Aedes aegypti African-related mitochondrial lineages distributed in Colombian’s cities with different eco-epidemiologic characteristics with regard to dengue virus (DENV). It has been proposed these lineages might indicate independent invasion sources. A previous phylogeographic study revealed two  Assessing to Colombian population structure and to support evidence of its probable source origin. We analysed a total of 267 individuals from cities of Bello, Riohacha and Villavicencio, which 241 were related to the West and East African mitochondrial lineages . Eight polymorphic microsatellite loci were analysed aiming population structure.Ae. aegypti is placed in Colombia. Likewise, genetic evidence indicates no significant differences among individuals related to WAL and EAL is placed. Results indicate substantial gene flow among distant and low-connected cities composing a panmictic population with incipient local differentiation of Ae. aegypti populations of Colombia, and lack concordance between mitochondrial and nuclear genealogies suggest that Colombian Ae. aegypti shared a common demographic history. Under this scenario, we suggest current Ae. aegypti population structure reflects a single origin instead of contemporary migration, which founding populations have a single source from a mitochondrial polymorphic African ancient. Minimal genetic differentiation in low-connected  Aedes aegypti, which also transmits other viruses such as yellow fever virus, chikungunya and Zika. Similar to other vector-borne diseases, arboviruses infection must be reduced mostly by controlling vector populations.Ae. aegypti are relevant to perform vector control and entomologic surveillance against these infections.Dengue is the arthropod-borne viral disease with the fastest propagation worldwide, having a dramatic increase around 30 fold its global incidence over the past 50 years, estimated in 390 million cases by year.Aedes aegypti population structure has been well studied around the world, and its genetic heterogeneity and historical migration routes have been unveiled at multiple several geographic scales including global scale,,,,,,Ae. aegypti microgeographic population structure in the Americas has disclosed diverse population dynamics  is placed in eclectic ecogeographical regions such as boat traffic driving migration in Peruvian Amazon,,,,,,In Colombia, a first study based on random amplified polymorphic DNA-polymerase chain reaction products analysis (RAPD) reported local genetic population substructure in city of Cali, which was suggested involved in differences of vector competence and insecticide resistance ratios.Ae. aegypti Colombian populations reported the presence of the two mitochondrial lineages related to East and West African ancient populations, circulating in distinct frequencies across cities of Bello (BE), Riohacha (RI) and Villavicencio (VI), which having different eco-epidemiologic characteristics . Cities of BE, RI and VI are roughly equidistant each other (~900 km by road), and they are separated by orographic barriers such as Central and East Colombia cordilleras, and not often huge human displacement is placed among them.Ae. aegypti mitochondrial lineages from different Africa sources to the New World and Latin American countries.,,,,Ae. aegypti populations demography, using nuclear genetic information such as polymorphic microsatellite loci and genome-wide single nucleotide polymorphisms (SNPs) approach, unveiled lack genetic concordance between differentiation patterns observed at nuclear genetic level and East, and West African mitochondrial related populations.,,,,,On the other hand, a more comprehensive work of Ae. aegypti in this study we analysed genetic differentiation of Colombian populations, as well as addressed for whether mitochondrial West Africa lineage (WAL) and East Africa lineage (EAL) represent structured populations. Our results indicate substantial gene flow between geographically distant cities composing a panmictic population with substantial recent gene-flow of Ae. aegypti in Colombia, and support lack concordance between mitochondrial and nuclear genetic information. Under this scenario we propose that Colombian Ae. aegypti compose a unique population with incipient local genetic differentiation originated from a single source from a mitochondrial polymorphic African ancient.Due no comprehensive evidence for population structure of Colombian Sample collection and sites of the study - Ae. aegypti adult mosquitoes were collected in cities of BE, RI and VI during 2012 and 2013 using entomological networks as described elsewhere.DNA extraction and microsatellite genotyping - Genomic DNA was extracted from each adult mosquito following an insect DNA extraction protocol.Supplementary data (Table I)]. PCR reactions for multiplex M1, M2 and M3 were conducted in a final volume of 25 µL using 3 µL of DNA template (~10 ng/µL), 1X PCR buffer , 1.5 mM MgCl2, 0.2 mM dNTP, 0.2 µM of each primer, and 1 U of Taq DNA polymerase . For M4 multiplex, PCR reaction was conducted in a final volume of 20 µL using 1.5 µL of DNA template (~10 ng/µL), 1X PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTP, 0.025, and 0.5 µM of forward and reverse primers respectively, and 0.75 U of Taq DNA polymerase . The fragments were amplified following thermal cycling conditions previously reported.Genetic diversity of Ae. aegypti populations - We used GenoDive v.3.0a), effective number of alleles (Ne), observed (Ho), and expected (He) heterozygosity, and inbreeding coefficient (Gis). Statistical significance was assessed with 1,000 permutations as implemented in GenoDive v.3.0.We used GenoDive v.3.0ficient Gis. StatiGenetic structure of Ae. aegypti populations - We test population genetic subdivision using the FST analogue GST ,FST calculation was restricted to visible allele sizes  in FreeNA software.t test using 1,000 iterations. A Mantel test was also performed in GenAlEx v.6.5 on geographic (at neighborhood) and genetic distance (pairwise phiPT) using 1,000 permutations.a priori assignation to a geographical origin was evaluated using the Bayesian assignment approach as implemented in STRUCTURE v.2.3.3.,Genetic structure of the complete data set without an Relationship inference and demographic analysis of Ae. aegypti populations - We estimate genetic relationship between individuals of Colombian populations as well as the relationship grade within Bello individuals belonging mitochondrial lineages, using the kinship (ks) coefficient,ks values were categorised according to Iacchei et al.ks < 0.0475); quarter-siblings (0.0475 < ks < 0.09375); half-siblings (0.09375 < ks < 0.1875); full-siblings (0.1875 < ks < 0.375); and nearly identical (ks > 0.375). We test normality of data by Anderson-Darling test, and statistical significance (p < 0.05) of ks values among geographical demes  as well as between lineages using Kruskal-Wallis test in dplyr package in R.Supplementary data ]: (i) full model bi-directional gene flow, (ii) one-directional gene flow out of BE to RI and VI, and bi-directional gene flow between them, (iii) one-directional gene flow from RI to VI and BE, and bi-directional gene flow between them (iv) one-directional gene flow from VI to BE and RI, and bi-directional gene flow between them (v) the null model of panmixia . We used standard microsatellite parameters, with uniform prior distribution set for both Theta  and M  similar for all populations, search strategy was performed in three independent runs and parameters were: 10,000 recorded steps [a], 10 sample increment [b], and 1 concurrent chains (replicates) [c] that summarises 1,000,000 of visited parameters [a*b*c] with a burn-in of 1,000 runs using a static heating scheme of four chains with default temperature parameters. We selected the suitability of parameters used by checking the convergence of posterior probabilities distribution over all loci and effective sample size (ESS) > 200. We selected the best model test according to the log Bayes factor (LBF) based on the accurate marginal likelihoods of the Bezier approximation score generated for the five models.We used a Bayesian coalescence-based algorithm in order to address the directionality of gene flow among cities according to migration rates using Migrate-n v.4.4.4.Microsatellite description of Ae. aegypti of Colombia - All eight loci showed less than 15% of missing data and any out 267 individuals showed above 50% of missing data, so all individuals were included in further analyses ]. No genotyping errors such as allele dropouts or stuttering were detected for any locus, but presence for possible null alleles having frequencies ranking between 2% to 18% were estimated in some loci across cities [Supplementary data (Table II)].a) per locus of 14.5, a mean effective number of alleles per locus (Ne) of 3.7, mean observed heterozygosity (Ho) of 0.53, and expected heterozygosity (He) of 0.67, and overall global inbreeding index (Gis) of 0.21 . Pairwise comparisons among cities indicated lower genetic differentiation between BE and VI , followed for a moderate between BE and RI , and RI and VI . The unbiased FST per locus based on ENA correction including null alleles was not significant different from the FST estimated , indicating possible null alleles present in sample do not affect the result observed [Supplementary data (Table III)]. A Mantel test on the whole dataset showed significant but weak correlation  between genetic (phiPT) and geographic distance (y = 11.54x + 236.85).Supplementary data ]. However, after assignment test according to a proportion of ancestry ≥ 80%, the highest number of individuals were grouped into main clusters ]. Otherwise, individuals did not cluster based on whether they are related to the WAL or EAL, even assuming 50% of genetic mixture per individual [Supplementary data (Table IV)].Genetic structure based on Bayesian clustering indicated that the optimal clustering number is K = 4 according to the highest ΔK value obtained . Median value for the entire dataset was ks = -0.01529, indicating most of individuals are unrelated (ks < 0.09375), as expected in a wide roughly panmictic population. Overall kinship coefficient for cities was not significant  in Kinship coefficient between individuals related to mitochondrial lineages were observed [Supplementary data ].g-cities B. No sigMoreover, according to the lowest LBF estimated from marginal likelihood of Bezier score , the best probability model (> 99.9%) test was for constant migration rate model (v), followed by similar values observed in those one-directional gene-flow based models (model (ii), marginal likelihood. = -107228.81; (iii), marginal likelihood = -108560.43, and (iv), marginal likelihood = -140668.89), and finally for the three cities for-bidirectional model (i) . According this result any number of migrants per generation can be accurate estimated from populations, which indicates strong evidence for possible past or recent gene-flow among the three cities.Ae. aegypti populations from three endemic cities for relevant arboviruses such as dengue, Zika and chikungunya. Our results indicate Ae. aegypti Colombian population composes a panmictic population with substantial recent gene-flow among distant cities with minimal evidence of isolation-by-distance process. Furthermore, result derived from Bayesian clustering analysis where four moderated mixed genetic cluster were found as well as low values of GST index between populations, and relatively low heterozygosity observed throughout in all loci analysed would indicate high inbreeding or possible Wahlund effect is placed within populations. Likewise, high within-cities Kinship values, and null model chosen as the best scenario, indicates local demographic dynamics could driving incipient genetic differentiation processes as observed in some individuals from the three cities.Here we disclose genetic population structure of Colombian Ae. aegypti dispersal between distant localities is passive migration associated to human movement, results raised here suggest at least two possible not mutually exclusive hypotheses can be drawn: (i) a sustained-continuous migration between intermediate populations exists without isolation by distance evidence (i.e. patchy metapopulation