Research Article |
Corresponding author: Jacek Urbaniak ( jacek.urbaniak@upwr.edu.pl ) Academic editor: Irina Belyaeva
© 2021 Jacek Urbaniak, Paweł Kwiatkowski, Paweł Pawlikowski.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Urbaniak J, Kwiatkowski P, Pawlikowski P (2021) Genetic diversity of Salix lapponum populations in Central Europe. PhytoKeys 184: 83-101. https://doi.org/10.3897/phytokeys.184.71641
|
Salix lapponum is a cold-tolerant relict species in Europe that occurs in several sites, probably reflecting previous migration routes of S. lapponum during the Pleistocene. However, only a few data are available on the genetic structures of populations of S. lapponum. In this study, we use PCR-ISSR markers to investigate genetic variation in 19 European populations of S. lapponum L. AMOVA analysis shows that most of the variation (55.8%) occurs within populations; variability among groups accounts for 19.7%. An AMOVA analysis based on four groups determined by STRUCTURE analysis shows similar results: variability of 54.1% within the population and variability of 18.9% between the four population groups, based on geographic regions. Within individual geographic groups, which are characterised by the studied populations, the lowest variability (as well as the highest homogeneity) was found in populations located in Belarus. The obtained results are consistent with our expectations that the European Lowland could be a significant geographic barrier for gene flow over large geographic distances for S. lapponum. Both the Scandinavian and Belarusian populations, as well as those coming from NE Poland, are characterised by significant genetic distinctiveness. However, some populations from NE Poland and the Sudetes show similarities with populations from other geographic regions, indicating existing genetic relationships between them. Moreover, the results suggest a fairly clear division of the population into 4 emerging geographic regions, although separated by a geographical barrier: the Polish lowland, which forms part of the larger geographic unit known as the European Lowland.
Europe, genetic variation, phylogeography, relict plant, Salicaceae, Salix
Quaternary glaciation, with numerous glaciations and deglaciations in Scandinavia and Central Europe, strongly influenced changes in the distributions of both specific plants and entire plant biomes. During Pleistocene climatic fluctuations, numerous arctic plants migrated southward, where they commonly colonised habitats primarily in European mountain ranges; in some cases, they also disappeared from these locations during warmer period called interglacials. Currently, various plant species growing in boreal areas in the Northern Hemisphere and in subalpine zones in lower latitudes in isolated mountains of the alpine system (subalpine zones) or on the lowlands – often grow on the edges of their ranges, presenting disjunct geographic distribution (
Disjunctive plant populations are often attributed to isolated localities in specific habitats where the climatic and edaphic conditions have allowed for their survival as relict species until the present postglacial period (
Numerous phylogeographic studies have described processes including colonisations or re-colonisations from refugial zones, extinctions, or migrations across thousands of kilometres in the Northern Hemisphere (
During the Last Glacial Maximum (LGM), all natural vegetation of temperate Europe changed substantially. This also applies to the development history of various Salix species, for which results have been obtained from numerous paleobotanical studies and from research using modern genetic methods (
For this study, we used samples collected from nineteen populations of species across the geographical range in Europe (Table
Populations of S. lapponum included in the study: location in geographical regions, coordinates and Nei’s gene diversity value. The populations’ abbreviations are given below the table.
Region | Population (number) / abbreviation1 | Longitude, Latitude | h = |
---|---|---|---|
Sudetes | (1) KWS | 50°45'22"N, 15°41'29"E | 0.144 |
(2) POL | 50°46'5"N, 15°42'35"E | 0.136 | |
(3) KMS | 50°44'51"N, 15°42'0"E | 0.147 | |
Scandinavia | (4) DOV | 62°11'29"N, 9°44'59"E | 0.143 |
(5) FUL | 61°29'58"N, 12°42'34"E | 0.144 | |
(6) NAT | 59°21'42"N, 15°8'52"E | 0.121 | |
NE Poland | (7) DWO | 53°56'31"N, 23°23'16"E | 0.172 |
(8) WIZ | 53°11'48"N, 22°23'45"E | 0.110 | |
(9) SZT | 54°7'55"N, 23°24'10"E | 0.139 | |
(10) PRU | 54°9'51"N, 22°55'24"E | 0.098 | |
(11) BKL | 53°17'9"N, 22°36'22"E | 0.114 | |
(12) BIA | 52°41'22"N, 23°44'46"E | 0.147 | |
(13) BLA | 53°16'32"N, 22°32'58"E | 0.162 | |
(14) JMO | 51°27'36"N, 23°7'14"E | 0.109 | |
Belarus | (15) GLU | 55°7'38"N, 27°43'5"E | 0.129 |
(16) DIK | 55°32'26"N, 27°50'1"E | 0.163 | |
(17) SIE | 55°47'25"N, 29°3'31"E | 0.140 | |
(18) WIT | 55°13'21"N, 29°0'11"E | 0.122 | |
(19) BEN | 55°33'37"N, 29°14'7"E | 0.121 |
Molecular analysis was conducted on plants from all populations. In the field, leaves of S. lapponum were randomly sampled in populations at distances that generally depended on the spatial extent of the populations (5–6 m). Fresh leaves were collected in plastic zip bags, dried in silica orange gel, transported to the laboratory, and stored until DNA extraction. In general, we studied eight plant samples per population and 152 specimens of S. lapponum in total. Whole plant material was collected in the months of June or July from 2015 to 2017. Herbarium voucher specimens that were used for DNA are stored in author collection (J. U.) deposited at WRSL Herbarium (Wrocław, Poland) and are available on request.
