PHYLOGENETICS OF LYTHRACEAE SENSU LATO

Int. J. Plant Sci. 163(2):215–225. 2002. 䉷 2002 by The University of Chicago. All rights reserved. 1058-5893/2002/16302-0002$15.00

PHYLOGENETICS OF LYTHRACEAE SENSU LATO: A PRELIMINARY ANALYSIS BASED ON CHLOROPLAST rbcL GENE, psaA-ycf3 SPACER, AND NUCLEAR RDNA INTERNAL TRANSCRIBED SPACER (ITS) SEQUENCES Ye-lin Huang* and Su-hua Shi1,*,† *Key Laboratory of Gene Engineering of the Ministry of Education, Zhongshan University, Guangzhou 510275, Guangdong, People’s Republic of China; and †School of Life Sciences, Zhongshan University, Guangzhou 510275, Guangdong, People’s Republic of China

Phylogenetic relationships of Lythraceae sensu lato (s.l.) were investigated by parsimony and likelihood analyses of 85 accessions representing 23 species, 16 genera that have been assigned to the family at various times. The three data sets, including the chloroplast rbcL gene, the psaA-ycf3 spacer, and the nuclear internal transcribed spacer (ITS; including the 5.8S ribosomal gene), are highly congruent on the basis of the partition homogeneity test. No significantly long branches are detected by relative apparent synapomorphy analysis. Phylogenetic analyses based on the combined data strongly support the monophyly of the Lythraceae s.l., in which the satellite genera Dubanga, Punica, Sonneratia, and Trapa were included, with Onagraceae and Combretaceae as outgroups. Paraphyly of subfamily Lythroideae (p Lythraceae sensu stricto) is proposed with the other four monotypic subfamilies nested within. The analysis further supports the sister relationship between Sonneratia and Trapa instead of Duabanga and Sonneratia, the latter of which have traditionally been treated as constituting the ditypic family Sonneratiaceae sensu Engl. & Gilg. The main clades within Lythraceae s.l. exhibit relatively weak support. Terminal relationships among genera, however, have strong bootstrap support. Keywords: Lythraceae sensu lato, phylogeny, rbcL, psaA-ycf3 spacer, ITS regions.

Introduction

Angiosperm Phylogeny Group 1998; Shi et al. 2000; Thorne 2000) suggests that Lythraceae s.str. and the small satellite families form a monophyletic group, which supports a broader delimitation of the family. The Lythraceae sensu lato (s.l.) comprise small to large trees, shrubs, and perennial and annual herbs adapted to a wide variety of vegetation types, including mangrove swamps, rain forests, seasonally dry savannas, coastal dunes, freshwater marshes, and shallow waters of ponds and rivers (Graham et al. 1993a). All members of the family share numerous specialized features with the rest of the order Myrtales: intraxylary phloem; vestured pitting of vessel elements; minute, divided, axillary stipular processes; brochidodromous leaf venation; ellagic acid–rich tissues; and a suite of embryological features. Cytological work has demonstrated a basic chromosome number of xp8 of the family (Graham et al. 1993a, 1993b). Four genera having xp5 (Diplusodon, Lythrum, Nesaea, and Peplis) are thought to be derived from an ancestral xp8 (Graham 1992; Graham et al. 1993b). Duabanga, Sonneratia, and Trapa differ from the remainder of the family in having xp12. Five genera (Decodon, Duabanga, Lagerstroemia, Pemphis, and Punica) are apparently ancient polyploids (for review, Graham et al. 1993a). Koehne’s (1903) monograph of Lythraceae, which proposed two tribes distinguished by complete septation of the ovary (Nesaeeae) versus incomplete septation (Lythreae), remains the definitive reference on the intergeneric classification of the Lythraceae s.str. Further studies, including morphology (Graham et al. 1993a), palynology (Lee 1979; Graham et al. 1987, 1990), and anatomy (Baas and Zweypfenning 1979), fail to

The Lythraceae as defined by Dahlgren and Thorne (1984) and Graham et al. (1993a) comprise 31 mostly highly distinctive genera and more than 600 species. This family is distributed worldwide, especially in mesophytic to wet habitats of the subtropics and Tropics. Lythraceae have traditionally been allied with Onagraceae, both of which were placed together in the order Myrtales on the basis of morphological and anatomical evidence (Dahlgren and Thorne 1984; Johnson and Briggs 1984), and this treatment has been largely supported by recent molecular studies (Conti et al. 1996, 1997; Angiosperm Phylogeny Group 1998). The delimitation of the Lythraceae has been problematic historically. Whereas a general consensus exists that the genera of the Lythraceae sensu stricto (s.str.) are closely related, there has been considerable disagreement as to whether to include the satellite genera Duabanga, Punica, and Sonneratia in the Lythraceae, all of which have been placed in separate families at various times (Koehne 1903; Melchior 1964; Hutchinson 1973; Dahlgren 1975; Takhtajan 1980, 1986; Cronquist 1981; Thorne 1981, 1983; Tobe and Raven 1983; Dahlgren and Thorne 1984; Johnson and Briggs 1984). Thorne also suggested the inclusion of a fourth satellite genus, Trapa, as a subfamily of Lythraceae. Recent morphological and molecular research (Graham et al. 1993a, 1998; Conti et al. 1996, 1997; 1 Author for correspondence; telephone 86-20-84113677; fax 8620-84113652; e-mail [email protected].

Manuscript received June 2001; revised manuscript received September 2001.

