The use of a non-coding region of chloroplast DNA in phylogenetic studies of the subtribe Sonchinae (Asteraceae: Lactuceae)

Transcription

1 Pl. Syst. Evol. 215:85-99 (1999) --Plant. Systematics and Evolution Springer-Verlag 1999 Printed in Austria The use of a non-coding region of chloroplast DNA in phylogenetic studies of the subtribe Sonchinae (Asteraceae: Lactuceae) SEUNG-CHUL KIM, DANIEL J. CRAWFORD, ROBERT K. JANSEN, and ARNOLDO SANros-GUERRA Received July 10, 1997; in revised version November 18, 1997 Key words: Sonchinae, Asteraceae. - cpdna, non-coding region (psba-trnhöu6), phylogeny. Abstract: The systematic utility of sequences from a non-coding region of chloroplast DNA (cpdna) between psba and trnh (6v6) was examined by assessing phylogenetic relationships in subtribe Sonchinae (Asteraceae: Lactuceae). Primers constructed against highly conserved regions of trna genes were used for PCR amplification and sequencing. The psba-trnh intergenic spacer contains several insertions and deletions (indels) in Sonchinae with the length varying from 385 to 450bp. Sequence divergence ranges from 0.00% to 7.54% within Sonchinae, with an average of 2.4%. Average sequence divergence in Sonchus subg. Sonchus is 2.0%, while the mean for subg. Dendrosonchus and its close relatives in Macaronesia (the woody Sonchus alliance) is 1.0%. Our results suggest that this region does not evolve rapidly enough to resolve relationships among closely related genera or insular endemics in the Asteraceae. The phylogenetic utility of psba-trnh sequences of the non-coding cpdna was compared to sequences from the ITS region of nuclear ribosomal DNA. The results suggest that ITS sequences evolve nearly four times faster than psba-trnh intergenic spacer sequences. Furthermore, the ITS sequences provide more variable and phylogenetically informative sites and generate more highly resolved trees with more strongly supported clades, and thus are more suitable for phylogenetic comparisons at lower taxonomic levels than the psba-trnh intergenic chloroplast sequences. Chloroplast DNA (cpdna) has been used extensively to investigate phylogenetic relationships at a wide range of taxonomic levels in plants. Chloroplast genes are now used routinely to infer phylogenies because direct sequencing of polymerase chain reaction (PCR) products makes it relatively easy to obtain sequence data. DNA sequences from several chloroplast genes, such as rbcl, matk, ndhf, atpb, and rps2, have been used to estimate phylogenetic relationships at higher taxonomic levels. For example, rbcl has been widely used at the family level or above (PrUCE & PAL~R 1993, SMrrn & al. 1993, SCOTLAND & al. 1995, SOLrIS & al. 1995, PLtmKErT & al. 1995). Several studies have also shown that rbcl can be used

2 86 S.-C. KIM & al.: at the generic and infrageneric levels in some groups (CONTI Æ al. 1993, GADEK & OUINN 1993, KRON & CI~ASE 1993, PRICE & PALM~R 1993, SOLTIS & al. 1993, PAX & al. 1997), but is sometimes too conserved to resolve phylogenetic relationships between closely related genera (DOEBLEY & al. 1990, GAIJT & al. 1992, KIM K.-J. & al. 1992, SMITH & al. 1993, SOLTIS & al. 1993, XIANa & al. 1993, KIM Y.-D. & JANSEN 1996). The maturase encoding gene matk, which is located in the intron of the transfer RNA gene for lysine (trnk), evolves faster (average of 2.5 times) than rbcl (NEUHAUS & LINK 1987; WOL~ & al. 1992; JOHNSON & SOLTIS 1995; XIAN~ Q.-Y., pers. comm.), and has been used successfully for generic-level phylogenetic reconstmction in several angiosperm families (JOHNSON SOLTIS 1994, STEELE & VILGALYS 1994). The ndhf gene, which encodes the ND5 protein of chloroplast NADH dehydrogenase, has also been shown to be useful for resolving phylogenies within and among plant families that have experienced recent and rapid radiafion (CLARK ~ al. 1995; KIM K.