Relationships among Genera in Saniculoideae and Selected Apioideae (Umbelliferae) Inferred from nrITS Sequences

Abstract

The internal transcribed spacers (ITS1 and ITS2) of 18S-26S nuclear ribosomal DNA were newly sequenced for eight species of Umbelliferae (six species from subfamily Saniculoideae: Actinolema macrolema, Astrantia minor, Eryngium giganteum, E. coeruleum, Hacquetia epipactis, and Lagoecia cuminoides, two species from subfamily Hydrocotyloideae: Dickinsia hydrocotyloides and Azorella trifurcata), as well as Hohenackeria exscapa, a species of uncertain position in the family. Phylogenetic analyses of new data, plus previously reported sequences of 52 other species using neighbor-joining, maximum parsimony, and maximum likelihood methods yielded similar results: (1) Actinolema is sister to Astrantia corresponding to Drude's treatments; (2) in Astrantia, molecular divergence is revealed between sects. Astrantiella (A. minor) and Astrantia (A. major, A. maxima); (3) Eryngium appears to be paraphyletic; (4) Hacquetia might be treated as a part of Sanicula; and (5) Lagoecia is very distant from all other Saniculoideae and close to some genera of Apioideae. Our results correspond to matK data previously published: (1) Hohenackeria forms a clade with Bupleurum, in a position near the base of the Apioideae tree; (2) Azorella is sister to a large cluster uniting all Saniculoideae and Apioideae, being slightly closer to them than to the Hydrocotyle-Araliaceae clade; (3) Dickinsia is very distant from phenetically similar Hydrocotyle, falling within a large cluster of Apioideae, but also including Lagoecia and Naufraga.

Full text

INTRODUCTION
Almost all molecular-phylogenetic studies of
Umbelliferae have dealt with the largest subfamily
Apioideae (Downie & Katz-Downie, 1996, 1999;
Plunkett & Downie, 1999; Katz-Downie & al., 1999;
Downie & al., 1999, 2000a). The second and smaller
subfamily Saniculoideae of Drude’s most widely accept-
ed classification has been treated only in connection with
a general molecular testing of the familial structure rela-
tive to monophyly of Saniculoideae and their sister rela-
tionship with Apioideae (e.g., Plunkett & al., 1996a, b,
1997; Valiejo-Roman & al., 1998). Intensive investiga-
tion also has been made in the genus Sanicula, with
results being partly analyzed biogeographically (Vargas
& al., 1998, 1999).
In the present study we elucidate in more detail the
relationships among the main genera of Saniculoideae
(Actinolema, Astrantia, Eryngium, Hacquetia, Lagoecia,
Sanicula). We also compare them with selected genera of
Apioideae, which presumably are basal or marking main
branches in previously published ITS and other gene
trees. In our analysis we also included a few genera of
Hydrocotyloideae, a newly sequenced Dickinsia, and
Araliaceae as outgroup.
Saniculoideae are the smallest subfamily of
Umbelliferae; they number nine genera and approxi-
mately 330 species, distributed over all continents
(Pimenov & Leonov, 1993). One genus of Saniculoideae,
Eryngium, is the largest of the family, with c. 250 species
(Pimenov & Leonov, 1993; Wörz, 1999a). One of the
most critical problems in the taxonomy of Saniculoideae
is the position of Lagoecia. Morphologically based taxo-
nomic conclusions (Drude, 1897) are in strong conflict
with previous molecular (matK) data (Plunkett & al.,
1996a).
MATERIALS AND METHODS
Pla nt accessio ns. — Newly examined for ITS1
and ITS2 of 18S-26S nuclear ribosomal DNAwere eight
species from seven genera of Umbelliferae (subfamily
Saniculoideae—Actinolema macrolema, Astrantia
minor, Eryngium giganteum, E. coeruleum, and
Lagoecia cuminoides; subfamily Hydrocotyloide ae—
91
Valiejo-Roman & al.  Relationships in Saniculoideae51  February 2002: 91–101
Relationships among genera in Saniculoideae and selected Apioideae
(Umbelliferae) inferred from nrITS sequences
Carmen M. Valiejo-Roman 1, Elena I. Terentieva2, Tagir H. Samigullin1& M ichael G. Pimenov2
1Department of Evolution ar y B ioch emistry, A. N. Be lozersky Institut e, Mosc ow State U niversity, Moscow
1198 99, Russia . E -mail: vall ejo@ ge ne be e. ms u.s u (a uthor f or c or res pondence)
2Bo tani ca l G ard en, M oscow State U nivers ity, M os co w 1198 99, Russia . E-m ai l: p im e nov@ 2.botg ard.
