High paraphyly of Swertia L.

(Gentianaceae) in the Gentianella-lineage as revealed by

nuclear and chloroplast DNA sequence variation
Author(s): P. Chassot, S. Nemomissa, Y.-M. Yuan and P. Küpfer
Source: Plant Systematics and Evolution, Vol. 229, No. 1/2 (August 2001), pp. 1-21
Published by: Springer
Stable URL: https://www.jstor.org/stable/23644058
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Plant Syst. Evoi. 229:1-21 (2001) Plant Systematics
and Evolution
1 Springer-Verlag 2001
Printed in Austria

High paraphyly of Swertia L. (Gentianaceae)

in the Gentianella-imeage as revealed by nuclear
and chloroplast DNA sequence variation
P. Chassot1, S. Nemomissa2, Y.-M. Yuan1, and P. Kiipfer1

'institut de Botanique, Laboratoire de botanique évolutive, Université de Neuchâtel, Suisse

2The National Herbarium, Biology Department, Addis Ababa University, Ethiopia

Received June 22, 2000

Accepted June 2, 2001

Abstract. The genus Swertia L., as currently occupy a more derived position. Two of the latter
defined, is polymorphic and mainly distributed clades
in show a close relation with species of
temperate regions of the northern hemisphere. Gentianella s. str., and one is included in a large
Phylogenetic relationships between Swertia and clade comprising Comastoma, Jaeschkea and Lo
the other genera of the Swertiinae sensu Struwe matogonium. Selected character states and their
et al. (unpubl. data) are discussed based on proposed polarity, such as number and structure
cladistic analyses of DNA sequence data. The of nectaries, stylar and seedcoat characteristics,
sequences used for this purpose include the trnL pollen morphology, fusion of floral parts and
(UAA) intron, the intergenic spacers (IGS) bechromosome number are discussed in the context
tween trnL (UAA) and trriF (GAA) exons, and of molecular data. Rugose, spinose, or winged
between trnS (UGA) and ycf9 exons of cpDNA,seeds are found mainly in basal lineages, while
as well as the ITS region of nrDNA. Although smooth ones are typical for derived species.
moderately resolved, the phylogenies resultingChromosome numbers follow a similar pattern
from the separate analyses of nuclear and chlowith x=13 restricted to basal lineages, while in
roplast data are congruent, and the incongruence more derived clades, x is always smaller than 13.
length difference test (Farris et al. 1995) detectedWith respect to the molecular phylogeny, taxo
no character incongruence. The phylogeny sug nomic circumscriptions in the Swertiinae sensu
gested by the analysis of combined data sets Struwe et al. (unpubl. data) does not seem to
defines Swertia as strongly paraphyletic in relation reflect phyletic relationships.
to the other genera. This taxon may have acted as
a stem group, giving rise to diverse lineages, someKey words: Gentianaceae, Swertia, phylogeny,
of which are morphologically distinct and have paraphyly, nectary, ITS, trnL, trnL-T, trnS, ycf9.
been recognised at the generic level. Latouchea and
Obolaria are closely related and occupy the
basalmost position in the molecular tree. Swertia Introduction
species are distributed in 9 different clades, three
of which share a basal polytomy with Bartonia, Since the last infrafamilial classification of
Frasera, Gentianopsis, Halenia, Megacodon, Gentianaceae published by Gilg (1895), the
Pterygocalyx and Veratrilla. Two lineages havecircumscription of the subtribe Gentianinae
an intermediate position. The remaining 4 clades has remained quite stable. Obolaria and

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 2 P. Chassot et al.: Phylogeny of Swertia

Bartonia were included in Grisebach's (1

tribe Swertieae by Bentham and Hooker
(1876) but were subsequently removed from
the Gentianinae by Gilg (1895). Both gene
were reincluded therein following the re
molecular results of Struwe et al. (1998). Th
Gentianella-lineage (Gillett 1957; Toyokun
1962, 1963; Ho and Liu 1990), in oppositi
to the Gentiana-lineage, was defined to inc
those genera of the subtribe Gentianinae sen
Gilg (1895) that have corolline nectaries,
corolla lobes with 5-9 vascular bundles, coro
las without plicae or folds and calyces with
an intracalycular membrane (present in Gen
tianopsis but of a different nature than i
Gentiana (Gillett 1957)). The genera that
into this category and that are dealt with i
this study are Comastoma, Frasera, Gentian
la, Gentianopsis, Halenia, Jaeschkea, Lom
togonium, Swertia and Veratrilla. The des
tion of the Gentianella-lineage was a first s
towards the recognition of two distinct e
tionary lineages in the Gentianinae Gilg. in honour of Emanuel Swert, a botanical
Previous molecular studies have confirmed author of the 17th century. The circumscrip
these two lineages (Yuan and Küpfer 1995, tion of the genus has often been debated,
Struwe et al. 1998). Pterygocalyx and Mega- resulting in disagreements among researchers
codon have whorls of nectaries at the base of of the family. Part of this debate is due to the
the ovary (Ho and Pringle 1995) but were morphological similarities (i.e. nectariferous
found to be distinct from Gentiana and its and rotate corolla lobes) of the species of
allied genera by the molecular studies of Yuan Swertia and related genera to one another,
and Küpfer (1995) and therefore also included namely, Halenia, Lomatogonium and Veratril
in the Gentianella-lineage. Latouchea also has la. Many species of these genera were
whorled nectaries at the base of the ovary but described under Swertia, e.g. Lomatogonium
has not been sequenced prior to this study. gamosepalum (as S. gamosepala), Veratrilla
Struwe et al. (unpubl. data) consider that baillonii (as S. mekongensis), Halenia cornicu
Gilg's subtribe Gentianinae should be raised lata (as S. corniculata). These genera are now
to a tribal status and further divided into two widely accepted as distinct from and perhaps
subtribes. These are subtribe Gentianinae more advanced in evolution than Swertia
(Crawfurdia, Gentiana, Tripterospermum) and (Allen 1933, Liu and Ho 1992). Lom
subtribe Swertiinae {Bartonia, Comastoma, was established as a distinct genu
Frasera, Gentianella, Gentianopsis, Halenia, (1830) based on Gentiana carinthi
Jaeschkea, Latouchea, Lomatogonium, Mega- on grounds of its decurrent stigma
codon, Obolaria, Pterygocalyx, Swertia, Verat- is dioecious and Halenia has sp
rilla). Therefore, the Gentianella-lineage, as lobes except in sect. Swertiella
used in the text and figures, is herewith defined where the corolla lobes have sh
to include all the aforementioned genera and ances.
corresponds to the subtribe Swertiinae sensu In addition to the similaritie
Struwe et al. aforementioned genera, the definition of

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P. Chassot et al.: Phylogeny of Swertia 3

Swertia proper has varied significantly s

was first described by Linnaeus. Many othe
genera were segregated from it, while ot
were redundantly described from Asia, Nor
America, Europe and Africa. The followin
taxa are now recognised as synonyms of
Swertia (Shah 1990, 1992; Pringle 1993; H
and Pringle 1995; Garg 1987): Frasera Walte
(1788), Tesseranthium Kellogg (1862) and
Leucocraspedum Rydb. (1917) from North f
America; Anagallidium, Ophelia, Agathotes
Griseb. (1839, 1845) and Kingdon-Wardia C.
Marquand (1929) from the Himalayas; Sczu
nia, Stellera and Rellesta Turcz. (1840, 18
from eastern Asia; Monobothrium Höchst. m
(1844) from Africa; Swertopsis Makino (1891
from Japan; Blepharaden Dulac (1867) fro
the Pyrénées. Henricea Lem. (1824) is not va
due to its earlier use in Asteraceae. For lo
Grisebach (1845), the generic concept of Sw
tia was narrow, that is, Anagallidium, Stell
and Ophelia were segregated from Swertia,
Frasera was accepted as a distinct genus.
Bentham and Hooker's (1876) generic conc
of Swertia was basically similar to that of i
Grisebach. Gilg (1895) listed the species of c
Frasera under his section Euswertia. Frasera
perhaps the most controversial of the variou
described synonyms of Swertia. Kuntze (
discussed its generic relationships and red
the taxon under Swertia. Card (1931) revise
Frasera and recognised it as a distinct ge
Frasera was restored to Swertia by St. Jo
(1941) and extensively dealt with by Pringl
(1979, 1990) where evidence was presented i
favour of its inclusion in Swertia. On the o
hand, Toyokuni (1965) even went beyond
recognising Frasera as a distinct genus an
moved some species of Swertia, e.g. S. tashi
S. pseudochinensis and S. bimaculata to Frase
and made new combinations of names acc
ingly. However, Toyokuni's systematic trea
ment of Frasera has not been widely accept
It is evident from this brief account that
generic concept of Swertia has never bee
stable, mainly because macromorphological
characters do not exhibit clear enough patte
to unambiguously justify taxonomic unit

