Genetic Divergence in the Natural Regeneration of Black Poplar Along the Vistula River in Poland

1 Title: Genetic divergence in the natural regeneration of black poplar along the Vistula River in 1 Poland 2 Authors: Dominika Robak*, Andrzej Lewandowski, Weronika Barbara Żukowska 3 Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, 62-035 Kórnik, Poland 4 Address: *corresponding author; Institute of Dendrology, Polish Academy of Sciences, 5 Parkowa 5, 62-035 Kórnik, Poland; email: [email protected] 6 Keywords: Populus nigra, genetic variation, natural regeneration, vegetative and generative 7 reproduction, clones, Vistula River 8 9 2 Abstract: Many years of land use transformation within river valleys have drastically changed 10 these ecosystems. Black poplar is a tree species characteristic of riparian habitats. 11 Unfortunately, due to specific environmental requirements, its populations have difficulties 12 with natural regeneration. Here, we genotyped 623 black poplar individuals from four 13 populations located along different sections of the Vistula River. This river, which is the largest 14 in Poland, is characterized by the variable degrees of regulation and transformation of its natural 15 environment. Each black poplar population consisted of a group of mature trees and a group of 16 naturally regenerated trees. The results showed that fully generative regeneration occurred only 17 along the least transformed middle section of the river. One example of regeneration comprised 18 only root suckers, and the remaining two appeared to be half generative and half vegetative in 19 origin. The genetic differentiation among the natural regeneration groups was almost twice as 20 high as that among the mature tree groups. It can be assumed that due to the lack of suitable 21 areas for seed germination, black poplar will reproduce mainly vegetatively, which may be a 22 way to ensure the survival of the species. However, the adaptive potential of the youngest 23 generations is unknown, especially in the face of progressive climate change. Our research 24 confirms the need to monitor seedlings and saplings along major rivers and to conduct 25 molecular analyses to assess their gene pools. We conclude that to preserve black poplar genetic 26 resources, ex situ protection in the form of local clone archives is necessary. 27 1. Introduction 28 It is assumed that as climate change progresses, many species, due to various limitations, will 29 lose habitat areas that are optimal for their growth and development. The most dire situation is 30 projected for species that will be unable to compensate for the shifts in their range due to an 31 inability to colonize new areas (Dyderski et al. 2018; Puchałka et al. 2023). Dyderski et al. 32 (2018) showed that among forest trees, the most endangered species are mainly pioneer and 33 coniferous species. Pioneer species have a better ability to spread, and in their case, the threat 34 3 greater than dispersal limitations would be the lack of areas suitable for colonization (Dyderski 35 et al. 2018). Black poplar is a species that has already experienced negative effects from human 36 activity and, in part, from climate change. It is the main forest tree species of riparian woodlands 37 that are considered centres of biodiversity (Pielech 2021; Rajpar, Rajpar, and Zakaria 2022). 38 Unfortunately, it is estimated that only 1% of the riparian forests that once existed in Europe 39 have remained (Lefèvre et al. 1998; Smulders et al. 2008). Black poplar still occurs throughout 40 Europe, Central Asia, North Africa, and Western Siberia (Bugała 1973). Nevertheless, the 41 number of trees is rapidly decreasing, especially in Western Europe (Cottrell 2004; Holub and 42 Procházká 2000; Lefèvre et al. 1998; Tylkowski 2010). Because there is a real problem with 43 natural regeneration, the population remnants consist mostly of trees that are close to terminal 44 age. 45 Black poplar has great ecological and economic value. It establishes on riverbanks, providing 46 natural flood protection and contributing to substantial improvements in the environment due 47 to its ability to absorb pollutants (Šiler, Skorić, and Mišić 2014). As a fast-growing tree, it is 48 not only an excellent source of biomass for energy uses but also an intermediate product for the 49 furniture and paper industries. Black poplar has been widely used in selective breeding for the 50 development of resilient, fast-growing poplar hybrids (Šiler et al. 2014; Vanden Broeck et al. 51 2005). As a pioneer species, black poplar plays an important role in the initial successional 52 stage of riparian ecosystems (Chenault et al. 2011; Rotach 2004). It is dioecious and reproduces 53 mainly generatively. Mature trees produce large amounts of seeds, but their lifespan under 54 natural conditions is quite short (Braatne, Rood, and Heilman 1996; Vanden Broeck et al. 2005). 