Molecular phylogeny of Lysimachia (Myrsinaceae) based on chloroplast trnL–F and nuclear ribosomal ITS sequences

MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution 31 (2004) 323–339 www.elsevier.com/locate/ympev

Molecular phylogeny of Lysimachia (Myrsinaceae) based on chloroplast trnL–F and nuclear ribosomal ITS sequences Gang Hao,a Yong-Ming Yuan,b,* Chi-Ming Hu,a Xue-Jun Ge,a and Nan-Xian Zhaoa a

South China Institute of Botany, The Chinese Academy of Sciences, Guangzhou 510650, China b Institut de Botanique, Universit
e de Neuch^ atel, Neuch^ atel CH-2007, Switzerland Received 12 May 2003; revised 16 July 2003

Abstract Both nuclear ribosomal ITS and chloroplast trnL–F sequences were acquired for 57 species (accessions) of Lysimachia and its close relatives, and were analyzed together with sequences retrieved from databases. The results of phylogenetic analyses based on these data (separately or combined) show that Lysimachia is paraphyletic, with the monotypic genus Glaux nested deeply inside. Previous suggestions that Anagallis and Trientalis could be ingroups of Lysimachia were not corroborated by our results. The molecular phylogenies do not support the current infrageneric divisions of Lysimachia. Subgenus Lysimachia contains at least five independent lineages. The Hawaii endemic subgenus Lysimachiopsis was shown to group with subgenera Palladia and Heterostylandra, instead of subgenus Idiophyton as previously suggested. The two North American representatives of Lysimachia, subgenus Seleucia and section Verticillatae of subgenus Lysimachia are group together as the most basal clade of the genus. Parallel and independent evolutions were inferred for morphological characters that were previously used as diagnostic criteria. Molecular phylogenies do not offer clear inferences on the overall historical biogeography of Lysimachia, but Southeast Asia origins of several clades, including the Hawaiian endemic clade and the Iberian Lysimachia ephemerum are strongly supported. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Anagallis; Asterolinon; Glaux; ITS; Lysimachia; Myrsinaceae; Primulaceae; Trientalis; trnL–F

1. Introduction With about 191 species of perennial and annual herbs, Lysimachia L. is one of the largest genera traditionally included in Primulaceae (e.g., Cronquist, 1981; Takhtajan, 1997), where it comprises the largest part of the tribe Lysimachieae together with some small genera including Anagallis L., Trientalis L., Glaux L., Asterolinon Hoffmanns. and Link, and Pelletiera A. St-Hil. Recent molecular and morphological studies have resulted in the re-evaluation of existing classifications and the re-delimitation of the families of the traditional Primulales, and the tribe Lysimachieae was transferred to the family Myrsinaceae (Anderberg and St ahl, 1995; Anderberg et al., 1998, 2002; K€ allersj€ o et al., 2000). The majority of species of Lysimachia occur throughout temperate and subtropical regions of the * Corresponding author. Fax: +41-32-718-3001. E-mail address: [email protected] (Y.-M. Yuan).

1055-7903/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1055-7903(03)00286-0

Northern Hemisphere. It reaches the tropics in southeastern Asia, and has several representatives in South America and Africa (Table 1). The genus as a whole is nearly cosmopolitan, and its highest diversity occurs in the southwest China, where several subgenera and sections are much more concentrated than in any other area of the world (Hu, 1994; Hu and Kelso, 1996). The first comprehensive taxonomic treatment of Lysimachia was conducted by Handel-Mazzetti (1928) in the course of a revision of the Chinese species. Significantly, he was the first to give due taxonomic emphasis to floral structure, especially to the nature of androecium. One hundred and forty seven species were recognized worldwide, and an infrageneric classification was proposed by subdividing the genus into the subgenera Idiophyton, Lysimachia (as Eulysimachia), Palladia, Lysimachiopsis, and Naumburgia. In RayÕs (1956) monograph of Lysimachia in the New World, section Seleucia subsection Seleucia (Steironema) and section Theopyxis, included in subgenus Lysimachia by

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Table 1 Classification, geographic distribution and number of species of the genus Lysimachia Subgenus Heterostylandra Idiophyton

Lysimachia

Lysimachiopsis Naumburgia Palladia

Seleucia Theopyxis

Section Idiophyton Apodanthera Oppositifoliae Alternifoliae Lerouxia Lysimachia Nummularia Rosulatae Verticillatae

Apochoris Candidae Chenopodiopsis Ephemerum Lubinia Miltandrae Palladia Pumilae Spicatae

Distribution

Number of species

Central China S.W. China, N. Vietnam S.E. Asia, Indo-China peninsula S.W. China S.W. China, N. Burma, Indo-China Europe Eurasia Eurasia, mainly China S. China N. America Hawaii Eurasia, N. America N. and Central China E. Asia, N. Vietnam Mainly W. China, N.W. India, Mediterranean, S.E. Africa Mediterranean Asian & American Pacific coast, Islands of Indian Ocean Central and S.W. China Mainly China, Europe, Africa S.W. China to N. India E. Asia N. America Central to S. America

1 1 32 3 3 3 4 59 2 6 16 1 1 4 7 1 1 5 22 2 6 7 4

Classification mainly follows Chen and Hu (1979), except for the concepts of subgenus Seleucia and Theopyxis that follow Ray (1956).

Handel-Mazzetti (1928), were elevated to subgenera. Chen and Hu (1979) re-appraised the Chinese species and presented a modified subdivisional system (Table 1). Although Handel-MazzettiÕs (1928) framework was adopted, the following changes were made: the transfer of section Apodanthera (ca. 36 species) from subgenus Lysimachia to subgenus Idiophyton; the transfer of section Rosulatae (two species) from subgenus Lysimachiopsis to subgenus Lysimachia; and the creation of a new subgenus for the monotypic section Heterostylandra. Bennell and Hu (1983), in a study of pollen morphology of 98 species and varieties of Lysimachia, found that pollen types corresponded closely to the major taxonomic groups proposed by Chen and Hu (1979). Although aimed at elucidating relationships at the family level, recent phylogenetic studies have indicated that Lysimachia as previously defined is heterogeneous and paraphyletic regarding the remaining genera of tribe Lysimachieae (Anderberg and St ahl, 1995; Anderberg et al., 1998; K€ allersj€ o et al., 2000; Martins et al., 2003). These studies involved only a limited number of taxa of Lysimachia, however, with the majority of species not included, and thus our understanding of the infrageneric phylogeny of this genus is incomplete. In a phylogenetic study of the generic circumscription and infrageneric relationships by Hao and Hu (2001) based on morphological data, Lysimachia was paraphyletic. The genera Glaux, Pelletiera, and Trientalis nested within Lysimachia. The monophyly of the subgenera Idiophyton, Lysimachiopsis, and Palladia were

supported, whereas subgenus Lysimachia was polyphyletic. Moreover, subgenus Seleucia grouped with Trientalis. Only some internal nodes were supported with moderate confidence, indicating that tree stability was low and the phylogeny of Lysimachia requires testing with additional data. Here we assess the relationships of Lysimachia and related genera with sequences from the chloroplast trn L(UAA)–trnF(GAA) region (trnL–F), and the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. Both of these regions have proven phylogenetically informative for inferring relationships in various plant groups, particularly at the intergeneric or interspecific levels (e.g., Chassot et al., 2001; Mast et al., 2001). The main objectives of this study are: (1) to evaluate the monophyly of Lysimachia; (2) to identify the major infrageneric lineages of Lysimachia and assess whether they corroborate existing classifications of the genus, and (3) to explore the biogeographic implications of the molecular phylogenies of Lysimachia.