-J. & JANSEN 1995; OLMSTEAD & REEVES 1995). It is widely recognized that molecular phylogenetic studies should include multiple markers to assure that the gene trees are an accurate representation of the species phylogeny (DoYLE 1992). The most widely used markers in plants are from chloroplast genes and the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (nrdna). Direct sequencing of the ITS has proven useful for elucidating phylogenetic relationships at or below the generic level (e.g. BALDWIN & al. 1995). Most cpdna coding regions do not evolve rapidly enough to resolve relationships at these lower taxonomic levels (DOEBLEY & al. 1990, GAUT & al. 1992). Several non-coding regions of cpdna have potential utility at lower taxonomic levels (BöHLE & al. 1994; EHRENDORF~R & al. 1994; GIELLY & TABERLET 1994; HAM & al. 1994; MANEN & al. 1994; MANEN & NATALI 1995; NATALI & al. 1995; GIELLEY & TABERLET 1996; KIM J.-H. & al. 1996). For example, GnaLLY & TABERLET (1994) showed that introns and intergenic spacers evolve 1.93 to times faster than rbcl and that insertions/deletions (indels) occur as often as nucleotide substitutions. Their later study (GIELLY & TABERLET 1996), based on intron sequences from the chroloplast trnl gene showed that the ITS sequences have two to three times higher sequence divergence than the trnl intron. They concluded that ITS sequences are more appropriate for estimating phylogenetic relationships at the intrageneric level. We sequenced the spacer between chloroplast genes psba and trnh ~aug~ to evaluate its phylogenetic utility at lower taxonomic levels in the Asteraceae. Phylogenetic trees based on ITS sequences for the Sonchinae (KIM S.-C. & al. 1996a, b; Fig. 1) enable direct comparisons of sequences of an intergenic cpdna spacer and the ITS region. In addition, the maternally inherited chloroplast sequences provide an independent assessment of phylogenetic relationships within subtribe Sonchinae (Asteraceae: Lactuceae). Materials and methods Total genomic DNA was isolated from leaf tissue using the CTAB method of DOYLE Æ DOYLE (1987), and purified in CsC1/ethidium bromide gradients. Double-stranded DNAs of the non-coding region between psba and trnh genes were amplified direcfly by 30 cycles

3 Non-coding sequences of Sonchinae 87 of symmetric PCR using two primers. The primers were designed with T. SANG (Michigan Sate University) using the alignment of previously published sequences of dicots (Fabaceae, Brassicaceae, and Asteraceae; SI~APIRO & TEWaRI 1986, REITH & STRAUS 1987, AMBROS~N~ & al. 1992). The primer sequences, which were used for both DNA amplification and sequencing, are: 5'-GTT ATG CAT GAA CGT AAT GCT C-3 / and 5 I- CGC GCATGG TGG ATT CAC AAT C-3'. Methods for PCR amplification, purification of PCR products, and sequencing reactions are given in SANG & al. (1994) and KIM S.-C. & al. (1996b). Sequences were aligned manually, which was feasible because of the small number of unambiguous indels (insertions/deletions). All sequences were deposited in GenBank (Table 1) and the aligned sequences are available from the first author upon Table 1. Source of plant materials for sequence comparisons of chloroplast intergenic spacer between psba and trnh. Voucher specimens are deposited in the Ohio State University Herbarium (OS) and the Herbarium of University of Texas (TEX). All sequences are deposied in Genome sequence Database (GSDB) Taxa Voucher GSDB accession numbers Aetheorhiza CASS. A. bulbosa (L.) CASS. Dendroseris D. DON D. litoralis SKOTTSB. D. marginata (BERT. & DCNE.) HOOK. & ARN. Embergeria BouLos E. grandifolia (T. KIRK) Botmos Kirkianella ALLAY K. novae-zelandiae (HooK. f.) ALLAN Lactucosonchus (SCH. Bin) SVENT. L. webbii (SCH. Bin) SVENT. Launaea CASS. L. arborescens (BATT.) MURB. Prenanthes L. P. altissima L. P. pendula ScH. Bw. P. purpurea L. Reichardia ROTH R. ligulata (VENT.) KUNDEL &; SUNDING R. picroides (L.) ROTH R. tinginata (L.) ROTH Sonchus L. Subg. Dedrosonchus SCH. BIP ex BOULOS S. acaulis DUM.-CouRs. S. canariensis (SCH. Bw.) BOULOS S. fruticosus L. f. S. gonzalezpadroni SVENT. S. gummifer L~NK JANSEY 1105 (TEX) STUESSY & al (OS) STtmSSY & al (OS) AZKINSON 118/85 (OS) D. GLENNY 4910 (OS) KIM & al (OS) KIM & al (OS) MEtIRHOFF s.n. (TEX) KIM & al (OS) KIM & al (OS) KIM & al (OS) Belgium Bot. Gard KEW KIM & al (OS) I~M & al (OS) KIM & al (OS) KIM & al (OS) KIM & al (OS) GSDB:S: GSDB:S: GSDB:S: GSDB:S: GSDB:S: GSDB ;S: GSDB:S: GSDB:S: GSDB:S: GSDB:S: GSDB:S: GSDB:S: GSDB:S: GSDB:S: GSDB:S: GSDB:S: GSDB:S:

