Abstract
Flow cytometry, using propidium iodide and 4′,6‐diamidano‐2-phenylindole staining, was used to estimate the nuclear DNA content (2C) and the proportion of A–T base pairs in 16 species of the Mediterranean genus Cistus. Genome sizes were shown to be constant within species, since no significant intraspecific variation in 2C DNA content was detected. At the genus level, up to about 1·5‐fold differences in absolute DNA amounts were observed, ranging from 3·92 pg in C. crispus to 5·88 pg in C. monspeliensis. The (AT) : (GC) ratio was close to 1, and was similar for all species examined, ranging from 47·87 % A–T content in C. clusii, to 50·67 % in C. populifolius. Pink‐flowered species (subgenus Cistus) had lower DNA amounts than white‐flowered species (subgenera Leucocistus and Halimioides). However, the distribution of DNA amounts in Cistus appeared to be continuous and did not permit a clear separation of infra‐generic ranks in the genus.
Key words: Flow cytometry, nuclear DNA content, genome size, A–T content, Cistus, Cistaceae
INTRODUCTION
The Cistaceae are a small family (approx. 175 taxa; Mabberly, 1997) that have a mostly holartic distribution. Shrubby species are not uncommon within the family, and many of them play a prominent role in the configuration of Old World Mediterranean xeric landscapes.
Cistus L., one of the seven genera into which the family has traditionally been divided (Grosser, 1903), is relatively small (19 Mediterranean and Macaronesic species are currently recognized; Dansereau, 1939; Greuter et al., 1984), but shows a noteworthy morphological diversification. Vegetative (presence or absence of petiole, leaf shape, hair morphology) and reproductive features (inflorescence architecture, sepal number, petal colour, pollen morphology, style length, type of capsule dehiscence and number of capsule valves) are so variable that several authors have proposed infra‐generic classifications to depict taxonomic relatedness better. However, there are conflicting views concerning the circumscription and systematic ranks of the segregates (Spach, 1836; Willkomm, 1856; Grosser, 1903). The most extreme classification (Spach, 1836) divided Cistus into five genera, but this view has not been followed by later authors, who included all the variants at the subgeneric (Cistus, Leucocistus, Halimioides) and sectional (Cistus, Macrostylia, Ledonella, Ledonia, Stephanocarpus, Ladanium, Halimioides) ranks within Cistus (Table 1).
Table 1.
Taxa, code numbers and origin of the accessions of Cistus analysed
| Taxa | Code number | Origin |
| Subgenus Cistus | ||
| Sect. Macrostylia Willk. | ||
| C. osbeckiaefolius Webb ex Pitard & Proust | 20025 | Canary Islands, Spain |
| C. symphytifolius Lam. | 20113 | Canary Islands, Spain |
| Sect. Cistus | ||
| C. albidus L. | 20116 | Gaucin, Spain |
| 20066 | Ibiza, Balearic Islands | |
| 20030 | Sorede, Spain | |
| 20067 | Valencia, Spain | |
| C. crispus L. | 20093 | Badajoz, Spain |
| 20070 | Barig, Spain | |
| 20220 | La Murta, Spain | |
| C. creticus L. | 20099 | Grossetto, Italy |
| 20022 | Florence, Italy | |
| 20090 | Valencia, Spain | |
| C. heterophyllus Desf. subsp. Carthaginensis (Pau) Crespo & Mateo | 20011 | Cartagena, Spain |
| Sect. Ledonella (Spach) Willk. | ||
| C. parviflorus Lam. | 20021 | Bot. Garden Valencia |
| Subgenus Leucocistus Willk. | ||
| Sect. Ledonia Dunal | ||
| C. populifolius L. | 20020 | Castelló, Spain |
| 20045 | Bot. Garden Coimbra | |
| C. psilosepalus Sweet | 20026 | Pontevedra, Spain |
| 20111 | Abrantes, Portugal | |
| C. salviifolius L. | 20038 | Huar Islands, Croatia |
| 20066 | Valencia, Spain | |
| C. albanicus E.F. Warburg ex Heywood | 20027 | Bot. Garden Jena |
| Sect. Stephanocarpus (Spach) Willk. | ||
| C. monspeliensis L. | 20014 | Ibiza, Balearic Islands |
| 20080 | Valencia, Spain | |
| Sect. Ladanium (Spach) Gren. | ||
| C. ladanifer L. | 20010 | Bot. Garden Valencia |
| 20224 | Sierra Morena, Spain | |
| C. laurifolius L. | 20079 | Valencia, Spain |
| 20027 | Granada, Spain | |
| Subgenus Halimioides (Willk.) Demoly & P. Monts | ||
| Sect. Halimioides Willk. | ||
| C. clusii Dunal | 20110 | Cofrentes, Spain |
| 20025 | Granada, Spain | |
| C. libanotis L. | 20228 | Cadiz, Spain |
Taxonomic relationships among Cistus species are based largely on morphological characteristics. However, the extent to which these distinguishing features truly indicate natural relationships, or whether they have originated by convergent evolution, is not currently known. Karyological stability of chromosome number has been repeatedly noted (Markova, 1975, and references therein) and precludes further systematic refinement.
