-- Pl. Syst. Evol. 215:209-227 (1999) Systematics and Evolution © Springer-Verlag 1999 Printed in Austria

Molecular phylogeny of and closely related : evidence from 5.8 S, ITS 1 and ITS 2 of the rDNA

ELINA LESKIN N and CECILIA ALSTRÖM-RAPAPORT

Received August 15, 1997; in revised version June 5, 1998

Key words: Salicaceae, Salbe, Populus, Chosenia, Flacourtiaceae, Idesia. - Phylogeny, ITS, rDNA. Abstraet: A ribosomal DNA region, including the entire 5.8S RNA gene and the internal transcribed spacers ITS 1 and ITS 2, was used for studying the phylogeny of Salicaceae and the relationship between Salicaceae and Flacourtiaceae. The length of the ITS regions within Salicaceae and Flacourtiaceae was similar to that found in other angiosperms. The GC content of both ITS regions was high, varying 62.7-72.2%. The most parsimonious clusters the wind-pollinated Chosenia bracteosa among the Salix species, suggesting that it should be included in the Salix. The grouping within Salix subg. Salix as paraphyletic, for which reason the subgeneric division is questionable. Populus was monophyletic and formed a sister group to Salix. The interspecific variation of the ITS sequences was very small in Salicaceae, which is in contradiction to the age of the group according to the evidence from fossil data. Idesia polycarpa from Flacourtiaceae shows great sequence similarity with Salicaceae, but the analysis of 5.8S rDNA supports monophyly of the four species of Flacourtiaceae sampled for this study.

Salicaceae is a family of dioecious woody and with a distribution primarily in the northern hemisphere. The family comprises c. 350 species of willows and poplars, which are classically divided into two genera, Salix and Populus. Salicaceae is the only family in the order Salicales, which belongs to the subclass Dillenidae under Magnoliophyta (BRUMMIrr 1992). HALLmR (1910, 1912) suggested that the species Idesia polycarpa in Flacourtiaceae (order Violales) is closely connected to Salicaceae. This idea was initially rejected, but has later gained ground (MEEtJSE 1975, MmLER 1975, CROYQt~ST 1988). The coupling is supported by that fact the salicin is produced both in Salicaceae and Idesia along with some other Flacourtiaceae, but not by any other (CRONQUIST 1988). Other characters shared by Idesia polycarpa and Salicaceae is the presence of salicoid teeth in the leaves (HICKEY • WOLFE 1975) and several similarities in their wood anatomy (MmLZR 1975). Within Salicaceae the genus Salix with its well developed nectaries has been thought to be predominantly insect-pollinated, while Populus is considered as 210 E. LESKINEN& C. ALSTRÖM-RAPAPORT: wind-pollinated (CRONQUIST 1988). Salix bracteosa was found to be wind- pollinated and this together with some morphological characters motivated NAKAI (1920) to assign it to a new genus Chosenia. More recently even some other Salix species have been shown to be at least partly wind-pollinated (AR~us 1974; Fox 1992; ALSTRöM-RAPAPORT& LASCOUX, pers. comm.). It is questionable whether the mode of pollination divides the species into two monophyletic groups, therefore the mode of pollination may not be an adequate character for dividing Salix further into two genera. The genus Salix is a complex group and its has continuously, from the days of LINNAEUS, been under revision. SKVORTSOV (1968), who has worked mainly with European and Asian willows, has divided Salix into three subgenera, Salix, Vetrix and Chamaetia, which are further divided into several sections. SKVOkTSOV points out, however, that the separation of Vetrix and Chamaetia is not clear, and this has led DoRN (1976), working mainly with American willows, to acknowledge only two subgenera, Salix and Vetrix. There are several taxonomical problems also at the species level. One of the main difficulties in the identification of species within Salix has been attributed to hybridization. MZIKJ~E (1984), however, considers the effect of hybridization exaggerated since there are several other problems contributing to the difficulties in species identification. The species of Populus appear to have a stable diploid genome, while the ploidy level of Salix species varies both interspecifically and intraspecifically. Wm~Jx'qsoN (1944) summarized the chromosome numbers of 27 Salix species. Nine were diploid, seven tetraploid and the rest displayed several ploidy levels. Polyploidy has been observed in different Salix species groups that have independent diploid ancestors. Polyploidization must therefore have occurred independently several times in Salix. It has even been suggested that poly- ploidization occurred more than once within the same species (ARcus & DORN 1976). Our investigation seeks to clarify some of the questions stated above using DNA sequence analysis. We have chosen to study the internal transcribed spacer (ITS) regions and the interlying 5.8S rDNA, which are known to be appropriate for systematic surveys at the generic and familial levels within the angiosperms (BALDWIN & al. 1995). This region is highly repeated within the plant genome, but undergoes rapid concerted evolution, which promotes intragenomic uniformity. Thirteen of the c. 300 Salix species, Chosenia bracteosa and four of the c. 50 Populus species were used in out analysis along with four species from Flacourtiaceae and Betula pendula. The Salbc species were chosen to represent the putative subgenera. Our aims are 1) to describe the ITS sequences and their divergence within Salicaceae, and to study 2) the taxonomic position of Chosenia bracteosa within Salicaceae, 3) the division of Salix into subgenera and 4) the taxonomic position of Idesia polycarpa.

