Chromosome numbers of selected species of Elatine L . ( Elatinaceae )

The paper reports chromosome numbers for 13 taxa of Elatine L., including all 11 species occurring in Europe, namely E. alsinastrum, E. ambigua, E. brachysperma, E. brochonii, E. californica, E. campylosperma, E. gussonei, E. hexandra, E. hungarica, E. hydropiper, E. macropoda, E. orthosperma, E. triandra originating from 17, field-collected populations. For seven of them (E. ambigua, E. californica, E. campylosperma, E. brachysperma, E. brochonii, E. hungarica, E. orthosperma) the chromosome numbers are reported for the first time. With these records, chromosome numbers for the whole section Elatinella Seub. became available. Although 2n = 36 was reported to be the most common and the lowest chromosome number in the genus, our data show that out of thirteen species analyzed, six had 36 chromosomes but five species had 54 chromosomes, and the lowest number of chromosomes was 18. These data further corroborates that the basic chromosome number in Elatine is x = 9.

The aim of this study was to count the chromosome numbers for 13 Elatine species, originated from 17 populations.This was done to support our ongoing work to unravel phylogenetic relationship within the European members of the genus.For seven of the taxa studied, namely: E. ambigua Wight, E. californica A. Gray, E. campylosperma Seub., E. brachysperma A. Gray, E. brochonii Clav., E. hungarica Moesz and E. orthosperma Dueb., the chromosome numbers have not been reported yet.

Material and methods
Plants studied were collected in field across Europe, in Asia and North America (Tab.1).The field-collected plants were cultivated at both the Center for Molecular Biology at the University of Szczecin and/or Department of Botany at the University of Debrecen.Once the plants reached maturity and produced seeds, these were either sent to Szczecin (in case of plants cultivated in Debrecen), or processed locally.Seeds were sown in 12.5 × 8.5 cm plastic boxes on sterilized soil, which was continuously wetted with distilled water.Plants were grown in climate controlled culture chamber with 12 h/day light and 30 000 LUX light intensity, temperatures: under light 22 ±2°C and under darkness 18 ±2°C.Roots were immersed in 0.05% colchicine solution (Sigma-Aldrich, St. Louis, USA) for 3 hours at 16°C, then washed in ice-cold distilled water for 10 min.Pretreated roots were fixed in Carnoy's solution (absolute ethanol : glacial acetic acid 3:1 v/v) for 24 hours at 4°C.Roots were washed in distilled water, and the root tips were dissected under a stereoscopic microscope.Each root tip was macerated directly on a microscope slide in a mixture of 4% (w/v) pectinase (Fluka, Buchs, Switzerland), 6% (w/v) hemicellulase (Sigma-Aldrich, St. Louis, USA) and 4% (w/v) cellulase (Sigma-Aldrich, St. Louis, USA) in 0.01 M citric acid -sodium citrate buffer (pH 4.8), for 4 hours at 37°C in a humidity chamber.Root tips were carefully washed with 45% acetic acid.Each preparation was covered with a cover glass, heated for 20 min at 47°C, and then the root tips were gently squashed.The cover slip was removed after freezing over dry ice, and the slides were air-dried overnight.Slides were dehydrated in a graded ethanol series (70%, 96%, and 99.8%) at room temperature, air-dried and stained with DAPI (1 μg/ml; Sigma-Aldrich, St. Louis, USA) for 15 min.The slides were rinsed briefly in distilled water, air-dried and mounted in Vectashield® Hard Set mounting medium for fluorescence (Vector Laboratories, Burlingame, USA) and analyzed with the epifluorescence microscope Axio Imager Z2 (Carl Zeiss, Oberkochen, Germany).The resulting images were captured and analyzed using the GenASIs software (Applied Spectral Imaging).From each species/ population about 50 slides were prepared and analyzed.In order to confirm the results chromosomes were counted in 30 well-spread metaphase plates.

