Gene flow is one of the main factors shaping genetic diversity within and among tree populations, and occurs through pollen and seed dispersal. Recent findings of pollen-release asynchronies in distant populations of Scots pine (
Genetic diversity provides the fundamental basis for the evolution of forest tree species and for their adaptation to change (
Neutral genetic diversity, which has little or no effect on the phenotype, is valuable for studying the effects of historical events such as population size changes, dispersal and vicariance, and of contemporary processes affecting gene flow, such as pollen and seed dispersal (
Gene flow occurs through pollen and seed dispersal, and in wind-pollinated trees is usually more extensive by pollen than by seeds,
Multiple ecological factors can restrict pollen-mediated gene flow among wind-pollinated tree populations, and hence lead to temporal or spatial increases in genetic drift. Physical barriers (
The fragmented natural Scots pine (
A recent study found mismatches in timing of pollen release between a Western and an Eastern population from Scotland. Pollen was released first in the west, between 9.8 to 15.8 days earlier than in the east (
Based on the previous evidence, we hypothesised that, despite extensive pollen dispersal by wind, the presence of an East-West asynchrony in pollen production might result in IBD due to a higher probability that more synchronous populations will mate with each other. Alternatively, the absence of identifiable IBD would suggest that despite the presence of pollen asynchrony, effective gene flow at the regional scale, might have prevented differentiation. To test this hypothesis we characterised genetic diversity along the full East-West gradient of Scots pine within Scotland with two sets of neutral molecular markers, nuclear and chloroplast microsatellites (SSR). The use of both markers can allow greater understanding of the factors driving differentiation than based on either alone (
Scots pine (
Eighteen populations were selected to cover the full native range of Scots pine within Scotland (
Total genomic DNA was extracted from 50 mg silica gel-dried needles using a QIAGEN DNeasy® plant extraction kit (QIAGEN Ltd. Crawley, UK) following the manufacturer’s protocol. All individuals from the eighteen Scottish populations were genotyped at six nuclear (nSSR) and five chloroplast (cSSR) microsatellite markers. We used six nSSR: PSAJ223770/SPAC11.14, PSAJ223766/SPAC11.8 (
Genetic diversity estimators within populations were estimated using FSTAT ver. 2.9.3.2 (
To estimate population differentiation, we calculated
We performed individual-based Bayesian assignment methods using data from nuclear loci in STRUCTURE ver. 2.3.4 (
For testing isolation by distance we used nuclear markers in SPAGeDi ver. 1.4 (
To reduce the number of sites to an analytically tractable set for estimating migration patterns, we grouped sites with their nearest neighbours to give seven site clusters, corresponding to the biochemical zones described by
Among the six nuclear loci analysed, the number of alleles per locus (
Among the five chloroplast loci analysed, the number of alleles per locus (c
When testing differences among populations, multilocus
Structure identified
Although weak, IBD was significant in the Scottish populations (slope = 0.0038,
The relative migration network (Fig. S3a in Supplementary material) shows all relative migration rates between site clusters of Scottish populations of Scots pine. Those rates indicate that gene flow was, in most cases, greater in the West-East direction than in the East-West direction, although it was not significantly asymmetric. In
Our study presents a detailed genetic survey of a subset of the remaining natural populations of Scots pine from Scotland using both bi-parentally inherited nuclear SSR and paternally inherited chloroplast SSR. Three main results were obtained: (i) high levels of genetic variation and low population differentiation; (ii) a weak pattern of isolation by distance; and (iii) an increase of nuclear diversity towards the East. While we detected some discrepancies (
In agreement with other molecular markers (
We detected low levels of population differentiation, which represented less than 2% of the genetic variation among populations for both nuclear and chloroplast markers (
Despite evidence of extensive gene flow and consequently weak overall genetic structure, some significant differentiation among populations was apparent. In particular, the most western population SHI was most differentiated from other populations (greatest
Our results showed a weak but significant pattern of IBD across Scotland. IBD occurs as a consequence of limited gene dispersal such that populations close to each other tend to be more genetically similar than populations farther apart (
Some of the Scottish populations showed significant levels of inbreeding (
Our results indicated increased nuclear genetic diversity towards the East. This West-East (W-E) trend does not fit theoretical expectations based on inferred patterns of post-glacial colonisation, which for Scots pine, and most other native species in Britain, has occurred from south to north (
While levels of diversity will be substantially dependent on past population sizes, asymmetric gene dispersal due to prevailing wind direction can also play an important role in shaping the current distribution of genetic diversity (
Native pine forest in Scotland suffered a very substantial historic reduction in abundance. Despite this reduction and the resulting geographical isolation of populations, high levels of genetic variation and low levels of population differentiation still persist, suggesting that effective population size, together with extensive gene flow, has been high enough to limit the effects of genetic drift. This finding highlights the importance of maintaining large effective population sizes, especially in geographically marginal populations, to increase the probability of forest persistence.