55 The light pappus of black poplar seeds promotes long-distance dispersal by wind and water. 56 Specific water and soil conditions, such as bare, moist and permeable soil, and a lack of 57 competition are necessary for the seeds to germinate, but even when this happens, most will not 58 survive the first year due to flooding or drought stress (Johnson 2000; Seiwa et al. 2008; Vanden 59 4 Broeck et al. 2005). Seedling density can vary from only 20 to, under favourable conditions, 60 more than 4,000 per square metre (Braatne et al. 1996). However, due to environmental 61 restrictions, seedling establishment is very rare, occurring on average every 10–20 years 62 (Vanden Broeck et al. 2005). Moreover, human interference has unfortunately led to a lack of 63 habitat areas that provide proper conditions for seed germination. 64 Under unfavourable environmental conditions, as an alternative reproductive strategy, black 65 poplar can reproduce vegetatively (Braatne et al. 1996). First, branches and twigs that are 66 broken during storms and floods are transported along the river and can then be buried in the 67 sediment, where they can resprout (Braatne et al. 1996; Smulders et al. 2008). Second, damage 68 to the apical meristem can lead to the growth of multiple trunks of the same genotype (Barsoum, 69 Muller, and Skot 2004; Del Tredici 2001; Żukowska, Wójkiewicz, and Lewandowski 2021). 70 Moreover, the root system can produce shoots known as root suckers. Trunk damage or root 71 disturbance due to fire, mowing or grazing promotes the development of root suckers; therefore, 72 we can presume that a stimulus is needed (Barsoum et al. 2004). Apart from the danger posed 73 by the limited possibility of generative reproduction, an additional threat to black poplar is that 74 it may cross with fast-growing hybrid poplar varieties that have been planted in great numbers 75 throughout Europe since the end of the 18th century (Bugała 1973; Vanden Broeck et al. 2005). 76 This poses a real risk to the integrity of the black poplar gene pool (Vanden Broeck et al. 2015). 77 High morphological similarity makes it difficult to distinguish pure black poplars from hybrids 78 based solely on phenotypic characteristics (Bugała 1973). 79 The black poplar is a noteworthy research object because, on the one hand, it has two different 80 reproductive strategies, and on the other hand, its natural ecological niche has clearly been 81 transformed. Furthermore, dioecious species such as black poplar might be particularly 82 susceptible to climate change due to their tendency to spatially segregate male and female 83 individuals. This segregation is often reinforced by physiological and morphological 84 5 specializations of each sex to distinct microhabitats (Hultine et al. 2016). Therefore, the 85 question arises whether black poplar will be able to survive and maintain genetic variability as 86 well as adaptive potential in the future or whether any actions should be taken to protect this 87 species and its gene pool. In Poland, black poplar is still common, but it should be noted that 88 many populations are rather old and small and occur in the form of small groupings. The 89 remaining river where there are suitable conditions for natural regeneration is the Vistula River 90 (Wójkiewicz et al. 2019), the largest river in Poland, with a length of ca. 1,047 km. The 91 environmental conditions and plant communities along individual river sections vary due to 92 river waterflow regulation works carried out in recent centuries. The waterflow of the upper 93 section has been regulated by approximately 60% (Wójkiewicz et al. 2019; Żelazo 2013). 94 Intensive work on regulating this part of the Vistula began after 1840. The riverbed was 95 subjected to numerous modifications, which resulted in its rapid deepening and narrowing. This 96 led to sedimentation of debris in the spaces between the transverse dams and in the floodplain. 