52 # November 2003: 783–801 Lihová & al.

# Cardamine pratensis group

The Cardamine pratensis (Brassicaceae) group in the Iberian Peninsula: tax-

onomy, polyploidy and distribution
Judita Lihová1 , Andreas Tribsch2 & Karol Marhold1,3

1 Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 14, SK-845 23 Bratislava, Slovak Republic.
judita.lihova@ savba.sk (author for correspondence); [email protected]
2 Department of Higher Plant Systematics and Evolution, Institute of Botany, University of Vienna, Rennweg
14, A-1030 Vienna, Austria. [email protected]
3 Department of Botany, Charles University, Benátská 2, CZ-128 01 Praha 2, Czech Republic

The Cardamine pratensis group is a taxonomically critical species complex with pronounced karyological and
morphological variation. In the present study, representatives from the Iberian Peninsula have been investigat-
ed using karyological, morphometric and molecular (amplified fragment length polymorphism) analyses. As a
result of this combined approach, three species are recognized in the area studied: C. pratensis s.str., C. cras-
sifolia, and a herein newly described species, C. castellana. For C. pratensis s.str., consistent with the pattern
known from other European regions, wide variation in chromosome numbers is revealed, consisting of diploid
to heptaploid populations in northern and central parts of the Peninsula. Little morphological and genetic dif-
ferentiation accompanies these cytotypes. Re-evaluation of populations up to now ascribed to C. crassifolia
reveal two separate taxa: (1) populations from the Eastern Pyrenees, representing typical C. crassifolia, and (2)
those from central Iberian mountains treated as C. castellana. These two diploid taxa differ in several quanti-
tative morphological characters, as well as in morphology of rhizome and basal parts of stem, and they are also
distinct in AFL P markers, indicating strong genetic differentiation.

KEYWORDS: AFLPs, Cardamine castellana, Cardamine crassifolia, chromosome numbers, morphometrics,

Portugal, Spain.

ably representing (pre)Pleistocene relicts, were revealed,

INTRODUCTION which corresponded to diploid species from the Balkan
The genus Cardamine L. (Brassicaceae) comprises (C. penzesii Anèev & Marhold, C. rivularis Schur) and
several taxonomically critical species complexes, among Iberian (C. crassifolia Pourr.) Peninsulas. All remaining
them the C. pratensis group, which shows complicated diploid to polyploid taxa formed a phylogenetically
patterns of morphological and karyological variation young, derived group showing poor phylogenetic resolu-
(Lövkvist, 1956). It includes several diploid and poly- tion: C. pratensis L. s.str. (2n = 2x–7x; including C. udi-
ploid closely related taxa distributed throughout most of cola Jord., C. nemorosa Lej., C. latifolia Lej., C. rivu-
Europe, occurring also in northern Africa, northernmost laris auct. non Schur), C. matthioli Moretti (2n = 2x), C.
North America, and Asia (Schulz, 1903; Lövkvist, 1956; majovskii Marhold & Záborský (2n = 4x), C. dentata
Jones & Akeroyd, 1993; Jalas & Suominen, 1994). The Schult. (2n = 7x–12x), C. nymanii Gand. (2n = 8x–10x),
group has been investigated by several authors, especial- and probably also C. granulosa All. (2n = 2x).
ly in northern (Lövkvist, 1956; Dale & Elkington, 1974), Cardamine pratensis s.str. is the most widespread
central (Urbanska-Worytkiewicz & Landolt, 1974; species of the group in Europe (Jalas & Suominen,
Landolt, 1984; Marhold, 1994a, b, 1996) and southeast- 1994), composed of several cytotypes and exhibiting
ern Europe (Marhold, 1994a, b, 1996; Marhold & Anèev, wide morphological variation (Lövkvist, 1956; Marhold,
1999). Various approaches including karyological analy- 1996). Despite its heterogeneity, morphological variation
ses, morphometrics, ecological studies, cultivation and is rather continuous, and taxa sometimes segregated and
crossing experiments have been used in these studies, recognized (e.g., C. pratensis subsp. picra De Langhe &
which helped develop taxonomic concepts for the group. D’ hose, C. pratensis subsp. major Tomšovic, C. udicola,
Most recently, a molecular study on a European- C. nemorosa, C. latifolia) can be hardly distinguished
wide scale was performed using several markers from C. pratensis s.str. It seems that diversification of the
(isozymes, RAPD, cpDNA and ITS sequencing) to eluci- C. pratensis group occurred relatively recently, possibly
date the evolutionary history of the C. pratensis group in postglacial times as proposed by Franzke & Hurka
(Franzke & Hurka, 2000). Two old lineages, most prob- (2000), and that hybridization and polyploidy played a

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Lihová & al. # Cardamine pratensis group 52 # November 2003: 783–801

major role in the evolution of this group.

From the Iberian Peninsula, two taxa belonging to MATERIALS AND METHODS
the C. pratensis group have been reported: C. pratensis Plant material. — Twenty-nine populations of C.
s.str. and C. crassifolia (Jones & Akeroyd, 1993; Rico, pratensis s.str. were sampled for morphometric evalua-
1993; both on the level of subspecies). The former is dis- tion originating from Portugal and several provinces of
tributed in northern and central parts of the Peninsula Spain, to represent wide range of morphological varia-
(Rico, 1993), and so far only two records on chromo- tion. For morphometric analyses, each populational sam-
some numbers, diploid and triploid, both from Portugal, ple consisted of 20–40 individuals; for chromosome
have been reported (Lövkvist, 1956; Queirós, 1973). numbers 2–7 plants per population (27 populations) were
Although Lövkvist (1956) indicated a distinct position of analyzed. Material of C. crassifolia used in morphomet-
the Portuguese diploid, no detailed comparative morpho- rics was represented by eight populational samples from
logical studies with non-Iberian representatives of C. the Eastern Pyrenees, and 13 samples from central
pratensis s.str. have been performed so far. Cardamine Iberian mountains (Sierra Segundera, Sierra de Gredos,
crassifolia, one of the assumed basal taxa of the group Sierra de Albarracín, Sierra de Gúdar; Table 1). As some
(Franzke & Hurka, 2000), is a diploid species described of them were of small population size, fewer individuals
from the Eastern Pyrenees (Pourret, 1788), but reported were sampled (12–35 plants); in the case of material
also from central and western parts of the Pyrenees, and from Sierra de Albarracín, individuals from three geo-
from isolated mountain ranges in central Spain graphically close sites (populations no. 25, 26, 27; alto-
(Lövkvist, 1956; Rico, 1993; Mateo Sanz & al., 1994). It gether 16 plants) were treated together in analyses per-
possesses several morphological traits unique within the formed at the populational level, as one populational
C. pratensis group (creeping rhizome, ascending stem, sample (see morphometric analyses). For each popula-
lack of basal rosette of leaves, presence of stolons) tion of C. crassifolia chromosome numbers were
(Lövkvist, 1956; Rico, 1993), although central Iberian checked in 2–4 individuals (21 populations). Professor E.
representatives and those from the Central Pyrenees have Landolt and Professor K. Urbanska (Zürich, Switzer-
been sometimes regarded as intermediate between C. land) provided us with their unpublished results of 15
crassifolia and C. pratensis s.str. (Rico, 1993). analyses of chromosome numbers from the Iberian
In this paper we present a combined karyological, Peninsula, together with voucher specimens. They are
morphometric and AFLP-fingerprinting (amplified frag- published in this paper in order to complete information
ment length polymorphism) study focusing on the acquired by us (Table 1).
Iberian populations of the C. pratensis group. The AFLP analyses included altogether 114 samples, and
emphasis was put on (1) exploring morphological and the following taxa were represented (Table 1): C. crassi-
karyological variation of Iberian populations of C. folia from the Eastern Pyrenees (7 populations/13 indi-
pratensis s.str., (2) elucidating their relationship to non- viduals) and from central Spain (Sierra de Gredos, Sierra
Iberian populations, i.e., to test their distinction proposed de Albarracín; 6/12), Iberian C. pratensis s.str. (12/31),
by Lövkvist (1956), and (3) on resolving the taxonomic C. pratensis s.str. from France, Italy, Slovakia and
position of central Iberian populations ascribed to C. Slovenia (7/18), C. matthioli (5/10) from Slovenia and
crassifolia. Slovakia, C. majovskii (5/10) from Slovenia, C. granu-
Morphometric analyses have been already success- losa from Piedmont (northern Italy; 2/5), as well as pop-
fully applied in previous studies on Cardamine species ulations of taxonomically uncertain position from central
with complex morphological variation, and enabled Italy, considered close to C. granulosa (4/12). Voucher
delimitation of taxa by using a combination of several specimens of all analyses performed in this study are
morphological characters (Marhold, 1992; Marhold, deposited in SAV; only those of chromosome analyses
1996; Lihová & al., 2000). AFLP-fingeprinting is a done by Urbanska and Landolt (see Table 1) are in ZT.
recently developed (Vos & al., 1995) and already widely For chorological study specimens from the follow-
used molecular method, suitable for studies on popula- ing herbaria were investigated: B, BC, BIO, COI, JACA,
tional and low taxonomic levels (e.g., Hedrén & al., M, MA, MACB, MAF, RNG, SALA, SALAF, SANT, ZT
2001; Zhang & al., 2001; Marhold & al., 2002; Tribsch (see Appendix).
& al., 2002). As the Iberian Peninsula has been consid- Karyological analyses. — Chromosome numbers
ered one of the major European Pleistocene refugia were determined from individuals collected in the field
(Comes & Kadereit, 1998), studies in this area might be and then cultivated at the Institute of Botany, Slovak
significant also from the point of view of the evolution- Academy of Sciences, Bratislava. Squashes were pre-
ary history of the C. pratensis group and related taxa. pared from root tips treated as described in Marhold & al.
(2002), and chromosomes were counted from mitotic
metaphase plates.

