana, IL 61801, USA
glabrous petals and fruits. The remaining African species
ally with several Eurasian species of Pimpinella and share
a chromosome base number of x = 9 and usually hairy
petals and fruits.
Keywords Cryptotaenia Cytology Frommia
Fruit anatomy nrDNA ITS Phellolophium
Pimpinelleae
Introduction
Pimpinella L., with about 150 species distributed
throughout much of the Old World (Pimenov and Leonov
1993), is one of the largest genera of the family Apiaceae.
However, because of its large size, wide geographical
range and high levels of infraspecific variation, the generic
boundary of Pimpinella and its infrageneric relationships
remain unclear (Abebe 1992; Spalik and Downie 2007).
The genus was last revised in its entirety by Wolff (1927),
who subdivided it into three sections based on petal colour,
fruit and petal vestiture and life history. In this system, the
African and Malagasy species were accommodated within
sections Tragium and Tragioselinum depending on whether
the petals and fruits are papillose to hairy or glabrous
respectively. More recent taxonomic studies of the African
representatives of Pimpinella have revealed several new
tropical African species (Townsend 1985, 1989; Abebe
1989, 1992) and, as a result, about 49 sub-Saharan (Van
Wyk and Tilney 2004) and 5 Malagasy (Sales et al. 2004)
species are generally recognised.
In a recent analysis of nuclear ribosomal DNA internal
transcribed spacer (nrDNA ITS) sequence data, Spalik and
Downie (2007) reported that the African species hitherto
classified in Cryptotaenia DC. [i.e. C. africana (Hook. f.)
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Drude, C. calycina C.C.Towns., and possibly C. polygama
C.C.Towns.] comprised a clade within the tribe Pimpinelleae, together with the Malagasy endemic species Phellolophium madagascariense Baker and Pimpinella
betsileensis Sales & Hedge, and the African endemic genus
Frommia H.Wolff. As this African clade was sister group
to a clade comprising the Eurasian members of Pimpinella,
Spalik and Downie highlighted the need for the inclusion
of additional African Pimpinella species in future molecular phylogenetic studies to ascertain whether these species
were more closely related to the Eurasian or to the Malagasy group. The present study is aimed at assessing the
phylogenetic relationships of the African and Malagasy
species of Pimpinella and related genera through comparisons of fruit anatomy, cytology and ITS sequence data,
using a representative sampling of Pimpinella species.
These results represent a contribution towards an ultimate
reassessment of the generic boundary and infrageneric
classification of the genus Pimpinella.
Materials and methods
Fruit anatomy
Mature fruits from a representative sampling of African
and Malagasy species of Cryptotaenia (2 spp.), Frommia
(1 sp.), Pimpinella (13 spp.) and Phellolophium (1 sp.) were
studied. These fruits were first rehydrated and then placed in
a mixture of formalin, acetic acid and alcohol (FAA) for a
minimum of 24 h and subsequently treated according to a
modification of the method of Feder and O’Brien (1968) for
embedding in glycol methacrylate (GMA). This modification involves a final infiltration in GMA for 5 days.
Transverse sections, about 3 lm thick, were cut using a
Porter-Blüm ultramicrotome and stained according to the
periodic acid Schiff/toluidine blue (PAS/TB) method of
Feder and O’Brien (1968). Voucher specimens for the fruit
anatomical study are listed in Appendix 1.
Molecular data
Total DNA was extracted from herbarium or silica dried
material using a PureLinkTM Plant Total DNA Purification
Kit (Invitrogen, Carlsbad, CA, USA). The entire ITS region
was PCR-amplified using the primer combinations of Sun
et al. (1994) and the amplification methods outlined in
Downie and Katz-Downie (1996). Successfully amplified
PCR products were purified according to the ExoSAP
protocol of Werle et al. (1994) using 5 units of Exonuclease I (New England Biolabs, Ipswich, MA, USA) and
0.5 units of Shrimp Alkaline Phosphatase (Promega,
Madison, WI, USA). Sequencing reactions were carried out
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using a BigDye Terminator version 3.1 Cycle Sequencing
Kit (Applied Biosystems Inc., Foster City, CA, USA) and
sequenced on either an ABI (Applied Biosystems) 3130 XL
or 3730 XL sequencer. Complementary strands were
assembled and edited using Sequencher version 3.1.2
(Gene Codes, Ann Arbor, MI, USA) and manually aligned
in PAUP* (Swofford 2002), positioning gaps so as to
minimise nucleotide mismatches. Sources of material used
in the study are provided in Appendix 2. All newly
obtained sequences have been deposited in GenBank.
Phylogenetic analyses
The phylogenetic analyses are based on a representative
sampling of the genus Pimpinella, which includes 16 species from Africa and Madagascar and 26 species from
Eurasia. The 19 ITS sequences of African and Malagasy
Pimpinella (of which 18 were newly obtained) were analysed with 29 accessions of Eurasian Pimpinella (Tabanca
et al. 2005) and a sampling of several other genera from the
tribe Pimpinelleae obtained from Spalik and Downie
(2007). Included was a poorly known and undescribed
Zambian species referred to by Cannon (1973) as Pimpinella sp. B. Two species from the closely related tribe
Apieae and four species from the tribe Pyramidoptereae
were included as outgroups, with the latter tribe used to
root the trees. In total, 68 ITS accessions were included in
the phylogenetic study. Data sets are available on request
from the corresponding author. Phylogenetic analyses of all
data sets were conducted using the maximum parsimony
(MP) algorithm of PAUP* with character transformations
treated as unordered and equally weighted (Fitch parsimony; Fitch 1971) and gaps treated as missing data. Tree
searches were performed using a heuristic search with
1,000 random sequence additions, tree bisection-reconnection (TBR) branch swapping, and the MULPARS
option in effect. A limit of ten trees per replicate was set to
reduce the time spent on swapping in each replicate.