97 Rich sediments that accumulated in floodplains quickly became overgrown with vegetation, 98 leading to a drastic reduction in the capacity of these places. After 1950, the debris was retained 99 by built retention reservoirs, leading to the stabilization of the bed (Żelaziński 2014). The 100 middle section is the least transformed and the most natural, having the greatest ecological 101 value. Plans for regulating the waterflow of the middle Vistula, developed at the end of the 19th 102 century, were partially implemented only after 1945. Cascading, planned in the 1970s, was 103 completely abandoned (Łajczak et al. 2021). The lower and longest section has numerous 104 regulatory structures and levees (Wójkiewicz et al. 2019; Żelazo 2013). On the basis of the 105 degree of transformations, it can be divided into four subsections, which are partially or 106 completely regulated. Regulatory work on the lower Vistula began in 1835–1855. Such 107 waterflow regulation works contributed to the transformation of the riverbed from a braided 108 6 bed with numerous sandbanks and tussocks into an almost rectilinear type of riverbed with an 109 alternating arrangement of sloping sandbars (Łajczak et al. 2021). 110 Natural regeneration capacity and genetic variation in black poplar are very important issues, 111 considering the progressive nature of climate change, as well as the differential levels of 112 transformation of the Vistula. Therefore, we analysed the gene pools of the remaining black 113 poplar populations where both mature trees and natural regeneration occurred. Specifically, we 114 wanted to answer the following questions: (1) Does the natural regeneration gene pool mirror 115 the gene pool of mature trees? (2) Does the chosen reproductive strategy vary depending on the 116 location and the degree of land transformation? (3) Do populations located in different sections 117 of the Vistula River differ genetically? (4) Are the gene pools of the populations in need of 118 protection, and if so, how should such protection be carried out? 119 2. Methods 120 2.1 Study sites and plant material 121 We sampled four populations of black poplar trees located along different parts of the Vistula 122 River (Table 1). From each site, we analysed the group of mature trees and the group of 123 naturally regenerated trees (623 individuals in total). We collected fresh leaves that were stored 124 at -20°C until extraction. Most groups of naturally regenerated trees (Vi2R–Vi4R) were 125 collected along a one-metre transect. However, the Vi1R group was collected along a five126 metre transect due to the large size of the analysed area. The Vi1 population is located in the 127 upper Vistula section. The entire area is covered with grass and small shrubs. Regarding the 128 Vi2 population from the middle, least regulated section of the Vistula, this area is exposed, 129 sandy, and lacking in grass cover. The Vi3 population is located partly in a nature reserve. The 130 area where the mature tree group is located is overgrown, while the natural regeneration group 131 is located slightly farther away, next to the railway bridge in an open sandy area. The Vi3 and 132 7 Vi4 populations are located in the regulated section of the Vistula River. Comprehensive full 133 regulation of this section of the riverbed was carried out in the years 1880–1892 after a series 134 of great floods. The riverbed was straightened and narrowed with stone spurs. There is an 135 almost straight-line bed in this part of the river, with a winding current at low water levels and 136 an alternating system of sloping bars and stream pools (Łajczak et al. 2021). 137 Additionally, samples of various poplar varieties were included in the present research as a 138 reference to identify potential hybrids in our dataset. They comprised five individuals marked 139 as Populus deltoides trees, 11 male Populus nigra cv. Italica trees and European hybrids such 140 as Populus ‘Grandis’, P. ‘Marilandica’, P. ‘Robusta’, and P. ‘Serotina’. These are the most 141 popular Euramerican hybrids planted in Poland. We also genotyped several hybrid individuals 142 that grew near the black poplar populations we analysed. 143 2.2 Molecular analyses 144 Total DNA was extracted from frozen leaves according to a modified cetyl145 trimethylammonium bromide (CTAB) isolation method (Dumolin, Demesure, and Petit 1995). 146 We used 100 to 120 mg of leaf tissue ground in liquid nitrogen. Five microlitres of RNAse A 147 (10 mg/mL) was added after the initial incubation to remove the RNA. Qualitative and 148 quantitative assessment of the isolated DNA was performed using a BioPhotometer plus 149 spectrophotometer (Eppendorf AG, Germany) as well as via agarose electrophoresis. 