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Table 1. Origin of plant material used in karyological (2n), morphometric (Morph) and AFLP analyses. Lu - Portugal, Hs
- Spain, It - Italy, Sk - Slovakia, Sl - Slovenia, Fr - France. Data on chromosome numbers labelled with asterisk are taken
from Lihová & Marhold (2003); all other data represent new records. Those labelled with pluses were counted by K.
Urbanska and E. Landolt, with voucher specimens deposited at ZT. Collectors: EL - E. Landolt, JL - J. Lihová, KM - K.
Marhold, KU - K. Urbanska, MP - M. Perný.
Taxon / locality 2n Morph AFLP
Cardamine crassifolia Pourr.
01 - Hs. Lérida: E Pyrenees, NW of Meranges, Riu Duran, near Refugi J. Folchi Girona, 2320 m, 2 Jul 2001, JL 16 ´ ´
02 - Hs. Lérida: E Pyrenees, N of Meranges, at the lake Estany de Malmiu, 2260 m, 1 Jul 2001, JL 16 ´ ´
03 - Hs. Gerona: E Pyrenees, Núria valley, torrent de Noufonts, 2000 m, 27 Jun 2001, JL 16 ´ ´
04 - Hs. Gerona: E Pyrenees, Núria valley, torrent de Noufonts, 2200-2300 m, 27 Jun 2001, JL 16 ´ ´
05 - Hs. Gerona: E Pyrenees, Núria valley, torrent de Fontalba, 2100 m, 28 Jun 2001, JL 16 ´ ´
06 - Hs. Gerona: E Pyrenees, Núria valley, torrent de Finestreles, 2200 m, 28 Jun 2001, JL 16 ´ ´
07 - Hs. Gerona: E Pyrenees, east of Núria valley, Refugi Manelic, 2000 m, 30 Jun 2001, JL 16 ´ ´
08 - Hs. Gerona: E Pyrenees, between Ribes de Fréser and Camprodon, Rierra d’ Abella, 1580 m, 6 Jun 2000, MP - ´
09 - Fr. 0.5 km SE Porta, 1504 m, 3 Jun 1974, EL & KU 898 16+
10 - Fr. Haute Aude, Vallée de l’ Aude, N of Capcir - La Forge, 1400 m, 3 Jun 1974, EL & KU 893 16+
11 - Fr. Haute Aude, Vallée de l’ Aude, Capcir, near Barrange de Matemols, ca. 1500 m, 3 Jun 1974, EL & KU 894 16+
12 - Fr. Aude, Foret de Barrés, près de la Pla de Barrés, 1700 m, 3 Jun 1974, EL & KU 896 16+
13 - Fr. En aval du Barrage, Les Bouillouse, dépression de la Bouillousette, 2000 m, 3 Jun 1974, EL & KU 16+
Cardamine castellana Lihová & Marhold
14 - Hs. Asturias: Cordillera Cantabrica, near Puerto de Leitariegos, 1470 m, 25 May 2001, MP & al. 16
15 - Hs. Zamora: Sierra Segundera, at the road from San Martin de Castañeda to the lake Laguna de los Peces, NW of
Mt.Gencianal, 1640–1660 m, 24 Jun 2002, KM, P. Bariego Hernández & E. Rico 16 ´
16 - Hs. Zamora: Sierra Segundera, near the lakes Lagunas Herbosas, SE slopes of mountains Moncalvillo and Moncalvo,
1830-1950 m, 29 Jun 2002, KM & P. Bariego Hernández 16 ´
17 - Hs. Zamora: Sierra Segundera, NW of the lake Laguna de los Peces, 1820 m, 25 Jun 2002, KM 16 ´
18 - Hs. Zamora: Sierra Segundera, NW of the lake Laguna de Cubillas (= Laguna de la Yegua), 1820-1880 m, 25 Jun 2002, KM 16 ´
19 - Hs. Zamora: Sierra Segundera, Arroyo del Fuego, 1620–1555 m, 27 Jun 2002, KM 16 ´
20 - Hs. Ávila: Hoyos del Espino, near Hoyos del Collado, 1500 m, 10 May 2000, KM; 10 May 2001, JL 16 ´ ´
21 - Hs. Ávila: Hoyos del Espino, road to Plataforma de Gredos, 1460 m, 10 May 2000, KM; 10 May 2001, JL 16 ´ ´
22 - Hs. Ávila: Hoyocasero, pinar de Hoyocasero, 1260 m, 10 May 2000, KM 16 ´
23 - Hs. Ávila: Santiago del Collado, on the way to Puerto de Peña Negra, 1700 m, 10 May 2000, KM; 10 May 2001, JL - ´ ´
24 - Hs. Ávila: Becedas, Peña Negra, 1800 m, 27 Jun 2002, M. Martínez Ortega MO1546 & Muñoz Centeno 16
25 - Hs. Guadalajara: Sierra de Albarracín, SW of Alustante, Fuente de los Valles, 1500 m, 14 May 2001, JL & G. Nieto Feliner 16 ´ ´
26 - Hs. Guadalajara: Sierra de Albarracín, S of Orea, Hoz Seca, 1500 m, 14 May 2001, JL & G. Nieto Feliner 16 ´ ´
27 - Hs. Teruel: Sierra de Albarracín, W of Noguera, Barranco de la Peña Aguda, 1400 m, 14 May 2001, JL & G. Nieto Feliner 16 ´ ´
28 - Hs. Teruel: Sierra de Gúdar, E of Puerto de Valdelinares, Cuarto del Prado, 1830 m, 19 Jun 2002, JL 16 ´
Cardamine pratensis L. s.str. (Iberian Peninsula)
29 - Lu. Beira Litoral: Fermentelos, near Aveiro, 6 m, 10 Apr 2000, JL & al. 16 ´
30 - Lu. Beira Litoral: Reserva Natural de Paul de Arzila, between Arzila and Pereira, 5 m, 10 Apr 2000, JL & al. 30 ´ ´
31 - Lu. Beira Litoral: between Cabecinhas and Calvão, 32 m, 10 Apr 2000, JL & al. 30 ´
32 - Lu. Beira Litoral: ca. 50 km N of Coimbra, at the road Mira - Vagos, 5 km of Vagos, 24 May 1972, EL 24+
33 - Lu. Beira Litoral: 2 km N of Vagos, 24 May 1972, EL 24+
34 - Hs. A Coruña: Brión, 80 m, 4 Apr 2000, JL & I. Pulgar 30 ´ ´
35 - Hs. A Coruña: Teo, Rariz, Bouñou, 180 m, 4 Apr 2000, JL & S. Ortiz 30 ´
36 - Hs. A Coruña: Portomuoro, river Tambre, 160 m, 6 Apr 2000, JL & I. Pulgar 30 ´ ´
37 - Hs. A Coruña, Ames, Ortoño, near Bertamiráns, 6 Apr 2000, JL & S. Ortiz 30 ´
38 - Hs. A Coruña: O Pino, San Miguel de Cerceda, O Castro, 400 m, 7 Apr 2000, JL & S. Ortiz 30 ´ ´
39 - Hs. Pontevedra: Vila de Cruces, Toiriz, river Arnego, 380 m, 5 Apr 2000, JL & I. Pulgar 30 ´
40 - Hs. León: Manzanal del Puerto, 1100 m, 9 May 2000, KM 30 ´
41 - Hs. Zamora: Pedralba de la Praderia, 980 m, 9 May 2000, KM 48 ´
42 - Hs. Segovia: San Miguel de Bernuy, river Duratón, 820 m, 8 May 2000, KM 56 ´
43 - Hs. Segovia: Prádena, Villar, 1050 m, 8 May 2000, KM 56 ´ ´
44 - Hs. Segovia: Matabuena, between Soria and Segovia (km. 156), 1150 m, 8 May 2000, KM - ´
45 - Hs. Segovia: Sierra de Guadarrama, between La Granja and Peñalara, 1100-1600 m, 13 May 2001, JL 56 ´
46 - Hs. Madrid: Sierra Guadarrama, El Paular, Monasterio de El Paular, 1160 m, 12 May 2001, JL 56 ´ ´
47 - Hs. Cantabria: Luena, Sel de la Carrera, N of Puerto del Escudo, at Emb. del Ebro, 500 m, 7 May 2001, JL & M. Herrera 56 ´ ´
48 - Hs. Cantabria: Pto. de la Magdalena, ca. 200 m from San Pedro del Romeral, 780 m, 7 May 2001, JL & M. Herrera 16 ´ ´
49 - Hs. Cantabria: between Campillo and S. Roque de Riomiera, 750 m, 7 May 2001, JL & M. Herrera 32 ´
50 - Hs. Cantabria: Soba, from Revilla to Puerto de Los Tornos, 700 m, 21 May 2001, MP & M. Herrera 32 ´
51 - Hs. Álava: Urcabustaiz, Beluntza, 640 m, 6 May 2001, JL & I. Biurun - ´
52 - Hs. Álava: Villarreal de Álava, Elosu, 560 m, 6 May 2001, JL & I. Biurun 48 ´

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Table 1. (continued).
Taxon / locality 2n Morph AFLP
53 - Hs. Guipuzcoa: Anzola, Puerto Descarga, 430 m, 6 May 2001, JL & I. Biurun 46 ´
54 - Hs. Guipuzcoa: Régil, between Régil and Pto. de Bidania, 480 m, 6 May 2001, JL & I. Biurun 48 ´
55 - Hs. Guipuzcoa: Segura, Otzaurte, 600 m, 22 May 2001, MP & I. Biurun - ´
56 - Hs. Guipuzcoa: 8 km SE of Irún, Erlaitz, 497 m, 20 May 2001, MP 32 ´
57 - Hs. Navarra: Larraín, Sierra de Aralar, 1 km of Baraibar, 600 m, 6 May 2001, JL & I. Biurun 46 ´ ´
58 - Hs. Navarra: Yerri, Sierra de Urbasa, Raso de Bidoiza, S of Pto. de Urbasa, 900 m, 6 May 2001, JL & I. Biurun 48 ´ ´
59 - Hs. Navarra: Quinto Real, 2200 m S of Irurita, 700 m, 8 May 2001, JL & I. Biurun 32 ´ ´
60 - Hs. Navarra: 500 m S of Venta Quemada, Puerta de Velote, 840 m, 5 Apr 1974, EL 911 40+
61 - Hs. Huesca: Central Pyrenees, Alta Ribagorza, NW of Aneto, between Aneto and Refugi de Llauset, 1700 m, 23 Jun 2002, JL 16 ´
62 - Hs. Huesca: Valle de Aran, Casau, W of Viella, 5 Jun 1974, EL & KU 903 16+
63 - Fr. Pyrenees, 1 km E of Gourette, Col d’Aubisque, ca. 1450 m, 5 Jun 1974, EL & KU 16+
64 - Fr. Hautes Pyrenees, 4 km N of Col de Peyresourde, 5 Jun 1974, EL & KU 904 16+
65 - Fr. Pyrenees, aux bonnes Crèpes, ca. 3 km E Col d’Aspin, ca. 1400 m, 5 Jun 1974, EL & KU 16+
66 - Fr. Pyrenees, road to Col du Souloz (7 km E of the pass), 5 Jun 1974, EL & KU 16, 32+
67 - Fr. 1.5 km E of La Mongie, at the road to Col du Tourmalet, ca. 1700 m, 5 Jun 1974, EL & KU 32+
68 - Fr. 8 km N of Col des Pourtalet, ca. 1250 m, 5 Jun 1974, EL & KU 910 40+
Cardamine pratensis L. s.str. (non-Iberian)
69 - Sl. Alpsko obmoèje: between Zg. Stranje and Godiè, at the Kamniška Bistrica stream, 430 m, 8 Apr 2001, JL & T. Baèiè 30* ´ ´
70 - Sl. Predalpsko obmoèje: Radomlje, 330 m, 18 Apr 2001, JL & T. Baèiè 30* ´ ´
71 - Sl. Dinarsko obmocje: Notranjsko, Cerkniško jezero, near Otok, 560 m, 17 Apr 2001, JL & B. Trèak 44* ´ ´
72 - Fr. Grenoble, Arboretum de l’ Université J. Fourier, at the river Isère, 5 Apr 2001, P. Mráz 30 ´
73 - Fr. Dept. Isère, Massif de la Chartreuse, St. Piere-Chartreuse, 1 Apr 2001, P. Mráz 30 ´
74 - It. Piedmont: Torino, Stupinigi, 250 m, 25 Apr 2001, JL & al. 30 ´
75 - Sk. Pova ský Inovec Mts., S of Trencianske Jastrabie, Patrovec, 320-340 m, 6 Oct 2001, MP 44 ´
76 - Sk. Slovenské rudohorie Mts., Mt. Kojšovská hola, 1160 m, 1 Aug 2000, JL 30 ´
Cardamine matthioli Moretti
77 - Sl. Subpanonsko obmoèje: Štajerska, between Ptuj and Placar, 240 m, 20 Apr 2001, JL & B. Frajman 16* ´
78 - Sl. Subpanonsko obmoèje: Trnovska vas, 250 m, 20 Apr 2001, JL & B. Frajman 16* ´
79 - Sl. Predalpsko obmoèje: vicinity of Celje, Šmartinsko jezero lake, 260 m, 20 Apr 2001, JL & B. Frajman 16* ´
80 - Sl. Subpanonsko obmoèje: vicinity of Slovenske Konjice, between Tepanje and Pobre , 280 m, 20 Apr 2001, JL & B. Frajman 16* ´
81 - Sk. Pova ský Inovec Mts., S of Trencianske Jastrabie, Patrovec, 320–340 m, 6 Oct 2001, MP - ´
Cardamine majovskii Marhold & Záborský
82 - Sl. Predalpsko obmoèje: Ljubljansko Barje, Blatna Brezovica, 300 m, 17 Apr 2001, JL & B. Trèak 32* ´
83 - Sl. Predalpsko obmoèje: vicinity of Ljubljana, Krumperk, 320 m, 18 Apr 2001, JL & T. Baèiè 32* ´
84 - Sl. Predalpsko obmoèje: vicinity of Ljubljana, Tacen, 320 m, 18 Apr 2001, JL & T. Baèiè 32* ´
85 - Sl. Predalpsko obmoèje: vicinity of Ljubljana, Topole near Mengeš, 340 m, 18 Apr 2001, JL & T. Baèiè 32* ´
86 - Sl. Dinarsko obmoèje: Sodra ica, 540 m, 17 Apr 2001, JL & B. Trèak 32* ´
Cardamine granulosa All.
87 - It. Piedmont: Giaveno, Pomeri, 17 May 2002, JL & al. ´
88 - It. Piedmont: San Francesco su Avigliana, 620 m, 17 May 2002, JL & al. ´
Cardamine pratensis s.l., central Italy
89 - It. Toscana: Stáffoli, 8 m, 26 Apr 2001, JL & al. ´
90 - It. Abruzzo: Gran Sasso, Sorgenti del Vomano, Passo delle Capannelle, 1257 m, 27 Apr 2001, JL & al. ´
91 - It. Abruzzo: Lago di Campotosto, Le Serre, 1340 m, 27 Apr 2001, JL & al. ´
92 - It. Abruzzo: Gran Sasso, Voltigno, under Mt. Fiore, 1360 m, 27 Apr 2001, JL & al. ´

Morphometric analyses. — Morphological (1) Iberian representatives of the C. pratensis group.

characters were measured on herbarium specimens col- The following ten quantitative characters were meas-
lected from the field, and the analyses were performed in ured: length of petals, width of petals, length of sepals,
four steps: (1) the whole Iberian material of the C. length of longer and shorter filaments, number of stem
pratensis group was evaluated in order to explore overall leaves, number of segments on the stem leaf nearest the
patterns of variation, (2) samples of C. crassifolia from midpoint of the leafy part of stem, number of segments
the Eastern Pyrenees and central Iberian mountains were on the middle stem leaf, number of lateral inflorescences
analyzed comparatively in more detail, (3) different cyto- (longer than 1 cm), and number of leaflets of basal
types found within Iberian C. pratensis s.str. were com- leaves. Floral organs were attached to adhesive tape
pared to examine the extent of their morphological dif- when fresh, and size characters were measured on them
ferentiation, and (4) Iberian populations of C. pratensis after drying. In addition, six ratios were computed (based
s.str. were examined relative to those from central on measurement on the stem leaf nearest the midpoint of
Europe. the leafy part of stem): length of stem up to the lowest