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 4 P. Chassot et al.: Phylogeny of Swertia

(i.e. 6 for Swertia s. 1., and even less for the mini
other genera), and these views were not widely an
adopted. Molecular markers. Five molecular markers
Previous molecular results (Yuan and were used in this study, i
Kupfer 1995, von Hagen and Kadereit 2001 ) sPacers of nrDNA <ITS 1
revealed possible polyphyly or paraphyly in reSions f ■WoroplM'
0 . f , f. . , . c ■ and trnS-ycß) intergenic spacers). Primers ITS5
Swertia bu the limited number of species (5'.GGAAGTAGAAGTCGTAACA
sampled prohibited estimating to which extent. (5'-TCCTCCGCTTATTG
In view of the summary of the systematic (White et al. 1990) were used for th
problems involved with regard to the generic Gf the internal transcribed spacers
concept of Swertia and the lack of a rigorous ribosomal DNA. The name an
molecular phylogeny of the members of the primers used for the amplificatio
Gentianella-lineage, the present study mainly tron and IGS trnL-F follow Taberl
addresses 1) the circumscription of Swertia, Primers "c" (5'-CGAAATCGG
i.e., to test the monophyly of Swertia in regard G-3'), "d" (5'-GGGGATAGA
to its current taxonomic definition, 2) the AC-3 ) were used for the amplif
systematic position and affinities of Swertia to (UAA) intron and e (5 -GGTTCA
, . , • ., n ,. „ TATCCCC-3 ) and f" (5 -ATTTGAACTGGTG
related genera in the Gentianella-hneage and 3) . _. ^ ^ ' , r
,, , , c . . . „ . „ ACACGAG-3 ) were used for the spacer between
the phylogeny of the genera of the Gentianella- T n T A * a -» j , t? \ w
^ J ° J & trnL- (UAA)- 3-exon and tr«F-(GAA) gene.
meage. The spacer between trnS- (UGA) and ycf9 genes
was amplified with primers trnS (5'-GAGAGAG
Materials and methods AGGGATTCGAACC-3') and trnfM (5'-CATA
ACCTTGAGGTCACGGG-3') (Demesure et al.
Taxon sampling. The species included in this study 1995).
are listed in Table 1. Those sequences retrieved DNA extraction. DNA was extracted from
from Genbank are indicated with an asterisk silica gel dried leaf tissue (Chase and Hills 1991).
following their accession numbers. All the genera Total DNA extraction was made using the CTAB
included in the Swertiinae sensu Struwe et al. procedure of Doyle and Doyle (1987).
(unpubl. data) are represented. Sampling of Swer- Polymerase chain reaction (PCR). Double
t/a species has taken the following parameters into stranded DNA was directly amplified by PCR of
consideration: geographical distribution, morpho- all markers. Reaction volumes were 25 pi and
logical variation such as number and features of contained 2.5 pi 10X PCR buffer, 1 pi 25mM
nectaries on corolla lobes, seed surface morphol- Mg ,-+, 0.5 pi lOmM dNTPs, 0.5 pi of lOmM
ogy (smooth and rugose), palynology, habit primers, 0.2 pi HotstarTaq polymerase (5u/pl)
(annual, monocarpic and polycarpic perennials) (QIAGEN, Basel), and 17.05 pi ddH20. About
and existing infrageneric classifications (Ho et al. 10-20 ng genomic DNA was added to the PCR
1994; Shah 1990, 1992). All the geographical and cocktail. PCR was performed in a Biometra®
morphological groups in this taxon are sampled, Tgradient thermal cycler and consisted of 15 min
except for a few monotypic groups for which at 95 °C for the activation of the Hotstar polymer
material was not available. A total of 48 opera- ase, followed by 30 cycles of 30 sec at 94 °C, 30 sec
tional taxonomic units (OTUs) are included in at 55 °C, 1 min 30 sec at 72 °C with a final
this study as representatives of the ingroup taxa. extension period of 4 min at 72 °C.
Three Gentiana species were used as outgroup, PCR purification and sequencing. The quality
following the latest molecular studies of the and quantity of the PCR products were checked on
Gentianaceae - Gentianinae (Yuan and Küpfer 0.8% agarose gel using a mini-gel applying a low
1995, Struwe et al. 1998). They were selected from voltage. There was always a single sharp band
26 other species of the Gentiana-lineage (including resolved on the gel for ITS, trnL intron and IGS
Crawfurdia and Tripterospermum) following a trnL-F after each polymerase chain reaction,
preliminary analysis of ITS sequences such as to PCR products were purified using QIAquick™

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 P. Chassot et al.: Phylogeny of Swertia

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 8 P. Chassot et al.: Phylogeny of Swertia

purification kit (QIAGEN AG, Basel) following

the manufacturer's protocol prior to sequencin
Cycle sequencing was performed using the A
dideoxy chain termination method using an ABI c
PRISM™ BigDye™ Terminator cycle sequencing fo
kit with AmpliTaq DNA polymerase FS (Applied
Biosystems) in a Biometra® Tgradient therma
cycler (5 pi reaction volumes). Cycling paramete
were 25 cycles of 20 sec at 96 °C for denaturat
10 sec at 54 °C for primer annealing and 4 min at
60 °C for primer extension. The cycle sequencing
products were cleaned by ethanol precipitation and
applied to the ABI 310 automated sequencer each individual data set, resulting in a more
(Applied Biosystems). Basecalling was checked on accurate estimate of the true phylogeny (Barrett
the chromatograms using Sequence Navigator et al. 1991, Mishler 1994, Olmstead and Sweere
(Applied Biosystems) and edited manually when 1994). However, heterogeneity among data sets
necessary. Sequences have been deposited in Gen- other than sampling error, such as different sto
bank with their accession numbers indicated in chastic processes acting on the characters or
Table 1. different branching histories, can lead to erroneous
Sequence alignment. The boundaries of ITS phylogenetic inferences. Under
and trnL intron in the study material were it is justified to keep the da
determined by comparison with Gentiana sequenc- a consensus method (B
es (Yuan and Küpfer 1995, Gielly et al. 1996). The et al. 1995). Following
limits of IGS trnS-ycf) and IGS trnL-¥ were bination approach (Huelse
established by comparison with sequences of have used the incongruenc
Arabidopsis thaliana, Nicotiana tabacum and Zea test of Farris et al. (1995
mais (Genbank numbers: AP000423, Z00044, same settings as for other a
X86563). ITS sequences were preliminarily aligned in PAUP* to assess t
with ClustalX (Tompson et al. 1997) and subse- between the different da
quently hand-adjusted. Chloroplast sequences trnL, IGS trnL-¥, IGS trn
were aligned manually in order to respect indel
events otherwise not recognised. Potentially infor
mative indels of more than 1 base were recoded (1 Results
for presence, 0 for absence) and added to the data
matrix. The data matrix is available from the Characteristics of IT
authors on request. length ranges from 173 to 237 bases for ITS1
Phylogenetic analyses. All data sets were ana- and from 226 to 232 bases for ITS2. When
lysed with heuristic parsimony searches using aligned, the consensus sequences have 268 and
PAUP*, v.4.0b4 (Swofford 2000). In all analyses 267 sites for ITS1 and ITS2, respectively. Out
characters were equally weighted and unordered of these, 6 were excluded from ITS1 and 16
(Fitch 1971). Gaps were treated as missing data. All from ITS2 because of alignment ambiguities,
searches consisted of 1000 random taxon addition leaving respectively 116 and 113 informative
sequences with TBR branch swapping, MULPARS sites. ITS1 sequences were found to be slightly
and ACCTRAN options on; branches with mini- les§ dive t (lo/o_25o/o) than ITS2 (1o/o_
mum lengths of zero ( amb- ) were collapsed to 00n/\ a • c n
e . , 28%). Among species of Swertia, sequence
form polytomies. ' n ^
For the results based on bo
combined data, the robustness
was evaluated using jackknife w
(Farris 1996) as implemented in
index (Bremer 1988, Donoghu
analyses were performed with