4 88 S.-C. K~ & al.: Table 1 (continued) Taxa Voucher GSDB accession numbers Subg. Origosonchus BOtJLOS S. luxurians (R. E. FRIES) C. JEFFV, EY S. schweinfurthii OHr. & HmRN Subg. Sonchus L. S. asper L. HmL S. bourgeaui SCH. BIe. S. kirkii (T. KIRK) AJ~LAN S. maritimus L. S. palustris L. S. tuberifer SWyT. Sventenia FONT QUER S. bupleuroides FONZ Qt;ER Taeckholmia BOULOS T. capillaris (SWNT.) BOULOS T. pinnata (L. f.) BOVLOS Taxaxacum WEBER T. officinale WEBER I~ox 2559 (OS) KNOX 2560 (OS) JANSEN 1109 (TEX) KIM 1035 (OS) SmBtJRY s.n. (OS) L. VILAR s.n. (OS) KIM 1050 (OS) KIM & al (OS) KIM & al (OS) I~M & al (OS) KIM & al (OS) JAYSZY 1107 (TEX) GSDB:S: GSDB:S: GSDB:S: GSDB:S: GSDB:S: GSDB:S: GSDB:S: GSDB:S: GSDB:S: GSDB:S: GSDB:S: GSDB:S: request. Complete sequences of the psba-trnh intergenic region were generated for 30 accessions representing nine genera and 24 species of subtribe Sonchinae (from a total of 11 genera and about 130 species; B~MER 1993, 1994) and one species of Taraxacum (Table 1). Two species of Dendroseris (subtribe Dendroseridinae of STEBBINS 1953) were also sequenced because this genus was clearly nested within subtribe Sonchinae in the ITS phylogeny (KIM S.-C. & al. 1996b; Fig. 1). Three species of Prenanthes were also sequenced to determine the taxonomic position of P. pendula (see Discussion). Fitch parsimony was performed using PAUP version (SwoFmPm 1993) with all changes weighted equally and ACCTRAN, MULPARS, and TBR options. Multiple islands of equally parsimonious trees (MAI~D~SON 1991) were searched for by performing 100 random entries in heuristic searches. Prenanthes purpurea was used as an outgroup based on the previous phylogenetic study of ITS sequences (KaM S.-C. & al. 1996a, b; Fig. 1). Indels were treated as missing data. Bootstrap analysis using HEURISTIC search, with simple addition, ACCTRAN, MULPARS, and TBR options, was conducted with 100 replicates (maxtree=100) (FEI~SENSTEIN 1985) to evaluate the amount of support for monophyletic groups. Decay analysis (B~MER 1988, DONOGHUE & al. 1992) was also performed using the same HEURISTIC search parameters and trees up to five steps longer were examined. Pairwise sequence divergences were calculated by the Kimura twoparameter method (KIMURA 1980) using PHYLIP version 3.52c (FEI~SEYSTE~N ). TO compare the phylogenetic utility of ITS and the psba-trnh intergenic spacer sequences in the Sonchinae, the original ITS data matrix for 49 taxa of Sonchinae (K~M S.-C. & al. 1996a, b; Fig. 1) was reduced to 30 taxa (same taxa as in cpdna data set). The same options were used to find the shortest trees for the reduced ITS data set. Parsimony analysis

5 Non-coding sequences of Sonchinae 89 was also performed on the combined data sets. The same options were used to find the shortest trees using the combined data. Results Length and sequence divergence of psba-trnh spacer. The length of the intergenic spacer between psba and trnh varies from 385bp (Reichardia picroides) to 450bp (Sonchus palustris) in the subtribe Sonchinae. The aligned sequences cover 506bp and contain 23 indels. The direction of evolutionary change of indels was hypothesized by outgroup comparison. A total of ten insertions and 13 deletions was inferred; approximately 50 % are shorter than four base pairs. The longest insertion, which was 25 bp, occurred only in S. palustris. Of the seven putative insertions longer than four base pairs, six were found to be direct repeats or part of direct repeat motifs. A five base pair deletion was shared by all three species of Reichardia. All genera of Sonchinae, except two basal genera Reichardia and Launaea, have a 15 bp insertion. Sequence divergence in the psba and trnh intergenic spacer ranged from 0.00 to 7.54 % (R. picroides and Embergeria grandifolia) in the Sonchinae, with an average of 2.4 %. Mean sequence divergence within subg. Sonchus was 2.0 %, while within the woody Sonchus alliance in Macaronesia it was 1.0 % (Table 3). Within Reichardia, sequence divergence ranged from 0.0 % to 3.5 %, with an average of 2.3 %. Phylogenetic analysis of the psba-trnh spacer sequences. A total of 105 variable sites was found among the 506 aligned nucleotides (21%) in subtribe Sonchinae and the outgroup, with 48 of them (about 10%) phylogenetically informative. Parsimony analyses identified 8952 equally parsimonious trees with a length of 118, a consistency index (CI) of (excluding autapomorphies) and a retention index (RI) of (Fig. 2). These trees do not support the monophyly of the Sonchinae as delimited by B~MER (1993, 1994) because Dendroseris and Prenanthes pendula are embedded within the subtribe (Fig. 2). The genus Dendroseris, endemic to the Juan Fernandez Islands in the Pacific, is closely related to the woody Sonchus alliance in Macaronesia. There are no synapomorphies supporting the monophyly of the woody Sonchus alliance in Macaronesia. The cpdna tree also suggests that Reichardia and Launaea are basal within the Sonchinae. Both Sonchus and Prenanthes are polyphyletic, whereas Reichardia and Sonchus subg. Origosonchus are monophyletic. Phylogenetic analysis of the reduced ITS data (30 taxa) and combined data sets. There are 252 variable sites (50 %) in the reduced ITS data set, 182 (36 %) of which are phylogenetically informative. Parsimony analysis found 90 equally parsimonious trees (one of which is shown in Fig. 3) with a length of 570 (CI:0.599, RI:0.748). These trees, like the one based on the original data set of K~ S.-C. & al. (1996b), suggest that the Sonchinae of BREMER (1993) is not monophyletic, and that the clade including Reichardia and Launaea represents the basal lineage in the subtribe. The monophyly of the woody Sonchus alliance in Macaronesia is still strongly supported in the reduced ITS tree (bootstrap value of 99 %, decay value > 5). Finally, the reduced ITS phylogeny suggests that Sonchus palustris is no longer the sole sister group to the alliance, and that several Pacific

6 90 S.-C. KIM & al.: Table 2. Summary of parameters calculated for each of the three data sets. NA not applicable; *values are based on strict consensus tree Aligned sequence length Number of variable sites Number of informative sites Tree length Number of the shortest trees Consistency index (excluding autapomorphies) Average bootstrap value* Average decay value Number of resolved nodes* Average sequence divergence Skewness of tree-length Distribution (gl) Reduced ITS psba-trnh Combined (30 taxa) (30 taxa) (30 taxa) (50%) 83 (16%) 335 (33.3%) 182 (36%) 48 (9.5%) 230 (22.8%) % 66.4% 79.3% NA % 2.4% NA genera, such as Dendroseris, Kirkianella, and Embergeria (i.e. clade B and C of Fig. 1), are more closely related to Aetheorhiza and several species of subg. Sonchus (clade D) than to the woody Sonchus alliance (clade A). These relationships are weakly supported by both bootstrap and decay values. Parsimony analysis of the combined data sets (ITS and psba-trnh spacer sequence) was also conducted and found 360 equally parsimonious trees (not shown). These trees are topologically almost identical to the orte based on the reduced ITS data set, except for the relationships in the woody Sonchus alliance (i.e. clade A). Comparison ofpsba-trnh and ITS. A summary of 11 parameters calculated for the three different data sets is shown in Table 2. The total lengths of aligned sequences in the reduced ITS and psba-trnh regions are nearly identical: 504 and 506 in ITS and psba-trnh, respectively. However, the ITS sequences provide approximately three times more variable sites (252 vs. 83) and almost four times more phylogenetically informative positions (182vs. 48) than the chloroplast intergenic spacer. Furthermore, the ITS trees provide more resolved nodes and higher decay and bootstrap values. All three data sets have high Ga values, indicating that there is a strong phylogenetic signal in the data. Discussion Phylogenetic comparisons among the reduced ITS, the psba-trnh, and combined trees. The phylogenetic trees derived from three data sets show both congruence and incongruence. First, trees generated from the reduced ITS, the psba-trnh, and combined data sets, strongly suggest that the Sonchinae as circumscribed by B~MER (1993, 1994) is not monophyletic, and that Dendroseris from the Juan Fernandez Islands and Prenanthes pendula from the Canary Islands