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support these tribal divisions. A morphological analysis of the Lythraceae s.l. (excluding Trapa) conducted by Graham et al. (1993a) provided the first phylogenetic estimate of the family based on modern cladistic methods. Two major clades, distinguished primarily by anthotelic/blastotelic inflorescences and wet/dry stigmas, were recognized on the basis of cladistic analyses using 26 characters from anatomy, floral morphology, pollen, and seed morphology. Internal branches, however, are weakly supported by only seven nonhomoplasious characters. The level of homoplasy in the family is higher than average for a plant group based on morphological characters (Graham et al. 1993a). To provide new sources of information for elucidating relationships in this family, we generated DNA sequence data from two cpDNA regions and one nuclear region: rbcL and PY-IGS (the intergenic spacer between the psaA and ycf3 genes) of chloroplast DNA and the internal transcribed spacer (ITS) of nuclear ribosomal DNA. The two noncoding regions (PY-IGS and ITS) were used to complement the weak resolution among species in the rbcL cladogram of Conti et al. (1997) because noncoding sequences tend to evolve faster than coding sequences and may provide more information for phylogeny reconstruction. We included representatives of all five subfamilies of Lythraceae defined by Graham et al. (1998) and Thorne (2000) (Duabangoideae, Punicoideae, Sonneratioideae, Trapoideae, and Lythroideae p Lythraceae s.str.) from Eurasia, Africa, and America in this study. Our main objectives were to (1) evaluate the relative utility of the different sequences for a preliminary phylogenetic analysis of Lythraceae s.l.; (2) reexamine the classification of several uncertain taxa in the light of our new sequence data, especially the status of the five subfamilies in Lythraceae s.l.; and (3) provide additional information for the assessment of relationships among the genera within the family.

Material and Methods We followed the classification scheme of Thorne (2000). Taxa sampled and voucher information, literature citations, and GenBank accession numbers for the three data sets are listed in table 1. Our sampling focused mainly on the Eurasian species, including those from the four genera that were formerly placed in the satellite families Duabangaceae, Punicaceae, Sonneratiaceae, and Trapaceae, and those from 14 genera of the traditionally recognized Lythraceae, four of which are endemic to the Americas or Africa. The phylogenetic hypotheses that have been proposed for the order Myrtales provide a basis for choosing appropriate outgroups for systematic analysis of the Lythraceae s.l. Sequences from the chloroplastencoded rbcL gene (Conti et al. 1996, 1997) suggest that Lythraceae s.l. is sister to Onagraceae, but, as the authors indicated, more extensive taxon and gene sampling was needed before detailed relationships could be inferred. Therefore, Ludwigia hyssopifolia and Fuchsia hybrida cultivar (Onagraceae) and Combretum wallichii and Quisqualis indica (Combretaceae) of Myrtales were used as outgroups for the parsimony analyses. Total genomic DNAs were extracted from fresh and silicadried leaves using the CTAB procedure (Doyle and Doyle 1987), which was followed by purification with a DNA purification system (DPS) kit made by our laboratory. In four

cases (Decodon, Nesaea, Pemphis, and Peplis), PCR products from herbarium specimens were obtained by following our modified method for preparation of template DNA from herbarium samples. The procedure basically follows the general glass powder purification protocol (GlassMax DNA Isolation Matrix System GibcoBRL, 15549-017). However, the final glass precipitate is directly used as the template for PCR. The same accessions of total DNA were used for sequencing ITS, rbcL, and PY-IGS, except for some sequences of Fuchsia, Lythrum, Punica, and Quisqualis, which were retrieved from GenBank (see table 1). The rbcL gene was amplified by using primers PR1f and PR2r (see table 2). Besides amplifying primers, two interior primers, PR3r and PR4f, were designed for sequencing on the basis of the rbcL gene sequence of Lythrum hyssopifolia (Conti et al. 1996). PR3r is located at positions 729–750 in the rbcL gene, and PR4f is located at positions 712–730. The intergenic spacer between the psaA and ycf3 genes (PYIGS) is located from positions 43,487 to 44,238 in the chloroplast DNA of tobacco (Nicotiana tabacum) (Shimada and Sugiura 1991). PCR was performed by using PG1f and PG2r (see table 2), both for amplifying and sequencing under the same conditions as those for ITS amplification. PG1f is located 48–78 bp downstream from the 5 end of the psaA gene. PG2r is 29–59 bp upstream from the 3 end of the ycf3 gene (K.-J. Kim, personal communication). Sequences of the primers used in this study are listed in table 2. The ITS region (including the ITS-1 and ITS-2 spacers and the 5.8S gene) were amplified by following Wen and Zimmer (1996). The primers for amplifying the ITS regions were ITS4 and ITS5. Sequencing primers were C5.8S, ITS4, N5.8S, and N18L18 (Wen and Zimmer 1996). A major concern when dealing with ribosomal DNA is whether the sequences employed are orthologous or paralogous. In this study, we conducted a pilot project to assess whether different ITS copies exist in our chosen taxa and also tried to assess the amount of infraspecific variation that might be expected for the ITS region. The PCR products of most samples were purified by using the QIAquick PCR purification kit (CN 28104, QIAGEN) and sequenced by using an ABI 377 Genetic Analyzer (Applied Biosystems). For partial samples, both DNA strands were sequenced manually with the Sequenase Version 2.0 DNA sequencing kit (US70770 Amersham) and alpha 35S-dATP as a radioactive tracer and compared with all taxa to ensure accuracy. Sequences were aligned with Clustal X software (Thompson et al. 1997) and further modified manually. In most cases, the placement of gaps was straightforward. Two regions, however, one in ITS-1 (bp 60–124) and the other in ITS-2 (bp 455–513), were difficult to align. The problematic regions were excluded or partitioned equally between the ingroup and outgroup as nonoverlapping separate indels. The latter was expected to reduce the probability of incorrect homology assessment among families while retaining potential phylogenetic information within each family (Karol et al. 2000). Although our results showed the same tree topologies in the above two alignment options with minor differences among their bootstrap values, we adopt the conservative treatment in the following

HUANG & SHI—PHYLOGENETICS OF LYTHRACEAE SENSU LATO analyses, i.e., the one that excludes the ambiguously aligned regions. Prior to phylogenetic analysis, relative apparent synapomorphy analysis (RASA 3.0 Turbo [Lyons-Weiler 2001]) was used to measure phylogenetic signal (tRASA) and perform a noise reduction with all gaps coded as question marks (Lyons-Weiler et al. 1996, 1998). Taxon-variance ratios (Lyons-Weiler and Hoelzer 1997) were examined to screen for potential longbranch attraction problems. All phylogenetic analyses were conducted by using PAUP* 4.0b5 (Swofford 1998) and TreePuzzle 5.0 (Strimmer and von Haeseler 1996, 1997). All sequences have been deposited in GenBank (table 1).