Materials and methods Eighteen species belonging to Salicaceae (order Salicales), four species belonging to Flacourtiaceae (order Violales) and one species of Betulaceae (order Fagales) were Molecular phylogeny of Salicaceae and closely related Flacourtiaceae 211

Table 1. List of species sequenced for ITS and the origin of samples, UBG Uppsala Botanical Garden, Sweden, UEF Ultuna Experimental field, Sweden, UWM University of Wisconsin- Madison, USA, GBG Gothenburg Botanical Garden, Sweden, HCG Helsinki City Gardens, Finland, CBG Copenhagen Botanical Garden, Denmark, MBG Melbourne Botanical Garden, Victoria: L&A-R LESrdNEN & ALSTRöM-RAPAPORT,A-R ALST~öM-RAPAPORT,L&P LESVdYEN & PAMmO; UPS Botänical Museum, Uppsala University Species, family Source Voucher information Salicaceae Salix alba L. UBG L&A-R S-1 (UPS), EMBL AJ006423 & amygdaloides A~ERSSON UEF L&A-R S-2 (UPS), EMBL AJ006424 S. dasyclados WIMMER UEF L&A-R S-3 (UPS), EMBL AJ006425 S. exigua NurT UWM SYTSMA, no voucher, EMBL AJ006426 S. fragilis L. UBG L&A-R S-4 (UPS), EMBL AJ006427 S. herbaceae L. UBG L&A-R S-5 (UPS), EMBL AJ006428 S. pentandra L. UBG L&A-R S-6 (UPS), EMBL AJ006429 S. purpurea L. UBG L&A-R S-7 (UPS), EMBL AJ006430 S. retusa L. UBG L&A-R S-8 (UPS), EMBL AJ006431 S. schwerinii E. WOLF UEF A-R S-9 (UPS), EMBL AJ006433 S. serpyllifolia ScoP. UBG L&A-R S-10 (UPS), EMBL AJ006432 S. triandra L. UBG L&A-R S-11 (UPS), EMBL AJ006434 S. viminalis L. UEF L&A-R S-12 (UPS), EMBL AJ006435 Chosenia bracteosa (TuRcz.) NAK. GBG L&A-R S-14 (UPS), EMBL AJ006436 Populus alba L. UBG L&A-R S-15 (UPS), EMBL AJ006437 P. deltoides BARTR. Æ MARSI~. HCG L&P S-16 (UPS), EMBL AJ006438 P. lasiocarpa OLIV. UBG L&A-R S-17 (UPS), EMBL AJ006439 P. trichocarpa TORR. & GREY UBG L&A-R S-18 (UPS), EMBL AJ006440 Flacourtiaceae Idesia polycarpa MAXlM. CBG L&A-R S-19 (UPS), EMBL AJ006441 Azara integrifolia RuIz & PAVON MBG SPENCER, no voucher, EMBL AJ006442 A. serrata Rurz & PAVON MBG SPENCER, no voucher, EMBL AJ006443 Dovyalis caffra HooK. f. GBG NBVENDOR~ no voucher, EMBLAJ006444 Betulaceae Betula pendula ROTH UBG L&A-R S-20 (UPS), EMBL AJ006445 sequenced for the 5.8S ribosomal DNA and the internal transcribed spacers on its either side, ITS 1 and ITS 2. The samples were obtained from the collections of the Agricultural University of Uppsala, Helsinki City Garden and the Botanical Gardens of Uppsala, Gothenburg, Copenhagen, Melbourne and University of Wisconsin-Madison (Table 1). The Salix species were chosen to represent the different subgenera within the genus and several sections (Table 2). DNA isolation and sequencing. The DNA was isolated from leaves. The isolation was made according to the CTAB protocol by HmLIS & al. (1990) for plants, fungi and algae. The DNA region including ITS 1, ITS 2 and the interlying 5.8S rDNA were amplified from the isolated DNA by a polymerase chain reaction (PCR) using an automatic thermocycler (PTC-100 TM Programmable Thermal controller, MJ Research, Inc.). Of the sample supernatant 10 gl was amplified in a final volume of 50 gl (containing 500mM KC1, 100mM Tris-HC1 and 1% Triton X-100) and 1.2mM Mg ++, 20 ng/gl BSA, 0.2mM dNTR 2.5 u Taq polymerase (Dynazyme) and 0.25 gM of each primer. The primers a and d (see Fig. 1 for primer information) annealed to the end of the 18S rDNA adjacent to ITS 1 and 212 E. LESVd~N & C. ALSTRöM-RAPAPORr:

Table 2. Division of Salix into subgenera and sections according to SKVOgTSOV (1968) and Dom,~ (1976) SKVORTSOV (1968) DORN (1976)

Species Subgenus Section Subgenus Section

Salix alba Salix Salix Salix - S. fragilis ...... - S. exigua " - " Longifoliae S. amygdaloides " Humboldtinae " Humboldtinae S. pentandtra " Pendtandrae " Salicaster S. triandra " Amygdalinae " - S. herbacea Chamaetia Retusae Vetrix Retusae S. retusa " " " " S. serpyIlifolia ...... S. dasyclados Vetrix Vimen " Vimen S. schwerinii ...... S. viminalis ...... S. purpurea " Helix " Helice

18S rRNA 5.8S rRNA 28S rRNA

a * c »

ITS 1 4 b ITS 2 4 d

a=5'-TCGTAACAAGGTTTCCGTAGG-3' b=5'-GCTGCGTTCTTCATCGWTG-3' c=5'-CAWCGATGAAGAACGCAGC-3' d =5'-TTCCTTCCG CTTATTGATATGC-3' Fig. 1. Approximate location of primers for amplifying the sequencing the ITS regions in the genome of Salicaceae. Primers a and d were used for amplifying and a-d were used for sequencing. W = 50 % of A and 50 % of T. Primer a is based on sequence comparisons from the Genbank, primers b-d are slight modifications from the primers of WHmE & al. (1990) the beginning of the 28S rDNA adjacent to ITS 2. Thermal cycling consisted of an initial denaturation step (6 min in 94 °C), followed by 35 cycles comprising a denaturation step (1 min 10 s in 94 °C), an annealing step (50 s in 54 °C) and an extension step (1 min 30 s in 72°C), followed by a final extension step (10min in 72°C). The amplified DNA was purified by filtration with Wizard TM PCR Preps DNA purification system (Promega), combining the products of separate reactions to produce a concentrated DNA solution enriched in the target sequence and ready for sequencing. PCR products were sequenced by the chain termination method as described by CASANOVA Æ al. (1990) using a T7 SequencingTM Kit (Pharmacia Biotech). Both strands of each sample were sequenced using four primers (Fig. 1) in order to gain good resolution over the whole DNA region. The sequenced region comprised a short sequence at the 3 r end of 18S rDNA, ITS 1, the entire 5.85 rDNA, ITS 2 and a short sequence at the 5 t end of 28S rDNA (Fiß. 2). Coding Molecular phylogeny of Salicaceae and closely related Flacourtiaceae 213

