# Genome Size Study in the Valerianaceae: First Results and  New Hypotheses

Authors: Oriane Hidalgo; Joël Mathez; Sònia Garcia; Teresa Garnatje; Jaume Pellicer; Joan Vallès
Journal: Journal of Botany
 (2010)

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## Abstract

The purpose of this study is to provide a new focus to contribute, from the perspective of genomic evolution, towards a better understanding of the Valerianaceae evolutionary history. Chromosome numbers were determined by Feulgen staining in 24 populations of 18 species (first count forValerianella multidentata, 2n=2x=14–16), and DNA contents were assessed by flow cytometry in 74 populations of 35 species (first assessments in all taxa but Centranthus ruber). A molecular phylogeny based on the trnL-trnF and including 41 new sequences was established, with the first DNA sequence for Centranthus nevadensis, Valeriana rotundifolia, V. saxatilis, Valerianella multidentata, and V. turgida. This work is the first large genome size study devoted to the Valerianaceae, showing a range of DNA amounts from 2C=0.39 pg (Valerianella turgida) to 2C=8.32 pg (Valeriana officinalis). At the family level, changes in basic chromosome number and genome size coincide with or precede major shifts in the evolutionary history of the group, such as those concerning stamen number and floral symmetry.


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## Body

## 1. Introduction

The family Valerianaceae (currently considered within the Caprifoliaceae s.l.; [1]) comprises ca. 400 species of which approximately 200 are included in Valeriana L., the biggest genus of the order Dipsacales. The Valerianaceae are cosmopolitan in their natural distribution, with the exceptions of Australia and the Pacific islands, where they were introduced, and can be nowadays considered as naturalized. Several of their representatives have economic interest as medicinal (e.g., V. officinalisL.), edible (e.g., Valerianella Mill., corn salad or lamb’s lettuce), or ornamental plants (e.g., Centranthus DC.). The family always roused important scientific interest, and its study has broadly benefited from the new molecular techniques, especially the phylogenetic reconstructions. Recent phylogenies based on DNA sequencing [2–7] have considerably modified the traditional classification of the family [8–12]. Molecular results recognize only six genera: Centranthus, Fedia Gaertn., Nardostachys DC., PatriniaJuss., Valeriana (includingAretiastrum DC., Astrephia Dufr., Belonanthus Graebn., Phyllactis Pers., Porteria Hook., and Stangea Graebn.), and Valerianella. Plectritis (Lindl.) DC. is nested amongst South American Valeriana [5, 6]. Moreover, Bell [7] suggested a possible further taxonomic treatment of the family considering Fedia as a synonym of Valerianella. New genera may also be described for some of the Valeriana species that do not group with their congenerics in the phylogenetic reconstructions and make the genus Valeriana paraphyletic in its current circumscription. These are Valeriana longiflora Willk., which appears closely related to the genus Centranthus, and alsoValeriana celtica L. and V. hardwickii Wall., branched somewhere between Nardostachys and Valerianella plus Fedia clades [4, 5].Morphology in Valerianaceae is of great interest for its impressive diversity of forms, mainly resulting in adaptations to a wide range of ecological conditions (from the sea board to Andean páramos at 4000 m), and concerning both vegetative and reproductive parts. This diversity has been especially well studied from the inflorescential structure  perspective, with a common basic type, the thyrse, and very different forms and levels of complexity [13, 14]. Family Valerianaceae stands out in Dipsacales by presenting four different stamen numbers in a series (mainly four stamens in Patrinia and Nardostachys, three in Plectritis, Valerianella and Valeriana, two in Fedia, and one in Centranthus), while the other families show one or two distinct stamen numbers [15, 16]. These features, along with some cases of strong corolla zygomorphy (in Centranthus, with 4 : 1 petals orientation; in Fedia, with 2 : 3 petals orientation) make the family very attractive for studying the genetic base of floral characters, which has been initiated through an evolutionary-developmental approach by [16, 17]. Evo-devo studies highlight the crucial importance of duplication events in the evolution of genes involved in developmental processes (e.g., [18, 19]). These events often correspond to whole duplication genome ([20], and references therein), making the karyological and cytogenetic data essential for understanding many evolutionary processes, as, for example, the floral morphological changes.Nevertheless, karyological and cytogenetic data in Valerianaceae are basically limited to chromosome counts. The Valerianaceae exhibit a dysploid series of five basic chromosome numbers [21]: x=15 in American Valerianella, x=13 in Nardostachys, x=11 in Patrinia and Valeriana celtica, x=8 in Centranthus, Fedia, Valeriana and Valerianella, and x=7 in Valeriana and Valerianella. Polyploidization events are common, some genera being exclusively polyploid, such as Centranthus (tetraploid) or Fedia (tetraploid and hexaploid), while species of Patrinia and Valeriana can exhibit various ploidy levels, from diploid to octoploid. Hence, both polyploidy and dysploidy  seem to have played a significant role in the differentiation and evolution of these plants. Cytogenetic data (such as banding, fluorescent in situ hybridization and genome size assessment) would certainly be of a great interest for understanding the evolution of the family. Nuclear DNA amount assessments constitute a fundamental complement to chromosome counts and, in addition, a powerful tool in order to establish the relationships between closely related taxonomical groups (e.g., in Echinops L.; [22], Orobanche L.; [23]), as well as to understand the evolution within related genera from a broader point of view (e.g., Liliaceae; [24], Orchidaceae; [25]). Furthermore, genome size is a useful tool to detect possible hybrid and polyploid origins of taxa (e.g., in Carthamus L.; [26], Nicotiana L.; [27], Artemisia L.; [28]), and intraspecific variation can reveal incipient speciation [29].The aims of the present work are to: (a) enlarge the knowledge concerning nuclear DNA amounts in the Valerianaceae, to date limited to only one report (“Kentranthus ruber Druce” 1C=0.42pg; [30]), (b) integrate these results with those of chromosome number and molecular phylogeny, and (c) provide a new focus that could contribute, from the perspective of genome evolution, to verify the hypotheses of previous works on the relationships among the above-mentioned genera.