bi o.m su.r u
Th e inte rna l tra ns cribe d spac ers (I TS 1 and ITS2) of 18S-26S nuclea r ri bosoma l DN A w er e newly se quence d
for ei ght species of Umbelli ferae (six s pecie s from s ubfamily Sanic uloid ea e: Ac tinolema m acrolem a, Astrantia
minor, E ry ngium gigan te um , E. coeruleum, H acqu et ia epipactis , and Lagoe cia cum inoid es , two spec ies f rom
subfamily Hydrocotyloideae: Dickinsia hydrocotyloides and Azorella trifu rcat a) , as w ell as H oh en ac keria e xs-
ca pa, a s pe cies of u nc er tain position in th e fam ily. Phy loge ne tic a na ly se s o f ne w data , plu s pr ev iousl y repor t-
ed seque nc es of 52 oth er species us ing ne ighb or- joining, maxim um parsim on y, and ma ximum likelihood m et h-
ods yiel ded similar r es ults: (1) Actin olema is sister to Astrantia cor re spondin g to Drude’s tr ea tmen ts; (2) in
Astrantia, m olec ular diverge nc e is re vealed be tw een sects. As trant iella (A. minor ) and Astrantia (A. m a jor, A.
maxima); (3) Eryng iu m appears to be paraphyletic; (4) Hacque tia might be t reated as a pa rt of San ic ula; and
(5) Lagoe c ia is very d istant fr om all other S anic uloid ea e and close to some genera of A pioide ae. Ou r results
co rre spond to m atK data previously published: (1) Hohe nackeria forms a clade with Bupl euru m, i n a pos it ion
near the base of the Apioide ae tree ; (2 ) Azorella is siste r to a la rge clu ste r un iting all San iculoidea e and
Apioide ae, being slig htly closer t o them than to t he Hydrocotyle -A ra lia ceae c lade; (3 ) Dickinsi a is very dis-
tant from phe ne tically sim ilar H yd roc otyle , falling w ithin a large clus ter of Apioi de ae , but also including
Lagoecia and Naufr aga.
KEYWORDS: Ap iaceae, Ap io ideae, IT S, nrDNA, ph yl ogen y, Sa niculoidea e.

Dickinsia hydrocotyloides and Azorella trifurcata) and a
species of uncertain position, Hohenackeria exscapa.
Data on the eight complete ITS1 and ITS2 sequences,
including GenBank accession numbers, their source and
voucher information, are presented in Table 1. Some of
these species were previously sequenced (not ITS):
Lagoecia cuminoides (matK; Plunkett & al., 1996a),
Hacquetia epipactis (Rpl16; Downie & al., 2000b),
Azorella trifurcata (chloroplast restriction sites; Plunkett
& al., 1999).
In total, 60 species of Umbelliferae (28 genera) were
included in the analysis. Complete ITS1 and ITS2
sequences were compared for 18 species (18 genera)
from Umbelliferae subfamily Apioideae, 35 species (six
genera) from subfamily Saniculoideae, seven species
(four genera) from subfamily Hydrocotyloideae and
three species (three genera) from the outgroup family
Araliaceae (Aralia, Eleutherococcus, and Hedera). Of
the 60 species of Umbelliferae examined, 24 species of
Apioideae were studied earlier (Downie & Katz-Downie,
1996; Valiejo-Roman & al., 1998; Katz-Downie & al.,
1999; Downie & al., 2000a). For GenBank accession
numbers and voucher information see Table 1.
We selected outgroup taxa (sequences) guided by the
following considerations: first, we included in our analy -
sis all genera marking main clades from previous publi-
cations (including ours) on molecular phylogeny of the
family; second, we included additional genera that are
critical in the context of previous taxonomic hypotheses
for Saniculoideae, and for which the ITS sequences are
available.
DNA e x t ra c t i on , a m pl ifi c a ti on, a n d s e q u e n c-
i n g . — Total genomic DNAs were extracted from silica
gel dehydrated leaves or herbarium specimens using the
modified CTAB procedure (Doyle & Doyle, 1987).