7 Non-coding sequences of Sonchinae 91 should be included in the subtribe. Both Dendroseris and P. pendula are clearly nested within the Sonchinae (Figs. 2, 3, and not shown). These results are in agreement with phylogenies generated from ITS sequences (Fig. 1) and cpdna restriction site (WHITTON & al. 1995) data. Therefore, the Dendroseridinae of STZBBINS (1953) should not be recognized as a distinct subtribe. All trees also indicate that Reichardia and Launaea are basal in the Sonchinae and are not part of the main radiation of the subtribe. The basal position of the two genera is shown in all trees (Figs. 2, 3, and not shown) and this relationship is further supported by the lack of the 15 bp insertion in the two genera in the cpdna tree (Fig. 2). In the cpdna tree, Launaea is sister to the clade containing Reichardia species, and these two genera are sister to the remainder of the Sonchinae (Fig. 2). However, the reduced ITS, combined data, and the original ITS trees suggest that Launaea is sister to the clade containing the remaining genera of the Sonchinae (Figs. 1, 3, and not shown). The phylogenetic relationship between the two genera needs to be investigated further with rauch broader sampling. Nevertheless, this study strongly suggests that Reichardia and Launaea represent old lineages within the Sochinae. All trees except cpdna indicate that the woody Sonchus alliance in Macaronesia, i.e. woody members of Sonchus and five allied genera, was derived from a common ancestor and a single dispersal event is responsible for the extensive morphological and ecological diversity of the alliance (Figs. 1, 3, and not shown). The monophyly of the woody Sonchus alliance is strongly supported by bootstrap (higher than 99 %) and decay values (at least more than five steps). However, the psba-trnh spacer tree (Fig. 2) suggests a close relationship between Dendroseris and the woody Sonchus alliance (albeit with weak support) and provides no support for the monophyly of the alliance in Macaronesia. This is probably due to the slow rate of substitution in the psba-trnh spacer sequence. Based on the reduced and combined data sets, it seems highly likely that the entire alliance is a monophyletic group and the original ITS trees also strongly supports this view (Fig. 1). The sister taxon (of taxa) of the woody Sonchus alliance in Macaronesia is uncertain. In the psba-trnh tree, the clade containing S. palustris and species of Sonchus endemic to Africa (i.e. subg. Origosonchus) is sister to the clade with the alliance and Dendroseris (Fig. 2). However, in both the reduced ITS and combined data trees, S. palustris is sister to the clade containing all genera of Sonchinae except Reichardia and Launaea. Furthermore, subg. Origosonchus is sister to clades B, C, and D (Fig. 3 and not shown). In the original ITS tree (Fig. 1), S. palustris is the only sister taxon to the entire alliance. Therefore, the sister taxa of the entire alliance are uncertain, but it seems likely that S. palustris, which occurs widely in Europe, or African Sonchus species may have been involved in the origin of the woody Sonchus alliance in the Macaronesia. All data sets support the close relationship of Aetheorhiza to subg. Sonchus sectt. Sonchus and Asperi (Figs. 1, 2, 3, and not shown) and these suggest that Aetheorhiza originated after the Sonchus group diverged from a common ancestor with the Reichardia and Launaea lineage. ITS data suggested that Prenanthes pendula, the only species in the genus endemic to the Canary Islands, is most closely related to the Macaronesian woody