Genome size, being fairly constant within a species, is especially important for taxa delimitation, can be correlated with evolutionary processes within taxonomic groups and can be used as additional evidence in plant systematics (Bennett and Leitch, 1995). Previous reports have shown the usefulness of standardized DNA C‐values to define infra‐generic boundaries (see Ohri, 1998, for references). There is just one previous report of nuclear DNA amounts in Cistaceae, on a Helianthemum species (Bennett and Smith, 1991). This is not surprising since it was estimated that only 1 % of angiosperm species had been analysed up to 1997 (Bennett et al., 1997), although it is expected that data will be available for an additional 1 % within the next few years (Hanson et al., 2001).
We report the use of flow cytometry to evaluate the nuclear 2C DNA content of a representative sample of Cistus species, to determine patterns of genome size variation and the relative proportion of A–T base pairs within the genus and to determine possible correlations with the existing infra‐generic classifications. As genome size is a key biodiversity character (Hanson et al., 2001) and has special relevance for evolutionary studies (Leitch et al., 1998), determination of the variation in nuclear DNA content in Cistus may significantly improve our knowledge of this poorly studied family.
MATERIALS AND METHODS
Plant material
This study included representatives of all of the subgenera of the genus Cistus. With the exception of Cistus chinamadensis A. Bañares & P. Romero (sect. Macrostylia), C. varius Pourret (sect. Stephanocarpoidea) and C. munbyi Pomel (sect. Halimioides), for which no living material was available, all currently recognized species of Cistus were studied. Usually, plant specimens were also grown from seeds of wild origin. However, in a few cases, seeds of plants cultivated in botanical gardens were also used. The list of accessions and the origin of the plant material is shown in Table 1. Voucher specimens are deposited in the herbarium VAL.
2C DNA content measurements
Three individuals from each accession were analysed by flow cytometry. A small amount of fresh leaf material, usually less than 100 mg, was chopped with a sharp razor blade in a nuclei isolation buffer. The samples were stained with propidium iodide (PI), which intercalates into double‐stranded DNA, or with 4′,6‐diamidino‐2‐phenylindole (DAPI), which binds at AT‐rich regions of DNA. For PI staining, the two‐step procedure originally used for DAPI staining by Otto (1990), modified by Doležel and Göhde (1995) and subsequently adapted to intercalating dyes, ethidium bromide (Baranyi and Greilhuber, 1996) and PI (Greilhuber and Obermayer, 1997), was used. Leaves were chopped in a glass Petri dish in 1 ml ice‐cold Otto buffer I, containing 0·1 m citric acid and 0·5 % (v/v) Tween 20. The material was filtered through a 40‐µm nylon mesh and centrifuged at 150 g for 8 min. The pellet was resuspended in 100 µl fresh Otto buffer I and incubated at room temperature for 60 min. Subsequently, 1 ml of Otto buffer II, consisting of 0·4 m Na2HPO4, was added to each sample. Nuclei were stained by adding a concentrated, RNase A‐containing PI solution to give final concentrations of 50 µg ml–1 PI and 50 µg ml–1 RNase A. Finally, after 15 min at room temperature, nuclei suspensions were analysed in the flow cytometer. For DAPI staining, leaf material was chopped at room temperature in 0·4 ml nuclei isolation buffer (‘high resolution DNA kit’, solution A: nuclei isolation; Partec GmbH, Münster, Germany) and incubated for 2 h at room temperature after adding 0·4 ml fresh isolation buffer containing 1 % (w/v) cellulase (Onozuka), 2 % (w/v) polyvinylpyrrolidone and 15 mm 2‐mercaptoethanol. Finally, the suspension was filtered through a 40‐µm nylon mesh and mixed with 2 ml of staining buffer (‘high resolution DNA kit’, solution B: DAPI staining; Partec) for a minimum of 5 min. For each sample, at least 10 000 nuclei were analysed and the ratio of G1 peak means of the sample and the internal standard was calculated. Various standards