Despite potential barriers to gene flow such as population fragmentation or phenological asynchrony, gene flow among populations can still be sufficient to counteract their genetic isolation. However a weak signal of isolation by distance was detectable among the Scottish populations, suggesting that some spatial limitation of gene dispersal occurs, although gene flow is extensive. The detected gene flow patterns and geographic distribution of genetic variation were consistent with gene dispersal limitation due to prevailing wind patterns. From a practical point of view, taking into account such landscape impacts on genetic diversity is important when designing afforestation strategies or determining priorities in conservation and management plans.
Although western populations had relatively lower nuclear diversity, and there was greater differentiation and directional bias of gene flow towards the East, there was no evidence to suggest that any of the populations analysed here are genetically at risk. However, over recent decades, there has been extensive establishment of Scots pine plantations throughout the country and it would be interesting to understand the impact such plantations might have on the diversity and structure of subsequent generations of native Scots pine.
We thank the Scottish Forestry Trust, University of Stirling and Centre for Ecology and Hydrology for co-funding this research. We are grateful to COST action FP1102 MaP-FGR (http://map-fgr.entecra.it/) for supporting researcher mobility during this project.
Study populations for nSSR and cSSR analyses.
Genetic diversity parameters for nSSR (above) and cSSR (below). (a) Genetic diversity for nSSR (
Isolation by distance (IBD). Black line represent the slope of the correlation of the natural log of the linear spatial distance [Ln(Spatial Distance (km)] against
Relative migration networks for nSSR with populations sorted in seed groups (see
Studies assessing neutral genetic variation of Scots pine in Scotland using variable molecular markers. (RFLP): Restriction Fragment Length Polymorphism; (SSR): Simple Sequence Repeat or microsatellite; (SNP): Single Nucleotide Polymorphism; (No. pop): number of studied populations; (No. ind): number of genotyped individuals.
Marker | Location | No. pop. | No. ind. | No. markers | Gst*/Fst**/ AMOVA#/ | Diversity | Reference | Main conclusions |
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Monoterpene | Scotland | 41 | 6705 | 11 | Similarity matrix: 0-24 over 30 | - |
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Variation between sites allowed divide the natural range into several areas of biochemical similarity, the most distinct being a north-western group of sites with Shieldaig as its most distinctive site. A trend of gradually decreased of similarity from the north east from Scotland in a south westerly direction. |
Monoterpene | Europe | 6 | 953 | 11 | Similarity matrix: 3-24 over 30 | - |
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Northern European populations were similar to each other but the three from middle and southern Europe showed large differences from them and from each other. Western region from Scotland showed similarity to middle Europe and south western populations to northern Europe. |
Scotland (from |
