Conserv Genet DOI 10.1007/s10592-015-0756-7

RESEARCH ARTICLE

A climatic relict or a long distance disperser: conservation genetics of an disjunct polyploid plant

1 1 2 3 Laura Kvist • Leila Aminian • Romuald Rouger • Marjut Kreivi • 4 5 1 1 Marika Laurila • Marko Hyva¨rinen • Jouni Aspi • Annamari Markkola

Received: 27 October 2014 / Accepted: 9 July 2015 Ó Springer Science+Business Media Dordrecht 2015

Abstract The Primula sibirica group is a set of approx- phryganodes is more likely a climatic relict in the Bothnian imately a dozen Arctic, taxonomically unrelated plant than a long-distance disperser and that the endangered species that share a similar disjunct distribution on the southern population should be considered as an evolu- shores of the Arctic and the northernmost part of the tionary significant unit rather than a mere representative of Baltic . The origin of this phylogeographic pattern is the main population. not known. It has been suggested, first, that the species arrived after the last glaciation from the , second, Keywords Puccinellia phryganodes Á Rear edge that they are relicts of once larger populations or, third, that population Á Microsatellites they arrived via jump dispersal. One of the species is the polyploid Creeping alkali grass, Puccinellia phryganodes, which is critically endangered in . Here we used Introduction microsatellite markers to study seven extant and three extinct populations from coasts of the Bothnian Bay and Many species are responding to the climate warming by the Arctic Sea (N = 297). We estimated the genetic shifting their distributions towards higher latitudes, but for diversity in the study populations and applied principal arctic terrestrial species, this is impossible as the Arctic component analysis and Bayesian and coalescence meth- Ocean forms a geographic barrier (Parmesan 2006). The ods to examine their population genetic structure and predicted change in the Arctic is, however, far more evolutionary history. We found that the endangered Both- complex, in addition to the changes in distribution there are nian Bay population still harbors a reasonably high amount now ample evidence of ongoing changes in community of genetic diversity and is differentiated from the geo- structure and function. In vegetation, this can be seen for graphically closest populations. We show that Puccinellia example as an increase in productivity, density and cover as shrubs and trees are becoming more frequent (Elmen- dorf et al. 2012; Miller and Smith 2012). In addition, & Laura Kvist simulations of climatic warming suggest an increase in laura.kvist@.fi total plant biomass for the Arctic and an increase in shrub 1 Department of Biology, University of Oulu, PO Box 3000, biomass at the expense of other plant functional types 90014 Oulu, Finland (Epstein et al. 2000). 2 Biological and Environmental Sciences, School of Natural During range shifts, some populations left behind can Sciences, University of Stirling, Stirling FK94LA, UK persist in refugia if favorable conditions are met. These 3 Faculty of Biochemistry and Molecular Medicine, University relict populations are not uncommon; many species pre- of Oulu, PO Box 5400, 90014 Oulu, Finland sently found at high latitudes have relict populations in the 4 Natural Resources Institute Finland, Tutkimusasemantie 15, , which they inhabited during the previous colder 92400 Ruukki, Finland climatic conditions of the last glacial (Hewitt 2004; Hampe 5 Botany Unit, Finnish Museum of Natural History, University and Jump 2011). These populations can be two or three of Helsinki, PO Box 7, 00014 Helsinki, Finland orders of magnitude older than the populations located at 123 Conserv Genet the current main range and retain information of their the species have arrived via jump dispersal aided by winds phylogenetic history and unique local adaptations lacking or birds (Havas 1961). Many of these species are currently from the main range, especially if they have been isolated endangered in Finland (Rautiainen 2006). for long (Hampe and Petit 2005). This makes them One such species is the perennial Creeping alkali important for the conservation of the genetic diversity and grass, Puccinellia phryganodes (Trin.) Scribn. and Merr., evolutionary potential of the species (Hampe and Petit which has a circumpolar, though discontinuous distribu- 2005) and highly valuable for local and regional biodi- tion mostly on shores of the Arctic Sea. In , P. versity. Management strategies for such species need to be phryganodes growsonlyontheNorwegiancoastofthe based on proper understanding of their history and current Arctic Sea and in the Bothnian Bay. It inhabits saline or state and on a realistic projection on their future (Hampe brackish meadows, marshes and seashores. The species is and Jump 2011). a polyploid, with variation of ploidy level from triploids The Bothnian Bay (Fig. 1) is the northernmost part of to tetraploid and even hexaploids. Subspecies phrygan- the of the . It is located at the odes in Russian and Alaska is proposed to be center of a post-glacial uplift area, with a maximum uplift tetraploid, neoarctica in Canadian arctic triploid, rate of about 10 mm/year (Steffen et al. 2009). The Baltic vilfoidea in Svalbard tetraploid and sibirica from Sea is a brackish inland sea, connected to the by to a mixture of triploids and tetraploids shallow straits. Salinity decreases towards the Bothnian (Aiken et al. 2007;Consauletal.2010;Elven2014). It is Bay, from 20 to 30 g/kg close to the straits to the North Sea stoloniferous, capable of extensive clonal growth and to about 3.5 g/kg in the Bothnian Bay (Samuelsson 1996). propagates mainly by producing easily detaching axillary Several plant species inhabiting the shores of the Bothnian shoots. It flowers, but it has been proposed that seeds are Bay belong to the so-called Primula sibirica-group (named not produced, at least not in (Siira 2011; according to the former scientific name of Primula nutans), Elven 2014). However, an isozyme study from Arctic which is a diverse group of circumpolar Arctic plant spe- Canada showed that variation within and between popu- cies that have disjunct distributions in the Baltic (Ericson lations is high, suggesting either a hybrid origin for the and Wallentinus 1979). The origin of the species belonging species, occurring a number of times at different regions to the Primula sibirica group in the Baltic Sea is not or, more likely, that sexual reproduction occasionally known. It has been suggested that they arrived after the last occurs (Jefferies and Gottlieb 1983). glaciation when the water connection from the White Sea The first record of Puccinellia phryganodes in Finland is was still open (Ericson and Wallentinus 1979; but see from 1926, but the inconspicuous species has likely been discussion), the species once inhabited a large area of present already before. Since then it was found at as many which the Baltic populations are remnants (Eurola 1999)or as 17 sites (Siira 2011). Currently, the species is present