The entire ITS1-5.8S-ITS2 region was PCR-ampli-
fied using a pair of primers corresponding to conserved
areas of the 3’ end of 18S (“ITSL” primer) and 5’ end of
26S (“ITS4” primer) rDNAs (White & al., 1990). Each
PCR reaction cycle proceeded as follows: 40 s at 95°C,
40 s at 58°C, 40 s at 72°C. The first cycle was preceded
by an initial denaturation step of 2 min at 95°C. To allow
completion of unfinished DNA strands and to terminate
the PCR reaction, 2 min 72°C extension period followed
completion of the 30 terminal cycles. Each PCR product
was purified with Qiagen purification columns [PCR
purification Kit (250), USA] and some PCR products
were electophoresed in 1% agarose gel (Large DNA Low
Melt, Biozym agarose) and stained with ethidium bro-
mide. DNA fragments were recovered from gels using
the Geneclean II Kit (USA). The purified DNA was
resuspended in sterile water.
Valiejo-Roman & al.  Relationships in Saniculoideae 51  February 2002: 91–101
92
Table 1. List of tax a of Apiaceae analysed, with voucher specimens and Genbank access ion numbers.
New sequence data
Actinolema macrolema Boiss., Armenia, between Azavan and Garny, 6 Jun 1977, Pimenov & al. 255 (MW), AF 337184; Astrantia minor
L., Switzerland, canton Valais, les Alpes Penines, col du Gd. St. Bernard, 8 Jul 1996, Pimenov & al. S96-6 (MW), AF 337191; Azorella
trifurcata (Gaertn.) Pers., Bot. Gard. Geneve, 15 Sep 1998, Pimenov s.n.(living material), AF 337185; Dickinsia hydrocotyloidesFranch.,
China, Sichuan, Dashu Nie 52 (NAS), AF 337188; Eryngium coeruleum L., Uzbekistan, Zeravschtan Mts., near village, Mehpatkan, 10
Jun 1990, Pimenov & al. 83 (MW), AF 337189; Eryngium giganteum M. Bieb., Turkey, prov. Artvin, Arsiansky Mts., Yalnichan pass, 6
Jul 1994, Pimenov & al. 287 (MW), AF 337190; Hohenackeria exscapa(Steven) Koso-Pol., Morroco, prov. Khenifra, Moyen Atlas Ajdir,
5 May 1983, Lewalle 11482 (MHA), AF 337186; Lagoecia cuminoides L., Turkey, Izmir, Bornova, hills near Ege Universitesi campus,
22 May 1995, Pimenov & E. V. Kljuykov T95-19 (MW), AF 337187.
Sequence data from GenBank
Aegopodium kashmiricumb, AF077872; Aethusa cynapium
d, U30582, U30583; Apium graveolensdU30552, U30553; Aralia elatab,
AF077875; Astrantia majorb, AF077876; Bunium eleganse, AF073543, AF073544; Bupleurum falcatum
b, AF077877; Carum carvi
b,
AF077878; Crithmum maritimumc, U30540, U30541; Eryngium billardie rib, AF077886; E. cervantesiia, AF031960; E. campestre
b,
AF077887; E. mexicanuma, AF031961; Elaeosticta allioidese, AF073547, AF073548; Eleutherococcus senticosusb, AF077885; Falcaria
vulgarisb, AF077888; Hydrocotyle bonariensis
b, AF077894; H. mexicanab, AF077893; H. novae-zeelandiaeg, U72382; H. vulgarisb,
AF077895; Hacquetia epipactis
b, AF077892; Hedera colchicab, AF077884; Heteromorpha arborescens
d, U27578, U30314; Naufraga
balearicae, AF073563, AF073564; Oedibasis platycarpaf, AF008632, AF009111; Oenanthe pimpinelloidesd, U78371, U78431;
Olymposciadium caespitosumd, U78379; Physospermum cornubiense
b, AF077904; Pyramidoptera cabulica
f, AF008631, AF009110;
Sanicula arctopoidesa, AF031974; S. argutaa, AF031977; S. bipinnataa, AF031982; S. bipinnatifidaa, AF031980; S. canadensisa,
AF031967; S. chinensisa, AF031965; S. crassicaulisa, AF031988; S. deserticol aa, AF031989; S. elataa, AF031966; S. europaeaa,
AF031964; S. graveolensa, AF031992; S. hoffmannii
a, AF031995; S. laciniataa, AF031998; S. mariversaa, AF031968; S. moraniia,
AF032011; S. orthacanthaa, AF031963; S. peckianaa, AF031999; S. purpureaa, AF031971; S. rubriflorab, AF077907; S. sandwicensis
a,
AF031970; S. saxatilisa, AF032004; S. tracyia, AF032006; S. tuberosaa, AF032010; Scaligeria moreana
e, AH008893; Trachyspermum
ammid, U78380, U78440.