Maximum Parsimony For each data set, phylogenetic reconstruction with maximum parsimony (MP) was conducted by using the heuristic search option in PAUP* (Swofford 1998) with TBR branch swapping, MULPARS, ACCTRAN, and 500 replicates of random-addition sequence order in effect. Characters were assigned equal weights at all nucleotide positions (Fitch 1971). Gaps were coded as missing data. For each analysis, strict consensus trees were constructed from all most parsimonious trees. Bootstrap analyses (1000 “full heuristic” replicates with the same search strategy described above) were conducted to examine the relative level of support for individual clades on the cladograms of each search (Felsenstein 1985). Sequence divergence in different regions was computed as the average number of nucleotide differences per site between two sequences according to Kimura (1980) (with gaps excluded) using PAUP* (Swofford 1998).

Maximum Likelihood Maximum likelihood (ML) analysis was preformed by using the quartet sampling and Neighbor-Joining (NJ) parameter estimation procedure of TreePuzzle 5.0 (Strimmer and von Haeseler 1996, 1997), which implements a fast tree search algorithm (quartet puzzling) that allows analysis of large data sets and automatically assigns estimations of support to each internal branch. Lythrum hyssopifolia (Onagraceae) was used as outgroup on the basis of the Onagraceae’s sister relationship to Lythraceae (Dahlgren and Thorne 1984; Conti et al. 1996). Three different models of nucleotide substitution, the TN model (Tamura and Nei 1993), the HKY model (Hasegawa et al. 1985), and the SH model (Schoeniger and von Haeseler 1994), were examined. Rate heterogeneity was taken into account by considering invariable sites and by introducing gdistributed rates for the variable sites. Three rate heterogeneity models: g-distributed rates, two rates (1 invariable + 1 variable), and a mixed model (1 invariable rate + g-distributed rates) were also tested. For each data set, a ML search was performed by using parameters estimated from the data with 1000 puzzling steps. The MP and ML analyses were performed for data sets (1) rbcL, (2) PY-IGS, (3) ITS, and (4) the combined data set. Before combining the data sets, data congruence was assessed with the partition homogeneity test (Farris et al. 1995; implemented with PAUP* 4.0b5 [Swofford 1998]). One thousand replicates were performed, and the resulting P value was used to determine whether the use of the combined data sets for phyloge-

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netic reconstruction was appropriate. Alternative phylogenetic hypotheses were evaluated by using MacClade 3.05 (Maddison and Maddison 1992) to prepare trees that reflected the alternate relationships. These were loaded into PAUP* (Swofford 1998) as constraint trees using the same search strategy as that described above, except that PAUP* was asked to find the shortest trees consistent with the topology in question. The difference between the length of these trees and the globally shortest trees provides an indication of the parsimonious cost involved in accepting the alternative hypothesis. We used topological constraint trees in PAUP* (Swofford 1998) to determine the monophyly cost (i.e., the number of steps beyond the most parsimonious necessary for monophyly) of Lythraceae s.str. (Lythroideae) and Sonneratiaceae sensu Engl. & Gilg, for which we sampled more than one species in combined analysis. The Templeton test (Templeton 1983; Mason-Gamer and Kellogg 1996) was used to test whether the alternative topologies obtained by moving branches in the family analysis were significantly longer than the most parsimonious trees.

Results The numbers and percentages of variable and potentially phylogenetically informative characters found in each data set are presented in table 3. The partial rbcL gene sequenced in this study includes 1258 nucleotides for the 22 operational taxonomic units (OTUs), which covers 88.1% of the total rbcL gene in Lythrum hyssopifolia (1428 bp, GenBank accession number L10218). There are no indels in the partial rcbL gene, and all 22 aligned sequences have the same length. Sequence divergence ranges from 1.28% to 5.28% among the ingroup and 2.84% to 6.33% between ingroup and outgroup. Our primers for the PY-IGS region covered the entire spacer region plus 48 bp of the 5 end of the psaA gene and 27 bp of the 3 end of the ycf3 exon. Within Lythraceae s.l., Cuphea hookeriana has the longest (789 bp) sequence and Lagerstroemia speciosa has the shortest sequence (736 bp) among the 26 OTUs. Among the outgroups, the PY-IGS sequences of Fuchsia hybrida cultivar and Ludwigia hyssopifolia are 757 bp long. Those of Combretum wallichii and Quisqualis indica are 737 and 728 bp, respectively. Sequence divergence ranges from 1.38% to 10.39% among sampled lythraceous species and from 7.79% to 16.36% between the ingroup and outgroup taxa. Divergence in PY-IGS exceeds that in rbcL (average 5.83% vs. 3.08%). The ITS spacers are the most length variable of the three regions analyzed. The sequence for this region in Lythraceae s.l. covers ITS-1 (179–241 bp), 5.8S gene (163–166 bp), and ITS-2 (195–242 bp). Sequence length ranges from 560 bp in Lythrum to 631 bp in Sonneratia. Sequence divergence ranges from 6.8% (between Ammannia baccifera and Nesaea luederithii) to 28.5% (between C. hookeriana and Trapa maximowiezii) (average 20.7%). ITS-2 sequence divergence values are lower than those for ITS-1. Divergence between ingroup and outgroup genera ranges from 24.9% (between Pemphis acidula and F. hybrida cultivar) to 35.8% (between Duabanga grandiflora and Q. indica). Multiple accessions from a variety of sources were sequenced whenever possible for 12 species representing 12 different genera, except for four genera for which only herbarium samples were available (table 1). From

Table 1 Accessions and Voucher Specimens of Lythraceae and Outgroups Sampled

Taxa

Family or subfamily

Accession number Voucher

Ammannia baccifera 1 Ammannia baccifera 2*

Lythroideae Lythroideae

Tang, S. Q. 99010301 (SYS) Jian, S. G. 200507 (SYS)

Cuphea hookeriana 1 Cuphea hookeriana 2*


Tang, S. Q. 99070501 (SYS) He, X. J. 99-769 (SYS)

Cuphea lanceolata*

Lythroideae

Shi, S. H. 99090201 (SYS)

Decodon verticillatus

Lythroideae

Hill, Steven R. 18871 (A)

Duabanga grandiflora 1

Duabangoideae

Huang, S. D. 990401 (SYS)

Duabanga grandiflora 2*

Duabangoideae

Huang, S. L. 9942101 (SYS)

Heimia myrtifolia 1 Heimia myrtifolia 2* Lagerstroemia speciosa1

Lythroideae Lythroideae Lythroideae

Tang, S. Q. 99070502 (SYS) Wang, Y. 0349 (SYS) Shi, S. H. 99060103 (SYS)