Barents Sea

Billefjord gian SeaTalvik Eahpárašcohkka Lakselv

Norwe

Tömppä Isomatala Väliteonkarit White Savilahti Sea Onega Bay Tauvo 10 km nBay a RUSSIA

SWEDEN FINLAND Bothni

Y A W Gulf of R O Bothnia N nland Gulf of Fi 0 100 200 300 400 500 km

Fig. 1 Locations of the sampled Puccinellia phryganodes populations 123 Conserv Genet only at two sites in the Bothnian Bay, on the shores of the population is now lost. The White Sea population was island of Hailuoto, which started to emerge from the sea sampled from a that contained suitable coastline about 2000 years ago (Alestalo 1979). It used to occupy habitat for the species of about 5 km2 (Lumme J, pers. also several sites on the shores of the mainland (see Fig. 1), obs.), but the whole area was not checked for the existence but those sites have now gone extinct. P. phryganodes is a of the species. The populations from Eahpa´rasˇcohkka and poor competitor with taller plants (Niemela¨ et al. 2008) and Billefjord were located at stretches of about 20 9 170 m grows only on a narrow zone of the lowest shore meadow and 150 9 230 m, respectively, between a road and the zone. It requires newly emerged open shore with fine- coastline. Both of the population samples consisted of particle-sized silty soil. Mechanical disturbance, i.e. ice- individuals collected from larger continuous and smaller scouring alleviate detrimental effects of competition. patchy growth sites. Samples from Talvik were from a Grazing by geese is able to slow down succession only population located in a bottom of a bay, extending about slightly (Laurila et al. unpublished). In Finland P. 300 m along the coastline. Population from Lakselv was phryganodes is listed as critically endangered, the most sampled from a 20 9 190 m area on a sandy river delta important threats are overgrowth and disturbance of below a riverbank. meadows and climate warming in addition to apparent DNA extraction followed the method described by stochastic threats (Rassi et al. 2010). Rogers and Bendich (1985). We selected 11 microsatellite The purpose of this study was to (1) examine if the loci originally developed for Puccinellia maritima by Bothnian Bay population is genetically distinct from the Rouger et al. (2014). Amplifications were performed in closest populations on the shores of the White and Nor- four multiplexes, combining Pm29 (forward primer label- wegian/Barents , (2) study the history of the popula- led with FAM), Pm26 (VIC) and Pm10 (PET) into the first, tion, i.e. find out how and when the Bothnian Bay was Pm61 (FAM), Pm65 (VIC) and Pm19 (PET) into the sec- colonized by the species and (3) define if the Bothnian Bay ond, Pm27 (FAM) and Pm25 (VIC) into the third and population forms a management unit and therefore inten- Pm12 (NED), Pm34 (VIC) and Pm39 (PET) into the fourth sified management is required. multiplex. All PCR reactions were performed in 10 ll volumes, including primers in variable concentrations according to Rouger et al. (2014), using 50–100 ng of

