Parmelina carporrhizans in the Mediterranean region. Using eight microsatellite and mating-type markers we showed that fungal populations are highly diverse but lack spatial structure. This is likely due to high connectivity and long distance dispersal of fungal spores. Consistent with low levels of linkage disequilibrium and lack of clonality, we detected both mating-type idiomorphs in all populations. Furthermore we showed that the Macaronesian Islands are the result of colonization from the Mediterranean Basin. The unidirectional gene flow, though, seemed not to be sufficient to counterbalance the effects of drift, resulting in comparatively allelic poor peripheral populations. Our study is the first to shed light on the high connectivity and lack of population structure in natural populations of a strictly sexual lichen fungus. Our data further support the view of the Macaronesian Islands as the end of the colonization road for this symbiotic ascomycete.The Mediterranean region, comprising the Mediterranean Basin and the Macaronesian Islands, represents a center of diversification for many organisms. The genetic structure and connectivity of mainland and island microbial populations has been poorly explored, in particular in the case of symbiotic fungi. Here we investigated genetic diversity and spatial structure of the obligate outcrossing lichen-forming fungus  Cryptic biodiversity is an essential component of biodiversity that will be considerably affected by global climate change in the next decades5Lichen-forming fungi are a successful group of nutritionally specialized fungi that form obligate symbioses with photosynthetic partners. Molecular studies of lichen-forming fungi have repeatedly indicated that numerous distinct lineages, i.e. cryptic species, can be hidden under a single taxon. This shows that species diversity of lichen-forming fungi might be vastly underestimated, mainly because of the plasticity of morphological and chemical characters and the frequency of homoplasy and convergence of characters78Lichen-forming fungi are interesting subjects for population genetics studies. One of the reasons for this is the vast range of reproductive strategies, i.e., from purely asexual to exclusively sexual, that could differentially shape their population structures111314Buellia frigida, a crustose lichen of continental Antarctica, an SSR-based study revealed high intra-population diversity, local population differentiation and limited dispersal, probably resulting from harsh habitat conditions and scarce spatial and temporal habitat availabilityLobaria pulmonaria, SSR-based studies have shown highly clonal and structured populations, resulting from low level of recombination and limited dispersal capacity of vegetative propagules20Parmotrema tinctorumParmelina tiliaceaLobaria pulmonaria is also the only lichen species for which population-level data on the distribution of mating-type idiomorphs is available so farFor investigating intra-population variability and inferring the role of reproductive strategies in shaping population dynamics, highly variable molecular markers such as microsatellites (SSRs) are one of the most appropriate tools, especially for highly clonal organisms such as lichens17Parmelina carporrhizans24. Parmelina carporrhizans is a foliose, epiphytic macrolichen with the center of distribution in the Mediterranean region. The Mediterranean region, comprising the Mediterranean Basin and the Macaronesian Islands, is an important centre of diversification for many plant and animal species2526In this study we analyzed intra-species genetic diversity, population structure, and mating-type distribution in natural populations of the obligate outcrossing lichen-forming fungal species P. carporrhizans is a result of migration from the Macaronesian Islands to the continent or from the continent to the Macaronesian Islands.To test these hypotheses, we used highly variable, species-specific microsatellite and mating-type specific markers. First, we analyzed the species population genetic diversity and evaluated how it is hierarchically and spatially partitioned. We assessed the extent to which alleles are geographically restricted or shared across regions. Second, we calculated the relative abundance of the two mating-type idiomorphs in the populations. Self-incompatible species require the presence of both mating types in the population for sexual reproduction. Third, we estimated migration rates among mainland and island groups. Combining genetic diversity and gene flow estimates we inferred whether the distribution of P. carporrhizans.We successfully amplified the MAT-genes fragments in 93% of the samples. In each sample we found a single band corresponding to one idiomorph . These rThe overall difference in frequency of the mating-type idiomorphs was not significantly different, with MAT1-1 found in 55% of the samples, and MAT1-2 in 45% of the samples. Only two populations (Morocco and Herbés) had a significantly skewed higher frequency of MAT1-1 idiomorphs .All eight microsatellite loci were highly polymorphic. The analysis of 220 samples with eight microsatellite markers resulted in a total of 220 microsatellite alleles, ranging from 16 alleles (Pcar1) to 46 alleles (Pcar7) . No idenOverall we found a high unbiased haploid diversity (mean uh = 0.846), ranging from 0.744 (Tenerife) to 0.898 (Sicily). Populations had high population allelic richness (mean = 5.244), ranging from 3.973 (Cádiz) to 6.047 (Gredos). Private allelic richness was moderately low in all populations, ranging from 0.447 (Tenerife) to 1.072 (Gredos). At the regional level, the Macaronesian Islands had lower allelic (11.486) and private (3.095) allelic richness than the mainland populations  .P = 0.014), Cádiz , Famalicão  and Herbés  (P. carporrhizans populations occurs within populations (92.1%), with lesser amounts among populations within regions (2.3%) and among regions (5.6%) (ST = 0.079) and regions (FCT = 0.056) are not significant .We detected low levels of linkage disequilibrium (LD) across populations (mean = 0.069). Low but significant LD values were found in four populations, namely Gran Canaria 1  . The AMOs (5.6%) . PermutaST values between populations were low, ranging from 0 to 0.023 (ST = 0.016) were statistically significant.As expected, pairwise Fto 0.023  and onlyBased on the discriminant analysis of principal components (DAPC), three genetic clusters were considered optimal to describe the data . The scatter plot of local densities of distances  that were significantly geographically restricted (P < 0.001). The overall observed mean distance between geographically restricted alleles was also significantly smaller than the expected mean , but higher than the overall observed mean geographic distance between all shared alleles thus indicating over-dispersion  of the Macaronesian Islands group (1.16) was considerably smaller than that of the mainland group (16.6). The migration analysis detected high, unidirectional gene flow from the mainland group to Macaronesia .P. carporrhizans in the Mediterranean region. Sexually reproducing fungal species are expected to show higher genetic diversity and weaker population structure than vegetatively reproducing species11P. carporrhizans exhibit high levels of genetic diversity and a complete absence of clonality. This is strikingly different from the trend reported for predominantly asexual lichen species, e.g. L. pulmonaria, whose populations harbor, almost always, a high number of clonal multilocus genotypes  is different from P. carporrhizans, which might also have contributed to the differences in population structures of the two species. As compared to vegetatively reproducing species however, as expected, P. carporrhizans displayed lower genetic structure. For example, a recent study based on sequencing data, showed high population differentiation in the closely-related vegetatively reproducing (isidiate) species, Parmelina tiliaceaP. carporrhizans, the Macaronesian populations of P. tiliacea were the most genetically diverse.Another species, for which the genetic variability and population structure have been analyzed using SSR markers, is P. carporrhizans indicates high frequency of sexual recombination resulting in novel genotypic combinations in each generation. We obtained evidence for this prediction by analyzing the distribution of mating-type idiomorphs in P. carporrhizans populations.The high genetic diversity of P. carporrhizans is a heterothallic, i.e. self-incompatible, species, as previously found using RAPD fingerprinting on cultured spore isolateset al.P. carporrhizans population seems to be sufficient to ensure panmictic populations and thus low levels of linkage disequilibrium.Our results based on mating-type ratios corroborate that P. carporrhizans suggested that core populations of the Mediterranean Basin and edge populations in the Macaronesian Islands are still highly connected via gene flow. To corroborate this hypothesis, we estimated migration rates between these two population groups. Our results indicate that populations across the southern range of the species are still highly connected. However, we found that gene flow is unidirectional, i.e., from core towards edge populations. This may well explain the smaller population size and lower allelic richness of the island populations. Moreover, on the Canary Islands, P. carporrhizans does not grow at low altitudes, where it is replaced mainly by Parmotrema species, but grows in the Erica–Myrica heath  communities on well-lit borders of the laurel forest (‘monteverde’), on sweet chestnut, elms, poplars, pines, etc. from 800 to 1500 m a.s.l. The composition of the forests (at least in Tenerife) has undergone major changes during the Late Holocene, in particular, suitable habitats such as arboreal species of the vegetation alongside laurel forest have considerably declined as a consequence of human activity and climatic variationGenetic diversity and population structure analyses of Based on these observations, we propose that migratory fluxes from core populations can only partially compensate for the detrimental effects of drift, likely caused by founder effects, by maintaining relatively high genetic diversity in peripheral populations. Several studies on different organisms  towards the Macaronesian Islands.In conclusion, we showed that the strictly sexual, heterothallic lichen-forming fungus Parmelina carporrhizans represents an ideal system to test these predictions for strictly outcrossing lichen-forming fungi. Additional studies in this direction can further shed light on the effect of dispersal mode on the co-phylogeographic dynamics of the lichen