Phylogeny of Veronica in the Southern and Northern Hemispheres based on plastid, nuclear ribosomal and nuclear low-copy DNA

Molecular Phylogenetics and Evolution 54 (2010) 457–471

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Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev

Phylogeny of Veronica in the Southern and Northern Hemispheres based on plastid, nuclear ribosomal and nuclear low-copy DNA Dirk C. Albach a,*, Heidi M. Meudt b a b

Institut für Spezielle Botanik, Johannes Gutenberg-Universität Mainz, Bentzelweg 9b, 55099 Mainz, Germany Museum of New Zealand Te Papa Tongarewa, P.O. Box 467, Wellington 6011, New Zealand

a r t i c l e

i n f o

Article history: Received 12 May 2009 Revised 17 September 2009 Accepted 22 September 2009 Available online 29 September 2009 Keywords: CYCLOIDEA Hebes Incongruence ITS Phylogeny Plastid DNA Veronica

a b s t r a c t The cosmopolitan and ecologically diverse genus Veronica with approximately 450 species is the largest genus of the newly circumscribed Plantaginaceae. Previous analyses of Veronica DNA sequences were in stark contrast to traditional systematics. However, analyses did not allow many inferences regarding the relationship between major groups identified, hindering further analysis of diversification and evolutionary trends in the genus. To resolve the backbone relationships of Veronica, we added sequences from additional plastid DNA regions to existing data and analyzed matching data sets for 78 taxa and more than 5000 aligned characters from nuclear ribosomal DNA and plastid DNA regions. The results provide the best resolved and supported estimate of relationships among major groups in the Northern (Veronica s. str.) and Southern Hemisphere (hebes). We present new informal names for the five main species groups within the Southern Hemisphere sect. Hebe. Furthermore, in two instances we provide morphological and karyological characters supporting these relationships. Finally, we present the first evidence from nuclear low-copy CYCLOIDEA2-region to compare results from the plastid genome with the nuclear genome. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction In its most recent circumscription, the genus Veronica is the largest genus of Plantaginaceae with about 450 species (Albach et al., 2005a). It is distributed worldwide with centers of diversity in western Asia and New Zealand. The species exemplify a large range of life forms (minute annuals to small trees) and occur in diverse habitats (semideserts to aquatics, tropical to polar, sea level to alpine). Correspondingly, they exhibit a remarkable diversity of different traits in their vegetative morphology. In order to understand the evolution of these characters and to distinguish between parallel evolution from common ancestry, it is necessary to base the interpretation on a robust phylogenetic hypothesis. The phylogeny of Veronica has been the subject of several recent studies using nuclear ribosomal internal transcribed spacer region (ITS) and plastid DNA (cpDNA) rbcL, trnL-trnL-trnF and rps16 intron sequences (Wagstaff and Garnock-Jones, 1998, 2000; Wagstaff et al., 2002; Albach and Chase, 2001, 2004; Albach et al., 2004a,b, 2005b,c). These studies provided a general framework which al-

* Corresponding author. Present address: AG Biodiversität und Evolution der Pflanzen, Institut für Biologie und Umweltwissenschaften, Carl von OssietzkyUniversität Oldenburg, Carl von Ossietzky-Strasse 9-11, D-26111 Oldenburg, Germany. Fax: +49 (0)441 7983331. E-mail addresses: [email protected] (D.C. Albach), heidim@tepapa. govt.nz (H.M. Meudt). 1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2009.09.030

lowed the establishment of a new infrageneric classification of the genus (Albach et al., 2004c). In addition, they provided evidence that characters such as life history and position of the inflorescence (i.e., stem ending in an inflorescence or in a vegetative shoot), formerly used for infrageneric classification, evolved several times in parallel. These studies have also led to the inference that the woody Southern Hemisphere species were derived from herbaceous Northern Hemisphere ancestors rather than the other way around (Albach and Chase, 2001; Wagstaff et al., 2002). Three major issues remain unresolved with respect to Veronica phylogeny and classification. First, the relationships among the 12 subgenera in Veronica are generally not well supported (e.g., Albach et al., 2004a,b). Such poor resolution may indicate that speciation in Veronica has occurred via a rapid species radiation, but it unfortunately also hinders the interpretation of evolutionary transitions on a genus-wide level. Phylogenetic analysis of more DNA sequence markers is warranted to further our understanding of morphological evolution and subgeneric relationships in Veronica. Second, several examples of incongruence were indicated in previous phylogenetic studies. For example, Albach and Chase (2004) demonstrated that the closest relative of the Southern Hemisphere species differed depending on which DNA region was used, but the phylogenetic signal in these regions was not strong enough to reject alternative positions. They suggested that additional, more informative chloroplast markers and other nuclear (low-copy) markers are necessary to shed light on such cases of incongruence.

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Finally, relationships among the Southern Hemisphere species have remained poorly resolved. Previous studies were based largely on ITS markers (Wagstaff and Garnock-Jones, 1998, 2000; Wagstaff et al., 2002; Albach et al., 2005b), because phylogenetic analyses of cpDNA markers lacked sufficient taxon sampling and/ or were unresolved due to the low number of informative sites (rbcL, Wagstaff et al., 2002; trnL-trnL-trnF, Albach et al., 2005b; Low, 2005). The ITS phylogeny with the best taxonomic sampling to date of the Southern Hemisphere species showed that although some of the segregate genera were monophyletic, few of the relationships among these groups received high bootstrap support (Wagstaff et al., 2002). In particular, the limits and evolutionary relationships of Chionohebe and Parahebe, which formed a grade along the backbone of the Southern Hemisphere clade, were not well resolved (Wagstaff et al., 2002). (Throughout this paper we refer to the main species groups within the Southern Hemisphere sect. Hebe by new informal names: semi-whipcord hebes, snow hebes, speedwell hebes, sun hebes, and hebes; see Table 4 and Section 4.) More DNA markers are needed to test the relationships found in the ITS phylogeny, and to understand more fully the phylogenetic history of Veronica in the Southern Hemisphere, especially in New Zealand. The main aim of this study is to reconstruct the phylogeny of Veronica using six DNA markers to address the subgeneric relationships, topological incongruence found in previous studies, and relationships among the main species groups in the Southern Hemisphere. To reconstruct the phylogeny of Veronica, we chose four cpDNA regions (trnL-trnL-trnF, rps16, rpoB-trnC, and psbAtrnH), the nuclear ribosomal ITS region, and the nuclear low-copy gene, CYCLOIDEA (more precisely CYCLOIDEA2; see below, in the following called CYC2). Of these six markers, four have been used previously in phylogenetic analyses of Veronica. For example, the ITS and trnL-trnL-trnF regions have been used extensively in many studies of Veronica (Wagstaff and Garnock-Jones, 1998, 2000; Wagstaff et al., 2002; Albach and Chase, 2001, 2004; Albach et al., 2004a,b, 2005b,c), whereas the rps16 intron (in the following called rps16) has only been used in the Northern Hemisphere species (Albach and Chase, 2004). In addition, the rpoB-trnC spacer region, ranked as one of the most variable plant markers of the plastid genome by Shaw et al. (2005), was found to be informative in an analysis of V. subg. Stenocarpon (von Sternburg, 2007; Albach et al., 2009) and was also used in a phylogeographic study of the Southern Hemisphere snow hebes (sensu Table 4; Meudt and Bayly, 2008). The psbA-trnH spacer is another highly variable cpDNA region (Sang et al., 1997; Shaw et al., 2005, 2007) that has been suggested as a potential DNA barcode marker in plants due to its high variability, short length, and consistent amplification across land plants (Kress et al., 2005; Chase et al., 2007; Edwards et al., 2008), but it has not yet been used for Veronica. We therefore chose psbA-trnH as a fourth cpDNA region to assess its variability and usefulness as a phylogenetic marker in Veronica. Finally, we wanted to compare the results of these ITS and cpDNA markers with a low-copy nuclear marker. A test of several nuclear markers (Albach, unpublished) revealed that the CYC2 gene is sufficiently variable to resolve relationships in the genus and at the same time can be confidently aligned across Veroniceae.

2. Materials and methods 2.1. Sampling and plant material A total of 78 individuals were included in this study representing 68 species (71 individuals) of Veronica, all 12 subgenera (sensu Albach et al., 2004c; Garnock-Jones et al., 2007) and all major clades within these subgenera. Seven individuals of six other gen-

era of Veroniceae (Lagotis, Paederota, Picrorhiza, Wulfeniopsis, Wulfenia, and Veronicastrum) were designated as outgroups. For all but eight terminals (Veronica abyssinica, V. campylopoda, V. catarractae, V. filiformis, V. javanica, V. planopetiolata, V. spicata, and V. triphyllos) the same DNA was used for all DNA regions (Table 1). Two individuals each of four species were sampled from different geographic localities (e.g., V. densifolia, New Zealand and Australia; V. salicifolia, New Zealand and Chile; and V. macrantha and V. thomsonii (incl. V. myosotoides) from different New Zealand locations). Due to the poor quality of herbarium extracted DNA, sequences of Veronica tubata could not be generated for the rps16, psbA-trnH spacer and rpoB-trnC spacer. ITS sequences of Veronica lanceolata and trnL-trnL-trnF-sequences of V. acuta, V. hulkeana, V. lavaudiana, and V. pentasepalae could not be sequenced cleanly in both directions and were not included in those data sets. Finally, the rpoB-trnC could not be reliably amplified in all species of Veronica and is lacking for 12 terminals. Sequences newly generated for this study include all 70 CYC2 sequences (from 60 individuals), all 77 psbA-trnH sequences, 21 ITS sequences, 25 trnL-trnL-trnF sequences, 50 rpoB-trnC sequences, and 54 rps16 sequences. The classification follows Albach et al. (2004c) and Garnock-Jones et al. (2007). Voucher specimens for all plants used in this study are listed in Table 1.