2. Materials and methods 2.1. Ingroup sampling and outgroup choice As far as possible, samples were selected to maximize representation of infrageneric groups and distributional range. Of the eight currently recognized subgenera, subgenus Theopyxis (four Neotropical species) were not

G. Hao et al. / Molecular Phylogenetics and Evolution 31 (2004) 323–339

available to us. Other related genera (both Lysimachieae and non-Lysimachieae), Aegiceras Gaertn., Anagallis, Ardisia Sw., Ardisiandra Hook. f., Cyclamen L., Glaux, Myrsine L., and Trientalis were also sampled. Multiple accessions were sampled for Lysimachia longipes Hemsl. and Glaux maritima L. to assess possible intraspecific variation. In total, 59 accessions of trnL–F sequences were acquired for Lysimachia (49 species) and related or outgroup genera such as Glaux, Anagalis, Ardisia, Cyclamen, and Myrsine (seven species). Twelve accessions of trnL–F sequences of Lysimachia or related taxa retrieved from GenBank database were also included in the analyses (Table 2). The trnL–F data set therefore consisted of 71 taxa. Four representative species of the closely related families of Ericales (Clavija spinosa Mez. and Theophrasta americana L. of Theophrastaceae, Maesa japonica Merrill of Maesaceae, and Soldanella pusilla Baumg. of Primulaceae) were used as outgroups based on K€ allersj€ o et al. (2000). For ITS, 57 accessions were newly acquired for 49 Lysimachia species and five species of other related genera; ITS sequences of three species were retrieved from GenBank database (Table 2). Our ITS data set therefore consisted of 60 taxa. The closely related taxa Ardisia crenata Sims, Cyclamen persicum Mill., and Myrsine faberi Pipoly and C. Chen of Myrsinaceae were used as outgroups based on K€ allersj€ o et al. (2000). 2.2. DNA extraction, PCR amplification, and sequencing Total DNA was extracted from fresh or silica-gel dried leaves with the method of Doyle and Doyle (1987). The trnL–F and ITS fragments were amplified by using standard double-strand polymerase chain reaction (PCR). The primers ‘‘c’’ and ‘‘f’’ (Taberlet et al., 1991) were used to amplify the trnL–F fragment comprising the trnL (UAA) intron, the trnL (UAA) 30 exon, and the intergenic spacer between trnL (UAA) and trn F (GAA) genes. The primers ‘‘ITS5’’ and ‘‘ITS4’’ (White et al., 1990) were used to amplify the ITS fragment comprising ITS1, the 5.8S gene, and ITS2. The PCR products were purified by using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA), following the manufacturerÕs protocol prior to sequencing. Sequencing reactions were performed by using the dye-terminator cycle-sequencing ready-reaction kit by following the manufacturerÕs protocol, and analyzed on an ABI 377 DNA Sequencer (Applied Biosystems, Foster City, CA). Each fragment was sequenced for both strands.

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F sequences required only minor manual adjustment for alignment. Three regions (80 base pairs (bp) total in the alignment) involved tandem repeats and alignment ambiguity, and were excluded from phylogenetic analyses. Potentially informative and unambiguously assessed indels of the trnL–F were scored as binary characters (presence versus absence) regardless of length and added to the data matrix (32 characters total). ITS sequences required a higher level of manual adjustment for alignment. Five regions ranging from 10 to 29 bp in length (91 bp total in the final alignment) involved high alignment ambiguity and were excluded from the analyses. The indels of ITS sequences were not scored as characters because they could not be unambiguously assessed. 2.4. Maximum parsimony analysis For maximum parsimony (MP) analysis, all data sets were analyzed with heuristic searches by using PAUP* v4.0b10 (Swofford, 2000). Characters were equally weighted and unordered, with gaps treated as missing data. Branch collapse option was set to collapse if minimum length as zero. Heuristic searches of 1000 replicates of random addition of sequences, with TBR branch swapping, MULTREES, ACCTRAN, and STEEPEST DESCENT options on. Relative clade support was evaluated by the bootstrap analyses (Felsenstein, 1985). Bootstrap values were calculated by using 1000 replicates of heuristic searches, with the random sequence addition, TBR branch swapping, MULTREES options on, the STEEPEST DESCENT option off. A monophyletic Lysimachia topology was used as a constraint in parsimony analyses, and the resultant maximally parsimonious trees were compared with the unconstrained trees using TempletonÕs significantly less parsimonious (SLPT ) test (Templeton, 1983). 2.5. Data congruence test For the assessments of congruence among the two data sets, we performed the character-based incongruence length difference test (or partition homogeneity test) of Farris et al. (1994), by using the Partition Homogeneity Test function of PAUP. The partition ITS vs. trnL–F was conducted on the combined data matrix with 1000 replicates of data partitions, a heuristic search with random sequence addition and TBR branch swapping.

2.3. Sequence alignment 2.6. Bayesian analyses Sequences of ITS and trnL–F were aligned with Clustal X applying the default parameters (Thomson et al., 1997) and then manually adjusted for indels otherwise not properly recognized by Clustal. The trnL–

To obtain a posterior probability distribution on trees, we performed the Bayesian analyses with a Markov-Chain Monte Carlo sampling method by using

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Table 2 Origin of samples, voucher information, and GenBank database accession numbers of DNA sequences of Lysimachia and related genera Subgenus

Section

Locality

Voucher

GenBank Accessions trnL–F

ITS

L. L. L. L. L. L. L. L.

alpestris Champ. ex Benth. candida Lindl. chenopodioides Watt ex Hook. f. christinae Hance ciliata L. circaeoides Hemsl. clethroides Duby (1) clethroides Duby (2)

Lysimachia Palladia Palladia Lysimachia Seleucia Palladia Palladia Palladia

Rosulatae Candidae Chenopodiopsis Nummularia

Hong Kong, China Hubei, China Yunnan, China Hubei, China Dublin, Ireland, cult. Hubei, China Kayashan, Korea n/a

Xing s.n. Hao 204 Han 3 Hao 203 Douglas s.n. Hao 201 Korea, Hao 228a n/a

AF547685 n/a AF547740 AF547690 AF547735 AF547689 AF547707 n/a

L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L.

confertifolia C.Y. Wu congestiflora Hemsl. crispidens Hemsl. davurica Ledeb. decurrens Forst deltoidea Wight engleri R. Knuth ephemerum L. fistulosa Hand.-Mazz. foenum-graecum Hance fordiana Oliv. fortunei Maxim glanduliflora Hanelt glutinosa Rock grammica Hance heterogenea Klatt inikii Marr insignis Hemsl. japonica Thunb. lancifolia Craib laxa Baudo lobelioides Wall. longipes Hemsl. (1) longipes Hemsl. (2) melampyroides R. Knuth microcarpa C.Y. Wu nemorum L.

Idiophyton Lysimachia Heterostylandra Lysimachia Palladia Lysimachia Idiophyton Palladia Lysimachia Idiophyton Lysimachia Palladia Palladia Lysimachiopsis Lysimachia Palladia Lysimachiopsis Idiophyton Lysimachia Idiophyton Idiophyton Palladia Lysimachia Lysimachia Lysimachia Idiophyton Lysimachia

Yunnan, China Sichuan, China Hubei, China Seoul, Korea, cult. Guangdong, China Yunnan, China Yunnan, China Dublin, Ireland, cult. Guangdong, China Yunnan, China Guangdong, China Guangdong, China Anhui, China Hawaii, USA Hubei, China Anhui, China Hawaii, USA Guangxi, China Guangdong, China Yunnan, China Yunnan, China Yunnan, China Anhui, China Anhui, China Hunan, China Yunnan, China Neuchatal, Switzerland

Hao 254 Hao 220 Hao 212 Hao 227a Ye 3980 Han 314 Peng s.n. Douglas s.n. Ye et al. 3561 Hao 250 Ye et al. 3940 Hao 279 Guo 20008 Marr 1304 Hao 209 Guo 20006 Marr s.n. Hao 245 Hao 278 Gong s.n. Han 6 Han 7 Guo 200012 Guo 200020 Deng 15945 Han 5 Yuan HG2