structure), (ii) the population structure is retained from an ancient population invasion occurred in the recent past, it means a large founding population that rapidly spread throughout Colombia. Thus, based on our results, we conclude that microsatellite data present here are probably mirroring the ancient event of single origin instead of contemporary migration among populations. However, although this is a plausible hypothesis, further studies involving broader populations and using more resolutive markers still need to be tested.The three cities analysed BE, RI and VI are distant, separated by northern Andean cordilleras, and low human displacement is placed among them. Because, the main source for ,Ae. aegypti populations show moderated population differences at spatiotemporal scales, and it was suggested as a consequence of local pressures.Microgeographic structure of Colombian populations has been slightly traced so far, but no clear picture of population demographic has been supported. Several studies at local level using dominant genetic markers such as RAPDs,,,,,A previous research reported heterogenous distribution of mtDNA haplotypes in those cities, which have distinct dengue incidences and vector control strategies, for instance in BE city not often chemical control is applied but community-based and biological control programs are major used strategies, unlike RI and VI where insecticide-based control is often performed.,,,Ae. aegypti using genome-wide and highly genetic resolutive markers such as polymorphic microsatellite loci and SNPs, have indicated a single source in the native range to the New World invasive range around the 16th-century, as consequence of slave trade between Africa and Americas.,,,Ae. aegypti founding population is coherent with a single invasion source of a large mitochondrial polymorphic African related population that rapidly spread throughout Colombia. This hypothesis does not exclude scenarios for multiple introductions nor colonisation routes followed by high contemporary gene flow in Colombian populations.West and East related African mitochondrial lineages have widely been reported throughout most of South American countries such as Peru,Ae. aegypti Colombian populations. Recent advances in next-generation sequencing technology show promising results in resolving population structure. Rašić et al.Ae. aegypti populations that originated from different locations compared to microsatellite markers, and analyses for genetic relatedness of individuals from same populations was more accurately estimated when RAD-based genetic data was employed. Additionally, Lee et al.Despite results agreeing with previous results suggesting lacking geographic structure of Colombian populations, it is worth notice since these studies were relied on relatively small genetic markers number , therefore we suggest further studies using more resolutive genetic markers must be conducted to assessing inter-population dynamics at a finer scale of


Tropical mountains harbor exceptional concentrations of Earth’s biodiversity. In topographically complex landscapes, montane species typically inhabit multiple mountainous regions, but are absent in intervening lowland environments. Here we report a comparative analysis of genome-wide DNA polymorphism data for population pairs from eighteen Indo-Pacific bird species from the Moluccan islands of Buru and Seram and from across the island of New Guinea. We test how barrier strength and relative elevational distribution predict population differentiation, rates of historical gene flow, and changes in effective population sizes through time. We find population differentiation to be consistently and positively correlated with barrier strength and a species’ altitudinal floor. Additionally, we find that Pleistocene climate oscillations have had a dramatic influence on the demographics of all species but were most pronounced in regions of smaller geographic area. Surprisingly, even the most divergent taxon pairs at the highest elevations experience gene flow across barriers, implying that dispersal between montane regions is important for the formation of montane assemblages. Islands and mountaintops are often considered evolutionary dead ends. Using whole genomic data of 18 bird species and demographic models, the authors show that populations become isolated at high elevations, but disjunct montane populations maintain gene flow and thus the capacity for further colonisation. Physical barriers that differ in their capacity to maintain the geographic separation of populations are likely to directly impact gene flow, population differentiation and ultimately, speciation5. Alternatively, local adaptation to particular environments across gradients can generate diversity in the absence of physical barriers12. Because the efficacy of a given barrier or gradient is mediated by the distribution of the species that encounters it, evaluating its overall influence on evolutionary processes requires comparative data from geographically isolated taxon pairs that occur across gradients in different environmental settings, and that possess a variety of ecological characters. Such data can provide estimates of when populations become geographically isolated from one another, the extent of gene flow between incipient species (diverging populations) and temporal changes in population demography. Here we integrate these estimates to ask: (i) Does barrier strength influence population differentiation? (ii) Does barrier strength affect population differentiation of species with different elevational distributions? (iii) To what extent do population sizes fluctuate over time, (iv) Is there evidence of population segregation along elevational gradients? (v) Are montane populations recruited from the lowlands or do they arise through colonization from mountaintop to mountaintop? (vi) How do levels and directionality of gene flow  vary between populations of species across elevational gradients?A fundamental goal of biology is to understand the mechanisms that underlie the spatio-temporal formation of species and communities. Divergence among geographically isolated populations is the common mechanism by which speciation is initiated in vertebrate taxa15. This would lead to a signature of populations of species that inhabit higher elevations being more differentiated compared to populations of species inhabiting lower elevation. Second, populations may colonize one montane region from another montane region15. For example, in New Guinea, montane taxa might repeatedly colonize uni-directionally from the dominant central mountain range to the many smaller outlying ranges. If this is the case, we expect to find evidence of gene flow even between montane populations. Third, high elevation populations may form by in situ parapatric speciation, i.e. through the break-up of one population into two along an elevational gradient for example through adaptation to specific elevational environments. This would be evidenced by high levels of differentiation between individuals of the same population that occur at high elevations and lower elevations.Answering these questions allow us to address three competing explanations of the evolutionary origin of montane populations. First, populations that currently live only at high elevations may have originated in the lowlands but shifted upslope over time16 and area (≈ 4000 km2 above 500 m and ≈1000 km2 above 1000 m). The two focal mountain ranges in New Guinea, on the other hand, are part of continental Australo-Papua. They are separated by a “soft” lowland barrier; and are asymmetric in terms of age and area. While estimates of the age of the New Guinean terranes vary significantly, the large Central Range  started to rise some 10 Mya and reached its present elevation about 5 Mya, while the Huon mountains  rose above sea-level some 4 Mya and reached their present elevation about 1.5 Mya18. The mountains of New Guinea reach > 4000 m a.s.l, while the mountains on Buru and Seram are lower, reaching 2500 m a.s.l. and 3000 m a.s.l., respectively. However, due to the Massenerhebung effect19, temperature drops more quickly with increasing altitude in the isolated Moluccan islands than in New Guinea, and both systems encompass environmental extremes from sweltering lowland swamps to chilly mountain cloud forests. Finally, while the geological history has affected the Indo-Pacific biota significantly, dramatic climatic oscillations during the Pleistocene are also expected to impact diversity build-up in the region21.For this study, we use explicit demographic models to analyse genomic data of discrete populations with different elevational distributions. We assembled 15 de novo genomes and generated whole-genome resequencing data for eighteen forest bird species with populations distributed along multiple elevational gradients, geographically separated by distinct barriers  Genetic differentiation is generally greater across the ocean barrier than the lowland barrier. (ii) Differentiation is greater between populations of montane species than between populations of lowland species in New Guinea but not in the Moluccas. (iii) There were marked fluctuations in population sizes during the Pleistocene. (iv) There is negligible differentiation along elevational gradients, hence little evidence for sympatric speciation. (v) Individuals of montane populations continuously disperse to other mountains. (vi) The direction of colonization is predominantly from a larger mountain range to a smaller mountain range.Melanocharis versteri to 1053.5 Mbp in Sericornis nouhuysi. The number of scaffolds ranged from 14,086 scaffolds in Melipotes ater to 87,957 scaffolds in Ficedula hyperythra and N50 ranged between ca. 40 Kbp to and 25 Mbp. Benchmarking Universal Single-Copy Orthologs (BUSCO) analyses of genome completeness ranged from a high proportion of complete BUSCOs in Melipotes ater, 86.8% to only 66.7% complete BUSCOs in Rhipidura albolimbata. For most species, the proportions of complete BUSCOs were 75–80%. Overall, the proportion of missing BUSCOs was low, ranging from 6.6% in Melipotes ater to 15.2% in Rhipidura albolimbata  showed a pairwise kinship coefficient of 0.144, indicative of being half-siblings. The two individuals were collected at the same locality on the same date. Similarly, two Ifrita kowaldi individuals (D116 and D117) showed a pairwise kinship coefficient of 0.135, also suggestive of being half-siblings. In this case, the individuals were collected on the same sampling locality on two consecutive days. To not bias downstream demographic analyses, one of the P. schlegelii (A118) and one of the I. kowaldi (D117) individuals were excluded from all subsequent analyses. For all other species, no closely related individuals were identified.Sampling of closely related individuals can dramatically bias estimates of population structure and demographics. Two ST (the fixation index), which quantifies the degree of genetic differentiation between populations based on the variation in allele frequencies, ranging between 0  and 1 (complete separation of populations)  in order to visualize population structure  species pairs in our study  calculation of FST) was high between islands ; Ceyx lepidus (FST = 0.16), Thapsinillas affinis (FST = 0.15), Ficedula buruensis (FST = 0.13) and Pachycephala macrorhyncha (FST = 0.09). In contrast, differentiation in Ficedula hyperythra was consistent with population-level differentiation (FST = 0.04). In all cases, individuals from Buru and Seram were clearly differentiated in the PCA and STRUCTURE plots  to determine any potential parapatric differentiation along the gradients. In all possible comparisons, FST values did not differ significantly from 0. Moreover, PCA plots showed that samples did not cluster according to elevation , Paramythia montium (FST = 0.09) and Ifrita kowaldi (FST = 0.07)  Fig.  with pop07) Fig. . By contParamythia montium (FST = 0.10 both between Mount Wilhelm and Mount Scratchley and between Huon and Mount Scratchley). In the case of Peneothello sigillata, the Mount Scratchley population appeared genetically well-differentiated from both the populations of Mount Wilhelm (FST = 0.06) and Huon (FST = 0.07). In both cases, STRUCTURE