2.2. DNA extraction, amplification and DNA sequencing For 12 of the sampled individuals, DNA was extracted using DNeasy Plant Mini Kits (Qiagen GmbH, Hilden, Germany) following the manufacturer’s instructions. The remaining individuals sampled in this study had been extracted for previous studies. The trnL-trnL-trnF region was amplified with primers c and f of Taberlet et al. (1991), ITS with primers ITSA (Blattner, 1999) or ITS1a (Downie and Katz-Downie, 1996) and ITS4 (White et al., 1990), the rpoBtrnC spacer with primers rpoB and trnC-R (Shaw et al., 2005), the psbA-trnH spacer using primers psbA (Sang et al., 1997) and trnH (Tate and Simpson, 2003), the rps16 with primers rpsF and rpsR2 (Oxelman et al., 1997), and CYC2 with primers (DYTVTCCATCGGCATTGC) and (GATGAAYTTRTGCTGATCCAAAATG) (C.N. Wang, pers. comm.). The PCR program of 95 °C 2 min, 95 °C 1 min, 50– 55 °C 1 min, 72 °C 1.5–2 min, 36 to 2, 72 °C 5 min, 10 °C hold was used for all markers except CYC2 (94 °C 2 min, five times (94 °C 30 s, 55 °C 1 min, 72 °C 2 min), 35 times (94 °C 30 s, 60 °C 1 min, 72 °C 2 min), 72 °C 5 min, 10 °C hold). PCR products were cleaned using QIAquick PCR purification and gel extraction kit (Qiagen GmbH, Hilden, Germany) following the manufacturer’s protocols, or by adding 5 U exonuclease I (EXO) and 1 U shrimp alkaline phosphatase (SAP), and returning the tubes to the PCR machine for 37 °C 30 min, 80 °C 15 min, followed by 10 °C hold. PCR fragments of CYC2 that proved to be heterogeneous after direct sequencing were cloned into the pGEM T-easy vector and processed following the manufacturer’s instructions (Promega GmbH, Mannheim, Germany). Up to six plasmids containing cloned products were sequenced. Cloning of CYC2 was not attempted for the Southern Hemisphere species. Sequencing reactions (10 lL) were carried out using 1 lL of the Taq DyeDeoxy Terminator Cycle Sequencing mix (Applied Biosystems Inc., Foster City, CA, USA) and the same primers as for PCR and run out on automated sequencers. Both strands were sequenced. Sequences were assembled and edited using SequencherTM4.7.2 (Gene Codes Corp., Ann Arbor, MI, USA) or Geneious (Biomatters Ltd., New Zealand, www.geneious.com). CYC2 sequences retrieved after cloning, which were markedly shorter (>100 bp shorter) and considered as pseudogenes, were excluded from the analysis. Heterogeneous sites were coded as polymorphic. Assembled sequences were manually aligned prior to analysis.

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D.C. Albach, H.M. Meudt / Molecular Phylogenetics and Evolution 54 (2010) 457–471 Table 1 Voucher information and GenBank Accession Nos. for specimens used in this study. Sequences newly sequenced for this study begin with FJ848 and are in bold. Taxon name

ITS

trnL-trnLtrnF

rps16

psbAtrnH

rpoB-trnC

CYC2

Voucher

AF313028

AF486416

AY218805

FJ848085

FJ848162

n.a.

Miehe et al., 98-31710 GOET

AF509814 AF313024 AF509813 n.a.

AF486415 AF486408 AF486414 n.a.

FJ848212 AY218807 AY218806 n.a.

FJ848086 FJ848091 FJ848090 n.a.

FJ848163 FJ848168 FJ848167 n.a.

BG BONN 10240-30, BONN Albach 209, WU R. McBeath 2214, K Lancaster 763, K; Albach 123, K

AF313030 AF313025 FJ848064

AF486412 AF486409 AF486411

AY218802 AY218804 AY218808

FJ848087 FJ848088 FJ848089

FJ848164 FJ848165 FJ848166

n.a. FJ848270 FJ848269 FJ848266, FJ848267 n.a. FJ848268 n.a.

Chase s.n., K Albach 74, WU Dickoree 13014, GOET

Veronica subg. Beccabunga (Hill) M.M. Mart. Ort., Albach and M.A. Fisch. Veronica acinifolia L. AF509798 AF486399 FJ848215 V. beccabunga L. AF313015 AF486403 AY218820 V. reuterana Iboiss. AY540866 AY486447 FJ848216 V. serpyllifolia L. AF313017 AF486400 AY218821

FJ848098 FJ848099 FJ848100 FJ848101

FJ848175 FJ848176 n.a. FJ848177

FJ848282 n.a. FJ848283 FJ848284

M. Fischer s.n. (Lefkas), WU Albach 122, K Albach 676, WU Albach 64, WU

V. subg. Chamaedrys (W.D.J. Koch) Buchenau V. arvensis L. AF313002 V. chamaedrys L. AF313003

FJ848115 FJ848116

EU282078 FJ848187

FJ848295 FJ848296– FJ848297

Albach 147, WU Albach 121, K

n.a. FJ848181

n.a. n.a.

Outgroups Lagotis angustibracteata P.C. Tsoong and H.P. Yang Lagotis stolonifera (K. Koch) Maxim. Paederota lutea Scop. Picrorhiza kurrooa Royle ex Benth. Veronicastrum stenostachyum (Hemsl.) T. Yamaz. Veronicastrum virginicum Farw. Wulfenia carinthiaca Jacq. Wulfeniopsis amherstiana (Benth) D.Y. Hong

AF486380 AF486377

FJ848224 AY218814

V. subg. Cochlidiosperma (Rchb.) M.M. Mart. Ort, Albach, and M.A. Fisch. (plus V. javanica) V. crista-galli Steven AF509799 AF486367 AY218816 FJ848109 V. javanica Blume AY540867 AY540872 FJ848219 FJ848106

V. sibthorpioides Deb., Degen and Hervier V. triloba Opiz

AY850099

AY540876

FJ848221

FJ848110

FJ848184

FJ848289

Dolmkanov 17.4.1983, TBS Fries et al. 2016, BR (ITS, trnL-trnL-trnF); Garnock-Jones s.n., cult from seeds of PGJ 2621, WELT SP086369 (rps16, psbA-trnH, rpoB-trnC) Martínez-Ortega 831, SALA

AF509804

AF513333

AY218815

FJ848111

FJ848185

FJ848290

Albach 242, WU

V. subg. Pellidosperma (E.B.J. Lehm.) M.M. Mart. Ort., Albach and M.A. Fisch. V. glauca Sibth. and Sm. AF313006 AF486395 FJ848220 V. triphyllos L. FJ848065 FJ848039 AY218817

FJ848108 FJ848107

FJ848183 FJ848182

n.a. FJ848288

M. Fischer 9, 7.4.1999, WU Albach 832, WU (ITS, trnL-trnL-trnF, psbAtrnH, rpoB-trnC), Albach 244, WU (rps16)

V. subg. Pentasepalae (Benth.) M.M. Mart. Ort., Albach and M.A. Fisch. V. cuneifolia subsp. isaurica P.H. Davis AF486354 AF486372 AY218804

FJ848121

FJ848191

FJ848302

L. Struwe 1409, WU

V. subg. Pocilla (Dumort.) M.M. Mart. Ort., Albach and M.A. Fisch. V. campylopoda Boiss. AF486364 AY673624

AY218811

FJ848117

FJ848188

FJ848298

V. filiformis Sm.

AF486363

AF486368

FJ848225

FJ848118

FJ848189

FJ848299

V. intercedens Bornm. V. polita Fr.

AY673609 AF509818

AY673628 AF486369

FJ848226 FJ848227

FJ848119 FJ848120

n.a. FJ848190

FJ848300 FJ848301

Schönswetter and Tribsch 4152, WU (ITS, rps16, CYC2); Albach 656, WU (trnL-trnLtrnF, psbA-trnH, rpoB-trnC) Albach 298, WU (ITS, trnL-trnL-trnF); Albach 858, WU (rps16, psbA-trnH, rpoB-trnC) Albach 666, WU Albach 146, WU

AF511479/ AF511480 AF486407 AF486405

FJ848217

FJ848102

n.a.

n.a.

Albach s.n, WU

AY218818 FJ848218

FJ848103 FJ848104

FJ848178 FJ848179

FJ848285 FJ848286– FJ848287

Albach 66, BONN Albach 65, BONN (ITS, trnL-trnL-trnF); Andy Jones s.n. K 5.3 3 (rps16, psbA-trnH, rpoBtrnC)

FJ848229

FJ848123

n.a.

n.a.

Wagstaff 94.105, CHR 512486

FJ848066

AY540890, AY540903 FJ848040

FJ848228

FJ848122

FJ848192

n.a.

NSW 502261

AY540870

AY540874

n.a.

n.a.

n.a.

n.a.

M.M.J. v. Balgooy 566, K

FJ848075

n.a.

FJ848238

FJ848132

FJ848194

FJ848312

Garnock-Jones 2593, WELTU 20205

AF229043

n.a.

FJ848239

FJ848133

n.a.

n.a.

Molloy 8.9.82, CHR 512487

V. subg. Pseudolysimachium (Opiz) Buchenau V. daurica Steven AF313023 V. longifolia L. V. spicata L.

V. subg. Pseudoveronica J.B. Armstr. V. sect. Labiatoides V. nivea Lindl. (Derwentia nivea (Lindl.) B.G. Briggs and Ehrend.) V. perfoliata R. Br. (Derwentia perfoliata (R. Br.) B.G. Briggs and Ehrend.) V. sect. Hebe Heath hebe V. tubata Diels (Albach) (Detzneria tubata Diels) Sun hebes V. hulkeana Muell. (Heliohebe hulkeana (F. Muell.) Garn.-Jones) V. lavaudiana Raoul (Heliohebe lavaudiana (Raoul) Garn.-Jones)

AF313021 AF313022

AF037382

(continued on next page)

