applyparastyle “fig//caption/p[1]” parastyle “FigCapt” Botanical Journal of the Linnean Society, 2019, 191, 164–187. With 6 Figures. Extended studies of interspecific relationships in Daucus Downloaded from https://academic.oup.com/botlinnean/article-abstract/191/2/164/5573335 by U S Dept of Agriculture user on 25 September 2019 (Apiaceae) using DNA sequences from ten nuclear orthologues Keywords=Keywords=Keywords_First=Keywords HeadA=HeadB=HeadA=HeadB/HeadA FERNANDO MARTÍNEZ-FLORES1,*, MANUEL B. CRESPO1, EMMANUEL GEOFFRIAU2, HeadB=HeadC=HeadB=HeadC/HeadB CHARLOTTE ALLENDER3, HOLLY RUESS4, CARLOS I. ARBIZU5,†, PHILIPP SIMON4 and HeadC=HeadD=HeadC=HeadD/HeadC DAVID M. SPOONER4 Extract3=HeadA=Extract1=HeadA REV_HeadA=REV_HeadB=REV_HeadA=REV_HeadB/HeadA 1Departamento de Ciencias Ambientales y Recursos Naturales (dCARN), Universidad de Alicante, P.O. REV_HeadB=REV_HeadC=REV_HeadB=REV_HeadC/HeadB Box 99, ES-03080 Alicante, Spain REV_HeadC=REV_HeadD=REV_HeadC=REV_HeadD/HeadC 2IRHS, Agrocampus Ouest, INRA, Université d’Angers, Angers, France REV_Extract3=REV_HeadA=REV_Extract1=REV_HeadA 3Warwick Crop Centre, School of Life Sciences, Wellesbourne Campus, University of Warwick, Warwick, BOR_HeadA=BOR_HeadB=BOR_HeadA=BOR_HeadB/HeadA CV35 9EF, UK BOR_HeadB=BOR_HeadC=BOR_HeadB=BOR_HeadC/HeadB 4 BOR_HeadC=BOR_HeadD=BOR_HeadC=BOR_HeadD/HeadC United States Department of Agriculture, Agricultural Research Service, University of Wisconsin- BOR_Extract3=BOR_HeadA=BOR_Extract1=BOR_HeadA Madison, Madison, WI, 53706-1590, USA 5 EDI_HeadA=EDI_HeadB=EDI_HeadA=EDI_HeadB/HeadA Dirección de Recursos Genéticos y Biotecnología, Instituto Nacional de Innovación Agraria, Av. La EDI_HeadB=EDI_HeadC=EDI_HeadB=EDI_HeadC/HeadB Molina 1981, Lima 12, Peru EDI_HeadC=EDI_HeadD=EDI_HeadC=EDI_HeadD/HeadC EDI_Extract3=EDI_HeadA=EDI_Extract1=EDI_HeadA Received 11 September 2018; revised 25 April 2019; accepted for publication 8 July 2019 CORI_HeadA=CORI_HeadB=CORI_HeadA=CORI_HeadB/HeadA CORI_HeadB=CORI_HeadC=CORI_HeadB=CORI_HeadC/HeadB CORI_HeadC=CORI_HeadD=CORI_HeadC=CORI_HeadD/HeadC Daucus has traditionally been estimated to comprise 21–25 species, but a recent study expanded the genus to c. 40 CORI_Extract3=CORI_HeadA=CORI_Extract1=CORI_HeadA species. The present study uses ten nuclear orthologues to examine 125 accessions, including 40 collections of 11 ERR_HeadA=ERR_HeadB=ERR_HeadA=ERR_HeadB/HeadA species (D. annuus, D. arcanus, D. decipiens, D. durieua, D. edulis, D. gracilis, D. minusculus, D. montanus, D. pumilus, ERR_HeadB=ERR_HeadC=ERR_HeadB=ERR_HeadC/HeadB D. setifolius and D. tenuissimus) newly examined with nuclear orthologues. As in previous nuclear orthologue studies, ERR_HeadC=ERR_HeadD=ERR_HeadC=ERR_HeadD/HeadC Daucus resolves into two well-defined clades, and groups different accessions of species together. Maximum likelihood ERR_Extract3=ERR_HeadA=ERR_Extract1=ERR_HeadA and maximum parsimony analyses provide concordant results, but SVD quartets reveals many areas of disagreement INRE_HeadA=INRE_HeadB=INRE_HeadA=INRE_HeadB/HeadA of species within these two major clades. With maximum likelihood and maximum parsimony analyses Daucus INRE_HeadB=INRE_HeadC=INRE_HeadB=INRE_HeadC/HeadB montanus (hexaploid) is resolved as an allopolyploid between D. pusillus (diploid) and D. glochidiatus (tetraploid), INRE_HeadC=INRE_HeadD=INRE_HeadC=INRE_HeadD/HeadC whereas with SVD quartets it is resolved as an allopolyploid between D. glochidiatus and an unknown Daucus sp. We INRE_Extract3=INRE_HeadA=INRE_Extract1=INRE_HeadA propose the new combination Daucus junceus (Durieua juncea) for a neglected species endemic to the south-western App_Head=App_HeadA=App_Head=App_HeadA/App_Head Iberian Peninsula often referred to as D. setifolius, and we place D. arcanus in synonymy with D. pusillus. Three BList1=SubBList1=BList1=SubBList lectotypes are also designated. BList1=SubBList3=BList1=SubBList2 KEYWORDS: carrot – Daucinae – germplasm – nomenclature – phylogeny – taxonomy – typification. SubBList1=SubSubBList3=SubBList1=SubSubBList2 SubSubBList3=SubBList=SubSubBList=SubBList SubSubBList2=SubBList=SubSubBList=SubBList SubBList2=BList=SubBList=BList INTRODUCTION drastically changing our understanding of the species boundaries (Arbizu et al., 2014b, 2016; Spooner et al., The latest comprehensive taxonomic monograph 2014) and ingroup and outgroup relationships of of Daucus L. by Sáenz (1981) recognized 21 species; Daucus (e.g. Spalik & Downie, 2007; Arbizu et al., Rubatzky, Quirós & Simon (1999) later estimated 25 2014a; Banasiak et al., 2016; Spooner et al., 2017). species. Recent morphological and molecular studies Banasiak et al. (2016) used DNA sequences from nuclear using plastid and nuclear DNA sequences have been ribosomal ITS and the three plastid markers (the rps16 intron, rpoC1 intron and rpoB-trnC intergenic spacer) to redefine and expand Daucus to include the following *Corresponding author. E-mail: [email protected] †Current address: Departamento de Horticultura, Facultad de genera and species into its synonymy: Agrocharis Agronomía, Universidad Nacional Agraria La Molina, Av. La Hochst. (four species); Melanoselinum Hoffm. (one Molina s/n, Lima 12, Peru. species); Monizia Lowe (one species); Pachyctenium Published by Oxford University Press on behalf of The Botanical Journal of the Linnean Society 2019, 191, 164–187 164 INTERSPECIFIC RELATIONSHIPS IN DAUCUS 165 Maire & Pamp. (one species); Pseudorlaya (Murb.) & Link [≡Pseudorlaya pumila (L.) Grande], D. setifolius Murb. (two species); Rouya Coincy (one species); Desf. and D. tenuissimus (A.Chev.) Spalik, Wojew., Tornabenea Parl. (six species); Athamanta della- Banasiak & Reduron [≡Tornabenea tenuissima cellae Asch. & Barbey ex E.A.Durand & Barratte (A.Chev.) A.Hansen & Sunding]. We lack vouchers for and Cryptotaenia elegans Webb ex Bolle. They made three accessions that grew as young plants sufficient to the relevant nomenclatural transfers into Daucus, obtain DNA, but failed to grow to flowering stage as is Downloaded from https://academic.oup.com/botlinnean/article-abstract/191/2/164/5573335 by U S Dept of Agriculture user on 25 September 2019 expanding the genus to c. 40 species. In addition, typical for some Daucus sp. pl. that are biennial or have they divided Daucus into four sections, three of which other problems relating to flowering in cultivation. [section Daucus, section Melanoselinum (Hoffm.) Spalik, Wojew., Banasiak & Reduron, section Anisactis DC.] we examine here; we do not examine section CHROMOSOME COUNTS Agrocharis (Hochst) Spalik, Banasiak & Reduron that We obtained chromosome counts of all the species is sister to the sections above and comprised of the examined in this study from the literature using former genus Agrocharis. This expansion of Daucus the Missouri Botanical Garden Index to Plant and new sectional classification renders Daucus Chromosome Numbers (IPCN; Goldblatt & Johnson, difficult to characterize morphologically, and it was 1979; (https://www.tropicos.org/Project/IPCN) (Table based solely on molecular data. 2) and other sources from the literature on Daucus. We Arbizu et al. (2014a) identified 94 nuclear found chromosome number references for all examined orthologues in Daucus, constructed a phylogenetic species except for D. bicolor Sm., D. conchitae Greuter tree with these and determined ten of them to provide and D. gracilis. essentially the same result as all 94, paving the way for additional cost-effective nuclear orthologue studies in Daucus. These ten nuclear orthologues were then DNA EXTRACTION AND SEQUENCE Generation successfully used in a focused study of the species New accessions were grown in a greenhouse at the boundaries of the D. guttatus Sibth. & Sm. complex University of Wisconsin-Madison, tissue harvested (Arbizu et al., 2016), which, in concert with data and freeze-dried leaves, and DNA extracted with from type specimens (Martínez-Flores et al., 2016), CTAB (Doyle & Doyle, 1990). For markers dc10366, redefined the species boundaries and nomenclature of dc10966, dc15347, dc16308, dc16577, dc3374, dc3902 this group. The focus of the present study is to expand and dc35097, 200 ng of DNA was amplified in 20 μL further these studies by adding 40 accessions of taxa volume containing 1× Buffer, 1.5 mM MgCl2, 0.2 mM from 11 species and from areas not examined with dNTP (Promega Corporation, USA), 0.2 μM each these ten nuclear orthologues. We provide suggestions forward and reverse primer and 1 U taq (GoTaq, on further taxonomic research on Daucus and new Promega Corporation, USA) with the following taxonomic decisions and synonymies. program steps: 94 °C for 5 min, 35 cycles of 94 °C for 30 s, 55 °C for 1 min and 72 °C for 2 min, followed by 72 °C for 10 min. For markers dc16645 and dc32914, MATERIAL AND METHODS and any unamplified product from the previous eight markers, the magnesium concentration was doubled to ACCESSIONS EXAMINED 3 mM and the annealing step lowered from 55 °C to Previously sequenced amplicons were obtained from 50 °C. The forward and reverse strands were Sanger Arbizu et al. (2014a, 2016) and are listed in those sequenced for each amplicon at the Biotechnology publications. The 40 new accessions (Table 1) were Center of the University of Wisconsin-Madison. obtained from (1) the Agrocampus Ouest – IRHS, Amplicons with SNP differences or an insertion or France, (2) the Warwick Crop Centre, UK and (3) an deletion, discovered during Sanger sequencing, were expedition in Spain in 2016. Newly examined taxa further processed with SSCP following