AF547742 AF547745 AF547775 AF547744 AF547781 AF547799 AF547762 AF396219* AF396220* AF547770 AF547749 AF547751 AF547761 AF547759 AF547777 AF547773 AF547798 AF547758 AF547771 AF547760 AF547763 AF547754 AF547785 AF547746 AF547753 AF547783 AF547767 n/a AF547772 AF547778 AF547776 AF547756 AF547757 AF547788 AF547774 AF547786

—

Miltandrae Spicatae Spicatae Apodanthera Nummularia —

Lysimachia Palladia Nummularia Oppositifoliae Ephemerum Nummularia Apodanthera Nummularia Spicatae Miltandrae —

Nummularia Miltandrae —

Idiophyton Nummularia Apodanthera Apodanthera Palladia Nummularia Nummularia Nummularia Apodanthera Lerouxia

AF547715 AF547694 AF547697 AF547706 AF547704 AF547719 AF547724 AF547720 AF547703 AF547716 AF547705 AF547688 AF547700 AF547738 AF547691 AF547699 AF547721 AF547712 AF547687 AF547717 AF547733 AF547741 AF547702 AF547723 AF547729 AF547732 AF547722

G. Hao et al. / Molecular Phylogenetics and Evolution 31 (2004) 323–339

Taxon

Lysimachia Idiophyton Lysimachia Lysimachia Lysimachia Idiophyton Lysimachia Idiophyton Lysimachia Lysimachia Lysimachiopsis Lysimachia Palladia Naumburgia Idiophyton Lysimachia

Nummularia Apodanthera Nummularia Nummularia Nummularia Apodanthera Nummularia Apodanthera Nummularia Verticillatum —

Nummularia Palladia —

Apodanthera Lysimachia

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

Cyclamen spinosa Mez. C. persicum Mill. C. purpurascens Mill. Glaux maritima L. (1) G. maritima L. (2) G. maritima L. (3) Maesa japonica Merrill Myrsine faberi Pipoly et C. Chen M. africana L. Pellitiera wildpretii B.Valdes

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

Soldanella pusilla Baumg. Theophrasta americana L. Trientalis europaea L.

—

—

—

—

—

—

Switzerland Guangxi, China Sichuan, China Sichuan, China Anhui, China Yunnan, China Sichuan, China Yunnan, China Anhui, China Dublin, Ireland, cult. Hawaii, USA Sichuan, China Yunnan, China Neuchatel, Switzerland Guangxi, China Dublin, Ireland, cult. n/a Neuchatel, Switzerland Guangxi, China Guangdong, China, cult. n/a Guangdong, China, cult. n/a n/a

Yuan s.n. Hao 239 Hao 224 Hao 218 Guo 20009 Hao 249 Hao 219 Hao 248 Guo 20007 Douglas s.n. Marr 283 Hao 226 Hu s.n. Yuan s.n. Hao 237 Douglas s.n. n/a Yuan HG3 Hao 235 Hao 231 n/a Hao 232 n/a n/a

AF547764 AF547765 AF547800 AF547747 AF547755 AF547768 AF547748 AF547769 AF547752 AF547779 AF547784 AF547750 AF547743 AF547787 AF547766 AF547782 AF402446* AF547792 AF547793 AF547796 AF402448* AF547795 AF402441* AF402442*

n/a Hubei, China, cult. n/a Zurich, Switzerland, cult. Ningxia, China n/a n/a Guangdong, China, cult. n/a n/a

n/a Hao 207 n/a Yuan s.n. Ge s.n. n/a n/a Hao 269 n/a n/a

AF402450* AF547794 AF402445* AF547789 AF547790 AF402444* AF402451* AF547797 AF402447* n/a

n/a n/a Quebac, Canada

n/a n/a Kupfer s.n.

AF402434* AF402449* AF547791

AF547708 AF547710 AF547695 AF547692 AF547701 AF547713 AF547693 AF547714 AF547698 AF547734 AF547737 AF547696 AF547686 AF547725 AF547711 AF547736 n/a AF547739 AF547709 AF547730 n/a n/a n/a AJ491416* AJ491660* n/a AF164011* n/a AF547726 AF547728 n/a n/a AF547731 n/a AJ491415* AJ491659* n/a n/a AF547727

G. Hao et al. / Molecular Phylogenetics and Evolution 31 (2004) 323–339

L. nummularia L. L. nutantiflora Chen et C.M. Hu L. omeiensis L. paridiformis Franch L. perfoliata Hand.-Mazz. L. petelotii Merr. L. phyllocephala Hand.-Mazz. L. pittosporoides C.Y. Wu L. pseudo-henryi Pamp. L. quadrifolia L. L. remyi Hillebr. ssp. kipahuluensis Marr L. rubiginosa Hemsl. L. taliensis Bonati L. thyrsiflora L. L. vittiformis Chen et C.M. Hu L. vulgaris L. Aegiceras corniculatum (L.) Blanco Anagallis arvensis L. f. arvensis Anagalis arvensis L. f. coerulea Baumg A. crenata Sims Ardisia speciosa Blume Ardisia gigantifolia Stapf Ardisiandra wettsteinii J. Wagner Asterolinon linum-stellatum (L.) Duby

Accession numbers marked with Ô*Õ were retrieved from GenBank, and the others were acquired during the present study (—, not applicable; cult., cultivated; n/a, not available).

327

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G. Hao et al. / Molecular Phylogenetics and Evolution 31 (2004) 323–339

MrBayes 2.01 (Huelsenbeck and Ronquist, 2001; Huelsenbeck et al., 2001) on the combined data matrix. Using one arbitrarily chosen MP tree, molecular evolution models were selected through the Hierarchical Likelihood Ratio Tests procedure as implemented in the software Modeltest (Version 3.06) (Posada and Crandall, 1998). Modeltest recommended the GeneralTime-Reversible model with invariable sites and rate heterogeneity (i.e. GTR + I + G model). One out of every 50 trees was sampled for one million generations of searches applying the recommended GTR + I + G model with six substitution rates, gamma shape, and invariable site proportion estimated during the search. No a priori assumptions about the topology of the tree were made. Three separate runs were performed to ensure that each of the searches were mixing well or searching randomly across the tree space landscape. Burn-in, the generation time for each parameter to reach the stationary state, was determined by visual inspection of the log-likelihood values. Posterior probabilities for each branch were calculated on the basis of the trees visited by the Markov chains well after the burn-in, by computing a consensus of all the post-burn-in trees with PAUP* v4.0b10 (Swofford, 2000).

these areas host only a minor part of the total number of species of Lysimachia, and previous studies suggest that these areas are less likely to be the ancestral areas for the majority of the genus (Chen and Hu, 1979; Hu, 1994). To provide a comparison with standard parsimony optimization, Dispersal-Vicariance analysis (DIVA) was also conducted to infer ancestral areas by using DIVA 1.1a (Ronquist, 1996, 1997). DIVA reconstructs ancestral areas by minimizing dispersal and extinction events needed to explain the observed distribution pattern based on an inferred fully resolved phylogeny, and vicariance is considered as the default mode of speciation. Terminal taxa were scored according to their distributions across the above five areas to generate the distribution data matrix, and this matrix was subsequently optimized onto the same tree used to illustrate the character optimizations. DIVA optimizations of both unconstrained and area constrained to a maximum of two were conducted following the reasoning of Ronquist (1996, 1997) and Donoghue et al. (2001).