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Table 1 (continued) Taxon name V. pentasepala (L. B. Moore) Garn.Jones (Heliohebe pentasepala (L.B. Moore) Garn.-Jones) V. raoulii Hook. f. (Heliohebe raoulii (Hook. f.) Garn.-Jones) V. scrupea Garn.-Jones (Heliohebe acuta Garn.-Jones) Semi-whipcord hebes V. cupressoides Hook. f. (Leonohebe cupressoides (Hook. f.) Heads) V. hookeri (Buchanan) Garn.-Jones (Leonohebe ciliolata (Hook. f.) Heads) V. quadrifaria T. Kirk (Leonohebe cheesemanii (Buchanan) Heads) V. tetrasticha Hook. f. (Leonohebe tetrasticha (Hook. f.) Heads) Speedwell hebes V. catarractae G. Forst. (Parahebe catarractae (G. Forst.) W.R.B. Oliver) V. cheesemanii Benth. (Parahebe cheesemanii (Benth.) W.R.B. Oliv.) V. decora (Ashwin) Garn.-Jones (Parahebe decora Ashwin) V. hookeriana Walp. (Parahebe hookeriana (Walp.) W.R.B. Oliver) V. lanceolata Benth. (Parahebe lanceolata (Benth.) Garn.-Jones) V. lilliputiana Stearn (Parahebe canescens (A. Wall) W.R.B. Oliver) V. linifolia Hook. f. (Parahebe linifolia (Hook. f.) W.R.B. Oliver) V. lyallii Hook. f. (Parahebe lyallii (Hook. f.) W.R.B. Oliver) V. melanocaulon Garn.-Jones (Parahebe martinii (Garn.-Jones) Garn.-Jones) V. senex (Garn.-Jones) Garn.-Jones (Parahebe senex Garn.-Jones) V. spathulata Benth. (Parahebe spathulata (Benth.) W.R.B. Oliv.) V. vandewateri Wernham (Parahebe vandewateri (Wernham) P. Royen) Snow hebes V. chionohebe Garn.-Jones (Chionohebe glabra (Cheeseman) Heads) V. ciliolata (Hook. f.) Garn.-Jones (Chionohebe ciliolata (Hook. f.) B.G. Briggs and Ehrend.) V. densifolia (F. Muell.) F. Muell. (Chionohebe densifolia (F. Muell.) B.G. Briggs and Ehrend.) V. densifolia (F. Muell.) F. Muell. (Chionohebe densifolia (F. Muell.) B.G. Briggs and Ehrend.) V. planopetiolata G. Simpson and J.S. Thomson (Parahebe planopetiolata (G. Simpson and J.S. Thomson) W.R.B. Oliver) V. pulvinaris (Hook. f.) Cheeseman (Chionohebe pulvinaris (Hook. f.) B.G. Briggs and Ehrend.) V. spectabilis (Garn.-Jones) Garn.Jones (Parahebe spectabilis Garn.Jones) V. thomsonii (Buchanan) Cheeseman (Chionohebe thomsonii (Buchanan) B.G. Briggs and Ehrend.) V. thomsonii (Buchanan) Cheeseman (Chionohebe thomsonii (Buchanan) B.G. Briggs and Ehrend.)

ITS

trnL-trnLtrnF

rps16

psbAtrnH

rpoB-trnC

CYC2

Voucher

FJ848076

n.a.

FJ848240

FJ848134

FJ848195

FJ848313

Garnock-Jones 2589, WELTU

AF037380

AY540885, AY540899 n.a.

FJ848241

FJ848135

FJ848196

FJ848314

Decourtye 22.2.86, CHR 512459

FJ848237

FJ848131

FJ848193

FJ848311

Garnock-Jones 2592, WELTU 20206

AF037378

AY540880, AY540894

FJ848251

FJ848145

FJ848202

FJ848322

Buxton 15.12.1989, CHR 512449

FJ848077

FJ848050

FJ848250

FJ848144

EU349506

n.a.

Bayly 1733, WELT SP083954

AF037377

AY540886, AY540900 FJ848051

FJ848249

FJ848143

FJ848201

FJ848321

FJ848252

FJ848146

FJ848203

FJ848323

Heenan and Garnock-Jones 1999, CHR 512472 Druce 20.10,1989, CHR 512451

AY034859

AY540887, AY540901

FJ848253

FJ848148

FJ848204

FJ848324

FJ848078

FJ848053

FJ848254

FJ848149

EU349510

n.a.

Garnock-Jones 2403, CHR (ITS, trnL-trnLtrnF); Maich s.n., WELT SP086368 (rps16, psbA-trnH, rpoB-trnC, CYC2) Garnock-Jones 2587, WELTU

AF229047

AY540877

FJ848255

FJ848150

FJ848205

FJ848325

Glenny and Wagstaff 95.08, CHR 512467

FJ848079

FJ848054

FJ848256

FJ848151

EU349511

FJ848326

Thorsen s.n., WELT SP086389

n.a.

FJ848055

FJ848257

FJ848152

FJ848206

FJ848327

Garnock-Jones 2632, WELT SP086371

AF037394

FJ848052

AY21813

FJ848147

n.a.

n.a.

Garnock-Jones Aug 1990, CHR 512446

FJ848080

FJ848056

FJ848258

FJ848153

FJ848207

FJ848328

Molloy and Druce, 22.02.1991, CHR 470347

FJ848081

FJ848057

FJ848259

FJ848154

EU349516

FJ848329

Bayly 585, WELT SP080859/A

AF037396

FJ848058

FJ848260

FJ848155

EU349509

FJ848330

Williams 15.04.1976, CHR 512456

FJ848082

FJ848060

FJ848262

FJ848157

FJ848208

FJ848332

Garnock-Jones 2360, WELT SP086370

AF229051

FJ848061

FJ848263

FJ848158

EU349517

FJ848333

Garnock-Jones 2263, WELTU 16860

FJ848084

AF486381

AY21813

FJ848161

FJ848211

n.a.

Barker 59, K

FJ848070

FJ848044

FJ848233

FJ848127

EU349550

FJ848307

Bayly 1843, WELT SP084042

FJ848067

FJ848041

FJ848230

FJ848124

EU349522

FJ848304

Bayly 1812, WELT SP084019

FJ848068

FJ848042

FJ848231

FJ848125

EU349533

FJ848305

Bayly 1779, WELT SP083990 (Australia)

FJ848069

FJ848043

FJ848232

FJ848126

EU349541

FJ848306

Bayly 1855, WELT SP084057 (New Zealand)

AF229050

FJ848059

FJ848261

FJ848156

EU349513

FJ848331

Simpson s.n., CHR 512626 (ITS); GarnockJones 2606a, WELTU 20207 (trnL-trnL-trnF, rps16, psbA-trnH, rpoB-trnC, CYC2)

FJ848072

FJ848046

FJ848235

FJ848129

EU349565

FJ848309

Glenny 9093, WELT SP084074

AF229044

FJ848062

FJ848264

FJ848159

FJ848209

FJ848334

Garnock-Jones and Malcolm 2039, CHR 470104

FJ848073

FJ848047

FJ848236

FJ848130

EU349570

FJ848310

Bayly 1651, WELT SP083901 (V. thomsonii 1)

FJ848071

FJ848045

FJ848234

FJ848128

EU349554

FJ848308

Bayly 1818, WELT SP084022 (V. thomsonii 2)

FJ848074

AY034866, AY034867

D.C. Albach, H.M. Meudt / Molecular Phylogenetics and Evolution 54 (2010) 457–471

461

Table 1 (continued) Taxon name V. trifida Petrie (Parahebe trifida (Petrie) W.R.B. Oliv.)

ITS

trnL-trnLtrnF

rps16

psbAtrnH

rpoB-trnC

CYC2

Voucher

FJ848083

FJ848063

FJ848265

FJ848160

FJ848210

FJ848335

Bayly 1841, WELT SP084041

AF037393

AY540883, AY540897 AY540878, AY540892

FJ848242

FJ848138

n.a.

n.a.

Heenan 12.9.89, CHR 512484

FJ848243

FJ848136

n.a.

FJ848315

Heenan 17.11.89, CHR 512444 (V. macrantha 1)

AY 034853

FJ848048

FJ848244

FJ848137

FJ848197

FJ848316

Glenny and Wagstaff 95.09, CHR 512468 (V. macrantha 2)

AF037388

AY540882, AY540896 AY540881, AY540895 FJ848049

FJ848245

FJ848139

n.a.

FJ848317

Garnock-Jones 4.12.90, CHR 512441

FJ848247

FJ848141

FJ848199

FJ848319

Wagstaff 95.10, CHR 512466 (New Zealand)

FJ848248

FJ848142

FJ848200

FJ848320

Buxton 1992, WELTU 16898 (South America)

FJ848246

FJ848140

FJ848198

FJ848318

Hair 26.10.6, CHR 512475

FJ848113 FJ848114

EU282076 EU282077

FJ848293 FJ848294

Albach 71, BONN Martínez-Ortega 713, SALA

AY218819

FJ848105

FJ848180

n.a.

Albach 124, K

n.a.

n.a.

n.a.

FJ848303

Albach 120, K

FJ848222

FJ848112

FJ848186

FJ848291– FJ848292

UA 174, SALA

Fischer 728/98, BONN (ITS), E.Fischer 8060, BONN (others) Albach 184, WU

hebes V. elliptica G. Forst. (Hebe elliptica (G. Forst.) Pennell) V. macrantha Hook. f. (Hebe macrantha (Hook. f.) Cockayne and Allan) V. macrantha Hook. f. (Hebe macrantha (Hook. f.) Cockayne and Allan) V. odora Hook. f. (Hebe odora (Hook. f.) Cockayne) V. salicifolia Forst. (Hebe salicifolia (G. Forst.) Pennell) V. salicifolia Forst. (Hebe salicifolia (G. Forst.) Pennell) V. salicornioides Hook. f. (Hebe salicornioides (Hook. f.) Cockayne and Allan)

AF037391

AF037385 AF037386 AF069465

AY540879, AY540893

V. subgen. Stenocarpon (Boriss.) M.M. Mart. Ort., Albach and M.A. Fisch. V. fruticulosa L. AF313004 AF486383 AY218812 V. mampodrensis Losa and P. Monts. DQ227331 DQ227337 FJ848223 V. subgen. Synthyris (Benth.) M. Mart. Ort., Albach, and M.A. Fisch. V. missurica subsp. major (Hook.) M.M. AF313019 AF486397 Mart. Ort. and Albach V. missurica subsp. stellata (Pennell) n.a. n.a. M.M. Mart. Ort. and Albach V. subgen. Triangulicapsula M.M. Mart. Ort., Albach and M.A. Fisch. V. chamaepithyoides Lam. AF509796 AF511477/ AF511478 V. subgen. Veronica L. V. abyssinica Fresen.

AF313009

AF513350

FJ848213

FJ848092

FJ848169

FJ848271

V. alpina L.

AF313013

AF486387

FJ848214

FJ848093

FJ848170

V. glandulosa Hochst. ex Benth.

AF313008

AF486394

AY218822

FJ848094

FJ848171

V. montana L.

AF313014

AF486388

AY218824

FJ848095

FJ848172

V. officinalis L. V. scutellata L.

AF313012 AF509805

AF486391 AF486393

n.a. AY218823

FJ848096 FJ848097

FJ848173 FJ848174

FJ848272, FJ848273 FJ848274– FJ848278 FJ848279– FJ848280 FJ848281 n.a.