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peduncle of flower or fruit/length of stem leaf most discriminating characters are presented in the form
(HP1/LL2), length of stem leaf/number of leaf segments of box-plots. Besides the quantitative characters meas-
(LL2/NS2), length of terminal leaf segment/width of ter- ured, also qualitative ones were observed and recorded
minal leaf segment (LTS/WTS), length of lateral leaf for each specimen. These included characters on rhizome
segment/width of lateral leaf segment (LLS/WLS), (thickness, growth orientation) and on basal parts of stem
length of terminal leaf segment/length of lateral leaf seg- (ascending vs. erect stem, presence of stolons, presence
ment (LTS/LLS), and length of stem leaf/length of ter- or absence of basal rosette), as the following character
minal leaf segment (LL2/LTS). As the size of vegetative combination, thin, horizontally creeping rhizome,
organs is largely influenced by environmental factors ascending stem and lack of rosette, has been considered
(see e.g., Lövkvist, 1956; Dale & Elkington, 1974), these diagnostic for typical C. crassifolia from the Pyrenees
characters were used only for computing ratios. The (Lövkvist, 1956; Rico, 1993).
characters evaluated in this study were selected follow- (3) Iberian C. pratensis s.str. CDA on both individu-
ing our previous studies on Cardamine species (Marhold, als and populations as OTUs and non-parametric classi-
1996; Lihová & Marhold, 2003) and initial field obser- ficatory discriminant analysis were performed, with var-
vations, aiming to include the characters that might have ious cytotypes revealed (see Results) as groups. The aim
discriminatory value. of these analyses was to explore whether particular cyto-
Cluster analysis (UPGMA, unweighted pair-group types can be distinguished morphologically. The charac-
method using arithmetic averages; Everitt, 1986) and ters evaluated were those included in (1), plus the num-
principal component analysis based on a correlation ber of segments on the third stem leaf (numbered from
matrix (PCA; Sneath & Sokal, 1973; Krzanowski, 1990) the stem base up), as this character was used in previous
were performed using populations characterized by mean studies on the C. pratensis group (Marhold, 1996;
values as OTUs (operational taxonomic units, i.e., Lihová & Marhold, 2003). Because basal leaves are
objects). Prior to clustering, data were standardized by sometimes lacking on plants at flowering stage, the char-
zero mean and unit standard deviation, and Euclidean acter number of leaflets of basal leaves had to be omitted
distance was used for computing pairwise similarities from analyses performed at the individual level, but was
between OTUs. included when evaluating populations. The values of
(2) Cardamine crassifolia. PCA, and subsequently measured characters (mean, interquartile range and
canonical discriminant analysis (CDA) and non-paramet- 10/90 percentiles) scored for different cytotypes were
ric classificatory discriminant analysis (Klecka, 1980) compared to obtain detailed insights into their variation.
were performed on individuals as OTUs and populations (4) Iberian vs. central European C. pratensis s.str.
of C. crassifolia from two main distributional areas, the For their morphological comparison, all quantitative
Eastern Pyrenees and central Iberian mountains, as characters (qualitative differences were not observed)
groups. The same character set was used as in part (1). used in previous studies on the C. pratensis group from
PCA was performed to reduce the overall variation in 16 the Carpathians and Pannonia (Marhold, 1996) and from
examined characters into three uncorrelated variables Slovenia (Lihová & Marhold, 2003) were included: size
(components), and to depict morphological relationships of flowers [for exact list of floral characters see (1)],
among individual specimens. CDA, which by weighting number of leaves, number of segments on the third stem
characters maximizes differences between groups, was leaf, and number of lateral inflorescences. Central
conducted to reveal the extent of morphological separa- European populations were represented by four ploidy
tion suggested by PCA and analyses in part (1). To levels: 2n = 30, 38, 44 and two diploid (2n = 16) mor-
determine characters mostly contributing to that separa- phological types called C. rivularis auct., and “ucranica
tion, total canonical structure expressing correlation of type” [for details and origin of samples see Marhold
the characters with the canonical axis was computed. In (1996) and Lihová & Marhold (2003)]. PCA and
the classificatory discriminant analysis, which assesses UPGMA cluster analysis were performed using 29
the percentage of OTUs classified correctly into predict- Iberian and 45 central European populations as OTUs.
ed groups, discriminant function was determined by the All morphometric analyses were done by using the
cross-validation procedure using k-nearest-neighbours (k SAS 8.2 package (SAS Institute, 2000), except for clus-
= 11). In this procedure, classification criterion is based ter analyses which were run using SYN-TAX 2000
on n-1 individuals (n = total number of individuals) and (Podani, 2001).
then applied to classify the individual left out. AFLP analyses. — Total DNA was extracted from
Descriptive statistical parameters of the measured char- silica gel-dried leaves following the CTAB extraction
acters (mean, median, standard deviation and per- protocol by Doyle & Doyle (1987) with minor modifica-
centiles) for populations from the Pyrenees and central tions. The AFLP procedure (Vos & al., 1995) was the
Iberian mountains were also computed, and those of same as described in detail in Schönswetter & al. (in

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Lihová & al. # Cardamine pratensis group 52 # November 2003: 783–801

press). DNA restriction with the endonucleases EcoRI

France
and MseI and ligation to double stranded adapters were
performed in one step. After that, preselective amplifica-
tion with primers with a one selective base extension fol-
lowed. On a basis of a primer test with 15 different
primer combinations, three primer pairs were chosen for pratensis
pratensis
2n = 16
2n = 24
selective amplification: EcoRI -AAG-(HEX), MseI - pratensis
pratensis
2n = 30
2n = 32
CTG; EcoRI -ATC-(6-FAM), MseI -CAG; EcoRI -AGC- Portugal
pratensis
pratensis
2n = 40
2n = 46
Spain
(NED), MseI -CTG. The amplified AFLP fragments were pratensis
pratensis
2n = 48
2n = 56
electrophoresed and detected in an ABI Prism 377 crassifolia
castellana
2n = 16
2n = 16
sequencer, and then analyzed with GeneScan® software
(PE Applied Biosystems). Presence or absence of frag- 0 100 200 300 400 km

ments ranging from 70 to 500 bp were scored for each

sample (only well scorable, unambiguous fragments
were analyzed) and transferred into a binary matrix using Fig. 1. Map showing distribution of different cytotypes of
GenoGrapher (version 1.6.0, © Montana State Univer- C. pratensis s.str. in the Iberian Peninsula, the investiga-
ted populations of C. crassifolia in the Eastern Pyrenees,
sity 1999; http://hordeum.msu.montana.edu/genograph- and C. castellana from the central Iberian mountains.
er/). Distribution of AFLP fragments across the studied
taxa was extracted from the binary data matrix. For each
taxon (or geographically isolated group of populations), 2n = 16 (Table 1). For C. pratensis s.str. remarkable kary-
the total number of AFLP fragments, mean number of ological variation was revealed (Table 1, Fig. 1). Diploid
fragments per individual, number of exclusive (present in to heptaploid populations, including also dysploid ones,
a given taxon only, but not necessarily in all its samples) were found across its distribution in the Iberian
and diagnostic fragments (present in all samples of a Peninsula. Diploids (2n =16) were recorded from Beira
taxon and absent from all other taxa) were calculated. Litoral region in Portugal, from the Basque country (cen-
Principal coordinate analysis (PCoA) based on pairwise tral part of northern Spain), and from the Central
similarities computed with Jaccard’s coefficient was per- Pyrenees. Two cytotypes at the tetraploid level, namely
formed using SYN-TAX 2000 (Podani, 2001). 2n = 30 and 2n = 32 were observed, the former one con-
Neighbour-joining tree using Nei & Li (1979) genetic centrated in Galicia (NW Spain) and Portugal (but found
distance was generated by the TREECON program (ver- also on the northern slopes of the Eastern Pyrenees), the
sion 1.3b; Van de Peer & De Wachter, 1994) with boot- latter in the Basque country and Central Pyrenees. In a
strap option (2,000 replications). In addition, analysis of geografically small area in Beira Litoral one diploid (2n
molecular variance (AMOVA; Arlequin, version 2.000, = 16) and one hypotetraploid (2n = 30) population was
Schneider & al., 2000) was calculated from a matrix of found by us, in addition to two triploid (2n = 24) counts
squared Euclidean distances to estimate population dif- recorded by Urbanska and Landolt (Table 1). Two penta-
ferentiation in C. pratensis s.str. and C. crassifolia ploid populations with 2n = 40 were recorded from the
(including populations from the Eastern Pyrenees and Western and Central Pyrenees. At the hexaploid level,
those from central Iberian mountains). Total genetic vari- two cytotypes with 2n = 46 and 48 were found. Popu-
ance was partitioned into levels of individuals within lations with 2n = 56 were determined from central (prov.
populations, among populations, and among groups of Madrid, Segovia) and northern Spain (prov. Cantabria).
populations. Various groupings of populations (with two Morphometric analyses. — (1) Iberian represen-
to nine groups), either based on geographic regions or tatives of the C. pratensis group. Both the PCA (Fig. 2)
ploidy level were tested. Population sample no. 44 (see and cluster analysis (figure not shown) performed on the
Table 1) of unknown ploidy level was omitted from the whole Iberian material gave similar results. Two main
AMOVAs. In all cases, 10,000 permutations were run to groups, corresponding to C. pratensis s.str. and C. cras-
obtain F-statistics. sifolia as previously classified, were resolved, clearly
separated along the first axis in the ordination graph.
Only one population of C. pratensis s.str. from the
Central Pyrenees (population no. 61, see Table 1) was
RESULTS unexpectedly placed among samples of C. crassifolia.
Karyological analyses. — All populations previ- All floral and leaf characters (excluding ratios) con-
ously classified as C. crassifolia from the Pyrenees and tributed almost equally to division along the first axis, as
central Iberian mountains were found to be diploid with seen from the eigenvector values (Table 2). Cardamine

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Table 2. Eigenvectors expressing correlation of charac- Table 3. Morphometric analyses of C. crassifolia from the
ters with principal components (axis 1, axis 2, axis 3) in Eastern Pyrenees and C. castellana from central Iberian
morphometric analysis of Iberian populations of the C. mountains. PCA - eigenvectors showing correlation of
pratensis group (C. pratensis s.str., C. crassifolia from characters with principal components (axis 1, axis 2);
the Eastern Pyrenees and C. castellana from central CDA - total canonical structure expressing correlation of
Iberian mountains). characters with the canonical axis.

Character Axis 1 Axis 2 Axis 3 PCA

length of petals 0.314 -0.226 0.014 Character Axis 1 Axis 2 CDA
width of petals 0.304 -0.221 0.451 length of petals 0.458 0.121 0.634
length of sepals 0.313 -0.157 0.020 width of petals 0.438 0.038 0.936
length of longer filaments 0.275 -0.309 0.015 length of sepals 0.388 0.124 0.487
length of shorter filaments 0.274 -0.299 0.050 length of longer filaments 0.442 0.034 0.702
number of stem leaves 0.266 0.265 0.064 length of shorter filaments 0.333 0.110 0.251
number of segments on the stem leaf number of stem leaves -0.116 0.295 -0.375
nearest the midpoint of stem 0.317 0.185 0.080 number of segments on the stem
number of segments on the middle leaf nearest the midpoint of stem -0.161 0.313 -0.241
stem leaf 0.327 0.110 0.093 number of segments on the middle
number of lateral inflorescences 0.175 0.177 0.160 stem leaf -0.109 0.404 -0.140
number of leaflets of basal leaves 0.304 0.224 0.008 number of lateral inflorescences -0.011 0.142 0.032
HP1/LL2 0.163 0.351 0.073 number of leaflets of basal leaves -0.226 0.261 -0.435
LL2/NS2 -0.017 -0.463 0.013 HP1/LL2 0.111 0.085 0.297
LTS/WTS 0.090 0.167 0.666 LL2/NS2 0.024 -0.265 -0.165
LLS/WLS 0.088 -0.175 0.634 LTS/WTS -0.038 0.433 -0.127
LTS/LLS -0.189 0.210 0.269 LLS/WLS -0.006 0.435 -0.092
LL2/LTS 0.284 0.227 -0.161 LTS/LLS -0.111 -0.032 -0.204
LL2/LTS -0.118 -0.252 -0.240
pratensis s.str. has larger flowers, and higher number of
leaves, leaf segments and leaflets on basal leaves when Pyrenees and central Iberian mountains, respectively,
compared to C. crassifolia. Populations of C. crassifolia were revealed, although with many specimens in over-
were placed in two partially overlapping groups, refer- lapping positions (Fig. 3). Floral characters showed the
ring to the samples from the Eastern Pyrenees on one highest correlation with the first axis (Table 3, PCA). In
side, and those from central Iberian mountains on anoth- CDA distinct separation between individuals from these
er. two areas was achieved (Fig. 4), based on floral charac-
(2) Cardamine crassifolia from the Eastern Pyrenees ters, as indicated by PCA, and on the number of leaflets
and central Iberian mountains. In PCA of individual of basal leaves (see total canonical structure in Table 3,
plants, two groups corresponding to the samples from the CDA). As can be seen also from the mean values and
interquartile ranges of these characters (Fig. 5), Pyrenean
plants have generally larger petals, longer filaments, and
axis 3
lower number of leaflets of basal leaves. Classificatory
2 . 91 discriminant analysis also showed good extent of separa-
tion, as 96–97% of the specimens were correctly classi-
fied. Most of the misclassified plants came from the pop-
1 . 06
ulation in Sierra de Gúdar (population no. 28; Table 1),
due to slightly longer filaments, fewer stem leaves and
- 0. 79
leaflets of basal leaves possessed by these individuals.
3. 17
Except for the differences in quantitative characters
0 . 84 recovered between Pyrenean and central Iberian popula-
- 2. 64
4. 73 - 1. 48
axis 2 tions, we observed also pronounced qualitative differ-
1 . 70

axis 1 - 1. 34
ences. While plants from the Pyrenees have an exclu-
- 4. 37 - 3. 81
sively thin, horizontally creeping rhizome, ascending
Fig. 2. Principal component analysis of Iberian popula- stem, and basal leaves not forming a rosette, those from
tions of the C. pratensis group based on 16 morphologi- central Iberian mountains differ in this respect. Both
cal characters (see Table 2). Circles, C. pratensis s.str.; plants with horizontal and erect rhizome (or intermediate
crosses, C. crassifolia from the Eastern Pyrenees; cubes, one) can be found, often at least the upper part of the rhi-
C. castellana from central Iberian mountains. First three
axes explain 51.78%, 18.28% and 10.30% of total varia-
zome is thickened (and sometimes bearing multiple
tion. flowering stems), basal leaves either form or do not form