Internal support was assessed with 1,000 bootstrap replicates using TBR swapping and holding ten trees per replicate. Only bootstrap percentage (BP) values greater than
50% are reported, and the following scale was used to
evaluate support percentages: 50–74%, weak; 75–84%,
moderate; and 85–100%, strong. Appropriate models of
nucleotide evolution were selected for each dataset using
MODELTEST version 3.06 (corrected Akaike information
criterion; Posada and Crandall 1998). Bayesian inference
(BI) was performed for 3 million generations of Monte
Carlo Markov Chains with a sampling frequency of 100
using MRBAYES version 3.1.2 (Huelsenbeck and
Ronquist 2001; Ronquist and Huelsenbeck 2003). Suboptimal trees were discarded as the ‘‘burn-in’’ phase (25% of
all trees obtained), and from the remaining trees a majority
Phylogenetic position of African and Malagasy Pimpinella species
203
Table 1 Chromosome numbers of Pimpinella species and other taxa included in the ITS analyses
Taxa
Chromosome numbers
Sources
Aphanopleura capillifolia
n = 11
Vasil’eva et al. 1991, 1993
Bunium bulbocastanum
n = 10; 2n = 20
Buttler 1985; Vasil’eva et al. 1985; Verlaque and Filosa 1992
Cryptotaenia africana
n = 11; 2n = 22
Auquier and Renard 1975; Morton 1993
Crithmum maritimum
n = 10; 2n = 20
Al-Bermani et al. 1993; Vasil’eva et al. 1993; Pimenov et al. 1998
Deverra burchellii
n = 11
Al-Eisawi 1989 (as Pituranthos triradiatus)
Frommia ceratophylloides
n = 11
Constance et al. 1971, 1976
Nothosmyrnium japonicum
2n = 20
Pan et al. 1995
Pimpinella affinis
n = 9; 2n = 16
Constance et al. 1976; Yurtseva 1988
P. anisum
2n = 18,19
Pimenov et al. 2003
P. buchananii
n = 11; 2n = 22
Constance and Chuang 1982; Abebe 1992
P. caffra
P. corymbosa
n=9
n = 10; 2n = 20
Constance and Chuang 1982
Al-Eisawi 1989; Pimenov et al. 1996
P. cretica
n = 10
Al-Eisawi 1989
P. diversifolia
n = 9; 2n = 18
Cauwet-Marc et al.1982
P. eriocarpa
n=8
Al-Eisawi 1989
P. favifolia
n = 22
Constance and Chuang 1982
P. hirtella
2n = 18
Abebe 1992 (as P. volkensii)
P. huillensis
n=9
Constance and Chuang 1982
P. ledermannii
n=9
Constance and Chuang 1982 (as P. engleriana)
P. lutea
2n = 20
Verlaque et al. 1992
P. oreophila
2n = 18
Hedberg and Hedberg 1977 (as P. kilimandscharica)
P. peregrina
n = 9; 2n = 16, 18, 20
Yurtseva 1988; Abebe 1992; Pimenov et al. 1996
P. puberula
2n = 18
Yurtseva 1988
P. rhodantha
n = 9, 10; 2n = 20, 36, 40
Constance et al. 1976; Pimenov and Vassilieva 1983;
Yurtseva 1988; Daushkevich et al. 1995
P. saxifraga
2n = 18, 20, 36, 40
Gawlowska 1967
P. sp. B
n = 11
Constance and Chuang 1982
P. tragium
2n = 18, 20, (22)
Yurtseva and Tikhomirov 1998; Pimenov et al. 2003
P. trifurcata
2n = 22
Abebe 1992
Psammogeton biternatum
2n = 18
Ahmad and Koul 1980
Psammogeton canescens
n = 11
Khatoon and Ali 1993
Stoibrax dichotomum
n = 10
Silvestre 1990
Taxa for which chromosome counts were unavailable were coded as missing in the reconstruction analyses (see Fig. 3)
rule consensus was produced using the ‘‘sumt’’ command.
Only posterior probability (PP) values greater than 0.5 are
reported, and the following scale was applied: 0.50–0.84,
weak; 0.85–0.94, moderate; and 0.95–1.0, strong.
Chromosome base numbers (Table 1) were reconstructed onto all maximally parsimonious trees using
Mesquite version 2.5 (Maddison and Maddison 2008).
Results and discussion
Phylogenetic relationships
The ITS matrix consisted of 623 unambiguously aligned
nucleotide positions of which 317 were variable and 231
parsimony informative. The MP analysis resulted in 36
equally most parsimonious trees with a tree length (TL) of
788 steps, consistency indices (CI) of 0.59 and 0.53 (with
and without uninformative characters respectively) and a
retention index (RI) of 0.81. MODELTEST selected the
SYM ? G model of evolution for use in the BI analysis.
The topologies of the MP strict consensus tree and the BI
majority rule consensus tree were congruent (with the
differences between these trees summarised in Fig. 1) and
retrieved similar groups to those shown by Spalik and
Downie (2007). The tribe Pimpinelleae was recovered as
monophyletic (PP 1.0, BP 98). Within this tribe, the largest
genus Pimpinella was rendered paraphyletic by the inclusion of African Cryptotaenia and the small African and
Malagasy endemic genera Frommia and Phellolophium.