2.3. Sequence analysis All data sets were analyzed separately (trnL-trnL-trnF, rps16, rpoB-trnC, psbA-trnH, ITS, and CYC2) and combined (combined cpDNA, combined cpDNA + ITS, hereafter cpDNA and combined data sets, respectively) using parsimony, maximum likelihood and Bayesian inference. The CYC2 data set was not combined with the other data sets due to the presence of multiple alleles for a single terminal and questionable orthology. Indels were coded for the parsimony analysis with SeqState v. 1.32 (Müller, 2005) using the modified complex indel coding method (Müller, 2006), which was shown to be the best indel coding method available (Simmons et al., 2007). Parsimony bootstrap analyses were conducted with and without gaps in PAUP* 4.0b10 (Swofford, 2002) using a heuristic search strategy with 1000 replicates, 10 runs of random taxon addition, TBR branch swapping and with a maxtree limit of 20 trees per replicate. Maximum likelihood and Bayesian analyses were conducted using a model estimated with Modeltest 3.06 (Posada and Crandall, 1998) based on the AIC. Likelihood analyses were conducted using GARLI (Zwickl, 2006) based on results by Morrison (2007) that GARLI is the most efficient likelihood program for data sets with few taxa and many sites. Five runs were conducted using the default conditions and model specifications inferred by Modeltest.

Fischer 713/98, WU Albach 151, WU Albach and Chase 114, K C. Dobes 7026, WU

Bayesian analyses were conducted for all data sets using MrBayes version 3.1 (Huelsenbeck and Ronquist, 2001) with the GTR model (all except CYC2) or the HKY model (CYC2) and uninformative priors. All analyses were run twice for 3 million generations. Four parallel Markov chain Monte Carlo chains were used for each replicate run, and one tree for every 100 generations was saved. For cpDNA and combined data set runs, separate models were allowed for each DNA region. After graphing all free parameters (except topology and branch lengths), we verified that they had reached stationarity around the 50,000th generation for all analyses, which were subsequently discarded as burn-in. For the rpoBtrnC spacer stationarity was not reached until after 200,000 generations and subsequently 250,000 generations were discarded as burn-in. A 50% majority rule consensus tree of the remaining saved trees was reconstructed in PAUP* version 4b10 for each run. We investigated eight relationships detected in one but not the other data set by constraining maximum likelihood analyses in GARLI to these relationships and running the analyses three (cpDNA), five (ITS), or eight (CYC2) times. The specific relationships are (1) V. chamaepithyoides as sister to V. subg. Pseudoveronica, (2) those two sister to V. subg. Stenocarpon, (3) V. subg. Chamaedrys sister to V. subg. Pocilla plus V. subg. Pentasepalae, (4) V. subg. Pocilla plus V. subg. Pentasepalae sister to V. subg. Pseudoveronica, (5) monophyly of V. subg. Cochlidiosperma, (6) V. javanica sister to a

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Table 2 Information on the DNA markers and data sets used in this study. Data set

ITS

rpoB-trnC

psbA-trnH

rps16

trnL-trnL-trnF

cpDNA

Combined

CYC2

No. individuals No. aligned characters No. coded gap characters No. variable sites No. informative sites % divergence Ti/Tv %GC No. steps in optimal tree CI/RI of optimal tree

77 722 53 403 (48.8%) 283 (35.0%) 10.4% 1.7 57.1% 1543 0.46/0.73

67 1682 54 569 (30.7%) 276 (15.3%) 4.9% 0.8 30.9% 896 0.61/0.84

77 673 21 278 (38.3%) 178 (24.4%) 8.4% 0.7 30.7% 672 0.48/0.76

76 1126 48 366 (28.2%) 202 (15.4%) 3.3% 1.2 34.4% 554 0.58/0.81

74 1169 28 381 (30.2%) 221 (17.3%) 3.8% 1.1 35.2% 600 0.64/0.86

78 4650 151 1594 (31.1%) 877 (17.1%) 4.5% 0.8 32.5% 2770 0.56/0.80

78 5372 204 1637 (33.4%) 1160 (19.5%) 5.7% 1.1 36.7% 4428 0.46/0.73

60 (70 sequences) 787 17 294 (37.3%) 202 (25.7%) 6.8% 1.4 42.3% 721 0.51/0.75

monophyletic V. subg. Cochlidiosperma, (7) V. subg. Synthyris sister to V. subg. Beccabunga, (8) monophyly of V. sect. Hebe. Significance of the results was tested in PAUP* 4.0b10 (Swofford, 2002) using the Shimodaira–Hasegawa-test with full optimization and 1000 bootstrap replicates. 3. Results 3.1. Molecular data sets Information on the six individual data sets (ITS, rpoB-trnC, psbAtrnH, rps16, trnL-trnL-trnF, and CYC2), and cpDNA and combined data sets, is summarized in Table 2. Whereas ITS, rps16, and trnLtrnL-trnF have all been used and characterized in previous Veronica studies (see above), psbA-trnH and CYC2 are novel markers. These latter two markers are thus further discussed here, along with rpoB-trnC, which has been used previously but not characterized (Meudt and Bayly, 2008; Albach et al., 2009). Of the four cpDNA markers we used here, the psbA-trnH region is the shortest (673 base pairs, bp, aligned) and most variable (38.3% variable sites), showing 8.4% divergence among species of Veronica (Table 2). Despite its high variability, psbA-trnH is known to contain several motifs that are conserved across angiosperms (Štorchová and Olson, 2007), two of which are also found in our data set. For example, the short TTAGTGTATA-motif in the 30 UTR is present in all Veroniceae studied except Veronicastrum virginicum, which has a 5 bp deletion in this region. A second example, a stem-loop motif, GGAGCAAT, is clearly recognizable in the alignment and with a few exceptions conserved in Veroniceae with insertions frequent just before this region. Individuals of the x = 8 clade, comprising the six subgenera Pseudoveronica, Pocilla, Pentasepalae, Triangulicapsula, Stenocarpon, and Chamaedrys and mostly sharing a chromosome base number of x = 8, differ in this region by a single substitution at the end of the motif (T ? C) with the respective compensatory change in the corresponding second strand of the stem. The stem is 2–5 bp longer in this clade than in other species of Veronica and in sect. Hebe may be even longer due to a stretch of A and T to a loop of potentially just 2 bp. The length of the rpoB-trnC spacer region is highly variable among sampled Veronica species, ranging from 938 bp to 1243 bp. This nearly equals the range found across angiosperms reported by Shaw et al. (2005). These authors reported some large gaps in the central part of the region of up to 81 bp, which is small compared with those in the sun hebe group (sensu Table 4; 300 bp) and V. vandewateri (116 bp). We were unable to sequence 10 individuals for this marker, due to inconsistent, non-universal PCR amplification, which has been noted previously (Mort et al., 2007). The region nevertheless proved to be more variable compared to the other three cpDNA regions, which is in contrast to comparisons in other studies (e.g., Goodson et al., 2006). The characteristics of the CYC2 gene (Table 2) in Veroniceae are comparable to those published for other groups (e.g., Smith et al.,

2004; Wang et al., 2004). All sequences correspond to CYC2 sensu Preston et al. (2009) and thus suggest that our primers are specific for this copy type. Several pseudogenes, which contain large deletions and high sequence divergence, were sequenced in the present study and were subsequently removed from the data set prior to analysis. All remaining sequences contained indels of 3 bp or multiples thereof with the exception of a 1 bp deletion in V. serpyllifolia, a 68 bp deletion in V. acinifolia and a 4 bp deletion in V. polita. All these deletions were autapomorphic changes not influencing the analyses and likely represent sequencing errors of non-proofreading Taq polymerase. 3.2. Phylogenetic analyses In general the gap characters were consistent with the nucleotide substitution data. For all regions, parsimony analyses without gap characters had the same topologies as the corresponding analyses with gaps coded as additional characters, or the resulting trees had minor differences among some relationships which nevertheless lacked significant support in either analysis. Bootstrap support values for relationships were generally identical or lower in the analyses without gap characters, and therefore only those with gaps are reported. Likewise, analyses of the four separate cpDNA data sets did not differ from the combined cpDNA analysis but were generally less resolved and less supported. Therefore, only the combined cpDNA analysis is reported. The most parsimonious trees had very similar topologies to the Bayesian and maximum likelihood trees, which suggests that our results are robust to the different assumptions associated with these approaches. Therefore, only the Bayesian 50% majority rule trees are shown with posterior probability values (PP) and parsimony bootstrap percentages (BS) (Figs. 1–3). In the ITS phylogeny (Fig. 1), Veronica is monophyletic but with PP and BS < 50%. Paederota is sister to Veronica with high support (97% BS, 100% PP). Of the nine subgenera with more than one species sampled, eight are monophyletic with high support in one or both analyses, whereas subgenus Cochlidiosperma is polyphyletic (Table 3). The most recognizable result of our ITS phylogeny is the presence of a clade comprising subgenera Pseudoveronica, Triangulicapsula and Stenocarpon (which received high support in our study, 74% BS, 98% PP), and another clade comprising subgenera Pocilla, Pentasepalae and Chamaedrys (83% BS, 100% PP). This result is consistent with almost all previous ITS analyses with broad but differing sampling across the genus (Albach and Chase, 2001, 2004; Albach et al., 2004a,b,c, 2005b,c; Wagstaff et al., 2002). In addition, subg. Pellidosperma is closely related to this latter clade. Subgenus Triangulicapsula is highly supported (83% BS, 100% PP) as sister to the Southern Hemisphere subgenus Pseudoveronica. As in previous ITS studies, relationships among other subgenera or groups of subgenera within Veronica are not highly supported. Within Veronica subg. Pseudoveronica, sect. Labiatoides is sister (98% BS, 99% PP) to sect. Hebe in the ITS analysis (100% PP including

D.C. Albach, H.M. Meudt / Molecular Phylogenetics and Evolution 54 (2010) 457–471

463

Fig. 1. ITS phylogeny of Veronica. Bayesian 50% majority rule tree showing bootstrap percentage values (BP) from parsimony analysis and posterior probability values (PP) from Bayesian analysis above/below branches, respectively. Subgeneric affiliation and informal names for species groups within subg. Pseudoveronica sect. Hebe are shown. Nodes with 100% support are indicated with an asterisk.

the monotypic sect. Detzneria which is nested within it; Fig. 1). Within sect. Hebe, five main species groups are resolved that correspond largely to previously recognized genera. Branches leading to these informal species groups, which we refer to by new informal

names (semi-whipcord hebes, snow hebes, speedwell hebes, sun hebes, and hebes; see Table 4 and Section 4), are very short with low support values. In addition, species that fall outside of the main species groups in unresolved places in the ITS tree include

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D.C. Albach, H.M. Meudt / Molecular Phylogenetics and Evolution 54 (2010) 457–471

Table 3 Subgeneric sampling and support values in this study. Subgenus

Total no. spp.

