Genome size and endoreplication in two pairs of cytogenetically contrasting species of Pulmonaria (Boraginaceae) in Central Europe

Abstract

Genome size is species-specific feature and commonly constant in an organism. In various plants, DNA content in cell nucleus is commonly increased in process of endoreplication, cellular-specific multiplication of DNA content without mitosis. This leads to the endopolyploidy, the presence of multiplied chromosome sets in a subset of cells. The relationship of endopolyploidy to species-specific genome size is rarely analysed and is not fully understood. While negative correlation between genome size and endopolyploidy level is supposed, this is species- and lineage-specific. In the present study, we shed light on this topic, exploring both genome size and endoreplication-induced DNA content variation in two pairs of morphologically similar species of Pulmonaria, P. obscura-P. officinalis and P. mollis-P. murinii. We aim (i) to characterize genome size and chromosome numbers in these species using cytogenetic, root-tip squashing and flow cytometry (FCM) techniques; (ii) to investigate the degree of endopolyploidy in various plant organs, including the root, stem, leaf, calyx and corolla using FCM; and (iii) to comprehensively characterize and compare the level of endopolyploidy and DNA content in various organs of all four species in relation to species systematic relationships and genome size variation. We have confirmed the diploid-dysploid nature of chromosome complements, and divergent genome sizes for Pulmonaria species: P. murinii with 2n = 2x = 14, 2.31 pg/2C, P. obscura 2n = 2x = 14, 2.69 pg/2C, P. officinalis 2n = 2x = 16, 2.96 pg/2C and P. mollis 2n = 2x = 18, 3.18 pg/2C. Endopolyploidy varies between species and organs, and we have documented 4C-8C in all four organs and up to 32C (64C) endopolyploid nuclei in stems at least in some species. Two species with lower genome sizes tend to have higher endopolyploidy levels than their closest relatives. Endoreplication-generated tissue-specific mean DNA content is increased and more balanced among species in all four organs compared to genome size. Our results argue for the narrow relationship between genome size and endopolyploidy in the present plant group within the genus Pulmonaria, and endopolyploidization seems to play a compensatory developmental role in organs of related morphologically similar species.

Keywords: Boraginaceae; Pulmonaria; endoreplication; flow cytometry; genome size; geophytes.

Figure 1.

Overview of morphological and cytological variation of four Pulmonaria species, P. mollis (2n = 18), P. murinii (2n = 14), P. obscura (2n = 14) and P. officinalis (2n = 16) as documented with whole habit of plants (first column of figures), inflorescence (second column), leaf shape (third column) and flow cytometry histograms documenting genome size variation with chromosome figures in insets (fourth column). Species relatedness is expressed with different colours, P. mollis and P. murinii in shades of red, P. obscura and P. officinalis in shades of blue, while genome size similarity is expressed with different colour intensity, P. murinii and P. obscura with smaller genomes in lower colour intensity (orange and pale blue, respectively), P. mollis and P. officinalis with larger genomes with higher colour intensity (red and blue, respectively). G₀/G₁, peak of embryo nuclei in G₀/G₁ cell cycle phase; G₂, peak of embryo nuclei in G₂ cell cycle phase; IRS, internal reference standard; P, Pulmonaria.

Figure 2.

Endopolyploidy level variation in four Pulmonaria species, P. mollis (2n = 18), P. murinii (2n = 14), P. obscura (2n = 14) and P. officinalis (2n = 16). (A) Variation in endoreduplication index, EI, as a measurement of endopolyploidy, calculated values as well as mean ± SD are depicted. Values for particular parameter followed by different letters are significantly different at P = 0.05 (Kruskal–Wallis and Dunn’s tests separately performed for each organ). (B) Principal component analysis for EI variation in four organs: root, stem, calyx and corolla. (C) Representative flow cytometry histograms. Species relatedness is expressed with different colours, P. mollis and P. murinii in shades of red, P. obscura and P. officinalis in shades of blue, while genome size similarity is expressed with different colour intensity, P. murinii and P. obscura with smaller genomes in lower colour intensity (orange and pale blue, respectively), P. mollis and P. officinalis with larger genomes with higher colour intensity (red and blue, respectively).

Figure 3.

Tissue-specific mean DNA content (meanDNA) variation in four Pulmonaria species, P. mollis (2n = 18), P. murinii (2n = 14), P. obscura (2n = 14) and P. officinalis (2n = 16). (A) Variation in meanDNA, calculated values as well as mean ± SD are depicted. Values for particular parameter followed by different letters are significantly different at P = 0.05 (Kruskal–Wallis and Dunn’s tests separately performed for each organ). (B) Principal component analysis for meanDNA variation in four organs: root, stem, calyx and corolla. Species relatedness is expressed with different colours, P. mollis and P. murinii in shades of red, P. obscura and P. officinalis in shades of blue, while genome size similarity is expressed with different colour intensity, P. murinii and P. obscura with smaller genomes in lower colour intensity (orange and pale blue, respectively), P. mollis and P. officinalis with larger genomes with higher colour intensity (red and blue, respectively).

Figure 4.

Comparative analysis of genome size and endoreduplication level (expressed as EI) in four species of Pulmonaria, P. mollis (2n = 18), P. murinii (2n = 14), P. obscura (2n = 14) and P. officinalis (2n = 16) and in four investigated organs: root, stem, calyx and corolla. Data were standardized to the zero mean and unit variance prior to plotting.