Materials and methods template DNA, 0.2 mM of dNTPs, 2 mM of MgCl2, and 0.3 U of DNA polymerase (Biotools) in 19 PCR-buffer. Sampling and laboratory work The amplification profile consisted of an initial denatura- tion for 2 min (94 °C), followed by 35 cycles of denatu- We used DNA samples extracted from 297 plants collected ration for 30 s (94 °C), annealing for 1 min (50 °C for first from ten different localities (Fig. 1) during the years and third multiplex and 55 °C for the second and fourth 2000–2002, using a sampling interval from 10 to 30 m. multiplex) and synthesis for 30 s (72 °C) with a final Sample sizes were: Norway, northern / synthesis in 60 °C for 30 min. The PCR products were (2002): Billefjord N = 32, Eahpa´rasˇcohkka analysed with ABI PRISM 3730 DNA Analyzer (Applied N = 35, Talvik N = 35, Lakselv N = 35, Finland, Both- Biosystems) and scored with GeneMapper v. 4.0. nian Bay (2000): To¨mppa¨ N = 2, Isomatala N = 30, Va¨liteonkarit N = 33, Savilahti N = 7, Tauvo, N = 45 Genetic diversity and Russia, White Sea, Onega Bay (2002): N = 43. Of the Finnish Bothnian Bay populations, all the populations, As the ploidy level of P. phryganodes in our sampling sites except Isomatala and the closely located recolonized small is not certain (2n = (3x) 21 or (4x) 28; Aiken et al. 2007), population at To¨mppa¨, are now extinct (Markkola 2013). special attention for the number of alleles was paid during Isomatala has the largest population, estimated to be spread the scoring. The R-package Polysat v. 1.3 (Clark and over 17 hectares (Markkola 2013). The population at Jasienuk 2011; http://openwetware.org/wiki/Polysat) was Va¨linteonkarit had been founded quite recently, as the used to assist the ploidy estimation with the function esti- species was not yet present there in the mid-1980. The first matePloidy, which shows the mean and maximum number records are from 1996, when 20 small patches were found; of alleles per sample. Based on several individuals having by 1999, the colonized area was about 52 m2 (Siira 2011), four alleles at several loci (1 in locus Pm10, 17 in Pm29, 1 but it was not found anymore in 2013. The small popula- in Pm12, 7 in Pm27, 21 in Pm34 and 10 in Pm39), all tion in Savilahti, found in 1999 on a bank of a ditch had individuals were set as tetraploids. disappeared by 2007 (Markkola J, pers. obs.). In Tauvo, Allele numbers were then calculated with the there were over a hundred small (0.005–2.925 m2) patches alleleDiversity function in Polysat. Identical genotypes at the time of sampling (Laurila M, pers. obs.), but also that (possible clones) were searched using the function 123 Conserv Genet assignClones. Allele frequencies were estimated using the data. STRUCTURE uses a Markov chain Monte Carlo deSilvaFreq function (De Silva et al. 2005), which iterates (MCMC) approach to infer the number of clusters (K)ina genotype frequencies based on allele frequencies and ‘al- data set without prior information of the sampling loca- lelic phenotype’ frequencies and then recalculates allele tions. It assumes that populations are in Hardy–Weinberg frequencies from genotype frequencies. For this function, equilibrium and linkage equilibrium. As our study popu- selfing and null-allele frequencies should be given, and we lations have unknown ploidy levels and histories and assumed a selfing rate of 0.9 (the species apparently has no reproduce perhaps mainly clonally, these assumptions are sexual reproduction in our populations) and null-allele likely violated, which can lead to an overestimation of K. frequency of 0.05. DeSilvaFreq function assumes even- However, this analysis can assist to find indications of the numbered ploidy, which might be violated here. Thus, the population structure. A model with population admixture allele frequencies were estimated also using the function and correlated allele frequencies within populations was SimpleFreq for comparison, which however, might assumed. We conducted a series of ten independent runs underestimate common allele frequencies and overestimate for each value of K between 1 and 13, with a burn-in period rare allele frequencies leading to an underestimation of FST of 10,000 iterations and collected data for 100,000 itera- (Clark and Jasienuk 2011, Polysat manual). We excluded tions. The likelihood of the data and following log proba- locus Pm12 from these and further calculations because it bilities for the different number of subpopulations were did not amplify in one population (see results) and com- calculated for each K. In addition, we used the results from bined samples from To¨mppa¨ (N = 2) with population STRUCTURE as an input to the ad hoc method by Evanno Isomatala, which are located only about 4 km from each et al. (2005), which estimates DK between the consecutive other. In addition, seven individuals scored with less than numbers of populations. The highest DK can be inferred as five loci were excluded from the analyses (one from the best estimator of the actual K (Evanno et al. 2005). To Billefjord, one from Eahpa´rasˇcohkka, one from Talvik and align and visualize the output from the STRUCTURE two from Isomatala, one from Va¨liteonkarit and one from analyses across the ten replicates for the identified K value, Tauvo). we used program CLUMPP v. 1.1.2 (Jakobsson and Rosenberg 2007) for alignments and program Distruct v. Population genetic structure 1.1 (Rosenberg 2004) for visualization of the CLUMPP output. Prior to that, a web-based program STRUCTURE An allele size permutation test (Hardy 2003) in program HARVESTER v. 0.6.93 (Earl and vonHoldt 2012) was SPaGeDi v.1.4. (Hardy and Vekemans 2002) was first used used to transform the STRUCTURE result files into to assess if stepwise mutations affect genetic differentiation CLUMPP formats. in our study populations. This test is based on the com- The program SPaGeDi was used to infer isolation by parison of observed pairwise RST values with the distri- distance using regression of the pairwise RST-values (RST/ bution of RST values (pRST) obtained by 10,000 (1-RST) and logarithms of geographic distances between permutations of allele sizes among allelic states. If the populations. We used nine spatial categories and 10,000 observed RST values are found to be significantly larger permutations of individual spatial locations. Standard than the pRST, there is indication that stepwise mutations errors were estimated by jackknifing over loci. contribute to genetic differentiation among populations and