18S rDNA ITS 1

I Salba GTGAACTGCG G~J~GGATCAT TGTCG~CC TGCCCC .... GGCAG~CGA CCCGCG~CT Samy Sdas Sexig Sfrag ...... C Sherb Spent ...... C Spurp Sret Sschw ...... Y ...... C Sserp ...... C Strian ...... ° ...... C Svim ...... C Cbrac ...... C Palba ...... T-.°°° A ...... C Pdelt ...... T-.... A ...... C Plasio Ptric Ipoly Azint ...... --...T A ..... G ...... T ..... Azser ...... TGCCT A ...... T ..... Dcaff ...... - ...... T ......

Salba CGTGGCATGA CAAGCTGGGC --TCG-GGGG GCACCCGCCC CT-CGCGTCC AC--GCGTGC Samy Sdas ...... T ...... Sexig ...... T ...... T ...... Sfrag Sherb ...... T ...... Spent Spurp ...... T ...... Sret ...... T ...... Sschw ...... T ...... Sserp ...... T ...... Strian ...... C ...... Svim ...... T ...... Cbrac ...... T ...... Palba ...... T ...... GC. Pdelt ...... T ...... T ...... G.. Plasio ...... T ...... T ...... G. Ptric ...... T ...... T ...... S. Ipoly ...... C ...... T ...... T...R..G. Azint T ...... GA. ..C...T..G G .... C ...... TT...G ...... C... C..AC..G. Azser T ...... GA. ..C...C..G GG...C ...... G.TT...G ...... C... C.CAC..G. Dcaff T ..... CGAG ..T...A..G CTG.A ...... T.TT.T.. .C.T ...... CT..TG.G.

Salba CGTGGAGGGA CGCG-TCCGC GCCCGACACG GCCCGCCAAC GAACCCCGGC GCGAGAAGCG Samy ...... T ...... Sdas ...... A...T ...... T ...... Sexig ...... A...T ...... T ...... Sfrag ...... T ...... Sherb ...... A...T ...... T ...... Spent ...... T ...... Spurp ...... A. .T ...... T ...... Stet ...... A. .T ...... T ...... Sschw ...... A. .T ...... T ...... Sserp ...... A. .T ...... T ...... Strian ...... A. .T ...... T ...... Svim ...... A. .T ...... T ...... Cbrac ...... Æ. .Œ ...... T ...... Palba ...... T ...... --TG. ° . .T°..A ...... £delt ...... A.C ...... --TG.. . .T. ° .A ...... Plasio ...... T ...... --.G.. .T...A ...... Ptric ...... A.C ...... --TG.. .T...A ...... Ipoly ...... A...T ...... A..G.. °T ...... Azint ...... C.T.. A..T...G.. .T ...... A ...... Azser ...... AAC.T ..... T...G.. .vB ...... A ...... Dcaff • C...C..G T..A...T.. A ...... G.A .T..AA... A ...... Fig 2 (continued) 214 E. L~SmNEN & C. ALSTRöM-RAPAPORT:

Salba CCAAGGAAAT GGAGTACCAG GAGCACGCCC TCGTAGCCTC GGTGTCGGGG ...... GC Samy Sdas ...... T ...... T ...... Sexig ...... T ...... T ...... Sfrag Sherb ...... T ...... T ...... Spent Spurp ...... T ...... T ...... Sret ...... T ...... T ...... Sschw ...... T ...... T ...... Sserp ...... T ...... T ...... Strian ...... T ...... T ...... R ...... Svim ...... T ...... T ...... M ...... Cbrac ...... T ...... T ...... Palba ...... T ...... T ...... G ..... C ...... C ...... Pdelt ...... T ...... T ...... G ..... C ...... C ...... Plasio ...... T ...... T ...... G ..... C..C ...... C ...... Ptric ...... T ...... T ...... G ..... C ...... C ...... Ipoly ...... C. T ...... TG ...... A.A ...... Azint ...... T. A ...... A.A .C.A..A... C...C.A.CT .... A.A... CGCATGGG.. Azser ...... T. A ...... G.A .C.A..A... C...C.A.CT .... A.A... CGCATGGG.. Dcaff ...... C. A ...... G.A .... G ..... C...C..TGA A.G..GCTCT GG .... GG.T

5.88 rDNA

I Salba GCGCCTTCTT TTT-GTGATA ATCT-TAACG ACTCTCGGCA ACGGATATCT CGGCTCTCGC Samy Sdas ...... G ...... Sexig ...... G ...... Sfrag Sherb ...... G ...... Spent Spurp ...... C ...... G ...... Stet Sschw Sserp Strian ...... G ...... Svim Cbrac ...... G ...... Palba ...... C.G ...... A ...... Pdelt ...... C.G ...... A ...... Plasio ..... C... C.G ...... A ...... Ptric ...... C.G ...... A ...... Ipoly • ..T ...... C. .G...A ...... Azint .T.T.G ...... C.C ..... G..GA ...... C ...... Azser .T.T.G ...... C.C ..... G...A ...... Dcaff ...T..C.AA ..... C ..... G...A ...... T ...... T..

