Distribution and Diversity of Cytotypes in Dianthus Broteri As Evidenced by Genome Size Variations

Annals of Botany 104: 965–973, 2009 doi:10.1093/aob/mcp182, available online at www.aob.oxfordjournals.org

Distribution and diversity of cytotypes in Dianthus broteri as evidenced by genome size variations

Francisco Balao*, Ramoń Casimiro-Soriguer, Marıá Talavera, Javier Herrera and Salvador Talavera Departamento de Biologıá Vegetal y Ecologıá, Universidad de Sevilla, Apdo. 1095, E-41080 Sevilla, Spain

Received: 20 April 2009 Returned for revision: 26 May 2009 Accepted: 15 June 2009 Published electronically: 25 July 2009 Downloaded from https://academic.oup.com/aob/article/104/5/965/138784 by guest on 30 September 2021 † Background and Aims Studying the spatial distribution of cytotypes and genome size in plants can provide valu- able information about the evolution of polyploid complexes. Here, the spatial distribution of cytological races and the amount of DNA in Dianthus broteri, an Iberian carnation with several ploidy levels, is investigated. † Methods Sample chromosome counts and flow cytometry (using propidium iodide) were used to determine overall genome size (2C value) and ploidy level in 244 individuals of 25 populations. Both fresh and dried samples were investigated. Differences in 2C and 1Cx values among ploidy levels within biogeographical provinces were tested using ANOVA. Geographical correlations of genome size were also explored. † Key Results Extensive variation in chromosomes numbers (2n ¼ 2x ¼ 30, 2n ¼ 4x ¼ 60, 2n ¼ 6x ¼ 90 and 2n ¼ 12x ¼180) was detected, and the dodecaploid cytotype is reported for the first time in this genus. As regards cytotype distribution, six populations were diploid, 11 were tetraploid, three were hexaploid and five were dodecaploid. Except for one diploid population containing some triploid plants (2n ¼ 45), the remaining populations showed a single cytotype. Diploids appeared in two disjunct areas (south-east and south-west), and so did tetraploids (although with a considerably wider geographic range). Dehydrated leaf samples provided reliable measurements of DNA content. Genome size varied significantly among some cytotypes, and also exten- sively within diploid (up to 1.17-fold) and tetraploid (1.22-fold) populations. Nevertheless, variations were not straightforwardly congruent with ecology and geographical distribution. † Conclusions Dianthus broteri shows the highest diversity of cytotypes known to date in the genus Dianthus. Moreover, some cytotypes present remarkable internal genome size variation. The evolution of the complex is discussed in terms of autopolyploidy, with primary and secondary contact zones.

Key words: Autopolyploidy, Caryophyllaceae, chromosome numbers, contact zones, dried leaves, dodecaploid, ﬂow cytometry, Mediterranean, propidium iodide, refugia.

INTRODUCTION Cytological races within polyploid species can be spatially/ ecologically segregated, which is explained on the basis of Polyploidy, defined as the possession of at least three complete minority cytotype exclusion (Levin, 1975) or varying ecologi- sets of chromosomes, is a widespread phenomenon in the flow- cal tolerances (Van Dijk and Bakx-Schotman, 1997; ering plants. It occurs in 30–35 % of the angiosperms accord- Mandakova and Munzbergova, 2006). Alternatively, several ing to Stebbins (1971), whereas Coghlan et al. (2005) cytotypes can coexist within a population, and this is often estimated that 50–70 % of all species could be polyploid. interpreted as suggestive of a variety of evolutionary/ecologi- Polyploidy has significant effects on the biochemistry, ecophy- cal process that operate simultaneously (e.g. Heuchera grossu- siology and morphology of plants (Levin, 1983; Thompson lariifolia, Segraves et al., 1999; Ranunculus adoneus, Baack, et al., 1997; Levin, 2002; Pearse et al., 2006; Buggs and 2004; Solidago altissima, Halverson et al., 2008). It follows Pannell, 2007), but the most obvious effect is increasing that knowledge on geographical distribution of cytotypes and overall DNA amount following doubling of chromosome sets. their ecological correlates can be useful to give insights on Knowledge on the DNA content of the non-replicated speciation. monoploid genome (i.e. the 1Cx value sensu Greilhuber Carolin (1957) studied ploidy levels in 91 species of the et al., 2005) may be useful in comparisons across polyploid genus Dianthus and found most (67 %) were diploid, even complexes (Smarda et al., 2008a). It seems monoploid though polyploidy was not uncommon: tetraploid species genome size can respond to polyploidization in different amounted to 18.7 %, hexaploids to 6.6 %, and the remaining ways, as some studies show decreased 1Cx values following (7.7 %) presented several cytotypes. In the section Plumaria polyploidization, others document enlarged values (Ozkan of Dianthus to which D. broteri belongs, a later study by et al., 2003; Leitch and Bennett, 2004; Bennetzen et al., Weiss et al. (2002) showed a majority of species containing 2005), and still others report constant 1Cx values within poly- two or three ploidy levels. Tetraploids and hexaploids were ploid series (Ohri, 1998; Ozkan et al., 2006). Therefore, most frequent in the section, whereas pentaploids, triploids detecting patterns of change in 1Cx values within a given taxo- and diploids were relatively rare. Furthermore, 2x cytotypes nomic group may allow for inferences about phylogenetic appeared mainly in isolated glacial refugia and were less history. common than 4x and 6x. Weiss et al. (2002) also reported a * For correspondence: E-mail [email protected] few cases of populations composed of cytotype mixtures. # The Author 2009. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] 966 Balao et al. — Ploidy levels in Dianthus broteri

Dianthus broteri is a subshrub with large, fragrant flowers MATERIALS AND METHODS pollinated by crepuscular hawkmoths (F. Balao, unpubl. Sampling res.). Endemic to the Iberian Peninsula, it invariably lives in locations close to the coast from the south of Portugal to the During the summers of 2006 and 2007, 244 individuals chosen east of Spain (Fig. 1). Although some authors (Boissier and at random were sampled in 25 populations of Dianthus broteri Reuter, 1852; Lemperg, 1936; Bernal et al., 1990) considered Boiss. & Reut. distributed around the distribution range of the D. broteri a close relative of North African D. serrulatus Desf., species (Fig. 1). This range covers four biogeographical pro- recent sequencing studies of the two taxa using ITS and vinces (Rivas-Martıńez et al., 2004) which present different cpDNA (P. Vargas et al., RJB, Madrid, Spain, unpubl. res.) types of the Mediterranean climate. For example, the Coastal indicate that they are only distantly related. In D. broteri Lusitan-Andalusian province is markedly influenced by the both tetraploid and octoploid populations are known Atlantic Ocean and as a result enjoys relatively benign eco-