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 P, Chassot et al.: Phylogeny of Swertia 9

divergence, and only one potentially inform

tive indel larger than 1 nucleotide was foun
ITS phylogeny. The parsimony analysis
carried out using PAUP* resulted in 70 equa
parsimonious phylogenetic trees of 980 step
(CI = 0.50, RI = 0.53 including autapomor
phies). Their strict consensus is presented in
Fig. la. The single possibly informative inde
situated near the end of ITS2 has no influen

Swertia crassiuscula , |*j

Swertia engleri
Swertia cuneata

Gentianella foliosa
Gentianella umbellata

Swertia punicea
Swertia binchuanensis
Swertia macrosperma
Swertia ciliata

Swertia pubescens
Jaeschkea microsperma
Swertia hispidicalyx
Swertia yunnanensis
Comastoma puimonarium
Comastoma traillianum

Lomatogonium brachyantherum
Lomatogonium macranthum
Lomatogonium perenne
Lomatogonium bellum
I Swertia aff. pseudohookeri
I Swertia chirayita
j Swertia decora
' Swertia delavayi
Halenla brevicornis

Halenla weddeliana
Halenla eiliptica
Swertia tetraptera
Swertia bimaculata
Veratrilla bailtoni
Swertia tashiroi
Bartonla virginica
Swertia kilimandscharica
Swertia volkensii
Swertia cordata

Swertia angustifolia
Swertia perennis
Swertia caiycina
Megacodon stylophorus

-I '■ Frasera albomarginata

1 Frasera speciosa
too i Gentianopsis grandis
J d12 ' Gentianopsis contorta
Pterygocalyx volubilis
Obolaria virginica
Latouchea fokienensis
Gentiana lutea
Gentlana phyllocalyx
Gentiana frigida

Fig. 1. Strict consensus trees of A 70 most parsimonious trees of 980 steps, CI = 0.50, RI = 0.53 (including
autapomorphies) from nuclear DNA analysis; B 24 most parsimonious trees of 659 steps, CI = 0.75, RI = 0.77
(including autapomorphies) from chloroplast DNA analysis. Above branches: jackknife values if greater than
50%. Below branches: decay index

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 10 P. Chassot et al.: Phylogeny of Swertia

random error and may in turn account for t

low ILD test value. trnL-F and trnS-ycf9, respectively. Out of
The ITS phylogeny presents an important these, 90 were excluded from tr
polytomy at its base, that consists of several trnL-F and 21 from trnS-yc
well supported clades. Latouchea forms a alignment ambiguities, leaving r
sister-group relationship with Obolaria. Gen- 83 and 76 informative sites. trn
tianopsis and Pterygocalyx are strongly asso- were found to be slightly more
ciated, as well as the two sampled species of 19%) than trnL (0%—12%) and
Frasera, that join in an independent clade (0%—11 %). When the three d
with high support. Megacodon forms an combined, the sequence divergen
isolated branch, as does Bartonia. Veratrilla 0% to 14%. As noticed in ITS
shows a sister group relationship to the sequence divergence between Sw
Halenia - Swertia clade with moderate jack- and other genera can be much lower than
knife value (66%) and decay index (d = 3). between two different species of Swertia. 32
Swertia is present pro parte in this basal potentially informative indels of more than 1
polytomy, notably in the well-supported clade nucleotide were recoded and added to the data
containing the type of the genus, S. perennis. matrix (5 from trnL, 14 from IGS trnL-F and
Two species (S. bimaculata and S. tetrapterd) 13 from IGS trnS-ycf9).
show a close link with Halenia. Swertia TrnL intron, trnL-F and trnS-ycf9 intergen
tashiroi does not group with any other ic spacer phylogeny. The parsimony analysis of
Swertia species present at the base of the the chloroplast data resulted in 24 trees of 659
tree. Swertia decora and S. delavayi also form steps (CI = 0.75, RI = 0.77 including autapo
a highly supported clade. morphies). The three chloroplast partitions
The upper part of the tree is better were found to be congruent with p = 0.42.
resolved, although jackknife support is below Figure lb presents the strict consensus of the
50% for deeper nodes, with at its base the 24 most parsimonious trees, which offers
moderately supported clade of S. aff. pseudo- a moderate degree of resolution. The basal
hookeri - S. chirayita. The Lomatogonium - polytomy suggested by the ITS data is partly
Comastoma clade lacks support as a whole, resolved by the chloroplast sequences. The
but L. brachyantherum is associated to Gentianopsis - Pterygocalyx clade and the
Comastoma with good jackknife and decay Obolaria - Latouchea clade appear at the base
values. Jaeschkea is placed in a polytomy with of the tree, with a jackknife value of 75% and a
the S. ciliata - S. pubescens clade and the S. decay index of 2 supporting their segregation
yunnanensis - S. hispidicalyx clade. Gentianel- from the remaining taxa. Also, the Swertia
la species are more closely related to Swertia decora - S. delavayi clade is excluded from this
than to Comastoma, Lomatogonium or Gen- polytomy with good support. For the remain
tianopsis. Within the uppermost Swertia ing taxa, the branching is identical to the one
clade, they formed a monophyletic sister found by the analysis of ITS data, with the
taxon to S. punicea and S. binchuanensis with exception of Veratrilla, that is not placed
a jackknife and decay index support of 67% inside the Swertia - Halenia clade.
and 2, respectively. The upper part of the tree consists of a large
Characteristics of chloroplast sequences. pentatomy, in which the individual
The chloroplast sequence length ranges from well supported. Here again, but with
361 to 393 bases for the trnL intron, from 234 to lution, the branching is similar
403 bases for the IGS trnL-F and from 271 to phylogeny, with the exception o
350 bases for the IGS trnS-ycf 9. This variation and the S. yunnanensis - S. hispidi
is mainly due to long A/T repeats in trnL, and that are included in a well supported
to indels in the two IGS's. When aligned, the with Lomatogonium and Comastoma.

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 P. Chassot et al.: Phylogeny of Swertia 11

ITS phylogeny, L.br achy antherum forms a s

group relationship with Comastoma species, (Yuan and Kiipfer 1995, von Hägen and
although this clade does not group with the Kadereit 2001). This idea has been further
remainder of Lomatogonium species. investigated by searching for trees compatible
Combined nuclear and chloroplast DNA with a constrained monophyletic Swertia
phylogeny. The ILD test probability was 0.54 clade. Such trees are 31 steps longer (ITS), 16
in favour of homogeneity between nuclear and steps longer (chloroplast data), and 46 steps
chloroplast data. All branches whose position longer (combined data) than the unconstrained
differs across the trees received low statistical most parsimonious trees, clearly departing
support in at least one tree, or formed a from the notion of sub-optimality (p < 0.1
polytomy. in SLP test for all data sets).
The analysis of equally weighted characters
yielded 48 trees of 1649 steps (CI = 0.60, „.
RI = 0.62). This represents only 10 more steps iscussion
than the sum of the most parsimonious trees Phylogeny of Swertiinae and Swertia. The
obtained by separate analysis of nuclear and potential difficulties inherent to the distinction
chloroplast data. Figure 2 presents their strict between a gene tree and species phylogeny
consensus. were explored in detail (e.g. Doyle 1992).
Statistical support is globally increased, not Combining data s
only for clades recognised independently from signal, which m
each data set, but also for the deeper nodes in the "correct" ph
the upper half of the tree. The phylogeny 1999, Hardig et al. 2000, von Hagen and
inferred from the combined data sets presents Kadereit 2001). The resolution of the internal
a different branching pattern than the ones clades and high jackknife values due to the
obtained from separate analysis. In the com- analyses of the combined data sets in the
bined tree, Obolaria and Latouchea are the Gentianella-lineage suggests that this is also
basalmost taxa, as suggested by the chloroplast the case in our study. The tree derived from the
tree, whereas Gentianopsis and Pterygocalyx analysis of combined data sets was thus used
are placed in the large basal polytomy. Data as final phylogenetic hypothesis,
combination has brought no further resolution Swertia, as currently defined in regional
of this polytomy, except for the grouping of floras or otherwise (Pringle 1990, 1993; Shah
Bartonia and S. tashiroi. The position retained 1990, 1992; Ho and Pringle 1995), is not a
for Veratrilla is the same as in the ITS tree. monophyletic taxon when considering the
Swertia decora and S. delavayi are placed molecular data. It is a strongly paraphyletic
according to the chloroplast tree, whereas the stem group, and the data suggest that different
position of S. chirayita and S. aff. pseudohook- ancestral taxa or lineages of Swertia account
eri follows the ITS tree. Jaeschkea and the for the origin of several morphologically dif
S. hispidicalyx - S. yunnanensis clade group ferent genera of the Gentianella-lineage. Nev
with Comastoma and Lomatogonium as it is the ertheless, despite the use of 5 different markers,
case in the chloroplast phylogeny. The position parsimony analysis of our data still leaves two
of Gentianella species as sister group to S. binch- major polytomies unresolved.
uanensis S. punicea remains unchanged. The Phylogenetic relationships in the Gentianel
S. crassiuscula - S. macrosperma clade, present /n-lineage have been addressed previously
but poorly supported in the ITS tree, and not (Yuan and Kiipfer 1995, Struwe et al. 1998,
detected in the chloroplast tree, has a good von Hägen and Kadereit 2001), yet, only few
jackknife support in the combined phylogeny. of the concerned taxa were included, and the
The phylogenetic relationships resolved in phylogenies were based on fewer markers,
these analyses confirm previous molecular These studies differ from our results notably