2.7. Character-state optimization and biogeographic analyses

3.1. Sequence characteristics

Selected morphological characters were mapped onto the molecular phylogeny by using MacClade version 3.08 (Maddison and Maddison, 1992). One arbitrarily chosen MP tree based on combined analyses was used to illustrate the tracing. The zero-length branches of the tree were arbitrarily resolved to obtain dichotomies; this was to accommodate DIVA (see below), and had no effect on character optimizations. Character-state reconstruction assumed Fitch parsimony and accelerated transformation optimization (ACCTRAN) to minimize the independent gains of traits. Geographic distribution was also considered as an unordered multiple-state character and traced in the same way to locate ancestral areas. Five areas of endemism were designated on the basis of the results of Chen and Hu (1979) and Hu (1994): (1) Southeast Asia, comprising the Southeast Asian islands, the mainland Southeast Asian countries, the area of the southern and southwestern China, and the adjacent eastern Himalayas, (2) eastern and northeastern Asia, comprising northern (north of the Yangtze River) and northeastern China, Korea, Japan, and Russian Far East, (3) Europe and the Mediterranean region, (4) North America, and (5) Hawaii. Wide distributions (over more than two areas) were counted as polymorphism. Several species occur in the western Himalayas, Central Asia, India, Africa, and central to South America, but were not available for our study. Thus, these areas are not considered here. Nevertheless,

3. Results

All the newly acquired sequences have been submitted to GenBank (Table 2). The length of the unaligned trnL–F fragments ranges from 748 to 822 bp. The aligned sequences comprise 1023 sites. The alignment was straightforward and unambiguous except for three tandem repeat regions (in total 80 bp in the aligned data matrix). These ambiguously alignable sites were excluded in subsequent analyses. Gaps between 1 and 28 bp were introduced, of which 32 potentially informative indels were scored as binary characters regardless of their sizes and were added to the sequence data. The final trnL–F data set consisted of 1058 characters, of which 80 (7.6%) were excluded, 715 (67.6%) are constant, 96 (9.1%) are variable but uninformative, and 167 (15.8%) are informative. These data resulted in uncorrected pairwise sequence divergence values ranging from 0 to 11.5% among all taxa, and 0 to 6.0% among the ingroup taxa. No evidence of paralogous sequences was found for ITS sequences, because all PCR products were resolved as a single band and no double peaks were encountered in sequencing. The length of the unaligned ITS fragments ranged from 598 to 630 bp. The aligned ITS sequences had 664 characters, of which 91 (13.7%) involving alignment ambiguity (five regions of 10–29 bp) were excluded, 266 (40.1%) are constant, 70 (10.5%) are variable but uninformative, and 237 (35.7%) are potentially informative. This data set resulted in uncorrected pairwise sequence divergence values ranging from

G. Hao et al. / Molecular Phylogenetics and Evolution 31 (2004) 323–339

0 to 22.2% among all taxa, and 0 to 19.5% among the ingroup taxa. The alignments of the sequences were deposited in TreeBASE, and are also available from the corresponding author. 3.2. TrnL–F phylogeny The MP analyses of the trnL–F data set resulted in 240 equally most parsimonious trees (411 steps, consistency index [CI] ¼ 0.75 including autapomorphies, CI ¼ 0.66 excluding autapomorphies, and retention index [RI] ¼ 0.85). The strict consensus of these trees and the relative supports of clades are shown in Fig. 1-I. The trnL–F consensus tree is moderately resolved with several main clades highly supported, whereas the relationships among closely related taxa (e.g., within clades A, B, and G) are poorly resolved or received poor support. Lysimachia is paraphyletic, as the genus Glaux (clade D) nested deeply inside it. The close relationship between Anagallis and Asterolinon is highly supported, whereas the relationships among the main lineages are unresolved or received only weak support. When the phylogenetic reconstruction was constrained such that Lysimachia is monophyletic, the shortest trees were seven steps longer than the unconstrained trees. TempletonÕs (1983) SLPT test showed that the topology of the constrained trees significantly to marginally depart from the notion of sub-optimality (P ¼ 0:008 to 0:052) (Table 3). 3.3. ITS phylogeny MP analysis of the ITS data set resulted in 368 equally most parsimonious trees (1009 steps, CI ¼ 0.49 including autapomorphies, CI ¼ 0.45 excluding autapomorphies, and RI ¼ 0.77). The strict consensus of these trees and bootstrap clade support are shown in Fig. 1-II. The topology of the ITS strict consensus tree is similar to that based on trnL–F data, supporting clades A–H with slightly higher resolution within each main clade but with only weak supports toward the base of the tree. The monophyly of Lysimachia is not supported, because Glaux is nested deeply inside it. The genera Anagallis, Asterolinon, Pelletiera, and Trientalis form a weakly supported group that is weakly supported as sister to the Lysimachia-Glaux lineage. When the phylogenetic reconstruction was constrained for a Lysimachia monophyly, the shortest trees are nine steps longer than the unconstrained trees, but TempletonÕs SLPT test showed that the topology of the constrained trees did not significantly depart from sub-optimality (P ¼ 0.061 to 0.221) (Table 3). Clades A–H maintained the same composition as in the trnL–F trees, and all received strong support. However, the relationships among those clades resolved by the ITS data (Fig. 1-II) differed from

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those derived from the trnL–F data (Fig. 1-I) in two aspects: (1) in ITS trees the well-supported AnagallisAsterolinon-Pelletiera lineage grouped with Trientalis, together standing as sister to Lysimachia-Glaux (albeit with poor support), whereas in trnL–F trees, AnagallisAsterolinon lineage and Trientalis fell in a polytomy; (2) L. longipes (clade C) and Glaux (clade D) switched their positions in the ITS and trnL–F trees. However, in both respects bootstrap values were 6 73%. 3.4. Data congruence and combined phylogeny The combined ITS + trnL–F data matrix consisted of 58 taxa and 1719 characters (1055 trnL–F characters and 664 ITS characters), of which 171 (10.0%) were excluded, 1047 (60.9%) are constant, 140 (8.1%) are variable but uninformative, and 361 (21.0%) are informative. The ITS and trnL–F data set were found combinable on the basis of incongruence length difference test (P ¼ 0:79). Therefore, we conducted the combined analyses. The MP analysis on the combined data set resulted in 48 equally most parsimonious trees (1296 steps, CI ¼ 0.55 including autapomorphies, CI ¼ 0.49 excluding autapomorphies, and RI ¼ 0.78). The strict consensus of these trees and the clade supports are shown in Fig. 2. The topology of the strict consensus based on the combined data is nearly the same as that based on ITS data alone, but the bootstrap support are higher. Clades A–H were recovered with strong support. Lysimachia is paraphyletic as Glaux nested deeply inside it. Anagallis, Asterolinon, and Trientalis group together with weak support as the first diverging ingroup. Clade H diverges first within Lysimachia, followed by clade G. Glaux (clade D) groups with clade B of Lysimachia. When the MP reconstruction was constrained for Lysimachia monophyly, the shortest trees are 14 steps longer than the unconstrained trees. TempletonÕs (1983) SLPT test also showed that the topology of the constrained trees significantly to marginally departed from sub-optimality (P ¼ 0:013 to 0:079) (Table 3). Bayesian analyses were also conducted on the combined data for comparison with the MP results. The GTR + I + G model was found fit the combined data best. Parameters estimated for this model and the 95% credibility intervals are shown in Table 4. The burn-in of the Markov chain Monte Carlo occurred around the 6000th generation. To buffer these estimates, the credible posterior probability distribution was created from all samples taken after 12,000 generations. The 95% majority consensus (not shown) of all the post-burn-in trees highly resembles the combined MP strict consensus in topology. Three repeated runs generated the same clade supports. The significant posterior probabilities are plotted on the MP consensus (Fig. 2). The clades A– H all received significant supports (P ¼ 1:0). Only three