## 2. Material and Methods

### 2.1. Plant Material

Table1 shows the provenance of the species investigated. The sampling includes representatives of all the six genera considered in the family in regard to the results of molecular phylogenies [4–6]. Studied plants come from germinated cypselas, collected in the field or obtained from Botanic Gardens. Due to difficulties in germinating seeds of Valeriana representatives, whole individuals were collected in the field and cultivated in the Institut Botànic de Barcelona.Table 1
Origin of the populations studied with indications of the herbaria where voucher specimens are deposited, chromosome counts, genome size data, and GenBank accession numbers (only for the new sequences).
Species (population number)Collection dataLife cycle12C (pg)22n3Ploidy level1Cx42C (Mbp)5Mean HPCV of sample (%)Mean HPCV of standard (%)trnL-trnF GenBank accession numberCentranthusFrance: Vaucluse, Mt.P1.30 ± 0.03324x0.331271.43.83 ± 0.631.25 ± 0.59HM162366angustifolius (Mill.)  DC. (1)Ventoux, Hidalgo 212 &Hidalgo (BC)CentranthusFrance: Hautes-Alpes,P1.35 ± 0.02324x0.341320.35.57 ± 0.863.77 ± 0.65HM162367angustifolius (Mill.)  DC. (2)VI-2005, Hidalgo &Romashchenko (MPU)CentranthusFrance: Hautes-Alpes,P1.41 ± 0.07324x0.3513793.35 ± 0.551.50 ± 0.43angustifolius (Mill.)  DC. (3)Les Infournas, Hidalgo 201 & Martin (BC)CentranthusSpain, Catalonia, PortA1.56 ± 0.04324x0.391525.75.22 ± 1.144.12 ± 0.95calcitrapae  (L.) Dufr. (1)d'Àger, Garnatje 229 &Vallès (BCN)CentranthusFrance: Gard, SaintA1.58 ± 0.02324x0.401545.23.28 ± 0.701.99 ± 0.62HM162368calcitrapae  (L.)   Dufr. (2)André de Majencoules,4-VI-2005, Hidalgo, Romashchenko &  Romo (BC)CentranthusGreece, Crete: Elafonissi,A1.61 ± 0.01324x0.401574.63.62 ± 0.961.06 ± 0.87calcitrapae  (L.)  Dufr. (3)Bot. Gard. MNHN  1996-08CentranthusGreece, Crete: Hania,A1.62 ± 0.03324x0.41584.43.16 ± 1.451.51 ± 0.56calcitrapae  (L.)  Dufr. (4)Bot. Gard. MNHN  1996-15Centranthuscf.Morocco: Hidalgo,A1.80 ± 0.03324x0.451760.43.46 ± 0.602.20 ± 0.70HM162369calcitrapae   (L.) Dufr. (5)Romo 13128 & Soriano (BC)Centranthus lecoqiiFrance: Lozère, LesP1.22 ± 0.05324x0.311193.23.62 ± 1.582.09 ± 0.48Jord. (1)Vignes, Bot. Gard.  MNHN 2002-39Centranthus lecoqiiFrance: Cons. Bot.P324xJord. (2)Porquerolles Ly815Centranthus lecoqiiFrance: Hérault, St.P324xHM162370Jord. (3)Guilhem le Désert, Mathez 1076 (MPU)Centranthus longiflorusLebanon: Les Cèdres,P1.42 ± 0.03324x0.361388.87.90 ± 0.687.71 ± 2.41Steven var.latifoliusBoiss.Bou Dagher-Kharrat, Hidalgo &  Romashchenko  409 (BC)CentranthusBot. Gard. HauniensisA1.97 ± 0.06324x0.491926.72.58 ± 1.191.64 ± 1.26HM162371macrosiphon  Boiss.2000-2315Centranthus nevadensisMorocco: Akchar, surP1.16 ± 0.02324x0.291134.54.23 ± 0.982.27 ± 0.77Boiss. (1)Tizi Ouzli, Hidalgo,Romo 13035 & Soriano (BC)Centranthus nevadensisMorocco: DjebelP1.19 ± 0.01324x0.31163.83.80 ± 0.371.87 ± 0.78HM162372Boiss. (2)Lechâab, Hidalgo, Romo 13147& Soriano (BC)Centranthus ruberFrance: Bouches-du-P1.14 ± 0.04324x0.291114.93.77 ± 1.590.81 ± 0.41(L.) DC. (1)Rhône, la Sainte Baume,VI-2005, Hidalgo, Romashchenko & Romo (BC)Centranthus ruberRumania: Valea Moril,P1.14 ± 0.02324x0.291114.94.56 ± 0.492.36 ± 0.22HM162373(L.) DC. (2)Distr. Cluj, Bot. Gard.Cluj-Napoca 2006-1820Centranthus ruberItaly, Sicily: Siracusa,P1.16 ± 0.04324x0.291134.53.67 ± 0.710.88 ± 0.44(L.) DC. (3)Buccheri Mt., Bot. Gard.Palermo 2006-750Fediacf.  cornucopiaeItaly, Sicily: Siracusa,A1.70 ± 0.02324x0.431662.62.68 ± 0.310.18 ± 0.08HM162374(L.) Gaertn. (1)Lauro-Buccheri Mt.,(asF. caput-bovis)Bot. Gard. Palermo 2006-751Fedia cornucopiaeWilld coll., Bot. Gard.A1.79 ± 0.04324x0.451750.65.28 ± 0.963.28 ± 1.16(L.) Gaertn. (2)Coimbra 2005-1321Fedia gracilifloraBot. Gard.A1.53 ± 0.04324x0.381496.34.48 ± 0.962.53 ± 1.17HM162375Fisch. & C.A.Mey.Montpellier (MPU)Fedia pallescensMorocco: Mehdyia,A1.14 ± 0.02324x0.291114.95.85 ± 1.542.71 ± 1.14HM162376(Maire) MathezEl-Oualidi s.n.-1998 (MPU)Nardostachys jatamansiBot. Gard. Lautaret,P3.49 ± 0.042.06 ± 0.721.25 ± 0.82(D. Don) DC.FrancePatrinia rupestrisRussia: Siberia, Bot.P2.56 ± 0.012.81 ± 1.011.87 ± 1.1Dufr.Gard. Lautaret, FrancePatrinia scabiosifoliaJapan: Bot. Gard. OfunaP1.50 ± 0.035.09 ± 0.714.08 ± 0.37Link (1)314-2006Patrinia scabiosifoliaJapan: Tsukiono-machi,P2.57 ± 0.07ca. 444x0.642513.53.7 ± 0.681.91 ± 0.58HM162377Link (2)Tone-gun, Bot. Gard.Chiba University2006-193Valeriana apulaSpain, Aragon: Huesca,P0.97 ± 0.02162x0.49948.665.58 ± 0.872.16 ± 0.54HM162378Pourr.Anglios, 2005, Vallès (BCN)Valeriana celticaFrance: Savoie, EvettesP2.11 ± 0.041.81 ± 1.371.29 ± 1.10L. (1)circus, Bot. Gard. Lautaret, FranceValeriana celticaItaly: Aosta, Cogne,PHM162379L. (2)Hidalgo 503 (MPU)Valeriana dioicaFrance: Hautes-Alpes,P2.87 ± 0.053.25 ± 0.310.72 ± 0.70L. (1)Lautaret pass, VI-2005Douzet, Hidalgo &Romashchenko (BC)Valeriana dioicaFrance: Gard, BonheurP3.01 ± 0.013.11 ± 0.210.72 ± 0.49HM162380L. (2)valley, 5-VI-2005,(♀)(♀)(♀)Hidalgo, Romashchenko3.08 ± 0.013.22 ± 0.141.78 ± 0.51& Romo (BC)(♂)(♂)(♂)Valeriana longifloraSpain, Aragon: SantaP0.96 ± 0.025.84 ± 0.992.91 ± 0.30Willk. (1)Anna dam, Garnatje 172& Vallès (BCN)Valeriana longifloraSpain, Aragon, SantaPHM162381Willk. (2)Anna dam, Garnatje,Hidalgo 500 &Luque (MPU)Valeriana montanaFrance: Hautes-Alpes,P1.23 ± 0.013.92 ± 1.321.59 ± 1.05L. (1)Les Infournas, Hidalgo202 & Martin (BC)Valeriana montanaSpain, Catalonia:P2.24 ± 0.045.98 ± 2.410.88 ± 0.62L. (2)Girona, Camprodon,Garnatje 179 (BC)Valeriana montanaItaly: Maritime Alps,P2.40 ± 0.052.79 ± 0.362.21 ± 0.83L. (3)Cuneo, 6-VIII-2005,Peccenini & Vallès(BCN)Valeriana montanaFrance: Hautes-Alpes,P2.42 ± 0.032.79 ± 0.492.17 ± 0.73HM162382L. (4)between the Galibier andLautaret passes, Garnatje& Hidalgo 220 (BC)Valeriana montanaSpain, Catalonia: Lleida,P2.50 ± 0.05324x0.6324452.08 ± 0.281.32 ± 0.80HM162383L. (5)Vall d'Aran, on the wayto the Restanca fromArties, Garnatje 234 &Vallès (BCN)Valeriana montanaFrance: Hautes-Alpes,P2.51 ± 0.032.81 ± 0.692.35 ± 0.84L. (6)spontaneous in theLautaret Bot. Gard.,Garnatje & Hidalgo221 (BC)Valeriana montanaSpain, Catalonia: Lleida,P2.54 ± 0.032.52 ± 0.980.87 ± 0.77HM162384L. (7)Vall d'Aran, on the wayto the Restanca fromArties, Garnatje 235 &Vallès (BCN)Valeriana montanaFrance: Hautes-Alpes,P2.60 ± 0.03324x0.652542.82.38 ± 0.831.36 ± 0.54L. (8)between Le Roy and Pisepass, Hidalgo 210 &Martin (BC)Valeriana montanaFrance: Hautes-Alpes,P2.64 ± 0.052.48 ± 1.001.82 ± 0.70HM162385L. (9)Gleize pass, Hidalgo 208& Martin (BC)Valeriana montanaFrance: Hautes-Alpes,PHM162386L. (10)Galibier pass,4-VIII-2001, Hidalgo &Hidalgo (BC)Valerianacf.Spain, Catalonia:P2.46 ± 0.033.65 ± 2.062.06 ± 0.67montana L. (11)Barcelona, Montserrat,2005, Casanova (BC)Valerianacf. montanaL. (12)France: Hautes-Alpes,P2.54 ± 0.023.3 ± 0.241.98 ± 0.60Gleize pass, VI-2005, Hidalgo & Romashchenko (BC)Valeriana officinalisCroatia: Dalmatia,P2.97 ± 0.04142x1.492904.74.07 ± 0.292.22 ± 0.49HM162387L. (1)Biokovo Mt., 2006,Siljak-Yakovlev (BC)Valeriana officinalisFrance: Gard, AigoualP3.16 ± 0.033.19 ± 0.241.44 ± 0.42HM162388L. (2)Mt., 3-VI-2005, Hidalgo,Romashchenko &Romo (BC)Valeriana officinalisSpain, Catalonia:P4.62 ± 0.082.76 ± 0.483.60 ± 0.66L. (3)Girona, Llanars,Garnatje 169, D. Roca &J. Roca (BC)Valeriana officinalisSpain, Catalonia: Lleida,P6.51 ± 0.111.49 ± 1.012.89 ± 1.24L. (4)Vielha, 2005,Vallès (BCN)Valeriana officinalisSpain, Catalonia: Lleida,P8.05 ± 0.081.78 ± 0.762.91 ± 0.64L. (5)Vall d’Aran, Garnatje175 & Vallès (BCN)Valeriana officinalisNorway: Trs, Storfjord,P8.15 ± 0.25ca. 568x1.027970.71.40 ± 0.922.52 ± 1.67HM162389L. (6)Rovijok, Bot. Gard.Oulu 2004/5-425Valeriana officinalisFrance: Gironde,P8.32 ± 0.10  (leaf)3.14 ± 1.26 (leaf)4.99 ± 0.49 (leaf)L. (7)Gradignan, V-2005,8.09 ± 0.08  (root)1.40 ± 1.11 (root)5.83 ± 0.47 (root)Hidalgo &Revel-Hidalgo (BC)Valeriana pyrenaicaSpain, Catalonia: Lleida,P1.26 ± 0.01162x0,61232.35.37 ± 0.642.81 ± 0.26HM162390L.Vall d’Aran, Garnatje176 & Vallès (BCN)ValerianaFrance: Hautes-Alpes,P2.52 ± 0.052.16 ± 0.301.41 ± 0.51HM162391rotundifoliaVill.Devoluy, La Cluse,Hidalgo 207 &Martin (BC)Valeriana saliuncaFrance: Savoie, GalibierP1.46 ± 0.03162x0.731427.94.21 ± 0.363.21 ± 0.25HM162392All.pass, Garnatje 166 &Vallès (BC)Valeriana saxatilisItaly: Apuan Alps,P1.05 ± 0.003.17 ± 0.282.21 ± 0.65L. (1)summit of Sagro Mt.,Hidalgo 213 (BC)Valeriana saxatilisItaly: Apuan Alps,P1.02 ± 0.013.62 ± 0.382.36 ± 0.24HM162393L. (2)Hidalgo 217 (BC)Valeriana tripterisFrance: Hautes-Alpes,P1.46 ± 0.03162x0.731427.93.28 ± 0.711.79 ± 0.69L. (1)Lautaret pass, Hidalgo203 & Martin (BC)Valeriana tripterisFrance: Gard, Mt.P1.51 ± 0.014.01 ± 0.382.24 ± 0.48L. (2)Aigoual, 3-VI-2005,(♀♂)(♀♂)(♀♂)Hidalgo, Romashchenko1.52 ± 0.023.75 ± 0.242.12 ± 0.53& Romo (BC)(♀)(♀)(♀)Valeriana tripterisFrance: Vaucluse, Mt.P1.51 ± 0.01182x0.761476.82.75 ± 0.351.34 ± 0.40HM162394L. (3)Ventoux, Hidalgo 211 &Hidalgo (BC)Valeriana tripterisSpain, Catalonia: Lleida,P1.54 ± 0.035.63 ± 1.004.86 ± 1.03HM162395L. (4)Espot, road to Espot Ski,17-VI-2007, Garnatje233 & Vallès (BCN)Valeriana tripterisSpain, Catalonia:P1.58 ± 0.063.88 ± 0.742.01 ± 0.81HM162396L. (5)Girona, Setcases,Garnatje 170, D. Roca &J. Roca (BC)Valeriana tripterisFrance: Gard, Mt.PHM162397L. (6)Aigoual, 14-VII-00,Hidalgo (BC)Valerianacf.Italy: Apuan Alps,P1.23 ± 0.06324x0.311202.93.93 ± 0.662.34 ± 0.98HM162398tripterisL. (7)Hidalgo 218 (BC)ValerianaFrance: Les Sièges,P3.74 ± 0.032.96 ± 0.993.66 ± 0.42HM162399tuberosaL.Mathez 1088 (MPU)Valerianella coronataItaly: Aymavilles,A0.48 ± 0.00142x0.24469.440.59 ± 0.230.99 ± 0.53(L.) DC. (1)Cerignan, Bot. Gard.Cogne 2006/7-838Valerianella coronataFrance: Vaucluse, “laA0.53 ± 0.01142x0.27518.348.90 ± 0.703.26 ± 0.65(L.) DC. (2)Bastide des Jourdans”,(asV. pumila)Cons. Bot. de Porquerolles1998-CO 2688Valerianella dentataFrance: Maine-et-Loire,A0.42 ± 0.01162x0.21410.7611.88 ± 2.540.55 ± 0.37(L.) Pollich (1)Montreuil-Bellay,(asV. rimosa)“Méron”, Bot. Gard.Nantes 2004-306Valerianella dentataBot. Gard. BonnA0.49 ± 0.01 (leaf)162x0.25479.029.64 ± 0.85 (leaf)1.09 ± 0.85 (leaf)(L.) Pollich (2)2004/5-236870.47 ± 0.02 (root)8.69 ± 0.83 (root)2.29 ± 0.74 (root)Valerianella dentataItaly, Sicily: Palermo,A162xHM162400(L.) Pollich (3)Rocca Busambra-Ficuzza, Bot. Gard.Palermo 2006-756Valerianella discoideaGreece, Crete: Hania,A142xLoiselBot. Gard. MNHN2005-96/14Valerianella echinataFrance: Aveyron, nearA0.60 ± 0.01162x0.3586.85.54 ± 2.242.16 ± 0.71DC.Brunas de Creissels(Larzac N.), BernardValerianella eriocarpaItaly, Sicily: Palermo,A0.47 ± 0.02162x0.24459.666.31 ± 0.500.92 ± 0.86HM162401Desv. (1)Rocca Busambra-Ficuzza, Bot. Gard.Palermo 2006-757Valerianella eriocarpaBot. Gard. NantesA0.54 ± 0.01162x0.29528.1210.07 ± 2.831.86 ± 0.61Desv. (2)2004-302Valerianella locustaFrance: Loire Atlantique,A0.44 ± 0.02162x0.22430.3211.37 ± 1.691.18 ± 0.84HM162402L. (1)cheméré "Le Moulin",Bot. Gard. Nantes2004-303Valerianella locustaL.  (2) (asV. carinata)France: Loire Atlantique,A0.49 ± 0.02162x0.25479.228.50 ± 1.570.20 ± 0.19HM162403Gard. Nantes 2004-301ValerianellaSpain, Catalonia:A0.46 ± 0.02162x0.23449.888.43 ± 1.290.22 ± 0.20microcarpaLoiselTarragona, Ebre delta,2004, Pyke (BC)ValerianellaSpain, Aragon:A0.52 ± 0.0214–162x0.26508.567.78 ± 0.770.97 ± 0.92HM162404multidentata LoscosZaragoza, 2004,& PardoPyke (BC)Valerianella ponticaUkraine: Crimea,A0.51 ± 0.022x0.26498.7811.47 ± 1.330.18 ± 0.10Velen.Boratinsky, Didukh,Romashchenko, Romo1029bis & Susanna (BC)Valerianella turgidaGreece: Oxia, Bot. Gard.A0.39 ± 0.01162x0.2381.4212.15 ± 1.540.65 ± 0.67HM162405Betcke (1)MNHN 2002-190Valerianella turgidaUkraine: Crimea,A0.47 ± 0.02162x0.24459.667.49 ± 2.661.97 ± 0.52HM162406Betcke (2)Boratinsky, Didukh,Romashchenko, Romo10171 & Susanna (BC)Valerianella vesicariaIsrael: Judean Mts. Bot.A0.61 ± 0.052x0.31596.589.58 ± 2.980.36 ± 0.33MoenchGard. Tel Aviv 20011Life cycle: A annual, P perennial. 2 2C nuclear DNA content (means ± SD of 10 measurements—two replicates for five individuals each). 32n somatic chromosome number. 2n and ploidy levels are estimated through bibliographical record (using the database http://www.tropicos.org/), except those in bold, which were verified in this study. 41Cx monoploid genome size (DNA content per basic chromosome set [31]). 52C [Mbp]: 1pg=978 Mbp [32].