RST statistics should be preferred over FST-statistics, Coalescence analyses of population history whereas nonsignificant differences indicate the opposite.

Population pairwise RST-values between the sampling The function SimpleFreq in Polysat package was used to locations were then estimated with SPaGeDi. For com- calculate allele frequencies for each locus and population. parison, also pairwise FST-values were estimated using the The obtained allele frequencies were then used with the calcFst function in Polysat from De Silva and Simple fre- function Sample in R to resample alleles, which were quencies. A principal component analysis (PCA) was per- randomly ordered to generate diploid multilocus genotypes formed with the Polysat package, first using the function for the input in the coalescence analyses with DIYABC meandistance.matrix to estimate a pairwise distance matrix (Cornuet et al. 2014) similarly to Lepais et al. (2013). between the individuals and then using the function cmd- These resampled populations included as many ‘individu- scale to perform the PCA on the matrix. The function als’ as originally sampled from each population. Based on meandistance.matrix uses the Bruvo distance (Bruvo et al. geographical proximity and low divergence (see results), 2004), which also takes mutations into account. sampling sites from the Norwegian and the Barents Seas A Bayesian approach in the program STRUCTURE were combined into one population and the same was done v.2.2 (Pritchard et al. 2000; see also Falush et al. 2003) was for the sampling sites from the Bothnian Bay. The mean applied to infer the best number of genetic clusters in our mutation rate was set to 5.05 9 10-4 based on mean 123 Conserv Genet mutation rates estimated for corn and wheat (Table 1 in Barents Sea shores, 44.5 % had maximum of two alleles, Marriage et al. 2009) and a stepwise mutation model was 36.5 % had a maximum of three alleles and 19.0 % had a assumed. Based on a number of preliminary runs, we set maximum of four alleles per locus. Corresponding per- priors for effective population sizes as follows: Norwegian centages for the Bothnian Bay populations were 21.4, 60.3 Sea = 104–3 9 107, Bothnian Bay = 100–3 9 104, White and 18.3 % and for the White Sea population 14.0, 76.7 Sea = 100–3 9 104 and ancient population = 104– and 9.3 %, respectively. The number of alleles per locus 3 9 107 and for divergence times: t1 = 10–10,000 and varied from 4 (Pm12) to 25 (Pm34) and per population t2 = 10–20,000. Six different scenarios were chosen to be from 26 (Savilahti) to 54 (Billefjord; Table 2). The mean tested in the final run (Fig. 2), following the preliminary number of alleles was the lowest in the Bothnian Bay runs. We simulated 6,000,000 data sets, which were then (31.25, N = 117) and the highest in the Norwegian/Barents compared to the observed data to choose the scenario that Seas (47.25, N = 137). One identical genotype was best explains the data. We compared the fit of the observed detected in all populations, except from the small sample and simulated summary statistics as recommended in the from Savilahti, Bothnian Bay. This genotype was found in DIYABC manual and used all the available summary 4.6–42.9 % of the individuals, depending on the popula- statistics. Four summary statistics are available for one tion. In addition, two other genotypes were shared between sample; the mean number of alleles, mean heterozygosity, populations from Tauvo and Va¨liteonkarit (Table 3). mean size variance and mean Garza-Williamson’s M and The allele permutation test with SPaGeDi suggested that seven for two samples; the mean number of alleles, mean stepwise mutations at microsatellite loci contributed to heterozygosity and mean size variance, FST, classification genetic differentiation among sampling sites significantly index, shared allele distance and (dl)2 distance. The con- compared with genetic drift and migration (observed

fidence in scenario choice was evaluated by estimating type RST = 0.4692, permuted pRST = 0.218351; P = 0.0007). I and II errors. Finally, the best scenario was used to Population pairwise RST-values (Table 4) show the largest estimate divergence times and effective population sizes. value between the Finnish population Va¨liteonkarit and the Norwegian population Talvik (about 700 km apart,

RST = 0.8097) and the lowest between the two Finnish Results populations Savilahti and Tauvo (about 25 km apart,