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Within the paraphyletic Pimpinella lineage (PP 1.0, BP
94), three major clades were recovered. The African species of Pimpinella were allied with both the P. hirtella
A.Rich. group in clade A and the P. buchananii H.Wolff
group in clade C. The majority of the Eurasian species of
Pimpinella sampled comprised a clade with P. saxifraga L.
(the type species) within a strongly supported (PP 1.0, BP
96) but poorly resolved clade B. Sister group to this Eurasian clade was a strongly supported clade A (PP 1.0, BP
98), comprising both African and Eurasian species of
Pimpinella. Within this clade the African species formed a
strongly supported group (PP 1.0, BP 99—the P. hirtella
group), sister to the Eurasian species. Clade C (PP 1.0, BP
80), which was the earliest to diverge, comprised only
African and Malagasy species. The African Cryptotaenia
was paraphyletic, and a clade comprising the two Malagasy
species of Pimpinella (PP 1.0, BP 100) was a weakly
supported sister group to the Malagasy genus Phellolophium (PP 0.84, BP 60). The monotypic African endemic
genus Frommia was placed within a group of tropical
African Pimpinella species (hereafter the P. buchananii
group) in which it was moderately to strongly supported as
a sister group to Pimpinella sp. B. (PP 1.0, BP 79).
African and Malagasy Pimpinella
The infrageneric classification of Pimpinella proposed by
Wolff (1927) does not correspond to any of the clades
recovered and, as such, it is clearly artificial (Fig. 1). The
main diagnostic characters of the three sections recognised
by Wolff (1927) are biennial or perennial plants with yellow
petals and variable fruits (sect Reutera); perennial (rarely
annual or biennial) plants with white petals and glabrous
petals and fruits (sect. Tragoselinum); or annual, biennial or
perennial plants with white petals and hairy to papillate
petals and fruits (sect. Tragium). In Wolff’s classification,
the African and Malagasy species were, with the exception
of P. hirtella which was placed in subsect. Eusaxifragae,
separated from the Eurasian species into either subsect.
Afrotragium or subsect. Afrotragoselinum. Although the
African and Malagasy species were placed into two of the
three major clades, they were found to ally within each. For
the African species, their separation based on the presence or
absence of hairs on the petals and fruits appears to be only
slightly useful. In general, the P. buchananii group and allied
Malagasy species have glabrous petals and fruits (although
they are variable in P. rigidistyla C.C.Towns.), while the
P. hirtella group has hairy petals and fruits (except for
P. kyimbilaensis H.Wolff and P. oreophila Hook.f., and in the
P. caffra D.Dietr. complex, where these features are variable). Although the presence or absence of hairs is a conservative, species-specific character for the tropical African
species, in the southern African members this character is
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well known to be of limited value because of its infraspecific
variation (Townsend 1985). However, the placement of the
southern African species P. caffra, P. krookii H.Wolff and
P. transvaalensis H.Wolff within the P. hirtella group suggests that the sporadic loss of hairs is likely to be a secondary
adaptation. While there are some useful fruit anatomical
differences among the species of Pimpinella (Fig. 2;
Table 2), fruit anatomy appears to be more useful for distinguishing species, as previously suggested by Abebe
(1992), than for the recognition of infrageneric groups.
While most of the African and Malagasy species have
multiple vallecular vittae between the ribs, in P. transvaalensis (Fig. 2d) and P. oreophila (Fig. 2c) only a single vallecular vitta between each rib was observed. This may prove
particularly useful to distinguish between the closely related
P. caffra and P. transvaalensis. However, fruits from many
more populations of both of these species need to be included
before any definite conclusions can be made.
Although no obvious morphological or anatomical
characters could be correlated with the results of the
molecular phylogenies, the reconstruction of cytological
data (Table 1) onto the MP trees revealed interesting results.
This is in contrast to the study of Abebe (1992) who could
not find any correlation between cytological variation and
the subgeneric divisions based on fruit and leaf morphology
within Pimpinella. In the subfamily Apioideae, the predominant chromosome base number is x = 11 (Moore
1971). Within Pimpinella, however, x = 8, 9, 10 and 11
have all been recorded, sometimes for the same species, with
x = 9 being the most prevalent. For all the species for which
counts are available within the early diverging clade C
(Fig. 3) comprising the P. buchananii group, including
Cryptotaenia africana, a count of x = 11 has been recorded,
while those species in the P. hirtella group consistently have
a count of x = 9, as found also in the Eurasian members of
this clade. Optimizations of chromosome base numbers
(Fig. 3) over all 36 minimal length trees differed only in the
reconstruction of P. sintenisii H.Wolff as having either a
base number of x = 10 or ambiguously reconstructed as
x = 9 or x = 10. Reconstruction of the cytological data
suggests x = 10 to be the plesiomorphic state for the genus.
A chromosome count of x = 11 found in the early diverging
clade C is recovered as an apomorphy for this lineage, while
x = 9 is apomorphic for clade A, with a subsequent decrease
to x = 8 in P. eriocarpa Sol. The ancestor to the entirely
Eurasian clade B, including the type species P. saxifraga,
appears to have had a basic chromosome number of x = 10,
although subsequent decreases to x = 9 have occurred in
many species. The possibility that x = 11 (the predominant
number in the Apioideae) is the plesiomorphic state within
the genus, with subsequent aneuploidy resulting in decreases
to x = 10, 9 and 8 in the more derived lineages, seems to be a
plausible hypothesis. However, counts for more species are
Phylogenetic position of African and Malagasy Pimpinella species
205
Fig. 1 Bayesian inference (BI) tree of ITS sequence data for
Pimpinella species and some related taxa (tribe Pimpinelleae) and
outgroups from tribes Apieae and Pyramidoptereae. Posterior probability (PP) values are presented above the branches. Bootstrap
percentage (BP) values are presented below the branches. Branches
supported only in the BI tree are indicated by dashed lines. The
current sectional classification of Pimpinella is indicated with both
coloured text and abbreviations alongside the species names. Those
species of Pimpinella not included by Wolff (1927) or Abebe (1992)
are indicated in black text. Tribal affinities and the placement of the
African species are indicated with brackets, while the circled letters
indicate the three major clades of Pimpinella discussed in the text
required, preferably from several populations, to assess
infraspecific variation, a feature already noted from several
Eurasian species (Table 1).
