Abstract
Populus and Salix species are key components of forest and riparian ecosystems and hold substantial economic value in forestry, bioenergy, and restoration practices. In Türkiye, these genera are widely distributed across diverse ecological zones, yet regional molecular data remain limited. In this study, I examined genetic relationships among six native species—Populus nigra L., Populus alba L., Populus tremula L., Populus euphratica Olivier, Salix alba L., and Salix caprea L.—using an integrative molecular approach. Twelve nuclear microsatellite (simple sequence repeats; SSR) markers and three plastid DNA barcode regions (matK, rbcL, trnH–psbA) were employed to assess marker performance, genetic variation, and species-level relationships. The analyses revealed clear genetic differentiation between Populus and Salix and substantial variation among Populus species. SSR loci detected both conserved and lineage-specific alleles, with limited allele sharing between genera, while plastid barcodes showed marker-specific patterns of sequence variation and phylogenetic resolution. Among the plastid regions, matK provided the strongest discriminatory signal, whereas rbcL was more conservative and trnH–psbA exhibited higher variability. P. tremula and P. nigra displayed higher levels of sequence and allelic variation compared with the other taxa, whereas P. euphratica showed a more distinct genetic profile. The combined use of nuclear SSRs and plastid barcodes provided complementary insights into genetic structure and evolutionary relationships among selected Populus and Salix species. Although limited in taxon and marker coverage, this study contributes regionally focused molecular data from a biogeographically underrepresented area and provides a foundation for future multilocus and genome-wide investigations of Salicaceae diversity in Türkiye.
Keywords: Populus, Salix, DNA barcode, Nuclear microsatellite, Phylogenetic
Introduction
The family Salicaceae, including the genera Populus and Salix, comprises fast-growing, predominantly dioecious trees and shrubs that play key ecological roles in temperate and boreal ecosystems (Dickmann & Kuzovkina, 2014). Species within this family contribute to soil stabilization, carbon sequestration, hydrological regulation, and habitat formation, particularly in riparian and floodplain environments (Karrenberg, Edwards & Kollmann, 2002; Kuzovkina & Volk, 2009). Owing to their rapid growth, high biomass production, and tolerance to environmental stress, many Populus and Salix species are widely used in forestry, phytoremediation, erosion control, and ecological restoration.
Beyond their ecological and economic importance, Populus and Salix have emerged as model systems in plant genetics, evolutionary biology, and ecological physiology. Both genera are characterized by extensive geographic distributions, high morphological plasticity, frequent hybridization, and diverse reproductive strategies (Tuskan et al., 2006; Smulders et al., 2008; Sanderson et al., 2023; Kaya et al., 2023). For example, Populus euphratica exhibits exceptional tolerance to salinity and drought (Rajput et al., 2016), whereas many Salix species are highly effective in riparian restoration due to rapid vegetative propagation and strong root development.
Despite these shared traits, Populus and Salix differ markedly in leaf morphology, genome structure, and evolutionary history, making them valuable systems for comparative phylogenetic research (Fig. 1). Recent phylogenetic and phylogenomic studies have substantially advanced understanding of relationships within and between the two genera. Genome-wide analyses and nuclear single nucleotide polymorphism (SNP) datasets have clarified species boundaries, detected hybridization and introgression, and revealed patterns of reticulate evolution (Wang et al., 2014; Liu et al., 2016, 2022; Sanderson et al., 2023; Ogutcen et al., 2024). These studies consistently support Populus and Salix as monophyletic sister genera (Wang et al., 2014; Lauron-Moreau et al., 2015; Liu et al., 2016). Nevertheless, species delimitation within each genus remains challenging due to incomplete lineage sorting, interspecific hybridization, and overlapping morphological traits.
Figure 1. Leaf pattern of four Populus and two Salix species.
POWO (2025).
Türkiye hosts four native Populus species (P. nigra, P. alba, P. euphratica, P. tremula) and 27 native Salix species distributed across diverse ecological zones, including riparian corridors, wetlands, montane regions, and semi-arid landscapes (Velioğlu, Bostancı & Akgül, 2020). Despite this diversity and ecological significance, regional genetic relationships and evolutionary patterns within Turkish Populus and Salix remain poorly resolved. Previous studies in Türkiye have largely focused on morphology or single-marker plastid analyses, offering limited resolution of species boundaries, marker transferability, and multilocus genetic structure (Çiftçi et al., 2017; Ciftci & Kaya, 2019; Acar et al., 2020, 2022; Değirmenci et al., 2022). To date, no study has applied an integrative multilocus framework combining nuclear simple sequence repeat (SSR) markers with plastid DNA barcodes across multiple native taxa and geographically distinct river systems.
This lack of regional multilocus data represents a significant knowledge gap, particularly for genera characterized by frequent hybridization and complex evolutionary histories, and is especially important given increasing habitat degradation and climate-driven stress across Türkiye. To address this gap, the present study employs an integrative approach using 12 nuclear SSR loci and three plastid barcode regions (matK, rbcL, trnH–psbA) to investigate phylogenetic relationships among six native Populus and Salix species. Specifically, the study aims to (i) assess genetic divergence within and between genera, (ii) evaluate SSR marker transferability across taxa, (iii) characterize plastid sequence variation among native species, and (iv) identify informative loci for molecular systematics and conservation genetics within the Salicaceae. By focusing on populations sampled from multiple river systems, this study provides the first multilocus, population-based molecular assessment of Turkish Populus and Salix, contributing regionally focused data that complement, rather than seek to redefine, existing global phylogenetic frameworks.