(Carolin, 1957; Coy et al., 1997), and the latter have been logical conditions with regard to rainfall and temperature (no Downloaded from https://academic.oup.com/aob/article/104/5/965/138784 by guest on 30 September 2021 described by Gallego (1986) as a ‘splinter’ species winter frosts). The adjoining Betic province is bioclimatically (D. inoxianus Gallego, with 2n ¼ 120). heterogeneous and composed of plains and mountain ranges In the present study, an analysis of the cytotypes of bordering the Guadalquivir River. With dry to perhumid D. broteri s.l. is carried out using chromosome counts and regimes, biodiversity is accordingly high in this region, flow cytometry. The latter technique is more and more used which also has high rates of paleoendemism. East of the in studies of genome size and polyploidy, its main advantage Betic province, the Murcian-Almeriensian region presents being that it allows the analysis of large numbers of randomly subarid to arid conditions and relatively cold winters. Lastly, chosen individuals (Kron et al., 2007). Furthermore, it can be the Balearic-Catalan-Provençal province (partly insular) has performed on dehydrated plant material in some species a coastal variety of the Mediterranean climate and rugged (although care must be used in applications other than ploidy topography. All four provinces share high rates of human dis- determination; Suda and Travnicek, 2006). Many recent turbance (e.g. wildfires are common), although the intensity of reports support flow cytometry as a valid technique to assess perturbations tends to decrease from lowlands to mountains. intraspecific genome size variations (e.g. Pecinka et al., Precise details of the localities and populations are provided 2006; Walker et al., 2006; Smarda et al., 2008b). The main in the Table 1. At each site, seeds and leaves were collected goals were: (a) to ascertain which and how many levels of from the plants and preserved in individual, numbered contain- ploidy exist, as well as their geographical distribution; (b)to ers. Leaves were then stored in silica gel, and seeds sown in estimate genome size and investigate any possible relation- individual pots in the greenhouse of the University of ships with environmental parameters; (c) to make reasonable Seville. Voucher specimens for all populations are deposited inferences on the evolutionary history of polyploidy in this at the Herbarium of the University of Seville (SEV, Spain). complex, based on monoploid genome size and geographic distribution data. Chromosome counts Seeds from 59 plants of nine populations (Table 1) were ger- minated on moist filter paper in a germination chamber at 25 8C, under a 16 : 8 light : darkness regime. Root tips 1–3 cm long were collected and treated with 0.002 M 8-hydro- xyquinoline for 3 h at 4 8C, then fixed in 3 : 1 ethanol : acetic acid at 4 8C for a minimum of 24 h. Following staining with alcoholic hydrochloric acid-carmine (Snow, 1963) at 30 8C for 3 d, root tips were squashed on a microscope slide. Photomicrographs of chromosomes at mitotic metaphase were taken with a 63 Carl-Zeiss Axiophot photomicroscope 25 23 24 (Carl-Zeiss Inc., Germany) equipped with Sony DXC-390P 22 21 Exwave HAD (Tokyo, Japan). Snapshots were exported and 20 studied using image analyser software. 19 1 2 18 Flow cytometry measures 6 17 7 3 4 5 8 10 13 Ploidy 14 15 16 The absolute nuclear DNA content of 244 individuals, 9 12 2x Atlantic Ocean (6)8–10(19) per population, was determined by flow cytome- 11 4x try (FCM hereafter) between February and May of 2008. Most 6x 0 50 100 150 200 250 300 km Mediterranean Sea often fresh leaf material taken from greenhouse-grown seed- 12x lings was used, but for a few populations in which there was no alternative (see Table 1) silica-dried leaves collected in F IG. 1. Distribution of 25 Dianthus broteri populations, and delimitation of the field were employed. biogeographical provinces. CLA, Coastal Lusitan-Andalusian province; BET, Betic province; MA, Murcian-Almeriensian province; BCP, Balearic- Nuclear suspensions for FCM were prepared following the Catalan-Provençal province. Symbols show diploids, tetraploids, hexaploids protocol of Dolezel et al. (1989). Approximately 100 mg of and dodecaploids as indicated. leaf tissue were chopped with a razor blade onto a Petri dish Downloaded fromhttps://academic.oup.com/aob/article/104/5/965/138784bygueston30September2021

TABLE 1. Geographical data of studied Dianthus broteri populations, and DNA content as estimated by ﬂow cytometry

2C DNA content (pg) REGION Biogeographic DNA ploidy Population number, name, elevation province* Co-ordinates Collector no. n Mean + s.d. Min–max level (n) 1Cx DNA (pg) Material Standard

PORTUGAL Pop. 1. Troia, 15 m CLA 388250N/88490W S.T. et al. 259/06 10 3.50 + 0.03 3.46–3.54 4x (3) 0.88 Fresh leaves Pisum Pop. 2. Comporta, 25 m CLA 388180N/88440W S.T. et al. 256/06 10 3.49 + 0.03 3.46–3.55 4x 0.87 Fresh leaves Pisum Pop. 3. Monte Cle´rigo, 43 m CLA 378200N/88510W S.T. et al. 232/06 14 1.93 + 0.04 1.86–1.99 2x (13) 0.97 Both Pisum Balao Pop. 3. Monte Cle´rigo, 43 m CLA 378200N/88510W S.T. et al. 232/06 5 2.90 + 0.04 2.86–2.96 3x 0.97 Silica-dried Pisum Pop. 4. SaõBra´s de Alportel, 218 m CLA 378090N/78500W S.T. et al. 213/06 10 1.83 + 0.03 1.78–1.86 2x 0.92 Fresh leaves Pisum tal. et SPAIN,GUADALQUIVIR VALLEY Pop. 5. Valverde, 250 m CLA 378300N/68400W S.T. et al. 239/07 10 9.47 + 0.22 9.20–9.86 12x 0.79 Fresh leaves D. broteri 4x Pop. 6. Moguer, 24 m CLA 378090N/68490W S.T. et al. 328/06 10 9.67 + 0.17 9.36–9.86 12x 0.81 Fresh leaves D. broteri 4x in levels Ploidy — Pop. 7. Hinojos, 118 m CLA 378180N/68260W S.T. et al. 848/05 10 9.74 + 0.12 9.55–9.92 12x (8) 0.81 Fresh leaves D. broteri 4x Pop. 8. Donãna, Acebroń, 11 m CLA 378070N/68310W S.T. et al. 845/05 10 9.66 + 0.18 9.40–9.96 12x 0.81 Fresh leaves D. broteri 4x Pop. 9. Donãna, Puntal, 6 m CLA 368580N/68260W S.T. et al. FA/05 8 9.76 + 0.07 9.66–9.84 12x 0.81 Fresh leaves D. broteri 4x Pop. 10. Donãna, Peladillo, 41 m CLA 378050N/68350W S.T. et al. 333/06 10 3.66 + 0.05 3.57–3.74 4x 0.92 Fresh leaves Pisum