The genomic DNA was isolated after cell disrupting in a Mixer Mill MM400 (Retsch, Haan, Germany), using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The quality of the isolated DNA was determined using 1% TBE agarose electrophoresis. The Inter Simple Sequence Repeat microsatellite markers were selected to study the genetic diversity of the populations of S. lapponum. These markers are known to be highly polymorphic and are useful in studies on genetic diversity and species relationships; they enable easy differentiation of closely related specimens. ISSR has also been successfully employed to assess hybridisation and to detect hybrid taxa (
The results were analysed using CLIQS software (
All scientific names are given following
The research markers selected by us for studying (ISSR) have some limitations in their application due to the lower number of generated markers than for example AFLP, but are still widely used in various types of phylogeographic research. Molecular variance analysis (AMOVA) indicates that most of the genetic variation in two groups of populations: Sudetes vs. NE Poland and Belarus occurs within populations (55.8%), while the variability among the groups accounts for 19.7% (Table
Groups/ populations | Partitioning | d.f. | Sum of Squares | Variance components | Percentage of variation | Fst-statistic |
---|---|---|---|---|---|---|
All populations | Among populations | 19 | 1245.5 | 7.3 | 43.8 | 0.43 |
Within populations | 134 | 1255.2 | 9.3 | 56.2 | ||
Sudetes | Among populations | 3 | 172.9 | 5.9 | 37.4 | 0.37 |
Within populations | 28 | 279.4 | 9.9 | 62.6 | ||
Scandinavia | Among populations | 2 | 151.3 | 8.3 | 48.2 | 0.48 |
Within populations | 21 | 188.2 | 8.9 | 51.8 | ||
NE Poland | Among populations | 7 | 358.7 | 5.4 | 37.2 | 0.37 |
Within populations | 54 | 495.1 | 9.2 | 62.8 | ||
Belarus | Among populations | 4 | 118.9 | 2.8 | 23.1 | 0.23 |
Within populations | 31 | 292.4 | 9.4 | 76.9 | ||
Sudetes and NE Poland vs. Belarus | Among groups | 1 | 158.4 | 3.4 | 19.7 | 0.44 |
Among populations | 7 | 291.8 | 4.2 | 24.5 | ||
Within populations | 59 | 571.8 | 9.7 | 55.8 | ||
Sudetes vs. Scandinavia vs. NE Poland vs. Belarus | Among groups | 3 | 443.6 | 2.6 | 18.9 | 0.46 |
Among populations | 16 | 801.9 | 5.3 | 30.6 | ||
Within populations | 134 | 1255.2 | 9.4 | 54.1 |
The AMOVA analysis based on four groups (Sudetes vs. Scandinavia vs. NE Poland vs. Belarus) shows a similar result: variability of 54.1% within the population and variability of 18.9% between the four groups of the population based on geographic regions. High genetic variability was also found with the analysis of all studied populations, which were treated as one group. The genetic variability value was 43.8% among populations and 56.2% within populations; the Fst value was 0.43 (p < 0.001). Within geographic groups, which are characterised by the studied populations, the lowest variability and also the highest homogeneity were found in populations located in Belarus. The genetic variation among Belarusian populations was low (23.1%). In contrast, the highest inter-population variability was found in Scandinavia (48.2%) and the variability in the Sudetes and NE Poland was similar: 37.4% and 37.2%, respectively.
High and statistically significant genetic differences between all geographical groups of the population were found; again, the greatest differentiation was noted between the Belarusian populations and the Scandinavian populations (0.5). The greatest differences were noted between the populations from Belarus and between the populations from the Sudetes and NE Poland (Table
Pairwise genetic differentiation in between group of populations S. lapponum studied using ISSR microsatellites. Number of studied populations in each groups are given in brackets in the first column.