suggested a scenario of K = 3, with individual assignments matching the three geographically circumscribed populations. For Pachycephala schlegelii, genetic differentiation was relatively high between Huon and Mount Scratchley (FST = 0.05), but low between Mount Wilhelm and Mount Scratchley (FST = 0.01). Accordingly, STRUCTURE suggested a scenario with K = 2 groups. For the remaining two species Sericornis nouhuysi showed some differentiation (FST = 0.03) between Mount Wilhelm and Huon and Aethomyias papuensis showed minor differentiation , genetic differentiation was low (FST = 0.01). In Huon, all samples were collected at the same elevation, except for Ifrita kowaldi, for which genetic differentiation of FST = 0.03 was found between individuals collected at 2300 m and 2950 m  between populations inhabiting higher elevations. We found no relationship between genetic differentiation (FST) and the altitudinal floor (the lowest elevation at which a species/population occurs) for the five Moluccan species, but for all New Guinean taxa with the exception of Melipotes fumigatus/ater we found a significant positive correlation . This relationship remained significant even when excluding the two lowland taxa .If lineages colonize mountains from the lowlands, followed by range contraction and differentiation in the highlands, we would expect a signature of larger genetic differentiation Ifrita kowaldi with ca. 200,000 individuals, and the highest being Toxorhamphus novaeguineae and Peneothello sigillata with ca. 1,000,000 individuals.The inferred demographic histories of the eighteen bird species encompassed the time period from ca. 10 Mya to 20 Kya  and Pliocene (2.6–5.3 Mya). During this period, most species showed a rather flat curve. The most dramatic demographic changes for the majority of species occurred in the Pleistocene (beginning around 2.6 Mya) but the timing of population increases and decreases vary between species. All PSMC results were consistent when bootstrapping the data but clearly become more dubious toward the present , populations from Mount Wilhelm and Huon shared the same demographic history over time. However, for all other New Guinean species, populations from Mount Wilhelm and Huon showed contrasting demographic patterns from the mid-late Pleistocene towards the present: populations from Huon experienced an important demographic decline, dropping to a historical low by the Late Pleistocene, while populations from Wilhelm remained stable and did not experience the decline observed in Huon. This contrasting demographic pattern can be mostly clearly observed in Origma robusta, Ifrita kowaldi, Rhipidura albolimbata, Ptiloprora guisei and Melipotes fumigatus/ater. Similarly, populations of the species occurring on Mount Wilhelm, Huon and Mount Scratchley showed comparable demographic histories up until the mid-late Pleistocene, at which point each population began to behave slightly differently, with Huon always showing the lowest population sizes.PSMC plots generated for the re-sequenced individuals Figs. –4 alloweFicedula hyperythra and Thapsinillas affinis showed a common demographic history on both Buru and Seram. The two Ceyx lepidus populations also appear to have a similar demographic history up until 1 Mya, when the effective population size spiked on Buru, but not on Seram , we conducted PSMC analyses with re-sequenced data mapped to the following non-conspecific reference genomes: Sericornis nouhuysi (for the closely related Aethomyias papuensis), Pachycephala schlegelii  and Ficedula hyperythra (for the closely related Ficedula buruensis). While the PSMC plots for these three species should be interpreted with some caution, we observed the same large population expansions during the Pleistocene, but the expansion occurred in the late- rather than the mid-Pleistocene.For the three species with no de novo genome  were suggested for those species with high genetic differentiation , higher migration rates (ca. 10−5) were suggested for the lowland species Toxorhamphus novaeguineae and Melilestes megarhynchus and for those species with low genetic differentiation . For a few species, migration rates were close to zero (10−11–10−18), but we found no clear directionality in migration rates between Mount Wilhelm and Huon.Models including migration always performed better than models without migration. Migration rates between Buru and Seram were low for the five species compared, ranging between proportions of 10Thapsinillas affinis to ca. 600,000 individuals in Ficedula buruensis  and between 0.1 and 1.8 My (mean = 1.2 My) for the New Guinean populations.Ancestral population sizes were similar across species, with an average of ca. 325,000 individuals, ranging from ca. 100,000 in ST = 0.09–0.16) on the two islands of Buru and Seram . Ficedula hyperythra appears to have undergone a broad and recent radiation from mainland Asia across the mountains of the Indonesian islands30. That F. hyperythra has a higher dispersal capacity  than the other focal Moluccan species  is supported by dispersal estimates using fastsimcoal2  than in those found on different New Guinean mountain ranges (separated by a “soft” lowland barrier). Four of the five species examined in the Moluccas have genetically well-separated populations , and mix freely across the intervening lowlands. The high gene flow predicted between populations of the lowland species is concordant with the dispersal estimates  in fastsimcoal2  and the smaller Saruwaged Range on the Huon Peninsula represent members of the island’s core passerine bird clades ; this was preceded by a long period of relative demographic stability during the Pliocene and Miocene.Ceyx lepidus), especially considering the significant genetic differentiation between them (FST = 0.09–0.16). This strongly indicates that shared regional climatic and environmental fluctuations, rather than island-specific community dynamics or stochasticity, have governed population sizes in the Moluccan islands during the past few million years.PSMC curves of populations of the same species on Buru and Seram show remarkable similarity  are highly similar for many species that are members of different families Figs.  and 4, a41. Many species underwent range shifts as the extent of suitable habitats changed, and it is hypothesized that montane species in the tropics may have descended to lowland regions during colder periods. Such elevational range shifts are also likely to affect population densities as more suitable habitat space becomes available41.It is well-known that climatic fluctuations in the Pleistocene have strongly influenced the demographic histories and distributions of species42. Hence, we suggest that the population size oscillations observed herein reflect the severe Pleistocene climatic oscillations45, in combination with the marked tectonic re-arrangements and uplift that have greatly affected the region17.While in general tropical regions were not as heavily affected as regions at higher latitudes, the effect of the Pleistocene climatic changes in the Indo-Pacific was significant, and is believed to have caused the extinction of many large vertebrate taxa, while a range of medium- and small-bodied species underwent major geographic range shiftsFor the New Guinean species, we find strikingly contrasting demographic patterns between populations from Mount Wilhelm and from Huon in the more recent past Fig. . In geneIfrita kowaldi for which individuals at 2300 and 2900 m a.s.l. show some genetic divergence  through time, and relatively small population sizes at present (reflecting the more limited available area). We do not find such patterns. Finally, under the lowland recruitment hypothesis, isolated populations at high elevations are expected to be highly sedentary. Fastsimcoal2 suggests low but non-negligible rates of migration for even the most highly diverged species in our study (in the order of 10−7 migrants per generation), implying that montane populations do, in fact, have the ability to disperse.Taxon pairs at higher elevations in New Guinea are significantly more genetically differentiated relative to taxon pairs at lower elevations. This pattern could suggest that populations are recruited from the lowlands and then progressively become more isolated from one another as they move into the highlands. However, our demographic analyses  suggest that the two most diverged taxon pairs, 47 suggested that montane distributions are driven by repeated colonization of the outlying ranges from the Central Range. The smaller populations of the outlying ranges have a higher probability of going extinct, so in the case considered here, colonization patterns would be predicted to become uni-directional from the Central Range to Huon. Under this hypothesis, highland species continuously colonize other highland regions via direct dispersal from mountaintop to mountaintop.Instead, our results emphasize the relative importance of dispersal between montane regions. Further, the patterns of dispersal that we recover in our demographic data support Diamond’s hypothesis that these dispersal events follow the logic of island biogeography theory. Because of the physical configuration of New Guinea’s mountains, with a dominating Central Range akin to a “continent” and several small outlying ranges that can be viewed as “islands”, DiamondWhile our data clearly demonstrate that population sizes of all species analyzed herein have fluctuated markedly due to climatic and geological dynamics, several lines of evidence indicate that the divergent patterns in demographic (PSMC) trajectories between populations of the Central Range and Huon populations are best interpreted as reflecting a net colonization of Huon from the Central Range. All favored models in the demographic analyses in fastsimcoal2 included migration, which suggests continuous gene flow between Mount Wilhelm and Huon , such that highland species are able to disperse between separated mountain regions. While dispersal rates are often similar in both directions between Huon and the Central Range Table , the smaFollowing dispersal, newly formed Huon populations separate from those of Mount Wilhelm, with population sizes (Ne) decreasing in these “founder” populations. Further support for this dispersal scenario is a visible increase in population sizes (Ne) for the majority of species preceding the split of demographic histories (PSMC curves). This increase either reflects the actual range expansion event prior to divergence, or a build-up of individuals  within the source range. While the Huon populations decrease due to the population bottleneck associated with a founder event and reduced inhabitable area, Central Range populations remain stable or increase after the split. If we assumed a lowland origin followed by upslope range contraction into separate mountain ranges and/or vicariant speciation, we would expect that Central Range populations would also decline post-divergence.Montane avifaunas might have had greater opportunity to disperse between their preferred habitat during glacial maxima, when elevational habitat belts shifted downslope and possibly connected previous isolated montane populations. However, many species in our study show appreciable divergences that significantly predate recent glacial maxima. Moreover, speciation via habitat connectivity and divergence is a vicariant process, and the demographic histories (PSMC curves) do not reflect range contractions following population divergence. This mechanism is not, however, mutually exclusive with direct mountain-to-mountain dispersal. If gaps between tracts of montane forest shrank during glacial maxima, this would have reduced distances between allopatric populations, and increased the probability of individuals dispersing between them. Consequently, dramatic climatic oscillations during the Pleistocene would favor both potential mechanisms.Our comparisons using whole genomic data of population pairs of eighteen Indo-Pacific bird species shed new light on the formation of montane diversity across tropical island settings. The genomic data generated in this study allow us to infer the underlying mechanisms leading to the formation of highland taxonomic diversity, revealing that levels of genetic