SPAIN,BETIC RANGES Pop. 11. Chiclana, 44 m CLA 368230N/68070W S.T. et al. 829/05 10 3.60 + 0.04 3.50–3.64 4x (5) 0.90 Fresh leaves Pisum Pop. 12. Ronda, 672 m BET 368450N/58100W S.T. et al. 827/05 10 3.52 + 0.09 3.44–3.73 4x (5) 0.88 Fresh leaves Pisum

Pop. 13. Zafarraya1, 1025 m BET 368590N/48110W S.T. et al. 335/06 10 1.77 + 0.03 1.70–1.80 2x 0 967 .89 Fresh leaves Pisum broteri Dianthus Pop. 14. Zafarraya2, 856 m BET 368560N/48070W S.T. et al. 336/06 10 1.76 + 0.02 1.72–1.78 2x (14) 0.88 Fresh leaves Pisum Pop. 15. Orgiva, 370 m BET 368530N/38240W S.T. et al. 338/06 9 1.78 + 0.02 1.75–1.82 2x (4) 0.89 Fresh leaves Pisum Pop. 16. Laroles, 1024 m BET 378000N/38010W S.T. et al. 341/06 10 1.78 + 0.03 1.74–1.83 2x 0.89 Fresh leaves Pisum Pop. 17. Cartagena, 210 m MA 378350N/08570W S.T. et al. 280/07 10 5.11 + 0.05 5.02–5.19 6x 0.85 Silica-dried Pisum Pop. 18. San Miguel de Salinas, 54 m MA 378580N/08450W S.T. et al. 276/07 9 5.15 + 0.07 5.00–5.22 6x (4) 0.86 Fresh leaves Pisum Pop. 19. Pen˜o´n de Ifach, 2 m MA 388380N/08040E S.T. et al. 268/07 8 5.50 + 0.10 5.32–5.61 6x 0.92 Silica-dried Pisum

SPAIN,IBERIC MOUNTAINS Pop. 20. Albufera de Valencia 1, 2 m BCP 398200N/08190W S.T. et al. 264/07 9 3.34 + 0.06 3.25–3.43 4x 0.84 Silica-dried Pisum Pop. 21. Albufera de Valencia 2, 1 m BCP 398230N/08190W S.T. et al. 267/07 10 3.41 + 0.06 3.35–3.51 4x 0.85 Silica-dried Pisum Pop. 22. Lliria, 175 m BCP 398370N/08360W S.T. et al. 247/07 9 3.42 + 0.06 3.33–3.55 4x 0.86 Silica-dried Pisum Pop. 23. Alcublas, 950 m BCP 398500N/08410W S.T. et al. 250/07 9 3.26 + 0.09 3.09–3.36 4x (3) 0.82 Fresh leaves Pisum Pop. 24. Azuebar, 336 m BCP 398500N/08200W S.T. et al. 254/07 8 3.40 + 0.04 3.36–3.48 4x 0.85 Silica-dried Pisum Pop. 25. Sierra de Espada´n, 580 m BCP 398530N/08200W S.T. et al. 259/07 6 3.12 + 0.03 3.07–3.16 4x 0.78 Fresh leaves Pisum

n is the number of plants analysed. DNA ploidy level is followed (in parenthesis) by the number of karyological preparations analysed. * Rivas-Martıńez et al. (2004): CLA, Coastal Lusitan-Andalusian province; BET: Betic province; MA, Murcian-Almeriensian province; BCP, Balearic-Catalan-Provençal province. 968 Balao et al. — Ploidy levels in Dianthus broteri containing 1 mL of LB01 buffer. The resulting suspension was greater than 5.5 % (either within a population, or within a filtered through 30-mm mesh CellTric disposable filter (Partec given ploidy level) were run simultaneously to confirm that GmbH, Mu¨nster, Germany), and stained with 50 mgmL21 of the divergences were not due to methodological artefacts. propidium iodide and 50 mgmL21 RNase. Until analysis, samples were kept on ice and protected from light. FCM measurements were taken at facilities of the Biology Statistical analyses Research Services (CITIUS, University of Seville), using a Differences in ploidy levels among populations and biogeo- Coulter CYTOMICS FC500-MPL (Beckman Coulter, graphical provinces were evaluated using analysis of variance Fullerton, CA, USA) equipped with a 20-mW argon-ion (ANOVA) and Tukey’s HSD post-hoc tests. Dependent vari- laser at 488 nm. ables for the analyses were absolute DNA content (2C The primary internal standard used to determine DNA value) and monoploid genome size (1Cx). Relationships amount was Pisum sativum ‘Ctirad’, a taxon with 9.09 pg of between relative genome size (population means) and geo- Downloaded from https://academic.oup.com/aob/article/104/5/965/138784 by guest on 30 September 2021 2C nuclear DNA. For dodecaploid material, and because graphic variables (longitude, latitude and altitude) were inves- peaks overlapped, a tetraploid specimen of D. broteri was tigated using Spearman’s rank correlations. A Mantel test used as a secondary standard which was calibrated against based in Spearman’s rank correlation with 1000 permutations P. sativum. The genome size for this tetraploid specimen was used to test the relationship between geographical distance was 2C ¼ 3.46 pg (nine replicate measurements performed and monoploid genome size. All analyses were performed with on different days). Peak means were established through R statistical software (R Development Core Team, 2008). manual gating using CXP software (Beckman Coulter). Overall, 732 FCM measurements were performed, with three replicates per plant. Replicate measurements were RESULTS taken on different days in order to minimize instrumental Chromosomes counts drift. Except for six readings from five individual plants which were discarded due to poor sample quality (i.e. Photomicrographs of chromosome complements are presented ,1000 nuclei per peak), all measurements were used for ana- in Fig. 2. Each population had a single level of ploidy, which lyses (i.e. within-plant extreme values were not dismissed). could range between 2n ¼ 30 (pop. 3; Fig. 2A), 2n ¼ 60 (pops Absolute DNA content was calculated for each sample as the 1, 11, 12, 23; Fig. 2B), 2n ¼ 90 (pop. 18; Fig. 2C) and 2n ¼ average of the three replicates, and monoploid genome size 180 (pop. 8; Fig. 2D). As the basic number in the genus (1Cx) estimated as the amount of nuclear DNA divided by Dianthus is x ¼ 15 (Carolin, 1957), these chromosome ploidy level. A few samples with genome size variation numbers would correspond, respectively, to diploid, tetraploid,

