Chromosome Numbers and Genome Sizes of All 36 Duckweed Species (Lemnaceae)

Usually, chromosome sets (karyotypes) and genome sizes are rather stable for distinct species and therefore of diagnostic value for taxonomy. In combination with (cyto)genomics, both features provide essential cues for genome evolution and phylogenetic relationship studies within and between taxa above the species level. We present for the first time a survey on chromosome counts and genome size measurement for one or more accessions from all 36 duckweed species and discuss the evolutionary impact and peculiarities of both parameters in duckweeds.


Introduction
A review about the aquatic monocot model family of duckweeds and its emerging economic importance has been published recently [1]. However, at this time, not for all duckweed species chromosome counts and genome sizes were known. Here, we compile corresponding data for at least one accession (clone) for each species, for some of them for the first time (see Figure 1, Results and Discussion). For previous data see [2] and references therein. Chromosome number (blue) and genome size (red) of duckweeds. When chromosome numbers and genome sizes refer to newly investigated species or were taken from [2] or [3] (Le. Figure 1. Chromosome number (blue) and genome size (red) of duckweeds. When chromosome numbers and genome sizes refer to newly investigated species or were taken from [2] or [3] (Le. minuta), they appear as numbers. For references to other chromosome counts (blue dots and ovals [for closely adjacent values]) see [2]. Other genome size estimations (red dots and ovals) were from [4].

Material and Methods
The duckweed accessions investigated in this paper are listed in the legend of Figure 1 and in Figure 3 and were kindly provided by Dr. K.-J. Appenroth, Friedrich-Schiller-University, Jena, Germany. For plant cultivation, chromosome preparation, super-resolution microscopy and genome size measurement, see [2].

The Genome Sizes
There are several approaches to determining genome size, defined as pg/2C or Mbp/1C (C means the unreplicated haploid [or meiotically reduced] genome). Older papers applied Feulgen densitometry of nuclei, later ones mostly use flow cytometry [9] which allows for a much larger number of nuclei to be measured. These direct measurements require an internal reference standard with a known genome size close to that of the target species. Recently, based on sequenced genomes, k-mer distribution, as a bioinformatic approach, also served for genome size estimation. The accuracy of flow cytometry depends on the quality of the prepared nuclei suspension and the suitability of the used buffer and reference species. For precise genome size measurements, it is recommended that the coefficient of variation (CV) does not exceed 3% [9]. The k-mer size results provide a useful estimation, which should be complemented by direct measurements [10,11]. For all 36 duckweed species of the five genera, the genome size has been measured ( Figure 1 and [2][3][4]12] [2]. The largest intrageneric variation was found in the genus Wolffia with 432 Mbp for Wolffia australiana to 2203 Mbp for Wo. arrhiza. For 11 species representing all duckweed genera, genome size was positively correlated with nuclear and cell volume of guard cells, and with progressive organ reduction, but negatively with frond size and phylogenetic age. No correlation was observed with the number of chromosomes or the rDNA loci [2]. The larger genomes of phylogenetically younger genera do not necessarily indicate a general evolutionary genome expansion in duckweeds. The high ratio of soloLTRs to intact retroelements of~8 in S. polyrhiza [13], and the respective value of 11-14 in Wo. australiana [14]-the species with the smallest genome in its genus-could rather be a hint that the oldest genus Spirodela, after early branching from the other duckweeds, experienced genome shrinking due to a deletion-biased DNA double-strand break repair pathway [15]. The same could be true for Wo. australiana within its genus. In these cases, the likely scenario would be a bi-directional evolution of genome size starting from an intermediate ancestral duckweed genome of~400 to 800 Mbp. Studies on DNA double-strand break repair in species with a high ratio of soloLTRs to intact retroelements could test this assumption. Genome size estimations for S. polyrhiza, S. intermedia and La. punctata from different laboratories are rather similar [2][3][4]. The other genomes measured by Hoang et al. [2] are in general larger than those measured for the same species by Wang et al. [4]. This might be caused by the use of different internal reference standards or different equipment, but most likely by assuming a different genome size of Arabidopsis thaliana as a basis for calculation (157 Mbp in [2], versus 147 Mbp in [4]). An initially unexpected finding was the strong variation of genome size estimation for clones of the same species. In case of clones of Le. aequinoctialis and La. punctata, the explanation is spontaneous, respectively induced, whole genome duplication (WGD) [2] (Figure 1). The genome sizes for two groups of Le. minor clones (~400 Mbp versus~600 Mbp), however, differ by >50% ( [4]; M. Bog, J. Fuchs, K.-J. Appenroth unpublished). For clone 5500, a genome size of 481 Mbp was estimated [16] and 409 Mbp for clone 8623 with 2n = 42 chromosomes [2]. For clone 8627, we counted 63 chromosomes and measured a genome size of >600 Mbp (PTNH & JF unpublished). Molecular fingerprinting for tubulin genes [17] suggested that clone 8627 belongs to Lemna japonica and is a hybrid of Lemna minor and Le. turionifera. Our data identified it as triploid. Further investigations are needed to uncover whether in other duckweed species with a high intraspecific variation of DNA content, clone-specific WGD with a fast subsequent genome size reduction occurred, or whether these groups of clones represent cryptic species or interspecific hybrids. The occurrence of interspecific hybrids in duckweeds is a surprise because they require sexual propagation-sometimes even involving unreduced gametes as in clone 8627 (see above)-which, according to laboratory observations, might occur very rarely in predominantly vegetatively propagating duckweed species. On the other hand, the maintenance of interspecific hybrids seems to be favored by asexual propagation.

