Biochemical Systematics and Ecology 33 (2005) 1087e1106

www.elsevier.com/locate/biochemsyseco

Veronica: Chemical characters for the support

of phylogenetic relationships based on nuclear
ribosomal and plastid DNA sequence data
Dirk C. Albach a,1,*, Søren R. Jensen b, Fevzi Özgökce c,
Renée J. Grayer d
a
Department of Higher Plant Systematics, Institute of Botany, University of Vienna,
Rennweg 14, A-1030 Vienna, Austria
b
Department of Chemistry, The Technical University of Denmark, DK-2800 Lyngby, Denmark
c
Department of Biology, Faculty of Science and Arts, Yüzüncü Yil University, 65080 Van, Turkey
d
Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK
Received 15 December 2004; accepted 6 June 2005

Abstract

Molecular phylogenetic analyses have revealed many relationships in Veronica (Plantagi-

naceae) never anticipated before. However, phytochemical characters show good congruence
with DNA-based analyses. We have analysed a combined data set of 49 species and subspecies
derived from the nuclear ribosomal ITS-region (18 new sequences) and the plastid trnL-F
region (12 new sequences) of Veronica emphasizing subgenera Chamaedrys and Pocilla and
separate analyses of subgenera Pentasepalae (ITS only) and Pseudolysimachium. Results for
subgenus Chamaedrys show that European and Asian perennial species are monophyletic
sister groups with the annual species consecutive sisters to them. All species of Veronica that
contain cornoside are found in this subgenus, although some species seem to have secondarily
lost the ability to produce this compound. Subgenera Pocilla and Pentasepalae are well
supported sister groups characterized by the occurrence of 8-hydroxyflavones. The traditional
subsection Biloba of subgenus Pocilla is biphyletic with Veronica intercedens being clearly

* Corresponding author. Tel.: C49 6131 39 23169; fax: C49 6131 39 23524.
E-mail address: albach@uni-mainz.de (D.C. Albach).
1
Present address: Institute of Special Botany, Johannes Gutenberg-Universität Mainz, Bentzelweg 9b,
55099 Mainz, Germany.

0305-1978/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bse.2005.06.002
1088 D.C. Albach et al. / Biochemical Systematics and Ecology 33 (2005) 1087e1106

separate from the rest of the group. This result is mirrored by the unusual phytochemical
arsenal of V. intercedens, which is the only species in the genus analysed to date to contain
melittoside and globularifolin. Subgenus Pentasepalae appears to be a clade of diverse lineages
from southwestern Asia and a single European clade. Species shown to have 6-
hydroxyflavones do not form a monophyletic group. Subgenus Pseudolysimachium seems to
have originated in Eastern Asia. 6-Hydroxyflavones acylated with phenolic acids are common
in this subgenus but may have originated only later in the evolution of the group. Possible
chemotaxonomic markers for other groups are discussed.
Ó 2005 Elsevier Ltd. All rights reserved.

Keywords: Veronica; Plantaginaceae; Chemosystematics; Phylogenetic analysis; ITS; trnL-F region

1. Introduction

Veronica is a genus of the Plantaginaceae sensu APG (1998) formerly placed in

Scrophulariaceae with about 500 species (Albach et al., 2004a). It is distributed over
most of the Northern Hemisphere and in many parts of the Southern Hemisphere, and
is ecologically highly diverse with species growing in aquatic to dry steppe habitats
from sea level to high alpine regions. This diversity and the fact that many species have
beautiful blue flowers may explain the interest Veronica has drawn for a long time.
Extensive breeding programs and morphological studies between 1900 and 1955, i.e. by
Watzl (1910) and Lehmann and his pupils (e.g., Lehmann, 1908, 1937; Römpp, 1928;
Härle, 1932; Riek, 1935; Schlenker, 1936; Riek-Häußermann, 1943) have been
followed by cytological and morphological analyses by M.A. Fischer (1967, 1972,
1975, 1987), Martı́nez-Ortega (Martı́nez-Ortega et al., 2000; Martı́nez-Ortega and
Rico, 2001) and floristic surveys (e.g., Borissova, 1955; Hartl, 1966e1968; M.A.
Fischer, 1978, 1981). Cladistic analyses incorporating much of the morphological data
have been published by Hong (1984) and Kampny and Dengler (1997). Phytochemistry
of Veronica and related species of the Northern Hemisphere has been studied by several
authors with an emphasis on iridoid compounds (e.g., Grayer-Barkmeijer, 1973;
Lahloub et al., 1993; Taskova et al., 2002, 2004) and flavonoid compounds (e.g.,
Grayer-Barkmeijer, 1978, 1979; Peev, 1982; Albach et al., 2003).
Recently, molecular techniques and phylogenetic analyses have been applied to
Veronica and related genera (Wagstaff and Garnock-Jones, 1998; Albach and Chase,
2001; Albach et al., 2004b,c). These studies have helped revolutionizing our ideas
about the evolution of the genus and led to a new, phylogenetic infrageneric
classification of Veronica (Albach et al., 2004a). Combined with the vast amount of
information from other aspects of their biology, we now have a much better
understanding of how major groups in Veronica are delimited and related, and how
they have evolved. This information combined in a phylogenetic framework opens
a new perspective for character evolution in the genus and helps focus on those
clades that have not been studied intensively in the past.
One area of the study of plant evolution that has shown strong correlations with the
results of DNA sequence analyses is chemotaxonomy (Grayer et al., 1999). The
 D.C. Albach et al. / Biochemical Systematics and Ecology 33 (2005) 1087e1106 1089