41 | 953 | 11 | Similarity matrix: 0-24 over 30 | - | |||
Monoterpene | Scotland | 40 | 5765 | 6 | 0.045* | 0.272-0.378 |
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High genetic diversity. Several populations in the Western region from Scotland were distinct from all others and each other. Scottish Scots pine forest originated from more than one refugium after the last glaciation. |
Isozymes | 14 | 2177 | 9 | 0.028* | 0.291-0.311 | |||
RFLP (mtDNA) | Scotland | 20 | 466 | Coxl mitochondrial gene | 0.37** | - |
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Two mitotypes were present: mitotype a is present at all sites, but that mitotype b is confined to three western populations |
SSR (cpDNA) | Scotland | 7 | 330 | 17 | 0.032# | 0.950-0.987 |
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Higher levels of diversity for the Scottish populations than those for European population. A mutation in one loci occurred in the Western region of Scotland. |
Europe | 8 | 185 | 17 | Scot |
0.908-0.976 | |||
SNP (nDNA) | Scotland | 21 | 42 | 16 | -0.017-0.023 | 0.754-0.819(0.831 at 8 loci) |
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High genetic diversity |
Europe | 7 | 40 | 10 | 0.071-0.079 | (0.795 at 8 loci) | |||
SSR (nDNA) | Scotland | 21 | 1680 | 3 | - | - |
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High levels of outcrossing in Scottish populations |
SNP (nDNA) | Scotland | 12 | 120 | - | 0.009** | 0.67 |
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High levels of nucleotide diversity within populations |
SNP (mtDNA) | Scotland | - | 0.81 | |||||
SSR (nDNA) | Scotland | 2 | 647 | 12 | 0.004** | 0.56-0.58 |
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High levels of genetic diversity and presence of moderate fine-scale spatial genetic structure |
Details of study sites. Population area was obtained from
Population name | Code | Seedzone | Pine area(ha) | Lat N(dec. deg.) | Long E(dec. deg.) | Altitude(m a.s.l.) |
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Cona Glen | COG | SW | 189 | 56.78 | 5.33 | 148 |
Glen Loy | GLL | SW | 74 | 56.91 | 5.13 | 170 |
Crannach | CRA | SW | 70 | 56.5 | 4.77 | 296 |
Coille Coire Chuilc | CCC | SC | 67 | 56.41 | 4.71 | 257 |
Meggernie | MEG | SC | 277 | 56.58 | 4.35 | 306 |
Black Wood of Rannoch | BWR | SC | 1011 | 56.67 | 4.32 | 275 |
Abernethy | ABE | EC | 2452 | 57.24 | 3.66 | 341 |
Rothiemurchus | ROT | EC | 1744 | 57.15 | 3.77 | 318 |
Allt Cul | ALT | NE | 13 | 57.11 | 3.33 | 476 |
Glen Tanar | GLT | NE | 1564 | 57.05 | 2.86 | 334 |
Glen Affric | GLA | NC | 1532 | 57.27 | 4.92 | 256 |
Amat | AMA | NC | 181 | 57.87 | 4.59 | 137 |
Loch Clair | LOC | NW | 126 | 57.56 | 5.34 | 132 |
Shieldaig | SHI | NW | 103 | 57.51 | 5.64 | 81 |
Beinn Eighe | BEE | NW | 182 | 57.63 | 5.35 | 63 |
Glen Einig | GLE | N | 27 | 57.95 | 4.76 | 55 |
Strath Oykell | STO | N | 14 | 57.95 | 4.64 | 103 |
Rhidorroch | RHI | N | 103 | 57.93 | 4.97 | 182 |
Genetic diversity estimators for nuclear (nSSR) and chloroplast (cSSR) markers. (
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COG | 30 | 6.1670(3.3120) | 6.0992(2.9791) | 1 | 0.4944(0.2352) | 0.5779(0.2402) | 0.1460*** | 28 | 3.2(1.095) | 13 | 4 | 0.9339(0.0227) |
GLL | 30 | 5.8330(2.9270) | 5.7860(2.6523) | 0 | 0.4944(0.1500) | 0.5801(0.2743) | 0.1500*** | 25 | 2.8(0.837) | 14 | 1 | 0.9400(0.0280) |