striking generic character is reflected in the epithet of its
only species, F. ceratophylloides Wolff. The leaves
resemble those of Ceratophyllum L. due to the sessile,
finely divided pinnae with linear ultimate leaf segments
(Cannon 1973). Although Cannon (1978) suggested a
possible affinity to the genus Carum L., the generic
placement of Frommia has remained unclear. In the analysis carried out by Spalik and Downie (2007), Frommia
Frommia
The genus Frommia is an anomalous African endemic
genus with a limited distribution in Malawi. The most
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A. R. Magee et al.
Fig. 2 Transverse sections through the fruits of African (a–j) and
Malagasy (k–n) species of Pimpinella, Phellolophium (o), Frommia
(p) and Cryptotaenia (q, r). a P. acutidentata (Fanshawe s.n., K);
b P. hirtella (Ash 2677, MO); c P. oreophila (Townsend 2428, K);
d P. transvaalensis (Miller 5910, K); e P. caffra (Hilliard & Burtt 9777,
K); f P. caffra subsp. conopodioides (Pawek 9268, K); g P. huillensis
(Milne-Redhead 10894, K); h P. buchananii (Milne-Redhead 9045A, K);
i P. trifurcata (Jean Pawek 13923, MO); j P. lindblomii (Gilbert 6531,
K); k P. tenuicaulis (Baron 3241, MO); l P. betsileensis (Humbert 28087,
P); m P. perrieri (Bosser 8622, P); n P. ebracteata (Baron 929, K); o Ph.
madagascariensis (Pettersen & Nilsson 359, K); p F. ceratophylloides
(Hooper et al 1844, K); q C. africana (Verdcourt 2481, PRE); r
C. canadensis (PE0866713). cv commissural vita; rd rib oil duct; rv
additional rib vita; vb vascular bundle; vv vallecular vita. Scale 0.4 mm
was placed sister group to a clade comprising the Malagasy
endemics species Phellolophium madagascariense and
Pimpinella betsileensis. In the current analysis, with a
broader sampling of African Pimpinella, the genus was
moderately to strongly placed as a sister group to the
undescribed Pimpinella sp. B., the latter known only from a
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Phylogenetic position of African and Malagasy Pimpinella species
207
Table 2 Summary of selected anatomical characters for the mericarps of those species of African Pimpinella and related taxa included in the
fruit anatomical study
Vestiture
Ribs
Number of
vascular
bundles
Rib oil ducts in
mature fruits
Number of
vittae
per vallecula
Additional
rib vittae
Commissural
seed surface
C. africana
Glabrous
±Prominent
5
Inconspicuous
2
-
Flat
C. canadensis
Glabrous
Inconspicuous
7
Inconspicuous
1
-
Flat
F. ceratophylloides
Glabrous
Inconspicuous
5
±Prominent
3
-
Concave
P. acutidentata
Glabrous
Inconspicuous
5
?
2–4
-
?
P. betsileensis
Glabrous
±Prominent
5
±Prominent
2 or 3
-
?
P. buchananii
Glabrous
Inconspicuous
5
Inconspicuous
3
-
Concave
P. caffra
Glabrous to pilose
±Prominent
5
Inconspicuous
3 or 4
-
Flat
P. ebracteata
Glabrous
±Prominent
5
±Prominent
3
-
Concave
P. hirtella
Pilose
Inconspicuous
5
Inconspicuous
3 or 4
-
Concave
P. huillensis
Pilose
±Prominent
5
Inconspicuous
3
-
Slightly concave
P. lindblomii
Glabrous
Inconspicuous
5
Inconspicuous
3
-
Concave
P. oreophila
Glabrous
±Prominent
5
Inconspicuous
1
-
Flat
P. perrieri
Glabrous
±Prominent
5
Inconspicuous
3
-
Flat
P. tenuicaulis
P. transvaalensis
Glabrous
Glabrous
Inconspicuous
±Prominent
5
5
±Prominent
Inconspicuous
3 or 4
1
-
Slightly concave
Flat
P. trifurcata
Glabrous
Inconspicuous
5
Inconspicuous
3 or 4
-
Concave
Ph. madagascariense
Glabrous
Very prominent
5
Inconspicuous
1
?
Flat
Question marks represent character states that were not visible in the material studied
few collections in Zambia (Cannon 1973), within the
P. buchananii group of clade C (Fig. 1). The latter species
also has finely divided, linear ultimate leaf segments like
Frommia, although without sessile pinnae. Pimpinella
lineariloba Cannon from Angola may form part of this
group as it also has linear ultimate leaf segments and,
according to Cannon (1973), a possible affinity to Pimpinella sp. B. In transverse section the fruits of Frommia
(Fig. 2p) appear similar to those of the P. buchananii group
(Fig. 2h–j), with multiple vallecular (three in each furrow)
and commissural (four, sometimes appearing confluent in
mature fruits) vittae, as well as a prominently concave
commissural seed surface (Table 2). Furthermore, a chromosome base number of x = 11, as found in Pimpinella sp.
