Zooming in onth elettuc e genome: species relationships inLactuca s.L, inferred from chromosomal and molecular characters WimJ .M .Koopma n , CENTRALE 3279 Promotoren: Prof. Dr.Ir . L. J.G .va nde rMaese n Hoogleraar Plantentaxonomie, Leerstoelgroep Biosystematiek Wageningen Universiteit Prof. Dr.Ir .E .Jacobse n Hoogleraar Plantenveredeling, Laboratorium voor Plantenveredeling Wageningen Universiteit Co-promotor: Dr. R.G .va nde nBer g Universitair Hoofddocent, Leerstoelgroep Biosystematiek Wageningen Universiteit Promotiecommissie: Prof.Dr .P .Baa s Universiteit Leiden Dr. J.H .d eJon g Wageningen Universiteit Dr. J.C .M .de nNij s Universiteit vanAmsterda m Prof. Dr.R .F .Hoekstr a Wageningen Universiteit Prof. Dr.A .Lebed a Palacky University Stellingen i L. sativa,L. serriola,L. saligna, enL. virosastamme na fva nee n gemeenschappelijke voorouder:L. sativa ishet produc t van selectie doord emens ,L. serriola ontstond als cultuurvolger,L. saligna ontstond doornatuurlijk e selectiee nL. virosaontston ddoo r hybridisatieme tee nno gonbekend emannelijk e ouder. Ditproefschrift,H.2,4. II DeZuid-Afrikaans e soortL. dregeanai see n 17eeeuws e ontsnapper uit deslateel t in de Compagnies Tuin van de VOC bij de Kaap, en conspecifiek met L. sativa, L. serriola,L. dregeanae nL. altaica. Ditproefschrift, H.5 . Ill Gezien de nauwe verwantschap van L. tatarica met L. sibirica (zoals blijkt uit hun morfologie, ITS-1sequenties ,kruisbaarheid ,AFL Ppatrone n enDN Agehaltes) ,i sd e indelingva ndez esoorte ni napart egener anie t gerechtvaardigd. Ditproefschrift , H.4,5,6 . ContraShih (1988) . IV The subsectie Cyanicaesoorte n L. tenerrima en L.perennis (en mogelijk L. graeca) zijn niet nauw verwant met de andere Europese Lactuca soorten, en moeten uit het geslachtLactuca worde n verwijderd. Ditproefschrift,H .4,5 . ContraFerakov a(1977) . V Inafwijkin g vanhet algemen ebeel di nhoger eplante n(Baro wan dMeister ,2002 ) zijn DNAhoeveelhei de nA Tgehalt ei nLactuca s.l .significan t (ennegatief ) gecorreleerd. Ditproefschrift, H.6 . VI Omdato pAFL Pdat agebaseerd e fenogrammen encladogramme nove rhet algemeen een hoge mate van congruence vertonen, leidt het interpreteren van UPGMA clusteringal sphylogenetisch e analyse(bijv . Sharmae tal. , 1996;Caiced oe tal. , 1999; Aggarwal et al., 1999) waarschijnlijk toch tot betrouwbare conclusies over evolutionaire verwantschappen. Koopman,ungepubliceerd egegevens . EjBppsqsijqndu n 'ucuidoo^i •sdiqsuopBpjAJBuopnjoA au o suoisnpuoa aiqBipiinsijnsaiupsXiquqojd(6661 'FI3\e/AieS3y-6661 ''U*J3opaoxuj!g66l ''F p Bmreqs -Sa) SISATBUBDpaua§o[Aq dB IO J Suuajsnp VWOdfl Suppisira 'sureiSopBp pasBq dldV PUB suraiSouaqd pasBq <nJV J° souatuSuoa jBjauaS aqi 8uuapisuo3 IA •9 113'sisamsiiu, pajBpUOO(XpAIJB§3 U puB) ApuBoyniSis 3JE sapads xs VDUIDVJ guoure /(ouanbaij XV PUB jusjuoo VNQ '(Z00£ '-tsJsiapMpu s AVOJBQ) siirejdaaqgi q in uopBtvus UOUIUIODaq i 01 ISBJIUODU\ A •gViO 'sisainsnij, voniovq snuaSaq ima g papnpxa aq pjnoqs puB 'sapads vomovj uBsdomg jaqio aqi jo AUB 01 pajBpi Apsop 1011ai B (noavuS •7 Ajqissod puB) smuduad j puB viuujaudj 7 sapads avDiuvA'j uopoasqns aqx AI '(886I)H!1SWJU03 •9' 5> H3 