F IG. 2. Metaphase chromosomes from Dianthus broteri s.l. root tips: photomicrographs of (A) a diploid (2n ¼ 30) from population 3 (the arrow indicates a satellited chromosome); (B) a tetraploid (2n ¼ 60) from population 12; (C) a hexaploid (2n ¼ 90) from population 24; (D) a dodecaploid (2n ¼ 180) from population 7. Scale bars ¼ 10 mm. Balao et al. — Ploidy levels in Dianthus broteri 969 hexaploid, and dodecaploid levels. Chromosomes were of The geographic distribution of cytotypes as estimated from small size (1–2.5 mm), metacentric or submetacentric. FCM is shown in Fig. 1. Two disjunct areas with diploid popu- Satellited chromosomes were observed in all of the cytotypes lations were apparent, one in south Portugal (pops 3 and 4) and observed. another along the Betic Ranges in S Spain (pops 13–16). Tetraploid cytotypes were widespread (11 populations, or 44 % of total) and occupied two disjunct areas in east and Flow cytometry and reliability of measurements south-west Iberia. In contrast, hexaploid and dodecaploid cyto- Genome size and ploidy levels in 25 populations of types were relatively rare and spatially restricted, the former to Dianthus broteri are shown in Table 1. In general, all ploidy an area east of the Betic Ranges (pops 17–19) and the latter to levels showed clear DNA peaks (Fig. 3), with observed the lower Guadalquivir River valley (pops 5–9). Except for ratios of 1.00 : 1.56 : 2.06 : 2.88 : 5.58, respectively, for 2x, one population with diploid and triploid plants (pop. 3), the

3x,4x,6x and 12x cytotypes. FCM measurements revealed remaining populations showed a single cytotype. Downloaded from https://academic.oup.com/aob/article/104/5/965/138784 by guest on 30 September 2021 that most nuclei were in G0/G1 phase, although in young leaf samples (i.e. growing very actively) a considerable proportion Genome size variation of nuclei in phase G2 existed. As the DNA content of G2 nuclei invariably doubled that of the G0/G1 phase, it seems safe to As expected, ploidy level was a major determinant of DNA assume that measurements of DNA content were linear. amount (Fig. 4A; ANOVA: F4,239 ¼ 22105; P , 0.001), and Measurement error (calculated as the standard deviation of the mean 2C value was significantly different among any three replicates per individual) was smaller than 0.015 in two cytotypes. For any ploidy level, the DNA content varied 95 % of cases, so this value was assumed to represent the more among than within populations (ANOVA: F25,218 ¼ true deviation of individual measurements at P ¼ 0.05. A 104.9; P , 0.001). negative correlation existed between the sample coefficient As listed in Table 1, the amount of 2C DNA varied . . . of variation (CV) and ploidy level (Spearman’s gs ¼ –0.79, 5 86-fold, from 1 70 pg in diploids (2n ¼ 30) to 9 96 pg in P , 0.001), but this was likely to be a methodological artefact, as samples were always run with the same instrument settings (see Discussion). A e The CV for peaks varied between 1.4 % and 4.5 % (mean + 10 standard deviation, 3.64 + 1.35 %) and, in general, fresh ¼ material yielded significantly better readings (average CV 8 2.8 %) than dehydrated samples (CV ¼ 4.5 %; Mann– U ¼ P , . Whitney 1727, 0 001). Nevertheless, drying did not d modify DNA amount estimations in any important ways: in 6 a subset of nine populations including dried and fresh 2C DNA (pg) 4 c samples that were analysed with ANOVA, the 1Cx value was b independent of tissue state (F1,68 ¼ 1.263, n.s.; for the popu- a lation effect, F8,68 ¼ 33.150, P , 0.001; note, however, that 2 the interaction term could not be tested in this case). For the only population which had both dried and fresh leaves analysed (pop. 3), means were statistically indistinguishable (two- B samples t test, t ¼ 0.605, d.f. ¼ 12, n.s.). a 1·00 b

Ploidy Channel CV 200 2x 2x 50 4·12 % 0·95 cc

12x 3x 78 3·42 % 150 4x 103 3·97 % 0·90 6x 144 3·25 %

4x 12x 279 2·20 % 500 1Cx DNA (pg) 0·85 d 100 3x * 6x 400 300 0·80

Number of nuclei 50 200 100 2x 3x 4x 6x 12x 0 100 200 300 400 500 0 Ploidy 0 50 100 150 200 250 300 350 400 450 500 Fluorescence (channel number) F IG. 4. Box-plot representation of total genome size variation (A) and monoploid DNA amount (B) in different cytotypes of Dianthus broteri. Means not F IG. 3. Histograms of fluorescence intensity of propidium iodide-stained significantly different at P ¼ 0.05 are indicated by the same letter (Tukey’s nuclei in Dianthus broteri cytotypes. For clarity, the histogram for triploids HSD test). Horizontal lines represent the median, and boxes and whiskers, and the standard (asterisk, Pisum sativum ‘Ctirad’) is presented as an insert. respectively, the interquartile range and the non-outlier ranges. Circles Coefficients of variation (CV) for each of the five ploidy levels are also given. denote outliers. 970 Balao et al. — Ploidy levels in Dianthus broteri

300 A B 335/06_4 232/06_2 333/06_5 259/07_1 Peak ratio 1·196 Peak ratio 1·046

200 Sample Channel CV Sample Channel CV

259/07_1 95·3 2·37 335/06_4 169·46 2·07

333/06_5 114·0 2·34 232/06_2 177·56 2·14 100 Number of nuclei Downloaded from https://academic.oup.com/aob/article/104/5/965/138784 by guest on 30 September 2021