objective of the present paper is therefore to investigate the distribution of

phytochemical compounds, which may be useful as an independent test of relation-
ships suggested in DNA sequence analyses. Taskova et al. (2004) have compared the
distribution of iridoid compounds in the genus Veronica with dendrograms based on
DNA sequences. However, interesting chemotaxonomic characters can also be found
among flavonoids and verbascoside-like compounds of Veronica. In two accompany-
ing studies, we investigate the phytochemistry of additional species focusing on
unusual flavonoids (acylated 6- and 8-hydroxylated flavone glycosides, Albach et al.,
2005) and the iridoid glucoside, ajugol, and on the non-iridoid compound, cornoside,
in the genus (Jensen et al., 2005). The distribution of these characters is discussed here
in the light of a phylogenetic hypothesis derived from DNA sequences from the nuclear
ribosomal internal spacer region (ITS 1, 5.8 S rDNA, ITS 2) and the plastid trnL-F
region (trnL intron, trnL 3# exon, trnL-F spacer).

2. Materials and methods

2.1. Plant sampling

For a larger analysis, 49 accessions were sequenced for the ITS- and trnL-F region
with an emphasis on subgenera Chamaedrys and Pocilla, which appeared as most
interesting in the phytochemical analyses (Albach et al., 2005). Taxa from all other
subgenera sensu Albach et al. (2004a) were added to the data matrix (Table 1).
Veronica montana and Veronica beccabunga were designated as outgroups based on
previous analyses (Albach and Chase, 2001; Albach et al., 2004b,c). Due to limited
variation in the trnL-F region of subgenus Pentasepalae, a smaller matrix of 15 ITS-
sequences including eight new sequences (Table 1) for species of subgenus
Pentasepalae was analysed separately and rooted with Veronica czerniakowskiana
based on previous analyses (Albach et al., 2004b,c). Finally, nine ITS- and five trnL-
F-sequences available from subgenus Pseudolysimachium (Table 1) were analysed in
separate analyses with Veronica glandulosa, V. beccabunga and Veronica gentianoides
as outgroups based on previous analyses (Albach et al., 2004b,c). Eleven of the trnL-F
and 19 of the ITS-sequences are reported here for the first time. Voucher specimens
were made for all plants used in this study (Table 1).

2.2. DNA extraction, amplification and sequencing

The protocol followed previous studies (Albach and Chase, 2001; Albach et al.,
2004b). Total genomic DNA was extracted from herbarium material and silica gel-
dried leaf samples according to the 2x CTAB procedure of Doyle and Doyle (1987).
The trnL intron, 3# exon, and trnL-F spacer (hereafter trnL-F ) were amplified with
primers c and f of Taberlet et al. (1991). ITS-sequences were amplified and sequenced
using the primers 17SE (Sun et al., 1994) and ITS4 (White et al., 1991). PCR
products were run on a 1.0% TBE-agarose gel, cut out of the gel, and cleaned using
QIAquickÔ PCR purification and gel extraction kit (Qiagen GmbH, Hilden,
1090 D.C. Albach et al. / Biochemical Systematics and Ecology 33 (2005) 1087e1106