### 2.2. Karyological and Cytogenetic Analyses

#### 2.2.1. Chromosome Counts and/or Determination of the Ploidy Level

Root tip meristems were obtained either by germinating seeds on wet filter paper in Petri dishes at room temperature, or from plants cultivated in pots in the greenhouse. They were pretreated with 0.002 M 8-hydroxyquinoline for 3 h at 16°C. Material was fixed in absolute ethanol, trichloromethane, and glacial acetic acid (6 : 3 : 1) and stored in the fixative at 4°C during at least two days before processing. Samples were hydrolysed in 1 N HCl for 5 min at 60°C, stained with 1% aqueous aceto-orcein for 1 h minimum, and squashed on slides into a drop of 45% acetic acid-glycerol (9 : 1). Metaphase plates were photographed with a digital camera (Zeiss AxioCam HRm) mounted on a Zeiss Axioplan microscope, and images were analysed with Axio Vision Ac version 4.2.


#### 2.2.2. Nuclear DNA Assessments

Leaf tissue of five individuals for each studied population was chopped in 600μl of LB01 isolation buffer [33] with a razor blade, together with the chosen internal standard. The speciesPetunia hybridaVilm. `PxPc6’ (2C = 2.85 pg) and Pisum sativum L. `Express long’ (2C = 8.37 pg) were used as internal standards [34] to cover the range of 2C-values found. Seeds of the standards were provided by the Institut des Sciences du Végétal (CNRS), Gif-sur-Yvette (France). For each individual, two independent samples were extracted and measured the same day. Samples were supplemented with 100 μg/ml of ribonuclease A (RNase A, Boehringer) and subsequently stained with 36 μl of propidium iodide (1 mg/ml) to a final concentration of 60 μg/ml (Sigma-Aldrich Química), kept on ice for 20 min and measured in an Epics XL flow cytometer (Coulter Corporation). The instrument was set up with the standard configuration. Excitation of the sample was performed using a standard 488-nm air-cooled argon-ion laser at 15 mW power. Forward scatter (FSC), side scatter (SSC), and red (620 nm) fluorescence for propidium iodide were acquired. Optical alignment was based on optimized signal from 10-nm fluorescent beads (Immunocheck, Epics Division, Coulter Corporation). Time was used as a control for the stability of the instrument. Red fluorescence was projected onto a 1,024 monoparametric histogram. Gating single cells by their area versus peak fluorescence signal excluded aggregates. Acquisition was automatically stopped at 8,000 nuclei. Measurements were made at the Serveis Cientificotècnics (Universitat de Barcelona).


#### 2.2.3. Effect of Valepotriates on Measurements

Valerianaceae contain,inter alia, valepotriates, a family of chemical compounds of great medicinal interest, thanks to which some representatives are considerably exploited by the pharmaceutical industry. Valepotriates are known DNA intercalators (they do have severe effects on DNA in e.g., PCR amplifications) and might thus possibly influence genome size measurements by altering the hydrodynamic diameter of the DNA. Furthermore, they could constitute endogenous staining inhibitors, which affect results by causing stoichiometric errors [35, and references therein]. Valepotriates are mainly stored in oil vesicles in the roots and rhizomes [36]. Therefore, we performed measurements on some roots and compared them with DNA C-values obtained for leaves of the same individuals to detect a potential effect of these chemical compounds on genome size assessments if any.


#### 2.2.4. Statistical Analyses

ANOVA and LSD test were performed with the Statgraphics Plus 5.1 program (Statistical Graphics Corp., USA).


### 2.3. Phylogenetic Analyses

#### 2.3.1. DNA Extraction, Amplification and Sequencing

Total genomic DNA was extracted from silica-dried, herbarium voucher or fresh leaves using the Nucleospin Plant extraction kit (Macherey-Nagel, GmbH & Co., Duren, Germany). PCRs were carried out with PTC100 (MJ Research, Inc.) research thermal cyclers in 25μl volume. The whole plastid trnL-trnF region [including the trnL intron, the 3′trnL (UAA) exon, and the intergenic spacer between trnL (UAA) and trnF (GAA)] was amplified and sequenced with the universal primers trnL-c, forward, and trnL-f, reverse, and, in some cases, trnL-d, reverse, and trnL-e, forward [37]. The PCR amplification conditions used were 94°C, 1 min 35 sec; 34x (93°C, 1 min; 58°C, 1 min; 72°C, 1 min); 72°C, 10 min, and storage at 4°C. PCR products were purified with QIAquick PCR Purification Kit (Qiagen, Valencia, California, USA) or with DNA Clean & Concentrator-5 D4003 (Zymo Research, Orange, California, USA). Direct sequencing of the amplified fragments was performed using the Big Dye Terminator Cycle Sequencing v3.1 (PE Biosystems, Foster City, California, USA). Nucleotide sequencing was carried out at the Serveis Cientificotècnics (Universitat de Barcelona) with an ABI PRISM 3700 DNA Analyzer (PE Biosystems, Foster City, California, USA).


#### 2.3.2. Sequence Assembly, Alignment, and Analyses

Nucleotide sequences were assembled and edited using MacClade 4.08 [38]. Bayesian inference (BI) was carried out with MrBayes version 3.1.2 [39]. The most appropriate nucleotide substitution models were chosen with MrModeltest version 2.3 [40]. Four Markov chains were run simultaneously for 1 × 106 generations, and these were sampled every 100 generations. Data from the first 1,000 generations were discarded as the burn-in period, after confirming that likelihood values were stabilized prior to the 1,000th generation. The 50% majority rule consensus trees and posterior probability (PP) of nodes were calculated from the pooled samples. We proceeded to an additional analysis, adding to the matrix the gap data codified with the Barriel method, as previously used for Valerianaceae in [3]. Partitioned dataset analysis was carried out for this dataset including the codified gaps. A gamma-shaped rate variation was stated for the codified data following the manufacturer’s protocol, applying the model selected with MrModeltest for the DNA data.