Materials and Methods
Sample collection and DNA extraction
A total of 149 individual trees representing six populations from four distinct river systems in Türkiye were sampled for this study. Specifically, leaf samples were collected from 26 individuals of P. alba (Tekirdağ River), 26 of P. nigra (Dicle River), 26 of P. euphratica and 26 of S. alba (Göksu River), 27 of S. caprea L. (Çoruh River), and 18 of P. tremula (Kızılırmak River). Healthy young leaves were collected in the field, immediately dried in silica gel, and transported to the laboratory for genomic DNA extraction.
The six river systems were selected to capture broad ecological and biogeographical variation across Türkiye. These rivers span contrasting environmental gradients—including coastal (Tekirdağ), semi-arid (Dicle), Mediterranean riparian (Göksu), montane (Çoruh), and Central Anatolian continental climates (Kızılırmak)—where Populus and Salix species experience distinct selective pressures. Several basins, particularly the Dicle and Göksu Rivers, also represent conservation priority areas due to hydrological alteration, habitat fragmentation, and increasing anthropogenic stress. Sampling across these diverse regions allowed for a more comprehensive assessment of genetic differentiation associated with ecological heterogeneity and geographically structured populations.
Total genomic DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) method following the protocol of Doyle & Doyle (1990), with minor modifications to optimize DNA yield and purity from woody plant tissues. The modification was carried out using chloroform instead of chloroform-isoamyl alcohol (24:1). Additionally, a mixture of 0.08 volumes of 7.5 M ammonium acetate and 0.54 volumes of isopropanol was used to remove the secondary metabolites that inhibit PCR amplification and to purify the DNA. DNA concentration and quality were assessed using a BioDrop μLITE spectrophotometer (BioDrop Ltd., Cambridge, UK) by measuring absorbance ratios at A260/280 nm. Only samples with optical density values between 1.7 and 2.0 were considered suitable for downstream PCR applications.
Microsatellite amplification
To investigate genetic diversity and structure, twelve highly polymorphic nuclear microsatellite loci were selected from the International Populus Genome Consortium (IPGC, 2016). Primer sequences and annealing temperatures were chosen based on established protocols optimized for interspecific transferability (Van Der Schoot et al., 2000; Smulders et al., 2001) (File S1, Table S1).
PCR reactions were performed in a 20 μL total volume containing 4 μL of 5× HOT FIREPol® Blend Master Mix (Solis BioDyne, Tartu, Estonia), 0.25 μM of each forward and reverse primer, 20 ng of template DNA, and nuclease-free water to reach the desired volume. Two thermal cycling protocols were used depending on primer requirements, both starting with an initial denaturation at 95 °C, followed by 30–35 cycles of denaturation, annealing, and extension, and ending with a final extension step at 72 °C (Van Der Schoot et al., 2000; Smulders et al., 2001). PCR products were initially visualized on 3% agarose gels to verify successful amplification prior to genotyping.
DNA barcoding amplification
To support species identification and phylogenetic reconstruction, three plastid barcode regions (trnH-psbA, matK and rbcL) were targeted (File S1, Table S1). Although the nuclear ITS region is frequently used in plant barcoding, it is known to present challenges in Populus and Salix due to paralogous copies, incomplete concerted evolution, and hybridization, which may result in ambiguous or non-homologous sequences (Hou et al., 2019). Preliminary amplification tests conducted in this study produced inconsistent and low-quality ITS products, particularly in Salix species. To ensure data reliability and avoid complications arising from multi-copy nuclear loci, ITS was excluded, and only plastid markers (matK, rbcL, trnH-psbA)—which showed high amplification success and sequence quality—were used for phylogenetic reconstruction (Hebert et al., 2003). PCR reactions were carried out in a 20 μL reaction volume containing 5 μL HOT FIREPol® Blend Master Mix, 0.5 μL each of forward and reverse primers (10 μM), 5 μL template DNA, and 9 μL deionized water. Thermal cycling conditions included an initial denaturation at 94 °C for 4 min, followed by 30 cycles of 94 °C for 40 s, annealing at the appropriate primer-specific temperature for 35 s (annealing temperature (Ta):57 for rbcL and trnH-psbA. Ta:58 for matK), and 72 °C for 40 s. A final extension was conducted at 72 °C for 5 min. PCR products were visualized on 3% agarose gels stained with ethidium bromide and run at 90 V for 30 min.
Data analysis
Samples that failed to amplify were reattempted up to two additional PCR runs. Only loci producing clear, reproducible bands were retained for downstream SSR genotyping. PCR products from the barcode regions were sequenced bidirectionally by Sanger dideoxy sequencing at BM Labosis (Çankaya, Ankara). Sequencing reactions were carried out in a total volume of 25 µL using an ABI 310 Genetic Analyzer and an ABI 3730XL 96-capillary automated sequencer (Applied Biosystems, Foster City, CA, USA). All chromatograms were manually inspected in MEGA11. Low-quality reads, sequences containing multiple ambiguous base calls, or those with Phred quality scores <20 were removed. Forward and reverse reads were assembled only when both directions provided consistent, high-quality base calls. Sequences failing quality criteria were excluded from alignment and phylogenetic reconstruction. For each species, plastid barcode regions were successfully sequenced from three individuals.
