Geographic variation in Festuca rubra L. ploidy levels and systemic fungal endophyte frequencies

RESEARCH ARTICLE Geographic Variation in Festuca rubra L. Ploidy Levels and Systemic Fungal Endophyte Frequencies Serdar Dirihan 1 , Marjo Helander 1,2 , Henry Va ¨re 3 , Pedro E. Gundel 4 , Lucas A. Garibaldi 5 , J. Gonzalo N. Irisarri 4 , Irma Saloniemi 1 , Kari Saikkonen 2 1Department of Biology, University of Turku, Turku, Finland, 2Natural Resources Institute Finland (Luke), Turku, Finland, 3Botanical Museum, Finnish Museum of Natural History, University of Helsinki, Helsinki, Finland, 4IFEVA, Facultad de Agronomı ´a, Universidad de Buenos Aires, CONICET, Buenos Aires, Argentina, 5Grupo de Investigacio ´n en Agroecologı´a (AGRECO), Sede Andina, Universidad Nacional de Rı ´o Negro (UNRN) and Consejo Nacional de Investigaciones Cientı ´ficas y Te ´cnicas (CONICET), San Carlos de Bariloche, Rı ´o Negro, Argentina *[email protected] Abstract Polyploidy and symbiotic Epichloe ¨fungal endophytes are common and heritable characteristics that can facilitate environmental range expansion in grasses. Here we examined geographic patterns of polyploidy and the frequency of fungal endophyte colonized plants in 29 Festuca rubra L. populations from eight geographic sites across latitudes from Spain to northernmost Finland and Greenland. Ploidy seemed to be positively and negatively correlated with latitude and productivity, respectively. However, the correlations were nonlinear; 84% of the plants were hexaploids (2n = 6x = 42), and the positive correlation between ploidy level and latitude is the result of only four populations skewing the data. In the southernmost end of the gradient 86% of the plants were tetraploids (2n = 4x = 28), whereas in the northernmost end of the gradient one population had only octoploid plants (2n = 8x = 56). Endophytes were detected in 22 out of the 29 populations. Endophyte frequencies varied among geographic sites, and populations and habitats within geographic sites irrespective of ploidy, latitude or productivity. The highest overall endophyte frequencies were found in the southernmost end of the gradient, Spain, where 69% of plants harbored endophytes. In northern Finland, endophytes were detected in 30% of grasses but endophyte frequencies varied among populations from 0% to 75%, being higher in meadows compared to riverbanks. The endophytes were detected in 36%, 30% and 27% of the plants in Faroe Islands, Iceland and Switzerland, respectively. Practically all examined plants collected from southern Finland and Greenland were endophyte-free, whereas in other geographic sites endophyte frequencies were highly variable among populations. Common to all populations with high endophyte frequencies is heavy vertebrate grazing. We propose that the detected endophyte frequencies and ploidy levels mirror past distribution history of F.rubra after the last glaciation period, and local adaptations to past or prevailing selection forces such as vertebrate grazing. PLOS ONE | DOI:10.1371/journal.pone.0166264 November 15, 2016 1 / 16 a11111 23(1 $&&(66 Citation: Dirihan S, Helander M, Va¨re H, Gundel PE, Garibaldi LA, Irisarri JGN, et al. (2016) Geographic Variation in Festuca rubra L. Ploidy Levels and Systemic Fungal Endophyte Frequencies. PLoS ONE 11(11): e0166264. doi:10.1371/journal.pone.0166264 Editor: Tzen-Yuh Chiang, National Cheng Kung University, TAIWAN Received: August 5, 2016 Accepted: October 25, 2016 Published: November 15, 2016 Copyright: 2016 Dirihan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Academy of Finland grants 137909, 281354 and 292732 Fund receiver author: KS, as well as INTERACT (Grant Agreement No. 262693) under the European Community’s Seventh Framework Programme. Competing Interests: The authors have declared that no competing interests exist. Introduction Biogeographic generalizations on the factors responsible for patterns of species’ ranges are largely based on comparisons of closely related species [1]. For