## 2.1. Plant Material

Table1 shows the provenance of the species investigated. The sampling includes representatives of all the six genera considered in the family in regard to the results of molecular phylogenies [4–6]. Studied plants come from germinated cypselas, collected in the field or obtained from Botanic Gardens. Due to difficulties in germinating seeds of Valeriana representatives, whole individuals were collected in the field and cultivated in the Institut Botànic de Barcelona.Table 1
Origin of the populations studied with indications of the herbaria where voucher specimens are deposited, chromosome counts, genome size data, and GenBank accession numbers (only for the new sequences).
Species (population number)Collection dataLife cycle12C (pg)22n3Ploidy level1Cx42C (Mbp)5Mean HPCV of sample (%)Mean HPCV of standard (%)trnL-trnF GenBank accession numberCentranthusFrance: Vaucluse, Mt.P1.30 ± 0.03324x0.331271.43.83 ± 0.631.25 ± 0.59HM162366angustifolius (Mill.)  DC. (1)Ventoux, Hidalgo 212 &Hidalgo (BC)CentranthusFrance: Hautes-Alpes,P1.35 ± 0.02324x0.341320.35.57 ± 0.863.77 ± 0.65HM162367angustifolius (Mill.)  DC. (2)VI-2005, Hidalgo &Romashchenko (MPU)CentranthusFrance: Hautes-Alpes,P1.41 ± 0.07324x0.3513793.35 ± 0.551.50 ± 0.43angustifolius (Mill.)  DC. (3)Les Infournas, Hidalgo 201 & Martin (BC)CentranthusSpain, Catalonia, PortA1.56 ± 0.04324x0.391525.75.22 ± 1.144.12 ± 0.95calcitrapae  (L.) Dufr. (1)d'Àger, Garnatje 229 &Vallès (BCN)CentranthusFrance: Gard, SaintA1.58 ± 0.02324x0.401545.23.28 ± 0.701.99 ± 0.62HM162368calcitrapae  (L.)   Dufr. (2)André de Majencoules,4-VI-2005, Hidalgo, Romashchenko &  Romo (BC)CentranthusGreece, Crete: Elafonissi,A1.61 ± 0.01324x0.401574.63.62 ± 0.961.06 ± 0.87calcitrapae  (L.)  Dufr. (3)Bot. Gard. MNHN  1996-08CentranthusGreece, Crete: Hania,A1.62 ± 0.03324x0.41584.43.16 ± 1.451.51 ± 0.56calcitrapae  (L.)  Dufr. (4)Bot. Gard. MNHN  1996-15Centranthuscf.Morocco: Hidalgo,A1.80 ± 0.03324x0.451760.43.46 ± 0.602.20 ± 0.70HM162369calcitrapae   (L.) Dufr. (5)Romo 13128 & Soriano (BC)Centranthus lecoqiiFrance: Lozère, LesP1.22 ± 0.05324x0.311193.23.62 ± 1.582.09 ± 0.48Jord. (1)Vignes, Bot. Gard.  MNHN 2002-39Centranthus lecoqiiFrance: Cons. Bot.P324xJord. (2)Porquerolles Ly815Centranthus lecoqiiFrance: Hérault, St.P324xHM162370Jord. (3)Guilhem le Désert, Mathez 1076 (MPU)Centranthus longiflorusLebanon: Les Cèdres,P1.42 ± 0.03324x0.361388.87.90 ± 0.687.71 ± 2.41Steven var.latifoliusBoiss.Bou Dagher-Kharrat, Hidalgo &  Romashchenko  409 (BC)CentranthusBot. Gard. HauniensisA1.97 ± 0.06324x0.491926.72.58 ± 1.191.64 ± 1.26HM162371macrosiphon  Boiss.2000-2315Centranthus nevadensisMorocco: Akchar, surP1.16 ± 0.02324x0.291134.54.23 ± 0.982.27 ± 0.77Boiss. (1)Tizi Ouzli, Hidalgo,Romo 13035 & Soriano (BC)Centranthus nevadensisMorocco: DjebelP1.19 ± 0.01324x0.31163.83.80 ± 0.371.87 ± 0.78HM162372Boiss. (2)Lechâab, Hidalgo, Romo 13147& Soriano (BC)Centranthus ruberFrance: Bouches-du-P1.14 ± 0.04324x0.291114.93.77 ± 1.590.81 ± 0.41(L.) DC. (1)Rhône, la Sainte Baume,VI-2005, Hidalgo, Romashchenko & Romo (BC)Centranthus ruberRumania: Valea Moril,P1.14 ± 0.02324x0.291114.94.56 ± 0.492.36 ± 0.22HM162373(L.) DC. (2)Distr. Cluj, Bot. Gard.Cluj-Napoca 2006-1820Centranthus ruberItaly, Sicily: Siracusa,P1.16 ± 0.04324x0.291134.53.67 ± 0.710.88 ± 0.44(L.) DC. (3)Buccheri Mt., Bot. Gard.Palermo 2006-750Fediacf.  cornucopiaeItaly, Sicily: Siracusa,A1.70 ± 0.02324x0.431662.62.68 ± 0.310.18 ± 0.08HM162374(L.) Gaertn. (1)Lauro-Buccheri Mt.,(asF. caput-bovis)Bot. Gard. Palermo 2006-751Fedia cornucopiaeWilld coll., Bot. Gard.A1.79 ± 0.04324x0.451750.65.28 ± 0.963.28 ± 1.16(L.) Gaertn. (2)Coimbra 2005-1321Fedia gracilifloraBot. Gard.A1.53 ± 0.04324x0.381496.34.48 ± 0.962.53 ± 1.17HM162375Fisch. & C.A.Mey.Montpellier (MPU)Fedia pallescensMorocco: Mehdyia,A1.14 ± 0.02324x0.291114.95.85 ± 1.542.71 ± 1.14HM162376(Maire) MathezEl-Oualidi s.n.-1998 (MPU)Nardostachys jatamansiBot. Gard. Lautaret,P3.49 ± 0.042.06 ± 0.721.25 ± 0.82(D. Don) DC.FrancePatrinia rupestrisRussia: Siberia, Bot.P2.56 ± 0.012.81 ± 1.011.87 ± 1.1Dufr.Gard. Lautaret, FrancePatrinia scabiosifoliaJapan: Bot. Gard. OfunaP1.50 ± 0.035.09 ± 0.714.08 ± 0.37Link (1)314-2006Patrinia scabiosifoliaJapan: Tsukiono-machi,P2.57 ± 0.07ca. 444x0.642513.53.7 ± 0.681.91 ± 0.58HM162377Link (2)Tone-gun, Bot. Gard.Chiba University2006-193Valeriana apulaSpain, Aragon: Huesca,P0.97 ± 0.02162x0.49948.665.58 ± 0.872.16 ± 0.54HM162378Pourr.Anglios, 2005, Vallès (BCN)Valeriana celticaFrance: Savoie, EvettesP2.11 ± 0.041.81 ± 1.371.29 ± 1.10L. (1)circus, Bot. Gard. Lautaret, FranceValeriana celticaItaly: Aosta, Cogne,PHM162379L. (2)Hidalgo 503 (MPU)Valeriana dioicaFrance: Hautes-Alpes,P2.87 ± 0.053.25 ± 0.310.72 ± 0.70L. (1)Lautaret pass, VI-2005Douzet, Hidalgo &Romashchenko (BC)Valeriana dioicaFrance: Gard, BonheurP3.01 ± 0.013.11 ± 0.210.72 ± 0.49HM162380L. (2)valley, 5-VI-2005,(♀)(♀)(♀)Hidalgo, Romashchenko3.08 ± 0.013.22 ± 0.141.78 ± 0.51& Romo (BC)(♂)(♂)(♂)Valeriana longifloraSpain, Aragon: SantaP0.96 ± 0.025.84 ± 0.992.91 ± 0.30Willk. (1)Anna dam, Garnatje 172& Vallès (BCN)Valeriana longifloraSpain, Aragon, SantaPHM162381Willk. (2)Anna dam, Garnatje,Hidalgo 500 &Luque (MPU)Valeriana montanaFrance: Hautes-Alpes,P1.23 ± 0.013.92 ± 1.321.59 ± 1.05L. (1)Les Infournas, Hidalgo202 & Martin (BC)Valeriana montanaSpain, Catalonia:P2.24 ± 0.045.98 ± 2.410.88 ± 0.62L. (2)Girona, Camprodon,Garnatje 179 (BC)Valeriana montanaItaly: Maritime Alps,P2.40 ± 0.052.79 ± 0.362.21 ± 0.83L. (3)Cuneo, 6-VIII-2005,Peccenini & Vallès(BCN)Valeriana montanaFrance: Hautes-Alpes,P2.42 ± 0.032.79 ± 0.492.17 ± 0.73HM162382L. (4)between the Galibier andLautaret passes, Garnatje& Hidalgo 220 (BC)Valeriana montanaSpain, Catalonia: Lleida,P2.50 ± 0.05324x0.6324452.08 ± 0.281.32 ± 0.80HM162383L. (5)Vall d'Aran, on the wayto the Restanca fromArties, Garnatje 234 &Vallès (BCN)Valeriana montanaFrance: Hautes-Alpes,P2.51 ± 0.032.81 ± 0.692.35 ± 0.84L. (6)spontaneous in theLautaret Bot. Gard.,Garnatje & Hidalgo221 (BC)Valeriana montanaSpain, Catalonia: Lleida,P2.54 ± 0.032.52 ± 0.980.87 ± 0.77HM162384L. (7)Vall d'Aran, on the wayto the Restanca fromArties, Garnatje 235 &Vallès (BCN)Valeriana montanaFrance: Hautes-Alpes,P2.60 ± 0.03324x0.652542.82.38 ± 0.831.36 ± 0.54L. (8)between Le Roy and Pisepass, Hidalgo 210 &Martin (BC)Valeriana montanaFrance: Hautes-Alpes,P2.64 ± 0.052.48 ± 1.001.82 ± 0.70HM162385L. (9)Gleize pass, Hidalgo 208& Martin (BC)Valeriana montanaFrance: Hautes-Alpes,PHM162386L. (10)Galibier pass,4-VIII-2001, Hidalgo &Hidalgo (BC)Valerianacf.Spain, Catalonia:P2.46 ± 0.033.65 ± 2.062.06 ± 0.67montana L. (11)Barcelona, Montserrat,2005, Casanova (BC)Valerianacf. montanaL. (12)France: Hautes-Alpes,P2.54 ± 0.023.3 ± 0.241.98 ± 0.60Gleize pass, VI-2005, Hidalgo & Romashchenko (BC)Valeriana officinalisCroatia: Dalmatia,P2.97 ± 0.04142x1.492904.74.07 ± 0.292.22 ± 0.49HM162387L. (1)Biokovo Mt., 2006,Siljak-Yakovlev (BC)Valeriana officinalisFrance: Gard, AigoualP3.16 ± 0.033.19 ± 0.241.44 ± 0.42HM162388L. (2)Mt., 3-VI-2005, Hidalgo,Romashchenko &Romo (BC)Valeriana officinalisSpain, Catalonia:P4.62 ± 0.082.76 ± 0.483.60 ± 0.66L. (3)Girona, Llanars,Garnatje 169, D. Roca &J. Roca (BC)Valeriana officinalisSpain, Catalonia: Lleida,P6.51 ± 0.111.49 ± 1.012.89 ± 1.24L. (4)Vielha, 2005,Vallès (BCN)Valeriana officinalisSpain, Catalonia: Lleida,P8.05 ± 0.081.78 ± 0.762.91 ± 0.64L. (5)Vall d’Aran, Garnatje175 & Vallès (BCN)Valeriana officinalisNorway: Trs, Storfjord,P8.15 ± 0.25ca. 568x1.027970.71.40 ± 0.922.52 ± 1.67HM162389L. (6)Rovijok, Bot. Gard.Oulu 2004/5-425Valeriana officinalisFrance: Gironde,P8.32 ± 0.10  (leaf)3.14 ± 1.26 (leaf)4.99 ± 0.49 (leaf)L. (7)Gradignan, V-2005,8.09 ± 0.08  (root)1.40 ± 1.11 (root)5.83 ± 0.47 (root)Hidalgo &Revel-Hidalgo (BC)Valeriana pyrenaicaSpain, Catalonia: Lleida,P1.26 ± 0.01162x0,61232.35.37 ± 0.642.81 ± 0.26HM162390L.Vall d’Aran, Garnatje176 & Vallès (BCN)ValerianaFrance: Hautes-Alpes,P2.52 ± 0.052.16 ± 0.301.41 ± 0.51HM162391rotundifoliaVill.Devoluy, La Cluse,Hidalgo 207 &Martin (BC)Valeriana saliuncaFrance: Savoie, GalibierP1.46 ± 0.03162x0.731427.94.21 ± 0.363.21 ± 0.25HM162392All.pass, Garnatje 166 &Vallès (BC)Valeriana saxatilisItaly: Apuan Alps,P1.05 ± 0.003.17 ± 0.282.21 ± 0.65L. (1)summit of Sagro Mt.,Hidalgo 213 (BC)Valeriana saxatilisItaly: Apuan Alps,P1.02 ± 0.013.62 ± 0.382.36 ± 0.24HM162393L. (2)Hidalgo 217 (BC)Valeriana tripterisFrance: Hautes-Alpes,P1.46 ± 0.03162x0.731427.93.28 ± 0.711.79 ± 0.69L. (1)Lautaret pass, Hidalgo203 & Martin (BC)Valeriana tripterisFrance: Gard, Mt.P1.51 ± 0.014.01 ± 0.382.24 ± 0.48L. (2)Aigoual, 3-VI-2005,(♀♂)(♀♂)(♀♂)Hidalgo, Romashchenko1.52 ± 0.023.75 ± 0.242.12 ± 0.53& Romo (BC)(♀)(♀)(♀)Valeriana tripterisFrance: Vaucluse, Mt.P1.51 ± 0.01182x0.761476.82.75 ± 0.351.34 ± 0.40HM162394L. (3)Ventoux, Hidalgo 211 &Hidalgo (BC)Valeriana tripterisSpain, Catalonia: Lleida,P1.54 ± 0.035.63 ± 1.004.86 ± 1.03HM162395L. (4)Espot, road to Espot Ski,17-VI-2007, Garnatje233 & Vallès (BCN)Valeriana tripterisSpain, Catalonia:P1.58 ± 0.063.88 ± 0.742.01 ± 0.81HM162396L. (5)Girona, Setcases,Garnatje 170, D. Roca &J. Roca (BC)Valeriana tripterisFrance: Gard, Mt.PHM162397L. (6)Aigoual, 14-VII-00,Hidalgo (BC)Valerianacf.Italy: Apuan Alps,P1.23 ± 0.06324x0.311202.93.93 ± 0.662.34 ± 0.98HM162398tripterisL. (7)Hidalgo 218 (BC)ValerianaFrance: Les Sièges,P3.74 ± 0.032.96 ± 0.993.66 ± 0.42HM162399tuberosaL.Mathez 1088 (MPU)Valerianella coronataItaly: Aymavilles,A0.48 ± 0.00142x0.24469.440.59 ± 0.230.99 ± 0.53(L.) DC. (1)Cerignan, Bot. Gard.Cogne 2006/7-838Valerianella coronataFrance: Vaucluse, “laA0.53 ± 0.01142x0.27518.348.90 ± 0.703.26 ± 0.65(L.) DC. (2)Bastide des Jourdans”,(asV. pumila)Cons. Bot. de Porquerolles1998-CO 2688Valerianella dentataFrance: Maine-et-Loire,A0.42 ± 0.01162x0.21410.7611.88 ± 2.540.55 ± 0.37(L.) Pollich (1)Montreuil-Bellay,(asV. rimosa)“Méron”, Bot. Gard.Nantes 2004-306Valerianella dentataBot. Gard. BonnA0.49 ± 0.01 (leaf)162x0.25479.029.64 ± 0.85 (leaf)1.09 ± 0.85 (leaf)(L.) Pollich (2)2004/5-236870.47 ± 0.02 (root)8.69 ± 0.83 (root)2.29 ± 0.74 (root)Valerianella dentataItaly, Sicily: Palermo,A162xHM162400(L.) Pollich (3)Rocca Busambra-Ficuzza, Bot. Gard.Palermo 2006-756Valerianella discoideaGreece, Crete: Hania,A142xLoiselBot. Gard. MNHN2005-96/14Valerianella echinataFrance: Aveyron, nearA0.60 ± 0.01162x0.3586.85.54 ± 2.242.16 ± 0.71DC.Brunas de Creissels(Larzac N.), BernardValerianella eriocarpaItaly, Sicily: Palermo,A0.47 ± 0.02162x0.24459.666.31 ± 0.500.92 ± 0.86HM162401Desv. (1)Rocca Busambra-Ficuzza, Bot. Gard.Palermo 2006-757Valerianella eriocarpaBot. Gard. NantesA0.54 ± 0.01162x0.29528.1210.07 ± 2.831.86 ± 0.61Desv. (2)2004-302Valerianella locustaFrance: Loire Atlantique,A0.44 ± 0.02162x0.22430.3211.37 ± 1.691.18 ± 0.84HM162402L. (1)cheméré "Le Moulin",Bot. Gard. Nantes2004-303Valerianella locustaL.  (2) (asV. carinata)France: Loire Atlantique,A0.49 ± 0.02162x0.25479.228.50 ± 1.570.20 ± 0.19HM162403Gard. Nantes 2004-301ValerianellaSpain, Catalonia:A0.46 ± 0.02162x0.23449.888.43 ± 1.290.22 ± 0.20microcarpaLoiselTarragona, Ebre delta,2004, Pyke (BC)ValerianellaSpain, Aragon:A0.52 ± 0.0214–162x0.26508.567.78 ± 0.770.97 ± 0.92HM162404multidentata LoscosZaragoza, 2004,& PardoPyke (BC)Valerianella ponticaUkraine: Crimea,A0.51 ± 0.022x0.26498.7811.47 ± 1.330.18 ± 0.10Velen.Boratinsky, Didukh,Romashchenko, Romo1029bis & Susanna (BC)Valerianella turgidaGreece: Oxia, Bot. Gard.A0.39 ± 0.01162x0.2381.4212.15 ± 1.540.65 ± 0.67HM162405Betcke (1)MNHN 2002-190Valerianella turgidaUkraine: Crimea,A0.47 ± 0.02162x0.24459.667.49 ± 2.661.97 ± 0.52HM162406Betcke (2)Boratinsky, Didukh,Romashchenko, Romo10171 & Susanna (BC)Valerianella vesicariaIsrael: Judean Mts. Bot.A0.61 ± 0.052x0.31596.589.58 ± 2.980.36 ± 0.33MoenchGard. Tel Aviv 20011Life cycle: A annual, P perennial. 2 2C nuclear DNA content (means ± SD of 10 measurements—two replicates for five individuals each). 32n somatic chromosome number. 2n and ploidy levels are estimated through bibliographical record (using the database http://www.tropicos.org/), except those in bold, which were verified in this study. 41Cx monoploid genome size (DNA content per basic chromosome set [31]). 52C [Mbp]: 1pg=978 Mbp [32].