recommended for plant DNA flow cytometry (Raphanus sativus, Allium cepa, Zea mays, Glycine max and Vicia faba), kindly provided by Dr J. Doležel (Institute of Experimental Botany, Olomouc, Czech Republic), were tested in preliminary experiments; Raphanus sativus ‘Saxa’ (2C = 1·1 pg; Doležel et al., 1992) was chosen as an optimal internal standard. Measurements of DNA content were performed with a PA‐II flow cytometer (Partec). A 20‐mW argon ion laser light source (488 nm wavelength) with an RG 590 longpass filter was used for PI‐stained nuclei. The 100 W Osram HBO 100/2 mercury arc lamp, TK420 dichroic mirror and GG 435 longpass filter of the equipment were used in the case of DAPI staining.
The percentage of A–T base pairs was calculated for each sample analysed as described by Ali et al. (2000), using the formula of Godelle et al. (1993):
where %(A + T) (ref.) is the AT content of the internal standard, Raphanus sativus (46·99 %; Doležel et al., 1992), peak ratios are calculated from DAPI histograms, and DNA content refers to the 2C values of each sample, previously determined by PI staining, and the internal standard.
ANOVA was performed using the SPSS program to evaluate differences in 2C DNA content and A–T values at several hierarchical levels.
RESULTS
Following PI or DAPI staining, nuclei suspensions suitable for flow cytometric analysis of nuclear DNA amounts proved to be rather difficult to prepare from the taxa included in this study. The use of standard protocols, such as those established by Doležel et al. (1989) or by Pfosser et al. (1995), resulted in very high backgrounds and, at best, DNA content histograms of very low, unacceptable resolution. The two‐step procedure described in Materials and Methods was finally chosen for PI staining; this produced sharp DNA peaks, with relatively low coefficients of variation (CV) (range 3·4–6·4 %, mean CV = 4·8) for nuclei isolated from young leaves of Cistus plants. Simplified versions of this protocol were also tested, as suggested by J. Doležel (http://www.ueb.cas.cz/Olomouc1/lcgcm/index.htm), but did not give satisfactory results. DAPI‐stained nuclei, prepared following the procedure recommended by the fluorometer’s manufacturer (Partec), modified to include an enzymatic digestion of cell walls, also produced sharp peaks in the DNA histograms. However, they showed higher backgrounds and slightly higher coefficients of variation (range 4·9–6·9 %, mean CV = 5·7) than the PI samples. Omission of the cellulase treatment generally resulted in much broader DNA peaks and even higher CVs.
The values of nuclear DNA content (2C) measured by PI staining are summarized in Table 2, and Fig. 1 shows representative histograms. Amounts of nuclear DNA were remarkably uniform within species. Thus, for a given species, differences among individuals and among accessions were not statistically significant, indicating the stability of genome size in Cistus. Mean 2C DNA content in Cistus was 4·75 ± 0·55 pg. Nuclear DNA content varied 1·52‐fold among accessions, ranging from 3·88 pg (C. crispus) to 5·88 pg (C. monspeliensis). These two species also showed the lowest (3·92 pg) and highest (5·77 pg) mean 2C DNA amounts (Table 2). Mean DNA contents were significantly different among subgenera (ANOVA, F = 18·1, P < 0·0001), sections (F = 22·75, P < 0·0001) and species (F = 114·23, P < 0·0001). Subgenus Cistus showed the lowest mean genome size (2C DNA amount 4·48 ± 0·39 pg), ranging from 3·92 pg in C. crispus (sect. Cistus) to 4·96 pg in C. parviflorus (sect. Ledonella). Subgenus Leucocistus showed higher mean values (4·88 ± 0·56 pg), ranging from 4·45 pg in C. ladanifer (sect. Ladanium) to 5·88 pg in C. monspeliensis (sect. Stephanocarpus), whereas subgenus Halimiodes showed the highest mean 2C DNA amounts (5·45 ± 0·26 pg), 5·28 pg in C. clusii and 5·77 pg in C. libanotis. The (AT) : (GC) ratio was close to 1 and was similar for all species examined (Table 3). The calculated mean of A–T base pairs fraction in Cistus was 48·91 ± 0·73 %, ranging from 47·87 % in C. clusii (sect. Halimioides) to 50·67 % in C. populifolius (sect. Ledonia).