aVargas & al. (1998); bValiejo-Roman & al. (1998); Downie & al. (1996c, 1998d, 2000ae); fKatz-Downie & al. (1999); gMitchell & al. (unpubl.).

We used the forward “ITSL” and reverse “ITS3”
(White & al., 1990) primers for sequencing ITS1 and
“ITS2” and reverse “ITS4” (White & al., 1990) primers
for sequencing ITS2. Purified PCR products were
sequenced using the Cyclist Exo Pfu DNA sequencing
kit (Stratagene, California, U.S.A.).
Reactions were separated electrophoretically in 6%
polyacrylamide gels in which the xylene cyanole dye
marker was run 30 cm (for a short gel) and 80 cm (for a
long gel), so the entire ITS1 or ITS2 region could be read
on both gels. Gels were dried onto Whatman 3MM paper
in a vacuum dryer and then exposed to X-ray film
(Retina, Germany) for 2–4 days at room temperature.
Only the ITS1 and ITS2 regions were included in the
analysis since sequence data for the 5.8S subunit were
incomplete for many taxa, and those that were available
were not sufficiently variable to warrant additional
sequencing. The obtained sequences were aligned using
the SED editor of the VOSTORG phylogenetic analysis
package (Zharkikh & al., 1990). The alignment is avail-
able from the authors upon request.
P h y l og e n e t ic a na ly sis. — The resulting data
matrix was analyzed by programs Tree-puzzle (Strimmer
& von Haeseler, 1996), fastDNAml (Olsen & al., 1994;
Felsenstein, 1981), PAUP* (Swofford, 2000), and
TreeCon package (Van de Peer & De Wachter, 1997).
For phylogenetic reconstruction, parsimony analysis
involved a heuristic search conducted with PAUP* ver-
sion 4.0b4a (Swofford, 2000) using TBR (tree-bisection-
reconnection) branch swapping, options Mulpars,
Steepest descent, Collapse, and Acctran selected, with
character states specified as unordered and equally
weighted. All searches were conducted with 10 random
addition replicates. The amount of phylogenetic informa-
tion in the parsimony analyses was estimated using the
consistency index (Kluge & Farris, 1969), retention
index, and homoplasy index (Farris, 1989). All most par-
simonious trees were saved.
Bootstrap (Felsenstein, 1985) analyses were per-
formed to assess the degree of support for particular
branches on the tree. Bootstrap values were calculated
from 100 replicate analyses with TBR branch swapping
and random addition sequence of taxa. One thousand
most parsimonious trees from each replicate were saved.
In the parsimony analyses all gaps were treated as miss-
ing data.
Distance trees were calculated using the neighbor
joining method (Saitou & Nei, 1987), implemented using
TreeCon (Van de Peer & De Wachter, 1997). Distance
matrices were calculated using the method of Jin & Nei
(1990). Insertions and deletions were taken into account.
Maximum likelihood phylogeny estimation was
explored using the fastDNAml program (version 1.2.2;
Olsen & al., 1994), based on the procedures of
Felsenstein (1981). Eight rate categories were used, cal-
culated by Tree-puzzle program (Strimmer & von
Haeseler, 1996). A maximum likelihood tree was inferred
using empirical base frequencies, a transition/transver-
sion ratio of 2.12, randomizing the input order of
sequences (jumble), and by invoking the global branch
swapping search option.
RESULTS
Among all 63 representatives of Umbelliferae and
Araliaceae examined, the length of the ITS1 region var-
ied from 222 to 263bp and ITS2 between 187 and 229bp.