differentiation varies predictably across taxa as a result of species distributions and ecology. Specifically, we find that (i) “hard” oceanic barriers generate greater differentiation than “soft” lowland barriers, (ii) a higher altitudinal floor is associated with greater differentiation between populations, (iii) physical barriers generate greater differentiation than environmental gradients and (iv) demographic fluctuations are greater in smaller mountain ranges than larger ones. These data further highlight the importance of dramatic Pleistocene climatic oscillations, which have influenced the extent of montane forests, to determine temporal patterns of population expansion and contraction along the elevational gradients. Intermittent dispersal between disjunct New Guinean montane regions is repeatedly documented across taxon pairs and is likely an important factor underlying the expansion and build-up of montane passerine bird diversity in this area. Dispersal is much less frequent between taxon pairs in the Moluccan islands. While it is often assumed that islands and mountaintops represent evolutionary dead ends, we show that disjunct montane populations of the same species can maintain gene flow, as well as their capacity to further colonize new regions. This has implications for how we view the formation of montane biotas, and how we approach the conservation of its present diversity.This research complies with all relevant ethical regulations in Indonesia and in Papua New Guinea. Research in New Guinea was approved by the State Ministry of Research and Technology . The PNG National Museum and art gallery and the Conservation and Environment Protection Authority (CEPA) of Papua New Guinea provided research permits and export permits. Research in Indonesia was approved by the State Ministry of Research and Technology , the Ministry of Forestry (Republic of Indonesia), the Research Center for Biology, Indonesian Institute of Sciences (RCB-LIPI) and the Bogor Zoological Museum. Collectively, these institutions provided permits to carry out fieldwork in Indonesia and to export select samples.Ceyx lepidus) and seventeen passerine birds (Passeriformes) representing the three superfamilies  , samples were obtained along elevational transects in two separate mountain ranges on New Guinea: the northeast slope of Mount Wilhelm (4509 m a.s.l.) in the Central Range and in the Saruwaged Range (4121 m a.s.l.) in the Huon Peninsula (hereafter “Huon”). Eleven of the thirteen species were obtained from medium or high elevations, and specimens were collected at different sampling stations between 1700 and 3700 m a.s.l. Two lowland species, T. novaeguineae and M. megarhynchus, were included, with samples obtained between 50 and 1200 m a.s.l. Because the widespread montane species M. fumigatus is not present in the Saruwaged Range, we compared populations of this species from Mount Wilhelm with the congeneric M. ater, which is endemic to the Huon Peninsula. As P. guisei, is replaced at higher elevations by its congener P. perstriata, we included samples of P. perstriata from Mount Wilhelm and Mount Scratchley (P. perstriata is not present in the Huon Peninsula) to assess the potential for admixture between these two species. The Central Range and the Saruwaged Range are separated by the comparatively arid Markham/Ramu River valley, which features extensive river floodplains, Imperata cylindrica grassland, and anthropogenic gardens. To assess the connectivity of populations of lowland species, we obtained samples from a third population from appropriate habitats in the biogeographic transition zone between Mount Wilhelm and Huon, north of the Markham/Ramu River. As the nearest forested corridor between the Central and Saruwaged ranges, these samples allow us to directly gauge the strength of the river barrier. For a subset of five of the New Guinean highland species , we additionally sampled individuals from a third population from Mount Scratchley, which is a continuation of the Central Range in south-eastern New Guinea. By doing so, we are able to contrast population demographic trajectories between mountains within the Central Range as well as our primary contrast with the well-isolated outlying mountains of the Huon Peninsula.Our study included populations pairs of eighteen bird species; one non-passerine species (Coraciiformes) the Moluccan dwarf kingfisher (Corvides  species s]) Fig. . For thiCeyx lepidus, Pachycephala macrorhyncha, Ficedula hyperythra, Ficedula buruensis and Thapsinillas affinis) were obtained from the Indonesian islands of Buru and Seram, within the Moluccan Islands to the west of New Guinea. Buru is 9505 km2 with a maximum elevation of 2428 m a.s.l., while Seram is 17,100 km2 with a maximum elevation of 3027 m a.s.l. The two oceanic islands are separated by ~75 km of ocean, but due to their close proximity share a substantial amount of bird species. On Buru, samples were collected in the northwest part of the island at elevations ranging between 227 and 1425 m a.s.l., while on Seram samples were obtained from the north coast and inland between elevations of 185–1300 m a.s.l. , no de novo genome was assembled since the study already included the de novo genome of another closely related species .To facilitate demographic analyses, a de novo genome was assembled for fifteen of the eighteen species using the 10X Chromium linked-reads platform at the Science for Life Laboratory in Stockholm, Sweden  was extracted using a Thermo Scientific KingFisher Duo magnetic particle processor (ThermoFisher Scientific) with the KingFisher Cell and Tissue DNA Kit. Only one individual was used as a template for each genome sequence. Following quantification on a Qubit Fluorometer (ThermoFisher Scientific) and validation of the extract quality on an agarose gel, the extracts were used to prepare Illumina sequencing libraries. Libraries were sequenced on an Illumina HiSeqX (HiSeq Control Software HD 3.5.0.7/RTA 2.7.7) with a 2 × 151 bp setup using HiSeq X SBS chemistry. The Bcl to FastQ conversion was performed using BCL2FASTQ v.2.19.1.403 from the CASAVA software suite.49 as incorporated in the nextflow-core Neutronstar workflow50. Standard assembly statistics were obtained including total assembly length, number of scaffolds, scaffold N50 and percentage of base assembly missing in scaffolds larger than 10 kb. These statistics give an idea of the contiguity of the assembly. To validate the assembly, independent contiguity metrics were also obtained using QUAST v5.0.151, a quality assessment tool for genome assemblies. In addition, BUSCO v4.0.252 was used to assess the assembly quality in terms of conserved gene recovery. The software inspects the de novo assemblies searching for Benchmarking Universal Single-Copy orthologs called BUSCOs. A set of 300 eukaryote orthologs was used. BUSCOs were classified as complete and single-copy, complete and duplicated, fragmented or missing. All de novo assembled genomes are deposited on NCBI under SRA accession: PRJNA 637995.All genomes were assembled de novo using Supernova v2.0.1Thapsinillas affinis to 18 in Ptiloprora guisei and Pachycephala macrorhyncha. Genomic DNA was extracted from either muscle tissue or blood samples stored at −80 °C using a Thermo Scientific KingFisher Duo magnetic particle processor (ThermoFisher Scientific) with the KingFisher Cell and Tissue DNA Kit. DNA was quantified using a Qubit Fluorometer (ThermoFisher Scientific). Libraries were made using either Illumina TruSeq DNA Library Preparation Kits (high DNA input) or Rubicon Thruplex DNA-seq Kits (low DNA input), with an average fragment size of 350 bp in both cases. Samples were sequenced on an Illumina HiSeqX (HiSeq Control Software HD 3.5.07/RTA v2.7.7) with a 2 × 151 bp setup using HiSeq X SBS chemistry. The Bcl to FastQ conversion was performed using BCL2FASTQ v.2.19.1.403 from the CASAVA software suite. All resequencing data are deposited on NCBI under SRA accession: PRJNA580350.A total of 226 individuals representing the eighteen species were re-sequenced at SciLifeLab, Stockholm (Supplementary Data http://www.bioinformatics.babraham.ac.uk/projects/fastqc) and the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx-toolkit), after which we trimmed the first 5 base pairs (bp) and the last 11 bp from the forward reads, and the first 5 bp and the last 21 bp of the reverse reads. Next, adapters and low quality bases were removed with Trimmomatic v0.3653, using a minimum Phred score of 15. Subsequently, paired and unpaired reads were aligned separately to the respective de novo genomes using BWA v0.7.154. Aligned data were pre-processed using Picard v2.6.055, which included merging (MergeSamFiles), coordinate-sorting (SortSam) and identifying duplicate reads that may arise during library preparation (MarkDuplicates).Data quality of the Illumina reads was initially assessed using FastQC  in order to visualize population structure using the princomp function in R60. Prior to the analysis, the PCAngsd software v0.9561 was used to remove related individuals based on a kinship matrix. Individuals with a pairwise kinship coefficient higher than 0.06 were considered to be closely related.Standardized pairwise F62, a model-based clustering algorithm that infers the most likely number of groups (K) in the data. The analysis was performed with K = 1–6, assuming an admixture model, correlated allele frequencies and without population priors. In order to choose appropriate burn-in and run lengths several test runs were run at different Ks and different lengths (from 105 to 106) and checked for consistent estimates of the parameters P, Q and lnPr(X | K). Ultimately, a burn-in of 10,000 steps followed by 100,000 additional Markov Chain Monte Carlo iterations were performed. For each K, 10 independent runs were conducted to check the consistency of the results. The most likely K was inferred using the method of Evanno et al.63, which measures the steepest increase of the ad hoc statistic ΔK based on the rate of change in the log probability of data between successive K values.Population structure was further investigated using the Bayesian assignment approach implemented in STRUCTUREST) values and elevational floor using a Spearman’s rank correlation. If lineages colonize mountains from the lowlands, followed by range contraction and differentiation in the highlands, we would expect a strong correlation between FST and elevational floor (the lower trailing edge of the distribution) coupled with no dispersal between populations. We obtained elevational information for New Guinea species . After excluding non-autosomal regions, variants were called with the mpile and call commands in BCFtools. The following filtering criteria were applied: we excluded sites for which read depth was less than 10 or more than 100; sites with Phred quality scores below 20; and sites in the proximity of indels. Subsequently, we used the ‘consensus’ command in BCFtools to incorporate all variants into a single sequence using IUPAC codes.For each de novo sequenced individual (one per species) and all re-sequenced individuals, a consensus sequence was generated using BCFtools v1.9The consensus sequence was then divided into non-overlapping 10 bp bins, which were scored as heterozygous if there was at least one heterozygote nucleotide position in the bin, and otherwise scored as homozygous. The total number of iterations was set to 25; T max (-t) was set to 15; the initial mutation/recombination ratio (-r) was set to 5; and the atomic time interval pattern (-p) was set to “4 + 25*2 + 4 + 6”. Bootstrapping was performed by splitting the data into shorter segments that were then randomly sampled with the replacement for a total of 100 rounds.25 to generate the expected AFS under different models of historical divergence using coalescent simulations, select the best-fit model using the composite multinomial likelihood, and infer demographic parameters. We were particularly interested in inferring current and ancestral effective population size, time of divergence, the occurrence of historical events allowing for population size changes  and the presence of gene flow and its directionality (symmetric/asymmetric). To this end, we maximized the likelihood of the observed AFS under a total