## 2.2. Karyological and Cytogenetic Analyses

### 2.2.1. Chromosome Counts and/or Determination of the Ploidy Level

Root tip meristems were obtained either by germinating seeds on wet filter paper in Petri dishes at room temperature, or from plants cultivated in pots in the greenhouse. They were pretreated with 0.002 M 8-hydroxyquinoline for 3 h at 16°C. Material was fixed in absolute ethanol, trichloromethane, and glacial acetic acid (6 : 3 : 1) and stored in the fixative at 4°C during at least two days before processing. Samples were hydrolysed in 1 N HCl for 5 min at 60°C, stained with 1% aqueous aceto-orcein for 1 h minimum, and squashed on slides into a drop of 45% acetic acid-glycerol (9 : 1). Metaphase plates were photographed with a digital camera (Zeiss AxioCam HRm) mounted on a Zeiss Axioplan microscope, and images were analysed with Axio Vision Ac version 4.2.


### 2.2.2. Nuclear DNA Assessments

Leaf tissue of five individuals for each studied population was chopped in 600μl of LB01 isolation buffer [33] with a razor blade, together with the chosen internal standard. The speciesPetunia hybridaVilm. `PxPc6’ (2C = 2.85 pg) and Pisum sativum L. `Express long’ (2C = 8.37 pg) were used as internal standards [34] to cover the range of 2C-values found. Seeds of the standards were provided by the Institut des Sciences du Végétal (CNRS), Gif-sur-Yvette (France). For each individual, two independent samples were extracted and measured the same day. Samples were supplemented with 100 μg/ml of ribonuclease A (RNase A, Boehringer) and subsequently stained with 36 μl of propidium iodide (1 mg/ml) to a final concentration of 60 μg/ml (Sigma-Aldrich Química), kept on ice for 20 min and measured in an Epics XL flow cytometer (Coulter Corporation). The instrument was set up with the standard configuration. Excitation of the sample was performed using a standard 488-nm air-cooled argon-ion laser at 15 mW power. Forward scatter (FSC), side scatter (SSC), and red (620 nm) fluorescence for propidium iodide were acquired. Optical alignment was based on optimized signal from 10-nm fluorescent beads (Immunocheck, Epics Division, Coulter Corporation). Time was used as a control for the stability of the instrument. Red fluorescence was projected onto a 1,024 monoparametric histogram. Gating single cells by their area versus peak fluorescence signal excluded aggregates. Acquisition was automatically stopped at 8,000 nuclei. Measurements were made at the Serveis Cientificotècnics (Universitat de Barcelona).


### 2.2.3. Effect of Valepotriates on Measurements

Valerianaceae contain,inter alia, valepotriates, a family of chemical compounds of great medicinal interest, thanks to which some representatives are considerably exploited by the pharmaceutical industry. Valepotriates are known DNA intercalators (they do have severe effects on DNA in e.g., PCR amplifications) and might thus possibly influence genome size measurements by altering the hydrodynamic diameter of the DNA. Furthermore, they could constitute endogenous staining inhibitors, which affect results by causing stoichiometric errors [35, and references therein]. Valepotriates are mainly stored in oil vesicles in the roots and rhizomes [36]. Therefore, we performed measurements on some roots and compared them with DNA C-values obtained for leaves of the same individuals to detect a potential effect of these chemical compounds on genome size assessments if any.


### 2.2.4. Statistical Analyses

ANOVA and LSD test were performed with the Statgraphics Plus 5.1 program (Statistical Graphics Corp., USA).


## 2.2.1. Chromosome Counts and/or Determination of the Ploidy Level

Root tip meristems were obtained either by germinating seeds on wet filter paper in Petri dishes at room temperature, or from plants cultivated in pots in the greenhouse. They were pretreated with 0.002 M 8-hydroxyquinoline for 3 h at 16°C. Material was fixed in absolute ethanol, trichloromethane, and glacial acetic acid (6 : 3 : 1) and stored in the fixative at 4°C during at least two days before processing. Samples were hydrolysed in 1 N HCl for 5 min at 60°C, stained with 1% aqueous aceto-orcein for 1 h minimum, and squashed on slides into a drop of 45% acetic acid-glycerol (9 : 1). Metaphase plates were photographed with a digital camera (Zeiss AxioCam HRm) mounted on a Zeiss Axioplan microscope, and images were analysed with Axio Vision Ac version 4.2.


## 2.2.2. Nuclear DNA Assessments

Leaf tissue of five individuals for each studied population was chopped in 600μl of LB01 isolation buffer [33] with a razor blade, together with the chosen internal standard. The speciesPetunia hybridaVilm. `PxPc6’ (2C = 2.85 pg) and Pisum sativum L. `Express long’ (2C = 8.37 pg) were used as internal standards [34] to cover the range of 2C-values found. Seeds of the standards were provided by the Institut des Sciences du Végétal (CNRS), Gif-sur-Yvette (France). For each individual, two independent samples were extracted and measured the same day. Samples were supplemented with 100 μg/ml of ribonuclease A (RNase A, Boehringer) and subsequently stained with 36 μl of propidium iodide (1 mg/ml) to a final concentration of 60 μg/ml (Sigma-Aldrich Química), kept on ice for 20 min and measured in an Epics XL flow cytometer (Coulter Corporation). The instrument was set up with the standard configuration. Excitation of the sample was performed using a standard 488-nm air-cooled argon-ion laser at 15 mW power. Forward scatter (FSC), side scatter (SSC), and red (620 nm) fluorescence for propidium iodide were acquired. Optical alignment was based on optimized signal from 10-nm fluorescent beads (Immunocheck, Epics Division, Coulter Corporation). Time was used as a control for the stability of the instrument. Red fluorescence was projected onto a 1,024 monoparametric histogram. Gating single cells by their area versus peak fluorescence signal excluded aggregates. Acquisition was automatically stopped at 8,000 nuclei. Measurements were made at the Serveis Cientificotècnics (Universitat de Barcelona).


## 2.2.3. Effect of Valepotriates on Measurements

Valerianaceae contain,inter alia, valepotriates, a family of chemical compounds of great medicinal interest, thanks to which some representatives are considerably exploited by the pharmaceutical industry. Valepotriates are known DNA intercalators (they do have severe effects on DNA in e.g., PCR amplifications) and might thus possibly influence genome size measurements by altering the hydrodynamic diameter of the DNA. Furthermore, they could constitute endogenous staining inhibitors, which affect results by causing stoichiometric errors [35, and references therein]. Valepotriates are mainly stored in oil vesicles in the roots and rhizomes [36]. Therefore, we performed measurements on some roots and compared them with DNA C-values obtained for leaves of the same individuals to detect a potential effect of these chemical compounds on genome size assessments if any.


## 2.2.4. Statistical Analyses

ANOVA and LSD test were performed with the Statgraphics Plus 5.1 program (Statistical Graphics Corp., USA).


## 2.3. Phylogenetic Analyses

### 2.3.1. DNA Extraction, Amplification and Sequencing

Total genomic DNA was extracted from silica-dried, herbarium voucher or fresh leaves using the Nucleospin Plant extraction kit (Macherey-Nagel, GmbH & Co., Duren, Germany). PCRs were carried out with PTC100 (MJ Research, Inc.) research thermal cyclers in 25μl volume. The whole plastid trnL-trnF region [including the trnL intron, the 3′trnL (UAA) exon, and the intergenic spacer between trnL (UAA) and trnF (GAA)] was amplified and sequenced with the universal primers trnL-c, forward, and trnL-f, reverse, and, in some cases, trnL-d, reverse, and trnL-e, forward [37]. The PCR amplification conditions used were 94°C, 1 min 35 sec; 34x (93°C, 1 min; 58°C, 1 min; 72°C, 1 min); 72°C, 10 min, and storage at 4°C. PCR products were purified with QIAquick PCR Purification Kit (Qiagen, Valencia, California, USA) or with DNA Clean & Concentrator-5 D4003 (Zymo Research, Orange, California, USA). Direct sequencing of the amplified fragments was performed using the Big Dye Terminator Cycle Sequencing v3.1 (PE Biosystems, Foster City, California, USA). Nucleotide sequencing was carried out at the Serveis Cientificotècnics (Universitat de Barcelona) with an ABI PRISM 3700 DNA Analyzer (PE Biosystems, Foster City, California, USA).