SSR genotyping
Fluorescently labeled SSR amplicons were subjected to capillary electrophoresis using an ABI PRISM® 310 Genetic Analyzer (Applied Biosystems Foster City, CA, USA) at BM Laboratory Systems (Ankara, Türkiye). Allele sizes were scored using Peak Scanner v2.0 software (Applied Biosystems, Foster City, CA, USA), with GeneScan 400 ROX used as the internal size standard. Population-level genetic statistics (e.g., He, Fst, Analysis of Molecular Variance (AMOVA)) were not computed because only three or four SSR loci amplified in several species, which does not provide sufficient marker coverage for reliable population genetic inference.
DNA sequence analysis and phylogenetics
Sequence chromatograms were manually inspected in MEGA v11 (Tamura, Stecher & Kumar, 2021) to ensure base-calling accuracy, and ambiguous sites were resolved by comparing forward and reverse reads. The resulting sequences for each locus were aligned using CLUSTALW (Thompson, Higgins & Gibson, 1994), and a consensus sequence was generated to represent each species for each barcode region. Sequence identity and coverage were verified using BLASTn searches against the NCBI GenBank database (Altschul et al., 1990), considering both query coverage and percentage identity. Consensus sequences obtained from Turkish populations were aligned and concatenated with publicly available reference sequences for the same species to enhance phylogenetic resolution from NCBI GenBank. Only sequences with verified species identification and complete locus coverage were included. Because the primary objective was to infer species-level relationships, population-specific variation within Türkiye was not analyzed separately. Phylogenetic tree was constructed using Maximum Likelihood (ML) method under the General Time Reversible (GTR) model with gamma distribution and invariant sites (G+I), as implemented in MEGA11. The Neighbour-Joining (NJ) method (Saitou & Nei, 1987) was also employed to compare topologies. The robustness of branches was assessed by 1,000 bootstrap replicates. Phylogenetic trees were drawn to scale, with branch lengths proportional to genetic distance estimated using the maximum composite likelihood method.
Results
Allelic diversity
A total of 12 SSR loci were analyzed across six species of Populus and Salix to examine allelic patterns. Although all primers were tested across species, amplification profiles revealed notable interspecific variation. The number of alleles per locus varied considerably among taxa. The loci WPMS14, WPMS15, and WPMS18 were successfully amplified in both Populus and Salix species. The PMGC2163 locus demonstrated broader amplification, being detected in S. alba and all Populus species. Amplification of WPMS03 and WPMS04 was restricted to P. tremula and P. nigra, whereas WPMS05, WPMS20, and PMGC93 consistently amplified across all Populus species, indicating their potential utility as robust cross-species markers. Additionally, WPMS09 and WPMS16 amplified in P. tremula, P. alba, and P. nigra, while WPMS07 was amplified in P. tremula, P. euphratica, and P. nigra. Among the amplified loci, P. tremula, P. nigra, and P. alba showed the highest allelic richness, particularly at WPMS05, PMGC2163, and PMGC93, respectively. In contrast, P. euphratica exhibited fewer alleles across all loci. A summary of allelic profiles per locus is presented in Table 1.
Table 1. DNA fingerprinting based on 12 microsatellite markers (Bold, italics and underlined alleles common among species).
| SRR loci | P. tremula (18 indv) | P. alba (26 indv) | P. euphratica (26 indv) | P. nigra (26 indv) | S. alba (26 indv) | S. caprea (27 indv) |
|---|---|---|---|---|---|---|
| WPMS03 | 210, 224, 230, 234, 236, 239, 242, 248, 251, 259 | – | – | 220, 224, 228, 232, 234 | – | – |
| WPMS04 | 258, 266, 274 | – | – | 263, 270, 272, 274 | – | – |
| WPMS05 | 274, 278, 280, 282, 285, 288, 290, 304 | 298, 306, 310, 312 | 286, 288, 290 | 232, 274, 276, 278, 282, 285, 301 | – | – |
| WPMS07 | 222, 228, 230, 236, 238, 250, 262 | – | 215, 219 | 222, 224, 228, 230, 232, 236 | – | – |
| WMPS09 | 245, 250, 256, 258, 260 | 224, 230, 236, 245, 248, 250 | – | 230, 245, 250, 252, 260, 274 | – | – |
| WMPS14 | 251, 263, 266, 269, 272, 280 | 204, 207, 219, 222, 228, 231 | 213, 219, 231, 237 | 210, 231, 237, 242, 244 | 192, 198, 204, 219 | 219, 222, 225, 228, 231, 234 |
| WPMS15 | 220, 226, 228, 233 | 195, 198, 218 | 182, 185 | 203, 210, 212, 218, 226 | 158 | 182, 185 |
| WPMS16 | 129, 134, 145, 150 | 166, 168, 172, 174, 178 | – | 145, 150 | – | – |
| WMPS18 | 210, 219, 225, 228, 231, 234, 243 | 216, 222, 225, 228, 231, 237 | 225, 228, 231 | 219, 225, 228, 231, 234 | 213, 219, 222, 225, 231 | 219, 274, 298 |
| WPMS20 | 214, 228, 232, 251, 256, 268, 270, 27 4 | 198, 200, 208, 210, 212 | 214 | 251, 263, 270, 272, 274, 280 | – | – |
| PMGC2163 | 224, 230, 236, 242, 248, 252, 258, 264 | 192, 194, 198, 200, 204, 206 | 194, 200, 204 | 194, 200, 202, 208, 212, 222, 224, 240, 248 | 194, 198, 200, 204, 206, 212, 216 | – |
| PMGC93 | 351, 354, 357, 363 | 351, 354, 357,360, 363, 366 | 348, 351, 357, 369 | 351, 357, 363 | – | – |
Conserved alleles among species
Shared alleles were identified at several loci. For instance, the 219 bp allele at the WPMS14 locus was found in P. alba, P. euphratica, S. alba, and S. caprea. Similarly, the 231 bp allele occurred in P. alba, P. euphratica, P. nigra, and S. caprea. Overall, WPMS14 showed allele sharing among at least two species, except P. tremula. At the WPMS15 locus, P. euphratica and S. caprea shared the 182 and 185 bp alleles. The 226 bp allele was common to P. tremula and P. nigra, while the 218 bp allele was shared by P. alba and P. nigra.