Salba ATCGATG~G PakCGTAGCGA AATGCGATAC TTGGTGTGAA TTGCAGAATC CCGTGPakCCA Samy Sdas Sexig Sfrag Sherb Spent Spurp Sret Sschw Sserp Strian Svim Cbrac Palba Pdelt Plasio Ptric Ipoly Azint Azser Dcaff Fig. 2 (continued) Molecular phylogeny of Salicaceae and closely related Flacourtiaceae 215

Salba TCGAGTCTTT GAACGCAAGT TGCGCCCGAG GCCTCCTGGT CGAGGGCACG TCTGCCTGGG Samy Sdas Sexig Sfrag Sherb Spent Spurp Sret Sschw Sserp Strian Svim Cbrac Palba Pdelt ...... Y ...... Plasio Ptric ...... T ...... Ipoly ...... A°..C T ...... Azint ...... A°..C ..... - ...... Azser ...... A...C ..... - ...... Dcaff .... T ...... A...C ..... - ......

ITS 2

--I Salba TGTCACGCAT CGTCGCCCCC GCTCCCC--T CGGCTCACGA GGGCGGGGG- CGGATACTGG Samy Sdas Sexig Sfrag Sherb Spent Spurp Sret Sschw Sserp Strian Svim Cbrac Palba ...... T ...... ~ ...... Pdelt Plasio ...... T ...... Ptric Ipoly ...... A ...... T...R ...... A ...... Azint ...... A.AG.A ...... GA... Azser ...... ATAG. G.C ...... G ...... GA... Dcaff ...... AAA .... TC ..... CA.G ...... G ......

Salba TCTCCCGCGC G--CTCCCGC CCGTGGTTGG CCTA/~kATCG AGTCCCCGGC GACGGTCGCC Samy Sdas ...... T ...... Sexig ...... T..C ...... T ...... Sfrag Sherb ...... T ...... Spent Spurp ...... T ...... Sret ...... T ...... Sschw ...... T ..... R .... T... Sserp Strian ...... T ...... Svim ...... T ..... G .... T... Cbrac ...... T ...... T ...... Palba ...... T..C ...... C ...... T ...... Pdelt ...... T..C..C ..... C ...... T ...... Plasio ...... G... T..C..C ..... C ...... T ...... Ptric ...... T..C..C ..... C ...... T ...... Ipoly C ...... T .... T..C ...... C ...... T ...... A ..... Azint C ...... T ...... T..C ...... C ...... TT ...... CC .... Azser ...... T ...... T..C ...... C ...... TT ...... CC .... Dcaff ...... T .TG..T .... T ...... C ..... T ...... T ...... CT... Fig. 2 (continued) 216 E. LESK1NEN & C. ALSTRÖM-RAPAPORT:

Salba ACGACAAGCG GTGGTTGAGA GACCCTCGGA CACGGTCGTG CGCGTGCTC- GTCGCCCCC- Samy Sdas ...... AT ...... Sexig ...... T ...... -. Sfrag ...... T ...... Sherb ...... T ...... Spent ...... T ..... T ..... Spurp ...... T ...... Sret ...... ST ...... Sschw ...... T ...... Sserp ...... T ...... Strian ...... T ...... T ...... -. Svim ...... T ...... Cbrac ...... T ...... Palba .... G ...... C..CT ...... Pdelt .... G ...... T ...... C° .C ...... Plasio .... G ...... T ...... C° .C ...... Ptric .... ~ ...... T ...... C. .C ...... Ipoly .... ~ ...... CT .CT ...... T.. Azint .T...G ...... A..A ...... C. .C ...... T.C Azser .T...G ...... A. A..A ...... C. .C ...... T.C Dcaff .... @ ...... T.A. AG ...... A.. .C.T ..T..G.TTT

I Salba GG-ACCTCCC GGACCCCCGA GCATT---GG C-TTTC~GG ATGCTCTCGT TGCGACCCCA Samy ..G ...... Sdas Sexig Sfrag ..G ...... Sherb Spent Spurp ..G ...... Sret ..G ...... Sschw Sserp Strian Svim ..G ...... Cbrac .°G ...... Palba • G.T .... T ...... T..G .... C. .A ~ .... T ...... Pdelt ..G.T .... T ...... T..G .... C. .A ..C...T ...... C ...... Plasio • G.T .... T ...... T..G .... C. .A ..C...T ...... Ptric • G.T ...... T..G .... C. .A • C...T ...... C ...... Ipoly • G ...... T ...... T..G .... C. T. .... ATG ...... Azint • G.T .... T ...... T ...... C. T. T ..... T ...... Azser ù G ...... T ...... T ...... C. T. T ..... C ...... Dcaff • G.TA...A A ..... T ...... CATC.. ùT..G.T ...... C ......

28S rDNA

Salba GGTCAGGCGG GACTACCCGT GAGTTTAA Samy Sdas Sexig Sfrag Sherb Spent Spurp Stet Sschw Sserp Strian ...... T ...... Svim Cbrac Palba Pdelt Plasio Ptric Ipoly Azint Azser Dcaff Molecular phylogeny of Salicaceae and closely related Flacourtiaceae 217 region termini were determined by comparison with Krigia spp., Asteraceae (KIM & JANSEN 1994) and rice, Oriza sativa, Poaceae (TAvOJWA & al. 1985). Analyses of data, Sequences were aligned using CLUSTAL V with both fixed and floating gap penalties of 10 (Hic¢3iNS & al. 1992). The species in Salicaceae and Flacourtiaceae were compared using the program MEGA 1.01 (KuMAR & al. 1993). Pairwise distances, based on estimating the number of nucleotide substitutions per nucleotide site, were calculated using KTMURA'S2-parameter model (K~u~ 1980). Gaps were treated as missing data. The phylogeny of Salicaceae and Flacourtiaceae was examined by the maximum parsimony method with the program PAUP 3.1. Published 5.8S rDNA sequences of several other species of dicots were also included in the analysis made using the heuristic options of 1000 random addition sequences and TBR branch swapping (Acer platanoides, Aceraceae, Geraniales, GenBank access no. U57773; Cucumis melo, Cucurbitaceae, Violales, GenBank access no. Z48805; Oxalis villosula, Oxalidaceae, Geraniales, GenBank access no. Z66547; Paeonia lutea, Paeoniaceae, Theales, GenBank access no. U27683; Rhododendron luteum, Ericaceae, Ericales, GenBank access no. X96814; Sinapis alba, Brassicaceae, Capparales, GenBank access no. X15915). The choice of species in the analysis was made with the help of classification by B~MER & al. (1997). A more detailed analysis including 22 species of Salicaceae and Flacourtiaceae was made on the entire region sequenced (the 3' end of 18S RNA gene, the entire 5.8S RNA gene, the 5 r end of the 28S RNA gene and the internal transcribed spacers ITS 1 and ITS 2) was also made using the heuristic options of 1000 random addition sequences and TBR branch swapping in PAUP 3.1, and a bootstrap analysis based on 500 replicates was used for estimation support. All changes were weighted equally. The multistate option was applied due to polymorphism in some species.