of 8 different demographic scenarios applied to each population pair across all species. The simplest model was (i) a null model with no growth and no migration between populations in isolated mountain ranges. We also tested (ii) a model with migration but no growth, (iii) a model with exponential growth but no migration and (iv) a model with both exponential growth and migration. Furthermore, we tested the likelihood of a demographic event in the past that led to the occurrence of a bottleneck both (v) with and (vi) without migration, and an event leading to a sudden expansion, again (vii) with and (viii) without migration. For those species in which the PSMC plots showed a marked difference in the demographic history of the two populations we compared, we additionally tested (ix) a model with a bottleneck in the first population and a sudden expansion in the second population and the reversed scenario, i.e. (x) a model with a sudden expansion in the first population and a bottleneck in the second population.In addition to PSMC, we also modeled demographic history with an approach based on the allele frequency spectrum (AFS). We used the program fastsimcoal2https://github.com/shenglin-liu/vcf2sfs. Exact models and priors for all parameters are described in Supplementary Methods. For every demographic model, 100 independent estimations with different initial parameter values were run and results for the estimation with the highest likelihood were reported. The best-fitting demographic model was identified using Akaike’s Information Criterion (AIC) score67.The AFS for all populations were directly generated from the final variant calls (vcf files) using the scripts in Further information on research design is available in the Supplementary figures and tablesPeer Review FileSupplementary Dataset 1Supplementary Dataset 2Reporting Summary


Dicranum scoparium, which has facultative male dwarfism, having both dwarf males (DMs) and normal-sized males (NMs). We retrieved 119 SNP markers from transcriptomes which were used to genotype 403 samples from 11 sites at seven localities in southern Sweden. Our aims were to compare the genetic variability and genetic structure of sexually reproducing populations at different geographic levels  and compare in particular the relative contribution of females, dwarf males and normal-sized males to the observed genetic diversity. The numbers of DMs differed strongly between sites, but when present, they usually outnumbered both females and NMs. Low genetic differentiation was found at locality level. Genetic differentiation was strongest between cushions for females and NMs and within cushions for DMs indicating small scale structuring and sometimes inbreeding. NMs were more clonal than either DMs or females. Genetic diversity was similar between females and DMs, but lower for NMs. Two haplotypes were shared between females and DMs and one haplotype was shared between a DM and a NM. In conclusion, our results show that DMs and NMs play different roles in reproduction, inbreeding may occur at cushion level, but gene flow is high enough to prevent substantial genetic drift.Among plants, gender dimorphism occurs in about 10% of all angiosperms and more than 50% of all moss taxa, with dwarf males (DM) found exclusively in some unisexual mosses. In this study, we explore the role of male dwarfism as a reproductive strategy in the widespread acrocarpous moss  Osedax or anglerfishes  share the trait of having a lifecycle dominated by the haploid gametophyte, the diploid sporophyte generation is initiated after sexual fertilization and remain connected to the maternal shoot throughout its short existence. Most mosses are capable of both asexual and sexual reproduction. Asexual reproduction is common in mosses, allowing clonal regeneration from gametophytic fragments or specialized vegetative diaspores (gemmae). Sexual reproduction depends on water for antherozoids (sperm cells) to swim to the archegonia . Due to Nannandry is unique to mosses among land plants and appears to have evolved as a means to increase the fertilization efficiency by decreasing the separation distance between genders and thus increasing the number of available mating partners for a female . Minute If females in a nannandric species are predominantly fertilized by dwarf-males we can expect that the system is driven toward higher specialization for nannandry  and eventually obligate nannandry. In a species with facultative nannandry, a dwarf male transmits its genes during one season, whereas a normal-sized male can transmit its genes over many years in the same patch and hence could slow down adaptation. However, if normal-sized males and dwarf-males are equally frequently fertilizing the females, the system may stay in a facultatively nannandric condition, without drift toward obligate nannandry. The relative frequency of dwarf males and normal-sized males and their associated haplotypic networks is therefore a key for understanding the development of a nannandric species.Homalothecium lutescens (Hedw.) H. Rob. have greatly extended our knowledge about nannandry in bryophytes . Our overall aims are to employ the molecular markers in a moss with facultative nannandry in order to (1) reveal the hierarchical genetic population structure (2) determine if the levels of genetic diversity and clonality compare between females and males and (3) assess the relative shares of dwarf males and normal-sized males to the total standing genetic diversity.The genus dwarfism . In thisDicranum scoparium is widely distributed in the Holarctic and is growing on various substrates, preferably on acidic soil, rotten logs, tree stems and sometimes in sympatry with other Dicranum species ( species . Both noD. bonjeanii and five of Dicranum majus) from 11 sites were collected from seven different localities, namely Snogeröd Lunnerna (Lu), Röan (Ro), Munkarp (Mu), Klöva Haller (KH), Skanörs Ljung (SL), Dalby (Da), Bjärsjölagård , and further observation of vegetative growth.Fresh bryophyte material was collected in August 2015 across Skåne, South Sweden. In total 54 fertile cushions  according to the protocol of the manufacturer and eluted in 100 μl AE buffer. All DNA extracts were stored at −20°C.Dicranum majus Turner female gametophyte. Each stem was first cleaned in deionized water and then flash frozen in liquid nitrogen before being stored at −80°C. Consequently, RNA from ten samples were obtained using the RNeasy Plant mini kit following the protocol of the manufacturer . The quality, quantity and integrity of the RNA extractions were measured on agarose gels, with Qubit® 2.0 fluorometer  and BioAnalyzer , respectively.We applied a strategy involving transcriptome analyses for selection of SNP markers, which requires RNA extracts of high quality and quantity. Fresh plant material was used for RNA extraction and one female stem per locality was randomly sampled in order to represent the genetic variability of the population. Three additional extraction was done for pooled normal-sized males, sporophytes (diploid generation) and one 1 using NEXTflex RNA-Seq kit .The SNP library was produced with the MiSeq system  using the 300 bp paired-end settings. The library construction was carried out at Lund University sequencing facilityDe Novo reference assembly using Trinity . Finally, to ensure the variants were still adequate markers of genetic diversity, we removed variants which were present in more than 8 (out of 10) samples. This resulted in a final selection of 119 SNPs.The sequencing of the moss transcriptomes resulted in about 1 million reads/sample . The sam Trinity . Reads f Trinity  with –m  Trinity  mpileup The genotyping of 738 DNA samples was carried out at the SNP&SEQ Technology Platform, Department of Medical Sciences of Uppsala University. It was performed using a multiplexed primer extension chemistry of the iPLEX assay . The allD. scoparium individuals only, excluding sporophytes data. The final number of individuals analyzed in this study is found in adegenet version 2.1.2  independently for each subset were excluded. Finally, we controlled again that the data set would contain no more than 10% missing information and eventually remove the individual or SNP that would not meet the criteria.mlg.filter function by collapsing unique MLH (multilocus haplotypes) utilizing Nei genetic distance given the farthest neighbor clustering algorithm. This method allows to incorporate genotypes that have missing data or genotyping error into a parent clusters. The clustering threshold was set for each dataset individually and missing data were replaced with the mean of the alleles for the entire data set.To remove the effect of genetic linkage due to clones and adjust for missing data and genotyping errors that may occur during sequencing large datasets c.f. , multilod, which accounts for the number of loci sampled (poppr.amova function and the ade4 implementation. Fixation indices (Φ statistics) were interpreted as a measure of subpopulation differentiation. The probability of retrieving the observed Φ under the null hypothesis of no differentiation between subpopulations was calculated using the randtest function of the ade4 package with 1000 iterations of the permutation test.Multilocus lineages were further used for the estimation of the haplotypic diversity G;  and Nei’ sampled . Conside sampled . A signi sampled ] consideAssuming that the diversity of DM haplotypes residing on a female is determined by the number male spores that germinated on the female, the proportion of DMs should covary with haplotypic diversity in a population. Therefore, the proportion of each sex type at locality was plotted against the haplotypic diversity G. Kruskal Wallis non-parametric test was used to examine the differences in G between sex types. Furthermore, an ANOVA was used to test for the differences of ĥ between sex types as residuals followed a normal distribution. We also compared Nei’s gene diversity ĥ against G in order to display the contribution of each haplotype to the total variability at cushion level.Finally, population structure was analyzed in two ways. First, a principal coordinate analysis (PCoA) was performed on female and male individuals. The analysis was based on a pairwise genetic distance matrix, with Prevosti’s distance, equivalent to dissimilarity distance. Then, a minimum-length spanning network (msn) of all unique MLL was constructed based on the Prevosti’s distance in order to assess how female and male haplotypes were related across different populations. Resulting graphs were edited with Inkscape v.1.0.0.From the 10 initial samples, 119 SNPs were identified for genotype screening . Out of 769 extracted DNA samples, 738 were suitable for genotyping, whereas 31 lacked enough DNA for genotyping. Of the 119 SNPs, 90 SNP markers were identified in all 738 genotyped DNA samples. The fraction of SNP markers with call rate >0 reached 75.63% and the average sample call rate per SNP reached 88.49%. The fraction of individuals with call rate >0 reached 95.80% while the average SNP call rate per sample reached 69.86%. Five out of 90 SNPs were invariable (5.56%). The 29 deficient SNPs had no allele signal cluster separation  which resulted in inaccurate genotype calls.D. scoparium haploid dataset contained 403 individuals and 68 loci  than both females (mean G = 6.9) and DMs (mean G = 8.2) . Furthermore, the haplotypic diversity for DMs varied a lot dependent on the number of individuals that were possible to genotype , although this is largely due to two localities with abundant population of dwarf males (Bj and Ro).Multilocus haplotypic diversity (G) differed between localities, from F = 4.83; P = 0.048). The DMs (mean ĥ = 0.24) were usually more variable than the NMs (mean ĥ = 0.15) and NMs had significantly lower gene diversity than both females and DMs . In most localities NMs had ĥ values ranging between 0 and 0.19, but one exception was found at Lu, with ĥ = 0.34 for both females and NMs  than males (mean ĥ = 0.23) . Clonality was strongest for NMs at all localities, with high IA values and r̄Almost 90% of the total variation was partitioned nearly equally within cushions and among cushions within localities, while only 11% of the variation occurred among localities : GAM. 25A PCoA was produced to visualize the genetic distance of female and male individuals . OverallThe first three axes of the male PCoA explained 28.13% cumulative variance . Similar to the females, the male PCoA displayed little structure at locality level , except To visualize relationships between female and male MLLs, a minimum-spanning network was constructed . The numDicranum scoparium at different scales. Dwarf males and normal-sized males often occurred together in a moss cushion. We showed that the overall levels of genetic diversity within females and dwarf males were relatively similar and much higher than among normal-sized males. The lower genetic diversity in NMs may therefore be a consequence of fewer haplotypes in the populations. Furthermore, clonality occurred in all three categories, yet at different degrees: level of clonality was high among NMs whereas it was much less so in females and DMs. Most of the genetic differentiation was partitioned within and among cushions and only a low fraction among localities. These results suggest that effective gene flow occurs regularly over long distances.In this study, we examined the genetic variability and structure of female and male populations of the facultatively nannandric moss Before we interpret the genetic diversity data, it is worth mentioning that (1) we have actively searched for loci that are variable and (2) that the regions are transcribed and therefore the SNP alleles are possibly under selection. This implies that absolute values of genetic diversity measures are not directly comparable with other studies, although our markers in several ways  resemble allozyme markers, for which much of the population level studies were done in the past.D. scoparium exists. Thus, designing a genetic diversity panel is challenging. Our approach, using RNA-seq and transcriptome assembly enables an affordable construction of transcribed portions of the genome that are likely evolutionarily relevant. Yet, the quality of such an assembly is highly dependent on the depth of sequencing, which is highly variable per transcript, and especially near the end of transcript. Furthermore, SNPs were identified using RNA-Seq reads aligned to the assembled transcriptome. This too is restricting and resulted in only 119 reliable SNPs. Then, due to the cost of RNA-Sequencing, we used only 10 samples  that would represent the total genetic diversity of each population. Hence, we selected variants that were relatively common and not entirely recurrent (in at least three and at most eight of the 10 samples). Furthermore, the extraction and genotyping steps themselves also lead to some losses. For example, DM for the populations in Lu were extracted but the genotyping was not successful. Finally, SNP data pose new challenges when compared to other markers, because they potentially contain more missing data, error in SNP calling due to genotyping errors or other sources of inaccurate allele calls . At locality levels genetic differentiation for females was particularly low (Φ = 0.06), which is indicative of a high gene flow. The corresponding values for und 0.10  and 0.3 und 0.10  from difDicranum populations are consistent with other moss studies that indicated limited population structure at larger scales but differentiation at cushion level  as well the highest index of association (r̄d = 0.97) among all female populations. These results indicate that vegetative growth of female cushions is predominant at this locality and are congruent with other moss species (The population of Bjärsjölagård (Bj) differs in several aspects; it is genetically divergent despite being geographically relatively close to sites Lu and Da . It prob 3.2–7.1 . The preyped DMs . In furt species . Moreove species , were clD. scoparium in Scania (Sweden) in a similar way as earlier done for the obligately nannandric species H. lutescens. The genetic differentiation at locality level was low indicating a strong gene flow. In the dense acrocarpous cushions of D. scoparium, the haplotypic diversity at patch level was higher for females (and NMs) than in the pleurocarpous and weft-forming H. lutescens, suggesting more frequent recruitment.The genetic influence of dwarfism on population structure has rarely been studied and only few comparable studies exist. Here, we used SNP markers to investigate the genetic structure of seven populations of the facultatively nannandric species D. scoparium, DMs and NMs often occurred together but the DMs were genotypically more frequent and divergent. The genotypic networks show that DM individuals at patch level represent both closely related haplotypes (suggesting inbreeding) and remotely related haplotypes . Nevertheless, NMs may regularly take part in fertilization as they are perennial and probably less sensitive to temporarily adverse climatic conditions. If so, the facultative nannandric system is prevented from transforming to obligate nannandry through sexual selection, contrasting to the nearly obligate or obligate nannandric conditions in H. lutescens and most Dicranum species.In Nannandry has probably developed to ensure fertilization and increase the number of male gamete donors. We see nannandry in mosses as a fertilization syndrome, a parallel to pollination syndromes in flowering plants, which ensure pollination and increases the available number of pollen donors. More studies on sexual reproduction and diversity in nannandric mosses are needed, especially in regards to facultative dwarfism. It would be particularly useful to determine the environmental condition for the recruitment and growth of normal-sized males to better understand their contribution to the population dynamics.https://www.ebi.ac.uk/ena/) under accessions PRJEB37280 and ERP119316 (secondary study accession).The raw dataset generated for this study can be found in the European Nucleotide Archive (AL and NC wrote the text, conducted the sampling of the specimens, and participated in the interpretation of the results. TG and NC commented and critically reviewed the manuscript. AL conducted the laboratory work and the data analyses. TG designed the scripts specifically designed for SNP filtering. All authors approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plethobasus cyphyus). Population structure analyses reveal five populations, three in the Upper Mississippi River Basin and two in the Ohio River Basin. Sampling locations exhibit a high degree of genetic diversity and contemporary migration estimates indicate that migration within river basins is occurring, although at low rates, but there is no migration is occurring between the Ohio and Mississippi river basins. No evidence of bottlenecks was detected, and almost all locations exhibited the signature of population expansion. Our results indicate that although anthropogenic activity has altered the landscape across the range of the Sheepnose, these activities have yet to be reflected in losses of genetic diversity. Efforts to conserve Sheepnose populations should focus on maintaining existing habitats and fostering genetic connectivity between extant demes to conserve remaining genetic diversity for future viable populations.North American freshwater mussel species have experienced substantial range fragmentation and population reductions. These impacts have the potential to reduce genetic connectivity among populations and increase the risk of losing genetic diversity. Thirteen microsatellite loci and an 883 bp fragment of the mitochondrial ND1 gene were used to assess genetic diversity, population structure, contemporary migration rates, and population size changes across the range of the Sheepnose mussel ( Population genetic analyses of the Sheepnose mussel reveal five populations. Sampling locations exhibit a high degree of genetic diversity and contemporary migration estimates indicate that migration between populations within river basins is occurring, although at low rates. But no migration is occurring between the Ohio and Mississippi river basins. North APlethobasus, all of which are listed as endangered. Of the three species, the Sheepnose currently occupies the broadest distribution   for DNA extraction were collected from seven different localities (Table ® Blood and Tissue Kit (Qiagen # 69506) according to the kit instructions. Cytology brush samples were stored in the lysis buffer provided with the Puregene Buccal Cell Core Kit B (Qiagen) and DNA was extracted following the kit instructions. Extracted DNA was quantified using a Nanodrop ND1000 spectrophotometer and stored at 4°C.A combination of microsatellite markers and mitochondrial DNA sequences was used to estimate genetic diversity, population structure, contemporary migration, and population size changes in the Sheepnose. Samples N = 64 for DNes Table  or by uses Table . Biopsy 2.1.220, 1 µl Biolase Buffer (10×), 0.3 µl MgCl2, 0.8 µl dNTP, 0.1 µl forward primer, 0.1 µl reverse primer, 0.05 µl M13 labeled oligo, 0.05 µl Biolase Taq polymerase, and 1 µl template DNA. Reactions were run in Eppendorf Master Cycler thermal cyclers under consistent conditions . Negative controls were performed with every reaction to detect potential contamination. PCR products were visualized on 1% agarose gels to confirm that the reaction was successful and that the negative control showed no contamination. PCR products were then sent to the Iowa State DNA Facility where they underwent capillary electrophoresis to determine allele sizes. The raw data were then scored using GeneMarker® v1.85  and LoGlyR (5′‐CCTGCTTGGAAGGCAAGTGTACT‐3′) primers , and 1 µl template DNA. Approximately ~1 ng of genomic DNA was used for the template. Reactions were run in Eppendorf Master Cycler thermal cyclers at the following conditions: . Negative controls were performed with every reaction to detect potential contamination. Products were then run on 1% agarose gel to assure that the reaction was successful and to verify that the negative control showed no contamination. Successful PCR products were prepared for sequencing using the ExoSAP‐IT . Template consisted of ~1 µl of genomic DNA in conjunction with forward and reverse sequencing reactions using Big Dye® ver. 3.1 cycle sequencing kit (Applied Biosystems # 4337454) and sent to the Iowa State DNA Facility for sequencing. The raw results were then analyzed and edited using GENEIOUS v8.1.6  were also generated for samples. DNA sequence data for the ND1 gene were generated through PCR with Leu‐uurF (52.22.2.1A), expected heterozygosity (He), observed heterozygosity (Ho), and fixation index (FST) were calculated using GenAlEx v6.5  . A permutation test was performed on the FST values using base RStudio to determine degree of genetic differentiation found among sampling locations that were located within versus between drainage basins. This test was permuted 100,000 times and p‐values were assessed at a.05 significance level. Microsatellite genotypes have been submitted to DRYAD (https://doi.org/10.5061/dryad.gxd2547mm).Each locus was tested for deviations from Hardy–Weinberg expectations, potential genotyping errors, and linkage disequilibrium at each collection site using MICROCHECKER , subsequent analyses to detect substructure were also conducted. STRUCTURE runs were conducted on the two populations identified in the initial run using the same procedures as previously, but with possible K values of 1–4 and 1–5. The web application POPHELPER  which can be interpreted as the fraction of migrants per generation in one population that is derived from a source population. These estimations are calculated using a Bayesian approach and MCMC sampling to generate values for m over the last few (<5) generations  and nucleotide (π) diversity for each sampling location. Pairwise FST values among sampling sites were calculated using ARELQUIN and tested for significance using 3000 permutations and a significance level of 0.05. A permutation test was performed on the FST values using base RStudio to determine degree of genetic differentiation found among sites that were located within versus between drainage basins. This test was permuted 100,000 times and p‐values were assessed at a 0.05 significance level. PopART  were assessed at a 0.05 significance level.A mismatch distribution