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Lihová & al. # Cardamine pratensis group 52 # November 2003: 783–801

Table 4. Total canonical structure expressing correlation Table 5. Distribution of AFLP fragments generated by
of morphological characters with canonical axes in dis- three primer combinations across the investigated taxa
criminant analysis of Iberian C. pratensis s.str. on the of the C. pratensis group. N IND = number of analyzed in-
populational level. Groups defined as different cytotypes. dividuals; NTF = total number of fragments; N AF = aver-
Character Axis 1 Axis 2 Axis 3 age number of fragments per individual (± standard devi-
ation); N EF = number of fragments present exclusively in
length of petals -0.343 0.114 0.489 the taxon (but not necessarily present in all its samples);
width of petals 0.417 0.215 0.272 N DF = number of fragments diagnostic for the taxon (i.e.,
length of sepals -0.022 0.421 0.427 fragments restricted to the taxon, present in all samples).
length of longer filaments 0.280 -0.017 0.452
Taxon NIND NTF NAF NEF NDF
length of shorter filaments -0.343 0.065 0.431
number of stem leaves 0.513 0.151 -0.186 C. pratensis s.str. 52 132 46 (±4.40) 39 -
number of segments on the third C. pratensis s.str., Iberian 31 106 46 (±4.79) 16 -
stem leaf 0.603 0.271 -0.026 C. pratensis s.str.,
number of segments on the stem leaf non-Iberian 21 107 46 (±3.88) 12 -
nearest the midpoint of stem 0.638 0.337 0.041 C. crassifolia 13 64 47 (±3.67) 14 -
number of segments on the middle C. castellana 12 63 44 (±3.01) 15 2
stem leaf 0.551 0.263 0.081 C. castellana
number of lateral inflorescences 0.230 -0.173 0.141 (Sierra de Gredos) 6 46 42 (±1.52) 7 3
number of leaflets of basal leaves 0.689 0.117 0.127 C. castellana
HP1/LL2 0.561 0.218 -0.337 (Sierra de Albarracín) 6 51 47 (±0.75) 6 5
LL2/NS2 -0.6645 -0.030 0.172 C. matthioli 10 59 42 (±1.85) 6 -
LTS/WTS 0.334 -0.254 0.314 C. majovskii 10 64 44 (±2.45) 4 -
LLS/WLS -0.254 -0.269 0.406 C. granulosa 5 58 45 (±3.96) 6 1
LTS/LLS 0.553 -0.440 0.106 C. pratensis s.l.
LL2/LTS 0.677 0.264 -0.056 (central Italy) 12 65 45 (±3.94) 15 4

a true basal rosette, and both ascending and erect stems leaves and ratios LL2/NS2, LL2/LTS; Table 4) on one
can be observed (Fig. 6). We found very few plants (12 side, and remaining populations on another, with diploids
out of 268 examined) having the same combination of in an intermediate position (Fig. 7). Heptaploid, hypo-
these characters, as is typical for plants from the tetraploid and tetraploid populations were separated from
Pyrenees. each other along the second axis, most correlated with
(3) Iberian C. pratensis s.str. In CDA performed on the length of sepals and ratio LTS/LLS. However, when
populations as OTUs and six cytotypes as groups, popu- CDA was performed on individual plants, different cyto-
lations with 2n = 48 and 46 were separated along the first types were found largely intermingled in one dense
canonical axis (most strongly correlated with the number group, showing only slight shifts along the axes (figure
of segments on stem leaves, number of leaflets of basal not shown). Similarly, in the classificatory discriminant

6 110

5
100

4
90
3

80
2
FREQU ENC Y

1 70
Axis 2

0
60

-1
50
-2

40
-3

-4 30

-5
20

-6
10
-4 -3 -2 -1 0 1 2 3 4 5 6

Axis 1 0

- 3. 6 - 2. 8 - 2. 0 - 1. 2 - 0. 4 0. 4 1. 2 2. 0 2. 8 3. 6 4. 4 5. 2 6. 0 6. 8

Fig. 3. Principal component analysis of individuals of C. Axis 1

crassifolia (crosses) from the Eastern Pyrenees and of C. Fig. 4. Histogram of canonical discriminant analysis per-
castellana (squares) from central Iberian mountains formed on individuals of C. crassifolia (hatched) from the
based on 16 morphological characters (see Table 3, Eastern Pyrenees and C. castellana (white) from central
PCA). First two axes explain 24.31% and 14.43% of total Iberian mountains based on 16 morphological characters
variation. (see Table 3, CDA).

790
 52 # November 2003: 783–801 Lihová & al. # Cardamine pratensis group

A B C D E
10 6 9 12
length of longer filaments (mm)

6 8
9 10
5 7

width of petals (mm)

length of petals (mm)

number of leaflets
5 8

number of leaves
6 8
7 4 5
4 6
6 4
3 3 4
5
3 2
4 2
2 1
2 3 0
castellana crassifolia castellana crassifolia castellana crassifolia castellana crassifolia castellana crassifolia

Fig. 5. Variation in selected morphological characters of C. crassifolia and C. castellana. Rectangles define 25 and 75
percentiles; horizontal lines show median; whiskers are from 10 to 90 percentiles; asterisks show extreme values. A,
max. length of longer filaments (mm); B, length of petals (mm); C, width of petals (mm); D, number of stem leaves; E,
number of leaflets of basal leaves.

analysis, the percentages of plants correctly classified (4) Iberian vs. central European C. pratensis s.str. In
into predicted groups (= cytotypes) were low, ranging the cluster analysis of Iberian and central European pop-
from 39.62% for plants with 2n = 32, to 87.76% for those ulations of C. pratensis s.str., Iberian populations did not
with 2n = 46. The latter cytotype, however, was repre- group together, but were found scattered in all three main
sented by only two populations, and thus the value can be clusters (Fig. 9). The population from the Central
somewhat biased. When comparing the values (mean, Pyrenees (no. 61, see Table 1), which showed a some-
median, percentiles) of the measured characters, increase what isolated position relative to other Iberian popula-
in length of petals and length of filaments can be tions, clustered here together with central European pop-
observed from diploid to heptaploid levels (Fig. 8). In ulations of 2n = 30 and 38, and the diploid C. rivularis
addition, hypotetraploids shifted with respect to lower auct. type. Similarly, in PCA no separation between
number of leaves and segments on leaves, and diploids Iberian and non-Iberian populations was seen (figure not
had the shortest sepals. Still, for all characters examined, shown). Also in the qualitative characters colour of petals
large overlap among cytotypes was evident (Fig. 8). and indumentum of rachis of basal leaves, no differences

A B

a b c d

2 cm
Fig. 6. Morphology of rhizome and basal parts of stem in the studied Iberian taxa. A, C. crassifolia (Eastern Pyrenees);
B, C. pratensis s.str.; C, C. castellana (central Iberian mountains), a–d show variation range in this species.

791
Lihová & al. # Cardamine pratensis group 52 # November 2003: 783–801

Table 6. Analysis of molecular variance (AMOVA) of AFLP phenotypes in C. pratensis s.str. (a) Partitioning of the over-
all variation into two levels (within and among populations), and (b)–(f) into three levels with different groupings; (b)
nine geographic regions: Madrid & Segovia provinces (central Spain), A Coruña province (NW Spain), Cantabria
province (N Spain), Navarra province (N Spain), central Portugal, SE France, Piedmont (NW Italy), Slovakia, Slovenia;
(c) cytotypes 2n = 16, 30, 32, 44, 46, 48, 56; (d) Iberian populations only, five geographic regions: Madrid & Segovia
provinces (central Spain), A Coruña province (NW Spain), Cantabria province (N Spain), Navarra province (N Spain),
central Portugal; (e) Iberian cytotypes only 2n = 16, 30, 32, 46, 48, 56; (f) Iberian and non-Iberian populations.
Population numbers as given in Table 1 are in brackets. d.f. = degrees of freedom; *** = P<0.001; ** = P<0.05.
Sum of Variance % of total
Grouping [population number] Source of variation d.f. squares components variance
(a) [30,34,36,38,43,46–48,57–59,69–76] among populations 18 432.43 7.20 56.65***
within populations 30 165.17 5.51 43.35***
(b) [43,46],[34,36,38],[47,48],[57–59],30,[72,73], among groups 08 274.64 3.39 26.14***
74,[75,76],[69–71] among populations 10 157.78 4.06 31.35***
within populations 30 165.17 5.51 42.51***
(c) 48,[30,34,36,38,69,70,72,73,74,76],59,[71,75], among groups 06 173.68 1.01 07.79**
57,58,[43,46,47] among populations 12 258.75 6.44 49.73***
within populations 30 165.17 5.51 42.48***
(d) [43,46],[34,36,38],[47,48],[57–59],30, among groups 04 134.05 3.64 30.55***
among populations 06 082.71 3.57 29.91***
within populations 17 080.17 4.72 39.54***
(e) 48,[30,34,36,38],59,57,58,[43,46,47] among groups 05 150.92 3.70 30.98***
among populations 05 065.85 3.52 29.51***
within populations 17 080.17 4.72 39.51***
(f) [30,34,36,38,43,46–48,57–59],[69–76] among groups 01 063.04 1.69 12.51***
among populations 17 369.38 6.32 46.75***
within populations 30 165.17 5.51 40.73***

Table 7. Analysis of molecular variance (AMOVA) of AFLP phenotypes in C. crassifolia and C. castellana. (a) Partitioning
of the overall variation into two levels (within and among populations), and (b)–(d) into three levels with different
groupings; (b) C. crassifolia (Eastern Pyrenees), C. castellana (Sierra de Gredos, Sierra de Albarracín); (c) C. crassifo-
lia Núria region, C. crassifolia Meranges region, C. castellana Sierra de Gredos, C. castellana Sierra de Albarracín; (d)
C. castellana Sierra de Gredos, C. castellana Sierra de Albarracín. Population numbers as given in Table 1 are in brack-
ets. d.f. = degrees of freedom; *** = P<0.001; ** = P<0.05; n.s. = not significant.
Sum of Variance % of total
Grouping [population number] Source of variation d.f. squares components variance
(a) [01–07,20,21,23,25–27] among populations 11 311.56 12.69 86.84***
within populations 13 25.00 01.92 13.16***
(b) [01–07],[20,21,23, 25–27] among groups 01 174.14 12.84 62.77**
among populations 10 137.42 05.69 27.83***
within populations 13 025.00 01.92 09.40***
(c) [03–07],[01,02],[20,21,23],[25–27] among groups 03 263.28 13.45 77.53***
among populations 08 048.28 01.97 11.38***
within populations 13 025.00 01.92 11.09***
(d) [20,21,23],[25–27] among groups 01 070.17 10.81 77.34n.s.
among populations 04 021.33 02.17 15.51**
within populations 06 006.00 01.00 07.16***

were found, as Iberian populations have reddish-violet Pontevedra, Guipuzcoa and Segovia, and in diploid to
flowers and rachis glabrous (mostly) or with patent hairs, heptaploid populations.
as is typical for C. pratensis s.str. Although some Iberian AFLP analyses. — In 114 individuals of the C.
populations had strongly thickened tuberous rhizomes, pratensis group investigated, 219 scorable fragments
not observed among central European populations, this were amplified by three primer combinations. Nine frag-
feature was neither correlated with any other morpholog- ments were monomorphic, and 19 fragments were
ical character, nor geographically, since such plants were restricted to single samples. The number of fragments
found in Portugal, in Spanish provinces A Coruña, amplified per taxon (or group of populations) varied