Complete length DNA sequences of ITS1 and ITS2
ranged between 419 and 467 bp. The number of constant ,
autapomorphic and parsimony-informative positions was
similar for both spacers (Table 2), and the ratio of termi-
nal taxa (63) to informative characters across both spac-
ers (355) was 1:5.6. Tree topologies were stable even if
several alternative alignments were considered, or ques-
tionable regions of alignment were excluded from the
analysis.
Phylogenies estimated using neighbor joining, and
maximum parsimony, and likelihood methods reveal
that, in the context of those species examined,
Umbelliferae ITS sequences are divided into three
clades: Apioideae, Saniculoideae, and Hydrocotyloideae.
Clades of Apioideae and Saniculoideae (without
Lagoecia) are monophyletic, whereas that of
Hydrocotyloideae is not. The position of some genera
does not correspond to their previous taxonomy.
Actinolema and three species of Astrantia form a
clade supported by a bootstrap value (BS) of 100% (Figs.
1, 3). The genus Eryngium demonstrates a paraphyletic
pattern. Four species of the Old World, E. coeruleum, E.
giganteum, E. campestre and E. billardierei, form a sep-
arate cluster supported by a bootstrap value of 100%, and
two species of the New World (E. mexicanum and E. cer-
vantesii) form another well-supported cluster (BS =
Valiejo-Roman & al.  Relationships in Saniculoideae
51  February 2002: 91–101
93
Table 2. S equence charac teristics of the two nuclear
rDNA internal transcribed spacers, separately and com-
bined, for 60 repr esentatives of Umbelliferae and three
genera of Araliaceae.
Sequence characteristic ITS1 ITS2 ITS1+ITS2
Spacer length variation (bp) 222–263 187–229 419–467
Number of sites
total aligned 326 303 629
constant 98 69 167
parsimony 172 183 355
autapomorphic 56 51 107

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Fig. 1. Neighbor-joining tree inferred from 63 aligned ITS1 and ITS2 sequences. Branch lengths are proportional to
distances estimated from the Jin & Nei (1990) model. Numbers at nodes indicate bootstrap e stimates for 100 replicate
analyses; values <50 % are not shown.

Valiejo-Roman & al.  Relationships in Saniculoideae51  February 2002: 91–101
95
Fig. 2. Strict co nsensus of 1976 minimal length 1767-step trees derived from equally weighted parsimony analysis of
combined ITS1 and ITS2 sequences from 63 taxa. CI (excluding parsimony uninformative characters) = 0.3979; RI =
0.7031; HI = 0.6021.

Valiejo-Roman & al.  Relationships in Saniculoideae 51  February 2002: 91–101
96
Fig. 3. 50% Majority rule consens us tree derived from equally weighted parsimony analysis of combined ITS1 and ITS2
sequences from 63 taxa. 100 bootstrap replicates were performed.

Valiejo-Roman & al.  Relationships in Saniculoideae51  February 2002: 91–101
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Fig. 4. Maximum likelihood tree constructed from 63 aligned ITS1 and ITS2 sequences from Umbelliferae using a tran-
sition/transversion rate ratio 2.12. Bran ch lengths are proportional to the number of the expected nucleotide substitu-
tions; scale bar corresponds to 1 substitution per 10 sites.

100%), which is closer to the large Sanicula -Hacquetia
clade (Figs. 1, 3). The genus Sanicula forms a large well-
supported clade (BS = 100%, Figs. 1, 3), Haquetia
belongs to this clade and forms with S. rubriflora a sep-
arate branch in neighbor joining (BS = 65%) and maxi-
mum likelihood trees (Figs. 1, 4). Apioideae form a mod-
erately supported clade (BS = 88%; 68%, Figs. 1, 3),
including Lagoecia from Saniculoideae (57%, 53%). The
most basal branches lead to Dickinsia from
Hydrocotyloideae (BS = 88%, 68%; Figs. 1, 3), and to
the Bupleurum and Hohenackeria clade (BS = 100%,
Figs. 1, 3), their branching pattern in maximum parsimo-
ny strict consensus tree is different from the other trees.
Azorella forms a separate branch (BS = 69%; 91%, Figs.
1, 3) between Apioideae-Saniculoideae and Hydro -
cotyloideae clades.