analysis using ARLEQUIN was performed on all sampling sites. This analysis estimates pairwise differences among all the sequences. A population that has not changed in effective size over a long period of time will display a ragged distribution of pairwise distances, whereas a population that has been growing generates distributions that are smoother  ranged from −0.052 at the TN site to 0.072 at the ALL site . The permutation test indicated a significant difference (p = .005) between samples found in the Upper Mississippi River Basin  and the Ohio River Basin .Two of the microsatellite loci (C109A and C115) amplified poorly and were subsequently dropped from the analysis. Only one locus (D113) exhibited an excess of homozygosity and potential null alleles at multiple sampling locations, and it was also dropped from analysis. Microsatellite analysis results described below are from the remaining 13 microsatellite loci Appendix . MICROCH Wright, , G’ST, a3.1.2K was 2 . Despite sampling sites clustering into major river basins, with few exceptions, each sampling location showed a high degree of differentiation from other sites  more migration occurring among sampling sites within the same drainage basin (Mississippi and Ohio basins) than sites found in different drainage basins. All instances of migration rates >0.1 were asymmetrical, with migrants moving in only one direction. Three of these were in the Upper Mississippi River Basin and two were in the Ohio River Basin  and the TIPP site having the fewest (n = 5). The number of unique haplotypes ranged from 3 to 9 per site and accounted for about 85% of detected haplotypes overall (33 of 39). Haplotype diversity (Hd) ranged from 0.4620 (TIPP) to 0.9286 (TN) and the nucleotide diversity (π) ranged from 0.0008 (TIPP) to 0.0039 (WIS). A comparison of pairwise FST values indicated that all sample sites were significantly different from each other except the TIPP and ALL sites and the WIS and ALL sites  (Table p = .01) and raggedness index   Table . The det5) Table   Table . However2) Table . Converg4This study found that despite the lack of gene flow and population isolation, there is still a high degree of genetic diversity among sampling sites at both the microsatellite and mitochondrial loci. Similar levels of genetic diversity have been found in other studies of both rare and common freshwater mussel species ; Formal analysis (lead); Methodology ; Writing – original draft (lead); Writing – review & editing . Kevin Roe: Conceptualization (lead); Funding acquisition (lead); Project administration (lead); Writing – review & editing .https://doi.org/10.5061/dryad.gxd2547mm.This article has earned an Open Data Badge for making publicly available the digitally‐shareable data necessary to reproduce the reported results. The data is available at Appendix S1Click here for additional data file.


Salixlapponum is a cold-tolerant relict species in Europe that occurs in several sites, probably reflecting previous migration routes of S.lapponum during the Pleistocene. However, only a few data are available on the genetic structures of populations of S.lapponum. In this study, we use PCR-ISSR markers to investigate genetic variation in 19 European populations of S.lapponum L. AMOVA analysis shows that most of the variation (55.8%) occurs within populations; variability among groups accounts for 19.7%. An AMOVA analysis based on four groups determined by STRUCTURE analysis shows similar results: variability of 54.1% within the population and variability of 18.9% between the four population groups, based on geographic regions. Within individual geographic groups, which are characterised by the studied populations, the lowest variability (as well as the highest homogeneity) was found in populations located in Belarus. The obtained results are consistent with our expectations that the European Lowland could be a significant geographic barrier for gene flow over large geographic distances for S.lapponum. Both the Scandinavian and Belarusian populations, as well as those coming from NE Poland, are characterised by significant genetic distinctiveness. However, some populations from NE Poland and the Sudetes show similarities with populations from other geographic regions, indicating existing genetic relationships between them. Moreover, the results suggest a fairly clear division of the population into 4 emerging geographic regions, although separated by a geographical barrier: the Polish lowland, which forms part of the larger geographic unit known as the European Lowland. Quaternary glaciation, with numerous glaciations and deglaciations in Scandinavia and Central Europe, strongly influenced changes in the distributions of both specific plants and entire plant biomes. During Pleistocene climatic fluctuations, numerous arctic plants migrated southward, where they commonly colonised habitats primarily in European mountain ranges; in some cases, they also disappeared from these locations during warmer period called interglacials. Currently, various plant species growing in boreal areas in the Northern Hemisphere and in subalpine zones in lower latitudes in isolated mountains of the alpine system  or on the lowlands – often grow on the edges of their ranges, presenting disjunct geographic distribution .Disjunctive plant populations are often attributed to isolated localities in specific habitats where the climatic and edaphic conditions have allowed for their survival as relict species until the present postglacial period . The hisNumerous phylogeographic studies have described processes including colonisations or re-colonisations from refugial zones, extinctions, or migrations across thousands of kilometres in the Northern Hemisphere . In receLGM), all natural vegetation of temperate Europe changed substantially. This also applies to the development history of various Salix species, for which results have been obtained from numerous paleobotanical studies and from research using modern genetic methods  Rouy, subgenera Vetrix Dumort. . For these reasons, S.lapponum L. like Andromedapolifolia L., Betulanana L., Carexbigelowii Torr. & Schwein., Dryasoctopetala L., Juncustrifidus L., Rubuschamaemorus L., Salixherbacea L. and Saxifragaoppositifolia L.– is considered a glacial relict  and its fairly narrow stenotypic habitats , S.lapponum constitutes a good model species for explaining the complex processes that lead to the formation of arctic-alpine disjunction. Thus, we pursue three aims in this study: 1) to determine the degree of genetic similarity between populations of S.lapponum; 2) to explain the origin of Central European populations situated in the contact zone with the main range and in isolated refugia; 3) to describe probable migration routes.During the Last Glacial Maximum . Fresh leaves were collected in plastic zip bags, dried in silica orange gel, transported to the laboratory, and stored until DNA extraction. In general, we studied eight plant samples per population and 152 specimens of S.lapponum in total. Whole plant material was collected in the months of June or July from 2015 to 2017. Herbarium voucher specimens that were used for DNA are stored in author collection (J. U.) deposited at WRSL Herbarium  and are available on request.Molecular analysis was conducted on plants from all populations. In the field, leaves of S.lapponum. These markers are known to be highly polymorphic and are useful in studies on genetic diversity and species relationships; they enable easy differentiation of closely related specimens. ISSR has also been successfully employed to assess hybridisation and to detect hybrid taxa , 0.5 mM ISSR primer, and 0.8 μl of total genomic DNA. The PCR cycle consisted of an initial denaturation at 95 °C for 6 min, according to a previous study , using a DNeasy Plant Mini Kit  according to the manufacturer’s protocol. The quality of the isolated DNA was determined using 1% TBE agarose electrophoresis. The Inter Simple Sequence Repeat microsatellite markers were selected to study the genetic diversity of the populations of rid taxa . These mus study , followeAMOVA  were performed using ARLEQUIN 3.5.1 with 1000 permutations to determine the distribution of genetic variation within and among the populations and to assess the importance of the main groups of populations  indicates that most of the genetic variation in two groups of populations: Sudetes vs. NE Poland and Belarus occurs within populations (55.8%), while the variability among the groups accounts for 19.7% (Table The research markers selected by us for studying (ISSR) have some limitations in their application due to the lower number of generated markers than for example AFLP, but are still widely used in various types of phylogeographic research. Molecular variance analysis  shows a similar result: variability of 54.1% within the population and variability of 18.9% between the four groups of the population based on geographic regions. High genetic variability was also found with the analysis of all studied populations, which were treated as one group. The genetic variability value was 43.8% among populations and 56.2% within populations; the Fst value was 0.43 (p < 0.001). Within geographic groups, which are characterised by the studied populations, the lowest variability and also the highest homogeneity were found in populations located in Belarus. The genetic variation among Belarusian populations was low (23.1%). In contrast, the highest inter-population variability was found in Scandinavia (48.2%) and the variability in the Sudetes and NE Poland was similar: 37.4% and 37.2%, respectively.The S.lapponum in Europe.High and statistically significant genetic differences between all geographical groups of the population were found; again, the greatest differentiation was noted between the Belarusian populations and the Scandinavian populations (0.5). The greatest differences were noted between the populations from Belarus and between the populations from the Sudetes and NE Poland Table ; these wDWO) (0.172) and the lowest value in the population located in Prudzieniszniki (PRU) (0.098). Correlation analysis of the relationship between geographic and genetic distances in the study area did not show any significant correlation between genetic variation and the geographic distance between the studied populations .Both the highest and the lowest index of genetic diversity, based on S.lapponum’s distribution  are divided into two groups, also with quite high bootstrap support, although these groups are partially mixed with each other and some representatives of the population are placed in both groups. A separate group is composed of individuals from the populations located in the Sudetes  that are grouped in one cluster on the figure. The exception is a population from Polana (POL), which seems to be genetically similar to the Scandinavian population gathered in Fuljalfleet (FUL). Both populations form a highly distinctive group with high bootstrap support  showing a high similarity with each other Fig.  were incort Fig. . The remS.lapponum population  was four, as reported by K = 5 . In general – not only for K = 2 but also for K > 2 the populations from Belarus appeared as most distinct in all runs. For K = 3, populations from the Sudetes grouped with populations from NE Poland, while Scandinavian populations formed a separate group, except for one population (FUL) that seems to be genetically closer to populations from the Sudetes. For K = 5, populations from Belarus and the Sudetes form nearly a consistent group; however, specimens from the Sudetes populations showed closer relations with several populations from NE Poland. Results obtained with the STRUCTURE program are almost identical to the results presented in Fig. For S.lapponum to survive in such unfavourable conditions in isolated regions, such as glacial refuges, was very important for its later recolonisation and the spread of genetic lines during the interglacial period. However, the reconstruction of the glacial and postglacial history of S.lapponum on the basis of palaeoecological data is quite difficult, because the record of macrofossils is not known  population. At the same time, the effects of between populations in Scandinavia and other regions are visible. In