792
 52 # November 2003: 783–801 Lihová & al. # Cardamine pratensis group

Table 8. Morphological characters distinguishing C. pratensis s.str., C. crassifolia (Eastern Pyrenees) and the newly
described C. castellana from central Iberian mountains. For quantitative characters, mean values with 5 and 95 per-
centiles in parentheses are given.
Character C. crassifolia C. castellana C. pratensis s.str.
Habitus always single stem (never sometimes with multiple stems sometimes with multiple stems
caespitose), often forming (caespitose), but not forming (caespitose), but not forming
cushions cushions cushions
Rhizome
length and width elongated, always equally thin elongated or shortened, mostly shortened, strongly thickened
thickened at least in the upper
part, very rarely entirely thin
growth orientation always horizontally creeping erect, oblique, rarely horizontal always erect, never creeping

branching usually branched along its length simple, if branched then in upper simple, if branched then
part only in upper part only
Stem
growth orientation always ascending ascending to erect always erect
Basal rosette absent (short internodes between present or absent (short internodes always present
basal leaves always retained) between basal leaves sometimes
retained)
Number of stem leaves (2–)3(–4) (2–)4(–6) (2–)5(–10)
Number of pairs of 1–2 1–4 2–9(–10)
leaflets on basal leaves
Petals
length (mm) (5.9–)8.1(–10.4) (5.0–)6.5(–8.3) (9.4–)13.3(–16.7)
width (mm) (3.5–)4.7(–5.9) (2.2–)2.9(–3.6) (5.9–)7.9(–9.7)
shape obovate to broadly obovate narrowly obovate obovate to broadly obovate

from 46 in populations from Sierra de Gredos (central found as well (Table 5). When comparing the average
Spain) to 132 in C. pratensis s.str. Except for C. praten- number of fragments amplified per individual in differ-
sis s.str. with the largest amount of samples analyzed, the ent taxa and cytotypes, only small differences were
highest numbers of exclusive fragments were recorded in found. With the exception of samples of Iberian diploid
C. crassifolia (14 fragments), central Iberian populations C. pratensis s.str. (no. 61), which showed only 36 (± 2.1)
(15), and in taxonomically uncertain populations from fragments per genotype, in all other samples, including
central Italy (15). In the latter two, and also C. granulosa diploid C. crassifolia and C. granulosa, 42–51 fragments
(Piedmont, northern Italy), diagnostic fragments were were scored. The number of fragments in C. pratensis
s.str. was correlated with ploidy level (Spearman correla-
axis 3
tion coefficient rs = 0.764, P = 0.05). However, the high-
est value was not found in the samples of 2n = 56, but in
4. 21
those of 2n = 44 (51 ± 1.9 fragments).
In PCoA based on Jaccard’s coefficient, several
0. 80
groups of related AFLP phenotypes can be recognized
(Fig. 10). Samples of C. pratensis s.str. both from the
Iberian Peninsula and other parts of Europe formed one
- 2. 61 dense group, without further division in the ordination of
8. 7 7
the first three coordinates. Diploid C. matthioli and
1. 4 2
tetraploid C. majovskii were placed close to each other
- 6. 03
- 5. 6 6
axis 1
and to C. pratensis s.str., even with some samples inter-
mingled. Cardamine crassifolia from the Eastern
- 2. 6 7 - 5. 9 3

axis 2 0. 31

3 . 30 - 1 3. 2 8 Pyrenees and central Iberian mountains formed two dis-

Fig. 7. Canonical discriminant analysis of Iberian C. pra- tinctly separated groups corresponding to their geo-
tensis s.str. with groups defined as different cytotypes graphic origin. Samples of C. granulosa from Piemont
and populations as OTUs, based on 17 morphological were different from those from central Italy, but this
characters (see Table 4). Hearts, 2n = 16; clubs, 2n = 30; Italian taxon will not be discussed here in more detail, as
circles, 2n = 32; crosses, 2n = 46; pyramids, 2n = 48; dia-
monds, 2n = 56. First three axes explain 73.93%, 10.90%,
it is being evaluated elsewhere, including also its mor-
and 8.83% of total variation. phology (Lihová & al., submitted b). In the PCoA ordi-

793
Lihová & al. # Cardamine pratensis group 52 # November 2003: 783–801

A 18 B C D E
9 14 22

length of longer filaments (mm)

7
8 12
16

length of sepals (mm)

7 17
length of petals (mm)

6 10

number of leaves

number of segments
14
6
5 8
12 5 12
6
4 4
10
4
3 7
8 3
2 2

6 2 1 2
16 30 32 46 48 56 16 30 32 46 48 56 16 30 32 46 48 56 16 30 32 46 48 56 16 30 32 46 48 56

Fig. 8. Variation in selected morphological characters compared among different cytotypes (2n = 16, 30, 32, 46, 48, 56)
of C. pratensis s.str. from the Iberian Peninsula. Rectangles define 25 and 75 percentiles; horizontal lines show medi-
an; whiskers are from 10 to 90 percentiles; asterisks show extreme values. A, length of petals (mm); B, length of sepals
(mm); C, length of longer filaments; D, number of stem leaves; E, number of segments on middle stem leaves.

nation including only C. pratensis s.str., C. matthioli and samples of C. pratensis s.str. was achieved, showing cor-
C. majovskii (graph not shown), slight separation among relation with ploidy level and geographic origin. Galician
hypotetraploids and central Iberian heptaploids (prov.
0 1 2
Dissimilarity
3 4
Segovia) formed two groups separated along the second
P M44-82
axis, with other Iberian and central European samples in-
59(32)
52(48) between. The neighbour-joining tree based on genetic
47(56)
58(48)
P44-78 distance of Nei & Li produced similar structure as PCoA
54(48)
57(46)
49(32)
(Fig. 11). The group consisting of C. pratensis s.str., C.
P44-75
53(46)
51(?)
matthioli and C. majovskii is rather heterogeneous, as is
P M44-83
P44-74
69(30)
evident from the bootstrap value of 57%, but at the same
45(56)
46(56)
43(56)
time it shows low resolution, only samples from the same
50(32)
39(30)
36(30)
population and those from geographically close localities
31(30)
41(48) clustering with higher support. Cardamine crassifolia
35((30)
PM44-81
P M44-80 from the Pyrenees formed one well supported group
P M44-84
P30-66
42(56)
(100% bootstrap value), as well as that from central
Iberian mountains (92%), but they both received only
P44-77
P44-73
P44-79
P44-72
56(32)
P30-59
moderate support as one group (69%). Such clustering is
P30-65
71(44)
70(30)
consistent with a rather high number of fragments exclu-
RI-26
45(56)
P38-69
sively recorded (although not present in all accessions)
P38-71
P38-70 for each of them, 14 fragments for the Pyrenean and 15
48(16)
P44-76
P30-57 for the central Iberian samples (Table 5). When consid-
ering both as one entity, only three such fragments were
40(30)
P30-61
P30-67
P30-56
P30-62
P30-63
scored.
P30-58
RI-27
RI-28
AMOVA in C. pratensis s.str. without subdividing
RI-31
61(16)
P38-68
populations into groups showed moderate differentiation
P30-64
U C-21 among populations, as 56.65% of total variation account-
UC-24
P30-60
UC-23
ed for among-population variation. Partitioning of popu-
RI-29
RI-32
RI-25
lations of C. pratensis s.str. into nine geographic regions
RI-30
34(30)
UC-20
revealed that 26.14% of the overall variation was
UC-22
37(30)
29(16)
explained by among-group variation, 31.35% by varia-
30(30)
38(30) tion among populations, and 42.51% by that within pop-
Fig. 9. Cluster analysis of Iberian and Central European
ulations (Table 6). When differentiating ploidy levels
populations of C. pratensis s.str. based on eight morpho- (seven groups), only 7.79% of the total variation was
logical characters. Central European samples: P30, 2n = assigned to variation among cytotypes, while most of the
30; P38, 2n = 38; P44, PM44, 2n = 44; UC, C. pratensis variation was among (49.73%) and within populations
"ucranica type" 2n = 16; RI, C. rivularis auct. non Schur, (42.48%). AMOVA including only Iberian populations of
2n = 16; labels are the same as in Marhold (1996). Iberian
samples are numbered as in Table 1, with chromosome
C. pratensis s.str. subdivided geographically (six groups)
numbers indicated in parentheses, and marked by verti- gave higher value for the among-group variation
cal bars. (30.55%) than the analysis including also non-Iberian

794
 52 # November 2003: 783–801 Lihová & al. # Cardamine pratensis group

axis 3
Lihová & Marhold. Morphological characters distin-
0. 288
guishing this species from C. crassifolia and C. pratensis
s.str. are summarized in Table 8.

0. 109
Cardamine castellana Lihová & Marhold, sp. nov.
– Holotype: Spain. prov. Ávila: Sierra de Gredos, Hoyos
del Espino, near Hoyos del Collado, 1500 m, 10 May
2001, J. Lihová s.n. (SAV). Isotypes: SALA, BC, MA.
- 0 . 0 71
0 . 24 1

- 0. 0 08
Descriptio: Herba perennis, (7–)13–23(–26) cm alta
- 0 . 2 50 axis 2
(ad infimum pedunculum). Rhizoma repens, obliquum
- 0. 25 7
vel erectum, plerumque incrassatum certe in parte supe-
0. 2 30
0. 0 21

riore, tantum raro omnino tenue, interdum ferens plures

axis 1 - 0. 188
- 0 . 3 9 7 - 0. 50 6

floriferos caules (caespitosus), raro prostratos usque

Fig. 10. Principal coordinate analysis of AFLP data of 114
samples of the C. pratensis group. Clubs, C. pratensis
ascendentes stolones. Caulis ascendens usque erectus,
s.str.; diamonds, C. crassifolia (Eastern Pyrenees); simplex vel raro ramosus. Folia basalia glabra, pinnata,
cubes, C. castellana (central Iberian mountains); circles, 1–4 jugata, rosulata vel congesta prope basin caulis; foli-
C. majovskii; crosses, C. matthioli; hearts, C. granulosa ola terminalia late ovata usque subrotundata, integra vel
(Piedmont); spades, samples from taxonomically uncer- in apice remote crenata, basi reniformia vel obtusa,
tain populations from central Italy. The first three axes
explain 13.52%, 11.14% and 7.88% of total variation.
majora foliolis lateralibus; foliola lateralia late elliptica,
ovata usque subrotundata. Folia caulina (1–)2–6(–7),
pinnatisecta, glabra, segmenta foliorum mediorum
samples. The Iberian cytotypes showed much higher dif- (4–)5–11(–13); segmentum terminale oblongum usque
ferentiation (30.98 % vs. 7.79% with the inclusion of ellipticum vel oblanceolatum usque obovatum, integrum
non-Iberian samples), but still, most variation was vel in apice remote crenatum, segmenta lateralia oblonga
explained by among- and within-population differentia- usque elliptica vel oblanceolata; numerus segmentorum
tion. In accordance with neighbour-joining and PCoA inflorescentiam versus decrescens. Inflorescentia race-
results, only 12.51% of the total variation can be attrib- mosa; petala saturata usque diluta rubelloviolacea, raro
uted to variation between Iberian and non-Iberian popu- paene alba, anguste obovata, raro obovata, apice sinuata,
lations, suggesting that there is hardly any genetic differ- (4.5–)5–8.3(–9) mm longa et (2–)2.3–3.6(–4.2) mm lata.
entiation between Iberian populations and those from Sepala elliptica usque anguste obovata, (2.4–)
other parts of Europe. 2.6–3.8(–4.2) mm longa. Staminum 6, tetradynama,
In the two-level AMOVA of populations of C. cras- antherae luteae, stamina filamenta longiora
sifolia from the Eastern Pyrenees and those from central (3.1–)3.5–4.9(–5.6) mm longa, filamenta breviora
Iberian mountains, strong differentiation among the pop- (1.3–)1.9–3.1(–3.5) mm longa. Stigma conspicuum,
ulations was revealed (86.84%; Table 7). As can be seen dilatatum. Pedunculi patentes usque erecto-patentes, sili-
from the three-level AMOVA, this is attributable mostly quae divergentes in eundem angulum ac pedunculi vel
to the regional differentiation (E Pyrenees vs. central erectae. 2n = 2x = 16.
Iberian mountains Sierra de Gredos and Sierra de Albar- Description: Perennial herb, (7–)13–23(–26) cm tall
racín; 62.77% of total variation), with a much lower (up to the lowest flower/fruit peduncle). Rhizome creep-
value for among-population variation within a region ing, oblique or erect, mostly thickened at least in the
(27.83%). When subdividing both Pyrenean and central upper part, only seldom entirely thin, sometimes with
Iberian populations into two geographic groups, even multiple stems (caespitose), and occasionally with pros-
higher proportion of the variation accounted for among- trate to ascending thin stolons. Stem ascending to erect,
region variation (77.53%). This is due to high genetic simple or rarely branched above. Basal leaves glabrous,
differentiation also between populations in Sierra de pinnate, with 1–4 pairs of leaflets, either forming a basal
Gredos and Sierra de Albarracín (77.34%), as also shown rosette or only congested near the stem base; terminal
in the neighbour-joining tree (Fig. 11). leaflet broadly ovate to roundish, entire or remotely cre-
Description of the new taxon. — Both mor- nate at apex, reniform or obtuse at base, larger than lat-
phological and molecular comparative analyses demon- eral ones, lateral leaflets broadly elliptic, ovate to
strate that populations from central Iberian mountains, roundish. Stem leaves (1–)2–6(–7), pinnatisect, glabrous,
traditionally classified within C. crassifolia, are different middle stem leaves with (4–)5–11(–13) segments; termi-
from typical C. crassifolia, and should be regarded as a nal segment oblong to elliptic or oblanceolate to obovate,
separate taxonomic entity. On a basis of this evidence, entire or remotely crenate at apex, lateral ones oblong to
we describe here a new species, Cardamine castellana elliptic or oblanceolate; number of segments gradually

795
Lihová & al. # Cardamine pratensis group 52 # November 2003: 783–801

96 pratensis-34(30)
pratensis-34(30)
42 pratensis-38(30)
pratensis-38(30)

Iberian Peninsula
40
pratensis-36(30)
49 pratensis-38(30)
99 pratensis-30(30)
pratensis-30(30)
91 pratensis-48(16)
78 pratensis-48(16)
pratensis-48(16)
99 pratensis-57(46)
pratensis-57(46)
pratensis-57(46)
pratensis-59(32)
pratensis-59(32)
pratensis-59(32)
90 pratensis-58(48)
pratensis-58(48)
89 pratensis-47(56)
96 pratensis-47(56)
pratensis-47(56)
99 pratensis-69(30)
pratensis-69(30) Slovenia
pratensis-70(30)
pratensis-70(30)
100 pratensis-72(30) France
pratensis-72(30)
96 pratensis-74(30)
99 pratensis-74(30)
pratensis-74(30)
Italy
61 pratensis-73(30)
pratensis73(30)
pratensis-73(30)
France
57 81 pratensis-46(56)
53

Peninsula
pratensis-46(56)
pratensis-46(56)