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 12 P. Chassot et al.: Phylogeny of Swertia

by proposing additional resolution for some

branches included in the polytomies of our t
tree, although these relationships receive only
low statistical support. Whether the lack of the

. Swertia crassiuscula 2 f f P1 20(1) Ophelia, perennes

- Swertia engieri 2 1 f p1 20(2) Ophelia, perennes
- Swertia cuneata 2 f f P1 Rugosa, -
■ Gentianeila ioKosa 1 t n P1
- Gentianeila umbellata 1 t n a 18(3)
- Swertia punicea 2 1 f a 20(19) Ophelia, ramosae
- Swertia binchuanensis 2 I f a 20(19) Ophelia, ramosae
• Swertia macrosperma 2 f 1 a
18(19) Ophelia, ramosae
2 12, 18 (4. 5)
0. Comastoma pulmonarium t n a

P Comastoma traillianum 2 t n a

Lomatogonium brachyantherum 2 f 1 a 16(19)

2 f 1 a 16(5)
Lomatogonium perenne 2 f 1 P1 -

ELomatogonlum macranthum
Lomatogonium bellum 2 1 1 a

Swertia hispidicalyx 2 1 f a +
Platynema, -
I Swertia yunnanensis 2 1 f a n=10 (19) •
Ophelia, ramosae
Jaeschkea microsperma 2 i n a
16(6)
r Swertia ciliata 1 1 n a n=10. 12(7, 8) +
Platynema, -
' Swertia pubescens 1 f n a 36(19) +
Platynema, -
1 f f P2 + Macranthos, -
I Swertia aff. pseudohookeri
I Swertia chirayita 2 f f a n=13 (7) Ophelia, ramosae
-

- Swertia decora 2 1 f a 26(19) + Ophelia, ramosae

-I—s
1 Swertia delavayi 2 f f a 26(19) +
Ophelia, ramosae
Haienia brevicornis 1 t s a n=11+2 (9)
Haienia weddeliana 1 t s P1 n=11 (10)
Haienia elliptica 1 t s a 22(5)
Swertia tetraptera 2 f 1 a 14(11) Heteranthos, -
Swertia bimaculata 2 f n a 26, 24. r>=11 -

Ophelia, maculatae
Veratrilla bailioni 2 f f pi - [(20.12,13)
Swertia iashiroi 1 f n a 52(19) Ophelia, maculatae
Bartonia virginica (1) t (n) a n=26 (14)
Swertia kilimandscharica 4a 1 I f pi 26(1) *■ Montana, -
Swertia volkensii 4 1 f n pi 26(1) Montana, -
Swertia cordata 4 1 f n a n=13 (7) + Ophelia, maculatae
Swertia angustifolia 2b 1 f f a 26 (7) +
Spinosisemina, -
Swertia perennis 3a 2 f 1 p1 28 (15) Swertia, -
Swertia calycina 2a 2 f 1 P1 -

Rugosa, -
Megacodon stylophorus 4 w t P2
Gentianopsis grandis 2b 1 t n a 52(5)
Gentianopsis contorta 2b t

Pterygocatyx volubilis 3ab w t -

P1 24 (18)
Frasera albomarginata 2 1 f I P2 26(16) + Frasera, -
Frasera speciosa 3a 2 1 f P2 n=39 (16) + Frasera, -
Obolaria virginica 1 w t P1 n=28 (17)
-d Latouchea fokienensis 4b w t -
P1
Gentiana lutea
Gentiana phyllocalyx
Gentiana f rigida

Fig. 2. Strict consensus tree of 48 most parsimonious trees of 1649 steps, CI = 0.60, RI = 0.62 (including
autapomorphies) from analysis of combined data. Above branches: jackknife values if greater than 50%. Below
branches: decay index. In regard: states for the principal characters discussed in the text. References for
chromosome numbers: (1) Hedberg and Hedberg (1977), (2) Nemomissa (1994), (3) Gagnidze et al. (1992), (4)
Krogulevitch (1978), (5) Yuan and Küpfer (1993), (6) Yuan et al. (1998), (7) Khoshoo and Tandon (1963), (8)
Mehra and Gill (1968), (9) Weaver and Rudenberg (1975), (10) Pringle (1981), (11) Ho et al. (1999), (12)
Shigenobu (1983), (13) Roy et al. (1988), (14) Rork (1949), (15) Favarger (1952), (16) Post (1956), (17) Kondo
(1970), (18) Yuan and Küpfer (unpublished), (19) present study, (20) Wada (1954)

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 P. Chassot et al.: Phylogeny of Swertia 13

clades consist of geographically isolated ta

and/or genera represented by only a few
species. Swertia tashiroi is an insular end
in Taiwan and the south-western islands of t
Japan. Its fleshy habit and chromosome n
ber (2n = 52) distinguish it from all the othe
Swertia species included in the basal polytom
Megacodon is a distinct genus comprising only
two species restricted to the eastern Himala-
yas. Veratrilla is morphologically very clos
Swertia but its two species are clearly dioe-
cious, a very rare trait in the Gentianella- inae (Meszaros 1994). According to Ho and
lineage. Latouchea (1 species) is endemic to Pringle (1995), nectaries are sometimes absent
south-eastern and south-western China. Bar- in Pterygocalyx. Megacodon, Latouchea and
tonia (4 species) and Obolaria (1 species) are Obolaria are different by bearing nectaries in a
North American saprophytes. Frasera (14 whorl at the base of the ovary like in Gentiana.
species) is also restricted to North America Regarding Obolaria, Holm (1897) states that
and, despite an indubitable resemblance with "the corolla bears nectaries", and describes a
Swertia, seems to have differentiated indepen- scale halfway between the base of the filament
dently from the remaining Swertia species. and the base of the corolla tube. He then adds
As suggested in Yuan and Küpfer's (1995) that "the grooves are very imperfect". Howev
ITS tree and von Hägen and Kadereit's (2001) er, Lindsey (1940) reports glandular material at
matK/lTS tree, Comastoma and Lomatogoni- the base of the ovary. Wood and Weaver (1982)
um also group together in our phylogeny, but believe that the latter are more likely the
in a broader clade including Swertia pro parte nectaries. Concerning Bartonia, neither Wood
and Jaeschkea. Lomatogonium was found to and Weaver (1982) nor Holm (1907) mention
be polyphyletic by von Hägen and Kadereit nectaries. A recent collection of B. Virginica
(2001) and our results also suggest its para- was examined by the authors. The basal part of
phyly in relation to Comastoma. Lomatogo- the corolla lobes was found to be thickened and
nium brachyantherum has none of the of brownish-green colour. It was not possible
characteristics of Comastoma (corolla lobes to assess if this tissue is glandular but it is
fused in a tube with fringed scale at its base). reminiscent of the nectariferous tissue found
The scenario that such an observed clustering in S. tashiroi or S. bimaculata.
of L. brachyantherum with Comastoma has its Corolline nectaries are either fimbriate or
root in an erroneous definition of the taxon is naked, with or without well-developed mar
ruled out. The Gentianella species form a clade ginal scales, or even sunken in spurs as in
corresponding to the Gentianella s. str. (uni- Halenia. The nectary features such as number,
nectariate) clade of von Hägen and Kadereit as well as their nakedness or fimbriation were
(2001) with the same Swertia species as sister used for the segregation of genera. But in
clade. Binectariate Gentianella species are ab- regard to the molecular phylogeny, nectaries
sent from our taxon sample but were placed cannot be considered as reliable synapomor
clearly outside of the Gentianella s. str. clade phic characters at the generic level. Their
and close to Lomatogonium species in von number may be identical between two distantly
Hägen and Kadereit's (2001) study. Note that related clades, and, similarly, naked nectaries
Jaeschkea microsperma is included in the characterise distantly related genera like Bar
broad Lomatogonium - Comastoma - Swertia tonia (?), Comastoma, Gentianella, Gentianop
clade in our combined phylogeny, whereas sis, Pterygocalyx as well as some species
J. oligosperma was found to be clearly sepa- of Swertia included in this study, e.g.