150 Genotyping of the examined individuals was carried out using 15 microsatellite markers 151 developed by van der Schoot et al. (2000) and Smulders et al. (2001). The amplification of the 152 WPMS markers 01, 03, 04, 07, 10-18, 20 and 22 was performed via three multiplex PCRs 153 according to the protocols described by Wójkiewicz et al. (2019). The size of the PCR products 154 was analysed using fluorescence detection in an ABI3130xl automatic capillary sequencer with 155 GeneScanTM 500 LIZTM (Thermo Fisher Scientific, USA) as an internal standard. The 156 genotypes were scored using GeneMapper ver. 4.0 (Thermo Fisher Scientific, USA) and 157 8 confirmed manually. Nuclear DNA win3 and chloroplast DNA trnS-trnfM markers were used 158 to screen hybrid individuals, and the reaction conditions were the same as those described by 159 Wójkiewicz et al. (2019). The identification of hybrid trees was also carried out on the basis of 160 the microsatellite marker data. To identify signals of introgression in the analysed groups of 161 black poplar individuals, the STRUCTURE 2.3.4 program was used (Falush, Stephens, and 162 Pritchard 2003; Pritchard, Stephens, and Donnelly 2000). Ten independent runs were 163 performed for each K from one to 14 with burn-in lengths of 50,000 and 500,00 iterations. We 164 also used GenAlEx ver. 6.5 software to identify clones (Peakall and Smouse 2006, 2012). All 165 hybrid individuals and clones were excluded from further analyses. 166 2.3 Data analyses 167 To estimate the frequency of null alleles at all loci, we used the maximum likelihood method 168 (Dempster, Laird, and Rubin 1977), available in the FreeNA program (Chapuis and Estoup 169 2007). The parameters of genetic variability were determined by calculating parameters such 170 as the mean number of alleles (A), mean effective number of alleles (AE), number of private 171 alleles (AP), and the mean observed (HO) and expected heterozygosity (HE). These parameters 172 were calculated for each of the analysed groups using GenAlEx ver. 6.5 software. The FSTAT 173 ver. 2.9.4. program (Goudet 2003) was used to determine allelic richness (AR). The values of 174 the inbreeding coefficient were determined, including null allele correction (FISNull), using 175 INEst ver. 2.2 software (Chybicki and Burczyk 2009). The calculations were performed using 176 two models – the 'nfb' model, which considers null alleles, inbreeding coefficients and 177 genotyping failures – and the 'nb' model, which considers null alleles and genotyping failures. 178 A comparison between these two models allowed us to determine the importance of inbreeding. 179 We set the parameters as follows: 200,000 MCMC iterations, updates performed every 10th 180 cycle, and a burn-in of 20,000. To determine the level of genetic variability within and among 181 the analysed populations, we applied the analysis of molecular variance (AMOVA) method, 182 9 with a p value calculated with 10,000 permutations, implemented in Arlequin ver. 3.5.2.2 183 (Excoffier and Lischer 2010). We also calculated population pairwise fixation indices (FST) and 184 Slatkin's analogues of FST (RST). We performed the principal coordinate analysis (PCoA) using 185 GenAlEx to assess the genetic relationships among the populations. To assign the studied black 186 poplar individuals to a given genetic group, we conducted a clustering analysis in the 187 STRUCTURE program. The calculation parameters were the same as those described in section 188 2.2. We used StructureSelector web-based software (Li and Liu 2018) to generate the results. 189 Because there were large differences in the number of individuals among the groups, the most 190 likely value of K was determined using the Evanno method (Evanno, Regnaut, and Goudet 191 2005) and the Puechmaille method (Puechmaille 2016). To estimate the effective size of 192 individual populations (Ne) with the linkage disequilibrium (LD) approach (Waples and 193 England 2011), we used the NeEstimator ver. 2.01 program (Do et al. 2014). We followed the 194 authors’ recommendation and set the allele frequency threshold (Pcrit) to 0.02. The 95% 195 confidence intervals (CIs) were calculated using the parametric option that implements the χ2 196 approximation (Waples 2005). In the last step, we used two bottleneck testing approaches. 197 Using the INEst program, the M-ratio (MR), defined as the ratio of the number of alleles to the 198 range of allele sizes, was calculated (Garza and Williamson 2001). We also performed a test 199 for excess heterozygosity (Cornuet and Luikart 1996) with the parameters described by Peery 200 et al. (2012). The analysis was carried out under the two-phase mutation model (TPM), which 201 includes single-step and multistep mutations. For both bottleneck tests, significance was 202 analysed using the Wilcoxon signed-rank test based on 1,000,000 permutations. 