RST =-0.0199). The mean RST of comparisons between Two (Pm26 and Pm19) of the tested 11 loci did not amplify the Bothnian Bay and Norwegian/Barents Seas was 0.5695, in P. phryganodes and one (Pm12) did not amplify in whereas within the regions it was only 0.1710 Eahpa´rasˇcohkka. We could not define the ploidy level; the (SD = 0.1628) and 0.0899 (SD = 0.0501) for the Both- number of alleles per individual per locus varied from two nian Bay and the Norwegian/Barents Seas, respectively. to four (Table 1). Among individuals at the Norwegian/ Comparisons involving the Russian population from Onega Bay were high when compared with the Finnish popula- Table 1 Number of Puccinellia phryganodes individuals having a tions (mean = 0.6773, SD = 0.0715) and low when maximum of two, three or four alleles, shown for each study compared with the Norwegian populations population (mean = 0.1017, SD = 0.0815). Signals of isolation by Population Two Three Four Total distance were detected with RST-statistics, as the regression alleles alleles alleles between ln(distance) and RST/(1-RST) was significantly positive (b = 0.1303, P = 0.0354). Norwegian/ Barents Sea Population pairwise FST-values from the De Silva fre- quencies (Table 4) show the smallest divergence between Billefjord 9 14 9 32 the Finnish populations Isomatala and Savilahti, which are Eahpa´rasˇcohkka 21 7 7 35 located about 16 km from each other. The correlation Lakselv 19 12 4 35 between the F -values from De Silva and the Simple Talvik 12 17 6 35 ST frequencies was high (r = 0.852, P \ 0.001), in general, White Sea the values from De Silva frequencies were smaller. The Onega Bay 6 33 4 43 highest F -value was found between a comparison of Bothnian Bay ST Norwegian and Finnish populations (Billefjord and Tauvo). Isomatala 16 15 1 32 In general, the FST values were much smaller than the RST Va¨liteonkarita 11 18 4 33 a values and they did not correlate (r =-0.0566, Savilahti –617P = 0.743). The PCA analysis revealed clear clustering of a Tauvo 140445individuals by origin; individuals from the Bothnian Bay a population was extinct by 2013 cluster together and individuals from the Norwegian/ 123 Conserv Genet

Scenario 1 Scenario 2 Scenario 3 Posterior probabilities: Posterior probabilities: Posterior probabilities: 0.0014 (Direct approach) 0.0019 (Direct approach) 0.9962 (Direct approach) 0.0025 (Logistic approach) 0.0175 (Logistic approach) 0.9800 (Logistic approach)

t2

t1

0 Norwegian White Sea Bothnian Norwegian White Sea Bothnian Norwegian White Sea Bay Sea Bay Sea Bay

Scenario 4 Scenario 5 Scenario 6 Posterior probabilities: Posterior probabilities: Posterior probabilities: 0.0000 (Direct approach) 0.0005 (Direct approach) 0.0000 (Direct approach) 0.0000 (Logistic approach) 0.0000 (Logistic approach) 0.0000 (Logistic approach) t2

t1

0 Norwegian White Sea Bothnian Norwegian White Sea Bothnian Norwegian White Sea Bothnian Sea Bay Sea Bay Sea Bay

Fig. 2 The six scenarios tested with DIYABC with posterior probabilities of each scenario shown

Barents Seas together. Individuals from the White Sea 0.980 through the logistic regression method. This scenario cluster among the Norwegian individuals (results not includes an ancient population that was split in lineages shown). leading to the coasts of the White Sea and to the coasts of Results of the STRUCTURE analysis showed an the Bothnian Bay. The White Sea lineage was further split increase of the ln probability for increasing values of to a lineage leading to the Norwegian Sea coast. Type I K (Fig. 3a). However, the Evanno’s method revealed the error of the scenario 3 (true scenario is rejected) was rel- largest change of the ln probabilities for DK = 3 (Fig. 3b). atively high (0.192 and 0.442), but type II error of this The barplot for K = 3 (Fig. 3c) showed that most of the scenario (false scenario is not rejected) was low (0.047 and Finnish samples are grouped into one cluster and most of 0.080 for direct and logistic regression methods, respec- the White Sea samples into the other. Samples from the tively). Two out of 36 summary statistics simulated Norwegian/Barents Seas are divided mostly between two according to scenario 3 and compared with the observed clusters. The first of these clusters is shared with the White statistics were significantly smaller (Garza Williamson Sea while the other is more specific to the Norwegian/ indexes for the Norwegian/Barents Seas and the White Sea, Barents Seas, though this latter cluster is present also in P \ 0.05) than observed. Estimates (modes) of effective Bothnian Bay, mainly in population Va¨liteonkarit. population sizes were 2.82 9 107 (95 % highest posterior The best scenario for the population history from the densities, 95 % HPD = 1.28 9 106 – 2.96 9 107) for the coalescence analysis was scenario 3 in Fig. 2, with a pos- Norwegian Sea, 1.97 9 104 (95 % HPD = 7.53 9 103 – terior probability of 0.996 through the direct method and 2.85 9 104) for the White Sea, 2.19 9 104 (95 % 123 Conserv Genet