B and the other species from the P. buchananii group
(Fig. 3h–j), has also been recorded for Frommia ceratophylloides (Constance et al. 1971, 1976).
Phellolophium
The Malagasy endemic genus Phellolophium was described
by Baker (1884) who proposed an affinity to Seseli L. and
Foeniculum Mill. Recently, another undescribed species
(Sales et al. 2004) was discovered, with an affinity to
Pimpinella speculated (Sales and Hedge 2010). In Spalik and
Downie (2007), Phellolophium was shown to be closely
related to the African members of the tribe Pimpinelleae,
together with Pimpinella betsileensis, the only Malagasy
species they sampled. In the current analysis, the sister group
relationship between Phellolophium and Malagasy Pimpinella was confirmed, with this group successively sister to the
P. buchananii group. While Wolff (1927) placed the Malagasy species of Pimpinella together with the African species
in his subsect. Afrotragoselinum, they were separated into the
series Madagassicae. Phellolophium shares the broad, pinnately divided leaves with some of the Malagasy species of
Pimpinella but differs in the presence of prominent involucral
and involucel bracts, fruits with thick corky ribs and four
vallecular vittae, and five slightly smaller vittae usually situated more or less below the ribs (Fig. 2o; Table 2).
Cryptotaenia
The geographically disjunct genus Cryptotaenia was
shown by Spalik and Downie (2007) to be polyphyletic,
with its species occurring in tribes Oenantheae (Cryptotaenia s.s.), Scandiceae (C. elegans Webb ex Bolle) and
Pimpinelleae (C. africana, C. calycina and possibly
C. polygama). As in the analyses done by Spalik and
Downie (2007), both the MP and BI analyses presented
herein place the African species of Cryptotaenia (C. africana, C. calycina) successively sister to the lineage comprising Phellolophium, the Malagasy species of Pimpinella,
and the P. buchananii group. The fruits of C. africana
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A. R. Magee et al.
Fig. 3 Parsimony-based reconstructions of chromosome base numbers (Table 1) in Pimpinella and related taxa when optimized over
one of the 36 minimal length trees inferred from MP analysis of ITS
data. The placement of the African species are indicated with
brackets, while the circled letters indicate the three major clades of
Pimpinella discussed in the text
(Fig. 2q) appear only superficially similar to those of
Cryptotaenia s.s. (Table 2). In C. canadensis (L.) DC.
(Fig. 2r) the fruits are diagnostically different with only
one vallecular vitta between the ribs and two additional
vascular bundles located near the commissure. The fruits of
C. africana, in contrast, have multiple vittae between the
ribs and only five vascular bundles (Fig. 2q), as found also
in most species of Pimpinella (Fig. 2a–n).
isolated position separate from the Eurasian species. These
authors, through biogeographical analyses, also suggested
that the African taxa were derived from a common ancestor
of Middle Eastern origin which extended into Africa
through a Middle East–East African tract and subsequently
into Madagascar. With the inclusion of additional African
and Malagasy species of Pimpinella in our study, it is now
apparent that these African and Malagasy taxa are not as
isolated as previously suggested. Although it is likely that
the species from Madagascar are derived from a single
ancestor of African origin, it would appear that more than
one dispersal event into Africa from Eurasia must have
taken place.
Biogeography
Spalik and Downie (2007) suggested that the African and
Malagasy members of tribe Pimpinelleae occupied an
123
Phylogenetic position of African and Malagasy Pimpinella species
Taxonomic implications
While it is tempting to simply include the anomalous African
genera Cryptotaenia, Frommia and Phellolophium into an
expanded and already rather poorly defined genus Pimpinella,
there remains a need for a broader sampling of the East Asian
species which, according to Zhou et al. (2008, 2009), are
placed within the ‘‘East Asia clade’’—a group that is distantly
related to Pimpinelleae. Formal taxonomic and nomenclatural
changes should only be implemented once the full range of
diversity within the genus has been studied. Chemotaxonomic
studies may also prove to be valuable in defining the genus, as
these studies indicate that Pimpinella is unique in the presence
of norsesquiterpenes and phenylpropanoids (Kubeczka 1997;
Tabanca et al. 2005). Although the morphological characters
traditionally used to define infrageneric groups within
Pimpinella appear to be of limited use, the results of this study
suggest that cytological data should be explored in more
detail as an important phylogenetic character.
Acknowledgments The authors thank the curators and staff of K,
MO, NBG, P and PRE who kindly made their specimens available for
study, and the University of Johannesburg, the South African Biosystematics Initiative, the National Research Foundation of South Africa
and the USA National Science Foundation (DEB 0089452) for funding.
Appendix 1: Voucher information for fruit material
studied in transverse section
Cryptotaenia DC.: C. africana (Hook. f.) Drude, Verdecourt
2481 (PRE); C. canadensis (L.) DC., PRE0866713 (PRE),
Howler 11047 (PRE). Frommia H.Wolff: F. ceratophylloides H.Wolff, Hooper et al. 1844 (K). Pimpinella L.:
P. acutidentata Norman, Fanshawe s.n. (K); P. betsileensis
Sales & Hedge, Humbert 28087 (P); P. buchananii H.Wolff,
Milne-Redhead 9045A (K); P. caffra D.Dietr. subsp. caffra,
Hilliard & Burtt 9777 (K); P. caffra subsp. conopodioides
C.C.Towns., Pawek 9268 (K); P. ebracteata Baker, Baron
929 (K); P. hirtella A.Rich., Ash 2677 (MO); P. huillensis
Welw. ex. Engl., Milne-Redhead 10894 (K); P. lindblomii
H.Wolff, Gilbert 6531 (K); P. oreophila Hook.f., Townsend
2428 (K); P. perrieri Sales & Hedge, Boissier 8622 (P);
P. tenuicaulis Baker, Baron 3241 (MO); P. transvaalensis
H.Wolff, Miller 5910 (K); P. trifurcata H.Wolff, Jean
Pawek 13923 (MO). Phellolophium Baker: P. madagascariense Baker, Pettersson & Nillssan 359 (K).