Table 1
Voucher specimens and GenBank accession numbers for sequences used in the phylogenetic analysis
Species Subgenus Voucher ITS trnL-F
GenBank GenBank
acc. no. acc. no.
V. agrestis Pocilla Albach 386, WU AF509784 AF513335
V. amoena Pocilla Schelkovnikov and AF509786 AF513334
Schmidt 5.4.1907, TBS
V. argute-serrata e Pocilla Rechinger 35177, WU AF509787 AF513337
Afghanistan
V. argute-serrata e Pocilla Albach 640, WU AY673605 AY673623
Turkey
V. armena* Pentasepalae Struwe 1404, WU AF313040 e
V. arvensis Chamaedrys Albach 147, WU AF313002 AF486340
V. beccabunga Beccabunga Albach 122, K AF313015 AF486403
V. biloba Pocilla Albach 636, WU AY673606 AY673625
V. bombycina Pentasepalae Struwe 1403, WU AF486353 AF486376
V. campylopoda e Pocilla Schönswetter and AF486364 AF486370
Jordan Tribsch 4152, WU
V. campylopoda e Pocilla Albach 656, WU AY673608 AY673624
Turkey
V. capillipes Pocilla Rechinger 51188, WU AY673607 AY673627
V. catarractae Hebe Garnock-Jones 2403, CHR AY034859 AY540887/
AY540901
V. caucasica* Pentasepalae Albach 326, WU AF486357 e
V. ceratocarpa Pocilla Öztürk 429, WU AY741514 AY673626
V. chamaedryoides Chamaedrys Albach 393, WU AY673611 AY673631
V. chamaedrys Chamaedrys Albach 121, K AF313003 AF486377
V. chamaepithyoides Triangulicapsula UA 174, SALA AF509796 AF511477/8
V. cinerea* Pentasepalae Albach and Chase 113, K AY144458 e
V. crista-galli Cochlidiosperma Dolmkanov 17.4.1983, TBS AF509799 AF486367
V. cuneifolia subsp. Pentasepalae Struwe 1409, WU AF486354 AF486372
isaurica
V. czerniakowskiana Pentasepalae Terme 39177E, EVIN AF486362 AF486371
V. dahurica Pseudolysimachium BG Jena, WU AF313023 AF411479/80
V. dichrus* Pentasepalae Struwe 1407, WU AF312998 e
V. filiformis Pocilla Albach 298, WU AF486363 AF486368
V. fruticulosa Stenocarpon Albach 71, WU AF313004 AF486383
V. gentianoides Beccabunga Albach 72, WU AF313018 AF486401
V. glandulosa Veronica Fischer 713/98, WU AF313008 AF486394
V. glauca Pellidosperma Chase s.n., K/M. A. AF313006 AF486395
Fischer 9, 7.4.1999, WU
V. incana Pseudolysimachium BG Bonn, WU e AY486449
V. nakaiana Pseudolysimachium Sun and Kim 12022, WU AF515210 AF486406
V. intercedens e 1 Pocilla Albach 667, WU AY673610 AY486439
V. intercedens e 2 Pocilla Albach 666, WU AY673609 AY673628
V. jacquinii Pentasepalae Albach 70, WU AF313000 AF513341
V. javanica unplaced Fries et al. 2016, BR AY540867 AY540872
V. krumovii Chamaedrys Albach 484, WU AY673612 AY673633
V. lanosa Stenocarpon Schickhoff 1377, GOET AY540868 AY486442
V. laxa Chamaedrys Dickoree 13042, GOET AY673613 AF486378
V. liwanensis* Pentasepalae Struwe 1411, WU AF312997 e
V. longifolia Pseudolysimachium Albach 66, WU AF313021 AF486407
 D.C. Albach et al. / Biochemical Systematics and Ecology 33 (2005) 1087e1106 1091

Table 1 (continued)
Species Subgenus Voucher ITS trnL-F
GenBank GenBank
acc. no. acc. no.
V. longifolia e UK Pseudolysimachium McSheahan 48, K AY673619 e
V. magna Chamaedrys Albach 360, WU AY673615 AY673634
V. micans Chamaedrys Schönswetter 2567, WU AY673616 AY673632
V. missurica Synthyris Chase s.n., K AF313019 AF486397
V. montana Veronica Albach 151, WU AF313014 AF486388
V. nivea Derwentia CHR512486 AF037382 AY540890/
AY540903
V. oltensis Pentasepalae Struwe 1405, WU AF312995 AF513339
V. opaca Pocilla Polatschek 8.7.90, W AY673617 AY673630
V. ovata Pseudolysimachium Matsumoto s.n., WU AY673618 e
V. paederotae Pentasepalae Klein 7901, WU AF509783 AF513340
V. peduncularis* Pentasepalae Albach 325, WU AF486356 e
V. persica Pocilla Fay 175, K AF509785 AF513336
V. polita Pocilla Albach 146, WU AF509818 AF486369
V. propinqua* Pentasepalae Albach 327, WU AF486361 e
V. rubrifolia Pocilla n.n., WU AF509788 AF513338
V. salicifolia Hebe CHR512466 AF037385 AY540881/
AY540895
V. schmidtiana Pseudolysimachium S. Umezawa 20130, WU AY673620 e
V. spicata e cult. Pseudolysimachium BG Bonn 09333, WU AF313022 AF486405
V. spicata e UK Pseudolysimachium Fay e Avon Gorge, K AY673621 e
V. spicata e UK Pseudolysimachium Fay e Avon Gorge, K AY673622 e
V. teucrium* Pentasepalae Albach 119, K AF312999 e
V. thessalica Stenocarpon Raus and Rogl 5072, SALA AF509792 AF513343
V. triloba Cochlidiosperma Albach 242, WU AF509804 AF513333
V. triphyllos Pellidosperma Albach 244, WU AF509795 AF486396
V. turrilliana Pentasepalae Albach 278, WU AF486360 AF486374
V. verna Chamaedrys Albach 149, WU AF509789 AF486379
V. vindobonensis Chamaedrys Fischer, cult. Wien, WU AY673614 AF510426
Species with an * were only used in the analysis of ITS-sequences for V. subgenus Pentasepalae.

Germany) following the manufacturer’s protocols. Sequencing reactions (10 ml) were
carried out using the Taq DyeDeoxy Terminator Cycle Sequencing mix (Applied
Biosystems Inc.). Reactions were run out on a Prism 377 automated sequencer
(Applied Biosystems Inc.), and both strands were sequenced. Sequences were
assembled and edited using Sequence NavigatorÔ (Applied Biosystems Inc.).
Assembled sequences were manually aligned prior to analysis. Aligned sequence
matrices are available from DCA by request.