No. sampled spp.

ITS (BS/PP)

cpDNA (BS/PP)

Combined (BS/PP)

Beccabunga Chamaedrys Cochlidiosperma Pellidosperma Pentasepalae Pocilla Pseudolysimachium Pseudoveronica Stenocarpon Synthyris Triangulicapsula Veronica

30–35 10–15 12 7 ca. 70 28–30 ca. 30 155–180 30–35 18 2 40–45

4 2 4 2 1 4 3 35 2 1 1 6

100/100 99/100 Not monophyletic —/100 n/a 98/100 100/100 98/99 —/96 n/a n/a 100/100

100/100 100/100 100/100 100/100 n/a 100/100 100/100 96/100 —/— n/a n/a 100/100

100/100 100/100 100/100 100/100 n/a 100/100 100/100 —/100 —/— n/a n/a 100/100

Totals

435–470

65

n/a

n/a

n/a

V. cheesemanii, V. cupressoides, V. linifolia, V. macrantha, V. senex, and V. vandewateri. In the cpDNA phylogeny (Fig. 2), there is high support in both parsimony and Bayesian analyses for a monophyletic Veronica (100% BS, 100% PP), as well as for eight subgenera—only subg. Stenocarpon is not highly supported (Table 3). Paederota is sister to Veronica with high support (100% BS, 100% PP), subg. Veronica is highly supported as sister to the rest of the genus, and subg. Beccabunga and Synthyris are highly supported as sister groups. In contrast to the ITS phylogeny, there is high support for a clade of subg. Pseudoveronica, Pentasepalae, Pocilla, and Triangulicapsula, as well as the x = 8 clade (subg. Pseudoveronica, Pocilla, Pentasepalae, Triangulicapsula, Stenocarpon and Chamaedrys) and the endospermpodium clade (named after the particular endosperm structure in its seeds; x = 8 clade plus Pellidosperma and Cochlidiosperma). These results agree with previous analyses of cpDNA based on the trnLtrnL-trnF region (Albach et al., 2004a,b, 2005b,c; Albach and Greilhuber, 2004) or trnL-trnL-trnF and the rps16 (Albach and Chase, 2004). Within subg. Pseudoveronica, sect. Labiatoides is polyphyletic with one species sister (96% BS, 100% PP) to sect. Hebe (100% PP), and the other sister to sect. Detzneria and nested within sect. Hebe in the cpDNA tree (Fig. 2). The five informal species groups are all monophyletic (Table 4), but as with the ITS analyses, relationships among these species groups are not highly supported. In contrast with the ITS analyses, V. macrantha and V. cupressoides are placed with the semi-whipcords (sensu Table 4; 73% BS, 100% PP), the hebes (sensu Table 4) are not monophyletic, and V. linifolia, V. senex, V. vandewateri and V. cheesemanii emerge together with the speedwell hebes (sensu Table 4). In addition, of the four species from subg. Pseudoveronica sampled twice in our analyses (V. densifolia, V. macrantha, V. salicifolia, V. thomsonii), all are monophyletic in the ITS tree, but only V. macrantha is monophyletic in the cpDNA tree. The combined (ITS + cpDNA) phylogeny (Fig. 3) gives nearly identical results to those noted above for the cpDNA phylogeny (Fig. 2), but the sister group to subg. Pseudoveronica is subg. Triangulicapsula (92% BS, 98% PP) and the verrucate clade, named after the verrucate seed surface of its members, is monophyletic (x = 8 clade plus Pellidosperma; 86% BS, 88% PP), both of which are congruent with the ITS topology (Fig. 1). Also, within subg. Pseudoveronica, sect. Labiatoides is sister to sect. Hebe, and all five informal species groups are highly supported (Table 4). As with the ITS analyses, the placement of V. macrantha, V. linifolia, V. planopetiolata, and V. tubata fall outside these main groups in unresolved positions. Constraining V. chamaepithyoides as sister to V. subg. Hebe in the cpDNA analysis did not provide a significant rejection in the Shimodaira–Hasegawa-test (p = 0.504). However, a position of V.

subg. Stenocarpon as sister to those two and V. subg. Chamaedrys as sister to V. subg. Pocilla plus V. subg. Pentasepalae was rejected (p = 0.045 and p = 0.022, respectively). The analysis of 70 CYC2 sequences from 60 individuals gave results that are similar to that of the other analyses but with notable differences (Fig. 4). For example, monophyly of Veronica (with the exception of V. stellata, see below) and the sister group relationship with Paederota were again recovered, the latter strongly supported (100% BS, 100% PP). By contrast, monophyly of several subgenera in Veronica was not supported, among them V. subg. Pseudoveronica, V. subg. Chamaedrys, V. subg. Pseudolysimachium and V. subg. Veronica. Constraining the analysis to produce a monophyletic section Hebe resulted in a significantly worse result (p = 0.013). Importantly, the analysis of CYC2 also retrieved a monophyletic endosperm-podium clade, although the x = 8 and verrucate clades are not monophyletic due to the inclusion of V. subg. Cochlidiosperma. 4. Discussion 4.1. Phylogenetic relationships among Veronica subgenera The individual and combined analyses of six DNA markers from nuclear ribosomal, chloroplast, and low-copy nuclear DNA presented here provide us with what is currently the best estimate of the phylogeny of Veronica. In addition, comparison of the individual and cpDNA analyses (Figs. 1 and 2) with the combined analysis (Fig. 3) and previous analyses (Albach and Chase, 2001, 2004; Albach et al., 2004a,b, 2005b,c; Wagstaff et al., 2002) highlights those subgeneric relationships that are highly supported and others that are still poorly resolved. In contrast with previous ITS analyses (e.g., Albach and Chase, 2001, 2004; Albach et al., 2004a), all our analyses except CYC2 (Fig. 4; see below) support the monophyly of Veronica, including our ITS analysis (albeit with weak support, Fig. 1) and cpDNA and combined analyses (maximum support, Figs. 2 and 3). Among the ITS and cpDNA tree topologies reported here, there is broad agreement with respect to the circumscription of the 12 major clades within Veronica (subgenera sensu Albach et al., 2004c), although there is little congruence in exact relationships among them. For example, there is strong support for subgenus Veronica as the sister to the rest of the genus in the cpDNA and combined trees (Figs. 2 and 3), which is consistent with most previous cpDNA phylogenetic studies (Albach et al., 2004a, 2005b). The fact that this early-branching position of subg. Veronica receives stronger support in the combined analysis than the individual ITS and cpDNA analyses demonstrates that there is some support for such a relationship in the ITS data set, as was first revealed by a previous ITS analysis that excluded C–T transitions (Albach et al., 2004a). Other subgeneric relationships found in most

465

D.C. Albach, H.M. Meudt / Molecular Phylogenetics and Evolution 54 (2010) 457–471 Table 4 Formal and informal groups within the Southern Hemisphere Veronica subg. Pseudoveronica. Formal sectional name

Proposed informal name (this study)

Previous segregate generic name(s)

No. spp.

Veronica sect. Labiatoides

n/a

Derwentia

ca. 20-25

V. sect. Hebe

n/a

n/a

ca. 130

Heath hebes

Detzneria

1

1

Sun hebes

Heliohebe

5

5

Semi-whipcord hebes

Leonohebe

4

4

Speedwell hebes

Parahebe (in part—groups A, C, and non-alpine B)

ca. 24

Snow hebes

Chionohebe + Parahebe (in part—group B alpine spp.)

10

8

Hebes

Hebe

88

5

No. sampled Notes spp. 2

33

11

A natural group (comprising V. perfoliata, V. nivea, and ca. 20 other Australian spp.*) that is sister to sect. Hebe A large monophyletic section that includes all species from the six informal groups outlined below Comprising the sole species V. tubata, it is consistently nested within sect. Hebe and therefore formal sectional recognition is not warranted A natural group (comprising V. hulkeana, V. lavaudiana, V. pentasepala, V. raoulii, and V. scrupea) that is monophyletic with high molecular and morphological support A natural group, with V. cupressoides sister to the remaining species V. hookeri, V. quadrifaria, and V. tatresticha A natural group, sometimes with high support, comprising V. brevistylis*, V. catarractae, V. cheesemanii?, V. decora, V. hookeriana, V. lanceolata, V. linifolia?, V. lyalli, V. lilliputiana, V. melanocaulon, V. senex, and V. spathulata (New Zealand), plus V. vandewateri and potentially 11 other New Guinea spp.* not yet sampled A natural group, but relationships within this group are complex; the cushions (V. chionohebe, V. ciliolata, V. pulvinaris, V. thomsonii) are likely monophyletic within this group that also contains V. birleyi*, V. laxa*, V. planopetiolata, V. spectabilis, V. trifida, and V. densifolia The largest species group, shown to be monophyletic (potentially excluding V. macrantha and V. petriei) in previous studies but only poorly sampled here

References

Albach and Briggs (unpublished) Wagstaff et al. (2002)

Garnock-Jones (1993b)

Bayly and Kellow (2006)

Garnock-Jones and Lloyd (2004), Albach et al. (2005b), van Royen (1972)

Meudt (2008), Heads (2003), Garnock-Jones and Lloyd (2004)

Bayly and Kellow (2006)

Asterisks designate species that have not yet been sampled in molecular phylogenetic studies.