example, polyploidy appears to be positively associated with latitude, altitude and recent deglaciations [2–5], and biotic interactions have been connected to adaptive radiation of plants [6–8]. Although different ploidy levels have commonly been documented within species as well, extensive studies on geographic species-specificploidy distributions and importance of biotic interactions are mostly lacking [9], and sporadic findings are often conflicting. Grasses are a perfect model for studies on geographic ploidy distributions and importance of biotic interactions because they cover higher area of land than any other group of plants across all the continents except Antarctica [10]. We selected red fescue (Festuca rubra L. sensu lato) as a model species for our study. First, it is a wild perennial Eurasian grass widely distributed and phenotypically variable in the Northern hemisphere. Plants falling into morphologically distinguishable categories are often inconsistently classified as both species and subspecies showing local adaptations [11,12]. Available literature suggests extensive hybridization between (sub)species, potentially resulting in observed natural polyploids (2n = 14, 21, 28, 42, 49, 56, 64 and 70) [11,12]. Interfertile plants exhibit striking morphological variation and part of the ecotypic diversity is suggested to be related to the polyploidization [13]. Second, red fescue is well known for its variable and occasionally high frequencies of systemic fungal endophytes [14–17]–plant associated fungi that are suggested to act as defensive plant mutualists and thereby expand distribution range of the host grass [7,18–21]. Both polyploidy and systemic, vertically in germline transmitted Epichloë endophytes are common grass characteristics that can be adaptive to various environmental conditions [2,4,6, 7,22–24]. Polyploidy, the multiplication of the complete set of chromosomes, can bestow adaptive potential and evolutionary flexibility on plants and thereby improve their competitive and invasive capacity into northern latitudes [4,23,25]. Possessing more than two sets of chromosomes can cause heterosis, shield polyploids from deleterious effects of mutations for example by reducing the incidence of homozygous recessives, and buffer against inbreeding depression and genetic drift [2,5,23]. Because polyploids are usually unable to interbreed with their diploid conspecifics, polyploidy is recognized as one of the major mechanisms of sympatric speciation [5] and in some species the number of chromosomes appears to be positively correlated with latitude or altitude [2–4,26]. Recently, the role of polyploidization as a modulator of adaptive symbiosis between plants and microbes has been recognized [27]. For example, polyploidization can affect biotic interactions through changes in the chemical profile of the plant [8]. However, the empirical evidence is variable and partly contradictory [28–30], and the question how ploidy-driven adaptations to environmental conditions and microbial interactions codetermine plant distribution is unknown. Similar to polyploidy, Epichloë endophytes examined in this study can also drive the geographic distribution of host grasses [31]. Grass endophytes of the genus Epichloë are common symbionts of cultivated and wild Pooideae grass species. In the symbiosis endophytic fungus grows systemically and asymptomatically throughout the aboveground plant parts, and depending on the species it may be transmitted either vertically via host seeds and/or horizontally by sexual spores [6,7]. In many species, the fungus is mostly asexual and is transmitted vertically from mother plant to its offspring. Vertical transmission is supposed to promote fidelity between partners and lead to mutualistic symbiosis because the fitness of the heritable fungus and the host grass is tightly linked [32–38]. Thus, the symbioses are commonly thought to be mutualistic. Numerous studies have demonstrated that Epichloë species can increase grass resilience to