2.3. Sequence analysis

Insertions and deletions are frequent in both DNA regions. We have scored those
gaps that are neither found in mononucleotide repeats nor are only 1 bp long as
present/absent to preserve the information included in them. All matrices were
analysed with PAUP* 4.0b10 (Swofford, 1998) using heuristic parsimony methods.
1092 D.C. Albach et al. / Biochemical Systematics and Ecology 33 (2005) 1087e1106

Ten runs of random taxon addition (10 replicates each) using tree bisection
reconnection (TBR) were conducted with MulTrees (keeping multiple shortest trees)
in effect and no tree limit. Bootstrap percentages were assessed using 1000 replicates
and TBR-branch swapping with a maximum of 100 trees per replicate.

3. Results and discussion

3.1. ITS

Aligned sequences of the ITS-region included 759 characters including 4 gaps

scored as present/absent (one having a CI ! 1). One hundred and ninety-seven
characters were potentially parsimony informative. We found 121 most parsimoni-
ous trees, which required 943 steps (Fig. 1; CI Z 0.46, RI Z 0.71). Relationships
among the subgenera are not well supported except for the clade consisting of
subgenera Chamaedrys, Pocilla and Pentasepalae (72 BP). However, monophyly of
subgenera is well supported. Within subgenus Chamaedrys (99 BP), the annual
species Veronica arvensis and Veronica verna are consecutive sisters to the perennial
species. Among the latter, the Asian species, Veronica laxa and Veronica magna, are
sister to the European species (75 BP). Veronica chamaedrys is most closely related to
Veronica micans (90 BP). Within subgenus Pocilla (100 BP), two clades are strongly
supported that resemble subsection Agrestes in the circumscription of Lehmann
(1908; 96 BP) and subsection Biloba sensu Römpp (1928; 100 BP). Within subsection
Agrestes, Veronica polita, Veronica persica and Veronica opaca form one moderately
supported clade (87 BP), as does the group of Veronica ceratocarpa, Veronica
filiformis, and Veronica agrestis. A position of Veronica amoena close to subsect.
Agrestes has hitherto not been suggested. Within subsection Biloba, Veronica argute-
serrata is not monophyletic, one sequence being more closely related than the other
to Veronica campylopoda. Veronica biloba and Veronica capillipes are strongly
supported sisters (99 BP). Veronica intercedens, usually considered part of subsection
Biloba, does not seem to belong to the group in a strict sense.

3.2. trnL-F

Aligned sequences of the trnL-F region included 1067 characters including 16 gaps
scored as present/absent with 136 characters potentially parsimony informative. We
stopped saving trees when 1000 most parsimonious trees that required 475 steps were
found (Fig. 2; CI Z 0.79, RI Z 0.83). Repeating the search five times did not result in
searches finding shorter trees. Relationships among subgenera do find some
bootstrap support above 50%. Within subgenus Chamaedrys (83 BP), the branching
pattern differs from that in the ITS-analysis by V. verna switching its place with
V. magna and V. laxa, which renders the perennial species of the subgenus non-
monophyletic. Veronica vindobonensis is sister to V. micans (59 BP) rather than found
in a clade together with Veronica krumovii and Veronica chamaedryoides as in the
ITS-analysis. Within subgenus Pocilla (90 BP), subsection Biloba is paraphyletic with
 D.C. Albach et al. / Biochemical Systematics and Ecology 33 (2005) 1087e1106 1093

Fig. 1. One of 121 most parsimonious trees from the analysis of the ITS-region for 49 taxa of Veronica.
Numbers above branch and before the slash are branch lengths, numbers behind the slash are bootstrap
percentages. Branches not present in strict consensus tree are dotted with branch lengths in italics.
1094 D.C. Albach et al. / Biochemical Systematics and Ecology 33 (2005) 1087e1106

Fig. 2. One of more than 1000 most parsimonious trees from the analysis of the trnL-F region for 49 taxa of
Veronica. Numbers above branch and before the slash are branch lengths, numbers behind the slash are
bootstrap percentages. Branches not present in strict consensus tree are dotted with branch lengths in italics.
 D.C. Albach et al. / Biochemical Systematics and Ecology 33 (2005) 1087e1106 1095

respect to subsection Agrestes and Veronica rubrifolia. Within subsection Agrestes,

V. amoena is sister to the rest of the subsection. Subgenus Pentasepalae (97 BP) is
characterized by a lack of resolution due to low sequence divergence except for the
sister-group relationship of V. czerniakowskiana to the rest of the subgenus (75 BP).