8 33~ 92 S.-C. KN & al.: 5O% (>5) % 12(4) 66% (2) / (>5), % 13(2) 72% 6(1) 56% 100' 21% ù2ù 2.29_ ~~t 77% ~ _6 7~ 43 n %1 «' 13 ~~~ ~78o~ 11oo : / 920/0 ""~'"1 5(,5/ I / I looo~ I ~ 4± 12(lo/I (2} ~4 95% Krigia montana Microseris laciniata Pyrrhopappus multicaulis Outgroups Lactuca perenis Lactuca sativa J Prenanthes purpurea m Prenanthes Taraxacum officinale ~ Taraxacum Prenanthes altissima ~ Prenanthes Reichardia picroides -- Reichardia tinginata Reichardia Reichardia ligulata Launaea arborescens -- Launaea nudicaulis _ Launaea Aetheorhiza bulbosa -- Sonchus kirkii Sonchus asper Clade D Sonchus oleraceus Sonchus bourgeaui -- Sventenia bupleuroides -- Babcockia platylepis Prenanthes pendula(n) Prenanthes pendula(s) Sonchus tuberifer Sonchus canariensis Sonchus congestus The woody Sonchus fruticosus Sonchus Sonchus gonzalezpadroni alliance in Sonchus ortunoi Macaronesia Taeckholmia pinnata (Clade A) Taeckholmia canariensis Taeckholmia heterophylla Taeckholmia arborea Lactucosonchus webbii -- Sonchus palustris Sonchus arvensis Sonchus maritimus Clade C Kirkianella novae-zelandiae Embergeria grandifofia -- Dendroseris fitoralis -- Dendroseris marginata Dendroseris macrantha Dendroseris micrantha Dendroseris pruinata Dendroseris neriifolia Dendroseris pinnata Dendroseris berteroana Dendroseris regia Sonchus luxurians Sonchus schweinfurthii q m Dendroseris (The Juan Fernandez Islands, Chile) (Clade B) Fig. 1. ITS sequence phylogeny of subtribe Sonchinae (modified from K]u S.-C. & al. 1996b). This is one of the 144 equally most parsimonious trees with a length of 898 (consistency index of excluding autapomorphies and retention index of 0.743). Dashed lines indicate branches that collapse in the strict consensus tree. The bootstrap support is shown below the nodes Sonchus alliance, especially to Sventenia and Babcockia (KN S.-C. & al. 1996a, b; Fig. 1). In the cpdna phylogeny (Fig. 2), P. pendula is clearly nested within a clade containing the woody Sonchus alliance and Dendroseris. The relationships indicated in the ITS and psba-trnh trees are congruent with the natural occurrence of intergeneric hybrids between Sventenia and P. pendula (SvEN'rENIUS 1960, HANSEN & S~DtNC 1985) in suggesting a close relationship between the taxa. The

9 Non-coding sequences of Sonchinae 93 is 51% 90% 1(? 2 45 Fo 1(1) 45% E Prenanthes purpurea ~ Outgroup Taraxacum officinale -- Taraxacum Prenanthes altissima ~ Prenanthes Reichardia tingitana 1 Reichardia ligulata Reichardia picroides t(9). 22% 3 6 Sonchus bourgeaui Sonchus maritimus 3(4) 1 62% 2(42 81N 3 Embergeria grandifolia Reichardia Launaea arborescens ~ Launaea Aetheorhiza bulbosa 1 1 (1) Sonchus kirkfi 66% Sonchus asper Clade D of Fig.1 Kirkianella novae-zelandiae] Clade C of Fig.1 / q --J 151: 4(4) 71% d P rtion 1(1) 4O% 2(1) 46%.110.) 15% 3(1) 7O% 5(5) 100% Sonchus tuberifer.1~o0o~.. " " 23 Yo 1~ 2 -- Sonchus canariensis 1(.0). 4 Sonchus fruticosus 27% Sonchus gummifer... 1.(0j... 12% Sonchus palustris m Sonchus subg. Sonchus Sonchus luxurians Sonchus subg. Sonchus schweinfurthfi - Origosonchus - - Sonchus gonzalezpadroni The woody Sonchus alliance in - - Sonchus acaulis Macaronesia Taockholmia pinnata (clade A of Fig.1) Prenanthes pendula Taeckholmia capillaris Sventenia bupleuroides Lactucosonchus webbii _ Dendroseris fitoralis "] Dendroseris (clade B of Dendroseris marginata J Fig.1 : The Juan Fernandez Islands) Fig. 2. One of the 8952 equally parsimonious trees of the subtribe Sonchinae (BREMER 1993, 1994) based on intergenic spacer sequences between psba and trnh of chloroplast DNA (consistency index of excluding autapomorphies and retention index of 0.787). Three taxa of Prenanthes are in bold. Dashed lines indicate branches that collapse in the strict consensus tree. Number above nodes represent base substimtions followed by the decay values in parentheses and the bootstrap support (%) is shown below the nodes psba-trnh spacer of cpdna does not, however, provide sufficient resolution to determine the phylogenetic relationship of P. pendula within the Macaronesian clade. Nevertheless, the occurrence of natural hybrids and the placement of P. pendula in the Macaronesian clade in both nuclear and cpdna trees suggests that P. pendula shares a more recent common ancestor with members of the woody Sonchus alliance than it does with other members of Prenanthes. Thus, Prenanthes as now delimited is not monophyletic. More detailed studies of Prenanthes,