### 2.3.2. Sequence Assembly, Alignment, and Analyses

Nucleotide sequences were assembled and edited using MacClade 4.08 [38]. Bayesian inference (BI) was carried out with MrBayes version 3.1.2 [39]. The most appropriate nucleotide substitution models were chosen with MrModeltest version 2.3 [40]. Four Markov chains were run simultaneously for 1 × 106 generations, and these were sampled every 100 generations. Data from the first 1,000 generations were discarded as the burn-in period, after confirming that likelihood values were stabilized prior to the 1,000th generation. The 50% majority rule consensus trees and posterior probability (PP) of nodes were calculated from the pooled samples. We proceeded to an additional analysis, adding to the matrix the gap data codified with the Barriel method, as previously used for Valerianaceae in [3]. Partitioned dataset analysis was carried out for this dataset including the codified gaps. A gamma-shaped rate variation was stated for the codified data following the manufacturer’s protocol, applying the model selected with MrModeltest for the DNA data.


## 2.3.1. DNA Extraction, Amplification and Sequencing

Total genomic DNA was extracted from silica-dried, herbarium voucher or fresh leaves using the Nucleospin Plant extraction kit (Macherey-Nagel, GmbH & Co., Duren, Germany). PCRs were carried out with PTC100 (MJ Research, Inc.) research thermal cyclers in 25μl volume. The whole plastid trnL-trnF region [including the trnL intron, the 3′trnL (UAA) exon, and the intergenic spacer between trnL (UAA) and trnF (GAA)] was amplified and sequenced with the universal primers trnL-c, forward, and trnL-f, reverse, and, in some cases, trnL-d, reverse, and trnL-e, forward [37]. The PCR amplification conditions used were 94°C, 1 min 35 sec; 34x (93°C, 1 min; 58°C, 1 min; 72°C, 1 min); 72°C, 10 min, and storage at 4°C. PCR products were purified with QIAquick PCR Purification Kit (Qiagen, Valencia, California, USA) or with DNA Clean & Concentrator-5 D4003 (Zymo Research, Orange, California, USA). Direct sequencing of the amplified fragments was performed using the Big Dye Terminator Cycle Sequencing v3.1 (PE Biosystems, Foster City, California, USA). Nucleotide sequencing was carried out at the Serveis Cientificotècnics (Universitat de Barcelona) with an ABI PRISM 3700 DNA Analyzer (PE Biosystems, Foster City, California, USA).


## 2.3.2. Sequence Assembly, Alignment, and Analyses

Nucleotide sequences were assembled and edited using MacClade 4.08 [38]. Bayesian inference (BI) was carried out with MrBayes version 3.1.2 [39]. The most appropriate nucleotide substitution models were chosen with MrModeltest version 2.3 [40]. Four Markov chains were run simultaneously for 1 × 106 generations, and these were sampled every 100 generations. Data from the first 1,000 generations were discarded as the burn-in period, after confirming that likelihood values were stabilized prior to the 1,000th generation. The 50% majority rule consensus trees and posterior probability (PP) of nodes were calculated from the pooled samples. We proceeded to an additional analysis, adding to the matrix the gap data codified with the Barriel method, as previously used for Valerianaceae in [3]. Partitioned dataset analysis was carried out for this dataset including the codified gaps. A gamma-shaped rate variation was stated for the codified data following the manufacturer’s protocol, applying the model selected with MrModeltest for the DNA data.


## 3. Results

### 3.1. Chromosome Counts

Chromosome numbers of the populations studied are indicated in Table1, and metaphase plates are presented in Figure 1. According to our data, we provide the first count for Valerianella multidentataLoscos & Pardo. Ploidy levels ranging from diploid to octoploid were detected although diploid and tetraploid were predominantly reported. Good metaphase plates are quite difficult to obtain for Valerianaceae. In fact, this is certainly the main reason why only few chromosome studies have been carried out in the family, and that especially few chromosome pictures have been published to present.Figure 1
(a)–(v). Somatic metaphases. (a)Centranthuscf.calcitrapae (5), 2n=32. (b)C. lecoqii (2), 2n=32. (c)C. macrosiphon, 2n=32.(d)C. ruber(3), 2n=32.(e)Patrinia scabiosifolia, 2n= ca. 44. (f)Valeriana apula2n=16.(g) V. montana(5), 2n=32.(h)V. officinalis (1),  2n=14.(i)V. officinalis(6), 2n=  ca. 56. (j)V. pyrenaica, 2n=16. (k) V. saliunca,2n=16. (l) V. tripteris (3),  2n=18.(ma-b)V. cf. tripteris(7), 2n= 32. (n) Valerianella coronata (1), 2n= 14. (o) V. coronata (2), 2n= 14. (p) V. dentata (1), 2n= 16. (q) V. dentata (2), 2n= 16. (r) V. discoidea, 2n= 14. Arrows indicate a chromosome pair much larger than the rest. (s) V. eriocarpa (1), 2n= 16. (t) V. locusta, 2n= 16. (u) V. multidentata, 2n= 14–16. (v) V. turgida (1), 2n= 16. Scale bars = 10 μm.


### 3.2. Genome Size Assessments

Data on nuclear DNA content are presented in Figure2 and Table 1. The present data are the first reports on nuclear DNA content in all the genera studied excepting Centranthus  and in all the species studied of this genus except for C. ruber (L.) DC., which was to date the only member of the family with a known genome size [30]. Genome size in Valerianaceae varies about 21.3-fold, from 2C = 0.39 pg (Valerianella turgida Betcke, 2x) to 2C = 8.32 pg (Valeriana officinalis, 8x). Although the half peak coefficients of variation (HPCV) are rather high for some species (in all cases but one of the genus Valerianella), their mean value is 4.62% for the target plants and 2.04% for the standards. More in-deep research is suitable in Valerianella to try and obtain measurements with better HPCV.(a)–(c). Histograms of nuclear DNA content obtained for some representatives of Valerianaceae. (a)Centranthus nevadensis (2) (2C = 1.19 ± 0.01). (b) Valeriana officinalis (7) (2C = 8.32 ± 0.10). (c) Valerianella coronata (2) (2C = 0.53 ± 0.01). Peak C: sample species nuclei. Peak H: standard species nuclei.
(a)(b)(c)The difference in genome size between leaves and roots measured in the same population has been addressed forValeriana officinalis (7) (leaves: 2C of 7.67–8.97 pg and roots: 2C of 8.04–8.14 pg, at 95% confidence interval) and Valerianella dentata (L.) Pollich (2) (leaves: 2C of 0.485–0.495 pg and roots: 2C of 0.464–0.476 pg, at 95% confidence interval). A significant difference has been detected for Valerianella dentata (ANOVA, P=0.0036), which may indicate a possible effect of valepotriates on genome size assessments in this species, also suggested by the especially high HPCV values found in this annual genus (Table 1).


### 3.3. Molecular Phylogeny

Both AIC (Akaike Information Criterion) and hLRT (hierarchical Likelihood Ratio Tests) selected GTR+G (General Time Reversible model with gamma distribution) as the best-fit model. The results of the Bayesian analyses are presented in Figure3. Phylogenetic trees obtained through the analyses of the two different datasets basically led to the same tree topology and supports. Nevertheless, three clades are only significantly supported when codified gap information is taken into account. These are (a) Valerianaclade I (PP=0.95, Figure 3), (b) Valeriana longiflora Willk. plus Centranthus clade (PP=1.00, Figure 3), and (c) the grouping of Fedia cornucopiae (L.) Gaertn. AF446986 with Fedia graciliflora Fisch. & C.A.Mey. (PP=0.96, Figure 3).Figure 3
Molecular phylogeny based on the Bayesian analysis of thetrnL-trnF region and the codified gap information, with posterior probability values ≥0.95 indicated on the branches. Wider branches are those significantly supported in the Bayesian analysis of the trnL-trnF region without including the information from the gaps. Taxonomic information, relevant floral characters, basic chromosome number, and data on ploidy levels are indicated. GenBank accession numbers are provided for the previously published sequences. Numbers in brackets differentiate populations of a same species (see Table 1).


## 3.1. Chromosome Counts

Chromosome numbers of the populations studied are indicated in Table1, and metaphase plates are presented in Figure 1. According to our data, we provide the first count for Valerianella multidentataLoscos & Pardo. Ploidy levels ranging from diploid to octoploid were detected although diploid and tetraploid were predominantly reported. Good metaphase plates are quite difficult to obtain for Valerianaceae. In fact, this is certainly the main reason why only few chromosome studies have been carried out in the family, and that especially few chromosome pictures have been published to present.Figure 1
(a)–(v). Somatic metaphases. (a)Centranthuscf.calcitrapae (5), 2n=32. (b)C. lecoqii (2), 2n=32. (c)C. macrosiphon, 2n=32.(d)C. ruber(3), 2n=32.(e)Patrinia scabiosifolia, 2n= ca. 44. (f)Valeriana apula2n=16.(g) V. montana(5), 2n=32.(h)V. officinalis (1),  2n=14.(i)V. officinalis(6), 2n=  ca. 56. (j)V. pyrenaica, 2n=16. (k) V. saliunca,2n=16. (l) V. tripteris (3),  2n=18.(ma-b)V. cf. tripteris(7), 2n= 32. (n) Valerianella coronata (1), 2n= 14. (o) V. coronata (2), 2n= 14. (p) V. dentata (1), 2n= 16. (q) V. dentata (2), 2n= 16. (r) V. discoidea, 2n= 14. Arrows indicate a chromosome pair much larger than the rest. (s) V. eriocarpa (1), 2n= 16. (t) V. locusta, 2n= 16. (u) V. multidentata, 2n= 14–16. (v) V. turgida (1), 2n= 16. Scale bars = 10 μm.


## 3.2. Genome Size Assessments

Data on nuclear DNA content are presented in Figure2 and Table 1. The present data are the first reports on nuclear DNA content in all the genera studied excepting Centranthus  and in all the species studied of this genus except for C. ruber (L.) DC., which was to date the only member of the family with a known genome size [30]. Genome size in Valerianaceae varies about 21.3-fold, from 2C = 0.39 pg (Valerianella turgida Betcke, 2x) to 2C = 8.32 pg (Valeriana officinalis, 8x). Although the half peak coefficients of variation (HPCV) are rather high for some species (in all cases but one of the genus Valerianella), their mean value is 4.62% for the target plants and 2.04% for the standards. More in-deep research is suitable in Valerianella to try and obtain measurements with better HPCV.(a)–(c). Histograms of nuclear DNA content obtained for some representatives of Valerianaceae. (a)Centranthus nevadensis (2) (2C = 1.19 ± 0.01). (b) Valeriana officinalis (7) (2C = 8.32 ± 0.10). (c) Valerianella coronata (2) (2C = 0.53 ± 0.01). Peak C: sample species nuclei. Peak H: standard species nuclei.
(a)(b)(c)The difference in genome size between leaves and roots measured in the same population has been addressed forValeriana officinalis (7) (leaves: 2C of 7.67–8.97 pg and roots: 2C of 8.04–8.14 pg, at 95% confidence interval) and Valerianella dentata (L.) Pollich (2) (leaves: 2C of 0.485–0.495 pg and roots: 2C of 0.464–0.476 pg, at 95% confidence interval). A significant difference has been detected for Valerianella dentata (ANOVA, P=0.0036), which may indicate a possible effect of valepotriates on genome size assessments in this species, also suggested by the especially high HPCV values found in this annual genus (Table 1).