drought, flooding,pathogens and herbivores, and thus promote their Polyploidy and Endophytic Symbiosis across Latitude PLOS ONE | DOI:10.1371/journal.pone.0166264 November 15, 2016 2 / 16 competitive ability in grass communities [14,18,21,39–41]. The defensive mutualism against herbivores due to mycotoxins appears to provide the most prevalent ground for mutualistic endophyte-grass interactions [6,7,20,21,42,43]. However, an increasing number of empirical studies has revealed that the symbiosis can range from antagonistic to mutualistic interactions, and mutualism is less frequent in wild grasses compared to agronomic grasses in nutrient-rich environments [20,21,38,44,45]. This study aims to reveal potential linkages between geographic patterns of polyploidy and plant-fungal endophyte symbiosis. We explore both ploidy levels and endophyte frequencies in red fescues over a wide range of environments and latitudes across Europe. The benefits from Epichloë species are found to be positively correlated with high nutrient availability and productivity [20,46], and polyploidy is believed to increase towards less productive higher latitudes and altitudes [4,23]. Thus, we studied primary productivity (the normalized difference vegetation index, NDVI) of our study sites [47]. We also hypothesize that ploidy levels and the frequencies of endophyte symbiosis would be correlated, because polyploidization can modulate plant-microbe interactions and both polyploidization and systemic grass endophytes can promote host fitness. Because benefits of endophytes are particularly obvious in high nutrient environments, we may assume that the positive correlation should be stronger in environments with the highest primary production. This assumption is supported by a recent broad-scale study [46] suggesting that primary production is positively associated with the occurrence of systemic grass endophyte symbiosis. Materials and Methods More than one thousand red fescue (Festuca rubra L.) plants in total were collected from 29 populations (10–70 plants/population) from eight geographic sites across Europe (Spain, Switzerland, southern Finland, Faroe Islands, Iceland, two areas in northern Finland and Greenland; Table 1). Plants were not collected from national parks or other protected areas requiring permissions. In Nordic countries “everyman’s right” gives everyone rights to access, enjoy for recreation and collect unprotected plants, berries and mushrooms in a way which does not damage the environment or disturb others regardless who owns or occupies the land. Plants from Spain and Switzerland were collected from public land. Geographical coordinates, altitude from the sea level and features of the site were recorded for each population (Table 1). To ensure the proper species identification of the plants and that collected plants represent individual genets, only flowering individuals growing at least 10 meters apart from each other were collected. Plants were dug up with a soil core and placed into plastic bags for transportation. All the collected grasses were planted in 250 ml pots with added peat and sand mix around the original soil core and kept in a greenhouse in Turku University Botanical Garden (60°26’N, 22°10’E) in ambient daylight and 20–24°C (summer time) and 4–8°C (winter time) temperatures. The collection sites represent a broad biogeographical region varying in terms of latitudes, altitudes, climatic zones (continental, oceanic), biological selection pressures such as grazing (Table 1) and seasonal changes in abiotic environmental conditions. For example, sites in Spain represent grassland and xerophytic forest, both in Mediterranean climate characterized by summer droughts and rainy winters. Sites located on higher latitudes are characterized by stronger seasonal changes in day length and associated light quality limiting primary production [48], short growing seasons in summer and long and cold winters. However, oceanic sites (Iceland and Faroe Islands) strongly affected by the Gulf-stream are characterized by high precipitation year-round, cool summers and relatively mild winters compared to the other sites on the same latitudes. Polyploidy and Endophytic Symbiosis across Latitude PLOS ONE | DOI:10.1371/journal.pone.0166264 November 15, 2016 3 / 16 Table 1. The collection sites of Festuca rubra plants and their attributes. N = number of collected plants. Mean NDVI = mean normalized difference vegetation index estimated from year 2000 to 2012. Inf. % = percentage of endophyte infected plants (endophyte frequency) in population. 