10 94 S.-C. KIM & al.: (>5~ r 100'~ 20(>5) 97% 33(>5) 100% 4(1) 38% I 4O% 6~14 I 9(>5) 1 100%1 5 4 Sonchus luxurians 5(3) I 94% 5 Sonchus schweinfurthfi 7 Sonchus tuberifer 3 Sonchus canariensis 3 Sonchus acaulis 7 Sonchus fruticosus 3 Sonchus gummifer 1 Prenanthes purpurea 40 Taraxacum officinale 133 Prenanthes altissima 5 Reichardia tinginata Reichardia ligulata 12 Reichardia picroides 20 Aetheorhiza bulbosa -- 4 Sonchus kirkii 5 Sonchus asper... ' 14 Sonchus bourgeaui 20 Sonchus maritimus 19 Kirkianella novae-zelandiae Embergeria grandifolia Dendroseris litoralis Dendroseris marginata Sonchus gonzalezpadroni Taeckholmia capillaris Taeckholmia pinnata 6 Sventenia bupleuroides 1(1) I 49% 5 Prenanthes pendula 3o Lactucosonchus webbfi Sonchus palustris Launaea arborescens Outgroup Taraxacum Prenanthes "] Reichardia Clade D Clade C 3 Dendroser~(The Juan Fernandez islands, clade B) -] Sonchus subg. Origosonchus The woody Sonchus alliance in Macaronesia (clade A) Launaea Fig. 3. Orte of 90 equally parsimonious trees based on reduced ITS sequence (Tree length = 570; CI=0.599; RI=0.748). Number above nodes represent base substitutions followed by the decay values in parentheses. Number below branches indicate bootstrap support (%) Dashed line indicates branch that collapses in strict consensus tree including African (i.e.p. subpeltata), Asian, and North American species, are needed for a more accurate circumscription of the genus. Based on the congruence among the trees discussed above, it seems reasonable to conclude that subtribe Sonchinae of Bp,~~R (1993, 1994) is not monophyletic and Dendroseris and P. pendula should be included in the Sonchinae. The results also suggest that Reichardia and Launaea represent old lineages in the subtribe and that the woody Sonchus alliance in Macaronesia is a monophyletic assemblage.

11 Non-coding sequences of Sonchinae 95 However, the sister group of the entire alliance remains uncertain. The genus Sonchus as a whole is highly polyphyletic and it needs to be revised. Prenanthes pendula is more closely related to members of the woody Sonchus alliance in Macaronesia than to congeneric species of Prenanthes. Finally, at this point, the relationships among clades A-D cannot be resolved clearly based on available data. Phylogenetic utility of the psba-trnh intergenic spacer. The summary of tree statistics for the psba-trnh spacer and the ITS region indicate that ITS sequences are more useful for resolving relationships in the Sonchinae (Table 2). The ITS sequences produced fully resolved trees at the inter- and infrageneric levels, while there are many unresolved nodes (due to lack of synapomorphies) at infrageneric (e.g. Sonchus) or intergeneric levels of insular endemics (e.g. the woody Sonchus alliance) in the cpdna trees (Fig. 2). Furthermore, ITS sequences produced fewer equally parsimonious trees (with slightly higher homoplasy) and thus resulted in trees with higher bootstrap and decay values. There are approximately three times as many variable sites and about four times as many informative sites in the ITS sequences than in psba-trnh sequences. This shows that the non-coding sequences of nrdna are more suitable than short cpdna noncoding sequences for resolving relationships in subtribe Sonchinae. Average sequence divergence of the intergenic spacer between psba and trnh is 2.4 % (Tables 2 and 3), whereas an average of 9.9 % sequence divergence was estimated for ITS sequences (KN S.-C. & al. 1996b). This indicates that the psbatrnh intergenic spacer of cpdna evolves approximately four times slower than the ITS region of the nuclear ribosomal DNA. In all cases, sequence divergence of non-coding regions of cpdna is lower than in the ITS (Table 3). Our results are congruent with the GIELLY & al. (1996) comparison between the trnl intron of cpdna and nrdna ITS in the genus Gentiana (Gentianaceae). They suggested that ITS sequences have two to three times higher sequence divergence than trnl intron sequences. They also found that the ITS-based phylogeny displays higher bootstrap values and slightly higher homoplasy, suggesting that the trnl intron is more useful at the intergeneric level. These studies indicate that the substitution rate of intergenic spacer of cpdna