0 50 100 150 200 250 50 100 150 200 250 300 350 400 Fluorescence (channel number) Fluorescence (channel number)

F IG. 5. Histograms of fluorescence intensity in tetraploid (A) and diploid (B) Dianthus broteri cytotypes. The graph depicts divergences in DNA content among populations (represented by bifurcated peaks) within each cytotype. For clarity in the representation, diploids were measured on higher channels. dodecaploids (2n ¼ 180). Yet this variation was not solely due a to ploidy level, as 2C DNA amount could also vary to some 1·00 a extent within cytotypes. For diploid plants, for example, the 2C value varied between 1.70 and 1.99 pg (i.e. a 1.17-fold 0·95 b b b . . b variation); triploids ranged from 2 86 to 2 90 pg (about 0·90 d 1.03-fold); tetraploids from 3.07 to 3.74 pg (1.22-fold); hexaploids from 5.00 pg to 5.61 pg (1.12-fold); and dodecaploids 0·85 c . . . 1Cx DNA (pg) from 9 20 to 9 96 pg (1 08-fold). 0·80 The monoploid DNA amount (1Cx) ranged between 0.78 pg and 1.00 pg and, according to ANOVA, was significantly 2x 3x 4x 12x 2x 4x 6x 4x dependent on ploidy level (F4,239 ¼ 81.65, P , 0.001). Post-hoc comparison tests failed to reveal any significant ian- Betic nsianearic- ençal Coastal Murc Bal Catalan- differences in 1Cx value between tetraploids and hexaploids Prov (Fig. 4B). In general, the amount of monoploid DNA was Almerie inversely related to ploidy level (i.e. 2x,3x,4x,6x and 12x Lusitan-Andalusian cytotypes, respectively, had mean 1Cx values of 0.91, 0.96, Biogeographic provinces 0.86, 0.87 and 0.81 pg). . F IG. 6. Box-plot graph of monoploid genome size (1Cx value) in Dianthus Variation in genome size was usually ,5 5 % within popu- broteri cytotypes from four biogeographical provinces. Means not significantly lations. In the few examples with higher variability (up to 8.7 different at P ¼ 0.05 are indicated by the same letter (Tukey’s test). Horizontal % in populations 3, 5, 12 and 24), simultaneous FCM analyses lines represent the median, boxes span the interquartile range, and whiskers the of the samples confirmed that these unusual values had been non-outlier ranges. Circles denote outliers. caused by methodological artefacts. Additionally, concurrent runs demonstrated substantial among-population differences ANOVA for an assessment of monoploid genome size vari- (within diploids and tetraploids) which resulted in distinct flu- ation (F7,237 ¼ 121.85, P , 0.001). According to post-hoc orescence peaks (Fig. 5). tests, Coastal Lusitan-Andalusian diploids and triploids were indistinguishable as regards monoploid DNA (overall mean . . Geographic scale of genome size variation (1Cx) 0 95 + 0 03 pg), and so were Coastal Lusitan-Andalusian tetraploids, Betic diploids and tetraploids, as well as In general, monoploid DNA content was unrelated to altitude, Murcian-Almeriensian hexaploids (0.89 + 0.03 pg). Balearic- latitude or longitude. Correlations between elevation and mono- Catalan-Provençal tetraploids (0.83 + 0.03 pg) and dodecaploid DNA amount were largely non-significant, although sig- ploids (0.81 + 0.02 pg) presented 1Cx values that were in nificant relationships were detected (for tetraploids only) with both cases significantly smaller than those of the remaining latitude (r ¼ –0.89, n ¼ 11) and longitude (r ¼ –0.69, n ¼ populations. 11). This implied increasing 1Cx values from north to south and from east to west. A Mantel test failed to detect any broad spatial autocorrelations for monoploid DNA amount (gM ¼ DISCUSSION 0.10, n.s.), although some structuring existed at smaller spatial Cytotype diversity scales (e.g. populations within 100 km of each other; Mantel correlogram gM ¼ 0.22, P ¼ 0.002). Many species in the subfamily Caryophylloideae have diploid Combining cytotypes and biogeographic provinces resulted and tetraploid cytotypes, although only Silene ciliata Pourret in eight groups (presented in Fig. 6) that were subject to was known previously to present a long polyploid series Balao et al. — Ploidy levels in Dianthus broteri 971

(2x,3x,4x,10x,14x,16x,18x,20x and 30x;Ku¨pfer, 1974). D. gratianopolitanus; table 1 in Suda and Travnicek, 2006). The present data document the most extensive polyploid If this condition is met, dried samples can be the most suitable series known to date for Dianthus, with five ploidy levels material to study absolute genome size in large numbers of (2x,3x,4x,6x and 12x). The complexity of this series is com- plants, or even herbarium vouchers. Suda and Travnicek parable to those reported in Senecio carniolicus (Suda et al., (2006) used DAPI in their study, but the present data indicate 2007) and Claytonia perfoliata (Miller, 1976). that propidium iodide can also be used to obtain reliable Dianthus broteri was previously considered a tetraploid measurements. taxon, and this was actually the most common cytotype in As regards spatial and ecological correlates, the effects of the present study (44 % of all populations). The relatively elevation and geographical location on genome size were in rare dodecaploid cytotype (2n ¼ 180) was restricted to five general weak. Size varied significantly with latitude/longitude populations west of the Guadalquivir Valley, and is described only in the tetraploids, although not continuously (rather, the