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 14 P. Chassot et al.: Phylogeny of Swertia

S. bimaculata, S. volkensii and S. tashiroi.

Fimbriate nectaries are found in Frasera
Veratrilla and Swertia, where the fimbr
can also occur in association with a more
less developed marginal scale. Spurs howev
are restricted to Halenia sect. Haleniastrum
(Allen 1933). At the infrageneric level howe
von Hägen and Kadereit (2001) have show
that nectary number and fimbriation are r
resentative of phylogenetic relationships
Gentianella. Such a pattern is not found f
Swertia species. rica clade are different from those of all other
Little has been published on the pollination species. They are either corrugate
biology of Swertia and allied genera. In their (S. volkensii, S. calycina, S. cordat
observations, Khoshoo and Tandon (1963) dral with spinose projections (S. ang
note that even in mixed populations of cross- or flat with an annular wing (S. pere
pollinated Swertia, nearly each studied species other species of Swertia have seeds m
has its specific pollinator and that the shape and rounded, with a smooth to warted te
fimbriation of the nectaries seem to play an small wings in the S. aff. pseudohooke
important role in the pollinator specificity. Future investigation of a broader
Under such pollinator-mediated selective près- pie will allow assessing with more prec
sure, both fimbriate and naked nectaries may reliability of this character,
have evolved several times and been acquired Molecular results were also comp
independently in the different lineages of the detailed pollen studies carried
the Gentianella-lineage from plesiomorphic Nilsson (1964, 1967, 1970), Nilsson and
whorled nectaries situated around the ovary as Skvarla (1969) and Jonsson (1973). According
is seen in Latouchea, Megacodon and Obolaria. to Nilsson, pollen morphological data are
To be noted, is that in a majority of cases, the difficult to interpret due to similar pollen types
nectary itself is devoid of fimbriae in those taxa in unrelated taxa throughout the Gentianella
where it is not readily accessible, i.e. when the lineage, and also to transitional sexine patterns
corolla lobes are fused {Bartonia (?), Comas- when more material is investigated. Only few
toma, Gentianella, Gentianopsis, Jaeschkea), or suggestions regarding inter-generic pollen mor
when hidden inside a spur as in Halenia sect. phological affinities were made. Nilsson's re
Haleniastrum (Allen 1933). Similarly, a parallel mark that the pollen structure of Comastoma
tendency towards conspicuous fimbriation can and Gentianopsis/Pterygocalyx is clearly dif
be seen with the vascularised fimbriae at the ferent from that of Gentianella is supported by
base of the corolla lobes in Gentianella and the our tree, as well as the affinity of Comastoma
comparable but nonvascularised structure in with Lomatogonium. Likewise, the pollen of
Comastoma. In Gentianopsis, fimbriae are ab- Frasera is different from that of most other
sent, but the petals are fringed in many species. Swertia species. On the other hand, the simi
When viewed in the context of the molec- larity between the pollen of Halenia and
ular phylogeny, the character state of seed Lomatogonium/Comastoma is not congruent
shape and ornamentation doesn't appear as with the molecular data. Concerning Swertia,
representative of relationships between genera. Nilsson treated the species according to their
Many unrelated taxa have smooth rounded geographic distribution and only made corn
seeds, and, similarly, Veratrilla bailloni, Swer- ments on a few interesting taxa. The African
tia perennis and Frasera speciosa for example species are divided in 3 groups. In the S. eras
all have flat annular winged seeds. Neverthe- siuscula type, the exine is beset with spinules.

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 P. Chassot et al.: Phylogeny of Swertia 15

The distinction between the other two gr

(5. kilimandscharica and S. volkensii types) is
less marked, and there are a number of speci
with transitional pollen morphology (Jonsso
1973), but both types are devoid of spinules.
This is congruent with the molecular tree.
Regarding the Asiatic species, such a clear
correlation between Nilsson's five pollen typ
and our tree cannot be found. Spinuliferou
exine seems to be a good character in discr
inating the two African clades, but in th
context of the whole Gentianella-lineage, t
trait appears to be homoplasious. Beside th
African species of the S. crassiuscula type
spinules are found in S. cuneata, the only
Himalayan species belonging to the African l
S. crassiuscula clade, S. tashiroi and Loma-
togonium p.p. same clade. Swertia calycina and related spe
The presence of a style has been used as a cies are all perennial and
distinguishing feature in the segregation of ecology. These species ar
Frasera from Swertia by Bentham and Hooker annual species of the S. an
(1876) and has been commonly retained by occur at lower elevations.
later authors as a diagnostic character of the valid for the S. aff. pseudo
taxon. At that time, little was known of the and S. chirayita (annual) gr
global variability of Swertia, but it is now discussed above, the charac
evident that styled stigmata are found in many or winged seeds doe
Swertia species. Besides, as Pringle (1990) synapomorphic with respec
mentions for Swertia perennis and Frasera, and proper to the basal S. calycina -
stylar differences are "a matter of relative S. kilimandscharica lineage of Swertia.
length rather than being qualitative". When Cytological data and taxonomy. As pointed
viewed in regard to the molecular tree, it is out by Pringle (1990), earlier taxonomic defi
clear that this trait does not reflect phyloge- nitions and phyletic hypotheses based on
netic relationships in the genus. chromosome counts of a restrained number of
Another morphological marker worth con- species of the Swertia complex have not been
sidering is the fusion of corolla lobes to form a confirmed by the increasing number of counts
tube. Although petals are fused in all the taxa of new taxa. Many species in the Gentianella
of the Gentianella-lineage, the tube is nearly lineage have been recently studied cytologically,
inconspicuous in Frasera, Lomatogonium, giving rise to new considerations (summarised
Swertia and Veratrilla, in contrast to the other by Yuan 1996 and Pringle 1990). Concerning
genera where it is well developed. This floral Swertia s. 1., Pringle notes that there are two
trait has significantly contributed to the con- prevailing base numbers, i.e. x= 13 and x = 10,
fusion in the taxonomy of the Gentianella- with a few aneuploid and polyploid species in
lineage. As shown by the phylogenetic tree, both groups. This is congruent with our results,
corolla tubes have appeared and disappeared where all species from S. pubescens upward in
several times in the group. Therefore, phylo- the tree have n = 9, 10, 18, all others sharing
genetic inferences and subsequent grouping n=13, 14, 26. Only S. tetraptera (n = 7) (Ho
based on the extent of corolla tube formation et al. 1999) departs from this picture. More
may be misleading. generally, and despite the lack of data for