Among the loci, the WPMS18 exhibited the highest number of shared alleles. The 225, 228, and 231 bp alleles were detected in all Populus species, and the 225 and 231 bp alleles also appeared in S. alba. The 219 bp allele was shared by S. caprea, P. tremula, P. euphratica, and S. alba. At the PMGC93 locus, the 351, 354, and 357 bp alleles were conserved in P. tremula, P. alba, P. euphratica, and P. nigra. The 363 bp allele was present in all Populus species except P. euphratica, but absent in Salix species. WPMS20 alleles 251, 270, and 274 were shared between P. tremula and P. nigra, indicating a close phylogenetic relationship. The 145 and 150 bp alleles at WPMS16 were exclusive to P. tremula and P. nigra.
DNA barcoding and phylogenetic analysis
DNA sequences were successfully obtained for all three barcode regions (matK, rbcL, and trnH-psbA) from all sampled Populus and Salix species (File S2). Among these, the matK region exhibited the greatest variation in sequence length, ranging from 556 base pairs in P. euphratica to 788 base pairs in other Populus species. In contrast, the trnH-psbA region yielded the shortest sequences, ranging from 242 base pairs in S. alba to 305 base pairs in P. tremula. The rbcL region showed uniform sequence lengths across all species (586 base pairs) (File S2). In the BLAST search for the rbcL region, P. tremula (KX163000.1) and P. alba (KX162988.1) demonstrated 100% sequence identity with previously published sequences. P. nigra, P. euphratica, and S. caprea showed 99.83% sequence identity with reference sequences P. nigra (JF429910.1), P. euphratica (MK267314.1), and S. caprea (OZ037764.1), respectively. All examined species, except P. nigra, exhibited 100% identity with their respective aligned reference sequences. For the trnH-psbA region, S. caprea displayed the highest sequence identity with its reference sequence (OZ037764.1), whereas P. tremula showed the lowest identity with its respective reference (LC806940.1) (File 1, Table S2).
Phylogenetic analyses were performed on three plastid barcode regions (matK, rbcL, and trnH–psbA) using sequences retrieved from GenBank for four Populus and two Salix species (File S3). The coding matK region exhibited sequence lengths between 824 bp (S. alba) and 846 bp (P. nigra), with P. nigra and P. alba showing the most conserved sequences, while P. tremula and P. euphratica displayed higher variability. P. tremula had 35 variable sites (33 parsimony-informative), and P. euphratica contained the highest number of singletons (n = 37). Salix species showed very low variation (two variable sites, one singleton per species), and no indels were detected. Nucleotide diversity (π) was generally low (maximum π = 0.01 in P. tremula), with stable GC content across taxa (33.5–34.0%) (Table 2). The rbcL gene region showed uniform sequence lengths (513 bp for Populus, 549 bp for Salix) and high sequence conservation, particularly in P. euphratica. P. tremula had 69 variable sites (54 singletons, eight parsimony-informative), while P. alba exhibited 59 singletons without informative positions. Among Salix species, S. alba contained five singleton sites and one indel, and S. caprea had two variable sites (one parsimony-informative). Nucleotide diversity was highest in P. alba (π = 0.03), and GC content ranged from 43.0% to 45.3% (Table 3). In contrast, the non-coding trnH–psbA spacer displayed the greatest variability, with sequence lengths from 233 bp (P. nigra) to 367 bp (P. alba and P. euphratica). P. tremula showed the highest variation (52 variable and 43 singleton sites) followed by P. nigra (42 variable, 36 singleton sites), with 10 and six parsimony-informative sites, respectively. S. caprea also exhibited moderate variability (20 variable, 12 singleton, eight informative sites), whereas S. alba remained more conserved. Indels were observed in all taxa, most notably in P. nigra (n = 8) and S. caprea (n = 7), suggesting structural divergence in this intergenic region. Nucleotide diversity was highest in P. tremula (π = 0.05) and P. nigra (π = 0.04), while Salix species showed relatively low but consistent diversity (π = 0.01). GC content varied widely among taxa (20.5–32.7%) (Table 4).