Table 2 Number of alleles found from each Puccinellia phryganodes population and locus Population Pm10 Pm29 Pm61 Pm65 Pm12 Pm25 Pm27 Pm34 Pm39 Total/population

Norwegian/Barents Sea Billefjord 4 5 2 5 4 3 6 19 6 54 Eahpa´rasˇcohkka 6 4 6 7 0 3 6 7 10 49 Talvik 5 5 2 3 4 4 4 10 4 41 Lakselv 5 5 4 8 3 2 5 7 6 45 White Sea Onega Bay 5 7 2 4 4 3 4 9 7 45 Bothnian Bay Isomatala 3 4 2 3 2 4 5 5 4 32 Va¨liteonkarita 46323442331 Savilahtia 33233226226 Tauvoa 44262438336 Total/locus 7 10 6 11 4 5 8 25 13 a population was extinct by 2013

Table 3 Number of identical and private genotypes found from the studied Puccinellia phryganodes populations Population Genotype Total genotypes/ population A B C D E F G H I J K L M N Private

Norwegian/Barents Sea Billefjord 2 2 28 30 Eahpa´rasˇcohkka 4 31 32 Talvik 15 2 18 20 Lakselv 5 2 3 25 28 White Sea Onega Bay 2 41 42 Bothnian Bay Isomatala 9 4 6 2 11 15 Va¨liteonkarita 6 1 1 3 22 26 Savilahtia 77 Tauvoa 3 1 2 5 3 2 29 35 Total genotypes 46 2 2 2 3 4 6 2 2 3 3 5 3 2 212 226 a population was extinct by 2013

HPD = 9.26 9 103 – 2.92 9 104) for the Bothnian Bay Discussion and 4.26 9 106 (95 % HPD = 1.80 9 106 – 2.80 9 107) for the ancient population. Estimates for the divergence The main finding of this study was that the Bothnian Bay times were 1.57 9 104 (95 % HPD = 6.77 9 103 – P. phryganodes populations are genetically distinct from 1.96 9 104) generations ago for the split between the the geographically closest populations in the White and White Sea—Norwegian/Barents Seas and Bothnian Bay Norwegian/Barents Seas. We also estimated that the and 5.90 9 103 (95 % HPD = 1.73 9 103 – 8.52 9 103) Bothnian Bay populations have diverged from the White generations ago for the split between the White and the and Norwegian/Barents Sea populations 7000–20,000 Norwegian/Barents Seas. generations ago.

123 Conserv Genet

Table 4 Pairwise RST- (below the diagonal) and FST- (above the diagonal) values between the studied Puccinellia phryganodes populations Billefjord Eahpar. Lakselv Talvik Onega Bay Va¨liteonk. Isomatala Savilahti Tauvo

Billefjord 0.0444 0.0739 0.0942 0.0325 0.0242 0.0333 0.0567 0.0970 Eahpa´rasˇcohkka 0.0616** 0.0283 0.0840 0.0544 0.0385 0.0390 0.0659 0.0733 Lakselv 0.0940*** 0.0470 0.1075 0.0759 0.0517 0.0656 0.0935 0.0867 Talvik 0.0384** 0.1389*** 0.1594*** 0.0697 0.0657 0.0304 0.0436 0.0726 Onega Bay 0.0417** 0.1542*** 0.1877*** 0.0232 0.0726 0.0290 0.0607 0.0790 Va¨liteonkarit 0.6039*** 0.7271*** 0.6105*** 0.8097*** 0.7818*** 0.0340 0.0570 0.0771 Isomatala 0.4586*** 0.6078*** 0.4765*** 0.6926*** 0.6648*** 0.3288*** 0.0071 0.0438 Savilahti 0.3853*** 0.5282*** 0.4306*** 0.6556*** 0.6289*** 0.3778 0.0618 0.0617 Tauvo 0.4642*** 0.5637*** 0.4695*** 0.6288*** 0.6337*** 0.2237** 0.0539 -0.0199

Significant RST-values are indicated with ** P \ 0.01 and *** P \ 0.001

In general, the genetic diversity was relatively high in

a t our study populations (Tables 2, 3). This was somewhat

(a) a -5500

d

f surprising, as Siira (2011) and Elven (2014) have recently o -6000

y

t

i

l

i suggested that the species reproduces only asexually. Yet,

b

a -6500

b high genetic variation in P. phryganodes in the Arctic

o

r p -7000 Canada has also been reported, indicating that either sexual

n

L

d -7500 reproduction occurs to some degree or multiple

e

t a hybridizations with several species have taken place (Jef-

m -8000

i

t

s

e feries and Gottlieb 1983). Based on our results, we cannot

f -8500 o exclude either of these possibilities.