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 16 P. Chassot et al.: Phylogeny of Swertia

several genera, x= 13 seems to be common

the basal lineages, although there are a few r
variations around this number. The paucity
species investigated in the more derived c
(S. pubescens upward and particularly Loma-
togonium and Comastoma), coupled with
greater variability of chromosome numbers
keeps us from understanding karyological
relationships. Yuan and Küpfer's (1993) sug
gestion that Comastoma (x = 8, 9) shows mor
affinity with Lomatogonium (x = 8) than
Gentianella (x = 9) from a karyological an
morphological point of view is also supported
by our phylogeny. The position of Jaeschk
(n = 8) close to Lomatogonium and Comastom
is also congruent with the karyology, althou
different chromosome numbers have been
reported for other Jaeschkea species (n = 9,
for J. oligosperma (as J. gentianoides) (Go
et al. 1981, Koul and Gohil 1973) and n= 10
for J. canaliculata (as J. latisepala) (Mehra
Vasudevan 1972, Vasudevan 1975)). earlier synonyms were published by authors
The importance of polyploidy associated to unaware of previous descriptions or unfamiliar
dysploidy and/or hybridisation has been with the global morphological variability
stressed in the explanation of the karyotype exhibited by species on other continents. He
evolution of Gentianopsis and particularly of further remarks that erroneous descriptions of
Gentiana (Yuan and Küpfer 1993, Yuan 1996). type material has also contributed to mislead
Several examples of polyploidy can be found in ing taxonomic treatments. Although most
our phylogeny (e.g. Bartonia, Frasera speciosa, present day authors agree to consider Swertia
Obolaria, Swertia tashiroi, S. pubescens). Cases as a large genus including all previous segre
of up dysploidy are found in S. perennis and gates, some synonyms are still debated and
Obolaria. Down dysploidy seems to be more pertinent to the molecular phylogeny. These
frequent, e.g. Pterygocalyx, S. bimaculata, are Anagallidium, Frasera and Ophelia, and
Halenia spp., S. pubescens. The case of shall be briefly discussed here. Grisebach
S. tetraptera illustrates the importance of this (1836) separated S. dichotoma and S. tetrapet
factor in the karyological evolution of the ala to the genus Anagallidium. Later, Ma
Swertiinae. Its position in the phylogeny sug- (1976) also placed S. tetraptera in this genus,
gests that it is derived from an ancestor with Even if there are no reliable characters i
n = 13 and that dysploidy may have affected up Grisebach's generic diagnosis, these three sp
to 12 chromosomes during the evolution of this cies are taxonomically closely related (Shah
taxon. In regard to the molecular phylogeny, it 1992). Embryological studies in S. tetraptera
seems that parallel evolution of karyotypes due (Xue et al. 1999) reveal more affinities with
to the aforementioned factors may have led to Halenia than with Swertia, and the position o
similar chromosome numbers in unrelated lin- S. tetraptera in our phylogeny suggests that
eages, e.g. between Gentianella spp. (n = 9, 18) Anagallidium might be a justified taxon
and S. pubescens (n = 18), or between Halenia although its proper rank and its relation
spp. (n=ll) and Gentianopsis ciliata (n = 22) S. bimaculata may be difficult to determin
(Favarger 1949), at different ploidy levels. The status of Frasera has been widely debated

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P. Chassot et al.: Phylogeny of Swertia 17

although no reliable character has ever be

brought up to justify its separation from
Swertia (Pringle 1990). Nilsson's studies in
cate that the species of Frasera may have a
monophyletic origin. Only two species are
included in our tree, but preliminary resu
including all 14 species (not shown) suppor
the monophyly of this lineage without a c
link to any other Swertia species, contrarily
Toyokuni's (1965) suggestions. At present, th
taxon is generally regarded as a section o
Swertia (Pringle 1990) but its position in
phylogeny at the same level as well differ
ated genera might favour its recognition at
generic rank, a position also adopted by e
Struwe et al. (unpubl. data). The definitio
and taxonomic rank of Ophelia have varie
depending on the authors. At present, th
character referred to for treating Ophelia
distinct taxon under Swertia is the annual
habit of the plants (excepted sect. Opheli
perennes in Ho et al. 1994). Our phylogeny
clearly suggests that the annual or perennial
habit of the species is not representative
relationships in Swertia, neither at a gene
level, nor at a sectional level. by Grisebach (1836). Garg (1987) proposed
A number of local infrageneric classifica- S. ciliata as lectotype. In the Chinese edi
tions have been attempted for Swertia (see of the Flora of China, Ho et al. (1988) g
Nemomissa (1994) for a review), but only S. angustifolia as type of sect. Ophelia. N
three publications offer a treatment of the that S. angustifolia perfectly fits the des
whole genus (Shah 1990, 1992; Ho et al. tion of sect. Spinosisemina published later b
1994). Shah divided Swertia into two sub- Ho et al. (1994).
genera (Swertia and Ophelia) and proposed In order to elaborate a satisfactory class
35 informal groups according to 1) geo- fication of Swertia and allied genera, and to
graphical distribution, and 2) morphological better understand the phylogeography of this
similarities. Ho et al. proposed 11 sections group, it seems necessary to first establish clear
and 16 series based on the hypothetical relationships between taxa and to identify
evolutionary polarity of morphological char- reliable morphological characters correlated
acters. When compared, these two classifica- with the paraphyletic Swertia clades identified
tions are contradictory in many ways, that in the phylogeny. Discussing biogeographic
is, species in one of Shah's groups are placed issues and character polarisation section by
in different series by Ho et al. and vice section as attempted by Ho et al. (1994) is not
versa. Figure 2 shows the position of species convincing in absence of the aforementioned
in the subdivisions of Shah and Ho et al. conditions. A phylogenetic analysis of a much
respectively. Both classifications are not wider taxon sampling (ca. 150 species) is
congruent with the molecular data. The underway in our laboratory and should
subdivisions of Swertia according to the allow proposing a comprehensive taxonomic
perennial versus annual habit of the species treatment reflecting evolutionary relationships

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 18 P. Chassot et al.: Phylogeny of Swertia

chloroplast DNA in plants.

within the Gentianella-Ymtàge asMol. Ecol. 4: as new
well
129-131.
phylogeographic hypotheses.
Don D. (1836) London Edinburgh Philos. Mag. &
The authors are indebted to Lena Struwe and
J. Sei. 8: 75-78.
Chris Hardy for their help to analyse the sequence
Donoghue M. J., Olmstead R. G., Smith J. F.,
data. The financial support of the Swiss National
Palmer J. D. (1992) Phylogenetic relationships of
Science Foundation (Grant N° 3100-052885) and of
Dipsacales based on rbcL sequences. Ann. Mis
the Aubert Foundation is very much appreciated. souri Bot. Gard. 79: 333-345.
We thank François Bretagnolle and Jason Grant for
Doyle J. J., Doyle J. L. (1987) A rapid DNA
their comments on the manuscript, and James isolation procedure for small quantities of leaf
Pringle for the constructive correspondence and
tissue. Phytochem. Bull. 19: 11-15.
for sending samples of Frasera caroliniensis. We
Doyle J. J. (1992) Gene trees and species trees:
also thank Dan Nicolson, Mark Strong and other
molecular systematics as one-character taxo
members of the Smithsonian Institution, Washing
nomy. Syst. Bot. 17: 144-163.
ton, D. C. for collecting and sending material of
Dulac J. (1867) Flore des Hautes-Pyrénées. F.
Obolaria and Bartonia, and Tom Schweich for
Savy, Paris, p. 449.
providing material of Frasera albomarginata.
Eriksson T. (1998) AutoDecay ver. 4.0 (program
References distributed by the author). Bergius Foundation,
Royal Swedish Academy of Sciences, Stock
Allen C. K. (1933) A monograph of the American holm.
species of the genus Halenia. Ann. Missouri Farris J. S. (1969) A successive approximations
Bot. Gard. 20: 119-222. approach to character weighting. Syst. Zool. 18:
Barrett M., Donoghue M. J., Sober E. (1991) 374-385.
Against consensus. Syst. Zool. 40: 486-493. Farris J. S., Källersjö M., Kluge A. G., Bult C.
Bentham G., Hooker J. D. (1876) Gentianeae. (1995) Testing significance of incongruence.
Genera Plantarum 2: 799-820. Londini. Cladistics 10: 315-319.
Braun A. ( 1830) Lomatogonium, ein neues GenusFarris
für J. S., Albert V. A., Källersjö M., Lipscomb
Gentiana carinthiaca Froehl. Flora 14: 221-224. D., Kluge A. G. (1996) Parsimony jackknifing
Bremer K. (1988) The limits of amino acid outperforms neighbor-joining. Cladistics 12:
sequence data in angiosperm phylogenetic re 99-124.
construction. Evolution 42: 795-803. Favarger C. (1949) Contribution à l'étude cary
Bremer B., Jansen R. K., Oxelman B., Backlund ologique et biologique des Gentianaceae. Bull.
M., Lantz H., Kim K.-J. (1999) More characters Soc. Bot. Suisse 59: 62-86.
or more taxa for a robust phylogeny - case study Favarger C. (1952) Contribution à l'étude cary
from the coffee family (Rubiaceae). Syst. Biol. ologique et biologique des Gentianaceae. II.
48: 413-435. Bull. Soc. Bot. Suisse 62: 244-257.
Bull J. J., Huelsenbeck J. P., Cunningham C. W.,
Favarger C. (1962) L'évolution parallèle du cary
Swofford D. L., Waddell P. J. (1993) Partition
otype. Rev. Cytol. Biol. Veget. 25: 277-286.
ing and combining data in phylogenetic analysis.
Fitch W. M. (1971) Toward defining the course of
Syst. Biol. 42: 348-397. evolution: minimum change for a specific tree
Card H. H. (1931) A revision of the genus Frasera.
topology. Syst. Zool. 20: 406-416.
Ann. Missouri Bot. Gard. 18: 245-282. Gagnidze R., Küpfer P., Yuan Y.-M. (1992)
Chase M. W., Hills H. H. (1991) Silica-gel: An ideal
Chromosome numbers of some Gentianaceae
from the Caucasus. Bull. Soc. Neuchâtel. Sei.
material for field preservation of leaf samples for
DNA studies. Taxon 40(2): 215-220. Nat. 115: 47-52.
De Queiroz A., Donoghue M. J., Kim K.-J. (1995) Garg S. (1987) Gentianaceae of the Northwest
Separate versus combined analysis of phylogeHimalaya. International Bioscience Monogrpah
netic evidence. Ann. Rev. Ecol. Syst. 26: 657-681.
17. Today & Tomorrow's Printers and Publish
Demesure B., Sodzi N., Petit R. J. (1995) A set ofers, New Delhi.
universal primers for amplification of polymorGielly L., Yuan Y.-M., Küpfer P., Taberlet P.
phic non-coding regions of mitochondrial and (1996) Phylogenetic use of noncoding regions in