Iberian
58 67 pratensis-43(56)
pratensis-43(56)
pratensis-43(56)
40 pratensis-44(?)
pratensis-44(?)
pratensis-44(?)
67 pratensis-75(44)
pratensis-75(44)
76 pratensis-76(30)
pratensis-76(30)
Slovakia
pratensis-76(30)
94 matthioli-81
matthioli-81
pratensis-71(44)
pratensis-71(44) Slovenia
pratensis-71(44)
pratensis-70(30)
61 matthioli-77
69 matthioli-77
50 majovskii-82
majovskii-82
48 majovskii-83
majovskii-84
43 majovskii-85
majovskii-85
48 majovskii-86
majovskii-86
Slovenia
53 matthioli-78
matthioli-78
52 matthioli-79
matthioli-79
majovskii-83
majovskii-84
45 matthioli-80
matthioli-80
91 granulosa-87
84
100
granulosa-87 Italy,
granulosa-87
89 granulosa-88 Piedmont
43 granulosa-88
pratensis s.l.-89
93 pratensis s.l.-89
99 pratensis s.l.-89
pratensis s.l.-91
40
pratensis s.l.-90
pratensis s.l.-90
100 pratensis s.l.-90 central
pratensis s.l.-91
pratensis s.l.-90 Italy
47
68 pratensis s.l.-92
87 pratensis s.l.-92
pratensis s.l.-92
95 castellana-25
41 castellana-25
58 castellana-26 Spain,
100 61 castellana-27 Sierra de Albarracin
castellana-27
92 castellana-26
97 castellana-21
99 castellana-21 Spain,
59 castellana-23
83 castellana-23 Sierra de Gredos
69 86 castellana-20
castellana-20
crassifolia-2
49 crassifolia-1
100 66 crassifolia-2
crassifolia-1
crassifolia-3
crassifolia-3 Spain,
96
crassifolia-4 Pyrenees
crassifolia-7
crassifolia-7
crassifolia-5
crassifolia-4
94 crassifolia-6
crassifolia-6

Fig. 11. Neighbour-joining tree of AFLP data of 114 samples of the C. pratensis group, including C. pratensis s.str. (with
chromosome numbers indicated in parentheses), C. matthioli, C. majovskii, C. crassifolia (samples from the Eastern
Pyrenees), C. castellana (samples from Sierra de Gredos and Sierra de Albarracín), C. granulosa (Piedmont) and indi-
viduals from taxonomically uncertain populations from central Italy. For numbers and origin of samples see Table 1.
Values above branches are bootstraps using 2,000 replicates. Branches with less than 40% bootstrap are shown as
unresolved.

796
 52 # November 2003: 783–801 Lihová & al. # Cardamine pratensis group

diminishing towards the inflorescence. Inflorescence Cardamine pratensis s.str., karyological and
racemose; petals deep to pale reddish-violet, seldom morphological diversity. — Cardamine pratensis
almost white, narrowly obovate, sinuate at the tip, s.str. is a highly polymorphic polyploid species wide-
(4.5–)5–8.3(–9) mm long and (2–)2.3–3.6(–4.2) mm spread in Europe. Although Lövkvist (1956) indicated
wide. Sepals elliptic to narrowly obovate, that in maritime districts of Portugal, NW Spain and SW
(2.4–)2.6–3.8(–4.2) mm long. Stamens 6, tetradyna- France, a diploid taxon, different from C. pratensis s.str.,
mous, anthers yellow, filaments of longer stamens could be recognized, we cannot support his view. In both
(3.1–)3.5–4.9(–5.6) mm long, filaments of shorter sta- morphological and molecular evaluation, Iberian popula-
mens (1.3–)1.9–3.1(–3.5) mm long. Stigma conspicuous, tions clearly fall within the variation of this species in
enlarged. Peduncles patent to erect-patent, siliquae diver- other parts of Europe (Fig. 9, Table 6). Previously, only
gent from infructescence-rachis at the same angle as diploid and triploid chromosome numbers for this taxon
peduncles or erect. 2n = 2x = 16. were reported from the Iberian Peninsula (both from
Distribution and habitat: Spain. Sierra de Gúdar, Portugal; Lövkvist, 1956; Queirós, 1973), but we
Sierra de Albarracín, Sierra de Gredos, Sierra Segundera, revealed a much wider karyological variation (Table 1,
Cordillera Cantabrica; wet meadows and pastures, Fig. 1). We recorded diploid populations in Portugal
stream banks, 1200–2000 m. (Beira Litoral region), in the Spanish province Cantabria,
and in the Central Pyrenees. Diploids of C. pratensis
Key to the Iberian representatives of the s.str. otherwise occur scattered across Europe; they have
Cardamine pratensis group been found in Central Europe (e.g., Lövkvist, 1956;
1a. Whole rhizome equally thin, as wide as stem . . . 2 Urbanska-Worytkiewicz & Landolt, 1974; Marhold,
1b. Whole or only upper part of rhizome thickened . . 3 1994, 1996, 1999), in Belgium (Vyvey & Stieperaere,
2a. Rhizome horizontally creeping, stem ascending; 1984), and France (Guinochet, 1946; Bernard, 1974;
basal leaves with 1–2 pairs of lateral leaflets; petals Vyvey & Stieperaere, 1984). In the Central Pyrenees
broadly obovate or obovate, 3.5–5.9 mm wide . . . . (province Huesca) we found one diploid population
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. crassifolia (Table 1, population no. 61) morphologically somewhat
2b. Rhizome horizontally creeping to erect, stem deviating from other Iberian populations. Due to small
ascending to erect; basal leaves with 1–4 pairs of floral organs and lower number of leaf segments in some
leaflets; petals narrowly obovate, 2.3–3.6 mm wide individuals, in morphometric analyses it was placed
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. castellana close to C. crassifolia (Fig. 2). Most of the plants had,
3a. Middle stem leaves with 9–21 segments; petals however, erect stems, short and thick rhizomes, and true
broadly obovate or obovate, 9–15 mm long, 5–9 mm basal rosettes with a high number of leaflets, all traits
wide . . . . . . . . . . . . . . . . . . . . . . C. pratensis s.str. characteristic of C. pratensis s.str. Still, in a few cases
3b. Middle stem leaves with 5–11 segments; petals nar- tendency to ascending stems typical for C. crassifolia
rowly obovate, 5–8 mm long, 2.3–3.6 mm wide . . and rhizomes resembling C. castellana was observed.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. castellana We have seen similar specimens also from other, adja-
cent localities (see Appendix, specimens marked by
Full list of studied specimens of the Cardamine asterisks), and morphological peculiarity of plants from
pratensis group in the Iberian Peninsula is given in the that area has been already noticed by Rico (1993). Bolòs
internet version of Taxon. & Vigo (1990) classified populations from the Central
Pyrenees as C. pratensis subsp. rivularis (Schur) Nyman
(º C. rivularis Schur), which in fact is a different taxon
restricted to the mountains of Bulgaria and the Southern
DISCUSSION Carpathians, having violet anthers and appressed hairs on
Combined karyological, morphological and molecu- the rachis of basal leaves (vs. yellow anthers and patent
lar studies suggest that three taxa of the Cardamine hairs present here; Marhold, 1994a; Franzke & Hurka,
pratensis group can be recognized in the Iberian 2000). Several authors, however, have misinterpreted
Peninsula: C. pratensis s.str., C. crassifolia, and the here- this taxon, and reported it erroneously from higher alti-
in described new species, C. castellana. Whereas C. tudes in the Eastern Carpathians, Eastern Alps,
pratensis s.str. is composed of several cytotypes, ranging Apennines, Central Massif, and also from the Pyrenees
from diploid to heptaploid, the latter two Iberian endem- (Lövkvist, 1956; Urbanska-Worytkiewicz & Landolt,
ic species are diploid. All three taxa show morphological 1974; Landolt, 1984). As conspicuous morphological
differences in both qualitative and quantitative charac- variability, broad ecological range of habitats and large
ters, and based on molecular AFLP data they are also plasticity exist in C. pratensis s.str., we assume that the
genetically clearly differentiated. central Pyrenean plants, although at marginal position,

797
Lihová & al. # Cardamine pratensis group 52 # November 2003: 783–801

still fall within the variation of C. pratensis s.str., and Schur, C. udicola, C. pratensis subsp. picra, C. pratensis
should be classified within this species. However, past or subsp. major; de Langhe & D’ hose, 1976; Urbanska-
more recent introgression from C. crassifolia, occuring Worytkiewicz & Landolt, 1974; Tomšovic, 1986).
more eastward, cannot be excluded. Detailed study is Resulting from previous studies (Landolt, 1984; Franzke
needed, including several populations from that area, and & Hurka, 2000) and our observations, these cannot be
evaluating them also with molecular markers. regarded more than ecotypes or plasticity responses,
Hypotetraploid populations (2n = 30) of C. pratensis without clear morphological and molecular differentia-
s.str., which we found to prevail in the Atlantic region of tion. Morphometric comparison of different cytotypes of
Galicia and Portugal, represent the most widespread C. pratensis s.str. provided very similar results to those
cytotype in Europe (Guinochet, 1946; Hussein, 1955; obtained by Marhold (1996). Although shifts in some
Lövkvist, 1956; Vyvey & Stieperaere, 1984; Marhold, characters at the populational level were found among
1994a, b). Because of two longer chromosomes in the the cytotypes, these, due to large overlap in the ranges,
karyotype, Lawrence (1931) assumed that this cytotype blurred at the level of individual plants. Several molecu-
originated by chromosome fusion. As a result, 7- and 8- lar markers used by Franzke & Hurka (2000) failed to
chromosome sets can then meet in the genome of poly- resolve relationships and origin of different cytotypes of
ploid plants, and contribute to the karyological complex- C. pratensis s.str. Neither the highly resolving AFLP-fin-
ity of the species (Lövkvist, 1956). Normal tetraploids gerprinting applied here has brought much more resolu-
with 32 chromosomes are less frequent in Europe (e.g., tion, and thus, supported its current taxonomic treatment
Vyvey & Stieperaere, 1984; Lippert & Heubl, 1988; as a single species. Genetic affinity among geographical-
Marhold, 1999), and in the Iberian Peninsula they seem ly close populations of the same ploidy level was
to be concentrated in the Basque country and Central revealed, most likely reflecting current extensive gene
Pyrenees (Fig. 1). Populations with 40 chromosomes, flow over short distances. As suggested by genetic parti-
here found in the province of Navarra, have been report- tioning shown by AMOVA (Table 6), gene flow is
ed so far solely from France (central Jura; Guinochet, restricted by both different ploidy levels and large geo-
1946). Interesting is the chromosome number 2n = 46, graphic distances. Very low among-cytotype differentia-
which we confirmed for several plants in two localities in tion on the European scale, in contrast to higher differ-
the provinces Guipuzcoa and Navarra. There are no other entiation among geographic regions, also indicates that
reports for such populations in Europe (only a single the polyploids in C. pratensis s. str. might not have a sin-
plant determined from France by Lövkvist, 1956), but gle origin, but could have evolved independently and
these two populations are morphologically and geo- polytopically. Alternatively, such pattern could be attrib-
graphically close to the normal hexaploid ones (2n = 48), uted to gene flow across ploidy levels within regions. To
thus they may have originated in them. Higher poly- support either of these hypotheses, however, much larg-
ploids with 48 and 56 chromosomes, which we found in er and detailed sampling across the whole distribution
several localities in central and northern Spain, occur range would be needed.
mainly in northern Europe (England, Finland, Denmark, Apart from C. pratensis s.str., populations resem-
Sweden; Hussein, 1955; Lövkvist, 1956; Dale & bling C. dentata have been reported from northern Spain
Elkington, 1974). Performing crossing experiments, (provinces Soria, Burgos, Navarra; Montserrat-Recorder,
Lövkvist (1956) revealed that C. pratensis s.str. is an 1967; Rico, 1993). Cardamine dentata is a high poly-
allogamous species, and that plants of different ploidy ploid (2n = 7x–12x) species distributed in central and
levels (except for diploids) are partially to fully interfer- northern Europe (Lövkvist, 1956). Although often misin-
tile. Especially at higher ploidy levels they often cross terpreted by some authors (see Marhold, 1994a), mor-
and produce viable offspring with various aneuploid phologically it is well characterized by having all leaves
chromosome numbers. Weak reproductive barriers and pinnate with distinctly stalked and deciduous leaflets,
existence of gene flow among individual cytotypes, and large white flowers (Marhold, 1994a). As we have
together with two basic chromosome numbers (x = 7, 8), not seen any herbarium specimens or plants in the field
seem to be the reason for the karyological complexity of which would correspond to this taxon, and bearing in
the species in Europe. mind also its distribution, we find its occurrence in Spain
Based on our morphometric evaluation, we can con- as highly improbable.
firm large morphological variation for this species in the Cardamine crassifolia, an endemic diploid
Iberian Peninsula, already noticed by Montserrat- species from the Eastern Pyrenees. — From our
Recorder (1967) and Rico (1993), and known from other herbarium and field studies, C. crassifolia should be
parts of Europe. Several attempts have been made to split regarded as a narrow endemic confined to the Eastern
this heterogeneous species and to recognize several sep- Pyrenees. The westernmost locality documented seems
arate taxa (e.g., C. nemorosa, C. rivularis auct. non to be in Andorra, since westward from that, in the Central

