DOI: 10.5061/dryad.jwstqjqjb
title: A Pleistocene legacy of gene pools, ecodemes and admixtures of Stuckenia pectinata (L.) Börner as evidenced from microsatellites, complete chloroplast genomes and ribosomal RNA cistron (Europe, Africa)

description: Stuckenia pectinata (L.) Börner is a widely distributed submerged plant
 well-studied for its ecology, distribution, and molecular diversity.
 Globally, various genotypic lineages and hybrids of Stuckenia species have
 been identified using nuclear rRNA (ITS) and chloroplast sequences
 (notably rpl20-5’rps12 and trnT-TrnL). These studies have shown
 intraspecific variability in S. pectinata, with two gene pools
 ('genotype 1a' and '1b') reported for Europe and
 Africa. Moreover, former isozyme research suggested distinct freshwater
 and brackish water gene pools. Therefore, our primary objective was to
 determine whether these ecodemes correspond to either 'genotype
 1a' or '1b'. Using fifteen nuclear microsatellite loci,
 complete chloroplast genome sequences (156,677 bp), and the rRNA cistron
 (7,178 bp), we analyzed the genetic identity of 313 S. pectinata samples
 (representing 124 unique clones) from 12 populations in Europe and Africa.
 Chloroplast genomes of three African Rift lake populations corresponded to
 ‘genotype 1b’, while those of nine European populations corresponded
 to ‘genotype 1a’. Microsatellites also clearly distinguished
 ‘genotype 1a’ from ‘1b’ in an individual PCoA and Structure analysis,
 whereas incomplete homogenization of 5S-rRNA sequences suggested either
 ongoing differentiation or intraspecific hybridization between ‘genotype
 1a’ and ‘1b’. Haplotype lineages, rRNA cistron mutations, and
 microsatellites revealed an additional subdivision within ‘genotype 1a’,
 distinguishing a freshwater gene pool from a brackish water one.
 Approximate Bayesian computation analyses using nuclear microsatellites
 supported a demographic expansion model and a time of divergence for the
 African Rift lake populations as well as for the European freshwater and
 brackish water populations, dating back to the Late Pleistocene. Divergent
 chloroplast lineages might refer to different refugia during the Last
 Glacial Maximum. Stuckenia pectinata from Lake Hora (Ethiopia), Lake
 Balaton (Hungary), and the Camargue (France) each harbored two distinct
 maternal haplotypes from Selinunte (Italy), indicating F1 hybrids, whereas
 those from Lake Peipsi (Estonia), amongst others, showed incomplete rRNA
 homogenization. It is hypothesized that contemporary S. pectinata
 populations, especially in Europe, retained a legacy of ancient gene pool
 differentiation despite a history of hybridization, admixture, and
 chloroplast capture. It is recommended that studies on the ecology and
 reproductive strategies of this seemingly common and widespread species
 should take into account its genetic identity.
description: <strong>2.1 Plant materials</strong>
 A total of 313 individual shoots were collected in twelve
 populations and ranged from 5-50 per population (Table 1). Collections
 from Estonia, the Netherlands, Belgium, France, Hungary, Spain, Italy,
 Ethiopia, and Kenya (Fig. 1) were built up from 2007 – 2018 by the authors
 using a similar methodology. Samples were taken at 2 m intervals along a
 transect and approximately 1 m depth, using a rake from a boat or by
 wading parallel to the edge. Leaves were dried and preserved in individual
 bags with silica gel until further analysis.
 <strong>2.2 Nuclear microsatellite
 analysis</strong> Primers were
 fluorescence-labelled with four different dye-labels (6FAM, VIC, NED, and
 PET). DNA concentration was 20–50µg/ml. A primer mix was made by mixing
 0.2 µM of each primer together. Multiplex PCRs consisted of 6.25 µl master
 mix (Qiagen Multiplex PCR kit), 1.25 µl primer mix, 2.5µl
 H<sub>2</sub>O, and 2.5µl of genomic DNA. PCR was performed in
 a thermal cycler (Bio-Rad MyCycler, Hercules, California, USA) with the
 following conditions: an initial denaturation of 95°C for 15 minutes (as
 indicated in protocol of Qiagen multiplex PCR kit manual) followed by 35
 cycles of: 30 seconds denaturation at 95°C, 90 seconds annealing at 57°C
 and 80 seconds elongation at 72°C, followed by a final extension of 30
 minutes at 60°C. PCR products were separated on an ABI3730XL sequencer
 (Applied Biosystems (Waltham, Massachusetts, USA), runs were carried out
 by Macrogen (Seoul, Korea), and allele sizes were determined with
 GeneMarker v.2.60 (SoftGenetics LLC, State College, USA).<strong>
 </strong>Two multiplex reactions, of eleven (SPEC in Bossaer et al.,
 2024) and nine primer pairs (Pp in Nies and Reusch, 2004), were performed.