Appendix 2: Voucher information of ITS sequences
with corresponding voucher specimens (for the new
accessions) and GenBank accession numbers
New accessions of Pimpinella L. P. alismatifolia
C.C.Towns., FM986448; Bidgood et al. 3564 (K).
209
P. buchananii H.Wolff, FM986455; Winter 4179 (PRE).
P. buchananii H.Wolff, FM986456; Winter 4188 (PRE).
P. caffra D.Dietr., FM986447; Van Wyk et al. 4233
(JRAU). P. favifolia C.Norman, FM986458; Winter 3992
(PRE). P. favifolia C.Norman, FM986453; Phillips 1612
(MO). P. huillensis Welw. ex. Engl., FM986443; Biegel
3461 (PRE). P. huillensis Welw. ex. Engl., FM986454;
Fanshawe 1011 (P). P. hirtella A.Rich., FM986444; De
Wide 6578 (PRE). P. krookii H.Wolff, FM986445; Hilliard
& Burtt 14439 (K). P. kyimbilaensis H.Wolff, FM986452;
Gereau et al. 3150 (MO). P. ledermannii H.Wolff subsp.
engleriana (H.Wolff) C.C.Towns., FM986457; de Witte
6205 (PRE). P. oreophila Hook.f., FM986450; Abebe 1124
(MO). P. perrieri Sales & Hedge, FM986460; Boissier
16.027 (P). P. rigidistyla C.C.Towns., FM986459; Gereau
& Kayombo 3987 (MO). P. transvaalensis H.Wolff,
FM986449; Winter s.n. (PRE). P. trifurcata H.Wolff,
FM986446; LaCroix 4284 (PRE). P. sp. B, FM986451;
Brummitt et al. 16990 (MO). Published GenBank accessions. Aphanopleura capillifolia (Regel & Schmalh.)
Lipsky, DQ516368h. Aphanopleura trachysperma Lipsky,
AF008629e, AF009108e. Arafoe aromatica Pimenov &
Lavrova, AF077874c. Arafoe aromatica Pimenov &
Lavrova, U78383b, U78443b. Bunium bulbocastanum L.,
DQ443722j, DQ443724j. Crithmum maritimum L.,
AH003474a. Cryptotaenia africana (Hook. f.) Drude,
DQ516370h. Cryptotaenia africana (Hook. f.) Drude,
DQ516371h.
Cryptotaenia
calycina
C.C.Towns.,
DQ516372h. Deverra burchellii (DC.) Eckl. & Zeyh.,
AM408887i.
Frommia
ceratophylloides
Wolff,
DQ647630h. Nothosmyrnium japonicum Miq., DQ516367h.
Oedibasis platycarpa Koso-Pol., AF008632d, AF009111d.
Phellolophium madagascariense Baker, DQ647627h.
Phellolophium madagascariense Baker, DQ647628h.
Phellolophium madagascariense Baker, DQ647629h.
P. affinis Ledeb., AY581780g. P. anisetum Boiss. & Balansa, AY581781g. P. anisum L., AY581782g. P. aromatica M.Bieb., AY581784g. P. aurea DC., AY581785g.
P. betsileensis Sales & Hedge, DQ647626h. P. cappadocica Boiss. & Balansa, AY581786g. P. corymbosa Boiss.,
AY581787g. P. cretica Poir., AY581789g. P. cretica Poir.,
AY581788g. P. diversifolia DC., DQ516369h. P. eriocarpa
Sol., AY581790g. P. flabellifolia (Boiss.) Benth. & Hook.
f., AY581791g. P. isaurica Matthews, AY581792g. P. kotschyana Boiss., AY581793g. P. kotschyana Boiss.,
DQ516373h. P. lutea Desf., DQ516374h. P. niitakayamensis Hayata, DQ516375h. P. nudicaulis Trautv.,
AY581794g. P. oliverioides Boiss. & Hausskn.,
AY581795g. P. paucidentata Matthews, AY581796g.
P. peregrina L., AY581797g. P. peucedanifolia Fischer ex
Ledeb., AY581798g. P. puberula (DC.) Boiss.,
AY581799g. P. rhodantha Boiss., AY581800c. P. saxifraga L., AY581801g. P. sintenisii H.Wolff, AY581802g.
123
210
P. tragium Vill., AY581803g. P. tragium Vill., AY581804g. P. tragium Vill., AY581805g. Psammogeton biternatum Edgew., AF164839f, AF164864f. Psammogeton
canescens (D.C.) Vatke, AF008630e, AF009109e. Pyramidoptera cabulica Boiss., AF008631d. Stoibrax dichotomum (L.) Raf., DQ516366h.
Downie and Katz-Downie (1996)a; Downie et al.
(1998)b; Valiejo-Roman et al. (1998)c; Katz-Downie et al.
(1999)d; Lee and Downie (1999)e; Downie et al. (2000)f;
Tabanca et al. (2005)g; Spalik and Downie (2007)h; Winter
et al. (2008)i; Degtjareva et al. (2009)j.
References
Abebe D (1989) New species of Pimpinella (Umbelliferae) from
Ethiopia. Kew Bull 44:341–348
Abebe D (1992) Systematic studies in the genus Pimpinella L.