203 3. Results 204 3.1 Identification of hybrids and clones 205 In the analysed populations, we detected a total of 258 clones, which constituted 41.41% of all 206 individuals (Table 2). We recorded high clonality in the Vi3R, Vi4M, and Vi4R groups 207 16 individuals were derived from root suckers, but some of them were produced via generative 356 regeneration. This may be because this group grew next to the railway bridge. During its 357 construction, the area was probably exposed, which meant that the conditions created by the 358 construction work partially enabled the germination of seeds and the survival of seedlings. 359 Regarding the Vi4 population, which is located closest to the influx of Vistula, the trees grew 360 relatively far from the present-day river current. They likely grew there because of the original 361 course of the Vistula River. There is currently no area favourable for seed germination or 362 seedling development, and all the young specimens we found were root suckers. Therefore, it 363 appears that under unfavourable environmental conditions, the only available reproductive 364 strategy is vegetative. Čortan and Tubić (2017), in their work carried out along the upper 365 Danube in Serbia, found that successful regeneration still occurs, but it is sporadic and mainly 366 vegetative. They concluded that 78% of individuals had good or decent regenerative potential, 367 13% had poor regenerative potential, and 10% had no regenerative capacity. The authors 368 emphasized, however, that natural regeneration was mainly vegetative. There was no specific 369 pattern of distribution of individuals with different natural regenerative strategies, and 370 individuals were distributed randomly in the analysed area. No particular environmental 371 conditions directly affected the ability to regenerate, but this issue requires more research on 372 environmental factors and competition with other taxa. 373 4.4 Genetic variation and differentiation of populations located in different sections of the 374 river 375 Our research showed that the analysed populations are characterized by high genetic diversity, 376 with the Vi2 population having the highest genetic variability, as indicated by the parameters 377 described in Table 2. Genetic diversity parameters such as the number of alleles, the effective 378 number of alleles, and allelic richness were comparable between individual groups of black 379 poplar. These results are in line with other studies on Polish or European populations of black 380 17 poplar (Čortan et al. 2016; Čortan and Tubić 2017; Pospíšková and Šálková 2006; Wójkiewicz 381 et al. 2021). The number of private alleles was also comparable between individual groups and 382 ranged from zero to six, except for the Vi2R group, where AP = 27. This high AP value may be 383 because at least some seeds were transported with the river current from different locations. 384 Furthermore, the seedlings grew in two long rows adjacent to the main riverbed, so we can 385 assume that they developed during different periods. Our results are similar to those obtained 386 by Wójkiewicz et al. (2019), where AP ranged from three to 19, or by Čortan et al. (2016), where 387 AP ranged from two to 40. The average level of heterozygosity of the analysed groups did not 388 differ from the level of heterozygosity of black poplar populations growing both in Poland 389 (Lewandowski et al. 2021; Lewandowski and Litkowiec 2017; Wójkiewicz et al. 2019, 2021) 390 and in other European countries (Čortan et al. 2016; Jelić et al. 2015; Mazal et al. 2022; 391 Smulders et al. 2008). 392 Despite the uniform level of genetic variation, the pairwise FST and RST values showed that the 393 Vi1M, Vi1R, and Vi4M groups were the most genetically different from the others. The results 394 of the PCoA confirmed the above results, as the above-mentioned groups clustered on opposite 395 sites of the first coordinate, which explained 52.08% of the total genetic variation. Genetic 396 clustering revealed that according to the Evanno method, the analysed populations from the 397 Vistula River belonged to two different genetic clusters, where the Vi1 population from the 398 upper section of the river constituted a separate genetic group. Considering the Puechmaille 399 method, which is thought to be more accurate when the studied groups differ in the number of 400 individuals, the most likely genetic structure of the analysed trees was represented by three 401 genetic groups, where the Vi1 and Vi3 populations constituted separate subgroups. This result 402 supports greater genetic structuring of black poplar populations and constraints to gene flow 403 along the Vistula River. 