3.3. Combined data set

The complete data set of both regions combined included 1826 characters
including 20 gaps scored as present/absent with 333 characters potentially parsimony
informative. We found 384 most parsimonious trees that required 1441 steps (Fig. 3;
CI Z 0.56, RI Z 0.73). The analysis differs from that of the single data sets in an
overall increase in bootstrap support in all relationships. Branching pattern among
subgenera Pocilla, Pentasepalae, and Chamaedrys resembles that in the analysis of
ITS. In subgenus Chamaedrys (100 BP), the annual species are paraphyletic with
respect to the perennials (71 BP). Branching pattern in subgenus Pocilla (100 BP) is
similar to that of the trnL-F analysis with subsection Biloba paraphyletic to
subsection Agrestes and V. rubrifolia and V. amoena sister to the rest of subsection
Agrestes. Due to the lack of variation in the trnL-F data set, relationships within
subgenus Pentasepalae (95 BP) are the same as in the analysis of ITS.

3.4. ITS-data set for subgenus Pentasepalae

The 15 sequences for subgenus Pentasepalae included 36 potentially parsimony-

informative characters but no potentially parsimony-informative indel. We found
three most parsimonious trees that required 133 steps (Fig. 4; CI Z 0.78, RI Z 0.66).
Bootstrap support is generally low with the exception of the European species from
subsection Pentasepalae and the association of Veronica peduncularis and Veronica
caucasica.

3.5. Analyses of subgenus Pseudolysimachium

The analysis of nine ITS-sequences from six species from subgenus Pseudoly-
simachium included 76 potentially parsimony-informative characters and resulted
in two most parsimonious trees (Fig. 5A) with 175 steps (CI Z 0.89, RI Z 0.86).
The analysis of five trnL-F-sequences from five species included 24 potentially
parsimony-informative characters and resulted in a single most parsimonious tree
(Fig. 5B) with 71 steps (CI Z 0.99, RI Z 0.97). The most parsimonious trees from
the analysis of ITS are reasonably well resolved, but the one from the analysis of
trnL-F is not, because of limited sequence variability. Both analyses agree in
Veronica nakaiana from Eastern Asia being sister to the rest of the subgenus. In
the analysis of ITS-sequences, V. nakaiana is consecutively followed by two other
East Asian species, Veronica dahurica and Veronica schmidtiana. However,
bootstrap support is relatively low except for the monophyly of the subgenus, of
those two species where more than one sequence was available, and of the clade
comprising the European Veronica spicata and Veronica longifolia together with
1096 D.C. Albach et al. / Biochemical Systematics and Ecology 33 (2005) 1087e1106

Fig. 3. One of 384 most parsimonious trees from the combined analysis of the ITS-region and the trnL-F
region for 49 taxa of Veronica. Numbers above branch and before the slash are branch lengths, numbers
behind the slash are bootstrap percentages. Branches not present in strict consensus tree are dotted with
branch lengths in italics. Branches with bootstrap support above 70% appear in double thickness, those
above 95% in triple thickness.
 D.C. Albach et al. / Biochemical Systematics and Ecology 33 (2005) 1087e1106 1097

Fig. 4. One of three most parsimonious trees. The other two show Veronica cuneifolia either as sister to
Veronica cinerea or as sister to V. subsect. Pentasepalae. Numbers above the branches indicate branch
lengths, those below the branch bootstrap support.
 1098
D.C. Albach et al. / Biochemical Systematics and Ecology 33 (2005) 1087e1106
Fig. 5. Phylograms for sequence analyses of subgenus Pseudolysimachium. Branch lengths (before the dash) and bootstrap percentages above 50% (after the
dash) are indicated. (A) Strict consensus and one of two most parsimonious trees of the analysis of the ITS-region (the other most arsimonious tree combines
V. spicata and V. longifolia with V. ovata as sister to both). (B) Most parsimonious tree of the analysis of the trnL-F region.
 D.C. Albach et al. / Biochemical Systematics and Ecology 33 (2005) 1087e1106 1099

Veronica ovata. Sequence variability within V. spicata is surprisingly high. Even

two individuals within one English population differ by three nucleotide
substitutions, which is also supported by strong differentiation in AFLP-fingerprint
patterns (Albach, unpubl.).