## 3.3. Molecular Phylogeny

Both AIC (Akaike Information Criterion) and hLRT (hierarchical Likelihood Ratio Tests) selected GTR+G (General Time Reversible model with gamma distribution) as the best-fit model. The results of the Bayesian analyses are presented in Figure3. Phylogenetic trees obtained through the analyses of the two different datasets basically led to the same tree topology and supports. Nevertheless, three clades are only significantly supported when codified gap information is taken into account. These are (a) Valerianaclade I (PP=0.95, Figure 3), (b) Valeriana longiflora Willk. plus Centranthus clade (PP=1.00, Figure 3), and (c) the grouping of Fedia cornucopiae (L.) Gaertn. AF446986 with Fedia graciliflora Fisch. & C.A.Mey. (PP=0.96, Figure 3).Figure 3
Molecular phylogeny based on the Bayesian analysis of thetrnL-trnF region and the codified gap information, with posterior probability values ≥0.95 indicated on the branches. Wider branches are those significantly supported in the Bayesian analysis of the trnL-trnF region without including the information from the gaps. Taxonomic information, relevant floral characters, basic chromosome number, and data on ploidy levels are indicated. GenBank accession numbers are provided for the previously published sequences. Numbers in brackets differentiate populations of a same species (see Table 1).


## 4. Discussion

We address here the analysis of genome size variation and its phylogenetic and taxonomical implications  in the taxa considered.


### 4.1. The Basal Grades

They consist of the taxa showing the most ancestral characters within the Valerianaceae, namely, the generaNardostachys andPatrinia, along with the clade grouping Valeriana celtica and Valeriana saxatilis L. (Valeriana clade III, Figure 3). Shifts in biogeography, flower morphology, and basic chromosome number occurred at this point of the evolutionary history of the family. From this moment on, the distribution area of the Valerianaceae, until that time restricted to Asia (Nardostachys andPatrinia), was enlarged to the other continents. At the same time the number of stamens decreased from four to three. These changes just preceded the change in basic chromosome numbers from x=11 to x=8, occurring at the arising of Valerianeae tribe (Figure 3).TheValeriana clade III (P=1.00, Figure 3) constituted by two species from the Alps, V. celtica and V. saxatilis, is branched between Patrinieae  and Valerianeae, a position that was previously found for V. celtica[4, 5], and stated for the first time here for V. saxatilis. Although their genome sizes fall within the range of the remaining Valeriana, these two valerian species present x=11 as basic chromosome number [41, 42], which had only been detected in Patrinia. Furthermore, V. celtica has a yellow corolla, also like species of Patrinia. Nevertheless, species of clade III differ from Patrinia by a pappus-like CentranthusandValeriana, and the number of stamens reduced to three as in Valeriana and Valerianella(whereas Nardostachys presents four and occasionally five stamens andPatrinia presents five or less stamens; [43, 44]). Therefore, species of clade III are from morphological, cytogenetic, and phylogenetic points of view in between Patrinieae and Valerianeae. These results raise numerous questions. Should this clade III form a new genus? Could any other Valeriana species be susceptible to join this group? Then, should this group be classified within Valerianeae, or as a new tribe (their inclusion in the Patrinieae being impossible under the monophyly criterion)?


### 4.2. Fedia Plus Valerianella Clade

These species have the smallest genome sizes known in the family (Table1) to present. Fediaand Valerianella are exclusively composed of annual herbaceous plants. All the species belonging to these two genera, excepted F. pallescens (Maire) Mathez, are common weeds in waste ground and cultivated land. Valerianella, which comprises around 50 species that are mainly diploid and with three stamens, is widely distributed in Eurasia, Africa and America. The genus Fedia includes only three tetraploid species and one hexaploid subspecies [45], with two stamens, and is restricted to the Mediterranean region. Molecular inferences suggest that Fedia species should have originated from Valerianella (Figure 3; [7]), in other words that autogamous strongly associate to anthropogenic environment (Valerianella) would have given rise to allogamous, polyploid (Fedia).Genome size in those genera shows thatFedia (1Cx=0.29 to 0.45 pg) has significantly higher DNA values than Valerianella (from 1Cx=0.20 to 0.31 pg) (P=0.0000), which corroborates the conclusions of Albach & Greilhuber [46] that allogamy is related to higher DNA values than autogamy. Furthermore, Fedia presents the “allogamy syndrome,” morphological adaptations linked to the breeding system (e.g., zygomorphic corolla, development of a nectar gibbosity, tube elongation, and occurrence of polychroic corolla). The intensity of this syndrome varies between the species and the subspecies of Fedia [47]. The tendency within the genus is toward an increasing genome size and degree of allogamy syndrome, from Fedia pallescens (1Cx=0.29 pg) to F. graciliflora(1Cx=0.38 pg) and F. cornucopiae (1Cx=0.43–0.45 pg).TheValerianella group splits in two major clades (Figure 3), one including V. turgida, V. locusta (L.) Laterr., and the Fediaspecies. The genusValerianella is revealed in this study as a relatively homogeneous group respecting genome size within Valerianaceae, with 2C values that range from 0.39 pg (V. turgida) to 0.61 pg (V. vesicariaMoench). Furthermore, some of Valerianella species, such as V. turgida, account for very small genomes, which fall into the range of the smallest genome size records in angiosperms [48], around the same scale than Arabidopsis thaliana (L.) Heynh. (1C=0.16 pg; [49]), for long considered as the lowest angiosperms C-DNA value, nowadays reported in the genus Genlisea (G. margaretae1C=0.064 pg; [50]).The chromosome number ofValerianella multidentata, 2n=2x=14–16, is reported here for the first time. This endemic restricted to a small area of Catalonia, Spain, is very close to V. discoidea Loisel (see [51]; 2n=2x=14–16), which has been in turn related to V. coronata (L.) DC. ([52]; 2n=2x=14). All these species belong to section Coronatae. The chromosome number of 2n= 14 is the smaller within the genus. Metaphase plates of V. discoidea (Figure 1(r)) show a chromosome pair much larger than others that may result from a chromosome fusion sparking off the descending diploidy observed in the section.


### 4.3. Centranthus Plus Valeriana  longiflora  Clade

Centranthus is a circum-Mediterranean genus of nine species, characterized by flowers with one stamen, related to Valeriana longiflora(PP=1.00, Figure 3; [5]). All the species show the same chromosome number, 2n=4x=32, both annuals (C. calcitrapae (L.) Dufr., C. macrosiphonBoiss.) and perennials (the other species), and both widespread (C. calcitrapae, C. ruber) and narrowly distributed species (the remaining). Centranthus is organized in three sections: Section  Calcitrapae (C. calcitrapaeand C. macrosiphon), sect. Centranthus (represented in our study by C. angustifolius (Mill.) DC., C. lecoqii Jord., C. longiflorus Stev. and C. ruber), and sect.Nervosa (not represented in our study). In this case, section Calcitrapae, annual (1Cx=0.39–0.49 pg), has higher genome size than section Centranthus, perennial (1Cx=0.29–0.36 pg). Annual plants have been usually reported to account for smaller genome sizes than perennials [53], as it is comprehensible that the transcriptional machinery would be more efficient in smaller genomes, in order to quickly complete the life-cycle. Otherwise, exceptions to this rule have been found in different plant groups [22, 54], and this seems to be the trend in the genus Centranthus.The common occurrence of hybridization events in the overlapping areas ofCentranthus species [55], and the fact that some species or infraspecific taxa show intermediate morphological characters, can indicate that several of these taxa could have a hybrid origin. As it has been reported in different plant groups, nuclear DNA content of hybrids corresponds to approximately the mean of both parental genome sizes, or is slightly smaller/larger than expected [26, 27, 56–64]. Within the section Calcitrapae, representatives of southern Spain and Morocco have a doubtful taxonomic assignation, somewhere between C. calcitrapae and C. macrosiphon ([55] for southern Spain; e.g., J Mathez, Université Montpellier II, France, “unpubl. res.” for Morocco). One population of Morocco was assessed, C.cf. calcitrapae (5), and the 1Cx value obtained of 0.45 pg turns out to be the average between the means of typical C. calcitrapae (1Cx=0.40 pg) and C. macrosiphon (1Cx=0.49 pg), this being consistent with the hypothesis of a hybrid origin for this Moroccan population. Also, C. lecoqii is morphologically intermediate between C. angustifolius and C. ruber [55]. The 1Cx amount of C. lecoqii (0.31 pg), the mean of those of C. angustifolius(0.34 pg) and C. ruber(0.29 pg), is compatible with the hypothesis of a hybrid origin for the former taxon.


### 4.4. Valeriana  montana  Clade

This group of alpine plants is constituted by species with hermaphrodite flowers and of restricted area (V. saliunca All. and V. supina Ard. from the Alps, V. pyrenaica L. and V. apula Pourr. from the Pyrenees), two gynodioecious species widely distributed, V. tripteris L. and V. montana L. (from the mountains of Southern Europe).  Valeriana rotundifolia Vill. is a gynodioecious taxon of obscure status, present in the Alps and Corsica, and morphologically close to V. tripterisand V. montana [65]. The molecular phylogeny shows a Valeriana tripteris complex constituted by the populations of V. montana, V. rotundifolia, V. supina and V. tripteris, who are forming a robust clade (PP=1.00, Figure 3), with the exception of V.cf.tripteris (7) that is sister to the remaining ones. This result raises the question of the monophyly of V. tripteris, but also shows a possible interesting biogeographical pattern as the population V.cf.tripteris (7) is the only sequenced that grows at the east of the Alps. Furthermore, this population is not only divergent in terms of DNA sequence, but also for its cytogenetic characters. Although V.cf.tripteris (7) is a tetraploid (Figure 1 ma-b), its genome size 2C=1.23 pg ranks low if compared with tetraploid V. montana (2C =2.50–2.60 pg), and even with diploid V. tripteris (2C =1.46–1.51 pg).Intermediate forms betweenV. tripteris and V. montana, such as V. cf.montana (11-12), V.cf.tripteris (7) or V. rotundifolia, occur throughout the range of distribution, which makes their taxonomic delimitation difficult. This is the reason why these taxa have been sometimes considered to represent a single species with various subspecific entities, and also as two closely related species with their intermediate forms as subspecies (see [65], and references therein). Cryptic hybrid and/or polyploid taxa may largely account for the taxonomic heterogeneity of V. tripteris complex and the genome size variation observed in the group, especially at intraspecific level. However, Briquet & Cavillier [65] pointed out that intermediate forms are found in isolated populations, without V. montana, which makes their hybrid origin improbable and their polyploid origin from V. tripteris maybe more likely. Should this polyploid hypothesis be sustained, the question remains as to why tetraploid V. tripterisare morphologically similar to V. montana. One explanation could be that the speciation of V. montana could have also been induced by a polyploidization event of V. tripteris, which would have arisen previously in evolutionary time. This would explain both the more notable morphological and ecological differentiation between the two species, and in some cases the more important decrease in monoploid genome size with respect to that of the intermediate forms [23].


### 4.5. Valeriana officinalis  Clade (Valeriana Clade II, Figure3)

Valeriana officinalis and relatives constitute a difficult complex of numerous taxa of specific and infraspecific ranges, with doubtful morphological delimitation. Valeriana officinalis presents an unusual basic chromosome number for the genus of x=7. This basic number is also found in V. wallrothii Kreyer, a species closely related to Valeriana officinalis in the molecular phylogeny [6], but not in other representatives of the group as, for example, V. dioica or V. tuberosa. The populations of V. officinalis measured exhibit different DNA amounts, suggesting four different DNA ploidy levels of 2x, 4x, 6x, and 8x (Table 1). Even so, it would be necessary to determine whether those differences really correspond to different ploidy levels, or if they are partially due to a high intraspecific variability within V. officinalis. This doubt concerns particularly the ploidy levels that are uncommon in the V. officinaliscomplex, like the hexaploid level (corresponding to 42 chromosomes), known only from V. coreanaBriq. [66] and V. transjenisensisKreyer [67]. The results compiled in the present study allow us to conclude that the complexity within V. officinalis, in which polyploidization events are largely implicated, is much more than previously considered.