404 4.5 Conservation implications 405 18 Our results indicated that all groups experienced a bottleneck effect in the more distant past; in 406 turn, the insignificance of all p values for the excess heterozygosity test showed that no 407 bottleneck occurred recently in any of the analysed groups. The estimated effective population 408 size was greater only for the mid-river Vi2 population (Ne = 140.40 for Vi2M and Ne = 233.00 409 for Vi2R). In the remaining groups, the Ne values ranged from 16.10 to 38.80. Franklin (1980) 410 proposed the 50/500 rule, which is an important indicator in conservation genetics. It assumes 411 that populations with Ne less than 50 are at risk of extinction due to reduced survival and 412 reproduction resulting from inbreeding. To ensure long-term viability, the population must have 413 a Ne greater than or equal to 500. Taking the above into account, we can conclude that the low 414 effective sizes of the analysed populations are alarming despite low and, in almost every case, 415 insignificant inbreeding and still high genetic variation. 416 The black poplar populations in Poland are mostly old, close to terminal age, and usually occur 417 in small groups. For example, due to significant transformations of the Warta River, there is no 418 area within its reach that is suitable for seed germination and seedling growth (Lewandowski 419 et al. 2021). Field observations conducted by Żukowska et al. (2021) showed that most 420 individuals along the Odra River are old and that the heavily transformed river valley does not 421 meet the required water and soil conditions for seed germination. For that reason, black poplar 422 also has little chance of generative regeneration along this river. Our research shows that black 423 poplar populations still maintain a high level of genetic variability along the Vistula River. The 424 groups of naturally regenerated trees are more diverse than the group of mature trees, which is 425 a good outcome because it increases diversity. On the other hand, the adaptive potential of 426 natural regeneration in the face of climate change is unknown. Moreover, low Ne values may 427 lead to the extinction of these populations within several generations. It can be said that the 428 Vistula River is the last place for the generative reproduction of black poplar in Poland. 429 Nevertheless, although natural regeneration still occurs, it is not known whether this species 430 19 will survive. The obtained results indicate that the gene pools are not preserved and are 431 diverging, moving away from the original dynamic. The fate of young individuals cannot be 432 predicted, especially with progressive climatic changes. Root suckers are a kind of lifeline for 433 the survival of this species, but they can have very negative consequences in terms of genetic 434 variation and perhaps adaptive potential. Studies indicate that clonality increases allelic 435 diversity, polymorphism, and heterozygosity in a population but reduces genotypic diversity 436 (Meloni et al. 2013; Żukowska et al. 2021). We conclude that to preserve black poplar gene 437 pools, ex situ protection in the form of local clone archives is needed. This type of protection 438 for the populations from the upper and lower sections of the Vistula River seems to be 439 necessary, and the middle section of the river should be monitored for regeneration and survival 440 of seedlings and saplings. 441 Our research showed that generative regeneration occurs quite rarely. Although natural 442 regeneration still occurs, it is vegetative in most places. Black poplar is a species that requires 443 open space for seed colonization, which is almost impossible in Poland due to the past 444 transformations of river valleys. Natural regeneration may only seem to be generative; to verify 445 this, it is necessary to carry out analyses using molecular biology techniques. Vegetative 446 regeneration may be a way for black poplar to survive, but root suckers usually grow only from 447 a few genotypes, as seen in our studies, where root suckers came from five, 10 or 11 trees. 448 However, the genetic basis for the formation of root suckers and the reasons why some 449 individuals are more predisposed to are unknown, and this topic requires further research. 450 Considering both our research and research previously conducted in Poland, taking action to 451 ensure the protection of black poplar in our country seems to be necessary. 