Table 2.
Nuclear 2C DNA content, in pg (mean ± s.d., n = 3), determined by flow cytometry with PI staining, of accessions, species, sections and subgenera of Cistus
| Taxon | Subgenus | Section | Species | (Ac. No.) | Accession |
| Subgenus Cistus | 4·48 ± 0·39a | ||||
| Sect. Macrostylia | 4·52 ± 0·43a | ||||
| C. osbeckiaefolius | 4·13 ± 0·06a,b | (20025) | 4·13 ± 0·06a,b,c,d | ||
| C. symphytifolius | 4·91 ± 0·10d | (20113) | 4·91 ± 0·10g,h,i | ||
| Sect. Cistus | 4·43 ± 0·38a | ||||
| C. albidus | 4·78 ± 0·08d | (20116) | 4·79 ± 0·09e,f,g,h | ||
| (20066) | 4·78 ± 0·15e,f,g,h | ||||
| (20030) | 4·76 ± 0·05e,f,g,h | ||||
| (20067) | 4·82 ± 0·02f,g,h,i | ||||
| C. crispus | 3·92 ± 0·09a | (20093) | 3·98 ± 0·08a,b,c | ||
| (20070) | 3·88 ± 0·13a | ||||
| (20220) | 3·90 ± 0·01a,b | ||||
| C. creticus | 4·34 ± 0·15b,c | (20099) | 4·24 ± 0·12b,c,d | ||
| (20022) | 4·38 ± 0·16d,e | ||||
| (20090) | 4·40 ± 0·18d,e,f | ||||
| C. heterophyllus subsp. Carthaginensis | 4·82 ± 0·02d | (20011) | 4·82 ± 0·02f,g,h,i | ||
| Sect. Ledonella | 4·96 ± 0·11a | ||||
| C. parviflorus | 4·96 ± 0·11d | (20021) | 4·96 ± 0·11h,i,j | ||
| Subgenus Leucocistus | 4·88 ± 0·56a | ||||
| Sect. Ledonia | 4·84 ± 0·43a | ||||
| C. populifolius | 4·29 ± 0·11b,c | (20020) | 4·27 ± 0·17c,d | ||
| (20045) | 4·31 ± 0·04c,d | ||||
| C. psilosepalus | 5·22 ± 0·15e | (20026) | 5·26 ± 0·22j,k | ||
| (20111) | 5·18 ± 0·06i,j,k | ||||
| C. salviifolius | 4·76 ± 0·12d | (20038) | 4·77 ± 0·11e,f,g,h | ||
| (20066) | 4·74 ± 0·15e,f,g,h | ||||
| C. albanicus | 5·34 ± 0·07e | (20027) | 5·34 ± 0·07k | ||
| Sect. Stephanocarpus | 5·88 ± 0·1b | ||||
| C. monspeliensis | 5·88 ± 0·14f | (20014) | 5·88 ± 0·17l | ||
| (20080) | 5·88 ± 0·14l | ||||
| Sect. Ladanium | 4·46 ± 0·15a | ||||
| C. ladanifer | 4·45 ± 0·17c | (20010) | 4·46 ± 0·16d,e,f | ||
| (20224) | 4·44 ± 0·22d,e,f | ||||
| C. laurifolius | 4·46 ± 0·15c | (20079) | 4·41 ± 0·08d,e,f | ||
| (20027) | 4·52 ± 0·20d,e,f,g | ||||
| Subgenus Halimioides | 5·45 ± 0·26b | ||||
| Sect. Halimioides | 5·45 ± 0·26b | ||||
| C. clusii | 5·28 ± 0·09e | (20110) | 5·30 ± 0·02j,k | ||
| (20025) | 5·27 ± 0·14j,k | ||||
| C. libanotis | 5·77 ± 0·14f | (20228) | 5·77 ± 0·14l |
Within each column, means followed by the same superscript indicate no significant differences according to the Student–Newman–Keuls method (P < 0·01).