Lagerstroemia speciosa 2*

Lythroideae

He, X. J. 99-767 (SYS)

Lagerstroemia villosa*

Lythroideae

Shi, S. H. 2000-01037 (SYS)

Lawsonia inermis 1

Lythroideae

Qiu, H. X. 99070201 (SYS)

Lawsonia inermis 2* Lythrum salicaria 1


Tang, S. Q. 99070511 (SYS) Lei, Y. B. 005 (SYS)

Lythrum salicaria 2 Nesaea luederithii


Tsang, W. T. 27844 (IBSC) Karibib 1056, 1957 3. 28 (A)

Pemphis acidula

Lythroideae

Liao, C.C. 1150, 1993.2.24 (A)

Peplis portula Punica granatum 1

Lythroideae Punicoideae

Montezuma s.n. (KE) Hao, G. 20000318 (SYS)

Punica granatum 2*

Punicoideae

Wang, J. B. 00-041602 (SYS)

Rotala indica 1 Rotala indica 2*


Tang, S. Q. 99070503 (SYS) Jian, S. G. 200508 (SYS)

Rotala rotundifolia*

Lythroideae

Gong, X. 2000-52303 (SYS)

Sonneratia alba*

Sonneratioiodeae

Chen, S. C. 990604 (SYS)

Sonneratia apetala*

Sonneratioiodeae

Qiu, X. Z. 974312 (SYS)

Geographical origin

rbcL

IGS

ITS

Guilin, Guangxi, China Cult. in South China Botanical Garden, Guangzhou, China Guilin, Guangxi, China Cult. in South China Botanical Garden, Guangzhou, China Cult. in Strybing Arboretum, San Francisco, USA Deposited in A, Washington Co., Rhode Island, USA Liangfengjiang natural reserves, Nanning, China Jianfenglin natural reserves, Hainan, China Guilin, Guangxi, China Yangsuo, Guangxi, China Cult. in Zhongshan University Campus, Guangzhou, China Cult. in South China Botanical Garden, Guangzhou, China Jianfenglin natural reserves, Hainan, China Cult. in South China Botanical Garden, Guangzhou, China Guilin, Guangxi, China Cult. in Wuhan Botanical Garden, Wuhan, China Lingshan, Guangxi, China Deposited in A, collected from Nemibia (Southwest Africa) Deposited in A, collected from Lanyu Island, Taiwan, China Portugal Cult in South China Botanical Garden, Guangzhou, China Cult. in Wuhan University Campus, Wuhan, Hubei, China Guilin, Guangxi, China Cult. in South China Botanical Garden, Guangzhou, China Cult. in Kunming Botanical Garden, Kunming, China Hainan Island, Hainan, China Futian natural reserves, Shengzhen, Guangdong, China

AY036145 na

AY035733 na

AY035754 AF420216

AY036137 na

AY035723 na

AF201691 AF420221

na

na

AY035763

AY036140

AY035728

AY035752

AY036150

AY035738

AF163695

na

na

AF208695

AY036147 na AY036149

AY035735 na AY035737

AF201693 AF420215 AF201689

na

na

AF420218

na

AY035739

AY035755

AY036146

AY035734

AF201690

na L10218a

na AY035727

AF420217 AY035749

AF421496 AY036144

AF421495 AY035732

AY035750 AY035753

AY036138

AY035725

AY035762

AY036139 L10223b

AY035726 AY035724

AY035751 AY035760

na

AY035742

AY035761

AY036148 na

AY035736 na

AY035758 AF420220

na

na

AY035759

na

AY035741

AF163701

na

AY035740

AF163697

HUANG & SHI—PHYLOGENETICS OF LYTHRACEAE SENSU LATO

219

Table 1 (Continued )

Taxa

Family or subfamily

Accession number Voucher

Sonneratia caseolaris1

Sonneratioiodeae

Huang, Y. L. 990435 (SYS)

Sonneratia caseolaris2*

Sonneratioiodeae

Huang, Y. L. 99051115 (SYS)

Woodfordia fruticosa 1 Woodfordia fruticosa 2*


Tang, S. P. 99070504 (SYS) He, X. J. 99-768 (SYS)

Trapa maximowiezii 1 Trapa maximowiezii 2

Trapoideae Trapoideae

Wang, J. B. 2000-041601 (SYS) Zhang, C. 2000-1010 (SYS)

Ludwigia hyssopifolia

Onagraceae

Yuan, C. C. 2000-72401 (SYS)

Fuchsia hybrida cultivar

Onagraceae

Jian, S. G. 20010207 (SYS)

Combretum wallichii

Combretaceae

Shi, S. H. 990703005 (SYS)

Quisqualis indica

Combretaceae

Ye, C. X. 99031301 (SYS)

Geographical origin

rbcL

IGS

ITS

Futian natural reserves, Shengzhen, Guangdong, China Leizhou mangrove natural reserves, Guangdong, China Guilin, Guangxi, China Cult. in South China Botanical Garden, Guangzhou, China Liangzhihu, Hubei, China Cult. in Wuhan Institute of hydrobiology, Wuhan, China Cult. in Zhongshan University Campus, Guangzhou, China Cult. in South China Botanical Garden, Guangzhou, China Cult. in Kunming Botanical Garden, Kunming, China Zhongshan University Campus, Guangzhou, China

AY036143

AY035731

AF208696

na

na

AF420219

AY036136 na

AY035722 na

AF201692 AF420222

AY036141 AY036142

AY035729 AY035730

AY035757 AY035756

AY036152

AY035745

AY035747

L10220c

AY035746

AY035748

AY036151

AY035743

AF208731

L01948d

AY035744

AF160470

Note. Asterisk denotes taxa not included in the final combined analysis. SYS p Zhongshan (Sunyatsen) University; A p Harvard University, Arnold Arboretum; IBSC p South China Institute of Botany; KE p Kent State University Herbarium. Classification based on Thorne (2000). a Lythrum hyssopifolia (Conti et al. 1993). b Punica granatum (Conti et al. 1993). c Fuchsia cyrtandroides (Conti et al. 1993). d Quisqualis indica (Albert et al. 1992).

