Variation in genome size, cell and nucleus volume, chromosome number and rDNA loci among duckweeds

Abstract

Duckweeds are small, free-floating, largely asexual and highly neotenous organisms. They display the most rapid growth among flowering plants and are of growing interest in aquaculture and genome biology. Genomic and chromosomal data are still rare. Applying flow-cytometric genome size measurement, microscopic determination of frond, cell and nucleus morphology, as well as fluorescence in situ hybridization (FISH) for localization of ribosomal DNA (rDNA), we compared eleven species, representative for the five duckweed genera to search for potential correlations between genome size, cell and nuclei volume, simplified body architecture (neoteny), chromosome numbers and rDNA loci. We found a ~14-fold genome size variation (from 160 to 2203 Mbp), considerable differences in frond size and shape, highly variable guard cell and nucleus size, chromosome number (from 2n = 36 to 82) and number of 5S and 45S rDNA loci. In general, genome size is positively correlated with guard cell and nucleus volume (p < 0.001) and with the neoteny level and inversely with the frond size. In individual cases these correlations could be blurred for instance by particular body and cell structures which seem to be linked to specific floating styles. Chromosome number and rDNA loci variation between the tested species was independent of the genome size. We could not confirm previously reported intraspecific variation of chromosome numbers between individual clones of the genera Spirodela and Landoltia.

Figure 1

Phylogenetic relationship, frond, stomata and nuclei morphology of duckweed species. (A) Phylogenetical position. (B, C) Differences in size and morphology of fronds and stomata. (D, E) Nuclei shape and distribution within the guard cells. Numbers indicate genome size (B), average cell (C) and nuclear volumes (D), and percentage of nuclear to cell volume (E). Scale bars = 200 µm (B) and 5 µm (C–E).

Figure 2

Variation in cell morphology (A), floating-style (B) and genome size (C) in duckweeds. (A) Epidermis cell walls are bent in S. intermedia, undulated in La. punctata, Le. minor and rather straight in Wa. hyalina and Wo. arrhiza. Stomata are spherical in S. intermedia and Le. minor, or elliptic as in La. punctata, Wa. hyalina and Wo. arrhiza. Varying epidermis cell sizes (a–c) in the different duckweed species. (B) Wa. hyalina: Free-floating, two-ovate fronds cohere together. The bent vertical appendage (arrow) is formed from the lower wall of a pouch. Wa. lingulata: Two tongue-shaped fronds cohere together with frond ends curved downward bringing most of the surface under water. Wo. microscopica: Free-floating, dorsoventral fronds with irregular polygonal flat dorsal surface and a ventral projection, the pseudo-root (arrow). Wo. columbiana: Nearly spherical fronds with most of their surface submerged. Stomata are present in the free-floating (Wa. hyalina, Wo. microscopica) and almost absent in the submerged (Wa. lingulata, Wo. columbiana) species. (C) Numbers indicate the deviation of genome size in % (our data relative to that of Wang et al.) in the same duckweed clone. Scale bars = 10 µm (A), 5 mm (B).

Figure 3

Guard cell and nuclear volume measurement (A) and linear regressions of duckweed cell parameters (B). (A) DIC and fluorescence microscopy image stacks (left) were applied separately (here merged images) to measure the guard cells and the nuclei inside, respectively. The x-y areas (µm²) and the z dimension (µm) were measured based on the black (guard cells) and red (nuclei) encircled regions via the ZEN software (spatial illustration, right). (B) Regressions between genome size and cell (1) and nucleus volume (2), and between nucleus and cell volume (3). ***p < 0.001 for the correlation coefficient r.

Figure 4

Equal and abnormal nuclei distribution in sister guard cells of Wa. hyalina (A–C) and Wo. australiana (D–F) and unusual nuclei shape of La. punctata (5562-A4 mutant) (G–I). (A,D) Normal situation (one nucleus per cell); (B,C,E) both nuclei in one sister guard cell; (F) the lower nucleus (arrow) is possibly migrating into the sister cell. (G) Overview of the nuclei shape in the epidermis of the tetraploid La. punctata clone 5562_A4 and enlarged frame (H,I) Scale bars = 5 µm.

Figure 5

Chromosome numbers of eleven duckweed species, identified in somatic metaphases. Scale bars = 5 µm.

Figure 6

5S and 45S rDNA loci (arrowheads) of eleven duckweed species. Two loci of 5S and one locus of 45S rDNA were detected on S. polyrhiza, S. intermedia, La. punctata, Wa. hyalina, Wo. australiana; one locus of 5S and 45S each were detected on Le. minor, Le. disperma, Le. aequinoctialis and Wo. microscopica; three loci of 5S and two loci of 45S rDNA were detected in Wa. rotunda and Wo. arrhiza. Framed: minor loci of 5S rDNA. Scale bars = 5µm