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I

60

trnL-F

61 95

61

86

A

95

93

55

B 52

53

57

56

C 62

D E F

99 63

95

94

93

85

98

60 82 68

G

84

91

H

100

94 100

99

53

59 100

91

100

L. chenopodioides L. decurrens L. lobelioides L. taliensis L. circaeoides L. glanduliflora L. ephemerum L. heterogenea L. clethroides (1) L. clethroides (2) L. fortunei L. candida L. glutinosa L. iniki L. remyi ssp. kip. L. crispidens L. christinae L. fordiana L. paridiformis L. congestiflora L. grammica L. deltoidea L. fistulosa L. phyllocephala L. rubiginosa L. japonica L. omeiensis L. melampyroides L. perfoliata L. pseudo-henryi L. longipes (1) L. longipes (2) Glaux maritima (1) Glaux maritima (3) Glaux maritima (2) L. davurica L. vulgaris L. thyrsiflora L. nemorum L. nummularia L. punctata L. confertifolia L. petelotii L. microcarpa L. pittosporoides L. vittiformis L. foerum-graecum L. nutantiflora L. insignis L. engleri L. alpestris L. lancifolia L. laxa L. ciliata L. quadrifolia Trientalis europaea Anag. arvensis f. a. Anag. arvensis f. c. Ast. linum-stellatum Pelletiera wildpretii Ardisia crenata Myrsine faberi Aegiceras corniculatum Myrsine africana Ardisia gigantifolia Ardisia speciosa Cyclamen persicum Cyclamen purpurascens Ardisiandra wettsteinii Theophrasta americana Clavija spinosa Maesa japonica Soldanella pusilla

86

II

76 59

ITS 100

97

99

A 74 100

100

B 73

82 99 57

100

100

C

D

100

97

74

E

99

F 53

60 66

95

G

100

H

100

56

100 98 100 100

Fig. 1. Comparison of strict consensus trees obtained from separate MP analyses of trnL–F (length ¼ 411, CI ¼ 0.75 including autapomorphies, CI ¼ 0.66 excluding autapomorphies, and RI ¼ 0.85) and ITS (length ¼ 1009, CI ¼ 0.49 including autapomorphies, CI ¼ 0.45 excluding autapomorphies, and RI ¼ 0.77). Numbers above the branches are bootstrap values supporting the corresponding branch when greater than 50%. Clades A– J are discussed in the text.

G. Hao et al. / Molecular Phylogenetics and Evolution 31 (2004) 323–339 Table 3 Results of TempletonÕs SLPT test on constrained monophyly of Lysiamchia against unconstrained optimal MP solutions. Data set

Additional steps required

P

trnL–F ITS Combined

7 9 14

0.008–0.052 0.061–0.221 0.013–0.079

P values are based on comparisons between all constrained trees with five randomly chosen unconstrained trees.

clades (as indicated by asterisks in Fig. 2) shown by the combined MP consensus with moderate bootstrap supports (68–73%) did not receive significant supports from Bayesian analyses (P < 0:95). Based on the high resolution and significant support of the main lineages, we considered the combined MP trees good estimations of the species phylogeny. We also conducted a MP analysis on the combined molecular data and 44 (out of an original 48) morphological characters used by Hao and Hu (2001). Fortyeight equally parsimonious trees of 1470 steps were recovered (CI ¼ 0.52 including autapomorphies and 0.46 excluding autapomorphies, RI ¼ 0.77). The strict consensus (tree not shown) was nearly identical to that from the combined molecular MP trees, the only difference being that Glaux fell within a polytomy consisting of clades A, B, and C, instead of grouping with clade B of Lysimachia. Bootstrap analyses did not provide any additional branch support compared to the analyses without the morphological characters. 3.5. Character-state mapping Four morphological characters, occurrence of colored gland on plant, flower merosity, filament fusion and position, and anther attachment, that were previously used to diagnose subdivisions in Lysimachia in combination with some other characters, were traced onto molecular trees based on the combined MP analyses. Shown in Fig. 3 is the optimization onto an arbitrarily chosen tree. Alternative tree topologies influence only the equivocal assignations of the character states. None of the characters examined exhibit unique synapomorphic character-state changes. All the four morphological characters show poor fit on the chosen molecular tree, with CI ranging from 0.17 to 0.33, RI 0.50 to 0.80, and the re-scaled CI 0.13 to 0.20 (Fig. 3). 3.6. Biogeographic analysis Optimizations of the five areas of endemism onto combined MP trees with standard Fitch parsimony analysis and DIVA revealed similar results (Fig. 4). The Fitch parsimony optimization infers Southeast Asia or Europe-Mediterranean area or North America as

331

equally possible ancestral state for the clades A–H. Clades A–D were inferred as being derived from Southeast Asia. Noticeably, the Hawaiian endemic clade and the Iberian Lysimachia ephemerum within clade A were both derived from Southeast Asia. The ancestral area was revealed as being either Europe-Mediterranean area or East and Northeast Asia for clade E, whereas it was revealed as Europe-Mediterranean area for clade F, Southeast Asia for clade G, and North America for clade H. Unconstrained DIVA optimization of ancestral areas required 30 dispersal events, whereas constrained optimization with the maximum area number allowed for each node as two required 31 dispersal events. The results of the constrained optimization are illustrated in Fig. 4. Because DIVA considers vicariance as the default model of speciation, it often reconstructs wide distribution for basal nodes (Ronquist, 1996, 1997). Here it optimizes Southeast Asia and North America together or both Europe-Mediterranean area and North America as possible ancestral areas for the most recent common ancestor (MRCA) of clades A–H. The divergence of the North American clade H from the rest clades was inferred as due to a vicariant event. The MRCA of clades A–D, and the MRCA of clade G are inferred as Southeast Asia origin. DIVA gives less certain inference on the ancestral areas of the MRCA of the clades A–F. Nevertheless, DIVA unambiguously infers Southeast Asia as the ancestral area for each of the clades A, B, C, and G. The divergences of the Hawaiian endemic clade and the Iberian L. ephemerum are inferred as vicariance between Southeast Asia and Hawaii and between Southeast Asia and Europe-Mediterranean area, respectively.

4. Discussion 4.1. Paraphyly of Lysimachia Previous studies based on both morphology and molecular data (although only very limited species of Lysimachia were sampled for molecular data) have suggested paraphyly of Lysimachia (Anderberg and St ahl, 1995; Hao and Hu, 2001; K€allersj€ o et al., 2000; Martins et al., 2003). Three species of Lysimachia included in the molecular phylogenetic study based on atp B, ndh F, and rbcL (K€allersj€ o et al., 2000) were shown to belong to two distinct clades, one of which grouped with Glaux and the other grouped with Anagallis. The phylogenetic analyses by Anderberg and St ahl (1995), Hao and Hu (2001), and Martins et al. (2003) also revealed grouping of the genera Anagallis, Glaux, and Trientalis with different species of Lysimachia. Our present data confirmed the paraphyly of Lysimachia with Glaux nested deeply inside it. Our trnL–F data alone failed to

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G. Hao et al. / Molecular Phylogenetics and Evolution 31 (2004) 323–339 section

90 1.00

72 1.00

100 1.00

55 100 1.00

100 1.00

A

62

95 1.00

100 1.00

L. chenopodioides L. decurrens L. lobelioides L. taliensis L. ephemerum L. circaeoides L. glanduliflora L. heterogenea L. clethroides L. fortunei L. glutinosa L. iniki L. remyi ssp. kip.

Palladia Ephemerum

73

B 100 1.00

82 1.00

59 99 1.00

73 *

71 1.00

78 1.00

D

Spicatae

Lysimachiopsis Heterostylandra

E F

77 1.00

100 1.00

97 1.00

100 1.00

100 1.00

L. davurica L. vulgaris L. thyrsiflora

99 1.00

L. nemorum L. nummularia L. punctata L. confertifolia L. petelotii L. microcarpa L. pittosporoides

91 1.00 92 1.00

61

100 1.00

L. nutantiflora L. insignis L. engleri L. alpestris

H

76 *

L. lancifolia L. laxa

100 1.00

90 1.00

Nummularia

Lysimachia

Nummularia Lysimachia Lysimachia Naumburgia Lerouxia Nummularia

L. ciliata L. quadrifolia

100 1.00 100 1.00

100 1.00

100 1.00

Lysimachia

Apodanthera Idiophyton

L. vittiformis L. foerum-graecum

93 1.00

75

G

L. omeiensis L. perfoliata L. pseudo-henryi L. phyllocephala L. rubiginosa

L. longipes (1) L. longipes (2)

69

68 *

L. christinae L. fordiana L. paridiformis L. congestiflora L. deltoidea L. fistulosa

L. melampyroides L. grammica Glaux maritima (1) Glaux maritima (2)

100 1.00

C

Palladia

Miltandrae

L. crispidens 67

subgenus

Chenopodiopsis

Idiophyton Oppositifoliae Rosulatae

Lysimachia

Apodanthera

Idiophyton

Verticillatae

Seleucia Lysimachia

Anagallis arvensis f. a. Anagallis arvensis f. c. Ast. linum-stellatum Trientalis europaea Ardisia crenata Myrsine faberi Cyclamen persicum

Fig. 2. Strict consensus of the trees generated from MP analyses on the combined trnL–F and ITS data (length ¼ 1296, CI ¼ 0.55 including autapomorphies, CI ¼ 0.49 excluding autapomorphies, RI ¼ 0.78). Numbers above the branches are bootstrap values supporting the corresponding branch when greater than 50%. Figures under the branches are the posterior probabilities supporting the corresponding branches obtained by Bayesian analyses. Asterisks indicate the three clades that did not receive significant Bayesian support (P < 0:95). At right are the subgenera and sections of the Lysimachia (see Table 1 and text for details). Clades A–J are the same as in Fig. 1.