4x %, 6x % and 8x % = percentage of tetraploids, hexaploids and octoploids, respectively, in population. Population code Geographic site Population N Geographic coordinates Altitude (m a.s.l.) Mean NDVI Inf. % 4x % 6x % 8x % Features of the site Grazing SP1 Spain Ca ´ceres 31 N 40˚12’1’’ W 5˚45’11’’ 768 0,59 81 100 0 0 Xerophytic forest High (cattle) SP2 Spain Salamanca 1 27 N 40˚56’20’’ W 6˚7’6’’ 863 0,57 67 78 22 0 Meadow High (cattle) SP3 Spain Salamanca 2 37 N 40˚58’24’’ W 5˚57’33’’ 812 0,51 59 81 3 16 Meadow High (cattle) SW1 Switzerland Andermatt 26 N 46˚32’19’’ E 8˚40’31’’ 1500 0,42 23 0 96 4 Meadow with natural grassland vegetation, slope facing east High (cattle) SW2 Switzerland Biez 25 N 46˚37’42’’ E 8˚35’26’’ 1600 0,41 36 0 100 0 Meadow with natural grassland vegetation, slope facing north-west High (cattle) SW3 Switzerland Piasca 23 N 46˚53’56’’ E 8˚42’9’’ 1850 0,19 22 0 100 0 Meadow with natural grassland vegetation, sawed every second year, slope facing south High (cattle) FI1 Southern Finland Hanko 1 42 N 59˚50’23’’ E 23˚13’40’’ 0 0,55 0 2 98 0 Meadow along the coast Low FI2 Southern Finland Hanko 2 44 N 59˚50’27’’ E 23˚13’15’’ 0 0,53 0 0 93 7 Meadow along the coast Low FI3 Southern Finland Hanko 3 40 N 59˚53’0’’ E 23˚5’52’’ 0 0,43 0 5 95 0 Meadow along the coast Low FO1 Faroe Sandoy 39 N 61˚50’11’’ W 6˚51’21’’ 69 0,42 21 3 97 0 Meadow High (sheep) FO2 Faroe Nolsoy 41 N 62˚1’15’’ W 6˚41’8’’ 55 0,33 5 0 98 2 Meadow High (sheep) FO3 Faroe Mykines 37 N 62˚5’51’’ W 7˚40’56’’ 125 0,16 68 0 100 0 Meadow High (sheep) FO4 Faroe Vagar 24 N 62˚6’59’’ W 7˚26’43’’ 246 0,30 25 0 83 17 Meadow High (sheep) FO5 Faroe Eysturoy 39 N 62˚17’24’’ W 7˚2’10’’ 316 0,34 54 5 87 8 Meadow High (sheep) FO6 Faroe Vidoy 32 N 62˚22’3’’ W 6˚32’32’’ 148 0,31 44 9 91 0 Meadow High (sheep) IC1 Iceland Iceland 1 44 N 64˚47’34’’ W 21˚32’0’’ 390 0,28 32 2 98 0 Meadow High (sheep) IC2 Iceland Iceland 2 34 N 64˚48’52’’ W 23˚23’14’’ 10 0,34 32 0 97 3 Meadow High (sheep) IC3 Iceland Iceland 3 42 N 66˚1’21’’ W 20˚23’39’’ 38 0,30 26 5 95 2 Meadow High (sheep) GR1 Greenland Greenland 1 70 N 69˚14’59’’ W 53˚31’15’’ 0 0,16 3 0 100 0 Meadow along the coast Low GR2 Greenland Greenland 2 10 N 69˚15’27’’ W 53˚32’40’’ 0 0,19 0 0 100 0 Meadow along the coast Low FI4 Northern Finland 1 Halti 1 22 N 69˚15’0’’ E 21˚24’36’’ 860 0,20 0 0 100 0 Meadows along rivulets above tree-line with patchy grass and sedge dominated vegetation Moderate (reindeer) FI5 Northern Finland 1 Halti 2 42 N 69˚15’0’’ E 21˚19’12’’ 900 0,11 0 0 0 100 Moderate (reindeer) FI6 Northern Finland 1 Halti 3 32 N 69˚16’12’’ E 21˚19’12’’ 920 0,28 0 0 100 0 Moderate (reindeer) FI7 Northern Finland 2 Kevo 1 34 N 69˚38’6’’ E 27˚5’1’’ 91 0,31 56 3 97 0 Meadow High (reindeer) (Continued) Polyploidy and Endophytic Symbiosis across Latitude PLOS ONE | DOI:10.1371/journal.pone.0166264 November 15, 2016 4 / 16 Ploidy determination Ploidy levels of the plants were determined by flow cytometry(FCM) [49]. We used known chromosome counts of F.rubra plants as references for the FCM results [49]. Plants from different populations (one to three plants for each population) were randomly chosen to microscopically determine cytotype. These reference plants were grown hydroponically to produce fresh root tips. Aseptically cut root tips were pretreated with 1% 1-alphabromonaphtalene and stained in 2% acetic orcein solution [50]. The root tips were then squashed in a drop of 45% acetic acid on the slides and analyzed