 Genotyping was done for fifteen polymorphic nuclear microsatellite loci:
 SPEC7 (Genbank accession OR592089), SPEC14 (OR592085), SPEC25 (OR592084),
 SPEC26 (OR592090), SPEC30, SPEC35 (OR592083), SPEC38 (OR592087), SPEC57
 (OR592082), SPEC59 (OR592088) and for Pp28, Pp34, Pp26, Pp37, Pp39 and
 Pp40). Five loci (Pp24, Pp32, Pp42, SPEC23 (OR592086), and SPEC55
 (OR592091)) did not amplify in every population and were
 omitted. <strong>2.3 Next-generation
 sequencing</strong> Genomic DNA extracts of 24
 samples (two of each population and designated a and b) were made at the
 Plant Biology and Nature Management (APNA) lab of the Vrije Universiteit
 Brussel (VUB) and processed for next generation sequencing analysis using
 the E.Z.N.A. SP plant DNA Mini Kit (Omega biotek, Norcross, GA, USA). The
 quantity and purity (260/280 and 260/230 ratios) of the DNA were
 determined using a Nanodrop one Spectrophotometer (Thermo Fisher
 Scientific, Waltham, Massachusetts, USA). Extractions were repeated for
 samples with a 260/280 ratio of less than 1.8 and/or a concentration lower
 than 5 ng/μL. If necessary, multiple DNA extractions of one sample were
 pooled and concentrated by ethanol precipitation. An Illumina paired-end
 library was constructed using the TruSeq nano DNA Kit. After passing
 quality inspection (DNA concentration between 5 to 15ng/ul), the
 constructed library (TruSeq Nano DNA Kit) was sequenced by 300 bp x 2
 paired-end sequencing in an Illumina MiSeq platform (Macrogen, Seoul,
 South Korea). <strong>2.3.1 Chloroplast genome
 assembly, alignment, and comparative analysis</strong>
 Raw data was filtered out to remove the joint sequence and
 low-quality reads to obtain high-quality clean data. The Illumina pair-end
 next-generation sequencing (NGS) product is used as the input file for
 <em>de novo</em> chloroplast assemblies. The <em>de
 novo</em> chloroplast assemblies were first done for two chloroplast
 genomes using NOVOPlasty assembly at Kmer = 33 (Dierckxsens et al., 2017).
 All assemblies were executed by taking a single read from the dataset that
 originates from the targeted plastid as seed (<em>rbcL</em>)
 and taking 30% as a subsample from the FASTA file with default parameters.
 These were compared to the existing annotated <em>S.
 pectinata</em> chloroplast genome (GenBank accession number NC057253
 of Lake Dianchi, China, in Tian et al., 2019) and appeared exactly similar
 in genome structure and perfectly aligned. Therefore, the remaining
 samples were assembled using the ‘assemble to reference’ function in
 Geneious Prime v 2022.2.2 (©Biomatters) software. Illumina 2 x 300 bp
 paired-end reads were processed in Geneious to obtain complete chloroplast
 genome sequences. All assemblies used NC057253 (<em>S.
 pectinata</em>) as a reference genome, and the mapping of reads of
 24 samples was performed with an average of 9,968 – 73,648 reads, with a
 mean depth of reads ranging from 8 – 141 coverage. The 24 consensus
 sequences and the fully annotated NC057253 were aligned with MAFFT
 v7.388<em> </em>(Katoh et al., 2002; Katoh and Standley,
 2013). <strong>2.3.2 Nuclear rRNA cistron
 assembly</strong> The nuclear ribosomal cistron
 (18S, ITS1, 5.8S, ITS2 and 26S) was assembled from a <em>S.
 pectinata</em> (Genbank MT784071, unpublished, submitted by
 Langkjaer and Leerhoei in 2020) 5,623 bp sequence containing an internal
 transcribed spacer 1 (partial sequence), 5.8S ribosomal RNA gene (complete
 sequence) and internal transcribed spacer 2 (partial sequence) and
 subsequently used three times as a seed in Geneious to progressively
 enlarge the flanking regions. A 7,178 bp nuclear ribosomal cistron was
 obtained for each sample, averaging 4,986 – 52,625 reads with a mean depth
 of reads ranging from 252 – 1,632 coverage. The generated consensus
 sequences of 7,178 bp length for a total of 24 samples were aligned for
 comparative description of mutated positions using MAFFT v7.388 (Katoh et
 al., 2002; Katoh and Standley, 2013).
description: # A Pleistocene legacy of gene pools, ecodemes and admixtures of Stuckenia
 pectinata (L.) Börner as evidenced from microsatellites, complete
 chloroplast genomes and ribosomal RNA cistron (Europe, Africa)
 [https://doi.org/10.5061/dryad.jwstqjqjb](https://doi.org/10.5061/dryad.jwstqjqjb) ## Description of the data and file structure * Original_data_Microsatellites.xlsx Stuckenia pectinata microsatellite scores are provided in GenAlex format :  A1 = number of microsatellite loci B1 = total number of individuals C1 = number of populations D1 to O1 = number of individuals within each population D2 to O2 = population codes C3 to AF3 = names of 15 microsatellite loci with colors  (green, yellow, blue, red) corresponding to the fluorescent labels used in multiplex PCR From row 4 onwards: Column A = individual sample codes; B = population codes; C to  AF = length of microsatellite allele (biallelic) for each microsatellite locus  * Stuckenia_chloroplast_sequences_TRIEST_ET_AL_2025_AqBot_196_10382512.zip Nucleotide sequences of complete chloroplast genomes are in FASTA format * Stuckenia_rRNAcistron_sequences_TRIEST_ET_AL_2025_AqBot_196_10382512.zip Nucleotide sequences of the full rRNA cistron (including ITS) are in FASTA format  Population codes are as in TRIEST et al. 2025 Aquatic Botany 196 10382512