these sequences, we observed two cases of intraspecific variation. The two Lythrum salicaria accessions differed by two nucleotide substitutions (divergence p 0.36%), and the two accessions representing T. maximowiezii differed by four nucleotide substitutions (divergence p 0.65%). All other accessions were identical within species. No indications of paralogous loci were observed in this analysis. The PCR products were present on the agarose gel as sharply delimited single bands. Further, few individual positions showed double peaks (as determined by automated sequencing) that could indicate occurrence of paralogous loci. The results of direct sequencing suggest that heterogeneity in the ITS region did not pose a major problem for phylogeny reconstruction. Inspection of the aligned sequences indicates that some indels may be informative in more narrowly circumscribed studies where alignments, and thus identification of indels, would be unambiguous. For example, Lythrum and Peplis apparently share a 54-bp deletion located just before the conserved region of ITS-1 (positions 70–123). Further, the parsimony analysis that treat gaps as fifth states based on the ITS data results in trees congruent with the “gaps as missing data” analysis except

for the enigmatic placement of Rotala, which receives weak support in both trees. Although the differences in position exist only in branches with weak bootstrap support when indels as present/absent characters are included/excluded in the analyses, it still poses a problem in evaluating the phylogenetic utility of indels in Lythraceae s.l. The topology of the tree based on this combined data is the same whether gaps are treated as missing data or score as a fifth state. The MP trees suggest large differences in rates of evolution among taxa. For example, Cuphea and Trapa show a relatively high rate of sequence variation for all DNA regions in comparison with other taxa. We sought to determine whether rate heterogeneities were large enough to produce misleading phylogenetic analyses because of potential long-branch attractions. No significant long branches were detected by the RASA regression analysis of the rbcL data set. When Trapa is removed from the data set, the tRASA test statistic drops from 12.69 (with all species included) to 7.876. Similar RASA analyses were performed for each of the other data sets. No significantly long branches were detected in the IGS, ITS, or combined threeregion data sets. Excluding the noise by using RASA, the align-


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Table 2 Base Composition of Amplification and Sequencing Primers Primer rbcL: PR1f a PR2ra PR3rb PR4f b ITS:c ITS4a ITS5a C5.8Sb N5.8Sb N18L18b PY-IGS: PG1f a PG2ra

Sequence 5-ATG TCA CCA CAA ACA GAG ACT-3 5-CCT TCA TTA CGA GCT TGC ACA C-3 5-CAT TTC TTC ACA TGT ACC TGC-3 5-ATT ACT TRA ATG CTA CTG-3 5-TCC TCC GCT TAT TGA TAT GC-3 5-GGA AGT AAA AGT CGT AAC AAG G-3 5-TGC GTT CAA AGA CTC GAT-3 5-ATC GAG TCT TTG AAC GCA-3 5-AAG TCG TAA CAA GGT TTC-3 5-CAT TCC TCG AAC GAA GTT TTT ACG GGA TCC-3 5-TCC CGG TAA TTA TAT TGA AGC GCA TAA TTG-3

a

For amplification and sequencing. For sequencing only. c Wen and Zimmer (1996). b

ment length of the combined data set was reduced from 2774 to 2410. Further, the parsimony analysis resulted in trees congruent with the original analysis without RASA treatment. The bootstrap values for some clades differed slightly. The numbers and lengths of most parsimonious trees obtained and the consistency, retention, and rescaled consistency indices of the various analyses performed are presented in table 3. The P values resulting from the partition homogeneity test indicate that the data partition is random (P values range between 0.770 and 0.980) and that the data sets are reasonably congruent (table 4). The separate analyses produced topologies that are roughly congruent with each other but generally do not have high bootstrap support (result not shown). All trees based on individual data exhibit four well-supported clades (Ammannia-Nesaea-Lawsonia, Cuphea-Woodfordia, LythrumPeplis, Pemphis-Punica), but most clades are unresolved with respect to each other in strict consensus trees. The parsimony analysis based on a combination of all data sets resulted in a single fully resolved tree with higher bootstrap support for most branches (fig. 1). Analysis of the combined sequences provides strong support for the monophyly of Lythraceae s.l. (bootstrap support p 98%). Two major deep clades are identified. One comprises Cuphea, Pemphis, Punica, and Woodfordia with 83% bootstrap support; the remaining genera form the other clade, which has weak bootstrap support (54%). Forcing the Lythraceae s.str. into a monophyletic group to the exclusion of Duabanga, Punica, Sonneratia, and Trapa requires 63 extra steps on the tree, and this new topology is significantly different (P ! 0.0001) from the most parsimonious tree on the basis of the Templeton test. When the constraint tree holding the monophyly of Sonneratiaceae sensu Engl. & Gilg was tested, 16 extra steps were needed, and the topology was significantly different from the most parsimonious trees (P p 0.0077). Moving Trapa to the base of Lythraceae s.l. results in a tree 29 steps longer than the most parsimonious trees (P p 0.0006). Similarly, placing the other previously recognized families apart from Lythraceae s.l. results in trees between 25 and 38 steps longer than the shortest trees. All these

alternative topologies require significantly more steps (P ! 0.0001 for Duabanga and Punica; P p 0.0008 for Sonneratia). TreePuzzle analyses based on separate and combined data using different nucleotide substitution and rate heterogeneity models result in quartet puzzling (QP) trees of similar topology to those from MP (shown with the combined data set and the highest likelihood only; fig. 2). ML analyses yield tree topologies that are less resolved than MP trees. Five well-supported clades (Ammannia-Nesaea-Lawsonia, Cuphea-WoodfordiaPemphis-Punica, Decodon-Lythrum-Peplis, Duabanga-Lagerstroemia-Sonneratia-Trapa, Heimia-Rotala) are found by QP searches. Although each clade is fully resolved, clades are unresolved with respect to each other.