Table 2. Phylogenetic analysis of four Populus and two Salix species based on matK barcode gene region.
| Species/Filogenetic parameters | Populus nigra | Populus tremula | Populus alba | Populus euphratica | Salix alba | Salix caprea |
|---|---|---|---|---|---|---|
| Accession no.* | PX101359 | PX101357 | PX101360 | PX101358 | PX101361 | PX101362 |
| Number of aligned individuals | 26 | 29 | 25 | 28 | 22 | 21 |
| Sequence length | 846 | 836 | 835 | 835 | 824 | 827 |
| Conserved region | 829 | 801 | 832 | 787 | 822 | 825 |
| Variable region | 17 | 35 | 3 | 37 | 2 | 2 |
| Singleton region | 14 | 2 | 0 | 37 | 1 | 1 |
| Parsimony region | 3 | 33 | 3 | 0 | 1 | 1 |
| Indel | 0 | 0 | 0 | 0 | 0 | 0 |
| Nucleotide diversity | 0.0 | 0.01 | 0.00 | 0.00 | 0.00 | 0.00 |
| GC content % | 33.5 | 33.8 | 33.8 | 34 | 33.9 | 34 |
Note:
Accession numbers refer to sequences deposited in GenBank.
Table 3. Phylogenetic analysis of four Populus and two Salix species based on rbcL barcode gene region.
| Species/Filogenetic parameters | Populus nigra | Populus tremula | Populus alba | Populus euphratica | Salix alba | Salix caprea | |
|---|---|---|---|---|---|---|---|
| Accession no.* | PX093670 | PX093668 | PX093671 | PX093669 | PX093673 | PX093672 | |
| Number of aligned individuals | 33 | 29 | 13 | 14 | 20 | 23 | |
| Sequence length | 513 | 513 | 513 | 513 | 549 | 549 | |
| Conserved region | 503 | 451 | 454 | 513 | 544 | 547 | |
| Variable region | 10 | 69 | 59 | 0 | 5 | 2 | |
| Singleton region | 3 | 54 | 59 | 0 | 5 | 1 | |
| Parsimony region | 7 | 8 | 0 | 0 | 0 | 1 | |
| Indel | – | – | – | – | 1 | – | |
| Nucleotide diversity | 0.00 | 0.01 | 0.03 | 0.00 | 0.00 | 0.00 | |
| GC content % | 45.3 | 43.5 | 43.5 | 43.4 | 43.7 | 43 | |
Note:
Accession numbers refer to sequences deposited in GenBank.
Table 4. Phylogenetic analysis of four Populus and two Salix species based on trnH-psbA barcode gene region.
| Species/Filogenetic parameters | Populus nigra | Populus tremula | Populus alba | Populus euphratica | Salix alba | Salix caprea |
|---|---|---|---|---|---|---|
| Accession no.* | PX101364 | PX101366 | PX101365 | PX101363 | PX101368 | PX101367 |
| Number of aligned individuals | 15 | 13 | 16 | 10 | 8 | 19 |
| Sequence length | 233 | 234 | 367 | 367 | 242 | 281 |
| Conserved region | 191 | 181 | 359 | 364 | 234 | 261 |
| Variable region | 42 | 52 | 8 | 3 | 8 | 20 |
| Singleton region | 36 | 43 | 2 | 0 | 6 | 12 |
| Parsimony region | 6 | 10 | 6 | 3 | 2 | 8 |
| Indel | 8 | 1 | 2 | 2 | 2 | 7 |
| Nucleotide diversity | 0.04 | 0.05 | 0.01 | 0.00 | 0.01 | 0.01 |
| GC content % | 20.5 | 26.4 | 32 | 32.7 | 29.7 | 30 |
Note:
Accession numbers refer to sequences deposited in GenBank.
To provide an integrated overview, the results from the three plastid DNA barcode regions (matK, rbcL, and trnH–psbA) were compared across Populus, Salix, and the combined dataset (Table 5). As detailed above, the matK region exhibited the highest number of parsimony-informative sites, the trnH–psbA spacer showed the greatest nucleotide diversity and indel frequency, and rbcL remained the most conserved marker with consistently high GC content. In Populus species, the average sequence lengths were 846 bp (matK), 513 bp (rbcL), and 235 bp (trnH–psbA), with conserved regions of 786, 444, and 170 bp, respectively. The highest number of singleton sites occurred in rbcL (55), followed by trnH–psbA (43) and matK (15). Parsimony-informative sites were most abundant in matK (45), with fewer in trnH–psbA (22) and rbcL (14). The trnH–psbA region exhibited 13 indels and the highest nucleotide diversity (π = 0.03), while matK and rbcL showed lower diversity (π = 0.00 and π = 0.01, respectively). GC content ranged from 26.1% in trnH–psbA to 34.5% in rbcL. In Salix species, average sequence lengths were 827 bp (matK), 513 bp (rbcL), and 265 bp (trnH–psbA), with conserved regions of 817, 507, and 248 bp, respectively. Variation across markers was limited: variable sites ranged from 6 in rbcL to 17 in trnH–psbA, and singleton counts were low (2–6 per region). Parsimony-informative sites were few (4–11 per marker). Indels were found in rbcL (n = 1) and trnH–psbA (n = 9). All markers displayed minimal nucleotide diversity (π ≤ 0.01), with rbcL showing the highest GC content (43.6%). trnH–psbA contributed the greatest sequence variation (variable sites = 93; π = 0.04) when all species were analyzed together, whereas matK and rbcL provided complementary phylogenetic signals at higher taxonomic levels (Table 5). These results highlight distinct marker-specific patterns in sequence variability and demonstrate the advantage of combining plastid loci for robust phylogenetic inference within Salicaceae.