Scandinavian populations, however, there are alleles common to populations from both the Sudetes and NE Poland, demonstrating close genetic relationships and similarities between them  is definitely most closely related to the populations of the Sudetes (K = 3–4) and NE Poland (K = 3–5). It is possible that the migrating individuals of S.lapponum remained in relatively small populations and, therefore, the combined effects of genetic drift and intra-population mutational processes increased differentiation between them. This is indicated by significant genetic diversity, as illustrated by the very high calculated coefficients of differentiation between the studied populations of S.lapponum presented in Tables The populations of hem Fig. , 3. The S.lapponum from Scandinavia to the south, which occurred during the favourable climatic periods of the Pleistocene through Poland, which was almost certainly favoured by the climatic conditions at the time. In the Sudetes, S.lapponum reaches its southern distribution limit, and the obtained results indicate closer relations between populations from the Sudetes and those from NE Poland. Certainly, in the past, isolated refugia existed in Central Europe. It is also possible that the separation of both population groups from the Sudetes and NE Poland took place relatively recently. Similar results have also been obtained for S.arbutifolia from the most NW locations in Japan. S.lapponum, but also of other species.There are also similarities between the analysed populations from the Sudetes and NE Poland, which may be attributed to the relatively small populations compared; this is confirmed by the results of the pairwise genetic differentiation analysis Table . The calIn the Northern Hemisphere, climatic oscillations during the Quaternary period caused significant changes in plant distribution, which resulted in the repeated expansion and fragmentation of species’ ranges and affected their patterns of genetic diversity. Cold-adapted plants  are believed to be more threatened during the Quaternary period than other plant groups .S.lapponum probably survived the glaciation in several isolated locations in Central Europe. Therefore, for example, populations from the Sudetes can be included in the populations found in the interglacial refugia for cold-adapted species (S.lapponum probably colonised the areas of Belarus and NE Poland, far away from the population in the Sudetes and relatively close to the Scandinavian sites.The currently observed decline in population sizes and geographical ranges, limited generative reproduction, and short-range spreading make boreal species more susceptible to loss of genetic diversity than, for example, plants in the temperate or Mediterranean zones. Moreover, climate scenarios  predict  species . During S.lapponum could survive the LGM in NE Poland and Belarus on the edge of the Scandinavian glacier in the area with various phylogenetic lines. It is also possible that this part of Europe was re-occupied by the migration waves of S.lapponum originating in Scandinavia and the Sudetes.The clearly low and similar level of variation, especially within Belarusian populations, may result from two different processes: the founding effect and genetic drift caused by small population sizes. However, in both the Scandinavian and Sudeten populations, probably there was increased genetic variation, represented in migrants who re-settled the newly available space. In many plant species, a correlation is found between population size and genetic variation , which iPopulustremula L. in Central Europe (Salixherbacea (S.lapponum.These two waves of migration probably met, creating a local suture zone with a mixed haplotype character. A similar scenario has also been proposed for l Europe  and for herbacea . HoweverSalixlapponum populations into several genetic groups. The populations from Belarus were the most genetically different. Populations from NE Poland and the Sudetes show similarities with populations from other geographic regions, indicating existing genetic relationships between them. It is possible that there was a meeting and exchange of genes between populations in southern Europe and Scandinavian populations and from NE Poland. However, to reach a more thorough understanding, additional studies are needed that include not only intra- and inter-population variability studies based on investigating phylogenetic lineage.The conducted research shows the division of the studied


Knautia arvensis and Plantago lanceolata.Restoration of species‐rich grasslands is a key issue of conservation. The transfer of seed‐containing local plant material is a proven technique to restore species‐rich grassland, since it potentially allows to establish genetically variable and locally adapted populations. In our study, we tested how the transfer of local plant material affected the species diversity and composition of restored grasslands and the genetic variation of the typical grassland plant species For our study, we selected fifteen study sites in southeastern Germany. We analyzed species diversity and composition and used molecular markers to investigate genetic variation within and among populations of the study species from grasslands that served as source sites for restoration and grasslands, which were restored by transfer of green hay and threshed local plant material.Knautia arvensis and Plantago lanceolata were comparable at source and restoration sites and genetic variation among populations at source and their corresponding restoration sites were only marginal different.The results revealed no significant differences in species diversity and composition between grasslands at source and restoration sites. Levels of genetic variation within populations of the study species Our study suggests that the transfer of local plant material is a restoration approach highly suited to preserve the composition of species‐rich grasslands and the natural genetic pattern of typical grassland plant species. In our study, we analyzed the impact of restoration on the genetic variation of typical grassland plant species. Our results indicate that the transfer of local seed‐containing plant material is a technique highly suitable to create genetically variable and locally adapted plant populations. Therefore, all species occurring in the grasslands were identified and registered while walking across the study sites in the vegetation period 2016  and Plantago lanceolata (L.). K. arvensis is a hemicryptophytic, perennial plant species, belonging to the Caprifoliaceae family  , MseI‐CTT/EcoRI‐AGG (D3), and MseI‐CTT/EcoRI‐ACT (D4). Primer combinations for P. lanceolata were MseI‐CTG/EcoRI‐AGC (D2), MseI‐CAA/EcoRI‐AGG (D3), and MseI‐CAG/EcoRI‐ACA (D4). EcoRI primers were labeled with fluorescent dyes for fragment detection . DNA fragments were separated by size with capillary gel electrophoresis performed on an automated sequencer . The results were exported as .crv files. AFLP fragment patterns were evaluated using the software Bionumerics 4.6 . Each strong and clearly defined DNA fragment was classified as present (1) or absent (0) to create a binary (0/1) matrix, which was the basis for further statistical analysis. We repeated about 10% of the samples and calculated a genotyping error rate  protocol by Rogers and Bendich  with ada2.5Based upon the species occurrence list, species diversity was calculated for each site as number of occurring species. We used a one‐way ANOVA to test whether species diversity differed significantly between source and restoration sites and Spearman's rank correlation coefficients to check whether species diversity depended on the year of restoration. All tests were done in IBM Statistics 24 for Windows, IBM Corporation.Furthermore, we estimated the degree of floristic (dis)similarity in vegetation composition between the source and restoration sites. We performed a nonmetric multidimensional scaling (NMDS) with presence–absence data based on Sorensen similarity index using PC‐ORD version 7 software  with the program AFLPsurv  and red list species . On average, we identified 63 plant species per grassland. At source sites, the number of plant species varied between 55 and 86 with a mean of 71 species (Table p = .178) and did not depend on the year of restoration . NMDS revealed differences in species composition between grasslands at source and restoration sites . Grasslands were more similar to each other at source sites than at restoration sites. However, the plots originating from the same source site were not grouped together.In total, we observed 165 plant species in the grasslands at all study sites Table . They cos Figure , but MRP3.2K. arvensis, AFLP analysis resulted in 127 fragments. 78.57% of the fragments were polymorphic. In populations at source sites, Nei's gene diversity (HKa) varied between 0.27 and 0.30 with a mean of 0.29. In populations at restoration sites, HKa ranged from 0.22 to 0.30 with a mean of 0.28 (Table p = .835). Population size (PS) of K. arvensis (PSKa) differed significantly between source and restoration sites , but we observed no significant correlation between HKa and population size (PS) or year (Y) of restoration .For P. lanceolata, 122 fragments could be detected and 90.35% of the fragments were polymorphic. In populations at source sites, Nei's gene diversity (HPl) ranged from 0.31 to 0.32 with a mean of 0.32 . Population size (PS) of P. lanceolata (PSPl) differed not significantly between source and restoration sites , and we observed also no significant correlation between Nei's gene diversity and population size (PS) or year (Y) of restoration .For 3.3K. arvensis, the principal coordinate analysis  values at K = 1–3, it is likely that K = 3 is over clustering these data, and only one genetic cluster is present. In the AMOVAs (Table PT = 0.04). Source and restoration sites differed only weakly from each other (ΦPT = 0.04). Molecular variance among populations at source sites was, however, slightly higher (ΦPT = 0.06) than among populations at restoration sites (ΦPT = 0.03). Comparing genetic variance between source and restoration sites for each transfer separately revealed ΦPT values between 0.004 and 0.07  also has a high value at K = 1, it is likely that all individuals also cluster into one group.Principal coordinate analysis Figure  also revs Figure . HoweverPT = 0.02) as well as between source and restoration sites, among populations from source sites or among populations from restoration sites . Comparing genetic variance between source and restoration sites for each transfer separately resulted again in very low levels of molecular variance varying from 0.002 to 0.05  or equivalent (P. lanceolata) to genetic differentiation among populations from source sites. These results support the assumption that—at least for the investigated populations in our study—grassland restoration by transfer of green hay and threshed plant material caused neither a decrease in genetic variation within nor an increasing divergence among populations at source and restored sites. This again supports our assessment that the transfer of local plant material seems to be an approach, which allows to restore genetically comparable grassland populations. Our study underpins, therefore, the perception that the transfer of local plant material is indeed the restoration approach most suitable to preserve the natural genetic pattern of plant species.Restoration by transfer of local plant material may not only reduce genetic variation within populations but also increase genetic differentiation among populations. Seed harvesting may comprise for seasonal or practical reasons not all individuals at the source site, which means that not the full gene pool available is transferred but only a subset. Moreover, seedling establishment at the restoration site may represent a filter selecting specific genotypes. Both could result in increased levels of genetic differentiation among populations from source and restoration sites. In our study, genetic differentiation among populations of both The authors declare that they have no conflict of interest.Franziska Kaulfuß: Investigation (lead); writing‐original draft . Christoph Reisch: Conceptualization (lead); writing‐original draft .