Discussion As in other work involving the combination of multiple molecular data sets (Soltis et al. 1998; Hoot et al. 1999), analyses based on the combined data sets of Lythraceae s.l. required shorter computer run times and resulted in trees with better resolution and improved bootstrap support than analyses of each data set separately. Congruence between independent data sets is one of the most fundamental ways to determine the reliability of phylogenetic inferences (de Queiroz et al. 1995; Miyamoto and Fitch 1995; Huelsenbeck et al. 1996). In this study, three individual data sets from different sources (chloroplast genome or nuclear genome, coding region or spacer region) are highly congruent on the basis of the partition homogeneity test (table 4). The most widely accepted concept of the Lythraceae during the last century has been the narrow one proposed in the only monograph of the family (Koehne 1903), which excluded Duabanga, Punica, Sonneratia, and Trapa. With more recent and extensive information on pollen morphology, chromosome numbers, wood and floral anatomy, molecular evidence, and new lythraceous genera described in recent years, the delimitation of the family has been altered to include one to four satellite genera in Lythraceae (Melchior 1964; Hutchinson 1973; Takhtajan 1980; Cronquist 1981; Tobe and Raven 1983; Dahlgren and Thorne 1984; Johnson and Briggs 1984; Graham et al. 1993a; Conti et al. 1996, 1997; Angiosperm Phylogeny Group 1998; Shi et al. 2000; Thorne 2000). Because members of the Lythraceae s.l. are morphologically distinct, placement of the monogeneric families Duabangaceae, Punicaceae, Sonneratiaceae, and Trapaceae has been difficult (Melchior 1964; Hutchinson 1973; Takhtajan 1980; Cronquist 1981). Johnson and Briggs (1984) inferred the monophyly of most Lythraceae s.l. but excluded Trapa based on the results of a cladistic analysis of the order Myrtales in which the monophyly of Lythraceae s.l. was established by the autapomorphy of the multiplicative outer integument in the seed. We agree with Graham et al. (1993a), however, in the interpretation of the seed coat of the Duabanga as not multiplicative but rather consisting of a two-layered testa and a two-layered tegmen (Graham et al. 1993a). Absence of a multilayered testa in Duabanga eliminates the only synapomorphy defining Lythraceae s.l. among the 77 characters used to describe the order Myrtales (Johnson and Briggs 1984; Graham et al. 1993a). Traditionally, affinities have been recognized between Onagraceae and Lythraceae s.str. through similarities in the teeth

HUANG & SHI—PHYLOGENETICS OF LYTHRACEAE SENSU LATO and cilia on the leaf margins (Hickey 1981), fibrous exotegmen of the seeds, and pinnate petal venation (Dahlgren and Thorne 1984). In contrast, Takhtajan (1980) proposed a close relationship between Combretaceae and Lythraceae s.str. In an rbcL tree of the order Myrtales (Conti et al. 1997), the sister group relationship of Combretaceae with a clade formed by the Onagraceae and Lythraceae lineage was weakly supported. The combined data here, however, provide strong bootstrap support for the monophyly of Lythraceae s.l. based on the MP and QP analyses (98% and 91%, respectively). The rbcL-IGSITS topology supports an expanded interpretation of familial boundaries for Lythraceae to include Duabanga, Punica, Sonneratia, and Trapa in the Lythraceae lineage. The terminal relationships among the genera generally show strong bootstrap support. Both MP and QP trees based on three data sets, however, show low bootstrap values at some deeper nodes. The intergenetic relationships reflected by the rbcL-IGS-ITS tree do not correspond to the morphologically based classifications (Koehne 1903; Graham et al. 1993a). Koehne’s tribes, distinguished primarily by complete or incomplete septation of the ovary, and the subtribes, distinguished primarily by presence or absence of wings on the seed, combine genera from different monophyletic groups as this study indicated. Paraphyly in subfamily Lythroideae was observed by Graham et al. (1993a) and is also observed here. However, the putative basal clade (Duabanga-Sonneratia-Punica-LagerstroemiaLawsonia), which was regarded as containing the primitive groups in the cladistic analysis based on morphological characters (Graham et al. 1993a), is not supported in our rbcLIGS-ITS tree. The most significant phylogenetic implications that emerge from our analyses are summarized below.

Duabanga-Lagerstroemia-Sonneratia-Trapa The MP analyses recover the following well-supported topology in the MP tree: {{{Duabanga, Lagerstroemia}, {Sonneratia, Trapa}}, {{Ammannia, Nesaea}, Lawsonia}} (fig. 2). In the QP tree, the big clade collapses into two parallel clades: {{Duabanga, Lagerstroemia}, {Sonneratia, Trapa}} and {{Ammannia, Nesaea}, Lawsonia} (fig. 2). All seven genera have in common a primarily Old World distribution. Trapa is traditionally treated as Trapaceae, which consists of one genus with three to 15 species of aquatic herbs distributed in temperate to tropical regions of the Old World (Wan and Li 2000). The

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Table 4 P Values from Partition-Homogeneity Test Data sets

P value

rbcL vs. PY-IGS rbcL vs. ITS PY-IGS vs. ITS rbcL/ITS vs. PY-IGS rbcL/PY-IGS vs. ITS PY-IGS/ITS vs. rbcL

0.770 0.960 0.980 0.780 0.960 0.980

Note. One thousand replicates for various partitions of data based on 22 common OTUs.

morphology of Trapa is too highly modified to suggest links with other families of Myrtales with any certainty, as is the case of many aquatics (Les and Haynes 1995; Barrett and Graham 1997). Some distinctive features of Trapa include a unique embryology and a characteristic double apex on the teeth of the leaf margins. Takhtajan (1980) suggested that Ludwigia (Onagraceae) was more closely related to Trapaceae than to Lythraceae. He later considered the Trapaceae to be sufficiently distinct to warrant placement in its own suborder, Trapineae (Takhtajan 1997). He suggested that the relationships of Trapaceae lie with Onagraceae and Lythraceae. Raven (1979), however, rejected any link between Onagraceae and Trapaceae. Although a relationship between Trapa and Lythraceae s.str. has been proposed several times in the past (Thorne 1981, 1992, 2000; Dahlgren and Thorne 1984; Johnson and Briggs 1984; Graham et al. 1993b, 1998; Takhtajan 1997), the Lythraceae lack any morphological autapomorphic character that could be used to support or refute the placement of Trapa (Graham et al. 1993a). Because Trapa is morphologically distinct, the evidence from DNA was critical to understanding its position within the Myrtales. Conti et al. (1997) recognized the sister group relationship between Trapa and Lythrum with weak support from an rbcL data set of the Myrtales without sampling Sonneratia. Our results indicate a sister group relationship between Trapa and Sonneratia (figs. 1, 2) in a derived position within the Lythraceae s.l. Sonneratia comprises five to 11 species of mangrove trees that extend from the Indian Ocean to the Pacific Ocean; Duabanga comprises two or three species of rain forest trees confined to southeastern Asia (Backer and van Steenis 1951; Jaya-

Table 3 Comparison of Indices for the Trees Analyzed

Data set (OTUs) rbcL (22) PY-IGS (26) ITS (37) rbcL/PY-IGS (22) rbcL/ITS (22) PY-IGS/ITS (26) rbcL/PY-IGS/ITS (22) Note.