Fig. 1. Histograms of relative nuclear DNA content obtained after simultaneous flow‐cytometric analysis of propidium iodide‐stained nuclei of Raphanus sativus (internal standard) and Cistus osbeckiaefolius (A), C. creticus (B) or C. albidus (C).
Table 3.
Percentage of A–T content (mean ± s.d.) of species, sections and subgenera of Cistus
| Taxon | Subgenus | Section | Species |
| Subgenus Cistus | 48·87 ± 0·49a | ||
| Sect. Macrostylia | 48·65 ± 0·66a,b | ||
| C. osbeckiaefolius | 48·82 ± 0·99a,b | ||
| C. symphytifolius | 48·47 ± 0·20a,b | ||
| Sect. Cistus | 48·96 ± 0·46a,b | ||
| C. albidus | 48·88 ± 0·27a,b | ||
| C. crispus | 48·65 ± 0·41a,b | ||
| C. creticus | 49·39 ± 0·54b,c | ||
| C. heterophyllus subsp. carthaginensis | 48·62 ± 0·31a,b | ||
| Sect. Ledonella | 48·73 ± 0·10a,b | ||
| C. parviflorus | 48·73 ± 0·10a,b | ||
| Subgenus Leucocistus | 49·18 ± 0·83a | ||
| Sect. Ledonia | 49·08 ± 0·87a,b | ||
| C. populifolius | 50·67 ± 0·15d | ||
| C. psilosepalus | 49·00 ± 0·33a,b | ||
| C. salviifolius | 48·89 ± 0·33a,b | ||
| C. albanicus | 48·02 ± 0·52a | ||
| Sect. Stephanocarpus | 49·11 ± 0·19a,b | ||
| C. monspeliensis | 49·11 ± 0·19a,b | ||
| Sect. Ladanium | 49·51 ± 0·91b | ||
| C. ladanifer | 48·81 ± 0·36a,b | ||
| C. laurifolius | 50·21 ± 0·68c,d | ||
| Subgenus Halimioides | 47·92 ± 0·32b | ||
| Sect. Halimioides | 47·92 ± 0·32a | ||
| C. clusii | 47·87 ± 0·25a | ||
| C. libanotis | 47·97 ± 0·44a |
Within each column, means followed by the same superscript indicate no significant differences according to the Student–Newman–Keuls method (P < 0·01).
DISCUSSION
Previously, there was only a single report of DNA C‐values in Cistaceae: 8·90 pg (4C) in Helianthemum nummularium (Bennett and Smith, 1991). This is a diploid perennial species (2n = 20) belonging to tribe Cistineae, which also includes Cistus. This value is very close to the mean DNA amount of Cistus (4·75 pg/2C) and falls well within the ranges detected in this study. However, it is premature to anticipate overall levels of genome size variation within Cistaceae, since the taxa surveyed (now covering about 10 % of the species) show identical life‐forms and have similar ecological requirements, two factors that have been correlated with DNA content variation in some plant groups (e.g. Bennett, 1976; Nandini et al., 1997; Naranjo et al., 1998; Dimitrova and Greilhuber, 2000). In fact, the data available for Cistaceae are in the lower range of the C‐values reported for the higher group, eurosids (range 0·2–33 pg/2C, mean 17 pg/2C; Leitch et al., 1998), in which it is phylogenetically included (Savolainen et al., 2000).