All Groups | Sudetes | Scandinavia | NE Poland | Belarus | |
---|---|---|---|---|---|
All Groups (19) | 0.46 | ||||
Sudetes (3) | - | ||||
Scandinavia (3) | 0.43 | - | |||
NE Poland (8) | 0.40 | 0.44 | - | ||
Belarus (5) | 0.47 | 0.50 | 0.49 | - |
Both the highest and the lowest index of genetic diversity, based on
The analysis using neighbourhood joining for reconstructing the relationships among populations, revealed differences between the population samples collected from different geographical regions of S. lapponum’s distribution (Fig.
Moreover, all of the selected subgroups were well-supported by bootstrap analysis. Populations from Belarus (GLU, DIK, SIE, WIT, BEN) showing a high similarity with each other (Fig.
Tests performed with the use of the STRUCTURE program, with which the clustering method was implemented, allowed for a more precise elucidation of the genetic variability of the S. lapponum population (Fig.
For K = 2, all of the populations studied were divided into two groups: populations from Belarus and all other geographical regions (Sudetes, Scandinavia, NE Poland). In general – not only for K = 2 but also for K > 2 the populations from Belarus appeared as most distinct in all runs. For K = 3, populations from the Sudetes grouped with populations from NE Poland, while Scandinavian populations formed a separate group, except for one population (FUL) that seems to be genetically closer to populations from the Sudetes. For K = 5, populations from Belarus and the Sudetes form nearly a consistent group; however, specimens from the Sudetes populations showed closer relations with several populations from NE Poland. Results obtained with the STRUCTURE program are almost identical to the results presented in Fig.
Climate fluctuations during the Pleistocene markedly changed distribution patterns for both plant and animal species. Consequently, full phylogenetic compatibility of species can be found only rarely, and usually only on a regional scale (
The results obtained from genetic analyses of the S. lapponum population show that genetic diversity between the studied populations in the area of the species occurrence is significant, while gene flow is clearly geographically limited. This fact may be explained by the lack of continuity in the occurrence of S. lapponum and by the large geographic areas that separate the studied populations, thus preventing the free flow of genes. For example, in central Poland, there are no S. lapponum sites – not even scattered sites. As can be seen on Fig.
The populations of S. lapponum in Scandinavia seem to be different from those in the rest of Europe, apart from the Fuljafleat (FUL) population. At the same time, the effects of between populations in Scandinavia and other regions are visible. In Scandinavian populations, however, there are alleles common to populations from both the Sudetes and NE Poland, demonstrating close genetic relationships and similarities between them (Fig.
There are also similarities between the analysed populations from the Sudetes and NE Poland, which may be attributed to the relatively small populations compared; this is confirmed by the results of the pairwise genetic differentiation analysis (Table
In the Northern Hemisphere, climatic oscillations during the Quaternary period caused significant changes in plant distribution, which resulted in the repeated expansion and fragmentation of species’ ranges and affected their patterns of genetic diversity. Cold-adapted plants (arctic and boreal) are believed to be more threatened during the Quaternary period than other plant groups (
The currently observed decline in population sizes and geographical ranges, limited generative reproduction, and short-range spreading make boreal species more susceptible to loss of genetic diversity than, for example, plants in the temperate or Mediterranean zones. Moreover, climate scenarios (
The clearly low and similar level of variation, especially within Belarusian populations, may result from two different processes: the founding effect and genetic drift caused by small population sizes. However, in both the Scandinavian and Sudeten populations, probably there was increased genetic variation, represented in migrants who re-settled the newly available space. In many plant species, a correlation is found between population size and genetic variation (
These two waves of migration probably met, creating a local suture zone with a mixed haplotype character. A similar scenario has also been proposed for Populus tremula L. in Central Europe (
The conducted research shows the division of the studied Salix lapponum populations into several genetic groups. The populations from Belarus were the most genetically different. Populations from NE Poland and the Sudetes show similarities with populations from other geographic regions, indicating existing genetic relationships between them. It is possible that there was a meeting and exchange of genes between populations in southern Europe and Scandinavian populations and from NE Poland. However, to reach a more thorough understanding, additional studies are needed that include not only intra- and inter-population variability studies based on investigating phylogenetic lineage.
We would like to thank Maryla Kwiatkowska for her help in conducting the research. The research and publication is financed under the Leading Research Groups support project from the subsidy increased for the period 2020–2025 in the amount of 2% of the subsidy referred to Art. 387 (3) of the Law of 20 July 2018 on Higher Education and Science, obtained in 2019.
Table S1
Data type: Docx file.
Explanation note: Parwise Nei’s genetic distances (Nei, 1978) for populations of S. lapponum based on ISSR analysis.
Table S2
Data type: Docx file.
Explanation note: Nucleotide sequences and annealing temperatures of ISSR primers used in study.
Figure S1
Data type: Jpg file.
Explanation note: Delta K values for Structure analysis.