plants
Communication
Chromosome Numbers and Genome Sizes of All 36 Duckweed
Species (Lemnaceae)
Phuong T. N. Hoang †,‡ , Jörg Fuchs † , Veit Schubert
, Tram B. N. Tran ‡ and Ingo Schubert *
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, D-06466 Stadt Seeland, Germany
* Correspondence: schubert@ipk-gatersleben.de
† These authors contributed equally.
‡ Permanent address: Biology Faculty, Dalat University, District 8, 66000 Dalat, Vietnam.
Abstract: 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.
Keywords: chromosome number; duckweeds; evolution; genome size; karyotype; Lemnaceae
1. Introduction
Citation: Hoang, P.T.N.; Fuchs, J.;
Schubert, V.; Tran, T.B.N.; Schubert, I.
Chromosome Numbers and Genome
Sizes of All 36 Duckweed Species
(Lemnaceae). Plants 2022, 11, 2674.
https://doi.org/10.3390/
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.
plants11202674
Academic Editors: Viktor Oláh,
Klaus-Jürgen Appenroth and K.
Sowjanya Sree
Received: 28 June 2022
Accepted: 8 October 2022
Published: 11 October 2022
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Plants 2022, 11, 2674. https://doi.org/10.3390/plants11202674
https://www.mdpi.com/journal/plants
Plants 2022, 11, 2674
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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].
The values for Le. perpusilla 8539 (519 Mbp), Le. tenera 9024 (526 Mbp), Le. turionifera 8693 (475 Mbp),
We. caudata 9165 (772 Mbp); We. denticulata 8221 (717 Mbp), We. neotropica 8848 (599 Mbp), We. oblonga
9391 (755 Mbp), 2n = 42), We. repanda 9062 (1190 Mbp, 2n = 40), We. welwitschii 9469 (780 Mbp),
Wo. cylindracea 9056 (2144 Mbp; 2n = 60), Wo. elongata 9188 (936 Mbp; 2n = 40), Wo. neglecta 9149
(1354 Mbp; 2n = 40) were obtained by flow-cytometry and determined in this paper. Species for
which we provide in this paper the first or new values are in bold and the corresponding values are
underlined. * indicates the values for the colchicine-induced tetraploid La. punctata clone 5562.
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2. Material and Methods
The duckweed accessions investigated in this paper are listed in the legend of Figure 1
and in Figure 2 and were kindly provided by Dr. K.-J. Appenroth, Friedrich-SchillerUniversity, Jena, Germany. For plant cultivation, chromosome preparation, super-resolution
microscopy and genome size measurement, see [2].
Figure 2. 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) [3] were used to identify overlapping chromosomes. Individual chromosomes
were enumerated arbitrarily. Scale bar: 5 µm.
3. Results and Discussion
3.1. The Chromosomes
At least for one accession of all 36 duckweed species, chromosome counts are now
available (for review see Figure 1 and [2]). For six species, Lemna tenera 9024 (2n = 40),
Wolffiella caudata 9165 (2n = 42), Wolffiella repanda 9062 (2n = 40), Wolffia cylindracea 9056
(2n = 60), possibly a triploid accession, Wolffia elongata 9188 (2n = 40) and Wolffia neglecta
9149 (2n = 40), chromosome counts (Figure 2) are presented for the first time in this paper.