3.6. Discussion of the DNA sequence analyses

The present analysis of DNA sequence data focuses on two subgenera (subg.
Pocilla and Chamaedrys), which appeared especially interesting regarding their
phytochemical arsenal. Relationships between subgenera have been the focus of
previous publications (Albach and Chase, 2004; Albach et al., 2004b,c). Subgenus
Pocilla is sister to subgenus Pentasepalae in analyses of both nuclear ITS and plastid
trnL-F sequences with weak support in the separate analyses (ITS: 57 BP; trnL-F: 68
BP) but moderate support in the combined analysis (84 BP). This sister-group
relationship has also been found in all previous publications (Albach and Chase,
2001, 2004; Albach et al., 2004b,c). The present analysis extends taxon sampling in
subgenus Pocilla, allowing for the first time a discussion of relationships in this
subgenus. As mentioned before, subgenus Pocilla includes species belonging to the
Agrestis-group sensu Lehmann (1908) and ‘‘Verwandtschaftsgruppe’’ Biloba sensu
Römpp (1928). The analysis supports subsection Agrestes in the circumscription of
Lehmann (1908) including V. agrestis, V. filiformis, V. ceratocarpa, V. polita,
V. persica, and V. opaca. Apart from the two strictly European tetraploid species
V. agrestis and V. opaca, all other species originate from the hyrcanian-caucasian
region with three of them (V. ceratocarpa, Veronica francispetae e both diploid;
Veronica siaretensis e unknown ploidy) restricted to this region. V. polita,
V. filiformis and V. persica (the first two diploid, the latter tetraploid) have become
cosmopolitan weeds at different times. The occurrence of V. filiformis in this group is
peculiar because it differs from all other species of the subgenus in being perennial and
self-incompatible (Lehmann, 1944), whereas all other species are annuals and at least
those of subsection Agrestes self-compatible (Lehmann and Schmitz-Lohner, 1954).
Self-incompatibility in subsection Biloba is unlikely. Morphological differences
between the species of subsection Agrestes have been discussed by Thaler (1951) and
Fischer (1987). Several publications have dealt with the origin of the polyploid species
in this subsection. Based on karyological and biogeographical evidence, Beatus (1936)
hypothesized that all three tetraploid species (V. agrestis, V. opaca, V. persica) are
autopolyploids, V. persica derived from V. filiformis and the other two derived from
V. polita. Lehmann (1940) contradicted Beatus with respect to V. persica, which he
considered to be also derived from V. polita. Fischer (1987) however, argued based on
morphological intermediacy that V. persica is an allopolyploid species derived from
a cross of V. polita and V. ceratocarpa. Evidence presented here from both
maternally- and biparently-inherited DNA sequences supports a contribution of
V. polita to the genomes of V. persica and V. opaca, although it cannot be confirmed
whether they are autopolyploid or allopolyploids, because ITS-sequence could be
converging towards the maternal parent. The position of V. agrestis as sister to
V. filiformis is rather surprising as a close relationship has not been proposed before
1100 D.C. Albach et al. / Biochemical Systematics and Ecology 33 (2005) 1087e1106

based on morphological evidence, although their flavonoid profiles are very similar
(Grayer-Barkmeijer, 1979). Further investigations are necessary to elucidate the
ancestry of the polyploid species in this group.
Subsection Biloba is characterized by pairwise fusion of the calyces, more or less
recurved pedicels when fruiting and in most cases strongly bilobed capsules. Our
analyses (Figs. 1e3) do not support this subsection to be monophyletic in its original
circumscription by Lehmann (1910; see also Römpp, 1928). All analyses indicate
V. intercedens to be more closely related to V. rubrifolia than to the rest of the
subsection. V. intercedens was split from subsection Biloba by Elenevsky (1977)
together with Veronica cardiocarpa to subsection Cardiocarpae based on the
verticillate cauline leaves in these species. This is also supported by the unusual
iridoid phytochemistry of V. intercedens, which is the only species in the genus to
contain melittoside and globularifolin (Albach et al., 2003). V. cardiocarpa has not
yet been investigated phytochemically. V. rubrifolia does not share any of the
characters of subsection Biloba or subsection Cardiocarpae mentioned above and its
position in the phylogeny remains doubtful until further annual species from
southwest Asia have been included in the analyses.
Analysis of the subgenus Pentasepalae supports the sister-group relationship of
V. czerniakowskiana to the rest of the subgenus (Figs. 1e4) found in previous
analyses (Albach et al., 2004b,c). However, using cpDNA, subgenus Chamaedrys is
sister to a larger clade of subgenera Pentasepalae and Pocilla, subgenus Stenocarpon
and the Hebe complex in analyses of the plastid trnL-F region (Fig. 2, Albach et al.,
2004b,c) and the plastid rps16 intron (Albach and Chase, 2004).
The association of the perennial V. chamaedrys with the annual V. arvensis was
first noted by Albach and Chase (2001). Both species have never been considered
closely related because the distinction between annuals with a terminal inflorescence
and those perennial species that only have lateral inflorescences was considered the
major division in the genus (Bentham, 1846; Römpp, 1928). Apart from this,
V. arvensis and V. verna have seeds that differ strongly from V. chamaedrys and
relatives (Martı́nez-Ortega and Albach, in preparation). Phytochemical similarity of
these species (absence of catalpol esters in both species, Taskova et al., 2004; Jensen
et al., 2005) is therefore valuable support for the results of DNA sequence analyses.
The results for subgenus Chamaedrys presented here (Figs. 1e3) show that the
perennial species are further divided into an Asian group (V. magna, V. laxa) and
a European group. The European group can be termed V. chamaedrys sensu lato
because all species have been included in V. chamaedrys at one time or another.
Apart from the tetraploid V. chamaedrys all other species sampled are diploid. No
definite answer can be given regarding the ancestors of V. chamaedrys but it is
noteworthy that in all analyses V. chamaedrys seems to be closer to the Central
European species V. vindobonensis and V. micans than to the strictly Balkanian
species V. krumovii and V. chamaedryoides. An origin of V. chamaedrys in Central
Europe seems therefore more likely. However further diploid populations of
V. chamaedrys in Central and Eastern Europe are known (Fischer, 1973; M.A.
Fischer, pers. comm.). Those populations so closely resemble the tetraploid
V. chamaedrys that they have not been separated from it at any taxonomic rank.
 D.C. Albach et al. / Biochemical Systematics and Ecology 33 (2005) 1087e1106 1101

