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.

2.1 Plant materials

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.

2.2 Nuclear microsatellite analysis

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 H₂O, 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). 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.

2.3 Next-generation sequencing

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).

2.3.1 Chloroplast genome assembly, alignment, and comparative analysis

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 de novo chloroplast assemblies. The de novo 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 (rbcL) and taking 30% as a subsample from the FASTA file with default parameters. These were compared to the existing annotated S. pectinata 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 (S. pectinata) 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 (Katoh et al., 2002; Katoh and Standley, 2013).

2.3.2 Nuclear rRNA cistron assembly

The nuclear ribosomal cistron (18S, ITS1, 5.8S, ITS2 and 26S) was assembled from a S. pectinata (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).