T G

0.204 0.186–0.223 0.207 0.188–0.225

C A

0.306 0.283–0.328 0.659 0.437–0.916

C I

0.394 0.296–0.473 1.066 0.792–1.454

A<>C A<>G

2.160 1.712–2.768 0.702 0.499–0.972

A<>T C<>G

0.548 0.352–0.789

C<>T

4.441 3.503–5.601

G<>T

1.000 1.000–1.000 Mean 95% credible interval

Base frequencies Site rates Substitution rate

Table 4 Estimated parameters of molecular evolution for the combined trnLF and ITS data

0.283 0.262–0.303

G. Hao et al. / Molecular Phylogenetics and Evolution 31 (2004) 323–339

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resolve the basal polytomy. However, our ITS and combined data suggest the rest of the genera except for Glaux, including Anagallis, Asterolinon, Pelletiera, and Trientalis, all nested outside of Lysimachia, although such resolutions are only weakly or moderately supported by MP bootstrap values (Figs. 1 and 2). Glaux is a widely accepted monotypic genus, distributed in northern temperate areas. It is distinctive from Lysimachia in having apetalous flowers. The petals of the genera Asterolinon (1 species in the Mediterranean region, and 1 in Africa) and Pelletiera (1 species in South America, and 1 species in Macaronesia) are also reduced to various extents. Our combined analyses, however, placed Glaux with the main part of section Nummularia of subgenus Lysimachia, and revealed that Asterolinon and Pelletiera are closely related to Anagallis instead of Lysimachia. Anagallis has ca. 20 species, occurring in Africa, Europe, and South America. In its gross morphology, Anagallis closely resembles Lysimachia, except for its circumscissile capsule versus the valvular capsule of Lysimachia. This led Anderberg and St ahl (1995) to suggest that Anagallis is a monophyletic ingroup in Lysimachia. In a recent molecular study of Primuloid families by K€allersj€ o et al. (2000), where three Lysimachia species were sampled, Anagallis joins Lysimachia nemorum in a group with 100% support, which in turn becomes the sister to a clade containing Glaux and another two Lysimachia species, with Trientalis in turn as their sister group. Our results did not suggest a close relationship between Anagallis and L. nemorum. Instead, Anagallis was confirmed to closely relate to Asterolinon, as revealed recently by Martins et al. (2003). The genus Trientalis comprises two species that are widely distributed in northern temperate areas. Its flowers are usually 7-merous and solitary in the axils of subterminal leaves. Anderberg and St ahl (1995) suggested that Trientalis is probably a monophyletic clade belonging to the paraphyletic Lysimachia. The 6–8merous corolla is also found in some species of Lysimachia, such as those of subgenera Lysimachiopsis and Naumburgia (Fig. 3). The pseudo-verticillate leaves also occur in several species of Lysimachia, (e.g., Lysimachia paridiformis, Lysimachia insignis, and Lysimachia sciadophylla). Our results did not definitively resolve the phylogenetic position of Trientalis: while trnL–F data failed to resolve its position, ITS and combined data suggested that it is closely related to Anagallis, Asterolinon, and Pelletiera although the bootstrap support is low (Figs. 1 and 2). 4.2. Implications of the molecular phylogenies on the infrageneric classification of Lysimachia Our data do not corroborate the current infrageneric classification of Lysimachia, because some core

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G. Hao et al. / Molecular Phylogenetics and Evolution 31 (2004) 323–339 steps 6 3 9 4 CI 0.17 0.33 0.22 0.25 RI 0.78 0.50 0.76 0.80 RC 0.13 0.17 0.17 0.20

plant with colored glands plant without colored glands flower 5-merous flower 6–8-merous filament free, adnate filament fused filament fused and connate with corolla anther dorsifixed anther basifixed

A

B

D C E F

L. chenopodioides L. decurrens L. lobelioides L. taliensis L. circaeoides L. glanduliflora L. ephemerum L. heterogenea L. clethroides L. fortunei L. glutinosa L. remyi ssp. kip. L. iniki L. crispidens L. christinae L. fordiana L. paridiformis L. congestiflora L. deltoidea L. perfoliata L. pseudo-henryi L. omeiensis L. fistulosa L. rubiginosa L. phyllocephala L. melampyroides L. grammica Glaux maritima (1) Glaux maritima (2) L. longipes (1) L. longipes (2) L. davurica L. vulgaris L. thyrsiflora L. nemorum L. nummularia L. punctata L. confertifolia L. petelotii L. microcarpa L. pittosporoides L. vittiformis L. foerum-graecum

G H

L. insignis L. nutantiflora L. engleri L. alpestris L. lancifolia L. laxa L. ciliata L. quadrifolia An. arvensis f. a. An. arvensis f. c. Ast. linum-stellatum Trientalis europaea Ardisia crenata Myrsine faberi Cyclamen persicum

Fig. 3. An arbitrarily chosen MP tree generated from the combined trnL–F and ITS data illustrating optimization of selected morphological characters. On the right side of the tree, the states of the four morphological characters are plotted for each species. Optimization of character state changes is indicated directly on the branches as short bars. Double bars separating two character states indicate equivocal solutions. Showing on the upper right of the figure are the measurements of fitness of the characters on the tree, including steps, consistency index (CI), retention index (RI), and re-scaled consistency index (RC).

G. Hao et al. / Molecular Phylogenetics and Evolution 31 (2004) 323–339

♥

♥ ♣ ♦ ♠

♥

SE Asia E & NE Asia

♥ ♥♦

Europe & Medit. N America
Hawaii

♦

♥ ♥ ♥





♥

A




♥ ♥ ♥ ♥

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♥♦♣

♥

♥

♥ ♥

♥

♥

♥♣ ♥♦ ♥♠

♥ ♥

♥♣ ♥♦

♦♣♠

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♦ ♥♦ ♣♦

♦♣

E

♦

F ♥♦

♣ ♦

♥

♥ ♥♦

♥

♥ ♥

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♥♦

♥ ♦ ♥♦ ♥♠ ♦♠

L. foerum-graecum

♥

♥♠ ♦♠

♥

G ♠ ♦ ♥♦ ♦♠

L. chenopodioides L. decurrens L. lobelioides ♥ L. taliensis L. circaeoides ♥ L. glanduliflora L. ephemerum L. heterogenea L. clethroides ♥ L. fortunei L. glutinosa
L. remyi ssp. kip. L. iniki L. crispidens L. christinae L. fordiana ♥ L. paridiformis L. congestiflora L. deltoidea L. perfoliata ♥ L. pseudo-henryi L. omeiensis L. fistulosa L. rubiginosa L. phyllocephala L. melampyroides L. grammica Glaux maritima (1) ♣ ♦♠ Glaux maritima (2) L. longipes (1) ♥ L. longipes (2) L. davurica ♣ ♥♣ ♣♦ L. vulgaris ♣♦ L. thyrsiflora L. nemorum ♦ L. nummularia L. punctata L. confertifolia ♥ L. petelotii L. microcarpa L. pittosporoides ♥ L. vittiformis