Due to this fact they should be considered in future analyses investigating the
ancestors of the tetraploid form of V. chamaedrys.
Subgenus Pentasepalae is a species-rich group with its 70 species mostly found in
Turkey and Iran. Its geographical outliers, subsect. Pentasepalae in Europe and
subsect. Petraeae in the Greater Caucasus, are the only well supported groups (97 BP
and 83 BP, respectively; Fig. 4) in this subgenus. A larger analysis with more
informative characters is needed to resolve the relationships of this group.
Subgenus Pseudolysimachium is a highly diverse group with approximately 25
species currently accepted despite approximately 400 names and combinations
available. This taxonomic complexity demonstrates the high morphological variability
within the subgenus and explains the difficulties for taxon sampling in molecular
analyses. Another problem in systematic analyses of this subgenus is the probably
widespread interfertility and hybridisation between taxa (e.g. Härle, 1932). The current
analyses are therefore only intended as broad guidelines in analyses of AFLP-
fingerprints (Albach, unpubl.). Nevertheless, one important result is the paraphyly of
East Asian species and the derived position of European species, which indicates an
East Asian origin of the subgenus. This result is important because it points to those
species that may resemble most the ancestral species of this subgenus. Among those
species is V. schmidtiana, which is the only species that was not associated with this
subgenus before 1968 (Yamazaki, 1968), because it has a flattened capsule, which is
common among other species of Veronica but only found in a few Eastern Asian species
of subgenus Pseudolysimachium. V. schmidtiana is also distinct from the rest of the
subgenus in pollen morphology (Hong, 1984) and its rosette habit, which is frequently
found among other species of Veronica (e.g. subgenus Synthyris) but only rarely among
the more derived members of subgenus Pseudolysimachium. V. schmidtiana, however,
shares with the rest of the subgenus the unusual chromosome number of x Z 17 (e.g.
Sakai, 1935). Its inflorescence is long and terminal but not as dense and spike-like as
those of most other species. The loosely flowered inflorescence is associated with short-
tubed corollas and is found in several Eastern Asian species of this subgenus but not in
its derived members. An emphasis of future studies should therefore be put on the East
Asian species and not only study its relationship based on DNA-markers but also study
its phytochemistry (see below), seed and pollen.

3.7. Review of chemotaxonomic markers in Veronica

Molecular systematics has often given results that were hitherto unsuspected,
which in turn has reinitiated research in other fields of systematics (e.g. Williams and
Friedman, 2004). Phytochemistry is a prime example for this. Sometimes
taxonomically useful characters were overlooked and only later appreciated (Grayer
et al., 1999) but much too often phytochemistry is only incompletely known, which
prevents a full assessment of its use in classifying plants. Veronica is a fortunate
exception to this. A comparison of our analyses of ITS- and plastid trnL-F sequence
data with published reports on iridoids, flavonoids and verbascoside-like compounds
reveals several compounds that are good indicators of relationships, several of them
previously not recognized. Due to the lack of information on iridoids in the Hebe
1102 D.C. Albach et al. / Biochemical Systematics and Ecology 33 (2005) 1087e1106

complex and the complexity of information on flavonoids in the Hebe complex

(Bayly et al., 2000, 2002), we focus here on species from the Northern Hemisphere.
Comparisons reveal not only synapomorphies for clades, so far exclusively
characterized by nucleotide differences but also reveal much about the evolution
of these phytochemical compounds. Convergence in phytochemical arsenal seems to
be widespread. The convergence in the production of acetylated 8-hydroxyflavone
allosyl glycosides between Veronica (Grayer-Barkmeijer, 1979; Tomás-Barberán
et al., 1988; Albach et al., 2003) and Stachys (Lamiaceae, Lenherr et al., 1984;
Tomás-Barberán et al., 1988) is just one example. Reversals to ancestral conditions
seem to be frequent despite long divergence from ancestors that also contained these
compounds. This supports the idea of Grayer et al. (1999) that those genes
responsible for the biosynthesis of such compounds may be retained in the genome
despite lack of expression. Alternatively, these reversals may occur if additional steps
in the synthesis of secondary compounds are secondarily lost again. Analyses of
genes underlying the biosynthetic pathways such as those in the flavonoid
biosynthetic pathway (Shirley, 1996; Quattrocchio et al., 1999; Zufall and Rausher,
2004) will be necessary to determine the fate of the underlying genes.
Among those compounds that are chemotaxonomically useful in Veronica are
iridoids, flavonoids and verbascoside-like compounds. Mussaenoside is one of the
few iridoid compounds in Veronica not derived from aucubin and catalpol. It was
first detected by Grayer-Barkmeijer (1973, 1979) and then identified by Afifi-Yazar
and Sticher (1981). Subsequent studies showed that it is almost exclusively found in
a clade comprising Veronica officinalis, Veronica aphylla and Veronica urticifolia
(axillary inflorescences) and Veronica alpina and Veronica bellidioides (terminal
inflorescences) (Grayer-Barkmeijer, 1979; Taskova et al., 2002, 2004), which used
to be classified in two different sections. Grayer-Barkmeijer (1973, 1979) already
noted that species producing mussaenoside also share the same basic chromosome
number (x Z 9) and have similar seeds. However, the clade was not formally
recognized until recently (subgenus Veronica, Albach et al., 2004a), because former
intrageneric classifications of Veronica relied on the presence or absence of
a terminal inflorescence, an unstable character in this group. Two other iridoids
that are likely to be chemotaxonomically useful are melittoside and globularifolin,
which are found in V. intercedens (Albach et al., 2003). Investigation of related
species is necessary to evaluate their distribution, but occurrence in Veronica
tenuissima and V. cardiocarpa seems likely because they also do not blacken while
drying, an indication that aucubin is lacking. Finally, ajugol has been found in
members of Veronica subsection Biloba (Jensen et al., 2005), and as a dominant
compound is a chemotaxonomic marker for this subsection, although it is also
found in trace amounts in V. arvensis (subgenus Chamaedrys) (Jensen et al., 2005).
Other species of Veronica contain aucubin, catalpol and its esters, whereas the
specific combination of esters may be significant for various groups at various
taxonomic levels (Taskova et al., 2002, 2004), no ester is confined to a group at
subgeneric level or below.
Among flavonoids, 8-hydroxyflavones are the most prominent chemotaxonomic
marker supporting the sister-group relationship of subgenus Pocilla and subgenus
 D.C. Albach et al. / Biochemical Systematics and Ecology 33 (2005) 1087e1106 1103