here for the first time. Gallego and Talavera (1993) reported a correlation was due to clustering in southern and eastern Downloaded from https://academic.oup.com/aob/article/104/5/965/138784 by guest on 30 September 2021 plant with 2n ¼ 8x ¼ 120 chromosomes at one of the dodeca- areas). Furthermore, biogeographical provinces did not show ploid sites (pop. 7), but since no octoploids were found at the clear-cut differences as regards genome size (Fig. 6), study sites their count might be incorrect. making unlikely a role of soil conditions and/or climate (cf. Bituminaria bituminosa, Walker et al., 2006; Ceratonia siliqua, Bures et al., 2004; Festuca pallens, Smarda and Genome size variation Bures, 2006). Lastly, genome size followed the general trend In general, the present study revealed considerable intraspe- (Leitch and Bennett, 2004) to become smaller with increasing cific variations in genome size, but these cannot be automati- ploidy levels (Fig. 4B), although differences were statistically cally assumed to be real. Greilhuber (2005) demonstrated that significant in a few cytotypes only (for example, dodecaploids methodological artefacts can contribute to variability, so one had the lowest 1Cx value whereas diploids showed the must be cautious with interpretations. We believe within- highest). cytotype variation reported in the present study for D. broteri (up to 1.22-fold in simultaneously run tetraploids; Genome size and cytotype evolution Fig. 5) should be considered genuine, as our samples were from young plants, grown in a common environment, repli- Information on genome size given in this study, combined cated well (standard deviation ¼ 0.015) and had low coeffi- with geographical distribution of cytotypes, can provide cients of variation (3.64 % on average, thus making any some insight on the origin and evolution of the polyploid effect of secondary metabolites on measurements unlikely). series. In short, there were five ploidy levels (including tri- In contrast, within-population variability (5.5 % on average) ploids) which also present some internal variability as regard could not be established firmly through simultaneous runs. DNA amount. The most parsimonious explanation for the The likely reason behind was insufficient sensitivity of the fact that diploid, tetraploid and hexaploid populations along method; as shown by Benson and Braylan (1994) and the Betic Ranges, as well as tetraploid populations of the Dolezel and Go¨hde (1995), two individual peaks can be distin- Atlantic coast were all alike in terms of monoploid DNA guished only if they differ in an amount that doubles their amount (Fig. 6) is that most of these cytotypes originated by coefficients of variation. In the present study this means differ- autopolyploidy. Furthermore, the differently sized genomes ences greater than 5.6 (CVs were around 2.8 at best in the of Coastal Lusitan-Andalusian diploids (i.e. Serra de present study), so within-population variability remained Monchique and Serra do Caldeiraõ) vs. those of Betic areas undemonstrated. (i.e. Sierra Nevada and Sierra Tejeda-Almijara) could be a Another point that requires interpretation in the present data symptom of prolonged separation among them. Confinement was the correlation between the coefficients of variation of of diploid cytotypes to southern areas has also been observed samples and levels of ploidy (see Results). This was certainly in other Dianthus species (Weiss et al., 2002) and suggests a by-product of the fact that low ploidy levels were read (as it these areas may have acted as refugia during Pleistocene gla- is often done) on lower channels (i.e. with lower voltage) of ciations. Actually, these two geographic areas have been the flow cytometer. These channels are invariably less recognized previously as refugia for other species including precise and, as instrumental settings were kept constant trees (e.g. Quercus spp., Olalde et al., 2002; Petit et al., throughout, relatively higher internal variation could not be 2002; Pinus sylvestris, Sinclair et al., 1999) and herbs (e.g. avoided in low polyploid samples. Senecio gallicus, Comes and Abbott, 1998; Armeria spp., The use of dry material to study ploidy levels is relatively Larena et al., 2006). infrequent, and has been subject to criticism. In their compara- It is also noteworthy that southern and eastern tetraploid tive study, Suda and Travnicek (2006) showed that results are populations had significantly different 1Cx values (Fig. 6). to a great extent species-dependent, and concluded that fresh This suggests they may have experienced different evolution- material should always be preferred over dried samples for ary histories and/or independent polyploidization events, a studies of absolute genome size (mostly because drying notion supported by results of experimental crosses decreases fluorescence and a parallel decline in measurement (F. Balao; unpubl. res.) between 4x plants from the Betic reliability; i.e. coefficients of variation get larger). However, Ranges (pop. 11) and eastern Spanish counterparts in taxa which do not experience strong decreases in fluor- (pop. 25). In these crosses, only 42.8 % of ovules were trans- escence (like D. broteri in the present study) measurements formed into mature seeds which either produced achlorotic of dehydrated leaves can still be valid (see also cotyledons or failed completely to germinate. A solid 972 Balao et al. — Ploidy levels in Dianthus broteri reproductive barrier seems to exist between tetraploid cyto- D. inoxianus, the splinter species described by Gallego types which, in addition, show a number of morphological (1986)]. In summary, results of the present study indicate differences: southern tetraploids have smaller flowers and that there is a diversity of processes in the evolution of less laciniated petals than eastern tetraploids (described in D. broteri cytotypes, but more research and molecular the past as a different species, D. valentinus; Willkomm, studies are still needed to get a clearer picture of the phylogeo- 1859). The different 1Cx values of eastern and southern tetra- graphy and evolution of the complex. ploids could also be due to introgression with other taxa, although this hypothesis has yet to be tested. Some authors (Bernal, 1989; Bernal et al., 1990) have suggested introgres- ACKNOWLEDGEMENTS sion between eastern D. broteri tetraploids and We thank Dr J. Dolezel for supplying the standard Pisum D. multiaffinis Pau, a species that also occurs in the Iberic seeds, and M. Carballo and A. Garcıá-Quintanilla for technical Mountains. Introgression is not uncommon in Dianthus assistance with the cytometry. Dr P. Gibbs and two anonymous Downloaded from https://academic.oup.com/aob/article/104/5/965/138784 by guest on 30 September 2021 (Andersson-Kotto¨, 1931; Carolin, 1957; Nimura et al., 2003, reviewers provided invaluable comments on a previous version 2008), and Gatt et al. (1998) reported that crossings of of the manuscript. This study was supported by a predoctoral D. caryophyllus (2x) and D. plumarius (6x), or D. plumarius grant to F. Balao from Spanish Ministerio de Educaciońy (6x) and D. knappi (2x) produce tetraploid hybrids. Ciencia (AP2005-4314); Junta de Andalucıá, Proyecto de Excelencia (2005/RNM848) and Ministerio de Educaciońy Cytotype distribution and contact zones Ciencia, Flora Iberica 7(2)[CGL2006-00817]. The Donãna National Park (Patronato del Parque Nacional de Donãna), As a rule, the populations studied contained a single cyto- gave permission to access to several sampling sites (project type. This may indicate an effect of minority cytotype exclu- 34/2004). sion (i.e. relatively rare cytotypes are prone to disappear in mixed populations; Levin, 1975) and/or a widespread lack of gene-flow between ploidy levels in nature. In Centaurea LITERATURE CITED jacea (Hardy et al., 2001) and Chamerion angustifolium (Husband and Sabara, 2004) plants with different levels of Andersson-Kotto¨ I. 1931. Interspecific crosses in the genus Dianthus. Genetica 13: 77–112. ploidy are effectively isolated reproductively, and this may Baack EJ. 2004. Cytotype segregation on regional and microgeographic scales also apply to D. broteri. in snow buttercups (Ranunculus adoneus: Ranunculaceae). American The above rule had two exceptions, namely (1) neighbour- Journal of Botany 91: 1783–1788. ing dodecaploid and tetraploid populations of the lower Bennetzen JL, Ma JX, Devos K. 2005. Mechanisms of recent genome size variation in flowering plants. Annals of Botany 95: 127–132. Guadalquivir valley, and (2) one population from Portugal Benson NA, Braylan RC. 1994. Evaluation of sensitivity in DNA aneuploidy (Monte Cle´rigo, pop. 3) which had a majority of diploids detection using a mathematical model. Cytometry 15: 53–58. along with some triploid plants. These two cases of cytotype Bernal M. 1989. Ma´s sobre el geńero Dianthus L. (Caryophyllaceae). Anales coexistence could be reasonably explained in terms of del Jardıń Botańico de Madrid 45: 574–575. ‘primary’ or ‘secondary’ contact zones. According to Petit Bernal M, Laıńz M, Munõz Garmendia F. 1990. Dianthus L. In: Castroviejo S. ed. Flora Iberica. Madrid: CSIC. et al. (1999), a primary contact zone is an area where a cyto- Boissier PE, Reuter GF. 1852. Pugillus plantarum novarum Africae borealis logical race gives rise to another, and then the two coexist Hispaniaeque australis. Genevae: ex typographia Ferd. Ramboz et socii. sympatrically. This seems the case of the Portuguese popu- Buggs RJA, Pannell JR. 2007. Ecological differentiation and diploid super- lation composed of a majority of diploids together with a iority across a moving ploidy contact zone. Evolution 61: 125–140. Bures P, Pavlicek T, Horova L, Nevo E. 2004. Microgeographic genome size few triploids which (we hypothesize) could have originated differentiation of the carob tree, Ceratonia siliqua, at ‘Evolution Canyon’, locally by fusion of (diploid) unreduced gametes. In accord- Israel. Annals of Botany 93: 529–535. ance with this view the amounts of monoploid DNA of the Carolin RC. 1957. Cytological and hybridization studies in the genus coexisting cytotypes were identical. Dianthus. New Phytologist 56: 81–97. In turn, neighbouring dodecaploid and tetraploid races in the Coghlan A, Eichler EE, Oliver SG, Paterson AH, Stein L. 2005. Chromosome evolution in eukaryotes: a multi-kingdom perspective. lower Guadalquivir valley could be best explained in terms of Trends in Genetics 21: 673–682. a secondary contact zone (sensu Petit et al., 1999; i.e. neither Comes HP, Abbott RJ. 1998. The relative importance of historical events and of the cytotypes can be claimed to have acted as a parent of the gene flow on the population structure of a Mediterranean ragwort, Senecio other and, due to genetic differences, plants become relatively gallicus (Asteraceae). Evolution 52: 355–367. Coy E, Tamayo MJ, Carrillo AF, Sańchez-Go´mez P. 1997. Nu´meros cromo- intersterile). Secondary contact zones have been described in soma´ticos de plantas occidentales, 746–750. Anales del Jardıń Botańico Aster amellus agg. (Mandakova and Munzbergova, 2006), de Madrid 52: 430. Knautia arvensis agg. (Kolar et al., 2009) and other polyploid Dolezel J, Go¨hde W. 1995. Sex determination in dioecious plants Melandrium complexes (Thompson and Lumaret, 1992). In the particular album and M. rubrum using high-resolution flow cytometry. Cytometry case of D. broteri, the dodecaploid and tetraploid races seem 19: 103–106. Dolezel J, Binarova P, Lucretti S. 1989. Analysis of nuclear-DNA content in largely unrelated to each other since they differ as regards plant-cells by flow-cytometry. Biologia Plantarum 31: 113–120. monoploid DNA amount (Fig. 4B). Furthermore, numerous Gallego MJ. 1986. Una nueva especie de Dianthus del litoral del SW de ex situ and in situ attempts at crossing dodecaploid and tetra- Espanã. Lagascalia 14: 71–72. ploid plants invariably failed (F. Balao; unpubl. res.), which Gallego MJ, Talavera S. 1993. Nu´meros cromosoma´ticos de plantas Occidentales, 696–707. Anales del Jardıń Botańico de Madrid 51: 280. suggests they are reproductively isolated. It is also remarkable Gatt MK, Hammett KRW, Markham KR, Murray BG. 1998. Yellow that dodecaploid D. broteri have scabrid stems, a feature not pinks: interspecific hybridization between Dianthus plumarius and found elsewhere [and used as a diagnostic character for related species with yellow flowers. Scientia Horticulturae 77: 207–218. Balao et al. — Ploidy levels in Dianthus broteri 973