analyses include the sister relationship of subgenera Pentasepalae and Pocilla and the monophyly of the x = 8 clade. Not surprisingly, these clades get maximum support in the combined analysis (Fig. 3). Relationships among subgenera Beccabunga, Pseudolysimachium, Synthyris and the endosperm-podium clade differed in the two analyses, therefore there is little support in the combined analysis except for the sister group relationship of subgenera Beccabunga and Synthyris (95% BS, 100% PP; Fig. 3), a relationship not found in the ITS analysis but also not rejected by an incongruence test. Such a relationship is found with similar support in the cpDNA analysis (Fig. 2) and has been found in one previous cpDNA analysis (Albach and Chase, 2004). The endosperm-podium clade, which includes practically all species with an endosperm podium (RiekHäußermann, 1943) in the genus, receives maximum support in the combined, cpDNA and CYC2 analyses (Figs. 2–4) consistent with previous cpDNA analyses. This clade has been found in ITS analyses before (Albach and Chase, 2001), although in most studies either part of subgenus Cochlidiosperma clustered with the other subgenera or the phylogeny was unresolved with respect to this clade. Given the structural synapomorphy and the high support in the cpDNA analysis, we have little reason to doubt the naturalness of the endosperm-podium clade. Subgenus Cochlidiosperma is the least supported subgenus in Veronica. In the combined analysis it gets maximum support including V. javanica, a position not supported by many morphological characters which rather suggest other affiliations. Despite being annual, V. javanica has a habit which resembles that of V. chamaedrys, with which it also shares the colliculate seed surface (Muñoz Centeno et al., 2006). Additionally, the distinguishing feature of the subgenus, the cymbi- to

cyathiform seeds, are lacking in V. javanica. The subgenus including V. javanica always receives strong support from cpDNA analyses but never in the ITS analyses, unless C–T transitions are excluded (Albach et al., 2004a), and is consequently also not rejected by an incongruence test. The analysis by Albach et al. (2004a) did not include V. javanica, but re-analyzing that data set with V. javanica leads to an association of V. javanica with V. crista-galli if C–T transitions are downweighted (and only then) but not to the monophyly of subgenus Cochlidiosperma (Albach, unpublished). The lack of significant incongruence indicates that the addition of more data is likely to stabilize the position of V. javanica as sister to V. subgen. Cochlidiosperma. The verrucate clade receives moderate support (86% BS, 88% PP) in the combined analysis (Fig. 3). It includes all species of the genus with a verrucate seed surface (Muñoz Centeno et al., 2006). The clade is also found in the ITS analyses (Fig. 1) and some previous ITS analyses (Albach et al., 2004a; Albach and Chase, 2004) and cpDNA analyses (Albach et al., 2005b,c). The case is therefore similar to that of the endosperm-podium clade, although several ITS and cpDNA analyses and structural synapomorphies support a sister group relationship of subgenera Cochlidiosperma and Pellidosperma. The x = 8 clade is one of the best divisions within the genus. It receives maximum support in the combined and cpDNA analysis (Figs. 2 and 3), was found consistently and strongly supported in previous cpDNA analyses, and was also found in a previous ITS analysis that excluded C–T transitions (Albach et al., 2004a). It will be important to increase taxon sampling in V. subgenus Cochlidiosperma and related subgenera for CYC2 to investigate its position using this marker.

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Fig. 2. cpDNA phylogeny of Veronica. Bayesian 50% majority rule tree showing bootstrap percentage values (BP) from parsimony analysis and posterior probability values (PP) from Bayesian analysis above/below branches, respectively. Subgeneric affiliation and informal names for species groups within subg. Pseudoveronica sect. Hebe are shown. Nodes with 100% support are indicated with an asterisk.

Within the x = 8 clade, the branching order in the combined analysis resembles that of the cpDNA analysis with the exception of subgenus Triangulicapsula being sister to subgenus Pseudoveronica. The greater affinity of this subgenus to subgenus Pseudoveron-

ica in ITS analyses has been noted in previous analyses of this marker. Remarkably, support for this sister group relationship is higher in the combined parsimony analysis (92% BS) than in the separate analysis (83% BS), indicating that the cpDNA data set con-

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Fig. 3. Combined (cpDNA + ITS) phylogeny of Veronica. Bayesian 50% majority rule tree showing bootstrap percentage values (BP) from parsimony analysis and posterior probability values (PP) from Bayesian analysis above/below branches, respectively. Subgeneric affiliation and informal names for species groups within subg. Pseudoveronica sect. Hebe are shown. Nodes with 100% support are indicated with an asterisk.

tains some signal for this relationship. This is also also supported by the insignificant Shimodaira–Hasegawa-test. The position of this enigmatic subgenus remains unresolved due to the morphological distinctiveness and the long branches in molecular analy-

ses. Comparing support for relationships between combined analysis and cpDNA analysis reveals one instance in which the combined analysis offers stronger support, i.e., for the monophyly of the clade without subgenus Stenocarpon (no support in cpDNA

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Fig. 4. CYC2 phylogeny of Veronica. Bayesian 50% majority rule tree showing bootstrap percentage values (BP) from parsimony analysis and posterior probability values (PP) from Bayesian analysis above/below branches, respectively. Subgeneric affiliation and informal names for species groups within subg. Pseudoveronica sect. Hebe are shown. Numbers after species represent different clones. Nodes with 100% support are indicated with an asterisk.

analysis, 96% PP in combined analysis). This particular branching order has been noted before in ITS analyses (Wagstaff et al., 2002; Albach and Chase, 2004) but not in cpDNA analyses.

To summarize, the specific cases of topological incongruence indicated before (e.g., Albach and Chase, 2004) are partly resolved. The addition of more data from the plastid genome has increased

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the support for relationships found previously with the trnL-trnLtrnF region. We therefore do not expect many changes in the relationships between subgenera of Veronica with the addition of more plastid markers. The question remains whether conflict between cpDNA and ITS indicates contrasting signal from plastid and nuclear genomes perhaps due to hybridization, or whether the conflict is caused by homoplasious characters and lack of phylogenetic signal for specific relationships in the ITS data set. Therefore, sequencing nuclear markers and identifying whether ITS is indeed representative for the nuclear genome is of prime concern in resolving the phylogeny of Veronica. We here provide the first data for a nuclear low-copy gene identified to be consistently amplifiable across the genus and sufficiently variable in a larger study investigating the phylogenetic utility of such markers in Veronica (Albach, unpublished). The CYC gene is best known for its effect on floral asymmetry (Carpenter and Coen, 1990; Luo et al., 1996; Preston et al., 2009) but has been used phylogenetically in various groups of angiosperms such as Gesneriaceae (e.g., Möller et al., 1999; Citerne et al., 2000; Smith et al., 2004, 2006; Wang et al., 2004) and Fabaceae (Ree et al., 2004; Hughes and Eastwood, 2006). Its phylogenetic utility in Plantaginaceae was shown by studies on Antirrhineae (Gübitz et al., 2003; Hileman and Baum, 2003), in which it was demonstrated to consist of a single copy per diploid genome in hexaploid Digitalis, one of the closest relatives of Veronica. A more recent analysis by Preston et al. (2009), however, revealed two functional copies in V. montana. Incorporating their sequences in our data set demonstrated that all our sequences are homologs of their CYC2 sequences. However, their sequence of V. montana is most similar to our sequence of V. serpyllifolia (Albach, unpublished), a relationship supported by the comparison of the voucher specimen of Preston et al. (2009; Hileman, pers. comm.). Direct sequencing of CYC2 was possible in the endosperm-podium clade but not in the earlier branching subgenera of Veronica and in related genera of Veroniceae, where direct sequences were polymorphic. This result coincides with the hypothesis of Albach and Chase (2004) that a polyploidy event occurred near the origin of Veroniceae, which would have led to the duplication of the CYC2 locus. Subsequently, a loss of the second locus in the ancestor of the endosperm-podium clade could explain the observed pattern. Unfortunately, our cloning efforts did not retrieve more than one sequence in the individuals that were cloned. However, similarity between the phylogenetic tree based on CYC2 (Fig. 4) and that of the combined tree (Fig. 3) suggests that paralogy at the tribal level does not seem to be a big problem. The position of Veronica stellata as sister to Picrorhiza kurrooa seems to be the only case that could be attributed to this particular hypothesized duplication event. Paralogy at higher levels are more obvious with the non-monophyly of V. glandulosa, V. subg. Pseudolysimachium and V. sect. Hebe as likely examples of this. 4.2. Phylogenetic relationships among Veronica species groups in the Southern Hemisphere Monophyly of the Southern Hemisphere subgenus Pseudoveronica and its two major sections, the Australian section Labiatoides and the New Zealand/New Guinea section Hebe (including monotypic sect. Detzneria) is strongly supported by most analyses (>89% BS and 99–100% PP support). The notable exception is the polyphyly of sect. Labiatoides in the cpDNA tree, in which only one of the two sampled species of sect. Labiatoides (V. perfoliata) is sister to sect. Hebe, whereas the other species, V. nivea, is nested within sect. Hebe and sister to V. tubata (sect. Detzneria; Fig. 2). Previous studies have also shown that sect. Hebe and sect. Labiatoides are reciprocally monophyletic in ITS analyses but not in cpDNA analyses (Wagstaff et al., 2002; Albach et al., 2005b). Given the

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high support in the combined analysis for the monophyly and sister relationship of sect. Labiatoides and sect. Hebe, we consider the position of V. nivea in the cpDNA analyses to be an artifactual branch attraction phenomenon due to poor sampling of sect. Labiatoides. This is supported by the fact that species of sect. Labiatoides are associated with different species of section Hebe in the cpDNA analyses published to date, i.e., with V. catarractae (or unresolved) in Wagstaff et al. (2002), with V. densifolia and V. tubata in Albach et al. (2005b) and with V. tubata or sister to sect. Hebe in the present study (Fig. 2). More detailed studies focussing on the ca. 25 species of sect. Labiatoides are currently in progress (Albach and Briggs, unpublished). Section Detzneria comprises the sole species Veronica tubata, which was formerly classified in the monotypic genus Detzneria and first analyzed phylogenetically by Albach et al. (2005b). Although low sequence variation in that study made interpretation difficult, analyses of ITS and trnL-trnL-trnF data showed that V. tubata was nested within sect. Hebe and was perhaps (ITS) one of its most basal branches. Because we were unable to sequence more cpDNA markers for V. tubata, our analyses are not able to further clarify its position, as it is variously sister to V. cheesemanii (ITS), V. nivea (cpDNA), or near the base of sect. Hebe (combined data set). Nevertheless, our analyses do not support sect. Detzneria as a stepping-stone species left over from the first immigration to Australasia by Veronica as suggested by Albach et al. (2005b) but rather support a dispersal event from New Zealand to New Guinea early in the diversification of sect. Hebe, which was later followed by a secondary dispersal and diversification of the ancestor of the ca. 12 New Guinea speedwell hebes (represented here by V. vandewateri). In our analyses, there are five main lineages within sect. Hebe that are highly supported by bootstrap and posterior probability values (especially the combined analysis, Fig. 3). In some cases, these lineages comprise groups that correspond to previously recognized genera. This result is largely consistent with early phylogenetic studies of this Southern Hemisphere group using mostly ITS (Wagstaff and Garnock-Jones, 1998, 2000; Wagstaff et al., 2002). Relationships among and within each of these five lineages are not well resolved or supported, and branch lengths are extremely short. Indeed the placement of some species is unresolved (e.g., V. macrantha, V. linifolia) or differs among markers (e.g., V. cupressoides, V. planopetiolata). To clarify these incongruent patterns and provide further support for species groups in future studies that build upon the backbone phylogeny presented here (Fig. 3), increased sampling of species within each species group and multiple individuals per species are warranted. For example, preliminary DNA data (J. Prebble, unpublished data) regarding the phylogenetic placement of a newly described New Zealand species, V. jovellanoides (Davidson et al., 2009), will be especially interesting given the morphology and chromosome number of this species, which make it difficult to place within sect. Hebe and in Veronica as a whole. In addition, three of the four species from sect. Hebe that were sampled twice are not monophyletic in the cpDNA tree (although they were placed in the correct species groups; Fig. 2). This finding, and also the topology of the snow hebe clade in the cpDNA tree (Fig. 2), are consistent with the finding of non-monophyly of species within the snow hebe group using the cpDNA marker rpoBtrnC (Meudt and Bayly, 2008). There was no evidence for chloroplast sharing among the snow hebe, speedwell hebe, or semiwhipcord hebe groups, except as expected for putative natural hybrid individuals sampled (Meudt and Bayly, 2008). DNA evidence supported the hybrid origin of these three individuals which represented two different crosses: Chionohebe glabra  Parahebe trifida (treated here as V. chionohebe  V. trifida, both belonging to the snow hebe group; Table 4) and C. pulvinaris  Leonohebe ciliolata