Table 5. Phylogenetic analysis of members of Salicaceae family based on matK, rbcL and trnH- psbA barcode gene regions.
| Populus species | Salix species | All species | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Species/Filogenetic parameters | matK | rbcL | trnH-psbA | matK | rbcL | trnH-psbA | matK | rbcL | trnH-psbA |
| Number of aligned species | 112 | 89 | 54 | 43 | 43 | 27 | 151 | 132 | 81 |
| Sequence length | 846 | 513 | 235 | 827 | 513 | 265 | 788 | 513 | 233 |
| Conserved region | 786 | 444 | 170 | 817 | 507 | 248 | 712 | 436 | 140 |
| Variable region | 60 | 69 | 65 | 10 | 6 | 17 | 76 | 77 | 93 |
| Singleton region | 15 | 55 | 43 | 2 | 2 | 6 | 15 | 55 | 45 |
| Parsimony region | 45 | 14 | 22 | 8 | 4 | 11 | 61 | 22 | 48 |
| Indel | 0 | 0 | 13 | 0 | 1 | 9 | 0.00 | 1 | 22 |
| Nucleotide diversity | 0.0 | 0.01 | 0.03 | 0.00 | 0.00 | 0.01 | 0.01 | 0.01 | 0.04 |
| GC content % | 33.8 | 34.5 | 26.1 | 33.9 | 43.6 | 30.5 | 32.3 | 43.5 | 26.1 |
When all species were analyzed together, 151 matK, 132 rbcL, and 81 trnH-psbA sequences were aligned. The average sequence lengths were 788 bp (matK), 513 bp (rbcL), and 233 bp (trnH-psbA). Conserved regions spanned 712, 436, and 140 bp, respectively. trnH-psbA exhibited the highest number of variable sites (93), parsimony-informative sites (48), the highest indel count (22). This region also showed the highest nucleotide diversity (π = 0.04) and the lowest GC content (26.1%), highlighting its strong resolution potential for closely related taxa. In contrast, matK and rbcL showed lower variability (π = 0.01) but provided complementary phylogenetic signals, particularly at higher taxonomic levels (Table 5).
A phylogenetic tree was reconstructed using a concatenated alignment of the three plastid barcode regions (matK, rbcL, and trnH–psbA). The combined plastid dataset clearly resolved Populus and Salix as two distinct and well-supported clades. Within Salix, S. caprea and S. alba formed a strongly supported clade (bootstrap support >99). Within Populus, P. nigra, P. alba, and P. tremula clustered together, while P. euphratica was resolved as a distinct lineage sister to this group with high bootstrap support. Overall, the concatenated plastid phylogeny provided stronger support for intergeneric relationships and major intrageneric groupings than individual barcode trees, while maintaining patterns consistent with marker-specific analyses (Fig. 2).
Figure 2. Phylogenetic tree reconstructed from a concatenated alignment of matK, rbcL, and trnH–psbA plastid sequences for Populus and Salix species.
Analyses were performed using the Maximum Likelihood method under the GTR + G + I model. Numbers on nodes indicate bootstrap support values (%) based on 1,000 replicates; values <50% are not shown. Branch lengths represent substitutions per site.
Discussion
This study integrates nuclear SSR markers and plastid DNA barcodes to evaluate genetic relationships among selected Populus and Salix species sampled from Türkiye. The regional setting is central to interpreting these results: Türkiye lies at the intersection of the Euro-Siberian, Irano-Turanian, and Mediterranean floristic zones, and its river corridors span strong climatic and ecological gradients. This biogeographic complexity is directly relevant to patterns of divergence, potential secondary contact, and local adaptation in riparian trees, yet Turkish populations remain underrepresented in many broader Salicaceae phylogenetic and phylogeographic syntheses. The present dataset therefore contributes regionally focused molecular evidence that can help improve the geographic representation of Populus and Salix lineages and inform conservation priorities in threatened riparian systems.
Nuclear SSR patterns and marker performance
SSR analysis revealed substantial interspecific polymorphism and clear differentiation between Populus and Salix, supporting the utility of nuclear fingerprinting for species-level comparisons in these genera. Among the loci tested, WPMS05, PMGC2163, and PMGC93 amplified consistently across all Populus species, suggesting their potential value as broadly transferable markers within the genus. By contrast, amplification success was lower and more variable in Salix, which likely reflects genomic divergence affecting primer binding sites or structural differences (Smulders et al., 2001; Tuskan et al., 2006; Perdereau et al., 2014; Tokdemir et al., 2023; Ogutcen et al., 2024), and may be further influenced by polyploidy and complex genome architecture that can reduce cross-species amplification efficiency (Wagner, Gramlich & Hörandl, 2018; Sanderson et al., 2023).
Across species, allelic richness was highest in P. tremula, P. nigra, and P. alba, consistent with their broader ecological ranges and adaptive flexibility (Fussi, Lexer & Heinze, 2010; Jansson & Douglas, 2007). In contrast, P. euphratica exhibited reduced allelic variation, which is plausibly associated with ecological specialization and demographic constraints in arid and saline habitats (Hampe & Petit, 2005; Zhang et al., 2019; Kansu & Kaya, 2020). These contrasts are compatible with a regional interpretation in which differing habitat breadth and landscape fragmentation contribute to divergent levels of nuclear diversity across Turkish lineages.