Genome size and A–T content appear to be fairly stable within Cistus, showing little variation within and among accessions belonging to the same species. This is a prerequisite for using DNA C‐values as an additional taxonomic character. However, the calculated values of DNA content and A–T percentage and the nature of the variation among species limit its use in Cistus. These findings are in accord with the stability of the ploidy level of the genus (all species investigated to date are diploid) and the relative homogeneity of the karyotypes among species (Markova, 1975). At low taxonomic levels, the most promising use of genome size could be as a useful marker for the detection of hybrids. Hybridization has been reported to be an active process in Cistus and many hybrid combinations within and among pink‐ or white‐flowered species have been recorded in the field, based on intermediate morphological characters (Grosser, 1903). Furthermore, extensive artificial hybridizations have shown the lack of crossing barriers between most Cistus species (Gard, 1910, 1912, 1914). However, the extent to which reticulation has played a significant role in the diversification of the genus has not been assessed. Two species have been suggested to be of hybrid origin. Cistus albanicus, a narrow endemic plant from south‐east Europe (Warburg, 1968), was originally described as a hybrid between C. monspeliensis and C. salvifolius (C. × florentinus Lamark nm. adriaticus Margraf), although it was not growing with either. Some studies have reported that diploid interspecific hybrids have a DNA content intermediate between values of the parents involved (Buitendijk et al., 1997). The mean 2C DNA content of C. albanicus (5·34 pg) is very close to the expected value (5·3 pg) of the F1 hybrid between C. monspeliensis and C. salvifolius. On this basis, its hybrid origin should be reconsidered and further studied using discrete and more powerful molecular markers. In either case, the DNA content of C. albanicus is significantly different (P < 0·001) from that of C. monspeliensis and C. salvifolius, the only two species in subgenus Leucocistus growing in south‐east Europe. Cistus varius is a narrow endemic Mediterranean species, and is the only representative of sect. Stephanocarpoidea (Dansereau, 1939). It has been suggested that it is also of hybrid origin, having C. crispus and C. monspeliensis (which belong to different subgenera) as putative parents (Grosser, 1903). Unfortunately, no living accession of this rare species was available for study. Determination of its nuclear DNA content should assist in the clarification of its taxonomic status, due to the genome size differences found between the proposed parents. Thus, it is hypothesized that if C. varius is an F1 hybrid between C. crispus and C. monspeliensis, its 2C DNA content should be close to 4·8 pg.
Characteristic genome sizes or skewed base pair compositions could not be associated with currently recognized infra‐generic units within Cistus in this study. In fact, variation in DNA content is mainly continuous, with overlapping values among sectional and even subgeneric taxa. This, together with the fact that the nuclear DNA amount in Cistus is relatively constant, with a variation of only 1·5‐fold, limits its use in delimiting infra‐generic divisions. In Cistus, patterns of variation in DNA amount do not parallel those of morphological variation. On this basis, few suggestions concerning the delimitation and rearrangement of its infra‐generic divisions could be made. Even one of the earlier and most followed divisions of the genus, the splitting of Cistus into pink‐ and white‐flowered species (Spach, 1836; Willkomm, 1856), is only loosely correlated with the DNA content or the proportion of A–T base pairs. Certainly, our results indicate that pink‐flowered species (subgenus Cistus) have significantly lower mean DNA content than white‐flowered ones (subgenus Leucocistus and Halimioides). However, the ranges are, to a large extent, overlapping. This makes flower colour an uncertain predictor of the DNA content in the genus. The critical systematic position of members of subgenus Halimioides has been reported several times (Dansereau, 1939; Démoly and Montserrat, 1991). These species (C. clusii, C. libanotis and C. munbyi) are closely related morphologically to taxa of sect. Commutata of the genus Halimium. Some authors have suggested the merging of Cistus and Halimium into a single genus (Löve and Kjellqvist, 1964), due to the existence of these intermediates and the sharing of a unique basic chromosome number (x = 9) in the family. DNA amounts in subgenus Halimioides range among the highest reported values in Cistus. Nevertheless, their genome size is very similar to that of C. monspeliensis, a species unanimously included within subgenus Leucocistus and with which no apparent taxonomic relatedness or unifying morphological features have been postulated. Interestingly, preliminary results on DNA content in two subspecies of Halimium umbellatum (sect. Commutata), closely related to C. clusii and C. libanotis, have shown significant lower mean C‐values (3·76–3·92 pg/2C) than those reported for species of Cistus subgenus Halimioides (5·28–5·77 pg/2C) (M. Boscaiu, P. Ellul and O. Vicente, unpubl. res.). Unfortunately, at present, there is no established morphological or molecular‐based phylogeny of Cistaceae and it is, therefore, difficult to interpret the variation detected in genome size from an evolutionary perspective.