(Umbelliferae) from tropical Africa. Bot J Linn Soc 110:3
27–372
Ahmad I, Koul AK (1980) IOPB chromosome number reports,
LXVIII. Taxon 29:543
Al-Bermani A-KKA, Al-Shammary KIA, Gornall RJ, Bailey JP
(1993) Contribution to a cytological catalogue of the British and
Irish flora, 3. Watsonia 19:169–171
Al-Eisawi DM (1989) Chromosome counts of Umbelliferae of Jordan.
Ann Bot 47:201–214
Auquier P, Renard R (1975) Nombres chromosomiques de quelques
Angiospermes du Rwanda, Burundi et Kivu (Zaire). Bull Jard
Bot État Bruxelles 45:421–445
Baker JG (1884) Flora of Madagascar. Bot J Linn Soc 21:349–350
Buttler KP (1985) Chromosomenzahlen von Gefässpflanzen aus
Hessen (und angrenzenden Ländern). Hess Florist Briefe 34:
37–42
Cannon JFM (1973) Studies in tropical African Umbelliferae I:
Frommia ceratophylloides and the Diplolophium buchananii
complex. Edinburgh J Bot 32:195–201
Cannon JFM (1978) Umbelliferae. In: Launert E (ed) Flora Zambesiaca vol 4. Royal Botanical Gardens, Kew, London
Cauwet-Marc A-M, Carbonnier J, Farille M (1982) Contribution à
l’étude caryologique des Ombellifères du Nepal. I Candollea
35:497–510
Constance L, Chuang T-I (1982) Chromosome numbers of Umbelliferae (Apiaceae) from Africa south of the Sahara. Bot J Linn
Soc 85:195–208
Constance L, Chuang T-L, Bell CR (1971) Chromosome numbers in
Umbelliferae IV. Am J Bot 58:577–587
Constance L, Chuang T-L, Bell CR (1976) Chromosome numbers in
Umbelliferae V. Am J Bot 63:608–625
Daushkevich JV, Alexeeva TV, Pimenov MG (1995) IOPB chromosome data 10. Int Org Plant Biosyst Newsl 25:7–8
Degtjareva GV, Kljuykov EV, Samigullin TH, Valieo-Roman CM,
Pimenov MG (2009) Molecular appraisal of Bunium and some
related arid and subarid geophilic Apiaceae-Apioideae taxa of
the Ancient Mediterranean. Bot J Linn Soc 160:149–170
Downie SR, Katz-Downie DS (1996) A molecular phylogeny of
Apiaceae subfamily Apioideae: evidence from nuclear ribosomal
DNA internal transcribed spacer sequences. Am J Bot 83:
234–251
Downie SR, Ramanath S, Katz-Downie DS, Llanas E (1998)
Molecular systematics of Apiaceae subfamily Apioideae:
123
A. R. Magee et al.
phylogenetic analyses of nuclear ribosomal DNA internal
transcribed spacer and plastid rpoC1 intron sequences. Am J
Bot 85:563–591
Downie SR, Watson MF, Spalik K, Katz-Downie DS (2000)
Molecular systematics of Old World Apioideae (Apiaceae):
relationships among some members of tribe Peucedaneae sensu
lato, the placement of several island endemic species, and
resolution within the apioid superclade. Can J Bot 78:506–528
Feder N, O’Brien TP (1968) Plant microtechnique: some principles
and new methods. Am J Bot 55:123–142
Fitch WM (1971) Towards defining the course of evolution: minimal
change for a specific tree topology. Syst Zool 20:406–416
Gawlowska M (1967) Pimpinella nigra Willd. in Poland part III.
Numbers of chromosomes in Pimpinella nigra Willd. and related
species. Diss Pharm 19:439–450
Hedberg I, Hedberg O (1977) Chromosome numbers of afroalpine
and afromontane angiosperms. Bot Not 130:1–24
Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference
of phylogeny. Bioinformatics 17:754–755
Katz-Downie DS, Valiejo-Roman CM, Terentieva EI, Troitsky AV,
Pimenov MG, Lee B, Downie SR (1999) Towards a molecular
phylogeny of Apiaceae subfamily Apioideae: additional information from nuclear ribosomal DNA ITS sequences. Plant Syst
Evol 216:167–195
Khatoon S, Ali SI (1993) Chromosome atlas of the angiosperms of
Pakistan. Department of Botany, University of Karachi, Karachi
Kubeczka KH (1997) The essential oil composition of Pimpinella
species. In: Başer KHC, Kirimer N (eds) The 28th international
symposium on essential oils, proceedings, 1–3 September,
Eskisehi Anadolu University, Eskisehir, Turkey, pp 35–56
Lee B-Y, Downie SR (1999) A molecular phylogeny of Apiaceae
tribe Caucalideae and related taxa: inferences based on ITS
sequence data. Syst Bot 24:461–479
Maddison WP, Maddison DR (2008) Mesquite: a modular system for
evolutionary analysis. Version 2.5. http://mesquiteproject.org
Moore DM (1971) Chromosome studies in the Umbelliferae. In:
Heywood VH (ed) The biology and chemistry of the Umbelliferae. Supplement 1 to the Botanical Journal of the Linnaean
Society. Academic Press, London
Morton JK (1993) Chromosome numbers and polyploidy in the flora
of Cameroon Mountain. Opera Bot 121:159–172
Pan Z-H, Sheh M-l, Liu X-T, Yao X-M (1995) On karyotypes and
geographical distribution of endemic genera in Umbelliferae
from China. J Plant Resour Environ 4:1–8
Pimenov MG, Leonov MV (1993) The genera of the Umbelliferae.