under microscope. Preparations were mounted with enthalan after the metaphases were photographed. We sampled a ca. 0.5 cm 2 leaf piece from each plant. The sample was chopped in a glass Petri dish with an aseptic razor blade in 1 ml ice-cold nuclei isolation buffer (LBO1 in one-step procedure). The suspension was mixed by sucking and discharging with the pipette several times and then filtered into an Eppendorf tube using a 50 μm nylon mesh. DNA fluorochrome stock solution with 50 μl ml -1 propidium iodide (PI) and 50 μl ml -1 ribonuclease (RNase) was added and incubated on ice one hour before FCM analysis [51]. The 96 well plate-based FCM procedure [49] was carried out using LSR II (Bechton Dickinson San Jose, USA) flow cytometer at the Turku Centre for Biotechnology, Finland. Pisum sativum L. ‘Ctirad’ (2C DNA value = 9.09 pg) plants obtained from the Institute of Experimental Botany (Laboratory of Molecular Cytogenetics and Cytometry, Olomouc, Czech Republic), were used as an external reference to determine DNA quantity in pictograms (pg). In addition, known tetraploid and hexaploid F.rubra plants were used for each FCM run. The FCM channel was determined as G 1 peak for each sample and DNA ploidy levels were estimated as follows: Ploidy level of sample ¼G1peak flourescence of sample ðmedianÞ  G1peak flourescence of reference ðmedianÞ Ploidy level of reference The flow cytometric data were measured with Flowing Software version 2.4.1 (Perttu Terho, Turku Centre for Biotechnology, Finland; www.flowingsoftware.com). Endophyte detection Fungal endophyte status (endophyte infected E+ / endophyte free E-) of each of the study plants was detected by plating three leaf sheaths ɖsurfacesterilized by incubation for 1 min in 90% ethanol, 4 min in 4% sodium hypochlorite and 30 s in 90% ethanol ɖcut into 5 pieces and placed on potato dextrose agar (5% PDA). The Petri dishes were monitored up to three weeks Table 1. (Continued) Population code Geographic site Population N Geographic coordinates Altitude (m a.s.l.) Mean NDVI Inf. % 4x % 6x % 8x % Features of the site Grazing FI8 Northern Finland 2 Kevo 2 40 N 69˚43’56’’ E 27˚12’0’’ 85 0,31 75 3 85 13 Meadow High (reindeer) FI9 Northern Finland 2 Kevo 3 34 N 69˚45’32’’ E 26˚59’19’’ 107 0,31 50 0 94 6 Meadow High (reindeer) FI10 Northern Finland 2 Kevo 4 42 N 69˚54’36’’ E 27˚1’48’’ 73 0,27 45 2 98 0 Riverbank High (reindeer) FI11 Northern Finland 2 Kevo 5 31 N 69˚56’11’’ E 26˚27’45’’ 106 0,28 23 0 94 6 Riverbank High (reindeer) FI12 Northern Finland 2 Kevo 6 35 N 69˚56’41’’ E 26˚43’22’’ 85 0,29 20 0 100 0 Riverbank High (reindeer) doi:10.1371/journal.pone.0166264.t001 Polyploidy and Endophytic Symbiosis across Latitude PLOS ONE | DOI:10.1371/journal.pone.0166264 November 15, 2016 5 / 16 for their systemic endophyte fungal growth. When typical white Epichloë fungal endophyte mycelia grew out from several leaf pieces, the plant was considered as endophyte infected [16]. The infection status of individual plants was verified later by staining and microscopic examination of several seeds of each plant. Systemic and vertically via host grass transmitted endophytic fungi are host species-specific.Similarly to other studies, we have identified the fungus associated with red fescue as E.festucae by comparing the rDNA sequences with Blast searches of GeneBank in our previous studies [16]. NDVI To compare vegetation productivity of the study sites quantitatively, we calculated the normalized difference vegetation index (NDVI) for each population separately using NASA MODIS satellite images (Fig 1,Table 1). The NDVI accurately estimates functional attributes of the ecosystem such as aboveground net primary production (ANPP), its inter-annual variation and vegetation phenology [47,52,53]. NDVI is closely and positively correlated with leaf area and the fraction of photosynthetically active radiation absorbed by green vegetation [54–56]. NDVI data uniquely allows site characterization because it represents the specific consequences of environmental