ACKNOWLEDGEMENTS
We are indebted to J. Doležel (Institute of Experimental Botany, Olomouc, Czech Republic) for providing seeds of several plant standards for flow cytometry, to M. Wessel (Johann Wolfgang Goethe University, Frankfurt, Germany) for seeds and herbarium material of Cistus albanicus, and to all botanical garden staff who supplied us with seeds of other Cistus taxa. We would also like to thank M. Steinberg (Partec GmbH, Münster, Germany) for technical advice on the PA‐II flow cytometer, G. Nieto‐Feliner for critical reading of the manuscript and James Houghton for reviewing the English grammar and style.
Supplementary Material
Received: 13 November 2002; Returned for revision: 6 March 2002; Accepted: 30 May 2002
References
- AliHBM, Meister A, Schubert I.2000. DNA content, rDNA loci, and DAPI bands reflect the phylogenetic distance between Lathyrus species. Genome 43: 1027–1032. [DOI] [PubMed] [Google Scholar]
- BaranyiM, Greilhuber J.1996. Flow cytometric and Feulgen densitometric analysis of genome size variation in Pisum Theoretical and Applied Genetics 92: 297–307. [DOI] [PubMed] [Google Scholar]
- BennettMD.1976. DNA amount, latitude and crop plant distribution. In: Jones K, Brandham PE, eds. Current chromosome research. Amsterdam: Elsevier North Holland Biomedical Press, 151–158. [Google Scholar]
- BennettMD, Leitch IJ.1995. Nuclear DNA amounts in angiosperms. Annals of Botany 76: 113–176. [Google Scholar]
- BennettMD, Smith JB.1991. Nuclear DNA amounts in angiosperms.Philosophical Transactions of the Royal Society of London, Series B 334: 309–345. [DOI] [PubMed] [Google Scholar]
- BennettMD, Cox AV, Leitch IJ.1997. Angiosperm DNA C‐values database. http://www.rbgkew.org.uk/cval/database1.html. [Google Scholar]
- BuitendijkJK, Boon EJ, Ramanna MS1997. Nuclear DNA content in twelve species of Alstroemeria L. and some of their hybrids. Annals of Botany 79: 343–353. [Google Scholar]
- DansereauP.1939. Monographie du genre Cistus L. Boissiera 4: 1–90. [Google Scholar]
- DémolyJP, Montserrat P.1991. Notes et nouveautés nomenclaturales sur le genre Cistus L. Anales del Jardín Botánico de Madrid 49: 150–152. [Google Scholar]
- DimitrovaD, Greilhuber J.2000. Karyotype and DNA‐content evolution in ten species of Crepis (Asteraceae) distributed in Bulgaria. Botanical Journal of the Linnean Society 132: 281–297. [Google Scholar]
- DoleželJ, Göhde W.1995. Sex determination in dioecious plants Melandrium album and M. rubrum using high‐resolution flow cytometry. Cytometry 19: 103–106. [DOI] [PubMed] [Google Scholar]
- DoleželJ, Binarova P, Lucretti S.1989. Analysis of nuclear DNA content in plant cells by flow cytometry. Biologia Plantarum 31: 113–120. [Google Scholar]
- DoleželJ, Sgorbati S, Lucretti S.1992. Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physiologia Plantarum 85: 625–631. [Google Scholar]
- GardM.1910. Recherches sur les hybrides artificiels de Cistes, obtenus par Ed. Bornet. I. Notes inédites et résultats experimentaux. Annales des Sciencies Naturelles, ser. 9, 12: 71–116. [Google Scholar]
- GardM.1912. Recherches sur les hybrides artificiels de Cistes, obtenus par M. Ed. Bornet. II. Les espèces et les hybrides binaires, avec notes inédites de Ed. Bornet. Beihefte zum Botanischen centralblat t29: 306–394. [Google Scholar]