♥

♥

♥

H ♦

♠

♠

♦ ♠ ♦♠

♦ ♦♠

♥ ♥♦

♦

♦ ♠

♥

L. insignis L. nutantiflora L. engleri L. alpestris L. lancifolia L. laxa L. ciliata L. quadrifolia An. arvensis f. a. An. arvensis f. c. Ast. linum-stellatum Trientalis europaea Ardisia crenata Myrsine faberi Cyclamen persicum

335

♥ ♥ ♥ ♥ ♥ ♥♣ ♦ ♥♣ ♥♣ ♥♣




♥ ♥♣ ♥ ♥ ♥♣ ♥ ♥ ♥♣ ♥ ♥ ♥ ♥ ♥♣ ♥ ♣♦♠ ♣♦♠ ♥ ♥ ♥♣ ♣♦♠ ♣♦♠ ♦ ♦ ♦ ♥ ♥ ♥ ♥ ♥ ♥ ♥ ♥ ♥ ♥ ♥ ♥ ♠ ♠ ♥♣♦♠ ♥♣♦♠ ♦ ♣♦♠ ♥ ♥ ♦

Fig. 4. An arbitrarily chosen MP tree generated from the combined trnL–F and ITS data illustrating the ancestral areas inferred from Fitch parsimonious analyses and DIVA. On the right side of the tree, the geographic distributions of each terminal taxon are plotted. Results based on Fitch parsimony are shown by symbols above the clades, and character-state changes are indicated directly on the branches as short bars. Results based on constrained DIVA are shown on the right of each internal node. Double bars separating two character states or combinations of states indicate that the character states at both sides of the bars are the equally parsimonious solutions.

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infrageneric groups, such as subgenera Lysimachia and Idiophyton, sections Nummularia and Apodanthera are para- or polyphyletic (Fig. 2). Subgenus Lysimachia was revealed as a heterogeneous assembly of at least five lineages. Subgenus Idiophyton was shown to be paraphyletic with respect to section Rosulatae of subgenus Lysimachia. The monotypic subgenera Naumburgia and Seleucia each grouped with a clade of subgenus Lysimachia. The Hawaiian endemic subgenus Lysimachiopsis is monophyletic. The sampled three species (out of 16) formed a highly supported clade (bootstrap 100%) sister to all sampled species of subgenus Palladia. The monotypic subgenus Heterostylandra is sister to the clade comprising of subgenera Lysimachiopsis and Palladia. Our phylogenies did not offer any evaluation on the monophyly of the North American subgenus Seleucia, for which we were able to sample only one species (out of seven). At sectional level, section Nummularia of subgenus Lysimachia and section Apodanthera of subgenus Idiophyton are paraphyletic. The former comprises at least three distinct lineages, and the latter comprises two. All the rest of the sampled sections are either monophyletic, monotypic, or with only one species sampled.

within the genus. In a previous phylogeny based on only morphological data (Hao and Hu, 2001), L. crispidens was sister to all the remaining Lysimachia species sampled and some other related genera, but with only poor support. However, our results place L. crispidens as sister to the clade comprising subgenera Palladia and Lysimachiopsis with strong support. L. crisipidens was found to have the same type of pollen as subgenus Palladia (Bennell and Hu, 1983). The heterostylandry shown by this species may have implications on the origin of the well-known heterostyly of Primulaceae (Handel-Mazzetti, 1928).

4.2.1. Clade A: subgenera Palladia, Lysimachiopsis, and Heterostylandra Subgenus Palladia is characterized by white flowers with filaments free and adnate halfway up the corollatube. Palynologically it is relatively homogeneous, with most of its species having strongly prolate pollen. The monophyly of this subgenus is supported with our results. The monophyly of the Hawaiian endemic subgenus Lysimachiopsis was suggested previously (Marr and Bohm, 1997, 1999). The apomorphic characters supporting the monophyly of this subgenus include shrub habit, red and 5–10-merous flowers, and tetracolporate pollen (Bennell and Hu, 1983; Marr and Bohm, 1997, 1999). Its closest relatives have been suggested to be Lysimachia alpestris (subgenus Lysimachia section Rosulatae) (Handel-Mazzetti, 1928) or Lysimachia laxa (cf. Marr and Bohm, 1997), hypotheses not supported by our study. Our phylogenies place the monotypic subgenus Heterostylandra as the first-diverging clade within clade A (Figs. 1 and 2). The sole species of this subgenus (Lysimachia crispidens) was previously considered as a section of subgenus Palladia by Handel-Mazzetti (1928). Chen and Hu (1979) elevated it to the level of subgenus, however, as it possessed many unique characters, such as rosulate leaves, scapiform stem, long corolla tube, and unique heterostylandry (with two types of flowers: short filament and style type versus long filament and style type). Chen and Hu (1979) considered this species as the most recently derived

4.2.3. Clade C and D: L. longipes and Glaux The two accessions of L. longipes (clade C) and the accessions of G. maritima (clade D) were resolved differently by trnL–F and ITS data: their positions relative to clades A and B reversed in these two phylogenies (Fig. 1). Such incongruence has been confirmed by sampling multiple accessions for both taxa, by repeated sequencing, and even by including in our analyses the independently acquired sequence data retrieved from GenBank. Whether this incongruence was due to random error or any biological consequence remains unknown. In both gross and pollen morphology, L. longipes shows high similarity to other members of subgenus Lysimachia section Nummularia (Bennell and Hu, 1983; Chen and Hu, 1979).

4.2.2. Clade B: subgenus Lysimachia section Nummularia p. p. Clade B comprises of 13 species of subgenus Lysimachia, all from section Nummularia, the largest section in the genus. The apomorphies that define this group include 5-merous flowers; anthers shorter than filaments, versatile, opening by lateral slits; pollen tricolporate, medium-sized, prolate-spheroidal to subprolate, with a smooth partial to fine reticulate tectum. Our results do not offer well-supported resolution of species relationships within this group.

4.2.4. Clade E: subgenus Lysimachia section Lysimachia and subgenus Naumburgia The two species sampled (out of four) of subgenus Lysimachia section Lysimachia (Lysimachia vulgaris and Lysimachia davurica), constituted a well-supported monophyletic group that is sister to the monotypic subgenus Naumburgia (Lysimachia thyrsiflora) (Fig. 2). The monotypic subgenus Naumburgia, widely scattered in north temperate regions, is distinct from others in gross morphology (including flowers usually 6–7 merous, in pedunculate, capitate or spike-like racemes). Nowicke and Skvarla (1977) elevated it to generic level. Bennell and Hu (1983) revealed that the pollen morphology of L. thyrsiflora was similar to that of L. vulgaris and L. davurica in being tricolporate, prolate-spheroidal to