is comparable to that of cpdna introns and that short non-coding sequences (e.g. about 500 bp) of cpdna are more suitable for phylogenetic study at the generic rather than at the interspecific levels. Rates of nucleotide substitutions appear to vary considerably among different plant groups as weil as among different non-coding regions of chloroplast DNA. At Table 3. Comparisons of average pairwise sequence divergence between psba-trnh intergenic spacer and ITS within several Sonchinae taxa (B~uER 1993, 1994) Reichardia Sonchus subg. Sonchus Sonchus subg. Dendrosonchus The woody Sonchus alliance in Macaronesia Subtribe Sonchinae psb A-trnH ITS 2.3% 4.7% 2.0% 9.7% 0.9% 1.7% 1.0% 2.5% 2.4% 9.9%

12 96 S.-C. KIM & al.: the infrageneric level, the psba-trnh noncoding region seems to be useful for several genera (Table 3). For example, the average sequence divergence of subg. Dendrosonchus in the Macaronesian islands is approximately 0.9 % and that of the woody Sonchus alliance is 1.0%. In subg. Sonchus, the pairwise sequence divergence ranges from 0.0 to 3.6 %, with an average of 2.0 %. These values are comparable to the average divergence for the trnt(u~u)-trnl (UAA) intergenic spacer of Echium species in Macaronesia (BöHLE & al. 1994). In subg. Sonchus, the psba-trnh intergenic region evolves much faster than the trnt-trnl or trnl-trnf non-coding regions (KIM S.-C., unpubl, data). However, this region alone may not be useful for resolving relationships among closely related insular endemic groups (Fig. 2), especially ones that apparenfly diverged rapidly by adaptive radiäfion. Restriction site analysis of the entire chloroplast genome is more suitable for congeneric species (JANSEN al. 1997) or recently radiated insular endemics (FRANCISCO-ORTECA & al. 1995) than are short intergenic spacer sequences. This approach provides more information because it examines more sequence variation, especially when many frequent-cutting enzymes are used (JANSEN & al. 1997). In conclusion, the intergenic spacer between psba and trnh seems to be relatively well-suited for inferring phylogenies at the intergeneric or in some instances at the infrageneric levels in the Asteraceae. The small size of this region requires the use of only two primers for amplification and sequencing. Long stretches of As or Ts, which may complicate sequencing, are relatively rare (GIELLEY & TABERLET 1994). However, this region may not be long enough to provide sufficient numbers of characters for resolving relationships among congeneric species or recenfly radiated groups in Asteraceae. Preliminary sequence comparisons should be performed before undertaking large-scale taxonomic surveys due to the heterogeneity of evolutionary rates of non-coding sequences of cpdna. Other intergenic spacers are currently being sequenced but evaluation of their phylogenetic utility awaits further study. We thank JAVIER FRANCISCO-ORTEGA, AGUEDO MARRERO, PEDRO ORTEGA-MAcH[N and FRANCISCO GONZÄLEZ ARTILES, for assistance during the field work in the Canary Islands; ERI KNox, TOM MYERS, DAVID GLENNY, and P. J. GARNOCK-JONES for providing plant and DNA materials. Special thanks go to JAVIER FRANCISCO-ORTEGA for bis continual encouragement and help, and introducing us to Macaronesian plants during the course of this study. We also thank JEANNETTE WHITTON, BILLIE TURNER, and an anonymous reviewer for comments that improved the manuscript. This study was done by the first author in partial fulfillment of the Ph.D. at The Ohio State University, and was supported by a NSF Doctoral Dissertation Improvement grant DEB to D. J. C. and S.-C. K. and NSF grant (DEB ) to R. K. J. References AMBROSINI, M., CECI, L. R., FIORELLA, S., GALLERANI, R., 1992: Comparison of regions coding for trna (His) genes of mitochondrial and chloroplast DNA in sunflower: a proposal concerning the classification of 'CP-like' trna genes. - Pl. Molec. Biol. 20: 1-4. BALDWIN, B. G., CAMPBELL, C. S., PORTER, J. M., SANDERSON, M. J., WOJC1ECHOWSKI, M. F., DONOGHUE, M. J., 1995: Utility of nuclear ribosomal DNa internal transcribed spacer

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