symbiosis.The apparent absence of dispersal barriers found in our study implies that the establishment of newly dispersed fungal spores is not limited by the availability of compatible photobionts17474849P. carporrhizans from 11 localities to extensively cover the south-western distribution range of the species, from the Macaronesian Islands and Northern Africa, to Southwestern Europe where the species is most abundant  of terminal lobes of freshly collected thalli or recent herbarium specimens were used for DNA isolation with the DNeasy Plant Minikit .Small pieces (0.5 cmWe genotyped each specimen at eight fungus-specific microsatellite markers (Pcar1–8). Details of the markers, PCR conditions and multiplexing are given in ref. P. carporrhizans-specific MAT-genes primers we obtained partial MAT1-1 and MAT1-2 sequences from the scaffolds of the P. carporrhizans genomeXanthoria elegans, Xanthoria polycarpaLobaria pulmonariaFor designing 2 tests.To identify the mating-type idiomorph of each sample, we performed multiplexed PCR reactions in a total volume of 25 μL with 0.65 μL of each MAT1-1 primer (10 μM), 0.6 μL of each MAT1-2 primer (10 μM), 0.4 μL dNTP’s  and 0.5 μL DNA polymerase  and 2.5 μL of 10–50 ng DNA. PCR reactions were performed with an initial denaturalization at 94 °C for 4 min, followed by 10 cycles of denaturalization step at 95 °C for 10 sec, annealing at 55 to 50 °C for 20 sec and extension at 72 °C for 30 sec, followed by 30 additional cycles with annealing temperature of 50 °C for 20 sec and an additional extension at 72 °C for 5 min. We tested if both idiomorphs were evenly distributed in the entire area and in each individual sampling locality with χWithin-population genetic diversity was calculated by estimating allelic richness and private allelic richness using a rarefaction approach, implemented in ADZEpoppr of the R package poppr v.2.1.159To analyze the level of linkage disequilibrium we estimated rBarD (unbiased index of associationST) were calculated using Arlequin v3.5.To quantify genetic differentiation among and within populations we carried out a global analysis of molecular variance (AMOVA) as a weighted average over loci using Arlequin v3.5adegenet 2.0.15363To detect population structure we used the multivariate, model-free method DAPC  analysis with the package et al.To further analyze the overall spatial pattern of alleles and test for areas with geographically restricted alleles  we calculated the observed distribution of geographic distances between allelic occurrences in the overall data set and per marker and compared it with the panmictic distribution. To test for significant deviations of the observed mean distances from the expected under panmixia, we performed 1,000 nonparametric permutations of the allele-by-location data sets.6 generations, recording every 100 Kth step after a burn-in period of 5 × 104 generations. Convergence was monitored with Tracer (http://beast.bio.ed.ac.uk). All effective sample sizes of the MCMC chain were larger than 104.To test for the presence and infer the directionality of gene flow between the Macaronesian Islands and the mainland populations, we used the coalescent-based method MIGRATE-N 3.2.6How to cite this article: Alors, D. et al. Panmixia and dispersal from the Mediterranean Basin to Macaronesian Islands of a macrolichen species. Sci. Rep.7, 40879; doi: 10.1038/srep40879 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


To study whether thyroid function was associated with risk of hip or knee replacement due to primary osteoarthritis.In a prospective cohort study, data from the second and third survey of the Nord-Trøndelag Health Study were linked to the Norwegian Arthroplasty Register in order to identify total hip or knee replacement as a result of primary osteoarthritis.Among 37 891 participants without previously known thyroid disease we recorded 978 total hip replacements (THRs) and 538 total knee replacements (TKRs) during a median follow-up time of 15.7 years. The analyses were adjusted for sex, age, BMI (body mass index), smoking, physical activity and diabetes. We did not find any association between TSH (thyroid stimulating hormone) and THR or TKR due to osteoarthritis. Neither were changes in TSH over time, or overt hypo- or hyperthyroidism, associated with incidence of THR or TKR.No association was found between thyroid function and hip or knee joint replacement due to osteoarthritis. Osteoarthritis in the hip and knee is a major health problem and leads to significant morbidity . SeveralNo prospective population studies have investigated the association between thyroid function and osteoarthritis. An older cross-sectional study did not find any association between radiological knee osteoarthritis and thyroid status measured by thyroid stimulating hormone (TSH) . In thisIn the Nord-Trøndelag Health Study (HUNT) all inhabitants of Nord-Trøndelag county ≥ 20 years of age were invited to participate in three surveys: HUNT1 (1984–1986), HUNT2 (1995–1997) and HUNT3 (2006–2008) . This stn = 3895), missing information on BMI (n = 364), missing information on smoking (n = 962), previous THR or TKR (n = 644), missing date of operation (n = 99), emigration during baseline measurements period (n = 1) or self-reported osteoarthritis at baseline (n = 4545). Thus, a total of 37 891 people (22 714 women and 15 177 men) were