- GardM.1914. Recherches sur les hybrides artificiels de Cistes, obtenus par Ed. Bornet. III. Les hybrides dérivés et les hybrides complexes. Beihefte zum Botanischen centralblat t31: 373–416. [Google Scholar]
- GodelleB, Cartier D, Marie D, Brown SC, Siljak‐Yakovlev S.1993. Heterochromatin study demonstrating the non‐linearity of fluorometry useful for calculating genomic base composition. Cytometry 14: 618–626. [DOI] [PubMed] [Google Scholar]
- GreilhuberJ, Obermayer R.1997. Genome size and maturity group in Glycine max (soybean). Heredity 78: 547–551. [Google Scholar]
- GreuterW, Burdet HM, Long G.1984. Med Checklist. A critical inventory of vascular plants of the circum‐mediterranean countries. Genève: Conservatoire et jardin botanique de la Ville de Genève. [Google Scholar]
- GrosserW.1903. Cistaceae. In: Engler A, ed. Das Pflanzenreich. Berlin: Wilhelm Engelmann, iv (193): 1–161. [Google Scholar]
- HansonL, McMahon KA, Johnson MAT, Bennett MD.2001. First nuclear DNA C‐values for 25 angiosperm families. Annals of Botany 87: 251–258. [DOI] [PubMed] [Google Scholar]
- LeitchIJ, Chase, MW, Bennett, MD.1998. Phylogenetic analysis of DNA C‐values provides evidence for a small ancestral genome size in flowering plants. Annals of Botany 82 (Suppl. A): 85–94. [Google Scholar]
- LöveÁ, Kjellqvist E.1964. Chromosome numbers of some Iberian Cistaceae. Portugalia Acta Biologica, series A8: 69–80. [Google Scholar]
- MabberleyDJ.1997. The plant‐book. A portable dictionary of the vascular plants. Cambridge: Cambridge University Press. [Google Scholar]
- MarkovaM.1975. Karyosystematische Untersuchungen an den Cistaceae Bulgariens. Plant Systematics and Evolution 123: 283–315. [Google Scholar]
- NandiniAV, Murray BG, O’Brien IEW, Hammett KRW.1997. Intra‐ and interspecific variation in genome size in Lathyrus (Leguminosae). Annals of Botany 125: 359–366. [Google Scholar]
- NaranjoCA, Ferrari MR, Palermo AM, Poggio L.1998. Karyotype, DNA content and meiotic behaviour in five south American species of Vicia (Fabaceae). Annals of Botany 82: 757–764. [Google Scholar]
- OhriD.1998. Genome size variation and plant systematics. Annals of Botany 82 (Suppl. A.): 75–83. [Google Scholar]
- OttoF.1990. DAPI staining of fixed cells for high‐resolution flow cytometry of nuclear DNA. In: Crissman HA, Darzynkiewicz Z, eds. Methods in cell biology. Volume 33. New York: Academic Press, 105–110. [DOI] [PubMed] [Google Scholar]
- PfosserM, Amon A, Lelley T, Heberle‐Bors E.1995. Evaluation of sensitivity of flow cytometry in detecting aneuploidy in wheat using disomic and ditelosomic wheat‐rye addition lines. Cytometry 21: 387–393. [DOI] [PubMed] [Google Scholar]
- SavolainenV, Chase MW, Hoot SB, Morton CM, Soltis DE, Bayer C, Fay MF, De Brujin AY, Sullivan S, Qiu Y‐L.2000. Phylogenetics of flowering plants based on combined analysis of plastid atpB and rbcL gene sequences. Systematic Biology 49: 306–362. [DOI] [PubMed] [Google Scholar]
- SpachE.1836. Conspectus monographie Cistacearum. Annals de Sciences Naturelles, series 2, 6: 357–375. [Google Scholar]
- WarburgEF.1968. Cistus L. In: Tutin TG, Heywood VH, Burges NA, Moore DM, Valentine DH, Walters SM, Webb DA, eds. Flora Europaea. Volume 2. Cambridge: Cambridge University Press, 282–282. [Google Scholar]
- WillkommM.1856. Icones et descriptiones plantarum novarum criticarum et rariorum Europae Austro‐occidentalis praecipae Hispaniae. Leipzig: A.H. Payne. [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