Greilhuber J. 2005. Intraspecific variation in genome size in angiosperms: Pecinka A, Suchankova P, Lysak MA, Travnicek B, Dolezel J. 2006. identifying its existence. Annals of Botany 95: 91–98. Nuclear DNA content variation among central European Koeleria taxa. Greilhuber J, Dolezel J, Lysak MA, Bennett MD. 2005. The origin, evolution Annals of Botany 98: 117–122. and proposed stabilization of the terms ‘genome size’ and ‘C-value’ to Petit C, Bretagnolle F, Felber F. 1999. Evolutionary consequences of describe nuclear DNA contents. Annals of Botany 95: 255–260. diploid-polyploid hybrid zones in wild species. Trends in Ecology & Halverson K, Heard SB, Nason JD, Stireman JO. 2008. Origins, distri- Evolution 14: 306–311. bution, and local co-occurrence of polyploid cytotypes in Solidago altis- Petit RJ, Brewer S, Bordaćs S, et al. 2002. Identification of refugia and post- sima (Asteraceae). American Journal of Botany 95: 50–58. glacial colonisation routes of European white oaks based on chloroplast Hardy OJ, De Loose M, Vekemans X, Meerts P. 2001. Allozyme segre- DNA and fossil pollen evidence. Forest Ecology and Management 156: gation and inter-cytotype reproductive barriers in the polyploid 49–74. complex Centaurea jacea. Heredity 87: 136–145. R Development Core Team. 2008. R: A language and environment for stat- Husband BC, Sabara HA. 2004. Reproductive isolation between autotetra- istical computing. R Foundation for Statistical Computing, Vienna, ploids and their diploid progenitors in fireweed, Chamerion angustifolium Austria. (Onagraceae). New Phytologist 161: 703–713.