798
 52 # November 2003: 783–801 Lihová & al. # Cardamine pratensis group

and Western Pyrenees, only diploid to pentaploid popu- & al., 1994). However, detailed morphological and
lations of C. pratensis s.str. occur (Table 1, Fig. 1, molecular analyses reveal that these populations differ
Appendix). Further records for C. crassifolia have been from C. crassifolia from the Eastern Pyrenees, and there-
reported from various central Iberian mountains (Mateo fore deserve formal taxonomic recognition. This new
Sanz & al., 1994); these, however, refer to the herein taxon, C. castellana, shows interesting patterns of mor-
described new species C. castellana (Table 1, Fig. 1, phological variation. In most quantitative characters it
Appendix). A similar distributional pattern as in C. cras- resembles more C. crassifolia, whereas when examining
sifolia occurs also in another Iberian taxon, namely C. morphology of rhizome and basal parts of the stem, it
amara subsp. pyrenaea Sennen (Lihová & al., 2000). exhibits mixed characteristics of C. crassifolia and C.
These two taxa, both diploids, have similar ecological pratensis s.str. (Fig. 6). In the ITS sequence tree (Franzke
requirements and co-occur in some localities. Most & Hurka, 2000, under C. crassifolia) it appeared closer
recently, even a sterile hybrid between them has been to C. crassifolia than to C. pratensis s.str., as did also in
documented from two sites (Marhold & al., 2002). the neighbour-joining tree of AFLP data (Fig. 11). From
A relic position of C. crassifolia within the C. a taxonomical point of view, the critical question is
pratensis group has already been proposed by Lövkvist whether to adopt a specific or subspecific rank for this
(1956) on the basis of its distinct morphology and distri- taxon. Both the genetic differentiation between C. cras-
bution. Recent molecular study combining several mark- sifolia and C. castellana (revealed by the high between-
ers (Franzke & Hurka, 2000), as well as our AFLP data taxon differentiation in AMOVA and documented also by
favour this view, as they reveal C. crassifolia as a genet- several exclusive AFLP fragments in each of these two
ically well-differentiated taxon, possibly representing taxa, as opposed to the few they share) and differences in
one of the basal lineages of the C. pratensis group. The morphology show that they represent two well defined
evolutionary history of this polyploid complex, however, and distinct diploid taxa. To keep balanced and consis-
might be even more complicated than presented by tent taxonomic treatment of the C. pratensis group
Franzke & Hurka (2000). Our preliminary molecular through the whole distributional range, and considering
studies on polyploid C. raphanifolia (sensu Flora also the fact that it, although always treated and accept-
Europaea) show that it stands very close to the C. praten- ed as a group, might not be monophyletic (Lihová et al.,
sis group (Lihová & al., submitted a). It seems that nei- submitted a), we find the species rank for C. castellana
ther the C. raphanifolia nor C. pratensis groups, as cur- appropriate. Strong genetic differentiation revealed
rently understood, are monophyletic, and their evolution between populations from Sierra de Gredos and Sierra de
and diversification have involved taxa from both groups. Albarracín most probably indicates long-term isolation
The name C. crassifolia was first published by and absence of gene flow between these two mountain
Pourret in 1788. A detailed description of this taxon was ranges, but without any morphological differentiation.
provided by Sennen, under the name C. nuriae Sennen Although morphological and molecular data current-
(Sennen, 1917), and later C. pratensis subsp. nuriae ly available do not provide sufficient resolution for infer-
(Sennen) Sennen (Sennen, 1929). Following Sennen’ s ring the origin and evolutionary history of C. castellana,
treatment, some authors have preferred subspecific level two hypotheses might be suggested. The first is that C.
for this taxon, and this status was adopted in Flora crassifolia and C. castellana represent two closely relat-
Europaea (Jones & Akeroyd, 1993) as C. pratensis ed lineages, which have diverged from a common ances-
subsp. crassifolia (Pourr.) P. Fourn., as well as in Flora tor. They might have undergone morphological and
Iberica (Rico, 1993) under C. pratensis subsp. nuriae, genetic diversification due to their long geographic iso-
having priority against the former name (for full nomen- lation and adaptation to different abiotic conditions.
clatural account see Marhold & al., 2002). However, on Alternatively, intermediacy in morphology may indicate
the basis of clear molecular and morphological differen- hybrid origin of C. castellana between diploid C. praten-
tiation of this taxon, we prefer to keep it at the level of sis s.str. and C. crassifolia. More insights into the
species, as also treated by previous authors studying the genomic constitution of C. castellana and its close rela-
group in more detail (Lövkvist, 1956; Urbanska- tives are obviously needed.
Worytkiewicz & Landolt, 1974; Landolt, 1984; Franzke
& Hurka, 2000).
Cardamine castellana, the newly described ACKNOWLEDGEMENTS
Iberian species. — Populations occurring at higher This study arose in cooperation between the Real Jardín
altitudes in the central Iberian mountains (Sierra de Botánico, Consejo Superior de Investigaciones Científicas, Spain,
Gúdar, Sierra de Albarracín, Sierra de Gredos, Sierra and the Institute of Botany of the Slovak Academy of Sciences.
Segundera, and also Cordillera Cantabrica) have been The financial support provided by project no. 7080 from the Grant
included within C. crassifolia (Rico, 1993; Mateo Sanz Agency VEGA, Bratislava, Slovak Republic, by project no. 1131-

799
Lihová & al. # Cardamine pratensis group 52 # November 2003: 783–801

Cambridge.
4 from the Ministry of Education, Youth and Sports of the Czech Klecka, W. R. 1980. Discriminant Analysis. Sage University
Republic, and by an InternationalAssociation for Plant Taxonomy Papers, Series: Quantitative Applications in the Social
(IAPT) Research Grant is acknowledged. We are grateful to: our Sciences, no. 19. Sage, Beverly Hills.
colleagues from Spain and Portugal for their assistance in the field, Krzanowski, W. J. 1990. Principles of Multivariate Analysis.
especially to Gonzalo Nieto Feliner, Mercedes Herrera, Idoia Clarendon Press, Oxford.
Biurrun, Iñigo Pulgar, Santiago Ortiz, Josep Vicens, Luis Delgado, Landolt, E. 1984. Über die Artengruppe der Cardamine
pratensis L. in der Schweiz. Diss. Bot. 72: 481–497.
Montserrat Martínez Ortega, Patricio Bariego Hernández and
Lawrence, W. J. C. 1931. The chromosome constitution of
Jorge Paiva; Marián Perný and Patrik Mráz for collecting several Cardamine pratensis and Verbascum phoeniceum.
samples in the field; Michael Barfuss for preparing polyacry- Genetica 13: 183–208.
lamide gels for AFLP analyses; Elias Landolt and Krystyna Lihová, J., Fuertes Aguilar, J., Marhold, K. & Nieto Feliner,
Urbanska for unpublishedchromosome numbers and for providing G. Submitted a. Origin of the disjunct tetraploid
their herbarium collection for study; Zlata Komárová for plant Cardamine amporitana (Brassicaceae) assessed with
details illustrations (Figure 6); and to Tod F. Stuessy, Marcus nuclear and chloroplast sequence data. Amer. J. Bot.
Lihová, J. & Marhold, K. In press. Taxonomy and distribu-
Koch, and an anonymous reviewer for useful comments on the
tion of the Cardamine pratensis group (Brassicaceae) in
manuscript. We thank also the curators of herbaria mentioned in Slovenia. Phyton (Horn) 43.
the Methods for allowing us to study herbarium specimens. Lihová, J., Marhold, K. & Neuffer, B. 2000. Taxonomy of
Cardamine amara (Brassicaceae) in the Iberian Peninsula.
Taxon 49: 747–763.
Lihová, J., Tribsch, A. & Stuessy, T. F. Submitted b.
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Aedo, C., Gómez-Campo, C., Laínz, M., Montserrat, P., Muñoz s.n. (MA 539474); Oviedo, Beas, Puerto de la Espina, 1976,
Morales, R., Muñoz Garmendia, F., Nieto Feliner, G., Galiano & al. s.n. (MA 201370); Entre Aviles y el Cabo de Peñas, 1973,
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Bot. Jahrb. Syst. 32: 281–623. Guinea s.n. (RNG); Villaviciosa, mt. Calvera, 1952, Guinea s.n. (RNG);
Sennen, frère. 1917. Flore de Catalogne Additions et com- prov. Burgos: Cordillera Cantábrica, N of Puerto del Escudo, 1980, Miles,
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Sennen, frère. 1929. La flore du Tibidabo. Monde Pl. 30 Fernández Diez s.n. (MA 204696); Cañon del rio Ubierna, 1984, Galán &
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Schneider, S., Roessli, D. & Excoffier, L. 2000. Arlequin, P. Montserrat s.n. (JACA 314669, ZT); Burgos, Puerto del Escudo, 1976,
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1984, Herrera s.n. (BIO 0030); Merón, San Vicente de la Barquera, 1984,
Analysis. Genetics and Biometry Laboratory, Univ.
Aedo s.n. (MA 622547); Ramales de la Victoria, 1995, Carrasco s.n. (MA
Geneva, Switzerland.
556225); Cabezón de la Sal - San Vicente de la Barquera, 1994, Pulgar s.n.
Schönswetter, P., Tribsch, A., Stehlik, I. & Niklfeld, H. In
(SANT 29853); Ojambre, El Tejo, 1980, Amich & Sánchez s.n. (SALA
press. Glacial history of high alpine Ranunculus glacialis 23479); Villacantid, 1989, Valle (SALAF 82384); Puerto de Palombera,
(Ranunculaceae) in the European Alps in a comparative 1926, Font Quer s.n. (BC 111461); prov. A Coruña: Carnota, A Curra -
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Sneath, P. H. A. & Sokal, R. R. 1973. Numerical Taxonomy. Carnota, A Curra, 1995, Louzán s.n. (MA 581174, SANT 35957);
Principles and Practice of Numerical Classification. W. Dumbria, Ezaro, 1994, Louzán s.n. (SANT 35956); Valle del rio Tambre,
H. Freeman and Company, San Francisco. 1966, Rivas Goday (MA 47529, MAF 17055); Oleiros, 1979, Fernández
Tomšovic, P. 1986. Two new taxa in the Cardamine pratensis Diez s.n. (MA 224241, SALA 19427, BC 630678); Santiago, 1944,
agg. from Bohemia and Moravia (Czechoslovakia). Folia Figueroa Agea s.n. (SANT 01410); Ribeira, Parque Natural “Complexo
Geobot. Phytotax. 21: 429–430. Dunar de Corrudedo e lagoas de Carregal y Vixán”, 1993, Pulgar s.n.
Tribsch, A., Schönswetter, P. & Stuessy, T. F. 2002. (SANT 38711); Muxia, entre Frixe e Nemiña, 1994, Louzán s.n. (SANT
Saponaria pumila (Caryophyllaceae)and the ice age in the 40435); Monfero, entas Fragas de Eume, 1992, Amigo & Nornan s.n.
European Alps. Amer. J. Bot. 98: 2024–2033. (SANT 28101); Carnota, Monfero, Taboada, Fraga de Caaveiro, 1994,
Urbanska-Worytkiewicz, K. & Landolt, E. 1974. Soñora Gómez s.n. (SANT 37623); Ortigueira Mera, Barranco Vilariño,
Biosystematic investigations in Cardamine pratensis L. 1994, Soñora Gómez s.n. (SANT 37602); Fistera, Rostiro, 1994, Louzán
s.l. I. Diploid taxa in central Europe and their fertility rela- s.n. (SANT 40606); prov. Guipuzcoa: Alkiza, 1989, Biurrun s.n. (BIO
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42–139. Oleabzu, 1895, Gandoger s.n. (Flora Hispanica exsiccata, No. 29) (MA
47528); Hernani, 1895, Gandoger s.n. (Flora Hispanica exsiccata, No. 41)
Van de Peer, Y. & De Wachter, R. 1994. TREECON for
(MA 47527, COI); Pasajes, 1895, Gandoger s.n. (Flora Hispanica exsicca-
Windows: a software package for the construction and
ta, No. 37) (MA 47515, COI); Azkoitia, Asunción Epelde, 1992 (SANT
drawing of evolutionary trees for the Microsoft Windows
27016); Vergara am Puerto des Carga, 1967, Scholz & Hiepko 1046 (B);
environment. Computer Applic. Biosci. 10: 569–570. Monte del Castillo de la Nola en S. Sebastián, 1875, Mansferrer s.n. (BC
Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., 03106); prov. Huesca: *Cotiella, 1969, Fernández Casas s.n. (MA
Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, 410447, 409900); *Liri, Solana del Gallinero, 1992, Fernández & Sesé s.n.
M. & Zabeau, M. 1995. AFLP: a new technique for DNA (JACA 331492); Benasque, Peña Blanca, 1994, P. Montserrat & Bevington
fingerprinting. Nucl. Acids Res. 23: 4407–4414. s.n. (JACA 96494); *Castanesa, Orillas del Lago de Bacibé, P. Montserrat
Vyvey, R. & Stieperaere, H. 1984. A numerical analysis of & Dussaussois s.n. (JACA 162382); *San Juan de Plan, San Mamés-Fene
some diploid and tetraploid Cardamine pratensis L. (s.l.) Mayor, 1981, P. Montserrat & al. s.n. (JACA R115267); *Cerler, Empriu,
populations from Belgium and northern France. Bull. Soc. 1989, P. Montserrat s.n. (JACA 146089); prov. León: Ponferrada, Montes
Roy. Bot. Belgique 117: 341–250. de Valdueza, 1979, Temprano 287ET (MA 314934);Tal des Rio Esla nördl.
Zhang, L.-B., Comes, H. P. & Kadereit, J. W. 2001. Cistierna, 1967, Scholz & Hiepko 955 (B); prov. Lérida: *Muntanya de
Phylogeny and quaternary history of the European mon- Llacs, pr. Boí, 1944, Bolòs & Font Quer s.n. (BC 94601); Vall d’ Aran, Lés,
tane/alpine endemic Soldanella (Primulaceae) based on 1954, Bolòs s.n. (BC 140163); prov. Lugo: Quiroga, 1981 (SANT 15195);
ITS and AFLP variation. Amer. J. Bot. 88: 2331–2345. Monforte, 1908, Gandoger s.n. (COI); prov. Madrid: Miraflores de la
Sierra, 1976, Fernández Diez s.n. (SALA 8627); Sierra Guadarrama, Pto.
de los Cotos, 1971, Merxmüller & Gleisner s.n. (M); Sierra de
Guadarrama, inter Puerto de Cotos & al Paular, 1976, Fernández Casas