eligible for follow-up in this study. Each participant contributed person-years from baseline (either between August 1995 and June 1997 or between October 2006 and June 2008) until a THR or TKR due to osteoarthritis, THR or TKR due to other causes, migration, death or end of follow-up , whichever occurred first.In HUNT2, TSH was measured in 35 269 persons; in all women over 40 years old, in a random 50% sample of men over 40 years old and in a random 5% sample of participants aged 20–40 years. In HUNT3, TSH was measured in all 49 179 participants. We included 35 269 participants from HUNT2 and 13 132 new participants with TSH measurements from HUNT3. In persons that participated in both HUNT2 and HUNT3, baseline measurements from HUNT2 were used in the main analyses. Among these 48 401 individuals, 10 510 were excluded from analysis  and total triiodothyronine (T3) in HUNT2 were measured at the Hormone Laboratory, Aker University Hospital, Oslo, using DELFIA hTSH Ultra , DELFIA fT4 , and AutoDELFIA T3 , all from Wallac Oy, Turku, Finland. In HUNT3, serum TSH and fT4 were measured at Levanger Hospital, Levanger, Norway, using Architect cSystems ci8200 , and Architect cSystems ci8200 , respectively, both from Abbott, Clinical Chemistry, USA. The measurement methods of TSH in HUNT2 and HUNT3 have previously been compared, with similar results , and agrThe covariates were chosen based on previous literature, and had to be associated with both thyroid function and osteoarthritis. We mapped possible casual pathways between all variables using a directed acyclic graph model (DAG)  to visuaOsteoarthritis at baseline was based on an affirmative answer to the question: “Has a doctor ever said that you have/have had osteoarthritis?” from HUNT2 participants, and “Have you had or do you have osteoarthritis?” from HUNT3 participants.In this study, primary total hip or knee replacement was considered to be a surrogate measure of severe osteoarthritis; and we used primary total hip or knee replacement due to primary osteoarthritis as outcome. The 11-digit identification numbers assigned to every Norwegian citizen enabled linkage to the Norwegian Arthroplasty Register (NAR). For each arthroplasty performed, the orthopaedic surgeon submits a standardized form containing information about the patient, the diagnosis that led to the arthroplasty, the procedure and the type of implant used . NAR wasThe participants were placed in five categories according to their TSH level: One category indicating hyperthyroid function (<0.50 mU/l); three categories within the clinical reference range ; and one category indicating hypothyroid function (≥3.5 mU/l) . Hazard n = 19 397). TSH levels at HUNT3 were then subtracted from the TSH levels in HUNT2 to estimate the change in TSH during the intervening decade. Information on the other baseline covariates was taken from HUNT3. Among these participants a total of 7740 were excluded due to self-reported thyroid disease (n = 2744), missing information on BMI (n = 108), missing information on smoking (n = 621), previous THR or TKR (n = 928), missing date of operation (n = 29) or self-reported osteoarthritis at baseline (n = 3310). Thus, a total of 11 657 people were eligible for this sub-analysis. This sub-population was also used in an analysis that investigated whether the results changed when those who had started on thyroid medication after baseline in HUNT2 were excluded.A sub-analysis included only persons that participated in both HUNT2 and HUNT3  hypothyroidism (defined as TSH >4.0 mU/L combined with fT4 < 8.0 pmol/L), and the other with overt hyperthyroidism . This classification by overt hypo- or hyperthyroidism was made possible by the fT4 measurements taken in people whose TSH levels were <0.20 mU/l or >4.0 mU/l in HUNT2, and in people whose levels were <0.10 mU/l or >3.0 mU/l in HUNT3. Total T3 was only available in HUNT2 and only measured if TSH levels were <0.20mU/L.Two additional analyses were performed on the baseline population: First, we investigated the association between TSH level  and self-reported osteoarthritis at baseline by using a logistic regression model, adjusting for sex, age, BMI, smoking, physical activity and diabetes. Second, we compared the incidence rate of THR or TKR in participants with and without self-reported thyroid disease at baseline using Fisher’s Exact test. After excluding participants with missing information on BMI and smoking, previous THR or TKR, missing date of operation or self-reported osteoarthritis at baseline, 2955 participants reported thyroid disease.p < 0.05. The analyses were performed using Stata 14.0/SE .Proportional hazards assumptions were evaluated by Schoenfeld residuals tests. They showed proportional hazards on all covariates, except for age. Thus we did an additional stratified analysis on age, but it did not show different results (data not shown). Age was therefore kept as a continuous variable in all our analyses. All statistical analyses were two-sided with a significance level of Of the 37 891 participants, 908 (2.4%) had low TSH (<0.50 mU/l) indicating hyperthyroid function, and 2307 (6.1%) had high TSH (≥3.6 mU/l) indicating hypothyroid function . At baseline, the mean age was 50.7 years (SD 15.8), and the mean ages at THR and TKR were 69.5 years (SD 8.9) and 69.4 years (SD 8.4), respectively.TSH level did