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subprolate, and with a partial tectum. Our results confirm this close relationship. 4.2.5. Clade F: subgenus Lysimachia section Lerouxia and section Nummularia p. p. Three European species from subgenus Lysimachia (L. nemorum in section Lerouxia, and another two [Lysimachia punctata and Lysimachia nummularia] in section Nummularia), form a well-supported monophyletic group. Section Lerouxia currently contains only four European species. Morphologically, species of clade F are very similar to those of the clade B, the major part of section Nummularia. The distinct position of clade F suggested here needs further studies for better understanding of its relationship with clade B. 4.2.6. Clade G: subgenus Idiophyton and part of subgenus Lysimachia In Handel-MazzettiÕs (1928) treatment, section Apodanthera, with ca. 36 species, was placed in subgenus Lysimachia. Chen and Hu (1979) transferred it to subgenus Idiophyton based on shared morphological characters such as apically clustered leaves, lateral racemes, ligneous stem, short filaments, and large basifixed anthers mostly dehiscent by apical pores. Our data support this treatment. Section Rosulatae consists of two species (L. alpestris and Lysimachia rupestris) indigenous to the coastal regions of southern China. Handel-Mazzetti (1928) allied them with the Hawaiian section Cilicina ( ¼ subgenus Lysimachiopsis) to form subgenus Lysimachiopsis based on its spirally aggregated leaves forming nearly rosettes, a feature unique in Lysimachia. Chen and Hu (1979) assigned this section to subgenus Lysimachia based on the presence of short filaments, and suggested that it could be derived directly from section Apodanthera of subgenus Idiophyton based on pollen morphology (Bennell and Hu, 1983). Our data confirm its close relationship with subgenus Idiophyton. Besides the shared pollen morphology as mentioned by Bennell and Hu (1983), this section does not bear colored glands, suggesting affinity with subgenus Idiophyton and distinction from most of subgenus Lysimachia (Fig. 3). If so, this could imply that the relative length of filament and anther is not always a good phylogenetic character. 4.2.7. Clade H: Lysimachia ciliata and Lysimachia quadrifolia Lysimachia ciliata is the only representative species (out of seven) sampled from subgenus Seleucia, and L. quadrifolia is the only representative species (out of six) sampled from subgenus Lysimachia section Verticillatae; both are endemic to North America. These species form a highly supported clade. Subgenus Seleucia is characterized by the presence of staminodia (sterile filaments), supervolute corolla lobes, each lobe enclosing a

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fertile stamen, and by opposite and glabrous leaves (Coffey and Jones, 1980). Largely due to the salient feature of staminodia, Seleucia was treated as a distinct genus by several earlier authors (e.g., Britton and Brown, 1897; Gleason, 1952; Gray, 1848; Small, 1933). The taxonomic importance of staminodia, however, has been a matter of controversy in the systematics of Lysimachia. Handel-Mazzetti (1928) argued that some Chinese species (e.g., Lysimachia deltoidea, Lysimachia remota, and Lysimachia fistulosa) show transition in all features, and therefore it is not reasonable to treat Seleucia as a distinct genus; he consequently reduced it to a section of subgenus Lysimachia, with two subsections: Seleucia (Steironema) (six species, including L. ciliata), and Verticillatae (five species, including L. quadrifolia). The latter subsection is characterized by flowers that are axillary or in terminal racemes, yellow, and 5-merous, and could be confidently placed in subgenus Lysimachia. Ray (1956) elevated subsection Seleucia to the subgeneric rank. It was noted that if the staminodia were ignored, species of subgenus Seleucia could naturally fall into subgenus Lysimachia (Douglas, 1936; Ray, 1956). Bennell and Hu (1983) revealed that species of subsection Seleucia have a distinct pollen type with a triangular ambit and unique colporoidate aperture in Lysimachia, which seems in favor of RayÕs (1956) treatment in treating this group of species as a separate subgenus. Species of subsection Verticillatae, which lack staminodes, however, have pollen morphology similar to subgenus Lysimachia section Lysimachia. This implies that Verticillatae should be excluded from subgenus Seleucia, and retained as a section of subgenus Lysimachia (Bennell and Hu, 1983). The sister relationship of the two North American species sampled is highly supported by both trnL–F and ITS data (Fig. 1), confirming Handel-MazzettiÕs (1928) suggestion that staminodia are not reliable phylogenetic characters in Lysimachia. 4.3. Character evolution None of the four characters we traced showed unique synapomorphic changes and all showed poor fit when optimized onto the molecular trees. Multiple gains and parallel evolution were conspicuous (Fig. 3). Floral morphology, particularly the structure of the androecium, was considered by Handel-Mazzetti (1928) as the most important criterion for classifying Lysimachia into subgenera. He suggested the following evolutionary trends: stamens with short and wide filaments, and large, basifixed anthers represent the ancestral primitive type, whereas stamens with slender filaments, and small, dorsifixed anthers were derived; and the further fusion of the filament tube with the corolla represented the most advanced stage. Most later authors (e.g., Chen and Hu, 1979; Marr and Bohm, 1997) accepted this

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interpretation. Whereas our data do not offer insight on which type of filaments, fused or fused and further connate with corolla, is plesiomorphic for the MRCA of clades A–H, free and adnate filaments are inferred as derived. Our data also inferred the dorsifixed anther as plesiomorphic state for the MRCA of clades A–H, and parallel gains from dorsifixed to basifixed anthers are apparent (Fig. 3). Whereas the 5-merous flower was shown to be plesiomorphic, 6–8-merous flowers were shown to develop independently in the Hawaii subgenus Lysimachiopsis clade (Lysimachiopsis iniki, Lysimachiopsis remyi, and Lysimachiopsis glutinosa), in L. thyrsiflora, and in Trientalis. The colored glands are inferred as an apomorphic state. However, multiple gains of colored glands and the reversals are also suggested (Fig. 3). 4.4. Biogeographic implications of the molecular phylogeny Lysimachia and Anagallis occur throughout temperate regions of the Northern Hemisphere and into the southeastern Asian tropics, with some representatives in Africa and South America. Glaux and Trientalis are also widespread in boreal temperate regions (Bennell and Hu, 1983; Chen and Hu, 1979; Hu, 1994; Hu and Kelso, 1996). In considering the extraordinarily high species diversity and the distribution of the morphologically primitive types in these areas, Chen and Hu (1979) and Hu (1994) suggested that southwestern China (the area covering the provinces of Yunnan, Sichuan, Hubei, Guangxi and Guizhou) and adjacent regions of northern Indochina form the center of origin and primary diversification of Lysimachia. This suggestion is not fully congruent with the paleobotanical evidence, however, as the only known fossil record of Lysimachia consists of the fossil seeds from the late Middle Miocene of Jutland, Denmark (Friis, 1985). These fossils have been considered most similar to the seeds of the extant L. vulgaris, which is widespread in northern Africa, central and southwestern Asia, and boreal Eurasia and North America. The lack of any fossil record from other regions limits the use of fossil data for reliable biogeographic inference. Our analyses based on Fitch parsimony and DIVA do not offer clear inference on the overall historical biogeography of Lysimachia. The biogeographic resolution for the MRCA of the clades A– H is less certain. Standard Fitch parsimony suggests that Southeast Asia, Europe-Mediterranean area, or North America are equally possible as the ancestral area, whereas DIVA suggests Southeast Asia plus North America or Europe-Mediterranean area plus North America as the possible ancestral area for the MRCA of clades A–H. Nevertheless, Southeast Asia is confirmed as the ancestral area for each of the clade A, B, C, and G. The single origin of the Hawaiian endemic species is supported by several lines of evidence, including our

data. Handel-Mazzetti (1928) suggested that L. alpestris from southern China was closely allied to the Hawaiian endemics. Wagner et al. (1990) and Marr and Bohm (1997, 1999) have suggested Malaysian affinities for HawaiiÕs endemic species, and the latter authors have further suggested a close connection between Hawaii endemics and species similar to L. laxa of the subgenus Idiophyton. Our results suggest the Southeast Asian origin of the Hawaiian clade and the sister relationship with subgenus Palladia instead of subgenus Idiophyton. The Iberian L. ephemerum is also confirmed as of Southeast Asian origin (Fig. 4).

Acknowledgments The authors thank Dr. Richard Saunders, for critical reading of the manuscript, and the following people for providing plant materials: Philippe K€ upfer, Kendrick Marr, Xinhu Guo, Gerry Douglas, Huagu Ye, Fuwu Xing, Ronglan Han, Xun Gong, and Yunfei Deng. We are very much indebted to anonymous reviewers for their comments and suggestions. This study was financially supported by the National Science Foundation of China (39970053), the Knowledge Innovation Project (kscxz-sw-101A), and Field Frontiers Project (Director Foundation of South China Institute of Botany) of Chinese Academy of Sciences (to G. Hao), and the Hundreds of Talents Program of Chinese Academy of Sciences (to Y.-M. Yuan).

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