CRA | 30 | 6.3330(3.1410) | 6.2860(2.8557) | 1 | 0.5151(0.1960) | 0.6389(0.1990) | 0.1960*** | 25 | 3.0(1.225) | 14 | 1 | 0.8967(0.0431) |
CCC | 30 | 6.6670(4.2270) | 6.5777(3.7589) | 0 | 0.5722(0.0460) | 0.5995(0.2201) | 0.0460 | 23 | 2.8(1.304) | 14 | 1 | 0.9565(0.0220) |
MEG | 30 | 5.5000(2.8110) | 5.4753(2.5497) | 0 | 0.5556(0.1570) | 0.6573(0.1552) | 0.1570*** | 28 | 3.4(1.673) | 15 | 3 | 0.9048(0.0420) |
BWR | 30 | 6.3330(2.9440) | 6.2638(2.6361) | 0 | 0.5860(0.0560) | 0.6200(0.1885) | 0.0560 | 23 | 3.2(1.304) | 14 | 3 | 0.9368(0.0331) |
ABE | 30 | 7.1670(3.7640) | 7.0532(3.3582) | 1 | 0.6111(0.0590) | 0.6490(0.2224) | 0.0590 | 27 | 2.8(1.304) | 13 | 0 | 0.8291(0.0684) |
ROT | 30 | 5.8330(2.3170) | 5.7767(2.0727) | 1 | 0.5935(0.0500) | 0.6240(0.2063) | 0.0500 | 27 | 3.4(1.140) | 17 | 3 | 0.9487(0.0257) |
ALT | 30 | 6.3330(3.0770) | 6.2532(2.7636) | 1 | 0.5937(0.0120) | 0.6006(0.2542) | 0.0120 | 25 | 2.8(0.837) | 14 | 0 | 0.9433(0.0240) |
GLT | 30 | 7.1670(2.9270) | 7.0615(2.5983) | 0 | 0.5556(0.1100) | 0.6228(0.2215) | 0.1100* | 25 | 3.4(1.140) | 13 | 1 | 0.9167(0.0349) |
GLA | 30 | 7.3330(4.2270) | 7.1470(3.7213) | 0 | 0.4925(0.1170) | 0.5565(0.2660) | 0.1170** | 22 | 3.4(0.241) | 15 | 2 | 0.9524(0.0291) |
AMA | 30 | 6.5000(3.6740) | 6.4505(3.3049) | 0 | 0.5444(0.1160) | 0.6149(0.2604) | 0.1160** | 21 | 3.2(0.837) | 15 | 1 | 0.9667(0.0236) |
LOC | 30 | 6.6670(3.4450) | 6.5968(3.0930) | 0 | 0.5803(0.0690) | 0.6226(0.2130) | 0.0690 | 27 | 3.0(1.000) | 13 | 0 | 0.9373(0.0220) |
SHI | 30 | 5.5000(3.2710) | 5.4198(2.9151) | 0 | 0.4944(0.0330) | 0.5110(0.2737) | 0.0330 | 29 | 3.0(0.707) | 13 | 2 | 0.9458(0.0173) |
BEE | 30 | 6.6670(3.7240) | 6.6082(3.3824) | 1 | 0.5500(0.1040) | 0.6128(0.2978) | 0.1040* | 29 | 3.2(1.304) | 14 | 0 | 0.9335(0.0228) |
GLE | 30 | 6.5000(2.8810) | 6.3923(2.5723) | 2 | 0.4571(0.2140) | 0.5794(0.2517) | 0.2140*** | 28 | 2.8(0.837) | 16 | 2 | 0.9497(0.0214) |
STO | 30 | 7.3330(3.5020) | 7.1848(3.1003) | 0 | 0.5222(0.1450) | 0.6095(0.2079) | 0.1450*** | 27 | 2.8(1.304) | 14 | 1 | 0.9145(0.0334) |
RHI | 30 | 7.6670(3.5020) | 7.5522(3.1469) | 1 | 0.5714(0.1130) | 0.6430(0.2847) | 0.1130* | 30 | 3.2(1.304) | 19 | 2 | 0.9586(0.0209) |
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Hierarchical analysis of molecular variance (AMOVA) for nuclear (nSSR) and chloroplast (cSSR) markers at the individual, population and cluster of populations. The degrees of freedom (df), percentage of variation explained by each level (Variation, %), and the relevant
Source of variation | nSSR | cSSR | ||||
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df | Variation (%) | df | Variation (%) | |||
Among cluster of populations | 6 | 0.17 | 0.25 | 6 | 0.25 | 0.11 |
Among populations | 11 | 1.79 | <0.001 | 11 | 1.89 | <0.001 |
Within populations | 1062 | 98.04 | <0.001 | 453 | 97.86 | <0.001 |
Tab. S1 - Pairwise population differentiation (FST) (below diagonal) and Jost’s differentiation index, D (above diagonal).
Fig. S1 - Percentage of differentiated sites within Scotland using nSSR, calculated as the percentage of total sites that were significantly differentiated based on
Fig. S2 - The number of genetic clusters (K=2) identified by STRUCTURE for nSSR.
Fig. S3 - Relative migration networks for nSSR with populations sorted in seed groups.