DISCUSSION
S a n i c ul o id e a e . — All genera of Saniculoide ae
(with the exception of Lagoecia) included in this study
(Actinolema, Astrantia, Eryngium, Hacquetia, and
Sanicula ) form a monophyletic cluster, without any alien
species (Figs. 1–4). This cluster corresponds to the tribe
Saniculeae. The monotypic Petagnaea Caruel (=
Petagnia Guss.), closely related to Sanicula , is con-
firmed by matK and rps16 sequencing (Plunkett & al.,
1996a; Downie & Katz-Downie, 1999; Downie & al.,
2000a), as very probably in the same alliance. The treat-
ment of Petagnaea as a synonym of Cryptotaenia (and P.
saniculif olia as a synonym of C. canadensi s), proposed
by Hiroe (1979), is not confirmed by molecular data.
Only the positions of two Southern Hemisphere genera,
Alepidea (South Africa) and Oligocladus (South
America), remain untested by ITS and other gene
sequencing.
Actinolema (A. macrolema), investigated by DNA
sequencing for the first time, shows an independent
generic position and a close relationship to Astrantia
(Figs. 1–4). It is a little-known genus of two annual xero-
phytic species from SW Asia. It differs from Astrantia
not only in annual habit, but also in heterophylly, sessile
umbels, very short pedicels, very large leaf-like, net-
veined bracts, and stylopod shape (similar, however, to
Astrantia in characters of fruit anatomy; Tamamschjan,
1933). Our result corresponds to treatments by Drude
(1897), Wolff (1913), and Tamamshjan (1933). Koso-
Poljansky (1916), however, included Actinolema in
Astrantia as a section. This alternative is also not contra-
dicted by our results.
The genera Sanicula and Astrantia, both represented
by more than one species, are monophyletic (Figs. 1–4),
Astrantia being most divergent of the five genera of the
Saniculeae cluster in the classifications of Drude,
Calestani and other authors. Within Astrantia, the
Western European A. minor, representing section
Astrantiella Calest. (Calestani, 1905), is the most diver-
gent species, forming a sister clade to two other species
belonging to the type section of the genus (Macraster
Calest. = sect. Astrantia). These two sections differ in
calyx teeth (sepal) shape, leaf dissection, and general
habit. According to a cladistic analysis by Wörz (1999b),
sect. Astrantiella is regarded as advanced, and sect.
Astrantia least derived. In Wörz’s analysis, the most
primitive species is A. maxima; A. major forms a sepa-
rate branch of a highly variable polyploid complex. Our
arrangement of three species is not completely congruent
with Wörz’s cladogram, as the first branch is that of A.
minor.
Eryngium, the largest genus of Umbelliferae, was
represented in our analysis by only six species. It demon-
strates a paraphyletic pattern. Four species of the Old
World, E. coeruleum, E. giganteum, E. campestre and E.
billardierei, representing three sections, form one cluster,
and two species of the New World (E. mexicanum and E.
cervantesii) form another cluster, closer to the large
Sanicula-Hacquetia clade (Figs. 1–4).
On the basis of our results Hacquetia might be treat-
ed as a part of Sanicula. Hacquetia epipactis nests
among species of Sanicula, being closest to S. rubriflora
(Figs. 1–4). It is also related to Sanicula in morphology,
differing mainly in large leaf-like bracteoles and in some
fruit anatomical characters, e.g., the fruits in Sanicula are
covered by uncinate prickles, squamae or tubercles,
whereas those of Hacquetia have no outgrowths.
Hacquetia has large solitary vittae (Klan, 1947), where-
as the fruit secretory system in species of Sanicula is
variable (consisting of large to small, regularly or irregu-
larly arranged vittae). Analysis of rpl16 intron sequences
also show close affinity of Sanicula and Hacquetia
(Downie & al., 2000b), but as Sanicula in that analysis
contained no infrageneric molecular diversity, inclusion
vs. exclusion of Hacquetia could not be resolved.
Our Sanicula cluster, with some shortening of the
trees, approximates those obtained by Vargas & al.
(1998, 1999). We additionally included only S. rubriflo-
ra sect. Erythrosana (Valiejo-Roman & al., 1998). In our
strict consensus tree (Fig. 2) S. rubriflor a + Hacquetia
epipactis form one of the most divergent lineages in the
genus, equally distant from the majority of species inves-
tigated as S. chinensis and S. canadensis, which form
separate lineages. In neighbor joining the tree topology is
slightly different: Hacquetia and S. rubriflora form two
of the most divergent branches, with S. chinensis adja-
cent (Fig. 1). These results are in partial accordance with
the classification of Shan & Constance (1951).