801
Lihová & al. # Cardamine pratensis group 52 # November 2003: XX–XY

1113 (MA 394490); Sierra de Guadarrama, Cercedilla, 1912, Vicioso & Aveiro, Águeda, Fermentelos, entre Porto d’ Asna, Sorgacal e Porto da
Beltrán s.n. (MA 47503); Sierra de Guadarrama, Cercedilla, 1914, Vicioso Minhoteira, 1977, Marques s.n. (COI); Eirol, entre Aveiro e Águeda, 1965,
(BC 03115, BC 03116, MA 47502); Somosierra, Cutanda s.n. (MA A. Fernandes & al. s.n. (COI); Eixo, próximo de Eirol, 1969 s.coll. (COI);
47505); Cercedilla, 1968, Irco Cadero s.n. (MA 315521); Sierra Ponte, da Azurva, 1965, A. Fernandes, R. Fernandes & Paiva (COI);
Guadarrama, Vicioso s.n. (MA 47517); Guadarrama, Lagasca s.n. (MA Aguada de Baixo, 1965, A. Fernandes, R. Fernandes & J. Matos s.n.
47504); Braojos (Madrid), 1918, Vicioso s.n. (MA 47501, BC 03111); El (COI); Águeda, Ribeira de Ferreiros, pr. Recardaes, 1958, Santos s.n.
Escorial (Madrid), 1843, Rodríguez s.n. (MA 47506); Los Peñascales, (COI); Rio Novo, 1892 (COI); Paul de Arzila, Fernandes Costa s.n. (COI);
1942, Guinea s.n. (MA 321384); Paular - los Cotos, 1968, P. Montserrat proximo do Paul d’ Arzila, 1932, de Lousa s.n. (COI); Miranda do Côrvo,
s.n. (ZT); Puerto Los Cotos, 1968, P. Montserrat s.n. (JACA 226868); Gudinhela, 1943, de Lousa s.n. (COI); Serra da Louza, 1883, Henriques
prov. Navarra: Pto. des Roncesvalles de Ibaneta, 1971, Merxmüller & s.n. (COI); Arredes de Coimbra, 1880, Ferreira s.n. (COI); Alfaleros,
Gleisner s.n. (M); Sierra Aralar, 1953, Merxmüller & Wiedmann 874/53 1929, Carrisso & Mendonca s.n. (COI); entre a Ponte de Varela e S.
(M); Idurrieta, cerca de Jaurrieta, 1994, Nieto Feliner 3375GN & al. (MA Yacinto, s.coll., 1980 (COI); Coimbra, Arzila, 1988, Cristina & Lopez s.n.
545035); Baztán, Pto. Artesiega, barranco Olazas, 1980, Aldezabal & al. (COI); Douro Litoral: Porto, Matosinhos, 1889 (COI); Leca do Bailio,
s.n. (MA 505704, BIO 7902); *Quintorreal, Collado de Sagardegui, 1982, 1883, Casimiro Barbosa s.n. (COI); Porto, Ribeiro d’ Avintes, 1888, Leite
Belmonte s.n. (MA 328131); Isaba, Túnel de Eraice, 1993, P. Montserrat & s.n. (COI); Vergas (próximo de Mira), 1970, A. Fernandes & al. s.n. (COI);
Cernoch s.n. (JACA 29093); Larra, Dolina, P. Montserrat s.n. (JACA Vergas Cabecinhas, Galsáz, 1973, A. Fernandes & al. s.n. (COI); Leca de
473367); Contrasario, Ochagavia, Irati, 1987, Villar & G. Montserrat s.n. Palmeira, Boa Nova, 1977, Malato-Beliz 13231 & Guerra (M);
(JACA 0116387); Ulzama, 1965, P. Montserrat s.n. (JACA 9165); Irurzun, Estremadura: Serra de Cintra, 1840 (COI); Minho: Braga, Pico de
río Araquil, 1970, P. Montserrat s.n. (JACA 71570); Huarte-Araquil, 1978, Begahados, 1909, Ferreira s.n. (COI); Arredes de Braga-Monte do Crasto,
P. Montserrat & Bascones s.n. (JACA 30578); valle de Ulzana supra 1883, Lequeira s.n. (COI); Serra de Geraz, 1918, Felgueiros s.n. (COI);
Pamplona, 1966, P. Montserrat 16 (ZT); Urbasa, Segatos, 1956, P. entre Valenca e Sao Pédro da Torre, Veiga da Mira, 1946, da Lilva s.n.
Montserrat s.n. (ZT); Echarri-Aranaz, 1990, Olano s.n. (BIO 21463); (COI); Vila Verde, Cabarelas, 1970, A. Fernandes, R. Fernandes & J.
prov. Orense: Verín. Rabal. Rio Tamega, 1987, Rico & Giráldez s.n. (MA Matos s.n. (COI); Póvoa de Lanhoso, 1894, Sampaio s.n. (COI); France.
466936, MACB 32820, SALA 47079); Parada do Sil, entre Casuia e Basses Pyrenees, Cambo les Bains, Arnaga - La Nive, 1972, P. Montserrat
Baledo, 1996, Amigo, Ortiz, Louzán 762 & Marisa (SANT 034648); s.n. (ZT); Pyrenees Occidentales, Pau, 1991, Nicol, Lys 64260 & Arudy
Allariz, 1981, Amich, Giráldez, Rico & Sánchez s.n. (SALA 32381); (ZT); Pyr. Atlantiques, Vers Taron, 1991, Nicol, Lys & Arudy s.n. (ZT);
Villaviciosa, 1989, Amor & Giráldez s.n. (SALAF 85566); prov. Pyrénées, Les Eaux Bonnes, Rowland, Davis, Hyan & al. s.n. (RNG);
Pontevedra: Villagarcía, 1972, Valdés Bermejo s.n. (MAF 82404); La Pyrenees centrales, Bagnéres, de Luchon, Bourg d’ Oueil, 1992,
Guardia, 1899, Merino s.n. (MA 47522); San Adrián, Vilaboa, 1970, Castroviejo 12050 & al. (MA 512382).
Castroviejo s.n. (MA 196798); Bueu, Monte Ermelo, 1986, García Cardamine crassifolia Pourr.: Spain. Catalogne, Pyrénées à Núria, 1913,
Martínez s.n. (MA 332066); Mirador de Catoira, 1969, Valdes s.n. (MA Sennen s.n. (BC-Sennen 805517); Gerona, Queralbs, vall de Núria, Nou
308739); El Grove, prope Pedras Negras, entre el Istmo de la Lanzada y El Creu, 1993, Monasterio & al. s.n. (MA 528891); Catalogne, Pyrénées à
Grove, 1994, Carrasco, Pajarón & Silva-Pardo s.n. (MA 621680); entre Núria, 1914 (MA 47513, BC-Cadevall 816244); Nuria, come de les
Cuntis y Portela, 1982, Valdés Bermejo & Silva s.n. (MA 529820); rio Mulleres, 1921, Barnades s.n. (BC 603665); Cerdanya, Pr. de La Llagona,
Deza, 1982, Horjales & Redondo s.n. (SANT); prov. Segovia: Cantalejo, 1960, A. de Bolòs & E. de Bolòs s.n. (BC 602596); In Cataloniae
1974, Segura Zubizarreta s.n. (M); Prádena, La Callejuela, 1983, Romero Pyrenaeis, Núria, Nou Fonts, 1943, A. de Bolòs & E. de Bolòs s.n. (BC
s.n. (SALA 37249); Cantalejo, Navacentella, 1983, Romero s.n. (SALA 111454); Núria, Vall de les Mollera, 1958, Vigo, Villar, Recabado & al. s.n.
37248); Frumales, en el Vado de la Vaca, río Cega, 1982, Bayón s.n. (MA (BC 601500); Núria, in Pyrenaeis, 1919, Larriga s.n. (BC 111460); In
321379); Sierra de Guadarrama, Pinar de Valsaín, 1913, Vicioso s.n. (MA Pyrenaeis Cataloniae, Núria, 1880, Mansferrer s.n. (BC 03107); In
47519); Valsain, Cutanda s.n. (MA 47508); Fuente el Olmo de Pyrenaeis Cataloniae, supra Meranges, 1950, Capell (BC 114241);
Fuentidueña, Romero s.n. (MA 567636); entre Cotos y Rascafría, 1974, France. Pyrénées Orbs., Val d’ Eyne, 1919, Sennen s.n. (BC-Sennen);
López & Valdés Bermejo s.n. (MA 315409); Puerto de Somosierra subien- Cerdagne, Font Romeu, 1928, Sennen s.n. (BC-Sennen); Pyrénées Orbs.,
do de Riaza, 1974, Bellot & al s.n. (MA 315500); La Granja, 1992, García Le Capcir à Camporreils, 1919, Sennen s.n. (BC-Sennen); Cerdagne aux
Adá & Lopéz s.n. (MA 562790); Santo Domingo de Pirón, 1987, Egido & Bouillouses, 1916, Sennen s.n. (BC-Sennen); Cerdagne, Vallée
García s.n. (MA 562791); prov. Soria: San Leonardo, 1935, Ceballos s.n. d’ Angoustrine, 1919, Jude-Marie s.n. (MA 47509); Cerdagne, Vallée
(MA 47507); prov. Teruel: Bronchales, Sierra Alta, 1962, Kjellqvist & d’ Angoustrine, 1919, Sennen s.n. (BC-Sennen); Cerdagne, Val de Llo,
Löve 511 (RNG); prope Teruel, López Seoanez s.n. (MA 47525); prov. 1923, Sennen s.n. (BC-Sennen); Espoussouille (Capcir, V. de Galve), 1983,
Valladolid: Rábano, 1983, Romero s.n. (SALA 41872); prov. Vizcaya: P. Montserrat & Villar (MA 257466); Ariège, Etang de Llaurenti, 1984,
Lekeitio, 1983, G. Montserrat & al. s.n. (BIO 1570); Mekoleta, Landolt s.n. (ZT); Pyrenees orient., Val de Galbe, P. del Pla de l’ Ouriet,
Ochandiano, 1983, Gomez s.n. (BIO 1569); Bilbao, 1906, Sennen s.n. (BC- 1984, Landolt s.n. (ZT); Dept. Pyrénees Orientales, Lac de Bouillouses, Et.
Sennen); prov. Zamora: Ribadelago, borde del Camino por Seoanez, del Vivé, 1971, Merxmüller & Zollitsch s.n. (M); Andorra. Andorra, 1992,
1987, Roa s.n. (MA 510717); Ribadelago, 1948, Losa & P. Montserrat s.n. Aedo & al. s.n. (MA 523560).
(BC 114205); Olleros de Tera, Pradera Redonda, 1989, García Río s.n. Cardamine castellana Lihová & Marhold: Spain. prov. Asturias: Puerto
(SALA 56243); Portugal. Beira Litoral: Eixo, Arrujo, 1965, A. de Somiedo entre León y Asturias, 1970, Rivas Goday & Izco s.n. (MA
Fernandes & al. s.n. (M, COI); Aveiro, Eixo, 1954, J. & al. s.n. (COI); 22424); prov. Ávila: Sierra de Gredos, Hoyos del Espino - Refugio del
Aveiro, Requeixo, Pateira de Fermentelos, 1978, Marques s.n. (COI); Rey 1971, Merxmüller & Gleisner s.n. (M); Piedrahita, 1983, Valle &
Ilhavo, zminta da Valenta, 1954, J. Matos, A. Matos & Marques s.n. (COI); Iglesias s.n. (SALAF 72684); Hoyo del Espino, 1983, Ladero & Iglesias
Arredores de Montemór-o Volho, Fôja, 1911, Carvalho s.n. (P, COI); Mata s.n. (SALAF 65070); Puerto de la Peña Negra, 1982, Rico s.n. (SALA
de Fôja, 1942, Lemos s.n. (COI); Aveiro, Azurara, 1954, J. Matos & al. s.n. 26083); Pinar de Hoyocasero, 1987, Giráldez & Rico s.n. (SALA 56244);
(COI, P); Vilarinho da Lousa, Soito, Águas de Albi, 1961, A. Fernandes & prov. Teruel: Orihuela del Tremedal, 1907, Pau s.n. (MA 47518); prov.
al. s.n. (P, COI); Arredores da Figueira da Foz, Villa Verde, 1890, Moller Zamora: Porto, Valle del río Bibey, 1989, Casaseca, Rico & Giráldez s.n.
s.n. (P); Serra de Nogueira, aldeia de Nogueira, 1990, Sequeira & Coelho (MA 466933); Porto, Sierra Segundera, Moncalvillo, 1993, Aldasoro,
68 (MA 557314); pr. Águeda, ponte de Perraes, 1953, Romariz & Mendes Martínez Ortega, Pérez & Rico s.n. (SALA 56245); San Martín de
s.n. (MA 268571); Aveiro, Águeda, em frente de Espinhel, Pateira de Castañeda, 1978, Fernández Diez, Rico & Sánchez s.n. (SALA 25150);
Fermentelos, 1978, Marques s.n. (COI); Aveiro, Águeda, Fermentelos, Porto, Valle del río Bibey, 1989, Casaseca & al. s.n. (SALA 47050).
arredores do Porto da Minhoteira, 1977, Marques & Pereira s.n. (COI);

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