Nuclear DNA amounts in European Callitriche species (Callitrichaceae)

SUMMARY Nuclear DNA amounts were determined for nine species, one subspecies and one hybrid of the European Callitrichaceae.The 2C DNA values ranged from 1 -82 pg to 8-30 pg due to variationin chromosomenumbers (2 n = 6-38) and loss (maximally 40-9%) or amplification (maximally 28-5%) of the DNA amount. Theresults are discussed in relation to phylogeny.


INTRODUCTION
There is some evidence that the submerged species are descendants of terrestrial taxa.
The former species show, for instance, an important reduction in their morphological and anatomical characters (Schotsman 1982a). The species with 2n -8 are either amphibious, aquatic or submerged, and species with 2n = 6 are submerged only (see Table I). Therefore a reduction in characters seems to be coupled with a decrease in chromosome numbers. It is not known how the karyotype evolution towards 2n = 6 took place. All the chromosomes of the species with In = 10,8 or 6 have distinct arms and intraspecific chromosomal polymorphism has been observed in some populations of C. obtusangula and C. stagnalis (Schotsman 1967) The species with 2n = 20 have several characters in common with the 2n-10 species.
C. platycarpa (2n = 20) arose by allopolyploidization according to Savidge (1960) (Callitrichaceae) presently comprises at least 14 species of which 13 species and 1 subspecies are well known (Schotsman 1954(Schotsman , 1967(Schotsman , 1969(Schotsman ,1972a(Schotsman ,b, 1982aDersch 1974;Schotsman & Andreas 1980;Schotsman & Haldimann 1981;Haldimann 1982;Cook 1983). The delimitation of the taxa is based on a combination of morphological and anatomical characters, floral biology, cytological data, ecology and geographical distribution. Two species and one subspecies live wholly submerged in water (called 'submerged'), two species live in water as submerged form or as form with floating or aerial rosettes ('aquatic') and nine species grow in water as submerged form or as form with floating rosettes and in humid soil as terrestrial form (called 'amphibious'). The intrageneric chromosome numbers range from 2« = 6 to 2n = 38. The most frequent number is 2« = 10 and the basic number (x) is 5 (see Table 1).
Species with 2n= 10 have characters in common with landplants (Schotsman 1982a) and most of these taxa can live wholly emerged. Because the species of this genus probably originated from terrestrial phanerogames (Arber 1920;Takhtajan 1959), 2« = 2jc =10 could be the most primitive chromosome number of the genus. autopolyploidization according to Schotsman (1967). Which of these processes gave rise to the polyploidy of C. palustris (2n = 20) is also unknown. C. hybrid (2n = 15) is the only hybrid sensu stricto in this genus and probably originated from a cross between C. cophocarpa (2n=I0) and C. platycarpa (2« = 20; Savidge, 1959;Schotsman 1961bSchotsman , 1967Dersch 1974;Schotsman & Haldimann 1981). At least one of the species with 20 chromosomes is closely related to a species with 10 chromosomes.
Further evidence for systematic relationships within the genus Callitriche can be obtained by hybridization studies. Gene exchange between taxa, however, is generally prevented by various isolating mechanisms, one of the most important being the strong tendency to geitonogamy (Schotsman 1967(Schotsman , 1982a (Jacobsen et al. 1983;cf. Bennet & Smith 1976). The root tips were fixed and processed in the same way as the test material. The interphase structures of this potato are rather similar to those of the species of Callitricheand thus suitable as reference.