Results ITS 1 and 2 sequences and 5.8S rDNA gene in Salicaceae and Flacourtiaceae. The length of 5.8S rDNA is almost invariant in flowering plants, and it was 162- 164 bp within the species investigated (Table 3). The 5.8S rDNA showed very little nucleotide väfiation within Salicaceae with only two variable sites, one being at the beginning of the 5.8S gene (Fig. 2). Salix alba and S. amygdaloides differed by one nucleotide ffom all other Salix species, and Populus trichocarpa differed at one site from the other Populus species. An alignment of both Salicaceae and Flacourtiaceae had ten variable sites, including one gap. The length of the ITS regions within the species investigated was similar to that found in other angiosperms (BALDWlN & al. 1995). The length of ITS 1 was 222 bp in all Salix species including Chosenia bracteosa, except 223 bp in S. triandra (Table 3). Within Populus the length of ITS 1 was 219 bp in all species. In Flacourtiaceae the length vafied from 221 bp in Idesia polycarpa to 241 in . Betula pendula had an ITS 1 length of 216 bp. The ITS 2 regions in

Fig. 2. The alignment of rDNA sequence region in Salicaceae and Flacourtiaceae. The genes for rRNA genes are shown in bold (sites 1-22 are the 3' end of 18S rRNA, nueleotides 266-429 5.8S rRNA, and 652-688 the 5 t end of 28S rRNA). The symbols .... indicate a nucleotide identical with that on the top row, and "-" a gap. The polymorphic nucleotide sites are indicated: Y T/C, R A/G and M A/C 218 E. LESKINEN & C. ALSTRÖM-RAPAPORT:

Table 3. The length (n number of nucleotides) and GC content of ITS 1, 5.8S RNA and ITS 2 of the species sequenced ITS 1 5.8 S RNA ITS 2 n GC % n GC % n GC % Salicaceae, Salicales Salix spp. 222-223 65.8-68.5 163 55.8-56.4 211-212 67.8-69.7 Chosenia bracteosa 222 66.2 163 56.4 212 68.4 Populus spp. 215-219 67.5-69.9 163 55.8 207-213 69.0-71.2 Flacourtiaceae, Violales Idesia polycarpa 221 67.1 163 55.8 212 67.9 Azara spp. 234-241 65.0-66.0 162 55.6-56.2 213-214 66.7-67.3 Dovyalis caffra 227 64.3 162 54.3 223 62.7 Betulaceae, Fagales Betula pendula 216 61.3 164 55.8 223 62.8

Salicaceae and Flacourtiaceae were slightly shorter than the ITS 1 regions. The length of ITS 2 was 211-213 bp in Salicaceae and ranged from 212 bp in I. polycarpa to 223 in Dovyalis caffra in Flacourtiaceae. In Betula pendula the ITS 2 region was longer than the ITS 1 region being 223 bp. In Salix schwerinii, S. serpyllifolia, S. triandra, Populus deltoides and Idesia polycarpa some nucleotide sites were polymorphic, which showed as intraspecific variation (Fig. 2). Interspecific nucleotide differences in the ITS sequences were very small within the genus Salix. Within the Salix species the number of variable sites was 25 (12 in both ITS 1 and ITS 2 and one in 5.8S rDNA). Of these 11 were phylogenetically informative. Alignment of the SaIix species and Chosenia bracteosa was simple with only four 1 bp gaps. A comparison within Salicaceae (Salix, Chosenia and Populus) showed 35 variable and 20 phylogenetically informative sites in ITS 1, 32 and 21 in ITS 2, and two and orte in 5.8S rDNA, respectively. An alignment assumed four gaps of the length of 1-2 bp in ITS 1 and seven gaps in ITS 2. The alignment of Salicaceae and Flacourtiaceae had 93 variable and 54 phylogenetically informative sites in ITS 1, 70 and 39 in ITS 2, and nine and three in 5.8S rDNA, respectively. The longest gap in alignment was eight bp and totally there were 11 gaps in ITS 1, one gap in 5.8S rDNA and ten gaps in ITS 2 (Fig. 2). We calculated pairwise distances using Kimura's 2-parameter model. Becanse of close sequence similarities within both Salix and Populus, we used the mean distances of these genera to compare with the other species (Table 4). The analysis showed similarities between Idesia polycarpa and Salicaceae. Based on the ITS 1 sequences Salix species were closer to Idesia than Populus. The ITS 2 sequences were more uniform within Salicaceae and Flacourtiaceae than the ITS 1 se- quences, and the difference between Idesia polycarpa and the other Flacourtiaceae was smaller. The GC content of both ITS regions was highest in Salicaceae, varying between 65.8-69.8 % in Salix and Chosenia and 67.5-72.2 % in Populus (Table 3). The highest GC content of 68.0-68.5 % in ITS 1 and 68.9-69.8 % in ITS 2 were Molecular phylogeny of Salicaceae and closely related Flacourtiaceae 219