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(V. pulvinaris  V. hookeri from the snow hebe and semi-whipcord hebe groups, respectively). Evidence exists for other instances of hybridization between and within species groups in sect. Hebe (discussed in Meudt and Bayly, 2008; Bayly and Kellow, 2006). Although ability (or inability) to cross is not necessarily linked to classification or phylogeny, comparing genetic distances to data from future greenhouse and field studies on experimental hybridization, phenology, and reproductive biology would be fruitful and complementary to the results provided here. Thus, cpDNA markers correctly place species of sect. Hebe in the corresponding species group, but species relationships within each of these groups should be viewed with caution until multiple individuals from many more species are sampled and hypotheses on hybrid origin or chloroplast capture in some of the species can be evaluated. Over the past 10–15 years, several other molecular and morphological studies on certain groups within sect. Hebe have greatly improved our understanding of evolutionary relationships, character evolution, biogeography and taxonomy of this large diverse, ecologically important group (Briggs and Ehrendorfer, 1992; Wagstaff and Garnock-Jones, 1998, 2000; Wagstaff et al., 2002; Garnock-Jones, 1993a,b; Garnock-Jones and Lloyd, 2004; Bayly and Kellow, 2006; Meudt and Bayly, 2008; Meudt, 2008). More recently, investigation of phytochemical compounds in the group has gained renewed interest (Johansen et al., 2007; Pedersen et al., 2007; Jensen et al., 2008) and is starting to reveal support for relationships discovered in phylogenetics analyses of DNA sequence data (S. Jensen, pers. comm.). However, there is currently no useful, explicit taxonomy (whether formal or informal) for species groups within sect. Hebe that adequately synthesizes this new data. For this reason, we propose the following informal names for the five main lineages within sect. Hebe (Table 4): semi-whipcord hebes (formerly Leonohebe, 4 spp.), snow hebes (Chionohebe plus some Parahebe, 10 spp.), speedwell hebes (New Guinea and New Zealand Parahebe, ca. 24 spp.), sun hebes (Heliohebe, 5 spp.), and hebes (Hebe, 88 spp.). In addition, because our analyses clearly show that V. tubata is nested within sect. Hebe, maintaining sect. Detzneria is not justified or necessary and this monotypic group can be considered a sixth informal group (heath hebes). We believe that this informal classification provides descriptive names for putatively natural groups within sect. Hebe and will allow clearer communication among botanists by retaining useful information from previous classifications, while incorporating new information from recent studies. This is especially important in the snow hebe and speedwell hebe groups, which are paraphyletic when traditionally circumscribed as Chionohebe and Parahebe. These informal groups should be treated as hypotheses that after further testing, may lead to a more formal naming system within sect. Hebe (and perhaps also within the large hebe species group) in future studies.

5. Conclusions and future directions In summary, the phylogeny of Veronica presented here comprises the best estimate of evolutionary relationships among the subgenera, and in particular within the Southern Hemisphere species of subg. Pseudoveronica. Our study sampled 68 species (ca. 15% of the genus) representing all 12 subgenera—with a well-balanced diversity of species within each subgenus—and provides several insights into the evolution of this large, worldwide genus. We demonstrate the utility of cpDNA markers rpoB-trnC and psb-trnH for phylogenetic reconstruction in Veronica, but also stress that as opposed to cpDNA markers, low-copy nuclear markers such as the one used here (CYC2) may be the key for future studies with increased sampling within the genus. CYC in particular also offers the perspective of studying the evolution of floral symmetry in

Veronica. Whereas ancestors of the tribe Veroniceae have bilaterally symmetrical flowers, Veroniceae and Plantago evolved radial symmetry (Reeves and Olmstead, 1998); this is especially evident among the species of snow hebes (Garnock-Jones, 1993a; Garnock-Jones in Bayly and Kellow, 2006). Thus, comparing CYC sequences across Plantaginaceae may offer insight into whether nucleotide substitutions in the gene may have been responsible for the observed phenotypic changes. With respect to the closelyrelated Southern Hemisphere species, we propose a new informal taxonomy that will help communication regarding natural groups within subg. Pseudoveronica, and that should be further tested with future studies that include the remaining species. The robust phylogenetic hypothesis for the relationships among the main Veronica clades presented here (Fig. 3) and Veroniceae (Albach and Chase, 2004), together with another study with increased taxon sampling (Müller and Albach, in preparation), offer the starting point to explore issues of dating the phylogeny and determining the lineage diversification rate in Veronica. The clarification of appropriate outgroups will assist further study of relationships and character evolution within sect. Hebe. Exploring these issues has been hindered by phylogenetic uncertainty (see Section 1) and considerable rate heterogeneity combined with variation in life history (Müller and Albach, in preparation). Local molecular clock analyses (e.g., Rutschmann, 2006; Britton et al., 2007) that rely on robust phylogenies may be the key to finally clarifying these issues in Veronica in the near future. Acknowledgments We thank Steve Wagstaff, Murray Dawson and Phil GarnockJones for supplying DNAs or specimens for some Southern Hemisphere individuals, Marion Kever for assistance in the lab at the University of Mainz, Peter Ritchie for use of the Molecular Ecology Lab at Victoria University (Wellington) for some DNA work, Patrick Brownsey for support of this project, Phil Garnock-Jones for suggesting the hebe informal species group names, and Montserrat Martínez-Ortega, Phil Garnock-Jones, Steve Wagstaff and two anonymous reviewers for helpful comments on the manuscript. This work was supported in part by the Forschungsfonds of the Johannes Gutenberg-University Mainz to D.C.A. and in part by the New Zealand Foundation for Research Science and Technology OBI Defining New Zealand’s Land Biota (Contract C09X0501). References Albach, D.C., Chase, M.W., 2001. Paraphyly of Veronica (Veroniceae; Scrophulariaceae): evidence from the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA. J. Plant Res. 114, 9–18. Albach, D.C., Chase, M.W., 2004. Incongruence in Veroniceae (Plantaginaceae): evidence from two plastid and a nuclear ribosomal DNA region. Mol. Phylogenet. Evol. 32, 183–197. Albach, D.C., Greilhuber, J., 2004. Genome size variation and evolution in Veronica. Ann. Bot. 94, 897–911. Albach, D.C., Martínez-Ortega, M.M., Fischer, M.A., Chase, M.W., 2004a. Evolution of Veroniceae: a phylogenetic perspective. Ann. MO Bot. Gard. 91, 275–302. Albach, D.C., Martínez-Ortega, M.M., Chase, M.W., 2004b. Veronica: parallel morphological evolution and phylogeography in the Mediterranean. Plant Syst. Evol. 246, 177–194. Albach, D.C., Martínez-Ortega, M.M., Fischer, M.A., Chase, M.W., 2004c. A new classification of the tribe Veroniceae—problems and a possible solution. Taxon 53, 429–452. Albach, D.C., Meudt, H.M., Oxelman, B., 2005a. Piecing together the ‘‘new” Plantaginaceae. Am. J. Bot. 92, 297–315. Albach, D.C., Utteridge, T., Wagstaff, S.J., 2005b. Origin of Veroniceae (Plantaginaceae, formerly Scrophulariaceae) on New Guinea. Syst. Bot. 30, 412–423. Albach, D.C., Jensen, S.R., Özgökce, F., Grayer, R.J., 2005c. Veronica: chemical characters for the support of phylogenetic relationships based on nuclear ribosomal and plastid DNA sequence data. Biochem. Syst. Ecol. 33, 1087–1106. Albach, D.C., von Sternburg, M., Scalone, R., Bardy, K., 2009. Phylogenetics, morphology and differentiation of Veronica saturejoides (Plantaginaceae). Bot. J. Linn. Soc. 159, 616–636.