and human effects on vegetation functioning. We obtained NDVI values from the MODIS project through the MODIS global subsets tool (http://daac.ornl.gov/cgibin/MODIS/GLBVZ1Glb/modissubsetorderglobalcol5.pl). We used the MOD 13 Vegetation Indices product, gridded, 16-day composite images with 250-m pixel size. We extracted two essential attributes of aboveground primary production dynamics by calculating the NDVI average annual integral and its inter-annual coefficient of variation from February 2000 to December 2012 (Fig 1). These traits are known to capture important features of ecosystem functioning [57]. Statistical analysis We used linear models (linear regression) to analyse the effects of mean NDVI and latitude on endophyte infection frequencies and mean ploidy of populations. Normality of residuals was checked graphically and using Kolmogorov-Smirnov test results for the models. We used a logistic regression model to estimate ploidy level, altitude (Alt), latitude (Lat) and their pair-wise interactions as fixed effects on endophyte status (two levels: E+ and E-) using binomial error distribution (logit link). We also included population as a random effect (random intercept model) to account for the fact that individual plants were spatially nested within populations (29 populations in total). We used AIC (Akaike Information Criterion) to select best-fitting models for all combinations of fixed-effect variables. AIC values were obtained based on maximum-likelihoodestimates of regression coefficients,because models differed in their fixed structure but shared the same random structure (random intercepts), whereas parameter estimates for final models presented in figures were obtained using the restricted maximum likelihood method [58]. Models were estimated using lmer function of the lme4 package [59] in the R software [60], and AIC and Akaike’s weight for each model of all possible models based on different combinations of the predictor variables were obtained with the dredge function of the MuMln package [61] in the R software. Results Overall 84%, 9% and 7% of the plants were hexaploids (2n = 6x = 42), tetraploids (2n = 4x = 28) and octoploids (2n = 8x = 56), respectively. More than one ploidy level was detected in 19 out of 29 populations (Table 1). Ploidy level seems to be partly linked with productivity and latitude; ploidy positively and negatively associated with latitude (p<0.0001) and productivity Polyploidy and Endophytic Symbiosis across Latitude PLOS ONE | DOI:10.1371/journal.pone.0166264 November 15, 2016 6 / 16 (p<0.0001), respectively (Fig 2). It is noteworthy, however, that majority of the plants were hexaploids (2n = 6x = 42), and the positive correlation between ploidy level and latitude was not linear, and the correlation is due to the influence of four deviant populations. In the southernmost geographic site, Spain, 86% of the plants were tetraploids (2n = 4x = 28), and in northernmost Finland all of the plants in one population at high altitude (in Halti, 900m) were Fig 1. Monthly mean normalized difference vegetation index (mean NDVI) values for the Festuca rubra collection sites (for population codes see Table 1) from February 2000 to December 2012. doi:10.1371/journal.pone.0166264.g001 Polyploidy and Endophytic Symbiosis across Latitude PLOS ONE | DOI:10.1371/journal.pone.0166264 November 15, 2016 7 / 16 octoploids (2n = 8x = 56) (Table 1,Fig 2). Outside these extremes of the latitudinal range, hexaploid plants were dominant. In Greenland, all the plants were hexaploids, and in Switzerland 98% of the plants were hexaploids and only 2% octoploids (Table 1). In the other populations tetraploids and octoploids were sporadically distributed (Table 1). Endophyte infections were detected in 22 out of 29 red fescue populations (Fig 3). The seven totally endophyte-free populations were the three populations from Hanko in southern Finland, the three populations from Halti in northern Finland and one population from Greenland. Furthermore, only