This content downloaded from 150.161.240.235 on Fri, 13 Dec 2019 17:43:45 UTC
All use subject to https://about.jstor.org/terms
 P. Chassot et al.: Phylogeny of Swertia 19

the genus Gentiana L.: Holm chloroplast

T. (1907) Bartonia Muehl. An anatomical
trnL (UAA)
intron versus nuclear ribosomal internal tran study. Ann. Bot. 20: 441-448. Pis. 33, 34.
scribed spacer sequences. Mol. Phylogenet. Huelsenbeck
Evol. J. P., Bull J. J., Cunningham C. W.
5: 460-466. (1996) Combining data in Phylogenetic analysis.
TREE 11(4): 152-157.
Gilg E. (1895) Gentianaceae. In: Engler A., Prantl
K. (eds.) Die Natürlichen Pflanzenfamilien. Jonsson L. (1973) Pollen morphology in African
Wilhelm Engelmann, Leipzig, 4(2): 50-108. species of Swertia L. (Gentianaceae). Grana 13:
Gillett J. M. (1957) A revision of the North119-128.
Kellogg A. (1862) Proc. Calif. Acad. Sei 2: 144.
American species of Gentianella Moench. Ann.
Missouri Bot. Gard. 44: 195-269. Khoshoo T. N., Tandon S. R. (1963) Cytological,
Gohil R. N., Raina R„ Ashruf M. (1981) IOPB morphological and pollination studies on some
chromosome number reports LXXII. Taxon Himalayan species of Swertia. Caryologia 16:
30: 697. 445-477.
Grisebach A. H. R. (1839) Genera et Species Kondo K. (1970) The chromosome number of
Gentianarum adjectis observationibus quibus Bartonia virginica L. (Gentianaceae). Rhodora
dam phytogeographicis. J. L. Cottae, Stuttgart, 72: 551-553.
Tübingen. Koul A. K., Gohil R. N. (1973). Cytotaxonomical
Grisebach A. H. R. (1845) Gentianaceae. In: De conspectus of the flora of Kashmir (1). Chro
Candolle, Prodromus 9: 38-141. Dyer, Parisiis. mosome numbers of some common plants.
Hardig T. M., Soltis P. S., Soltis D. E. (2000) Phyton (Horn) 15: 57-66.
Diversification of the North American Shrub Krogulevich R. E. (1978) Karyological analysis of
genus Ceanothus (Rhamnaceae): conflictingthe species of the flora of eastern Sayana. In:
phylogenies from nuclear ribosomal DNA Malyshev L. I., Reshlcova G. A. (eds.) Flora of
and chloroplast DNA. Amer. J. Bot. 87: the Prebaikal. Novosibirsk, pp. 19-48.
108-123. Kuntze O. (1891) Reviso Generum Plantarum 2.
Hedberg I., Hedberg O. (1977) Chromosome Leipzig.
numbers of afroalpine and afromontane angio Lemaire C. (1824) In Bull. Soc. Philom. Paris 171.
sperms. Bot. Not. 130: 1-24. Lindsey A. A. (1940) Floral anatomy in Gentian
Ho T.-N., Liu S.-W., Wu C.-J. (1988) Gentiana aceae. Amer. J. Bot. 27: 640-652.
ceae. In: Flora reipublicae popularis sinicae. Linnaeus C. (1753) Species Plantarum, ed. 1.
Science Press. 62: 378. Holmiae, Laurentii Salvii, Stockholm.
Ho T.-N., Liu S.-W. (1990) The infrageneric Löve A., Löve D. (1953) Cytotaxonomical remarks
classification of Gentiana (Gentianaceae). Bull. on the Gentianaceae. Hereditas 39: 225-235.
British Mus. Nat. Hist., Bot., 20: 169-192. Löve A., Löve D. (1956) Cytotaxonomical con
Ho T.-N., Xue C.-Y., Wang W. (1994) The origin, spectus of the Icelandic flora. Acta Hort. Gotob.
dispersal and formation of the distribution 20: 40.
pattern of Swertia L. (Gentianaceae). Acta Liu S.-W., Ho T.-N. (1992) Systematic study on
Phytotax. Sin. 32: 525-537. Lomatogonium A. Br. Acta Phytotax. Sin. 30(4):
Ho T.-N., Pringle J. S. (1995) Gentianaceae. In: 289-319.
Flora of China. Missouri Botanical Garden, St. Ma Y. C. (1976) in Ho T.-N., Shih W.-L. (1976)
Louis. 16: 1-139. Qing xie dan and Dau da: Chinese medicinal
Ho T.-N., Wang W„ Xue C.-Y. (1999) A karyo herbs for treating hepatitis. Acta Phytotax. Sin.
morphological study on 5 species of Swertia 14(2): 65.
(Gentianaceae). Acta Bot. Boreal.-Occid. Sin. Makino T. (1891) 111. Fl. Japan. 1(11): 66.
19(3): 546-551. Marquand C. V. B. (1929) A botanical collection
Hochstetter C. F. F. (1844) Genera nova africana a from eastern Himalaya and Tibet. The botanical
me vel ab aliis auctoribus proposita, quae collection made by captain F. Kingdon-Ward in
reducenda videtur. In: Flora 27: 27. the eastern Himalaya and Tibet in 1924-25.
Holm T. (1897) Obolaria virginica L.: A morphoJ. Linn. Soc., Bot. 48: 207.
logical and anatomical study. Ann. Bot. (Lon Mehra P. M., Gill L. S. (1968) IOPB chromosome
don) 11: 369-383. number reports XVI. Taxon 17: 199-204.