Discussion
According to literature data [15,24,25] the basic number of chromosomes for the genus Elatine is x = 9.This study strongly supports this view as all of the analyzed species had chromosome number dividable by 9.Moreover, we report for the first time the existence of a diploid species, E. campylosperma, in the genus.No other diploid species in Elatine was reported so far.It was claimed [26] that different ploidy levels, such as tetraploids, octoploids and dodecaploids can exist within Elatine.In general, there are some dubious reports for chromosome numbers in the literature.One of the first chromosome number counts for an Elatine species was reported in 1974 [15], determining the chromosome number of E. hydropiper as equaling to 2n = 40.However, subsequent studies showed that chromosome number in this taxon is 2n = 36 [16].Also, our study confirmed that the number of chromosomes of E. hydropiper counted in two populations is 2n = 36.We also confirmed the same chromosome number (2n = 36) for E. alsinastrum, in line of what was previously reported [22].Actually, it was suggested that 2n = 36 is the most common number and the lowest number of chromosomes in species of Elatine genus, as it was also found in the recently described species E. gratioloides [21], and the species with the widest distribution range in the genus, E. triandra [17].In this study we showed that E. triandra can also have 2n = 54 chromosomes, thus the tetraploid and hexaploid level can coexist in the genus.Examples of plant species with different ploidy-levels (i.e., coexistence of different cytotypes) is numerous [27][28][29][30][31][32], especially when they are autopolyploids.
Similarly doubtful is the record of Contandriopoulos et al. [19], who reported 2n = 40 chromosomes in E. macropoda.Although this record much deviates from our counting, as we found 2n = 54 in this species (Tab.1), we cannot exclude that the material analyzed by the above authors was in fact 2n = 36.Another example of coexistence of different ploidy-level cytotypes is E. hexandra, for which two different chromosome numbers are reported, 2n = 72, 108 [20].In this paper we found that our Spanish and Polish material of E. hexandra had 2n = 12x = 108, but we cannot rule out that some populations of the species have different chromosome number.This view is further corroborated by the finding of Probatova and Sokolovskaya [18], who reported that other species, i.e., E. americana can also have 2n = (70-)72.The differences in the chromosome number counts in Elatine clearly demonstrate how difficult is to determine the number of such small chromosomes, and that there is a need to perform a number of tests and optimization to obtain accurate results.It should be noted that when the chromosomes are small in size and there are many of them in one cell, it is extremely difficult to obtain well-spread metaphase plates as, depending on the procedure of preparation, chromosomes may overlap or the metaphase plate may be incomplete.Both cases can lead to counting errors with a tendency to underreporting the number of chromosomes.
In our previous study we reported that E. gussonei collected from the Maltese Archipelago has the number of 2n = 8x = 54 chromosomes [23].Here, we have confirmed this result by adding two more populations from geographically distant locations.It is important to analyze the chromosome number from different population as some chromosome rearrangements may lead to the change of the chromosome number in genetically isolated populations.Unfortunately, we were not able to distinguish the types of chromosomes because of their minute size.Karyotyping could shed some light on the possibilities of chromosome rearrangements, which can be the reason for varying chromosome number.Consequently, we cannot totally exclude the existence of some populations of the same species with different chromosome number (e.g., 2n = 40 in case of a 2n = 36 species), but there is a slightly higher likelihood of incorrectly counting more chromosomes due to their small size.
Another aspect of chromosome number changes is the phenomenon of polyploidization itself.This aspect may explain different number counts in different reports.Most of the Elatine species are polyploids and it is a well-known fact that meiosis in newly formed polyploids is unstable [33,34].For example, several studies found that 30-40% of the progeny of autotetraploid maize is aneuploid [35].If aneuploids became isolated, they might give rise to a population with different chromosome number.Polyploid meiosis frequently produces aneuploid gametes, although their frequency varies between species or ploidy level.There is a relationship between polyploidy and aneuploidy: eupolyploids produce frequently aneuploids, which in turn can produce euploids [33].
It is believed, that polyploidization is a major driving force of plant evolution [36,37].Obtained chromosome number data clearly indicate that polyploidization was one of the most important phenomena accompanying the evolution of the genus Elatine.Further studies should bring the answer to the question if E. campylosperma is one of the ancestors of polyploids.Another question is whether these species are auto-or allopolyploids, which originated through hybridization event between different species, accompanied or followed by genome duplication.Also interesting is to correlate ploidy level in Elatine to geographic latitude the species most commonly inhabit.As it is demonstrated in Europe and North America [38], polyploids are more frequent at high latitudes.If we examine our list of chromosome numbers, we found that the European species found exclusively in the temperate climate zone (E.hungarica, E. hydropiper and E. orthosperma) have 2n = 4x = 36.Two of three Mediterranean species (E.gussonei, E. macropoda) have 2n = 6x = 54.This is an apparent contradiction to the above rule, and requires the reconstruction of phylogenetic relationships for a better understanding.Whatever the pattern behind this finding, the large chromosome number differences between different Elatine species suggests that the genome of Elatine is very tolerant to redundancy of genetic information, and its evolution was accompanied by multiple polyploidization events.