--Plant Pl. Syst. Evol. 215:209-227 (1999) Systematics and Evolution © Springer-Verlag 1999 Printed in Austria Molecular phylogeny of Salicaceae and closely related Flacourtiaceae: 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 tree clusters the wind-pollinated Chosenia bracteosa among the Salix species, suggesting that it should be included in the genus Salix. The grouping within Salix leaves 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 trees and shrubs 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 plants (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 taxonomy 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.