## 4.1. The Basal Grades

They consist of the taxa showing the most ancestral characters within the Valerianaceae, namely, the generaNardostachys andPatrinia, along with the clade grouping Valeriana celtica and Valeriana saxatilis L. (Valeriana clade III, Figure 3). Shifts in biogeography, flower morphology, and basic chromosome number occurred at this point of the evolutionary history of the family. From this moment on, the distribution area of the Valerianaceae, until that time restricted to Asia (Nardostachys andPatrinia), was enlarged to the other continents. At the same time the number of stamens decreased from four to three. These changes just preceded the change in basic chromosome numbers from x=11 to x=8, occurring at the arising of Valerianeae tribe (Figure 3).TheValeriana clade III (P=1.00, Figure 3) constituted by two species from the Alps, V. celtica and V. saxatilis, is branched between Patrinieae  and Valerianeae, a position that was previously found for V. celtica[4, 5], and stated for the first time here for V. saxatilis. Although their genome sizes fall within the range of the remaining Valeriana, these two valerian species present x=11 as basic chromosome number [41, 42], which had only been detected in Patrinia. Furthermore, V. celtica has a yellow corolla, also like species of Patrinia. Nevertheless, species of clade III differ from Patrinia by a pappus-like CentranthusandValeriana, and the number of stamens reduced to three as in Valeriana and Valerianella(whereas Nardostachys presents four and occasionally five stamens andPatrinia presents five or less stamens; [43, 44]). Therefore, species of clade III are from morphological, cytogenetic, and phylogenetic points of view in between Patrinieae and Valerianeae. These results raise numerous questions. Should this clade III form a new genus? Could any other Valeriana species be susceptible to join this group? Then, should this group be classified within Valerianeae, or as a new tribe (their inclusion in the Patrinieae being impossible under the monophyly criterion)?


## 4.2. Fedia Plus Valerianella Clade

These species have the smallest genome sizes known in the family (Table1) to present. Fediaand Valerianella are exclusively composed of annual herbaceous plants. All the species belonging to these two genera, excepted F. pallescens (Maire) Mathez, are common weeds in waste ground and cultivated land. Valerianella, which comprises around 50 species that are mainly diploid and with three stamens, is widely distributed in Eurasia, Africa and America. The genus Fedia includes only three tetraploid species and one hexaploid subspecies [45], with two stamens, and is restricted to the Mediterranean region. Molecular inferences suggest that Fedia species should have originated from Valerianella (Figure 3; [7]), in other words that autogamous strongly associate to anthropogenic environment (Valerianella) would have given rise to allogamous, polyploid (Fedia).Genome size in those genera shows thatFedia (1Cx=0.29 to 0.45 pg) has significantly higher DNA values than Valerianella (from 1Cx=0.20 to 0.31 pg) (P=0.0000), which corroborates the conclusions of Albach & Greilhuber [46] that allogamy is related to higher DNA values than autogamy. Furthermore, Fedia presents the “allogamy syndrome,” morphological adaptations linked to the breeding system (e.g., zygomorphic corolla, development of a nectar gibbosity, tube elongation, and occurrence of polychroic corolla). The intensity of this syndrome varies between the species and the subspecies of Fedia [47]. The tendency within the genus is toward an increasing genome size and degree of allogamy syndrome, from Fedia pallescens (1Cx=0.29 pg) to F. graciliflora(1Cx=0.38 pg) and F. cornucopiae (1Cx=0.43–0.45 pg).TheValerianella group splits in two major clades (Figure 3), one including V. turgida, V. locusta (L.) Laterr., and the Fediaspecies. The genusValerianella is revealed in this study as a relatively homogeneous group respecting genome size within Valerianaceae, with 2C values that range from 0.39 pg (V. turgida) to 0.61 pg (V. vesicariaMoench). Furthermore, some of Valerianella species, such as V. turgida, account for very small genomes, which fall into the range of the smallest genome size records in angiosperms [48], around the same scale than Arabidopsis thaliana (L.) Heynh. (1C=0.16 pg; [49]), for long considered as the lowest angiosperms C-DNA value, nowadays reported in the genus Genlisea (G. margaretae1C=0.064 pg; [50]).The chromosome number ofValerianella multidentata, 2n=2x=14–16, is reported here for the first time. This endemic restricted to a small area of Catalonia, Spain, is very close to V. discoidea Loisel (see [51]; 2n=2x=14–16), which has been in turn related to V. coronata (L.) DC. ([52]; 2n=2x=14). All these species belong to section Coronatae. The chromosome number of 2n= 14 is the smaller within the genus. Metaphase plates of V. discoidea (Figure 1(r)) show a chromosome pair much larger than others that may result from a chromosome fusion sparking off the descending diploidy observed in the section.


## 4.3. Centranthus Plus Valeriana  longiflora  Clade

Centranthus is a circum-Mediterranean genus of nine species, characterized by flowers with one stamen, related to Valeriana longiflora(PP=1.00, Figure 3; [5]). All the species show the same chromosome number, 2n=4x=32, both annuals (C. calcitrapae (L.) Dufr., C. macrosiphonBoiss.) and perennials (the other species), and both widespread (C. calcitrapae, C. ruber) and narrowly distributed species (the remaining). Centranthus is organized in three sections: Section  Calcitrapae (C. calcitrapaeand C. macrosiphon), sect. Centranthus (represented in our study by C. angustifolius (Mill.) DC., C. lecoqii Jord., C. longiflorus Stev. and C. ruber), and sect.Nervosa (not represented in our study). In this case, section Calcitrapae, annual (1Cx=0.39–0.49 pg), has higher genome size than section Centranthus, perennial (1Cx=0.29–0.36 pg). Annual plants have been usually reported to account for smaller genome sizes than perennials [53], as it is comprehensible that the transcriptional machinery would be more efficient in smaller genomes, in order to quickly complete the life-cycle. Otherwise, exceptions to this rule have been found in different plant groups [22, 54], and this seems to be the trend in the genus Centranthus.The common occurrence of hybridization events in the overlapping areas ofCentranthus species [55], and the fact that some species or infraspecific taxa show intermediate morphological characters, can indicate that several of these taxa could have a hybrid origin. As it has been reported in different plant groups, nuclear DNA content of hybrids corresponds to approximately the mean of both parental genome sizes, or is slightly smaller/larger than expected [26, 27, 56–64]. Within the section Calcitrapae, representatives of southern Spain and Morocco have a doubtful taxonomic assignation, somewhere between C. calcitrapae and C. macrosiphon ([55] for southern Spain; e.g., J Mathez, Université Montpellier II, France, “unpubl. res.” for Morocco). One population of Morocco was assessed, C.cf. calcitrapae (5), and the 1Cx value obtained of 0.45 pg turns out to be the average between the means of typical C. calcitrapae (1Cx=0.40 pg) and C. macrosiphon (1Cx=0.49 pg), this being consistent with the hypothesis of a hybrid origin for this Moroccan population. Also, C. lecoqii is morphologically intermediate between C. angustifolius and C. ruber [55]. The 1Cx amount of C. lecoqii (0.31 pg), the mean of those of C. angustifolius(0.34 pg) and C. ruber(0.29 pg), is compatible with the hypothesis of a hybrid origin for the former taxon.


## 4.4. Valeriana  montana  Clade

This group of alpine plants is constituted by species with hermaphrodite flowers and of restricted area (V. saliunca All. and V. supina Ard. from the Alps, V. pyrenaica L. and V. apula Pourr. from the Pyrenees), two gynodioecious species widely distributed, V. tripteris L. and V. montana L. (from the mountains of Southern Europe).  Valeriana rotundifolia Vill. is a gynodioecious taxon of obscure status, present in the Alps and Corsica, and morphologically close to V. tripterisand V. montana [65]. The molecular phylogeny shows a Valeriana tripteris complex constituted by the populations of V. montana, V. rotundifolia, V. supina and V. tripteris, who are forming a robust clade (PP=1.00, Figure 3), with the exception of V.cf.tripteris (7) that is sister to the remaining ones. This result raises the question of the monophyly of V. tripteris, but also shows a possible interesting biogeographical pattern as the population V.cf.tripteris (7) is the only sequenced that grows at the east of the Alps. Furthermore, this population is not only divergent in terms of DNA sequence, but also for its cytogenetic characters. Although V.cf.tripteris (7) is a tetraploid (Figure 1 ma-b), its genome size 2C=1.23 pg ranks low if compared with tetraploid V. montana (2C =2.50–2.60 pg), and even with diploid V. tripteris (2C =1.46–1.51 pg).Intermediate forms betweenV. tripteris and V. montana, such as V. cf.montana (11-12), V.cf.tripteris (7) or V. rotundifolia, occur throughout the range of distribution, which makes their taxonomic delimitation difficult. This is the reason why these taxa have been sometimes considered to represent a single species with various subspecific entities, and also as two closely related species with their intermediate forms as subspecies (see [65], and references therein). Cryptic hybrid and/or polyploid taxa may largely account for the taxonomic heterogeneity of V. tripteris complex and the genome size variation observed in the group, especially at intraspecific level. However, Briquet & Cavillier [65] pointed out that intermediate forms are found in isolated populations, without V. montana, which makes their hybrid origin improbable and their polyploid origin from V. tripteris maybe more likely. Should this polyploid hypothesis be sustained, the question remains as to why tetraploid V. tripterisare morphologically similar to V. montana. One explanation could be that the speciation of V. montana could have also been induced by a polyploidization event of V. tripteris, which would have arisen previously in evolutionary time. This would explain both the more notable morphological and ecological differentiation between the two species, and in some cases the more important decrease in monoploid genome size with respect to that of the intermediate forms [23].


## 4.5. Valeriana officinalis  Clade (Valeriana Clade II, Figure3)

Valeriana officinalis and relatives constitute a difficult complex of numerous taxa of specific and infraspecific ranges, with doubtful morphological delimitation. Valeriana officinalis presents an unusual basic chromosome number for the genus of x=7. This basic number is also found in V. wallrothii Kreyer, a species closely related to Valeriana officinalis in the molecular phylogeny [6], but not in other representatives of the group as, for example, V. dioica or V. tuberosa. The populations of V. officinalis measured exhibit different DNA amounts, suggesting four different DNA ploidy levels of 2x, 4x, 6x, and 8x (Table 1). Even so, it would be necessary to determine whether those differences really correspond to different ploidy levels, or if they are partially due to a high intraspecific variability within V. officinalis. This doubt concerns particularly the ploidy levels that are uncommon in the V. officinaliscomplex, like the hexaploid level (corresponding to 42 chromosomes), known only from V. coreanaBriq. [66] and V. transjenisensisKreyer [67]. The results compiled in the present study allow us to conclude that the complexity within V. officinalis, in which polyploidization events are largely implicated, is much more than previously considered.


## 5. Concluding Remarks

At the family level, changes in basic chromosome number and genome size coincide with or precede major shifts in the evolutionary history of Valerianaceae. One interesting example is the arising of strong zygomorphic flower inCentranthus and Fedia, which is in both cases consecutive to a polyploidization event. Therefore, cytogenetic studies are essential for understanding the family, and, in this sense, we will follow our effort for providing new data of this type, especially in those genera poorly studied or unknown at different levels (i.e., genome size) such as Plectritis.



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*Source: 797246-2010-08-18.xml*