Table 4. Proportions of nucleotide differences between the Salix and Populus species and Chosenia bracteosa, Idesia polycarpa, Azara intermedia, A. serram and Dovyalis caffra. In the comparison mean values were used for Salix and Populus species. The upper right triangle shows the Kimura 2-parameter values for the ITS 1 sequence and the lower left traingle for ITS 2 sequence Salix spp. C. bracteosa Populus spp. L polycarpa A. intermedia A. serrata D. caffra Salix spp. - 0.01 0.09 0.08 0.25 0.24 0.33 C. bracteosa 0.01 - 0.09 0.07 0.25 0.23 0.33 Populus spp. 0.09 0.09 - 0.09 0.25 0.25 0.34 I. polycarpa 0.11 0.11 0.09 - 0.21 0.21 0.33 A. intermedia 0.15 0.15 0.12 0.11 - 0.03 0.31 A. serrata 0.15 0.15 0.13 0.13 0.03 - 0.28 D. caffra 0.17 0.17 0.18 0.18 0.19 0.19 -

found within the "Salix alba group" of S. alba, S. amygdaloides, S. fragilis and S. pentandra. The overall variation within Flacourtiaceae was 62.7-67.9 %, being highest in Idesia polycarpa. The GC content in the other families studied here was clearly lower varying between 50.6-59.5 %, while in Betulaceae it was up to 61.3- 64.0 %. The 5.8S rDNA had a smaller nucleotide bias, the GC content ranging from 54.3 % to 56.4 % in the species sequenced in this study. Phylogeneti« analyses. 5.8S rDNA sequences from Salicaceae , Flacourtia- ceae and several other species representing different orders were used for maxi- mum parsimony analysis in order to solve the phylogenetic status of Salicaceae and Flacourtiaceae. The heuristic search produced two most parsimonious trees with a concistency index of 0.714 and requiring 49 steps. A strict consensus tree showed Salicaceae and Flacourtiaceae as monophyletic groups, although both of them were supported by only one nucleotide change (Fig. 3).

Rhododendrum luteum 5 Paeonia lutea 4 6 Sinapis alba

1 0 Acer platanoides

5 ~ Oxalis villosula 5 5 Betula pendula Cucumis melo 6 4 Dovyalis caffra 1 Azara integrifolia 1 A. serrata Fig. 3. The strict consensus tree based 0 Idesia polycarpa on 5.8S rDNA sequences of Salicaceae, 1 2 Flacourtiaceae and other species repre- 0 Populus alba senting different orders in the subclass P. trichocarpa Dillenidae. Betula pendula was used as 1 1 Salix amygdaloides an outgroup. Numbers below branches S. viminalis 1 are nucleotide differences 220 E. LESKINEN & C. ALSTRÖM-RAPAPORT:

Salix alba

S. amygdaloides S. fragilis S. pentandra S. dasyclados S. herbaceae S. purpurea S. retusa S. serpyllifolia S. schwerinfi

S. viminalis S. triandra S. exigua Chosenia bracteosa Populus alba P. deltoides

P. trichocarpa P. lasiocarpa Idesia polycarpa Azara integrifolia

A. serrata Dovyalis caffra

Salix alba

S. amygdaloides S. fragilis S. pentandra S. dasyclados S. herbaceae

52 S. purpurea --"2"-" S. retusa 2 S. serpyllifolia S. schwerinfi "--ry--lO0 803 [ 0 0 S. viminalis S. triandra Chosenia bracteosa Fig. 4a. The strict con- S. exigua sensus tree based on 5.8S RNA gene and Populus alba 4 ITS 1 and 2 sequences 98 P. deltoides 0 94 12 of Salicaceae and Fla- P. trichocarpa 3 courtiaceae. 4b. Boot- P. lasiocarpa strap analysis. Numbers 5 Idesia polycarpa above branches indicate 18 Azara integrifolia bootstrap % values and numbers below bran- 100 7 A. serrata 28 ches are nucleotide dif- Dovyalis caffra 64 ferences Molecular phylogeny of Salicaceae and closely related FIacourtiaceae 221

We hext used the whole sequence for analysing Salicaceae and Flacourtiaceae by rooting the tree between these families as suggested by the above analysis. The heuristic search with a consistency index of 0.834 required 241 evolutionary steps and produced 82 most parsimonious trees (Fig. 4a). In the strict consensus tree Idesia polycarpa was basal within Flacourtiaceae, and as already indicated by its close similarity with Salicaceae, its lineage had rar fewer substitutions than those leading to the other species in Flacourtiaceae. The genera Populus and Salix (including Chosenia) formed monophyletic sister groups. Chosenia bracteosa fell among the Salix species. Two groups remained in the consensus tree with Salix. One included S. alba, S. amygdaloides, S. fragilis and S. pentandra, which all belong to Do~'s subgenus Salix (Table 2) along with S. exigua and S. triandra. Another cluster was formed by the species pair S. schwerinii and S. viminalis from the section Vimen (Table 2). A bootstrap analysis gave high support to the results above, with the exception that S. exigua fell outside the rest of the genus Salix with a low bootstrap support of 52 % (Fig. 4b).