Evolutionary Impact of Genome Size and Karyotype Studies
Chromosome numbers and genome size, varying in parallel by the same wholenumber multiple, suggest a recent WGD (neopolyploidy) usually yielding autotetraploids in asexual clones. However, a nearly doubled genome size (or its absence) as well as a (nearly) doubled chromosome number (or its absence) alone are not sufficient to decide whether or not a WGD took place. Dysploid chromosome number reduction and/or fast reduction of genome size may blur real WGD. Therefore, in such cases, additional independent approaches are mandatory for arriving at conclusive statements. For instance, in Australian Brassicaceae with n = 4 to 6, evidence for mesopolyploidy (descendent dysploidy after WGD) was found through multicolor cross-FISH with bacterial artificial chromosome (BAC) pools representing the genome of A. thaliana [18]. All regions labeled by BAC pools from Arabidopsis, which represent the eight ancestral Brassicaceae chromosomes, appeared duplicated within the only four to six meiotic bivalents of the tested species. On the other hand, a genome can expand to double and more without WGD just by insertion-biased double-strand break repair (mainly retroelement insertion). In general, confirmed WGD leading to neopolyploidy seems to be more frequent between clones of distinct duckweed species than being responsible for genome size differences between species of the same or different genera. Of the natural species studied, only We. rotunda (clone 9072: 2n = 82; 1914 Mbp/1C), Wo. arrhiza (clone 8872: 2n = 60; 2203 Mbp/1C) and Wo. cylindracea (clone 9056: 2n = 60; 2144 Mbp/1C) are, so far, candidates for neopolyploids.
Karyotype studies employing molecular cytogenetics may help to elucidate the evolutionary origin of polyploid species. Genomic in situ hybridization (GISH) can identify the parental species if species-specific repetitive sequences are present in the genomes of the suspected parental species. For 'quasi-diploid' or paleopolyploid species (in fact all land plants experienced at least one very remote WGD), FISH with anchored unique sequences is an independent and direct approach to confirm or correct genome assemblies which are based on probabilistic methods. In this way, a robust chromosome-scale genome map has been achieved in several steps for S. polyrhiza [19][20][21]. Cross-FISH with single copy sequences can uncover chromosome homeology and chromosome rearrangements between related species. The two Spirodela species (S. polyrhiza, 2n = 40 and S. intermedia, 2n = 36) were studied by sequential multicolor cross-FISH with different pools of 96 BACs anchored in the genome of S. polyrhiza [22] and compared with genome assemblies for both species [21]. Eight chromosome pairs did not reveal rearrangements between both species. The other twelve chromosome pairs of S. polyrhiza correspond to the remaining ten pairs of S. intermedia and display (in part multiple) rearrangements ( Figure 2). The direction of the evolution cannot be determined with certainty, because the genus comprises only these two species. However, because most duckweed species tend to have 40 or more chromosomes, 40 might be the ancestral and 36 the derived chromosome number. No rearrangements were found between seven clones from different geographic origins of S. polyrhiza [20] and two clones of S. intermedia [21], respectively. These studies showed clearly that S. polyrhiza and S. intermedia are different species, despite similar genome size and overlap of most morphological features. In contrast to the situation described for the Brassicaceae family (e.g., ref. [23]), cross-FISH with S. polyrhiza BACs yielded only weak and dispersed signals but did not recognize chromosome homeology when applied to species of other duckweed genera (La. punctata, Le. aequinoctialis, Wolffiella hyalina, Wo. arrhiza), independently of stringency conditions [24]. So far, cross-FISH with oligo probes derived from chromosome assemblies of S. polyrhiza or S. intermedia also did not reveal chromosome homeology in other duckweed genera [25]. The probes yielded either dispersed signals in La. punctata and Le. aequinoctialis or no signals at all as in We. hyalina and Wo. australiana. Apparently, these genomes are too diverse and probe densities are too low to generate reliable signals across the duckweed genera. In future, homologous single copy sequences, selected assuming synteny between the genomes in question, should be designed for oligo-FISH to unassembled genomes [25]. Metaphases with chromosome counts reported for the first time in this paper. To count clearly the chromosome number, image stacks from spatial super-resolution structured illumination microscopy (3D-SIM) [26] were used to identify overlapping chromosomes. Individual chromosomes were enumerated arbitrarily. Scale bar: 5 µm.   To count clearly the chromosome number, image stacks from spatial super-resolution structured illumination microscopy (3D-SIM) [26] were used to identify overlapping chromosomes. Individual chromosomes were enumerated arbitrarily. Scale bar: 5 µm.