Locus-specific allele distributions also provide insight into evolutionary dynamics. Conserved alleles at WPMS14, WPMS15, and WPMS18 observed across Populus and S. alba may represent evolutionarily stable regions maintained by purifying or stabilizing selection (Hou et al., 2019), or ancestral polymorphisms retained through lineage sorting. Shared alleles between closely related taxa within Populus are consistent with recent divergence and/or gene flow, such as allele sharing at WPMS20 between P. tremula and P. nigra. By contrast, lineage-specific patterns at WPMS16—conserved in P. nigra and P. tremula but distinct in P. alba—suggest differential evolutionary trajectories among Populus species (Cicatelli et al., 2012; Bonthala & Stich, 2022). The overlap between alleles reported for P. deltoides (Uluğ, 2022) and alleles observed here further supports shared genetic patterns among closely related Populus taxa (Çiftçi et al., 2017). Allele sharing between P. euphratica and S. caprea at WPMS15 is notable, but it should be interpreted cautiously. SSR loci are prone to homoplasy (Selkoe & Toonen, 2006), and technical factors such as null alleles, size-calling inconsistencies, or differential amplification can generate apparent similarity unrelated to shared ancestry (Dakin & Avise, 2004). Thus, while such patterns may be compatible with retained ancestral polymorphism or convergence (Chen et al., 2010; Liu et al., 2018), robust inference would require sequence-based validation or genome-wide nuclear markers. Overall, the SSR results highlight pronounced divergence between Populus and Salix and meaningful differentiation among Populus species, potentially shaped by selective pressures and drift following lineage divergence (Slavov & Zhelev, 2010; Değirmenci, Acar & Kaya, 2019; Hou et al., 2019; Ciftci & Kaya, 2019; Uluğ, 2022; Porth, Goessen & Heinze, 2024; Uzan Eken et al., 2024).
Plastid barcoding, marker-specific resolution, and phylogenetic discordance
The marker-specific patterns observed among the three plastid barcode regions are consistent with their known evolutionary dynamics and reported performance in Salicaceae and other angiosperms. The coding regions matK and rbcL exhibited relatively low nucleotide diversity and high sequence conservation, particularly in Salix, reflecting their slower evolutionary rates and suitability for resolving higher-level relationships rather than closely related taxa (Newmaster, Fazekas & Ragupathy, 2006; Fazekas et al., 2008; CBOL Plant Working Group, 2009; Hollingsworth, 2011). In contrast, the non-coding trnH–psbA spacer showed substantially higher levels of sequence variation, indel frequency, and nucleotide diversity—especially within Populus—highlighting its greater resolution potential at lower taxonomic levels (Kress et al., 2005; Shaw et al., 2007). The elevated variability detected in P. tremula and P. nigra across plastid markers is consistent with their broader ecological amplitudes and complex demographic histories, whereas the lower plastid diversity observed in Salix species aligns with previous reports of plastid uniformity and limited discriminatory power in willows (Chen et al., 2010; Percy et al., 2014). Collectively, these results emphasize the complementary nature of plastid barcode regions and support the use of multiple loci to balance phylogenetic resolution across taxonomic scales within the Salicaceae.
The concatenated plastid phylogeny recovered Populus and Salix as two well-supported and clearly separated clades, reinforcing the genus-level distinction consistently observed in the individual marker trees. Within Salix, S. caprea and S. alba formed a strongly supported clade (bootstrap support >99), consistent with results from all single-marker analyses and reflecting their placement in different subgenera. This concordance indicates that plastid signal across loci is sufficient for resolving deeper splits within Salicaceae.
Within Populus, the concatenated tree recovered P. nigra, P. alba, and P. tremula as a closely related group, while P. euphratica was resolved as a distinct lineage sister to this cluster with high bootstrap support. This topology is broadly consistent with the dominant signal observed across the individual plastid markers and provides a more stable representation of plastid relationships than any single locus alone. Importantly, the concatenated analysis reduces some of the marker-specific topological instability observed in the individual trees, particularly those associated with the lower phylogenetic resolution of rbcL, while still reflecting the underlying plastid history.
Nevertheless, the concatenated plastid phylogeny should be interpreted cautiously. As a maternally inherited genome, plastid DNA represents a single evolutionary history and may be influenced by processes such as chloroplast capture, incomplete lineage sorting, and historical hybridization, all of which are well documented in Populus and Salix (Tsitrone, Kirkpatrick & Levin, 2003; Hamzeh & Dayanandan, 2004; Wang et al., 2014; Jelic et al., 2015). The placement of P. euphratica as sister to the remaining Populus species in the concatenated tree is consistent with plastid-based ambiguities reported in previous studies (Chen et al., 2010; Wang et al., 2014; Lauron-Moreau et al., 2015) and may reflect retained ancestral polymorphism or historical introgression rather than true species relationships.
When considered together, the individual and concatenated plastid phylogenies demonstrate that combining barcode regions improves support for deeper nodes and genus-level relationships but does not fully resolve species-level relationships within Populus. This outcome underscores the limitations of plastid data alone for closely related and hybridizing lineages and highlights the importance of integrating plastid barcodes with nuclear markers. In this study, congruence between the concatenated plastid topology and nuclear SSR patterns—particularly the distinctiveness of P. euphratica and the close relationship between P. nigra, P. alba, and P. tremula—supports the value of a multilocus framework while reinforcing the need for genome-wide nuclear data to achieve robust species-level phylogenetic inference in Salicaceae (Sanderson et al., 2023).