Lagoecia is very distant from all other Saniculoideae
Valiejo-Roman & al.  Relationships in Saniculoideae 51  February 2002: 91–101
98

and evidently does not belong to the subfamily (Figs.
1–4). In our trees it positions near Crithmum
(Apioideae), but this placement is regarded as tentative.
The taxonomic affinity of Lagoecia is controversial. The
genus was described by Linnaeus (1753) in Pentandria-
Monogynia (not in Digynia, into which the majority of
present Umbelliferae were positioned), perhaps due to
reduction to only one carpel in Lagoecia. Drude (1897)
recognized the tribe Lagoecieae with three genera:
Lagoecia, Petagnia (Petagnaea), and Arctopus, and
Wolff (1913) followed suit. Calestani (1905) divide d
Umbelliferae (s.l., including Araliaceae) into four subdi-
visions: Aralineae, Lagoecineae (with the single tribe
Lagoecieae), Eryngineae, and Ferulineae (Apioideae of
Drude). Koso-Poljansky (1916) proposed another affini-
ty for Lagoecia as a member of Ligusticoideae-Careae-
Carinae (“grex Cari”), i.e., among presently adopted
Apioideae. In the system of Cerceau-Larrival (1962),
Lagoecia was not regarded as a relative of Sanicula,
Astrantia, and Eryngium (Eryngioideae), but rather
placed in a monotypic tribe in Endressioideae.
Sequencing of matK of L. cuminoides (Plunkett & al.,
1996a), the only previous molecular study of the genus,
plus subsequent analysis by Plunkett & Downie (1999),
refer Lagoecia to the Aegopodium-group together with
Crithmum, Aegopodium , Carum, Cyclospermum,
Falcaria, Olymposciadium and Trachyspermum. Our
data show affinity of Lagoecia with Crithmum,
Trachyspermum, Scaligeria , Bunium, Elaeosticta,
Pyramidoptera, and Oedibasis, as well as a more distant
relationship with Aegopodium, Carum, Falcaria,
Olymposciadium and Aethusa. In general, however, the
position of Lagoecia outside of Saniculoideae, and
among Apioideae, seems confirmed (Figs. 1–4).
Ot he r su b f a m il i es. — Some previously uninves-
tigated genera of other subfamilies were also included in
the present analysis, being presumably of basic phyloge-
netic position in the family. These genera, all with
unclear taxonomic positions, are Dickinsia and Azorella
(Hydrocotyloideae) as well as the genus Hohenackeria
(Apioideae).
Hohenackeria was described by Fischer & Meyer
(1835) as monotypic with H. bupleuroi des. Earlier the
species was known as Valerianella exscapa
(Valerianaceae; Steven, 1812), its unusual habit being the
reason for classification of the species in another, unre-
lated, family. In Umbelliferae its position was a matter
for discussion. The authors of the genus put it in
Saniculoideae on the basis of umbel structure; they were
followed by Cosson (1856) who described a second
species, H. polyodon, from Algeria. Wolff (1913) was
inclined to put Hohenackeria in Saniculoideae. Bentham
& Hooker (1867), Boissier (1872), and Drude (1898)
regarded Hohenackeria as a relative of Bupleurum
(Ammineae, i.e., modern Apioideae) on the basis of leaf
similarity, and Cauwet-Marc & al. (1978) noted an affin-
ity between these two genera in pollen morphology.
Calestani (1905) separated Hohenackeria and its segre-
gate genus Keracia (H. polyodon, presently returned to
Hohenackeria) into a new tribe Hohenackerieae (cf.
Pimenov & Leonov, 1993). Koso-Poljansky (1914,
1916) placed this genus near Oenanthe (in his
Ligusticoide ae-Oenantheae), based on similar structures
in fruit anatomy (sclerified layer and aerophorous
parenchyma in mesocarp); he was followed by
Tamamschjan (1946) who presented the most complete
analysis of the morphology and systematics of
Hohenackeria.