'sissinsiuj, •psijpsnf }OUS TBI9U9 8SJBJBdS SU ISapad s 3S3qjJOUOpBOIJTSSB p3q j'(SJU91UO D VNQ PUB 'sujsijBd <njv 'A4HiqBssoio 'saouanbas i-sn 'XSopqdtoui jpqi uiaa luajBddB SB) vouiqis 7 puB vouvwt '7 uaaA\pq diqsuopBpj asop aqi 8uuapisuc>3 III •j-iO'sissqisnij, •vjivip 7puB'vuvaSdup j'vpujas J'VAIJVS •7qii M oijpadsuoo puB 'sde^ aqj IB 30 A 3U.lJ ° irap-reQs,XuBdui0 3aq i ui uopBApjna aanpaj AVmiuao /j UIOIJ adBosa UB SI DuvaSaup 7 sapads UBDUjv-mnoS 3i|l II Vr HD'sisaqjsiqx 'jiraiB dajBi uiiAvoiDpi npA "B qii MuopBzipuqA q paAjOAui VSOMA 7jo uopnjoAa aqipu B 'uopaaps TBITPBUA qpaAjoA a vuStjvs j 'paaM SuiAvojiqj-duiBO pajBpj aqi SB V\OUJL3S J 'UBUI Aq uopaaps qgncuqi paAjoAa VAIJVS •7 uojsaouB UOUIUIOD B UIOXJ aiBUiSuo VSOMA J puB 'vuSi]vs j 'vjoiuuas j 'VAIJVS 7 I suoijisodojj ^94O27JO\^^^\ Zooming in on the lettuce genome: species relationships inLactuca s.l., inferred from chromosomal and molecular characters WimJ . M. Koopman Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit Prof. dr. ir. L. Speelman in het openbaar te verdedigen op vrijdag 21 juni 2002 des namiddags te vier uur in de Aula *v 3 Zooming in on the lettuce genome: species relationships inLactuca s. 1.,inferre d from chromosomal and molecular characters / Wim J. M. Koopman / Ph.D. Thesis Wageningen University. ISBN 90-5808-676-3 Key words: AFLP, Asteraceae, base composition, chromosome banding, cladistics, Compositae, chromosomes, DNA content, flow cytometry, gene pool, genome evolution, herbarium collection, Internal Transcribed Spacer (ITS),karyotypes , Lactuca, Lactuceae, lettuce, molecular phylogeny, phenetic relationships, phylogenetic relationships, phylogenetic signal, plant breeding. Contents Introduction 1 Part I:Herbariu m systematics Chapter 1 A format for Asteraceae model voucher collections, developed in Lactuca L. 9 Part II:Zoomin g in on the lettuce genome Chapter 2 Chromosome banding patterns in Lettuce species {Lactucasect . Lactuca, Compositae) 21 Chapter 3 A numerical analysis of karyotypes and DNA amounts in lettuce cultivars and species (Lactuca subsect. Lactuca, Compositae) 33 Chapter 4 Phylogenetic relationships amongLactuca (Asteraceae) species and related genera based on ITS-1 DNA sequences 49 Chapter 5 Species relationships inLactuca s.l.(Lactuceae , Asteraceae) inferred from AFLP fingerprints 79 Chapter 6 Evolution of DNA content and base composition in Lactuca (Asteraceae) and related genera 97 Part III:Cladisti c theory Chapter 7 Phylogenetic signal in AFLP data sets 125 Part IV: Practical applications Chapter 8 Identifying lettuce species (Lactuca subsect. Lactuca, Asteraceae): A practical application of flow cytometry 145 Chapter 9 Plant systematics as a useful tool for plant breeders: examples from lettuce 161 Discussion 175 Summary 184 Samenvatting 186 Acknowledgements 188 Curriculum vitae 192 Publications 194 Introduction Introduction The genusLactuca L .an d its relatives According to Bremer (1994), the genus Lactuca can be classified in the Asteraceae subfamily Cichorioideae, tribe Lactuceae Cass., subtribe Lactucinae Dumort. Tribe Lactuceae is subdivided into 11 subtribes, together comprising more than 1550 species in 