Consistency with broader phylogenomic evidence and regional contribution
The broader literature consistently supports Populus and Salix as monophyletic sister genera (Wang et al., 2014; Lauron-Moreau et al., 2015; Sanderson et al., 2023; Ogutcen et al., 2024), despite earlier morphology-based interpretations suggesting overlap or convergence (Eckenwalder, 1996). The present study does not aim to revise these well-established relationships; rather, it contributes regionally focused molecular data from Türkiye. The plastid and SSR results are consistent with these multilocus and phylogenomic studies in recovering clear separation of Populus and Salix. Patterns of SSR allele sharing across some loci, alongside the absence of shared alleles at others, further support close relatedness as sister lineages while reflecting post-divergence differentiation.
Implications, limitations, and future directions in a Turkish context
From a conservation perspective, the results are relevant to Türkiye’s riparian ecosystems, which are increasingly affected by hydrological alteration and climate-driven stress. Populus and Salix function as foundation taxa in riverbank stabilization, hydrological regulation, and habitat formation, making the maintenance of genetic diversity a practical management priority. The pronounced distinctiveness of P. euphratica highlights the importance of conserving populations that may harbor adaptive traits associated with drought and salinity tolerance under intensifying aridity. More broadly, the lineage-specific alleles and genetically cohesive clusters observed here can inform seed sourcing, restoration planning, and germplasm conservation by emphasizing locally adapted gene pools.
At the same time, the scope of inference remains limited. Only six of the more than thirty Salicaceae species native to Türkiye were included, and uneven SSR amplification across taxa reduced marker comparability. The sampling strategy targeted species-level representation rather than population-level structure, precluding robust population genetic analyses and making population-level phylogenies methodologically inappropriate for the current dataset. Broader taxonomic and geographic sampling within Türkiye—combined with genome-wide markers and ecological correlates—will be essential for evaluating the generality of these patterns and for resolving the roles of hybridization, historical demography, and environmental gradients in shaping Salicaceae diversity. In this context, Anatolia’s recognized role as a major Pleistocene refugium and secondary contact zone for temperate trees (Albayrak et al., 2024) underscores the value of incorporating Turkish populations into future Eurasian-scale evolutionary analyses.
Conclusions
This study presents a regionally focused assessment of species-level phylogenetic relationships among native Populus and Salix taxa occurring in major river systems of Türkiye. By integrating nuclear SSR markers with plastid DNA barcodes, the analysis evaluates marker transferability, identifies conserved and lineage-specific alleles, and reveals patterns of genetic similarity among locally adapted species. Although the findings are consistent with established phylogenetic frameworks for the Salicaceae, they contribute novel molecular data from a biogeographically underrepresented region and emphasize the importance of incorporating local populations into broader systematic and conservation-oriented research. The study does not aim to address global phylogenetic relationships within the family but instead helps fill geographic and molecular gaps in existing datasets. By highlighting regional genetic differentiation and marker-specific performance, the results provide a foundation for future investigations. Expanded taxonomic sampling and the application of genome-wide markers across Türkiye will be essential to further elucidate patterns of diversification, adaptive variation, and evolutionary dynamics in these ecologically and economically important riparian tree lineages.
Supplemental Information
Acknowledgments
I would like to thank Prof. Zeki Kaya for kindly providing access to his laboratory facilities, which enabled the experimental work conducted in this study.
Funding Statement
The authors received no funding for this work.
Additional Information and Declarations
Competing Interests
The authors declare that they have no competing interests.
Author Contributions
Funda Özdemir Değirmenci conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.
Field Study Permissions
The following information was supplied relating to field study approvals (i.e., approving body and any reference numbers):
The plant specimens used in this study were collected under official field collection permits issued by the Republic of Türkiye Ministry of Agriculture and Forestry. These permits were obtained during earlier nationally funded research projects supported by TÜBİTAK (The Scientific and Technological Research Council of Türkiye), a national funding agency for scientific research.
All fieldwork was conducted in accordance with national regulations. Although these projects included large-scale sampling efforts, only a subset of the collected specimens relevant to this study were selected and used in the current manuscript.
The samples used here were collected within the scope of the following TÜBİTAK projects, all of which included official field permit approval:
• TÜBİTAK TOVAG 110O570 (2011–2014)
• TÜBİTAK TOVAG 213O154 (2014–2017)
• TÜBİTAK KBAG 117Z018 (2017–2020)
For this reason, I have acknowledged Prof. Dr. Zeki Kaya, the principal investigator of all three TÜBİTAK projects, in the Acknowledgments section of the manuscript for his valuable support and for facilitating access to the permitted field materials.
DNA Deposition
The following information was supplied regarding the deposition of DNA sequences:
The matK (PX101357–PX101362), trnH-psbA (PX101363–PX101368), and rbcL (PX093668–PX093673) sequences described in this study are available at GenBank.
Data Availability
The following information was supplied regarding data availability:
The clean sequence data for the matK, rbcL, and trnH-psbA regions used in phylogenetic analyses are available in the Supplemental Files.
The matK (PX101357–PX101362), trnH-psbA (PX101363–PX101368), and rbcL (PX093668–PX093673) sequences described in this study are available at GenBank.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The following information was supplied regarding data availability:
The clean sequence data for the matK, rbcL, and trnH-psbA regions used in phylogenetic analyses are available in the Supplemental Files.
The matK (PX101357–PX101362), trnH-psbA (PX101363–PX101368), and rbcL (PX093668–PX093673) sequences described in this study are available at GenBank.