452 20 Table 1. Description of black poplar populations analysed in the study. N – number of individuals 453 Pop Group Study site River section Location N Age (yrs) Vi1 Vi1M Tarnobrzeg, mature trees upper N 50.55 E 21.63 14 30-130 Vi1R Tarnobrzeg, natural regeneration 56 1-20 Vi2 Vi2M Wysoczyn, mature trees middle N 51.90 E 21.25 61 80-150 Vi2R Wysoczyn, natural regeneration 199 1 Vi3 Vi3M Toruń, mature trees lower N 53.00 E 18.61 37 30-150 Vi3R Toruń, natural regeneration 59 1-20 Vi4 Vi4M Mątowy Wielkie, mature trees lower N 54.01 E 18.85 41 20-40 Vi4R Mątowy Wielkie, natural regeneration 156 1-10 454 Table 2. Genetic diversity parameters (average values), where C is clonality, A is the number of alleles, 455 AE is the number of effective alleles, AR is the allelic richness, AP is the number of private alleles, Ho is 456 the observed heterozygosity, HE is the expected heterozygosity, FISnull is the mean inbreeding coefficient 457 corrected for the presence of null alleles (a – inbreeding is the significant component of the model), Ne 458 is the effective population size, MR is the M-ratio (MR significantly < MReq at ***p < 0.001; **p < 0.01; 459 *p < 0.05), and MReq are MRs under mutation-drift equilibrium. 460 C [%] A AE AR AP HO HE FISnul l Ne Ne 95%C Is MR MRe q Vi1 M 35.7 1 5.867 4.18 2 6.07 1 0 0.80 0 0.74 9 0.00 8 16.10 9.2038.10 0.536* ** 0.717 Vi1 R 16.0 7 9.933 3.75 9 5.22 5 4 0.75 1 0.68 8 0.00 2 30.10 25.9035.40 0.595* ** 0.797 Vi2 M 12.8 6 12.46 7 6.85 1 7.48 3 6 0.75 4 0.78 9 0.00 8 140.4 0 105.40 - 204.30 0.682* 0.783 Vi2 R 0 15.20 0 7.38 4 7.61 6 27 0.75 2 0.79 8 0.01 9a 233.0 0 204.00 - 269.50 0.691* * 0.833 Vi3 M 10.8 1 10.00 0 5.92 1 7.05 4 2 0.79 2 0.79 2 0.01 1 38.80 32.0048.30 0.608* ** 0.701 Vi3 R 74.5 7 7.867 4.71 7 6.76 7 0 0.72 1 0.73 4 0.01 1 30.30 21.2049.10 0.600* ** 0.732 Vi4 M 75.6 1 6.467 4.55 4 6.65 0 2/ 0 0.71 3 0.69 6 0.00 9 ∞ 66.70- ∞ 0.478 0.663 Avg 40.9 4 9.686 5.33 8 6.69 5 5 0.75 4 0.74 9 0.01 0 162.9 0 Table 3. Results of the AMOVA of (I) the groups of mature trees Vi1M-4M and (II) the groups of natural 461 regenerations Vi1R-4R. All values are significant at p < 0.001. d.f. – degrees of freedom. 462 21 Source of variation d.f. FST RST I Among populations 3 0.0307 0.0397 Within populations 204 0.9693 0.9603 II Among populations 2 0.0616 0.0707 Within populations 523 0.9384 0.9293 463 Table 4. Genetic diversity coefficients among the studied black poplar populations: FST (below diagonal) 464 and RST (above diagonal). Statistically insignificant values are marked in italics (p > 0.05). 465 Vi1M Vi1R Vi2 Vi2R Vi3 Vi3R Vi4M Vi1M -0.004 0.133 0.119 0.078 0.066 0.129 Vi1R 0.017 0.093 0.085 0.060 0.077 0.096 Vi2M 0.040 0.074 0.001 0.010 0.035 0.010 Vi2R 0.037 0.075 0.004 0.013 0.046 0.025 Vi3M 0.052 0.102 0.021 0.018 0.009 0.005 Vi3R 0.071 0.119 0.032 0.035 0.007 0.019 Vi4M 0.041 0.092 0.002 0.003 0.010 0.042 466 22 Figure 1. Principal coordinate analysis (PCoA) carried out at the (A) population level and (B, C, D) 467 individual level. 468 469 23 Figure 2. (A) Bar plots showing STRUCTURE results for the two most adequate K values, K=2 470 (according to the Evanno method) and K=3 (according to the Puechmaille method). (B) Distribution of 471 delta K over K=1-14 according to the Evanno method. (C) The MedMeanK, MaxMeanK, MedMedK, 472 and MaxMedK estimators used in the Puechmaille method to determine the most adequate K value. 473 474 24 Ethics and Integrity statements: 475 • data availability statement 476 The data that support the findings of this study are available from the corresponding author, D. 477 Robak, upon reasonable request. 478 • ethics and permit approval statement 479 The authors declare that they obtained the approval of the Regional Directorate for 480 Environmental Protection for conducting the study in Kępa Bazarowa Nature Reserve in Toruń, 481 Poland. 482 • funding statement 483 This research was financially supported by the Polish National Science Centre 484 (2021/41/B/NZ9/00722). Additional support was provided by the Institute of Dendrology, 485 Polish Academy of Sciences. 486 • conflict of interest disclosure 487 The authors declare that there is no conflict of interest regarding the publication of this paper. 488 • permission to reproduce material from other sources 489 Not applicable. 490 25 References 491 Arens, P., Coops, H., Jansen, J. & Vosman, B. (1998). Molecular genetic analysis of black 492 poplar (Populus nigra L.) along Dutch rivers. Molecular Ecology, 7(1), 11–18. 493 https://doi.org/10.1046/j.1365-294x.1998.00316.x 494 Barsoum, N., Muller, E. & Skot, L. (2004). Variations in levels of clonality among Populus 495 nigra L. stands of different ages. Evolutionary Ecology, 18(5–6), 601–624. 496 https://doi.org/10.1007/s10682-004-5146-4 497 Bordács, S., Borovics, A. & Bach, I. (2002). Genetic diversity of natural populations and gene 498 bank of black poplar in Hungary. In: Van Dam, B.C. & Bordács, S. 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