96 genera (excluding microspecies of Taraxacum Weber in F. H. Wigg., Hieracium L., and Pilosella Hill). Subtribe Lactucinae comprises ca. 270 species in 17 genera. Tribe Lactuceae is unequivocally characterized by its ligulate capitula and its milky latex. The circumscription of the subtribe Lactucinae is less straightforward, involving terms such as "often", "frequently", "usually", and "sometimes" (Bremer, 1994, p. 183). To make things worse, the generic boundaries within the tribe are even more obscure, causing genera to be repetitively synonymized and resurrected (Cronquist, 1985). In this thesis, I will refer to Lactuca and related genera according to the genus concept of Ferakova (1977). Her concept represents a view somewhere between splitting and lumping, including genera such as Mulgedium Cass., Lactucopsis Schultz-Bip. ex Vis. et Pane, and Phaenixopus Cass, in Lactuca, while for example the genera Mycelis Cass, and CicerbitaWallr . are considered separate. Lactuca according to Ferakova (1977) is subdivided into four sections: Phaenixopus (Cass.) Benth., Mulgedium (Cass.) C. B. Clarke, Lactucopsis (Schultz-Bip. ex Vis. et Pane.) Rouy, and Lactuca. Section Lactuca is subdivided into two subsections: Cyanicae DC. and Lactuca (including L. sativa L., the cultivated lettuce). L. sativa will be referred to as "lettuce". In addition toLactuca sens u Ferakova (1977),Lactuca sens u lato(s.l. )i suse d for easy reference to Lactuca sensu Ferakova and a group of closely related genera. I define Lactuca s.l. approximately according to Stebbins(1937) . In his concept,Lactuca no tonl y includesMulgedium, Lactucopsis,an d Phaenixopus, but Mycelis and part of Cicerbita (excluding C. alpina(L. ) Wallr. and C.pancicii (Vis.)Beauverd. ) as well.Lactuca s.l. asuse d inthi s thesis includesLactuca sensu Stebbins (1937), supplemented with C. alpina (C. pancicii was not examined), and Steptorhamphus tuberosus. The latter was not mentioned by Stebbins (1937), but fits his description of the genus Lactuca.Th e genus concepts of Ferakova (1977), Stebbins (1937), and othersar ediscusse d inmor e detail inchapte r4 . In the course of my research, living material from 18specie s of Lactuca s.l. became available, and all species were included. The species represent all sections and subsections of Ferakova (1977). In order to properly evaluate the relationships in Lactuca s.l., I also included four outgroup species from genera less closely related to Lactuca, viz. Prenanthes purpurea L., Chondrillajuncea L., Taraxacumofficinale Weber in F. H. Wigg., and Sonchus asper (L.) Hill (Stebbins, 1937; Bremer, 1994). Among these species, P. purpurea is considered most closely related to Lactuca s.l. Both Lactuca and Prenanthes are in subtribe Lactucinae (Bremer, 1994), and according to Stebbins (1953) they are connected in the Prenanthes-Lactuca line. The remaining species are classified in subtribe Crepidinae Dumort. (C.juncea and T officinale),an d Introduction in subtribe Sonchinae K. Bremer {Sonchus asper). Apart from P. purpurea and species from Lactuca s.l., I also included Cichorium intybus L. The subtribal affinities of Cichorium L. are unclear,