This content downloaded from 150.161.240.235 on Fri, 13 Dec 2019 17:43:45 UTC
All use subject to https://about.jstor.org/terms
 20 P. Chassot et al.: Phylogeny of Swertia

Mehra P. M., Vasudevan K. N. (1972) IOPB Roy S. C., Ghosh S., Chatterjee A. (1988) A
chromosome number reports XXXVI. Taxon 21: cytological survey of eastern Himalayan plants.
333-346. II. Cell Chromosome Res. 11: 93-97.
Mészaros S. (1994) Evolutionary significance Rydberg
of P. A. (1917) Flora of the Rocky Moun
xanthones in Gentianaceae: a reappraisal. Biotains. New York, The author. 665, 1065.
chem. Syst. Ecol. 22: 85-94. St. John H. (1941) Revision of the genus Swertia
Meusel H., Jäger E., Rauschert S., Weinert E. (Gentianaceae) of the Americas and the reduc
(1978) Vergleichende Chorologie der Zen tion of Frasera. Amer. Midi. Naturalist. 26:
traleuropäischen Flora II (Karten). VEB Gustav 1-29.
Fischer Verlag, Jena, Germany. Shah J. (1990) Taxonomic studies in the genus
Mishler B. D. (1994) Cladistic analysis of molecular Swertia L. (Gentianaceae): monograph. Part I.
and morphological data. Amer. J. Physical SCI Khyber 3: 17-114.
Anthrop. 94: 143-156. Shah J. (1992) Taxonomic studies in the genus
Nemomissa S. (1994) Swertia in North-East Africa. Swertia L. (Gentianaceae): monograph. Part II.
Doctorate thesis, University of Vienna, Austria. SCI Khyber 5: 127-231.
Nilsson S. (1964) On the pollen morphology in Shigenobu Y. (1983) Karyomorphological studies
Lomatogonium A. Br. Grana Palynol. 5: 298 in some genera of Gentianaceae II. Gentiana and
329. its allied four genera. Bull. Coll. Child Devel-.
Nilsson S. (1967) Pollen morphological studies in opm., Kochi Women's Univ. 7: 65-84.
the Gentianaceae-Gentianinae. Grana Palynol. Struwe L., Thiv M., Kadereit J. W., Pepper A. S.
7: 46-145. R., Motley T. J., White P. J., Rova J. H. E.,
Nilsson S., Skvarla J. J. (1969) Pollen morphology Potgieter K., Albert V. A. (1998) Saccifolium
of saprophytic taxa in the Gentianaceae. Ann. (Saccifoliaceae), an endemic of Sierra de la
Missouri Bot. Gard. 56: 420-438. Neblina on the Brazilian-Venezuelan border, is
Nilsson S. (1970) Pollen morphological studies in related to a temperate-alpine lineage of Gentian
the Gentianaceae. Acta Univ. Upsal. 165: aceae. Harvard Pap. Bot. 3(2): 199-214.
1-18. Struwe L., Hagen K. B. von, Kadereit J. W.,
Olmstead R. G., Sweere J. A. (1994) Combining Klackenberg J. Nilsson J. S., Thiv M., Albert V.
data in phylogenetic systematics: an empirical A. (2001) Systematics, character evolution, and
approach using three molecular data sets in the biogeography of Gentianaceae, including a new
Solanaceae. Syst. Biol. 43: 467-481. tribal and subtribal classification. In: Struwe L.,
Page R. D. M. (1996) TREEVIEW: An application Albert V. A. (eds.) Gentianaceae - systematics
to view phylogenetic trees on personal comput and natural history. Cambridge University
ers. CABIOS 12: 357-358. Press, Cambridge (in press).
Post D. M. (1956) Studies in the Gentianaceae: Swofford D. L. (2000) PAUP*. Phylogenetic Anal
Frasera and Swertia of North America. Unpub ysis Using Parsimony (*and Other Methods).
lished Ph.D. dissertation, University of Califor Version 4. Sinauer Associates, Sunderland, Mas
nia, Berkeley. 310 p. sachusetts.
Pringle J. S. (1979) New combinations in Swertia Taberlet P., Gielly L., Pautou G., Bouvet J. (1991)
(Gentianaceae). Phytologia 41(3): 139-143. Universal primers for amplification of three non
Pringle J. S. (1981) Nomenclatural transfers and coding regions of chloroplast DNA. Pl. Molec.
taxonomic notes on some South American Biol. 17: 1105-1109.
Gentianaceae. Phytologia 48: 281-285. Templeton A. R. (1983) Phylogenetic inference
Pringle J. S. (1990) Taxonomic notes on Western from restriction endonuclease cleavage site maps
American Gentianaceae. Sida 14: 179-187. with particular reference to the evolution of
Pringle J. S. (1993) Gentianaceae. In: Hichman J. humans and the apes. Evolution 37: 221-244.
C. (ed.) The Jepson Manual: higher plants Thompson
of J. D., Gibson T. J., Plewniak F.,
California. Univ. California Press, Ltd., Berke Jeanmougin F., Higgins D. G. (1997) The
ley, USA, pp. 666-672. ClustalX windows interface: flexible strategies
Rork C. L. (1949) Cytological studies in the for multiple sequence alignment aided by quality
Gentianaceae. Amer. J. Bot. 36: 687-701. analysis tools. Nucl. Acids Res. 24: 4876-4882.

This content downloaded from 150.161.240.235 on Fri, 13 Dec 2019 17:43:45 UTC
All use subject to https://about.jstor.org/terms
 P. Chassot et al.: Phylogeny of Swertia 21

Toyokuni H. (1962) Further Innis M. A.,remarks

Gelfand D. H., Sninsky toJ. the
J., Whitegenus
Comastoma. Acta Phytotax. T. J. (eds.) PCRGeobot.
protocols. Academic 20:Press,136—
San
138. Diego, CA, pp. 315-322.
Toyokuni H. (1963) Conspectus Wood, C. E. Jr., Weaver R. E. Jr. (1982) The
Gentianacearum
Japonicarum, a general genera
view of Gentianaceae
of the in the Gentianaceae
southeastern
indigenous to Japan. J. Fac. Sei. Hokkaido United States. J. Arnold Arbor. 63: 441-487.
Univ., Ser. V, 7(4): 137-259. Xue C.-Y., Ho T.-N., Liu J.-Q. (1999) Embryology
Toyokuni H. (1965) Systema Gentianarum Noviss of Swertia tetraptera Maxim. (Gentianaceae)
imum - facts and speculation relating to the and its systematic implication. Acta Phyto tax.
phylogeny of Gentiana, sensu lato and related Sin. 37(3): 259-263.
genera. Symb. Asahikawenses 1: 147-158. Yuan Y.-M., Küpfer P. (1993) Karyological studies
Turczaninow N. (1840) Description de deux nou of Gentianopsis Ma and some related genera of
veaux genres de la famille des Gentianées. Bull. Gentianaceae from China. Cytologia 58: 115
Soc. Imp. Naturalistes Moscou 2: 164-168. 123.
Turczaninow N. (1849) Flora Baicalensi-Dahurica. Yuan Y.-M., Küpfer P. (1995) Molecular phylo
Bull. Soc. Imp. Naturalistes Moscou 22: 283 genetics of the subtribe Gentianinae (Gentiana
338. ceae) inferred from the sequences of internal
Vasudevan K. N. (1975) Contribution to cytotax transcribed spacers (ITS) of nuclear ribosomal
onomy and cytogeography of the flora of the DNA. Plant Syst. Evol. 196: 207-226.
Western Himalayas (with an attempt to compareYuan Y.-M. (1996) Molecular Systematics and
it with the flora of the Alps). Part I. Ber. Karyological Evolution of Gentiana (Gentiana
Schweiz. Bot. Ges. 85: 57-84. ceae). Doctorate thesis, University of Neuchâtel,
von Hagen K. B., Kadereit J. W. (2001) The Switzerland.
phylogeny of Gentianella (Gentianaceae) and its Yuan Y. M. Küpfer P., Zeltner L. (1998) Chrom
colonization of southern hemisphere as revealed osomal evolution of Gentiana and Jaeschkea
by nuclear and chloroplast DNA sequence (Gentianaceae), with further documentations of
variation. Org. Divers. Evol. 1: 61-79. chromosome data for 35 species from Western
Wada Z. (1954) Cytological studies in some species China. Plant Syst. Evol. 210(3): 231-237.
of Swertia. Jap. J. Genet. 29: 180.
Walter T. (1788) Flora Caroliniana. Cambridge,
Mass.: Photolithographed by the Murray Print. Addresses of the authors: Philippe Chassot (cor
Co. for the Arnold Arboretum, 1946. (original respondence), Yong-Ming Yuan, Philippe Küpfer,
pub. J. Fraser, London), p. 87. Institut de Botanique, Laboratoire de Phanéroga
Weaver R. E., Rüdenberg L. Jr. (1975). Cytotax mie, Université de Neuchâtel, Emile-Argand
onomic notes on some Gentianaceae. J. Arnold 11, CH-2007 Neuchâtel 7, Suisse (E-mail: phi
Arbor. 56: 211-222. lippe.chassot@bota.unine.ch). Sileshi Nemomissa,
White T. L., Bruns T., Lee S., Taylor J. (1990) The National Herbarium, Biology Department,
Amplification and direct sequencing of fungalAddis Ababa University, P.O. Box 3434, Addis
ribosomal RNA genes for phylogenetics. In: Ababa, Ethiopia (E-mail: sileshin@hotmail.com).

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