one infected grass was found in the other population in Greenland (Table 1,Fig 3). Overall infection frequency of the study area was 29% but frequencies varied irrespective of ploidy (p = 0.14) (Table 2) among geographic sites, populations within geographic sites and among habitats (Fig 3,Table 1). The highest overall infection frequencies were found in Spain (69%) where occurrence of infections was high in all populations (Fig 3). At Kevo in northern Finland, on average 45% of grasses were infected but infection frequencies were much higher in meadows (60%) compared to nearby riverbanks (29%). The occurrence of infections in Faroe Islands, Iceland and Switzerland were 36%, 30% and 27%, respectively. In these geographic sites variation in endophyte occurrence among populations was considerable only in Faroe Islands varying from 5% to 68% (Table 1,Fig 3). Latitude appears not to be linked to the detected variation in endophyte occurrence (p = 0.65) (Table 2,Fig 4). Instead, infection frequencies appear to be associated with altitude (p = 0.04) but interactively with latitude (p = 0.03) (Table 2). The sampling was not, however, designed to test the importance of altitude and thus, these results remain inconclusive. In populations collected from low latitudes, in Spain and Switzerland, the endophyte frequencies were lowest in Switzerland where all collected populations were from high altitude (Table 1). In contrast, elsewhere infection frequencies varied irrespective of altitude. For example, populations collected from Greenland and Hanko, both situated at sea level, and Halti situated at 900 m above sea level were endophyte-free (Table 1) suggesting the altitude cannot account for patterns of endophyte occurrence. Endophyte frequencies of the grass populations were not associated with productivity (p = 0.16) (Fig 5). Normalized difference vegetation index (NDVI) values varied among geographic sites rather than along latitude (Table 1,Fig 6). The highest geographic site specificmean-NDVI values Fig 2. The effect of latitude (a) and mean normalized difference vegetation index (mean NDVI) (b) on mean ploidy of Festuca rubra populations. Fitted regression line in (a) is for illustrative purpose since statistical tests are suggestive due to problems of normality in the data. doi:10.1371/journal.pone.0166264.g002 Polyploidy and Endophytic Symbiosis across Latitude PLOS ONE | DOI:10.1371/journal.pone.0166264 November 15, 2016 8 / 16 0.51, 0.56 and 0.31 were estimated for southern Finland, Spain and Iceland-Faroe Islands, respectively (Table 1). Corresponding endophyte infection frequencies were 0% (southern Finland), 69% (Spain), 30% (Iceland) and 36% (Faroe Islands) demonstrating that overall productivity of the geographic site is unlikely to be linked with endophyte infection frequencies in red fescue populations. Monthly mean NDVI estimates, however, clearly demonstrate that primary production is seasonally limited in all the other study sites except in Spain (Fig 1). Discussion Our results do not support the hypotheses that polyploidization and the occurrence of systemic fungal endophytes in red fescue show latitudinal gradients, or that they are correlated with Fig 3. Collection sites and endophyte infection frequencies of Festuca rubra populations. Infection frequency circles are shown with the population codes (Table 1). doi:10.1371/journal.pone.0166264.g003 Table 2. Effects of ploidy, latitude, altitude and interaction between latitude and altitude (Lat x Alt) on endophyte status of Festuca rubra plants. Estimate Std. Error z value Pr (!|z|) Intercept -4.78 5.08 0.94 0.35 Ploidy 0.03 0.02 1.48 0.14 Latitude 0.04 0.08 0.44 0.65 Altitude 0.01 0.00 2.03 0.04* Lat x Alt -0.00 0.00 -2.14 0.03* *, p0.05 **, p0.01 ***, p0.001 doi:10.1371/journal.pone.0166264.t002 Polyploidy and Endophytic Symbiosis across Latitude PLOS ONE | DOI:10.1371/journal.pone.0166264 November 15, 2016 9 / 16 51. Dolez ˇel J, Greilhuber J, Suda J. 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