Rivas-Martıńez S, Penas A, Dıáz T. 2004. Biogeographic map of Europe. Downloaded from https://academic.oup.com/aob/article/104/5/965/138784 by guest on 30 September 2021 Kolar F, Stech M, Travnicek P, et al. 2009. Towards resolving the Knautia Leoń, Spain: Cartographic Service, University of Leoń. arvensis agg. (Dipsacaceae) puzzle: primary and secondary contact zones Segraves KA, Thompson JN, Soltis PS, Soltis DE. 1999. Multiple origins of and ploidy segregation at landscape and microgeographic scales. Annals polyploidy and the geographic structure of Heuchera grossulariifolia. of Botany 103: 963–974. Molecular Ecology 8 Kron P, Suda J, Husband BC. 2007. Applications of flow cytometry to evol- : 253–262. utionary and population biology. Annual Reviews of Ecology, Evolution Sinclair WT, Morman JD, Ennos RA. 1999. The postglacial history of Scots and Systematics 38: 847–876. pine (Pinus sylvestris L.) in western Europe: evidence from mitochondrial Ku¨pfer P. 1974. Liens de parente´ entre les flores Alpienne et Pyreńeéne. DNA variation. Molecular Ecology 8: 83–88. Boissiera 23: 1–322. Smarda P, Bures P. 2006. Intraspecific DNA content variability in Festuca Larena BG, Aguilar JF, Feliner GN. 2006. Dispersal across southern Iberian pallens on different geographical scales and ploidy levels. Annals of refugia? Integrating RAPDs, sequence data and morphometrics in Botany 98: 665–678. Armeria (Plumbaginaceae). Folia Geobotanica 41: 305–322. Smarda P, Bures P, Horova L, Foggi B, Rossi G. 2008a. Genome size and Leitch IJ, Bennett MD. 2004. Genome downsizing in polyploid plants. GC content evolution of Festuca: ancestral expansion and subsequent Biological Journal of the Linnean Society 82: 651–663. reduction. Annals of Botany 101: 421–433. Lemperg F. 1936. Studies in the perennial species of the genus Dianthus Smarda P, Bures P, Horova L, Rotreklova O. 2008b. Intrapopulation L. I. Meddelanden fra˚nGo¨teborgs Bot. Tra¨ga˚rd 11: 71–134. genome size dynamics in Festuca pallens. Annals of Botany 102: Levin DA. 1975. Minority cytotype exclusion in local plant populations. 599–607. Taxon 24: 35–43. Snow R. 1963. Alcoholic hydrochloric acid-carmine as a stain for chromo- Levin DA. 1983. Polyploidy and novelty in flowering plants. The American somes in squash preparations. Stain Technology 38: 9–13. Naturalist 122: 1–25. Stebbins GL. 1971. Chromosomal evolution in higher plants. London: Levin DA. 2002. The role of chromosomal change in plant evolution. Addison-Wesley. New York, NY: Oxford University Press. Suda J, Travnicek P. 2006. Reliable DNA ploidy determination in dehydrated Mandakova T, Munzbergova Z. 2006. Distribution and ecology of cytotypes tissues of vascular plants by DAPI flow cytometry – new prospects for of the Aster amellus aggregates in the Czech Republic. Annals of Botany plant research. Cytometry Part A 69: 273–280. 98: 845–856. Suda J, Weiss-Schneeweiss H, Tribsch A, Schneeweiss GM, Travnicek P, Miller JM. 1976. Variation in populations of Claytonia perfoliata Schonswetter P. 2007. Complex distribution patterns of di-, tetra-, and (Portulacaceae). Systematic Botany 1: 20–34. hexaploid cytotypes in the European high mountain plant Senecio carnio- Nimura M, Kato J, Mii M, Morioka K. 2003. Unilateral compatibility and licus (Asteraceae). American Journal of Botany 94: 1391–1401. genotypic difference in crossability in interspecific hybridization Thompson JD, Lumaret R. 1992. The evolutionary dynamics of polyploid Dianthus caryophyllus Dianthus japonicus between L. and Thunb. plants: origins, establishment and persistence. Trends in Ecology & Theoretical and Applied Genetics 106: 1164–1170. Evolution 7: 302–307. Nimura M, Kato J, Mii M, Ohishi K. 2008. Cross-compatibility and the Thompson JN, Cunningham BM, Segraves KA, Althoff DM, Wagner D. polyploidy of progenies in reciprocal backcrosses between diploid carna- 1997. Plant polyploidy and insect/plant interactions. The American tion (Dianthus caryophyllus L.) and its amphidiploid with Dianthus japo- Naturalist 150 nicus Thunb. Scientia Horticulturae 115: 183–189. : 730–743. Ohri D. 1998. Genome size variation and plant systematics. Annals of Botany Van Dijk P, Bakx-Schotman T. 1997. Chloroplast DNA phylogeography and 82: 75–83. cytotype geography in autopolyploid Plantago media. Molecular Ecology Olalde M, Herrań A, Espinel S, Goicoechea PG. 2002. White oaks phylo- 6: 345–352. geography in the Iberian Peninsula. Forest Ecology and Management Walker DJ, Mon˜ino I, Correal E. 2006. Genome size in Bituminaria bitumi- 156: 89–102. nosa (L.) C.H. Stirton (Fabaceae) populations: separation of ‘true’ differ- Ozkan H, Tuna M, Arumuganathan K. 2003. Nonadditive changes in ences from environmental effects on DNA determination. Environmental genome size during allopolyploidization in the wheat (Aegilops- and Experimental Botany 55: 258–265. Triticum) group. Journal of Heredity 94: 260–264. Weiss H, Dobes C, Schneeweiss GM, Greimler J. 2002. Occurrence of tetra- Ozkan H, Tuna M, Galbraith DW. 2006. No DNA loss in autotetraploids of ploid and hexaploid cytotypes between and within populations in Arabidopsis thaliana. Plant Breeding 125: 288–291. Dianthus sect. Plumaria (Caryophyllaceae). New Phytologist 156: 85–94. Pearse IS, Kru¨gel T, Baldwin IT. 2006. Innovation in anti-herbivore defense Willkomm M. 1859. Icones et descriptiones plantarum novarum criticarum et systems during neopolypoloidy – the functional consequences of instan- rariorum Europae austro-occidentalis praecipue Hispaniae. Leipzig taneous speciation. The Plant Journal 47: 196–210. Lipsiae: Sumtibus A. H. Payne.