n

a -9000 e We cannot say anything definite based on the ploidy

M 0 2 4 6 8 10 12 14 levels either, because we merely counted the number of K non-identical alleles for the microsatellite loci. No indi- (b) vidual was found with more than four alleles, but several 500 individuals in all populations had only two or three dif- ferent alleles per locus. This leaves the question of the 400 ploidy level open, the populations can be mixtures of

300 diploid, triploid and tetraploid individuals, mixtures of K triploids and tetraploids or all tetraploids. Polyploidy in 200 angiosperms occurs frequently and most species retain

100 traces of past duplications of their genomes (Cui et al. 2006). It has been suggested that polyploidy is advanta- 0 2 4 6 81012 geous for species in exploiting new niches by, for example, K increasing genetic variation and allowing individuals to (c) evolve novel biochemical pathways (Leitch and Leitch 2008). Thus, polyploidy can help a species like P. phryganodes to establish in highly disturbed habitats. Genetic variation in the Bothnian Bay populations was clearly lower than in the Norwegian/Barents Seas and the y la White Sea in terms of numbers of alleles and genotypes ta nkarit a Ba vo (Tables 2, 3). The estimated effective population sizes for Eahparaš. Lakselv Billefjord Talvik Isoma äliteo Savilahti Tau Oneg V Bothnian Bay (22,000) and White Sea (20,000) populations were about of the same magnitude and much lower than Fig. 3 Results from STRUCTURE analysis of Puccinellia phrygan- odes populations. a Means and variances for estimated Ln probabil- estimated for the Norwegian/Barents Sea population (about ities from 10 runs b DK values and c barplot for K = 3 30 million). The lower diversity of the Bothnian Bay