D.C. Albach, H.M. Meudt / Molecular Phylogenetics and Evolution 54 (2010) 457–471 Bayly, M.J., Kellow, A.V., 2006. An Illustrated Guide to New Zealand Hebes. Te Papa Press, Wellington, New Zealand. Blattner, F.R., 1999. Direct amplification of the entire ITS region from poorly preserved plant material using recombinant PCR. Biotechniques 27, 1180–1186. Briggs, B.G., Ehrendorfer, F., 1992. A revision of the Australian species of Parahebe and Derwentia (Scrophulariaceae). Telopea 5, 241–287. Britton, T., Anderson, C.L., Jacquet, D., Lundqvist, S., Bremer, K., 2007. Estimating divergence times in large phylogenetic trees. Syst. Biol. 56, 741–752. Carpenter, R., Coen, E.S., 1990. Floral homeotic mutations produced by transposonmutagenesis in Antirrhinum majus. Genes Dev. 4, 1483–1493. Chase, M.W., Cowan, R.S., Hollingsworth, P.M., van den Berg, C., Madrinan, S., Petersen, G., Seberg, O., Jorgensen, T., Cameron, K.M., Carine, M., Pedersen, N., Hedderson, T.A.J., Conrad, F., Salazar, G.A., Richardson, J.E., Hollingsworth, M.L., Barraclough, T.G., Kelly, L., Wilkinson, M., 2007. A proposal for a standardised protocol to barcode all land plants. Taxon 56, 295–299. Citerne, H.L., Moller, M., Cronk, Q.C.B., 2000. Diversity of cycloidea-like genes in Gesneriaceae in relation to floral symmetry. Ann. Bot. 86, 167–176. Davidson, G.R., de Lange, P.J., Garnock-Jones, P.J., 2009. Two additional indigenous species of Veronica (Plantaginaceae) from northern New Zealand: V. jovellanoides, a new and highly endangered species, and V. plebeia R.Br. N. Z. J. Bot. 47, 271–279. Downie, S.R., Katz-Downie, D.S., 1996. A molecular phylogeny of Apiaceae subfamily Apioideae: evidence from nuclear ribosomal DNA internal transcribed spacer sequences. Am. J. Bot. 83, 234–251. Edwards, D., Horn, A., Taylor, D., Savolainen, V., Hawkins, J.A., 2008. DNA barcoding of a large genus, Asphalathus L. (Fabaceae). Taxon 57, 1317–1327. Garnock-Jones, P.J., 1993a. Phylogeny of the Hebe complex (Scrophulariaceae: Veroniceae). Aust. Syst. Bot. 6, 457–479. Garnock-Jones, P.J., 1993b. Heliohebe (Scrophulariaceae—Veroniceae), a new genus segregated from Hebe. N. Z. J. Bot. 31, 323–339. Garnock-Jones, P.J., Albach, D., Briggs, B.G., 2007. Botanical names in Southern Hemisphere Veronica (Plantaginaceae): sect. Detzneria, sect. Hebe, and sect. Labiatoides. Taxon 56, 571–582. Garnock-Jones, P.J., Lloyd, D.G., 2004. A taxonomic revision of Parahebe (Scrophulariaceae—Antirrhinoideae) in New Zealand. N. Z. J. Bot. 42, 181–232. Goodson, B.E., Santos-Guerra, A., Jansen, R.K., 2006. Molecular systematics of Descurainia (Brassicaceae) in the Canary Islands: biogeographic and taxonomic implications. Taxon 55, 671–682. Gübitz, T., Caldwell, A., Hudson, A., 2003. Rapid molecular evolution of CYCLOIDEAlike genes in Antirrhinum and its relatives. Mol. Biol. Evol. 20, 1537–1544. Heads, M., 2003. Hebejeebie (Plantaginaceae), a new genus from the South Island, New Zealand, and Mt. Kosciuszko, SE Australia. Bot. Soc. Otago Newsl. 36, 10– 12. Hileman, L.C., Baum, D.A., 2003. Why do paralogs persist? Molecular evolution of CYCLOIDEA and related floral symmetry genes in Antirrhineae (Veronicaceae). Mol. Biol. Evol. 20, 591–600. Huelsenbeck, J.P., Ronquist, F., 2001. MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754–755. Hughes, C., Eastwood, R., 2006. Island radiation on a continental scale: exceptional rates of plant diversification after uplift of the Andes. Proc. Natl. Acad. Sci. USA 103, 10334–10339. Jensen, S.R., Gotfredsen, C.H., Grayer, R.J., 2008. Unusual iridoid glycosides in Veronica sects. Hebe and Labiatoides. Biochem. Syst. Ecol. 36, 207–215. Johansen, M., Larsen, T.S., Mattebjerg, M.A., Gotfredsen, C.H., Jensen, S.R., 2007. Chemical markers in Veronica sect. Hebe. Biochem. Syst. Ecol. 35, 614–620. Kress, W.J., Wurdack, K.J., Zimmer, E.A., Weigt, L.A., Janzen, D.H., 2005. Use of DNA barcodes to identify flowering plants. Proc. Natl. Acad. Sci. USA 102, 8369– 8374. Low, M.L.E., 2005. Evolution of gender dimorphism in Hebe (Plantaginaceae). Ph.D. Thesis, Victoria University of Wellington, New Zealand. Luo, D., Carpenter, R., Vincent, C., Copsey, L., Coen, E., 1996. Origin of floral asymmetry in Antirrhinum. Nature 383, 794–799. Meudt, H.M., 2008. Taxonomic revision of Australasian snow hebes (Veronica, Plantaginaceae). Aust. Syst. Bot. 21, 387–421. Meudt, H.M., Bayly, M.J., 2008. Phylogeographic patterns in the Australasian genus Chionohebe (Veronica s.l., Plantaginaceae) based on AFLP and chloroplast DNA sequences. Mol. Phylogenet. Evol. 47, 319–338. Möller, M., Clokie, M., Cubas, P., Cronk, Q.C.B., 1999. Integrating molecular and developmental genetics: a Gesneriaceae case study. In: Hollingsworth, M.L., Bateman, R.J., Gordall, R.J. (Eds.), Molecular Systematics and Plant Evolution. Taylor and Francis, London, pp. 375–402. Morrison, D.A., 2007. Increasing the efficiency of searches for the maximum likelihood tree in a phylogenetic analysis of up to 150 nucleotide sequences. Syst. Biol. 56, 988–1010. Mort, M.E., Archibald, J.K., Randle, C.P., Levsen, N.D., O’Leary, T.R., Topalov, K., Wiegand, C.M., Crawford, D.J., 2007. Inferring phylogeny at low taxonomic levels: utility of rapidly evolving cpDNA and nuclear ITS loci. Am. J. Bot. 94, 173–183.

471

Müller, K., 2005. SeqState: primer design and sequence statistics for phylogenetic DNA datasets. Appl. Bioinformatics 4, 65–69. Müller, K., 2006. Incorporating information from length-mutational events into phylogenetic analysis. Mol. Phylogenet. Evol. 38, 667–676. Muñoz Centeno, L.M., Albach, D.C., Sánchez-Agudo, J.A., Martínez-Ortega, M.M., 2006. Systematic significance of seed morphology in Veronica (Plantaginaceae). A phylogenetic perspective. Ann. Bot. 98, 335–350. Oxelman, B., Liden, M., Berglund, D., 1997. Chloroplast rps16 intron phylogeny of the tribe Sileneae (Caryophyllaceae). Plant Syst. Evol. 206, 393–410. Pedersen, P., Gotfredsen, C.H., Wagstaff, S.J., Jensen, S.R., 2007. Chemical markers in Veronica sect. Hebe. II. Biochem. Syst. Ecol. 35, 777–784. Posada, D., Crandall, K.A., 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817–818. Preston, J.C., Kost, M.A., Hileman, L.C., 2009. Conservation and diversification of the symmetry developmental program among close relatives of snapdragon with divergent floral morphologies. New Phytol. 182, 751–762. Ree, R.H., Citerne, H.L., Lavin, M., Cronk, Q.C.B., 2004. Heterogeneous selection on LEGCYC paralogs in relation to flower morphology and the phylogeny of Lupinus (Leguminosae). Mol. Biol. Evol. 21, 321–331. Reeves, P.A., Olmstead, R.G., 1998. Evolution of novel morphological and reproductive traits in a clade containing Antirrhinum majus (Scrophulariaceae). Am. J. Bot. 85, 1047–1056. Riek-Häußermann, C., 1943. Vergleichend-anatomische und entwicklungsgeschichtliche Untersuchungen über Samen in der Gattung Veronica. Beih. Bot. Centralbl. 62, 1–60. Rutschmann, F., 2006. Molecular dating of phylogenetic trees: a brief review of current methods that estimate divergence times. Divers. Distrib. 12, 35–48. Sang, T., Crawford, D.J., Stuessy, T.F., 1997. Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae). Am. J. Bot. 84, 1120– 1136. Shaw, J., Lickey, E.B., Beck, J.T., Farmer, S.B., Liu, W., Miller, J., Siripun, K.C., Winder, C.T., Schilling, E.E., Small, R.L., 2005. The tortoise and the hare II: relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis. Am. J. Bot. 92, 142–166. Shaw, J., Lickey, E.B., Schilling, E.E., Small, R.L., 2007. Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: the tortoise and the hare III. Am. J. Bot. 94, 275–288. Simmons, M.P., Müller, K., Norton, A.P., 2007. The relative performance of indelcoding methods in simulations. Mol. Phylogenet. Evol. 44, 724–740. Smith, J.F., Draper, S.B., Hileman, L.C., Baum, D.A., 2004. A phylogenetic analysis within tribes Gloxinieae and Gesnerieae (Gesnerioideae: Gesneriaceae). Syst. Bot. 29, 947–958. Smith, J.F., Funke, M.M., Woo, V.L., 2006. A duplication of gcyc predates divergence within tribe Coronanthereae (Gesneriaceae): phylogenetic analysis and evolution. Plant Syst. Evol. 261, 246–256. Štorchová, H., Olson, M.S., 2007. The architecture of the chloroplast psbA-trnH noncoding region in angiosperms. Plant Syst. Evol. 268, 235–256. Swofford, D.L., 2002. PAUP* Phylogenetic Analysis Using Parsimony (*and Other Methods). Sinauer Associates, Sunderland, MA. Taberlet, P., Gielly, L., Pautou, G., Bouvet, J., 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Mol. Biol. 17, 1105–1109. Tate, J.A., Simpson, B.B., 2003. Paraphyly of Tarasa (Malvaceae) and diverse origins of the polyploid species. Syst. Bot. 28, 723–737. Van Royen, P., 1972. The Scrophulariaceae of the alpine regions of New Guinea. Bot. Jahrb. Syst. 91, 383–437. von Sternburg, M., 2007. Phylogeographie und Differenzierung von Veronica saturejoides. Diploma Thesis, Institut für Spezielle Botanik. Johannes Gutenberg-Universität, Mainz. Wagstaff, S.J., Garnock-Jones, P.J., 1998. Evolution and biogeography of the Hebe complex (Scrophulariaceae) inferred from ITS sequences. N. Z. J. Bot. 36, 425– 437. Wagstaff, S.J., Bayly, M.J., Garnock-Jones, P.J., Albach, D.C., 2002. Classification, origin, and diversification of the New Zealand hebes (Scrophulariaceae). Ann. MO Bot. Gard. 89, 38–63. Wagstaff, S.J., Garnock-Jones, P.J., 2000. Patterns of diversification in Chionohebe and Parahebe (Scrophulariaceae) inferred from ITS sequences. N. Z. J. Bot. 38, 389– 407. Wang, C.-N., Möller, M., Cronk, Q.C.B., 2004. Phylogenetic position of Titanotrichum oldhamii (Gesneriaceae) inferred from four different gene regions. Syst. Bot. 29, 407–418. White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M., Gelfand, D., Sninsky, J., White, T. (Eds.), PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, pp. 315–322. Zwickl, D. J., 2006. Genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion. Ph.D. Thesis, University of Texas, Austin.