Pentasepalae (Albach et al., 2005). Whereas subgenus Pocilla contains annual

species, subgenus Pentasepalae is exclusively perennial. Life history (annuals versus
perennials) has been considered an essential character for the classification (e.g.
Römpp, 1928), but annuals have been shown to be derived in parallel multiple times
within Veronica (Albach et al., 2004b). Within these two subgenera, a reversal to
the production of 6-hydroxyflavones is also inferred several times (Albach et al.,
2005).
Less important chemotaxonomic markers among the flavonoids of Veronica are
6-hydroxyflavones acylated with phenolic acids (Albach et al., 2005). These
compounds, which we called spicosides AeF (Albach et al., in press), occur in five
out of eight investigated species of subgenus Pseudolysimachium. They are lacking
in V. dahurica and V. nakaiana, inferred to be the earliest diverging species in the
subgenus (Fig. 5), which contrasts with a possible presence of these compounds in
the ancestor of the subgenus. Two of the spicosides have also been found in
V. thymoides ssp. pseudocinerea (Saracoglu et al., 2004) belonging to subgenus
Pentasepalae, where this character is thought to have originated independently
(Albach et al., 2005). Other flavonoid compounds that may be chemotaxonomic
markers still need closer investigation. Grayer-Barkmeijer (1979) noticed the
restricted occurrence of luteolin 7,3#-O-glucuronide in Veronica fruticans, Veronica
fruticulosa and Veronica saturejoides. Previously these species were thought to
belong to two different subsections, but they are now considered more closely
related (Albach et al., 2004b). Scutellarein-glycosides may be a fourth flavonoid
chemotaxonomic marker in Veronica being only found in members of section
Beccabunga with the exception of Veronica peregrina (Grayer-Barkmeijer, 1979).
The position of V. peregrina in section Beccabunga was unrecognised until recently
(Albach and Chase, 2001; Albach et al., 2004c). Although the absence of
scutellarein-glycosides may argue against its inclusion, the overall similarity of the
flavonoid arsenal (Grayer-Barkmeijer, 1979) is clearly in support of the DNA-
based results. This highlights the trend already observed among iridoids that while
specific chemotaxonomic markers may be lacking, the overall similar combination
of compounds is generally supporting results from DNA-based molecular
systematics.
Finally, cornoside, a compound related to verbascoside, has been found in both
annual and perennial species of subgenus Chamaedrys, but not outside this subgenus
in Veronica (Jensen et al., 2005). It is therefore an important character supporting
a clade that until now has only been distinguished by DNA sequence data (Figs. 1e3;
Albach et al., 2004b,c).
Phytochemical characters may also be important as chemosystematic markers at
a lower taxonomic level in Veronica, e.g. the presence or absence of a diosmetin
glycoside in the species-complex of Veronica hederifolia (Peev, 1982). Further
examples have been given by Grayer-Barkmeijer (1973, 1978, 1979) and Taskova
et al. (1997, 2002), but many more may be found after closer inspection. Much
chemotaxonomic work is currently being carried out on the Hebe complex in New
Zealand, where several examples of chemosystematic markers useful at the species
level have been found (e.g. Bayly et al., 2002).
1104 D.C. Albach et al. / Biochemical Systematics and Ecology 33 (2005) 1087e1106

Acknowledgments

We thank the Studienstiftung des deutschen Volkes for a doctoral scholarship to

D.C.A. and financial support by FWF (Fonds zur Förderung der wissenschaftlichen
Forschung) project P15336.

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