not influence the risk of THR or TKR in the unadjusted analysis or the analysis adjusted for gender, age, BMI and smoking Table . NeitherIn a separate analysis studying the change in TSH (delta-TSH) during the time between HUNT2 and HUNT3, 11 657 participants were included. Of these, 200 received THR and 102 received TKR during a mean follow-up time of 6.1 years. No association was seen between changes in TSH and risk of THR or TKR  and  respectively. Also, no association was found between overt hypothyroid function and TKR . There were no cases of TKR amongst the overtly hyperthyroid, so no analysis could be performed in this subgroup.In additional analyses on the baseline population, there was no association between TSH levels and self-reported osteoarthritis at baseline (data not shown). Those with self-reported thyroid disease at baseline had an incidence rate of 0.0027 THR per person-year and 0.0015 TKR per person-year. Our main study population (excluding those with self-reported thyroid disease) had an incidence rate of 0.0021 THR per person-year and 0.0012 TKR per person-year. This gave and incidence rate ratio of 1.28 (95% CI 1.04–1.56) for THR, and 1.27 (95% CI 0.95–1.95) for TKR, and indicated a slightly higher incidence rate for THR in those with self-reported thyroid disease, but no significant difference in the incidence rate for TKR, compared to participants without self-reported thyroid disease.In this large prospective study we did not find any association between thyroid function and the risk of THR or TKR due to osteoarthritis. Neither were changes in TSH over time, or overt hypo- or hyperthyroidism, associated with incidence of THR or TKR.Few previous population studies have investigated the association of thyroid function with risk of osteoarthritis. In 1996, a cross-sectional study of 577 men and 798 women found no evidence of a significant association between current thyroid status and either chondrocalcinosis or osteoarthritis . HoweverPrevious or current thyroid disease at baseline was an exclusion criterion in our study. Nonetheless, all participants with TSH levels suggesting hypothyroid or hyperthyroid function may have received medical treatment for thyroid disease during the follow-up period, as participants with biochemical indication of pathological thyroid function were recommended to contact their general practitioners . This coOur findings must be interpreted in relation to recent genetic studies on intracellular T3 availability in joint cartilage. There has been an increased interest in the effect of deiodinase polymorphisms on osteoarthritis . IodothyOur study included over 37 000 persons without known thyroid disease at baseline, and in most cases thyroid function was measured many years prior to joint replacement. To the best of our knowledge, this is the first prospective population study addressing the association between thyroid function and joint replacement due to primary osteoarthritis. The prospective design and longitudinal data on TSH measurements are strengths of this study. By excluding participants with self-reported osteoarthritis at baseline it was possible to differentiate between risk of osteoarthritis development and progression.Our study used joint replacement due to primary osteoarthritis as a surrogate measure of severe osteoarthritis. Validation of the osteoarthrosis diagnosis from the Norwegian Arthroplasty Register has not been done in an unselected population . HoweverPrevious injuries increase the risk of knee osteoarthritis , 35, butThe interrelationship between BMI and thyroid function is complex and BMI could be treated as either a confounder or a mediator in our model . The reaThe participation rate in the HUNT-surveys was fairly high compared to most other surveys, but there is always a potential risk of selection bias that cannot be adjusted for in the statistical analysis . Blood sThis study focused on the relationship between thyroid function and osteoarthritis. However, there may also be an association between autoimmune thyroid disease and osteoarthritis . As thyrThe exclusion of participants who reported hypo- or hyperthyroidism in their answers regarding use of treatment or medication might have caused misclassification since radioiodine is used in the treatment of cancer and T4 in the medical treatment of goitre. However, these treatments are infrequent, and it is unlikely that they substantially altered our results.In the analysis comparing incidence rate of THR in people with and without self-reported thyroid disease we found a small, but significant, increased risk of THR in people reporting thyroid disease. And since we excluded participants with self-reported thyroid disease, this might have led to an underestimation of the effect on THR. We therefore did an additional analysis including those with self-reported thyroid disease: This showed no association between TSH levels and THR in a Cox-regression model (data not shown), and thus confirmed the findings from the primary analysis.In this prospective study of 37 891 participants without previously known thyroid disease, we did not find that thyroid function was associated with risk of hip or knee joint replacement due to osteoarthritis. Neither did we find any association between TSH development over time and risk of hip or knee joint replacement due to osteoarthritis.