Variable characters 222 288 355 508 569 639 855

(17.6%) (30.4%) (62.4%) (23.0%) (31.1%) (42.2%) (30.8%)

Informative characters 121 177 319 292 418 481 582

(9.60%) (18.7%) (56.1%) (13.2%) (22.9%) (31.7%) (21.0%)

Trees

Tree length

Consistency index

Retention index

Rescaled consistency index

4 27 3 6 2 1 1

378 438 1369 809 1651 1742 2081

0.5551 0.7397 0.4913 0.6516 0.5108 0.5468 0.5476

0.6071 0.7670 0.7207 0.6864 0.5558 0.6306 0.5836

0.4128 0.6234 0.3664 0.5159 0.3117 0.3747 0.3581

The ambiguous aligned ITS regions are excluded; consistency index excludes uninformative characters.

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INTERNATIONAL JOURNAL OF PLANT SCIENCES coideae and Lythroideae. The rbcL-IGS-ITS trees presented here indicate a strong sister group relationship between Sonneratia and Trapa, with 89% bootstrap support in the MP and 96% in the QP combined trees and Sonneratia-Trapa sister to the clade Duabanga-Lagerstroemia (70% in MP and 96% in QP). Our analyses suggest a close relationship between the clades {Sonneratia, Trapa} and {Duabanga, Lagerstroemia} (92% bootstrap value support in the MP tree [fig. 1] and 89% in the QP tree [fig. 2]). Although by now the available molecular data clearly suggest the placement of Trapa as closest to Sonneratia, the evolution of its unique morphology remains to be explained. The Sonneratia-Trapa clade suggested by molecular evidence needs to be tested further by sequencing of additional generic samples and inclusion of additional taxa (S. Graham, personal communication). A close relationship among Duabanga, Lagerstroemia, and Sonneratia, however, has been generally recognized on the basis of the shared anthotelic inflorescences and wet stigma surface (Weberling 1988; Graham et al. 1993a; Hoch et al. 1993). Although Duabanga and Sonneratia share many morphological similarities that have led many authors to accept their membership in the ditypic family Sonneratiaceae, Duabanga has been considered to be related more closely to Lagerstroemia than to Sonneratia on the basis of numerous specialized palynological characters (Corner 1976; Graham et al. 1990), wood anatomy (Baas and Zweypfenning

Fig. 1 Maximum parsimony tree of Lythraceae s.l. based on combined molecular data. Numbers above branches represent bootstrap values based on 1000 replicates. For tree parameters, see table 3.

weera 1967; Ko 1983; Duke and Jackes 1987). The two genera have traditionally been treated as constituting the ditypic family Sonneratiaceae (Melchior 1964; Jayaweera 1967; Hutchinson 1973; Cronquist 1981, 1988; Ko 1983). Most recently, they have been recognized as the separate monogeneric subfamilies Duabangoideae and Sonneratioideae in Lythraceae s.l. (Dahlgren and Thorne 1984; Graham et al. 1993a, 1998; Thorne 2000) or as separate families, Duabangaceae and Sonneratiaceae (Takhtajan 1986, 1997). Duabanga and Sonneratia are distinguished by branched foliar sclerids (Graham et al. 1993a) and by a chromosome number of xp12, whereas the rest of the family has chromosome numbers ranging from xp5 to xp8 (except for another satellite genus, Trapa) (Graham et al. 1993b). In addition, if included in the Lythraceae s.l., the two genera would be the only genera with triporate pollen (Graham et al. 1990). However, their overall wood anatomical variation has been regarded as well within that of the Lythraceae (Rao et al. 1987). A relationship to the Lythraceae in particular was indicated in both genera by the presence of a multicellular archesporium, otherwise restricted to Lythraceae s.str. (Tobe and Raven 1983). Tobe et al. (1986) thought that it appeared likely that the Sonneratiaceae sensu Engl. & Gilg, which comprises Duabanga and Sonneratia, were a relatively early offshoot of the branch leading to Puni-

Fig. 2 Quartet puzzling tree based on the combined data. Numbers above clades indicate support values, which can be interpreted in much the same way as bootstrap values. ⫺ ln likelihood p 12,032.89 based on SH substitution model with rate heterogeneity (1 invariable rate + g-distributed rates).

HUANG & SHI—PHYLOGENETICS OF LYTHRACEAE SENSU LATO 1979; Rao et al. 1987), and embryology (Tobe and Raven 1983). The monophyly of the Sonneratiaceae sensu Engl. & Gilg is strongly rejected by the Templeton Test (+16, P p 0.0077). The present results also agree with the conclusions of a study of Sonneratiaceae based on ITS data (Shi et al. 2000).

Ammannia-Nesaea-Lawsonia A clade comprising Ammannia, Nesaea, and Lawsonia is strongly supported (figs. 1, 2). This clade consistently appears in all analyses, no matter which of the three DNA regions are employed. The three genera are united by some pollen characters such as the presence of six pseudocolpi, moderately developed to distinct mesocolpal ridges, and faint to distinct annuli (Graham et al. 1990; Graham et al. 1993a). The close relationship of Ammannia and Nesaea, indicated by strong morphological similarity between the two genera, including a unique synapomorphic interlocking striate pollen exine (Graham et al. 1993a), is strongly supported by our results. The genus Lawsonia, however, appears to be closer to Lagerstroemia than it is to Ammannia-Nesaea based on morphological characters (Koehne 1903; Graham et al. 1993a).