RESULTS AND DISCUSSION
The results of the DNA measurements of interphase nuclei are summarized in Table I.
The amounts of DNA in absolute units are presented at the 2C level and per chromosome.
A 2C amount of about 2-8 pg DNA was found in three out of four amphibious species with 10 chromosomes, namely C. regis-jubae, C. stagnalis and C. cophocarpa. If, as mentioned in the Introduction, 10 chromosomes are the ancestral diploid chromosome number (basic number x = 5), then it may be assumed that 2-84 pg DNA (mean amount) is the original 2C amount of the species of Callitriche. Starting from the correctness of this assumption, the observed values represent valuable information (for reviews see Price 1976; Bennett & Smith 1976;Flavell 1982;Walbot & Cullis 1985).
The original amount is still present in C. cophocarpa , from which the values of C. stagnalis and C. regis-jubae do not differ significantly (/-tests: P> 0 05). These species differentiated (cf. Schotsman 1967Schotsman , 1977 without changing their nuclear DNA amounts. C. obtusangula is the only species with 10 chromosomes with a significantly different, namely a 28-5% higher, amount of DNA (/-test: RcOOl). Increase or decrease in nuclear DNA content has been recorded before for related plant species with similar chromosome numbers and has been explained as being due to amplification or deletion of base sequences, respectively, by unknown programs (Price 1976;Flavell 1982;Walbot & Cullis 1985). It is not known where the amplification took place in the chromosomes of C. obtusangula. This species differs from C. regis-jubae, C. stagnalis and C. cophocarpa in pollen shape and in a rare anatomical character of the pericarp (Schotsman & Andreas 1974;Schotsman 1977). Moreover, the pollen grains of C. obtusangula are not recognized by the stigmata of C. stagnalis and vice versa (inhibition of pollen germination and pollen tube growth), though these plants have a similar pollination (Schotsman, unpublished).
These facts provide evidence for a remarkable phylogenetic distance which is sustained by and may have occurred through the increase in nuclear DNA.   Flavell 1982;Walbot & Cullis 1985). The evolution from species with terrestrial characters with In = 10 towards submerged species with lower chromosome numbers, as noted in the Introduction, is thus accompanied by a complex karyotype evolution.
C. platycarpa (2n = 20) has double the original quantity of nuclear DNA, which is in accordance with the presence of polyploidy. This species is considered to be an allotetraploid of C. cophocarpa and C. stagnalis by Savidge (1960), but may equally be an autotetraploid of C. cophocarpa according to Schotsman (1967). The DNA amount (per chromosome) indicates that the ancestral parents were among the species with 10 chromosomes and about 2-8 pg DNA, to which C. cophocarpa as well as C. stagnalis belong. It Not available: C. hermaphroditica r*. submerged, 2« = 6; C. pulchra, submerged, 2« = 8; C. cribrosa, amphibious, 2n =8; C. lenisulca, aquatic, 2 n= 10.
Dutch species; C. cophocarpa very rare, last record from 1930. The sterile natural hybrid C. hybrid {2n = 15), collected in the Jura, probably originated from a cross between C. cophocarpa {2n= 10) and C. platycarpa (2n = 20), according to Schotsman (1967) and Schotsman & Haldimann (1981). The DNA measurements do not conflict with this assumption, for the DNA amount per chromosome (0-28 pg) of the hybrid is similar to that of the parental species.
Compared with C. platycarpa, C. palustris (2 n = 20) has a much lower amount of DNA.
The content per chromosome (0-17 pg) is the lowest of all species measured. This species is probably tetraploid because the chromosomes are acrocentric and rather similar to those of the 2n = 10 species (Schotsman 1954(Schotsman , 1967. If so and if the ancestor(s) had 10 chromosomes with about 2-8 pg DNA, the genomes of this species lost 40-9% DNA without loss of chromosomes. C. palustris also differs in some traits, e.g. in the pollination processes, from the other species with 2n= 10 and 20 chromosomes (Schotsman 1954(Schotsman , 1982a and thus is clearly separated phylogenetically. Hedberg & Hedberg (1977) suppose that the chromosome numbers of C. brutia (2 n = 28) and C. hamulata (2n = 38) arose through hybridization between an afromontane species with 2n=18 and a species with 2n= 10 and 2n = 20, respectively, followed by doubling of the chromosomes, but Schotsman (1982b) doubts such an alloploid origin.
The total quantities of DNA point to polyploidy. The DNA content per chromosome of these species is low and a loss of 36 0% and 27 0% DNA took place at the hexaploid level for C. brutia and at the octoploid level for C. hamulata, respectively. In both species it is likely that diminutionof DNA is due to Robertsonian fusion or meiotic missegregation, as described above, because of the presence of a pair of metacentric chromosomes and hypoploidy for two chromosomes (Schotsman 1961a(Schotsman ,b, 1967Haldimann 1982). Because the metacentrics are found in both species and have a normal meiotic behaviour, the interchanges apparently took place in one of the ancestral parents and not since the origin of these species. Whatever may have happened, the quantity of DNA lost in this way equals the amount of two non-essential translocation chromosomes in both species. The high percentages of loss indicate that somewhere in the genomes DNA diminutionnot due to chromosomal rearrangements has taken place. Because C. brutia and C. hamulatahave several characters in common, some authors (Clapham et al. 1962;Perring & Walters 1962) concluded that these callitriches are subspecies (of C. intermedia Hoffm.). However, Schotsman (1954Schotsman ( , 1958Schotsman ( , 1967 and Haldimann (1982) considered them as species, which is supported by the differences in loss of DNA content per genome.
The species C. lusitanica, C. palustris and C. brutia have to pass a short generation time for climatological and ecological reasons (Schotsman 1982a). They are equipped with a low DNA content per chromosome. The nuclear DNA content of a plant is correlated with the minimum generation time of that plant according to Bennett (1972).
Consequently, these three taxa may have lost DNA as an adaptation to a rapid development (cf. Grime & Mowforth 1982). C. antarctica Engelm. (2/? = 40) from the sub-Antarctic island South Georgia has a lower DNA content (0-23 pg DNA per chromosome) in order to survive in a cold environment (Bennett et al. 1982). This may also apply to C. hamulata which prefers a cold water habitat (Schotsman 1967).
The present study demonstrates that the European taxa of Callitriche have a more complex evolutionary history than previously supposed. The evolutionary relationships between the species can be explored further if the karyotypes are analysed first with chromosome banding techniques.