123 Conserv Genet populations might result from their history; past population In the light of the presented background, it is unlikely bottlenecks, genetic drift, as the populations are small and that halophilic P. phryganodes could have inhabited the isolated, or founder events, perhaps repeated over the shores of the present Baltic Sea prior to the Yoldia Sea centuries. Still, low genetic variation in the Bothnian Bay is stage. However, as proven by the present distribution in the hardly the main reason of the local extinctions, more likely Bothnian Bay, the species is also able to grow in low the populations have suffered from overgrowth of the salinity environments (about 3.5 g/kg in the Bothnian Bay meadows by taller graminoids in some areas and from compared to around 35 g/kg in the Norwegian and 25 g/kg overgrazing and trampling by cattle and geese in others in the White Sea; Swift and Koltermann 1988; Berger and (Niemela¨ 2009). Naumov 2000). This makes it possible that the species has Analyses with the program STRUCTURE found three spread to the Baltic already during the Yoldia Sea stage, groups. The main division was between the Bothnian Bay survived over the Anchylus Lake period and has been populations and others (also supported by the PCA), but an present ever since. Another possibility is that the species additional third cluster included individuals found mainly spread along the emerging coastline of the in the Norwegian/Barents Sea populations Billefjord, following the edges of the melting glacier at the coast of Eahpa´rasˇcohkka and Lakselv and in the Bothnian Bay present Norway and colonized the Baltic Sea from south- population Va¨liteonkarit. These individuals might repre- west. The time of divergence of the Bothnian Bay popu- sent migrants, be representatives of ancient shared poly- lation might actually favor this scenario; divergence of the morphism or erroneously classified due to deviation from present Bothnian population might have evolved ‘en route’ the Hardy–Weinberg equilibrium. The rest of the individ- from the populations that existed before the formation of uals from the Bothnian Bay were grouped into a cluster the Baltic Ice Lake east of the Fennoscandian ice sheet, specific for the Bothnian Bay, showing again that the where habitats for the species have most likely been pre- population is genetically distinct. sent. In this case, the population size and distribution in the The coalescence analyses indicated that the population Baltic might have been larger before, even covering the in the Bothnian Bay has diverged from other close popu- Atlantic shores of Norway. It is possible that as the tem- lations over 16,000 generations ago. The generation time of perature increased and the Fennoscandian ice sheet melted, P. phryganodes is unknown, but given that the minimum the southern populations went extinct, leading to disjunct generation time in family Poaceae is just one to two years distributions and a relict population at the Bothnian Bay. (Table 1 in Gaut et al. 1997), this would directly translate One explanation for the colonization of the Bothnian to over 16,000–32,000 years ago. Keeping in mind that Bay population has been that the species has spread only these estimates were obtained by resampling alleles for recently through jump dispersal by birds or wind (Havas coalescence analysis, which breaks the possible linkage 1961). The time of divergence and the large differentiation between alleles, the divergence time estimates should be of the Bothnian Bay population from the geographically regarded as the maximum times. closest populations, however, render this rather unlikely. During the Weichselian glaciation, the area of the pre- Even though genetic drift due to founder events and pos- sent Baltic Sea was repeatedly covered by the sible population bottlenecks can cause fast differentiation, Fennoscandian ice sheet. The ice sheet has had a complex the divergence time estimates are too large for a recent history, advancing and retreating several times during the colonization from nearby populations. In addition, genetic Weichselian, with lakes of fresh meltwater forming on the variation in the Bothnian Bay population, though being edges during the periods of retreat. The final melting began lower than in the other study populations, is still fairly about 15,000 years ago and formed a freshwater Baltic Ice high. This is contrary to what would be expected after Lake around 14,000 years ago (Vassiljev and Saarse 2013 occurrence of population bottlenecks and founder effects, and the references therein). Several authors have suggested which would have reduced genetic variation. that prior to that period, there had been a connection Regardless the history of the Bothnian Bay population, between the White Sea and the Baltic Sea, but later evi- it is evident that there are genetic differences between this dence have more or less undermined this possibility (e.g. population and the populations from the Norwegian/Bar- Saarnisto et al. 1995). The first marine stage of the Baltic ents Seas and the population from the White Sea. Fur- Sea after the last glacial maximum, the Yoldia Sea, was thermore, also ecological differences are likely, as the formed around 11,600 years ago (Hyttinen et al. 2013). Bothnian Bay population occupies shores of brackish Even after this period, the Baltic Sea went through one water, whereas the White and especially Norwegian Sea freshwater stage, when the Anchylus Lake was formed populations occupy habitats with higher salinity. These about 9500 years ago. The predecessor of the modern facts suggest that the Bothnian Bay population deserves to Baltic Sea, the Litorina Sea, was formed just around be treated as a separate conservation unit. Whether the unit 7500 years ago. of conservation is a management unit (MU) or an 123 Conserv Genet evolutionary significant unit (ESU), is a matter of defini- Crandall KA, Bininda-Emonds ORP, Mace GM, Wayne RK (2000) tion. Most commonly, an ESU is defined as a population Considering evolutionary processes in conservation biology. Trends Ecol Evol 15:290–295 showing high genetic and ecological distinctiveness and is Cui L, Wall PK, Leebens-Mack JH, Lindsay BG, Soltis DE, Doyle JJ, based on an idea that maintenance of different ESUs will Soltis PS, Carlson JE, Arumuganathan K, Barakat A, Albert VA, preserve the evolutionary potential of a species in changing Ma H, dePamphilis CW (2006) Widespread genome duplications environment (Crandall et al. 2000). The MUs are often throughout the history of flowering plants. Genome Res 16:738–749 considered as smaller units within an ESU, that are De Silva HN, Hall AJ, Rikkerink E, McNeilage MA, Faser LG (2005) genetically described by being reciprocally monophyletic Estimation of allele frequencies in polyploids under certain for mtDNA and showing significant divergence of allele patterns of inheritance. Heredity 95:327–334 frequencies at nuclear loci or ecologically distinct, as they Earl DA, vonHoldt BM (2012) STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and imple- are demographically independent from other units (Moritz menting the Evanno method. Conserv Genet Resour 4:359–361 1994; see Funk et al. 2012 for a recent review). The Elmendorf SC, Henry GH, Hollister RD, Bjo¨rk RG, Bjorkman AD, Bothnian Bay population is definitely demographically Callaghan TV, Collier LS, Cooper EJ, Cornelissen JH, Day TA, independent; it occupies a unique habitat on the shores of a Fosaa AM, Gould WA, Gre´tarsdo´ttir J, Harte J, Hermanutz L, Hik DS, Hofgaard A, Jarrad F, Jo´nsdo´ttir IS, Keuper F, brackish water body and is clearly genetically differenti- Klanderud K, Klein JA, Koh S, Kudo G, Lang SI, Loewen V, ated. All these features indicate that this population should May JL, Mercado J, Michelsen A, Molau U, Myers-Smith IH, be treated as an evolutionary significant unit. Oberbauer SF, Pieper S, Post E, Rixen C, Robinson CH, Schmidt NM, Shaver GR, Stenstro¨m A, Tolvanen A, Totland O, Troxler Acknowledgments We would like to thank all the people who T, Wahren CH, Webber PJ, Welker JM, Wookey PA (2012) travelled to fetch the samples and were important for the initiation of Global assessment of experimental climate warming on tundra the project especially Kari Koivula, Jaakko Lumme, Juha Markkola vegetation: heterogeneity over space and time. Ecol Lett and Pirjo Rautiainen. We owe much to Jouko Siira, who started the 15:164–175 studies on the ecology of P. phryganodes in the Bothnian Bay already Elven R (ed) (2014) Annotated checklist of the panarctic flora (PAF) in the 1960s. We also wish to acknowledge staff at Oulanka Research vascular plants. 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