both 5′ and 3′ end were removed. Reads shorter
than 36 bases were discarded. In a second step,
the reads were mapped to the Lemna minor
genome (Van Hoeck et al. 2015) using the
BWA-MEM algorithm of the Burrows-Wheeler
Alignment Tool (Li and Durbin 2009). All bam
files were merged, sorted, and all reads that had
low mapping quality (q < 10) were removed
using SAMtools v1.3.1 (Li et al. 2009). Indels
were called using GATK v3 (McKenna et al.
2010), and the reads around the indels were
realigned. SNPs were annotated and called from
the realigned bam file. VCFtools v0.1.13
(Danecek et al. 2011) was used to filter the SNP
data.
For tree reconstruction, we removed all SNP
loci from the final dataset that had more than
40% missing data. In total, 3005 loci were kept.
Among them, 2233 homozygous sites were used
to build the FASTA sequence alignment for
reconstructing the phylogenetic tree. Tree
reconstruction was done using MrBayes v3.2.6
(Huelsenbeck and Ronquist 2001). We used the
General Time Reversible substitution model with
gamma-distributed rate variation across sites
since this was the best model found by jModeltest2 v.2.1.5 (Darriba et al. 2012). We made two
independent runs with four chains each. The
temperature of the heated chains was set to 0.2.
The programme was run for 2,000,000
Table 11.1 Species and
clones (ID and accession
numbers) of the genus
Lemna used for
genotyping-by-sequencing.
The two species in bold
were used as an outgroup
for the genus
Species
Clone
ID
Accession
number
Origin
L. aequinoctialis
6746
a1258832
USA, Virginia
L. disperma
7269
a1258835
USA, California
L. gibba
8682
a1258833
Saudi-Arabia
L. japonica
8695
a1258836
Japan, Kyoto
L. minor
5500
a1096785
Ireland
L. minuta
6717
a1433919
Guatemala, Chinaltenango
L. minuta
7612
a1433922
Peru, Cutco
L. minuta
8669
a1258827
Japan, Kyoto
L. minuta
9484
a1433921
Greece
L. obscura
9342
a1258834
Venezuela, Maracaibo
L. perpusilla
8539
a1258826
USA, Virginia
L. tenera
9020
a1258830
Australia, Northern
Territory
L. trisulca
9529
a1258837
Germany
L. turionifera
9434
a1258828
Russia, Baikal Area
L. valdiviana
9401
a1433927
Venezuela, Sucre
L. valdiviana
9228
a1433926
Brazil, Bahia
L. valdiviana
9442
a1258831
Brazil, Mato Grosso
L. yungensis
9207
a1433919
Bolivia, La Paz
L. yungensis
9208
a1258829
Bolivia, La Paz
L. yungensis
9209
a1433924
Bolivia, La Paz
L. yungensis
9210
a1433925
Bolivia, La Paz
Landoltia
punctata
9589
a1096784
India, New Delhi
Spirodela
polyrhiza
7498
a1096783
USA, North Carolina
11 Genotyping-by-Sequencing for Species Delimitation …
117
than 36 bases were discarded. In a second step,
the reads were mapped to the Lemna minor
genome (Van Hoeck et al. 2015) using the
BWA-MEM algorithm of the Burrows-Wheeler
Alignment Tool (Li and Durbin 2009). All bam
files were merged, sorted, and all reads that had
low mapping quality (q < 10) were removed
using SAMtools v1.3.1 (Li et al. 2009). Indels
were called using GATK v3 (McKenna et al.
2010), and the reads around the indels were
realigned. SNPs were annotated and called from
the realigned bam file. VCFtools v0.1.13
(Danecek et al. 2011) was used to filter the SNP
data.
For tree reconstruction, we removed all SNP
loci from the final dataset that had more than
40% missing data. In total, 3005 loci were kept.
Among them, 2233 homozygous sites were used
to build the FASTA sequence alignment for
reconstructing the phylogenetic tree. Tree
reconstruction was done using MrBayes v3.2.6
(Huelsenbeck and Ronquist 2001). We used the
General Time Reversible substitution model with
gamma-distributed rate variation across sites
since this was the best model found by jModeltest2 v.2.1.5 (Darriba et al. 2012). We made two
independent runs with four chains each. The
temperature of the heated chains was set to 0.2.
The programme was run for 2,000,000
Table 11.1 Species and
clones (ID and accession
numbers) of the genus
Lemna used for
genotyping-by-sequencing.
The two species in bold
were used as an outgroup
for the genus
Species
Clone
ID
Accession
number
Origin
L. aequinoctialis
6746
a1258832
USA, Virginia
L. disperma
7269
a1258835
USA, California
L. gibba
8682
a1258833
Saudi-Arabia
L. japonica
8695
a1258836
Japan, Kyoto
L. minor
5500
a1096785
Ireland
L. minuta
6717
a1433919
Guatemala, Chinaltenango
L. minuta
7612
a1433922
Peru, Cutco
L. minuta
8669
a1258827
Japan, Kyoto
L. minuta
9484
a1433921
Greece
L. obscura
9342
a1258834
Venezuela, Maracaibo
L. perpusilla
8539
a1258826
USA, Virginia
L. tenera
9020
a1258830
Australia, Northern
Territory
L. trisulca
9529
a1258837
Germany
L. turionifera
9434
a1258828
Russia, Baikal Area
L. valdiviana
9401
a1433927
Venezuela, Sucre
L. valdiviana
9228
a1433926
Brazil, Bahia
L. valdiviana
9442
a1258831
Brazil, Mato Grosso
L. yungensis
9207
a1433919
Bolivia, La Paz
L. yungensis
9208
a1258829
Bolivia, La Paz
L. yungensis
9209
a1433924
Bolivia, La Paz
L. yungensis
9210
a1433925
Bolivia, La Paz
Landoltia
punctata
9589
a1096784
India, New Delhi
Spirodela
polyrhiza
7498
a1096783
USA, North Carolina
11 Genotyping-by-Sequencing for Species Delimitation …
117