Discussion Polymorphic nucleotide sites were found in Salix triandra, S. schwerinii, S. serpyllifolia and Idesia polycarpa. The ITS sequence of S. schwerinii was polymorphic at three nucleotide sites. One of the polymorphic nucleotides at each polymorphic sites was identical to that in S. viminalis and in the most parsimonious tree the two grouped together. Salix species are known to hybridize frequently (REcnnY~ER 1992), and it seems possible that our sample 'S. schwerinii' is a hybrid between, S. viminalis and another Salix species not included in out investigation. Salix schwerinii and S. viminalis are both classified in the section Vimen (Table 2). Ribosomal DNA is present in the nuclear DNA often in thousands of copies (RO~ERS & BEYDICH 1987). Normally sequence homogenization corrects the nucleotide differences that might arise in the complementary rDNA copies due to mutation or hybridization. In the polymorphic cases, concerted evolution has not yet led to complete homogenization, and they might even indicate recent hybridization. The ITS sequences of flowering plants are rather short (187-298 bp) compared with those in many other organisms (VENKATESWARLU& NAZAR 1991, BALDWlY & al. 1995). Salicaceae with its ITS sequences of 210-223 bp falls well within the range. There were two characteristics of interest for the ITS region in Salicaceae. First, the GC content of both ITS regions in Salicaceae was high, varying between 65.8-71.2%. It was roughly similar in both ITS sequences (Table 3), which is presumably due to coevolution, since both spacers are involved in the maturation of the large subuuit rRNAs (BALDWrN & al. 1995). The GC% in the ITS areas of the angiosperm genome is most often about 50 % or slightly more (BALDW~ & al. 1995), although it has been shown to range ffom c. 31% in Viscaceae (Niedrer & al. 1994) to as high as 77 % in Poaceae (TAKAIWA & al. 1985). It is to be noted that although the GC content was high in the ITS areas, it was close to 50 % in the interlaying 5.8S RNA gene. Light (low GC content) and heavy (high CG content) isochores are known from the DNA of warm-blooded vertebrates. The formation of isochores has been explained either 222 E. LESKINEN Æ C. ALSTRÖM-RAPAPORT: to be a result of selection (BERNARDI & al. 1985, 1988) or mutation pressure (Gu & LI 1994). In humans most genes are concentrated in the GC-rich isochores, which even replicate earlier in the ce11 cycle than the GC-low isochores (BERNARDI & al. 1985, 1988). In the ITS 1 and ITS 2 and 5.8S rDNA sequences of Salicaceae it is the reverse: GC content is high in the non-coding areas and lower in the coding area. Another interesting feature is that the sequence divergence and the number of phylogenetically informative sites were very low in Salicaceae, especially among Salix and Chosenia species. Such low values are rare in angiosperms (BALDWIN & al. 1995, WEN & ZIMMER 1996). It is tempting to assume that Salicaceae has undergone a recent evolution and that the c. 300 extant species are the outcome of this.However, in the fossil record Populus is known from as early as Late Paleocene, which ended about 65 MYA ago according to the geological time scale by HARLA~ & al. (1982), and Salix from Eocene, 55-65 MYA ago. These oldest willow fossils belong to subg. Salix found in North American Early Eocene formations in Wyoming and North Dakota (COLLINSON 1992). The earliest occurrences of Vetrix in Alaska, North Amefica date back to Oligocene, 38-55 MYA (WoL~ 1988). In Europe both Salix and Populus occur later in the fossil record. Of those species that we have used in our analysis, fossil evidence exists from Salix alba, S. fragilis, S. herbacea, S. pentandra, S. purpurea, S. triandra and S. viminalis (KOMAROV 1936, HUNrLEY & HUNTLEY 1992), which proves that Salicaceae is an old, well established plant group. DONOaHtrE & BALDWlN (1993) have presented similar results of low sequence divergence in the ITS region of Vibumum with a fossil record from Paleocene and Cretaceous periods. BALDWIN & al. (1995) conclude that some ancient woody groups evolve more than ten times slower than herbaceous lineages. If the separation of Salix and Populus dates 60 MYA and the diversification of Salix 40 MYA, the genetic distances of the ITS sequences suggests substitution rates as low as 0.75 x 10 -9 and 0.4 x 10 -9 substitutions per site and per year. Wood anatomy (TAKUTAJAN 1969 in MmLER 1975), floral morphology and similarity of the host-parasite relationships of Salicaceae and Flacourtiaceae show that they are closely related (MEEUSE 1975). It has even been suggested that Salicaceae should be included in the Violales (MEEUSE 1975, MmLER 1975) and lately this suggestion has gained more general acceptance (THom'~ 1992, B~MER & al. 1997). The 5.8S rDNA sequences in our study suggest that Flacourtiaceae and Salicaceae may have shared a common ancestor within the order Violales. MEEUSE (1975) even thought it possible that Salicaceae may be placed within Flacourtiacaeae as a tribe, which has recently gained support by CHASE & al. (1996). A coupling between Salicaceae and Idesia has been suggested earlier, partly because salicin is produced by both, along with some other Flacourtiaceae, but not by any other plants (CRONQUIST 1988). Another character shared by Idesia and Salicaceae is the salicoid teeth in the leaves (HICKEY & WOLFE 1975). The close similarity of the ITS sequences supports the relatedness of Idesia polycarpa to Salicaceae as the substitution distance between Idesia and Salix is even smaller than that between Populus and Salix (Table 4). This could result from slow evolution in Idesia, as the analysis of the shorter and more conserved 5.8S rDNA Molecular phylogeny of Salicaceae and closely related Flacourtiaceae 223 sequence presented Salicaceae and Flacourtiaceae as monophyletic sister groups (Fig. 3). Both groups were supported by a single substitution, and we conclude that the position of Idesia remains unclear. The maximum parsimony analysis placed Chosenia bracteosa among the Salix species, rendering Salix a paraphyletic group if Chosenia would be kept as a separate genus. We conclude that C. bracteosa should be included in the genus Salix. Although SKVORTSOVaccepts Chosenia as a separate genus in Salicaceae he admits that it is impossible to deny its similarities to Salix. For example, the buds are very similar to the buds of S. cardiophylla and the anatomy resembles that of the species in the section Longifoliae. The ITS sequences give no clear support to any specific affiliation of C. bracteosa within Salix. Its bark resembles that of the section Amygdalinae (SKVORTSOV 1968), and other morphological character resemble much those of S. amygdaloides. S~:VORTSOV (1968) divided Salix into three subgenera Salix, Chamaetia and Vetrix, while DORN (1976) divided the species only into two subgenera Salix and Vetrix, uniting SKVORTSOV'SChamaetia and Vetrix into one. In our material there are samples of each putative subgenus (Table 2). The maximum parsimony analysis of the ITS sequences and 5.8S rDNA produced a separate subgroup of four species S. alba, S. amygdaloides, S. fragilis and S. pendula, with tree-like growth forms, all from the subgenus Salix. According to the division of DORN and SKVORTSOV, S. exigua and S. triandra also belong to subgenus Salix. In our strict concensus analysis the positions of S. triandra and S. exigua were unresolved. However, in the bootstrap analysis S. exigua fell outside all other Salix species with a support of 52%, suggesting that S. exigua may have diverged from other Salix species earlier in the evolution. This conclusion is supported by CnON~ & al. (1995), who suggested that the North American S. exigua should be separated from all other willows, since it did not group with any of the American species of Salix and Vetrix on a study based on isozyme data. MOSSELER (1990) on the other hand found that hybrid crosses between species of the subgenera Salix and Vetrix did not produce with the notable exception of S. exigua. It produced seeds, when crossed to species in Vetrix, although with poor F1 viability, but did not produce any, when crossed to species of the subgenus Salix. According to SKVORTSOV(1968) and DORN (1976) those characters in Salix that resemble Populus most are 'primitive', and the subgenus Vetrix has more derived characters. Primitive characters in Salix include tree-like growth forms and high number of stamens (Table 5). It is possible, in the light of both morphological traits and our sequence data, that Vetrix is monophyletic and Salix paraphyletic, and our results give no positive support for the subgeneric division. DORN (1976) states polyploidy as a derived character. Within Salix polyploidization has apparently occurred several times, even within the subgenera (DORN 1976). The diploid chromosome number in Populus is 38. DiploidY appears ancestral in Salix as S. exigua also has 2n = 38. Among the species studied by us, the group of tree-form species (S. alba, S. fragilis and S. pentandra) has tetraploid numbers (2n = 76). Salix amygdaloides, which clustered with S. alba is diploid indicating that it might have reversed from tetraploidy. Salix triandra and S. retusa have varying ploidy levels (Table 5), and polyploidization seems to have occurred independently in them. 224 E. LESKINEN & C. ALSTRÖM-RAPAPORT:

