section Uninerves has morphological diagnostic
features, i.e. a single nerve compared to 3–7
nerves in the other Lemna sections. Landolt
(1986) originally reported two species under this
section, Lemna minuta Kunth (in that time as
L. minuscula Herter) and L. valdiviana Phil., but
had later added the species L. yungensis Landolt
(Landolt 1998), which was discovered growing
on wet rocks in the province Nor-Yungas, Bolivia. The delimitation of these species on a morphological basis has been always challenging
(Crawford et al. 1996), especially after L. yungensis had been added, which is even more
similar to L. valdiviana (Landolt 1998). The
separation of all three species as section by barcoding using plastidic/morphological (Les et al.
2002) and nuclear (Tippery et al. 2015) markers
was indicated by bootstrap values of 100%. Bog
et al. (2010) used manually scored amplified
fragment length polymorphism (AFLP) to analyse the genus Lemna. Lemna section Uninerves
could be separated by this method from other
clades (bootstrap value 86%). Although L. minuta formed a defined clade (bootstrap value
100%), the other two species did not form any
defined group. Moreover, one of the authors of
the aforementioned studies (Elias Landolt) had
doubts concerning this clear definition of
L. minuta, owing to the reason that the biogeographical coordinates of the five analysed clones
might not represent the actual diversity of this
species in nature. Similar to the study by Wang
et al. (2010), who investigated L. minuta and
L. valdiviana using plastidic DNA markers and
found no separation between them, Borisjuk
et al. (2015), who used the markers psbK-psbI
and atpH-atpF on all the three species, also
considered that the differentiation of species in L.
section Uninerves was not completely reliable.
Using a large number of clones and the plastidic
marker psbK-psbI, Bog et al. (2020) found that
L. minuta could be separated from L. valdiviana
and L. yungensis, but the latter two could not be
distinguished.
Recently, next-generation sequencing made
large-scale sequencing of genomes cheaper and
faster. Therefore, we aimed to apply
genotyping-by-sequencing (GBS; c.f. Elshire
et al. 2011; Poland et al. 2012; Wendler et al.
2014) in order to address the question whether
the species of the section Uninerves can be distinguished as (A) three independent species
(L. minuta, L. valdiviana, and L. yungensis) or
(B) L. valdiviana and L. yungensis should be
synonymised and treated as one species next to
L. minuta. Moreover, this also represents the first
investigation of using GBS on the genus Lemna
(Lemnaceae) to address the taxonomic challenge
within L. section Uninerves.
11.2 Materials and Methods
11.2.1 Plant Material
All plants were taken from the stock collection of
the University of Jena, Matthias Schleiden
Institute-Plant Physiology supplemented by
some clones from the Landolt Duckweed Collection, Zürich. The clones and their origin are
given in Table 11.1. Plants were cultivated in
N-medium (Appenroth et al. 1996) as described
before (Bog et al. 2010), shock frozen after
harvest in liquid nitrogen and freeze-dried.
11.2.2 Molecular Methods
DNA extraction based on silica columns was
done according to Valledor et al. (2014), and
subsequent construction of barcoded libraries for
the GBS approach was essentially performed
as described in Wendler et al. (2014).
Sequencing-by-synthesis (single read, 1 Â 107
cycles, index read 8 cycles) using the Illumina
HiSeq 2500 device was done according to protocols provided by the manufacturer (Illumina
Inc.).
11.2.3 Data Analysis
Raw reads were trimmed and cleaned using
AdapterRemoval v2.1.7 (Schubert et al. 2016).
All stretches of Ns and consecutive stretches of
low-quality bases (minimum quality of 30) on
116
M. Bog et al.
features, i.e. a single nerve compared to 3–7
nerves in the other Lemna sections. Landolt
(1986) originally reported two species under this
section, Lemna minuta Kunth (in that time as
L. minuscula Herter) and L. valdiviana Phil., but
had later added the species L. yungensis Landolt
(Landolt 1998), which was discovered growing
on wet rocks in the province Nor-Yungas, Bolivia. The delimitation of these species on a morphological basis has been always challenging
(Crawford et al. 1996), especially after L. yungensis had been added, which is even more
similar to L. valdiviana (Landolt 1998). The
separation of all three species as section by barcoding using plastidic/morphological (Les et al.
2002) and nuclear (Tippery et al. 2015) markers
was indicated by bootstrap values of 100%. Bog
et al. (2010) used manually scored amplified
fragment length polymorphism (AFLP) to analyse the genus Lemna. Lemna section Uninerves
could be separated by this method from other
clades (bootstrap value 86%). Although L. minuta formed a defined clade (bootstrap value
100%), the other two species did not form any
defined group. Moreover, one of the authors of
the aforementioned studies (Elias Landolt) had
doubts concerning this clear definition of
L. minuta, owing to the reason that the biogeographical coordinates of the five analysed clones
might not represent the actual diversity of this
species in nature. Similar to the study by Wang
et al. (2010), who investigated L. minuta and
L. valdiviana using plastidic DNA markers and
found no separation between them, Borisjuk
et al. (2015), who used the markers psbK-psbI
and atpH-atpF on all the three species, also
considered that the differentiation of species in L.
section Uninerves was not completely reliable.
Using a large number of clones and the plastidic
marker psbK-psbI, Bog et al. (2020) found that
L. minuta could be separated from L. valdiviana
and L. yungensis, but the latter two could not be
distinguished.
Recently, next-generation sequencing made
large-scale sequencing of genomes cheaper and
faster. Therefore, we aimed to apply
genotyping-by-sequencing (GBS; c.f. Elshire
et al. 2011; Poland et al. 2012; Wendler et al.
2014) in order to address the question whether
the species of the section Uninerves can be distinguished as (A) three independent species
(L. minuta, L. valdiviana, and L. yungensis) or
(B) L. valdiviana and L. yungensis should be
synonymised and treated as one species next to
L. minuta. Moreover, this also represents the first
investigation of using GBS on the genus Lemna
(Lemnaceae) to address the taxonomic challenge
within L. section Uninerves.
11.2 Materials and Methods
11.2.1 Plant Material
All plants were taken from the stock collection of
the University of Jena, Matthias Schleiden
Institute-Plant Physiology supplemented by
some clones from the Landolt Duckweed Collection, Zürich. The clones and their origin are
given in Table 11.1. Plants were cultivated in
N-medium (Appenroth et al. 1996) as described
before (Bog et al. 2010), shock frozen after
harvest in liquid nitrogen and freeze-dried.
11.2.2 Molecular Methods
DNA extraction based on silica columns was
done according to Valledor et al. (2014), and
subsequent construction of barcoded libraries for
the GBS approach was essentially performed
as described in Wendler et al. (2014).
Sequencing-by-synthesis (single read, 1 Â 107
cycles, index read 8 cycles) using the Illumina
HiSeq 2500 device was done according to protocols provided by the manufacturer (Illumina
Inc.).
11.2.3 Data Analysis
Raw reads were trimmed and cleaned using
AdapterRemoval v2.1.7 (Schubert et al. 2016).
All stretches of Ns and consecutive stretches of
low-quality bases (minimum quality of 30) on
116
M. Bog et al.