Decodon-Lythrum-Peplis A sister group relationship between Lythrum and Peplis is strongly supported (figs. 1, 2), and both appear to be closely related to Decodon, with 64% bootstrap support in the MP and 91% in the QP tree. The genera Lythrum and Peplis have been regarded as congeneric by Webb (1967). The same derived base chromosome number, xp5, was also reported in the two genera (Tobe et al. 1986). The genus Peplis is distinguishable from Lythrum by its lack of a floral nectary (Graham et al. 1987), and pollen dissimilarity is great enough to maintain the two genera (Graham et al. 1990). In our molecular analyses, a 54-bp deletion located in ITS-1 (bp 70–123) appears only in Lythrum and Peplis. The low divergence (3.8%) of the combined sequence data among the genera within the family is also found between these two genera. Both morphological and molecular data therefore confirm a very close relationship between the genera Lythrum and Peplis, but further studies with both molecular and morphological characters are needed in more species of both genera to determine whether the genera are indeed distinct.

Heimia-Rotala Heimia and Rotala form another well-supported group with 76% bootstrap support in the MP (fig. 1) and 84% in the QP trees (fig. 2). The two genera share such characters as seed compressed, internal epidermal straight hairs, and stamens in one whorl. None of them, however, can be regarded as unique synapomorphies to diagnose this clade on the basis of morphological cladistic analyses (Graham et al. 1993a). Heimia was placed in a clade comprising Haitia, Ginoria, and Tetrataxis and Rotala in a clade with Didiplis, Hionanthrea, and Peplis on the basis of several homoplasious characters in cladistic analysis. Both clades, with several other genera (Ammannia, Lythrum, Nesaea, and others), formed a major clade (Graham et al. 1993a). Because we could not sample the other

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five genera except for Peplis, the detailed relationships within the group were not able to be resolved in this study.

Cuphea-Woodfordia-Pemphis-Punica Cuphea and Woodfordia form a monophyletic group (with 96% bootstrap support in the MP tree and 97% in the QP tree) that is sister to a clade of Pemphis and Punica (85% in the MP tree and 78% in the QP tree). A close relationship between Cuphea and Woodfordia was also found in a morphological cladistic analysis (Graham 1995). All four genera share several derived characters, such as multicellular glandular hairs, globose glands, small-diameter pollen grains, stipitate ovaries, and spiral seed coat hairs. The same base chromosome number has also been shown in all four genera (Tobe et al. 1986). No morphological evidence, however, supports the close relationship between Pemphis and Punica based on previous cladistic analyses (Johnson and Briggs 1984; Graham et al. 1993a). Punica is traditionally treated as Punicaceae, a monogeneric family of two species found natively from the Balkan Peninsula to the western Himalayas (Punicia granatum) and on the island of Socotra (Punicia protopunica), the latter represented by only four individuals (Lucas and Synge 1978). There is common agreement that Punica is closely related to, and probably derived from, Lythraceae, from which it is distinguished by several specialized features, such as fruits with leathery pericarp and pulpy seeds with edible sarcotesta (Dahlgren and Thorne 1984; Graham et al. 1990). These unique features induced some authors to treat Punicaceae as a separate family. In contrast, the ovules of Punica, with their thick, multilayered outer integument and unicellular archesporium, differ from those of Lythraceae s.str. Wood anatomy (Bridgewater and Baas 1978; Graham et al. 1993a), chromosome data (Tobe et al. 1986), and pollen morphology (Patel et al. 1984; Graham et al. 1990), however, suggested the inclusion in, or at least a close relationship with, Lythraceae s.l. The combination of all these features leads Tobe and Raven (1983) to suggest Punica as being a distinct archaic offshoot within Lythraceae s.l. This placement was also indicated by the rbcL gene tree but with weak support (Conti et al. 1997). The present DNA analysis strongly supports the inclusion of Punica within Lythraceae (figs. 1, 2). In the combined MP and QP trees of three genes, Punica consistently appears as sister to Pemphis with relatively high bootstrap support (figs. 1, 2). In contrast, morphological character analysis placed Punica closest to Lagerstroemia (Graham et al. 1993a). The combined analyses yield a strongly supported hypothesis regarding the delimitation of the Lythraceae s.l. Lythroideae, as the largest and most diverse subfamily, is paraphyletic. Apart from the sequence data, there appear to be no morphological synapomorphies that unite all members of the family (Johnson and Briggs 1984; Graham et al. 1993a). Support for the basal split of Lythraceae s.l. into two major lineages is weak, which possibly reflects an early pattern of rapid radiation in the family as indicated by morphology (Graham et al. 1993a). Most likely, the plasticity of the morphological characters has contributed substantially to the lack of congruence between the cladograms based on molecular evidence and morphology. Because of the morphological diversity and the difficulty of

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determining homologous structures among members of the Lythraceae s.l., evidence from the nucleotide sequences offers the most promising sources of phylogenetic information. The molecular analyses presented here provide a new framework for the interpretation of morphological characters in these assemblages. Although the problem of homoplasy is certainly not resolved with the use of sequence data in phylogenetic analyses, the chance that patterns of homoplasy would have similar distributions in both morphological and nucleotide sequence data is negligible (Kron and Chase 1993). So molecular data can be regarded as a new and independent information source to complement the phylogeny inferred from morphological characters. We advocate and maintain a fair degree of suspicion concerning these results in weakly supported portions of the topology. The molecular data presented here support recognition of a more broadly defined Lythraceae s.l. These results should be considered as a starting point for further studies in which both morphological and molecular data are reexamined. Comparing the very generalized morphology of the family and the

distinctive genera, the suggestion of great age for the family (Graham et al. 1993a) may be reflected in these results.

Acknowledgments We thank David E. Boufford, Shirley A. Graham, Andrea Schwarzbach, Peter W. Fritsch, Susan Remer, and an anonymous reviewer for helpful comments on the manuscript; Xingjin He, Feng-xiao Tan for laboratory assistance; Ki-Joong Kim for providing the primer sequences of PY-IGS; and Ting Wang for providing the sequences of rbcL primers. We are grateful to Hua-xing Qiu, Xue-jun Ge, Xun Gong, Shao-qing Tang, Peter W. Fritsch, and Harvard Herbaria for kindly providing leaf tissue. This study was supported by grants from the National Science Funds for Distinguished Young Scholars (39825104), the National Natural Science Foundation of China (39970057, 30070053), the Natural Science Foundation of Guangdong Province (001223), the National Ministry of Education Foundation for Key Member Teachers, and Qiu Shi Science and Technologies Foundation.

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