Table 5. Life form, geographic distribution, stamen number and chromosome number of the Salicaceae species included in the phylogenetic analysis. Unmarked information is based on SKVORTSOV(1968). Geographic distribution: a western Africa, b eastern Europe, c western Europe, d former Soviet Union, e western China, fnorth-east China, g Korea, h Mongolia, i Asia Minor, j Japan, k North America, l South America, m Greenland, n Arctic Species Geographic Life form Stamen Chromosome distribution number number Salix alba a, b, c, d, i large tree 2 7611 S. fragilis b, c, d. medium tree 2 7611 S. triandra b, c, d, e, f, g, i, j high 311 38, 44, 8811 S. pentandra b, c, d medium tree 3-10 7611 S. amygdaloides large tree 3-8 (12) 4 384 S. herbacae b, c, d, m, n small shrub 2,8, 11 3811 S. retusa b, c, d small shrub 2 74, 76, 1112'11 S. serpyllifolia b, c small shrnb 2 37, 38, 393 S. dasyclados b, c, d large shrub 8 29 38 S. viminalis b, c, d large shrub 8' 11 29 3811 S. schwerinii b, c, d large shrub 2 3813 S. purpurea shrub 12 111 382 S. exigua k z shrub 24 3813 Chosenia bracteosa g, j8 large tree 8 55,8 3813 Populus alba tree 8 5-91 3811 P. deltoides k 8, 8 large tree8 30-607 3711 P. trichocarpa tree 40-601 P. lasiocarpa tree 3-61 (-110) 6 1BOES & STRAUSS 1994, 2BRUNSFELD8Z al. 1992, 3BuCHLER 1986, 4DORN 1976, 5HÄI~NSSON 1955, 6HON~ & al. 1987, 7KAUL 1995, 8KoMAROV 1936, 9LARSSON& BREMER 1991, 10LöVE & LöVE 1961, 11MEIrmE 1984, 12REcmN~ER 1992, 13yURTZEV& ZHUKOVA1982

The genus Populus consists of approximately 30 species divided into six sections. Out study includes four Populus species from different sections. The results from the ITS sequence support the phylogenetic tree produced by FAIVRE- RAMPANT (1992) based on the physical map of nuclear rDNA units for ten Populus species in four different sections. In both analyses P. alba, which has five to ten stamens, appears to be basal to the other Populus species. This agrees with the hypothesis that an increased number of stamens is a derived state in Populus (KAtm 1995).

We thank URBANGULLBERG for all support during the work, and KÄRE BREMER,BIRGITTA BREMER, PEKKAPAMILO and MARTINLASCOUX for their valuable comments in the manuscript. U~AN PETTERSSON, Ultuna Experimental Field, MATTIAS IWARSSON,Botanical Garden in Uppsala, MAGNUSNEVENDORF, Botanical Garden in Gothenburg, PE~R KaOGSTRUe, Botan- ical Garden in Copenhagen, SAMI'O SAIY~O, Gardens of the Helsinki City, ROGER SeENCER, Royal Botanical Garden in Melbourne, and KENNET~ SYXSMA, University of Wisconsin- Madison are greatly acknowledged for their help in collecting the material. The work was financed by NUTEK. Molecular phylogeny of Salicaceae and closely related Flacourtiaceae 225

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Addresses of the authors: ELINA LESKIYEN (e-mail: [email protected]), Department of Genetics, Uppsala University, Box 7003, S-750 07 Uppsala, Sweden. - CECILIA ALSTRöM-RAPAPORT, (e-mail: [email protected]), Depart- ment of Plant Breeding, Swedish University of Agricultural Sciences. Box 7003, S-750 07 Uppsala, Sweden. Present address: Department of Genetics, Uppsala University, Box 7003, S-750 07 Uppsala, Sweden.

Accepted June 5, 1998 by K. B~ME~