Most frequent are chromosome counts around 2n = 40. However, in some papers [2,5,6]
(and in older references in [2,4]), highly variable chromosome numbers were reported for
the same species, e.g., for Lemna aequinoctialis 20, 40, 50, 60 and 80 [5]; 40, 50, 66, 72, 78,
84 and 65–76 [7]; 42, 84 [6]; 40, 60, 80 [4]; and 42 [2]. Despite the predominantly asexual
propagation of many duckweed species, such a high variability is strange. Moreover,
in cases where previously variable chromosome numbers were reported and the corresponding clones are still available, deviating numbers could not be confirmed [2]. In all
tested clones of Spirodela polyrhiza (2n = 40) and Spirodela intermedia (2n = 36), we constantly
found the same chromosome number, even for clones for which deviating numbers were
reported before (see [2]). Nevertheless, smaller deviations in chromosome number between
asexual clones of the same species cannot be excluded due to the potential presence of B
chromosomes. Some of the samples apparently indicate autotetraploidy (Le. aequinoctialis,
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clone 2018 2n = 42, and clone 6746 2n = 84, refs. [2,6]) which occurred spontaneously, or was
induced [8] as in Landoltia punctata with 2n = 46 and 2n = 92 (clone 5562 [2]). However, the
quite high number of rather small chromosomes, which barely show any morphological differentiation (primary constrictions = centromeres, and secondary constrictions = nucleolus
organizing regions [NOR], are mostly not discernible), and which are difficult to prepare
from the small meristems, can lead to counting errors. For instance, for Wolffiella rotunda
clone 9072 (2n = 82), fluorescent in situ hybridization (FISH), with 45S rDNA as a probe,
revealed chromosome satellites, distally flanking the NOR, which could have easily been
miscounted as small separate chromosomes [2] (Figure S3 therein). Furthermore, small and
poorly structured chromosomes often make it difficult to decide whether they are monoor holocentric.
3.2. 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 [2–4,12]). The values for Lemna perpusilla 8539 (519 Mbp), Le. tenera 9024 (526 Mbp),
Lemna turionifera 8693 (475 Mbp), We. caudata 9165 (772 Mbp); Wolffiella denticulata 8221
(717 Mbp), Wolffiella neotropica 8848 (599 Mbp), Wolffiella oblonga 9391 (755 Mbp), 2n = 42), We.
repanda 9062 (1190 Mbp, 2n = 40), Wolffiella welwitschii 9469 (780 Mbp), Wo. cylindracea 9056
(2144 Mbp; 2n = 60), Wo. elongata 9188 (936 Mbp; 2n = 40) and Wo. neglecta 9149 (1354 Mbp;
2n = 40) were obtained by flow-cytometry in this paper. The results yielded a ~14-fold
range from 160 Mbp in the phylogenetically oldest genus, comprising S. polyrhiza and
S. intermedia, up to 2203 Mbp of Wolffia arrhiza of the phylogenetically youngest genus [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 [2–4]. 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
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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.
3.3. 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 [19–21]. 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 3). The direction of the
evolution cannot be determined with certainty, because the genus comprises only these two
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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].
Figure 3. Chromosome homeology and rearrangements between S. polyrhiza (n = 20) and S. intermedia
(n = 18) as revealed by cytogenomics, see [21,22]. The double arrows indicate that either direction of
evolution could have happened. Dark blue are chromosomes not involved in rearrangements; other
colors indicate rearranged chromosomes. Dark grey boxes: regions present only in S. polyrhiza; light
grey boxes: regions present only in S. intermedia. Enumeration indicates distinct chromosomes of the
respective species.
4. Conclusions
Having now compiled chromosome counts and genome sizes for at least one accession
of all 36 duckweed species (for some species these are the first data), a basis is available
upon which to investigate the evolution of duckweed genomes as well as the reasons for
the apparent intraspecific variation of both parameters. A future combination of genomic
data, genome size, chromosome number, GISH and FISH data will clarify the phylogenetic
position and taxonomic status of intrageneric duckweed accessions which are difficult to
assign to distinct species on the basis of morphological criteria.
Author Contributions: Conceptualization and funding aquisition, I.S.; data analysis P.T.N.H., J.F.,
V.S. and T.B.N.T., composition and writing, I.S., P.T.N.H., J.F. and V.S. All authors have read and
agreed to the published version of the manuscript.
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Funding: This work was supported in part by the German Research Foundation to IS (SCHU
951/18-1). PTNH was supported by the Vietnam National Foundation for Science and Technology
Development (NAFOSTED) grant # 106.01-2020.33.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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