Royal Botanic Gardens, Kew, London
Pimenov MG, Vassilieva MG (1983) IOPB chromosome number
reports LXXXI. Taxon 32:663–664
Pimenov MG, Dauschkevich JV, Vasil’eva MG, Kljuykov EV (1996)
Mediterranean chromosome number reports 6. Fl Medit 6:
288–307
Pimenov MG, Alexeeva TV, Kljuykov EV (1998) Mediterranean
chromosome number reports 8 (905–935). Fl Medit 8:221–245
Pimenov MG, Vasil’eva MG, Leonov MV, Daushkevich JV (2003)
Karyotaxonomical analysis in the Umbelliferae. Science Publishers, Enfield
Posada D, Crandall KA (1998) MODELTEST: testing the model of
DNA substitution. Bioinformatics 14:817–818
Ronquist F, Huelsenbeck JP (2003) MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:
1572–1574
Sales F, Hedge IC (2010) Ombellifères. In: Flora de Madagascar et
des Comores. Fam. 157. Muséum National d’Histoire Naturelle,
Paris
Sales F, Hedge IC, Coutinho AXP, Marques A (2004) Apiaceae
subfamily Apioideae in Madagascar. S Afr J Bot 70:446–448
Phylogenetic position of African and Malagasy Pimpinella species
Silvestre S (1990) Números cromosómicos para la flora Española.
592–602. Lagascalia 15:283–288
Spalik K, Downie SR (2007) Intercontinental disjunctions in Cryptotaenia (Apiaceae, Oenantheae): an appraisal using molecular
data. J Biogeogr 34:2039–2054
Sun Y, Skinner DZ, Liang GH, Hulbert SH (1994) Phylogenetic
analysis of Sorghum and related taxa using internal transcribed
spacers of nuclear ribosomal DNA. Theor Appl Genet 89:26–32
Swofford DL (2002) PAUP*: phylogenetic analysis using parsimony
(*and other methods), version 4.0b10. Sinauer Associates,
Sunderland, MA
Tabanca N, Douglas AW, Bedir E, Dayan FE, Kirimer N, Başer
KHC, Aytac Z, Khan IA, Scheffler BE (2005) Patterns of
essential oil relationships in Pimpinella (Umbelliferae) based
on phylogenetic relationships using nuclear and chloroplast
sequences. Plant Genet Resour 3:149–163
Townsend CC (1985) Notes on Pimpinella (Umbelliferae) in Central
and East Africa. Kew Bull 40:759–780
Townsend CC (1989) Umbelliferae. In: Polhill RM (ed) Flora of
Tropical East Africa. A.A. Balkema, Rotterdam
Valiejo-Roman CM, Pimenov MG, Terentieva EI, Downie SR,
Katz-Downie DS, Troitsky AV (1998) Molecular systematics of
Umbelliferae: using nuclear rDNA internal transcribed spacer
sequences to resolve issues of evolutionary relationships. Bot
Zhurn 83:1–22
Van Wyk B-E, Tilney PM (2004) Diversity of Apiaceae in Africa.
S Afr J Bot 70:433–445
Vasil’eva MG, Kljujkov EV, Pimenov MG (1985) Karyotaxonomic
analysis of the genus Bunium (Umbelliferae). Plant Syst Evol
149:71–88
Vasil’eva MG, Alexeeva TV, Pimenov MG, Kljuykov EV (1991)
IOPB chromosome data 3. Int Org Plant Biosyst Newsl 17:10–13
211
Vasil’eva MG, Daushkevich JV, Alexeeva TV, Pimenov MG (1993)
IOPB chromosome data 5. Int Org Plant Biosyst Newsl 20:7–9
Verlaque R, Filosa D (1992) Mediterranean chromosome number
reports 2 (107–117). Fl Medit 2:264–272
Verlaque R, Contandriopoulos J, Aboucaya A (1992) IOPB chromosome data 4. Int Org Plant Biosyst Newsl 18(19):9–10
Werle E, Schneider C, Renner M, Volker M, Fiehnet W (1994)
Convenient single-step, one tube purification of PCR products
for direct sequencing. Nucleic Acids Res 22:4354–4355
Winter PJD, Magee AR, Phephu N, Tilney PM, Downie SR, Van
Wyk B-E (2008) A new generic classification for African
peucedanoid species (Apiaceae). Taxon 57:347–364
Wolff H (1927) Umbelliferae-Apioideae-Ammineae-Carinae, Ammineae Novemjugatae et Genuinae. In: Engler A (ed) Das
Pflanzenreich, Heft 90 (IV. 228). W. Engelmann, Berlin
Yurtseva OV (1988) The cytologic study of some species of the genus
Pimpinella L. (Umbelliferae–Apioideae). Biol Nauki 11:78–85
Yurtseva OV, Tikhomirov VN (1998) Morphological diversity and
taxonomy of the Pimpinella tragium VIll. group (Umbelliferae–
Apioideae) in the Mediterranean. Feddes Repert 109:479–500
Zhou J, Peng H, Downie SR, Liu Z-W, Gong X (2008) A molecular
phylogeny of Chinese Apiaceae subfamily Apioideae inferred
from nuclear ribosomal DNA internal transcribed spacer
sequences. Taxon 57:402–416
Zhou J, Gong X, Downie SR, Peng H (2009) Towards a more robust
molecular phylogeny of Chinese Apiaceae subfamily Apioideae:
additional evidence from nrDNA ITS and cpDNA intron (rpl16
and rps16) sequences. Mol Phylogen Evol 53:56–68
123