We included in our comparative analysis the genera
and higher groups to which Hohenackeriawas previous-
ly regarded as closely related (with the exception of
Valerianaceae). Results clearly show a close relationship
with Bupleurum (Figs. 1–4). Neither Saniculoideae nor
Oenanthe are seen as relatives of Hohenackeria. The
Bupleurum-Hohenackeria clade is positioned near the
base of the Apioideae subtree. Heteromorpha, treated by
Downie & Katz-Downie (1996, 1999, etc.) as a more
basal taxon, does not have such a position in the present
cladogram (nor as in previously published ITS-based
molecular phylogenies of Umbelliferae; Valiejo-Roman
& al., 1998).
Two genera of traditional Hydrocotyloideae,
Dickinsia and Azorella , were also investigated.
Molecular evidence has suggested that the subfamily
may be polyphyletic or even belonging outside
Umbelliferae (Plunkett & al., 1996, 1998). The mono-
typic Dickinsia, endemic to China, was described by
Franchet (1886), and later independentl y as Cotylonia by
Norman (1922). Dickinsia hydrocotyl oides was placed
by Drude (1897) in Hydrocotyloideae-Hydrocotyleae-
Hydrocotylinae. Koso-Poljansky (1924) positioned the
genus in Azorelleae and moreover “Affinitas Azorellae”.
Sheh & Su (1992) showed similarity of Azorella and
Hydrocotyle in pollen morphology. Peng & Sheh (1991)
concluded that Dickinsia was most similar to members of
Mulineae from South America, and remote from two
other Chinese genera of Hydrocotyloideae, Hydrocotyle
and Centella.
The mainly South American genus Azorella with 26
species (Martinez, 1993) was classified by Drude (1897)
in Hydrocotyliodeae-Mulineae-Azorellinae. Koso-
Poljansky (1924) placed it in Azorelleae, Cerceau-
Larrival (1962) in Azorelloideae-Azorelleae, and Tseng
(1967) in an informal group of genera with Schizeilema,
Laretia, Huanaca, Spananthe, and Hermas. As was pre-
viously shown (Plunkett & al., 1996a, b; Downie & al.,
1996; Plunkett & Downie, 1999; Downie & al., 2000a,
b), all molecular-phylogenetic analyses (with different
Valiejo-Roman & al.  Relationships in Saniculoideae
51  February 2002: 91–101
99

genes) reveal the polyphyletic, or at least paraphyletic,
nature of Hydrocotyloideae sensu Drude. Hydrocotyle,
Centella, Micropleura, Spananthe and Trachymene are
closer to Araliaceae, and some genera (Eremocharis,
Bowlesia and Naufraga ) approach the true Apioideae.
With the inclusion of Dickinsia and Azorella this pattern
remains basically the same. Azorella is a sister genus of
a large cluster uniting all Saniculoide ae and Apioideae,
being slightly closer to them than to the Hydrocotyle-
Araliaceae clade. Analysis of cpDNA restriction site data
(Plunkett & Downie, 1999) showed that Azorella and
Bolax form a lineage with Centella , and rps16 intron
sequencing (Downie & Katz-Downie, 1999) reveals a
group consisting of Eremocharis, Azorella and Bolax.
Dickinsia is very distant from the phenetically similar
Hydrocotyle. It appears in a large cluster of Apioideae
including Lagoecia, as noted above, and Naufraga, as
shown previously by Downie & al. (2000a). As for
Naufraga, this genus appears close to Apium, receiving
some support from morphology. The genus was
described by Constance & Cannon (1967) without
detailed anatomical analysis of mature fruits. Some of
the described carpological features, such as absence of
carpophore and a secretory system without commissural
vittae, can also be found in some Apioideae. A sclerified
(“woody”) endocarp, peculiar to Hydrocotyleand its rel-
atives, is not detected. The “stipules” of the cauline
leaves can be interpreted as modified sheaths (at least,
the radical leaves are of a form usual for Apioideae: pin-
natifid lamina, petiole and enlarged sheath). In any case,
molecular analysis recommends further investigations on
the proper affinities of Naufraga, as well as of Azorella
and Dickinsia.
ACKNOWLEDGEMENTS
The authors thank Prof. A. S. Antonov for helpful critica l
comments and Dr. C. Jeffrey for comments on the manuscript and
linguistic corrections. This work was supported in part by the
Russian Foundation for Basic Research (projects 00-15-97905, 98-
04-48264 and 00-04-48266a).
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